The Study Of PERK-eIF2α-ATF4 Signaling Protein Expression In Rat Liver Fibrosis

To observe the expression of endoplasmic reticulum stress-related molecules p-PERK、p-eIF2a and ATF4 in the process of liver fibrosis induced by carbon tetrachloride (CCl4), and explore the important roles of the PERK-eIF2a-ATF4 signal transduction pathways in hepatic fibrosis. METHODS Forty male Wistar rats,180-200g, were divided into normal control group and liver fibrosis group randomly. The two groups were established by hypodermic injection of 40% CCl4 (0.3ml/100g) per three days or peanut oils respectively. Rats were sacrificed on 2、4、8 and 12 weeks respectively. Liver index was analyzed, and liver fibrosis degree and morphological change of liver were detected by HE staining. The protein expressions of p-PERK、p-eIF2a and ATF4 in hepatic tissue of two groups on 2、4、 8 and 12 weeks were detected by immunohistochemistry and Western blotting.The RNA expressions of ATF4 and CHOP were measured by real-time PCR. RESULTS Liver index and hepatic pathological examination showed fibrous connective tissue increased significantly since hypodermic injection of CCl4 for 4 weeks, the liver fibrosis degree of 8 weeks liver fibrosis group was higher than 4 weeks liver fibrosis groups significantly,12 weeks liver fibrosis group was formed pseudo-lobule. Through immunohistochemistry, we found p-PERK, p-eIF2a and ATF4 expressed mainly in the cytoplasm of liver cells. Semi-quantitative analysis found the expressions of ATF4 protein was increased in liver tissue of 2 weeks fibrosis group(P<0.01),while there were no change of p-PERK and p-eIF2a protein in rat liver between 2 weeks fibrosis group and 2 weeks normal control group (P>0.05). In 4 weeks、8 weeks and 12 weeks liver fibrosis group, the expression of p-PERK、 p-eIF2a and ATF4 protein were elevated obviously(P<0.01);8 weeks liver fibrosis group’s positive rate of p-PERK、p-eIF2a and ATF4 protein expression were higher than 4 weeks liver fibrosis group(P<0.01); 12 weeks liver fibrosis group’s positive rate of p-PERK protein expression was higher than 4 weeks and 8 weeks liver fibrosis group(.P<0.01),12 weeks liver fibrosis group’s positive rate of p-eIF2a and ATF4 protein expression were higher than the 4 weeks liver fibrosis group(P<0.01), but there was no significant difference compared with 8 weeks liver fibrosis group (P>0.05). Western blot detection, we also found there was no change of p-PERK, p-eIF2a and ATF4 protein in rat liver between 2 weeks liver fibrosis group and normal control group. In 4 weeks、8 weeks and 12 weeks liver fibrosis group, the expression of p-PERK protein was elevated obviously(P<0.01). In 4 weeks liver fibrosis group, the expression of p-eIF2a protein was higher than 2 weeks liver fibrosis group; in 4 weeks and 8 weeks liver fibrosis group, the expression of p-eIF2a protein was higher than the 2 weeks and 4 weeks liver fibrosis group; but in 12 weeks liver fibrosis group, the expression of p-eIF2a protein was no significant difference compared with 8 weeks liver fibrosis group. Further research, in 4 weeks liver fibrosis group, the expression of ATF4 protein was no change with the 2 weeks liver fibrosis group, ATF4 protein expression increased significantly since hypodermic injection of CCl4 for 8 weeks and 12 weeks. The results of Real-time PCR revealed that there were no difference in expression of ATF4 and CHOP between 2 weeks fibrosis group and normal group, while expressions of ATF4 mRNA was increased obviously in liver tissue of 4 weeks>8 weeks and 12 weeks fibrosis group(P>0.05). We also found CHOP mRNA expression increased obviously since hypodermic injection of CCl4 for 8 weeks, which was higher than 2 weeks and 4 weeks liver fibrosis groups (P<0.01). CONCLUSION:Endoplasmic reticulum stress was induced in the process of liver fibrosis. Activation of PERK-eIF2a-ATF4 signaling pathway in the state of endoplasmic reticulum stress may play an important role in apoptosis induction of hepatocytes.