The Characteristic Analysis Of MiRNA Network Imbalance In Bile Duct Cells By Biliary Tract Inflammationin Involved In Intrahepatic Bile Duct Stone Formation And Its Regulation Effect On The Formation Of DHLX

Objective:In recent years,China's economic is developing rapidly,the quality of people's living standards leap,medical technology level has also been greatly improved.The primary intrahepatic bile duct calculus(Hepatolithiasis,HL),which is common benign biliary tract disease in China,with complex causes,long-term recurrent bile duct inflammation is the main pathological process,would lead to biliary cirrhosis and bile duct cell carcinoma.It is of great significance to explore effective treatment and intervention mechanism.The surgical operation still is the main method that the disease treats at present,the postoperative stone recurrence is the biggest difficulty which the present disease treatment faces,especially for younger patients.So that it has to be performed again,or even multiple operations,bringing heavy psychological pressure and financial burden to patients and their families.Therefore,in-depth exploration of the pathogenesis of the disease and the formation of effective treatment means for key targets are the key issues that need to be solved for patients themselves and even for social development.For this reason,on the one hand,this study takes chronic calculous cholecystitis and choledochal secondary stone patients as the research object,through the analysis of the efficacy and safety of postoperative patients,and blood correlation test,and the bile metabonomics analysis and feature detection,to investigate the clinical effect and mechanism of DHLX on patients with chronic calculous cholecystitis and choledochal secondary stone.On the other hand,high-throughput miRNA chip technology,qRT-PCR technology,Northern Blot technology and KEGG enrichment analysis technology were adopted to clarify the characteristics of miRNA network imbalance of bile duct cells induced by biliary tract inflammation and the intervention target of DHLX,so as to provide more profound scientific basis for DHLX to treat primary intrahepatic bile duct stones.Methods:1.Clinical research:The diagnosis of patients with a clear chronic calculous cholecystitis and choledochal secondary stone in 142 cases,according to a randomized and control principle,is divided into DHLX treatment Group 72 cases and Xiaoyan Lidan tablet control group 70 cases.Before and after the treatment,the total scores of TCM syndromes,the B-ultrasound improvement of cholecystitis and gallstones,the blood laboratory test after treatment,and the change of bile endogenous metabolites(GC-MS)were observed,followed by the safety and curative effect of the two groups.2.Experimental research:(Experimental One)To clarify the characteristics of the imbalance of the bile duct cell Mi RNA network and the intervention target of the DHLX based on the NF-?B signaling pathway.From the large cholinergic direct injection of LPS,in 48 h,72h separation of large cholinergic tube cells,using high-throughput Mi RNA chip technology,qRT-PCR Technology,Northern Blot Technology,KEGG Enrichment analysis technology,clear bile duct inflammation induced by biliary cell Mi RNA.The characteristics of network imbalance and the intervention target of tuihuangling capsules.(Experimental Two)Based on LPS stimulation bile duct cells build model,gives DHLX intervention,draw lessons from modern chip technology and biological information analysis technology,miRNA isolation and culture cells of large dept,transfection miRNA expression plasmid or inhibit expression plasmid,qRT-PCR test target mRNA expression,Mi RNA chip technology combines with multivariate bioinformatics analysis methods to explore the multivariate regulatory mechanism of DHRLX through miRNAs for inflammatory bile duct cells.Results:1.Clinical research:(1)Compared with control group,the treatment group was slightly more effective than the control group(p=0.04);(2)The improvement of Syndrome score was significantly higher than that in control group(p<0.001);(3)The improvement of calculus in gallbladder was better than that in control group(p=0.03),and the improvement of bile permeability and wall coarse condition was better than control group(p=0.04,p=0.03);(4)Treatment before and after the control,both groups of patients with blood test indicators have improved,compared with the control group,the treatment group WBC,ALT,AST,TBIL,Dbil,TBA Improvement degree is greater(both p<0.001);(5)Two groups of patients before and after the treatment of bile endogenous metabolites in addition to LDL and other metabolites have a significant change(all p<0.001),and compared with the control group,the treatment group of alanine,citric acid,lactic acid,glycine,cholesterol,glycerin,malic acid,choline,Bezoar,the concentration of metabolites of the change of the difference is statistically significant(all p< 0.05);(6)No significant adverse reactions and special events were seen in both groups of patients with.2.Experimental research:LPS(5ug/ml)+DHLX(1mg/ml)concentration combined bile duct cell proliferation efficiency maintained in 95%.Compared to the blank group,mRNA expressions of Myd88?TRAF6?TAK1?IKKa?and NF-?B were all significantly increased in LPS control group ?LPS + PDTC group?LPS +SB203580 group ? LPS + PTDC + SB203580 group,which were reduced significantly in LPS + Chinese traditional medicine group ?LPS + PDTC+Chinese traditional medicine group ?LPS + SB203580+ Chinese traditional medicine group ?LPS + PTDC + SB203580+ Chinese traditional medicine group.The difference was statistically significant(p<0.05).Compared to LPS control group,mRNA expressions of Myd88?TRAF6?TAK1?IKKa?and NF-?B were a little bit lower in LPS + PDTC group?LPS + SB203580 group?LPS + PTDC +SB203580 group,but no significant difference(p>0.05).In the other hand,mRNA expressions of Myd88?TRAF6?TAK1?IKKa?and NF-?B were all significantly reduced in LPS + Chinese traditional medicine group ?LPS + PDTC+ Chinese traditional medicine group ?LPS + SB203580+ Chinese traditional medicine group ?LPS + PTDC + SB203580+ Chinese traditional medicine group,and the difference was statistically significant(p<0.05).Compared to LPS + Chinese traditional medicine group,mRNA expressions of Myd88?TRAF6?TAK1?IKKa?and NF-?B were a little bit lower in LPS + PDTC+ Chinese traditional medicine group ?LPS + SB203580+ Chinese traditional medicine group ?LPS + PTDC +SB203580+ Chinese traditional medicine group,but no significant difference(p>0.05).DHLX can be lowered in the bile duct cells IL-6 and tnf-?mRNA expression,compared with LPS group is statistically significant(p<0.05).DHLX can regulate the expression of 30 miRNAs in the inflammatory conditions of bile duct,affect the biological processes of signaling transduction of bile duct cells,organic compound reaction and hypoxia stress,regulate cell mass,endoplasmic reticulum and membrane raft,regulate signal transduction activity.The DNA binding and the activity of voltage-gated potassium channel in the transcriptional regulatory region,such as molecular biological functions,affect HIF-1 signal path,cgmp-pkg signal path,TNF signal pathway,chemokine signal transduction pathway,apoptotic signaling pathway,pi3k-akt signaling pathway and toll-like receptor signaling pathway.Conclusions: 1.Both DHLX and Xiaoyan Lidan tablet can effectively prevent and treat chronic calculous cholecystitis,but DHLX has better effect.2.DHLX can effectively improve the inflammatory state of gallbladder,work for discharging of sediment-like stones,regulate blood lipid and liver function related indicators,improve the characteristics of bile metabolism in patients,prevent the secretion of lithogenic bile from the cause of stone formation,and has no obvious toxic and side effects,which with high safety.3.DHLX can regulate the expression of IL-6 and TNF-alpha mRNA in bile duct cells,relieve the inflammatory stress of bile duct cells,and slow down the disease progression.4.DHLX capsule can realize the multidimensional regulation of inflammatory reaction to the cells of bile duct through regulating the miRNAs impact signal pathway,HIF-1 cGMP-PKG signaling pathways,TNF-? signaling pathways,chemokines and signal transduction pathways,apoptosis signaling pathways,PI3K/Akt signaling pathway and Toll like receptor signaling pathway and so on many biological processes and molecular signaling pathways.