Study On The Effect Of HSC-derived Exosomes Transporting Let-7b-5p On Promoting Hepatocyte Apoptosis And The Protective Effect Of Tiaogan Lipi Decoction In ALD

Alcoholic liver disease(ALD)is a chronic liver disease with a long course and complicated pathogenesis caused by long-term excessive intake of alcohol.In China,the incidence and mortality of ALD are increasing at an alarming rate,and it has become one of the most common and important chronic liver diseases in my country.The pathogenesis of ALD has not been fully elucidated,but a variety of cells such as hepatocytes and hepatic stellate cells have been confirmed to participate in it.The apoptosis and necrosis of hepatocytes as well as the activation and transdifferentiation of hepatic stellate cells run through the whole process of ALD.A large number of studies have found that cell communication in the liver is closely related to the progression of various liver diseases.For instant,exosomes play an critical role in hepatocellular carcinoma,non-alcoholic fatty liver,liver fibrosis and other diseases by transmitting information.Exosomes are extracellular vesicles with a diameter of 30-150nm,which can carry a variety of small molecules including microRNA(miRNA),act as a medium for information transmission between cells,and perform important biological functions.A lot of studies have confirmed that exosomes secreted from hepatocytes and hepatic stellate cell can be transported to neighboring cells in the liver,aggravating liver damage.In previous studies,it was found that the activated hepatic stellate cell-derived exosomes in the ALD model can induce hepatocyte apoptosis.The traditional Chinese medicine Tiaogan Lipi Decoction has a significant protective effect on ALD.However,the mechanism that the hepatic stellate cell-derived exosomes induce liver cell apoptosis as well as how does Tiaogan Lipi Decoction relief ALD is still unclear.This study aims to explore the mechanism of miRNA transported by hepatic stellate cell exosomes in ALD on hepatocytes.Besides,we hope to explore the molecular mechanism of Tiaogan Lipi Decoction in treating ALD,especially from the perspective of intrahepatic cell communication.Methods:(1)Hepatic stellate cells promote hepatocyte apoptosis in alcoholic liver disease(ALD).1)Construction of ALD model of HSC.Different concentrations of acetaldehyde were used to stimulate HSC for 48 hours,the cell survival rate was measured by CCK-8 method,and the levels of smooth muscle actin ?(?-SMA),collagen 1(Coll)and fibronectin(FN)were measured by WB.2)Co-culture experiment of activated HSC and hepatocytes.Transwell co-culturiing(activated/quiescent)HSC and hepatocytes.The rate of hepatocyte apoptosis was detected by flow cytometry.Real-time PCR and WB was using to detect the expression levels of hepatocyte apoptosis-related molecules Caspase3,Bax and Bcl-2.(2)The differential expression profile of miRNA in activated HSC exosomes and the screening of target genes.1)Separately treating HSC with exosome-free medium/acetaldehyde-free exosome-free medium.Collecting the exosomes in the supernatant by ultracentrifugation.Use electron microscopy,Western Blot,and particle size analysis to identify HSC exosomes(HSC-exo).2)Extract quiescent/activated HSC-exo,then using RNA-seq to analyze differential miRNA in exosomes.|log2(FC)|?2 was used as the standard to screen differentially expressed miRNAs.At last,using TargetScan database to predict the target gene of Let-7b-5p,and using the dual luciferase reporter gene experiment to verifie the binding of miRNA and target gene mRNA.(3)Activating HSC-exo transporing Let-7b-5p to promote hepatocyte apoptosis-1)After staining with PKH26,HSC-exo was incubated with hepatocytes for 4h,and then hepatocytes uptaking HSC-exo was observed and photographed by fluorescence microscope.The hepatocytes were incubated with HSC-exo(activated/quiescent group),and the apoptosis of hepatocytes was detected by flow cytometry,and the levels of Let-7b-5p,EZH2,Bax,and Bcl-2 mRNA in the hepatocytes were detected by real-time PCR.The Western Blot method was used to measure the expression levels of EZH2,Bax,and Bcl-2 in hepatocytes to verify the effect of HSC-exo in the activation group on hepatocyte apoptosis.2)Let-7b-5p mimics and Let-7b-5p mimic-NC are transfected into hepatic stellate cells.Real-time PCR was used to detect Let-7b-5p in hepatic stellate cell exosomes;Let-7b-5p mimic-modified hepatic stellate cell exosomes were co-cultured with hepatocytes,and hepatocyte apoptosis was detected by flow cytometry.Real-time PCR to detect the level of Let-7b-5p,EZH2,Bax,Bcl-2 mRNA in liver cells,Western Blot to detect the level of EZH2,Bax,Bcl-2.(4)Studying on the protective effect of Tiaogan Lipi decoction on Let-7b-5p-mediated hepatocyte apoptosis.1)Firstly,treating liver cells with different concentrations of Tiaogan Lipi decoction for 48 hours,and useing the CCK-8 method to determine the optimal concentration of Tiaogan Lipi decoction.2)Hepatocytes were divided into three group:NC group,Let-7b-5p overexpression group,medicine group.The medicine group was firstly treated with Tiaogan Lipi Decoction for 48 hours.After 48 hours,the NC group was transfected with Let-7b-5p mimic-NC.The Let-7b-5p overexpression group and medicine group were transfected with Let-7b-5p mimic to overexpress Let-7b-5p.Flow cytometry was used to detect the apoptosis levels of the three groups,the real-time PCR was used to detect the mRNA levels of Let-7b-5p,EZH2,Bax,and Bcl-2,and the Western Blot was used to detect the expression levels of EZH2,Bax,and Bcl-2.Results:(1)After treating HSC with acetaldehyde for 48 hours,the HSC survival rate of 300 ?M group was the highest--1.15 times that of the 0 ?M group.Western Blot results showed that the expression levels of ?-SMA,Coll and FN were significantly up-regulated compared with control group,which suggested that ALD modeling of HSC was successful constructed.(2)The results of real-time PCR and Western Blot results showed that expression of Caspase3 and Bax mRNA and protein in hepatocytes was up-regulated,and the expression of Bcl-2 mRNA and protein was down-regulated.It inferred that after activated by acetaldehyde,HSC can induce hepatocytes apoptosis.(3)The results of transmission electron microscopy,WB and particle size analysis showed that the ultracentrifugation extract of the cell supernatant was exosomes.RNA-seq results showed that the expression of Let-7b-5p in HSC-exo in the acetaldehyde-activated group was up-regulated compared with the control group.TargetSacn predicted that Let-7b-5p can target binding to the 3'UTR region of the mRNA of EZH2,and the dual luciferase reporter gene experiment suggested that Let-7b-5p can target binding to the 3'UTR region of the mRNA of EZH2 and inhibit it expression.(4)Fluorescence microscopy results showed that hepatocytes can endocytose HSC-exo labeled with PKH26,and the fluorescent label is located on the liver cell membrane.The results of real-time PCR and Western Blot showed that the co-culture of HSC-exo and hepatocytes in the activation group could significantly down-regulate the target gene EZH2 expression in hepatocytes,reduce the survival rate of hepatocytes,and significantly increase the rate of hepatocyte apoptosis.After transfection of Let-7b-5p mimics,the results of real-time PCR showed that the expression of Let-7b-5p in HSC and its exosomes was up-regulated.The results of real-time quantitative PCR and Western Blot showed that co-culture with mimic-exo could down-regulate expression of EZH2 and Bcl-2,and up-regulate the expression of Bax.At the same time,the apoptosis rate of hepatocytes was significantly increased.(5)Compared with the NC group,the expression of Let-7b-5p and Bax in the Let-7b-5p overexpression group was significantly up-regulated,the expression of EZH2 and Bcl-2 was significantly down-regulated,and the apoptosis rate of hepatocytes was significantly increased.Compared with the Let-7b-5p overexpression group,the expression of Let-7b-5p and Bax of medicine group was significantly down-regulated,the expression of EZH2 and Bcl-2 was up-regulated,and the apoptosis rate of hepatocytes was significantly decreased.Conclusion:In ALD,the exosomes of activated hepatic stellate cell are related to hepatocyte apoptosis.The activated hepatic stellate cells can induce liver cell apoptosis by transferring Let-7b-5p to hepatocytes through exosomes,and then regulating the expression of the target gene EZH2 in hepatocytes.The traditional Chinese medicine Tiaogan Lipi Decoction can reduce the apoptosis of liver cells by down-regulating Let-7b-5p of liver cells.