Study On The Efficacy And Mechanism Of Jianpihuoxue Decoction In Treating Atrophic Gastritis With Precancerous Lesions Based On TLR4 Pathway

Objective:To observe the clinical effect of Jianpihuoxue decoction on atrophic gastritis with precancerous lesions and to explore its therapeutic mechanism.A malignant transformed gastric epithelial cell model was established to observe the effect of drug-containing serum of Jianpihuoxue decoction on it and to explore the mechanism.To explore a new method of treating atrophic gastritis with precancerous lesions by Traditional Chinese Medicine,and to provide theoretical support for clinical drug use of this disease.Methods:1.Search the Chinese medicine treatment of atrophic gastritis with precancerous lesions clinical literature among Chinese Knowledge Network Database(CNKI),Wanfang Journal Database(WAN FANG),Chinese Biomedical Literature Service System(Sino Med),Weipu Chinese Science and Technology Journal Database(VIP),Cochrane Library,Pubmed Database,Embase Database during recent 20 years and screening these literatures,running the TCM Inheritance Assistance System(V2.5),Input prescription treatment data,Establish a database.Using "statistical report "," data statistics" and other modules,Using statistical methods such as association rules,entropy clustering for complex systems.Analysis the prescription among precancerous lesions,separately intestinal metaplasia,atypical hyperplasia,indicated dysplasia,the contents include drug four-qi five-flavor meridian statistics,frequency statistics,treatment statistics,prescription analysis,new prescription analysis.2.Search the Chinese and English databases for nearly 20 years of clinical researches on the treatment of atrophic gastritis with precancerous lesions based on the method of invigorating the spleen and activating blood,and screen these literatures.Use the Cochrane System Evaluation Manual to evaluate the quality of each literature.Utilize the Revman5.3 to carry out the meta-analysis of total effective rate,gastroscopy improvement rate,pathological improvement rate,the Hp eradication efficiency,and observe the course of treatment side effects.3.Using GEO databases(https://www.ncbi.nlm.nih.gov/geo/),Searching for the correlation between GLGD and CG,Extracting gene data sets,using PERL software,utilizing the "pheatmap"?biconductor "limma" package" in R language,extracting differential genes,then making the heatmap and volcano plot,Using the "Venn" package in R software,extracting the intersection genes of the gene data sets and draw the Venn map;Using the "colorspace "?"stringi "?" ggplot2" packages,the bioconductor "DOSE"?"cluster Profiler"?"enrichplot" packages in R software,obtaining GO enrichment analysis and KEGG enrichment analysis of the selected differential genes;Using the string databases(https://www.string-db.org/),Building a network of protein protein interaction network(PPI)for differential genes,and screening out the core genes.4.Clinical research.62 patients were randomly divided into observation group and control group using the table of random number,the observation group was given the Jianpihuoxue decoction(morodan and panax notoginseng powder),the control group was given folic acid tablets,both for 24 weeks.then observe and compare the clinical efficacy,TCM symptom scores,gastroscopy scores and pathological scores before and after treatment,and at the same time,do the clinical safety surveillance.Using the immuno fluorescence assay to detect the expression of and toll-like receptor 4(TLR4),P53,E-cadherin,vimentin in gastric mucosa before and after the treatment.5.In vitro experiment.Using 1-Methyl-3-nitro-1-nitrosoguanidine(MNNG)to induce malignant transformation in human gastric epithelial cells(GES-1)to construct a malignantly transformed gastric epithelial cells(T-GES-1)model.Taking drug containing serum of Jianpihuoxue decoction and folic acid tablets as interventions,GES-1 cells were divided into six groups: A group: GES-1 cells+serum of normal rats;B group: GES-1cells+MNNG+serum of normal rats;C group: GES-1 cells+MNNG+drug-containing serum of folic acid tablets;D group:GES-1cells+MMNG+drug-containing serum of Jianpihuoxue decoction;E group:GES-1 cells+MNNG+drug-containing serum of Jianpihuoxue decoction+TLR4 agonist;F group: GES-1 cells+MNNG+serum of normal rats+TLR4 agonist.Observe cell morphology of each group,and use CCK-8method to detect the cell viability after intervention of 24 hours,48 hours,and72 hours.use cell scratch test to detect the migration rate of cells in each group;use flow cytometry test to detect the apoptotic rate of cells in each group;use the polymerase chain reaction(PCR)technology to detect the expression of Vimentin,N-cadherin,E-cadherin m RNA;use the western blot technology to detect the pretein expression of TLR4,My D88,NF-?B,p-NF-?B;use immunofluorescence method to detect the expression of P53,Ki67,Muc2.at last make statistical analysis of these results.Results:1.A total of 232 articles were screened,Including 252 prescriptions,233 drugs.The drug quality is generally flat,the drug is mostly related to the spleen,stomach,liver.nourishing spleen and disinhibiting dampness is the most frequent method.And then the method of removing turbid and detoxifying,nourishing stomach yin,soothing liver and stomach,different pathological types have their own emphasis.the most frequently PLGC drugs are licorice,atractylodes macrocephala,curcuma,astragalus,radix astragali,radix glycyrrhizae,etc.The commonly used treatment groups are“Atractylodes macrocephala,Glycyrrhiza”,“Astragalus,Glycyrrhiza”,“Astragalus,Atractylodes macrocephae”,“Curcuma,Astragalus”,“Glycyrrhizae,Radix Paeoniae Alba”,“Salvia miltiorrhizae,Glycyrrhiza” etc.Extract 16 core durg groups,8 new prescriptions for PLGC treatment.extract 8 core drug groups,4 new prescriptions for intestinal metaplasia,atypical hyperplasia,dysplasia treatment respectively.and form a new square network map.2.Finally,39 RCT articles were included,with a total of 3786 patients,including 1994 in the spleen-invigorating and blood-activating treatment group and 1792 in the control group.The literature quality evaluation showed that patients were divided randomly by the random number table method in 21 articles.patients were divided randomly in the other 18 articles,but the specific random method was not described.2 of 30 articles were double-blind and double-simulated,and 1 was single-blind with simulated drugs.In 6articles,the number of cases in the description of the outcome observation index was inconsistent with the original number of cases,and the reason for the loss to follow-up was not explained;meta-analysis showed that the clinical effective rate in spleen-invigorating and blood-activating treatment group was better than that of the control group [RR=1.25,95%CI(1.21,1.30)];the improvement rate of Gastric endoscopy was better than that of control group[RR=1.39,95%CI(1.28,1.52)];the improvement rate of Gastric mucosal pathology was better than that of control group[RR=1.48,95%CI(1.38,1.60)];TCM symptom relief rate is higher than that in the control group [RR=1.25,95%CI(1.13,1.38)];Hp eradication efficiency rate is better than that in the control group [RR=1.25,95%CI(1.14,1.37)].Among the 39 articles,14 described the observation of side effects during the study,12 of 39 described observing side effects and 2 of them had side effects,including 6 cases of constipation(control group 2/60,observation group 4 /60),1 case of abdominal pain(control group 1/60),3 cases of dry mouth(control group 1/60,observation group 2/60),1 case of diarrhea(control group 1/90),1 case of headache(control group)1/90),1 case of skin rash(control group 1/90).3.Selecting GSE55696 ? GS87666 E data set as the research objects,finally screening out 121 differential genes,Of which 42 were upregulated,79 were downregulated in GLGD.The differential genes were mainly distributed in cell granule cavity,cell vesicles and high density lipoprotein granules;The biological processes involved in these genes include hormone metabolism,leukocyte chemotaxis,c GMP signaling pathways;The molecular functions of these genes include potassium channel operation,redox and signaling pattern recognition receptor activity.the pathways involved include interleukin17(IL17)pathway,hypoxia-inducible factor-1(HIF-1)pathway,arachidonic acid metabolism pathway,and gonadotropin-releasing hormone(Gn RH)secretion pathway.4.Clinical study results show that,by analysis of Chi-Square test,the clinical efficacy rate of observation group is higher than that of control group(Chi-square value=6.613,P<0.05).after treatment,the total TCM symptom scores are significantly declined in observation group(P<0.01),and lower than that in control group(P<0.01),of which the cardinal symptoms,including distended abdomen,fullness,hiccough,anorexia scores declined(P< 0.01),and lower than that in control group(P<0.05),of which the secondary symptoms scores,including fatigue,poor sleep,noise,acid regurgitation scores declined(P < 0.01),and lower than that in control group(P<0.05).after treatment,the gastroscope scores are significantly declined in observation group,including mucosa color scores,vascular permeability scores,mucosal eminence scores,mucosa erythema scores and the total gastroscope scores(P<0.05),of which,mucosa color scores and mucosa erythema scores are lower than that in control group(P <0.01).the pathological scores are significantly declined in observation group,including intestinal metaplasia scores,dysplasia scores(P <0.01),of which,dysplasia scores are lower than that in control group(P<0.01).For safety surveillance,morodan and panax notoginseng powder treatment is safe for PLGC patients.After treatment,the expression of TLR4?P53?Vimentin on gastric mucosa in observation group decreased than before(P<0.01),and lower than that in control group(P<0.01),the expression of E-cadherin on gastric mucosa in observation group increased(P<0.01),and higher than that in control group(P<0.01).5.In vitro study results showed that,normal GES-1 grows adherently,the cells are spindle-shaped and have regular morphology.After MNNG treatment,the cell morphology is gradually irregular and presents a polygonal shape.CCK-8 test showed that MNNG has a certain cytotoxicity to GES-1 cells in a concentration-dependent manner.Time has little effect on it.When the concentration of MNNG is 40?mol/L,the activity of GES-1 cells decrease sharply,and when the concentration is 20?mol/L,The inhibition rate was about 30%,so we choose MNNG 20?mol/L,24 h as the method of modeling,the effect of drug-containing serum on cell activity was concentration-dependent,and there was no obvious time-dependent,15%,24 h intervention was selected.After MNNG treatment,the migration rate of GES-1 cells increased,the apoptosis rate decreased,the EMT condition aggravated(P<0.05),the expression of P53,Ki67,and Muc2 increased(P<0.05),the protein expression of TLR4,My D88,NF-?B,p-NF-?B increased(P<0.05);after administration of the drug-containing serum of Jianpihuoxue decoction,the above situation was alleviated(P<0.05),and it was better than the drug-containing serum of folic acid tablets group(P<0.05);in MNNG induced T-GES-1 cells,add TLR4 agonist,the above situation aggravated(P<0.05),when we additionally add drug-containing serum of Jianpihuoxue decoction at the same time the above situation was better than the former(P<0.05).Conclusion:1.Jianpihuoxue decoction is effective in treating atrophic gastritis with precancerous lesions of spleen deficiency and blood stasis type,and has good safety,which is worthy of clinical promotion.2.Jianpihuoxue decoction can improve the expression of P53 and TLR4 in gastric mucosa of patients with atrophic gastritis with precancerous lesions,and can alleviate EMT,which may be the mechanism of its therapeutic effect.3.The drug-containing serum of Jianpihuoxue decoction can inhibit the malignant characteristics of malignantly transformed gastric epithelial cells,which is better than the drug-containing serum of folic acid tablets.Its potential mechanism is to regulate TLR4/My D88/NF-?B signal path and so as to inhibit EMT to realize its effect.