荣筋拈痛方调控软骨细胞NLRP3/caspase-1/GSDMD通路改善骨关节炎炎性病变的机制

Mechanism of Rongjin Niantong Formula in regulating chondrocyte NLRP3/caspase-1/GSDMD and improving inflammatory lesions of osteoarthritis

目的:探讨荣筋拈痛方调控NOD样受体热蛋白结构域相关蛋白3(NLRP3)/半胱氨酸天冬氨酸蛋白酶1(caspase-1)/消皮素D(GSDMD)通路减轻软骨细胞焦亡,改善骨关节炎炎性病变的作用机制。方法:将二代软骨细胞随机分为正常组、模型组[脂多糖(LPS)+三磷酸腺苷(ATP)]、荣筋拈痛方组(LPS+ATP+荣筋拈痛方)和抑制剂组(LPS+ATP+MCC950)。Hoechst 33342/PI双染检测软骨细胞膜的完整性;双转盘活细胞共聚焦实时成像分析系统观察NLRP3和caspase-1的定位与表达;ELISA检测白细胞介素(IL)-1β和IL-18的水平;RT-PCR检测NLRP3、caspase-1、GSDMD、Ⅱ型胶原蛋白(collagenⅡ)和聚集蛋白聚糖(aggrecan)mRNA水平;Western Blot检测NLRP3、pro-caspase-1、caspase-1、GSDMD、GSDMD-N、collagenⅡ、aggrecan、基质金属蛋白酶(MMP)-3和MMP-13蛋白表达。结果:与模型组比较,荣筋拈痛方可降低因细胞膜完整性丧失而染上的PI红色荧光强度;降低NLRP3和caspase-1蛋白表达的绿色荧光强度(P<0.01);降低软骨细胞上清液中炎症因子IL-1β和IL-18水平(P<0.01);降低NLRP3、caspase-1、GSDMD mRNA水平(P<0.05,P<0.01),提高collagenⅡ和aggrecan mRNA水平(P<0.01);降低NLRP3、pro-caspase-1、caspase-1、GSDMD、GSDMD-N、MMP-3和MMP-13蛋白表达(P<0.05,P<0.01),提高collagenⅡ和aggrecan蛋白表达(P<0.05)。结论:荣筋拈痛方可通过调控软骨细胞NLRP3/caspase-1/GSDMD途径,减轻因细胞焦亡而导致的炎症反应,改善骨关节炎炎性病变。

Objective: To discuss the mechanism of Rongjin Niantong Formula(RJNTF) in regulating pathways of NLRP3/caspase-1/GSDMD, reducing the pyroptosis of chondrocytes, and improving the inflammatory lesions of osteoarthritis.Methods: Second-generation cartilage cells are selected and divided into normal groups, model groups(LPS+ATP), RJNTF groups(LPS+ATP+RJNTF), and inhibitor groups(LPS+ATP+MCC950). Hoechst 33342/PI double staining to detect the integrity of chondrocytes membrane. NLRP3 and caspase-1 positioning and expressions of cartilage cells are observed under the laser scanning confocal microscope with dual plates. IL-1β and IL-18 expressions are tested by ELISA. NLRP3, caspase-1, GSDMD as well as collagen Ⅱ and aggrecan mRNA are analyzed by RT-PCR. Western Blot was used to analyze the protein expressions of NLRP3,pro-caspase-1, caspase-1, GSDMD, GSDMD-N, collagen Ⅱ, aggrecan, MMP-3 and MMP-13. Results: Compared with the model group, RJNTF reduced the PI red fluorescence intensity due to the loss of cell membrane integrity. NLRP3 and caspase-1 green fluorescence intensity were decreased(P<0.01). The levels of inflammatory factors IL-1β and IL-18 in the supernatant were decreased(P<0.01);The mRNA levels of NLRP3, Caspase-1 and GSDMD were decreased(P<0.05, P<0.01), collagen Ⅱ and Aggrecan m RNA were increased(P<0.01). The expression levels of NLRP3, pro-caspase-1, caspase-1, GSDMD, GSDMD-N, MMP-3 and MMP-13 were decreased(P<0.05, P<0.01), and the expression levels of collagen Ⅱ and aggrecan were increased(P<0.05). Conclusion:RJNTF regulates the NLRP3/caspase-1/GSDMD pathway, thereby reducing the inflammatory response caused by chondrocytes pyroptosis and improving the inflammatory lesions of osteoarthritis.