Study On The Notch-Hif-1? Signaling Pathway In Rat Liver Regeneration

Primary hepatic carcinoma(Hepatocellular carcinoma, HCC) is one of the most common malignant tumors in digestive system. Along with the development and progress of surgical techniques, hepatic lobectomy has become the only cure for cancer treatment. But the complications of liver regeneration after hepatectomy, which lead to liver function failure, have became a major problem for the surgeon. To further research on the mechanism of liver regeneration has a very important guiding significance to the clinical treatment. Previous studies have found that Notch signaling pathway and Hif-1? signaling pathway play a role in the regulation of liver regeneration. It is not clear whether there is a regulatory relationship between the two signaling pathways in liver regeneration. It is very important to elucidate the mechanism of this mechanism for the study of the mechanism of liver regeneration. ObjectiveIn this study, we used the gamma secreting enzyme inhibitor Fli-06 blocking Notch signaling pathway to study the liver regeneration after partial hepatectomy in rats.Meanwhile, we extracted primary hepatocytes to study the relationship between the Notch-Hif-1? signaling pathway. Methods1. SD rats were divided into control group(with intraperitoneal injection of normal saline, n=32) and experimental group(with intraperitoneal injection of gamma secretase inhibitor, n=32). The two groups were treated by partial hepatectomy, and each group was divided into four subgroups according to the time point when remnant liver tissues were preserved, namely, 0 d, 1 d, 3 d and 5d. The liver weight / body weight ratio was detected.2. The expression of Ki-67 and PCNA in the two groups of rats was detected by immunohistochemical method. The expression of HGF and VEGF protein was detected by the method of serum ELISA.3. The expression of Hif-1 alpha liver tissues of two groups were detected by immunohistochemistry method. The expression of Notch1, Hes1, VEGF, Hif-1 alpha HGF mRNA in regenerated liver tissues were detected by RT-PCR. NICD and Hif-1 alpha protein of the liver tissues during the liver regeneration process were analyzed and compared by Western-Blot methods.4. The primary hepatocytes were cultured by the classical two step perfusion methods, until the cells were successfully cultured and adherent.5. The primary cultured hepatocytes were divided into 2 groups, one group was control group(DMSO), and another group was the inhibitor group(dissolved DMSO of FLI-06). The expression of Notch1, Hes1, VEGF, Hif-1 alpha mRNA in hepatocytes were detected by RT-PCR. The expression of NICD, Hif-1 alpha and VEGF protein in liver cell proliferation was detected by Western-Blot method. Results1. Notch signaling inhibition in rat liver resection model was successfully established. The liver weight/body weight ratio in inhibitor group was significantly lower than the control group. There was a significant difference between the two groups(P?0.05). Immunohistochemical results suggested that the percentage of Ki-67 and PCNA positive cells in the regenerating liver in the inhibitor group after 1 d, 3 d, 5 d were significantly lower than the control group(P<0.05).2. Western Blot results suggested that the expression levels of NICD and Hif-1 alpha in 1 d, 3 d,5 d were significantly lower than the control group(P<0.05). RT-PCR results indicated that Hif-1 alpha, Hes1 HGF mRNA in inhibitor group were lower than the control group after 1 d, 3 d. There was a significant difference between the two groups(P<0.05). VEGF mRNA in inhibitor group were lower than the control group after 3 d, 5 d. There was a significant difference between the two groups(P<0.05).3. Primary rat hepatocytes were isolated and cultured successfully. Western Blot results suggested that the expression levels of NICD, Hif-1 alpha and VEGF were significantly lower than the control group(P<0.05). RT-PCR results indicated that Hif-1 alpha, Hes1 VEGF mRNA in inhibitor group were lower than the control group(P<0.05). ConclusionThe ability of the liver regeneration of rats after hepatectomy decreased significantly after gamma secretase inhibitors inhibited Notch signaling pathway.It was found that Hif-1 alpha signaling was downregulated in primary hepatocytes after gamma secretase inhibitors inhibited Notch signaling pathway. The results showed that the Notch-Hif-1 alpha signaling pathway may be involved in the regulation of rat liver regeneration.