Department of Orthopaedics, Minhang Hospital, Fudan University, Shanghai, China.
Orthop Surg. 2020 Apr;12(2):609-616. doi: 10.1111/os.12644. Epub 2020 Mar 8.
Endoplasmic reticulum stress (ERS) is present in chondrocytes of osteoarthritis, and the intensity of ERS is related to the degree of cartilage degeneration. In vitro intervention strategies can change the status of ERS and induce the inhibition of ERS-related pathway. Therefore, this study is designed to explore the role and molecular mechanism of cartilage stem cells (ACSCs) of ERS in chondrocytes after hip replacement.
Human cartilage cell lines C28/I2 were cultured as the control group. The ERS inducer was added into C28/I2 as ERS group. The third ERS + stem cells group was formed by adding cartilage stem cells into ERS group, and further transfection of si-PERK was defined as si-PERK + ERS + stem cells group. Cell cycle and apoptosis in the four groups were determined by flow cytometry. The protein expression of GRP78, PERK, ATF4, TMEM119, CDK4, Cyclin D, and BMP6 in chondrocytes in the four groups were investigated by western blot, and the distribution of PERK, TMEM119, and BMP6 in chondrocytes were observed by immunofluorescence assay. In addition, the transcriptional levels of Bcl2, Bax, and Caspase 3 were also determined by RT-PCR.
In cell cycle assay, ERS increased the accumulation of cells in G /G and G /M, while cartilage stem cells weakened the effects. The apoptosis rates in control group, ERS, ERS + stem cells, si-PERK + ERS + stem cells were 0%, 21.3%, 18.9%, and 15.9%, respectively, and the difference of apoptosis rate between the latter three groups and control group was statistically significant (P < 0.01). Stem cells could weaken the ERS-induced cell apoptosis, especially reducing the number of cells in the late stage of apoptosis from 5.4% to 1.1%. The protein level of GRP78, PERK, ATF4, TMEM119, and BMP6 in the group of ERS, ERS + stem cells, and si-PERK + ERS + stem cells were all significantly higher than those in control group, and the group of ERS + stem cells was the highest, all of the differences were significant (P < 0.01). However, the protein level of CDK4 and Cyclin D presented an absolutely opposite trend and the difference was still significant (P < 0.05). The group of si-PERK + ERS + stem cell was lower than those in the group of ERS + stem cell but higher than those in the group of ERS (P < 0.05). The level of Caspase 3 in the latter three groups was significantly higher than those in the control group, and the group of ERS was the highest (P < 0.01). Besides, the relative level of Bcl-2/Bax in control group was 1, but the group of ERS was about 0.5, and there was significant difference (P < 0.01). The ratio of Bcl-2/Bax in the group of ERS + stem cells was more than 2 and significantly higher than those of other groups.
ACSCs could reduce ERS-induced chondrocyte apoptosis by PERK and Bax/Bcl-2 signaling pathway.
内质网应激（ERS）存在于骨关节炎的软骨细胞中，其强度与软骨退变程度有关。体外干预策略可以改变 ERS 的状态，并诱导 ERS 相关途径的抑制。因此，本研究旨在探讨 ER 应激后髋关节置换术对软骨干细胞（ACSCs）的作用及分子机制。
培养人软骨细胞系 C28/I2 作为对照组。将 ERS 诱导剂加入 C28/I2 作为 ERS 组。将第 3 个 ERS+干细胞组加入 ERS 组，进一步转染 si-PERK 定义为 si-PERK+ERS+干细胞组。采用流式细胞术检测四组细胞的细胞周期和凋亡情况。采用 Western blot 法检测各组软骨细胞中 GRP78、PERK、ATF4、TMEM119、CDK4、Cyclin D 和 BMP6 的蛋白表达水平，并用免疫荧光法观察 PERK、TMEM119 和 BMP6 在软骨细胞中的分布。此外，还通过 RT-PCR 法测定 Bcl2、Bax 和 Caspase 3 的转录水平。
细胞周期检测显示，ERS 增加了 G/G 和 G/M 期细胞的积累，而软骨干细胞则减弱了这种作用。对照组、ERS 组、ERS+干细胞组和 si-PERK+ERS+干细胞组的细胞凋亡率分别为 0%、21.3%、18.9%和 15.9%，后 3 组与对照组比较差异有统计学意义（P<0.01）。干细胞可以减弱 ERS 诱导的细胞凋亡，特别是使晚期凋亡细胞数量从 5.4%减少到 1.1%。ERS 组、ERS+干细胞组和 si-PERK+ERS+干细胞组的 GRP78、PERK、ATF4、TMEM119 和 BMP6 蛋白水平均明显高于对照组，其中 ERS+干细胞组最高，差异均有统计学意义（P<0.01）。然而，CDK4 和 Cyclin D 的蛋白水平则呈现出完全相反的趋势，差异仍有统计学意义（P<0.05）。si-PERK+ERS+干细胞组的蛋白水平低于 ERS+干细胞组，但高于 ERS 组（P<0.05）。后 3 组的 Caspase 3 水平明显高于对照组，ERS 组最高（P<0.01）。此外，对照组的 Bcl-2/Bax 相对水平为 1，但 ERS 组约为 0.5，差异有统计学意义（P<0.01）。ERS+干细胞组的 Bcl-2/Bax 比值大于 2，明显高于其他组。
ACSCs 可通过 PERK 和 Bax/Bcl-2 信号通路减少 ERS 诱导的软骨细胞凋亡。
