Bushen Zhuangjin decoction inhibits TM-induced chondrocyte apoptosis mediated by endoplasmic reticulum stress.
Bushen Zhuangjin decoction inhibits TM-induced chondrocyte apoptosis mediated by endoplasmic reticulum stress.
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DOI：
10.3892/ijmm.2015.2387
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发表时间：
2015-12
影响因子：
5.4
通讯作者：
Li X
中科院分区：
文献类型：
--
作者：
Lin P;Weng X;Liu F;Ma Y;Chen H;Shao X;Zheng W;Liu X;Ye H;Li X
内质网（ER）应激引起的软骨细胞凋亡在骨关节炎（OA）的发病机制中起着重要作用。补肾壮筋汤在OA的治疗中应用广泛。然而，BZD抑制软骨细胞凋亡的细胞和分子机制仍有待阐明。在本研究中，我们研究了BZD对ER应激诱导的软骨细胞凋亡的影响，使用软骨细胞在体外模型的OA。取SD大鼠膝关节软骨细胞，用免疫组化法检测Ⅱ型胶原的表达。用4-苯基丁酸（4-PBA）检测衣霉素（TM）刺激软骨细胞后内质网应激介导的凋亡。我们发现，4-PBA抑制TM诱导的软骨细胞凋亡，这证实了成功诱导软骨细胞凋亡。MTT法显示，BZD以剂量和时间依赖的方式增强TM刺激的软骨细胞的活力。4′，6-diamidino-2-phenylindole（DAPI）染色、Annexin V-FITC结合试验和JC-1法显示，与未处理的软骨细胞相比，BZD处理的软骨细胞凋亡率和线粒体膜电位（Δ Δ m）损失明显降低。为了进一步阐明BZD抑制TM诱导的由ER应激介导的软骨细胞凋亡的机制，检测了BZD对TM诱导的软骨细胞凋亡的抑制作用，包括免疫球蛋白结合蛋白（Bip）、X-box结合蛋白1（Xbp 1）、转录激活因子4（Atf 4）、C/EBP同源蛋白（Chop）、caspase-9、caspase-3、逆转录-聚合酶链反应（RT-PCR）和Western blot检测B细胞淋巴瘤2（Bcl-2）和Bcl-2相关X蛋白（Bax）。BZD处理组软骨细胞Bip、Atf 4、Chop、caspase-9、caspase-3和Bax的mRNA和蛋白表达水平均显著低于对照组，而Xbp 1和Bcl-2的mRNA和蛋白表达水平显著高于对照组。此外，我们的所有研究结果表明，有TM刺激的软骨细胞与BZD处理和4-PBA处理之间没有显着差异。总之，我们的研究结果表明，BZD抑制TM诱导的软骨细胞凋亡介导的ER压力。因此，BZD可能是用于治疗OA的潜在治疗剂。
Chondrocyte apoptosis triggered by endoplasmic reticulum (ER) stress plays a vital role in the pathogenesis of osteoarthritis (OA). Bushen Zhuangjin decoction (BZD) has been widely used in the treatment of OA. However, the cellular and molecular mechanisms responsible for the inhibitory effects of BZD on chondrocyte apoptosis remain to be elucidated. In the present study, we investigated the effects of BZD on ER stress-induced chondrocyte apoptosis using a chondrocyte in vitro model of OA. Chondrocytes obtained from the articular cartilage of the knee joints of Sprague Dawley (SD) rats were detected by immunohistochemical staining for type II collagen. The ER stress-mediated apoptosis of tunicamycin (TM)-stimulated chondrocytes was detected using 4-phenylbutyric acid (4-PBA). We found that 4-PBA inhibited TM-induced chondrocyte apoptosis, which confirmed the successful induction of chondrocyte apoptosis. BZD enhanced the viability of the TM-stimulated chondrocytes in a dose- and time-dependent manner, as shown by MTT assay. The apoptotic rate and the loss of mitochondrial membrane potential (ΔΨm) of the TM-stimulated chondrocytes treated with BZD was markedly decreased compared with those of chondrocytes not treated with BZD, as shown by 4′,6-diamidino-2-phenylindole (DAPI) staining, Annexin V-FITC binding assay and JC-1 assay. To further elucidate the mechanisms responsible for the inhibitory effects of BZD on TM-induced chondrocyte apoptosis mediated by ER stress, the mRNA and protein expression levels of binding immunoglobulin protein (Bip), X-box binding protein 1 (Xbp1), activating transcription factor 4 (Atf4), C/EBP-homologous protein (Chop), caspase-9, caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were measured by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. In the TM-stimulated chondrocytes treated with BZD, the mRNA and protein expression levels of Bip, Atf4, Chop, caspase-9, caspase-3 and Bax were significantly decreased, whereas the mRNA and protein expression levels of Xbp1 and Bcl-2 were significantly increased compared with the TM-stimulated chondrocytes not treated with BZD. Additionally, all our findings demonstrated that there was no significant difference between the TM-stimulated chondrocytes treated with BZD and those treated with 4-PBA. Taken together, our results indicate that BZD inhibits TM-induced chondrocyte apoptosis mediated by ER stress. Thus, BZD may be a potential therapeutic agent for use in the treatment of OA.