熊果酸对肝星状细胞NADPH氧化酶-Hedgehog信号通路的影响  

Ursolic acid suppressed NADPH oxidase-Hedgehog signaling pathway in hepatic stellate cells in vitro

作　　者：陈璐[1] 何文华[1] 朱萱[1] 李弼民[1] 张焜和 施凤[1] 张新华[1] 

机构地区：[1]南昌大学第一附属医院消化内科,南昌330006 [2]江西省消化疾病研究所

出　　处：《第三军医大学学报》2014年第5期427-431,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金(81260082);江西省自然科学基金(20122BAB205019)~~

摘　　要：目的探讨熊果酸(ursolic acid,UA)对大鼠肝星状细胞(HSC-T6)的NADPH氧化酶(NOX)-Hedgehog(Hh)信号通路的影响。方法将处于对数生长期的HSC-T6细胞分为6组:正常对照组、瘦素组(100 ng/mL)、熊果酸自身对照组(50μmol/L)、DPI自身对照组(20μmol/L)、熊果酸干预组(瘦素+熊果酸)、DPI干预组(瘦素+DPI)。在药物作用12 h后,提取总RNA,采用RT-PCR法检测Shh、Smo、Gli1/2的表达;药物作用HSC-T6细胞24 h后,提取总蛋白,采用Western blot分别检测Rac1和Gli2的表达;药物作用12、24、48 h后,采用MTT法检测HSC-T6细胞的增殖情况。结果RT-PCR分析显示,瘦素刺激HSC-T6细胞12 h后Smo mRNA表达较正常对照组升高(P<0.05);Shh、Gli2 mRNA表达稍高于正常对照组,但差异无统计学意义(P>0.05);熊果酸干预后Shh、Smo和Gli2 mRNA表达明显低于瘦素组及正常对照组(均P<0.05);瘦素对HSC-T6细胞Gli1 mRNA的表达无影响,而熊果酸及DPI干预也不影响HSC-T6细胞Gli1mRNA的表达。瘦素刺激HSC-T6细胞24 h后,Rac1和Gli2蛋白的表达较正常对照组升高(P<0.01);熊果酸干预后Rac1和Gli2蛋白的表达明显低于瘦素组(P<0.01)。MTT分析显示瘦素促进HSC-T6细胞增殖,熊果酸干预12 h后HSC-T6细胞的生长抑制率均显著高于瘦素组(P<0.01),随着作用时间延长,熊果酸对细胞生长的抑制作用进一步增强。结论熊果酸能抑制瘦素诱导的HSC-T6细胞Hh信号通路Shh、Smo、Gli2 mRNA和Gli2蛋白表达。熊果酸通过抑制NOX亚基Rac1蛋白表达进而抑制Hh信号通路可能是它抑制肝星状细胞生长增殖的机制之一。Objective To determine the effect of ursolic acid （UA） on NADPH oxidase （NOX）- Hedgehog （Hh） signaling pathway in hepatic stellate cells （HSCs）. Methods Culture-activated HSC-T6 cells were divided into 6 groups： normal control group, leptin group （100 ng/mL）, UA group （50 μmol/L）, NOX inhibitor DPI （20μ mol/L） group, and the 2 intervention groups pretreated with UA or DPI followed by stimulation with leptin for different times. The mRNA expression of Shh, Smo and Glil/2 was detected in above ceils after 12 hours＇ treatment by RT-PCR. The protein expression of Racl and Gli2 were analyzed in the cells in 24 h of treatment by Western blotting. The proliferation of HSC-T6 cells was detected in 12, 24 and 48 h after treatment by MrlT assay. Results RT-PCR analysis showed that Smo mRNA expression was increased when leptin stimulated HSC-T6 cells for 12 h （P 〈 0.05 ）. The expression of Shh and Gli2 mRNA was also increased compared with normal control group, but without significant difference. UA pretreatment significantly down-regulated the mRNA expression of Shh, Smo and Gli2 compared with control and leptin group （ all P 〈 O. 05 ）. Leptin, UA and DPI had no effect on Glil mRNA expression. The protein expression of Racl and Gli2 was increased when leptin stimulated HSC-T6 cells for 24 h （ P 〈 0.01 ）. UA pretreatment significantly down- regulated Racl and Gli2 protein expression compared with leptin group （ all P 〈 0.01 ）. MTT assay showed that leptin promoted the proliferation in HSC-T6 ceils. After 12 hours＇ pretreatment with UA, the cell growth was inhibited significantly than in leptin treated cells （P 〈 0.01 ） in a time-dependent manner. Conclusion UA inhibits the expression of Shh, Smo, Gli2 mRNA and down-regulates the expression of Gli2 protein in HSC-T6 cells. One mechanism of UA inhibiting cell proliferation is probably via its inhibiting NOX subunit Racl and then NOX-Hh signaling pathway.

关 键 词：熊果酸 肝星状细胞 HEDGEHOG信号通路 NADPH氧化酶 RAC1 

分 类 号：R285.5[医药卫生—中药学] R322.47[医药卫生—中医学]