摘要
目的:观察丹参素对白介素-1β(Iinterleukin-1β,IL-1β)刺激的肝星状细胞(hepatic stellate cell,HSC)增殖、凋亡、Ⅲ型胶原合成和分泌,以及磷酸化核因子κB抑制蛋白α(Phosphorylated Inhibitor ofκBα, p-IκBα)、核转录因子κB蛋白65(Nuclear transcription factor-kappa B proteinum 65, NF-κB p65)的影响,探讨丹参素抗肝纤维化的分子机制。
方法:用改良的胶原酶消化-原位循环灌流-密度梯度离心法提取HSC,α-SMA免疫细胞化学染色法鉴定分离HSC。以不同浓度的丹参素作用于IL-1β刺激的HSC,MTT法、AO/EB免疫荧光和AnnexinⅤ-FITC/PI分别检测HSC的增殖、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核转位及细胞浆p-IκBα、细胞核NF-κB p65的表达。
结果:1.原代分离的HSC细胞得率3.5×107/只鼠,纯度大于95%,存活率大于96%. 2.与对照组相比,IL-1β刺激组HSC增殖明显增加(P<0.01),凋亡率减低(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24h后HSC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或P<0.01),以上作用呈浓度依赖性(P<0.05或P<0.01),且以0.25mmol/L丹参素作用为著;Western Blot结果显示IL-1β刺激组细胞浆p-IκBα、细胞核NF-κB p65蛋白表达明显增加(P<0.01),细胞浆p65表达明显减低(P<0.05),出现明显核转位,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HSC的NF-κB活性;不同浓度丹参素组细胞浆p-IκBα、细胞核NF-κB p65蛋白表达明显减低(P<0.05或P<0.01),上述蛋白减低作用呈浓度依赖性(P<0.05),0.25mmol/L丹参素组细胞浆p65蛋白表达明显增加(P<0.05),该组细胞核内p65阳性染色细胞数减少淡染,核转位减少,提示丹参素可以浓度依赖性地抑制HSC的IκBα的磷酸化,减少细胞核NF-κB p65表达,阻止NF-κB核转位,从而抑制NF-κB活性。
结论:1.改良的HSC分离法能分离出高产、高纯度的HSC。原代分离的大鼠HSC经过鉴定具有活化HSC的生物学特性,是体外研究肝纤维化的理想模型。2.丹参素抑制IL-1β诱导的HSC增殖,促使HSC凋亡,减少Ⅲ型胶原的合成和分泌,同时伴随磷酸化IκBα、细胞核NF-κBp65表达和NF-κB核转位减低,其机制可能与丹参素抑制Rel/NF-κB/IκB信号通路有关。
Objectives:To investigate the effects of Danshensu on proliferation、apoptosis、synthesis and secretion of collagen tapeⅢand expression of p-IκBαand NF-κB p65 induced by IL-1βin rat hepatic stellate cells(HSCs), and to elucidate the molecular mechanisms of Danshensu against hepatic fibrosis.
Methods:The rat HSC was isolated with improved collagenase digested in situ-liver recirculating perfusion and cultured in vitro. It was identified byα-SMA immunocytochemical stain .Then it was treated with different concentration of Danshensu. MTT colorimetric assay was used to detect proliferation of HSC. AO/EB immunoflurorescence micropy and combination Annexin-V-FITC/PI double-labelimmunofluorescence with flow cytometry were used to examine apoptosis of HSC. Synthesis and secretion of collagen tapeⅢwere detected by the quantitative immunocytochemical assay and ELISA method espectively. The amounts of cytoplasm p-IκBα、NF-κB p65 and of nuclear NF-κB p65 in HSC were determined by Western blot. Immunocytochemical stain and Western blot were used to observe nuclear translocation of NF-κB p65.
Results: 1. The yield of HSC was about 3.5×107 per rat.The purity of HSC exceed 95%,and the survival rate of HSC exceed 96%. 2. Compared with normal control group,IL-1βcould increase proliferation of HSC (P<0.01),reduce apoptosis of HSC(P<0.05), and facilitate synthesis and secretion of collagen tapeⅢin HSC(P<0.01). After treated with different concentration of Danshensu, HSC’s proliferation prominently reduced(P<0.01) and its apoptosis increased(P<0.01), especially of early apoptosis. Danshensu could also degrade synthesis and secretion of collagen tapeⅢin HSC (P<0.05 or P<0.01). Danshensu had a concentration-
dependent effect on avove parts (P<0.05 or P<0.01), especially in 0.25mmol/L Danshensu treated group.Western Blot results showed that IL-1βelevated amounts of cytoplasm p-IκBαand nuclear NF-κBp65(P<0.01), decreased amounts of cytoplasm p65 markedly (P<0.05), then facilitated nuclear translocation of p65. Immunocytochemical stain results signed that the number of nuclear NF-κB p65 positive HSC increased and the colour deepened. It mained that IL-1βcould induce NF-κB activation in rat HSC. Danshensu had a concentration-dependent effect on reducing amounts of cytoplasm p-IκBαand nuclear NF-κB p65(P<0.05 or P<0.01), and 0.25mmol/L Danshensu could decrease nuclear translocation of p65 markedly(P<0.05). In above group,immunocytochemical stain results showed that the number of nuclear NF-κB p65 positive HSC decreased and the colour grew pallid. Above results indicated that Danshensu could inhibit IκBαphosphorylation,reduce expresstion of nucear NF-κB p65, and prevent nuclear translocation of NF-κB,and then inhibit NF-κB activation in rat HSC.
Conclusions: 1.Improved isolation method of HSC has the character of high productin and high purity. Primary identified and cultured rat HSC has biological character of activated HSC and it was an important experimental object to investigate liver fibrosis in vitro.2.Danshensu could inhibit the proliferation of HSC, facilitate the apoptosis of HSC, and reduce the synthesis and secretion of collagen tapeⅢ. Meanwhile, Danshensu could inhibit IκBαphosphorylation,reduce expresstion of NF-κB p65, and prevent nuclear translocation of NF-κB,and then inhibit NF-κB activation in rat HSC. So Danshensu could inhibit Rel/NF-κB/IκB signal transduction pathway .It might be the mechanism of Danshensu against hepatic fibrosis.
引文
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