| AIMS: This article is to explore related gene expression of the IRE-1α-XBP-1 signal pathway during endoplasmic reticulum stress in the pathogenesis of nonalcoholic liver disease(NAFLD)and discuss the effect of metformin on IRE-1α-XBP-1 signal pathway during endoplasmic reticulum stress and investigate the mechanism of its therapeutic effect on NAFLD.METHODS:40 8-week-old male C57BL/6J mice were divided into three groups: Healthy control group,high-fat group and metformin group.The healthy control group received conventional feeding,while the high-fat group and metformin group were fed with a high-fat diet.The metformin group was treated with metformin for 4 weeks as soon as the model was successfully established.Pathological changes in the liver of mice were examined to assess the degree of fatty degeneration of liver.Fatty changes in liver examples of high-fat group and metformin group were graded,liver cell apoptosis index was calculated,and the amount of Bcl-2m RNA,Bimm RNA,Bcl-2 protein and Bim protein were detected to explore the relationship between fatty degeneration and liver cell apoptosis in NAFLD.Levels of GRP78 m RNA,IRE1 ɑ m RNA and IRE1 ɑ protein as biomarkers of endoplasmic reticulum stress were measured in liver tissue.Expressions of cell apoptosis related genes including JNKm RNA,Bcl-2m RNA,Bimm RNA were detected by means of RT-PCR.The difference of cell apoptosis related protein expression of Bcl-2 was analyzed by Western blot.Liver cell apoptosis index was determined using TUNEL method.RESULTS:Compared with healthy control group,the degree of fatty changes in the liver of mice in high-fat-group increased significantly[[(3.12± 0.83)vs(0.73± 0.13)](P<0.01).While there was a remarkable decrease in the fatty changes of liver in mice of the metformin group in compare with that of the high-fat-group[(1.63± 0.74)vs(3.12± 0.83)](P<0.05).Significant differences were found in liver cell apoptosis index[(0.40±0.53)vs(0.21±0.36)](P<0.01)、Bimm RNA[(0.0036±0.0012)vs(0.0036±0.0012)](P<0.01)、Bim protein [(0.31±0.06)vs(0.17±0.02)](P < 0.01),the amount of BCL-2m RNA[(0.0082±0.0011)VS(0.0128±0.0011)]、BCL-2 protein[(0.33±0.09)vs(0.50±0.09)](P<0.05)(P<0.05)between F2 grade fatty liver and F1 grade fatty liver.As for F3 grade fatty liver,there were also significant differences in liver cell apoptosis index[(0.57±0.37)vs(0.40±0.53)](P<0.05),the amount of Bimm RNA[(0.0050±0.0007)vs(0.0036±0.0012)](P<0.05)、BCL-2protein[(0.20±0.06)vs(0.33±0.09)](P<0.05)when compared with F2 grade fatty liver.Significant differences were found in liver cell apoptosis index[(0.40 ± 0.53)vs(0.21 ± 0.36)](P < 0.01)、BCL-2m RNA[(0.0046±0.0008)vs(0.0064±0.0003)](P < 0.05)、Bimm RNA[(0.0076±0.0013)vs(0.0050±0.0007)](P<0.05)、Bim protein [(0.57±0.06)比(0.40±0.06)](P<0.05))between F4 grade fatty liver and F3 grade fatty liver.In high-fat group,a significant increase was found in the levels of GRP78 m RNA[(0.0033±0.0011)vs(0.0014±0.0006)](P<0.05),IRE1 ɑ m RNA [(0.0169±0.0028)vs(0.0137±0.0032)](P<0.05)and IRE-1ɑ protein[(0.41±0.08)vs(0.32±0.04)](P<0.05)compared with healthy control group.There was also a remarkable increase in the expressions of ERS mediated apoptosis related genes JNKm RNA[(0.0014±0.0003)vs(0.0007±0.0003)(P<0.05)],Bimm RNA[(0.53±0.11)vs(0.25±0.21)](P<0.05),while the level of anti-apoptotic gene Bcl-2m RNA has decreased markedly[(0.17±0.06)vs(0.33±0.13)](P<0.05).Moreover,the liver cell apoptosis index[(0.61±0.55)vs(0.34±0.27)](P<0.01)has risen evaluated by TUNEL method.Conversely,after 4 weeks of intervention with metformin,the expressions of GRP78 m RNA[(0.0023±0.0005)vs(0.0033±0.0011)](P < 0.05)and IRE1 ɑ m RNA[(0.0127±0.0037)vs(0.0169±0.0028)](P<0.05)have significantly decreased in metformin group as compared with high-fat group.In metformin group,the levels of JNKm RNA[(0.0010±0.0003)vs(0.0014±0.0003)](P<0.05),JNK protein [(0.28±0.24)vs(0.53±0.11)](P<0.05),Bimm RNA[(0.0028±0.0018)vs(0.0056±0.0019)](P<0.05)、Bim protein [(0.24±0.29)vs(0.45±0.18)](P<0.05)have significantly reduced with a notable increase in the level of anti-apoptotic gene Bcl-2m RNA[(0.0102±0.0031)vs(0.0063±0.0018)](P<0.01)and its protein expression[(0.43±0.10)比(0.17±0.06)](P<0.05).The liver cell apoptosis index[(0.32±0.24)vs(0.61±0.55)](P<0.01)has decreased using the TUNEL method.CONCLUSION:(1)Establishment of NAFLD mouse model can be induced through high-fat feeding.(2)Endoplasmic reticulum stress is found in mouse model with fatty liver.(3)The severity of fatty liver in mouse model has a positive relationship with liver cell apoptosis.(4)ERS mediated IRE-1α-XBP-1 signal pathway can attribute to liver cell apoptosis through upregulating the expression of JNK protein,which may be involved in the process of NAFLD.(5)Metformin may depress the IRE-1α-XBP-1 signal path way during endoplasmic reticulum stress in NAFLD,so as to decrease the degree of liver cell apoptosis and lighten fatty degeneration or slow down the process of NAFLD. |
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