Preliminary Study On The Protective Mechanism Of Hepatocyte Growth Promoting Factor On Hepatocytes After Blockage Of Hepatic Blood Flow In Rats

Objective: To observe the protective effect of Promoting Hepatocyte Growth Factor(PHGF)on hepatocytes of RNAi rats,a rat model of hepatic inflow blockade was established by targeting mitochondrial transcription factor A(TFAM)and nuclear Respiratory Factor-1(NRF-1).To preliminarily study whether PHGF affects mitochondrial DNA(mtDNA)synthesis by regulating TFAM and/or NRF-1,thereby protecting mitochondrial function of hepatocytes.Methods: The first step is to establish a rat model of RNA targeting NRF-1 and TFAM.RNAi template sequence was designed and small interfering RNA(siRNA)was synthesized by chemical synthesis.Lipofectamin 2000 was used to transfect siRNA into HSC-T6 cells.Real Time PCR and Western blot were used to detect the effect of RNAi.According to the results,siRNA with the best interference effect was screened out.The lentivirus was packaged with the RNA sequence with the best inhibitory activity.Lentivirus granules were injected into rats via tail vein.The contents of TFAM and NRF-1 in hepatocytes were measured,and the effect of RNAi was observed compared with that of normal rats.The second step was to establish a rat model of RNAi blocked by hepatic blood flow and observe the effect of exogenous PHGF on the expression of NRF-1 and TFAM.In the experiment,SD rats,RNA rats targeting TFAM and RNA rats targeting NRF-1 were used.Rats in each group were randomly divided into experimental group and control group.The experimental group received intraperitoneal injection of PHGF 30 minutes before operation and daily after operation.The control group was given intraperitoneal injection of normal saline at the same time.Pringle maneuver was performed in both groups for 20 minutes.The changes of liver function,liver pathology and the expression of TFAM and NRF-1 in liver tissue at different time points(1d,3d,7d)after operation were observed.Statistical software SPSS18.0 was used to process the data obtained from each test.Results:(1)The interference effects of T2 sequence and N3 sequence targeting NRF-1 were the best among the three interference sequences targeting the target gene TFAM;(2)After the successfulpackaging of the two effective interference sequences,the rat liver was effectively infected by the two viruses,and the expression of target genes was downregulated;(3)Liver function was significantly impaired(ALT,AST,TB increased significantly)1 day after operation,Liver pathology showed congestion,structural damage and cell degeneration in different degrees,The ALT,AST and TB values in gene silencing group(group B and C)were higher than those in normal group(group A).There was no significant difference between the experimental group and the control group.With the recovery after operation,The ALT,AST and TB values of liver function decreased in all groups,On the 7th day after operation,the ALT,AST and TB values in the experimental group decreased more significantly than those in the control group.(4)The expression of TFAM in liver tissue of RNA rats targeting TFAM decreased more significantly than that of normal rats on the first day after operation;The expression of NRF-1 in liver tissue of RNAi rats targeting NRF-1 decreased more significantly than that of normal rats on the first day after operation;There was no significant difference between the three experimental groups and the control group.With the recovery after operation,The expression of TFAM/NRF-1 in each group increased after 3 and 7 days.The expression level in the experimental group was higher than that in the control group.At the last time point,the increase of target gene expression in gene silencing group was lower than that in normal group.Conclusion: PHGF has protective and restorative effects on the liver of RNAi rats targeting TFAM/NRF-1 after blockade of portal vein blood flow.Promoting the expression of TFAM and NRF-1 is an important way to play a protective role.