The Roles Of NF-κB In Liver Fibrosis

Liver fibrosis is characterized by an accumulation of extracellular matrix (ECM), which is mainly secreted by hepatic stellate cells (HSCs). Upon injury, the activated HSCs migrate to the necroinflammatory areas and proliferate there, secret collagen type I, III and tissue inhibitor of metalloproteinase 1 (TIMP-1). All of these HSC products cause ECM accumulation and finally liver fibrosis that was previously believed to be irreversible. However, recent animal study and clinical evidence indicated that the process of fibrogenesis is potentially reversible. One of the therapeutic strategies of fibrosis is to eliminate the activated HSCs. Any method inducing HSC apoptosis helps to eradicate activated HSCs. In experimental model, Oakley and colleagues found that HSC apoptosis reduced the expression of liver collagen, TIMP-1 and enhanced recovery from liver fibrosis. Therefore, an understanding of HSC apoptosis may provide a novel therapeutic approach to hepatic fibrosis. As is well known, fibrosis is frequently associated with inflammation, which is accompanied by the activation of transcription factor NF-kB. Classical NF-kB is a heterodimer of p50 and p65. Activation of NF-kB occurs after exposure of cells to LPS. Activated NF-κB translocates to the nucleus and functions as a transcription factor, inducing expression of anti-apoptotic Bcl-2 family proteins and A1. Activated HSC survival is dependent upon the NF-κB target gene A1, as siRNA targeted knockdown of A1-induced HSC apoptosis. Inhibition of NF-κB activation has been associated with apoptosis induction in many cancer cells. However, the studies of NF-κB inhibition on HSC apoptosis are still inconclusive. Some studies indicated that the inhibition of NF-κB induce HSC apoptosis, others did not. The present study studied the roles of NF-κB in liver fibrosis.Part1 Short interfering RNA targetting NF-kappa B induces apoptosis of hepatic stellate cells and attenuates extracellular matrix production Objective: The aim of the present study is to explore the effects of siRNA targetting NF-κB on the apoptosis and extracellular matrix production in hepatic stellate cells.Methods: The immortalized HSC cell line HSC-T6 was transfected with siRNA 72 h later, cells were stimulated by LPS for 1h; these cells were collected for further use. HSC apoptosis was determined by fluorescence activated cell sorter (FACS) analysis, TUNEL assay and caspase-3 activity measurement. Matrix metalloproteinase 2 (MMP-2) activity was evaluated with Gelatin zymography. The quantities of mRNA transcriptions of NF-κB p65, type I collagen, tissue inhibitor of metalloproteinases-1 (TIMP-1),α-smooth muscle actin (α-SMA) and transforming growth factor beta 1 (TGF-β1) and antiapoptotic protein A1 were evaluated with quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR).Reults:①siRNA knockdown of NF-κB in HSCs The siRNA knockdown of NF-κB p65 in HSCs was examined using western blot analysis and qRT-PCR. The expression of NF-κB p65 protein in siRNA group was decreased by (85.12±5.10)% compared with the scramble siRNA group (P<0.01). NF-κB p65 mRNA in siRNA group was decreased by (86.34±1.01)% at 72 h as compared with the scramble siRNA group (P<0.01). These results indicated that NF-κB siRNA effectively inhibited the expression of NF-κB p65 in HSCs.②NF-κB siRNA induced apoptosis in HSCs It has been reported that flow cytometry is the most sensitive method to examine apoptosis. We first tested the effect of NF-κB siRNA on DNA fragmentation by FACS analysis after propidium iodide staining of the cells. Apoptosis cells were significantly increased in siRNA transfected cell (15.40±0.72)% as compared with the scramble siRNA group (1.69%±0.55)% (P<0.05). TUNEL study also demonstrated the similar results. Caspase-3, a key enzyme in various forms of apoptosis and a biochemical hallmark of apoptosis, was also evaluated and the results showed that the activity of caspase-3 in NF-κB siRNA group was significantly increased compared with the scamble siRNA group.③NF-κB siRNA inhibited Bcl-2 protein and A1 mRNA expressions in HSCs Apoptosis is regulated by Bcl-2 family and A1 protein. To determine if knockdown of NF-κB by siRNA regulates Bcl-2 and A1 expressions in HSCs, the expression of Bcl-2 was examined by western blot analysis and qRT-PCR for A1 mRNA expression. The results showed that Bcl-2 protein (0.31±0.14 vs 0.71±0.06, P<0.050) and A1 mRNA expressions (0.60±0.06 vs 0.9±0.10, P<0.05) were significantly decreased in the activated HSCs transfected by NF-κB siRNA compared with those by scramble siRNA.④NF-κB siRNA increased MMP-2 activity, reduced the mRNA expression of type I collagen,α-SMA, TGF-β1, and TIMP-1 To investigate the effect of siRNA on the ECM production in HSCs, we detected MMP-2 activity, mRNA expression of TIMP-1, type I collagen,α-SMA, and TGF-β1. The results demonstrated that HSCs transfected by NF-κB siRNA significantly enhanced MMP-2 activity compared with those by scramble siRNA group. More than that, qRT-PCR indicated that type I collagen, TIMP-1,α-SMA, and TGF-β1 mRNA expressions were significantly decreased in HSCs transfected by NF-κB siRNA compared with those by scramble siRNA.Conclusions: siRNA targeting NF-κB eliminates NF-κB expression, inhibits anti-apoptotic factors such as Bcl-2, A1, stimulates proapoptotic factor such as caspase-3, and induces apoptosis. Another effect of NF-κB siRNA is to inhibit ECM-related genes such as type I collagen,α-SMA, TGF-β1. NF-κB knockdown also reduces TIMP-1, augments MMPs activity; the final consequence is the degradation of ECM.Part 2. Effect of bicylol on apoptosis, mitochondrial membrane potential, and Caspase 3 activity of rat Hepatic Stellate cellObjective: To study the role of bicylol on apoptosis, mitochondrial membrane potential, and Caspase 3 activity of HSC-T6 and inhibition of activity of NF-κB induced by H2O2.Methods: Rat HSCs was cultured in vitro and divided into 6 groups①blank control group,②0.01mM bicylol group,③0.1mM bicylol group,④0.5mM bicylol group,⑤without DMSO group,⑥AC-DEVD-CHO group. The cell proliferation of HSC-T6 were measured by 3H-TdR incorporation assay; apoptosis rate of HSC-T6 were studied With Hoechst3325 staining by fluorescence microscope; Mitochondrial membrane potential of HSC were detected under laser confocal microscope and flow cytometry after JC-1 dye staining.Results:①Bicyclol induced apoptosis of HSC-T6 time- and dose- dependently. After bicyclol treatment 24h, staining HSCs with Hoechest3325, imaging under fluorescence microscopy, we find that the nuclear enrichment and the formation of apoptotic bodies, find there were some HSCs apoptosis. The difference of each group apoptosis rate overall mean was statistically significant (P<0.05), it was show that bicyclol have effect on the rate of apoptosis. 0.5 mM bicyclol Group apoptosis rate overall mean was significantly higher than that of 0.1 mM bicyclol Group, 0.1 mM bicyclol apoptosis rate overall mean was significantly higher than that of control group, no DMSO group and 0.01mM bicyclol group (P<0.05). 0.5 mM bicyclol can significantly increase HSCs apoptosis.②Bicyclol had inhibition effect on HSCs proliferation in dose dependently.③The effect of bicyclol on the activity of NF-κB The expression of NF-κB p65 in HSC-T6 induced by H2O2were reduced in the bicyclol group compared with control group. The subcellular localization of the NF-κB p65 protein was assessed by immunocytochemistry and confocal microscopy. Nuclear fluorescence characteristics of translocated p65 protein were observed under basal condition consistent with the constitutive activated state of these cells. In contrast, p65 immunofluorescence was cytoplasmic and excluded from the nucleus in bicyclol -treated cells.④The effect of bicyclol on mitochondrial membrane potential By JC-1 confocal laser scanning microscopy detecting mitochondrial membrane potention, by analysis of variance, the difference of each group overall mean of mitochondrial membrane potential was statistically significant (P<0.05). The mitochondrial membrane potentials of 8h Group, 16h group and the positive control group have no difference; all was lower than that of the negative control group. it was show that 0.5mM bicyclol can reduced aHSCs mitochondrial membrane potentials , reaching the peak at 8h; after 24h, the effect begin decline. By JC-1 flow cytometry assaying HSCs mitochondrial membrane potention, the mitochondrial membrane potentials of 0.5mM Bicyclol group and the control group in after interval 8h and 16h,by T test, P<0.05, were statistically significant, were lower than the control group, showing that 0.5mM bicyclol can reduced aHSCs mitochondrial membrane potentials. By analysis of variance, the difference of the mitochondrial membrane potential of 8h, 16h, was statistically significant(P<0.05), by SNK multiple comparisons, of the mitochondrial membrane potential of 24h group is higher than 8h, 16h group. 8, 16 h two groups are of non-discriminatory.⑤The effect of bicyclol on Caspase3 activity detection Show that, by analysis of variance, the difference of each group Caspase3 overall mean was statistically significant (P<0.05), by SNK multiple comparisons, except the control group between 0.01mM group have no difference(P>0.05), 0.1mM group and Ac-EDVE - CHO group are of non-discrimination, the other the overall mean difference between the two groups were statistically significant (P<0.05), 0.5mM group Caspase3 have highest activity, followed by 0.1mM group and Ac-EDVE-CHO group, the control group and 0.01mM group are the lowest. It was show that 0.5mM bicyclol can increase the activity of the aHSCs.⑥The effect of bicyclol on ECM To investigate the effect of bicyclol on the ECM production in HSCs, we detected mRNA expression of TIMP-1, type I collagen,α-SMA, A1, and TGF-β1. The results demonstrated that bicyclol significantly decreased type I collagen, TIMP-1,α-SMA, and TGF-β1 mRNA expressions compared with those control group.Conclusion: Bicyclol inhibits anti-apoptotic factors such as A1, stimulates proapoptotic factor such as caspase-3, and induces apoptosis. Another effect of Bicyclol is to reduce mitochondrial membrane potention and inhibit ECM-related genes such as type I collagen,α-SMA, TGF-β1. Part 3 Protective Effect of bicyclol on CCL4-induced Acute Liver Injury via inhibition NF-κB in MiceObjective: To study the protective efflect of bicyclol on CCL4 induced liver injury and efflect on the expression of NF-κB, ICAM-1, COX-2 andα-SAM in mice.Methods: Sixty male mice were divided into six growps at random namely control group, modle group, bicyclol low dosage group (0.5 mg/ml /10g), bicyclol mild dosage group (1 mg/ml/10g) and high dosage group (5mg/ml/10g) and NF-κB inhibition group. The mouse of the bicyclol groups received 0.04ml/10g with different dose bicylol by oral administration, while other mouse received saline 0.04ml/10g. At the third day, mouse were injected introperitoneally by 1% CCL4, 0.1ml/10g except control group. Two hours after, the bicyclol groups recieved bicyclol treatments 0.04ml/10g with different dose bicylol. Other group received saline 0.04ml/10g. At fifth day, all animals were sacrificed. The serum were stored -20℃until ALT and AST assay. The liver specimens were stored at -70℃until MDA and SOD assay or fixed in 10% phosphate-buffered formaldehyde and embedded in parflin. Tissue section were stained with hematoxylin-eosin (HE).Other tissue section were stained immunohistochemstrically to detect ICAM-1, COX-2,α-SAM, and NF-κB and positive area were messured.Results: The acute liver inyury was induced successfully by CCL4. It is characterized by a necrosis of pericentral hepatocytes. Bicyclol could decrease ALT, AST in serum and MDA in hepatic homogenate and increase SOD compaired model group dose denpendly (P<0.01). ICAM-1 immunoreactivity accumulated in large amounts in the necrosis areas of model group. The expression of ICAM-1, COX-2,α-SMA and NF-κB were significantly reduced in bicyclol groups dose denpendly comparesed with model group.Conclusions: Bicyclol may protect liver lesions induced by CCL4 and inhibited the activity of NF-κB and the expression of ICAM-1, COX-2,α-SMA.