肝星状细胞论文：熊果酸对大鼠肝星状细胞NOX亚基p47(Phox)及其调控的下游信号通路的影响.doc

肝星状细胞论文：熊果酸对大鼠肝星状细胞NOX亚基p47(Phox)及其调控的下游信号通路的影响【中文摘要】肝纤维化是肝脏慢性损伤后的一种普遍现象,最终导致细胞外基质(extracellular matrix, ECM)在肝脏中过度沉积。瘦素是一个重要的促肝纤维化因子,它与HSC上的Ob受体结合后,引起Janus激酶(Janus kinase,JAK)磷酸化及JAK-信号转导及转录激活因子(signal transducers and activators of transcription factor, STAT)的活化。JAK还通过激活【英文摘要】Background:Hepatic fibrosis is a universal phenomenon of chronicity liver injury which eventually lead to the extracellular matrix deposition in the liver. Leptin is an important factor that can promote the liver fibrosis and cause phosphorylation of janus kinase (JAK) and JAK-signal transducers and activation of transcription factor (STAT) by binding with ob acceptor in HSCs. JAK activates reduced form of nicotinamide-adenine dinucleotide phosphate oxidase (NADPH oxidase, NOX) and the reduction-oxidation-sensitive cells signaling pathways Protein kinase B(AKT) and extracellular signal-regulated kinase (ERK) by generating reactive oxygen species (ROS). leading to the signaling cascade and gene expression related to the fibrosis and inflammation.Previously, we performed the study that Ursolic Acid (UA) could significantly improve liver tissue structure of hepatic fibrosis in vivo, and better than colchicine. It could inhibit the proliferation of HSCs and induce the apoptosis of HSCs in vitro. Pretreatment with UA could inhibit NOX subunit p22Phox and Racl mRNA and ROS induced by leptin and activation of nuclear factor-KB, and subsequently induce the apoptosis of HSCs. In this study, well observe effects of UA on NOX subunit p47pho and its downstream signaling pathway ERK1/2 induced by leptin in HSCs, and expression of collagen 1.:To explore the effects of UA on NOX subunit p47phox and its downstream signaling pathway ERK1/2 induced by leptin in HSCs(HSC-T6), and proliferation of HSCs and expression of ECM.Methods:HSC-T6 in the exponential growth phase were devided:normal control group; leptin (100ng/ml) treated; leptin treated together with UA (50M); leptin treated together with JAK inhibitor AG490 (50M); leptin treated together with NOX inhibitor DPI (20M) and leptin treated together with ERK inhibitor PD98059 (30M). HSC-T6 were treated with medicine for 30 minutes and membrane protein and phosphorylation protein expression of p47phox and ERK 1/2 were analyzed with western blotting; HSC-T6 were treated with medicine for 12 hours and mRNA expression of collagen 1 was analyzed with RT-PCR; HSC-T6 were treated with medicine for 12 hours,24 hours,48 hours and HSC-T6 proliferation was analyzed with MTT.Results:1. The effect of UA on aversion of NOX subunit p47phox induced by leptinHSC-T6 were treated for 30 minutes with Leptin, the membrane protein expression of p47phox was significantly increased compared with normal control group (P0.05); Treatment with AG490 and DPI, the data were presented as relative reduce compared with leptin treatment (both P0.05). Here we demonstrated that UA decreases the aversion of p47phox induced by leptin.2. The effect of UA on p-ERK1/2 expression induced by leptin in HSC-T6HSC-T6 were treated for 30 minutes with Leptin, the p-ERK1/2 levels were increased compared with normal control group (P0.05); Treatment with AG490,DPI and PD98059 treatment, the data were presented as relative reduce compared with leptin treatment (P0.05). Here we demonstrated that UA decreases the phosphorylation levels of ERK 1/2 induced by leptin.3. The effect of UA on mRNA expression of collagen 1 induced by leptin in HSC-T6 HSC-T6 were treated for 12 hours with Leptin, the mRNA expression of collagen 1 was increased compared with normal control group (P0.05); Treatment with AG490,DPI and PD98059 treatment, the data were presented as relative reduce compared with leptin treatment (P0.05). Here we demonstrated that UA decreases the mRNA expression of collagen 1 induced by leptin.4. The effect of UA on the HSC-T6 proliferation induced by leptinHSC-T6 were treated for 12 hours with Leptin, the HSC-T6 proliferation was increased compared with normal control group; Treatment with UA, the data were presented as relative reduce compared with leptin treatment (P0.05). HSC-T6 were treated for 24 hours with Leptin, the HSC-T6 proliferation was increased compared with normal control group; Treatment with UA, the data were presented as relative reduce compared with leptin treatment (P0.05). HSC-T6 were treated for 48 hours with Leptin, the HSC-T6 proliferation was increased compared with normal control group; Treatment with UA, the data were presented as relative reduce compared with leptin treatment (P0.05). Treatment with DPI. the data were presented as relative reduce compared with UA, AG490 and PD98059 treatment (P0.05). Here we demonstrated that UA inhibits the HSC-T6 proliferation induced by leptin.Conclusions: 1. Leptin can induce aversion of NOX subunit p47phox in HSC-T6, and activate NOX and ERK1/2. Treatment with UA can inhibit p47phox aversion and phosphorylation levels of ERK1/2.2. UA can inhibit HSC-T6 proliferation and mRNA expression of collagen 1. The mechanism may be related to inhibting activation of NOX-ERK1/2 signal net.【关键词】肝星状细胞 熊果酸 NOX亚基 ERK1/2 型胶原【英文关键词】hepatic stellate cells ursolic acid NOX subunit extracellular signal-regulated kinase1/2 Collagen 1【目录】熊果酸对大鼠肝星状细胞NOX亚基p47(Phox)及其调控的下游信号通路的影响摘要3-6ABSTRACT6-9第1章 引言13-161.1 肝纤维化的研究现状131.2 NADPH氧化酶在肝纤维化发病中的作用13-141.3 熊果酸的生物学活性及抗肝纤维化的作用14-151.4 本课题研究熊果酸抗肝纤维化的分子机制15-16第2章 材料与方法16-292.1 材料16-182.1.1 细胞株162.1.2 主要试剂16-172.1.3 主要仪器17-182.2 研究方法18-282.2.1 主要液体的配制18-202.2.2 肝星状细胞(HSC-T6)的培