The Expression Of Th17Cells And Their Cytokines In A Rat Model Of Non-Alcoholic Fatty Liver Disease

Objective To establish a rat model of NAFLD with high-fat diet,and observe theexpression of Th17cells,RORγtmRNA,IL-6and IL-17during the development ofNAFLD； So that we can investigate the immunomodulatory effect of Th17cells and itsrelated cytokines in NAFLD.Methods Sixty-four male SD rats weighed160±10g were randomly divided into normaldiet group(NG) and fat-rich diet group(FG) after adaptive feeding one week. Every grouphad thirty-two rats. NG was fed with normal diet while FG fed with high fat diet which wasmixed with88%normal diet,2%cholesterol and10%lard. At the end of feeding for2weeks,6weeks,10weeks and14weeks, eight rats in NG and FG were selected randomlyand given a fasting for12hours,then weighted. Blood samples in inferior venas werecollected for AST,ALT,TG,TC. The proportion of peripheral blood Th17cells in CD4+Tcells were tested by flow cytometry. Livers were dissociated and recorded, then calculatedtheir liver index. Liver tissue were sunk into10%formaldehyde solution and prepared todo pathological observation. The expression of RORγt gene was detected by realtime-PCR.Enzyme-linked immunosorbent assay(ELISA) was used to measure the levels of IL-6,IL-17in serum and liver homogenate.SPSS11.5was used to process all data. Quantitative datawas analyzed by independent-samples t test and counting data analyzed by rank sum test.Simple correlation and linear-regression analysis were used for correlation analysis.Difference were considered significant at p<0.05.Results1、The weights of rats in FG increased faster than NG in total. The liver weight and liverindex were higher than normal group’s from the end of2week(p=0.000),while the bodyweight in two groups appeared significantly difference at the end of6week (p=0.001).2、Hematoxylin-eosin staining indicated that in NG there wasn，t hepatological steatosis andinflammatory. However, the steatosis and inflammatory were gradually aggravated with the rearing time extended in FG. At the second weekend, FG appeared low-grade fatty liver,which mainly contained microvesicular hepatic steatosis, At the end of6week, themicrovesicular steatosis continued to increase and appeared macrovesicular steatosis,which up to grade2-3, then intrahepatic inflammatory lesions began to appear, which up tograde0-1. Steatosis and inflammatory lesions were increased, respectively up to grade3-4and grade2-3at the10thweekend. Until the14thweekend the liver of steatosis up to grade4which mainly with bullous, meanwhile, inflammation had reached grade3. Comparedwith NG, FG of steatosis all had significant differences(p=0.002, p=0.000, p=0.000andp=0.000respectively) and inflammation was significantly difference at the6thweekend(p=1.000, p=0.010, p=0.000and p=0.000respectively)3、All FG rats had a significantly elevated levels of serum ALT,AST,TG,TC in comparison with NG at the homologus week.The levels of TC in serum of FG all increased significantly at the end of2week(p=0.023).We observed ALT increased at the6thweekend(p=0.046),and at the10thweekend AST and TG was significantly increased(p=0.001and p=0.000respectively).4、We observed that the level of IL-6and IL-17in FG were higher than NG. In serum, the level of IL-6, IL-17were increased respectively at the6thand10thweekend(p=0.000and p=0.003respectively).And in liver, the concentration of IL-6was significantly difference at the second weekend while IL-17at the6thweekend(p=0.003,p=0.000respectively).5、 The proportion of peripheral blood Th17cells in CD4+T cells in NG was almostidentical while the rate in FG increased gradually. In comparison with NG, there was asignificantly increased from the end of10week (p=0.397, p=0.900, p=0.000, p=0.000respectively).6、Compared with control group, the expression of RORγt mRNA in all FG was increased(p=0.122,p=0.192,p=0.002,p=0.000respectively).7、 Correlation analysis in FG indicated that IL-6, IL–17had a obviously positivecorrelation between peripheral blood and liver(r=0.921, p=0.000; r=0.877, p=0.000respectively), and the proportion of peripheral blood Th17cells in CD4+T cells positivelyrelated to the level of IL-6and IL-17(r=0.824and r=0.734respectively). All these factors positively correlated with the degree of hepatocyte steatosis(r=0.693, r=0.898, r=0.801respectively)and inflammatory(r=0.664,r=0.907,r=0.823respectively).However, IL-6,IL-17coefficient correlation most heavy among them.Conclusion1、 Continuing high fat diet lead to liver steatosis and inflammation gradually aggravate,thus forming NASH.2、 The expression of Th17cells,IL-17and IL-6in peripheral blood were closely relatedeach other and all of them are positively correlated with the degree ofliver lesion.They may participate in the development of NAFLD. Th17cells may play aindirect role in the development of NAFLD through IL-6, IL-17.3、Measuring the concentration of IL-6、IL-17in serum may help to diagnose NAFLD andassess its severity.