塞来昔布对离体人膝骨关节炎细胞凋亡及EGFR/MAPK信号通路的影响

Effects of celecoxib on apoptosis of and EGFR/MAPK signal pathway in human knee osteoarthritis chondrocytes in vitro

摘要:

目的 探究塞来昔布对离体人膝骨关节炎细胞凋亡及表皮生长因子受体/丝裂原激活蛋白激酶（EGFR/MAPK）通路的影响。方法 体外分离及培养膝关节软骨细胞，并以甲苯胺蓝染色鉴定软骨细胞。将细胞分为空白对照组（人正常膝骨关节软骨细胞不做特殊处理）、模型组（人膝骨关节炎软骨细胞不做特殊处理）及塞来昔布低、高剂量组（人膝骨关节炎软骨细胞中分别加入含终浓度为50 μmol/L和200 μmol/L塞来昔布溶液的培养液）。采用CCK-8法、AnnexinV/PITC双染法分别检测各组细胞增殖、凋亡能力，Western blotting检测细胞增殖、凋亡及EGFR/MAPK通路蛋白的表达。结果 甲苯胺蓝染色结果显示，人正常膝骨关节软骨细胞、人膝骨关节炎软骨细胞内见蓝紫色异染颗粒，细胞核呈深蓝色，细胞质呈浅蓝色；与人膝骨关节炎软骨细胞形态比较，人正常膝骨关节软骨细胞体积较大，形态较规则，蓝紫色异染颗粒较多。与空白对照组比较，模型组，塞来昔布低、高剂量组细胞存活率降低（P <0.05），凋亡率升高（P <0.05）；与模型组比较，塞来昔布低、高剂量组细胞存活率升高（P <0.05），凋亡率降低（P <0.05）；与塞来昔布低剂量组比较，塞来昔布高剂量组细胞存活率升高（P <0.05），凋亡率降低（P <0.05）。与空白对照组比较，模型组，塞来昔布低、高剂量组EGFR、MMP-9、Caspase-3蛋白相对表达量升高（P <0.05），p-P38 MAPK/P38 MAPK蛋白相对表达量降低（P <0.05）；与模型组相比，塞来昔布低、高剂量组EGFR、MMP-9、Caspase-3蛋白相对表达量降低（P <0.05），p-P38 MAPK/P38 MAPK蛋白相对表达量升高（P <0.05）；与塞来昔布低剂量组比较，塞来昔布高剂量组EGFR、MMP-9、Caspase-3蛋白相对表达量降低（P <0.05），p-P38 MAPK/P38 MAPK蛋白相对表达量升高（P <0.05）。结论 塞来昔布能促进离体人膝骨关节炎细胞增殖，抑制细胞凋亡。其作用可能是通过抑制EGFR表达，促进P38 MAPK通路活化实现的。

Abstract:

Objective To investigate the effects of celecoxib on apoptosis of and epidermal growth factor receptor (EGFR)/mitogen activated protein kinase (MAPK) pathway in human knee osteoarthritis (OA) chondrocytes in vitro.Methods Human knee OA chondrocytes were isolated and cultured in vitro, and were identified via toluidine blue staining. Three groups, including control group (normal knee articular chondrocytes, no special treatment), model group (knee OA chondrocytes, no special treatment) and low- and high-dose celecoxib group (knee OA chondrocytes, treated with celecoxib at the final concentration of 50 μmol/L and 200 μmol/L, respectively). The cell proliferation and apoptosis were detected by CCK-8 assay and Annexin V/PITC staining, respectively. The protein expressions of molecules associated with cell proliferation, apoptosis, and EGFR/MAPK signaling pathway were detected by Western blotting.Results The toluidine blue staining showed bluish violet metachromatic granules in normal knee articular chondrocytes and knee OA chondrocytes, and the nucleus was stained dark blue while the cytoplasm was stained light blue. Compared with the knee OA chondrocytes, the size of normal knee articular chondrocytes was larger and regularly they are shaped, with more bluish violet metachromatic granules. Compared with the control group, the survival rate of chondrocytes was lower and the apoptosis rate was higher in the model group and low- and high-dose celecoxib group (P < 0.05). The survival rate of chondrocytes was higher and the apoptosis rate was lower in the low- and high-dose celecoxib group than those in the model group (P < 0.05). The survival rate of chondrocytes was higher and the apoptosis rate was lower in the high-dose celecoxib group compared with the low-dose celecoxib group (P < 0.05). The protein expressions of EGFR, MMP-9, and caspase-3 were higher, and the protein expressions of p-P38 MAPK/P38 MAPK were lower in the model group and low- and high-dose groups than in the control group (P < 0.05). Compared with the model group, the protein expressions of EGFR, MMP-9, and caspase-3 were lower, and the protein expressions of p-P38 MAPK/P38 MAPK were higher in the low- and high-dose celecoxib groups (P < 0.05). In addition, the protein expressions of EGFR, MMP-9, and caspase-3 were lower, and the protein expressions of p-P38 MAPK/P38 MAPK were higher in the high-dose celecoxib group compared with the low-dose celecoxib group (P < 0.05).Conclusions Celecoxib can promote the proliferation and inhibit the apoptosis of human knee OA chondrocytes in vitro, which may be achieved by inhibiting the expression of EGFR and activating the P38 MAPK pathway.