| Purpose: In order to obtain the ideal prescription for the treatment of obstructive cholestasis liver injury by data mining of the medical records of traditional Chinese medicine.Through network pharmacology research,in order to clarify the mechanism of action and the gene target and signal pathway of drug action on this disease.For the purpose to clarify the pathogenesis of obstructive cholestasis liver injury,and look for the core pathway and key target of drug pathogenesis.we combined with animal experiments.Through clinical experience and the professor’s experience in treating the disease,we added and subtracted the classic prescription.In order to clarify its efficacy,judge its safety,and verify its impact on the presumed pathway and target,We used a randomized controlled clinical trial method to carry out clinical observation,clarify its efficacy.Provide both theoretical and clinical foundation for popularize the application of using addition and subtraction of ‘Dachaihu Decoction’ to cure obstructive cholestasis liver injury in order to promote the rapid recovery of liver function after operation and reduce the pain of patients treated with cholecystectomy.Methods:This study consists of four parts: data mining of traditional Chinese medicine,network pharmacology,animal experiment and clinical observation.1.In the first part,the ancient and modern medical record cloud platform is used to conduct data mining on the treatment of obstructive cholestasis liver injury by traditional Chinese medicine,explore the medication rules of obstructive cholestasis liver injury,search the effective medical records of Chinese medicine in the treatment of obstructive cholestasis liver injury in CNKI,WIP,Wanfang and Chinese biomedicine,screen according to the standards,and establish a standardized medical record database.Through frequency,clustering,correlation analysis and other methods of the ancient and modern medical record cloud platform,the network is constructed and visualized,and the medication rules are analyzed.According to the analysis results,the core drug composition for the treatment of this disease is obtained,and the classic prescription for the treatment of obstructive cholestasis liver injury is deduced.2.In the second part,the mechanism of the ideal prescription in the treatment of cholestasis liver injury is clarified through network pharmacology,and the active ingredients of the classic prescription for the treatment of this disease are screened through searching the TCMSP database and TCMID database,and the matching rake points are standardized by Uni Prot.Through searching Dis Ge NET and OMIM databases and Venny2.1,we found the corresponding gene target of cholestasis liver injury.We obtain and visualize the PPI interaction map of common genes through STRING and analyze their key genes and core genes.Importing DAVID,we obtained KEGG signal map and core genes,matching their core components,and perform the three visualization operations.And carry on the docking between the drug core component and the core gene protein molecule.3.In the third part,the animal experiment part,according to the results of the first two parts of the study,taking SD rats as the test object,according to the principle of random grouping,they were randomly divided into seven groups,including blank control group,sham operation group,model control group,high-dose treatment group,medium-dose treatment group,low-dose treatment group,and positive control group.Among them,rats in the treatment group,positive control group and model control group were made models by obstructing the common bile duct of rats through surgical catheterization.The modeling steps in the sham operation group were the same as before,but the common bile duct was not obstructed.The third day after the operation,blood was collected from caudal tip to confirm the success of modeling.From the first day of successful modeling,each group was given a daily gavage,and the equivalent dose of corresponding drug was converted according to the ratio of body surface area of human and rat,and the drug was administered continuously for 7 days.All treatment groups were given traditional Chinese medicine granules by gavage,the blank control group,the sham operation group and the model control group were given equal volume of normal saline by gavage,and the positive control group were given Simetail normal saline suspension by gavage.Seven days after administration,blood was collected from abdominal aorta of rats in each group to prepare serum samples;Rat liver was taken and liver tissue samples were prepared.The contents of serum ALT,AST,ALP,GGT,TBIL and DBIL were determined by automatic biochemical analyzer,and serum TNFα,IL-6 and IL-1β were detected by Elisa detection method.The content of NLRP3 in liver tissue and the content of NLRP3 in liver tissue were determined by Elisa detection method.NF-κB-P65 protein expression and m RNA level in liver tissue were determined by Elisa detection method by Western blot and PCR,all data were statistically analyzed and experimental results were obtained.4.In the fourth part,through clinical experience and the professor’s experience in treating the disease,we added and subtracted the classic prescription.The randomized controlled clinical observation was used to determine its efficacy,judge its safety and verify its impact on the presumed pathway and target.The patients with obstructive cholestasis liver injury were taken as the study object,and 60 patients who met the criteria were included in strict accordance with the inclusion criteria.The patients were randomly divided into the observation group and the control group with 30 cases each.Both groups were treated with laparoscopic cholecystectomy+common bile duct incision and exploration+choledochoscopy+T-tube drainage to ensure the smooth drainage of the biliary tract.The control group was treated with routine liver protection,anti-infection and water and electrolyte balance after operation.On the basis of the basic treatment of the control group,the observation group began to take the classic Dachaihu decoction plus and minus granules 200 mL warm water twice a day(once in the morning and evening)from the first day after operation,Take it continuously for 7 days as a course of treatment.In the third and seventh day after operation after operation,liver function indexes were rechecked:ALT,AST,GGT,ALP,bilirubin indexes: DBIL,TBIL,and serum inflammatory factor indexes detected by enzyme-linked immunosorbent assay: IL-6,IL-1β,TNFα and the level of serum NLRP3 for statistical analysis,and improve the change of TCM syndrome score,judge its curative effect,and determine the occurrence of adverse reactions.Results:1.Data mining of TCM:The frequency statistics of 136 traditional Chinese medicine records for the treatment of obstructive cholestasis liver injury were carried out and it was concluded that the core drug was Dachaihu decoction plus or minus,with Banxia,Dahuang,Yinchen,Shengjiang,Chaihu,Zhishi,Huangqin,Baishao,Dazao,and pungent drugs returning to the spleen and stomach meridians.Cluster analysis suggested that the higher drug pairs were " Dahuang-Banxia"," Banxia-Shengjiang" and " Chaihu-Banxia".2.Network Pharmacology:The network pharmacology of Dachaihu Decoction,the core drug obtained from data mining,was studied and 151 active components of Dachaihu Decoction were retrieved,with 274 corresponding drug genes and 694 gene targets for cholestasis liver injury.The common targets of the effective components of Dachaihu Decoction and the target genes of cholestasis liver injury are 109.The key targets of its drug action are TNF,AKT1,IL6,IL-1β,MAPK3,MAPK14,TP53,APP,FN1,CASP8 according to the PPI protein interaction network.The top three key target KEGG signal pathways were Toll receptor signal pathway,TNF signal pathway and P13K-AKT signal pathway.NF-κB signal pathway is the common downstream pathway of the three,and the core genes are AKT and TNFα,IL6 and IL-1β.According to the core gene,the core effective components are identified as wogonin,quercetin and luteolin.And in molecular docking,it was confirmed that the chemical composition of the core was stable to the protein of the core gene.3.Animal experiment: SD rats were used as the experimental objects.The intervention was carried out with the classic prescription Dachaihu Decoction.Compared with blank control group,there was no significant change in serum ALP,GGT,ALT,AST,TBIL,DBIL,IL-6,IL-1β,TNFα and NLRP3 in liver tissue of sham operation group(P>0.05).Compared with the sham operation group,the serum ALP,GGT,ALT,AST,TBIL,DBIL,IL-6,IL-1β and TNFα of the model control group were significantly higher(P<0.01).In liver tissue,the content of NLRP3 was significantly increased(P<0.05).Western blot of liver tissue showed that the protein level of NF-κB-p65 was significantly increased(P<0.05).Real-time PCR results showed that the m RNA level of NF-κB-p65 was up-regulated after modeling(P<0.05).Compared with the model control group,the serum ALP,GGT,ALT,AST,TBIL,DBIL,IL-6,IL-1β,TNFα and NLRP3 in liver tissue of the middle-dose treatment group were significantly decreased(P<0.01).Western blot results of liver tissue showed that the level of NF protein decreased significantly(P<0.05).Real-time PCR results showed that NF-κB-p65 m RNA level decreased(P<0.05).Compared with the low-dose treatment group and the high-dose treatment group,the serum ALP,GGT,AST,TBIL,DBIL,IL-6,IL-1β in the middle-dose treatment group decreased(P<0.01).Meanwhile,serum ALT,TNFα and NLRP3 in the liver tissue decreased(P<0.05).Western blot results showed that NF-κB-p65 was significantly lower(P<0.05).Real-time PCR results showed that NF-κB-p65 m RNA decreased(P<0.05).Compared with the positive control group,the serum ALP,ALT,AST,TBIL,DBIL,IL-1β,TNF-α and NLRP3 in liver tissue in the middle dose treatment group was lower(P<0.01),and the level of serum GGT and TIL-6 was lower(P<0.05).Western blot results showed that NF-κB-p65 significantly decreased(P<0.05).Real-time PCR showed that NF-κB-p65 m RNA decreased(P<0.05).4.Clinical Observation: After the obstruction was removed by surgically,the addition and subtraction of ‘Dachaihu Decoction’ was used to treat patients with obstructive cholestasis liver injury.There was no statistically significant difference in gender,age and onset time between the two groups in the statistical analysis of baseline materials(P>0.05).There was no significant difference in serum ALT,AST,GGT,ALP,TBIL,DBIL,IL-6,IL-1β,TNFα and NLRP3 between the two groups before treatment(P>0.05).Compared with the control group in the third day after treatment,the serum ALT,AST,ALP,GGT,TBIL,DBIL of the observation group decreased significantly,and the difference was statistically significant(P<0.01).In the seventh day after treatment,compared with the control group,AST,GGT,ALP,in the observation group decreased significantly(P<0.01),and ALT,TBIL,DBIL decreased significantly(P<0.05).The recovery rate of ALT,AST,GGT,ALP and total indexes in the observation group was better than that in the control group(P<0.05).The recovery rate of TBIL and DBIL was the same.Compared with the control group in the third day after treatment,the serum IL-6,IL-1β,TNFα of the observation group decreased significantly,and the difference was statistically significant(P<0.01).In the seventh day after treatment,compared with the control group,IL-1β,TNFα in the observation group decreased significantly(P<0.01),and IL-6 decreased significantly(P<0.05).Compared with the control group in the third day after treatment,the NLRP3 of the observation group decreased significantly,and the difference was statistically significant(P<0.01).In the seventh day after treatment,compared with the control group,NLRP3 in the observation group decreased significantly(P<0.05).In the 30 cases in the control group,cured cases were 33.33%,effectual cases were23.33%,effective cases were 10.00%,and ineffective cases were 33.33%.In the 30 cases in the observation group,cured cases were 43.33%,effectual cases were 26.67%,effective cases were 10%,and ineffective cases were 20.00%.The total effectual rate of the control group was 56.67% while the rate of the observation group was 70.00% which could indicate that the total effectual rate of the observation group was higher than the rate of the control group,and the difference was statistically significant(P <0.01).The total effective rate of the control group was 66.67% while the rate of the observation group was 80.00% which could indicate that the total effective rate of the observation group was higher than the rate of the control group,and the difference was statistically significant(P <0.05).Comparing the total effectual and effective rate,the observation group was obviously higher than the control group.Therefore,the overall effect of the observation group was better than the effect of the control group.Conclusion:1.In the treatment of obstructive cholestasis liver injury by traditional Chinese medicine,pinellia ternate,rhubarb,wormwood,ginger,bupleurum Chinense,fructus aurantii,scutellaria baicalensis,white peony,jujube are the main drugs,and the fundamental principles of clearing heat,removing dampness,promoting the gallbladder and detoxicating are the basic principles,revealing that the classic prescription Dachaihu decoction is the ideal prescription for the treatment of obstructive cholestasis liver injury.2.Based on network pharmacology,it is speculated that Dachaihu Decoction has effect on FNTα,IL-1β and IL-6 expression through NF-κB signal pathway to treat cholestasis liver injury.3.Animal experiments confirmed that the pathogenesis of obstructive cholestasis liver injury is the activation of NF-κB pathway,releasing inflammatory factors IL-6 and IL-1β、TNFα and increasing the content of NLRP3 body,leading to jaundice and liver injury.Dachaihu Decoction can release jaundice and liver injury and reduce inflammatory factors including IL-6,IL-1β,TNFα and Inhibit NF-κB pathway and reduce NLRP3 body.Because NLRP3 body is a marker product of cell death,its role in regulating cell death is inferred.NLRP3 body is a marker product of cell death,so it can be inferred that it has the function of regulating pyroptosis.4.Applying the addition and subtraction of ‘Dachaihu Decoction’ to treat obstructive cholestasis jaundice of damp-heat type of liver and gallbladder can reduce liver enzymes,serum total bilirubin and direct bilirubin,reduce the release of inflammatory factors in the blood,and reduce the content of NLRP3 body in the blood.Therefore,it has been proved that it has the effect of protecting the liver and anti-inflammation and improving liver cell scorching by reducing the release of NLRP3 body.Based on the above conclusions,it is confirmed that the addition and subtraction of‘Dachaihu Decoction’ can inhibit NF-κB pathway and alleviate the inflammatory reaction of the liver,reduce the content of NLRP3 corpuscles,regulate the apoptosis of liver cells,and achieve the effect of protecting the liver and reducing jaundice.By reducing the level of NLRP3 and regulating the apoptosis of hepatocytes,the effect of protecting liver and reducing jaundice can be achieved,and the rapid recovery of liver function after operation can be promoted.Overall,addition and subtraction of ‘Dachaihu Decoction’ has lasting effect on obstructive cholestasis liver injury.This medicine is safe and reliable.It is worthy to further popularize and apply this medicine clinically when it also provides new thinking of studying using traditional Chinese medicine to cure obstructive cholestasis liver injury. |
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