Bicyclol In Proliferation And Apoptosis In Rat Hepatic Stellate Cells

Chronic liver disease is a kind of serious harm to human health worldwide,Hepatic fibrosis is middle and key element of this process.The main pathological characteristic of hepatic fibrosis is the increased deposition of newly formed extracellular matrix(ECM),resulting from a shifted balance between collagen synthesis and degradation.Though the pathogenesis of diverse hepatic diseases is different,the ultimate approach to liver fibrosisis similar.The activation of hepatic stellate cells(HSCs) plays a pivotal role in this process.Hepatic stellate cell(HSC) is the beginning of whole thing and play an important role in the process of the hepatic fibrosis. Inducing the apoptosis of the activated hepatic stellate cell can reverse the process of hepatic fibrosis. The increasing research has indicated that the activation and proliferation of HSC were related with the inhibition of apoptosis. Nuclear factor-κB (NF-κB) has a close relation with apoptosis. NF-κB, a protein that binds specifically to an enhancerκB sequence ofκimmunoglobin light chain is generated via homodimeric and heterodimeric interactions of Rel subunits. Mammalian Rel proteins found by now include p65 (RelA,NF-κB3), p50 (NF-κB 1) ,Rel (c-Rel),RelB和p52 (NF-κB 2), and p65/p50 are found earliest, distribute most widely, and are the NF-κB that play chief physiological functions. NF-κB can induce transcription and expression of multiple cellular cytokines by sequence specifically binding to their promoter or enhancer region, thus promotes inflammation, immunological response, and has been closely correlated with certain important pathological and physical process including cell proliferation, transformation and apoptosis.Bicyclol is the Chinese academy of medical sciences institute of drug resistance hepatitis drug development,the chemical name for a _l,5 a carbonyl group, which was a 2,3,2, 3 pair of methylene 2 oxygen radicals 4,4 Dimethyl oxygen radicals. Previous research shows,many experiments bicylol on acute and chronic liver injury were significantly protective effect. Clinical treatment for chronic hepatitis b, c viral hepatitis have achieved good effect,And the characteristics of good oral absorption and less adverse reactions. In addition, bicyclol still can make liver fibrosis rats ALT, AST, total bilirubin, hyaluronic acid (HA) andⅢtype (PⅢbefore collagen peptide level decreased significantly , albumin levels significantly increased. Hint us bicyclol may cut through the NF-κB gene expression in HSC, thereby increasing the apoptosis, slow HSC hepatic fibrosis. The main purpose of this paper is to study bicyclol experimental HSC whether antiap- optotic, thus to expand bicyclol provides the theory basis for clinical application.Objective: Through different concentration by design in vitro bicylol on the hepatic stellate cells, the test investigates the effects on proliferation and apoptosis of the tumor necrosis factor (TNF - a ) stimulated HSC.Methods: Rat HSC was cultured in vitro and divided into 4 groups①b lank control group;②T NF-a group: dealing with TNF-a;③bicyclol group: dealing with bicyclol;④bicyclol conbined TNF-a group: dealing with bicyclol and TNF-a .The expression levels of NF-κBp65 Nuclear proteins were detected by electrophoretic mobility shift assay; Determination of the distribution of NF-κB in HSC by immunofluorescence staining method ; the expression level of Matrix metalloproteinases inhibitory factor - 1 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR),;MTT method for determining proliferation rate of HSCs ;and the change of apoptosis of HSC was detected by terminal deoxynucleotidy1 transferase-mediated dUTP nick end labeling, TUNEL.Results:1.The result of the EMSA displayed that blank control group can be detected combination of high activity.TNF-a stimulation,combined with activity of NF-κB increased obviously( P < 0.01 ) .Bicyclol intervention,combined with activity of NF-κB bicyclol group decrease obviously.Bicyclol plus TNF-a group can be detected combination of high activity,compared with TNF - a group without apparent shrinkage(P>0.05). 2.Immunohistochemistry technique displayed that:the positive cells of NF-κB relatively increased in control and TNF-a groups, with normal configuration and in fluorescence microscopy can be observed red fluorescence . TNF-a group observed the fluorescence intensity of red was more than control group. The positive cells of NF-κB decreased obviously in groups treated by Bicyclol ,with densestained apoptotic cells.3.The result of the RT-PCR displayed that Matrix metalloproteinases inhibitory factor - 1 mRNA expression of the 0.5mmol·L-Bicyclol group in the cell decreased obviously(P< 0.01 ) ,compared with blank control group,TNF-a group,bicyclol combined TNF-a group,displayed that after the intervention of the bicyclol,the mRNA expression of Timp1 in reduced.4.MTT method displayed:compared with the control group, TNF-a group at 24h, 48h and 72h are significant differences, group bicyclol 48h (P <0.05) have significant differences. compared with TNF-a group, bicyclol Group 24h, 48h and 72h are significant differences, TNF-a combined bicyclol group at 24h, 48h and72h are also significant differences5.The TUNELdisplayed.:after by Bicyclol,24h, 48h, 72h three groups 0.5mmol L bicyclol apoptosis than TNF-a increased obviously (P <0.01), and 48h, 72h of apoptosis rate between the two groups had no significant difference(P>0.05)Conclusion:1.TNF-a could stimulate the proliferation of HSCs,increase protein expression and binding activity of NF-κB in vitro;Bicyclol could inhibit protein expression and binding activity of NF-κB in vitro.2.Bicyclol might inhibit the proliferation of TNF-a-stimulated HSCs and induce HSC apoptosis in vitro;3.The apoptosis of HSC increased after Bicyclol interfere,and the expression of Matrix metalloproteinases inhibitory factor - 1 mRNA decreased ,which can delay the process of hepatic fibrosis.