摘要
目的 基于Piezo1/NF-κB/NLRP3轴探讨机械应力介导膝关节滑膜炎的效应机制.方法 动物实验:将40只SD大鼠分为空白组、ACLT组、运动组、运动+抑制剂组4组,每组各10只.ACLT组行前交叉韧带横断手术,建立KOA大鼠模型;剩余除空白组外均采用动物跑台构建运动大鼠模型;运动+抑制剂组在运动模型的基础上,行腹腔注射GsMTx4,每日1 mg/kg.造模完成后对大鼠滑膜组织及血清进行HE染色、Western Blot、qRT-PCR及ELISA实验,检测Piezo1、NF-κB、NLRP3、ASC、Caspase-1、IL-1β、IL-6、TNF-α等相关蛋白及mRNA的表达.②细胞实验:将成纤维样滑膜细胞分为空白组、LPS组、拉力组、拉力+抑制剂组4组.空白组正常培养,LPS组使用含5µg/mLLPS的全培干预,剩余两组采用机械牵张仪器构建拉力模型,拉力+抑制剂组使用含5 µmol/L GsMTx4的全培干预.造模完成后的细胞通过免疫荧光、WB、PCR等技术进行上述指标的检测.结果 动物实验:运动组较空白组呈现明显的滑膜炎症,IL-1β、IL-6、TNF-α的表达明显升高(P＜0.05),并显著激活Piezo1、NF-κB、NLRP3、ASC、Caspase-1(P＜0.05).Piezo1 抑制剂 GsMTx4 可以部分减弱这些作用(P＜0.05).②细胞实验:拉力组滑膜细胞较空白组相比,各项指标表达同动物实验.GsMTx4同样可以部分减弱这些作用.结论 Piezo1蛋白能够感受机械应力,经NF-κB活化NLRP3炎症小体,诱发膝关节滑膜炎.
Abstract
Objective To investigate the mechanisms by which mechanical stress induces synovitis in the knee joint through the Piezo1/NF-κB/NLRP3 signaling pathway.Methods Animal experiment:Forty Sprague-Dawley rats were divided into four groups:blank/control group,ACLT group,exercise group,and exercise+inhibitor group,with ten rats per group.The ACLT group underwent ACL transection surgery to establish a rat model of knee osteoarthritis;remaining groups,excluding the control group,were subjected to a treadmill exercise model;the exercise+inhibitor group received intraperitoneal injections of GsMTx4 at 1 mg/kg per day on top of the exercise model.After the modeling was complete,rat synovial tissue and serum were analyzed using Hematoxylin and Eosin staining,Western Blot,qRT-PCR,and ELISA to detect the expression of proteins and mRNA related to Piezo1,NF-κB,NLRP3,ASC,Caspase-1,IL-1β,IL-6,and TNF-α.② Cell experiment:Fibroblast-like synoviocytes were divided into four groups:blank/control group,LPS group,tensile stress group,and tensile stress+inhibitor group.The control group was cultured normally;the LPS group was treated with 5 μg/mL LPS-containing culture medium;the remaining two groups were subjected to a mechanical stretching model,and the tensile stress+inhibitor group was treated with culture medium containing 5 μM GsMTx4.After modeling was complete,the cells were analyzed using immunofluorescence,WB,PCR,etc.,for the indicators mentioned above.Results Animal experiment:The exercise group displayed significant synovitis compared to the control group,with markedly increased expression of IL-1β,IL-6,and TNF-α(P＜0.05),and significant activation of Piezo1,NF-κB,NLRP3,ASC,and Caspase-1(P＜0.05).The Piezo1 inhibitor GsMTx4 was able to partially mitigate these effects(P＜0.05).② Cell experiment:The tensile stress group had increased expression of the indicators similarly to the animal experiment when compared to the control group.GsMTx4 was also able to partially reduce these effects.Conclusion Piezo1 protein can sense mechanical stress and activate the NLRP3 inflammasome via NF-κB,which leads to synovitis in the knee joint.
维普
万方数据