| UP to now,there are more than 1.3 billions people suffering from liver diseases worldwide,which is one of the most important causes of disease burden and death in the world.There are 10%-15% of chronic liver disease patients that would develop into liver fibrosis after 5-10 years,and then may develop to liver cirrhosis,even liver cancer.Hepatic fibrosis is a reversible pathological change in the imbalance of liver extracellular matrix(ECM)and abnormal deposition in the liver,leading to abnormal changes in liver structure and function.It is difficult to reverse when it develops further into cirrhosis and other diseases.Therefore,elucidating the mechanism of hepatic fibrosis,reversing or blocking the progression of hepatic fibrosis,and finding susceptibility factors for hepatic fibrosis are beneficial to the prevention of hepatic fibrosis and further targeted therapy.It is well known that alcohol is a susceptibility factor for liver fibrosis.Ethanol is first oxidized by alcohol dehydrogenase(ADH)to form acetaldehyde,which is then oxidized by aldehyde dehydrogenase(ALDH)to produce acetic acid.Acetaldehyde has mutagenic and carcinogenic properties and plays an important role in the development of alcoholic liver diseases such as liver fibrosis or even liver cancer.In addition to metabolism of ethanol,ADH is mainly involved in the metabolism of retinol.About 80% of the retinol(vitamin A)in the human body is stored in resting HSC.Retinol is oxidized to retinal by ADHⅠ,then retinal dehydrogenase(RALDH)is oxidized to generate retinoic acid(RA).In recent years,there is increasing evidence that RA involved in the progression of liver fibrosis.The ADH isoenzymes in the human body can be classified into seven subtypes based on their dynamic characteristics and amino acid sequences.Ethanol is mainly metabolized by ADHⅠ in the liver,while retinol is mainly metabolized by ADHⅠ and ADHⅠII in the liver.The expression and the activity of ADHⅠ that metabolite retinol and ethanol is significantly higher than that of ADHⅠII.Previous results of our research group showed that the activity of ADHⅠ in human liver fibrosis tissues was significantly higher than that of normal tissues by approximately 3 times.In addition,it was found that the level of basal activity of ADH was significantly positively correlated with the degree of hepatic fibrosis induced by diethylnitrosamine(DEN).It is suggested that ADH may be involved in hepatic fibrosis induced by ethanol and non-alcohol activation of HSC,but its mechanism has not yet been elucidated.The steady-state imbalance in the synthesis and degradation of ECM is the main pathological change in liver tissue during hepatic fibrosis.Abnormally activated hepatic stellate cells(HSCs)are the main source of ECM,and HSC activation is the key to the development of hepatic fibrosis.HSCs are activated and differentiate into myofibroblast(MFB),which secretes a large amount of collagen fibers and forms hepatic fibrosis.To further confirm the relationship between ADHⅠ activity and hepatic fibrosis,we first established a rat model of hepatic stellate cell line(HSC-T6)with overexpress and silence of ADHⅠ.Then the HSC-T6 model were activated by ethanol and non-alcoholic factors acting as LPS,TGF-β1.Real-time PCR and Western-blot were used to detect ADHⅠ expression,mRNA levels of fibrosis markers include type I collagen(COL1A1),α-smooth muscle actin(α-SMA).The expression of ADHⅠ was analyzed during the activation of HSC-T6 under exogenous stimulation,and whether the HSC activation-related factors were influenced by the changes of ADHⅠ expression companied with the exogenous stimulation factors,and related mechanisms were explored.This study intends to provide anti-hepatic fibrosis targets with important basic and clinical significance.Methods 1 Constructing expression vectors of ADHⅠ1.1 Amplication of ADHⅠ gene Using the rat liver c DNA library as a template,specific primers with Hind III(5’A|AGCTT3’)and Eco RI(5’G|AATTC3’)at the 5’ end were designed,and the ADHⅠ gene was amplified by PCR.1.2 Construction and identification of p EGFP-ADHⅠ recombinant plasmid The amplified gene fragment and p EGFP-N1 plasmid were digested by Hind III and Eco RI enzymes,then connected by T4 DNA ligase and transformed into E.coli DH5α competent cells,and kanamycin was screened for positive clones.Extracting plasmid for enzyme digestion and sequencing identification.2 Construction of HSC-T6 cell lines over-express ADHⅠ and silencing ADHⅠ The constructed PEGFP-ADHⅠ recombinant plasmid or the synthesized si ADHⅠ fragment was transfected into HSC-T6 cells by liposome transient transfection,and the transfection efficiency was observed under microscope at 24 h and 48 h.Further,real-time PCR and Western-blot determined the expression levels of ADHⅠ.3 Effect of ADHⅠ expression on activation of HSC-T6 cell line The mRNA of COL1A1 was measured by Real-time PCR in RNA extracted from control group,ADHⅠ over express group and ADHⅠ-silenced HSC-T6 cell line.4 Effects of exogenous stimuli on ADHⅠ expression Ethanol,TGF-β1 and LPS stimulated the HSC-T6 cell line of p EGFP-N1 control group,the extraction of RNA and protein in the cell at 24 h、48 h,by Real-time PCR and Western-blot determination of ADHⅠ mRNA and protein expression levels.5 The effect of ADHⅠ expression on the exogenous stimulation of HSC-T6 cell line activation Ethanol,TGF-β1,and LPS were used to stimulate stimulation with p EGFP-N1 control group,ADHⅠ over express group,and ADHⅠ-silenced HSC-T6 cell line,respectively.The mRNA expression levels of COL1A1 and α-SMA at 24 h and 48 h were detected.Result 1 Constructing expression vectors of ADHⅠ 1.1 Amplication of ADHⅠ gene The RNA extracted from rat liver was reversely transcribed into c DNA as a template.The length of the amplified ADHⅠ gene fragment was approximately 1148 bp,which was the same as the length of the sequence encoding ADHⅠ.1.2 Construction and identification of p EGFP-ADHⅠ recombinant plasmid Positive clones were screened with kanamycin resistance and the plasmids were extracted and digested with Hind III and Eco R I to obtain fragments of approximately 4700 kb and 1148 kb,respectively.The sequencing confirmed that the reading frame was correct.2 Construction of HSC-T6 cell lines overexpress ADHⅠ and silencing ADHⅠ Inverted fluorescence microscopy revealed that the overexpress ADHⅠ group emitted green fluorescence,and the mRNA and protein expression levels of ADHⅠ further demonstrated successful construction of cell lines that highly expressed ADHⅠ and silenced ADHⅠ.3 Effect of ADHⅠ expression on activation of HSC-T6 cell line In the HSC-T6 of over-express ADHⅠ,the mRNA level of COL1A1 was significantly increased(P<0.05).After silence of ADHⅠ,the mRNA level of COL1A1 was significantly decreased(P<0.0001).4 Effects of exogenous stimuli on ADHⅠ expression After 24 h and 48 h of ethanol-stimulated control group,the expression of ADHⅠ mRNA and protein was significantly increased(P<0.01);after 24 h and 48 h stimulation of TGF-β1 stimulated control group,the expression of ADHⅠ mRNA was not obvious.The ADHⅠ protein levels were significantly increased at 48h(P<0.05);after 24 h and 48 h of LPS-stimulated control group,ADHⅠ mRNA levels did not change significantly,and ADHⅠ protein levels increased significantly at 48h(P<0.05).5 The effect of ADHⅠ expression on the exogenous stimulation of HSC-T6 cell line activation 5.1 The effect of ADHⅠ expression on the exogenous stimulation of HSC-T6 cell line activation After 24 h and 48 h ethanol-stimulated of overexpress ADHⅠ group,the mRNA level of COL1A1 was significantly higher than that of control group(P<0.01),and the α-SMA mRNA level was significantly increased at 24h(P<0.001).There was no significant change at 48 h.After 24 h and 48 h of ethanol-stimulated ADHⅠ-silenced group,the mRNA level of COL1A1 was significantly lower than that of the control group(P<0.001),and the level of α-SMA mRNA was significantly lower at 24 h(P<0.01).There was no significant change at 48 h.After TGF-β1 stimulated of the overexpress ADHⅠ group for 24 h and 48 h,the mRNA level of COL1A1 was significantly higher than that of the control group(P<0.05),and the mRNA level of α-SMA was significantly increased at 24 h(P<0.001).No significant difference was observed at 48 h.Changes;TGF-β1 stimulated ADHⅠ silence group 24 h,48 h,compared with the control group,COL1A1 mRNA levels decreased significantly(P<0.05),α-SMA mRNA levels decreased significantly at 24 h(P<0.01),48 h no significant change.After 24 h and 48 h of LPS stimulation of the overexpress ADHⅠ group,the mRNA level of COL1A1 was significantly higher than that of the control group(P<0.01),and the mRNA level of α-SMA was significantly increased at 24 h(P<0.05).There was no obvious change at 48 h.After LPS stimulation of ADHⅠ- silenced group,the expression of COL1A1 mRNA was significantly lower than that of control group(P<0.001),and the level of α-SMA mRNA was significantly decreased at 24 h(P<0.001).There was no significant change at 48 h.5.2 Effect of different exogenously stimulated activation of HSC-T6 cell line Compared with non-alcoholic stimulated ADHⅠ over-expressing cell lines,the mRNA levels of COL1A1 increased significantly(P<0.05).Conclusion 1.Both alcohol and non-alcohol promote the expression of ADHⅠ,and alcohol stimulates the increase of ADHⅠ more than non-alcoholic factors.2.Increased ADHⅠ is a susceptibility factor for hepatic fibrosis.The mechanism may be related to ADHⅠ promoting HSC activation. |
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