Study On The Mechanism Of Action Of Danbai Granules In Treating Damp-heat Stasis Type SPID Based On TLR4/p38 MAPK And NF-κB Signaling Pathwa

Objectives1.To objectively evaluate the current status of research on the sequelae of pelvic inflammatory disease(SPID)and explore research hotspots and development trends.2.To investigate the clinical efficacy and safety of Danbai granules in the treatment of damp-heat stasis type of SPID.3.To explore the mechanism of action of Danbai granules in the treatment of SPID through network pharmacology and molecular docking.4.To explore the molecular mechanism of Danbai granules for the treatment of SPID and to validate the predicted results of network pharmacology.Methods1.Literature review:based on the literature data of Peking University core journals,CSCD journals,and CSSCI journals in the three major databases of China Knowledge Network,Wanfang,and Wipu from January 2000 to December 2022,relevant articles with the theme of Chinese medicine for the treatment of SPID were screened,and VOSviewer and CiteSpace software were used to visualize and analyze the literature,so as to analyze the current status of SPID research objectively and explore the future academic frontier and development trend.2.Clinical study:A prospective,multicenter,single-arm,pre-and post-treatmentcontrolled study was conducted.208 patients with SPID who met the inclusion criteria collected from outpatient clinics were treated with Danbai pellets for 2 consecutive menstrual cycles(stopping the medication during menstruation),and the patients’ TCM syndrome scores,local sign scores,visual analog scores(VAS),and DASS-21 scores,vaginal discharge cleanliness,adverse reactions and safety indexes such as liver and kidney function and blood examination before and after treatment were observed and analyzed and judged by statistical methods.3.Network pharmacology analysis:Network pharmacology was used to analyze the mechanism of action of Danbai granules for the treatment of SPID,and the results were initially validated using molecular docking techniques.Firstly,we collected the bioactive compounds and their corresponding targets of Danbai granule formula from the public database and the disease-related targets of SPID,then,we obtained the potential targets of Danbai granule for the treatment of SPID by R language,constructed PPI network with String database,and completed the gene GO and KEGG enrichment analysis by using clusterProfiler package.The potential biological processes and pathways of Danbai pellets for SPID were found,and finally molecular docking was performed with Autoduck software to calculate the binding activity between the first six targets and the first ten compounds.4.Experimental study:SD female rats were used as experimental subjects to prepare a damp-heat stasis type SPID animal model by using phenol plasma injection+mechanical injury+high-fat and high-sugar diet+damp-heat climate.Forty-two rats were randomly divided into normal control group,model group,Danbai granules low,medium and high dose group,and Kangwanyan capsule group.After 14 days of intervention,the rats were morphologically observed,and the uterine tissues were pathologically observed by HE staining.The levels of IL-6,IL-10,IL-4 and TNF-α in the serum of each group of rats were measured by ELISA,and the expression levels of TLR-4/p38 MAPK/NF-κB pathway-related proteins and mRNA in the uterine tissues were detected by Western Blot and RT-PCR,and the above data were statistically analyzed.Results1 Knowledge mapping analysis of Chinese medicine for SPID1.1 Analysis of annual publication volume:the overall publication volume in the past five years is not high,indicating that high-quality academic research still needs to be explored.1.2 Author cooperation network analysis:a research team represented by Wei Shaobin,Zhou Li,Wei Lihui,Gong Yun and Zeng Qian was formed among the core authors.1.3 Keyword co-occurrence analysis:the top 30 keywords such as sequelae of pelvic inflammatory disease,Chinese medicine reserved enema,Chinese medicine therapy,antibiotics,Chinese and Western medicine combination,inflammatory factor,clinical observation,etc.were obtained.1.4 Keyword cluster analysis:The research hotspots of TCM for pelvic inflammatory diseases were roughly divided into 7 categories,mainly in disease,mechanism research,treatment modality,research type,TCM treatment method,efficacy evaluation,etc.1.5 Keyword time-line graph analysis:Research hotspots in the past decade included Tolllike receptor 4,oxidative stress,signaling pathways,uterine microcirculation,randomized controlled trials,VAS scores,and T lymphocyte subpopulations.1.6 Key words emergent analysis:emergent words in recent years include blood rheology,T lymphocyte subpopulation,damp-heat stasis,efficacy,inflammatory factors,Kangwanyan capsule,quality of life,chronic pelvic pain,etc.,which are future research directions and hotspots.2 Clinical observation of Danbai granules on damp-heat stagnation type of SPID2.1 Total effective rate:the total effective rate reached 91.35%,which indicates significant clinical efficacy.2.2 TCM evidence:208 patients’ TCM evidence points after 1 month of treatment and after 2 months of treatment were compared with those before treatment,and the results were significantly different by t-test(P<0.01).2.3 Points of the sub-categories of symptoms:14 sub-categories of symptoms,including lower abdominal pain,lumbosacral pain,excessive amount of discharge,yellow color of discharge,odor of discharge,stuffiness and dullness,low fever,irregular menstruation,abdominal pain during menstruation,abdominal pain after exertion or intercourse,dry stool,sticky stool,tongue texture and tongue coating,were compared with those before treatment,and the differences were statistically significant by t-test(P<0.01);the differences were statistically significant(P<0.01);The differences between the two symptoms of loose stools and yellow urine were not significant before and after treatment(P>0.05).2.4 Local signs:208 patients’ local signs scores after treatment were compared with those before treatment,and the differences were statistically significant by t-test(P<0.01).2.5 VAS scores:the VAS scores of lower abdominal pain and lumbosacral pain in 208 patients after treatment were compared with those before treatment,and the differences were statistically significant by t-test(P<0.01).2.6 DASS-21 scores:the difference between the severity of DASS-21 score after treatment and before treatment in 208 patients was statistically significant by t test(P<0.05).2.7 Cleanliness of vaginal secretions:208 patients showed improvement in the cleanliness of vaginal secretions after treatment,and the difference was statistically significant(P<0.01)compared with that before treatment by t-test.2.8 Adverse reactions:During the medication period,4 patients had dizziness and headache symptoms,and 2 patients had nausea symptoms,which disappeared after adjusting the medication,and there were no patients who were forced to discontinue the medication in the middle of the treatment period.2.9 Recent follow-up recurrence evaluation:In 146 patients with SPID who were cured and apparently effective,after 8 weeks of drug discontinuation,8 patients had vague pain in the abdomen again after staying up late or exerting themselves,and 3 patients had a high amount of hypochondrium again,but the rest did not have any symptoms or signs related to SPID.3 Network pharmacology and molecular docking to explore the mechanism of action of Danbai granules in the treatment of SPID3.1 46 potential compounds and 220 action targets of Danbai granules were collected.3.2 952 SPID disease targets of SPID were obtained after screening and de-duplication.3.3 Common target screening and interactions network construction:106 overlapping targets were obtained by drawing the Venn diagram.3.4 Construction of PPI network and extraction of key targets:the network consists of 106 nodes and 1968 edges,IL-6,IL-1β,VEGFA,CASP3,PTGS2,JUN are the core proteins.3.5 GO and KEGG enrichment analysis:2978 GO entries were obtained,closely related to biological processes such as responses to lipopolysaccharides,molecules of bacterial origin,oxidative stress,chemical stress,and nutrient levels,152 signaling pathways were obtained by KEGG enrichment,involving AGE-RAGE,IL-17,TNF,HIF-1,Toll-like receptor Th17,PI3KAkt and other pathways.3.6 Molecular docking:both core compounds and potential target molecules docking scores were satisfactory.4 Discussion on the effect and mechanism of Danbai granules on rats with pelvic inflammatory sequelae of damp-heat and stagnation type.4.1 Construction of a combined pathological-evidence animal model:a compound method of phenol plasma injection+mechanical injury+high-fat and high-sugar diet+ damp-heat environment was used to prepare a damp-heat stasis type SPID rat model.4.2 General characterization observation of rats:the model group showed significantly damp-heat stasis evidence state compared with the NC group rats.The general condition and damp-heat stagnation symptoms of rats in each treatment group were alleviated.4.3 Verco score of pelvic adhesions in rats:compared with the NC control group,Verco score of rats in the model group was increased(P<0.05);compared with the model group,each treatment group showed different degrees of decrease(all P<0.05),DBG-H was the most obvious.4.4 Histomorphological observation of the rat uterus:the endometrial luminal wall of the NC group had clear structure,no congestion and edema,no necrosis,no inflammatory cell infiltration,occasional mild hyperplasia,and clear muscle fiber texture.In the model group,the endometrial luminal wall structure was altered,with endometrial congestion and edema,massive or laminar inflammatory cell infiltration,fibrous tissue proliferation,epithelial cell degeneration and necrosis,and atrophy and deformation of glands.All treatment groups showed varying degrees of remission.4.5 Serum IL-6,TNF-α,IL-4 and IL-10 levels in rats:compared with the NC group,the expression of IL-6 and TNF-α was significantly higher(P<0.05)and the expression of IL-4 and IL-10 was significantly lower(P<0.05)in the Model group;compared with the Model group,the expression of IL-6 and TNF-α was significantly lower in each treatment group(P<0.05),and IL-4 and IL-10 expression were significantly increased in all treatment groups(P<0.05),but still differed from the NC group(P<0.05).This trend of change was dose-dependent in all groups of Danbai pellets,with the most significant change in the DBG-H group.4.6 TLR4,p38 MAPK and NF-κB p65 mRNAs and proteins expression results:TLR4,p38 MAPK and NF-κB p65 mRNAs and proteins expression were significantly higher in the uterine tissues of rats in the Model group compared with the NC group(P<0.05);compared with the Model group,the above mRNAs and proteins in the uterine tissues of rats in each treatment group Compared with the Model group,the expression of these mRNAs and proteins in the uterine tissues of the treated rats was significantly lower(P<0.05),but still differed from the NC group(P<0.05),and this trend was dose-dependent in all groups of Danbai pellets,with the most obvious change in the DBG-H group.Conclusions1.Research on Chinese medicine for SPID was carried out earlier,but there is a relative lack of high-quality research,and clinical efficacy assessment is diversified,but basic research is still weak and needs further exploration,and immune modulation is one of the research hot spots.2.Danbai granules can significantly improve several clinical symptoms and signs,improve the cleanliness of vaginal secretions,relieve patients’ pain,improve patients’ anxiety,depression and stress emotions in patients with SPID with damp-heat stasis type,and have high safety and objective long-term efficacy.3.Through network pharmacology and molecular docking,it is clear that Danbai granules have multi-component,multi-target and multi-pathway characteristics for the treatment of SPID,and Toll-like receptor signaling pathway is one of the possible mechanisms of action.4.Danbai granules can effectively improve the general status of damp-heat stasis type SPID rats and improve pelvic adhesions,probably through regulating TLR-4/p38 MAPK/NFκB signaling pathway,affecting inflammatory factor expression and maintaining immune homeostasis for therapeutic purposes.