解剖学报

解剖学报 ›› 2015, Vol. ›› Issue (3): 342-347.doi: 10.16098/j.issn.0529-1356.2015.03.009

• 细胞和分子生物学 • 上一篇    下一篇

筋骨痹痛汤药物血清对白细胞介素-1β诱导的兔软骨细胞凋亡的保护作用

肖龙 袁海洲 钟沁 黄剑 冼华 黄英如*   

  1. 重庆医科大学中医药学院骨伤学科教研室, 重庆 400016
  • 收稿日期:2014-12-09 修回日期:2015-02-09 出版日期:2015-06-06 发布日期:2015-06-06
  • 通讯作者: 黄英如 E-mail:hyr12678@126.com
  • 基金资助:

    国家自然科学基金;重庆市卫生局中医药科技项目;重庆市九龙坡区科技项目

Protective effect of the serum of Jinggu Bitong decoction on the rabbits chondrocytes’apoptosis induced by interleukin-1β

XIAO Long YUAN Hai-zhou ZHONG Qin HUANG Jian XIAN Hua HUANG Ying-ru*   

  1. Department of Orthopaedics and Traumatoloy, Traditional Chinese Medicine College of Chongqing Medical University， Chongqing 400016, China
  • Received:2014-12-09 Revised:2015-02-09 Online:2015-06-06 Published:2015-06-06
  • Contact: HUANG Ying-ru E-mail:hyr12678@126.com

摘要：

目的 观察筋骨痹痛汤药物血清对白细胞介素-1β（IL-1β）诱导的兔软骨细胞Bcl-2、Bax表达及细胞凋亡的影响，探讨筋骨痹痛汤治疗骨性关节炎的机制。
方法 随机取雄性成年新西兰兔8只分成4组，分别灌服生理盐水、补肝肾中药（骨碎补、续断和淫羊霍）、独活寄生汤和筋骨痹痛汤，制得空白血清和药物血清。分离制备幼兔软骨细胞，采用甲苯胺蓝染色和Ⅱ型胶原免疫荧光法鉴定软骨细胞。取第3代软骨细胞，随机分5组实验：空白组（A组）、模型组（B组）、补肝肾中药对照组（C组）、独活寄生汤对照组（D组）、筋骨痹痛汤组（E组）。A组用含10%空白血清的DMEM培养36h，其余各组均先采用含IL-1β的DMEM诱导培养12h，然后B、C、D、E组分别给予含10%空白血清、10%补肝肾中药药物血清、10%独活寄生汤药物血清、10%筋骨痹痛汤药物血清的DMEM培养24h。实时定量聚合酶链反应（Real-time PCR)和免疫印迹法检测兔软骨细胞Bcl-2和Bax的mRNA和蛋白表达，流式细胞术检测细胞凋亡比例。结果 经鉴定分离制备的细胞为软骨细胞。软骨细胞培养24h后，C、D、E组Bcl-2 mRNA和蛋白表达高于B组，D、E组高于C组，E组高于D组，差异均有统计学意义（P＜0.05）；Bax mRNA和蛋白表达，以及软骨细胞凋亡率，C、D、E组低于B组，D、E组低于C组，E组低于D组，差异均有统计学意义（P＜0.05）。结论 筋骨痹痛汤药物血清对IL-1β诱导的兔软骨细胞凋亡具有明显保护作用，其机制可能是通过促进Bcl-2表达和抑制Bax表达，调节Bcl-2与Bax的比例来实现。

Abstract:

Objective To investigate the effect and the mechanism of the Jinggu Bitong decoction on osteoarthritis(OA) by observing the effects of the serum of Jinggu Bitong decoction on the rabbits chondrocytes ? apoptosis and the expression of Bcl-2 and Bax. Methods Eight adult male New Zealand rabbits were randomly divided into four groups which were gavaged with normal saline (NS), prescription for nourishing liver-kidney (PNLK, drynaria rhizome, dipsacus asperoids and herba epimedii), Duhuo Jisheng decoction and Jinggu Bitong decoction, respectively. The blank serum and the medicated serum were obtained. We identified chondrocytes by the toluidine blue staining and type Ⅱ collagen immunofluorescent staining. The 3th passage rabbit chondrocytes were randomly assigned to 5 groups: blank group (group A), model group (group B), PNLK group (group C), Duhuo Jisheng decoction group (group D) and Jinggu Bitong decoction group (group E). The chondrocytes were cultured with 10% blank serum 36 hours in group A, with IL 1-β 12 hours in group B, group C, group D and group E. Then the chondrocytes were cultured with 10% blank serum 24 hours in group B, with 10% PNLK serum in group C, with 10% Duhuo Jisheng decoction serum 24 hours in group D and with 10% Jinggu Bitong decoction serum 24 hours in group E. Real-time quantitative PCR (Real-time PCR) and Western blotting were used to detect the expression of Bcl-2’s and Bax’s mRNA and protein. The apoptosis rate was analyzed by flow cytometry (FCM). Results All the cells were identified as chondrocytes. After the 24 hours, for the expressions of Bcl-2’s mRNA and protein in group C, group D and group E was higher than group B, group D and group E was higher than group C, and group E were higher than group D. The results were statistically different (P＜0.05). For the expressions of Bax’s mRNA and protein and the apoptosis rate, group C, group D, and group E were lower than group B, group D and group E were lower than group C, and group E were lower than group D. The results were statistically different (P＜0.05). Conclusion The Jinggu Bitong decoction has a significant protective action to the rabbit chondrocytes’s apoptosis induced by IL-1β, and the possible mechanism is to promote the Bcl-2’s expression and control the Bax’s expression and control proportional of Bcl-2 and Bax.