Inhibition Of Liver Fibrosis By Silencing Intracellular Oxygen And Oxidative Stress Sensors In Hepatocytes

Liver fibrosis is a common pathological process resulting from chronic liver damage,many kinds of stress such as hypoxia,oxidative stress involved in this process.The prolylhydroxylase-hypoxia-inducible factor(PHD-HIF)pathway mitigates hypoxia whereas the suppression of PHD enzyme activity allows stabilization HIF-1 which activates kinds of reactions against hypoxia.As the same,inhibiting the intracellular oxidative stress sensor Keapl(Kelch-like ECH-associated protein 1)upregulates the expression of Nrf2(Nuclear factor-erythroid 2 p45-related factor 2)which activates cellular antioxidant response to defence oxidative stress.As a central part of the liver,hepatocytes consist of more than 70%,which is highly susceptible to hypoxia.Damaged hepatocytes release profibrogenic cytokines,that activate hepatic stellate cells(HSCs),and further aggravating the development of liver fibrosis.So the present study we used shRNAs to knockdown the expression of PHD 1 and Keapl simultaneously in vitro and in vivo,expecting to attenuate the hepatocytes'response to hypoxia and oxidative stress injuries,and furthermore exploring new target therapies for liver fibrosis.Part oneAnalysis of hypoxia and oxidative stress in liver fibrosisObjectives To conduct the CCl4-induced liver fibrosis model,investigating the hypoxia and oxidative stress in hepatocytes of liver fibrosis.Methods The rats were injected with 40%CCl4 olive oil suspension(1.5ml/kg)twice a week for up to 8 weeks.To estimate the severity of liver fibrosis,HE staining,Masson staining,and immunocytochemical staining technique for measuring the expression of a-smooth muscle actin(a-SMA)were used.Meanwhile,by detecting the expression of HIF-la,HIF-2? s Keap1,Nrf2 using immunocytochemical staining,the hypoxia and oxidative stress in hepatocytes of liver fibrosis were evaluated.Results We successfully established the CCl4-induced liver fibrosis model,and immunohistochemistry staining indicated that the expression of HIF-1?,HIF-2? and Keapl were markedly increased in rats treated with CCl4 as compared with vehicle controls.However,Nrf2 expression was decreased correspondingly,PHD 1 showed no obvious change.Conclusions These results suggested that hypoxia and increased oxidative stress implicated in hepatocytes of hepatic fibrogenesis.Part twoEffects of double-knockdown PHD1 and Keapl on the functions of hypoxic hepatocytesObjectives To evaluate the effect of double-knockdown PHD1 and Keapl on the functions of hypoxic hepatocytes.Methods Four kinds of PHD 1 shRNA and Keap 1 shRNA were respectively designed,and the transfection efficiency were measured by fluorescence microscope.The expression of PHD 1 and Keapl was analyzed by quantitative real-time polymerase chain reaction(qRT-PCR).In this way,the best sequence of gene silencing was to choose out.Then the PHD1shRNA and/or Keap1shRNA transfected mouse AML12 liver cells were exposed to 1%hypoxia,The lipid peroxidation and antioxidant enzymes activity were carried out by malondialdehyde(MDA)and reduced glutathione(GSH)assay.The analysis of apoptosis were assessed using Annexin V-APC/DAPI.And the CCK8 and lactate dehydrogenase(LDH)assays were performed to evaluate the potential cellular activity,and the expression of alpha-1 type I collagen(COL1A1),transforming growth factor-?1(TGF-? 1),and vascular endothelial growth factor-A(VEGF-A),insulin-like growth factor 1(IGF-1)were investigated via qRT-PCR and western blot.The Enzyme-linked immunosorbent assay(ELISA)was employed to determine the COL1A1,type ? collagen(COL3A1)levels in supernatant of co-transfected cells.Results Compared with the expression in single-transfected cells,co-transfected of these shRNAs displayed antioxidant effects,reduced hypoxia-induced cell apoptosis and higher cellular activity.In addition,co-transfected with PHD1shRNA and Keap1shRNA significantly further declined the mRNA and protein levels of TGF-?1,VEGF,IGF-1 in an hypoxia-treated AML12 hepatocytes compared with that in single-transfected group.ELISA also revealed the decreased COL1A1,COL3A1 expression in co-transfected cells,which is consistent with Western blot and qRT-PCR results.Conclusions The double-knockdown PHD1 and Keap1 attenuated the hepatocytes'response to hypoxia and oxidative stress injuries.Part threeEffects of hypoxic hepatocytes with double-knockdown PHD1 and Keapl on the functions of HSCsObjectives To evaluate the effect of hypoxic hepatocytes with double-knockdown PHD1 and Keapl on the functions of HSCs.Methods Conditioned medium(CM)from hypoxic co-transfected AML 12 cells was to culture the rat hepatic stellate cells(HSC-T6),then the expression of COL1A1,a-SMA,VEGF-A,TGF-?1 and IGF-1 were detected.Annexin V-APC/PI was used to evaluate the cells apoptosis,and CCK8 assay were to measure the cells proliferation.Results Compared with the expression in single-transfected CM,co-transfected CM further inhibited the expression of MMP-2,?-SMA,TGF-?1,VEGF-A and IGF-1 in HSC-T6 cells.Cellular proliferation were suppressed whilst apoptosis was enhanced when cultured in hypoxic double-knockdown CM.Conclusions Double-knockdown CM further alleviated HSCs activation commared with that in single-knockdown.Part fourEffects of double-knockdown PHD1 and Keapl in mice on CCl4-induced hepatic fibrosisObjectives To investigate whether double-knockdown PHD1 and Keapl in mice can enhance the resolution of CCl4-induced liver fibrosis.Methods The liver fibrosis model was made by intraperitoneal injection 25%CCl4 olive oil suspension(4ul/g)twice a week for about 8 weeks.From the beginning(preventive group)or the fifth week since the initiation of CCl4 injection(therapeutic group),PHD1shRNAand Keap1shRNA were given by hydrodynamic-based vein injection.Fluorescence microscope was used to observe the red and green fluorescence,and western blot to measure the expression of PHD1 and Keapl.HE,sirius red and Masson staining were applied to define the pathological stage of fibrosis.Immunocytochemical staining technique were used to evaluate the expression of a-SMA.Results The fluorescence of RFP and GFP were observed under a microscope.Compared with that in CCl4 group,downregulation of PHD1 and Keapl protein expression in liver was detected by western blot.Double-knockdown PHD1 and Keapl in mice relieved the degree of liver fibrosis via HE,sirius red and Masson staining,and meanwhile,immunocytochemical staining indicated that the expression of a-SMA was decreased when PHD1 and Keapl were both knockdown.Conclusions PHD1 and Keapl expression in liver could be knocked down through hydrodynamic-based vein injection of PHD1shRNA and Keap1 shRNA,and thus alleviating hepatic fibrogenesis.