基于TLR4-NF-κB的柴胡黄芩水煎液抑制CCl4诱导大鼠肝星状细胞激活作用机制研究  

Inhibitory effects of Bupleurum chinense-Scutellaria baicalensis decoction on activation of rat HSC induced by CCl_4

作　　者：李敏[1] 韩艳[1] 王华林[1] 王義雯 李静[1] 王斌[1] LI Min HAN Yan WANG Hua-lin WANG Yi-wen LI Jing WANG Bin(College of Pharmacy, Shanxi University of Chinese Medicine,Xian 712046,China)

机构地区：[1]陕西中医药大学药学院,陕西西安712046

出　　处：《中国药理学通报》2017年第5期729-732,共4页Chinese Pharmacological Bulletin

基　　金：陕西省科技厅项目(No 2013jk4023);陕西省中医药管理局项目(No zy06);陕西省教育厅项目(No 12JK1016)

摘　　要：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。Aim To investigate the inhibitory effect of Bupleurum chinense-Scutellaria baicalensis decoction（CQ）on activation of rat HSC induced by CCl4 and the mechanism.Methods Cells in logarithmic growth phase were cultured in culture medium without FBS for 24 h.After disassociated using 0.25% EDTA-trypsin, the cells were seeded into respective plates at the density of 1.5×109· L^-1 and cultured overnight.The cells were divided into the following groups：control group（no treatment）,model group（treated with 6 mmol·L^-1 CCl4 for 24 h）,CQ groups（pretreated with 6 mmol·L^-1 CCl4 for 24 h and 400,500,600 mg·L^-1 CQ for 24 h）.Concentrations ofhyaluronidase（HA）, laminin（LN）, procollagen Ⅲ（PCⅢ）,collagen type Ⅳ（Ⅳ-C）were assayed by ELISA kits.Gene expressions of Toll-like receptor 4（TLR4）and NF-κB were examined by RT-PCR analysis.Protein expression of TLR4 and NF-κB was examined by Western blot.Results CQ could significantly inhibit cell proliferation induced by CCl4.Furthermore, CQ at 600 mg·L^-1 significantly downregulated gene and protein expressions of TLR4 and NF-κB.And CQ reduced the secretion of HA,LN,PCⅢ,Ⅳ-C.Further studies disclosed that the TLR4 inhibitor TAK-242 and NF-κB inhibitor BAY 11-7082 could significantly inhibit the gene and protein expressions of NF-κB,but could not change gene and protein expression of TLR4,and reduced the secretion of HA,LN,PCⅢ,Ⅳ-C.Conclusion CQ could inhibit inflammatory responses in HSC induced by CCl4 probably by inhibiting the transcription activity and protein expression of TLR4-NF-κB, which indicates its possible therapeutic effect on liver fibrosis.

关 键 词：肝纤维化 柴胡-黄芩水煎液 HSC CCL4诱导 TLR4 NF-ΚB 

分 类 号：R-332[医药卫生] R282.71