白屈菜碱对活化后大鼠肝星状细胞CFSC-8B的增殖、胶原合成及TGF-β1受体的影响

Effects of Chelidonine on Proliferation,Collagen Synthesis and TGF-β1Receptor of Activated Hepatic Stellate Cells CFSC-8B in Rats

目的:考察白屈菜碱对活化后大鼠肝星状细胞CFSC-8B的增殖、胶原合成及转化生长因子β(1 TGF-β1)受体的影响。方法:取对数生长期的CFSC-8B细胞,分为正常对照组、模型组、溶剂组(乙醇)、阳性对照组(1μg/mL秋水仙碱乙醇溶液)和白屈菜碱低、中、高浓度组(2.1、4.2、8.4μg/mL白屈菜碱乙醇溶液)。除正常对照组外,其余各组细胞利用20μg/L的TGF-β1作用24h进行活化,后5组细胞给予相应药物干预24h。采用CCK-8法检测各组细胞增殖情况,酶消化法检测细胞上清液中羟脯氨酸(Hyp)含量,酶联免疫吸附测定法(ELISA)检测细胞上清液中Ⅰ型胶原蛋白(Col-Ⅰ)和Ⅲ型胶原蛋白(Col-Ⅲ)水平,Western blot法检测细胞中TGF-β1Ⅰ型受体(TβR-Ⅰ)和TβR-Ⅱ蛋白的表达,实时荧光定量-聚合酶链式反应法检测细胞中α-平滑肌肌动蛋白(α-SMA)、TβR-Ⅰ和TβR-ⅡmRNA的表达。结果:与正常对照组比较,模型组细胞增殖率、Hyp含量、Col-Ⅰ和Col-Ⅲ水平、TβR-Ⅰ和TβR-Ⅱ的蛋白表达水平以及α-SMA、TβR-Ⅰ、TβR-Ⅱ的mRNA表达水平均显著升高(P<0.05)。与模型组比较,溶剂组细胞的上述指标均无明显差异(P>0.05);白屈菜碱低浓度组细胞增殖率无明显差异(P>0.05);阳性对照组和白屈菜碱高浓度组细胞的上述指标均显著降低(P<0.05);白屈菜碱中浓度组细胞除Hyp和Col-Ⅲ水平降低不显著外,上述其余指标均显著降低(P<0.05)。与白屈菜碱中浓度组比较,白屈菜碱高浓度组细胞增殖率、Col-Ⅰ水平、TβR-Ⅰ和TβR-Ⅱ的蛋白及mRNA表达水平均显著降低(P<0.05)。结论:白屈菜碱能抑制活化后CFSC-8B细胞的增殖、胶原合成以及TβR-Ⅰ、TβR-Ⅱ蛋白和mRNA的表达。

OBJECTIVE:To investigate the effects of chelidonine on proliferation,collagen synthesis and TGF-β1 receptor of activated hepatic stellate cells CFSC-8B.METHODS:CFSC-8B cells in logarithmic phase were collected and then divided into normal control group,model group,solvent group(ethanol),positive control group(1μg/mL colchicine ethanol solution),chelidonine low,medium and high concentration groups(2.1,4.2,8.4μg/mL chelidonine ethanol solution).Except for normal control group,other groups were activated with 20μg/L TGF-β1 for 24 h;the latter 5 groups were intervened with relevant medicine for 24 h.Cell proliferation of activated cells was assayed by CCK-8 assay.Hydroxyprolin(Hyp)content was assayed by enzyme digestion;the levels of typeⅠcollagen(Col-Ⅰ)and typeⅢcollagen(Col-Ⅲ)were assayed by ELISA;the expressions of TβR-Ⅰand TβR-Ⅱprotein were assessed by Western blot;mRNA expressions ofα-SMA,TβR-Ⅰand TβR-Ⅱin hepatic stellate cells were assessed by RT-PCR.RESULTS:Compared with normal control group,cell proliferation rate,Hyp content,the levels of Col-Ⅰand Col-Ⅲ,the protein expressions of TβR-Ⅰand TβR-Ⅱas well as mRNA expressions ofα-SMA,TβR-Ⅰand TβR-Ⅱwere increased significantly(P<0.05).Compared with model group,there were no significant difference in above indexes of hepatic stellate cells in solvent group(P>0.05);there were no significant difference in the proliferation rate of hepatic stellate cells in chelidonine low concentration group(P>0.05),above indexes of hepatic stellate cells were decreased significantly in positive control group and chelidonine high concentration group(P<0.05).The decrease of Hyp and Col-Ⅲlevels were not significant in chelidonine medium concentration,but other above indexes were decreased significantly(P<0.05).Compared with chelidonine medium concentration group,the rate of cell proliferation,Col-Ⅰlevel,protein and mRNA expressions of TβR-Ⅰand TβR-Ⅱwere decreased significantly in chelidonine high concentration group(P<0.05).CONCLUSIONS:Chelidonine can inhibit the proliferation,collagen synthesis as well as the protein and mRNA expressions of TβR-Ⅰand TβR-Ⅱin activated CFSC-8B cells.