桃仁-红花药对抑制IL-1β诱导椎间盘软骨终板细胞发生退变的体外实验研究

doi:10.5246/jcps.2016.08.066

中国药学(英文版) ›› 2016, Vol. 25 ›› Issue (8): 590-597.DOI: 10.5246/jcps.2016.08.066

桃仁-红花药对抑制IL-1β诱导椎间盘软骨终板细胞发生退变的体外实验研究

牛凯^1,2,4, 李晨光^3,4, 袁松¹, 张雷², 施杞^3,4, 王拥军^3,4, 郑为超^2*

1. 舟山市第二人民医院, 浙江舟山 316000
2. 安徽理工大学医学院, 安徽淮南 232001
3. 上海中医药大学附属龙华医院, 上海 200032
4. 上海中医药大学脊柱病研究所, 上海 200032

收稿日期:2015-09-21 修回日期:2015-10-13 出版日期:2016-09-01 发布日期:2015-10-27
通讯作者: Tel./Fax: +86-0554-6668808, +86-0580-2363011, E-mail: nk342896716@126.com, wzheng@aust.edu.cn
基金资助:
National Natural Science Foundation of China (Grant No. 81273777, 81102606, 81072831), the National Basic Research Program of China (Grant No. 2010CB530400), the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT, IRT1270).

TRHH-herb pair prevents IL-1β-induced degeneration of endplate chondrocytes in vitro

Kai Niu^1,2,4, Chenguang Li^2,3, Song Yuan¹, Lei Zhang², Qi Shi^3,4, Yongjun Wang^3,4, Weichao Zheng^2*

1. Second People’s Hospital of Zhoushan, Zhoushan 316000, China
2. Medical College, Anhui University of Science and Technology, Huainan 232001, China
3. Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China
4. Institute of Spine, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China

Received:2015-09-21 Revised:2015-10-13 Online:2016-09-01 Published:2015-10-27
Contact: Tel./Fax: +86-0554-6668808, +86-0580-2363011, E-mail: nk342896716@126.com, wzheng@aust.edu.cn
Supported by:
National Natural Science Foundation of China (Grant No. 81273777, 81102606, 81072831), the National Basic Research Program of China (Grant No. 2010CB530400), the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT, IRT1270).

摘要/Abstract

摘要：

IL-1β作为炎性细胞因子, 在椎间盘退变的过程中起着至关重要的作用。本研究的主要目的是观察评价桃仁-红花药对含药血清在体外对IL-1β诱导退变的椎间盘软骨终板细胞的作用, 并探讨其机制。桃仁和红花作为药对灌胃给大鼠, 收集、准备相应的含药血清。经大鼠椎间盘分离、鉴定的第三代软骨终板细胞作为实验细胞, 随机分组为正常组(Group NC)、IL-1β 诱导组(Group IL)、桃仁红花药对含药血清组(Group TRHH), 正常组仅用标准的培养基培养, IL-1β诱导组加用IL-1β, 桃仁-红花药对含药血清组加用IL-1β、桃仁-红花药对含药血清。CCK-8法检测细胞增殖, 流式检测细胞凋亡, Real-time PCR(RT-PCR)检测Aggrecan, Col2α1, Col10α1, IL-6和SOX9 mRNA的表达。细胞免疫组化检测II型胶原、X型胶原的表达。臧红-O染色用于鉴定各组细胞中细胞外基质的表达。与正常组相比较, IL-1β诱导组软骨终板细胞增殖降低, 细胞凋亡增加(P<0.05), 下调Aggrecan, Col2α1和SOX9 mRNA的表达, 上调Col10α1和IL-6 mRNA的表达(P<0.05); 同时免疫组化和臧红-O染色示II型胶原蛋白的表达降低、X型胶原蛋白的表达升高。与IL-1β诱导组相比较, 桃仁-红花药对含药血清组在抑制软骨终板细胞凋亡的同时促进软骨终板细胞的增殖(P<0.05), 上调Aggrecan, Col2α1和SOX9 mRNA表达的同时下调Col10α1和IL-6 mRNA的表达(P<0.05); 促进II型胶原蛋白的表达增强、X型胶原蛋白的表达降低。桃仁-红花药对含药血清能够抑制IL-1β诱导椎间盘软骨终板细胞发生退变。

关键词: 含药血清, 桃仁-红花药对, 退变, 软骨细胞, 椎间盘

Abstract:

The inflammatory cytokine interleukin-1 beta (IL-1β) plays a key role in the process of intervertebral disc degeneration (IVDD). In the present study, we aimed to evaluate the effect of pharmaco-serum of "Taoren-Honghua-herb pair" on IL-1β-induced chondrocyte degeneration in vitro. Taoren (Semen persicae) and Honghua (Safflower carthamus) were administered to the rats, and the pharmaco-serum was collected and prepared. Chondrocytes of the third passage, isolated from the rat’s vertebral endplates, were treated by standard medium only (Group NC), IL-1β (Group IL) or combination of IL-1β and pharmaco-serum (Group TRHH). Cell proliferation and apoptosis were determined, and the expression of aggrecan, Col2α1, Col10α1, IL-6 and SOX9 at the mRNA level in chondrocytes was quantified by real-time PCR. Immunohistochemistry staining of type II and X collagen and Safranine O staining were also used to evaluate the chondrocytes. Compared with the Group NC, IL-1β treatment inhibited the cell proliferation and induced the cell apoptosis (P<0.05), and the expression of aggrecan, Col2α1 and SOX9 at the mRNA level was down-regulated. In contrast, the expression of Col10α1 and IL-6 was up-regulated after IL-1β treatment (P<0.05). Meanwhile, the immune-staining of type II collagen and Safranine O staining were decreased, while the staining of type X collagen was increased. Compared with the Group IL, cell proliferation was increased, and apoptosis of chondrocytes was decreased when cells were treated with the pharmaco-serum of TRHH-herb pair (P<0.05). The expression of aggrecan, Col2α1 and SOX9 at the mRNA level was up-regulated, while that of Col10α1 and IL-6 was down-regulated (P<0.05). Safranine O staining also showed increased positive staining (P<0.05). Taken together, the treatment of pharmaco-serum of TRHH-herb pair could prevent endplate chondrocyte degeneration induced by IL-1β.

Key words: Pharmaco-serum, TRHH-herb pair, Degeneration, Chondrocyte, Intervertebral discs

中图分类号:

R962.1

Supporting:

牛凯, 李晨光, 袁松, 张雷, 施杞, 王拥军, 郑为超. 桃仁-红花药对抑制IL-1β诱导椎间盘软骨终板细胞发生退变的体外实验研究[J]. 中国药学(英文版), 2016, 25(8): 590-597.

Kai Niu, Chenguang Li, Song Yuan, Lei Zhang, Qi Shi, Yongjun Wang, Weichao Zheng. TRHH-herb pair prevents IL-1β-induced degeneration of endplate chondrocytes in vitro[J]. Journal of Chinese Pharmaceutical Sciences, 2016, 25(8): 590-597.

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桃仁-红花药对抑制IL-1β诱导椎间盘软骨终板细胞发生退变的体外实验研究

TRHH-herb pair prevents IL-1β-induced degeneration of endplate chondrocytes in vitro

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