| Background:Cholecystitis is a common surgical disease in China.In China,with the improvement of people’s living standards,diet and environmental sanitation,the incidence of cholecystitis is also increasing and younger,the prevalence rate is more than 10%,the causes of cholecystitis can be mainly divided into two categories: on the one hand,bacterial invasion,on the other hand,gallbladder duct obstruction caused by stone incarceration.Therefore,cholecystitis can be divided into calculous cholecystitis and non-calculous cholecystitis.In recent years,90% to 95% of patients with calculous cholecystitis are caused by stone obstruction and incarceration.Stone obstruction and incarceration will cause mucosal damage to the compressed site,which in turn will cause repeated stimulation of bile esters and aggravate the inflammatory response.At present,the treatment of calculous cholecystitis is mainly surgery,supplemented by medical treatment.The main surgical methods are open or laparoscopic cholecystectomy,gallstone-preserving lithotomy,etc.Due to the risks of surgery for elderly or comorbidities,the combination of traditional Chinese medicine and Western medicine has become a new direction for the treatment of calculous cholecystitis.Qingrelidan granules are composed of rhubarb,gardenia,citrus husk,woody fragrance and money grass,which have the effect of moisture,yellowing and clearing heat,and are widely used in the treatment of cholecystitis,cholelithiasis and other diseases in clinical practice,with good clinical efficacy,but the mechanism of action of clear heat choleretic granules in the treatment of calculous cholecystitis is unclear.This study aims to analyze the mechanism of action of clear heat choleretic granules in the treatment of calculous cholecystitis by network pharmacology and metabolomics,and provide a reference for clinical use.Aim:1.Discover the target of clear heat choleretic granules in the treatment of calculous cholecystitis.2.To explore the mechanism of action of clear heat choleretic granules in the treatment of calculous cholecystitis.3.Potential mechanisms of action were validated using animal experiments and clinical studies.Methods:1.Use the Batman website database to screen the active ingredients of Qingmei choleretic granules,integrate the traditional Chinese medicine system database and TCMSP online data platform to predict the targets of the active ingredients of traditional Chinese medicine,and then combine the Sring database and Gene Cards database to obtain the relevant targets of calculous cholecystitis,and then use Cytoscape software to obtain the candidate therapeutic targets of Qinghei choleretic granules for calculous cholecystitis.The core therapeutic targets of choleretic granules on calculous cholecystitis were screened by Venn diagram and protein interaction analysis,and finally KEGG pathway enrichment analysis was used to predict the therapeutic pathways regulated by the core targets.2.Model of calculous cholecystitis mice by high-fat diet feeding.Twenty-one C57 BL mice were randomly divided into three groups(n=7): normal group(normal diet),calculous cholecystitis group(high-fat diet feeding for 8 weeks + pure water gavage),and Qingrelidan granules group(after model construction,treatment with clear heat choleretic choleretic granules).After the modeling was completed,the success of the modeling was verified by observing whether there were gallstones and white flocs in the gallbladder and detecting the inflammatory indexes in the serum amylase of mice.Finally,the animal serum was detected by targeted metabolomics to verify the therapeutic pathway predicted by network pharmacology.3.Clinical studies were self-controlled clinical trials.A total of 81 clinical samples were collected,which were divided into four groups: healthy control group(n=27),calculous cholecystitis group(n=27),Qingre Lidan Granule group(n=11),and operation group(n=17).Patients in the Qingre Lidan Granule group were all treated with surgery and the combination treatment of Qingre Lidan Granule.The patients in the operation group only received operation treatment.This study was a self-control experiment.The patient samples in the Qingre Lidan Granule group and the operation group were all from the calculous cholecystitis group.Human blood samples from all subjects were centrifuged(3000g,10min)after 1h of rest for serum.Clinical indicators including renal function,blood lipid,liver function,blood routine tests,postoperative dietary recovery time,postoperative pain relief time,and postoperative exhaust time were determined in the Clinical Laboratory of The First Affiliated Hospital of Dalian Medical University.According to these indicators,the clinical effects of the Qingre Lidan Granule group and the operation group were evaluated.After the evaluation,targeted metabolomics was measured in the patient’s serum to further verify the mechanism of Qingre Lidan Granules.Results:1.40 related targets of Qingrelidan granules for the treatment of calculous cholecystitis were obtained through network pharmacological analysis,and two signaling pathways related to bile acid secretion were screened out by combination with PPI analysis and KEGG pathway enrichment analysis.2.The results of biochemical index measurement showed that the inflammatory factors TNF-α and IL-4 in the calculous cholecystitis mice were significantly higher than those in the normal group,and the Qingrelidan granules group was significantly lower than that in the calculous cholecystitis group.The above pathological changes were significantly improved by clear heat and Qingrelidan granules,and the difference was statistically significant(p<0.05).The results of targeted metabolomics showed that the concentrations of total bile acids and secondary bile acids in mice with calculous cholecystitis were higher than those in the normal group,while the Qingrelidan granules group was significantly lower than that in the calculous cholecystitis group,and the difference was statistically significant(p<0.05).The concentration of taurine-bound bile acids in the calculous cholecystitis group was significantly higher than that in the normal group,and the concentration of taurine-bound bile acids in the Qingrelidan granules group was significantly lower than that in the calculous cholecystitis group,and the difference was statistically significant(p<0.05).In animal experiments,TCA(taurocholic acid),DHy LCA(dehydrosylcholic acid),and Iso-LCA(isolithocholic acid)were significantly higher than that in the normal group,and the Qingrelidan granules group was significantly lower than that in the calculous cholecystitis group,and the difference was statistically significant(p<0.05)3.In this clinical study,there were no significant differences in the general clinical data and preoperative liver function between the postoperative group and the Qingrelidan granules group(p<0.05),and the diet recovery time,pain relief time and postoperative exhaust time of the postoperative choleretic granule group were lower than those in the surgical group(p<0.05).Compared with the healthy group,the proportion of primary bile acids decreased in the calculous cholecystitis group and the proportion of taurine-bound bile acids increased.Compared with the calculous cholecystitis group,the proportion of primary bile acids gradually increased,and the proportion of taurine-bound bile acids decreased(p<0.05).Patients with calculous cholecystitis had decreased activity of enzymatic reactions associated with taurine-bound bile acids in the intestine compared with healthy groups.The enzymatic reaction of taurine-bound bile acids in the intestine was more active in the integrative treatment group than in the calculous cholecystitis group,and there was a statistically significant difference in p<0.05.Compared with the disease group,TCA(taurocholic acid)and TCDCA(taurodeoxycholic acid)had significant differences after clear heat and surgical treatment.and statistically significant(p<0.05).Conclusion:1.Treatment of calculous cholecystitis with clear choleretic granules may be through associated bile acid pathways.2.Animal experiments have shown that the improvement of calculous cholecystitis by clearing heat choleretic granules is indeed through the bile acid pathway,possibly by inhibiting the production of taurine-bound bile acids in secondary bile acids,and inhibiting TGR5 signaling by inhibiting the concentration of TCA.3.Clinically targeted metabolomics has found that the treatment of calculous cholecystitis with clear choleretic granules may be related to the decrease in the concentration of taurine-bound bile acids,especially TCA. |
