I2 = 21.4%; p = 0.282), DCR , PD , and QOL  and higher proportions of CD3+ T , CD4+ T , and the CD4+/CD8+ T ratio . The number of adverse events (AEs) was significantly lower in the TCM + chemotherapy group. The active components and targets of 19 high-frequency Chinese medicines obtained from the meta-analysis were screened and explored in network pharmacology analysis. Also, a regulatory network of active components and targets, a core network and key genes, a diagram of protein interaction, network topology analysis, and gene body GO function and KEGG pathway enrichment analysis were performed. A total of 120 active components were identified. NPM1 and HSPA8 are the most critical target proteins in the core network of protein interaction. HSP90AA1 is the most important target protein in the TCM group. KEGG enrichment analysis showed that it was highly significant in the lipid and atherosclerotic pathways. Therefore, moderate evidence revealed that TCM plus chemotherapy has obvious advantages over chemotherapy alone in terms of tumor responses, QOL, peripheral blood lymphocyte levels, and fewer AEs in the treatment of GC. The potential important targets and core genes were displayed.This meta-analysis plus network pharmacology aimed to investigate whether traditional Chinese medicine (TCM) combined with chemotherapy is associated with more beneficial efficacy data in the treatment of gynecological cancer (GC). A total of 11 randomized controlled trials (RCTs) consisting of 863 GC patients were included. Results showed a better ORR (RR: 1.42, 95% CI: 1.18–1.71; www.crd.york.ac.uk/PROSPERO/, identifier CRD42021252500. More than 8.6 million global women are diagnosed with cancer per year, with approximately 1.3 million cases of gynecological cancer (GC) . DespiteThe main treatment methods for gynecological tumors include surgery, chemotherapy, radiotherapy, or a combination of them. Chemotherapy is an important part of the treatment of gynecological tumors, but it will inevitably induce adverse effects such as deterioration, myelosuppression, and reduction. Take ovarian cancer (OC), for example. OC is the most common malignant tumor in gynecology, and the current standard treatment plan of which is postcytoreductive surgery combined with “paclitaxel + carboplatin” therapy. Also, emerging treatments such as poly(ADP-ribose) polymerase (PARP) inhibitor  and neoaIn China, traditional Chinese medicine (TCM) has gained more and more attention for malignant tumors because of its beneficial effects, such as low toxicity and enhanced efficiency , 8. FurtTherefore, we aimed to drive this meta-analysis to investigate the efficacy and safety of combination therapy by using TCM plus chemotherapy compared with chemotherapy alone, in which way we can spot suitable prescriptions to improve patients’ survival rates. High-frequency Chinese herbs will be extracted from those beneficial prescriptions. Whereas, there are still large gaps in our understanding of TCM, such as what are the essential effective components in TCM, what are the key potential targets, and whether interplays exist between these herbal components. As a result, we performed network pharmacology analysis to explore the potential pharmacological mechanisms and targets of TCM in the treatment of GC.This study adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. The protocol for this review was registered with PROSPERO (CRD42021252500).Three investigators independently searched several databases: PubMed, EMBASE, Cochrane, Chinese biological medicine (CBM), China Wanfang database, China National Knowledge Infrastructure databases (CNKI), Chinese Clinical Trial Registry (ChiCTR), ClinicalTrials.gov, and the World Health Organization International Clinical Trials Registry Platform from inception to September 26, 2021. Moreover, we also obtained the data sources from the abstracts and presentations recorded at some annual meetings, symposiums, or congresses such as ASCO, ESMO, ESGO, and so on, to ensure the relevant minutes were not overlooked. The complete search strategy was introduced in the Studies were included if they met the following criteria: if the people enrolled were GC patients ; if the subjects included had no contraindication to chemotherapy and no other systemic disease and were expected to live longer than 3 months; if TCM was in combination with chemotherapy for GC in the intervention group; if patients were treated with no TCM, or placebo, or conventional chemotherapy alone in the control group; if tumor response rate, QOL, AEs, serum tumor marker, and peripheral blood lymphocyte levels were reported (at least one of these outcomes); and if they were randomized controlled trials (RCTs).We excluded studies if they had not met our baseline requirements; if they employed the methods of acupuncture, massage, and other TCM treatment; if they did not offer the outcomes we needed; and if they were case reports, case series, observational studies, or other types of articles.+ T, CD4+ T, CD8+ T, and CD4+/CD8+ T ratio. Serum tumor markers were evaluated by CEA, AFP, and other common tumor markers.Three independent investigators finished this part following the requirements of PRISMA according to the above inclusion and exclusion criteria. All disagreements about selection were discussed and resolved by all investigators. The data extraction included the author, year, country, research type, number of cases, age of patients, and the indicators for demonstrating outcomes. Tumor response was the primary outcome, including complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD). The CR rates combined with PR rates were equal to the ORR; the CR, PR, and SD rates were equal to the DCR. Quality of life (QOL) and the incidence of AEs were also the main outcomes. Immune function , KPS score, and serum tumor marker were the secondary outcomes. Peripheral blood lymphocytes were evaluated by the levels of CD3Three researchers independently carry out the literature quality evaluation by applying the Cochrane evaluation handbook of RCTs (5.1.0), which includes the following characteristics covering randomization sequence generation (selection bias), allocation concealment (selection bias), blinding of participants and personal (performance bias), blinding of outcome assessment (detection bias), incomplete outcome data (attrition bias), selective reporting (reporting bias), and other bias. We used the Review Manager 5.3 software risk assessment tool to finish this part, and we decided through discussion or consultation with a third party when opinions were inconsistent.RevMan  and STATA  were utilized to perform statistical analyses including pooling the data and producing the forest plots.p-value less than 0.05 to be statistically significant. The I² test was also used to assess the between-study heterogeneity. If the heterogeneity test is p ≥ 0.1 and I2 ≤ 50%, it indicates that studies are homogenous between studies, and the fixed-effects model is used for combined analysis. Otherwise, we used a random‐effects model to pool effect size. I² regarded an estimated value applied to three fixed knots at 25%, 50%, and 75% as an indicator of mild, moderate, and high heterogeneity. Sensitivity analysis was also performed for examining the robustness of included trials to different aspects from methodological bias. In addition to this, publication bias was also estimated by an inverted funnel plot.The combination of relative ratio (RR) and mean difference (MD) with a 95% confidence interval (CI) was used to assess outcomes and considered a https://www.ncbi.nlm.nih.gov/), we selected the database GEO datasets. GMS mainly includes vulvar neoplasms (VN), vaginal cancer (VC), uterine cervix cancer (UCC), endometrial carcinoma (EC), OC, and fallopian tube carcinoma (FTC).VC and FTC were too few to have reliable disease samples in the GEOncbi database. Targets of VN, EC, and OC were mainly obtained, while the reliable disease samples of VC and FTC were too few to acquire. We searched for “vulvar neoplasm,” “uterine neoplasms,” and “ovarian neoplasms,” respectively. On the left, only series and expression profiling by an array of human tags are retained to narrow the screening range and obtain the corresponding samples of three tumor diseases  was used for obtaining the effective components of each TCM. The medicinal DL ≥0.18 condition played an important role in screening the effective components of TCM. Related targets were entered, and the corresponding targets of the selected active ingredients were derived. Perl has been used as an important tool for gene standardization, converting full names to genetic symbols.Section 2.7. The intersection of the two was taken, and a Venn diagram was drawn.A variety of active ingredients and corresponding targets of TCM were retrieved from the TCMSP database. Perl was used to form the “component-target” database of the active ingredients and GC disease targets in https://string-db.org/), the species was limited to humans, and a score of >0.9 was set to construct the protein interaction network diagram of the TCM group-GC. The data were then saved as TSV files, and the information of Node1, Node2, and combined score are imported into Cytoscape V3.7.2. Finally, the proximity centrality and intermediary centrality of each node are calculated by using the CytoNCA function package and a reference degree value. Finally, the core gene targets with the highest significance in topology analysis were selected.The intersected genes were imported into the STRING database  and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis should be performed for the intersection genes of TCM group-GC using the R language. First, the culsterProfiler package is imported, and the enrichGO function is used to import the human gene database through org.Hs.eg. and db function packages according to the intersection gene ID obtained previously. Under the conditions of “pvalueCutoff=0.05” and “qvalueCutoff=0.05,” the ID is converted into gene names and output to BP, CC, and MF groups for the GO enrichment analysis. Finally, the enrichPLOT and GGploT2 function packages are used to draw visual diagrams. Secondly, the KEGG enrichment analysis is carried out using the enrichKEGG function, and then the pathway—gene files and node files obtained by Perl are imported into CytoscapeV3.7.2 to build a KEGG network. Based on the above enrichment analysis results, we can predict the action mechanism of GC treated by various TCM.As shown in The main characteristics of the 11 included studies are shown in All of the 11 studies referred to randomization. Only 2 trials , 21 usedI2 = 21.4%; p = 0.000)   , and a meta-analysis was conducted through a random-effects model. The pooled results show that the quality of life of the combination therapy group is significantly better than that of the chemotherapy alone group  , CD4+ T , and CD4+/CD8+ T ratio  of the combination therapy group were significantly higher than those in the chemotherapy-alone group. While the level of CD8+ T cells of the combination therapy group didn’t show an obvious increase    Figure 8Sensitivity analysis eliminates each included study one by one and performs a summary analysis on the remaining studies to assess whether a single included study has an excessive impact on the results of the entire meta-analysis. The result of the sensitivity analysis is shown in The funnel plot is shown in To discern the commonality of the TCM formulae for combined chemotherapy in the included studies, we analyzed the occurrence frequency and compatibility of the compounds in those Chinese herbal prescriptions.We analyzed the characteristics of the 10 different TCM prescriptions mentioned in the included trials. Ordered according to their frequency of use, the TCM whose counts were more than three are listed in In addition, 4 of these studies (40%) used a combination of Dangshen/Renshen, Gancao, Baizhu, and Fuling, which is the most representative prescription for nourishing qi and is known as Sijunzi decoction. Based on these findings, it can be inferred that in the treatment of GC by TCM + chemotherapy, using basic qi tonic TCM to invigorate qi and strengthen the spleen can be an effective method, which was also found in some experiments. A new effective prescription was composed of these 19 herbs for subsequent network pharmacology analysis.Through the above meta-analysis, 19 kinds of TCM  have good effects on GC treatment. Due to the large number of Chinese medicines, we took Counts4 as the screening standard, and took the first 9 Chinese medicines as the TCM group.Using the TCMSP database, the TCM group was entered into the database, and the obtained ingredients were screened according to oral bioavailability (OB) of ≥30% and drug like (DL) of ≥0.18. Through searching the TCMSP database, there is no effective ingredient in Shengdi that meets ADME standards. Therefore, the remaining 8 TCM in the TCM group are used to screen the effective ingredients. Finally, there were 6 active components in Fuling, 88 active components in Gancao, 4 active components in Baizhu, 2 active components in Danggui, 16 active components in Huangqi, 8 active components in Baishao, 3 active components in Mudanpi, and 12 active components in Dangshen. After weight removal, there were 120 active components in the TCM group.Section 2.7. The genes with a high correlation with GC in |log FC|>1 adj.p-val<0.05 were screened by the method in Section 2.7. A total of 626 high correlation targets in VN, 3,884 high correlation targets in EC, and 5,665 high correlation targets in OC were obtained. As there were few highly correlated targets in VN, we expanded the screening conditions and screened |log FC|>0.5 p-val<0.05 as a highly correlated gene of VN. A total of 2,144 highly correlated targets in VN were obtained. After deleting the overlapping targets in the three groups of data, 8,949 high correlation targets of GC disease were obtained.The GEO database difference analysis was performed on the whole gene matrix of VN, EC, and OC obtained in In total, 120 active components were retrieved from the TCMSP database, and 225 directly corresponding targets were obtained after weight removal. A total of 8,949 highly correlated targets of GC disease were retrieved from the GEO and GeneCards databases, including 139 drug-disease common targets. After standardizing the gene, the full name of the gene is transformed into the gene symbol. Finally, Cytospace3.7.2 software was used to construct the regulation network of the TCM group  network was then required. The data in According to the results of topological parameter analysis, genes NPM1 and HSPA8 were the most critical genes in GC. Through the PPI core network, HSP90AA1, HSPA5, CDK2, and ESR1 were found to be the core genes directly associated with the effective components of the TCM group, among which HSP90AA1 ranked the highest.An enrichment analysis was carried out for 139 intersection genes of the screened TCM group and GC disease targets, and the analysis results of biological process (BP), cell composition (CC), and molecular function (MF) involved in this core target could be obtained in From the analysis of Whether the Chinese traditional medicine group—the intersection of GC disease gene KEGG enrichment analysis, gets the core targets in lipid and atherosclerosis, Kaposi’s sarcoma-associated herpesvirus infections, hepatitis B, diabetes complications of AGE-RAGE signaling pathways, fluid shear stress and atherosclerosis, and the human cytomegalovirus pathway is salient. It indicates that the components in the TCM group may achieve the purpose of curing diseases by acting on the above signal pathways  or progression-free survival (PFS); they only illustrated the curative effects of factors including tumor responses, QOL, peripheral blood lymphocyte levels, and AEs in treating GC. Thus, more newly offered evidence needs to be stated to confirm whether TCM plus chemotherapy could prolong the long-term survival rates of GC patients. Among those trials, lacking the data on the applications of injection-based, pill, and external administration of TCM, the included formulas were all oral administration. Also, almost all TCM were used after the chemotherapy cycle ended. The utilization of TCM before or during the chemotherapy cycle still needs more data to support. More studies to determine the best dosage form and use time of TCM are needed in the future.Myelosuppression, hepatic and renal dysfunction, and gastrointestinal reactions were common chemotherapy-related side effects , 31, whiWe also perform network pharmacology to explore the specific effect of the TCM group on the GC treatment process. According to the results of the PPI and KEGG parts, the TCM group plus three core genes mentioned above and the relevant signaling pathway obtained by GO and KEGG enrichment analysis have more to do with the occurrence and development of malignant tumors. Current studies demonstrate that the level of NPM1 is associated with the promotion of endometrial cancer by circWHSC1 . In addiNPM1 is a protein widely expressed in the nucleoli, which can travel between the nucleus and cytoplasm, participate in the assembly and transportation of ribosomal proteins, regulate centrosome replication, and regulate the expression of tumor suppressor ARF. Mutations in NPM1 push the protein to transfer from the nucleus to the cytoplasm, causing acute myeloid leukemia. However, Fan et al.  showed tMoreover, HSPA8 is a component of the HSP70 family expressing homologous proteins and is crucial to many cellular processes. In particular, its regulatory role in autophagy is decisive . FunctioFurthermore, the HSP90AA1 gene is a core gene directly associated with GC. Functioning as a homologous dimer, the protein it encodes is an inducible molecular chaperone. The encoded proteins contribute to the correct folding of specific target proteins through the cochaperone-regulated ATPase activity. There is evidence  that HSPThe strengths of our study include our wide-ranging online searches of literature in both English and Chinese databases. We applied broad criteria for pooling studies. We conducted a meta-regression analysis to determine if specific preparation yielded differing effects over the broad group, and in several cases they did.in vivo and in vitro. So we still need to take these compelling results with caution and this study should be carefully disseminated for clinical application. Our findings need to be further confirmed via more RCTs conducted in a rigorous manner.Some limitations may affect the drawn conclusion. First, there was a lack of large, multicenter, standardized RCTs, and the sample sizes of our included studies were mostly small or of moderate size. More RCTs with larger sample sizes are required to confirm these outcomes. Second, TCM’s individualized treatment and self-formulated prescription for inconsistent treatment with Chinese medicine may have led to heterogeneity in the meta-analysis, and none of them were able to provide definitive options for clinical treatment. Moreover, randomization methods, allocation concealment, and blinding are not clearly described in those included trials, which may result in inherent bias and exaggeration of the efficacy of the treatment group. In addition, our study induced a certain degree of heterogeneity due to the different classifications of tumors, pathologic stage of the tumor, the application of adjuvant therapy, and duration of treatment among included trials. Furthermore, almost all studies were conducted in China, which may lead to unavoidable regional bias. All these limitations may have resulted in insufficient evaluations of the outcome indicators. There are also some shortcomings in network pharmacology analysis. Due to the incomplete research on drug components and targets, the relevant data in the disease database are incomplete, leading to certain limitations and defects in the statistical results of network pharmacology in this paper. Also, the method of screening components of TCM by DL and OB values did not take the active ingredients into full consideration, which may affect the results. Furthermore, network pharmacology studies ignored the influence of component content of TCM on experimental results, and subsequent studies should focus on the relationship between content and curative effect. Moreover, network pharmacology is based on computer simulation analysis and molecular network data, whose statistical and predictive results need to be confirmed by specific experiments All in all, we hope the findings of this analysis will provide a shred of helpful evidence for clinicians to formulate the best treatment strategy for patients with GC and also provide scientific clues for researchers in this field. Moreover, the methodological ideas of this paper are shown in In summary, our study indicates that TCM plus chemotherapy is highly effective and safe in the treatment of people with GC. TCM plus chemotherapy has the characteristics of multiple pathways, multiple components, and multiple targets in the treatment of GC and diseases. Our study will provide valuable evidence for further evaluation of TCM. Moreover, some limitations increased the risk of bias, which, to some extent, affects the credibility of the satisfactory result. Thus, to confirm these finding, more rigorous randomized controlled trials are needed.The original contributions presented in the study are included in the article/LZ, LS and JY contributed to the design and conception of the study. JY and NR carried out the collection and processing of data. LQ and GY finished the figure diagramming. ZZ and LY performed the data analysis and interpretation. NR and LY wrote the manuscript. LZ, LS and JY revised the manuscript, then LZ gave the final approval of manuscript.This study was financially subsidized by the Project funded by Plan Project of Zhejiang Province Medical and Health Science and Technology ; China Postdoctoral Science Foundation ; Young Elite Scientists Sponsorship Program by CAST ; Research Project of Zhejiang Chinese Medical University ; The Construction Fund of Medical Key Disciplines of Hangzhou (OO20200385); Research Project of Zhejiang Chinese Medical University .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Yifei Jianpi Tongfu formula (YJTF) in CRC lung metastasis in a comprehensive and systematic way by network analysis, molecular docking, and experimental verification.  The lung is the second most common site of colorectal cancer (CRC) metastasis. This study aims to investigate the therapeutic effects and potential action mechanisms of  The main ingredients in YJTF were screened from the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP) and Traditional Chinese Medicine Integrated Database (TCMID), and the disease-related targets from the Online Mendelian Inheritance in Man (OMIM) and GeneCards and the compound-related targets from SwissTargetPrediction were collected. Then, Metascape was used for pathway annotation and enrichment analysis, and meanwhile, a protein-protein interaction (PPI) network was constructed. Molecular docking was carried out to investigate interactions between the active compounds and the potential targets. The in vivo effect of YJTF on CRC lung metastasis was observed in a tail vein injection mouse model.  A total of 243 active compounds and 81 disease-related targets of YJTF were selected for analysis. The results of multiple network analysis showed that the core targets of YJTF were enriched onto various cancer-related pathways, especially focal adhesion and adherens junction. The results of molecular docking demonstrated that all core compounds  were capable of binding with AKT1, EGFR, SRC, ESR1, and PTGS2. Experimental validation in vivo demonstrated that YJTF combined with oxaliplatin could significantly reduce the number of lung metastases and improve the quality of life in mice. Further research suggested that YJTF inhibited CRC lung metastasis probably by modulating epithelial-to-mesenchymal transition (EMT).  According to the analysis, YJTF can be considered as an effective adjuvant therapy for CRC lung metastasis. Colorectal cancer (CRC) is one of the most common diagnostic cancers and the fourth leading cause of cancer-related deaths worldwide . Due to The lung is the second most common distant metastatic site for primary CRC, and about 10–25% CRC patients eventually develop pulmonary metastasis . The surYifei Jianpi Tongfu formula (YJTF), a TCM prescription composed of diverse medicinal herbs, has proven effective for the treatment of various respiratory diseases, such as chronic obstructive pulmonary disease (COPD) and other diseases related to airway inflammation [Astragalus mongholicus Bunge (Fabaceae)), Radix Codonopsis ), Rhizoma Atractylodis Macrocephalae (Asteraceae) (RAM), Poria cocos ), Semen Coicis ), Radix Ophiopogonis ), Akebia Trifoliata Koidz ), Ampelopsis sinica ), Sargentodoxa cuneate  Rehd. et wils. ), Radix Asteris Tatarici ), Radix Platycodonis ), and Radix Glycyrrhiza ). Then, we tried to verify its curative effect and explore the potential mechanism by using the network analysis method in combination with in vivo experiments, attempting to propose new strategies for the adjuvant treatment of CRC lung metastasis.ammation –11. Whatammation –14. Knowhttp://lsp.nwu.edu.cn/tcmspsearch.php) [http://www.megabionet.org/tcmid/) [YJTF compounds were obtained from the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform 2.3   and the /tcmid/) . The TCM/tcmid/) . Compounhttps://pubchem.ncbi.nlm.nih.gov/) [http://www.swisstargetprediction.ch/) [https://www.uniprot.org/)  was used to normalize the naming of the drug targets.To identify the corresponding targets of the main compounds of YJTF, all chemical structures were prepared and converted into canonical SMILES using PubChem (ih.gov/) , then thion.ch/) , and thehttps://www.genecards.org/) and Online Mendelian Inheritance in Man (OMIM) (https://www.omim.org/). GeneCards is a comprehensive database that provides concise genome, proteome, transcriptome, disease, and functional data of all known and predicted human genes [https://www.uniprot.org/)  was used to normalize the naming of the disease-related targets.Potential genes associated with CRC lung metastasis were collected from GeneCards (an genes . The OMIan genes . UniProthttps://string-db.org/cgi/input.pl), a weighted interaction database with physical and functional interactions that are integrated from multiple data sources. To construct a PPI network with high confidence edges, we filtered the STRING with threshold 0.4 and only interactions with weight above the threshold were selected for the newly constructed PPI network.PPI data in human genomes were extracted from version11.0 of STRING (http://www.cytoscape.org/), an open-source software platform for visualizing complex networks [The visual network graphs were created by Cytoscape (version 3.7.2) (networks . The tarhttp://metascape.org/) [p value, the more the enrichment would be.To reveal the potential biological functions of YJTF in the treatment of CRC lung metastasis, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the Metascape database (pe.org/) , where tTo demonstrate the multicompound therapeutic features of YJTF, network construction was performed as follows: (1) the herb-compound-target network (H-C-T network) was constructed to explore the active compounds and their potential targets, and the core compounds were obtained through the H-C-T network; (2) the PPI network was built to analyze target interactions, from which Hub targets involved in YJTF treatment of CRC lung metastasis were selected; and (3) the target-pathway (T-P) network was constructed to show the functional pathways of YJTF for the treatment of CRC lung metastasis.All these networks were constructed and analyzed in Cytoscape 3.7.2 software. Then, four topological properties  were calculated to screen the putative targets for topological importance, knowing that larger the node degree, betweenness, or closeness centrality, the more important the node would be in the network .http://www1.rcsb.org/) [Molecular docking was conducted to validate whether the compounds in YJTF could bind to the potential targets. The 2D structures of the top five core compounds were downloaded from the TCMSP database  and PubCsb.org/) . Water m2.HCT-116 cells  were cultured in RPMI1640 medium  supplemented with 10% fetal bovine serum  and kept in a 37°C incubator with 5% COThe HCT 116 cell line was isolated from the colon of an adult male, colon cancer patient, which is commonly used to study cancer biology including the research of treatment schemes and drug screening. This is a growth factor-independent cell line that has been shown to be invasive and highly motile in in vitro studies. Subcutaneous xenograft experiments have demonstrated it to be highly tumorigenic. Therefore, it is currently widely used in the study of colorectal cancer metastasis , 27.The components of YJTF are as follows: RA 18 g, RC 20 g, RAM 15 g, Po 12 g, SC 15 g, RO 15 g, AK 9 g, Am 30 g, Sa 30 g, RAT 12 g, RP 9 g, and RG 5 g. The herbs were prepared into fluid extracts according to the standard operating procedures by the pharmacy of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine . All the herbs were extracted with boiling water, and then, the extracts were concentrated to obtain the decoction (2.47 g/mL).6 cells in 200 μl) via the tail vein into male BALB/c nude mice aged 4–5 weeks . Twenty-four mice were equally randomized into four groups: a control (Con) group, an oxaliplatin (Oxa) group, a YJTF (YFT) group, and a YJTF + Oxaliplatin (YJT + Oxa) group. Mice in YJT and YJT + Oxa groups were intragastrically administered with 200 μl YJTF decoction daily for 6 weeks, and mice in Con and Oxa groups were administered with 200 μl PBS. Mice in Oxa and YJT + Oxa groups were intraperitoneally injected with 5 mg/kg oxaliplatin twice a week for 6 weeks, and mice in Con and YJT groups were injected with an equal amount of glucose solution.All the animal experiments were approved by the Institutional Review Board of Shanghai Pulmonary Hospital, Tongji University School of Medicine , and performed in accordance with the national guidelines for the care and use of laboratory animals. The lung metastasis mouse model was established by intravenous injection of HCT116 cells  staining with E-cadherin, N-cadherin, and vimentin antibodies. The primary organs including the heart, liver, spleen, kidney, skin, and skeletal muscle were collected for hematoxylin and eosin (H&E) staining. Changes in the body weight of the animals were also recorded during the experiment.P < 0.05 was considered statistically significant.The SPSS software version 18.0  was used for statistical analysis. Data are expressed as the means ± standard deviation (SD). For comparisons of the means of multiple groups, one-way ANOVAs followed by LSD tests were performed. Altogether 935 components in YJTF were obtained from the TCMSP and TCMID databases, 87 of which belonged to RA, 134 to RC, 55 to RAM, 34 to Po, 38 to SC, 55 to RO, 12 to AK, 22 to Am, 25 to Sa, 91 to RAT, 102 to RP, and 280 to RG. According to the screening conditions of OB ≥ 30%, DL ≥ 0.18, and Lipinski's rule, 243 active components from 12 herbs of YJTF were collected, comprising 20 components extracted from RA, 21 from RC, 7 from RAM, 15 from Po, 9 from SC, 33 from RO, 6 from AK, 8 from Am, 5 from Sa, 19 from RAT, 7 from RP, and 93 from RG. After removing the redundancy, 214 components were selected as candidate bioactive components. Then, 872 protein targets were harvested based on the target prediction performed using the SwissTargetPrediction website tool. After eliminating the overlapping targets, 131 targets remained.The H-C-T network of YJTF was visualized in Cytoscape , which cThe targets for CRC lung metastasis were integrated from multisource databases including GeneCards and OMIM, and finally, a list of 2,047 disease-related targets was obtained after eliminating the duplicates  . The cluPP values<0.01. The top 10 significantly enriched terms were selected in the BP, MF, and CC categories, which are listed in To further excavate the significance of the 81 hub targets, gene ontology enrichment analysis was used, which consisted of three parts: BP , CC (cellular component), and MF (molecular function). As shown in the results of the enrichment, a total of 1,344 GO enrichment results were obtained, including BP , MF (126 terms), and CC (63 terms). We set the level of statistical significance at P < 0.01). The top 21 pathways at a significance threshold of LogP < -7 are displayed in The 81 putative targets of active compounds were mapped onto 102 KEGG pathways  were sectioned and stained with H&E for histological evaluation to validate the drug safety. After staining, the nucleus presented as blue and the cytoplasm as red by microscopy, and the tissue morphology can be clearly observed. H&E analysis of the major organs and tissues in each group indicated that there were no obvious abnormal changes (not shown) in the HE slices. It can be considered that the drug treatment  is safe and have no obvious toxic side effects .During the experiment, the body weight of the mice was measured at 4-day intervals . It was P < 0.01), and the number of lung metastases in the YJT group was slightly reduced (P < 0.05).After 6-week treatment, all mice were sacrificed and their lungs were collected and fixed in 4% PFA. Then, visible lung metastases were counted in each group of mice. The representative lung of each group and the quantitative mean number of visible lung metastases are shown in We performed IHC staining to evaluate the expression of E-cadherin, N-cadherin, and vimentin in the metastatic tumor tissues. Brown staining indicates positive expression areas, the shade of the color represents the expression level of the target protein, and the cell nuclei were stained in blue color by hematoxylin. As the picture shows, we found that the expression of E-cadherin was significantly upregulated in YJT and YJT + Oxa groups, while it was significantly downregulated in YJT and YJT + Oxa groups , suggestDistant metastasis is the main cause of cancer-related death and remains one of the focuses of cancer research. Metastasis is known as a multistep complex process involving multiple factors. EMT is an embryonic program that loosens cell-cell adherence complex and endows cells with enhanced progression. Cancer cells undergoing EMT are more aggressive and often display increased invasiveness, stem-like features, and resistance to apoptosis , 37. EMTβ-catenin, and MAPK/ERK1/2 pathways [In this study, we applied YJTF in the treatment of CRC lung metastasis and verified its therapeutic efficacy through in vivo experiments. To provide further insights into the underlying mechanisms, we used the systematic pharmacology method and molecular docking to explore the potential molecular mechanisms of the 243 bioactive compounds and 81 putative targets of YJTF obtained from the TCMSP and TCMID by overlapping the targets between YJTF-associated targets and the targets of CRC lung metastasis. According to the H-C-T network topological analysis, we found that the core compounds included quercetin, kaempferol, luteolin, apigenin, and isorhamnetin. Previous studies have demonstrated that all these compounds have therapeutic effects on various cancers. As the compound with the highest degree, quercetin is ubiquitously present in fruits and vegetables, being one of the most common dietary flavanols. The anticancer effects of quercetin include its ability to promote the loss of cell viability, apoptosis, and autophagy through the modulation of PI3K-Akt-mTOR, Wnt/pathways . Quercetpathways . Both kapathways . In addipathways . FurtherFurthermore, according to PPI analysis, we determined the interactions between the targets and their importance. AKT1, EGFR, SRC, ESR1, and PTGS2 are the top five targets in the PPI network, and they were reported as critical regulatory factors in cancer metastasis and EMT –49. ThroAlthough the incidence and mortality rate of CRC have decreased due to the advent of effective cancer screening measures, there has been an increase in the number of young patients diagnosed with CRC. Therefore, how to improve the quality of life of cancer patients has also become one of the focuses of comprehensive tumor treatment. In recent decades, several forms of complementary and alternative medicines have been utilized to defeat cancer worldwide. Traditional Chinese herbal medicines have been widely accepted as a mainstream form of complementary and alternative therapy with beneficial effects for cancer patients in China , 54. PreIn this study, we used the network analysis approach to investigate the active compounds and potential targets of YJTF, and explored their underlying molecular mechanisms in treating CRC lung metastasis. The results demonstrated that YJTF participated in various biological processes related to cancer, probably by regulating pathways related to cell adhesion, thus inhibiting CRC invasion and migration. Our subsequent molecular docking showed that each core compound of YJTF had a favorable binding ability with AKT1, EGFR, SRC, ESR1, and PTGS2. More importantly, our in vivo experiments validated the therapeutic effect of YJTF on CRC lung metastasis by regulating EMT. All these findings may provide a new thought and reference for the comprehensive treatment of CRC lung metastasis.


Objective: Schisandrae Sphenantherae Fructus (SSF) is a traditional Chinese medicine used to treat coughs and pulmonary inflammatory diseases. However, the pharmacodynamic material basis and mechanisms for SSF in asthma treatment remain unclear. This study aims to screen the anti-asthmatic fraction and verify the pharmacodynamic material basis, predict the potential mechanism, and verify the interaction ability between compounds and core targets.Methods: First, three fractions from SSF were compared in terms of composition, comparison, and anti-asthmatic effects. Then, the ultra-performance liquid chromatography-quadrupole/time-of-flight-mass spectrometry/mass spectrometry (UPLC-Q/TOF-MS/MS) strategy was used to identify the compounds from the active fraction, and the anti-asthmatic efficacy of the active fraction was further studied by the ovalbumin (OVA)-induced asthma murine model. Finally, network pharmacology and molecular methods were used to study the relationships between active compounds, core targets, and key pathways of PEF in asthma treatments.Results: The petroleum ether fraction (PEF) of SSF showed better effects and could significantly diminish lung inflammation and mitigate the level of serum immunoglobulin E (IgE), interleukin (IL)-4, IL-5, IL-6, IL-13, and IL-17 in mice. A total of 26 compounds from the PEF were identified, among which the main compounds are lignans and triterpenes. Moreover, 21 active compounds, 129 overlap-ping targets, and 10 pathways were screened by network pharmacology tools. The top five core targets may play a great role in asthma treatment. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested that the PEF can treat asthma by acting on multiple asthma pathological processes, including the IL-17 signaling pathway, T helper (Th) 17 cell differentiation, and the calcium signaling pathway. Molecular docking was performed to evaluate the interactions of the protein–ligand binding, and most docked complexes had a good binding ability.Conclusion: The present results might contribute to exploring the active compounds with anti-asthmatic activity. Asthma is a chronic disease that affects all age groups, and the prevalence of asthma has been increasing annually . It has Schisandra sphenanthera Rehd. et Wils. (Schisandraceae), is commonly used for treatment of coughs and pulmonary and inflammatory diseases and has been officially listed in the Chinese Pharmacopoeia (Schisandrae Sphenantherae Fructus (SSF), sourced from acopoeia . Accordiacopoeia . Howeverin silico network pharmacology method was applied to predict the active compounds, core targets, and potential pathway of the active fraction against asthma. Finally, molecular docking was applied to verify the results of network analysis.In this study, three fractions of SSF, namely, the petroleum ether fraction (PEF), ethyl acetate fraction (EAC), and a higher polar fraction (HPF) were compared in terms of UPLC chromatography and anti-asthmatic effects in an ovalbumin (OVA)-induced allergic asthma model. The pharmacodynamic material basis of the active fraction was studied by the ultra-performance liquid chromatography-quadrupole/time-of-flight-mass spectrometry/mass spectrometry (UPLC-Q/TOF-MS/MS) system, and the anti-asthmatic efficacy of the active fraction was further studied. Then, an SSF samples were collected from Hanzhong city , Shaanxi province of China. The authentication of the voucher specimens was identified by Yaodong Qi and Xueping Wei, researchers of the Institute of Medicinal Plant Development (IMPLAD), Beijing, China. The specimens were deposited in the Medical Plant Resource Research Center of IMPLAD.Male C57BL/6 mice (20–22 g) were purchased from SPF Biotechnology Co., Ltd. (license number: SCXK [Beijing] 2019-0010) . All animal experiments were approved by the Institutional Animal Care and Use Committee of IMPLAD  and were carried out in agreement with the Guide for the Care and Use of Laboratory Animals . All mice were housed under a 12-h light–dark cycle in a temperature- and humidity-controlled room with free access to tap water and irradiated food.n = 6). A control group (CON) was treated with vehicle  every other day from days 27–48 and saline sensitization/challenge. A model group (MOD) was treated with vehicle  every other day from days 27–48 and OVA sensitization/challenge. A positive control group (POS) was treated with 1 mg/kg dexamethasone every other day from days 27–48 and OVA sensitization/challenge. Also, four administration groups  were treated with 200 mg/kg SSF, PEF, EAC, and HPF every other day from days 27–48 and OVA sensitization/challenge.In the animal experiment of active fraction screening of SSF, mice were randomly divided into the following seven groups (n = 6). The control group (CON) was treated with vehicle  every other day from days 27–48 and saline sensitization/challenge. The model group (MOD) was treated with vehicle  every other day from days 27–48 and OVA sensitization/challenge. The positive control group (POS) was treated with 1 mg/kg dexamethasone every other day from days 27–48 and OVA sensitization/challenge. Also, three PEF groups  were treated with 100, 200, and 400 mg/kg PEF every other day from days 27–48 and OVA sensitization/challenge, respectively.In the anti-asthmatic animal experiment of the PEF, mice were randomly divided into the following six groups  staining to assess inflammation of the lungs. Pathological assessment of lung inflammation was graded blindly on a scale of 0–4 (least to most severe) based on an assessment of the quantity and quality of peribranchial infiltration of inflammatory cells, perivascular infiltration of inflammatory cells, and interstitial inflammation. The degree of inflammation and lesions of each slide was graded as follows: 0, no inflammation or lesion; 1, minimal; 2, mild; 3, moderate; and 4, severe. To measure the inflammatory responses, sera from the mice were collected. The levels of total IgE and inflammatory cytokines in serum were determined using the Elisa kits according to the manufacturer’s instructions.18 column . The column and autosampler were maintained at 30 and 10°C, respectively. The flow rate was set at 0.3 ml/min, and the injection volume was 1 μl. The mobile phase consisted of water (A) and acetonitrile (B), and the gradient conditions were as follows: 0–4 min, 42–50% B; 4–6 min, 50–48% B; 6–12 min, 48% B; 12–16 min, 48–62% B; 16–21 min, 62% B; 21–22 min, 62–85% B; 22–29 min, 85–95% B; 29–34 min, 95% B. The online UV spectra were recorded in the range of 200–400 nm.UPLC separation was achieved on a Waters ACQUITY I-Class system  using a Waters ACQUITY BEH Cm/z 100–1,200 Da and with a 1 s scan time. Nitrogen was used as a nebulizer and auxiliary gas. In positive ion mode, the following parameters were found: capillary voltage, 3 kV; sampling cone voltage, 20 V; source temperature, 100°C; desolvation temperature, 350°C; cone gas flow, 50 l/hr; desolvation gas flow, 600 l/hr, and collision energy, 6 eV. Leucine enkephalin was used as a lock mass with a reference mass value at m/z 556.2771. MS/MS fragment information was obtained using a collision energy ramp from 10 to 45 eV. In the negative ion mode, the following parameters were found: capillary voltage, 3 kV; sampling cone voltage, 40 V; source temperature, 100°C; desolvation temperature, 350°C; cone gas flow, 50 l/hr; desolvation gas flow, 600 l/hr, and collision energy, 32 eV. The lock mass solution was Leucine enkephalin with a reference mass value at m/z 554.2615.Mass spectrometry was operated on the Waters Xevo G2-XS Q/TOF mass spectrometer  equipped with an electrospray ionization (ESI) source controlled by MassLynx 4.2 software . A full scan was run in positive and negative modes, with a mass range from The raw data were imported into Progenesis QI . After automatic processing, alignment, peak picking, normalization, and deconvolution, the ingredients of the PEF were tentatively identified on the grounds of the in-house database we constructed before . Then, thttp://www.swissadme.ch, accessed on 11 December 2021) to assess the properties of pharmacokinetics and drug-likeness of compounds. Then, the SwissTargetPrediction  was employed to predict the putative targets of active compounds (Compounds from the active fraction were identified by the UPLC-Q/TOF-MS/MS method. The SMILES (Simplified Molecular Input Line Entry System) was entered in the SwissADME (ompounds .http://db.idrblab.net/ttd/), OMIM (https://www.omim.org/), DrugBank (http://www.drugbank.ca/), and GeneCards (https://www.genecards.org/)  (https://www.ncbi.nlm.nih.gov/geo/) (accessed on 12 December 2021). All asthma-related targets were carried out by jvenn online tools  (Asthma-related targets were searched from the following databases: Therapeutic Target Database (er 2021) . In addier 2021) .https://string-db.org, accessed on 12 December 2021) was then applied for PPI analysis (The overlapping targets between the active fraction and asthma were carried out by jvenn online tools (accessed on 12 December 2021). The STRING database   (The Gene Ontology (GO) function enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were constructed using the WebGestalt database (er 2021) . For GO er 2021) . Then, Chttp://www.rcsb.org/) (accessed on 13 December 2021) . Then, PDB formats were obtained by Open Babel 3.1.1, and energy minimization was employed by an MMFF94 force field. AutoDockTool 4.2.6 was used to convert the PDBQT formats . The strer 2021) . AutoDocThe molecular docking processes were carried out using AutoDock vina 1.2., and each docking task was repeated nine times with the exhaustiveness parameter of 16 and energy thresholds of ±2 kcal/mol . The lowp < 0.01 indicated significant differences.Data were expressed as mean (SD). Statistical analysis was performed using Prism 8.4.0. . Statistical comparisons were carried out based on was assessed by one-way analysis of variance (ANOVA). UPLC chromatograms of SSF, PEF, EAC, and HPF could be seen in p < 0.01)  . Howeverp < 0.01) (p < 0.01). Therefore, the staining results showed that the PEF is the most potent fraction. Serum IgE and interleukin (IL)-4, IL-5, and IL-13 were all elevated markedly in the model mice (p < 0.01), and these characteristics were substantially improved in the PEF group (p < 0.01)  . Compare < 0.01) . All thep < 0.01) (p < 0.01) (p < 0.01). The high-dose group (400 mg/kg) showed the lowest peribranchial, perivascular, and interstitial inflammation scores, indicating a better effect of the anti-asthmatic effect. Then, the expressions of serum IgE, IL-4, IL-5, IL-6, IL-13, and IL-17 was significantly increased in the model group compared with those in the control group (p < 0.01). After the administration of the PEF, the expression of inflammation cytokines aforementioned was markedly decreased (p < 0.01). Among the three PEF groups, the mice in the 400 mg/kg PEF group demonstrated better therapeutic effects  . Then, t < 0.01) . In the  effects . These fP value (<5), molecular weight (<500), number of hydrogen bonds for the donor (≤5), and acceptor (≤10)  . Duplicaor (≤10) .A total of 696 genes related to asthma were obtained from OMIM, DRUGBANK, Therapeutic Target Database, and GeneCards after removing the duplicate genes. On the other hand, four GEO datasets  were retrieved from the GEO database using asthma as the keyword. The distribution of differentially expressed genes (DEGs) was illustrated by volcano plots and hierarchical clustering analysis . We obtaA total of 129 overlapping targets between the PEF and asthma were obtained by jvenn online tools , and theThen, the cytohubba tool was applied to obtain key genes. The top five genes by degree ranking in the PPI were visualized, namely, mitogen-activated protein kinase (MAPK) 14, JUN, signal transducer and activator of transcription (STAT)3, MAPK3, and MAPK1 .The Gene Ontology annotation analysis and KEGG pathway analysis were carried out by webgestalt. The top ten pathways ranked by FDR were as follows: the AGE-RAGE signaling pathway in diabetic complications, the calcium signaling pathway, Th17 cell differentiation, pathways in cancer, hepatitis B, the IL-17 signaling pathway, the relaxin signaling pathway, the HIF-1 signaling pathway, neuroactive ligand–receptor interaction, and human cytomegalovirus infection .The top ten GO BPs were as follows: response to stimulus, biological regulation, metabolic process, multicellular organismal process, cell communication, localization, developmental process, cellular component organization, multi-organism process, and cell proliferation . The top2O2, janus kinase-signal transducer and activator of transcription proteins (JAK-STAT), mitogen-activated protein kinase-phosphoinositide-3-kinase (MAPK-PI3K), p53, and Notch were inferred upregulated with overlapping targets, and peroxisome proliferator-activated receptors (PPAR), Hippo, Wnt, hypoxia, and estrogen pathways were inferred downregulated upstream , toll-like receptor (TLR), tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), insulin, Hupstream . Based oupstream .in silico molecular docking approach was performed to test the affinity between the compounds and the top five targets, namely, MAPK14 (PDB ID: 5ETF); JUN (PDB ID: 5J41); STAT3 (PDB ID: 6SM8); MAPK3 (PDB ID: 4QTB); and MAPK1 (PDB ID: 4ZZN). A total of 15 compounds that have relationships with the top five targets were selected for molecular docking analysis. In total, 75 docking simulations were executed. The RMSD values were stable, indicating stable interaction and accurate results. Also, according to the binding affinity scores, most complexes show negative results, indicating good interactions between the ligands and the receptors. Unexpectedly, lack of good interaction between MAPK3 (PDB ID: 4QTB) and sphenadilactone E was observed, and the docking result was a positive affinity value  and sphenadilactone E   and exhibited the strongest binding energy  . Schinalcal/mol) . The resIn this study, we investigated the pharmacodynamic material basis and predicted the mechanisms of the active fraction from SSF in asthma treatment. The PEF may be the most potent anti-asthmatic fraction of SSF. A total of 26 compounds of the PEF were identified through the UPLC-Q/TOF-MS/MS technology, among which 21 components may be the active ingredients of asthma. A total of 129 overlapping targets between the PEF and asthma were obtained by jvenn online tools, among which five targets might play important roles: MAPK14, JUN, STAT3, MAPK3, and MAPK1. Then, the enrichment analysis revealed that the PEF might reduce inflammation by modulating Th2 cells, IL-17 signaling pathway, Th17 cell differentiation, and the calcium signaling pathway.In the experiment of active fraction screening of SSF, a UPLC chromatography strategy and an OVA-induced allergic asthma murine model were constructed. The UPLC chromatograms showed that the PEF has more common compounds with SSF than other fractions. The OVA allergen-caused asthma is a typical airway inflammatory disease, and intraperitoneal sensitization with OVA/alum adjuvant is frequently used in experimental allergic asthma . Pharmac2+  pathway . Schisan2+ .Then, in the experiment of PEF against asthma, our findings showed that the expression of IgE, IL-4, IL-5, IL-6, IL-13, and IL-17 were significantly higher in the model group. The oral administration of the PEF can reduce the production of the cytokines aforementioned. IgE is considered to be the first line of defense against pathogens and plays a significant role in the occurrence and development of asthma . During + T cells and regulatory T cell populations . IL-17 is considered to be one of the significant cytokines of the Th17 pathway and is involved in neutrophilic inflammation and airway remodeling in severe asthma . Th17 ceMolecular docking has become an important tool in computer-aided drug design over the last few years. Here, we used molecular docking to verify the interaction affinities between the top five targets  and related compounds. Most compounds demonstrated good affinities toward the five targets, which validated the network pharmacology results. It is noteworthy that nowadays, there have been few studies on the treatment of asthma with lignans and triterpenes. More attention might be paid to these compounds in asthma treatment in future studies.In this study, combined approaches were applied to clear the pharmacodynamic substances and explain the anti-asthmatic effects of SSF. More comprehensive experimental validation and clinical studies are required to verify these results in the future. In addition, further optimization of structures and dosage forms may be required to increase the anti-asthmatic activity and oral bioavailability for its clinical applicability. Taken together, our findings might provide a pharmacodynamic substance basis and potential mechanisms for the use of lignans and triterpenes of SSF for asthma treatment.In conclusion, this study may be the first to systematically study the pharmacodynamic material basis and potential mechanisms of the anti-asthmatic fraction of Schisandrae Sphenantherae Fructus. Our results have shown that the most potent anti-asthmatic fraction of SSF is the PEF. Subsequently, we found that the 21 significant active compounds identified from the PEF may attenuate the pathological and inflammatory changes in asthma through 129 overlapping targets and 10 pathways. Furthermore, MAPK14, JUN, STAT3, MAPK3, and MAPK1 might play important roles during anti-asthmatic progress, and the PEF might reduce inflammation by modulating Th2 cells, IL-17 signaling pathway, Th17 cell differentiation, and the calcium signaling pathway. This study highlights new insights into discovering active compounds with specific anti-asthmatic activity and provides a scientific basis for further experimental studies.


Background: Varicocele (VC) is a relatively common and treatable cause of male infertility. Mailuoshutong pill (MLST), a traditional Chinese patent medicine, is widely used for treating varicose vein disease, but the underlying mechanism of MLST on varicocele-associated male infertility is unclear.Objective: To reveal the phytochemical characterisation and multitarget mechanism of MLST on varicocele-associated male infertility.Methods: The components in MLST were determined using UHPLC-MS/MS. Through network analysis, we constructed the “Drug-Components-Targets-Disease” network and predicted the potential biological functions and signaling pathways of MLST. Finally, the therapeutic effects and potential mechanisms of MLST were discovered by pharmacological experiments.Results: By network analysis, the “Drug-Components-Targets-Disease” network was constructed, 62 components such as apigenin, limonin, kaempferol, and obacunoic acid may be the main active components of MLST for varicocele-associated male infertility, 28 targets such as VEGFA, PIK3CA, AKT1, and MTOR are considered as hub targets, signaling pathways such as HIF-1, Estrogen, PI3K/Akt, and mTOR may be key pathways for MLST against varicocele-associated male infertility. Through pharmacological experiments, we found that MLST ameliorated VC-induced testicular atrophy. Further histomorphology showed that MLST reduced VC-induced damage to testicular spermatogonia and seminiferous tubule, while MLST reduced ROS and MDA levels and increased antioxidant enzymes  levels. TUNEL staining and immunofluorescence showed that MLST reduced VC-induced apoptosis in testicular tissue, decreased BAX, and increased BCL2. Western blot results showed that MLST decreased the phosphorylation of PI3K, AKT, and mTOR proteins, and decreased the expression of HIF1α.Conclusion: The phytochemical characterisation and multitarget mechanism of MLST on varicocele-associated male infertility were discovered using network analysis and pharmacological experiments. We verified that MLST can inhibit the activation of the PI3K/Akt/mTOR signaling pathway, reduce the expression of HIF1α, and further attenuate VC-induced oxidative stress and apoptosis in the testis. These findings provide evidence for the therapeutic role of MLST in varicocele-associated male infertility. Varicocele (VC), one of the leading causes of male infertility, is characterized by abnormally enlarged veins within the pampiniform plexus . Venous Astragalus mongholicus Bunge , Lonicera japonica Thunb , Phellodendron amurense Rupr , Atractylodes lancea DC , Coix lacryma-jobi L , Scrophularia ningpoensis Hemsl , Angelica sinensis Diels , Paeonia lactiflora Pall , Glycyrrhiza glabra L , Hirudo , Scolopendra , Scorpio . Pharmacological studies of TCM have shown that the active components of these herbs have biological activities of antioxidant, anti-inflammatory, blood circulation promotion, hormone regulation, and immune enhancement (Mailuoshutong pill (MLST), a traditional Chinese herbal formula, each Gram of MLST is made from ancement . Clinicaancement . The effIn this study, we used a combination of ultra-high-pressure liquid chromatography-mass spectrometry/mass spectrometry (UHPLC-MS/MS), network analysis, and pharmacological experiments to discover the phytochemical characterisation and multitarget mechanism of MLST against varicocele-associated male infertility. UHPLC-MS/MS was used to identify the main active components of MLST, and network analysis was used to construct the “Drug-Components-Targets-Disease” network to predict the potential drug components, drug targets, and drug mechanisms of MLST. Finally, after the prediction of network analysis, we conducted an experimental pharmacological evaluation based on the pathogenesis of varicocele-related male infertility to elucidate its therapeutic effects and possible mechanisms of action, which can help guide the further development and clinical application of MLST. The flowchart of this study is shown in MLST was provided by Shandong Lunan Pharmaceutical Group . Methanol and acetonitrile were supplied by Merck KGaA at LC-MS purity, and formic acid was supplied by Sia Reagents at LC-MS purity. Malondialdehyde (MDA) colorimetric assay kit , superoxide dismutase (SOD) activity assay kit , catalase (CAT) activity assay kit , glutathione peroxidase activity assay kit , reduced glutathione colorimetric assay kit , reactive oxygen species (ROS) , TUNEL test kit , BAX antibody , BCL2 antibody , PI3K antibody , p-PI3K antibody , AKT antibody , p-AKT antibody , mTOR antibody , p-mTOR antibody , HIF1α antibody , ECL kit . Ultra-performance liquid chromatography , high-resolution mass spectrum , column , inverted fluorescence microscope , panoramic scanner , scanning electron microscopy (SEM) .http://www.swissadme.ch/)  using SwissADME online system (dme.ch/) . The comhttp://www.swisstargetprediction.ch/) (SwissTargetPrediction online platform (ion.ch/)  is a webhttps://www.genecards.org/) (https://www.disgenet.org/) (https://www.pharmgkb.org/) (GeneCards (ds.org/)  is a comet.org/)  is a genkb.org/)  is the mhttps://cn.string-db.org/) (Duplicate data of drug targets and disease targets were removed separately and imported into origin software (version 2021) to plot Venn diagrams, and the intersection targets were considered to be effective targets for MLST treatment of varicocele-associated male infertility. STRING (db.org/)  can be udb.org/) . Accordidb.org/) , the hubp < 0.05, these targets were found to be significantly enriched in GO terms or KEGG pathways. The GOplot package (Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of hub targets were performed to reveal the role of identifying hub targets in varicocele-associated male infertility progression and treatment. Enrichment analysis of hub targets was performed using the clusterProfiler package  in R sof package  of R sofhttps://pubchem.ncbi.nlm.nih.gov/) (https://www.pdbus.org/) (The structures of the ligands were obtained from the PubChem database (ih.gov/) , and theus.org/) . The prous.org/) . Finally36 SD rats, aged 8 weeks , were purchased from Beijing Viton Lever Laboratory Animal Technology Co., . The rats were placed in an animal room under 22 ± 1°C and 60% ± 2% interior design conditions with a light/dark cycle for 12 h. All experimental procedures involving animals were approved by Institutional Animal Care and Use Committee of Shandong University of Traditional Chinese Medicine and Animal Ethics Committee of Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, China . The animal care and use system and guidelines of Shandong University of Traditional Chinese Medicine were followed. Rats needed at least 1 week to adapt to the environment before doing the experiments.In this study, the classical way of establishing a VC rat model through a narrow left renal vein created by Turner  was usedOne week after modeling, 36 rats were randomly divided into the following six groups (6 rats in each group): a normal control group (Ct), a sham-operated group (Sham), a VC-induced model group (Model), a VC-induced group administrated with low-dose MLST, a VC-induced group administrated with medium-dose MLST, and a VC-induced group administrated with high-dose MLST. The dose of MLST intervention in rats was determined based on the human equivalent dose, which was calculated from the body surface area equivalent dose . The lowHE staining was used to evaluate the degree of testicular and epididymal tissue damage and sperm quality in each group. The left testis and epididymis tissues of each group of rats were fixed with 4% paraformaldehyde, dehydrated in alcohol, transparently treated with xylene, embedded in paraffin, and serially sectioned on a microtome with a slice thickness of 4 μm. The 4 μm sections were dewaxed, dehydrated, stained with hematoxylin, and sealed with neutral gum. Images of testicular and epididymal tissue structures were observed and acquired under an inverted fluorescence microscope and panoramic scanner.Observation ofSpermatogenic Tubule by SEMTesticular tissues were taken and gently rinsed with PBS, fixed at room temperature for 2 h with electron microscopy fixative, fixed at room temperature for 2 h with 0.1 M phosphate buffer PB (PH 7.4) prepared with 1% osmium acid, and rinsed 3 times with 0.1 M phosphate buffer PB. The tissues were sequentially dehydrated with alcohol and isoamyl acetate. The samples were dried and placed tightly on the double-sided adhesive of conductive carbon film on the sample stage of the ion jetting instrument for the 30 s. Finally, the structure of the spermatogenic tubule was observed by SEM.The levels and activities of MDA, GSH, SOD, CAT, and GSH-Px were measured with kits according to the instructions. Determination of ROS content by DHE fluorescence staining. Fresh testicular tissue was rewarmed in frozen sections, and the tissue was incubated with DHE diluted in PBS for 30 min at 37°C under light-proof conditions, then the slides were washed in PBS (PH 7.4) on a decolorized shaking table, and the sections were shaken dry, and then the nuclei were stained with DAPI staining solution for 10 min at room temperature and light-proof. The slides were then placed in PBS and washed three times for 5 min each time on a decolorized shaking table with no light, and the sections were shaken dry and sealed with an anti-fluorescence quenching sealer. The slides were placed under an inverted fluorescence microscope and panoramic scanner, and images were collected to determine the amount and variation of ROS content in the cells according to the red fluorescence in the cells.The testicular tissue was fixed with 4% paraformaldehyde, dehydrated, embedded, sectioned, dewaxed, added with fluorescence quenching agent for 5 min, washed with running water for 10 min, and the slides were treated according to the instructions of the TUNEL test kit (fluorescence method). After operating according to the instructions, wash with PBS (PH 7.4). After the slices are slightly dried, they are sealed with anti fluorescence quenching sealing agent (including DAPI). Slices were observed in the panoramic scanner and images were collected.Testicular tissue was fixed in 4% paraformaldehyde, dehydrated, embedded, sliced, and washed with PBS (10X). The slices were placed in the repair box of EDTA (PH 8.0) antigen repair buffer, and the antigen was repaired in the steamer. The fluorescent quenching agent was added, washed with running water, and incubated with 5% BSA at room temperature. After discarding the serum, the primary antibody (1:100) was incubated overnight. After the slices were washed and dried with PBS, they were covered with a suitable kind of secondary antibody, dripped with DAPI dye, and sealed with anti fluorescence quenching sealant. Bax and BCL2 were selected for the primary antibody The staining of Bax and BCL2 in testicular tissue was observed by the panoramic scanner.Rat testicular tissue samples were ground into a powder with liquid nitrogen, lysed at 1 mg plus 10 μl of lysis solution for 30 min on ice, and the supernatant was collected by centrifugation at 4°C for 10 min after lysis. Protein (20 µg) was electrophoresed on 8%–12% SDS-PAGE gels, transferred to PVDF membranes, blocked with 5% BSA for 1 h at room temperature, and incubated with the following primary antibodies: PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR, HIF1α, diluted according to the antibody instructions. The primary antibody was incubated overnight at 4°C. The membrane was washed 5 times with TBST buffer for 5 min each time, and the goat anti-rabbit antibody was diluted with 5% BSA  according to the antibody instructions, mixed well, and the secondary antibody was incubated at room temperature for 1 h. The membrane was washed 5 times with 1 × TBST buffer for 5 min each time, and the ECL assay was performed using the ECL kit. Band intensity was analyzed by ImageJ software.p < 0.05 was considered statistically significant.All quantitative data were expressed as mean ± standard error of mean and statistically analyzed using GraphPad Prism 9 , and one-way analysis of variance (ANOVA) was used for comparison between groups. A difference of Through UHPLC-MS determined, a total ion flow spectrum of the components in MLST was obtained , and a tPrediction of the targets of the included components by SwissTargetPrediction yielded a total of 62 components acting on 917 targets. Targets for varicocele and male infertility were searched in the GeneCards database, DisGeNET database, and PharmGKB database, and a total of 4,418 male infertility targets and 295 varicocele targets were obtained after removing duplicate entries. Finally, 64 intersecting targets were obtained by making a Venn diagram . These 6The information of components and hub targets was imported into Cytoscape to construct the “Drug-Components-Targets-Disease” network, as shown in p-values. These signaling pathways included HIF-1 signaling pathways, IL-17 signaling pathways, TNF signaling pathways, PI3K/Akt signaling pathways, and mTOR signaling pathways. The top 10 entries in each category of biological process, cellular component, molecular function, and KEGG signaling pathways were screened based on p-value and plotted in a circle diagram in GO enrichment analysis yielded 1,485 significant results, including 1,422 BPs, 17 CCs, and 46 MFs. In the BP category, the main biological processes in which the core targets are involved are cellular responses to chemical stress, responses to oxidative stress, and regulation of apoptotic signaling pathways. In the CC category, the core targets were mainly enriched in cellular components such as membrane rafts, membrane microdomains, and organelle outer membranes. In the MF category, the core targets mainly play molecular functions such as transcriptional cofactor binding, transcriptional coregulator binding, and nuclear receptor activity. A total of 120 pathways were obtained by KEGG enrichment analysis. Signaling pathways for cancer and other diseases were excluded, and the top 10 pathways of high significance were selected based on their p < 0.05), while the testis index of the rats with MLST intervention was better than that in the model group (p < 0.05)  .Histopathological changes in testes and epididymis were observed by HE staining. As shown in Furthermore, the damage of VC on the seminiferous tubule was further observed by scanning electron microscopy, and the protective effect of MLST on the seminiferous tubule was observed. As shown in p < 0.05). MLST reduced the levels of ROS and MDA in testicular tissues of rats with VC, and the higher the dose, the more significant the effect. The levels of testicular antioxidant enzymes  were significantly decreased in the model rats compared with the control and sham-operated rats (p < 0.05). The levels of these enzymes were significantly increased in the rats with VC treated with MLST (p < 0.05), and the effect was more pronounced with higher doses. In terms of SOD parameters, the low-dose group showed increased values compared with the model group, but the results were not significantly different (p > 0.05).The results of ROS, MDA, and antioxidant enzymes  in testicular tissues are shown in In addition, ROS fluorescence analysis showed that the percentage of ROS expression in testis tissue was significantly higher in the model group than in the control and sham-operated groups, and MLST significantly reduced the ROS content in the testes of VC rats . These rTUNEL staining was used to detect apoptosis in testicular tissue. The number of TUNEL positive cells (green fluorescence) in the model group was significantly higher than that in the control group, while the number of TUNEL positive cells was significantly reduced by MLST intervention . The expHypoxia-inducible factor-1alpha (HIF1α) is crucial in varicocele-induced oxidative stress and apoptosis, and we found that PI3K/Akt, mTOR, and HIF-1 signaling pathways play an important role in HIF1α expression by enrichment analysis and review of related literature , and by p < 0.05), and its upstream PI3K/Akt/mTOR signaling pathways-related protein  expression was increased (p < 0.05), indicating that VC activates the PI3K/Akt/mTOR signaling pathways and induces the expression of HIF1α. In contrast, HIF1α protein expression was reduced in MLST-treated rats compared with the model group (p < 0.05), and the expression of upstream PI3K/Akt/mTOR signaling pathways-related proteins  was reduced (p < 0.05), indicating that MLST inhibited the activation of PI3K/Akt/mTOR signaling pathways and reduced the expression of HIF1α.As shown in VC is the main known cause of male infertility, and among male infertility patients with VC, there are varying degrees of abnormalities in total sperm count, sperm motility, and sperm morphology . CurrentFirstly, we identified 62 components in MLST by UHPLC-MS/MS and ADME screening principles to provide component-based information on MLST. By constructing a “Components-Targets-Pathways” network and molecular docking, we screened the components with the strong binding ability to the pathway targets related to varicocele-associated male infertility: apigenin, kaempferol, limonin. Apigenin, as a biologically active flavonoid, has various biological properties  and can reduce oxidative stress in the testis . KaempfeFurthermore, the PPI network analysis was used to screen 28 hub targets of MLST against varicocele-associated male infertility. Among them, HIF1α, a highly specific nuclear transcription factor, is usually overexpressed in hypoxic environments and is an important hub for regulating oxygen homeostasis, and overexpression of HIF1α can induce testicular tissue damage . VasculaFurthermore, enrichment analysis of the hub targets revealed that MLST may regulate regulates oxidative stress and apoptosis to treat varicocele-associated male infertility. Oxidative stress is one of the main causes of testicular tissue damage induced by VC . OxidatiFurthermore, we established a VC rat model to study the effect of VC on testicular spermatogenesis and the therapeutic effect of MLST on varicocele-associated male infertility. In this study, we found that VC can cause the decline of the testicular index, previous studies have also confirmed this phenomenon , and MLSFinally, HIF1α was overexpressed in VC-induced rat testes by western blot analysis, and excessive HIF1α expression could lead to impairment of testicular spermatogenesis, which was similar to previous reports , and MLSThe present study found that MLST was effective in alleviating VC-induced testicular tissue damage. The components in MLST were identified by UHPLC-MS/MS, which provided an important basis for the clarification of the material basis and subsequent network analysis. By predicting and elucidating the multi-component, multi-target, and multi-pathway therapeutic effects of MLST on varicocele-associated male infertility through network analysis, it was experimentally verified that MLST can inhibit the activation of the PI3K/Akt/mTOR signaling pathway, reduce the expression of HIF1α, and further attenuate VC-induced oxidative stress and apoptosis in the testis. These findings provide evidence for the therapeutic role of MLST in varicocele-associated male infertility, as well as an idea for the study of other herbal compounding.


Methamphetamine (METH) addiction and its induced mental disorders have become a severe worldwide problem. A candidate Chinese herbal medicine (CCHM) in our lab had therapeutic effects on METH-induced locomotor sensitization, however, its chemical and pharmacological profiles remain to be elucidated. The current study aimed to investigate the effect of CCHM on conditioned place preference (CPP) induced by METH and screen the main active ingredients and key targets by using network pharmacology and molecular docking methods. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, Gene ontology (GO) analysis and protein-protein interaction (PPI) network were performed to discover the potential mechanisms. Results showed that CCHM could significantly inhibit METH-induced CPP behaviors in mice. A total of 123 components and 43 targets were screened. According to the network pharmacology analysis, ten hub targets including D(2) dopamine receptor (DRD2) and 5-hydroxytryptamine receptor 3A (HTR3A) were screened. GO analysis and KEGG enrichment indicated that mechanisms of CCHM treatment of METH addiction were related to multiple pathways such as dopaminergic synapse and serotoninergic synapse. Western blot results showed that the protein expressions of DRD2 in nucleus accumbens and prefrontal cortex were significantly decreased in METH group, while the protein expressions of HTR3A were significantly increased. These changes caused by METH could be prevented by CCHM pretreatment. The results of molecular docking displayed that the five active ingredients such as (S)-Scoulerine, Hyndarin, and Beta-Sitosterol had good affinities with DRD2 and HTR3A. In conclusion, this study constructed the CCHM’s pharmacologic network for treating METH addiction based on the method of network analysis and experimental verification, and analyzed its major active ingredients and potential targets, indicating a new direction for further revealing its mechanisms of effect on METH addiction. Conditioned place preference (CPP) test was used to assess the potential therapeutic effects of CCHM on METH addiction. The active components and hub targets, which were screened by the network pharmacology analysis, were verified by further experiments. Drug addiction is a severe worldwide social problem, which has attracted great attention. Compared with drugs extracted from plants such as heroin and cocaine, the proportion of people who abuse amphetamine-type synthetic drugs is increasing year by year. According to reports released by United Nations Office on Drugs and Crime and the Ministry of Public Security of China, methamphetamine (METH) has become the most commonly abused amphetamine-type stimulant in Southeast Asia and North America. METH can easily enter the central nervous system through the blood-brain barrier, so its effect on brain is more evident than its peripheral effect . StudiesCorydalis yanhusuo and Codonopsis, could significantly improve the learning and memory impairment induced by METH, and inhibit the acquisition and expression of METH-induced locomotor sensitization  were purchased from Beijing Charles River Laboratories . Mice were housed in a temperature-controlled room with a humidity of 45∼55% and subjected to a 12-h cycle of alternating light and dark. All mice had free access to food and water, and were pre-adapted to the environment for at least 3 days before the experiment. All experiment procedures were approved by the Ethics Committee of Jianghan University. As shown in The conditioned place preference (CPP) facility was brought from Shanghai Xinruan Information Technology Co., Ltd.  and comprised three compartments. One chamber had a grid floor and black walls (L × W × H: 35 cm × 35 cm × 35 cm). Another same-sized chamber had a mesh floor and white walls. The middle chamber had a flat, gray floor and gray walls (L × W × H: 15 cm × 35 cm × 35 cm) and is connected to the other two compartments by removable doors. The white and black chamber had a ceiling light and camera, respectively. The general activity in the facility was monitored by video, and the time spent in each chamber was recorded using XR-XT401 software.The CPP procedure used in this experiment was performed as previously described . The preC. yanhusuo, Poria cocos, Codonopsis pilosula, Licorice, Atractylodes macrocephala Koidz, Astragalus membranaceus, Angelica sinensis, and American ginseng. The components of CCHM were retrieved from Traditional Chinese medicine system pharmacology (TCMSP) database.1 Oral Chinese medicine must overcome the obstacles of absorption, distribution, metabolism, and excretion (ADME) process in order to be effective, so oral bioavailability (OB) is one of the most important pharmacokinetic parameters in ADME. High OB is usually an important index for determining drug similarity (DL) of active substances  network. The network was visualized with Cytoscape (3.8.0) and CytoHubba, a plug-in in Cytoscape, to filter the modules from the PPI network and to obtain the most important hub genes based on the degree score.Potential targets of CCHM on METH addiction were analyzed by online STRING 11.09 was used to conduct the GO analysis and KEGG enrichment of hub targets. Top terms of GO analysis and KEGG enrichment with thresholds of p-values ≤ 0.05 were chosen in functional annotation clustering.To further characterize the molecular mechanism of CCHM on METH addiction, the DAVID websiteLiterature retrieval was used to excavate and analyze the active components and possible mechanisms of CCHM in the treatment of METH addiction. This may provide literature verification for prediction results of the network pharmacology.Mice were sacrificed by cervical dislocation under anesthesia after behavioral testing. Protein extracts in nucleus accumbens and prefrontal cortex were isolated, lysed, and the supernatant was collected after centrifugation. BCA Protein Assay Kit was used to detect the concentration of protein extracts. Protein samples were separated and electroblotted onto the PVDF membrane. After blocking, membranes were incubated with primary antibodies followed by a horseradish peroxide conjugated secondary antibody. Enhanced chemiluminescence was used to visualize the band, and the intensity of each sample was determined quantitatively using Image J. Actin was used as loading control, and the results were normalized to the control group.10 Furthermore, AutoDock v.4.2.6 was used to add the hydrogens, calculate charges, and merge the non-polar hydrogens of protein structure (11 The SDF format of main active compounds was converted to PDB format using OpenBabel 2.4.1 (The amino acid sequence of the two hub targets D(2) dopamine receptor (DRD2) and 5-hydroxytryptamine receptor 3A (HTR3A) were downloaded from the PDB protein structure database.tructure . Next, tel 2.4.1 . AutoDocpost-hoc test. Differences between groups were considered to be statistically significant if values of p < 0.05.Statistical analysis was processed with SPSS 23.0 and Prism 8 software. Data were shown as the mean ± SE and analyzed using one-way ANOVA and LSD p < 0.01). As shown in p < 0.05), and CPP scores decreased with the increase of CCHM dose. Taken together, the results suggested that 2 mg/kg of METH was sufficient to induce CPP behaviors, and the co-administration of CCHM attenuated the acquisition of METH-induced CPP in mice.Conditioned place preference, a classic model widely used to assess drug-seeking behaviors, was used in this study to assess the preference for visual and tactile cues associated with METH after being administered with varying doses of CCHM. As shown in C. yanhusuo, six to P. cocos, 16 to C. pilosula, 69 to Licorice, four to A. macrocephala Koidz, 12 to A. membranaceus, two to A. sinensis, and six to American ginseng. After eliminating the overlaps, 151 compounds were chosen as candidate bioactive compounds for further analyses, and the detailed information was shown in A total of 163 compounds in CCHM obtained from TCMSP database met the requirement of OB ≥ 30%, DL index ≥ 0.18 and BBB ≥ −0.3, and 48 of which belong to Sixty METH addiction-related targets were collected from OMIM, 356 targets were collected from the Genecards database, and 11 targets were obtained from Drugbank database. After eliminating the overlaps, 392 targets were obtained for further analyses. The detailed information was shown in O-Methylisomucronulatol, 7-Methoxy-2-Methylisoflavone, Formononetin, Beta- Sitosterol, S-Scoulerine, Isocorypalmine, Leonticine, Medicarpin, Licochalcone, and Hyndarin. The detailed information was shown in Cytoscape software was used to construct the of “herb-compound-target” network, as shown in via the functional annotation tool of DAVID Bioinformatics Resources 6.8. There were 116 terms of biological process (BP), 20 terms of cellular component (CC), and 31 terms of molecular function (MF) in total, which met the requirements of Count ≥ 2 and p-value ≤ 0.05. The detailed information was shown in via adrenergic receptor signaling pathway, dopamine neurotransmitter receptor activity, dopamine binding to exert its therapeutic effects on METH addiction. To explore the underlying involved pathways of CCHM on METH addiction, KEGG pathway analysis of involved targets was conducted. The detailed pathway information of CCHM on METH addiction was shown in To identify the biological characteristics of putative targets of CCHM on METH addiction in detail, the GO and KEGG enrichment of involved targets were conducted p-value of PPI enrichment was less than 1.0e-16. To obtain the hub genes in the PPI network, these node pairs were entered into the Cytoscape software. The scores of nodes were calculated by cytoHubba, and the top 10 hub genes were shown in The PPI network of possible targets involved in the treatment of CCHM on METH addiction was constructed using STRING . There wThe hub gene symbols, full names, and functions were shown in via DAVID 6.8. There were respectively 51 BP, 5 CC, and 10 MF terms in total, which met the requirements of Count ≥ 2 and p-value ≤ 0.05. The detailed information was shown in p-values ≤ 0.05. The detailed pathway information of hub genes was shown in via dopaminergic synapse, cocaine addiction, neuroactive ligand-receptor interaction, amphetamine addiction, and serotonergic synapse.The GO analyses and KEGG enrichment of hub genes were conducted L-tetrahydropalmatine, had significant inhibitory effects on locomotor sensitization, CPP behavior, self-administration behavior, and relapse behavior induced by METH . Numerous studies have identified the MLDS as a common reward circuit and main neuroanatomical basis for the rewarding or positive reinforcement of addictive drugs, involving in drug-induced reinforcement, pathological memory, craving, and relapse . Addictiehaviors . Michaelehaviors , and redehaviors . These sehaviors ,c. Afterehaviors . Dopaminehaviors . DRD2 anehaviors . METH abehaviors . Animal ehaviors , suggestI5–HT) in oocytes expressing HTR3A receptor, while high dose METH inhibited I5–HT, and HTR3A receptor antagonist MDL72222 had an inhibitory effect on the acquisition and expression of METH locomoter sensitization  can be found in the article/The animal study was reviewed and approved by the Ethics Committee of Jianghan University.QR, LC, and YW designed the study, analyzed the data, and prepared the manuscript together. QR, XT, CL, and QX performed the experiments. All authors have read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.