丹参素对大鼠肝星状细胞增殖、凋亡及NF-κB活性的影响  

Tanshinol induces proliferation,apoptosis and NF-κB activation in cultured rat hepatic stellate cells after IL-1β inducement

作　　者：李冬[1] 戴立里[1] 余斌斌[1] 姜中华[1] 

机构地区：[1]重庆医科大学第二附属医院消化内科,重庆400010

出　　处：《第三军医大学学报》2009年第8期724-728,共5页Journal of Third Military Medical University

摘　　要：目的观察丹参素对IL-1β刺激的肝星状细胞增殖、凋亡及NF-κB活性的影响,探讨丹参素抗肝纤维化的分子机制。方法用原位-密度梯度离心法提取肝星状细胞(hepatic stellate cell,HSCs),以不同浓度的丹参素作用于IL-1β刺激的HSCs,MTT法、AO/EB免疫荧光和Annexin V-FITC/PI分别检测HSCs的增殖和凋亡;免疫细胞化学、ELISA法、Western blot分别检测Ⅲ型胶原的生成、NF-κB核转位及细胞质p-IκBα、细胞核NF-κB p65的表达。结果与对照组相比,IL-1β刺激组HSCs增殖明显增加(P<0.01),凋亡率明显减低(P<0.05),Ⅲ型胶原的合成和分泌明显增加(P<0.01);不同浓度丹参素作用24 h后HSCs增殖明显减低(P<0.01),凋亡明显增加(P<0.01),以早期凋亡为主,Ⅲ型胶原的合成和分泌亦明显减少(P<0.05,P<0.01);Western blot结果显示IL-1β作用30 min时细胞质p-IκBα、细胞核NF-κB p65蛋白表达明显增加(P<0.01),出现核转位;免疫细胞化学显示细胞核内p65阳性染色细胞增多浓染,提示IL-1β可以诱导HSCs的NF-κB活性;丹参素组细胞质p-IκBα、细胞核NF-κB p65蛋白表达明显减低(P<0.05,P<0.01),核转位减少,细胞核内p65阳性染色细胞数减少淡染,提示丹参素可以抑制IL-1β诱导的HSCs NF-κB活性。结论丹参素抑制IL-1β诱导的HSCs增殖,促使HSCs凋亡,减少Ⅲ型胶原的合成和分泌,其机制可能与抑制NF-κB活性有关。Objective To investigate the effects of tanshinol on the proliferation, apoptosis and NF-κB activation in rat hepatic stellate cells （HSCs） after IL-1β inducement, and to elucidate the anti-fibrotic molecular mechanisms of tanshinol. Methods The rat HSCs was isolated with collagenase in situliver recirculation perfusion and cultured in vitro. The cells were divided into 5 groups： normal control, IL-1β treatment group （ 10 ng/ml）, and tanshinol group 1, 2 and 3. The later 3 groups were pretreated with tanshinol at the concentrations of 0.062 5, 0. 125 and 0.25 mmoL/L respectively followed by 10 ng/ml IL-1β treatment 1 h later. MTT colorimetrie assay was used to detect the proliferation of HSCs. AO/EB immunoflurorescence microscopy and combination Annexin-V-FITC/PI double-labelimmunofluorescence with flow cytometer were employed to examine the apoptosis of HSCs. Synthesis and secretion of collagen m were detected by the quantitative immunocytochemical assay and ELISA respectively. The amounts of cytoplasm p-IκBα and NF-κB p65, and nuclear NF-κB p65 in HSCs were determined by Western blotting. Immunocytochemical staining and Western blotting were used to observe nuclear translocation of NF-κB p65. Results IL-1β increased the proliferation of HSCs （P 〈 0. 01 ） , reduced the cell apoptosis （ P 〈 0.05 ） , and facilitated the synthesis and secretion of collagen Ⅲ in HSCs （P 〈0.05, P 〈 0. 01 ） in comparison with normal control. After treated with different concentration of tanshinol, HSC＇s proliferation was prominently reduced （P 〈 0.01 ） and its apoptosis was increased （P 〈 0.01 ）, especially for early apoptosis. Tanshinol also degraded the synthesis and secretion of collagen Ⅲ in HSCs, especially in 0.25 mmol/L concentration group （P 〈0. 01）. Western blotting showed that IL-1β elevated amounts of cytoplasm p-IκBa and nuclear NF-κB p65 （P 〈 0. 01 ）, decreased amounts of cytoplasm p65 markedly （P 〈 0. 05）, then facilitated nuclear translocatio

关 键 词：肝纤维化 丹参素 肝星状细胞 增殖 凋亡 NF-ΚB IL-1B 

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