熊果酸(UA)对大鼠活化型肝星状细胞(HSC)的NADPH氧化酶(NOX)亚基及PI3K/Akt、P38MAPK信号通路活化的影响

 目的  观察熊果酸(ursolic acid,UA)对大鼠活化型肝星状细胞-6 (hepatic stellate cell T6,HSC-T6) NADPH氧化酶(NAPDH oxidase,NOX)亚基及其调控的磷脂酰肌醇-3-羟激酶/蛋白激酶(phosphatidyl inositol 3-kinase/Akt,PI3K/Akt)、P38丝裂原活化蛋白激酶(P38 mitogen activated protein kinase,P38MAPK)信号通路及其对基质金属蛋白酶组织抑制剂-1 (tissue inhibitor of metalloproteinase-1,TIMP-1)、基质金属蛋白酶-1(metal matrix proteinase -1,MMP-1)蛋白表达的影响,并探讨其机制。方法  取对数生长期的HSC-T6细胞,随机分成6组:空白对照组、瘦素组(100 ng/mL)、UA干预组(UA 50 μmol/L+瘦素)、NOX抑制剂DPI干预组(DPI 20 μmol/L+瘦素)、P38MAPK抑制剂SB203580干预组(SB203580 10 μmol/L+瘦素)及PI3K抑制剂LY294002干预组(LY294002 10 μmol/L+瘦素)。采用Western blot法分别检测NOX亚基gp91phox、p22phox、p67phox、Rac1蛋白表达;信号通路PI3K、Akt、P38MAPK的活化及TIMP 1、MMP 1蛋白表达。结果  UA能显著抑制瘦素诱导的gp91phox、 p22phox、p67phox、Rac1蛋白表达(P均<0.01),但UA对gp91phox、p67phox蛋白表达的抑制作用不及DPI和LY294002。UA能抑制瘦素诱导的PI3K蛋白表达及Akt、P38MAPK蛋白磷酸化(P均<0.01),与DPI及LY294002作用的差异无统计学意义。UA能抑制瘦素诱导的TIMP-1蛋白表达,同时促进MMP-1蛋白表达(P均<0.01)。结论  UA能抑制瘦素诱导的大鼠HSC T6细胞NOX亚基gp91phox、p22phox、p67phox、Rac1表达及PI3K/Akt、P38MAPK信号通路的活化;其下调TIMP-1蛋白及上调MMP-1蛋白表达的机制可能与UA抑制NOX调控的PI3K/Akt及P38MAPK信号通路有关。

Effects of ursolic acid (UA) on NADPH oxidase (NOX) subunit and its regulation on downstream signaling pathways in rat activated hepatic stellate cells (HSC)

Objective  To observe the effects of ursolic acid (UA) on the express ion of NADPH oxidase (NOX) subunit and its regulation on phosphatidyl inositol 3-kinase/Akt (PI3K/Akt) and P38- mitogen-activated protein kinase (P38MAPK) pathways and tissue inhibitor of metalloproteinase-1 (TIMP-1),metal matrix proteinase -1 (MMP-1) protein expression in rat activated hepatic stellate cells (HSC-T6),and explore the underlying mechanism.Methods   HSC-T6 cells in the exponential growth phase were randomly divided into six groups:Normal control group;Leptin (100 ng/mL) group;UA treatment group (Leptin treated together with UA50 μmol/L);DPI treatment group (Leptin treated together with DPI 20 μmol/L);SB203580 treatment group (Leptin treated together with SB203580 10 μmol/L) and LY294002 treatment group (Leptin treated together with LY294002 10 μmol/L).Protein expression of NOX subunit gp91phox,p22phox,p67phox,Rac1 and PI3K,p Akt,p P38MAPK and TIMP-1,MMP-1 were analyzed with western blotting.Results  UA inhibits the protein expression of gp91phox,p22phox,p67phox,Rac1 induced by leptin (all P<0.01);and effects of UA on gp91phox and P67phox were weaker than DPI and LY294002.UA inhibits the protein expression of PI3K and phosphorylation levels of Akt,P38MAPK induced by leptin (all P<0.01),but had no difference compared with DPI and LY294002 treatment (P>0.05).UA inhibits the protein expression of TIMP-1 and promotes the protein expression of MMP-1 induced by leptin (both P<0.01).Conclusions  UA could inhibit the protein expression of NOX subunit gp91phox,p22phox,p67phox,Rac1 and the phosphorylation levels of PI3K,Akt,P38MAPK induced by leptin in rat HSC T6.The mechanism of UA inhibits protein expression of TIMP-1 and promotes MMP-1 protein expression might be related to inhibiting activation of NOX mediated PI3K/Akt and P38MAPK signaling pathways.