| IntroductionLiver fibrosis and its end-stage disease (cirrhosis) are major world health problems. These liver diseases arise from chronic liver injury by a variety of etiological factors. Liver fibrosis causes liver failure and portal hypertension, which often require liver transplantation in later stages. Therefore, an understanding of the pathogenesis of liver fibrosis is crucial for the development of treatments that reverse its progression.The activation of hepatic stellate cells (HSCs) plays a pivotal role in the process of liver fibrosis. HSCs are nonparenchymal, quiescent cells that store vitamin A and maintain the normal basement membrane-type matrix in the normal liver. However, liver injury induces an "activation" process in HSCs that results in vitamin A loss, proliferation, and the synthesis of a ’fibrotic’ matrix that is rich in type I collagen. Activated-HSCs release proinflammatory and profibrogenic cytokines and produce tissue inhibitors of metalloproteinases (TIMPs), which shifts the balance of the extracellular matrix towards the deposition of collagen and fibrosis.The fibrosis process was considered to be reversible in recently years. The apoptosis of activated-HSCs is key to this reversal. Therefore, activated-HSCs are major cellular targets for the prevention of liver fibrosis progression.The differentiation of HSCs is similar to that of adipocytes. Therefore, adipocyte-specific genes might also coordinately regulate alterations in HSCs. The CCATT/enhancer binding protein family (C/EBPs) plays a key role in the differentiation of adipocytes, and C/EBP-alpha (C/EBP-α) controls preadipocyte maturation and cellular growth. In our previous research we found that C/EBP-α could induce HSCs apoptosis in vitro and in vivo. But the effect of C/EBP-α on hepatocytes and the differences between hepatocytes and HSCs in vivo remain unknown. Therefore, this study investigated the different apoptotic effects of C/EBP-α on hepatocytes and HSCs in vitro and in vivo. In addition, in order to get a better understand of hepatic fibrosis, we further investigated whether acetylation was involved in C/EBP-α during hepatic fibrosis. Part oneInvestigations on whether Ad-C/EBP-a exerts different apoptotic effects on hepatocytes and HSCs in vitroObjectives To investigate the different apoptosis effect of C/EBP-α in cultured hepatocytes and HSCs.Methods An adenovirus vector-expressing C/EBP-a gene was constructed, and a rat hepatic stellate cell line (HSC-T6) and hepatocyte cell line (BRL-3A) were treated with50,100,200MOI of Ad-C/EBP-a and Ad-EGFP respectively. After48h C/EBP-a expression was detected by Western Blot. DNA ladder, flow cytometry, Annexin V stain and PI uptake were applied to detect apoptosis of the two kinds of cells. Western Blot was used to analyze apoptosis pathways, which contains caspse3,8,9,12.Results Treatment with C/EBP-α could induce apoptosis of hepatocytes and HSCs with significant difference. Compared with controls, HSC-T6showed "DNA ladder" by DNA fragmentation from50MOI, but BRL-3A didn’t even at200MOI. Apoptosis rates of HSC-T6infected with Ad-C/EBP-α were markedly increased from50MOI by flow cytometry and AnnexinV/Pl detection in a dose-dependent manner, while BRL-3A have little change until200MOI. Western Blot shows caspase3was activated from50MOI in HSC-T6,200MOI in BRL-3A. Caspase9but not caspase8or12was involved in the apoptosis of the two kinds of cells. Caspase9inhibtor could inhibit apoptosis of HSC-T6significantly, but only partly inhibit BRL-3A.Conclusions There is significant difference in apoptosis of HSC-T6and BRL-3A induced by Ad-C/EBP-α, but they are all involved with mitochondrial pathway.Part Two Investigations on whether Ad-C/EBP-α exerts different apoptotic effects on hepatocytes and HSCs in vivoObjectives To investigate effect of C/EBP-α in CCl4induced mice liver fibrosis. Methods Liver fibrosis model of mice was made by intraperitoneal injection of CCl4. Mice were administrated with Ad-C/EBP-a (week1for early intervention or week6for late intervention) through the tail vein. Mice were scarified at week10. Immunohistochemical staining and Western Blot was applied to confirm C/EBP-a expression. Sinusoidal endothelial structure was observed by transmission electron microscope(TEM); Sera were collected for biochemical analysis of liver functions include total protein, albumin, y-GT and ALT. Hematoxylin and eosin (H&E) staining, Sirius red and hydroxyproline content measurement were applied to examine the collagen content in livers. Immunohistochemical staining was applied to detect α-SMA, PCNA expression. TUNEL was applied to detect apoptosis of hepatocytes and HSCs.Results Compared to controls, Ad-C/EBP-α injection group showed high C/EBP-α expression proved by immunohistochemistry and western blot, C/EBP-α positive cells are mainly HSCs.Sinusoidal endothelial cells lost their fenestrate and formed continuous basement membrane gradually; In mice of C/EBP-α-transfected group, no matter early intervention or late intervention group, collagen content decreased significantly which was confirmed by HE, Sirius red stain and hydroxyproline content of liver. γ-GT was down-regulated in Ad-C/EBP-α injection groups, while other seral biochemical assays showed no significant difference. The numbers of α-SMA positive cells were much lower in Ad-C/EBP-α injection groups by immunohistochemistry; TUNEL showed apoptosis cells were mainly HSCs.Conclusions In vivo Ad-C/EBP-a injection can attenuate the extent of liver fibrosis in mice, greatly reduce α-SMA positive cells, and induce HSCs apoptosis with little harmful effect on hepatocyte and liver function.Part Three Investigations on acetylation of C/EBP-α-induced-apoptosis in cultured HSCsObjectives To investigate whether acetylation was involved in C/EBP-α-induced-apoptosis in HSCs and the probable mechanism involved.Methods Three HDACIs include TSA. SAHA and nicotinamide were chosed. CCK-8was applied to detect proliferation inhibition rate after treated with different dosage of the three HDACIs in HSC-T6and BRL-3A cells. Western Blot was used to detect caspase3,8,9,12in HSC-T6treated with Ad-C/EBP-α alone or combined with TSA. Combination of Realtime RT-PCR and Western Blot were applied to detect inherent C/EBP-α mRNA and protein expression in HSC-T6at different times after treated with TSA; HATs TIP60was detected by Western Blot. Co-IP was applied to detected acetylation of C/EBP-α and its interaction with TIP60.Results(1) TSA could inhibit HSC-T6proliferation inhibition rate much higher than BRL-3A in the three HDACIs, while SAHA and nicotinamide couldnot.(2) TSA could induce HSC-T6apoptosis itself, and could enhance apoptosis of C/EBP-a in HSC-T6.(3) After TSA treatment, acetylation of C/EBP-α increased. C/EBP-α protein expression level increased and varied with different treated time.(4) After treated with TSA, TIP60protein expression had a same trend as C/EBP-α.Conclusions TSA could enhance C/EBP-α expression level in HSC-T6by promoting its acetylation, no matter inherent or exogenous. Inherent C/EBP-α expression was modulated by acetylation.TIP60was involved in C/EBP-α acetylation. |
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