They are highly promising producers of molecular hydrogen. PNSB can be cultivated in organic waste waters, such as wastes after fermentation. In most cases, wastes mainly contain acetic acid. Therefore, understanding the anaplerotic pathways in PNSB is crucial for their potential application as producers of biofuels. The present review addresses the recent data on presence and diversity of anaplerotic pathways in PNSB and describes different classifications of these pathways.Purple non-sulfur bacteria (PNSB) are anoxygenic photosynthetic bacteria harnessing simple organic acids as electron donors. PNSB produce  In most chemotrophic, aerobic, and facultative anaerobic bacteria, the central carbon metabolism uses the following pathways : tricarb2. The TCA cycle then oxidizes acetyl-CoA, some organic acids, and acetyl-CoA-metabolized substrates into CO2. The reducing equivalents derived from the degradation of carbohydrates and TCA cycle substrates initiate the ATP synthesis via aerobic and anaerobic respiration. This is the catabolic function of carbon metabolism pathways.These pathways take part in catabolism and anabolism. Sugars are oxidized via a glycolytic pathway (and similar reactions) with pyruvate (PA) production. The reversible pyruvate-oxidoreductase oxidizes PA into acetyl-CoA and COThe intermediates of these crucial metabolic pathways include 12 major precursor metabolites that are used to synthesize 70–100 cell building blocks . The mosRps. palustris using acetate transforms 22% of acetate into CO2, 67% of which is then fixed into the cellular material via ribulose bisphosphate carboxylase/oxygenase (Rubisco) [In purple bacteria, TCA cycle is active during the phototrophic anaerobic growth and dark aerobic respiration. In case of anaerobic phototrophic growth, when the bulk of ATP is a product of the light-dependent electron transport, the TCA cycle performs cellular biosynthesis and participates in the constructive metabolism  . TCA cycle activity is varied depending on the carbon substrate type of the medium  and on ta and b and molecular hydrogen. During the anoxygenic photosynthesis, PNSB use organic acids as electron donors. Taking into account the fact that many organic waste waters contain acetate as the key organic acid, scrutinizing anaplerotic pathways in PNSB is essential.The PNSB produce polyhydroxyalcanoates, a-aminolevuline acid, bacteriochlorophylls Here, we overview the latest data on the presence and diversity of anaplerotic pathways in PNSB, and describe different classifications of these pathways.2 fixation [2-fixing reactions is believed to be maintaining the redox balance of the cell [For cells using acetate as a sole organic substrate, the TCA cycle should be sustainable. An anaplerotic pathway replenishing the oxaloacetic pool should function due to the outflow of the TCA cycle intermediates. Evidently, the existence of a single anaplerotic pathway is necessary and sufficient. However, there is evidence on several anaplerotic pathways functioning simultaneously in one bacterium ,7. Currefixation . Under tthe cell ,15. 2-fixation pathways in the synthesis of TCA cycle intermediates is discussed below.Currently, seven pathways of autotrophic fixation of carbon dioxide are known: the 3-hydroxypropionate bi-cycle ,17,18; tThe glyoxylate cycle is the most common pathway for replenishing the OAA pool in the TCA [ the TCA . In a fuOne full turn of the glyoxylate cycle produces a TCA cycle intermediate (succinate) and a precursor molecule (glyoxylate) following the malate synthesis from glyoxylate and acetyl-CoA . As a result, many strains were renamed. In this review, we use the names after the authors of the publications with a new name in parentheses at the first mention. Some of them do not have malate synthase activity  do not m fulvum ,7,32,33.Rba. capsulatus [Rsp. palustris [In psulatus ,35,36,37alustris , ICL is alustris ,38 reducRba. capsulatus [Rba. capsulatus B10 grown with acetate and showing the active glyoxylate shunt enzymes [Rba. capsulatus St. Luis [Rba. capsulatus B10 culture were shifted to medium with acetate [Non-deactivation of ICL in lactate presence could arise from acetyl-CoA produced from the pyruvic acid by the pyruvate dehydrogenase and pyruvate:oxidoreductase. Acetyl-CoA is then involved in TCA cycle and glyoxylate cycle reactions. Pyruvate:oxidoreductase transcripts were not found in some of the samples of phototrophically acetate-grown psulatus . However enzymes . NotablySt. Luis . No lag  acetate .+ bacteria Rba. capsulatus [Paracoccus denitrificans [Rba. capsulatus, this happens during transience from a single lactate to a single acetate assimilation if the inoculum was more than 5 h in the stationary growth phase after lactate exhaustion [+ bacterium Rps. palustris has been reported to exhibit TCA cycle and glyoxylate cycle enzyme activity when growing in the acetate medium [Rps. palustris after growing under chemoheterotrophic, photoheterotrophic and photolithotrophic conditions [− and ICL+ PNSB without ICL activity possess other anaplerotic pathways.ICLpsulatus ,37,41 sirificans  were ablhaustion ,42 when e medium . TCA cycnditions . The glyThe ethylmalonyl-CoA pathway was discovered in some methylotrophs [2 molecules into two glyoxylate molecules, CoA  and H+ . Genomialustris . The enzalustris . This mialustris .Rs. rubrum strain S1H uses the ethylmalonyl-CoA pathway as the main pathway for assimilating acetate during photoheterotrophic growth with acetate as the only organic source. Notably, the acetate-grown cultures contained detectable enzymes of the CBB cycle, pyruvate synthase/pyruvate:oxidoreductase, and enzymes of citramalate synthase-involving pathway [Rs. rubrum strain S1H with a knocked-out key gene of the ethylmalonyl-CoA pathway (crotonyl-CoA reductase/carboxylase) grew aerobically or anaerobically using the succinate medium [The purple non-sulfur bacterium  pathway ,51. The Paracoccus, allowing the replenishment of the oxaloacetate pool [P. denitrificans when the cells grew with acetate as the only organic substrate [P. denitrificans apparently improves the biomass yield, whilst the glyoxylate cycle enables a faster growth on a medium with acetate being the only organic substrate. Utilizing the succinate, both strains with deletions grew similarly to the wild type. However, the strain without ethylmalonyl-CoA pathway showed a longer lag phase.The genomic analysis shows that the ethylmalonyl-CoA pathway is the main anaplerotic pathway for the genus ate pool . Of noteubstrate . The quaccr and icl genes  in P. denitrificans grown with succinate or acetate. In the bacterial cells grown with succinate, only the ccr preserved their quantity at the low basal level.The authors  comparedRba. capsulatus, which also has both the glyoxylate cycle and ethylmalonyl-CoA pathway genes, the ICL activity had patterns similar to the ccr in P. denitrificans when growing on acetate at an uncontrolled pH [icl in P. denitrificans [In olled pH . Howeverrificans .+ PNSB as in other bacteria with the glyoxylate cycle.The recent data show that this particular cycle can be combined in the ICLThe methylaspartate cycle ,52,53. The reaction sequence of this metabolic pathway was first demonstrated for the haloarchaeon Haloarcula marismortui  production. Mesaconate is then activated to mesaconyl-CoA (2-methylfumaryl-CoA), which is hydrated to β-methylmalyl-CoA, and β-methylmalyl-CoA is finally cleaved to propionyl-CoA and glyoxylate. Propionyl-CoA carboxylation leads to the methylmalonyl-CoA and subsequently to succinyl-CoA production, thus completing the cycle, whereas the condensation of glyoxylate with another molecule of acetyl-CoA yields malate, the final product of the methylaspartate cycle . The enzH. marismortui was determined as 26 ± 5 mM, and the equilibrium of the preceding glutamate mutase reaction is not on the side of methylaspartate (Km = 0.093 at 30 °C) [H. marismortui cells was 35 ± 5 mM  [A functioning methylaspartate cycle is expected to require a high intracellular glutamate concentration, because the affinity for substrate (Km) of methylaspartate ammonia-lyase for methylaspartate in t 30 °C) . Indeed,olcanii) .It was shown that methyaspartate and glyoxylate cycles are evenly distributed in haloarchaea . InteresRba. capsulatus SB1003 and 9 strains of Rsp. palustris did not reveal any genes necessary for this pathway in bacteria [There has been no evidence in favor of the methylaspartate cycle functioning in PNSB. Analysis of genomes of bacteria ,39.The citramalate cycle  or a novel alternative anaplerotic acetate assimilation pathway through PA and part of the ILV biosynthesis pathway [Rs. rubrum even in mutant strains with an inactivated CBB cycle [Rs. rubrum CBB cycle mutant growth when a terminal electron acceptor was absent [Rps. palustris with an inactivated CBB cycle, mutant Rs. rubrum was not capable of growing with succinate.Leroy  reported pathway ,51,57. TBB cycle . In thiss absent . The fluRs. rubrum.Later, additional reactions of conversion of acetyl-CoA to propionyl-CoA, involving pyruvate synthase and part of the pathway of biosynthesis/degradation of IVL, were revealed . The autRps. palustris [Rba. capsulatus have not been clearly defined, since the data on the presence of enzymes catalyzing 2-methylfumarate formation from (S)-citramalate and (S)-citramalyl-CoA conversion into (S)-citramalate are not available [The citramalate pathway has no potential for functioning in the studied strains alustris . The posvailable . AdditioThe reversiblepyruvate:ferredoxin oxidoreductase (orpyruvate synthase). In strict anaerobes, acetyl-CoA is generally transformed into PA by pyruvate:ferredoxin oxidoreductase, the same enzyme that functions in the reversed TCA cycle (rTCA cycle), the DC/HB cycle, and the reductive acetyl-CoA pathway [2 and acetyl-CoA produced through acetate uptake and the rTCA cycle during mixotrophic growth of green sulfur bacteria [Rs. rubrum S1H for direct absorption of acetyl-CoA through PA [ pathway . With no pathway . Pyruvatbacteria ,61,62. Tbacteria  since through PA .Since this enzyme is sensitive to oxygen, the acetate assimilation via the above route is not possible under the aerobic conditions; thus, alternative pathways exist in (micro)aerobic organisms. Furdui and Ragsdale  measuredRps. palustris via the reaction of pyruvate:ferredoxin oxidoreductase. Meanwhile, this pathway is transcriptionally active in acetate cultures of Rps. palustris CGA009 and some acetate cultures of Rba. capsulatus [Rps. palustris strains BisB18 and BisA53, the genes coding proteins for these reactions were not expressed in the Rba. capsulatus cultures grown with acetate.It has been shown that PA could be synthesized from acetate in nine examined strains of psulatus . WhereasThe CBB cycle. In this cycle, carbon dioxide is fixed to form C6-compounds. It can serve as a substrate for carboxylases; 3-PGA formed in this metabolic pathway under the glycolytic pathway enzymes can be further converted into PEP and PA. They can be subsequently carboxylated to malate or oxaloacetate the replenishing oxaloacetate pool of TCA cycle. The Rubisco, phosphoribulokinase and sedoheptulose bisphosphatase [sphatase  are uniq2 in the CBB cycle is Rubisco. Currently, different forms of Rubisco have been found in microorganisms. Four main forms of Rubisco are distinguished [The key enzyme that fixes COnguished . RubiscoRps. palustris strains display high protein sequence identity with the Rubisco-like proteins rlp1 and rlp2 [Rba. sphaeroides has two forms of Rubisco. Form I consists of eight large and eight small subunits that encode rbcL and rbcS genes, respectively. Form II is a hexamer that consists only of large subunits encoded by either the rbcL gene or the rbcR gene [Rba. capsulatus [Rps. palustris [cbbLS (form I Rubisco) and cbbM (form II Rubisco). Unlike these bacteria, Rsp. rubrum has only form II Rubisco, which consists of two subunits encoded by the cbbM gene [It should be noted that part of genes annotated as Rubisco subunits in the recently sequenced and rlp2 . Rba. spbcR gene . Rba. capsulatus  and Rps.alustris  also havbbM gene ,69. SurpbbM gene ,71.Rba. sphaeroides, Rba. capsulatus, and Rps. palustris genes of form I and form II Rubisco are located in cbbI and cbbII operons, respectively [cbb operons of these bacteria has been thoroughly studied [cbb operons has been shown to be activated by the Lys-R-type transcription regulator  is the CbbR—positive transcription regulator [ectively . The reg studied ,74,75,76or  instead of carbon dioxide (CO2) [2 is released. Thus, during the functioning of Rubisco as oxygenase, oxygen uptake and carbon dioxide release occur. The combination of the light-dependent absorption of oxygen and the release of CO2 is commonly called photorespiration [Rubisco can catalyze a reaction of ribulose-1,5-bisphosphate and molecular oxygen (Ode (CO2) . This exde (CO2) . In cyande (CO2) . In the piration . Howeverpiration .2 in the medium: the amount of enzyme in cells increases with an increase in CO2 [2 to the synthesis of other significant intermediates from the acetyl-CoA . In some cases, PNSB fix significant amounts of CO2 in the CBB cycle regardless of the organic compounds. For example, 22% of acetate carbon is converted to carbon dioxide and 67% of this carbon dioxide is fixed in the cell material through Rubisco during photoheterotrophic growth of the Rps. palustris using acetate medium [Rps. palustris was repressed by chemoheterotrophic growth but was not decreased during photoheterotrophic growth [Rba. sphaeroides [Rba. capsulatus [Rs. rubrum, Rs. fulvum, renamed as Magnetospirillum fulvum [Rps. palustris CBB cycle phosphoribulokinase mutants that cannot produce ribulose-1,5-bisphosphate demonstrate an inability to grow photoheterotrophically on succinate unless an electron acceptor is provided or H2 production is permitted [Rs. rubrum, but not of Rps. palustris, grew photoheterotrophically on malate [2 production.The synthesis and activity of Rubisco depends on the concentration of COe in CO2 . The leve in CO2 ,97. The e medium . Rubiscoc growth . It has aeroides  and Rba.psulatus . The CBBm fulvum ,100,101 n malate  without 2 fixation, or with the maintenance of the redox balance in cells but not with TCA cycle activity.Wang D. and coauthors suggested an alternative model wherein disrupting Rubisco activity prevents photoheterotrophic growth due to the accumulation of toxic ribulose-1,5-bisphosphate . It was The reductive TCA cycle. Some rTCA cycle reactions can be considered as anaplerotic pathways replenishing TCA intermediates. They start from the reductive carboxylation of acetyl-CoA to PA catalyzed by ferredoxin-dependent pyruvate synthase. PA is converted to oxaloacetate [oacetate  or PEP aoacetate . In PNSBoacetate  and the oacetate .The 3-hydroxypropionate bi-cycle (Chloroflexus aurantiacus [S)-malyl-CoA is formed from acetyl-CoA -malyl-CoA is cleaved (reaction 11a) into acetyl-CoA (it is returned to the cycle) and glyoxylate. The latter is further converted in the second cycle reactions of the 3-hydroxypropionate pathway to form (3S)-citramalyl-CoA -citramalyl-CoA is finally cleaved to PA and acetyl-CoA. As a result of the complete turnover of 3-hydroxypropionate bi-cycle PA molecule is formed from three bicarbonate molecules. Reactions 1–6 of 3-hydroxypropionate bi-cycle . Only a fraction of acetate during photoheterotrophic growth on medium with acetate and bicarbonate was oxidized to CO2, probably in the course of the assimilation process.The 3-hydroxypropionate bi-cycle is a way to coassimilate organic substrates such as glycolate, acetate, propionate, 3-hydroxypropionate, lactate, butyrate, or succinate . The ratChloroflexi phylum evolved late in the Earth’s history as a result of a series of horizontal gene transfer events. This explains the lack of geological evidence for this pathway based on the carbon isotope record [Rba. capsulatus [Rps. palustris [Rps. palustris is necessary.The 3-hydroxypropionate bi-cycle of the e record . Data onpsulatus . Genes oalustris . HoweverThe 3-hydroxypropionate/4-hydroxybutyrate cycle was found in Metallosphaera sedula [a sedula . This meOne complete turn of this cycle under the autotrophic growth leads to the formation of two acetyl-CoA molecules. One molecule is recycled in the biosynthesis of the cell material. In the presence of exogenous acetate, the functioning of enzymes of the 3-hydroxypropionate bi-cycle  includes pathways of glyoxylate formation. It consists of part of glyoxylate cycle reactions, photorespiration and glyoxylate formation pathway from glycine formed at one stage of the reductive glycine pathway.Group (II) includes pathways of propionyl-CoA and glyoxylate simultaneous formation: namely, part of methylaspartate cycle reactions, part of citramalate cycle reactions and the set of reactions of ethylmalonyl-CoA pathway.Group (III) includes pathways of propionyl-CoA synthesis (some of the reactions of 3-hydroxypropionate and of 3-hydroxypropionate/4-hydroxybutyrate cycles).2), subgroup B (including pathways of PA/PEP formation from the stored carbohydrates (glycogen)), and subgroup C (consisting of PEP/PA formation using CO2).Group (IV) includes pathways of PA/PEP formation. This group consists of subgroup A  from glycine synthesized in the reductive glycine pathway and PEP/PA formation from the CBB cycle intermediates.I.S)-citramalyl-CoA-lyase (EC: 4.1.3.24/4.1.3.25) and (3S)-malyl-CoA-thioesterase (EC: 3.1.2.30)); the glycerate pathway for PEP formation from glyoxylate; and the pathways of glyoxylate conversion into OAA through the β-hydroxyaspartate cycle.Pathways of glyoxylate conversion: the conversion into malate ; or malyl-CoA/-malate formation from PA; and cis-aconitate formation from PA and acetyl-CoA.Pathways of PA/PEP conversion into TCA cycle intermediates: PA/PEP carboxylating enzymes; the pathway of fumarate or -2-hydroxy-acid oxidase  in combination with catalase  or catalase-peroxidase ; NADH-dependent glyoxylate reductase ; and NADPH-dependent glyoxylate reductase (EC: 1.1.1.79) due to a reversible formation of glyoxylate from glycolate  [Glyoxylate can be used for OAA pool replenishment after being converted to malate (hus H16) .d-fructose-6-phosphate ; glycine transaminase (EC: 2.6.1.4); or alanine-glyoxylate aminotransferase (EC: 2.6.1.44)). This was not shown in purple bacteria. However, according to the complete genome sequencing data [Rba. capsulatus ).yoxylate  and furting data , there ierythro-3-methylmalyl-CoA (reaction 67) is catalyzed by 2-methylfumaryl-CoA hydratase (EC: 4.2.1.148). Then, L-erythro-3-methylmalyl-CoA is split into glyoxylate and propionyl-CoA by L-malyl-CoA lyase (EC: 4.1.3.24) or L-malyl-CoA/(S)-citramalyl-CoA lyase (EC: 4.1.3.24/4.1.3.25) -3-hydroxybutanoyl-CoA from acetoacetyl-CoA (S)-3-hydroxyacyl-CoA dehydrogenase (EC: 1.1.1.35), followed by crotonyl-CoA formation (S)-3-hydroxyacyl-CoA hydratase (EC: 4.2.1.17). Moreover, 3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase/3-hydroxybutyryl-CoA epimerase , an enzyme that commonly catalyzes a reversible conversion of (S)-3-hydroxybutanoyl-CoA to (R)-3-hydroxybutanoyl-CoA (R)-3-hydroxybutanoyl-CoA is the substrate for the polyhydroxyalkanoate synthase . However, crotonyl-CoA formation could be performed by other enzymes possessing substrate specificity to S)-ethylmalonyl-CoA from crotonyl-CoA  and NADPH-dependent (EC 1.4.1.3 and EC 1.4.1.4) glutamate dehydrogenases exist). Reaction 58 is catalyzed by glutamate mutase -citramalate synthase (EC: 2.3.1.182), catalyzing acetyl-CoA and PA condensation to (R)-citramalate with a release of coenzyme A.The first reaction of this pathway is citramalate production from PA and acetyl-CoA -citramalyl-CoA formation by reversible (R)-citramalyl-CoA lyase (EC: 4.1.3.46) followed by cleavage by succinyl-CoA:(R)-malate/(R)-citramalate CoA transferase (EC: 2.8.3.20), giving (R)-citramalate. The second indirect pathway of citramalate synthesis consists of two reactions -citramalyl-CoA lyase (EC: 4.1.3.25) synthesizes (S)-citramalyl-CoA, which is cleaved further to (S)-citramalate by (S)-citramalate-CoA transferase (EC: 2.8.3.11) or succinyl-CoA:(S)-malate/(S)-citramalate CoA-transferase (EC: 2.8.3.22). The authors of the citramalate cycle [R)-citramalate is known to be a substrate of the alternative threonine-independent isoleucine synthesis pathway [S)-citramalate is a substrate of the mesaconate pathway [S)-citramalyl-CoA to reactions of the 3-hydroxypropionate cycle has also been shown [S)-enanthiomer as a substrate [S)-citramalate formation.Other reaction sequences resulting in citramalate formation from acetyl-CoA and PA are possible as well. The first indirect pathway of citramalate production begins  from (R)te cycle ,10 did n pathway ,114 or i pathway . Whereas pathway , the conen shown . In addiubstrate ,117. TheS)-citramalate. Alternatively reversible class I fumarases (EC: 4.2.1.2) could perform this reaction, since their properties described in the literature allow it [Subsequent synthesis of 2-methylfumarate from citramalate  carboxylates acetyl-CoA to malonyl-CoA (reaction 69). Malonyl-CoA reductase  produces malonate semialdehyde from malonyl-CoA. Malonate semialdehyde synthesis could be realized via another pathway .Formation of 3-hydroxypropionate from malonate-semialdehyde (reaction 71) requires 3-hydroxypropionate reductase (EC: 1.1.1.298). Reaction 71 could also be realized by 3-hydroxypropionate dehydrogenase (EC: 1.1.1.59) or malonic acid semialdehyde reductase  could be catalyzed by 3-hydroxypropionyl-CoA synthase (EC: 6.2.1.36), 3-hydroxybutyryl-CoA hydrolase (EC: 3.1.2.4.) or enoyl-CoA hydratase . Reaction 73, conversion of 3-hydroxypropionyl-CoA to acryloyl-CoA, could be catalyzed by enoyl-CoA hydratase (EC: 4.2.1.17) instead of 3-hydroxypropionyl-CoA dehydratase (EC: 4.2.1.116) due to its broad substrate specificity. Acryloyl-CoA could be converted to propionyl-CoA (reaction 73) by a multifunctional enzyme, medium-chain acyl-CoA dehydrogenase (EC: 1.3.99.3). According to a new classification, it should be named acyl-CoA dehydrogenase (EC: 1.3.8.7). In β-alanine metabolism pathways, this enzyme plays the role of acrylyl-CoA dehydrogenase instead of the lacking analogs ; EC: 2.8.3.12, glutaconate CoA-transferase; EC: 1.3.1.95, acrylyl-CoA reductase (NADH)).Group IV includes PA/PEP formation pathways and subdivided by three subgroups by substrates for PA/PEP synthesize.Subgroup A consists of two pathways of PA formation from exogenous acetate and CO2  or formate:NADP+ oxidoreductase (formate dehydrogenase (NADP+), EC: 1.17.1.10). Condensation of formate and acetyl-CoA is catalyzed by reversible formate C-acetyltransferase .2 production by E. coli [− 2 + H+ CO2 + H2. The ‘forward’ reaction (CO2 and H2 production from formate) under physiological fermentative conditions is observed [2 fixation [The formate hydrogenlyase  enzyme complex is the key element of fermentative H E. coli , which cobserved ,124. Theobserved ,126. An fixation .E. coli mutant strains unable to assimilate acetate through a glyoxylate shunt. In these experiments, the entire cellular pool of PA is derived from acetate and formate supporting the activity of PFL in H2 uptake reaction.Pyruvate formate-lyase (PFL) is an oxygen-sensitive enzyme widespread in nature. This enzyme typically produces an additional ATP molecule (3 ATP molecules instead of two) during glucose fermentation . Despite+-dependent formate dehydrogenase from Rba. capsulatus catalyzes the reduction of CO2 to formate [Rba. capsulatus has attracted a lot of attention [The oxygen-tolerant and NAD formate . The biottention ,131.Subgroup B includes pathways of PA/PEP formation from the stored carbohydrates (glycogen). The use of storage compounds is possible in periods of shift from the growth using TCA cycle intermediates to acetate as the sole organic substrate . The duration of the transition period is determined by the amount of storage compounds and the formation of the enzymes that are to be synthesized in sufficient quantity to adapt to new growth conditions. For example, the lag phase characteristic of the growth of Rs. rubrum with acetate with low bicarbonate concentration is also characterized by the mobilization of internal glycogen by the cell acting as a source of PA/PEP. Therefore, it may substitute the ethylmalonyl-CoA pathway as an anaplerotic pathway supporting the viability of bacteria [the Entner–Doudoroff pathway , the Embden–Meyerhof–Parnas pathway , bacteria . Many ba pathway  along wiSubgroup C includes the PA/PEP formation reaction using CO2. This consists of PEP/PA formation from the CBB cycle intermediates and the PA/PEP formation pathway (through serine) from glycine synthesized in reductive glycine pathway.PEP/PA formation from the CBB cycleintermediates. This cycle includes reactions 21–32 shown in During the photoheterotrophic growth using acetate, lactate, malate, and succinate, PNSB produce carbon dioxide, since the carbon atom in the biomass is more reduced than in these compounds . HoweverIn some cases, especially when the acetate cultures grow at the maximum growth rate, the role of the cycle might be shifted to the anaplerotic pathway for the replenishment of TCA cycle intermediates. This suggestion needs further experimental verification.The PA/PEP formation pathway (through serine) from glycine synthesized in reductive glycine pathway -malate formation.S)-malate involving malate synthase (EC: 2.3.3.9) -citramalyl-CoA-lyase (EC: 4.1.3.24/4.1.3.25), which catalyzes the formation of L-malyl-CoA, and (3S)-malyl-CoA-thioesterase (EC: 3.2.1.30), thus catalyzing the formation of (S)-malate from L-malyl-CoA . Tartronate semialdehyde reductase catalyzes the formation of d-glycerate from tartronate semialdehyde (reaction 33). d-glycerate is converted to 2-phospho-d-glycerate under the action of the glycerate kinase (reaction 34). It is then cleaved by enolase to PEP and H2O (reaction 35). PEP is carboxylated to oxaloacetate phosphoenolpyruvate carboxylase -β-hydroxyaspartate (2O are formed (reaction 30). Iminosuccinate reductase catalyzes the formation of an aspartate molecule from iminosuccinate (reaction 31), which enters the first cycle reaction (reaction 28). One of its products is an oxaloacetate molecule synthesized from two glyoxylate molecules; in addition, glycine is formed as described above (one of the substrates of the second reaction of the cycle). The β-hydroxyaspartate cycle is present in the genomes of some Proteobacteria that also encode the CBB cycle [ century , and yet century . In thisBB cycle , but theThere are three pathways of propionyl-CoA conversion into TCA cycle intermediates replenishing OAA pool known up to date. The methylmalonyl-CoA pathway leads to the formation of succinyl-CoA from propionyl-CoA ). In othMethylmalonyl-CoA Pathway. Succinyl-CoA is formed in this pathway from propionyl-CoA using three reactions (S)-methylmalonyl-CoA formation from propionyl-CoA (reaction 4) can be catalyzed by two enzymes, propionyl-CoA carboxylase (EC: 6.4.1.3) and methylmalonyl-CoA carboxytransferase (EC: 2.1.3.1). Methylmalonyl-CoA/ethylmalonyl-CoA epimerase (EC: 5.1.99.1) isomerizes (S)-methylmalonyl-CoA into (R)-methylmalonyl-CoA (reaction 5). Methylmalonyl-CoA mutase (EC: 5.4.99.2) catalyzes the formation of succinyl-CoA from (R)-methylmalonyl-CoA (reaction 6).eactions . (S)-metMethylcitrate Cycle. The sequence of this pathway reactions is given in cis-aconitate formation is achieved via two reactions  and 2-methylaconitate isomerase (EC: 5.2.1.-).Rps. palustris strains and Rba. capsulatus [Salmonella enterica [The gene of aconitate hydratase/2-methylisocitrate hydratase (EC: 4.2.1.3/EC: 4.2.1.99) is absent in the genomes of all studied psulatus . Howeverenterica . Such poPathway of Propionyl-CoA Oxidation via Lactate to PA. The scheme of this pathway is shown in PA/PEP Carboxylating Enzymes (Anaplerotic Carboxylases). Synthesis of the TCA cycle intermediates from PA or PEP requires their carboxylation, which could lead to (S)-malate or oxaloacetate formation  reaction is decarboxylation. However, some malic enzymes demonstrated a pH-dependence of this favorable reaction direction  . In addixylation . Among ts SB1003 . HoweverRps. palustris strains and Rba. capsulatus SB1003 [Rl. eutropha [Among the enzymes catalyzing the reversible PEP carboxylation to OAA -malate can occur (EC: 1.1.1.83), but (R)-malate cannot be directly metabolized in the TCA cycle. (R)-malate is converted into maleate by maleate hydratase . Maleate can be further converted into fumarate by maleate isomerase . The latter is involved into TCA cycle reactions. pathway , malic eMethanocaldococcus jannaschii encoded by leuC gene [A multifunctional enzyme discovered in euC gene  is curreeuC gene .R)-malate to (S)-malate could be provided by enzyme having racemase activity , are considered to be reversible nowadays. As mentioned above, some carboxylyases (decarboxylases) direct the reaction depending on the environmental conditions  [tabolism  could hyte level . This pate level . The ita terreus , in the o maydis  via tranH, etc.) ,147. TheH, etc.) . Howevercis-aconitate formation pathway except for the condensation of PA and acetyl-CoA (S)-citramalyl-CoA lyase (EC: 4.1.3.25), and itaconyl-CoA conversion to itaconate is performed by succinyl-CoA synthetase -citramalyl-CoA into itaconyl-CoA  further carboxylates itaconate to cis-aconitate.Conversion of .St. Luis . AnotherSt. Luis . The CBBSt. Luis . AnotherSt. Luis . This syExcessive illumination is a stress for purple bacteria grown with acetate. An increase in the light intensity results in an excess of the proton motive force. This brings about an excessive NADH synthesis via a reverted electron flow by NADH dehydrogenase ,167. A dRs. rubrum S1H after a prolonged cultivation with acetate produced an “acetate-competent” strain [Anaplerotic pathways acceleration could eliminate strong light growth inhibition. For example, ” strain . It appeRs. rubrum wild type and “acetate-competent” strains showed that the “acetate-competent” strain could resist strong light much better than a wild strain [Changes in the synthesis of storage compounds are yet another mechanism to adapt to the stress of elevated light and acetate assimilation ,169. Purd strain . During Thus, the genetically determined anaplerotic pathways may take part in various critical processes underlying the adaptation of purple bacteria to environmental stresses.The data reviewed in this paper reveal that our understanding of anaplerotic pathways replenishing the TCA cycle intermediates in microorganisms and, particularly, in purple bacteria has expanded significantly in the last decade. This success results from advances in genomic sequencing and proteomic, transcriptomic, and metabolomic research. We have already known that the simultaneous functioning of more than one pathway is not an exception but rather a rule for numerous microorganisms . The intOur overview of possible anaplerotic pathways show that many reaction chains/cycles able to replenish TCA cycle intermediates have some additional functions. Furthermore, some chains were described previously as having another function . Thus, central carbon metabolism is a sophisticated system supporting the lives of microbials life, and our classification of different chains for anaplerotic function, inorganic carbon acquisition, synthesis of different blocks for overall metabolism or energetic reactions is just our simplification of their nature.


Shenzhi Jiannao (SZJN) prescription is a type of herbal formula adopted in the management of cognitive impairment and related disorders. However, its effects and related regulatory mechanisms on vascular dementia (VD) are elusive. Herein, network pharmacology prediction was employed to explore the pharmacological effects and molecular mechanisms of SZJN prescription on VD using network pharmacology prediction, and validated the results through in vitro experiments.Through a search in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database, chemical composition and targets for SZJN prescription were retrieved. The potential targets for VD were then obtained from the GeneCards and DisGeNET databases. The network was constructed that depicted the interactions between putative SZJN prescription and known therapeutic targets for VD using Cytoscape 3.7.1. Analysis of protein-protein interaction was achieved via STRING 11.0 software, followed by Gene Ontology (GO) functional enrichment and Kyoto Gene and Genome Encyclopedia (KEGG) pathway analyses. To validate the computer-predicted results, in vitro experiments based on an excitotoxic injury model were designed using glutamate-exposed PC12 cells, and treated with varying concentrations  of SZJN prescription. Cell viability and cell death were detected using the IncuCyte imaging system. Moreover, the expression profiles of Caspase-3 were analyzed through qRT-PCR.Twenty-eight potentially active ingredients for SZJN prescription, including stigmasterol, beta-sitosterol, and kaempferol, plus 21 therapeutic targets for VD, including PTGS2, PTGS1, and PGR were revealed. The protein-protein interaction network was employed for the analysis of 20 target proteins, including CASP3, JUN, and AChE. The enrichment analysis demonstrated candidate targets of SZJN prescription were more frequently involved in neuroactive ligand-receptor interaction, calcium, apoptosis, and cholinergic synaptic signaling pathways. In vitro experiments revealed that SZJN prescription could significantly reverse glutamate-induced cell viability loss and cell death, and lower the levels of Caspase-3 mRNA in glutamate-induced PC12 cells.Collectively, this study demonstrated that SZJN prescription exerted the effect of treating VD by regulating multi-targets and multi-channels with multi-components through the method of network pharmacology. Furthermore, in vitro results confirmed that SZJN prescription attenuated glutamate-induced neurotoxicity. Vascular dementia (VD), as the main type of dementia in Asia, is mainly the result of vascular risk factors such as atherosclerosis, hypertension and micro-strokes . VD accoPanax ginseng C.A.Mey. (Ginseng), Anemarrhena asphodeloides Bunge (Anemarrhenae), and Paeonia veitchii Lynch (Paeoniae rubra). In previous pharmacological study, it has been found that SZJN prescription improved cerebral ischemia in VD rats, reduced the levels of glutamate and γ - aminobutyric acid in the anterior cortex, and increased acetylcholine content [Shenzhi Jiannao (SZJN) prescription, the national new formula in China (ZL201010249665.8), is a classical herbal formula for the treatment of VD based on the clinical practice. This prescription is mainly composed of  content . The eff content . HoweverNetwork pharmacology approaches have been proven to be a powerful way for mechanistic exploration of Chinese medicine from the perspective of multicomponents, multitargets, and multichannels, which is consistent with the holistic view of Chinese medicine , 14. Thehttp://ibts.hkbu.edu.hk/lsp/tcmsp.php) manually. Subsequently, compound names and their molecular structures were verified in the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). Oral bioavailability (OB) ≥ 30% and drug-like (DL) ≥ 0.18 parameters were incorporated in the TCMSP database as drug screening conditions for the compounds.To determine and collect the chemical components of the three herbs contained in SZJN prescription, a search was performed from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform database , then target species were selected as “humans”. Targets related to VD were collected from two databases. One was the GeneCards (https://www.genecards.org/), which is a searchable, integrative database that provides comprehensive, user-friendly information on all annotated and predicted human genes [http://www.disgenet.org/), which is a comprehensive gene-disease association (GDA) database that provides the current knowledge of human genetic diseases [The main chemical components in SZJN prescription were determined and individually matched with potential targets according to TCMSP. First, names of the predicted target proteins were transformed into gene names via the UniProt database (an genes . The othdiseases . A generhttps://string-db.org) software.Using the Cytoscape 3.7.1 software, prediction results of potential targets for SZJN prescription were acquired, and a network of herbs-putative active components-putative therapeutic targets was constructed. The network topology analysis was achieved using a plugin in Cytoscape 3.7.1, after which a protein-protein interaction (PPI) network of the SZJN prescription was constructed via the STRING 10.5 (https://david.ncifcrf.gov/) to explore gene functions and uncover potential therapeutic targets of the active components of SZJN prescription. Analysis of the pathways provided scientific connotation for SZJN prescription.GO functional enrichment and KEGG pathway enrichment analyses were conducted using the David v 6.8 database  and RPMI-1640 basic culture medium were purchased from Procell Life Science and Technology Co. Ltd. ; Caspase-3 primers were synthesized by Sangon Biotechnology Co. Ltd. ;. L-glutamic acid (glutamate) was purchased from Sigma (USA); SZJN prescription, constituted from Ginseng, Anemarrhenae, and Paeoniae rubra, was acquired from the Chinese Medicine Preparation Department of China-Japan Friendship Hospital; Memantine hydrochloride (MH) was purchased from H. Lundbeck A/S (Denmark). SZJN prescription and glutamate were dissolved in RPMI 1640 complete medium, while MH was dissolved in dimethyl sulfoxide (DMSO) to obtain a final concentration of less than 0.5% for use in the experiments.2), and a temperature of 37 °C. Cells were treated with or without SZJN prescription , MH (10 μM), and glutamate (22.5 mM) for 24 h. Each experiment was performed in triplicate.PC12 cells were cultured in RPMI-1640 complete medium at 95% humidity, 5% carbon dioxide  were seeded into Corning 96-well tissue culture plates and then placed on the IncuCyte instrument. Results for cell viability and cell death were recorded for over 36 h. Dead cells were identified using fluorescent nuclear dye YOYO-1, a cell impermeable dye that enters cells via compromised membranes. To calculate the average percentage of cell death, the green fluorescent area (YOYO-1 positive) was divided by the total phase area.Apoptotic cells were quantitated using the Annexin V-FITC/propidium iodide (PI) apoptosis assay kit . Briefly, PC12 cells were digested with 0.25% trypsin, centrifuged, and resuspended in 200 μL 1 × binding buffer. Then, cells were stained with 5 μL Annexin V-FITC in the dark for 10 min. This was followed by another staining with 5 μL PI for 5 min. Samples were analyzed using a FACScan flow cytometer . At least 10,000 events were recorded. Apoptotic cells were expressed as a percentage of the total number of cells.−∆∆CT method.The effect of SZJN prescription on Caspase-3 was established through quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Briefly, total RNA from cells was extracted using the RNA Extraction Kit  according to the manufacturer’s instructions. The concentration and purity of the extracted RNA were determined using a Q5000 nucleic acid protein analyzer  . Then a reverse transcription kit  was used to reverse transcribed the RNA to cDNA. qRT-PCR was performed using the SYBR Green Master Mix on a CFX Connect Real-Time PCR System , with Caspase-3: Forward: 5′-GTACAGAGCTGGACTGCGGTATTG-3′; Reverse: 5′-AGTCGGCCTCCACTGGTATCTTC − 3′ and β-actin: Forward: 5′-GCAGTTGGTTGGAGCAA-3′; Reverse: 5′-ATGCCGTGGATACTTGGA-3′ primer pairs. qRT-PCR conditions were as follows: Initial denaturation step at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 1 min. β-actin served as the internal reference gene. The relative expression levels of target genes were calculated using the 2P < 0.05.All statistical analyses were performed via SPSS version 25.0. Data were presented as means ± standard deviations (SD). Statistical comparisons were achieved using one-way analysis of variance (ANOVA) for multiple groups with variance depicting homogeneous normal distribution data. Least—Significant Difference (LSD) test was applied for mean separations to reveal statistically significant differences, , Anemarrhenae, and Paeoniae rubra, respectively , including 70 items related to biological processes, such as drug reaction, transcription initiation of RNA polymerase II promoter, and steroid mediated signaling pathway; 15 items related to cell composition, such as nuclear and plasma membrane components, and membrane raft; 21 items related to molecular function,  were identified , and the unrelated pathways including those for cancer and hepatitis B were eliminated. Results demonstrated that the targets of SZJN prescription for treating VD treatment were mainly associated with neuroactive ligand-receptor interaction, calcium, cholinergic synapse, cardiac myocyte adrenergic signal transduction, adipocyte lipolysis regulatory, apoptosis, cAMP, salivary secretion, tumor necrosis factor, 5-hydroxy color Aminergic synaptic, neurotrophin and AMPK signaling pathways . However, SZJN prescription treatment  and MH significantly decreased the number of apoptotic cells to 15.87% ± 6.37%, 16.47% ± 4.13%, 9.47% ± 0.67% and 12.30% ± 1.67%, respectively (P < 0.05). The results demonstrated that SZJN prescription inhibited glutamate-induced apoptosis of PC12 cells.Annexin V-FITC/PI double staining was employed to examine apoptotic cells and verify the preventive effects of SZJN prescription on glutamate-induced neurotoxicity through inhibition of cell apoptosis Fig. a. In FigP < 0.01). Caspase-3 expression in the SZJN prescription group showed significantly varying profiles as follows: low , middle  and high  relative to the glutamate group. The expression levels in the SZJN-L group exhibited the highest decreasing trend, whereas the expression of Caspase-3 mRNA in the MH group decreased significantly   compared to the control group  Fig. c.According to the theory of traditional Chinese medicine (TCM), VD is described as “dementia” or “stroke combined with dementia”. The pathogenesis of VD in TCM is related to “a deficiency of essential Qi with toxin damaging brain collaterals”. Since VD lasts longer, blockage of the Qi and blood may induce cognitive impairment because brain neurons get limited nutrients . TherefoPrevious studies have revealed that several TCM components exert their therapeutic effects by acting on multiple targets –21. For A total of 28 active compounds after eliminating the duplicates were identified, particularly, stigmasterol, beta-sitosterol (β-sitosterol), and kaempferol exhibited obvious bioactivities. Pharmacological studies have confirmed that stigmasterol, β-sitosterol and kaempferol are active components of Ginseng , 30. MorWith regards to target points, PTGS2, PTGS1, PGR, ADRB2, ADRA1B, and CHRM1 recorded degrees≥10. However, PTGS2 had a maximum degree of 17, an implication that it is a potential SZJN prescription target vital in the management of VD. PTGS2, also known as cyclooxygenase-2 (COX-2), is a subtype of prostaglandin G/H synthetase (PGHS), and a vital enzyme in fatty acid metabolism . PTGS2 oThe topology of the PPI network demonstrated that CASP3, JUN, ACHE, PPARG, PTGS2, ADRB2, CHRM1, ADRA1B, AR, PGR (degree≥4.7) are potentially important targets for SZJN prescription in VD treatment. This is based on its multi-target, multi-path, and multi-system characteristics. However, all the targets in the network, except for PTGS2, were consistent with the PPI network. This controversy in results may be attributed to the scarcity of information regarding whether the herbs were processed or decocted, as well as the specificity of target action. It was found that CASP3 recorded the largest degree (8), therefore it acted as the main target for SZJN prescription. Caspase-3, a member of the cysteine protease family, is a downstream protein in the apoptosis pathway and an apoptotic enzyme that functions through the death receptor and mitochondrial pathways . The apoIn the present study, following GO functional enrichment analyses, it was revealed that target genes were associated with a drug reaction, transcription initiation of RNA polymerase II promoter, cell signal transduction, and cell proliferation, among others. These observations demonstrated that SZJN prescription may promote cell proliferation and reduce cell apoptosis. Results from related experiments indicate that SZJN prescription mediates the repair of damaged nerve cells via different mechanisms , 67.Moreover, the KEGG metabolic pathway analysis revealed that the targets were associated with neuroactive ligand-receptor interaction, calcium, and cholinergic synapse signaling pathways. Of note, the neuroactive ligand-receptor interaction signaling pathway incorporates all receptors and ligands related to intracellular and extracellular signaling pathways in the plasma membrane. These include adrenergic, muscarinic acetylcholine receptor (mAChR), and dopamine receptors, among others . Studies2+ signaling pathway and signal transmission of neuron synapses, it was found that an open voltage-gated Ca2+ channel could activate NMDA receptors and drive the release of neurotransmitters. Also, an imbalance in the Ca2+ signaling pathway potentially activated apoptosis signaling molecules, such as Caspase-3 and Bax. This process inhibits the expression of Bcl-2 and consequently activates the apoptosis pathway [Due to the close relationship exists between the Ca pathway . Another pathway . In the Furthermore, the central cholinergic system coordinates synaptic plasticity, learning, and memory ability. Acetylcholine (ACh) is a neurotransmitter of the cholinergic pathway, and functions as a nicotinic (N) or muscarinic cholinergic receptor (CHRM1–5). Notably, excess hydrolysis of ACh by AChE has been implicated in the loss of cholinergic neurons, prolonged synaptic signal transmission disorder, and cognitive dysfunction . InhibitHowever, there are still some limitations for this research. For example, the network pharmacology research relies more on various existing databases, but the screening criteria of compounds in databases may not be accurate enough. Moreover, the screened active ingredients might be inconsistent with the ingredients actually absorbed in blood of the patients with VD. As the accuracy and reliability of data has a great impact on the prediction results, it is necessary to further verify the reliability and quality of the predicted results, optimize the screening criteria of TCM databases, and improve the network pharmacology research. Therefore, further experimental and clinical verification of the efficacy of SZJN prescription on cerebrovascular microenvironment and neuroprotection is demanded to validate theoretical predictions.In this study, a systematic analysis for the characteristic of “multicomponents, multitargets, and multichannels” of SZJN prescription in treating VD was successfully performed through the method of network pharmacology and in vitro experiments. The experimental results were in good accordance with the results predicted from the network analysis of the SZJN prescription, especially the anti-apoptosis properties of the prescription. In the future, more experimental evidence should be provided for the neuroprotective and cerebral vascular protective effects of SZJN prescription against VD according to the results of network pharmacology research.


Context: Baiying Qinghou as a traditional Chinese medicine decoction shows anticancer property on laryngeal squamous cell carcinoma. However, little is known about the precise mechanism of Baiying Qinghou detection against laryngeal squamous cell carcinoma.Objective: This study was aimed to explore potential mechanism of therapeutic actions of Baiying Qinghou decoction on laryngeal squamous cell carcinoma.Materials and Methods: The active chemical components of Baiying Qinghou decoction were predicted, followed by integrated analysis of network pharmacology and molecular docking approach. The network pharmacology approach included target protein prediction, protein-protein interaction network construction and functional enrichment analysis.TP53, EGFR, PTGS2, NOS3 and IL1B as the key nodes in PPI network were cross-validated, among which EGFR, IL1B, NOS3 and TP53 were significantly correlated with the prognosis of patients with laryngeal squamous cell carcinoma. Finally, the binding modes of EGFR, IL1B, NOS3 and TP53 with quercetin were visualized.Results: Sitosterol and quercetin were predicted to be the overlapped active ingredients among three Chinese herbs of Baiying Qinghou decoction. The target proteins were closely associated with response to chemical, response to drug related biological process and cancer related pathways such as PI3K-Akt signaling, HIF-1 signaling and Estrogen signaling pathway. The target proteins of TP53, EGFR, NOS3 and IL1B involved with drug resistance and PI3K-AKT signaling pathway. TP53, EGFR, NOS3 and IL1B may be the candidate targets for the treatment of laryngeal squamous cell carcinoma.Discussion and Conclusion: Quercetin of Baiying Qinghou decoction showed therapeutic effect against laryngeal squamous cell carcinoma by regulating  Head and neck cancer (HNC) is the seventh most common malignant tumor worldwide and occurs in nasopharynx, larynx, and thyroid , 2. HeadCurrently, several mainstream options against LSCC include surgery, radiotherapy, chemotherapy and chemo-radiotherapy . HoweverNetwork pharmacology is proposed by Hopkins et al. and aims to explore the multilevel interactions of diseases, genes, and drugs as a whole . This syIn this study, we attempted to explore the mechanism of BYQH decoction in the treatment of LSCC by network pharmacology analysis. First, the key active chemical constituents and their target proteins of BYQH decoction were screened. Then, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of gene targets were carried out. Besides, protein-protein interaction (PPI) and target proteins were cross-validated followed by survival analysis. Finally, molecular docking was used to identify the binding mode of key active ingredients and target proteins of BYQH decoction.The traditional Chinese medicine systems pharmacology database (TCMSP) is an herbal repository and provides the system information about Chinese herbal medicines . The abshttps://www.drugbank.ca/) databases can predict the relationships between drugs and corresponding targets. Moreover, DrugBank database provides the detailed information on experimental and investigational drugs [TCMSP and DrugBank  functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were carried out. There are three categories for GO terms, including biological process (BP), cellular component (CC) and molecular function (MF). Gene symbols were input into string database. The parameters were set as the default values. The significantly enriched GO and pathway terms with The underlying relationships among target proteins were analyzed based on the string database according to the default parameters . The CytDisGeNET is a versatile platform that comprehensively collects human gene-disease associations  for the validation of computationally predicted genes for human diseases , 23. In https://xenabrowser.net/datapages/). Based on the clinical information, the HNSCC samples were selected according to following criteria: 1) samples were primary tumor tissues and the primary site was at the larynx; 2) the clinical information of samples with regard to TNM stage, clinical stage, grade and follow-up were complete; 3) the samples without survival time were deleted. Finally, a total of 105 laryngeal cancer samples were retained for the next prognostic analysis.Firstly, expression profiles of HNSCC samples deposited in the Cancer Genome Atlas (TCGA) dataset were downloaded from UCSC Xena (2 (count+1). After that, the optimal cutoff was determined using survminer package in R language based on the expressions of candidate genes. Subsequently, the samples were divided into high- and low- expression group according to the optimal cutoff. Following this, the survival analyses of gene targets were respectively preformed with survival package in R language. P < 0.05 was considered significant.To investigate whether key candidate genes are associated with prognosis of patients with laryngeal cancer, the survival analyses of these gene targets were carried out. In brief, the raw data of 105 laryngeal cancer samples were firstly extracted and then standardized by logThe Protein Data Bank (PDB) offers the experimental data for determined three-dimensional (3D) biological macromolecules structure . Herein,This study was approved by Ethics Committee of Provincial Hospital Affiliated to Shandong First Medical University and Shandong Provincial Hospital affiliated to Shandong University.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.TP53, EGFR, NOS3 and IL1B may be therapeutic targets of LSCC.The therapeutic mechanism of BYQH decoction on LSCC is analyzed by network pharmacology. Sitosterol and quercetin were key active ingredients BYQH decoction. The active chemical compounds of BYQH were retrieved from TCMSP database by using five keywords of “Solanum lyratum thumb” or “Scutellaria barbata” or “Duchesnea indica” or “Solanum nigrum” or “Actinidia chinensis Planch”. Totally, 174 active ingredients were obtained: 15 in Solanum lyratum thumb, 39 in Solanum nigrum, 94 in Scutellaria barbata and 26 in Actinidia chinensis Planch, respectively. Notably, the active components of Duchesnea indica were not included in TCMSP database. Subsequently, 41 key active compounds  were further screened according to the ADME parameters of OB ≥ 30%, DL ≥ 0.18 and Caco-2 ≥ −0.4 . InteresTP53, EGFR, NOS3 and IL1B et al.), response to drug and response to chemical . The membrane raft, plasma membrane region and plasma membrane raft were the three GO-CC terms enriched by gene targets. For GO-MF category, these genes were significantly involved in protein binding, enzyme biding and molecular transducer activity. In addition, the KEGG enrichment analysis revealed that the target genes were enriched in 172 pathways. Moreover, we found they were primarily correlated with several cancer-related pathways, such as PI3K-Akt signaling pathway , estrogen signaling pathway  and HIF-1 signaling pathway . These findings suggested that BYQH decoction may have essential effect on the treatment of LSCC.Totally, 137 gene targets of the active ingredients of BYQH decoction were predicted according to the methods mentioned above . AfterwaIL6 (interleukin-6 degree = 67), VEGFA , TP53 (degree = 61), JUN (degree = 58), EGF (degree = 57), MAPK1 (degree = 54), EGFR (degree = 53), PTGS2 (degree = 52), ESR1 (degree = 51), CAT (degree = 49), NOS3 (degree = 48), IL1B (degree = 47), HSP90AA1 (degree = 44), CCL2 (degree = 44), and AR (degree = 43).The potential interactions among target proteins of BYQH decoction was constructed and visualized by STRING database. As shown in PTGS2, NOS3, BCL2, ADH1C, TP53, MPO, EGFR, GSTP1, IL1B and GSTM1  served as the hub genes in PPI network. Therefore, these genes were considered to be key targets of BYQH decoction against LSCC.To further narrow the range of potential target genes of BYQH decoction, the candidate target genes were cross-validated. The genes related to LSCC were respectively acquired from Phenopedia and DisGeNET database, which provided information on gene-disease interactions. Then, the intersecting target genes were obtained between predicted genes and gene targets of BYQH decoction. Finally, a total of 10 intersection-associated genes were extracted, including nd GSTM1 . Among tADH1C, 7.03 for BCL2, 15.13 for EGFR, 13.45 for GSTM1, 16.06 for GSTP1, 10.53 for IL1B, 5.29 for MPO, 8.5 for NOS3, 9.36 for PTGS2, and 10.95 for TP53  were significantly associated with the prognosis of HNSCC patients  were investigated. Herein, we focused on analyzing the binding modes between two overlapped active compounds (sitosterol and quercetin) among three Chinese herbs and four target proteins. Our analysis indicated that the corresponding protein targets of sitosterol were PGR, NCOA2, NR3C2, which were not targets studied. For quercetin, 75 target proteins were obtained, including four key protein targets. Subsequently, the chemical structure formula of quercetin was acquired from TSMCP database, and four target proteins were also searched from PDB database, including TP53 (PDB ID: 3ZME), EGFR (PDB ID: 1XKK), NOS3 (PDB ID: 6PP1) and IL1B (PDB ID: 5R86). Finally, the molecular docking was carried out using LeDock tool. Notably, a closer binding between proteins and small biological molecules indicated more energy released and a lower ΔG value. The binding characteristics of key active ingredients of BYQH decoction and four key protein targets  were considered as hub nodes in PPI network. Among them, five proteins  were cross-validated. Additionally, survival analysis showed the expressions of ADH1C, EGFR, GSTM1, GSTP1, IL1B, NOS3 and TP53 were markedly correlated with the prognosis of HNSCC patients. Finally, the binding modes of quercetin and four target proteins  were constructed.TCM is an accepted medical practice and has been used for the treatment of complicated diseases such as cancers over past few decades in China , 28. BYQQuercetin , a common flavonol, is widely distributed in plant species, such as vegetables and grains. Our results showed that quercetin was a key active chemical compound of BYQH decoction. Extensive evidence suggests that quercetin exerts diverse biological functions, such as antioxidant, anti-inflammatory, anti-bacterial, antiviral and anticarcinogenic properties –31. NumeTP53, EGFR, NOS3 and IL1B were key hub genes in PPI network of target proteins of BYQH decoction and cross-validated in two databases (Phenopedia and DisGeNET). More notably, these four proteins were also target proteins of quercetin. p53 protein encoded by TP53 gene functions as a tumor-suppressive factor by regulating multiple cellular processes such as cell-cycle progression and apoptosis [TP53 can influence the progression of HNSCC and clinical treatment response [EGFR is frequently reported to be associated with HNSCC [EGFR by down-regulating the expression of MMP-2 and MMP-9 [EGFR copy number in HNSCC, which acts as a poor predictor for the prognosis of HNSCC patients [IL1B is an inflammatory cytokine gene and acts as a therapeutic target for the treatment of head and neck cancer [NOS3 such as single nucleotide polymorphism and insertion or deletion [NOS3 polymorphisms play crucial roles in the molecular mechanism of cancers and clinical survivals of patients undergoing cancers, including laryngeal cancer [NOS3, which was consistent with our finding [poptosis . Overwheresponse , 39. Cleresponse . Our molth HNSCC . Chan etnd MMP-9 . Similarpatients . IL1B isk cancer , 45. NOSk cancer . Previoudeletion , 48. Morl cancer –51. Guo  finding . BesidesTP53, EGFR, NOS3 and IL1B) were significantly enriched in cellular response to chemical stimulus, response to chemical, response to drug related biological processes. TP53 is a short-lived protein that plays a central role in mediating cellular response to stressful and genotoxic stimuli, such as anticancer drugs exposure [EGFR has been proposed to be the prognostic marker for HNSCC that closely related with radiation sensitivity, tumor size and recurrence [TP53, EGFR, NOS3 and IL1B in HNSCC patients exhibited a poor prognosis, which was consistent with the previous reports mentioned above. Thus, we speculated that the quercetin might reduce drug resistance of HNSCC by targeting TP53, EGFR, NOS3 and IL1B.Besides, in this study, GO enrichment analysis showed that the four genes of interest (currence . AdditioTP53, EGFR, NOS3. Our findings supported the significant role of PI3K-AKT signaling pathway in LSCC and proposed the target role of the key proteins identified in this study.Furthermore, PI3K-AKT signaling is considered to be a classical pathway related with various cancers , 56. TheAlthough our study identified several key components and target proteins of BYQH decoction, relevant experimental assays are needed to validate our findings. Although were considered as the prognostic indicators for patients with LSCC, The expression patterns of the four candidate prognostic indicators in LSCC have not been investigated in clinical samples. Moreover, the relationships of these prognostic genes and some clinical parameters are also not analyzed. Additionally, many chemical compounds are possibly not identified because the limited data is available in public databases, which may lead to some false positive results.TP53, EGFR, NOS3 and IL1B involved in drug resistance and PI3K-AKT signaling pathway. Moreover, the four genes of TP53, EGFR, NOS3 and IL1B exerted promising prognostic potential for LSCC. Our results offered an important clue for uncovering the underlying mechanism of BYQH decoction in the treatment of LSCC. However, more experimental evidences about the effects of quercetin on LSCC in vivo and in vitro by targeting TP53, EGFR, NOS3 and IL1B are warranted in the near future.Network pharmacology analysis was carried out to elucidate the therapeutic role of BYQH decoction on LSCC. Quercetin was predicted to be the active compounds of BYQH decoction by targeting Supplementary Figure 1Supplementary Table 1


Persistent proteinuria is associated with psoriasis risk, and the proteinuria amount significantly affects psoriasis development.Psoriasis, a chronic inflammatory dermatosis, has been associated with chronic kidney disease or end-stage renal disease. However, the association of the changes or amount of proteinuria with psoriasis development has not been evaluated. Using the Korean National Health Screening database, we assessed psoriasis development until 2018 in 6,576,851 Koreans who underwent health examinations in 2009 and 2011. Psoriasis was defined using the International Classification of Diseases, 10th revision (ICD-10) code L40. The risk of psoriasis was evaluated according to change in proteinuria (never [Neg (no proteinuria)/Neg], new [Neg/Pos (proteinuria present)], past [Pos/Neg] and persistent [Pos/Pos] proteinuria) and the proteinuria amount. During a median 7.23-year follow-up, 162,468 (2.47%) individuals developed psoriasis. After adjustments, the hazard ratio (HR) for psoriasis was higher in the persistent proteinuria group (1.32 [1.24–1.40]) than in the never proteinuria group. The past proteinuria group showed better renal outcome (1.03 [1.00–1.07]) than the new (1.05 [1.01–1.07]) and never proteinuria  groups did. The amount of random urine proteinuria was associated with increased HR for psoriasis. Subgroup analyses for age, sex, estimated glomerular filtration rate (eGFR), hypertension and diabetes showed that the persistent proteinuria group had a higher risk of psoriasis than the never proteinuria group, especially at eGFR < 60 mL/min/1.73 m Psoriasis is a chronic inflammatory skin disease that affects 1–3% of the general population . The preInflammation plays a pivotal role in the pathogenesis of chronic kidney disease (CKD). Proteinuria is not only a marker but also a risk factor for CKD. A previous study has shown that proteinuria is significantly associated with psoriasis . In addiIn this large population-based study, we aimed to investigate whether the changes in proteinuria and the amount of proteinuria are associated with a subsequent risk of psoriasis development. This study was conducted using data from the Korean National Health Insurance System (NHIS), which includes > 6.7 million Koreans with at least two proteinuria measurements.Owing to the confidentiality of the data used in this study and the strict privacy policy of the data holder, the data were kept strictly among the designated research personnel and cannot be provided to others regardless of anonymity.The Korean National Health Insurance Service manages a complete set of health information pertaining to 50 million Koreans and includes an eligibility database, a medical treatment database, a health examination database and a medical care institution database ,16,17. Tn = 223,016) /Neg) group included participants with no proteinuria both in 2009 and 2011. The persistent proteinuria (Pos [proteinuria present]/Pos) group comprised participants with proteinuria both in 2009 and 2011. The past proteinuria (Pos/Neg) group consisted of participants with proteinuria in 2009 and without proteinuria in 2011. The new proteinuria group (Neg/Pos) consisted of participants without proteinuria in 2009 but with proteinuria in 2011. Blood pressure (BP) was measured during the health examination by trained medical staff using auscultatory or oscillometric methods. The BP measurement protocol recommended at least 5 min of rest in a seated position followed by two repeated measurements with 5-min intervals . Body maThe primary end point of the study was newly diagnosed psoriasis, which was defined as a new record of ICD-10 code L40. in the database for the registered participants during the follow-up period.p-value < 0.05 was considered to indicate statistical significance. SAS version 9.4 software and SAS survey procedures  were used for all statistical analyses.Data are reported as mean ± standard deviation with intervals for continuous variables and as number (percentage) for categorical variables. To assess the risk of psoriasis related to proteinuria, we calculated the hazard ratios (HRs) with 95% confidence intervals (CIs) and analyzed these data using Cox proportional hazard regression models. We analyzed the associations between proteinuria and psoriasis development using three models: model 1, non-adjusted model; model 2, adjusted for age and sex; and model 3, adjusted for model 2 plus smoking, alcohol drinking, physical activity, BMI, low income, dyslipidemia, diabetes mellitus (DM) and eGFR. We also performed subgroup analysis for clinically important variables. A p < 0.001). The proportions of participants with male sex (59.83%), obesity (BMI ≥ 25 kg/m2) and abdominal obesity (waist circumference ≥ 90 cm in men and ≥ 85 cm in women) were higher in the incident psoriasis group than in the non-psoriasis group. Comorbidities such as DM, hypertension and dyslipidemia were more prevalent in the psoriasis group than in the non-psoriasis group. eGFR was lower, whereas BP, total cholesterol and glucose levels were higher in the psoriasis group than in the non-psoriasis group  were included in the never proteinuria group, 119,728 (1.82%) in the past proteinuria group, 101,695 (1.62%) in the new proteinuria group and 31,950 (0.48%) in the persistent proteinuria group. Compared to the other groups, the persistent proteinuria group was older and had more comorbidities such as DM, hypertension and dyslipidemia. The persistent proteinuria group also had higher BMI, waist circumference, fasting glucose levels, diabetes and dyslipidemia incidence and triglyceride levels; further, it included more male participants and current smokers than the never proteinuria group did .With the never proteinuria group as the reference, the multivariable adjusted HRs (95% Cis) for psoriasis outcome were 1.05 (1.01–1.09) for the past proteinuria group, 1.03 (1.00–1.07) for the new proteinuria group and 1.32 (1.24–1.40) for the persistent proteinuria group . We also2, showed an increased psoriasis risk in the past, new and persistent proteinuria groups compared with that in the never proteinuria group. However, in the case of eGFR ≥ 60 mL/min/1.73 m2, only the persistent proteinuria group showed a higher HR for psoriasis. The HR for psoriasis increased according to the amount of proteinuria only for participants with eGFR < 60 mL/min/1.73 m2.Subgroup analyses for CKD, which was defined as eGFR < 60 mL/min/1.73 m2, only the 4+ proteinuria group showed a significantly increased risk of psoriasis  . IncreasAs mentioned earlier, although a few studies have focused on the prevalence of proteinuria in patients with psoriasis, our study differs from these studies in that we investigated the occurrence of psoriasis in participants with or without proteinuria. The exact mechanism of how proteinuria affects psoriasis development is not fully understood. A possible explanation is that proteinuria is a biomarker and a risk factor for CKD, which is increased in inflammatory conditions such as psoriasis. Recently, emerging evidence has been reported that the role of gut microbiota, which closely interacts with the inflammation, kidney, cardiovascular and endocrine systems via metabolic, humoral and neural signaling pathways, causes chronic systemic inflammation, proteinuria, hypertension, diabetes and kidney disease . ProteinOur study showed that participants with persistent proteinuria had an obviously increased risk of psoriasis; however, new proteinuria development was also associated with a higher risk of psoriasis than that seen with past proteinuria occurrence. These results suggest the possibility that treatment of proteinuria using anti-proteinuric drugs such as renin-angiotensin-aldosterone system inhibitors might reduce psoriasis development. However, more systematic studies are needed to confirm this hypothesis.This study had several limitations. First, the data source of this study was the NHIS database, which lacks relevant clinical variables such as laboratory data and pathologic findings. Second, co-medications are not coded in the database and anti-proteinuria drug-related effects could have an influence on our study results. Third, we did not consider the severity of psoriasis, but only assessed its incidence.Persistent proteinuria is associated with psoriasis risk, and the proteinuria amount significantly affects psoriasis development.


Drug resistance is always a great obstacle in any endocrine therapy of breast cancer. Although the combination of endocrine therapy and targeted therapy has been shown to significantly improve prognosis, refractory endocrine resistance is still common. Dysregulation of the Hippo pathway is often related to the occurrence and the development of many tumors. Targeted therapies of this pathway have played important roles in the study of triple negative breast cancer (TNBC). Targeting the Hippo pathway in combination with chemotherapy or other targeted therapies has been shown to significantly improve specific antitumor effects and reduce cancer antidrug resistance. Further exploration has shown that the Hippo pathway is closely related to endocrine resistance, and it plays a “co-correlation point” role in numerous pathways involving endocrine resistance, including related pathways in breast cancer stem cells (BCSCs). Agents and miRNAs targeting the components of the Hippo pathway are expected to significantly enhance the sensitivity of breast cancer cells to endocrine therapy. This review initially explains the possible mechanism of the Hippo pathway in combating endocrine resistance, and it concludes by recommending endocrine therapy in combination with therapies targeting the Hippo pathway in the study of endocrine-resistant breast cancers. The incidence of breast cancer ranks first among all female malignancies . Most brThe complex reasons of endocrine resistance involves the estrogen receptor (ER) pathway, the growth factor receptor (GFR) pathways, and the CDK 4 and 6 pathway, as well as epigenetic modification. In recent years, the interdependence between BCSCs and drug resistance has emerged, and it often involves BCSC-related pathways, offering new insight into endocrine resistance.+CD24−/low [Nanog [Sox2 [Sox9 [ESR1 gene (estrogen receptor-gene), including Y537N, Y537S, and D598G, which are involved in ligand-independent activation of ER [BCSC-related markers, such as CD44D24−/low , ALDH [1D24−/low , CD133 [D24−/low ,Nanog [1w [Nanog , Sox2 [1og [Sox2 , and Soxx2 [Sox9 , were fox2 [Sox9 –16, letrx2 [Sox9 , and fulx2 [Sox9 . The prox2 [Sox9 . On the x2 [Sox9 . On the on of ER . Moreoveon of ER , ERβ [22on of ER  are all on of ER , giving on of ER  or diffeon of ER . TherefoBCSC-related pathways include the Hippo pathway, and those that signal via Hedgehog (Hh) signaling, transforming growth factor β (TGF-β) signaling, Notch signaling, PI3K/Akt/mTOR pathway, Wnt pathway, epidermal growth factor receptor (EGFR) pathway, ER signaling, and mitogen-activated protein kinase (MAPK) pathway. GFR signaling includes human epidermal growth factor receptor 2 (HER2) signaling, fibroblast growth factor receptor 1 (FGFR) signaling, and PI3K/Akt pathway, and they are three BCSC-related pathways. And other GFR signaling pathways are insulin-like growth factor 1 receptor (IGF-1R) signaling, MAPK signaling, and EGFR pathway. ER signaling is considered to be the main source of driving forces for the growth of ER + breast carcinomas. Changes in ER signaling are the first step for acquiring endocrine resistance, and this can include downregulation of ERα expression or loss of ERα expression caused by mutation or methylation of ERα-related genes . These dThe Hippo pathway is also called the Salvador/Warts/Hippo pathway, and can precisely control the number of cells and stop organism growth in a timely manner during the development of mammals.When the phosphorylation cascades of the Hippo pathway become blocked, these cells will differentiate abnormally and gradually develop into malignancies . Then, iIt has been confirmed that TAZ, YAP, LATS, and TEA domain family members (TEAD) are involved in the regulation of BCSCs. Cordenonsi et al.  first liBreast cancer can rely on ER signaling to promote cell proliferation, which is different from other tumors. The Hippo pathway is indeed associated with almost all pathways related to endocrine resistance, including ER signaling. Researchers have found partial cross talk between the Hippo pathway and ER signaling. Recent studies have found that targeting and activating the Hippo pathway can negatively regulate BCSCs and overcome endocrine resistance. Moreover, it also seems to be involved in the integration of GFR signaling pathways and Hippo pathway. This indicates that the Hippo pathway may make an unexpected contribution to endocrine therapy in ER + breast cancers.Preliminary non-canonical roles between the Hippo pathway and ER signaling in breast cancer is described in Fig. hi breast cells. GPER activated YAP/TAZ, suggesting that blockage of GPER by knockdown of YAP/TAZ was a great strategy for overcoming tamoxifen resistance in GPERhi breast cancers. Moreover, Zheng et al. [Studies in the MCF7 cell line (ER-positive breast cancer) have confirmed that correcting dysregulation of the Hippo pathway is a feasible scheme to inhibit breast cancer and overcome acquired drug resistance. The roles of the Hippo pathway in ER + breast carcinoma are described in Fig. g et al.  found thg et al.  confirmeThe Hippo pathway plays a “co-correlation point” role in several networks of BCSC-related pathways, including Notch, Wnt, EGFR, PI3K/Akt, MAPK/ERK1, Hh/GLI2,TGF-β pathway. Clara et al.  first prThe rest of GFR signaling pathways and Cyclin-dependent kinase 4 and 6 pathways involved in endocrine resistance also exert cross talk on the Hippo pathway. With regards to patients with luminal B subtypes, samples with low TAZ resulted in higher pathological complete response rates after trastuzumab-based neoadjuvant therapy, suggesting that HER2 linked with TAZ expression in a consistent manner . YAP/TAZThese roles of Hippo pathway in the integration of ER signaling are unique to breast cancers. The regulation of Hippo pathway on cancer stem cells is also affirmed, including BCSCs. Hippo pathway can not only combat BCSCs, but also can integrate these multiple pathways involve in endocrine resistance, including GFR ,BCSC-related pathways , and CDK4/6 pathway. The “co-correlation point” role of Hippo pathway in the multiple mechanism of endocrine resistance as described in Fig. In the process of endocrine therapy, the main driving forces for tumor growth were related to GFR signaling and even BCSC-related pathways. A dysregulated Hippo pathway did exert cross talk on these endocrine resistance-related pathways. A new therapeutic scheme that could be proposed to combat the “co-correlation point” of these pathways would focus on targeting the Hippo pathway. The combination of endocrine therapy and targeted therapy focusing on the Hippo pathway is expected to significantly improve the prognosis of patients with ER + breast cancers. There are many agents that can target the Hippo pathway, including Pevonedistat, Verteporfin, stains, and Metformin , 132. StTargeting the Hippo pathway has been researched in a variety of tumors and has been shown to have remarkable results. The combination of targeted therapy for the Hippo pathway and chemotherapy or other targeted therapies has also achieved initial results in breast cancers. Although its dysregulation can promote the occurrence and progression of tumors, reasonable regulation of this pathway can effectively inhibit tumors and combat endocrine resistance. However, the Hippo pathway is powerful, diverse, and complex. Breast cancers differ from other tumors, in that the former is derived from more of the non-canonical roles of the Hippo pathway via hormone receptors. More studies are needed to verify the feasibility and risk of regulation of the Hippo pathway in endocrine-resistant breast cancer. Moreover, our laboratory has shown that aromatase inhibitors such as Formestane can rely on ER-independent but androgen receptor-dependent roles to suppress ER + breast cancer, suggesting that aromatase inhibitors may be highly recommended as promising agents combined with targeting the Hippo pathway to significantly overcome endocrine resistance stimulated by estrogen-deprivation therapy in postmenopausal women . Targeti


This study aimed to determine the active ingredients of Huangqi Sijunzi Decoction (HQSJZD) and the targets in treating cancer-related fatigue (CRF) so as to investigate the treatment mechanism of HQSJZD for CRF.  This study adopted the method of network pharmacology. The active ingredients and targets of HQSJZD were retrieved, and the targets of HQSJZD in treating CRF were obtained using a Venn diagram. Next, a protein-protein interaction (PPI) network was constructed using the String database. The core targets of HQSJZD in treating CRF were identified through topological analysis, and functional annotation analysis and pathway enrichment analysis were carried out. Subsequently, a compound-disease-target regulatory network was constructed using Cystoscape 3.8.0 software.  A total of 250 targets of HQSJZD ingredients, 1447 CRF-related genes, and 144 common targets were obtained. Through topological analysis, 61 core targets were screened. Bioinformatics annotation of these genes identified 2366 gene ontology (GO) terms and 172 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.  The active ingredients in HQSJZD, that is, quercetin, luteolin, kaempferol, and naringenin, may act on AKT1, IL-6, VEGFA, MAPK3, CASP3, JUN, and EGFR to regulate the PI3K-Akt, TNF, and IL-17 signaling pathways, thereby suppressing inflammatory response, tumor gene expression, and tumor angiogenesis to treat CRF. This study investigated the pharmacological basis and mechanism of HQSJZD in the treatment of CRF using systematic pharmacology, which provides an important reference for further elucidation of the anti-CRF mechanism and clinical applications of HQSJZD, and also provides a method protocol for similar studies in the future. Cancer-related fatigue (CRF) refers to the painful, continuous, and subjective feeling of fatigue or exhaustion resulting from malignant tumor or tumor-related treatments, which is accompanied by symptoms such as limb heaviness, insomnia or somnolence, difficulty in activities, and abnormal emotional responses, but is not related to the amount of exercise . CRF hasAccording to traditional Chinese medicine (TCM), CRF belongs to the “consumptive disease” category. When cancer toxins have invaded the body for a prolonged period, the blood and Qi of the patients are impaired, which may lead to the impairment of kidney functions and the failure of the five organs, resulting in the occurrence of CRF. Moreover, surgery, radiotherapy, and chemotherapy aggravate blood and Qi impairment, thereby exacerbating fatigue symptoms. Therefore, the pathogenesis of CRF in TCM involves the impairment of the five organs and Qi/blood deficiency. In this regard, CRF is mainly treated by strengthening the body's resistance and tonifying the body to compensate for the deficiency.Codonopsis pilosula owing to the rareness of ginseng), Atractylodes, poria cocos, and radix glycyrrhizae. This is a classical and representative TCM preparation for Qi tonifying. The addition of Astragalus further enhances the Qi- and spleen-tonifying, body-resistance-strengthening, and consolidating effects. Pharmacological research [In recent studies, the therapeutic effects of Huangqi Sijunzi Decoction (HQSJZD) on treating CRF have been recognized by numerous doctors. HQSJZD is modified from Sijunzi Decoction, with the addition of Astragalus, which has Qi-tonifying, blood-replenishing, body-resistance-enhancing, and deficiency-tonifying effects. The Sijunzi Decoction consists of ginseng , with oral bioavailability (OB) ≥30% and drug likeness (DL) ≥0.18 used as screening criteria to screen for targets of corresponding ingredients. Next, the uniprot database (https://www.uniprot.org/) [Homo sapiens,” and the target proteins were transformed into corresponding genes.The components of HQSJZD, Atractylodes, database  (https:/ot.org/)  was usedhttps://www.genecards.org), NCBI database [https://www.ncbi.nlm.nih.gov), and OMIM database [https://www.omim.org) for CRD-related genes. Targets in the GeneCards database were screened according to the median value, so more related targets were obtained.Using “cancer-related fatigue” as keyword, human genes were screened in the GeneCards database  , with the common drug-disease targets screened. The biological species was set to “Homo sapiens,” and the minimum interaction score was set at medium confidence (0.400). The NetworkAnalyzer tool of the Cystoscape 3.8.0 software [The PPI network was constructed using the String database  , 6 CC terms, and 27 MF terms were retrieved. The top 10 GO terms of each category were plotted in the bubble chart . The top 20 pathways are shown in P values were selected: phosphatidylinositol 3 kinase/protein kinase B (PI3K-Akt) signaling pathways, prostate cancer, bladder cancer, pancreatic cancer, IL-17 signaling pathway, non-small cell lung cancer, TNF signaling pathway, and small cell lung cancer.The 144 common targets were significantly enriched into 172 pathways (The ingredient-disease pathway-target network was constructed using Cytoscape3.8.0 software . The netβ and IκB, and suppress the invasion and progression of malignant tumor cells [α by macrophages, and inhibit cancer cell invasion by controlling tumor cell growth factor production and regulating kinase activities [In this study, 144 active ingredients of HQSJZD in treating CRF were obtained; the most promising ones were quercetin, luteolin, kaempferol, naringenin, formononetin, and isorhamnetin. All of them are flavonoids with pharmacological activities such as enzyme regulation, blood circulation improvement, apoptosis regulation, and stem cell signal transduction regulation. Therefore, they have anti-inflammation, antioxidation, and antitumor effects . Modern or cells . Therefoor cells  and is ctivities . In additivities  and indutivities . It has tivities . Kaempfetivities . Isorhamtivities . Naringetivities , and redtivities . TherefoIn our analysis, the degree values of the targets, AKT1, IL6, VEGFA, MAPK3, CASP3, JUN, EGFR, MAPK1, and MYC, were greater than 90, suggesting that they are important core targets with great biological importance and close involvement in the treatment of CRF. AKT1 is a serine/threonine protein kinase and the key member of the PI3K pathway. Activated AKT1 can activate or suppress a series of downstream effectors, thus participating in the regulation of multiple biological processes, such as cell metabolism, proliferation, differentiation, apoptosis, and migration . Researcα and IL-17 [GO functional enrichment analysis and KEGG pathway enrichment analysis identified important functional terms and pathways that are closely related to HQSJZD and CRF targets, and further construction of the ingredient-disease pathway-target network established the connections between drug ingredients and pathways and revealed the molecular mechanisms of HQSJZD in treating CRD. The PI3K-Akt signaling pathway, TNF signaling pathway, and IL-17 signaling pathway were among the top pathways identified in KEGG analysis. The PI3K-Akt pathway plays an important role in tumor cell differentiation and proliferation in prostate cancer and ovarian cancer. Quercetin, the main active ingredient of HQSJZD, can suppress the overexpression of oncogene PTEN, downregulate PIP3 expression, inhibit AKT activity, and block the activation of the PI3K/Akt pathway to regulate tumor progression –43 and ind IL-17 , thus imIn conclusion, the active ingredients of HQSJZD, that is, quercetin, luteolin, kaempferol, and naringenin, may act on multiple targets like AKT1, IL6, VEGFA, MAPK3, CASP3, JUN, and EGFR and regulate multiple signaling pathways like PI3K-Akt, TNF, and IL-17 to suppress inflammatory responses, tumor gene expression, and tumor angiogenesis, thereby achieving the treatment of CRF. This study employed the method of network pharmacology and clearly revealed the “multicomponent, multitarget, and multipathway” mechanism of HQSJZD in treating CRF through network construction. Considering the limitation of network pharmacology, experimental studies are required to further analyze and validate the mechanism of HQSJZD in treating CRF identified in this study.


Eucommia ulmoides Oliver (EUO)-Tribulus terrestris L. (TT) drug pair in the treatment of hypertension-associated neurovascular lesions and IS via the ferroptosis pathway.In this study, we used the network pharmacology approach to explore the potential disease targets of the We used the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform to search for the key active compounds and targets of the drug pair. Based on the GeneCards database, the relevant targets for the drug pair were obtained. Then, we performed the molecular docking of the screened core active ingredients and proteins using the DAVID database and the R AutoDock Vina software. Based on the GSE22255 dataset, these screened target proteins were used to build random forest (RF) and support vector machine (SVM) models. Finally, a new IS nomogram prediction model was constructed and evaluated.There were 36 active compounds in the EUO-TT drug pair. CHRM1, NR3C1, ADRB2, and OPRD1 proteins of the neuroactive ligand-receptor interaction pathway interacted with the proteins related to the ferroptosis pathway. Molecular docking experiments identified 12 active ingredients of the drug pair that may tightly bind to those target proteins. We constructed a visual IS nomogram prediction model using four genes . The calibration curve, DCA, and clinical impact curves all indicated that the nomogram model is clinically applicable and diagnostically capable. CHRM1, NR3C1, ADRB2, and OPRD1, the target genes of the four effective components of the EUO-TT drug pair, were considered as risk markers for IS.The active ingredients of EUO-TT drug pair may act on proteins associated with the neuroactive ligand-receptor interaction pathway to regulate ferroptosis in vascular neurons cells, ultimately affecting the onset and progression of hypertension. Ischemic stroke (IS), one of the neurovascular disorders, is a common complication of hypertension, the second most common cause of death worldwide, and has become a major public health problem. Stroke affects approximately 15 million people worldwide each year, resulting in 5 million deaths and 5 million disabilities . A prospEUO is a living fossil plant. Recent studies have found more than a hundred compounds that can be extracted from EUO. Monomeric compounds and extracts of EUO have a wide range of pharmacological effects in the treatment of several diseases, including hypertension, hyperlipidemia, diabetes, and osteoporosis , 5. The Another Chinese medicine, TT, has been shown by modern pharmacological studies to be anti-hypertensive, diuretic, hypolipidemic, and anti-atherosclerotic. It also reduces left ventricular remodeling and improves cardiac function in the early post-myocardial infarction period –15. PhilRecent studies have found that treatment of ferroptosis may be an important means of preventing and improving the prognosis of patients with IS , 22. FerIn previous study, the ferroptosis of neuronal cells in hypertension is important for neurovascular diseases progression . This maIn this study, we used a network pharmacology approach to construct a “compound-target-disease” network through bioinformatics analysis to unravel the pharmacological basis and mechanism of action of the drug pair of EUO-TT in the treatment of hypertension-associated neurovascular lesions through the ferroptosis pathway. We believe that the findings of our study will provide a pharmacological basis for the clinical treatment of hypertension using Chinese medicine.In this study, we searched for the chemical composition of EUO and TT through the pharmacology platform of the Chinese medicine network. We extracted the main active compounds of each medicine with the screening conditions of oral bioavailability ≥30% and drug-likeness ≥0.18 .The hypertension-related targets were searched by MOL ID numbers (expressed as protein name) of compounds using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Further, the gene names corresponding to each target were searched by the Uniport database . Based oAfter importing the intersecting targets into the STRING database and restricting the study species to humans, we obtained the PPI network of EUO-TT drug pair acting on neuroactive ligand-receptor interaction pathway and ferroptosis pathway . The topThe active compounds and their targets in the neuroactive ligand-receptor interaction pathway were imported into Cytoscape 3.7.1 software, and the “STRING database” function was used to analyze the neuroactive ligand-receptor interaction pathway-ferroptosis pathway target network. The key compounds of EUO-Tribulus terrestris L drug pair mediating neuroactive ligand-receptor interaction pathway to coordinate ferroptosis pathway for the treatment of hypertension were screened based on compound-target connectivity.p < 0.05 was set for GO function enrichment and KEGG pathway analysis, of which results were visualized by R programming software . We found 36 active compounds after de-weighting. Further, 30 compounds were found to correspond to 114 genes, and the gene names of each target were obtained from Uniprot .P < 0.05 for GO functional analysis and KEGG pathway enrichment analysis. The pathways and functional information with the top P-values in each type of process and pathways were selected, where smaller P-values and longer bar lengths indicated the strongest enrichment significance  was higher than that of the SVM model (AUC = 0.845) .To create a visual model for IS prediction, we created a nomogram  using thThis study unraveled the mechanism of action of EUO-TT drug pair in the treatment of hypertension related endothelial injury through the ferroptosis pathway, providing a pharmacological basis for the clinical treatment of hypertension related cerebrovascular endothelial injury using Chinese medicine. CHRM1, NR3C1, ADRB2, and OPRD1, the target genes of the four active ingredients of the EUO-TT drug pair, were considered as risk markers for IS and a visual IS nomogram prediction model was constructed.Network pharmacology was used to screen the active compounds of EUO-TT L. drug pair and found that the target genes were mainly enriched in the neuroactive ligand-receptor interaction pathway in this research. By constructing the PPI network of neuroactive ligand-receptor interaction pathway-ferroptosis, we found that CHRM1, NR3C1, ADRB2, OPRD1, FTH1, TP53, and PCBP2 were the main target genes of the drug pair. Through the regulatory network of the drug's active ingredients and their key targets in the neuroactive ligand-receptor interaction pathway, we found 15 active ingredients of the drug pair that were involved in the regulation of the key target proteins. Further, molecular docking experiments verified that 12 active ingredients of the drug pair were tightly bound to the target proteins. The expression profiles of CHRM1, NR3C1, ADRB2, and OPRD1 were used to construct IS prediction models. The residual distribution of the RF model was found to be lower than that of the SVM model, and the ROC curve suggested a higher prediction accuracy of the RF model. The RF model rated the genes in ascending order of importance: OPRD1, CHRM1, NR3C1, and ADRB2. Following that, the nomogram prediction model was constructed using the four selected genes . However, only CHRM1, NR3C1, and OPRD1 were eventually included in the prediction model, as inclusion of these three components was sufficient to reliably predict prognosis in the logistic model. The calibration curve, DCA, and clinical effect curves all indicated that this nomogram model is clinically applicable and diagnostically capable. The enrichment of dendritic cell differentiation and protein serine/threonine/tyrosine kinase activity and manganese ion binding pathways was found to be the most correlated with the prediction results based on nomogram risk scores.SLC3A2, FTH1, TP53, and PCBP2 are directly or indirectly associated with ferroptosis. SLC3A2 is a SystemXc- heavy chain subunit involved in the maintenance of a redox steady state , 57. WheSimilar to previous studies, we found that SLC3A2, FTH1, TP53, and PCBP2 were the ferroptosis pathway-associated proteins that interacted with CHRM1, NR3C1, ADRB2, and OPRD1. The NR3C1 protein regulates the genetic features involved in the P53 signaling pathway . FurtherIn this study, CHRM1, NR3C1, ADRB2, and OPRD1, the target genes of the four EUO-TT L. drug pair active ingredients, were considered as risk markers for IS. CHRM1 was found to be associated with muscarinic receptor-mediated anti-inflammatory mechanisms . FollowiIn the present study, the active ingredients of EUO-TT L., namely cyclopamine, beta-sitosterol, mairin, tabernemontanine, moupinamide, epiquinidine, and 3-Hydroxymethylenetanshinquinone, were found for the first time to target and bind neuroactive ligand-receptor interaction pathway-related proteins, which in turn regulated the process of cellular ferroptosis and ultimately affected the onset and progression of hypertension. There is a lack of previous studies confirming the association of the alkaloids tabernemontanine and epiquinidine, and the terpenoids cyclopamine, and mairin of EUO with hypertension, ferroptosis, and the neuroactive ligand-receptor interaction pathway. Hence, these pathways can be a new therapeutic target for the prevention and treatment of hypertension.However, this study also has its shortcomings. The network pharmacology is based on the bioinformatics analysis of the database of herbal targets and disease genes, and thus lacks experimental validation. Hence, further experimental validation is needed to explore the mechanism of action of the drugs or their active ingredients. In addition, this study did not consider the selection of dosage in drug combination and the possible chemical reactions and changes of active ingredients in the process of concoction and decoction of herbal medicines. Furthermore, we also acknowledge that the current research in TCM for the treatment of endothelial injury in hypertension-related cerebrovascular disease is still limited, and therefore we suggest to provide more confirmation and discussion in the future.The active ingredients of EUO-TT L. drug pair can act on proteins related to the neuroactive ligand-receptor interaction pathway, which may affect the process of cellular ferroptosis and potentially affect the progression of hypertension. CHRM1, NR3C1, ADRB2, and OPRD1, the target genes of the four EUO-TT L. drug pair active ingredients, were considered as risk markers for IS.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.QZ: software, validation, formal analysis, data curation, and writing-original draft. JY: conceptualization, methodology, supervision, funding acquisition, and writing-review and editing. CY: methodology, conceptualization, project administration, and funding acquisition. XY: data curation and writing-review and editing. YC: software and data curation. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (No. 81804061), Shandong Province Taishan Scholar Construction Project Funds No. 2018-35, and Ji'nan Science and Technology Project (No. 201805078).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.