【作者】 宋顺宗；

【导师】 南极星；

【作者基本信息】 延边大学 ， 药物化学， 2011， 博士

【摘要】 肝脏受到外界因素刺激,造成急性肝损伤。肝脏损伤后可以通过临床药物治疗进行一定的修复、促再生作用,减轻或控制病情进展。造成的肝损伤可由肝纤维化进展为肝硬化,可以说肝纤维化是向肝硬化发展的必经阶段,所以及时治疗肝损伤,临床早期诊断肝纤维化,并及时加以控制,可以有效阻断疾病的进一步发展和转归,具有重要的临床治疗意义。随着分子生物学与药物研究的发展,急性肝损伤以及抗肝纤维化的研究有许多突破性的进展。目前临床多用N-乙酰半胱氨酸、拉米夫啶或恩替卡韦治疗,临床疗效甚微,尚无有效的治疗方法。阻止肝纤维化的进展或促进纤维化的逆转是治疗肝纤维化的关键。为此,积极寻找有效的治疗药物及药物作用靶点已成为研究者们的重要课题。本研究对山茱萸新苷和人参皂苷的肝保护作用和抗肝纤维化作用进行深入探讨,旨在探讨是否有效阻断肝细胞凋亡,抑制炎症发展进程,是否能缓解肝损伤以及肝纤维化。本研究采用四氯化碳(CCl4)诱导肝损伤作为动物研究模型,并用肿瘤坏死因子(TNF-α)刺激肝星状细胞(HSC),探讨山茱萸新苷肝保护和人参皂苷czh-A-69抗肝纤维化作用机制。(1)四氯化碳(CCl4)诱导肝损伤作为动物研究模型,探讨山茱萸新苷肝保护作用。大鼠腹腔注射CCl4诱导肝损伤16h后血清转氨酶、TNF-α和脂质过氧化物显著升高,而肝脏抗氧化酶降低。给予山茱萸新苷后各项指标得以改善。组织学研究表明山茱萸新苷抑制CCl4诱导肝损伤。此外CCl4诱导肝损伤后诱发肝脏一氧化氮(NO)、诱导型一氧化氮合成酶(iNOS)以及环氧化酶-2(COX-2)升高,细胞色素P4502E1(CYP2E1)表达降低。给予山茱萸新苷后可以显著抑制肝脏NO形成、降少iNOS和COX-2蛋白表达,修复CYP2E1水平。研究结果表明山茱萸新苷通过调节CYP2E1水平、抑制炎症应答以及减少氧化应激作用保护CCl4诱导的肝损伤。(2)研究人参皂苷czh-A-69对活化肝星状细胞的诱导凋亡作用。肝纤维化以Ⅰ型胶原为主的细胞外基质蛋白(ECM)在肝脏的过度沉积作为表征。ECM包括Ⅰ型胶原主要来源于活化的肝星状细胞(HSC)。在纤维化进程中,HSC由大量储存维生素A的静息状态转化为仅含少量维生素A的纤维形成细胞,同时大量表达α-肌动蛋白(α-SMA)和ECM。蛋白印迹结果表明人参皂苷czh-A-69通过介导MAPK/Akt/TLR4/TAK-1、bcl-2/bax比值和线粒体细胞色素G水平诱导活化HSC凋亡,从而逆转肝纤维化。人参皂苷代谢物czh-A-69(3.125μM)作用于活化HSC细胞24h后或6.25μM作用1h可以显著调节caspase-3和PARP表达；显著抑制TIMP-1、TLR4及TAK-1磷酸化；调节bcl-2/bax比值和线粒体细胞色素C水平；增加JNK/ERK/p38/MAPK磷酸化；显著抑制Akt磷酸化。因此,人参皂苷czh-A-69通过介导MAPK/Akt/TLR4/TAK-1信号系统调节肝纤维化,数据表明人参皂苷czh-A-69对肝纤维化的干预机制为将人参皂苷czh-A-69开发用于临床肝纤维化治疗提供了一定的理论基础。更多还原

【Abstract】 The present study elucidates the effects of cornuside on carbon tetrachloride (CC14)-induced hepatotoxicity. Rats were treated intraperitoneally with0.5ml/kg CCI4. At16h after the CC14treatment, the levels of serum aminotransferases, TNF-a, and lipid peroxidation were significantly elevated, whereas the hepatic antioxidative enzyme activities were decreased. These changes were attenuated by cornuside. The histological studies also showed that cornuside inhibited the CC14-induced liver damage. Furthermore, the contents of hepatic nitrite, inducible nitric oxide synthase (iNOS), cyclooxygenase-2(COX-2) were elevated after CC14treatment, while the cytochrome P4502E1(CYP2E1) expression was suppressed. Cornuside treatment inhibited the formation of liver nitrite, reduced the over-expression of iNOS and COX-2proteins, but restored the liver CYP2E1content compared with the CC14-treated rats. Our data demonstrate that cornuside protects the liver from CC14-induced acute hepatotoxicity, which might be due to its ability to restore the CYP2E1function and suppress the inflammatory responses, in combination with its capacity to reduce oxidative stress.The hepatic stellate cell (HSC) is the primary cell type in the liver responsible for excess collagen deposition during fibrosis. HSC changes from a quiescent to an activated situation undergoing a fibrogenic stimulus associated with increased extracellular matrix synthesis and increased cell proliferation. It’s investigated how Ginsenosides czh-A-69mediated MAPK/Akt/TLR4/TAK-1and mitochondria pathways in the balance of HSC activation and apoptosis in hapatic stellate cells. An activated HSC cell line was used in the present study. Western blot was applied to detect the expression of MAPK/Akt/TLR4/TAK-1and bcl-2/bax ratio and cytochrome c in the mitochondria. Ginsenosides czh-A-69significantly inhibited the proliferation of activated HSC in a dosage or time manner. Ginsenosides czh-A-69regulated caspase-3or PARP expression at24h after cultuted with czh-A-693.125μM or1h with6.25μM. Ginsenosides czh-A-69also significantly inhibited TIMP-1, TLR4and TAK-1phosphorylation at1h after adiministation with czh-A-696.25μM. Ginsenosides czh-A-69regulated bcl-2/bax and cytochrome c expression at24h after cultuted with czh-A-693.125and1.56μM, respectively, and at12h or1h with6.25μM. Ginsenosides czh-A-69increased JNK/ERK/p38/MAPK phosphorylation at1h after cultuted with czh-A-696.25μM. Furthermore, ginsenosides czh-A-69significantly inhibited Akt phosphorylation at15min after cultuted with czh-A-696.25μM. Therefore, ginsenosides czh-A-69may inhibit the proliferation of activated HSC cell and mediated MAPK/Akt/TLR4/TAK-1and mitochondria pathways in activated murine hepatic stellate cells. Ginsenosides czh-A-69regulated MAPK/Akt/TLR4/TAK-1signaling in HSCs during active fibrogenesis resulting in inhibition of extracellular matrix deposition and reduce of profibrogenic factors expression. These data suggest that ginsenosides czh-A-69may represent an effective therapeutic medicine for hepatic fibrosis.更多还原