| Objective: To observe the clinical efficacy and safety of Modified Chai Shao Liu Jun Zi Tang(MCLD)in the treatment of chronic atrophic gastritis(CAG),to predict the potential mechanism of MCLD in the treatment of CAG with the help of network pharmacology,and to carry out in vitro experiments to verify the regulatory mechanism at the cellular and molecular level,in order to provide theoretical and experimental support for the clinical treatment of CAG in MCLD.Methods:1.The study included 69 patients with CAG,who were randomly divided into two group,with 28 individuals in each group.The observation group received treated with TCM MCLD granules,while the control group received conventional symptomatic treatment with Western medicine,both for a duration of 24 weeks.After the course of treatment,the clinical symptom score(single symptom score,total score)and pathological score of the two groups of patients before and after treatment were statistically analyzed.Safety assessments were also conducted.2.The active effective ingredients,targets and standardized treatment of MCLD were used in TCMSP,Pub Chem and Uniprot databases.The targets of CAG and PLGC were obtained through Gene Card,OMIM,DrugBank,DisGeNet and SoFDA databases,and Wayne maps were drawn to obtain intersecting targets.STRING database and Cytoscape3.9.1 used software to build a network to screen the core targets and analyze the main target active ingredients,and GO enrichment analysis and KEGG pathway enrichment analysis were analyzed by Metascape platform.Finally,the potential mechanism of MCLD intervention in the treatment of CAG was predicted.3.CCK-8 was used to detect the effects of different concentrations of MNNG on the activity of EGS-1 cells.The mobility,clone formation ability andapoptosis rate of cells were detected by scratch healing assay,plate clone formation assay and flow apoptosis assay.RT-q PCR and Western blot were used to detect the EMT-specific marker Vimentin,the expression levels of E-cadherin protein and mRNA in order to determine the successful construction of the malignant transformation model.The protein expression levels of JAK2,p-JAK2,STAT3,p-STAT3,Vimentin and E-cadherin were detected by Western blot,and the inflammatory factors TNF-a and IL-6 were detected by ELISA to clarify the effect of MCLD on JAK2/STAT3 pathway and EMT process.Results:1 Clinical research(1)The clinical research results showed that the observation group had a significant decrease in TCM symptom scores after treatment compared to before treatment,and was better than the control group in improving stomach pain,bloating,bilateral stuffiness,irritability,and depression(P<0.05).The TCM symptom scores in the control group also showed a significant decrease compared to before treatment(P<0.05),and there was no significant difference between the two groups(P>0.05).(2)In terms of histopathology,the observation group had a better effect on the improvement of gastric mucosal pathological atrophy and chronic inflammation scores after treatment than the control group(P<0.05),while there was no significant difference in the pathological intestinal metaplasia,dysplasia and activity score of gastric mucosa between the two groups(P>0.05).(3)The detection results of serum indexes showed that PGI,G-17 levels were increased and PGII levels were decreased in both the observation group and the control group,and the observation group was superior to the control group,the difference was statistically significant(P<0.05).2 Network pharmacologyThe network pharmacology prediction results show that there are a total of 132 effective active ingredients in MCLD,821 action targets,615 CAG disease targets,1715 targets for PLGC diseases,762 IM disease targets,461 test targets,461 candidate targets,and 18 targets for traditional Chinese medicine ingredients,TCM symptoms,CAG and Pl GC.The top 10 targets screened by the DEGREE value through Cytoscape3.9.1 software plug-in are IL-6,TP53,EGFR,STAT1,CXCL8,MYC,CDKN2 A,IL-10,TGFB1,CDKN1 A.Based on the "component target pathway" network,it is speculated that the main effective ingredients of MCLD intervention in CAG include quercetin,kaempferol,isorhamnosum,and luteolin.Through GO annotation and KEGG signaling pathway enrichment analysis,it was found that the mechanism of action of MCLD in treating CAG may be related to regulating the JAK/STAT signaling pathway,PI3K/AKT signaling pathway,and 178 signal channels such as Fox O and HIF-1.Ultimately,it is speculated that MCLD may intervene in CAG through the JAK2/STAT3 pathway.3 In vitro experiment(1)The experimental research results showed that normal GES-1 adherent growth and regular morphology after MNNG;it suggested that MNNG is cytotoxic to GES-1 cells,and cell viability decreased with increasing concentration,and time had no effect on cell activity.When the MNNG concentration is 40 μmol/L,the cell vitality is inhibited at about30%,which is the most suitable modulus concentration,therefore,choosing 40 μmol/L induce malignant transformation of GES-1 cells for 24 hours.The MCLD concentration of600μg/mL and below has no effect on MC cell viability while concentration at 600μg/mL or above has an inhibitory effect on the viability of MC cells,therefore 100μg/mL and600μg/mL were selected as the low-dose and high-dose groups for subsequent experiments.(2)After MCLD treatment,the mobility and proliferation ability of GES-1 cells,Vimentin protein and mRNA increased(P<0.05;P<0.001),E-cadherin protein and messenger RNA decreased(P<0.001;P<0.001),p-JAK2 and p-STAT3 activated(P<0.05),inflammation factor TNF-a and IL-6 increased(P<0.01).(3)After MCLD treatment the mobility reduced(P<0.01)and apoptotic increased rate of cell were(P<0.001),the Vimentin protein and the mRNA decrease(P<0.05),E-Cadherin protein and mRNA elevation(P<0.05),Lower activation levels of p-JAK 2 and p-STAT 3(P<0.001),and the MCLD low dose group compared with the inhibitor group,it also significantly reduced the expression level of the protein above(P<0.05),decreased the inflammatory factor TNF-a and IL-6(P<0.05).Conclusion:1.Clinical researches have shown that MCLD can reverse the atrophy and intestinal metaplasia of gastric mucosa in some patients with CAG,and effectively improve the symptoms of traditional Chinese medicine.It has good clinical efficacy and safety,and is worthy of,have significant clinical effects,good safety,and is worthy of clinical promotion.2.The prediction of network pharmacology results suggest that the potential mechanism of action of MCLD in treating CAG may be related to the JAK2/STAT3 pathways.3.MCLD can inhibit the malignant transformed of gastric epithelial cells,and its mechanism may be to inhibit the EMT process by inhibiting the JAK2/STAT3 signaling pathways. |
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