| Objective: To observe the genes expression of IRE-1alpha–chop、caspase12 pathway i n NAFLD,to investigate the effect of IRE-1alpha –chop、caspase12 signal pathway in the pathogenesis of NAFLD,to observe the effects of metform in on hepatic cell apoptosis in IRE-1alpha-chop,caspase12 pathway,to inve stigate effects of metformin intervention NAFLD mechanism.Method: 30 male C57BL/6J mices aged 8 week were divided into three g roups: normal control group(A),model group(B)and metformin group(C),10 rats in each group.Control group of mice fed with normal diet;model group and metformin group were given high fat diet for 8 weeks,a fter successfully established selection 2mices is only observed after the fatt y liver model,the metformin group was given metformin(150mg/(kg.d))by gavage intervention for 4 weeks,the other two groups were given an e qual volume of saline.The last 12 hours after eating,under anesthesia,we igh the mice body weight,liver weight,fat tissue around the epididymis.And calculate the liver index,fat index;serum ALT,TG blood sample coll ection;collect liver tissue,one part of tissue for pathological sections,other tissuesaved in-80 C refrigerator to measure GRP78,IRE-1alpha,CHOP,Cas pase12,Bcl-2,Bim m RNA gene and protein expression.Hepatocyte apoptosis index was measured by TUNEL method.Results:1.The comparison of body weight,liver weight,fat weight,liver index,fat index of the three groups mice: compared with normal control group,afte r the high fat diet after 12 weeks,the model control group body weight o f mice was significantly increased,there was significant difference(2.3675±2.2568vs29.6975±2.1513,p=0.022),liver weight increased slightly,but the difference was not statistically significant(1.5488±0.1537vs1.4188±0.1193,p=0.073),also no significant differences in liver index(0.0464±0.0027vs0.0478±0.0025,p=0.302),fat weight was significantly increased,and there were significant differences(1.0038±0.4294vs0.5163±0.1761,p=0.002),fat index was significantly increased(p=0.002).Compared with the model group,metformin group in mice fed with high-fat diet for 8 weeks after the start of metformin on body weight after 4 weeks of drug interve ntion,metformin group mice decreased significantly,the difference there w as significant(28.9913±2.0693vs32.3675±2.2568,p=0.005),liver weight was slightly decreased,no significant difference(1.5038±0.1378vs1.5488±0.1537,p=0.52),on the contrary,liver index increased,and the differen ce was statistically significant,which may be associated with metformin on weight improvement significantly related to fat the weight was significantl y decreased,the difference was significant(0.0533±0.0024vs0.0464±0.0027,p=0.000),fat index decreased significantly,the difference was statistica lly significant(0.0145±0.0042vs0.0305±0.0106,p=0.000).2.Comparison of transaminase and lipid levels in plasma of three groups m ice: compared with normal control group,the high fat diet after 12 weeks,ALT level of the model control group significantly increased(61.35±16.43vs27.6±7.62,p=0.000);TG increased significantly(105.67±17.44vs65.84±18.77,p=0.000);compared with the model group,metformin group mi ce fed with metformin after 4 weeks of drug intervention,the metformin g roup ALT level decreased significantly(43.55±13.77vs61.35±16.43,p=0.013),metformin group TG level decreased significantly(88.69±6.42vs105.67±17.44,p=0.037).3.three groups of mice liver tissue pathological and histological score result s: liver tissue pathological sections,HE staining,and the normal control gr oup,liver tissue of mice in control group showed fatty degeneration of the liver after high-fat feeding model(p<0.01);compared with the model group,metformin group reduced the degree of hepatic steatosis in mice and th ere was a significant difference(p<0.05).4.quantitative PCR test results: compared with normal control group,in m odel group,after high fat diet,IRE-1am RNA,CHOPm RNA,Caspase12 m R NA,Bimm RNA expression levels increased significantly(0.0033±0.0011 v s0.0014±0.0006,p=0.000;0.0161±0.0026vs0.0129±0.0025,p=0.040;0.043±0.0181vs0.0192±0.0078,p=0.003;0.021±0.0091vs0.007±0.0031,p=0.000;0.0056±0.0019vs0.0018±0.001,p=0.000),the expression leve l of Bcl-2m RNA was decreased,but the difference was not statistically sig nificant(0.0058±0.0014vs0.0078±0.0014,P=0.07).Compared with the model control group.In metformin grou,after 4 weeks of metformin in liv er tissue the IRE-1am RNA,CHOPm RNA,Caspase12 m RNA,Bimm RNA ex pression level decreased,the differences were statistically significant(0.0024±0.0006vs0.0033±0.0011,p=0.038;0.0127±0.0037vs0.0161±0.0026,p=0.033;0.028±0.0141vs0.043±0.0181,p=0.045;0.012±0.0057vs0.021±0.0091,p=0.011;0.0028±0.0018vs0.0056±0.0019,p=0.003),the expr ession level of Bcl-2m RNA increased,the difference was statistically signif icant(0.0102±0.0031vs0.0058±0.0014,p=0.000).5.western blot test results: compared with normal control group,in liver t issue of high fat model group expression of GRP78 protein levels increase,there was significant difference(0.4475±0.0591vs0.2771±0.0514,p= 0.000),compared with the model group,metformin group liver tissue GRP78 protein expression level decreased,and there was significant(0.3621±0.1131vs0.4475±0.0591,p=0.043)compared.With the normal control group,IRE-1 protein,liver tissue of the high fat model group CHOP protein,Cas pase12 protein,Bim protein expression level increased,there was a signific ant difference(0.4089±0.088vs0.3183±0.0416,p=0.024;0.5217±0.101 v s0.2496±0.0793,p=0.000;0.4489±0.1153vs0.1988±0.1104,p=0.000;0.3457±0.1039vs0.2443±0.1092,p=0.033).model control group,the ex pression level of BCL-2 protein decreased,there was significant difference(0.1748±0.0604vs0.3277±0.1347,p=0.007).compared with the model gr oup,the liver the organization in the metformin group IRE-1 protein expre ssion level decreased,although there was no significant difference compare d with the model group(0.3397±0.0844vs0.4089±0.088,p=0.077).CH OP,Caspase12 in liver tissue of metformin group,the expression level of Bim protein decreased(0.2907±0.1449vs0.5217±0.101,p=0.000;0.2423±0.0894vs0.4489±0.1153,p=0.001;0.2422±0.0308vs0.3457±0.1039,p=0.030).compared with the control group,the level of expression of BC L-2 protein decreased in liver tissue of hyperlipidemia model group,there was significant difference(0.1748±0.0604vs0.3277±0.1347,p=0.007);co mpared with the model group,the level of expression of BCL-2 protein in creased significantly in liver tissue of metformin group(0.4312±0.1009vs0.1748±0.0604,p=0.000).6.TUNEL results: compared with normal control group,hyperlipidemia mo del group liver cell apoptosis was increased,there was significant differenc e(58.32±4.16vs30.63±50.25,p=0.011)and metformin group were also s een a few apoptotic hepatocytes but significantly decreased compared with the model group(20.26±5.41vs58.32±4.16,p=0.005).Conclusion:1.High fat diet can induce the formation of fatty liver in mice,increase t he weight of mice and the accumulation of fat in the epididymis.2.The expression of GRP78 and IRE-1a in the liver tissue of the mice w ith nonalcoholic fatty liver disease increased,suggesting that the liver tissu e of the fatty liver mice was in the endoplasmic reticulum stress.3.Metformin can reduce the body weight of mice,reduce the accumulatio n of epididymal adipose tissue,reduce the level of ALT,TG in mice,redu ce the fatty degeneration of the liver,and have a therapeutic effect on fatt y liver in mice4.Metformin can reduce the expression of GRP78 and IRE-1a in liver tiss ue of mice,which suggests that metformin can improve ER stress.5.Metformin may through the IRE-1a-CHOP,Caspase12 pathway,down-reg ulation of the pro apoptotic gene Bim expression of anti apoptosis gene B cl-2,thereby reducing the liver cell apoptosis,alleviate hepatic steatosis an d delay of steatohepatitis. |
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