This hypothesis is widely supported by modelling studies, but experimental tests are missing. Mixing of C4 components to generate artificial intermediates can be achieved via crossing, and the grass Alloteropsis semialata represents an outstanding study system since it includes C4 and non‐C4 populations. Here, we analyse F1 hybrids between C3 and C4, and C3+C4 and C4 genotypes to determine whether the acquisition of C4 characteristics increases photosynthetic performance. The hybrids have leaf anatomical characters and C4 gene expression profiles that are largely intermediate between those of their parents. Carbon isotope ratios are similarly intermediate, which suggests that a partial C4 cycle coexists with C3 carbon fixation in the hybrids. This partial C4 phenotype is associated with C4‐like photosynthetic efficiency in C3+C4 × C4, but not in C3 × C4 hybrids, which are overall less efficient than both parents. Our results support the hypothesis that the photosynthetic gains from the upregulation of C4 characteristics depend on coordinated changes in anatomy and biochemistry. The order of acquisition of C4 components is thus constrained, with C3+C4 species providing an essential step for C4 evolution.C 3 and C4, and C3+C4 and C4 accessions of the grass Alloteropsis semialata. While hybrids have upregulated C4 features, these do not consistently increase photosynthetic efficiency, highlighting the importance of the order of acquisition of components during C4 evolution.We analyse hybrids between C The C4 y Hatch, . The C4 4 and non‐C4 plants have been reported particularly for the eudicot genera Atriplex , and C3+C4 and C4 (C3+C4 × C4) accessions of A. semialata and analyse their phenotype to test the hypotheses that components of the C4 trait are additive, and that hybrids rank between their non‐C4 and C4 parents in terms of photosynthetic performance. Our study confirms that crosses between photosynthetic types in A. semialata are viable in experimental conditions, and shows that the C4 metabolism is disrupted in the hybrids despite the significant upregulation of anatomical and biochemical components of the C4 trait.Here we generate hybrids between C22.1Alloteropsis semialata (R. Br.) Hitchc. (Poaceae) from distinct geographical origins were generated from parental plants collected in the wild as seeds or cuttings  were obtained. Subsequent genotyping of the seedlings identified multiple F1 hybrids between parents with distinct photosynthetic types, including 14 C3 × C4 and 6 C3+C4 × C4 individuals  that were growing alongside them were added to increase the phenotypic diversity within photosynthetic types (Table −2 s−1 at bench level), 25/20°C day/night temperature, ambient CO2 concentration and relative humidity between 30% and 60%.Crosses between accessions of gs Table  and growls Table . The remon Table . Five C3es Table . For thees Table . All pla2.2A. semialata  in a total volume of 25 µl. The PCR mixtures were initially incubated in a thermocycler for 2 min at 94°C followed by 35 cycles consisting of 30 s at 94°C (denaturation), 1 min at 48°C  and 1 min at 72°C (elongation). Amplicons were cleaned using Exo‐SAP‐IT (Affymetrix), and Sanger‐sequenced at the Core Genomic Facility at the University of Sheffield. Sequencing chromatograms were individually inspected for heterozygous sites that are polymorphic among genetic groups corresponding to distinct photosynthetic types, since the presence of such heterozygous sites indicates an F1 hybrid between photosynthetic types. Because the maternal origin was known in advance , the information from the alternative bases was used to narrow down the pollen parent to a genetic lineage (Table We used three genetic markers to determine the genetic lineage of the pollen parent of each plant. These markers correspond to regions of the nuclear genome (~500 bp long) with sufficient variation to distinguish between different photosynthetic types and geographical groups, and were selected from a set of markers previously assembled to determine the origin of allopolyploids in ge Table . The hyb2.313C, in ‰) were reported relative to the standard Pee Dee Belemnite (PDB). Values of δ13C higher than −16‰ indicate that the plants grew using C4 photosynthesis . Carbon isotope analysis was conducted on 1–2 mg of leaf sample using an ANCA GSL preparation module coupled to a Sercon 20‐20 stable isotope ratio mass spectrometer (PDZ Europa). Carbon isotopic ratios  following the manufacturer's instructions. Cross sections 7–10‐μm‐thick were obtained using a microtome , and stained with Toluidine Blue O (Sigma‐Aldrich). An Olympus BX51 microscope coupled to an Olympus DP71 camera (Olympus Corporation) was used to photograph the cross sections. Leaf anatomical traits were measured using ImageJ v.1.51q  with an on‐column DNA digestion step . A total of 20 RNA‐Seq libraries  were prepared with the TruSeq RNA Library Preparation Kit v2 (Illumina) using 0.5 μg of starting RNA and aiming at a median insert size of ~155 bp (standard fragmentation protocol). Libraries were paired‐end sequenced (read length = 100 bp) on 1/24 of a single lane of an Illumina HiSeq 2500 flow cell in rapid mode  at the Sheffield Diagnostic Genetics Service. Raw sequence reads were filtered to remove adaptor contamination and low‐quality reads  using NGSQCToolkit v. 2.3.3 , each group containing all paralogs detected in the transcriptomes of A. semialata . We used this data set as it includes manually curated sets of co‐orthologs of 23 gene families known to have a function in the C4 biochemistry  known to be highly expressed in other A. semialata accessions  for 20 additional accessions  retrieved from two previous RNAseq studies .To explore the global differences in gene expression between hybrids and the parental types, we performed a differential expression analysis on the data set that was generated using the complete genome of A. semialata. Here we also included individuals of A. semialata from the RNAseq studies of Dunning et al. (https://broadinstitute.github.io/picard/), and variants were called on all individuals combined using BCFtools v. 1.9 , we identified SNPs that were common to all individuals of the same photosynthetic type and exclusive to them. Sites with more than 25% missing data within each photosynthetic group were removed, and a total of 114 SNPs were retained  was measured using two portable gas‐exchange systems Li‐6400XT (Li‐Cor). A/Ci curves were measured within the first 6 h of the photoperiod on the widest fully expanded leaf at a block temperature (Tblock) of 25°C, flow rate = 300 μmol s−1, photosynthetic photon flux density (PPFD) = 1500 μmol m−2 s−1, and were started after both net CO2 uptake (A) and stomatal conductance (gS) reached steady state at reference CO2 = 400 μmol mol−1. Reference CO2 was then changed in a stepwise manner to 250, 150, 120, 100, 85, 70, 50, 35, 400, 600, 800, 1000 and 1200 μmol mol−1. Readings were automatically logged after 2–3 min of leaf acclimation to each CO2 level. The CO2 compensation point (CCP) and maximum carboxylation efficiency (CE) were calculated following Bellasio et al.  was applied to the central portion of fully expanded leaves, and clear nail varnish was applied to the set resin impression. Images were captured from the nail varnish impressions at 20× using an Olympus BX51 microscope coupled to an Olympus DP71 camera, and stomatal density was quantified on 0.38 mm² fields using ImageJ.33.113C) of all hybrids between C4 and non‐C4 accessions were below the range of C4 values in A. semialata  and increased abundance of bundle sheath tissue (IS and OS) relative to the mesophyll , width of inner bundle sheath cells  and epidermis area , but these variables do not differentiate photosynthetic or cross types. The proportion of inner bundle sheath relative to mesophyll (IS/M) was on average doubled in C3 × C4 hybrids compared to C3 accessions, and was within the range of C3+C4, but the values were still 62% lower than the C4 average , with no significant effects of OS or IS widths. The IS/M ratio, on the other hand, was not significantly associated with vein density, but with both OS and IS width . Overall, C3 × C4 hybrids were not significantly different from C3+C4 in relation to the quantitative anatomical traits analysed here, except for BSD, which was significantly reduced in C3 × C4 as a result of the presence of some minor veins in most accessions. In C3+C4 × C4 hybrids, a quantitatively distinct phenotype was generated, with trait values ranking between C3+C4 and C4 plants. Overall, our results show that leaf anatomical traits in A. semialata are mostly inherited in an additive manner.Cls Table . The prek Figure . The firk Figure . C4 indil Figure . C3+C4 ae Figure . The disy Figure . The meat Figure . OS cells Figure . In the 3.33+C4 × C4 plants  from the other individuals, and accounted for 24.7% of the variation. Photosynthetic types were clearly separated by the second principal component, which accounted for 16.8% of the variation and placed the hybrids as intermediate to the photosynthetic types of their parents. Similar clustering patterns were observed when transcript abundance was quantified for the complete gene set extracted from the genome of A. semialata , NADP‐malic enzyme  and pyruvate phosphate dikinase  were 4‐ to 17‐fold higher in C4 than in C3 plants, with the transcript abundances of the C3+C4 plant slightly above those of C3 plants  that is highly expressed in C4 and C3+C4 accessions  that have been identified in A. semialata accessions  that is the pollen parent of two C3 × C4 hybrids , and it is highly expressed (>10 000 RPM) in four C3+C4 × C4 individuals that are offspring from the C4 accessions from Burkina Faso or Madagascar .Out of the nine genes encoding the key Cs Figure . Finallyglo‐1P2) and glutamate:glyoxylate aminotransferase  in C3+C4 × C4 hybrids relative to C3 and C3+C4 accessions, with average values that are similar to or below those of C4 accessions  had on average 63% lower transcript levels in C4 than in C3 accessions; in C3 × C4 and C3+C4 × C4 hybrids, transcript levels of these four genes were on average 12% and 33% lower than in C3, respectively, suggesting additivity  had higher average transcript abundance in C4 than in C3 accessions. The chloroplast genes glycerate 3‐kinase  and 2‐phosphoglycolate (2‐PG) phosphatase  had overlapping transcript abundance ranges across hybrid and photosynthetic types. Finally, a gene encoding a GOLDEN2‐LIKE transcription factor that has been previously demonstrated to be involved in the redistribution of organelles among cell types , out of which 21 were related to C4 photosynthesis and 4 to photorespiration , NADP‐malate dehydrogenase  and soluble inorganic pyrophosphatase . While nadpmdh‐1P2 was downregulated in the C4 group, the two others had increased transcript abundance in C4 relative to both hybrid types , P‐ (gldp1P) and L‐  was downregulated in both C3 × C4 and C3+C4 × C4 hybrids relative to C4 accessions. Gene ontology (GO) analyses did not detect any significantly enriched cellular processes for any of the full DE gene sets. However, the reduced DE gene set from the comparison between hybrid types was significantly enriched with genes related to the photosystems I and II, oxidoreductase activity, and 1,3‐beta‐d‐glucan synthesis , transmembrane transport, and photosystems I and II  between hybrid types, and between these and Cns Table . We founon Table . This coon Table . Among tn Figure . The inded Table . In thiss Figure . Finallyis Table . In the II Table .4 genes and those involved in the photorespiratory pathway is largely additive. In almost all cases, the transcript abundance of the hybrids ranks between those of the parental lineages.Overall, the analyses of expression profiles show that the inheritance of expression levels of C3.42 response curves (A/Ci) and steady‐state measurements at ambient CO2  was highest in two C4 accessions, but the differences between C3, C3+C4 and C4 plants were not significant  had the highest median values in C3 accessions, but the differences between groups were not significant , but only after excluding the outlier C3 accession ZIM1503, which had the highest stomatal density, but one of the lowest gs. The correlation between stomatal density and vein density was only marginally significant . The intrinsic water use efficiency (iWUE) at ambient CO2 was on average 38% higher in C4 than in other accessions, although a few C3, C3+C4 and C3+C4 × C4 individuals also showed C4‐like values  could explain the observed patterns, we inspected the variation in Tleaf during the A/Ci curves. Although Tleaf values up to 3°C above the median (26.3°C) were observed in a few accessions, 81% of all data points were collected within a 2°C interval  completely suppress Rubisco expression and/or activity in the mesophyll cells and/or (2) create an effective C4 cycle. However, the CCP of the C3 × C4 hybrids is substantially reduced compared to the C3 parents , the degree of C4 activity is higher in C3+C4 × C4 than in C3 × C4 hybrids. Our findings therefore recapitulate what has been shown for interspecific hybrids between photosynthetic types in several eudicot and monocot genera  and the distance between consecutive bundle sheaths (BSD). These traits are mostly determined by the presence of minor veins in C4 leaves, which has been previously shown to be the major anatomical difference between C4 and non‐C4 A. semialata . This in turn supports the idea that the changes towards a full C4 physiology must be built sequentially upon pre‐existing characteristics that enable the next change to provide a fitness advantage (Heckmann, 4 components independently, particularly under conditions expected to favour C4 plants, including long‐term exposure to low CO2 levels, should provide further insights on whether C4 evolution must follow a particular sequence of events for it to be viable.The current model of C Figures , despite5A. semialata to investigate how the upregulation of components of the C4 trait affects photosynthetic performance. We show that the hybrids have in most cases anatomical traits and gene expression patterns that are intermediate between those of the parents, and this leads to C4 activity that is equally intermediate between the two parents. The physiological benefits of a partial C4 metabolism in the hybrids appear only at low CO2 levels; at ambient CO2, there is no evidence of enhanced photosynthetic performance in the hybrids relative to their non‐C4 parents. Some hybrid individuals in fact perform worse than both parents at ambient CO2, and this possible hybrid depression could explain the lack of a clear hybrid zone in regions where the distributions of C4 and non‐C4A. semialata lineages overlap. Overall, our results support the hypothesis that photosynthetic gains arising from the upregulation of C4 features are conditional on coordinated changes in leaf anatomy and biochemistry. Future studies with the F2 offspring, where the different C4 components can be segregated, will be able to pinpoint the key genetic and phenotypic changes that lead to fitness gains, providing a unique opportunity to experimentally test long‐standing hypotheses about the evolution of C4 photosynthesis.In this study, we characterize the phenotype of hybrids between different photosynthetic types in the grass The authors declare no conflicts of interest.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.


P < 0.01) and 159 Kyoto Encyclopedia of Genomes and Genomes (KEGG) pathways (P < 0.01), mainly including the AGE-RAGE signaling pathway in diabetic complications and the PI3K-AKT signaling pathway. In animal experiments, the results of an ELISA assay showed that QYDP could regulate the expression levels of kidney function-related indexes and reduce the expression of inflammatory factors. qRT-PCR and western blot results showed that QYDP regulated the expression of hub genes. In conclusion, this study shows that QYDP could treat DN by antioxidative and antiinflammatory activity and inhibiting the PI3K-AKT signaling pathway.This study aimed to explore the potential mechanisms of Qishen Yiqi dripping pill (QYDP) against diabetic nephropathy (DN) through network pharmacology and animal experiments. The components and targets of QYDP and DN-related targets were retrieved from public databases. A total of 116 compounds and 160 targets of QYDP anti-DN were obtained. The top 10 hub targets including AKT1, TNF, ALB, INS, PPARG, IL-6, IL-1B, VEGF-A, JUN, and MAPK3 were screened by Cytoscape software. Then, the key targets of QYDP were enriched in 1815 Gene Ontology (GO) entries ( Diabetic nephropathy (DN) is the end-stage renal disease of type 2 diabetes mellitus (T2DM) . In 2021Astragalus penduliflorus Lam.), redroot sage , pseudoginseng , and fragrant rosewood . Studies have shown that the urinary serum excretion rate of diabetic patients is reduced by QYDP administration [Qishen Yiqi dropping pill (QYDP) is a traditional Chinese medicine (TCM) consisting of Radix Astragali  [https://www.megabionet.org/tcmid/) [https://lifecenter.sgst.cn/hit/) [Active ingredients of QYDP were extracted from the traditional Chinese medicine systems pharmacology database and analysis platform  , Traditi/tcmid/) , and Hercn/hit/) . We collhttps://stitch.embl.de) [The potential targets of QYDP were predicted by TCM-ID, TCMSP, HIT, and Search Tool for Interacting Chemicals   databaseembl.de) .https://omim.org) [https://www.disgenet.org) [https://www.genecards.org/) [Homo sapiens, and targets with a correlation score above the median (≥1.81388) were selected in the GeneCard database. The results of the above 3 databases are merged and duplicate targets are removed.DN-related targets were derived from three databases: Online Mendelian Inheritance in Man  , DisGeNEnet.org) , and Gends.org/) . For filUsing the STRING database , the pothttps://david.ncifcrf.gov) were used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the potential targets. GO enrichment analysis includes biological processes (BP), cellular components (CC), and molecular functions (MF). The enrichment results of GO and KEGG were obtained by setting P < 0.01.The Database for Annotation, Visualization, and Integrated Discovery  for 5 days. Mice with blood glucose concentrations of 11–18 mM were considered successful mouse models of DN. Then, all mice were randomly divided into six groups (n = 6 per group): control group, STZ-induced DN model group, QYDP administered groups ; and a high-dose QYDP + 740Y-P  group . After 5 weeks of administration, all mice were anesthetized with sodium pentobarbital  and then sacrificed by cervical dislocation. The blood and kidney samples were obtained for further analysis.The animal experiments were approved by the Ethics Committee of Taizhou Hospital of Zhejiang Province. Eight-week-old male C57BL/6J mice were obtained from Beijing HFK Bioscience Co., Ltd. (China), housed in a controlled room with a 12-hour light/dark cycle at 23 ± 2°C, and received food and water After fasting for 6 hours in the morning, the body weight and fasting blood glucose (FBG) levels were measured once a week using a blood glucometer . In addition, at the end of the experimental period, 24-hour urine volumes were collected from all mice in the metabolic cages.β, and tumor necrosis factor-α (TNF-α)) in the serum of all mice were also measured by ELISA kits.ELISA kits  were used to measure the levels of albumin (ALB), blood urea nitrogen (BUN), serum creatinine (Scr), total cholesterol (TC), and triglyceride (TG) in the urine or serum of all mice according to the protocol of the manufacturer. Similarly, the levels of inflammatory cytokines (interleukin 6 (IL-6), IL-1μm). Next, the slices were stained with hematoxylin and eosin for 3 min using a light microscope  to observe the histological images.HE staining was performed according to the protocols previously reported . FirstlyμL per well, including 10 μL of SYBR Green Mix, 1 μL of cDNA, 2 μL of primer pair mix, and 7 μL of DNAse/RNAse-free H2O. The cycling conditions for qRT-PCR are as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 12 sec and 62°C for 40 s. Gene expression was assessed by the 2−ΔΔCT method and GAPDH as the house gene. The primers are listed in Supplementary Total RNA was extracted from kidney tissues using TRIzol reagent  and then reverse-transcribed into cDNA using PrimeScript RT Master Mix  according to the manufacturer's instructions. PCR was performed using an SYBR Green kit , and the reaction volume was 20 #AP20705a, abcepta, China), VEGF-A , PI3K , MA, USA), p-PI3K , AKT , and p-AKT  overnight at 4°C, followed by incubated with secondary antibodies  for 1 h. The bands were detected in an enhanced chemiluminescence detection system .Proteins were extracted from kidney tissues using RIPA lysis buffer (Beyotime), loaded into a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, and transferred into polyvinylidene fluoride membrane. After blocking in the 5% skim milk, membranes were incubated with primary antibodies against PPARG  and expressed as mean ± standard deviation. One-way ANOVA followed by Tukey's tests was used to compare multiple groups. A total of 170 active ingredients were obtained from TCMSP, TCM-ID, and HIT databases. Then, 152 active ingredients were further screened by QED ≥0.2. A total of 6,828 DN-related targets were collected from TCMSP, TCM-ID, HIT, and STITCH databases. Then, through the binomial statistical mode algorithm, we obtained 578 potential targets and 133 active ingredients.A total of 312, 109, and 1,189 DN-related targets were extracted from GeneCard, OMIM, and DisGeNET databases, respectively. And, 1,333 DN-related targets remained followed by deleting the duplicate values.As shown in The STRING database was used to acquire PPI relationships of 160 key targets of QYDP anti-DN. The PPI network constructed by Cytoscape and consisted of 160 nodes and 3362 edges . The aveP < 0.01). As shown in To further explore the biological functions of the 160 key targets, GO enrichment analysis was performed on the DAVID database and obtained 84 terms of MF, 1679 terms of BP, and 52 terms of CC  network showed the associations of the key targets with the top 15 BP and top 15 pathways . The netTo investigate the effect of QYDP on the physiological and serological changes in DN mice, we measured physiological and serological indicators including body weight, FBG, ALB, Ucr, BUN, TC, TG, and Scr. As shown in Figures For the urine examination of DN mice, we detected the changes in ALB, Ucr, and BUN. As shown in Figures Next, we observed histopathologic changes in kidney tissues using HE staining . The resβ, IL-6, and TNF-α. Compared with the control group mice, the expressions of IL-6, IL-1β, and TNF-α in the serum of the model group mice were increased. In addition, the intervention of QYDP attenuated the renal inflammatory response and the secretion of these cytokines was significantly reduced, while the addition of 740Y-P attenuated the antiinflammatory effect of QYDP  in the serum of the DN mice and the western blotting suggested that QYDP could inhibit the PI3K-AKT signaling pathway. These results suggest that QYDP could treat DN by inhibiting the PI3K-AKT signaling pathway.Furthermore, the GO enrichment analysis revealed QYDP anti-DN may be associated with antioxidant activity. Studies have shown that oxidative stress plays a crucial role in DN, and excessive oxidative stress can lead to glomerulosclerosis and renal fibrosis, ultimately leading to DN , 29. Thehropathy , 31. Sevhropathy . It can hropathy . The PI3hropathy . PI3K-AKhropathy , 36. In In conclusion, the network pharmacology analysis showed that QYDP has 116 active compounds and 160 targets against DN. GO enrichment analysis revealed that QYDP anti-DN may be associated with antioxidant activity. KEGG enrichment analysis found that multiple pathways involved in QYDP alleviated DN, such as the AGE-RAGE signaling pathway in diabetic complications and the PI3K-AKT signaling pathway. Animal experiments suggest that QYDP may treat DN by reducing inflammation and oxidative stress levels and inhibiting the PI3K-AKT signaling pathway. Notably, our findings partially elucidate the complex anti-DN mechanisms in QYDP and provide insights into the efficacy and drug activity of QYDP.


Qinggan Huoxue Recipe (QGHXR) is originated from Xiao Chaihu Decotion. Many experimental studies have confirmed that QGHXR can significantly alleviate the symptoms of alcoholic liver disease (ALD), but the detailed mechanism is still unclear. Using traditional Chinese medicine network pharmacology analysis system database and animal experiments, we found that 180 potentially chemical compositions and 618 potential targets were screened from the prescription, which shared 133 signal pathways with ALD. Through animal experiments, it was found that QGHXR could reduce the liver total cholesterol (TC), serum TC, alanine aminotransferase, aspartate aminotransferase of ALD mice, reduce the lipid droplets and inflammatory injury of liver tissue. Meanwhile, it can also increase PTEN, decrease PI3K and AKT mRNA levels. In this study, we obtained the targets and pathways of QGHXR in the treatment of ALD, and preliminatively verified that QGHXR may improve ALD through PTEN/PI3K/AKT signaling pathway. Alcohol consumption is a major global health burden and a key cause of the development of alcohol liver disease (ALD) A growing number of studies have shown that traditional Chinese medicine (TCM) is useful in the treatment of ALD. Several studies have reported the health-beneficial effects of dietary phytochemicals, namely polyphenols, to prevent various diseases Bupleurum abchasicum Manden and Scutellaria baicalensis Georgi can clear liver and remove dampness, and are used as the main drugs. The second main drugs are Salvia miltiorrhiza Bge and Trionyx sinensis Wiegmann, which can activate blood circulation and remove blood stasis. As an auxiliary drug, Pueraria lobate (Willd.) Ohwi can relieve muscle soreness, thirst and promote the circulation of Yang Qi. The prescription is used for clearing heat and removing dampness, promoting blood circulation and removing blood stasis For example, astragalus saponins and astragalus polysaccharides were found to reduce liver injury in mice with ALD in vitro to improve alcoholic liver fibrosis Many experimental studies have confirmed that QGHXR can significantly alleviate the symptoms of ALD. Clinical randomised controlled trial study confirmed that it can significantly improve the symptoms and signs of anorexia, nausea, vomiting and jaundice in patients with ALD in vivo. It is the first time to collect the active components and gene targets of QGHXR and use network pharmacology to explore the mechanism of QGHXR improving ALD. It was found that 180 potentially chemical compositions and 618 potential gene targets were screened from the QGHXR and the all genes shared 133 signal pathways with ALD. Through animal experiments, we found that QGHXR could reduce the liver total cholesterol (TC), serum TC, alanine aminotransferase (ALT), aspartate aminotransferase (AST) of ALD mice, reduce the lipid droplets and inflammatory injury of liver tissue and we preliminarily verified that QGHXR may improve ALD through PTEN/PI3K/AKT signaling pathway. The detailed flow chart of the study is shown in Figure In previous studies, we have found that QGHXR can improve ALD and liver fibrosis through LXR-LPCAT3-ERS and TGF-β1/Smad signaling pathway, respectively, but all the mechanisms have not been explored clearly. In the present study, we first collect the active components of QGHXR using network pharmacology, and then collect the gene targets of these active components by the database TCM Network Pharmacology Analysis System (TCMNPAS). At the same time, the related gene targets of ALD were enriched, and then the common genes of both were enriched by KEGG pathway from the database. Finally, the pathway was verified by animal experiments http://54.223.75.62:3838/). It is mainly used for network pharmacological analysis of TCM compound prescription and its chemical ingredients, and provides one-stop solution for complex data analysis. The five herbs of QGHXR, Bupleurum abchasicum Manden., Scutellaria baicalensis Georgi, Salvia miltiorrhiza Bge, Pueraria lobate (Willd.) Ohwi, and Trionyx sinensis Wiegmann were input into the retrieval module and HIT, TCMID, STITCH, TCMSP and CUSTOM databases were selected. The QED value was set to 0.2, and the compound-target association score was set to 400. .TCMNPAS, a network pharmacological analysis system of TCM, was independently developed and designed by Professor Yang Ming in Longhua Hospital, Shanghai University of Traditional Chinese Medicine  to obtain the disease ID, and then inputs the obtained ID into the disease gene retrieval module of TCMNPAS to automatically obtain the related gene targets of ALD.The names  of the disease related to ALD are inputted into the disease ID retrieval module of TCMNPAS, which automatically connects with the GeneCard database  to construct PPI network.The potential targets of QGHXR and ALD targets information obtained from TCMNPAS database were sorted out and imported into STRING  adjusted P value (P. adjust) was used to reflect the association strength between targets and gene ontology, and P. adjust <0.05 was considered as a significant association, and GO enrichment and KEGG signaling pathway were obtained.The gene targets of QGHXR and ALD related gene targets obtained from TCMNPAS database were imported into the GO and KEGG database modules respectively, and the hypergeometric distribution model Thirty male C57BL/6 mice, SPF grade, 8 weeks old, were provided by Shanghai SLAC Laboratory Animal Co., Ltd, and reared in the Animal Experiment Center of Shanghai University of TCM . All animal experiments were approved by the Ethics Committee of Shanghai University of TCM (Ethics No: PZSHUTCM200110003). Feeding environment of mice is as below: indoor standard clean, standard temperature 22 ± 2 °C, humidity 55% ~ 65%, light/dark cycle 12h/12h, ventilation cage regular autoclave, regular replacement of pad material.QGHXR consists of Bupleurum abchasicum Manden., Scutellaria baicalensis Georgi, Salvia miltiorrhiza Bge, Trionyx sinensis Wiegmann and Pueraria lobate (Willd.) Ohwi . QGHXR were concentrated and dissolved to 7.41 g/kg by using double distilled water. In our previous study, the dose has been explored and verified through experiments There were 10 mice in each group . According to the mouse model of chronic and binge ethanol feeding (the NIAAA model) reference Mice were given 1% pentobarbital intraperitoneally and blood was collected by removing the eyes. The whole blood was kept at 4 °C for 3 hours for 2775 g/min, 15 min, and the supernatant was used to detect serum TG, TC, ALT and AST by Japanese Toshiba TBA-40FR automatic biochemical analyzer.After animal blood collection, the mice were sacrificed by cervical isolation. The fresh liver was removed and then stored in the -80 °C refrigerator. 50 mg of liver tissue from each liver tissue sample of the three groups were cut into a 1.5 ml centrifuge tube, and then adding 2-3 magnetic beads and 500 μl of tissue homogenate (ethanol: acetone =1:1) were added. Next, 1.5 ml centrifuge tubes were placed in an ultrasonic homogenizer at 60 Hz, 60s, and then 3 times, in 4 °C refrigerators overnight. The next day, after centrifugation at 2775g/min for 15 min, 150 μl of supernatant was used for assays. The supernatant was detected by Toshiba TBA-40FR automatic biochemical analyzer. Two pieces of 1cm * 1cm liver tissue were cut and fixed in 4% paraformaldehyde, dehydrated and embedded into paraffin sections for hematoxylin-eosin staining (HE). Frozen liver sections were stained with oil red. The images were collected by an optical microscope .According to the RNA extraction kits, the total RNA was extracted from 50mg liver tissue of each sample , and the total mRNA was reverse transcribed by one-step reverse transcription kit to synthesize cDNA. The expression level of each target gene was detected by fluorescence quantitative PCR amplification with SYBR Green Master Mix. The primer sequence is mPTEN: Forward Primer 5'-AAGACCATAACCCACCACAGC-3', Reverse Primer 5'-CCAGTCCGTCCCTTTCCAG-3', 124 bp; mPI3K: Forward Primer 5'-AAGCCATTGAGAAGAAAGGACTG-3', Reverse Primer 5'-ATTTGGTAAGTCGGCGAGATAG-3', 176 bp; mAKT: Forward Primer 5'-TGTCTGCCCTGGACTACTTGC-3', Forward Primer 5'-GGCGTTCCGCAGAATGTC-3', 166 bp; m β-actin: Forward Primer 5'-GAGACCTTCAACACCCCAGC-3', Reverse Primer 5'-ATGTCACGCACGATTTCCC-3', 263bp. The reaction conditions are as follows: 95 °C predenatured for 5 min, cycling once; 95 °C denatured for 10s, 60 °C annealed for 20s, 72 °C extended for 20s, and 40 cycles.t test. The p < 0.05 was considered statistically significant.All data are expressed as mean ± SEM. GraphPad Prism 6 was used for statistical analysis. Statistical comparisons of multiple groups were performed by one-way analysis of variance (ANOVA). Comparisons between two groups were performed using two-tailed unpaired Student's Supplementary Table S1).According to the results of TCMNPAS database, there were 101 active compounds in Bupleurum abchasicum Manden., 55 active compounds in Scutellaria baicalensis Georgi, 47 active compounds in Salvia miltiorrhiza Bge and 9 active compounds in Pueraria lobate (Willd.) Ohwi. But Trionyx sinensis Wiegmannis was not included in the database. Different Chinese herbs had common gene targets. In order to count how many kinds gene targets of the four Chinese herbs, the same gene targets were counted once. The results showed that there were 180 active compounds and 29 active compounds overlapped in different drugs. A total of 618 genes were obtained in QGHXR after the repetition was removed  238 compound GO pathways and 170 disease GO pathways Figure , and based on cellular components (CCs) 112 compound GO pathways and 93 disease GO pathways Figure . Comprehensive GO enrichment analysis showed that the potential targets of QGHXR and disease targets showed co-correlation at the GO level, and the GO similarity between QGHXR and ALD genes was 74.23%.GO enrichment analysis and KEGG pathway analysis were performed for relevant gene targets of QGHXR and ALD obtained from TCMNPAS database, with p <0.05. A total of 2592 compound GO pathways and 1814 disease GO pathways were obtained based on biological processes (BPs) Figure -S9. The top ten common signal pathways were obtained according to GeneRatio Table , and 4 related signal pathways were screened. These were Neuroactive ligand-receptor interaction, Lipid and atherosclerosis, Alzheimer's disease, and PI3K/AKT signaling pathway Figure B. The similarity of KEGG signal pathway associated with QGHXR and ALD was 89.1%.KEGG pathway analysis showed that there were 133 common signal pathways between QGHXR and ALD  Figure A-F.It was shown that serum ALT, AST, TG, TC, liver TG levels were significantly increased in EtOH-fed group mice compared with pair-fed group mice. QGHXR significantly reduced serum ALT, AST, TC and liver TC in the EtOH-fed group mice, but had no significant effect on serum TG and liver TG levels Figure G.HE staining showed that compared with pair-fed group, steatosis with large vesicular fat droplets and hepatocyte necrosis appeared in EtOH-fed group, and more inflammatory cell infiltration could be seen in hepatic sinusoid, while EtOH-fed+QGHXR group had obvious improvement effect. Oil red O staining showed that there were much lipid droplets in the liver tissue of EtOH-fed group mice with hepatocyte expansion, indicating the existence of liver injury. Compared with EtOH-fed group, the pathological morphology of liver tissue in EtOH-fed+QGHXR group was obviously improvedFigure H-J.Compared with the pair-fed group, the PTEN mRNA expression had a downward trend in EtOH-fed group and the mRNA level of PI3K and AKT had an increasing trend, but there was no significant statistical significance. Compared with EtOH-fed group, the level of PTEN mRNA was higher in EtOH-fed+QGHXR group, while the mRNA of PI3K and AKT obviously decreased, but PTEN did not show statistical significance Excessive and chronic alcohol intake can lead to many problems that affect a variety of physiological systems, including the immune, nervous, cardiovascular and digestive system Bupleurum abchasicum Manden., can protect the liver in alcoholic hepatitis mice with anti-inflammatory and anti-oxidation effects Scutellaria baicalensis Georgi, alleviates alcoholic liver injury through inhibiting oxidative stress, inflammatory response and regulating sonic hedgehog pathways Salvia miltiorrhiza Bge, can inhibit oxidative stress and inflammatory response, and alleviate liver fibrosis mainly by regulating Nrf2/HO-1 and NF-κB/IκBα signaling pathways Pueraria lobate (Willd.) Ohwi, can improve liver glucose and lipid metabolism by inhibiting oxidative stress and inflammation and activating AMPK signaling QGHXR is composed of five traditional Chinese drugs, among which saikosaponin D, the main active ingredient of Figure .In this study, the mechanism of action between QGHXR and ALD was explored by network pharmacology. A total of 180 active components and 618 potential targets of QGHXR and 316 potential targets of ALD were obtained by using TCMNPAS database. Network maps and PPI maps of drugs-active components-targets-disease were constructed using STRING, Cytoscape and other tools. In addition, QGHXR and ALD were enriched by GO and KEGG signal pathways, and the results were based on BP 2592 compound GO pathway and 1814 disease GO pathways, MF 238 compound GO pathways and disease GO pathways, CC 112 compound GO pathways and 93 disease GO pathways. KEGG pathway analysis showed that there were 133 common signal pathways between QGHXR and disease, and the top 10 common signal pathways were obtained according to Gene Ratio, and 4 related signal pathways were screened. They are neuroactive ligand-receptor interaction, lipid and atherosclerosis, Alzheimer's disease and PI3K/AKT signaling pathway. Then, the PI3K/AKT signal pathway was verified in ALD mice. The results indicated that QGHXR could obviously reduce serum ALT, AST, TC and liver TG level, but had no obvious influence in serum TG and liver TC level. At the same time, it can reduce liver lipid droplets, inflammation and hepatocyte injury in ALD mice. More importantly, compared with EtOH-fed group, the mRNA level of PTEN was higher, while the mRNA level of PI3K and AKT apparently decreased in EtOH-fed+QGHXR group in vitro cell experiments, and it is better to further verify other signal pathways. Finally, we are temporarily unable to select the active components of QGHXR and carry out efficacy verification and mechanism exploration from in vivo or in vitro experiments.However, there are some limitations in our present study. First of all, because the database did not collect the research data of Trionyx sinensis Wiegmann, we failed to enrich the active components and gene targets of Trionyx sinensis Wiegmann. Secondly, we did not further verify this pathway at the protein expression level and in vivo, which may be carried out through PI3K/AKT signal pathway, which provides a research basis for subsequent work. In the future research, we will focus on the following aspects. First, we will continue to verify the protein expression of PI3K/AKT pathway. Second, in vitro cell experiments will be performed in order to make the research more scientific and rigorous. Third, it would be continued to further explore other signal pathways of QGHXR and ALD. Finally, it is suggested to further analyze the active components or mnonomers of QGHXR and explore their efficacy and mechanism. It is hoped that through more research, we can understand the clear mechanism and develop new treatments for ALD.To sum up, this study revealed the mechanism of QGHXR improving ALD through multi-pathway and multi-target way, and proved that QGHXR can improve ALD through animal experiments Supplementary tables.Click here for additional data file.


Toxoplasma gondii is poorly understood.Adenine nucleotide translocase (ANT) is a protein that catalyzes the exchange of ADP/ATP across the inner mitochondrial membrane. Beyond this, ANT is closely associated with cell death pathways and mitochondrial dysfunction. It is a potential therapeutic target for many diseases. The function of the ANT in T. gondii. We constructed T. gondii ANT transgenic parasite lines, including endogenous tag strain, knockout strain and gene complement strain, to clarify the function and location of TgANT. Mitochondrial morphology was observed by immunofluorescence and transmission electron microscopy.The CRISPR/CAS9 gene editing tool was used to identify and study the function of the ANT protein in Toxoplasma gondii was found to encode an ANT protein, which was designated TgANT. TgANT localized to the inner mitochondrial membrane. The proliferation of the Δant strain was significantly reduced. More important, depletion of TgANT resulted in significant changes in the morphology and ultrastructure of mitochondria, abnormal apicoplast division and abnormal cytoskeletal daughter budding. In addition, the pathogenicity of the Δant strain to mice was significantly reduced.T. gondii. Depletion of TgANT inhibited parasite growth and impaired apicoplast and mitochondrial biogenesis, as well as abnormal parasite division, suggesting TgANT is important for parasite growth.Altogether, we identified and characterized the ANT protein of The online version contains supplementary material available at 10.1186/s13071-022-05295-7. Toxoplasma gondii is an obligate intracellular parasite known to infect humans and all other warm-blooded animals  of TgANT = 0.885)  in the ToxoDB database (ToxoDB), which we named TgANT. The prediction of Pfam database (Pfam: Search Pfam [xfam.org])showed that TgANT contained three mitochondrial carrier protein domains  and actin as controls. CytC was located in the intermembrane space of mitochondria and was fully protected from PK digestion without Triton X-100 permeabilization, whereas actin was digested by PK even without the addition of detergent. TgANT, like CytC, had to be treated with detergent to be digested by PK, indicating that TgANT was likely to be associated with the IMM  by double homologous recombination in the RHΔku80 strain  and broken (approx. 27%)  had been used in previous studies to examine daughter scaffold formation in T. gondii [ku80, daughter cell budding was asynchronous in some parasitophorous vacuoles (PVs)in Δant (approx. 45%), suggesting that deletion of the TgANT led to abnormal cell division . The survival curve showed that all mice infected with 100 RHΔku80 tachyzoites died within 7 days, while mice receiving 100 Δant tachyzoites survived for 21 days. Importantly, the complementation of TgANT rescued the pathogenicity to mice  system [In mammals, ANT has been reported to be involved in regulation of the cell death pathway and to be a critical target of apoptosis induction by NO . The inheviously , 26. We ) system , but becS. cerevisiae, the mutation of ANT results in disruption of mitochondrial morphology [T. gondii, depletion of TgANT led to defects in mitochondrial morphology. To further explore the ultrastructure of mitochondria in the Δant strain, we used transmission electron microscopy. The results showed that depletion of TgANT resulted in loss of density and a reduced number of mitochondrial cristae. However, we could not determine whether the morphological changes of mitochondria in the Δant strain was an indirect effect or merely due to ant gene deletion. Further studies are required to determine how TgANT affects mitochondrial morphology.In rphology . In T. gT. gondii also harbors another endosymbiotic organelle, the apicoplast, which is closely related to the mitochondrion [Toxoplasma gondii.In addition to the mitochondrion, hondrion . We susphondrion , 20. We T. gondii encodes an ANT protein (TgANT), which localizes to the IMM. TgANT is crucial for the proliferation of T. gondii and is required to maintain the morphology of mitochondria. Depletion of TgANT impaired apicoplast and mitochondrial biogenesis, as well as abnormal parasite division.In conclusion, we found that Additional file 1: Table S1. Primers used for this study.Additional file 2: Figure S1.a protein sequence alignment of TgANT using MEGA7 from Toxoplasma gondii, Saccharomyces cerevisiae, Plasmodium falciparum, Cyclospora cayetanensis, Arabidopsis thaliana, Cardiosporidium cionae, Homo sapiens and other species. b Prediction results of TMHMM Server v.2.0 showed that TgANT has four transmembrane domains.Additional file 3: Figure S2. Identification of the iΔant gene complementation strain. a PCR1 and PCR2 were used to amplify the 5′ homologous recombination fragment (1890 bp) and the 3′ homologous recombination fragment (1218 bp), respectively. Lanes 2 and 5 are the experimental group; lanes 1 and 4 are the negative control group; lanes 3 and 6 are the blank control groups. b IFA assay. GAP45 (green) marks the outline of the parasite; red indicates the target protein TgANT; DPAI (blue) marks the nucleus. Scale bar: 2 μm.


Bushao Tiaozhi Capsule (BSTZC) is a novel drug in China that is used in clinical practice and has significant therapeutic effects on hyperlipidemia (HLP). In our previous study, BSTZC has a good regulatory effect on lipid metabolism of HLP rats. However, its bioactive compounds, potential targets, and underlying mechanism remain largely unclear. We extracted the active ingredients and targets in BSTZC from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and literature mining. Subsequently, core ingredients, potential targets, and signaling pathways were determined through bioinformatics analysis, including constructed Drug-Ingredient-Gene symbols-Disease (D-I-G-D), protein–protein interaction (PPI), the Gene Ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Finally, the reliability of the core targets was evaluated using in vivo studies. A total of 36 bioactive ingredients and 209 gene targets were identified in BSTZC. The network analysis revealed that quercetin, kaempferol, wogonin, isorhamnetin, baicalein and luteolin may be the core ingredients. The 26 core targets of BSTZC, including IL-6, TNF, VEGFA, and CASP3, were considered potential therapeutic targets. Furthermore, GO and KEGG analyses indicated that the treatment of HLP by BSTZC might be related to lipopolysaccharide, oxidative stress, inflammatory response and cell proliferation, differentiation and apoptosis. The pathway analysis showed enrichment for different pathways like MAPK signaling pathway, AGE-RAGE signaling pathway in diabetic, IL-17 signaling pathway and TNF signaling pathway. In this study, network pharmacology analysis, and experiment verification were combined, and revealed that BSTZC may regulate key inflammatory markers and apoptosis for ameliorating HLP. S), type II diabetes mellitus, hypertension, and atherosclerosis2. The concentrations of lipids, such as triglycerides (TG), total cholesterol (TC) and low-density lipoprotein (LDL) increase, or the levels of high-density lipoprotein (HDL) decrease in the blood3.Hyperlipidemia (HLP) is a major risk factor for different cardiovascular diseases (CVDMicrocos paniculata L.), Paeoniae Radix Rubra , Curcumae Rhizoma , and Andrographis Herba  Nees). Our previous clinical studies have shown that BSTZC was effective and relatively safe in the treatment of HLP, with no obvious adverse events. Compared with the rat models with HLP of group, BSTZC significantly reduced the serum TC, TG, LDL-C and ApoB levels, improved the HDL-C and ApoA1 levels and ApoA1/ApoB ratio, reduced the hepatic TC and TG levels and promoted hepatic LCAT and LXR-α gene expression . BSTZC has a good regulatory effect on lipid metabolism of HLP rats4. Moreover, the chemical profile of BSTZC has been fully investigated using a UPLC–TOF–MS/MS method, a total of 53 chemical constituents were identified by literature comparison, and high mass spectrometry data analysis. The chemical constituent cluster was composed of 21 flavonoids, 10 phenolics, 5 monoterpene glycosides, 7 diterpene lactones and 10 sesquiterpenes, the identified chemical components mostly cover the main constituents of each medicinal material in the formula5. BSTZC has a significant effect on HLP, similar to other TCM formulae, BSTZC involves multiple components, targets, and pathways treatment of HLP. However, its core active ingredients and their potential mechanism of action have not been fully elucidated.Bushao Tiaozhi Capsule (BSTZC) is composed of Microctis Folium . Fenofibrate was purchased from Abbott Laboratories Limited (Lot#27232). Quercetin , kaempferol , baicalein , isorhamnetin , luteolin , and wogonin  were purchased from Ruifensi. Ltd.Chengdu, China.10. The 54 male C57BL/6 mice weighing between 18 and 22 g were obtained from the Guangdong Medical Laboratory Animal Center . All the mice were fed normal diet and housed in barrier system at standard room temperature and a 12 h light/dark cycle conditions. The experiment mice were divided into the following nine groups: control group, model group, fenofibrate group (26 mg/kg), wogonin group (25 mg/kg), baicalein group (25 mg/kg), kaempferol group (25 mg/kg), luteolin group (25 mg/kg), quercetin group (25 mg/kg) and isorhamnetin group (25 mg/kg). The administration groups were given corresponding drugs by gavage, once a day, for 5 days. On the third day of administration, except for the normal control group, all the mice in the other groups were intramuscularly injected with triton-1339  to induce acute hyperlipidemia model. At the end of the experimental period, all mice were anesthetized using isoflurane and sacrificed by inner canthus artery exsanguination, then the organs were reserved for analysis.All experiment procedures were compiled with the NIH recommendations for the use and care of animals. The animal experimental protocols were reviewed and approved by the animal ethics committee of Guangdong Provincial Engineering Technology Institute of Traditional Chinese Medicine , and all animal experiments were performed in accordance with relevant ARRIVE guidelineshttps://tcmspw.com/tcmsp.php) and literature mining were searched to collect the related active compounds of BSTZC. Oral bioavailability (OB) is one of the most important pharmacokinetic parameters, it represents the ability of a drug to enter the circulation. Drug-likeness (DL) indicates the similarity between a molecule and known drugs. OB and DL were used as the main parameters to screen the active ingredients, according to absorption, distribution, metabolism, excretion (ADME) criteria OB ≥ 30% and DL ≥ 0.18 were screened for eligible ingredients13.TCMSP (https://go.drugbank.com/) of active components of BSTZC were predicted on TCMSP platform and transformed the target name to standard gene name on Uniport (https://www.uniprot.org/) database, and then removed the duplications.TCMSP database was applied in this work. Related targets  and GeneCards (https://www.genecards.org/) database, with “hyperlipidemias” and “hyperlipidemia” as keyword. The genes from the above databases merged and removed the duplications.HLP related genes were collected from CTD (https://bioinfogp.cnb.csic.es/tools/venny/) was used to find out the overlapped targets between compound targets and disease targets. To explore the relationship between D-I-G-D more reasonably, Cytoscape 3.7.1 (https://cytoscape.org/) with visualized tool and all node degrees of networks were calculated. The color and node size scale were used to explain the whole network based on the number of edges. The node with the maximum number of edge count was indicated with large node.Venny 2.1.0 (https://string-db.org/), the screening condition used was “Homo sapiens”, and the results were saved. The resulting file was imported into Cytoscape v3.7.1 software, and BisoGenet and CytoNCA plug-in in Cytoscape were used to calculate the degree centrality (DC), betweenness centrality (BC), and closeness centrality (CC). The core target of the protein–protein interaction (PPI) network was filtered14.Overlapping targets of compound -disease were added into STRING (https://cran.r-project.org/src/base/R-4/) software to conduct conducted the Gene Ontology (GO) biological process enrichment analysis and Kyoto Encyclopedia of Genes and Genomes data obtained (KEGG) pathway enrichment with p < 0.05 and q < 0.05 as the thresholds17.We imported the core targets on the Bioconductor ClusterProfiler, org.Hs.eg.db and DOSE packages of R 4.0.2  with commercial kits from Nanjing Jiancheng .−ΔΔCt method, with 18 s as a reference in the mRNA analysis. The primers were shown in Table Liver samples of animal experiments were obtained by mechanically homogenized in ice water bath and centrifuged for 10 min at 2500 rpm, then the supernatant was collected. Total RNA from liver tissues was extracted using Trizol reagent  and reverse transcribed. RT-qPCR reactions was performed on iQ5 Multicolor Real-Time PCR detection system  with SYBR Green Dye detection . The data were analyzed using the 2p < 0.05 was considered statistically significant. All the results were expressed as mean ± SEM.All the grouped data were statistically evaluated with the SPSS 22.0 software. Kruskal–Wallis test and one-way ANOVA followed by LSD post hoc test were used to determine the significance of the differences between the groups. The value of 20, the molecular structure of each active compound was confirmed by TCMSP database. According to the screening threshold , BSTZC was found to be comprised of 43 compounds  Fig. . PPI net21. In total, there were 1715 GO biological process and 145 KEGG pathway enrichment results. The results showed that GO biological processes were related to the treatment of HLP, and included response to: cellular response to chemical stress(GO:0062197), response to oxidative stress (GO:0006979), regulation of DNA-binding transcription factor activity (GO:0051090), cellular response to oxidative stress(GO:0034599), response to reactive oxygen species(GO:0000302), and response to lipopolysaccharide (LPS) (GO:0032496). The bubble chart of the top 20 significant enrichment results in the GO analysis is shown in Fig. To further investigate the effector mechanism of BSTZC in the treatment of HLP, the 26 core target genes screened in PPI network were analyzed by GO biological process enrichment and KEGG pathwayressGO:004599, resTo investigate the effect of core active ingredients on lipid metabolism and hepatoprotective, we analyzed the biochemical profiles of serum. The result was shown in Fig. Excessive lipid accumulation in liver was an important factor inducing the vascular inflammation. To further elucidate the mechanism of BSTZC on hyperlipidemia, the inflammation and apoptosis related genes were detected. Figure 24. We established triton-1339W-induced hyperlipidemia mice model, supplied with core active ingredients of BSTZC, in order to confirm its anti-HLP effect. The results showed that TC, TG, LDL-C were significantly reduced and HDL-C was significantly increased by the quercetin and isorhamnetin groups. The other four core active components also had a certain effect on lowering blood lipid level, which indicated that BSTZC can improve the lipid level of riton-1339W-induced hyperlipidemia mice. Quercetin has been studied for antioxidant, anti-inflammatory, lowering blood lipid, and anti-atherogenic properties effect, after quercetin treatment, the content of TC, TG, LDL and free fatty acid in HLP rats can be decreased26. Kaempferol is a flavonol that has been shown to regulate cellular lipid and glucose metabolism27. Isorhamnetin can protect vascular endothelial cells, inhibit adipose differentiation, inhibit vascular endothelial cell proliferation, anti-inflammatory and other effects. Moreover, it could significantly inhibited LPS-induced TNF-α, IL-1β and IL-6 secretion both in vitro and in vivo28. Baicalin had anti-inflammatory effects on chronic inflammation-related diseases such as CVDs and inflammatory bowel disease29. Luteolin can restore vascular endothelial NO availability in high fat diet mice, and prevent obesity-associated systemic metabolic alterations and vascular dysfunction through antioxidant and anti-inflammatory mechanisms30. Consequently, they may probably be core active compounds of BSTZC against HLP.In previous studies, it has been shown that BSTZC possessed beneficial effects in the treatment of HLP, however, due to multiingredient and multitarget characteristics of Chinese medicine, the specific underlying mechanism of the pharmacodynamic effects is still unclear. Therefore, a network pharmacology approach was used to investigate the underlying mechanism of action of BSTZC in the treatment of HLP. Based on the D-I-G-D network combined with literature studies, we found that quercetin, kaempferol, wogonin, isorhamnetin, baicalein, and luteolin played major roles in this network. Meanwhile, we found the six core active compounds were all flavonoids, which have been reported to have lipid-lowering, anti-inflammatory, and antioxidant effects33. IL-6, VEGFA, TNF, and IL-1β are common proinflammatory factors. Overexpression of inflammatory mediators (such as cytokines and chemokines) leads to increased vascular permeability and a series of cascade reactions. Inflammatory responses can stimulate the oxidative stress response of adipocytes, affect their biological processes, such as proliferation and differentiation, and insulin sensitivity, and then lead to dyslipidemia and further accumulation of fat. IL-6, as a proinflammatory factor, affects the expression of other proinflammatory cytokines and chemokines in the early stage of inflammation, amplifying the inflammatory response35. IL-6 and IL-1β, as important mediators of inflammatory response, can aggravate dyslipidemia36. Vascular endothelial growth factor A (VEGFA) is an angiogenic factor in adipose tissue, and plays a key role in the regulation of angiogenesis in adipose tissue, when VEGFA is down-regulated expression, it inhibits adipose tissue angiogenesis, thereby reducing adipose tissue formation37. IL-6 could induce the up-regulation of VEGFA expression, accelerate the migration of vascular endothelial cells, increase vascular permeability, and induce angiogenesis38. TNF-α is an important regulator of inflammatory response and immune function39, which could induce the production of IL-1 and IL-6, and participate in systemic inflammatory response41. Furthermore, studies have shown that CASP3 plays a crucial role in the execution of cell apoptosis42. When HLP occurs, pancreatic cell damage activates CASP3 and leads to pancreatic cell apoptosis. At the same time, considering that HLP is often accompanied by fatty liver, oxidative stress increases the production of reactive oxygen species (ROS), leading to lipid peroxidation, and then leads to the production of TNF-α, IL-8 and other factors, which destroys the normal function of cells and leads to inflammation and apoptosis44. Enrichment analysis of GO and KEGG pathways on 26 core targets was performed, and we obtained 1715 GO biological processes and 145 KEGG pathways. Among them, GO function enrichment results were mostly related to oxidative stress response, ROS response, and lipopolysaccharide response. We speculated that the response to lipopolysaccharide and oxidative stress may be the most important biological process of BSTZC in the treatment of HLP. KEGG pathway enrichment results showed explicated HLP effect were involved in pathways including inflammatory response according to MAPK signaling pathway, AGE-RAGE signaling pathway in diabetic, IL-17 signaling pathway and TNF signaling pathway, and inflammation was highly related to blood lipid45. HLP plays a causal role in the development of CVDs due to endothelial dysfunction46, it may be associated with inflammatory response47. Meanwhile, dyslipidemia can be significantly improved by ameliorating inflammation48. MAPK signaling pathway played a crucial role in inflammation49, and participate in various biological processes and the regulation of nuclear factor κB (NF-κB) transcriptional activity. The stimulation of NF- κB increased the levels of downstream inflammatory factors such as TNF-α, IL-1β, and IL-6. MAPKs cascade can transfer extracellular signal to intracellular, thereby causing biological changes such as cell apoptosis53. AGE-RAGE signaling pathway plays a critical role in recruiting macrophages in inflammation and inducing oxidative stress. Hyperglycemia can activate age-rage signaling pathway, accelerate the synthesis of AGEs, affect the integrity of vascular wall by changing the interaction between matrix and cells, and promote the occurrence and development of vascular and neuropathy. Furthermore, it could lead to many complications of nonalcoholic fatty liver disease, including inflammation, fibrosis and insulin resistance55. IL-17 signaling pathway is related to the accumulation of proinflammatory chemokines and neutrophils, and plays a role in immune and autoimmune diseases56. It has been confirmed that IL-17 can stimulate cell production of TNF-α and IL-6, and TNF-α and IL-6 activate NF-κB by down-regulating TLR2-mediated ERK1/2 phosphorylation, resulting in further effects58. TNF signaling pathway was an important pathway in inflammation response, TNF activates the canonical NF-κB by regulating TNF receptors and its downstream signaling molecules60. Hence, we speculated that BSTZC may treat HLP through multiple targets and routes through regulating the level of oxidative stress, inhibiting inflammation and cell apoptosis.The PPI network showed that 26 core targets including IL-6, TNF, VEGFA, CASP3, and IL-1β were probably the most relevant targets for BSTZC in the treatment of HLPP < 0.05, P < 0.01) in the model group. These results indicated that HLP can cause the activation of these 4 factors, and their overexpression can lead to inflammatory response and promote cell apoptosis. However, compared with the model group, The quercetin and isorhamnetin groups significantly reduced the elevated levels of IL-6, IL-1B, VEGFA and CASP3 mRNA in model mice (P < 0.01). These results indicate that BSTZC can reduce the release of inflammatory mediators and inhibit cell apoptosis, thereby ameliorating HLP. The results of validation test are also consistent with the prediction results of network pharmacology, and it shows that this method has certain accuracy in screening the action target of BSTZC.Therefore, IL-6, IL-1β, VEGFA and CASP3 were selected as candidate targets of BSTZC against HLP. In vivo experiment, compared with the control group, the contents of IL-6, IL-1β, VEGFA and CASP3 mRNA were significantly increased (In the current study, an integrated strategy was first developed to explore the core active compounds and molecular mechanisms of BSTZC treats of HLP by network pharmacology analysis and in vivo experimental validation. Quercetin, kaempferol, wogonin, isorhamnetin, baicalein, and luteolin are the core active ingredients of BSTZC, which can regulate most targets related to HLP. Through PPI network screening, we found that 26 core targets including IL6, TNF, VEGFA, CASP3, and IL-1β may provide new ideas for the treatment of HLP. The MAPK signaling pathway, AGE-RAGE signaling pathway in diabetic, IL-17 signaling pathway and TNF signaling pathway were identified as the potential mechanism of BSTZC against HLP. The present work offered the convincing evidence that BSTZC may treat HLP by inhibiting inflammation and apoptosis, and provided the experimental basis for the clinical application of BSTZC in treating HLP (Supplementary Supplementary Table S1.


Type 2 diabetes (T2D) is a common chronic disease with many serious complications. Celastrol can prevent and treat type 2 diabetes by reversing insulin resistance in a number of ways. However, the specific mechanisms by which celastrol prevents and treats T2D are not well understood. The aim of this study was to explore the key gene targets and potential signaling pathway mechanisms of celastrol for the treatment of T2D.via the ClueGo plugin in Cytoscape and validated using Gene Set Enrichment Analysis. The therapeutic targets of celastrol were then analyzed by pharmacophore network pharmacology, intersected to identify the therapeutic targets of celastrol, enriched for all targets, and intersected to obtain the signaling pathways for celastrol treatment. The protein structures of the therapeutic targets were predicted using the artificial intelligence AlphaFold2. Finally, molecular docking was used to verify whether celastrol could be successfully docked to the predicted targets.GSE184050 was downloaded from the Gene Expression Omnibus online database. Blood samples from patients and healthy individuals with T2D were analyzed to identify differentially expressed genes (DEGs), and a protein−protein interaction network (PPI) was constructed. Key gene analysis of DEGs was performed using the MCODE plugin in Cystoscope as well as the Hubba plugin, and intersections were taken to obtain hub genes, which were displayed using a Venn diagram. Enrichment analysis was then performed 618 DEGs were obtained, and 9 hub genes for T2D were identified by the MCODE and Hubba plug-ins, including ADAMTS15, ADAMTS7, ADAMTSL1, SEMA5B, ADAMTS8, THBS2, HBB, HBD and HBG2. The DEG-enriched signaling pathways mainly included the ferroptosis and TGF-beta signaling pathways. A total of 228 target genes were annotated by pharmacophore target analysis, and the therapeutic targets were identified, including S100A11, RBP3, HBB, BMP7 and IQUB, and 9 therapeutic signaling pathways were obtained by an intersectional set. The protein structures of the therapeutic targets were successfully predicted by AlphaFold2, and docking was validated using molecular docking.Celastrol may prevent and treat T2D through key target genes, such as HBB, as well as signaling pathways, such as the TGF-beta signaling pathway and type II diabetes mellitus. Type 2 diabetes (T2D) is a chronic disease with a high prevalence worldwide , and theThe Gene Expression Omnibus (GEO)  is the mChinese medicine has been an important part of clinical practice in China for thousands of years. Numerous studies have shown the effectiveness of Chinese medicine in the treatment of various chronic diseases with minimal side effects . CelastrIn this study, we first screened the blood samples for DEGs using the Limma package and obtained the hub genes using 2 key gene analysis methods to obtain intersections. GO and KEGG enrichment of DEGs was performed to identify the signaling pathways enriched by DEGs, and the results were further validated using Gene Set Enrichment Analysis (GSEA) . PharmacThe GEO database is the largest comprehensive online database of gene expression, and the GSE184050 dataset was identified for download . The datFirst, two groups of samples were grouped according to the GSE184050 dataset, and analysis of variance was carried out in R (4.1.0) software using the “Limma” package, which is a commonly used analysis of variance package. Thresholds of |Log2(Fold Change)| > 0.5, p < 0.05 were used as screening conditions to identify significant DEGs.The STRING online database is a database for predicting protein−protein interactions. “Multiple protein” was selected from the STRING online database , and theCytoscape is a software program that graphically displays networks and performs analysis and editing. The ClueGo plug-in for Cytoscape (v3.9.0)  was usedThe 116 samples were divided into two groups, diabetic blood samples and healthy blood samples, which were analyzed for GO and KEGG enrichment by GSEA to enrich all genes expressed in the samples. The corresponding datasets were set up from the Molecular Signature Database. The T2D-related signaling pathways were validated against the enrichment results, and the validation results were graphically presented.The top 600 highest scores were used as screening criteria to predict celastrol targets using the pharmMapper database , the mosThe 228 target genes successfully analyzed by pharmacophore analysis and annotation were subjected to GO and KEGG enrichment analyses using Cytoscape’s ClueGo plugin to obtain the target signaling pathway for celastrol, which was then intersected with the signaling pathway enriched by DEGs and displayed using a Venn diagram to obtain the signaling pathway for celastrol in T2D.AlphaFold2 is an artificial intelligence program that predicts protein structures with an accuracy that is at the level of predictions observed by humans using sophisticated instruments such as cryoelectron microscopy.Five therapeutic target genes were obtained by taking the intersection of target genes of celastrol and DEGs of T2D, and the protein codes of the human species corresponding to the target genes were downloaded from the UniProt database , which iThe 2D structure SDF file of celastrol was downloaded from the PubChem database as a small-molecule ligand drug. The receptor was hydrogenated by AutoDocktools software  to conveR (4.1.0) was used for bioinformatics analysis, and the R package was used for statistical analysis. P < 0.05 was considered statistically significant.Dataset GSE184050 was downloaded from the GEO database. A total of 618 DEGs were obtained by differential analysis and displayed using volcano plots Figure 2The MCODE plug-in identified 5 important modules  and funded by the Outstanding Leaders Training Program of Pudong Health Bureau of Shanghai (Grant No. PWRL2019-01).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.


IGF1R appears to be an ideal therapeutic target in many cancers because it is a membrane protein and has receptor tyrosine kinase activity. Two principal types of inhibitors were initially used in trials: monoclonal antibodies IGF1R and small molecule tyrosine kinase inhibitors. However, outcomes in clinical trials were disappointing due to an overall infrequency of objective responses and a lack of predictable biomarkers. Thus, biomarkers should be identified to classify the patient response to anti-IGF treatment.The insulin and insulin-like growth factor (IGF) pathway have been documented to play an important role in physiological and pathological biological processes including many types of cancer. Typically, the IGF family has several types of proteins, such as receptors , ligands , ligand-binding proteins (IGFBP1-6), and IGFBP-specific proteases. Increased expression of many components of the IGF family has been invoked in carcinogenesis. The IGF axis has been implicated in many oncogenic processes through activating PI3K/AKT, and MAPK pathways. Moreover, IGF1R has also been associated with cancer metastasis through interacting with the integrin pathway in vivo, the overall change of substances and molecules were comprehensively analyzed, including the analysis of original pathways and the construction of new pathways, which can reflect the function and metabolic state of tissues and organs, and provide new ideas for exploring the molecular mechanism of biological regulation, key makers and action targets.Omics, including genomics, transcriptomics, proteomics and metabolomics, can provide a prerequisite for a comprehensive understanding of biological systems and mechanisms based on its high-throughput methods leading to a large amount of data. However, the single study for the changes of biomolecules in one certain level has been difficult to meet the rising expectations of systems biology. Therefore, the scientists propose to study the interaction and system mechanism systematically among multi-omics level through biological pathway analysis. In this method, the information of stress disturbance, pathophysiological state or changes after drug treatment can be obtained from genome, transcriptome, proteome, and metabolome experiments and enriched to trace the pathway with the largest and most concentrated changes. Through the analysis of genes, mRNA, proteins, and small molecules van der Velden et al. have identified a set of small molecules that reduces the activities of the growth hormone (GH)/IGF1 axis and its downstream effectors Jak2 and STAT5 by screening a 38480 compounds library. These small molecules also have the potential to inhibit GH-driven cancer growth and anti-cancer drug resistance in breast and colon cancer. Liu et al. used genetics and proteomics method and found that IGF2 receptor mRNA was remarkably upregulated in laryngeal cancer (LC). Moreover, a significant positive correlation between IGF2 receptor expression and tumor microenvironment, immunity, and prognosis. Liu et al. provided a summary analysis and a theoretical basis for the biological role and clinical value of the IGF binding protein (IGFBP) family in low-grade glioma (LGG), and they also found that IGFBP can be considered as the potential drug therapy targets of LGG. Lv et al. have found the association between IGF and lung cancer through large-scale clinical metabolomics. The relevant small molecule metabolites and metabolic pathways were also elucidated.In this Research Topic, we aim to encourage the authors finding new mechanisms of the IGF regulating the development of tumors from a clinical and/or molecular biological perspective especially under the integrating analysis of different omics. In the papers accepted after peer review in this Research Topic, the associations between IGF axis and multiple tumorigenesis, prognosis, and drug resistance have been found also with several possible mechanisms. In these papers, All authors listed have made a substantial, direct and intellectual contribution to the work and approved it for publication.


Cardiac injury induced by myocardial ischemic reperfusion (MI/R) is still an intractable question in clinical, and it has been confirmed as a major reason for the development of cardiovascular disease. Bioinformatics analysis has been widely used for revealing the pathogenic mechanism of diseases. This study attempted to identify the biomarkers and reveal the regulation mechanism of MI/R injury via bioinformatics analysis. p value <0.05 were identified as the differentially expressed genes (DEGs). The enrichment analysis of the DEGs was performed with the DAVID database and R language. Moreover, the protein-protein interaction (PPI) network of DEGs was performed with the STRING database and then visualized with Cytoscape.  The GSE67308 and GSE74951 were obtained from the GEO database. The datasets were analyzed with GEO2R tool, and the genes with |logFC| > 2 and κB, and TNF pathways. Besides, mmu-miR-92a-3p and mmu-miR-27b-3p were identified as the hubs miRNAs, and TNF, IL1B, and IFG1 were screened as the key nodes.  The results showed that 195 downregulated mRNAs and 240 downregulated mRNAs were found in GSE67308, and 11 miRNAs were found in GSE7495. 152 common genes were screened in DEGs of GSE67308 and the targets of 11 miRNAs in GSE7495. Moreover, the enrichment analysis showed that the common genes were related with inflammatory response, immune response, PI3K/AKT, NF- This study established a miRNA-mRNA network for cardiac injury induced by MI/R and provided the evidence concerning the molecular mechanism of MI/R injury, which provided some reference for MI/R treatment. Cardiovascular disease is a main risk of the human health in the world, and ischemia has been confirmed as a key reason leading the abnormal injury and apoptosis of cardiomyocyte , 2. MyocRecently, the difference in the RNA profiling of the patients suffered I/R injury and normal persons has been revealed by increasing studies, and some genes including microRNA (miRNA) and messenger RNA (mRNA) have also been confirmed as the key factors which are associated with the development of the symptom , 8. MicrThis study attempted to explore the effect of I/R surgery on the difference of the RNA profiling of mice, and the data of the RNA sequences were searched and obtained from the GEO database. Finally, the biomarkers and molecular mechanism of IR-myocardial injury were also revealed with bioinformatics methods.https://www.ncbi.nlm.nih.gov/). For GSE67308, the data of GSM1644168–GSM1644171 and the data of GSM1644172–GSM1644175 were selected for next analysis. For GSE74951, the data of 6 sham mice groups and 9 IR mice group were used for next analysis.The datasets including GSE67308 tested on GPL7202 and GSE74951 tested on GPL21136 were downloaded from Gene Expression Omnibus database .GSE67308 and GSE74951 were analyzed with GEO2R online tool of the GEO database, and the matrix file of the genes was obtained for subsequent analysis. Moreover, the genes with |logFC| > 2 and https://david.ncifcrf.gov/). After that, the packages including topGO, clusterProfiler, and org.Mm.eg.db of R language were performed to search the related functional modules of ENTREZIDs. Finally, modules with p value < 0.05 were visualized by R language. For GSE74951, the target of miRNAs with |logFC| > 2 and p value < 0.05 were obtained from miRWalk . After that, the potential targets were used for GO enrichment analysis.Gene ontology enrichment analysis was performed for investigating the functions of DEGs in GSE67308 and GSE74951. Briefly, ENTREZIDs of DEGs in GSE67308 were obtained from the DAVID database  enrichment analysis was performed to observe the related pathways of DEGs in GSE67308 and GSE74951. Simply, DEGs of GSE67308 were enriched by the online tool of DAVID database, and then, pathway modules with https://www.string-db.org/). After that, the key nodes of the datasets were visualized with Cytoscape software. For the miRNA-mRNA network, the common genes of DEGs in GSE67308 and the targets of GSE74951 were visualized with Cytoscape.The protein-protein interaction (PPI) network analysis was performed to select the key node genes in GSE67308 and GSE74951; DEGs of GSE67308 and the targets of GSE74951 were analyzed with STRING  can promote the inflammatory reactions and mediate abnormal apoptosis of cardiomyocytes and finally induce the injury or necrosis of myocardial tissues . MoreoveκB, chemokine signaling pathway, and so on, and the common genes of DEGs in GSE67308 and the targets of miRNAs GSE74951 also had connection with PI3K/AKT, NF-κB, and TNF pathways. The PI3K/AKT pathway serves as a classical avenue for cell survival and proliferation, and inhibited NF-κB, TNF, and toll-like receptor pathways have been also found to alleviate the MI/R injury [MI/R can mediate the succinate dehydrogenase activity which may promote the rapid oxidation of succinate and then induce the disorder of reactive oxygen species by reverse electron transport at mitochondrial complex I . ImpaireR injury . Thus, tβ can induce the inflammation to enhance the progression of MI/R [In this study, the genes including STAT1, IL1B, CCL2, TNF, TLR2, CXCL10, IL6, MMP9, CCL4, CXCL19, CXCL1, CD274, and CXCL2 were selected as the key nodes. For common genes, TNF, IL1B, and IFG1 were selected as the key nodes. TNF and IL-1 of MI/R . Accordi of MI/R . Moreove of MI/R .This study suggests mmu-miR-92a-3p, mmu-miR-27b-3p, and mmu-miR-370-3p as the biomarkers for MI/R injury, and mmu-miR-27b-3p promotes the progression of MI/R injury via targeting IFG1.


Noncoding RNAs play a critical role in various diseases such as diabetes, obesity, and liver diseases including MAFLD. However, there is no adequate knowledge about the translation of using these ncRNAs to the clinics, particularly in MAFLD conditions. The aim of this study was to identify ncRNAs in the etiology of MAFLD as a novel approach to the therapeutic targets.  Metabolic associated fatty liver disease (MAFLD) is a complex disease that results from the accumulation of fat in the liver. MAFLD is directly associated with obesity, insulin resistance, diabetes, and metabolic syndrome. PPAR We collected human and mouse MAFLD gene expression datasets available in GEO. We performed pathway enrichment analysis of total mRNAs based on KEGG repository data to screen the most potential pathways in the liver of MAFLD human subjects and mice model, and analyzed pathway interconnections via ClueGO. Finally, we screened disease causality of the MAFLD ncRNAs, which were associated with PPARs, and then discussed the role of revealed ncRNAs in PPAR signaling and MAFLD.  We found 127 ncRNAs in MAFLD which 25 out of them were strongly validated before for regulation of PPARs. With a polypharmacology approach, we screened 51 ncRNAs which were causal to a subset of diseases related to MAFLD.  This study revealed a subset of ncRNAs that could help in more clear and guided designation of preclinical and clinical studies to verify the therapeutic application of the revealed ncRNAs by manipulating the PPARs molecular mechanism in MAFLD. In hepatic steatosis, fat in the form of triglycerides and cholesterol esters accumulate in hepatocytes . NAFLD i disease –10.The cellular and molecular mechanisms involved in steatosis and MAFLD are not yet fully understood. However, evidence suggests that various factors, including inflammation of adipose tissue, hepatic lipogenesis, IR, mitochondrial abnormalities in the liver cells, and oxidative stress are involved in the progression of steatosis to fibrosis and cirrhosis –13.The liver controls fat homeostasis through complex interactions between hormones, transcription factors, and nuclear receptors. In MAFLD, fat is stored as triglycerides due to molecular pathways. Due to an imbalance between lipid absorption and lipid excretion, fat accumulates in hepatocytes. Pathways such as circulating lipid uptake, fatty acid (FA) oxidation, de novo lipogenesis, and fat export in very-low-density lipoprotein (VLDL) are impaired in MAFLD .In recent studies, a group of noncoding RNAs such as long noncoding RNA (lncRNAs), microRNAs (miRNAs), and circular RNAs (circRNAs) has attracted the attention of many researchers due to the regulation of gene transcription. These noncoding RNAs play a critical role in various diseases such as diabetes , obesitylncRNAs as a subgroup of ncRNAs with more than 200 nt lengths that modulate the post-transcriptional stages of genes of degradation, splicing, and translation of target genes . The othp-value<0.05 in the Benjamini & Hochberg test .Articles were searched on PubMed and Google Scholar from 2010 to 2022. The search was performed with the keyword fatty liver disease. We collected human and mouse fatty liver disease gene expression datasets available in Gene Expression Omnibus (GEO) , and reav6.8 (https://david.ncifcrf.gov/) based on Kyoto Encyclopedia of Genes and Genomes (KEGG) repository data to screen the most potential pathways in NAFLD mice liver. Then, we constructed and visualized the network via the ClueGO app, along with analyzing for protein-protein interactions by Cytoscape software, using interaction scores from STRING DB v11.0 set the threshold to 0.4 interaction score. The flowchart of the study is shown in We performed pathway enrichment analysis of total mRNAs using Database for Annotation, Visualization, and Integrated Discovery (DAVID) In this study, data mining and bioinformatics analyses were used to evaluate if noncoding RNAs associated to MAFLD also target PPARs. We collected and integrated a list of strongly validated associations of ncRNAs and MAFLD. Then, LncRRIsearch database was used to analyze data of lncRNA-mRNA interaction prediction . With a https://www.cuilab.cn/hmdd) and LncRNADisease v2.0 (http://www.rnanut.net/lncrnadisease). We set screening criteria including experimental detection method, strong validation assays , a score of 0.5 to 1.0, and excluded noncausal associations. These results could help in more clear and guided designation of preclinical and clinical studies to verify the therapeutic application of the revealed ncRNAs.Finally, we screened disease associations of the common ncRNAs between MAFLD and PPARs using HMDD v3.2 (https://circinteractome.nia.nih.gov/), and lncRNAs-mRNAs interactions in LncRRIsearch databases. We selected a subset of top 100 interactions among those that showed above 75% of total score. Then, we applied CytoScape 3.6.1 software to construct and analyze the interactions network based on Degree Centrality parameter.Finally, the resulted causal ncRNAs were used to construct a network for representation of their interactions with the analyzed genes of fatty liver disease. We collected all mRNAs interacting with the causal ncRNAs by the seed region complementarity algorithm of miRNAs-mRNAs interactions in TargetScan Human 7.0 database, CircRNAs-mRNAs interactions in Circ-Interactome  compared to low-fat (LF) diet groups. Moreover, GEO data analysis revealed 444 differentially expressed genes (DEGs) in human MAFLD, as well as 350 DEGs in mice MAFLD.We screened mRNAs from datasets GSE63067 and GSE85439, with differential expression in the liver using Limma package in GEO2R tool, based on criteria of log FC > 0.5, and adj The most powerfully enriched KEGG pathway in the human MAFLD data was PPAR signaling pathway. Also, this pathway was significantly enriched in mouse MAFLD data, demonstrating its significance in MAFLD molecular pathobiology. The top three KEGG pathways enriched in human MAFLD data included hsa03320 PPAR signaling pathway, FDR = 0.034; hsa04668 TNF signaling pathway, FDR = 0.0458; and followed by hsa04920 the adipocytokine signaling pathway, FDR = 0.0458 which are all associated with the pathobiology of MAFLD. Nevertheless, ClueGO analysis results of human data revealed that pathways corresponding to adipocytokine signaling pathway, insulin resistance, MAFLD, and T2DM have significant association via an Ontology gene connection with the PPAR signaling pathway as a hub node in the ClueGO pathway. Figures Moreover, ClueGO analysis results of mouse data revealed that pathways corresponding to peroxisome, AMPK signaling, Glycolysis/Gluconeogenesis, Prolactin signaling, and Biosynthesis of unsaturated FAs have major association via an Ontology gene connection with the central axis of the PPAR signaling pathway.A list of noncoding RNA associated with MAFLD were identified as 79 miRNAs , resistin, leptin, and adiponectin are secreted from adipose tissue. These molecules act as chemokines for macrophages to accumulate in the adipose tissue. Accordingly, obesity makes a chronic low-grade inflammatory condition that is considered a risk factor for liver diseases [Adipocytokines, including tumor necrosis factor-diseases . Adipocyα and PPARγ vastly function to reduce inflammation, while PPAR-beta(β) is a potential target for the treatment of insulin resistance [Recent studies revealed that there is a crosstalk between adipocytokine and PPAR signaling, in such a way that hepatic lipo-inflammation is modulated by adiponectin-activated PPARs and PPAR-induced adiponectin. The PPARsistance .γ agonists, rosiglitazone, and pioglitazone, which are reported to improve IR in diabetic patients [γ in adipocytes modifies target genes involved in fat uptake, lipid storage, the release of insulin-sensitive adipokines, and the production of inflammatory cytokines. PPARγ activation increases fat storage in adipose tissue and enhances insulin sensitivity [γ in obese ob/ob mice reduced hepatic triglyceride contents compared to control mice. In this condition, excess fat delivery to other tissues, including the striated muscles, leads to increased insulin resistance and the development of T2DM [γ expression in the liver of patients with MAFLD increases and activates the expression of adipogenic genes and exacerbates hepatic steatosis. It is noteworthy that clinical studies have shown that treatment of fatty liver with TZD reduces hepatic steatosis. The reduction in steatosis is due to the effects of TZD on adipose tissue, which prevents excess body fat from entering the liver and prevents the formation of dysfunctional fat cells. Increased adipose tissue formation following TZD is seen with weight gain in patients with TZ treated with fatty liver [Adiponectin activates hepatic adiponectin Receptor 2, then downstream AMP-activated protein kinase (AMPK) pathway is initiated, and triggers PPAR transcription factors. This results in the upregulation of genes responsible for ameliorating oxidative status, inflammation, and high levels of triglycerides . Furtherpatients . PPARγ isitivity . The res of T2DM . On the ty liver .α is expressed in tissues such as the liver and controls FA transport, hepatic glucose production, and FA metabolism. PPARα is a nutritional sensor that controls lipid metabolism in response to feeding and fasting [α regulates the transcription of genes involved in beta-oxidation, FA transport, and gluconeogenesis [α activation improves inflammation, fibrosis, and hepatic steatosis in MAFLD model mice. Therefore, PPARα agonists and their modulators are used as a strategy for the management and treatment of fatty liver [PPAR fasting . PPARα rogenesis . It alsoty liver .γ and PPARα before. Nevertheless, we constructed and analyzed a network consisting of liver disease causal ncRNAs interacting with MAFLD mRNAs.In this study, we also focused on the ncRNA network regulating the PPAR pathway. Interestingly, several ncRNAs associated with MAFLD have been validated by qRT-PCR for regulation of PPARMAFLD is a prevalent disorder and refers to a group of conditions where there is an accumulation of excess fat in the liver. MAFLD is directly associated with obesity, metabolic syndrome, and diabetes mellitus. Noncoding RNAs play a critical role in several diseases such as diabetes, obesity, and liver diseases, including MAFLD. The results of this study show that a subset of causal noncoding RNAs associated with PPARs can be used to treat MAFLD. This study could help in more clear and guided designation of preclinical and clinical studies to verify the therapeutic application of the revealed ncRNAs.