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目的探讨柴胡黄芩水煎液(CQ)对CCl_4诱导肝星状细胞(hepatic stellate cell,HSC)激活的作用及机制。方法取对数生长的HSC,在无血清培养基中培养24 h后,以1.5×10~9·L~(-1)浓度接种到培养板中过夜。实验分组如下:空白对照组(无药物处理),CCl_4诱导组(6 mmol·L~(-1)CCl_4作用6 h),给药组(6 mmol·L~(-1)CCl_4作用6 h后,再用400、500、600 mg·L~(-1)的CQ作用24 h),TLR4阻断剂TAK-242、NF-κB阻断剂BAY 11-7082组(6 mmol·L~(-1)CCl_4作用6 h后,再用TAK-242 10 mol·L~(-1)、BAY 11-7082 2 mol·L~(-1)作用24h)。处理之后,应用ELISA法检测培养基中透明质酸酶(hyaluronidase,HA)、层黏连蛋白(laminin,LN)、Ⅲ型前胶原(procollagenⅢ,PCⅢ)、Ⅳ型胶原(collagen typeⅣ,Ⅳ-C)浓度,RT-PCR方法检测细胞中Toll样受体4(Toll-like receptors4,TLR4)、核转录因子κB(NF-κB)基因的表达,应用Western blot法检测细胞中TLR4、NF-κB蛋白的表达。结果研究发现,与CCl_4干预组比较,CQ量效相关性降低CCl_4诱导的HSC增殖。600 mg·L~(-1)的CQ可以明显降低HA、LN、PCⅢ、Ⅳ-C细胞释放水平及TLR4、NF-κB基因、蛋白的表达。NF-κB抑制剂TAK-242、BAY 11-7082也可降低CCl_4造成的HSC增殖,HA、LN、PCⅢ、Ⅳ-C细胞释放水平及NF-κB基因、蛋白表达,不能下调TLR4基因、蛋白表达。结论 CQ可以抑制CCl_4诱导的炎症因子基因、蛋白表达,减轻肝纤维化相关指标的含量,其作用机制可能与TLR4-NF-κB转录活性及蛋白活性相关,提示其在抗肝纤维中的作用及机制。
Objective To investigate the effect and mechanism of decoction of Radix Bupleuri and Radix Scutellariae on activation of hepatic stellate cells (HSC) induced by CCl_4. Methods HSC cultured in logarithmic growth medium were cultured in serum - free medium for 24 h and then inoculated into culture plate at the concentration of 1.5 × 10 ~ 9 · L ~ (-1) overnight. The experimental groups were as follows: blank control group (without drug treatment), CCl 4 induction group (6 mmol·L -1 CCl 4 for 6 h) and administration group (6 mmol·L -1 CCl 4 for 6 h) , Then treated with 400, 500, 600 mg · L -1 CQ for 24 h), TLR4 inhibitor TAK-242 and NF-κB blocker BAY 11-7082 (6 mmol·L ~ (- 1) After 6 hours of CCl_4 treatment, TAK-242 10 mol·L -1 and BAY 11-7082 2 mol·L -1 for 24 h. After treatment, the expression of hyaluronidase (HA), laminin (LN), procollagen III (PCⅢ), collagen type Ⅳ (Ⅳ-C ), And the expressions of TLR4 and NF-κB were detected by RT-PCR. The expressions of TLR4 and NF-κB expression. Results The study found that, compared with the CCl_4 intervention group, the dose-effect relationship of CQ decreased the CCl_4-induced HSC proliferation. 600 mg · L -1 CQ can significantly reduce the release of HA, LN, PCⅢ, Ⅳ-C cells and TLR4, NF-κB gene and protein expression. The effects of NF-κB inhibitors TAK-242 and BAY 11-7082 on HSC proliferation, the release of HA, LN, PCⅢ and Ⅳ-C cells and the expression of NF-κB gene and protein were not significantly down-regulated . Conclusion CQ can inhibit the expression of inflammatory cytokines and proteins induced by CCl_4 and reduce the related indexes of liver fibrosis. The mechanism may be related to TLR4-NF-κB transcriptional activity and protein activity, suggesting its role in anti-hepatic fibrosis mechanism.