Th17Pathway In The Pathogenesis Of Alcoholic Liver Disease And Preliminary Study On The Mechanism Of Resveratrol

Objective: To elucidate the mechanisms of Th17pathway in acute andchronic alcoholic liver disease and the function of resveratrol.Methods:(1)①Chronic group,50C57BL/6mice were fed adequateLieber-DeCarli Control diet for5days, then divided into five groups randomly,the Control+CCL4(number=10), the Control+olive oil(number=10), theAlcohol+CCL4+CMC(number=10), the Alcohol+olive oil(number=10) andthe Alcohol+CCL4+Res(number=10). The Alcohol+olive oil， theAlcohol+CCL4+CMC, the Alcohol+CCL4+Res mice were fed Lieber-DeCarlialcohol diet during the first8weeks and the others were pair-fedLieber-DeCarli control diet. Lieber-DeCarli alcohol diet must be addedalcohol gradually from1%to5%. The Alcohol+CCL4+CMC and theAlcohol+CCL4+Res mice were given intragastric administration with CMC,resveratrol (45mg/kg/d) from the eighth week to the end, respectively. TheControl+CCL4, the Alcohol+CCL4+CMC and the Alcohol+CCL4+Res micewere given an intraperitoneal injection of CCL4(2.5%4ml/kg, twice)dissolved in olive oil at the ninth week，while the others received anintraperitoneal injection of oil(2.5%4ml/kg, twice). Mice continued ondrinking the same diets. All were killed at the tenth week.②A cutegroup,40C57BL/6mice were divided into four groups randomly， theControl(number=10), the Alcohol(number=10), the Alcohol+CMC(number=10) and the Alcohol+Res(number=10). The Control mice were fedLieber-DeCarli Control diet for10days followed by single gavage of maltose(5g/kg). The Alcohol, Alcohol+CMC and Alcohol+Res mice were fedLieber-DeCarli diet for10days followed by single gavage of ethanol (5g/kg).The Alcohol+CMC and the Alcohol+Res mice were given intragastric administration with CMC, resveratrol (45mg/kg) before gavage of ethanolrespectively. All were killed10hours after gavage.(2)The general conditionsof the mice were noted，such as hair gloss，appetite，behavior and so on．Thebody weight was measured weekly.(3)The changes of liver morphology orhistopathology, liver weight, the Hepatosomatic indices, alanineaminotransferase (ALT), aspartate aminotransferase (AST) and Triglyceride(TG) were detected in each group.(4)The lymphocytes in the liver and spleenfrom each group were isolated and CD4~+CD29~+, CD4~+CD49~+were identifiedby fluorescence activated cell sorting.(5) The expressions of IL-6, TGF-β,α5β1, IL-17, RORγt, STAT3, Socs3, and IRF4mRNA were observed byReal-time fluorescent quantitation PCR.Results:(1)①C hronic group: there was no morphological changebetween the livers of the Control+CCL4and Control+olive oil group. On theother hand the livers in the Alcohol+CCL4+CMC and the Alcohol+olive oilgroup grew more swelling, heavier and turned yellow; the change was themost significant in the Alcohol+CCL4+CMC group; the change was reducedin the Alcohol+CCL4+Res group. Further more, the Hepatosomatic indices ofthe Alcohol+CCL4+CMC and Alcohol+olive oil group elevated significantlycompared with the other three groups. It reached6.33±0.47in the group ofAlcohol+CCL4+CMC, Compared with Control+CCL4, Control+olive oil，andAlcohol+CCL4+Res group,(P<0.05). There showed no significance (P>0.05)between Alcohol+CCL4+CMC and Alcohol+olive oil group.②Acute group:the livers of the Alcohol and the Alcohol+CMC group grew swelling, heavier,turned little yellow and the Hepatosomatic indices increased significantlycompared with the control; the change of the Alcohol+Res group was reduced.(2)①C hronic group: the histopathology displayed normal in theControl+CCL4and Control+olive oil group. On the contrary, there was moreinflammation, fatty and vacuolar degeneration in the Alcohol+CCL4+CMCand Alcohol+olive oil group. Sirius red staining showedAlcohol+CCL4+CMC induced liver fibrosis. Compared with theAlcohol+CCL4+CMC group, the severity and scope were reduced in the Alcohol+CCL4+Res group.②Acute group: there was mild fatty and vacuolardegeneration in the Alcohol and the Alcohol+CMC group, and the lesionswere improved in the Alcohol+Res group．(3) Chronic group①There was nochange in the serum levels of ALT, AST, and TG between the Control+oliveoil and the Control+CCL4; Compared with Control+olive oil group, theAlcohol+oil group had a higher serum levels of ALT, AST and TG(P<0.05);②T he serum levels of ALT, AST and TG didn’t change in theAlcohol+CCL4+CMC group compared with the Alcohol+olive oilgroup(P<0.05);③Compared withthe Alcohol+CCL4+CMC group, the serumlevels of ALT, AST, and TG in the Alcohol+CCL4+Res group decreasedsignificantly(P<0.05). Acute group:①ALT, AST and TG in the Alcohol groupand the Alcohol+CMC group increased significantly compared with theControl group(P<0.05), and there was no significance between the Alcoholand the Alcohol+CMC group;④There was no significant change in theAlcohol+Res group.(4)The percentage of CD4~+CD29~+and CD4~+CD49~+inintrahepatic lymphocytes showed no differences between the Control+CCL4and the Control+olive oil group; Compared with the Control+olive oil group,the Alcohol+olive oil group showed a significantly higher expression ofCD4~+CD29~+in intrahepatic lymphocytes (P<0.05), and theAlcohol+CCL4+CMC group also showed upregulated expression ofCD4~+CD29~+(P<0.05). There was no changes of CD4~+CD49~+both in theAlcohol+olive oil and Alcohol+CCL4+CMC group; The percentage ofCD4~+CD29~+decreased in the Alcohol+CCL4+Res group(P<0.05); While thereshowed no signifcances among all the groups in spleen.(5)①C hronic group:There were no significance among the expressions of IL-17, TGF-β, IL-6,α5β1, RORγt, STAT3, IRF4, Socs3mRNA between the Control+olive oil theControl+CCL4group; Compared with Control+oil group, the Alcohol+oliveoil group had a higher expressions of IL-17(P<0.05), but the others didn’tchange;②Compared with the Alcohol+group, the expressions of IL-17,TGF-β, α5β1, STAT3, IRF4, Socs3mRNA in the Alcohol+CCL4+CMC groupincreased(P<0.05), but there was no difference in the expressions of IL-6and RORγt mRNA between the two groups;③Compared with theAlcohol+CCL4+CMC group, the Alcohol+CCL4+Res group had asignificantly decreased expressions of IL-17, TGF-β, α5β1and STAT3mRNA(P<0.05), but IRF4and Socs3mRNA didn’t change.②A cute group:there was no difference in the expressions of IL-17, IL-6, α5β1, RORγt,STAT3, Socs3mRNA among the Control, the Alcohol and the Alcohol+CMCgroup; Compared with the Control group, the Alcohol and the Alcohol+CMCgroup had a higher expressions of TGF-β and IRF4mRNA, and there was nodifference between the two groups; the expressions of TGF-β and IRF4mRNA significantly decreased in the Alcohol+Res group.Conclusions:1Chronic alcoholic hepatitis, liver fibrosis model andacute alcoholic hepatitis model were established through along-termLieber-DeCarli alcohol diet in combination with low-dose intraperitonealinjection of carbon tetrachloride and short-term Lieber-DeCarli alcohol dietfollowed by single gavage respectively.2CD4~+CD29~+expression on theintrahepatic lymphocyte is closely related to the infiltration and fibrosis ofTh17in liver.3Th17pathways may play an important role in the progressionof ALD, but there showed no relationship with acute alcoholic liver disease.4TGF-β may play an important role both in acute inflammatory response andliver fibrosis induced by alcohol.5α5β1may play an important role in theALF formation.6The expression of IL-17may be regulated by STAT3andSocs3in the development of ALD, but the mechanism of Socs3is not clear.7Res may inhibit the Th17pathway by downregulating CD29expression inlymphocytes of liver and STAT3expression, which plays an important role inanti-fibrosis.8Res may play an important role in acute and chronic alcoholicliver diseases by inhibiting the expression of TGF-β.9Probably, Res plays anantifibrotic effect by downregulating α5β1expression.10The effects of Res on IRF4in the different stages of alcoholic liver disease suggest that themechanism is complex, which need further study.