P < .001) were identified for the Runyan Mixture. The main action targets were EGFR, MAPK1, AKT1, PIK3CA, NFKB1, SRC, TNF, MAPK8, MET, and PTGS2. Among the identified signaling pathways, 20 were associated with microbial infection and 24 were related to the immune-inflammatory response. Experimental results in vitro showed that Runyan Mixture could significantly inhibit the expression of interleukin-1, interleukin-6, and tumor necrosis factor-α (P < .05) in macrophages by lipopolysaccharide stimulation. Based on the results of the protein-protein interaction network analysis and the anti-inflammatory effect in vitro, the efficiency of the Runyan Mixture in pharyngitis treatment could be attributed to the inhibition of the inflammatory response.This study aimed to elucidate the mechanism of action of Runyan Mixture in treating pharyngitis using a network pharmacological approach. The active components of the Runyan Mixture were obtained from the traditional chinese medicine systems pharmacology database and evaluated using Lipinski’s rules. The SwissTargetPrediction database was used to predict the action targets of the Runyan Mixture, and a protein-protein interaction network was constructed using the STRING database. Moreover, the anti-inflammatory effect of Runyan Mixture was validated in vitro using the lipopolysaccharide induced inflammation in macrophages. The Runyan Mixture was the liquid preparation from 8 traditional Chinese medicine. A total of 89 types of active components, 53 core targets, and 98 signaling pathways ( Clinically, pharyngitis is mainly categorized into acute pharyngitis and chronic pharyngitis. The former is mainly caused by a bacterial infection or secondary bacterial infection after a viral infection, predominantly presenting local symptoms. The latter is a chronic inflammation of the pharyngeal mucosa, submucosal tissue, and lymphoid tissue, with the main manifestations of pharyngeal pain, itching, burning sensation, dryness, irritation, hoarseness, and sometimes nausea and vomiting. Antibiotics are generally used to treat acute pharyngitis, which makes it easy to develop drug resistance. Studies have shown that the use of antibiotics did not improve the symptoms; therefore, researchers in China and other Asian countries–6 are trying to find alternative therapies such as using traditional Chinese medicine (TCM), which is effective in treating pharyngitis.Pharyngitis is an inflammatory immune disease. Due to air pollution, climatic changes, and other factors, the incidence rate of pharyngitis is as high as 20% to 50%, making it a common disease with a rising trend.Solidaginis herba, which is also assisted by Scrophulariae radix, Forsythiae fructus, and Chrysanthemi indici Flos. The main medicines for relieving dry cough and sputum are Asparagi radix, Ophiopogonis radix, and Rehmanniae radix, which have the common therapeutic effect. Glycyrrhizae radix et rhizome, play the role of moderating the property of the TCMS, so that all medicines of this prescription together to relieve the symptoms of pharyngitis, such as dry cough and swelling and pain in pharynx. Single or combined usages of these TCMs have been described efficient in the treatment of inflammation related diseases.–9 Runyan Mixture has been used for the treatment of pharyngitis at our hospital for several years, and clinical observation suggested that Runyan Mixture has a significant alleviating effect (data not published).Runyan Mixture is a hospital liquid preparation that is obtained via boiling extraction from composed of 8 TCMs. The main medicine against inflammation of pharynx is –12 Therefore, we adopted network pharmacology in the current study to clarify the mechanism of action of Runyan Mixture in the treatment of pharyngitis and verify the anti-inflammatory effect using in vitro inflammation model.However, the compounds of the Runyan Mixture are relatively complex, making it difficult to explain the specific mechanism of action, thereby affecting the promotion and application of TCM. Network pharmacology is a new model in drug research proposed by Andrew L Hopkins in 2007, which can be used to build and analyze complex networks of “component-target-disease” based on systems biology, multidirectional pharmacology, network biology, computer technology, and various omics technologies, to explore the relationship between drugs and diseases comprehensively.https://old.tcmsp-e.com/tcmsp.php). The Lipinski’s Rule of Five, namely molecular weight (MWV500), lipid-water partition coefficient (AlogP < 5), number of hydrogen bond donors (Hdon < 5), number of hydrogen bond acceptors (Hat < 10), and rotational bonds (RBN < 10), was used to preliminarily screen the active components of Runyan Mixture.In this study, the name of the active medicinal material in the Runyan Mixture was used as the keyword, and the chemical components of each medicinal material were obtained from the traditional chinese medicine systems pharmacology database (http://www.swisstargetprediction.ch/) database to list the targets of the Runyan Mixture. SwissTargetPrediction is an online tool for predicting small molecule targets. In this study, the probability value for evaluating the accuracy of target prediction was set as >0.The chemical structures of the active ingredients were illustrated using ChemBioDraw Ultra 14.0, saved in SMILES format, and uploaded to the SwissTargetPrediction . The unconnected nodes in the network were hidden, and the other parameters were set to default values. The analysis result file was downloaded in CSV format and imported into Cytoscape 3.7.2 software. The PPI network was analyzed using the Network Analyzer tool, and the core targets of the Runyan Mixture were screened according to degree value.The disease targets were retrieved from GeneCards database by the keyword of pharyngitis. The common targets between disease and Runyan Mixture were imported into the STRING database to build the PPI network. The specific parameter settings were as follows: The species was https://david.ncifcrf.gov/summary.jsp) to perform the gene ontology (GO) function and kyoto encyclopedia of genes and genomes (KEGG) signaling pathway enrichment analyses and P < .001 was considered as the criterion for screening the signaling pathways of the Runyan Mixture. Cytoscape 3.7.2 software was used to establish the network of chemical components of Runyan Mixture-their targets-signaling pathways. The nodes in the network represented the chemical components of the Runyan Mixture, their targets, and signaling pathways, which were connected by edges if the corresponding nodes showed an interaction.The core targets were imported into the DAVID database . It was a brown to black-brown liquid with a slightly fragrant odor and sweet and slightly bitter taste. The drug liquid was centrifuged at 3000 rpm for 30 minutes, and the supernatant was filtered using a 0.22 μm filter for sterilization, followed by dilution for 5, 50, 500, 5000, and 50,000 times in 1640 complete medium  and storage at 4 °C for subsequent usage.6 cells/well. After differentiation induced by 50 ng/mL phorbol-12-Myristate-13-Acetate for 24 hours, the cells were cultured in fresh 1640 complete medium overnight. Inflammatory stimulation was performed with 1 μg/mL lipopolysaccharide (LPS), and cells were collected without the action of drug concentration for 12 hours.THP-1 cells were seeded into 6-well plates at a density of 1 × 10http://links.lww.com/MD/I204. The quantitative PCR system (20 μL) included 10 μL TB Green Premix Ex Taq II, 1 μL upstream and downstream primers, 2 μL cDNA, and 6 μL H2O. Quantitative PCR was performed using a QuantStudio 5  amplification instrument, and the conditions were pre-denaturation at 95 °C for 30 seconds, and 40 cycles of amplification (95 °C for 3 seconds and 60 °C for 30 seconds). GAPDH was used as an internal reference to calculate 2−ΔCT. The expression of cytokines in the experimental group was compared with that in the LPS stimulation group as the control group.The total RNA of the collected THP-1 cells was extracted using the Trizol  method and measured using nanodrop. About 1 μg of total RNA was reverse-transcribed to cDNA using the reverse transcription kit (Takara). The reverse transcription system of 20 μL included 4 μL 5X PrimeScript Buffer, 1 μL PrimeScript RT Enzyme Mix I, 1 μg RNA, 50 pmol Oligo dT primer, and 100 pmol random primers. The reverse transcription conditions were as follows: 37 °C for 15 minutes and 85 °C for 5 seconds. The interleukin (IL-1), IL-6, and tumor necrosis factor-α (TNF-α) mRNA levels were detected using real-time quantitative PCR (Takara) and the primer sequences are shown in Table S1, Supplemental Digital Content, X ± S). The T test was used for pairwise comparison, and a P value < .05 was considered to be statistically significant.SPSS 18.0 software  was used for the statistical analysis of experimental data. The data were expressed as mean ± standard deviation , including 307 terms related to biological process, 32 terms related to cellular component, and 61 terms related to molecular function, accounting for 76.75%, 8.00%, and 15.25% of the terms, respectively. The top 10 terms of biological process, cellular component, and molecular function are shown in Figure P < .001) by enrichment, including 44 signaling pathways related to microbial infection   could significantly inhibit transcriptional expressions of IL-1, IL-6, and TNF-α (05) Fig. . The inhThe Runyan Mixture can effectively improve the symptoms of acute and chronic pharyngitis in clinical practice. However, its mechanism of action is not clear. Here, a combination of network pharmacology and in vitro cell experiments was performed to investigate the possible mechanisms of action of the Runyan Mixture in pharyngitis treatment.Solidago decurrens,14 and Chrysanthemum Indici Flos, hispidulin is the main active component of Chrysanthemum Indici Flos, and kaempferol and 7-hydroxycoumarin are the main active components of S decurrens.–17 The anti-inflammatory effect of kaempferol has been confirmed at the cellular level, characterized as reducing the production of pro-inflammatory cytokines such as thymic stromal lymphopoietin, TNF-α, and IL-8.,19 Caffeic acid can clear the reactive oxygen species released by inflammatory neutrophils and macrophages, and enhance the anti-inflammatory effect of the body by reducing the expression of proinflammatory cytokines.,21The results of network pharmacology of Runyan Mixture showed that there were several active components in Runyan Mixture. Among them, caffeic acid is a common major active component of Phlomis brevidentata H.W.Li Radix, which is efficient in treating pharyngitis. PTGS2, NFKB1, and TNF have been essential in chronic pharyngitis treatment by herba Sarcandrae. Though the roles of other targets in pharyngitis have not been reported, they are significant in the modulation of inflammatory cytokines.,25 The expression levels of IL-1, IL-6, and TNF-a were inhibited by Runyan Mixture in vitro results, evidently indicating its therapeutic role for pharyngitis by inhibiting the inflammatory response.A total of 228 targets were obtained after the intersection analysis of compound-disease targets, including 53 core targets, suggesting that the Runyan Mixture has a synergistic effect in the treatment of pharyngitis. According to the analysis results of the PPI network, the main targets of the Runyan Mixture are EGFR, MAPK1, AKT1, PIK3CA, NFKB1, SRC, TNF, MAPK8, MET, and PTGS2. Among the key signaling pathways, AKT1, MAPK1, and EGFR are also the targets of 3 iridoid glycosides from –28 Therefore, further studies are required for structure identification using different methods, such as mass spectrometry molecular network. Also, further pharmacodynamic experiments are required in the future to validate the mechanism of action of the Runyan Mixture in pharyngitis treatment.The present work also had certain limitations. The network pharmacology studies of TCM preparations do not consider the complex compounds changes and the ingredient losses from herbs during the extraction process.This study predicted the targets, mechanism of action, and signaling pathways related to the Runyan Mixture in the treatment of pharyngitis using network pharmacology. The results revealed that the mechanisms of action of the Runyan Mixture against pharyngitis were mainly attributable to pathways related to inflammatory responses. Moreover, the key active ingredients in the Runyan Mixture were caffeic acid, medicarpin, hispidulin, kaempferol, and 7-hydroxycurmarin. The in vitro experiments confirmed the anti-inflammatory effect of the Runyan Mixture by inhibiting the transcription levels of proinflammatory cytokines. These results might help clinicians understand the mechanism of action of Runyan Mixture in pharyngitis treatment.Conceptualization: Huihui Zhang, Yinzhi Jin, Zhenxin Li, Xinling Pan.Data curation: Huihui Zhang, Yingpeng Tong.Formal analysis: Guoyun Cai, Zhenxin Li.Funding acquisition: Huihui Zhang.Investigation: Zhenxin Li, Xinling Pan.Methodology: Yingpeng Tong, Guoyun Cai, Xinling Pan.Software: Huihui Zhang, Yingpeng Tong, Guoyun Cai.Supervision: Yinzhi Jin, Zhenxin Li.Validation: Guoyun Cai, Zhenxin Li, Xinling Pan.Writing – original draft: Huihui Zhang, Xinling Pan.Writing – review & editing: Yingpeng Tong, Yinzhi Jin, Guoyun Cai, Zhenxin Li, Xinling Pan.


In the first sections of this review, we provide a comprehensive description of the Hedgehog signalling pathway in mammals and the main general models of pathway activation. Subsequently, the review focuses on the oncogenic role played by this pathway in rhabdomyosarcoma and the inhibitors developed to date, as well as the clinical trials available in sarcomas. Finally, we provide a discussion and critical review of the results obtained in the clinical setting and their strong dependency on the type of tumour. In some cases, strong discrepancies between encouraging preclinical data and clinical trial results are clearly evident. Aberrant activation of the Hedgehog (Hh) signalling pathway is known to play an oncogenic role in a wide range of cancers; in the particular case of rhabdomyosarcoma, this pathway has been demonstrated to be an important player for both oncogenesis and cancer progression. In this review, after a brief description of the pathway and the characteristics of its molecular components, we describe, in detail, the main activation mechanisms that have been found in cancer, including ligand-dependent, ligand-independent and non-canonical activation. In this context, the most studied inhibitors, i.e., SMO inhibitors, have shown encouraging results for the treatment of basal cell carcinoma and medulloblastoma, both tumour types often associated with mutations that lead to the activation of the pathway. Conversely, SMO inhibitors have not fulfilled expectations in tumours—among them sarcomas—mostly associated with ligand-dependent Hh pathway activation. Despite the controversy existing regarding the results obtained with SMO inhibitors in these types of tumours, several compounds have been (or are currently being) evaluated in sarcoma patients. Finally, we discuss some of the reasons that could explain why, in some cases, encouraging preclinical data turned into disappointing results in the clinical setting.  Our understanding of the roles played by these pathways in paediatric tumours is progressing; however, it is far from that of better-known adult malignancies. Nevertheless, recent findings supported these pathways and some of their components as very promising and interesting putative therapeutic targets. Childhood cancers differ from adult malignancies owing to their different aetiology, biology, response to treatment, and outcome. However, despite the small number of cases of childhood cancers compared with the adult population, the understanding of the molecular biology of paediatric tumours has improved considerably in recent years. The pathways often known as FOXO1 and PAX3 or PAX7 genes), a stratification criterion that has been recently demonstrated to be a powerful predictor of prognosis ,5028,50]tivation ,51, whictivation   and GLI1 in malignant rhabdoid tumours is particularly suggestive, as about 25% of RMS tumours share the SNF5 mutational inactivation characteristic of rhabdoid tumours [Finally, in addition to the interactions with the better-known pathways described above, many other tumour-specific GLI dependencies have been described. For example, the EWS/FLI translocation driver of Ewing’s sarcoma directly transactivates de GLI1 promoter . Additio tumours ,68.The involvement of the Hh pathway in the genesis of RMS was first described in the patched knockout mouse by Hahn et al. in 1998, who reported that mice with heterozygous inactivation of PTCH1 had an increased incidence of an embryonal subtype of RMS (ERMS) . CurrentPTCH mutations nor activating SMO mutations appeared to be implicated in pathway activation [PTCH region 9q22 in one third of ERMS, and loss of SUFU has also been reported in 18% of ERMS [PTCH1, one in SUFU) [GLI1 gene was identified in a very small subset of ARMS tumours [Moreover, a minor but non-negligible percentage of patients bear genetic alterations that can trigger pathway activation in a ligand-independent manner. However, despite all authors agreeing that is not found in the majority of tumours, some controversy exists regarding the importance of this mutation-driven constitutive activation. On the one hand, some works reported that neither tivation ,74; on t of ERMS ,76. In ain SUFU) . Additio tumours ,75. Desp®, Genentech, San Francisco, CA, USA) and Sonidegib , for the treatment of adult BCC [®, Pfizer, New York, USA), employed in the treatment of newly-diagnosed acute myeloid leukaemia (AML) [The extensive involvement of the Hh pathway in the oncogenesis and progression of several cancers, including RMS, suggested a marked potential for pathway inhibitors as a putative molecularly-targeted anticancer therapy . These edult BCC ,81 and Gia (AML) . SMO inhibitors have shown encouraging results in the treatment of BCC and MB ,85,86,87Other compounds developed later, also demonstrated effectivity in preclinical models but their studies in clinical trials have been quite limited. Thus, despite promising preclinical results, TAK-44 was suspended in 2013 due to project prioritization, BMS-833923 (XL139) was withdrawn from the company pipeline in 2014 without disclosing the reason, and the CUR61414 clinical trial was halted due to unsatisfactory results . FinallyThe possibility of using the novel antifungal agent Posaconazole was recently pointed out; it can downregulate some targets of the Hh pathway . This work in vitro and in a murine model provided a theoretical basis that may have important clinical implications in developing Posaconazole as a promising agent against ERMS by targeting the Hedgehog pathway .cancersGLI inhibitors were highlighted as an interesting alternative to SMO inhibitors , as theyHedgehog inhibitors are thought to be a potential therapeutic alternative in about one third of human cancers (those with the pathway active), and, very often, preclinical data appeared to also corroborate their potential to treat them; however, clinical studies dramatically lowered expectations. In fact, the only approved indications for SMO inhibitors are BCC, MB, and AML, where Hh pathway inhibitors showed clear clinical responses. It may be too simplistic, but as a general principle, it could be stated that SMO inhibitors work acceptably well in solid tumours when they harbour pathway activating mutations, but have not yet been shown to be sufficiently effective in tumours in which the pathway is activated in a ligand-dependent manner or by non-canonical activation. The case of RMS, a type of tumour that does not stand out for harbouring mutations in Hh pathway genes does not constitute an exception to this general rule. Thus, despite evidence rendering the relationship between the Hedgehog pathway and RMS clearly manifest ,71,112, There are many possible explanations for the clinical failure of SMO inhibitors outside mutation-driven cancers. One reason may be the high concentrations used in preclinical experiments. For instance, even when using in vitro concentrations up to 300-fold higher than the levels required to block SMO in MB, the study in RMS of Hahn et al. concluded that the downregulation of GLI1 was very modest . Moreover, in the same study, the effects seen on cell proliferation did not correlate with the degree of GLI1 downregulation  and wereBeyond the use of SMO inhibitors, GLI inhibitors (such as GANT61), may have potential in tumours that express high levels of these proteins and may have a broader range of indications since they may function regardless of the presence of Hh-activating mutations. GANT61 has shown to inhibit GLI activity in preclinical models that also leads to tumour growth impairment . GANT61 Another promising therapeutic strategy is the combination of Hedgehog inhibitors with other agents. Potentially, combined approaches could act on crosstalk compensatory mechanisms with other pathways, which presumably could benefit not only patients with initially resistant tumours but also tumours that develop resistance after receiving targeted Hh monotherapy. However, selecting the ideal combination is highly challenging and requires a deeper understanding of the complex mechanisms involved. Sometimes, although there is biological evidence of interaction, some combinatory strategies are not effective, such as Hedgehog and Notch inhibition in patients with sarcomas ,94. In tPTCH1). In contrast, SHH MB patients with molecular aberrations of genes downstream of SMO (GLI2 and SUFU) did not respond to therapy. Notably, based on a strong diffuse P53 immunohistochemistry pattern, the authors suggested a link between TP53 DNA-binding domain mutations, which would favour gene amplification downstream of SMO, and the consequent lack of efficacy of Vismodegib. Therefore, genome sequencing and copy number analysis to identify Hh pathway mutations and potential cooperating mutations were proposed for future trials [GLI1, SHROOM2, SPHK1, and PDLIM3 and down-regulation of OTX2) strongly correlated with response in Sonidegib-treated BCC and MB patients [GLI1 in tumour and normal skin biopsies. However, GLI1 inhibition didn’t correlate with tumour responses, pointing at GLI1 as a good pharmacodynamic marker for SMO inhibitors but not for tumour response [PTCH1 loss in clinical trials of SMO inhibitors could potentially benefit patients and provide valuable new molecular data.In addition, the complexity of the aberrant involvement of Hh in cancer emphasises the need for predictive biomarkers to optimise treatment selection for each patient. This need has already been addressed with SMO inhibitors in MB, where the variable clinical responses correlate to the genomic heterogeneity of the SHH subgroup ,120,121.e trials . Howevere trials . This fipatients . Additioresponse ,123. UnfPathological activation of the Hh signalling pathway is known to play an oncogenic role in a wide range of solid tumours. Among them, those that are often associated with oncogenic mutations in several components of the pathway (MB and BCC) clearly stand out. In this particular cancer types, SMO inhibition has demonstrated strong clinical potential and, specifically for these tumours, three SMO inhibitors have already reached the clinical setting, rendering encouraging results. In these cases, the in vitro doses of SMO inhibitors required to inhibit the pathway are relatively low. On the other hand, in the tumour types in which pathway activation strongly relies on the presence of Hh ligands, as in RMS, the results are far less promising. Thus, the in vitro concentrations required to achieve anti-oncogenic effects in cell lines are very high (up to 600 times higher than in MB) and, very often, the specific Hh pathway target downregulation observed (such as in GLI proteins) are very disappointing, thereby suggesting an off-target toxicity that may explain why the positive effects observed in cell lines are not reproducible in the clinical setting. The development of GLI inhibitors is still incipient, with the majority of specific GLI compounds in preclinical stages. Only two naturally occurring compounds, ATO and Genistein, have reached clinical trials; but, in this case, the compounds were not designed to specifically bind GLI proteins, and can downregulate GLI proteins, along with many other targets. Another interesting point is the non-canonical activation, which very often is difficult to be distinguished from the canonical ligand-dependent. The non-canonical activation is thought to have an important role in RMS, since there is solid evidence that several pathways  can promote GLI protein activation in this particular type of cancer, thereby suggesting the possibility of using combined therapies which putatively may benefit patients with refractory tumours or even tumours that develop resistance after receiving Hh monotherapy. In this sense, the implementation of predictive biomarkers to personalize treatment for each patient would enormously help. This need has already been partially addressed with SMO inhibitors in MB , but we are still far away from this scenario in the majority of tumours (including RMS), since no predictive Hh-pathway biomarkers have been identified to date. Finally, the less characterised part of the pathway, the binding of ligands to the PTCH receptor and co-receptors CDO, BOC, and GAS1, may harbour strong potential for the development of future therapies, but these upstream elements, located in or related to the plasma membrane, remain still largely unexplored.


Blattabacterium cuenoti (hereafter Blattabacterium). In contrast to other obligate symbiotic systems, we found no clear evidence of “collaborative pathways” for amino acid biosynthesis in the genomes of these taxa, with the exception of collaborative arginine biosynthesis in 2 taxa, Cryptocercus punctulatus and Mastotermes darwiniensis. Nevertheless, we found that several gaps specific to Blattabacterium in the folate biosynthetic pathway are likely to be complemented by their host. Comparisons with other insects revealed that, with the exception of the arginine biosynthetic pathway, collaborative pathways for essential amino acids are only observed in phloem-sap feeders. These results suggest that the host diet is an important driving factor of metabolic pathway evolution in obligate symbiotic systems.Many insects harbor bacterial endosymbionts that supply essential nutrients and enable their hosts to thrive on a nutritionally unbalanced diet. Comparisons of the genomes of endosymbionts and their insect hosts have revealed multiple cases of mutually-dependent metabolic pathways that require enzymes encoded in 2 genomes. Complementation of metabolic reactions at the pathway level has been described for hosts feeding on unbalanced diets, such as plant sap. However, the level of collaboration between symbionts and hosts that feed on more variable diets is largely unknown. In this study, we investigated amino acid and vitamin/cofactor biosynthetic pathways in Blattodea, which comprises cockroaches and termites, and their obligate endosymbiont IMPORTANCE The long-term coevolution between insects and their obligate endosymbionts is accompanied by increasing levels of genome integration, sometimes to the point that metabolic pathways require enzymes encoded in two genomes, which we refer to as “collaborative pathways”. To date, collaborative pathways have only been reported from sap-feeding insects. Here, we examined metabolic interactions between cockroaches, a group of detritivorous insects, and their obligate endosymbiont, Blattabacterium, and only found evidence of collaborative pathways for arginine biosynthesis. The rarity of collaborative pathways in cockroaches and Blattabacterium contrasts with their prevalence in insect hosts feeding on phloem-sap. Our results suggest that host diet is a factor affecting metabolic integration in obligate symbiotic systems. Many insects have established intimate relationships with bacterial symbionts that have helped them to exploit new niches. In many cases, intracellular symbionts provide nutrients to their insect host, while the host provides a stable environment favorable to symbiont growth , 2. ThesBuchnera aphidicola (hereafter Buchnera). Aphids depend on Buchnera to supply many essential amino acids (EAAs) deficient in their phloem-sap diet (Buchnera lacks the genes coding for branched-chain amino acid aminotransferase (ilvE), the enzyme involved in the last step of BCAA biosynthesis (–One example is branched-chain amino acid (BCAA) biosynthesis in aphids and their endosymbiotic sap diet , 4. Howeynthesis , which iynthesis . The genynthesis –9.Such intimate metabolic interactions, which we term “collaborative pathways” , are notBlochmannia, has been proposed based on the gene content of the symbiont genome (Blattabacterium cuenoti (hereafter Blattabacterium). Blattodea have evolved a wide range of feeding habits and are sometimes known as “garbage collectors,” recycling dead plants, dead animals, and excrement  located in their fat body. Blattabacterium appears to have been lost in the lineage leading to all termites other than M. darwiniensis, as it has not been found in these taxa. The association between Blattodea and Blattabacterium is ancient and was established >200 million years ago , to which Escherichia coli belongs, the enzyme repertoire of the order Flavobacteriales (phylum Bacteroidota), to which Blattabacterium belongs, is not well-characterized. Unknown enzymes and pathways are still frequent in lineages that do not include model organisms, affecting prokaryote genome annotation . We then inferred the metabolic pathways of Blattodea using the genome and transcriptome of 2 Blattodean species harboring Blattabacterium  and the genome of 1 Blattodean species devoid of Blattabacterium (Zootermopsis nevadensis). Using this approach, we obtained robust inference of the pathways common across Blattodea. We compared the inferred metabolic pathways of Blattodea and Blattabacterium and identified potential collaborative pathways. Finally, we compared the profile of collaborative pathways in Blattabacterium with those of other host-symbiont systems with variable diets to see how diet affects the profile of collaborative pathways.In this study, we conducted a careful reconstruction of the metabolic pathways involved in amino acid and vitamin/cofactor biosynthesis of Blattabacterium Flavobacteriales bacteria and the genome of a representative of the genus Bacteroides of the Bacteroidales order, which we used as an outgroup (Table S1). We compared these with the metabolic pathways found in 7 Blattabacterium genomes. The presence/absence of each enzyme involved in amino acid and vitamin/cofactor biosynthesis is summarized for all genomes in Blattabacterium strains is the presence of urease (EC 3.5.1.5) in their genomes, a gene that is generally absent from the genomes of other strains of Flavobacteriales. Phylogenetic analysis of the urease large subunit (UreC) revealed that the sequences of Blattabacterium form a monophyletic cluster which is sister to sequences from members of the genus Paenibacillus (Firmicutes)  .Blattabacterium  and Mastotermes darwiniensis (MADAR), which have lost several of these pathways . On the contrary, specific gaps found only in Blattabacterium are likely to be complemented by host-derived metabolic intermediates, assuming that the host genome encodes genes complementing these gaps. Missing genes in the methionine, arginine, and riboflavin  biosynthetic pathways were assigned to common gaps. On the other hand, missing genes in the pyridoxine (B6), riboflavin , folate (B9), and menaquinone  biosynthetic pathways were assigned to the specific gap category. In addition, the enzyme involved in the final step of arginine biosynthesis, argininosuccinate lyase , and 2 enzymes involved in the synthesis of para-aminobenzoate  from the folate biosynthetic pathway, were absent from the genomes of the Blattabacterium strains CPUbr and MADAR, and were therefore considered as specific gaps , glutamine, asparagine, glycine, and proline, and two vitamins/cofactors, pantothenate (B5) and protoheme, were lost almost exclusively in acterium . In contacterium ; hereaftpathways . At the fic gaps . A gap iBlattabacterium-host metabolic collaboration, we compared the reconstructed metabolic pathways of Blattabacterium to those of their hosts, with the assumption that gaps common across Flavobacteriales and present in Blattabacterium probably do not need to be compensated by their hosts. We inferred the common metabolic pathways of Blattodea representatives by reconstructing consensus pathways from the cockroaches P. americana and C. punctulatus and the termite Z. nevadensis. The metabolic relationships between Blattabacterium and their hosts were classified into five categories  derived from the host genome. However, SerB is also absent from the genomes of several Flavobacteriales strains and an alternative enzyme catalyzing the same reaction, HisB (EC 3.1.3.-) , 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase (EC 4.1.1.97), and allantoicase (EC 3.5.3.4)  , is presuggested . We ther3.5.3.4) .Blattabacterium CPUbr and MADAR have undergone further genome reduction, being ~50kbp smaller than other strains , based on transcriptomic evidence. Therefore, we hypothesize that the arginine biosynthetic pathways of CPUbr and MADAR are collaborative , we identified a host enzyme, alkaline phosphatase (EC 3.1.3.1), which can bypass these reactions  and TrpE (EC 4.1.3.38), which are involved in the p-aminobenzoate (PABA) synthesis. We found no enzymes in the host genomes compensating for the loss of the PABA branch. We therefore hypothesize that the folate biosynthetic pathway is doubly-absent in CPUbr and MADAR.Among cofactor biosynthetic pathways, the riboflavin (B2), pyridoxine (B6), and menaquinone (K2) pathways were ependent . The proeir host . The thieir host . Althougeactions . Althougltonella . We thusBlattabacterium. Note that pathways present in secondary symbionts are omitted due to high variability in genome reduction levels. The data shown in Sulcia. Both Blattodea and carpenter ants have predicted collaborative arginine biosynthetic pathways; however, the enzymes involved are different. Carpenter ants require ornithine carbamoyltransferase (ArgI: EC 2.1.3.3) from their Blochmannia endosymbionts to complement their arginine biosynthetic pathway (part of the urea cycle), while in Blattodea, CPUbr and MADAR require argininosuccinate lyase (ASL: EC 4.3.2.1) from their hosts to complement their de novo arginine biosynthetic pathway. In phloem-sap feeders, collaborative EAA, and in particular BCAA biosynthetic pathways, are especially common, regardless of the symbiont's taxonomic position. The commonness of collaborative BCAA biosynthetic pathways in these insects is likely due to the evolutionary stability of the interaction between host and symbiont. This stability is also found in standard biosynthetic pathways. For example, Sulcia strain OLIH, which has the genome among Sulcia strains used in this study, has lost genes for all essential amino acids and vitamins/cofactors except for those for BCAA biosynthesis  and folate (B9) biosynthesis were detected  gaps shared by Blattabacterium and other Flavobacteriales strains, and (ii) gaps specific to Blattabacterium. We then analyzed the metabolic pathways of 3 species of Blattodea and compared them to those of Blattabacterium in order to examine potential metabolic collaborations in this symbiotic system. The product-level complementation (exchange of amino acids and vitamins between the host and its symbiont) shown in this study is consistent with results from previous studies, which performed genome-based flux balance analysis on Blattabacterium  and Blautrients , 23 and Blattabacterium lack biosynthetic pathway of B12, the reported higher cobalamin concentration in the hindgut of Blattodea is likely due to the presence of B12-producing microbes in their hindgut, such as Shimwellia blattae  in their hindgut , 75. Sin blattae . Unlike  blattae . We searC. punctulatus and M. darwiniensis and psyllids, which have another type of collaborative arginine biosynthesis pathway  phloem-sap feeders, which commonly have collaborative BCAAs, phenylalanine, and methionine biosynthetic pathways; (ii) carpenter ants, which have a collaborative arginine biosynthetic pathway; (iii) and the 2 blattodeans  pathway . Blood-f pathway .A previous study suggested that host gene content can be a constraint for collaborative pathway evolution . In the Sulcia systems lack collaborative pathways based on GS/GOGAT cycle. In this context, Blattodea do not need to use the GS/GOGAT cycle system because they use uric acid to store nitrogen wastes, and Blattabacterium utilizes host-provided urea as a nitrogen source  . The pron source . Because in ants . This po in ants . This maBlattabacterium and several gaps common across Flavobacteriales. However, since our approach focused on interpreting gaps rather than reconstructing whole system metabolic networks, it is unclear how the metabolic collaborations affect the total metabolic flux within the symbiotic system. Although multi-organism scale modeling methods are needed to answer such questions, these modeling methods require experimental evidence, such as metabolite analyses, chemically-defined diets, or differential gene expression data, to make accurate predictions. Future studies are needed to determine the precise mechanisms and adaptive significance of pathway complementation in insect-symbiont systems and to provide a clear picture of the evolution of their genomes.Our study has revealed several general trends of metabolic pathway evolution within and across insects and their symbionts. We found genomic evidence that symbiont partnerships vary in their amino acid and vitamin/cofactor biosynthetic collaborations. Experimental evidence, such as differential gene expression analysis or enzymatic assays, is required to identify the precise mechanisms by which these collaborations occur. For example, in the case of gaps unique to one or several strains of symbionts, complementation by unknown host enzymes or the emergence of new multifunctional enzymes should be considered as potential replacements . Our anaPeriplaneta americana  were determined using Proteinortho5  evolutionary stability; (ii) lineage-specificity, implying they are reactions which are complemented by alternative/unknown enzymes (Blattabacterium when shared by the 7 Blattabacterium genomes analyzed here but present in less than 10% of other Flavobacteriales. We refer to these gaps as “specific gaps”. Specific gaps were assumed to be complemented by the host if enzymes catalyzing the same reaction were found in the host genome. In case no such enzymes were found in the host genome, we assume the presence of alternative enzymes specifically found in Blattabacterium.We investigated whether gaps or missing enzymes in pathways –107. In  enzymes , 108; an enzymes . The for


Sijunzi decoction (SJZD), a classic Chinese formula, has been clinically used for the treatment of gastrointestinal disorders. However, few studies have uncovered its antitumor effects and its potential mechanisms against gastric cancer (GC). Therefore, this work aimed to identify the active compounds and putative targets of the SJZD and to further explore the potential mechanisms involved in the treatment of GC.  The active compounds and potential targets of the SJZD and related genes for GC treatment were collected from a public database. Traditional Chinese medicine (TCM)-compound-target-disease networks, Venn diagrams, protein–protein interactions (PPIs), gene ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to obtain the bioactive compounds, key targets, and potential pathways. Next, the human gastric adenocarcinoma cell line NUGC-4 was inoculated subcutaneously into the right flank of NCG mice to build a tumor-bearing mouse model to further verify the findings.  There were 117 compounds in the SJZD in total. The SJZD and GC had 161 and 3288 potential targets, respectively, among which 123 targets overlapped. The network analysis showed that quercetin, kaempferol formononetin, ginsenoside, atractylenolide III, etc., were bioactive molecules. The tumor necrosis factor (TNF), interleukin-6 (IL-6), cellular tumor antigen p53 (TP53), transcription factor AP-1 (JUN), and vascular endothelial growth factor A (VEGFA) were potential targets. A KEGG pathway enrichment analysis revealed 110 pathways involved in the pathways for cancer, including the PI3K-AKT signaling pathway. Validation experiments showed that the SJZD inhibited tumor growth and induced apoptosis in tumor cells. In addition, the SJZD downregulated expressions of VEGFA, iNOS, COX-2, and Bax/Bcl2 and inhibited the expressions of p-PI3K and p-AKT.  The SJZD treats GC by inhibiting blood vessel hyperplasia and inducing cell apoptosis by regulating the PI3K/AKT pathway. Helicobacter pylori infections, etc., can still significantly increase the risk of developing GC [With its heterogenous nature, gastric cancer (GC) is a highly aggressive malignancy, and it is the fourth most common cancer in the world, with more than 70% of the cases occurring in the developing world , 2. Due oping GC , 6. GC poping GC . Howeveroping GC . Traditioping GC , 12.Glycyrrhiza uralensis) [The Sijunzi decoction (SJZD) is a classic Chinese formula for “reinforcing asthenia qi” and nourishing the spleen; it consists of Renshen (Codonopsis pilosula), Baizhu , Fulin (Poria cocos), and Gancao  . In clinalensis) . Zhao Aialensis) . HoweverNetwork pharmacology is an integrated discipline that combines systems biology, multidirectional pharmacology, computational biology, and network analysis to investigate the relationships between drugs and diseases to find drug targets . ChineseGlycyrrhiza uralensis Fisch of legumes) were selected, and the herbs' compounds and targets were retrieved from the Traditional Chinese Medicine System Pharmacology Database . The compounds were screened according to the pharmacokinetic parameters “oral bioavailability (OB) ≥ 30%” and “drug likeness (DL) ≥ 0.18”. Then, the targets of GC were obtained from the GeneCards (https://www.genecards.org/), Online Mendelian Inheritance in Man , and Therapeutic Target Database  databases with “Gastric Cancer” as the keyword. Next, the corresponding gene names were found in the UniProt Knowledgebase (UniProtKB) (https://www.UniProt.org/) by searching for “Homo sapiens” [The keywords “Renshen” (Renshen is the root of the acanthaceae plant), “Baizhu” , “Fulin” (Fulin is the dried sclerotia of Poria cocos (Schw.)Wolf), and “Gancao”  with the following limiting condition: species (Homo sapiens). Then, the PPI network was analyzed by using Cytoscape 3.6.1, which obtained “Nodes” representing the target protein molecules and “Edges” representing the mutual relationships.The intersection targets of the SJZD and GC were uploaded to the STRING database (https://david.ncifcrf.gov/home.jsp) by inputting the above targets. Then, the top 10 terms were selected to make graphical structures in GraphPad Prism 8.0. The results of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were obtained from the DAVID database, and a bubble chart was made on imageGP (http://www.ehbio.com) to explore the correlated functional pathways of the selected genes.A gene ontology (GO) analysis including biological process (BP), cellular component (CC), and molecular function (MF) was performed in the DAVID database . The study was approved by the Medical Research Ethics Review Committee of the Beijing Shijitan Hospital of the Capital Medical University . When the tumor size reached 100 mm3, the tumor-bearing mice were randomly divided into 4 groups: model group (n = 6), SJZD-L group , SJZD-H group , and Urelumab group . Mice in the SJZD groups were administered SZJD once a day, and mice in the Model group were given an equal volume of distilled water. The mice in the Urelumab group were injected with Urelumab twice a week. The tumor size was measured every week. After the administration of the drugs for 21 days, the mice were sacrificed, and the tumors were isolated, photographed, and weighed. The tumor growth inhibition weight value (TGIW) and tumor growth inhibition volume value (TGIV) were statistically analyzed. TGIW% = (1-Treatment group weight/Model group weight) × 100%; TGIV% =  (1-Treatment group RTV/Model group RTV) × 100%; Relative tumor volume (RTV) = V21 day/V0 day; Tumor volume = 0.5 × major axis × minor axis2.The human gastric adenocarcinoma cell line NUGC-4 was maintained in a RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere containing 5% COL of PBS . The celμm sections using a microtome. After dewaxing and rehydration, the sections were stained with hematoxylin & eosin solution . Tumors were identified on sections. These sections were sealed using neutral gum and photographed under a microscope  at  × 200.Tissues were fixed with 10% neutral buffered formalin overnight, embedded in paraffin, and cut into 4.5 β-actin  was used as an internal control.Tumors were lysed using a radioimmunoprecipitation assay (RIPA) buffer to obtain the proteins. The protein concentrations were detected by a BCA protein assay kit. The proteins were separated by SDS/PAGE and blotted onto polyvinylidene fluoride (PVDF) membranes. Next, the membranes were blocked in skim milk for 2 hours and then incubated with primary antibodies against Bax , Bcl2 , p-PI3K , p-Akt , and VEGFA  at 4°C overnight, followed by HRP-conjugated secondary antibodies. Protein bands were detected using an ECL chemiluminescence system . After dehydration, hydration, and antigen recovery, tumor biopsies were incubated with the primary antibodies at the following dilution ratios: iNOS  and COX-2 . Subsequently, the sections were incubated with a secondary antibody for 2 hours at room temperature. Finally, these sections were sealed using neutral gum and photographed under a microscope at a magnification  × 200.t-test. P < 0.05 was considered to indicate a statistically significant difference.Data are presented as the mean ± standard deviation. Differences between the two groups were investigated using Student's As is shown in the Supplement To perform a comprehensive analysis, a TCM-compound-target-disease network was constructed , including pathways in cancer and the PI3K-AKT signaling pathway . The H&E results showed that the tumor cells in the model group were closely arranged and grew well, with hyperchromatic nuclei and normal morphologies. After treatment with a high dose of the SJZD, the cells showed necrosis and exhibited diffuse and ambiguous distributions . The results suggested that the SJZD regulated the PI3K/AKT/VEGF pathway to treat GC.According to the results of the PPI network and KEGG pathway enrichment analyses, specific targets, including VEGFA, Bax, Bcl2, and the PI3K/AKT pathway, were involved in the SJZD treatment of GC. Therefore, we used WB to measure the expression of these proteins. As shown in Figures P < 0.01). These results demonstrated that SJZD treatment downregulated the iNOS and COX-2 levels in the gastric tumor models.Furthermore, the expression levels of iNOS and COX-2 were evaluated by immunohistochemistry. Figures In this study, network pharmacology and in vivo experiments were performed to reveal the bioactivity and mechanism of SJZD action for treating GC. The TCM-compound-target-disease network showed connections between multiple compounds and targets, which is consistent with the holism of TCM . PPI resThe SJZD, a traditional Chinese prescription, has been used for the clinical treatment of gastrointestinal disorders for over 2000 years . Due to The PPI network and KEGG results indicated that VEGFA was the main target in the treatment with SZJD against GC. VEGF is a well-known angiogenic factor and plays an important role in the proliferation, migration, and invasion of vascular endothelial cells . The angTo further explore the mechanisms of SJZD action for the treatment of GC, KEGG enrichment analysis was performed, which showed that the PI3K-AKT signaling pathway was the most enriched gene involved in the SJZD treatment of GC. The PI3K-AKT signaling pathway participates in many cellular activities, including cell growth, differentiation, apoptosis, and cytoskeletal rearrangement . PI3K isIn summary, our study combined network pharmacology-based predictions and in vivo experiments to verify the antineoplastic efficacy and the potential mechanisms of SJZD action for the treatment of GC. We found that the SJZD exerted its anti-GC effects by downregulating the expression of VEGFA, iNOS, and COX-2 to inhibit blood vessel hyperplasia and by upregulating the Bax/Bcl2 ratio to induce cell apoptosis by regulating the PI3K/AKT pathway. However, there were still deficiencies in this study. The potential compounds involved in the SJZD treatment of GC need to be identified and validated in vivo or in vitro. In addition, the related targets should be further explored to provide a theory for the SJZD treatment of GC.


This study elucidates the potential therapeutic targets and molecular mechanisms of KTC in the treatment of PCOS.  Using the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP), the active ingredients and potential targets of KTC were obtained. The Gene Expression Omnibus (GEO) database was used to find differentially expressed genes (DEGs) related to PCOS. Search the CTD, DisGeNet, genecards, NCBI, OMIM, and PharmGKB databases for therapeutic targets related to PCOS. The intersection of potential targets, DEGs, and therapeutic targets was submitted to perform bioinformatics analysis by R language. Finally, the analyses' core targets and their corresponding active ingredients were molecularly docked. Discussion and Conclusions. We explored the active ingredients and molecular mechanisms of KTC in the treatment of PCOS and found that quercetin may be the core ingredient of KTC in the treatment of PCOS. 88 potential therapeutic targets of KTC for PCOS were discovered by intersecting the potential targets, DEGs, and therapeutic targets. According to bioinformatics analysis, the mechanisms of KTC treatment for PCOS could be linked to IL-17 signaling route, p53 signaling pathway, HIF-1 signaling pathway, etc. The minimal binding energies of the 5 core targets and their corresponding ingredients were all less than -6.5. Further research found that quercetin may replace KTC in the treatment of PCOS.  Polycystic ovary syndrome (PCOS) is one of the most common endocrine and metabolic diseases in gynecology. The main symptom of PCOS is excessive androgen, which also affects ovarian function and causes infertility . At presIn recent years, the curative effect of many classic prescriptions of traditional Chinese medicine (TCM) in the treatment of PCOS has been recognized by more and more people . AccordiAs one of the cutting-edge methods to explore the mechanisms of drug therapy, network pharmacology has achieved remarkable results in exploring the therapeutic mechanisms of TCM prescriptions and screening the active ingredients and therapeutic targets of TCM  based on the conditions of drug-like properties ≥ 0.18 and bioavailability ≥ 30% [https://www.uniprot.org/). Finally, the Cytoscape 3.7.2 software was used to construct the relationship network between the active ingredients and target genes of KTC.We searched the active ingredients of KTC through the TCMSP database .We merged the three PCOS-related datasets  found in the Gene Expression Omnibus (GEO) database and further used the R language “sva” and “limma” packages for batch correction and differentially expressed genes (DEGs) screening |log2 (foldchange)| > 1 and Therapeutic targets obtained from CTD, DisGeNet, genecards, NCBI, OMIM, and PharmGKB databases were combined with DEGs from the GEO database and screened for targets appearing in at least two databases. These targets were then intersected with KTC therapeutic targets to identify prospective KTC therapeutic targets for PCOS.We obtained the interactions between potential therapeutic targets of KTC through the STRING database. The protein-protein interaction (PPI) network was constructed by the Cytoscape software, and the core therapeutic targets were further screened according to the degree value. To investigate the probable molecular mechanisms of KTC in the treatment of PCOS, R language was used to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis.Firstly, identify the active ingredients that correlate to KTC's core targets in the treatment of PCOS. The active ingredient's two-dimensional structure was retrieved via the PubChem website and translated into the three-dimensional structure with the lowest free energy by using the ChemBio3D software. Then, the 3D structure of the core target was obtained through the PDB database, and the water molecules and small molecule ligands were deleted through the “PyMOL” software. Next, the “AutoDockTools” software was used to convert the protein and drug ingredient into PDBQT format files and identify active pockets. Finally, we used the “vina” software for molecular docking.We intersected the therapeutic targets of all active ingredients in KTC with the therapeutic targets of KTC. The active ingredient with the most overlapping targets was considered to be the core ingredient of KTC. Further bioinformatics analysis of the potential therapeutic targets of the core ingredients was performed.According to the screening conditions, 80 active ingredients and 204 therapeutic targets of KTC were obtained through the TCMSP database , NCBI, OMIM, and PharmGKB databases, respectively, by using keywords “polycystic ovary syndrome” and “PCOS” . In mammals, the MAPK family participates in a variety of biological processes in the human body. Currently, the 14 MAPK family members that have been identified played important roles in transforming extracellular stimuli into cellular responses . MAPK8 aα and TNF-β secreted by macrophages and T lymphocytes, respectively. As an adipokine of systemic inflammation, TNF-α is highly expressed in obese PCOS patients . Insuliysiology . Recent atients (; Wang D atients .By molecular docking of the 5 core targets and their corresponding drug ingredients, we found that wogonin-TP53, kaempferol-MAPK8, quercetin-TP53, and quercetin-MAPK1 have excellent binding efficiency. Wogonin, a naturally occurring flavonoid compound, has anti-inflammatory, antioxidant, anticancer, and antiviral effects . WogoninIn this study, we uncovered the targets and molecular mechanisms of KTC in the treatment of PCOS and confirmed that quercetin may replace KTC for the treatment of PCOS patients through network pharmacology, bioinformatics, molecular docking, and other methods. These results may provide evidence for the clinical application of KTC in the treatment of PCOS.


Biheimaer (BHM) is a hospital formulation for clinical treatment of dyspepsia and acid reflux, based on Compatibility Theory of Traditional Chinese Medicine. This study anticipated to elucidate the molecular mechanism of BHM against Functional dyspepsia via combined network pharmacology prediction with experimental verification.  Based on network pharmacology, the potential active components and targets of BHM in the treatment of functional dyspepsia were explored by prediction and molecular docking technology. The results of protein–protein interaction analysis, functional annotation, and pathway enrichment analysis further refined the main targets and pathways. The molecular mechanism of BHM improving functional dyspepsia mice induced by L-arginine + atropine was verified on the basis of network pharmacology. in vivo experiments showed that BHM could regulate intestinal smooth muscle contraction to play a therapeutic role in functional dyspepsia by reducing the expression of NOS3, SERT, TRPV1, and inhibiting the inflammatory cytokine  to intervene the inflammatory response in mice.  In this study, 183 effective compounds were screened from BHM; moreover, 1007 compound-related predicted targets and 156 functional dyspepsia-related targets were found. The results of enrichment analysis and  This study revealed the molecular biological mechanisms of the Traditional Chinese Medicine formulation of BHM in functional dyspepsia by network pharmacology and experimental verification, meanwhile provided scientific support for subsequent clinical medication. Helicobacter pylori eradication therapy, proton pump inhibitors, histamine-2 receptor antagonists, central neuromodulators, prokinetics, etc [Functional dyspepsia is one of the most common gastrointestinal diseases. Studies have shown that the prevalence of functional dyspepsia in Western populations is 9.8% to 40%, and the Eastern population is lower at 5.3% to 28%. , 2. The Artemisia rupestris (L.), Mentha longifolia (L.), Cuminum cyminum (L.), dried ginger, Elettaria cardamomum (L.) Maton, pomegranate seed, and other components, clearing away heat and toxic materials, regulating gastrointestinal function, reducing hiccups, and belching. Our group's previous studies have confirmed that Artemisia rupestris (L.), as a principal drug, has anti-inflammatory effects, improves immune function, promotes gastrointestinal digestion, and reduces reflux esophagitis and functional dyspepsia symptoms [Mentha longifolia (L.) on reducing food stagnation and flatulence has been confirmed by clinical studies, which reveals that Peppermint Oil in Mentha longifolia (L.) has a dose-related effect on gastrointestinal motility [Cuminum cyminum (L.) has the effect of treating indigestion and abdominal pain, which is recorded in the famous traditional Chinese medicine monograph “Puji Fang.” The role of dried ginger in resisting Helicobacter pylori has been widely reported [Biheimaer (BHM) is a hospital formulation for clinical treatment of dyspepsia and acid reflux, based on Compatibility Theory of Traditional Chinese Medicine, composed of symptoms . The effmotility . Cuminumreported , 9. Eletreported –12. Howein vivo experiments [Network pharmacology was proposed by Hopkins in 2007. With the help of network analysis, the relationship between drugs and disease targets is studied through “multi-component, multi-target, and multi-pathway.” Network pharmacology believes that the essence of disease is genes or encoded proteins, represented by one or more nodes in the network, are in an unbalanced state, which coincides with the law of occurrence and development of disease in the theory of Chinese medicine. Traditional Chinese Medicine believes that the essence of the disease is the deviation of the yin and yang attributes of the human body, and the deviation is corrected through multiple angles and multiple channels of traditional Chinese medicine compounds. In this study, network pharmacology was used to predict key targets that have a critical function in BHM treatment of functional dyspepsia, and pathway enrichment was used to predict the critical mechanisms. Ultimately, these mechanisms were verified through molecular docking and eriments . A briefhttps://old.tcmsp-e.com/tcmsp.php). When herbs were listed in TCMSP, active compounds were screened with thresholds value of oral bioavailability (OB) ≥ 30% and drug similarity (DL) ≥ 0.18 [https://www.swissadme.ch/).The compounds in BHM were identified from literature search and Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP)  ≥ 0.18 . The comhttps://www.swisstargetprediction.ch/). The identified targets of functional dyspepsia were collected from Gene Cards database (https://www.genecards.org/); the targets were searched by making “Functional dyspepsia” as key word. In order to understand the relationship between disease targets and BHM-related targets more intuitively, two sets of targets were input into Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/), respectively, to obtain the intersection targets.Potential targets associated with candidate compounds are predicted in Swiss Target Prediction System  (https:/https://string-db.org/) to explore the relations of protein–protein interaction (PPI) [https://metascape.org/) for the enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomics (KEGG) pathways. The “Input as species” and “Analysis as species” set as “H. sapiens.” Pathways satisfying P < 0.05 were considered as significant.The above intersection target proteins were imported into String platform (on (PPI) , and thehttps://www.rcsb.org/pdb/) and PubChem database (https://pubchem.ncbi.nlm.nih.gov/), and waters and ligands were removed by PyMOL. The protein structure was hydrogenated by imported into AutoDock 1.5.6. Finally, docking the processed protein and active compound in AutoDock to verify the molecular binding ability and calculate the minimum binding energy. The docking results were visualized using PyMOL.The molecular structures of the ligand and the target protein were downloaded from The Protein Data Bank (PDB) ; Polyclonal antibodies β-actin, SERT, NOS3 , TRPV1 , electrogenerated chemiluminescence (ECL) and color liquid .Biheimaer ; L-arginine , atropine , mosapride , IL-1n = 40), weight 18–22 g, were obtained from the Animal Experiment Center of Xinjiang Medical University. The animals were bred in the animal room of the Xinjiang Institute of Traditional Uygur Medicine with the room temperature of 20–26°C and the relative humidity of 40–70%. Before the experiment, all animals were adapted to rearing for 3 days and quarantined. Animals with abnormal growth were excluded and randomly divided into groups. The protocols for the experiments were approved by the Xinjiang Institute of Traditional Uygur Medicine Ethics Committee on animal experimentation  and conducted in accordance with internationally accepted principles for the use and the care of laboratory animals.Male Kunming (KM) mice , model group (group II), Mosapride group (group III), and BHM group (group IV). The positive drug mosapride (5.0 mg/kg/d) (group III), and the BHM (3.41 g/kg/d) (group IV) is given daily by oral gavage for 7 days. Except for the control group, the other groups were given L-arginine (2 g/kg) orally for 7 consecutive days. Before sacrifice, all of the mice were fasted without water restriction for 16 hours. On the 8th day, 10 minutes after atropine was given (3 mg/kg) by subcutaneous injection (SC), carbon powder semi-solid paste  was fed into the stomach, and sacrificed after another 10 minutes.After the mice were sacrificed, the stomach and intestine were dissected. After weighing the total weight of the stomach, cut the stomach body along the greater curvature of the stomach to obtain the net weight of the stomach. The gastric content is calculated by the total weight of the stomach minus the net weight. At the same time, the small intestine was quickly cut from the pylorus to the ileocecal area. After gently peeling the mesentery, the overall length between the pylorus and the ileocecal area and the distance from the pylorus to the front of the carbon paste were measured. Intestine was fixed in 4% PFA fix solution at room temperature for 24 h. The rate of gastric residual and intestinal propulsion rate are calculated by this formula:β and TNF-α were quantified by ELISA kits following manufacture instruction separately, and measured OD values by Spark microplate reader , and the corresponding concentrations were calculated.The cytokines levels of IL-1The duodenum tissues were stained by hematoxylin-eosin (HE) with paraffin section method. The pathological change was estimated by a third-party pathologist who did not participate in the experiment .μL RIPA buffer, and determined by BCA assay. The protein samples were separated by 8% SDS-polyacrylamide gel. The PVDF membranes were blocked with 5% skim milk for 2 h, and primary antibodies including TRPV1, SERT, eNOS (NOS3), and β-actin were added overnight (4°C). The membranes were incubated with the secondary antibody for 1 h and visualized using an ECL developer and multifunctional imaging system . The gray value of the target bands was analyzed using Image J, and the relative expression of proteins was expressed as target protein/β-actin.50 mg of the duodenum tissue of mice were extracted protein with 300 P < 0.05 was considered significant in statistics.All values are expressed as mean ± standard deviation. SPSS software (version 20.0) was used for the statistical analysis. One-way Analysis of Variance (ANOVA) was used for the comparison between groups. Through screening, 183 active components were identified, and verified chemical structures subsequently from the PubChem database. Based on that, 1007 compound-related targets were obtained after eliminating the duplicates. Moreover, 156 functional dyspepsia-related targets were confirmed from the GeneCards database. Depending on the 1007 putative targets of the BHM and the 156 functional dyspepsia-related targets, 54 integrated targets were identified as potential therapeutic targets of BHM against functional dyspepsia . A Venn By comparing the information in compounds and targets, it was found that rupestric acid, aciphyllic acid, isorupestonic acid, beta-pinene, and eucalyptol in BHM should be the most relevant active component related to functional dyspepsia, and the above 5 components were selected for the following efficacy verification .54 integrated targets were uploaded to the String database to determine interaction relationship. Then, the protein isolated outside the network was eliminated and the protein interactions (confidence level: 0.4) were imported into Cytoscape v3.8.0 to PPI networks, which consisted of 51 nodes and 258 edges, as shown in P < 0.05 were visualized by dot plot , molecular function (MF), cellular component (CC), are shown in dot plot , includiβ, CXCL8, NOS2, NOS3, TRPV1, and SLC6A4) was simulated and analyzed by molecular docking software  and core proteins .ELISA results  showed tP < 0.05), and the expression of TRPV1, NOS3, and SERT in the duodenum tissue of mice in BHM group was significantly lower than that in model group (P < 0.05). The results of in vivo experiments were consistent with the conclusions of network pharmacology.As shown in in vivo or in vitro experiments [The current research road of network pharmacology is to establish a target prediction network model of specific disease and its prevention or treatment drug based on public and published data. The model predicts the target of the drug being studied and thereby constructs a drug-target-disease network to explore the potential pharmacological mechanism. Ultimately, the potential mechanism is verified by corresponding experiments such as molecular docking, eriments . BHM conIn the present study, 1007 potential targets of the 183 components in BHM and 156 related targets of functional dyspepsia were identified. By comparing the information of quantity, contribution degree, and function of compounds and targets, the most potent compound is rupestric acid, aciphyllic acid, isorupestonic acid, beta-pinene, and eucalyptol. Relevant pharmacological studies in recent years have shown that they may have the effect of improving functional dyspepsia symptoms, involving gastroprotective, anti-inflammatory, and analgesic, promoting gastrointestinal motility , 20. ConAmong the overlapping targets between BHM and functional dyspepsia, IL1B, TNF, PTGS2, BDNF, TLR4, CXCL8, CASP3, CNR1, TRPV1, and NOS3 showed the highest degree values (top10) suggesting that they may be the main target of BHM for functional dyspepsia. These 10 target genes are divided into five directions related to inflammatory response, regulation of nitric-oxide synthase activity, neuroactive ligand-receptor interaction, and regulation of secretion according to their functions. This also reflects the characteristics of BHM's comprehensive regulation and coordinated restoration of body balance. With the largest degree values, IL1B and TNF were regarded as the more dominant of the overlapping targets. IL1B and TNF are important potent pro-inflammatory cytokines that play a key role in inflammatory response . These pThe biological process of GO enrichment analysis was significantly enriched in inflammatory response, regulation of secretion, sensory perception of pain, and regulation of ion transport. The neuroimmune regulation, especially the NO synthase, in functional dyspepsia participates in the regulation of secretion and regulation of ion transport, which play important roles in the pathogenesis of functional dyspepsia –34. NO sIn vivo experiments have confirmed that BHM could significantly promote gastrointestinal motility, and decrease the expression of TRPV1, NOS3, and SERT. However, other genes and biological pathways may also have a particular function in the treatment of functional dyspepsia by BHM, as demonstrated in our PPI network, GO and KEGG pathway analyses, which require more in-depth study.KEGG pathway annotation analysis obtained a number of related clustering pathways, which mainly include the neuroactive ligand-receptor interaction, calcium signaling pathway, serotonergic synapse, and inflammatory mediator regulation of TRP channels. This suggests that BHM can comprehensively regulate multiple signaling processes relevant to the pathophysiology of functional dyspepsia. The neuroactive ligand-receptor interaction pathway is composed of cell membrane receptors, such as capsaicin receptor, adrenergic receptor, muscarinic acetylcholine receptor, and 5-hydroxytryptamine receptor, which are involved in the transduction of intracellular and extracellular signals –38. In ain vivo experiments in mice. However, the research methods of network pharmacology still have some limitations. For example, the study on the chemical constituents of some herb and animal medicine is not through enough, resulting in the absence in some chemical components or targets and affecting the accuracy of study results.Using a series of predictive network model methods, this study analyzed the targets and pathways of BHM in the treatment of functional dyspepsia. Furthermore, in order to make the results of this study more reliable, we verified the main results in the underlying mechanism by


Scutellaria baicalensis Georgi (SBG) may relieve bone cancer pain (BCP) by regulating cell proliferation, angiogenesis, and apoptosis.The mechanism of SBG in the treatment of BCP remains to be further explored.The active compounds and targets of SBG were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and SwissTargetPrediction databases. BCP-related targets were screened from NCBI and GeneCards databases. Additionally, Cytoscape software was applied to construct network diagrams, and OmicShare platform was used to enrich Gene Ontology (GO) and pathways. Finally, the verification of active compounds and core targets was performed based on quantitative real-time PCR (qRT-PCR).Interestingly, we identified baicalein and wogonin as the main active components of SBG. A total of 41 SBG targets, including VEGFA, IL6, MAPK3, JUN and TNF, were obtained in the treatment of BCP. In addition, pathways in cancer may be an essential way of SBG in the treatment of BCP. Experimental verification had shown that baicalein and wogonin were significantly related to BCP core targets.The active components of SBG have been clarified, and the mechanism of the active components in treating BCP has been predicted and verified, which provides an experimental and theoretical basis for the in-depth elucidation of the pharmacodynamics material basis and mechanism of SBG. Common cancers, such as breast cancer and lung cancer, are prone to bone metastasis, which increase the morbidity, mortality, and more severe pain of patients . It is cScutellaria baicalensis Georgi (SBG), a bitter taste and cold nature, is the main herb of the famous prescription of Xiao Chai Hu Tang  (https://www.uniprot.org/). The complex relationships between active compounds and their targets were visualized based on Cytoscape software (version 3.7.2).The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) was used to screen the components of SBG. In this study, the range of oral bioavailability (OB) greater than or equal to 30% and drug-likeness (DL) greater than or equal to 0.18 were used to screen active ingredients . Subsequion.ch/)  were apphttps://www.ncbi.nlm.nih.gov/) (https://www.genecards.org/)   and Geneds.org/)  were usehttp://bioinfogp.cnb.csic.es/tools/venny/). The STRING database (https://string-db.org/) was used to construct a protein-protein interaction network (PPI). Furthermore, R-3.6.3 software was used for the visualization of core targets, and Cytoscape 3.7.2 software was used to construct the component-target-disease (C-T-D) network diagram of the active ingredient and the intersection target.To better investigate the complex relationship between components and diseases, the intersection targets of ingredients and diseases were obtained based on Venny 2.1.0 (http://www.UniProt.org/), and uploaded to the OmicShare platform (https://www.omicshare.com/) for GO and KEGG enrichment analyses and visualization.In order to explore the biological information and signal pathways involved in the treatment of bone cancer pain by SBG , intersehttp://biogps.org/), the mRNA expression data of the intersection targets in different tissues can be harvested. In order to investigate the potential relationship of the active ingredients between SBG and BCP at the organ level. In this study, a triple value of the median mRNA expression was used to determine the tissue distribution of the targets. Similarly, targets without corresponding mRNA expression data were also excluded from the analysis. Finally, a component-target-organ (C-T-O) network was constructed through Cytoscape to more intuitively reflect the distribution of targets in the organization.According to the BioGPS database  and wogonin  were added respectively. After 24 h of incubation, cell viability was detected with CCK8, and the OD value (450 nm) of each well was measured with a SpectraMAX M3 microplate reader , which was used to screen out the safe concentration range of the drug.Baicalein (purity ≧ 98%) and wogonin (purity ≧ 98%) were purchased from MedChemExpress . The cytotoxicity control test of the drug was detected using Cell Counting Kit-8 (CCK-8) . HT22 cells were seeded in a 96-well plate at a density of 3 × 10−ΔΔCt method. The experiment was repeated three times.Total cellular RNA was extracted using Trizol reagent  after 24 h incubation with various concentrations of baicalein  or wogonin . RNA purity and concentration were determined using a Nano Drop 2000 spectrophotometer . A reverse transcription kit  was applied to reverse transcribe RNA to cDNA. After reverse transcription, qRT-PCR was performed using the ABI 7500 Real-Time PCR System . The primer sequences were shown in P < 0.05 was considered to be statistically significant.The results of cell viability and target expression experiments were analyzed by one-way ANOVA. Data normalization was performed to control for the effect of changes in baseline parameters. GraphPad Prism 9.0 software  was used for analysis and visualization of study results. ) was generated and indicates that 33 active components and 448 drug targets had close relationships. In the component-target network diagram, the ellipses of different colors represent the targets of different components. Among them, baicalein (MOL002714) obtained more targets (Degree = 133) than other components, which were represented by a red ellipse.According to the range of OB and DL, the 33 active components of SBG were obtained , such asFigure S1Based on the above results, 41 common targets were obtained through the intersection of SBG and BCP targets . The PPIIn the C-T-D network , targetsGO:0010941), the regulation of cell migration (GO:0030334), the pathways in cancer (ko05200), IL-17 signaling pathway (ko04657), and TNF signaling pathway (ko04668). Results indicated that the active compounds of SBG alleviate BCP by regulating core targets of angiogenesis-related targets VEGF, Transforming growth factor beta-1 (TGFB1), Apoptosis-related targets Caspase-9 (CASP9), TNF, JUN, Estrogen receptor (ESR1), and MAPK3.The intersection targets were analyzed by GO and KEGG enrichment,  and S2. The tissue distribution of 41 intersection targets has shown in P > 0.05. Similarly, wogonin did not affect the viability of HT22 cells in the concentration range of 0–80 μM (P > 0.05). Subsequent experiments will select 0–160 µM baicalein and 0–80 µM wogonin to explore the expression of core targets.In order to more accurately examine the therapeutic effect of baicalein or wogonin on BCP, this study screened the safe concentration of baicalein or wogonin on the HT22 hippocampal neuron cell line . The resP < 0.05). In general, the qRT-PCR results indicated that baicalein and wogonin were involved in hippocampal neurogenesis by regulating VEGFA, IL6, MAPK3, JUN and TNF target genes, thereby exerting anti-BCP effects.The mRNA expression results of VEGFA, IL6, MAPK3, JUN and TNF target genes in HT22 cells after treatment of baicalein or wogonin were evidenced in Our research indicated that 33 active ingredients of SBG were related to 41 targets of BCP. The main flavonoids of SBG were baicalein, wogonin, oroxylin A, skullcapflavone II, and norwogonin. Pharmacokinetic studies had claimed that baicalin, baicalein, wogonin, wogonoside, and oroxylin A can be detected in rat plasma after administration, but baicalin and wogonoside absorption and elimination were quite slow in plasma , which iAccumulating studies have illustrated that the active ingredients of SBG have a good activity and can exert anti-cancer effects by regulating cell apoptosis, death, metastasis, and autophagy. Baicalein can reduce the number of the differentiated osteoblasts by regulating the proliferation and apoptosis of preosteoblasts, impairing the angiogenesis of endothelial cells, and inhibiting the proliferation of synovial cells . BaicaleBCP involves peripheral, central, and glial-mediated nociceptive and neuropathic components . NeuropaAt present, the pathological and therapeutic mechanisms of BCP have been reported. Chanling Gao exerted the analgesic effects in the cancer-induced bone pain (CIBP) rat model by inhibiting the IKKβ/NF-κB signaling pathway and the synthesis and release of TNF-α, IL-1β, and IL6 . AitongxRecently, baicalein and wogonin have been reported in the literature on pathway regulation. Studies have found that baicalein can target TLR4/HIF-1α/VEGF signaling pathway in the treatment of colorectal cancer . Baicalein vivo and in vitro verification experiments are still needed. Only in this way, we can fully reveal the mechanism of SBG in treating BCP and provide a theoretical basis for its targeted therapy.In summary, this study only predicted and preliminarily validated the mechanism of SBG in treating BCP. Although research has discovered key targets and mechanisms for regulating and treating BCP, a large number of in vitro were used to verify that baicalein and wogonin regulate the expression of key targets  in HT22 hippocampal cells. This study provides new ideas and theoretical basis for the mechanism of action of SBG in the treatment of BCP.In conclusion, the network pharmacology of this study was used to screen out the active compounds of SBG , 41 intersection targets , and 20 main pathways . Finally, experiments 10.7717/peerj.14394/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.14394/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.14394/supp-3Supplemental Information 3Click here for additional data file.


Due to hyperglycemia, patients receive long-term treatment. However, side effects and drug tolerance usually lead to treatment failure. GuaLouQuMaiWan (GLQMW), a common traditional Chinese medicine (TCM) prescription, has positive effects on controlling blood sugar and improving quality of life, but the mechanism is still unclear. To decipher their molecular mechanisms, we used a novel computational systems pharmacology-based approach consisting of bioinformatics analysis, network pharmacology, and drug similarity comparison. We divided the participants into nondisease (ND), impaired glucose tolerance (IGT), and type 2 diabetes groups according to the WHO's recommendations for diabetes. By analyzing the gene expression profile of the ND-IGT-T2D (ND to IGT to T2D) process, we found that the function of downregulated genes in the whole process was mainly related to insulin secretion, while the upregulated genes were related to inflammation. Furthermore, other genes in the ND-IGT (ND to IGT) process are mainly related to inflammation and lipid metabolic disorders. We speculate that 17 genes with a consistent trend may play a key role in the process of ND-IGT-T2D. We further performed target prediction for 50 compounds in GLQMW that met the screening criteria and intersected the differentially expressed genes of the T2D process with the compounds of GLQMW; a total of 18 proteins proved potential targets for GLQMW. Among these, RBP4 is considerably related to insulin resistance. GO/KEGG enrichment analyses of the target genes of GLQMW showed enrichment in inflammation- and T2D therapy-related pathways. Based on the RDKit tool and the DrugBank database, we speculate that (-)-taxifolin, dialoside A_qt, spinasterol, isofucosterol, and 11,14-eicosadienoic acid can be used as potential drugs for T2D via molecular docking and drug similarity comparison.The main pathophysiological abnormalities in type 2 diabetes (T2D) include pancreatic  After an irregular diet, an imperceptible and cumulative increase in fasting and 2 h postprandial blood glucose levels occurs [β-cell dysfunction and senescence. Moreover, compared with the loss of β-cells, supplementation through the differentiation of islet stem/progenitor cells [β-cells [β-cell content of T2D and IGT patients showed a downward trend [Type II diabetes T2D) is a chronic disease characterized primarily by abnormally high blood sugar levels. The number of patients, a high proportion of whom are young adults, suffering from T2D is increasing annually . The WorD is a chs occurs , which ior cells  and self[β-cells  is insigrd trend . Insulinrd trend . Insulinrd trend . Similarrd trend . In insurd trend . Moreoverd trend . Second,rd trend , 16. TheTrichosanthis Radix (THF), Aconiti Lateralis Radix Praeparata (FZ), Poria Cocos (Schw.) Wolf. (FL), Dianthi Herba (QM), and Rhizoma Dioscoreae (SY), can invigorate the spleen and kidney and eliminate dampness and diuresis, according to the principles of TCM for T2D. SY and FL proved to be effective in intervening blood glucose and blood lipid levels in animal models [In China, GuaLouQuMaiWan (GLQMW), a traditional prescription, synergizes with antidiabetic drugs in T2D treatment by reducing drug resistance  and stabl models , 20, andl models . AlthougT2D is a nonmutated disease, hence, its progression is laboriously illustrated through the change of a single gene. However, the changes in gene expression profiles during disease progression can be identified and visualized by transcriptome sequencing analysis , providiThe overall process of the research is shown in https://www.ncbi.nlm.nih.gov/geo). Compounds of GLQMW and its' target proteins origin from TCMSP (https://tcmspw.com/) and PharmMapper server, respectively.RNA-seq data of T2D (including 411 islet tissue samples) and the corresponding clinical data were downloaded from GEO dataset. By filtering out the sample missing diagnosis, the expression data for 326 islet tissue were kept for downstream analysis. Four cohort used in this study as validation datasets  were downloaded from the GEO database  which is recommended by WHO, we distinguished the T2D process of diabetic patients via glycosylated hemoglobin (HbA1c), and divided the samples into 3 groups .p.adj < 0.05. Heatmaps of differential genes were drawn using the R package “pheatmap”. For process-specific genes, only genes with significant differences and consistent trend in expression  in all three possible comparisons were considered T2D-specific genes.Differential expression analysis was conducted using the R package “limma”. The screening conditions for the differential genes were |log2FoldChange| > 0.3, β = 4 , we convert the adjacency matrix to a topological overlap matrix (TOM), and set the type of TOM to a signed network. Next, we perform hierarchical clustering to identify modules which contains at least 30 genes (minModuleSize = 30), calculated feature genes, hierarchically clustered the modules, and merged similar modules.We use the R package “WGCNA” to construct a gene-weighted coexpression network, when R package “limma” has processed the gene expression data of removing the batches. First, compared to the Pearson method, we chose the biweight midcorrelation method to construct the adjacency matrix to describe the correlation strength between nodes. Subsequently, choosing the soft threshold The module feature gene (ME) is the first principal component of the module and represents the expression pattern of the module in each sample. The degree of module membership (MM) refers to the correlation coefficient between genes and the characteristic genes of the module, which is used to describe the reliability of the gene belonging to the module. Based on ME and MM, we calculated the correlation between modules and clinical data to determine important clinical modules. Finally, we selected the modules whose expression changes were in line with the process of ND-IGT-T2D, and extracted the hub gene and core genes which were further analyzed.p and q value of < 0.05 were considered significantly enriched. For T2D, GO and KEGG enrichment analyses were based on upregulated and downregulated expressed genes. For GLQMW, we used “http://org.hs.eg.db/” to convert the target protein into a gene, and then performed GO and KEGG enrichment analysis.GO and KEGG enrichment analyses were performed with the aid of R packages “clusterProfiler,” “enrichplot,” and “ggplot2.” Only terms with both All of the chemical ingredients of GLQMW were obtained from TCMSP. The active compounds of GLQMW were mainly filtered with oral bioavailability (OB), drug-likeness (DL), and Caco-2 permeability (Caco-2), while those compounds in GLQMW with OB ≥ 30%, DL ≥ 0.18, and Caco − 2 ≥ −0.4 were preserved [z-score is an important basis for target fit. We choose 1.0 as the threshold, and extract all protein targets greater than the threshold for subsequent analysis. Based on cytoscape, we construct an active compound-target network, and select compounds with a larger number of nodes for key analysis.Download the 3D structure of the GLQMW compound from TCMSP. The PharmMapper server recognizes the pharmacophore of the compound, and uses the pharmacophore as the recognition group of given probe molecule to predict potential target proteins. PharmMapper server output results, including target name and protein number, among which https://go.drugbank.com/) [Download the “full database” document from the DrugBank database (nk.com/) , which cRDkit generates compound descriptors and compound fingerprints, and calculates the similarity of compound structures, based on the 2D and 3D structure of compounds.Our study compares active compounds in GLQMW and small molecule drugs in the DrugBank database, and outputs information on the top 10 drugs with drug similarity.https://www.rcsb.org/). The ligand and water macromolecule in these targets were removed, and the hydrogen atoms were added with pymol2.3. The targets were set to rigid and saved as pdbqt file format by AutoDock Tools 1.5.6. Finally, molecular docking was performed using Vina. Based on the top one minimal binding energy of each target, compounds in GLQMW were selected for further analysis of their binding mode, binding affinity, and critical interactions using PyMOL2.3 and LIGPLOT2.2.The 3D structure of protein: RBP4 , PLAT , MET , KIT , C1S , and HSD11B  were downloaded from the protein data bank (PDB) database  and SLC2A2 , respectively. In the ND-IGT, the most significantly upregulated and downregulated genes were PTGS2  and TMED6 , respectively. In IGT-T2D, the most significantly upregulated and downregulated genes were CEACAM7  and SLC2A2 , respectively.In ND-T2D, the most significantly upregulated and downregulated genes were APOD , IGT-T2D (226), and ND-IGT 100), 00, 1). Tp < 0.01). For GO enrichment results, a total of 409 GO entries of upregulated genes were obtained, including 354 BP, 33 MF, and 22 CC, and 115 GO entries of downregulated genes were obtained, including 72 BP, 12 MF, and 31 CC.The degrees of ND-T2D progression were grouped according to upregulated and downregulated expression, after which GO enrichment analysis was performed (p.adj < 0.01) of upregulated genes in ND-T2D mainly included regulatory response to injury, regulation of inflammatory response, regulation of cell adhesion, and acute inflammatory response regulation (β-cells. The BP entries enriched by downregulated genes (p.adj < 0.01) were mainly related to peptide hormone secretion, insulin secretion, protein secretion, etc.  were mainly mapped to glycosaminoglycan binding, cytokine binding, cytokine activity, integrin binding, and other pathways related to immune stress, which matched the BP results (p.adj < 0.01) are shown in Figures GO-MF enrichment results showed that upregulated genes  was found in cytokine-cytokine receptor interaction, MAPK signaling pathway, Hippo signaling pathway, TGF-beta signaling pathway, TNF signaling pathway, diabetes AGE-RAGE signaling pathway, IL-17 signaling pathway, ECM-receptor interaction, NF-kappa B signaling pathway, Toll-like receptor signaling pathway, and NOD-like receptor signaling pathway . Finally, 45 gene coexpression modules were constructed  and searched for the most significant associations. The results of this analysis showed that the turquoise and purple modules were markedly correlated with T2D .We conducted GO and KEGG analyses of genes in the turquoise and purple modules  over 0.2 and an MM over 0.8 as the cut-off criteria, 158 genes were identified as hub genes , FZ 16 Z-score was used as the basis for judging the strength of the correlation between the compound and the target protein, and the predicted targets that met the standard (Z − score > 1.0) were included in further studies. The PharmMapper platform was used to input the 3D structure of the drug-like compound and predict the human target protein by matching the pharmacophore. In this study, the p.adj < 0.05). The top 30 significantly enriched GO-BP terms are listed in GO enrichment analysis was performed on the target genes of GLQMW (p < 0.05), κB signaling pathway, NOD-like receptor signaling pathways, natural killer cell-mediated cytotoxicity, IL-17 signaling pathway, relaxin signaling pathway, and T-cell receptor signaling pathway. These pathways, as per our clinical observations, regulate the levels of related inflammatory factors in pancreatic islet tissues. Pathways related to glucose and lipid metabolism, such as those of type II diabetes, diabetic cardiomyopathy, endocrine and insulin resistance, insulin signaling, atherosclerosis, and adipocytokine signaling, play an important role in insulin resistance.KEGG analysis of the GLQMW target  binds to MET, activating signal transduction and the RAS and PI3K/Akt signaling pathways  to regulIn our research, small-molecule compounds  such as γ agonist properties, and PPARγ is the specific target of thiazolidinedione in the treatment of T2D [β and Toll-like receptors, exerting anti-inflammatory effects [γ/AMPK/ACC1 pathway, and therefore can reduce local inflammation [α-amylase and α-glucosidase activity by binding to their inactivation sites and has been shown to rapidly reduce the blood glucose concentrations 2 h after a meal in animal models [β and glucose 6-phosphatase [γ DNA-binding activity, activate the PPAR-γ pathway to inhibit IL-6 and iNOS gene expression, and achieve the purpose of slowing down local inflammation [Based on the RDKit tool, the small-molecule compounds obtained were compared with all small-molecule drugs released from the DrugBank database, and the top 10 drug compounds with similarity were output. The drug similarities of (-)-taxifolin, dihydromyricetin, silibinin, hesperetin, epicatechin, and epigallocatechin gallate were 0.9958, 0.8417, 0.8119, 0.7952, and 0.7227, respectively . Dihydrot of T2D . Hesperet of T2D . Epicatet of T2D . Epigallt of T2D . The simt of T2D , inhibitt of T2D , which it of T2D  and inhi effects . Gamolen effects . Drugs w effects . Madecasl models . AA redusphatase . Particusphatase . In the sphatase . Androstammation . 20-hydrThe docking results of the active compounds with C1S, HSD11B, KIT, MET, PLAT, and RBP4 are presented in Supplementary Table β cells, which mainly include a significant decrease in the number of pancreatic β-cells and a decrease in the function of β-cells to secrete and synthesize insulin. The decrease in the number of pancreatic β-cells is related to the disorder of the β-cell cycle [β-cell function is associated with islet β-cell dedifferentiation, glucotoxicity caused by high concentrations of glucose [With societal development and improvements in living standards, T2D has become a major chronic disease that threatens human health because of its gradually increasing incidence. Currently, physicians tend to use drugs for T2D control, supplemented by diet and rhythmic exercise. However, due to the continuous increase in insulin resistance in the body, weakening of drug sensitization, and increase in drug resistance in the body, it is often difficult for T2D patients in the later stage to maintain normal blood glucose ranges. The patient's quality of life significantly deteriorates due to constant medication and insulin injections, resulting in decreased treatment efficacy. Therefore, it is necessary to develop a new clinically effective treatment strategy. TCM is a medical discipline that has been summarized and developed by the Chinese nation in long-term practice. GLQMW, which are briefly recorded in the “JinGuiYaoLue,” have the function of treating T2D, but no scholars have summarized and discussed the effectiveness and treatment mechanism of TCM prescription. It is generally believed that the developmental trend of type 2 diabetes is ND-IGT-T2D, in which IGT is an intermediate state and not a stage of irreversible diabetes damage . Therefoll cycle , but the glucose , lipotox glucose , and chr glucose  to a cerκB and MAPK signaling pathways [β-cells. Therefore, the results in this study further explain the pathogenesis of T2D from the molecular mechanism and provide new strategies for treatment, such as specifically reducing the degree of inflammation in the pancreatic islets. According to the results of the Connectivity map (CMap) database -taxifolin, have potential therapeutic functions in T2D. We can isolate compounds for further experimental validation to determine whether they can be used as parents for new T2D drugs. Therefore, in the future, purified natural compounds can be prepared with a composition ratio similar to that of TCM prescriptions to replace existing decoction application methods [In the results of network pharmacology, the main targets of the active ingredients of GLQMW were HSD11B1, VDR, TGR5, FXR, and RBP4, all of which mainly related to T2D. Such finding may be the basis for the therapeutic effectiveness of GLQMW. According to the GO/KEGG results of GLQMW targets, in addition to inflammation-related and diabetes phenotype-related pathways, GLQMW was shown to be involved in pathways related to cell proliferation, such as the MAPK, JAK-STAT, Ras, and PI3K-Akt signaling pathways. GLQMW may restore the number of  methods . AlthougGLQMW treat T2D by anti-inflammatory and restoring islet cell function, and its potential therapeutically active compounds are (-)-taxifolin, dianoside A_qt, isofucosterol, 11,14-eicosadienoic acid, and spinasterol.