降钙素介导CD36、IL-17的表达对创伤性骨关节炎的影响

Effect of Calcitonin-Mediated Expression of CD36 and IL-17 on Traumatic Osteoarthritis

目的 探讨降钙素对白介素-1β(interleukin-1β,IL-1β)诱导的软骨细胞的保护作用并进一步研究其可能的机制。方法 2022年7月21日—11月10日以人软骨细胞为研究对象,使用不同浓度降钙素处理后,采用CCK-8试剂盒(cell counting kit CCK 8,CCK-8)实验筛选出降钙素最佳作用浓度进行后续实验。将人软骨细胞分为对照组、疾病组、治疗组,采用CCK-8法检测细胞活性,流式细胞术分析CD36表达和活性氧簇(reactive oxygen species,ROS)产生情况,酶标法检测各组细胞超氧化物歧化酶(superoxide dismutase,SOD)和丙二醛(malondialdehyde,MDA)水平,实时荧光定量聚合酶链反应(quantitative real-time polymerase chain reaction,q RT-PCR)检测降钙素、白介素-17(interleukin-17,IL-17)、基质金属蛋白酶13(matrix metalloproteinase 13,MMP13)、Ⅱ型胶原(type Ⅱcollagen,Col Ⅱ)的m RNA相对表达量,蛋白印迹法(Western blot)检测NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor heat protein domain associated protein3,NLRP3)、消皮素D(gasdermin-D,GSDMD)、GSDMD-N的蛋白表达水平。结果 人软骨细胞的活力对降钙素呈浓度依赖性,后选择50 nM浓度的降钙素进行细胞实验。疾病组细胞伴随ColⅡ的m RNA相对表达量为(0.47±0.06)、CD36阳性率为(0.17±0.02)%、SOD为(151.14±12.26)U/mL,低于对照组的(1.00±0.09)、(1.50±0.16)%、(242.33±21.17)U/mL(P <0.05);与对照组比较,疾病组细胞中MMP13和IL-17m RNA相对表达量、ROS的平均荧光强度(mean fluorescence intensity,MFI)及MDA含量升高;同时,NLRP3和GSDMD的蛋白表达水平上升,而GSDMD-N蛋白表达水平下降。然而,降钙素的使用能够逆转IL-1β诱导软骨细胞引起的上述一系列指标变化。Pearson分析显示,降钙素与CD36表达呈正相关(r=0.922,P=0.001),与IL-17呈负相关(r=-0.881,P=0.002),CD36与IL-17的表达水平呈负相关(r=-0.650,P=0.023)。结论 降钙素对IL-1β诱导的软骨细胞损伤具有保护作用,其作用机制与介导CD36、IL-17的表达,缓解软骨细胞的应激炎症反应有关。

Objective To investigate the protective effect of calcitonin on interleukin-1β(IL-1β)-induced chondrocytes,and further to study its potential mechanisms.Methods From July 21 to November 10,2022,human chondrocytes were treated with different concentrations of calcitonin,and the optimal concentration of calcitonin was selected by cell counting kit CCK 8(CCK-8) assay for follow-up experiments.Human chondrocytes were divided into control group,disease group and treatment group.The cell activity was detected by CCK-8 assay.The expression of CD36 and the production of intracellular reactive oxygen species(ROS) were analyzed by flow cytometry.The levels of superoxide dismutase(SOD) and malondialdehyde(MDA)were detected by enzyme labeling method.The mRNA relative expression levels of calcitonin,interleukin-17(IL-17),matrix metalloproteinase 13(MMP13) and type Ⅱ collagen(Col Ⅱ)were detected by quantitative real-time polymerase chain reaction(qRT-PCR).Western blot was used to detect the expression levels of NOD-like receptor heat protein domain associated protein 3(NLRP3),gasdermin-D(GSDMD) and GSDMD-N.Results It was found that the activity of human chondrocytes was concentrationdependent on calcitonin.Therefore,50 nM concentration of calcitonin was selected for subsequent experiments.In the disease group,the relative expression of Col Ⅱ mRNA was(0.47±0.06),the positive rate of CD36 was(0.17±0.02) %,and SOD was(151.14±12.26) U/mL,lower than control group(1.00±0.09),(1.50±0.16)%,(242.33±21.17) U/mL(P < 0.05).Compared with the control group,the relative mRNA expression of MMP13 and IL-17,mean fluorescence intensity(MFI) of ROS and MDA content of the disease group were increased.Meanwhile,the expression levels of NLRP3 and GSDMD were increased,while the expression levels of GSDMD-N were decreased.However,the administration of calcitonin reversed the IL-1β-induced changes in chondrocytes.Pearson analysis showed that calciton in was positively correlated with CD36 expression(r = 0.922,P = 0.001),negatively correlated with IL-17 expression(r =-0.881,P =0.002),and negatively correlated with CD36 expression level of IL-17(r =-0.650,P =0.023).Conclusion All those results suggested that calcitonin could protect IL-1β-induced chondrocytes damage,and its mechanism is related to mediating the expression of CD36 and IL-17,and alleviating the stress and inflammation of chondrocytes.