Effects of Gan-fu-kang on TNF-α Mediated NF-kB Signaling Pathway in Liver Fibrotic
|School||Dalian Medical University|
|Course||Pathology and Pathophysiology|
|Keywords||Hepatic fibrosis TNF-α Gan-Fu-Kang HSC NF-kB|
Objective:Tumor necrosis factor-alpha is the regulation of cell growth, differentiation,inducing inflammation and regulation of apoptosis, is an important cytokine. Studieshave shown that TNF alpha plays a very important role on liver fibrosis in thedevelopment process. The activation of hepatic stellate cells is the key link of thedeveloping process of liver fibrosis, Promoting hepatic stellate cell apoptosis has beenconsidered to be the initial factor of liver fibrosis. Nuclear factor-kB is an importanttranscription factor. In hepatic cells,endothelial cells, HSCs can be detected its activity,NF-kB has multiple adjusting function, can regulate a variety of gene expression, canenhance a variety of molecules, thereby preventing the apoptosis of HSCs or promotingthe survival and proliferation of HSCs. This experiment is to explore in vivo and invitro Effects of traditional Chinese medicine on TNF-α mediated NF-kB signalingpathway in liver fibrotic and discusses the mechanism. Treatment of liver fibrosissupplies a new feasibility theory basis and experimental base.Methods：In vivo: All healthy SD rats were randomly divided into five groups: normalcontrol group(C group), model group(M group),GFK treatment group(high,middle, lowdose group).Except normal control group,all groups rats were subcutaneously injectedwith10％CCl4(5ml/kg) two times each week for12weeks to induce liver fibrosis.Normal control group rats were injection the same dose of saline solution with the samemethod. Twelfth weekend, normal control group and model group rats were executedand removed liver tissue quickly. The beginning of the9th week, the HT, MT, and DTgroups rats were treated respectively by3215mg/kg/day,312.5mg/kg/day,31.25mg/kg/day for12weeks. The remaining rats were killed at the end of the20th weekby the same way and removed liver tissue standby. In vitro:A line of hepatic stellate cell (HSC-T6) was cultured in Dulbecco’smodified Eagle medium (DMEM). At37℃with5%CO2incubator in cultured andpassaged. When cells are grown in good condition, cells were treated with serum-freeDMEM for24hours and randomly assigned to three groups: normal controlgroup(treated only with DMEM contains10％normal rats serum),acetaldehydegroup(DMEM contains10％normal rats serum and acetaldehyde200μmol/L),and GFKgroup(DMEM contains10％serum of medium-dose GFK treated and acetaldehyde200μmol/L).The histopathological changes of liver tissues were observed by HEstaining.Liver tissue and hepatic stellate cells expression of TNF alpha was detected byimmunohistochemistry and Western blotting method. The mRNA expression of TNF-α,IKK-α, IkB-α, NF-KBp65were observed by reverse transcriptase polymerase chainreaction (RT-PCR).Results：1.The histopathological changes of liver tissues: HE staining of liver biopsiesshowed, compared with the C group, M group rats liver tissue were damaged severely,liver cells occurs serious fatty degeneration, necrosis, apoptosis, and inflammatory cellinfiltration; hepatic lobule structure is destroyed, the pseudolobule formation, fiberspacing increases. Compared with the M group, treatment groups were improveddifferently and MT group apparently2. Immunohistochemistry and Western blotting method assay for TNF-α proteinexpression: Compared with the C group, the positive expression of TNF-α wereincreased significantly in the M group (P<0.01). Compared with the M group, thepositive expression of TNF-α were decreased evidently in the MT group (P<0.05).3.RT-PCR results show: Compared with the C group, The mRNA expression ofTNF-α, IKK-α, IkB-α, NF-KBp65in M group were increased(P<0.05), compared withthe model group, treatment group GFK of gene expression levels were reducedsignificantly(P<0.01), in which the expression of IkB-α is the opposite tendency. Theexpression of TNF-α and NF-KBp65is positive correlation, and IkB-α is negativecorrelation.Conclusion1. TNF-α mediated NF-kB signaling pathway may be playing a promoting role inthe occurrence and progression of liver fibrosis.2. Ganfukang on liver fibrosis has significantly therapeutic effects; the role of the mechanism may be inhibit TNF-α mediated NF-kB signal transduction pathway.