| 康飞科,李鹏辉,朱 澍,窦丹丹,陈晓超.TMED10对骨关节炎中TGF-β/Smads信号通路的影响[J].,2024,(10):1825-1832 |
| TMED10对骨关节炎中TGF-β/Smads信号通路的影响 |
| Effect of TMED10 on TGF-β/Smads Signaling Pathway in Osteoarthritis |
| 投稿时间：2023-12-08 修订日期：2023-12-31 |
| DOI：10.13241/j.cnki.pmb.2024.10.005 |
| 中文关键词: 骨关节炎 跨膜P24转运蛋白10 转化生长因子-β 软骨细胞 TGF-β/Smads信号通路 |
| 英文关键词: Osteoarthritis Transmembrane p24 trafficking protein 10 Transforming growth factor-β Chondrocyte TGF-β/Smads signaling pathway |
| 基金项目:陕西省重点研发计划项目（2023-ZDLSF-14） |
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| 中文摘要: |
| 摘要 目的：探究跨膜P24转运蛋白10（TMED10）对骨关节炎中转化生长因子-β（TGF-β）/Smads信号通路的影响。方法：将大鼠软骨细胞分为Control组、IL-1β组、NC-sh+IL-1β组、TMED10-sh+IL-1β组、NC-OE+IL-1β组和TMED10-OE+IL-1β组。Control组和IL-1β组细胞不进行转染处理，NC-sh+IL-1β组、TMED10-sh+IL-1β组、NC-OE+IL-1β组和TMED10-OE+IL-1β组细胞分别转染NC-sh、TMED10-sh、NC-OE和TMED10-OE。转染后，除Control组之外，其他组软骨细胞均用白介素-1β（IL-1β）（10 ng/mL）处理24 h模拟OA软骨细胞的病理微环境。通过MTT法检测细胞增殖，通过TUNEL法检测细胞凋亡。采用前交叉韧带切断加内侧半月板部分切除法建立OA大鼠模型，将OA大鼠分为OA组、OA+NC-sh组和OA+TMED10-sh组（n=12），Sham组（n=12）大鼠进行假手术操作。Sham组和OA组大鼠关节腔内注射40 μL生理盐水，OA+NC-sh组和OA+TMED10-sh组大鼠关节腔内分别注射40 μL的NC-sh和TMED10-sh（滴度为1×109 TU/mL），每周注射1次，共4周。采用番红O/固绿染色法评价膝关节软骨形态，采用TUNEL法检测软骨细胞凋亡。采用RT-qPCR检测软骨细胞和大鼠关节软骨中TMED10的mRNA水平，采用Western blot检测软骨细胞和大鼠关节软骨中TMED10、TGF-β1、Smad2、p-Smad2、Smad3、p-Smad3、Collagen II和MMP-13的蛋白表达水平。结果：与Control组比较，IL-1β组TUNEL阳性率、TMED10 mRNA和TMED10、MMP-13蛋白水平均升高（P<0.05），相对细胞活力以及TGF-β1、p-Smad2/Smad2、p-Smad3/Smad3和Collagen II的蛋白水平均降低（P<0.05）。与IL-1β组和NC-sh+IL-1β组比较，TMED10-sh+IL-1β组TUNEL阳性率、TMED10 mRNA水平和TMED10、MMP-13的蛋白水平均降低（P<0.05），相对细胞活力以及TGF-β1、p-Smad2/Smad2、p-Smad3/Smad3和Collagen II的蛋白水平均升高（P<0.05）。与IL-1β组和NC-OE+IL-1β组比较，TMED10-OE+IL-1β组TUNEL阳性率、TMED10 mRNA水平和TMED10、MMP-13的蛋白水平均升高（P<0.05），相对细胞活力以及TGF-β1、p-Smad2/Smad2、p-Smad3/Smad3和Collagen II的蛋白水平均降低（P<0.05）。与Sham组比较，OA组大鼠的OARSI评分、TUNEL阳性率、TMED10 mRNA水平以及TMED10和MMP-13的蛋白水平均升高（P<0.05），TGF-β1、p-Smad2/Smad2、p-Smad3/Smad3和Collagen II蛋白水平均降低（P<0.05）。与OA组和OA+NC-sh组比较，OA+TMED10-sh组大鼠的OARSI评分、TUNEL阳性率、TMED10 mRNA水平以及TMED10和MMP-13的蛋白水平均降低（P<0.05），TGF-β1、p-Smad2/Smad2、p-Smad3/Smad3和Collagen II蛋白水平均升高（P<0.05）。结论：本研究表明TMED10可能通过抑制TGF-β/Smads信号通路导致软骨细胞丢失和软骨退变，TMED10/TGF-β/Smads信号通路可能是治疗OA的新靶标。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the effect of transmembrane p24 trafficking protein 10 (TMED10) on transforming growth factor-β (TGF-β) /Smads signaling pathway in osteoarthritis. Methods: The rat chondrocytes were divided into Control group, IL-1βgroup, NC-sh+IL-1βgroup, TMED10-sh+IL-1βgroup, NC-OE+IL-1βgroup and TMED10-OE+IL-1βgroup. Control group and IL-1βgroup were not transfected, and NC-sh+IL-1βgroup, TMED10-sh+IL-1βgroup, NC-OE+IL-1βgroup and TMED10-OE+IL-1βgroup were transfected with NC-sh, TMED10-sh, NC-OE and TMED10-OE, respectively. After transfection, chondrocytes in all groups except Control group were treated with interleukin-1β(IL-1β) (10 ng/mL) for 24 h to simulate the pathological microenvironment of OA chondrocytes. Cell proliferation was detected by MTT assay. Apoptosis was detected by TUNEL method. The OA model was established by anterior cruciate ligament amputation plus medial meniscectomy. The OA model rats were divided into OA group, OA+NC-sh group and OA+TMED10-sh group (n=12). The rats in Sham group (n=12) were undergoing sham operation. The rats in Sham group and OA group were injected 40 μL normal saline into the joint cavity. The rats in OA+NC-sh group and OA+TMED10-sh group were injected with 40 μL NC-sh and TMED10-sh (titer 1×109 TU/mL), respectively. The injection was given once a week for 4 weeks. The shape of knee cartilage was evaluated by saffranine O/ solid green staining. Chondrocyte apoptosis was detected by TUNEL method. The mRNA level of TMED10 in chondrocytes and rat articular cartilage was detected by RT-qPCR. Protein expression levels of TMED10, TGF-β1, Smad2, p-Smad2, Smad3, Collagen II and matrix metalloproteinase-13 (MMP-13) in chondrocytes and rat articular cartilage were detected by Western blot. Results: Compared with Control group, the positive rate of TUNEL, the TMED10 mRNA, TMED10 and MMP-13 protein levels of IL-1βgroup increased (P<0.05), the relative cell viability and the protein levels of TGF-β1, p-Smad2/Smad2, p-Smad3/Smad3 and Collagen II of IL-1βgroup decreased (P<0.05). Compared with IL-1βgroup and NC-sh+IL-1βgroup, the positive rate of TUNEL, the TMED10 mRNA, TMED10 and MMP-13 protein levels of TMED10-sh+IL-1βgroup decreased (P<0.05), the relative cell viability and the protein levels of TGF-β1, p-Smad2/Smad2, p-Smad3/Smad3 and Collagen II of TMED10-sh+IL-1βgroup increased (P<0.05). Compared with IL-1βgroup and NC-OE+IL-1βgroup, the positive rate of TUNEL, the TMED10 mRNA, TMED10 and MMP-13 protein levels of TMED10-OE+IL-1βgroup decreased (P<0.05), the relative cell viability and the protein levels of TGF-β1, p-Smad2/Smad2, p-Smad3/Smad3 and Collagen II of TMED10-OE+IL-1βgroup increased (P<0.05). Compared with Sham group, the OARSI score, the positive rate of TUNEL, the TMED10 mRNA, TMED10 and MMP-13 protein levels of OA group increased (P<0.05), the protein levels of TGF-β1, p-Smad2/Smad2, p-Smad3/Smad3 and Collagen II protein in OA group decreased (P<0.05). Compared with OA group and OA+NC-sh group, the OARSI score, the positive rate of TUNEL, the TMED10 mRNA, TMED10 and MMP-13 protein levels of OA+TMED10-sh group decreased (P<0.05), and the protein levels of TGF-β1, p-Smad2/Smad2, p-Smad3/Smad3 and Collagen II in OA+TMED10-sh group increased (P<0.05). Conclusion: This study suggests that TMED10 may lead to chondrocyte loss and cartilage degeneration by inhibiting TGF-β/Smads signaling pathway, and TMED10/TGF-β/Smads signaling pathway may be a new target for the treatment of OA. |
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