壮骨健膝方含药血清中药物成分骨碎补柚皮苷对IL-1β诱导兔退变软骨细胞“caveolin-p38MAPK”信号通路的影响

Effect of Naringin of Drynaria Rhizome,a Chinese Medical Component of Zhuanggu Jianxi Recipe Containing Serum on Caveolin-p38MAPK Signal Pathway in IL-1β Induced Rabbit Degenerated Chondrocytes

目的观察壮骨健膝方含药血清中药物成分骨碎补柚皮苷(简称柚皮苷)对IL-1β诱导退变软骨细胞中'caveolin-p38MAPK'通路相关信号因子小窝蛋白-1(caveolin-1)、磷酸化p38(p-p38)、磷酸化核转录因子-2(p-ATF-2)、IL-1β及TNF-α等的影响,进一步探讨该方保护关节软骨的机制。方法 (1)通过高效液相-飞行时间质谱联用(HPLC-TOF/MS)技术分析并获得壮骨健膝方含药血清中的原组方药物成分柚皮苷;(2)取4周龄新西兰兔双侧膝关节软骨,建立兔关节软骨细胞体外培养体系,取第二代软骨细胞用于后续实验。MTT法检测柚皮苷对软骨细胞增殖的影响;免疫组化方法检测柚皮苷对IL-1β诱导后的软骨细胞中Ⅱ型胶原表达的影响;Western blot法检测柚皮苷对IL-1β诱导后软骨细胞中caveolin-1、pp38与p-ATF-2蛋白表达的影响;RT-PCR检测柚皮苷对IL-1β诱导后软骨细胞中IL-1β及TNF-αmRNA表达的影响。结果 (1)柚皮苷标准品溶液及壮骨健膝方含药血清离子流图中柚皮苷的出峰时间均为23.5 min,分子量均在581.0~581.5 m/z之间;(2)柚皮苷能促进软骨细胞的增殖,抑制IL-1β对软骨细胞Ⅱ型胶原表达的影响;(3)与模型组比较,柚皮苷能降低IL-1β诱导后软骨细胞中caveolin-1、p-p38、p-ATF-2等蛋白及IL-1β、TNF-αmRNA的表达(P<0.05),并接近正常组水平。结论壮骨健膝方含药血清中原组方药物成分柚皮苷不仅能够促进软骨细胞的增殖,还可保护软骨细胞的功能,其机制可能与柚皮苷降低诱导退变软骨细胞中caveolin-1、p-p38与p-ATF-2的表达,抑制'caveolin-p38MAPK'通路的活动,进而减少通路下游IL-1β及TNF-αmRNA表达有关。

Objective To observe the effect of naringin of Drynaria Rhizome,a Chinese medical component of Zhuanggu Jianxi Recipe( ZJR) containing serum on caveolin- p38 MAPK signal factors( such as caveolin- 1,p- p38,p-ATF- 2,IL- 1β,and TNF- α) in IL- 1β induced rabbit degenerated chondrocytes,and further to explore its mechanism for protecting articular cartilages. Methods Naringin of Drynaria Rhizome was obtained and analyzed by HPLC- TOF / MS.Four weeks old New Zealand rabbits were killed and their bilateral knee joints were isolated aseptically. CDs were isolated and then culturedin vitro. The second generation of CDs were used for later experiment. The effect of naringin on CDs proliferation was detected by MTT assay. The effect of naringin on the expression of IL- 1β- induced collagen Ⅱ in CDs was detec-ted by immunohistochemical method. The effect of naringin on caveolin- 1,p- p38,and p- ATF- 2 protein in IL- 1β-induced CDs was detected by Western blot. The effect of naringin on mRNA expression of IL- 1β and TNF- α in IL- 1β-induced CDs was detected by RT- PCR. Results The appearance time of naringin in flow graphs of naringin standard solution and ZJR containing serum was 23. 5 min,and the molecular weight ranged between 581. 0 and 581. 5 m / z. Naringin could promote the proliferation of CDs,and inhibit the effect of IL- 1β on collagen Ⅱ in CDs. Compared with the model group,naringin could reduce the expression of caveolin- 1,p- p38,p- ATF- 2,IL- 1β,and TNF- α in IL- 1β induced CDs(P < 0. 05),which was approximate to the level of the normal group. Conclusions Naringin could not only promote the proliferation of CDs,but also protect IL- 1β- induced CDs. Its mechanism might be associated with decreasing the expression of caveolin- 1,p- p38,and p- ATF- 2 proteins,inhibiting caveolin- p38 MAPK signal pathway,and further reducing mRNA expression of IL- 1β and TNF- α in the downstream of caveolin- p38 MAPK signal pathway.