The heme pathway is essential for most of cellular life. In eukaryotic phototrophs, the entire pathway is plastid localized. Despite that, the enzyme responsible for the synthesis of hydroxymethylbilane, porphobilinogen deaminase, shows α-proteobacterial instead of expected cyanobacterial origins in rhodophytes, chlorophytes, plants, and most algae with complex plastid. However, no such enzyme has been found in the supposed partners of plastid endosymbioses, the heterotrophic eukaryotes, and cyanobacteria. I propose two scenarios explaining this phenomenon by either endosymbiotic gene transfer from the ancestor of mitochondria or a non-endosymbiotic lateral gene transfer from unspecified α-proteobacterium. Phylogenetic analysis of porphobilinogen deaminases does not reject any of the two proposed evolutionary scenarios.In most eukaryotic phototrophs, the entire heme synthesis is localized to the plastid, and enzymes of cyanobacterial origin dominate the pathway. Despite that, porphobilinogen deaminase (PBGD), the enzyme responsible for the synthesis of hydroxymethybilane in the plastid, shows phylogenetic affiliation to α-proteobacteria, the supposed ancestor of mitochondria. Surprisingly, no PBGD of such origin is found in the heme pathway of the supposed partners of the primary plastid endosymbiosis, a primarily heterotrophic eukaryote, and a cyanobacterium. It appears that α-proteobacterial PBGD is absent from glaucophytes but is present in rhodophytes, chlorophytes, plants, and most algae with complex plastids. This may suggest that in eukaryotic phototrophs, except for glaucophytes, either the gene from the mitochondrial ancestor was retained while the cyanobacterial and eukaryotic pseudoparalogs were lost in evolution, or the gene was acquired by non-endosymbiotic gene transfer from an unspecified α-proteobacterium and functionally replaced its cyanobacterial and eukaryotic counterparts. Phytomonas serpens, and several bacteria—that can live without heme [Paulinella chromatophora), all the enzymes of the pathway are encoded in the nucleus, expressed in the cytosol, and post-translationally targeted to their places of action. In phototrophic eukaryotes, enzymes of the original exosymbiont (eukaryotic host) pathway have mostly been lost, and the pathway has almost entirely been replaced by the plastid-located endosymbiont-derived metabolic route [The heme pathway is one of the metabolic routes essential for life as we know it. So far, we know of only a few organisms—the kinetoplastid parasite of plants, out heme . It has out heme ,3,4,5,6.out heme , chromerout heme , colpodeout heme , and theout heme . Likewisout heme ,3,8. In ic route ,4,5.Rodelphis [C. paradoxa (this work), a γ-proteobacterial enzyme in the euglenophyte Euglena gracilis [L. chlorophorum [P. chromatophora (this work).Porphobilinogen deaminase (PBGD) (syn. hydroxymethylbilane synthase) is the enzyme that catalyzes the third (eukaryotic heterotrophs) or fourth (eukaryotic phototrophs) step of the heme biosynthesis pathway. It converts porphobilinogen to hydroxymethylbilane, and this reaction is located in the cytosol of heterotrophs or the plastid of eukaryotic phototrophs, apicomplexan parasites , and Rododelphis . Plants,odelphis ,4,5. Thegracilis , an enzyA thorough inspection of the origins of PBGDs in the participants of plastid endosymbioses, particularly the heterotrophic eukaryotic exosymbiont (primary host) and cyanobacterial endosymbiont, found no gene encoding PBGD of α-proteobacterial origin. The enzyme from the primary host originates in an ancient eukaryotic nucleus . HoweverTM and aligned using Muscle [The amino acid sequences of PBGD from various organisms were downloaded from GenBankg Muscle  in SeaVig Muscle . The alig Muscle  and manug Muscle  . With a reduced dataset of 169 OTUs, the backbones of the Bayesian  trees weSeeking the origin of plastid-located PBGD in phototrophic eukaryotes requires an investigation of the endosymbiotic event preceding the acquisition of cyanobacteria, the supposed ancestor of primary plastids. It is evident that a eukaryotic cell participating in the plastid endosymbiosis in the role of an exosymbiont (primary host) passed, before the acquisition of the plastid, through the primary endosymbiotic event (or series of events) with a proteobacterium that subsequently evolved into the mitochondrion. Since heme is generally indispensable for the survival of any cell, participants in the “mitochondrial” endosymbiosis, a pre-eukaryotic cell and an α-proteobacterium, are presumed to have possessed their own tetrapyrrole biosynthetic routes. The first common precursor of the pathway, δ-aminolevulinate (ALA), can be synthesized by two different routes. The C4 pathway, synthesis of ALA by condensation of succinyl-CoA and glycine, catalyzed by ALA synthase, is present only in α-proteobacteria and the mitochondria of primary eukaryotic heterotrophs. All other organisms use the C5 pathway (synthesis of ALA from glutamate) as their ALA source ,4,5. We However, I should note here that no single enzyme involved in the heme pathway in eukaryotes shows direct phylogenetic affiliation to an archaeal counterpart . It partPaulinella chromatophora, which has the most recently acquired cyanobacterial endosymbiont, and still encodes 9 of the 10 enzymes involved in the autotrophic heme pathway in the “plastid” genome. Surprisingly, in the glaucophyte C. paradoxa only PBGD of likely cyanobacterial origin was found. The position of the glaucophyte gene in the tree was unstable when I constructed the tree by different methods  appeared equally related to mitochondria. In other words, some proteins of each α-proteobacterial lineage show close relationship to mitochondrial homologs. Similarly, work published by Abhishek et al. [Rhodospirillum rubrum and Rhodopseudomonas palustris. A recently published complex network approach [Rickettsiales.The second possible scenario interprets the presence of α-proteobacterial PBGD in eukaryotic phototrophs to be the result of a non-endosymbiotic transfer of the gene encoding PBGD from an α-proteobacterium B, insteadatasets ,3,4,8. Abacteria ,24,25,26bacteria  based onlleagues  suggestek et al.  demonstrapproach  suggestsRhizobiales and Rhodospirillales invariably represented the α-proteobacterial groups most closely related to the plastid-targeted PBGDs , shows α-proteobacterial origin in rhodophytes, chlorophytes, plants, and most algae with complex plastids. This origin is unexpected because there was no α-proteobacterial PBGD in the supposed partners of plastid endosymbiosis. Phylogenetic analysis cannot reject the mitochondrial origin of the gene and confirm the alternative hypothesis. The gene was either transferred from an ancestor of mitochondria when multiple redundant heme pathways were present in the endosymbiotic cell assembly, or it was obtained in a non-endosymbiotic gene transfer from α-proteobacteria. A single group of primary eukaryotic phototrophs, glaucophytes, uses the cyanobacterial pseudoparalog of PBGD, suggesting that the gene replacement occurred after the divergence of glaucophytes.


Scutellaria barbata D. Don (SBD) is a perennial herb that has been reported to protect against radiation-induced pneumonitis. However, the exact molecular mechanism is not known. The objective of this research was to investigate the against radiation pneumonitis ingredients and their functional mechanisms in SBD.Herbal medicines combined with radiotherapy significantly reduced the incidence of radiation pneumonitis (RP), and the SBD against RP through Traditional Chinese Medicine System Pharmacology (TCMSP) Database, and the “Herb–Ingredients–Target Genes–Disease” Network was constructed by using of Cytoscape. STRING analysis was performed to reveal the protein-protein interactions, and then we applied enrichment analysis on these target proteins, gene function, and pathways.Based on the network pharmacology approaches, we collected active ingredients and target genes in SBD regulate 65 RP related target proteins, which show that quercetin, luteolin, baicalein, wogonin may be the key active ingredients, while IL6, AKT1, VEGFA, MMP9, CCL2, prostaglandin-endoperoxide synthase 2 (PTGS2) (cyclooxygenase-2 [COX-2]), CXCL8, IL1B, mitogen-activated protein kinase (MAPK1), and IL10 were identified as critical targets. Besides, the results of Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that predicted targets of SBD are mostly associated with the pathological process of oxidative stress and inflammation. AGE- Receptor of Advanced Glycation Endproducts (RAGE) signaling pathway in diabetic complications, IL-17 signaling pathway, hypoxia-inducible factor-1 (HIF-1) signaling pathway, NF-kappa B signaling pathway might serve as the principal pathways for RP treatment.A total of 18 ingredients in SBD against RP was predicted from a holistic perspective, and the results provided theoretical guidance for researchers to explore the mechanism in further research.In our study, the pharmacological and molecular mechanism of  As the meta-analysis showed that herbal medicines combined with radiotherapy significantly reduced the incidence of radiation pneumonitis. Traditional Chinese Medicine (TCM) is widely used in China for thousands of years, and also practiced outside of China in later years. It is characterized by “multiple ingredients,” “multiple targets,” and “multi-pathway” in disease treatment.,5 Based on the traditional Chinese and Korean medicine theory, radiation therapy is regarded as a heat toxin pathogen.Radiation therapy may be curative in many types of cancer and also be used as part of adjuvant therapy to prevent tumor recurrence after surgery to remove a primary malignant tumor. However, the lung is more sensitive to the effects than other organs, and radiation pneumonitis is the significant side effect of thoracic radiation therapyScutellaria barbata D. Don (SBD) is a perennial herb which is known in TCM as Ban-Zhi-Lian, and the efficacy is mainly in heat-clearing and detoxifying properties (Qingre Jiedu in Chinese).SBD extraction and functional components were shown to possess vital biological activities like “antioxidant activity,” “reducing apoptosis and oxidative stress,” “anti-inflammatory activity,”–9 furthermore, some components have been reported to protect against radiation-induced pneumonitis and enteritis.–12 However, the pharmacological mechanisms have not yet been clearly explored.,14 has been widely applied to elucidate the function mechanisms for various diseases, such as cancer, cardiovascular disease, and metabolic disease.–21 In this research, we used the network pharmacology approach to systematically explored the mechanism of SBD in RP treatment by analyzing the active ingredients, potential target genes, and critical pathways.The TCM network pharmacology that integrates the systems biology and in silico technologies provides a systematic research strategy that conforms to the systematic and holistic perspective of the TCM theory,22.1SBD, as well as the small molecular structure information of the active ingredients, were collected from the Traditional Chinese Medicine System Pharmacology (TCMSP) Database (http://tcmspw.com/tcmsp.php 2020.03.16). The database is a unique systems pharmacology platform of Chinese herbal medicines that captures the relationships between drugs, targets, and diseases. It collected all the 499 herbs registered in Chinese pharmacopeia (2010), with a total of 12144 chemicals.All ingredients of SBD. As recommended in several articles,–25 the ingredients with OB ≥30% and DL ≥0.18 are considered to have better pharmacologic effects and are selected as candidate ingredients for the next step. According to the literature, scutellarin (OB 2.64% and DL 0.79) has been researched and proved to be one of the primary ingredients in SBD,,26,27 so we incorporated it into our study for further analysis.“Oral bioavailability” (OB) is an essential criterion for evaluating the quality of medicines. It reflects the proportion of the dose about an orally administered drug that enters the systemic circulation. “Drug likeness” (DL) refers to the similarity of a compound to a known drug. “Drug-like” compounds are not drugs, but they have the potential to become drugs. In this study, the 2 crucial absorption, distribution, metabolism, excretion-related pharmacokinetics parameters were used to screen the active ingredients of SBD were collected from TCMSP Then we use the UniProt knowledge database (https://www.uniprot.org/) with the selected species as Homo sapiens to transform the targets and obtain gene symbols.All the protein targets of bioactive ingredients in 2.2https://www.genecards.org/,2020.03.16) database provides genomic, proteomic, transcriptomic, genetic, and functional information on all known and predicted human genes.,29 The Online Mendelian Inheritance in Man  is a continuously updated catalog of human genes and genetic disorders and traits, with a particular focus on the gene-phenotype relationship. We set the keyword as “Radiation pneumonia” and then retrieved the detailed information of the target genes for RP.The target genes of radiation pneumonitis (RP) were obtained from the following 2 databases. Genecards (2.3SBD), ingredients, target genes, and disease (RP), a network of complex information was generated by Cytoscape 3.7.2 : an open-source bioinformatics software platform for visualizing molecular interaction networks and integrating with gene expression profiles and other state data. In the network plot, nodes represent the RP /SBD/ingredients/ target genes, while edges stand for that they are linked with each other.To illustrate the interactions among herb  is a biological database and web resource of known and predicted protein-protein interactions.,33 In STRING, we set the common target proteins with species as “Homo sapiens” and confidence score >0.4, then exported the PPI results as an image. The network nodes represent proteins, while the edges represent protein-protein associations.STRING  to perform it.GO The KEGG pathway enrichment analysis of overlapping target genes was executed by the R Project for Statistical Computing (R 3.6.3 https://www.r-project.org/).KEGG is an effort to link a set of genes in the genome with a network of interacting molecules in the cell, such as a pathway or a complex, representing a higher-order biological function.33.1SBD .We imported the 65 common target genes into the STRING database to generate the PPI network   The smaller the value of p. adjust, the greater correlation and importance. The x-axis represents the number of enriched genes (Barplot) and the ratio of the gene (Dotplot), while the y-axis represents GO terms. As the result shown, numerous biological processes associated with the research mechanism, then we intercepted the top 30 terms based on the P value   on the common targets shared by the SBD ingredient targets and RP-related targets, of which the first 30 enriched are presented in Figure We performed KEGG enrichment analysis (4 The primary mechanisms of tissue injury include direct DNA damage and the generation of reactive oxygen species (ROS), while cellular injury leads to inflammatory cell infiltration and cytokine release.,38 After radiation, the injured cells lead to the release of chemoattractant molecules, which can stimulate neutrophils arriving into the irradiated lung, and then promote local inflammation through the release of IL-1, IL-6, and reactive oxygen species.,40 As previous researches reports, radical-induced oxidative stress following thoracic irradiation participate in RP,,42 and the COX-2 is considered to be a pro-inflammatory enzyme during the oxidative stress.Histopathologically, radiation pneumopathy is characterized by “loss of epithelial cells; edema; inflammation; occlusions airways; air sacs and blood vessels; fibrosis tissue injury”.SBD against RP is closely related to oxidative stress and inflammation.In the present study, according to the PPI network, some important targets exhibit the therapeutic effects, which mainly concentrated in cytokine and protein kinase like IL6, AKT1, CCL2, PTGS2, COX-2, CXCL8, IL1B, MAPK1, IL10. GO and KEGG enrichment analysis together display that the potential key targets participating functions and pathways reflected in this aspects: inflammation; immunity; antioxidant; cell proliferation, differentiation; and apoptosis. In combination with analyses of the constructed network and enrichment results, we anticipate that the pharmacologic mechanism of  Interaction of RAGE with advanced glycation end products can also trigger spurs a surge of reactive oxygen species,,45 then provoke activation of the p21Ras and MAPKs. In this study, the IL-17 signaling pathway involving IL-17A and IL-17F signals via correspondent receptors to activate downstream pathways that include NF-kappaB, MAPKs and then induce the expression of cytokines and chemokines, which is also shown in the TNF signaling pathway. Besides those, the HIF-1 signaling pathway is noteworthy. HIF-1 acts as a master regulator of numerous hypoxia-inducible genes under hypoxia conditions, while it is induced not only in response to reduced oxygen availability but also by other stimulants, such as nitric oxide, or various growth factors. In reference previous study, the mRNA levels of HIF-1a, as well as of HIF-1 target genes vegfa, cxcl12, pgk1, were found to be up-regulated upon administration of bleomycin and the development of pulmonary inflammation and fibrosis. As shown in the HIF-1 signaling pathway, growth factor can synthesize HIF-1a via phosphoinositide 3-kinase or mitogen-activated protein kinase (MAPK) pathways. Radiation pneumonitis and radiation fibrosis are 2 closely related pathological processes of radiation-induced lung injury. Therefore, the intervention of HIF1 is important for both pneumonitis and later fibrosis.As shown in Figure SBD acting on key targets and pathways that may be the key research objects for further experimental studies. Refer to past literature, related studies have been conducted. Baicalein inhibited the expression of NF-κB p65 and the phosphorylation of p38 MAPK, extracellular signal-regulated kinase via dampening the NF-κB and MAPK signaling pathways to exerts its anti-inflammatory effects, and it also inhibited the expression of IL-6, IL-1β, Icam-1, and Vcam-1 in the irradiated intestine. Luteolin suppresses the expression of IL-6, iNOS, COX-2, and ROS by blocking the activation of MAPK and NF-κB pathways in acute lung injury induced by lipopolysaccharide.–50 Quercetin liposomes were shown to protect against radiation-induced and Lipopolysaccharide (LPS) induced acute pneumonitis by reducing the MDA content, increasing SOD activity in the lung tissues, and reducing the total cell counts and inflammatory cell proportions in the bronchoalveolar lavage fluid.,51 Wogonin reduced LPS-induced neutrophils infiltration, pro-inflammatory cytokines generation, adhesion molecules expression, Akt (PKB) phosphorylation, RhoA activation, and also prevented LPS-induced acute lung injury via regulating the PPARγ-involved NF-κB pathway.,53 However, the network pharmacology results and above literature reports partly reflects the effectiveness and mechanism, while the exact key ingredients and pharmacological mechanism of SBD acting on RP should be validated by further experimental studies.It is faster and also accurately predict potential active ingredients like quercetin, luteolin, baicalein, wogonin in 5SBD is widely used for inflammation and cancer, the pharmacological mechanism of interfering with lung cancer has been described, but RP has not. Our research applied a network approach to display that active ingredients in SBD probably play an essential part against RP by participating in the regulation of oxidative stress and inflammation, and the selected key targets, as well as signal pathways, can provide a reference for further experiments to clarify the precise mechanism.In conclusion, Conceptualization: Xin Zheng.Data curation: Pingyi Sun, Ai-Shuai Wang, Zhen-Fei Zhang, Yan-Li Zhang.Methodology: Pingyi Sun.Visualization: Pingyi Sun.Writing – original draft: Pingyi Sun.


The incidence of ulcerative colitis (UC) is increasing steadily in developed countries, it is plaguing nearly 1 million people in the United States and European countries, while developing countries have had a rapidly increased incidence over the past decades. Curcuma is widely used in treating malaria, UC, Crohn's disease, and colon cancer, which lead to diarrhea and bloody stool. However, the systemic mechanism of curcuma in treating UC is still unclear. Our work was supposed to expound how does curcuma alleviate UC in a comprehensive and systematic way by network pharmacology, molecular docking, and experiment verification. α and STAT3 before and after the treatment of curcuma which was verified by immunoblotting.  Traditional Chinese Medicine System Pharmacology Database (TCMSP), Shanghai Chemistry & Chemical Industry Data Platform (SGST), and papers published in Chinese Network Knowledge Infrastructure (CNKI) and PubMed were used to collect the chemical constituents of curcuma based on ADME . And effective targets were predicted by Swiss Target Prediction to establish the curcuma-related database. The disease targets of UC were screened by GeneCards and DrugBank databases, and Wayne (Venn) analysis was carried out with curcuma targets to determine the intersection targets. AutoDock software and TCMNPAS system were used to dock the core chemical components of curcuma with key UC targets. Protein interaction (PPI) network was constructed based on the STRING database and Cytoscape software. Gene function GO analysis and KEGG pathway enrichment analysis were carried out by using Metascape database. Finally, HE staining was performed to identify the inflammatory infiltration and expression difference in TNF-α and STAT3.  Twelve active components containing 148 target genes were selected from curcuma. Potential therapeutic targets of curcuma in the treatment of UC were acquired from 54 overlapped targets from UC and curcuma. Molecular docking was used to filter the exact 24 core proteins interacting with compounds whose docking energy is lower than −5.5 and stronger than that of 5-aminosalicylic acid (5-ASA). GO and KEGG analyses showed that these targets were highly correlated with EGFR tyrosine kinase inhibitor resistance, PI3K-Akt signaling pathway, JAK-STAT signaling pathway, MAPK signaling pathway, and inflammatory bowel disease (IBD). Experiments verified curcuma relieved pathological manifestation and decreased the expression of TNF-Helicobacter pylori infection pathway, probably by binding to STAT3 and TNF-α. Curcuma relieved the colon inflammation of ulcerative colitis via inactivating TNF pathway, inflammatory bowel disease pathway, and epithelial cell signaling in  UC is characterized by abdominal pain, diarrhea, and bloody stool . GloballTCM is gaining its popularity in the ameliorating subhealth state and treating disease. Much more attention has been focused on the treatment of UC by Chinese herb; curcuma is one of the popular ones. In ancient China, curcuma was widely used to invigorating the circulation of blood in clinical applications; nowadays, the function of the positive regulation of inflammatory cytokines in inflammatory diseases  and its It is well known that TCM is guided by the theory of TCM and characteristics of being multicomponents, multitargets, and multipathways in the treatment of diseases, which meets the requirements of systematically tackling complex diseases such as colorectal cancer. Guo et al. established the model of colorectal cancer and predicted the traditional Chinese medicine components of inhibiting inflammation-induced tumorigenesis by using network pharmacology method . Gupta ehttp://lsp.nwu.edu.cn/tcmsp.php), Shanghai Chemistry & Chemical Industry Data Platform , and papers published in Chinese Network Knowledge Infrastructure  and PubMed were used to collect the chemical constituents of curcuma. We screened curcuma compounds based on absorption, distribution, metabolism, and excretion (ADME) [https://pubchem.ncbi.nlm.nih.gov/) to finish Swiss ADME prediction [Traditional Chinese Medicine System Pharmacology Database   , and phan (ADME) . We furtediction , which wediction .http://www.swisstargetprediction.) [PubChem was used to search the chemical structures of the active compounds. Potential targets of curcuma were predicted by Swiss Target Prediction  . Probabihttps://www.genecards.org/) [https://www.drugbank.ca/) with the keyword “ulcerative colitis.” In addition, articles published in CNKI and PubMed about the known targets of its active compounds were counted [Data of UC-associated target genes were gathered from GeneCards (ds.org/)  and Drugds.org/)  (https:/ counted . Genes f counted .http://www.bioinformatics.com.cn/), and the overlapped targets of compound-disease were obtained as the potential targets for further analysis.The screened curcuma targets and UC targets were imported into Bioinformatics  (http://https://string-db.org/), which covered almost all functional interactions between the expressed proteins [Protein-protein interaction (PPI) network was derived based on the STRING database (proteins . Specieshttps://metascape.org/) [P value less than 0.05.The biological process (BP), molecular function (MF), cell component (CC), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were carried out using Metascape system (pe.org/) . In thisA visual network was constructed through Cytoscape software to reflect the complex relationship between active compounds, filtrated targets , and patUsing TCMNPAS system  and Autoβ-actin, anti-STAT3 antibody, anti-TNF-α antibody , HE dyeing , and neutral gum  were used.Curcuma , DSS , absolute ethyl alcohol, Tween-20, xylene substitute , RIPA Lysis buffer, PMSF, BSA, BCA Protein Quantitation Kit (Beyotime), PAGE gel rapid preparation kit, Multicolor Restrained Protein Ladder , Homogenized tube, ceramic beads, frozen storage tube , centrifuge tube , PVDF membrane , and 96-well plate (Eppendorf Life Sciences Corporation) were used.H2050r high-speed refrigerated centrifuge (Hunan Xiangyi Company), MIX-S vortex mixer, shaker oscillator , TGear mini centrifuge (Tiangen), heating magnetic agitator , SIM-F140 ice maker , electronic balance , enzyme labeling instrument , tissue grinding homogenizer , electric constant temperature blast drying oven , electrophoretic system, transfer system, glue rack, ultralow-temperature freezer , microtome , and TKY-BMB, electrothermostatic water bath .Healthy male Sprague Dawley (SD) rats, weighing 180 ± 20 g, were provided by Charles River Experiment Technology Co., Ltd., and the certificate number is SCXK (Hu) 2017-0005. The rats were housed in the animal room of Shanghai University of Traditional Chinese Medicine.The native herb was selected, and standard decoction pieces were prepared with reference to the Chinese Pharmacopoeia (2015 edition). Extract was prepared by boiling the samples in 8 times amount of water for 30 min. The procedure was repeated 3 times.SD rats were accepted to the laboratory for 7 days before the experiments. According to the random number table, the rats were divided into 3 groups of 4 rats each: control, model, and curcuma. Except for the control group, the UC model was prepared with 5% DSS, and the intervention was given according to the group after 7 days.The curcuma group was administered continuously by gavage with 2 ml 0.1 g/ml curcuma suspension for 7 days. The other two groups were given saline 1.08 g/kg.The steps to stain the samples are as follows: xylene I and xylene II, 10 min for each; 100% alcohol I, 100% alcohol II, 95% alcohol, and 85% alcohol, 5 min for each; water washing for 20 s, hematoxylin for 7 min, water washing for 1 min, 1% hydrochloric acid alcohol for 10 s, 50°C water washing for 5 min, eosin stain for 2 min, and water washing for 10 s; 85% alcohol, 95% alcohol, 100% alcohol II, and 100% alcohol I, each for 2 min; and xylene II and xylene I, 3 min for each. Neutral balsam was added after xylene was passerillaged.μL of RIPA, and 5 μL PMSF were added to each tube. After five times' homogenization, the colon tissue was basically broken. The supernatant was extracted after centrifugation to test the protein concentration and to collocate protein solution. Equivalent amounts of protein (200 μl) were denatured at 98°C for 10 min in sample loading buffer, then separated by electrophoresis in 15% gel, and electrotransferred onto 0.45 μm polyvinylidene difluoride membranes for 60 min at 350 mA. Subsequently, the membranes were blocked in blocking buffer  at 25 ± 5°C, followed by incubation with primary antibodies against STAT3 (1 : 5000) and TNF-α (1 : 5000) at 4°C overnight. After being washed with Tris-buffered saline containing Tween-20 (TBST) for 5 min three times, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. After the membranes were washed three times in TBST for 20 min each time, the bands were visualized on X-ray film using an enhanced chemiluminescence western blotting (WB) detection system. The Image Lab™ software was used for quantitative analysis.For protein extraction, tissues were placed in homogenized tubes; 5 porcelain beads, 500 The workflow of this study is summarized in TCMSP database, SGST, and articles published in CNKI and PubMed were used to gather the active components and targets, and 13 potential ingredients were discovered from curcuma . We exclWe obtained the curcuma-related targets from GeneCards (whose relevance score is above the median point) and the ones from DrugBank, CNKI, and PubMed . Then thhttp://www.rcsb.org/) to query their PDB ID [http://zinc.docking.org/). Molecular docking was progressed in TCMNPAS to calculate the docking score. The interaction strength between compounds and gene corresponding proteins can be expressed by docking fraction, and the lower the score is, the higher the interaction intensity is. Further, we docked the 54 potential targets with 5-ASA. As shown in We input 54 potential genes into the PDB database  was established through the STRING database. As shown in P < 0.05, such as cellular response to lipid, response to oxygen levels, and regulation of inflammatory response  are closely related to the inflammatory reaction process. p53 signaling pathway is included in the pathways in cancer. Moreover, MAPK signaling pathway and TGF-beta signaling pathway play a crucial role in immunological stress. In addition, we found some other pathways such as endocrine resistance, hepatitis B, serotonergic synapse, and longevity regulating pathway, which revealed that curcuma has a potential application in other related diseases. The KEGG pathways verified that curcuma cured UC by regulating gut microbiota, inflammatory process, immunization, and inflammatory reaction.To further reveal the potential mechanism of curcuma on the effect of UC, we conducted KEGG pathway enrichment analysis on 24 targets and screened out 95 pathways based on the threshold of P < 0.05. NumerouTo view the relationship between the components, common targets, and their corresponding pathways, a target-pathway network was constructed . Twenty According to the HE staining , DSS couα in the colon samples of the model group was significantly increased (P < 0.01). Compared with the model group, the curcuma group can reduce the expression of STAT3 and TNF-α (P < 0.05)  .α and STAT3 are two research hotspots. It was reported that curcumin and semibionic extraction of compound turmeric can inhibit the proinflammatory signaling by STAT3 and TNF-α in experimental colitis [κB expression by regulating tumor necrosis factor-α (TNF-α) in humans [UC is one of the autoimmune diseases affecting northern Europe, Canada, and Australia . The occ colitis , 31. Simn humans . Furthern humans .H. pylori later [α (TNF-α) through inhibiting NF-κB expression [The 12 found core compounds not only have anti-immune and anti-inflammation effects but also have the effect of regulating intestinal flora. Scholars verified that the extension of UC is important for the positivity rate of ri later . Moreovepression . Meanwhipression . Previoupression .α (degree = 35), STAT3 (degree = 33), and PTGS2 (degree = 32) might be the core targets of the PPI network, through whom curcuma may come into play.Molecular docking verified that the binding energy of 24 key targets was better than that of 5-ASA and lower than −5.5, meaning the interaction between the compounds and targets of UC can bind tighter than that of 5-ASA, indicating better therapeutic effects. Common target PPI network showed that the targets were cocontrolled by curcuma and UC, which revealed that curcuma could regulate the expression of UC-regulated targets and alleviate UC symptoms. AKT1 (degree = 43), EGFR (degree = 35), TNF-P < 0.05) indicated that the major hubs were significantly involved in multiple biological processes, including cellular response to lipid, cellular response to organic cyclic compound, and response to steroid hormone. Furthermore, molecular function enrichment analysis showed nuclear receptor activity, transcription factor activity, and steroid hormone receptor activity were contained in the healing process. The active targets involved were AKT1, EGFR, TNF, STAT3, and PTGS2, which mainly concentrate on the molecular processes of immunization, inflammatory reaction, gut microbiota, etc. Some of the genes and mechanisms have been verified in curcumin, while PTGS2 was proposed connecting with the treatment of UC by curcuma firstly as a gene with high possibility. Meanwhile, cellular components consist of RNA polymerase II transcription factor complex, transcription factor complex, nuclear transcription factor complex, and membrane raft, and EGFR, DPP4, ADAM17, BCL2, and CDK2 were contained in the cellular components. The verified impact of EGFR on UC by regulating epithelial barrier function could identify the reliability of this GO analysis [In order to predict the mechanism of curcuma in the treatment of UC, we analyzed the key candidate targets by performing GO enrichment results, such as biological processes, molecular functions, and cellular components. The GO terms  endocytosis the invading bacteria and secrete TGF-β and IL-6. Then, TGF-β and IL-6 stimulate Th17 activating STAT3 with RORγt and RORα for further inflammatory cytokines. The activated expression of STAT3 participates in the gene transcription and protein expression of a variety of inflammatory factors such as TNF-ammation –39. Besiammation . HoweverIn summary, consistent with clinical experience in the long history, experimental verification, and our HE staining, as well as our WB results, curcuma has significant advantages in the treatment of UC. At the same time, the uncovered targets and pathways were excavated for a better research of how curcuma relieves UC. It has a significant value to provide theoretical basis for clinical treatment of UC and a potent evidence for further study of the mechanism of curcuma in the treatment of UC.


Personalized breast cancer treatment with targeted therapy  requires identification of responder patients. Phenotypical heterogeneity within the primary, and between primary tumor and metastases, may however interfere with response to therapy, if based on a single primary tumor biopsy. In this study, we investigated this heterogeneity, using novel assays to measure activity of tumor-driving signal transduction pathways, e.g., estrogen receptor and PI3K pathways, in multiple samples distributed across the tumor and in metastases. Within the primary tumor, heterogeneity was dominant at microscale (biopsy block) and not at macroscale (across tumor), suggesting that a single biopsy is generally representative for the whole primary tumor. The differences found between pathway activity in primary tumor and metastases suggests that it is recommendable to analyze pathway activity in metastatic samples for treatment selection for late stage patients.n = 35), to micro-scale variation in activity on four adjacent samples of a single PT tissue block (n = 17), showed that macro-scale variation was not larger than micro-scale variation, except possibly for the PI3K pathway. Simulations using a “checkerboard clone-size” model showed that multiple small clones could explain the higher micro-scale variation in activity found for the TGFβ and Hedgehog pathways, and that intermediate/large clones could explain the possibly higher macro-scale variation of the PI3K pathway. While within PT, pathway activities presented a highly positive correlation, correlations weakened between PT and lymph node metastases (n = 9), becoming even worse for PT and distant metastases (n = 9), including a negative correlation for the ER pathway. While analysis of multiple sub-samples of a single biopsy may be sufficient to predict PT response to targeted therapies, metastatic breast cancer treatment prediction requires analysis of metastatic biopsies. Our findings on phenotypic intra-tumor heterogeneity are compatible with emerging ideas on a Big Bang type of cancer evolution in which macro-scale heterogeneity appears not dominant.Targeted therapy aims to block tumor-driving signaling pathways and is generally based on analysis of one primary tumor (PT) biopsy. Tumor heterogeneity within PT and between PT and metastatic breast lesions may, however, impact the effect of a chosen therapy. Whereas studies are available that investigate genetic heterogeneity, we present results on phenotypic heterogeneity by analyzing the variation in the functional activity of signal transduction pathways, using an earlier developed platform to measure such activity from mRNA measurements of pathways’ direct target genes. Statistical analysis comparing macro-scale variation in pathway activity on up to five spatially distributed PT tissue blocks ( Cancer can be described in terms of abnormal functioning of one or more signal transduction pathways that control major cellular functions, e.g., cell division, differentiation, migration, and metabolism. Around 10–15 signal transduction pathways can drive growth and metastasis of breast cancer . In cancA novel analysis method was described before to quantify signaling pathway activity in cancer. Based on Bayesian models, the method infers a pathway activity score from transcription factor-specific target gene mRNA levels ,20,21,22n = 33) and 9 PT with distant metastatic sites (n = 12) were analyzed. The goal of this study was to investigate phenotypic heterogeneity within primary and between primary and metastatic breast cancer lesions, using a novel mRNA-based assay platform to measure the functional activity of relevant signal transduction pathways on multiple samples pertaining to the same patient. Within the primary tumor, heterogeneity was estimated at macro-scale and at micro-scale. Macro-scale heterogeneity analysis was performed on up to five spatially distributed PT tissue blocks from 35 primary breast cancers of various subtypes; micro-scale heterogeneity analysis was performed on four adjacent samples of a single tissue block from 17 matched PT. To investigate phenotypic heterogeneity between primary tumor and metastatic breast cancer lesions, samples from 9 PT with matched lymph node . Breast cancer molecular subtyping was performed on surgically resected PT in a standard manner using the surrogate definitions from the 13th St Gallen International Breast Cancer Conference , based oA variable number of tissue blocks, taken from different locations, was available per PT to investigate heterogeneity in pathway activity at the macro-scale. To investigate heterogeneity at the micro-scale, four adjacent “quadrant” samples from the same tissue block (one block per tumor) were made available for a subgroup of PT. To investigate variation in pathway activity between primary tumor and metastases, two separate patient sample sets were analyzed: one with multiple PT blocks matched with a variable number of LN metastasis samples, the other containing a single PT block and a variable number of metastasis samples from different DS.Tissue slides were cut from the FFPE blocks at 10 μm thickness. The tumor area was annotated by an experienced pathologist and macrodissected, aiming at similar tumor tissue volumes for RNA extraction . RNA wasFunctional activity of signal transduction pathways was assessed using biologically validated computational pathway models as described before ,20,21,25The pathway activity assays were originally developed on data from Affymetrix HG-U133Plus2.0  microarrays but have been converted to qPCR mRNA measurements as input, to enable use of FFPE material . TypicalIn our study, we used qPCR pathway assays for the AR, ER, PI3K, HH, TGFβ and Wnt pathways, available on the FIPA Pathway Plate 1.0 . As such, these represent relevant hormone-driven pathways (AR and ER), growth factor pathways (PI3K), and stem cell-related pathways , which play a role across different breast cancer subtypes. PI3K pathway activity is based on the inverse activity of the measured FOXO transcription factor activity score, on the premise that no cellular oxidative stress is present; for this reason, the FOXO activity score is interpreted in combination with SOD2 target gene expression level to distinguish between growth control- and oxidative stress-induced FOXO activity, as described before ,27.Due to the heterogeneous sample set, we used linear mixed models ,29 to enpqy is the pathway activity score of patient p for tumor block q; tμ is the average of scores in this subtype classification group t ; pα is a random contribution of the patient tumor as a whole, compared to the group average ; and pqβ is the random contribution of block q which is used to accommodate tumor heterogeneity for patient p . The value σ,tblock describes the spread of scores within a single patient tumor (the tumor heterogeneity) for patients in a specific breast cancer subtype t and σpat describes the variation between individual patients, regardless of the breast cancer subtype they belong to.Here, σ,tblock or σpat. Since they are fitted parameters to the data, the models provide a confidence interval for these quantities. For the analysis performed with quadrant measurements, the model was extended to accommodate and estimate additional micro-scale heterogeneity. The model used has an additional layer that enables taking both the tissue block and the quadrant sample measurements into account  using nlme and emmeans packages [Macro-scale heterogeneity (from blocks) and micro-scale heterogeneity (from quadrants) are defined in terms of parameters of this model. Analysis was performed in STATA  and R  was comparable for all breast cancer subtypes  for each subtype. Instead, a simpler model could be used in our subsequent analysis, in which the estimate for the spread of scores within a single tumor is the same regardless of subtype . Further analysis, using this simpler model (σpat) is larger than the variance within a single tumor (σblock) for the WNT and ER pathways , while both variances were comparable for the remaining pathways , as well as in all tissue blocks , were analyzed  or at micro-scale (block quadrants), we calculated the ratio between the standard deviation of pathway activity score at the macro-scale and the standard deviation of pathway activity score at the micro-scale for each signaling pathway. The standard deviations were estimated using the model illustrated in  < 0.001 . For the < 0.001 E. The meExtensive noise analysis was performed to identify technical noise, which might have biased the pathway activity scores, and therefore, the ratio between variation at macro-scale and micro-scale . High qPSince the observed higher variation in HH and TGFβ pathway activity on micro-scale compared to macro-scale seemed counter-intuitive, a hypothetical computational checkerboard model was developed to help explain observed results . In thisp = 0.2, for the TGFβ pathway to 0.825, 95% CI , p = 0.006, for the HH pathway  the delta in pathway activity score between each lymph node metastasis and the average score of the respective PT samples, p,ΔLN, and (b) the delta in score between each PT tissue block and the respective PT block averagesp,, Δblock  matched with a variable number of DS metastatic samples was available (sample group III). Except for the TGFβ pathway, correlations between DS metastasis and matched PT were worse than correlations between LN metastasis and matched PT. Correlations remained positive for FOXO and the HH and TGFβ pathways, but became negligible for the AR and WNT pathways, and negative for the ER pathway, ranging from positive correlation of 0.601, 95% CI , p = 0.09 for the HH pathway to a negative correlation of −0.359, 95% CI , p = 0.3, for the ER pathway . Here again, the spread in q,Δmeta was larger than the spread in p,Δblock for all pathways, indicating that, as with LN metastasis, differences in pathway activity scores between PT and DS metastases are larger than the variation within the PT  was larger than variation between lymph nodes  and ileum (n = 1); HH pathway activity was highest in ovarian, ER pathway activity highest in a brain, and TGFβ highest in a skin and a bone metastatic lesion  staining within an ER positive primary tumor have been reported, but not related to pathway activity . ER-actiOur observation that, with the possible exception of the PI3K-FOXO pathway, the variation in pathway activity was generally not larger at the macro-scale than micro-scale, and for the HH and TGFβ pathways, even higher at the micro-scale, is compatible with currently emerging ideas on a Big Bang type of cancer evolution based on genomic analysis of PT, in which macro-scale heterogeneity appeared to be not the dominant form . CompatiSubsequently, we investigated whether pathway analysis of the primary tumor could predict pathway activity in metastatic tumors. While pathway activity scores between quadrant samples of a single block, and between blocks of one PT showed highly significant correlations, correlations decreased between PT and LN metastases and significance was lost. The lowest correlation was found between PT and DS metastases. These results provide interesting new information about breast cancer metastasis. Since LN metastatic samples were spatiotemporally close to the PT, a closer relation to the PT was expected compared to the DS metastases that are spatiotemporally further away. The PI3K pathway is generally thought to be an important metastasis-driving pathway in addition to its general role as a tumor “survival” pathway, explaining the relatively strong correlation in activity between PT and LN metastases . The obsInterestingly, a negative correlation was present for ER pathway activity between PT and DS metastases. Since only ER-positive luminal patients were included in this part of the study, a number of patients probably had received adjuvant hormonal therapy, potentially resulting in selection for ER pathway-inactive metastases . UnfortuObvious limitations of the study were the relatively limited patient and sample numbers, and lack of clinical treatment and outcome data. Despite this, to the best of our knowledge, the study is unique in allowing a quite detailed comparison between signaling pathway activity scores within a PT and between PT and metastases.The clinically relevant conclusions that can be drawn suggest that heterogeneity with respect to pathway activity within a single biopsy may generally be representative for the whole tumor, with the possible exception of the PI3K pathway, while variation in ER pathway activity is relatively small within the PT compared to that seen between luminal breast cancers from different patients. We cannot exclude that for the PI3K pathway, multiple spatially separated biopsies may be required to obtain a sufficiently complete picture of this pathway’s activity pattern across the tumor. Pathway activity scores found in the PT predicted activity in LN metastases to some extent, especially with respect to the PI3K-FOXO pathway. Prediction of pathway activity in DS metastases is no longer realistic, also in view of the large variation between metastases in different organ locations. If possible, taking biopsies from multiple metastatic locations may, therefore, be preferred when considering targeted therapy for metastatic disease, to choose the most effective (targeted) drug or drug combination.In view of its high clinical relevance, especially with respect to targeted treatment in (neo)adjuvant and metastatic settings, the current findings should be confirmed in subsequent clinical studies. We recommend that a confirmation study should: (1) optimize standardization of tissue sampling; (2) note size of the tumor and location of the sample taken in the 3D space of the tumor, to reduce the influence of variable microenvironment, e.g., oxygen concentration, immune cell infiltrate, etc.; (3) use similar amounts of cancer cells to extract RNA from. Importantly, application and further development of the statistical models that we introduced should help to improve quantification of cancer heterogeneity, necessary to objectively compare clinical studies and bring the field forward. We believe that such studies will further complement knowledge on tumor evolution and the resulting tumor heterogeneity, enabling improvement in choosing the most effective therapy for patients with breast cancer.


Respiratory diseases, especially the pandemic of respiratory infectious diseases and refractory chronic lung diseases, remain a key clinical issue and research hot spot due to their high prevalence rates and poor prognosis. In this review, we aimed to summarize the recent advances in the therapeutic effects and molecular mechanisms of key common bioactive compounds from Chinese herbal medicine. Based on the theories of traditional Chinese medicine related to lung diseases, we searched several electronic databases to determine the high-frequency Chinese medicines in clinical application. The active compounds and metabolites from the selected medicines were identified using the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) by analyzing oral bioavailability and drug similarity index. Then, the pharmacological effects and molecular mechanisms of the selected bioactive compounds in the viral and bacterial infections, inflammation, acute lung injury (ALI), chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, asthma, and lung cancer were summarized. We found that 31 bioactive compounds from the selected 10 common Chinese herbs, such as epigallocatechin-3-gallate (EGCG), kaempferol, isorhamnetin, quercetin, and β-sitosterol, can mainly regulate NF-κB, Nrf2/HO-1, NLRP3, TGF-β/Smad, MAPK, and PI3K/Akt/mTOR pathways to inhibit infection, inflammation, extracellular matrix deposition, and tumor growth in a series of lung-related diseases. This review provides novel perspectives on the preclinical study and clinical application of Chinese herbal medicines and their bioactive compounds against respiratory diseases. Respiratory diseases include respiratory infectious diseases, asthma, chronic obstructive pulmonary disease (COPD), interstitial pulmonary disease (ILD), and lung cancer. These diseases are characterized by the injuries of bronchial and alveolar tissue to cause respiratory dysfunction and even respiratory failure. Respiratory infectious diseases mainly caused by viruses or bacteria and often contagious, remain a major global public health problem. For example, since the outbreak of coronavirus disease (COVID-19) at the end of 2019, there have been nearly 180 million confirmed cases, including 3.9 million deaths by June 25, 2021 . PneumonTraditional Chinese medicine (TCM) has a history of more than 3,000 years and has been used for the prevention and treatment of many respiratory diseases. The ancient medicine books named “Shennong Ben Cao Jing” and “Shanghan Lun” clearly recorded the theories of traditional Chinese medicine, such as reducing phlegm and relieving cough and asthma, and many prescriptions for the prevention and treatment of lung-related diseases. Based on thousands of years of clinical application and the modernization of TCM research, hundreds of Chinese medicines have been shown to be effective in the current clinical applications for treating respiratory infections, asthma, chronic lung diseases, and lung cancer; these effects are based on the therapeutic and improved effects for acute respiratory symptoms and lung dysfunction . ImportaMorus alba L., Moraceae family, Chinese name: Sangbaipi, peel; Ginkgo biloba L., Ginkgoaceae family, Chinese name: Baiguo, seed; Aster tataricus L.f., Compositae family, Chinese name: Ziwan, root) and relieving cough and asthma (Perilla frutescens (L.) Britton, Lamiaceae family, Chinese name: Suzi, seed; Tussilago farfara L., Compositae family, Chinese name: Kuandonghua, flower; Datura metel L., Solanaceae family, Chinese name: Yangjinhua, flower; Ardisia japonica (Thunb.) Blume, Primulaceae family, Chinese name: Aidicha, leaf; Lepidium apetalum Willd., Brassicaceae family, Chinese name: Tinglizi, seed; Eriobotrya japonica (Thunb.) Lindl., Rosaceae family, Chinese name: Pipaye leaf; Prunus mandshurica (Maxim.) Koehne., Rosaceae family, Chinese name: Kuxingren, seed) were selected.According to the theories of TCM involving lung-related diseases, we searched Chinese or English electronic databases including CNKI database, Wanfang Data Knowledge Service Platform, VIP Chinese Science and Technology Journal database, PubMed database, and Web of Science database with keywords such as “traditional Chinese medicine,” “Chinese medicine,” or “respiratory diseases.” After the literature retrieval, the Chinese medicines widely used in clinical applications for reducing phlegm . The active compounds of each herb were sorted out by the screening criteria with  for the ADME  evaluation system. After sorting, we identified 165 bioactive compounds from these 10 herbs, such as epigallocatechin-3-gallate (EGCG), kaempferol, apigenin, ellagic acid and resveratrol for further analysis. Then, we searched the databases  using the keywords for one of the ingredients from the TCMSP and a type of disease, such as respiratory infection, COVID-19, inflammation, ALI, PF, COPD, asthma, or lung cancer to obtain articles published from January 2000 to May 2021.The effective components and their metabolites of the selected 10 medical plants were searched in the Traditional Chinese Medicine Systems Pharmacology Database . We further excluded the irrelevant records for the subject (n = 416), target herbs (n = 216), Chinese medicinal formulae/mixture compounds (n = 60), targeting drug delivery system (n = 25), or computational study without experimental validation (n = 60). Moreover, 74 reports for component analysis were added to obtain 349 full-text articles for eligibility assessment. Finally, 129 articles for the therapeutic effects and molecular mechanisms of 31 bioactive compounds from the selected 10 herbs were enrolled in the final analysis, after excluding similar studies or those not relevant to our topic of this review (n = 234). The detailed flow chart of the published articles collection is shown in Articles that included both components and disease terms, excluding review articles were identified as reference lists . Titles and abstracts of all the records were screened to exclude irrelevant studies  Vahl, Lonicera japonica Thunb., and Prunus mandshurica (Maxim.) Koehne., Rosaceae) (Forsythia suspensa (Thunb.) Vahl, Strobilanthes cusia (Nees) Kuntze, and Bupleurum chinense DC.) (Pogostemon cablin (Blanco) Benth., Platycodon grandiflorus (Jacq.) A.DC., and Pinellia ternata (Thunb.) Makino)  Koehne., and Atractylodes macrocephala Koidz.) can improve clinical symptoms, such as fatigue, cough, and fever, reduce the usage rate of antibiotics, and prevent the progression to severe COVID-19 (Eriobotrya japonica (Thunb.) Lindl., and Ginkgo biloba L., and isorhamnetin found in Lepidium apetalum Willd., Eriobotrya japonica (Thunb.) Lindl., Ginkgo biloba L., and Aster tataricus L.f. exhibit the ability to prevent SARS-CoV-2 from entering into ACE2+ cells (Tussilago farfara L. and Lianhuaqingwen granules inhibit the ACE2 enzyme activity (Eriobotrya japonica (Thunb.) Lindl. show antiviral and anti-inflammatory effects against H1N1 virus  signaling pathway to improve interferon sensitization  , Shufengnse DC.) , Huoxian Makino)  and HaisCOVID-19 . Current2+ cells . Neochloactivity . HesperiN1 virus . In H9N2N1 virus . β-sitostization . As for tization . SARS-CoALI is common in pulmonary infection, lung contusion, pulmonary embolism, and near-drowning, it can lead to acute respiratory distress syndrome (ARDS) . The morin vivo and in vitro experiments. Other inflammatory substances and harmful chemicals such as N-methyl-d-aspartate, methamphetamine, and paraquat (PQ) are also used in ALI studies. TNF-α and other cytokines are commonly used in the in vitro model construction of ALI. The changes in pulmonary function, lung wet/dry ratios, the morphology of lung tissue, and inflammatory factors in alveolar lavage fluid and serum are generally used to evaluate the inflammatory response. Canonical NF-κB pathway directly induces proinflammatory cytokines such as TNF-α, IL-1β, and IL-6. Subsequently, the activation of IL-1R1 and TNFR1 can make a positive feedback to activate the crucial pathway of inflammation via the NF-κB pathway  Lindl., and Lepidium apetalum Willd. can reduce the production of inflammatory cytokines and oxidative stress to prevent LPS-induced ALI in mice through the NF-κB pathway (Aster tataricus L.f. and Eriobotrya japonica (Thunb.) Lindl., which have anti-inflammatory activities and protective effects against ALI by downregulating cytokines and chemokines (Eriobotrya japonica (Thunb.) Lindl. and Morus alba L. improve ALI through a crosstalk of the MAPK and the NF-kB signaling pathways (Prunus mandshurica (Maxim.) Koehne, Eriobotrya japonica (Thunb.) Lindl., and Morus alba L. amygdalin and resveratrol suppress NF-κB activity and ROS production via inhibiting NLRP3 inflammasome  Britton, Eriobotrya japonica (Thunb.) Lindl. and Morus alba L. reduce PTEN and NF-κB acetylation through the activation of SIRT1 (Prunus mandshurica (Maxim.) Koehne inhibits the expression of TRPV1 and TRPA1 thereby providing anti-inflammatory and anticough effects (The model of ALI is mainly based on the induction by lipopolysaccharide (LPS) in  pathway . Ellagic pathway . TLR4/MYemokines . Furtheremokines . Rutin apathways . The NLRpathways . The bioammasome . SIRT1, e stress . Resveraof SIRT1 . Quercetof SIRT1 . Luteoliof SIRT1 . Accordiof SIRT1 . Liquiri effects . Taken tin vivo and in vitro models of COPD  Lindl., and Morus alba L. can inhibit the progression of COPD. Tussilagone and EGCG from the herbs mentioned above enhance the antiproliferative activity through the inhibition of the NF-κB pathway , the signaling pathways (NF-κB and MAPK pathways), and various stress-related molecules  participate in the different pathological stages of COPD . IL-8 re of COPD . Many st pathway . Amygdal pathway . Ursolic pathway . Liquiri pathway . Resvera pathway  or regul pathway . Alveola pathway . Resvera pathway . Other r pathway . The abo pathway . Togethe pathway .via the crosstalk of NF-κB and TGF-β pathways  promote the pathogenesis of asthma . Naïve Cpathways . Glabridpathways . Luteolipathways . Taken tpathways .in vitro and in vivo models of A549 and H1975 cells  Lindl. and Ginkgo biloba L. can suppress the levels of Axl and Tyro three to reduce the resistance to platinum  Lindl. and its compounds  may be considered an effective Chinese herbal medicine for the treatment of viral infections. Flavonoids, including apigenin, galangin, isorhamnetin, rutin, moracin M, amygdalin, hesperidin, quercetin, formononetin, luteolin, and liquiritin, exhibit good bioactivity against ALI. As for inflammation, bioactive compounds from Aster tataricus L.f. and Eriobotrya japonica (Thunb.) Lindl. have potential anti-inflammatory activity, suggesting that apigenin, quercetin, luteolin, and isorhamnetin are effective anti-inflammatory compounds. In the studies of COPD, bioactive compounds have mainly attenuated cigarette smoke-induced emphysema, airway remodeling, and inflammation through the NF-κB, MAPK, and TGF-β/Smad pathways, and resveratrol is one of the important and effective bioactive substances against COPD. Multiple components, including β-sitosterol, ferulic acid, quercetin, hesperidin, EGb761, and resveratrol, are directly or indirectly related to TGF-β/Smad, which is a crucial target for PF. These components can effectively suppress biological process of EMT and ECM. In asthma, rosmarinic acid, tussilagone, formononetin, and galangin targeting the MAPK and NF-κB pathways to reduce OVA-specific IgE, and ameliorate airway hyperplasia and hypertrophy. Importantly, these active components such as organic acids and flavonoids can inhibit the proliferation and migration of lung cancer and increase its sensitivity to radiotherapy and chemotherapy. Hyperoside, resveratrol, glabridin, luteolin, and kaempferol are considered potential candidates for the treatment of lung cancer based on a large number of studies. Collectively, ECCG, kaempferol, isorhamnetin, quercetin, and β-sitosterol are important bioactive compounds for prevention and treatment of ALI, PF, and lung cancer. Taken together, multiple bioactive compounds from the 10 different herbs have potential therapeutic effects against respiratory diseases by regulating various molecular pathways (According to the theories of TCM and commonly used Chinese medicines in the clinical application against respiratory diseases, this review summarizes the pharmacological effects and molecular mechanisms of 31 active compounds of 10 Chinese herbal medicines in six main lung-related diseases, including pulmonary infection, ALI, PF, COPD, asthma, and lung cancer. Many studies have demonstrated that bioactive compounds can ameliorate bacterial, virus, and LPS-induced pulmonary infection by targeting the NF-κB, MAPK, Nrf2/HO-1, and NLRP3 pathways, reducing the release of cytokines and chemokines, and suppressing inflammation by pathological reaction, oxidative stress, and ROS production. pathways , 3.However, in the studies of different bioactive compounds on respiratory diseases, three important aspects should be considered. Firstly, only key and common active ingredients in each herb are summarized, which might not be fully representative of the herb. More active components should be further identified to explore their pharmacological effects against respiratory diseases. Secondly, multiple compounds In a herb can target similar or different signaling pathways to play the potential roles in those diseases. The network of various targets of different components might be used to explain the combined effect of the formula. Thirdly, different signaling pathways or pathological procedures in infection, inflammation, COPD, or lung cancer are potential targets for these active ingredients. However, the potential targets and the binding role of these active compounds still remain unclear. It should be a future direction for most researchers to confirm specific targets of those potential drug candidates using multiple modern techniques. Finally, the quantitative analysis for the biological activity, toxicity and selectivity of 31 bioactive compounds should be performed in a kind of respiratory diseases to predict the promising candidates for drug development using systematic review and meta-analysis. Overall, this review provides novel perspectives on the preclinical study and clinical application of herbal medicines and their bioactive compounds against respiratory diseases.In summary, 10 Chinese herbal medicines were selected based on the theories of TCM and high-frequency use of Chinese medicines in clinical application. The pharmacological effects and molecular mechanisms of 31 bioactive compounds from these 10 Chinese herbs in infection, ALI, PF, COPD, asthma, and lung cancer were summarized. The bioactive compounds, such as epigallocatechin-3-gallate, kaempferol, isorhamnetin, quercetin, and β-sitosterol, can mainly regulate the NF-κB, Nrf2/HO-1, NLRP3, TGF-β/Smad, MAPK, and PI3K/Akt/mTOR pathways to inhibit infection, inflammation, extracellular matrix deposition, and tumor growth in a series of lung-related diseases. This review provides novel perspectives on the preclinical study and clinical application of Chinese herbal medicines and their bioactive compounds against respiratory diseases.


Cardio-renal syndrome (CRS) is a rapidly recognized clinical entity which refers to the inextricably connection between heart and renal impairment, whereby abnormality to one organ directly promotes deterioration of the other one. Biological markers help togain insight into the pathological processes for early diagnosis with higher accuracy of CRS using known clinical findings. Therefore, it is of interest to identify target genes in associated pathways implicated linked to CRS. Hence, 119 CRS genes were extractedfrom the literature to construct the PPIN network. We used the MCODE tool to generate modules from network so as to select the top 10 modules from 23 available modules. The modules were further analyzed to identify 12 essential genes in the network. These biomarkersare potential emerging tools for understanding the pathophysiologic mechanisms for the early diagnosis of CRS. Ontological analysis shows that they are rich in MF protease binding and endo-peptidase inhibitor activity. Thus, this data help increase our knowledge onCRS to improve clinical management of the disease. The incidence of cardiac and chronic renal dysfunction gave a term Cardiorenal Syndrome (CRS), which has been widely used without well-known definition. It is classified within five subtypes and reflection of its pathophysiology includes time-frame, and combinedcardiac-renal failure . It can The manual retrieval of key genes was done from the available resources like databases, literature relevant to CRS. Several important keywords was used throughout the search such as "cardiorenal syndrome", "renocardio syndrome", "Cardiovascular and chronic/acutekidney disease", "heart and kidney disease", "renal and myocardial/heart/congestive failure", "acute kidney injury and coronary artery disease", "CRS biomarkers", "CRS Type 1-5", "CKD and CVD", "AKI and MI", "CAD and CRF", "diagnosis" and "prognosis" in numerousdatabases and literature based search engine are PubMed database NCBI , Google Scholar and so on, and I came to find some potential studies published so far .We custThe Protein-Protein Interaction Network (PPIN) was built by submitting the Cardiorenal syndrome associated genes in STRING version 9.1 databases . It provides an integrative and critical assessment of PPInetworks with the diversity of organisms. STRING is a biological database of known as well as predicted protein-protein interactions, that can offer deep insight of cellular processes . The datFor the cluster/module/community formation in the PPI network of genes, Cytoscape was used with plugin Molecular Complex Detection (MCODE), to identify highly interconnected regions in the network. The plugin provides tools for investigating and visualizingprotein interactions on the detailed molecular level of modules and interaction sites. And default statistical parameters were used as "Degree cutoff = 2", "node score cutoff = 0.2", "k-core = 2 (default more than 1)" and "max. Depth = 100". The protein consistingmodules involved cellular processes are functional while binding each other. Epochal modules can be identified as highly interconnected subgraphs and several computational approaches are now inevitable to extract from complex networks. Moreover, MCODE was employedto the highly dense modules at the level of network stability .Gene ontology (GO terms) provides a dynamic, comprehensive, and standardized vocabulary that can be annotated in all eukaryotes, mostly including 3 independent categories, biological process (BP), molecular function (MF), and cellular components/localizations(CC). The Enrichr database (http://amp.pharm.mssm.edu/Enrichr/) was utilized to perform GO functional annotation P value < 0.05 was considered as statistically significant. Enrichment analysis is a popular method for analyzing gene sets generated by genome-wideexperiments. Enrichr currently contains 332911 annotated gene sets from 164 gene set libraries. Enrichr, an integrative web-based and mobile software application that includes new gene-set libraries, an alternative approach to rank enriched terms, and variousinteractive visualization approaches to display enrichment results. The software can also be embedded into any tool that performs gene list analysis .Pathway enrichment analysis (PEA) is a more potent approach for gene analysis in genomics, mostly applied to gene expression and support an interactive evaluation of the possible effects of variations on function, regulation or interaction of gene (mRNA) products.Genomic data are increasingly being used to identify biological pathways offers the potential of greater power for discovery with natural connections to biological mechanisms and networks underlying complex diseases . EnrichrPeer-reviewed articles published in reputed journals were screened for key genes/proteins that are directly or indirectly associated with CRS. More than hundred (approx-104) publications published in between 2008 to 2020 covered a set of 119 non-redundant geneslinked with CRS as described in Table 1 (see PDF) . Out of Reported genes were used to make a PPI network, which was constructed using the STRING database, the network consisted of 1149 nodes and 25548 interaction pairs The functions of clusters/subnetworks were evaluated by GO and pathway enrichment analyses (PEA). The results demonstrated that a total of 765 GO terms (MF and BP), 108 KEGG pathways and 114 TFs were enriched. Based on the most significant P values, the topfifteen GO terms, KEGG pathways and TFs were selected. For better understanding these 12 target genes, Enrichr database was utilized to perform GO function and KEGG pathway enrichment analysis. GO terms (MF and BP), KEGG pathways and TFs are described in Table 3 (see PDF),Table 4 (see PDF) and Table 5 (see PDF). Respectively. GO analysis indicated that the key genes were significantly enriched in protease binding, cytokine activity, mitogen-activated protein kinase kinase binding, hormone activity, transition metal ion binding,regulation of blood vessel diameter, regulation of cell migration, positive regulation of macromolecule biosynthetic process, negative regulation of blood vessel diameter, regulation of systemic arterial blood pressure by hormone, and Pathway information isinherently redundant, as genes often participate in multiple pathways, and databases may organize pathways hierarchically by including general and specific pathways with many shared genes, AGE-RAGE signaling pathway in diabetic complications, HIF-1 signalingpathway, Chagas disease (American trypanosomiasis), Relaxin signaling pathway, Renin secretion. Pathway enrichment analysis in to provide functional insight into the identified network marker. The interaction between transcription factor (TF) proteins and DNA iselementary to the regulation of transcription, a coordinated process that responds to environmental factors to achieve temporal and tissue specificity. Therefore, the ability to predict and identify TF binding sites throughout genomes is integral to understandingthe details of gene regulation and for inferring regulatory networks. The NOS3 gene has many polymorphisms among them; the evidence showed that the coding region 4b/4a, the G894T, and the T786C variants is significantly implicated in CRS etiopathogenesis. Variationsin the number of genes are the leading cause of increasing risk of the disease. A key gene plays a crucial role in the regulation of reduced blood vessel relaxation and NOS3; these are two main risk factors. REN genes found significantly involved in the progressionof CV and disease independent of the classical renin-angiotensin-aldosterone-system. Downstream of ANG I and not targeting the activity of REN or their concentration, since the relation of heart failure and kidney problems with increased renin levels, mainly withthe availability of ACEi or ARB. Loss of negative response of ANG II while renin release leads to growth of renin level secretion by ACEi and ARB. Analysis of multiple variations shows that greater levels of CRP significantly predict LV dysfunction and cardiachypertrophy. Finally, increased levels of CRP in the patients having hemodialysis are linked with a higher risk of death that indicates some values towards prognosis for this inflammatory mediator. As a biological marker of systemic inflammation, CRP may playrole in endothelial dysfunction by enhancing the expression level of endothelial cell adhesion molecules, lower level of nitric oxide and prostaglandin secretion from cells of endothelial tissues, augmenting low-density lipoprotein uptake by macrophages, andinducing complement-mediated inflammatory reaction. Chronically inhibiting the synthesis of NO may lead to upregulation of cardiac ACE and Ang II receptors, possibly mediating inflammatory changes. It has been previously reported that the stimulus for the sympathetichyperactivity found in renal dysfunction generated from kidney failure and that growing sympathetic outflow in CRF possibly could be controlled with the inhibition of ACE. MPO plays a role as a master enzyme in the ROS generation through catalyzing the conversionof hydrogen peroxide. Increasing levels of MPO were able to observe higher risk of CAD development in normal candidates. MPO has been considered to be an important oxidative stress pathway in ESRD. Hypomethylation of EDN1 gene in collected duct cells of renalinner medullary that express high levels of ET-1. In contrast, fibroblasts have a hypermethylated edn1 gene and express only a lower amount of ET-1. In renal epithelial cells EDN1 gene has a major role for calcium, whereas Vezf1 has a special response of EDN1gene activity in endothelial cells. Insulin resistance in endothelial cells promote the progression of prothrombotic factors level, proinflammatory markers, and ROS, that may cause to increase in the intracellular levels of adhesion molecule 1 (ICAM-1) andvascular cell adhesion molecule 1 (VCAM-1). In order to generate CVD two independent pathways play crucial roles through the contribution of Insulin resistance, (1) atheroma plaque formation and (2) ventricular hypertrophy and diastolic abnormality. Both pathwayscause heart dysfunction. TGF-β1 found in both myocardial fibroblasts and cardiomyocytes. Higher expression of TGF-β1 is seen in heart during the process of cardiac development and pathology. Angiotensin II (Ang II) has potential for hypertrophic stimulus,responsible for progression of TGF-β1 gene expression. TGF-β1 is a principal mediator of the hypertrophic growth response of the heart to Ang II. Epidermal growth factor receptor (EGFR) (or ERBB1), a membrane tyrosine kinase receptor expressed in thekidney, showed the activity after renal failure, and many preclinical reports have shown it is a potent target in CKD therapy. Regulation of many cellular responses is associated with activity of EGFR signaling pathway, such as cell proliferation, inflammation,and regulation of extracellular matrix, all processes and mechanisms functionally involved in the onset and renal failure progression. CCL2 considerably reduces hypoxia-induced cell death in cultured cardiac myocytes. In all the modulating processes of chemokines,CCL2 and their receptor CCR2 found to be most important for the shift from physiological conditions to pathological conditions in both heart and vessels. CCL2 is chemotactically attractive for mononuclear cells that are a source of fibrogenic mediators likeTGF-β and Fibroblast Growth Factor. Additionally, it causes synthesis of macrophages of both TGF-β1 and collagen. (SERPINE1), that has also been considered a mediator with potency of diabetic nephropathy and glomerulosclerosis. The study concluded thatthe cell cycle arrest biomarkers, urinary IGFBP-7, and TIMP-2, which may be responsive to take benefit towards clinical ways to predict the progression of type 1 CRS development after renal ischemia in patients having decompensated heart dysfunction. In thispilot study, ILGF-7 and TIMP-2 have been evaluated in the CRS development.We report 12 essential genes in the network through module analysis as potential emerging tools for understanding the pathophysiologic mechanisms for the early diagnosis of CRS. In this study we have established a complex potent CRS-related mRNA regulatorynetwork, which gives a deep understanding of molecular mechanisms and it provides key points in seeking novel therapeutic options for CRS. Although the findings will be required to validate through experimentation. A computational system based approach providesa systematic framework to find a hope in terms of the connection of a single biomarker candidate towards driving functional dependencies between clinically interlinked diseases. With the gigantic amount of present data generated from heart and kidney clinicalstudies, a uniformed pipeline is required for data curation that will facilitate researchers in retrieval of potential biomarkers from existing literature. To deepen understanding of the genes mechanisms, functions and their involvement, Enrichr database searchGene Ontology and KEGG analysis was also performed. Our study shows a comprehensive picture of molecular features related to the functional interplay between renal and the cardiovascular system. In this study, a literature mining approach to curated genesreported in the context of the CRS, and analyzed their features on the level of protein interaction networks.