电针对膝骨关节炎大鼠膝关节滑膜组织细胞焦亡的影响

Effect of electroacupuncture on pyroptosis of synovial tissues in rats with knee osteoarthritis

目的:观察电针对膝骨关节炎(KOA)大鼠膝关节滑膜组织中细胞焦亡相关蛋白的影响,探究电针治疗KOA的作用机制。方法:40只SD大鼠随机分为对照组、模型组、电针组和药物组,每组10只。用4%木瓜蛋白酶0.2mL关节腔注射建立KOA大鼠模型。电针组予右膝关节“内膝眼”“犊鼻”电针治疗,15min/次,1次/d,6d/周,连续4周。药物组予塞来昔布24mg/kg灌胃,1次/d,6d/周,连续4周。干预前后采用Lequesne评分评估大鼠KOA功能障碍程度;ELISA法检测血清白细胞介素(IL)-1β和IL-18的含量;HE染色法观察右膝关节滑膜组织的形态学变化并进行滑膜炎病理评分;免疫组织化学法观察右膝关节滑膜组织中NOD样受体蛋白3(NLRP3)的表达;实时荧光定量PCR法检测右膝关节滑膜组织中NLRP3、凋亡相关斑点样蛋白(ASC)、Gasdermin D(GSDMD)、半胱氨酸天冬氨酸蛋白酶1(Caspase-1)、IL-1β、IL-18 mRNA的表达;Western blot法检测右膝关节滑膜组织中NLRP3、ASC、Caspase-1、GSDMD、GSDMD-N、IL-1β、IL-18蛋白的表达。结果:与对照组比较,模型组右膝关节Lequesne评分和滑膜炎病理评分显著上升(P<0.01),血清中IL-1β、IL-18的含量显著增加(P<0.01),NLRP3在滑膜内衬层和血管、炎性细胞中大量表达,滑膜中NLRP3、ASC、GSDMD、Caspase-1、IL-1β、IL-18的mRNA和蛋白表达水平及GSDMD-N的蛋白表达水平均显著升高(P<0.01)。与模型组比较,电针组和药物组右膝关节Lequesne MG评分和滑膜炎病理评分显著降低(P<0.01),血清中IL-1β、IL-18的含量显著降低(P<0.01),NLRP3在滑膜内衬层和血管、炎性细胞中有少量阳性表达,滑膜中NLRP3、ASC、Caspase-1、IL-1β、IL-18的mRNA和蛋白表达水平及GSDMD-N的蛋白表达水平均显著降低(P<0.01),GSDMD的mRNA表达水平降低(P<0.05),药物组GSDMD蛋白表达水平降低(P<0.05)。与药物组比较,电针组血清中IL-1β、IL-18的含量升高(P<0.05),滑膜组织中IL-1β的mRNA和蛋白的表达显著升高(P<0.01,P<0.05)。结论:电针能减轻KOA大鼠膝关节滑膜组织的炎性反应,其机制可能与下调NLRP3、ASC、Caspase-1、GSDMD、GSDMD-N、IL-1β和IL-18的表达水平,减少滑膜组织中细胞焦亡的发生有关。

Objective To observe the effect of electroacupuncture(EA)on pyroptosis-related proteins in synovium of knee joint in rats with knee osteoarthritis(KOA),in order to explore its mechanism underlying improvement of KOA.Methods Forty male SD rats were randomly divided into control,model,EA and medication groups,with 10rats in each group.The KOA model was established by injecting 0.2mL 4%papain solution into the right intra-articular cavity,followed by repeating the injection again on day 4and 7after the first injection.After successful modeling,rats of the EA group received EA stimulation of“Neixiyan”(EX-LE4)and“Dubi”(ST35)on the right limb for 15min,once every day,6days per week,for a total of 4weeks,and those of the medication group received gavage of celecoxib 24mg/kg,once every day,6days per week,for a total of 4weeks.The se-verity of dysfunction of the right knee was assessed by using Lequesne’s score.Serum interleukin(IL)-1βand IL-18contents were detected by ELISA.Histopathological changes of the synovium tissue of the right knee joint were observed to give score(synovial pathological score)after H.E.staining.The expression position and intensity of Nod-like receptor pyrin domain 3(NLRP3)in synovial tissue were observed by immunohistochemistry.The expression levels of NLRP3,apoptosis-associated speck-like protein containing card(ASC),Caspase-1,Gasdermin D(GSDMD),IL-1βand IL-18mRNAs and proteins(including GSDMD-N)in the synovial tissue of the right knee joint were detected by real-time fluorescence quantitative PCR and Western blot,separately.Results Compared with the control group,the model group had a significant increase in the Lequesne’s score,synovial pathological score,serum IL-1βand IL-18contents,and the expression levels of NLRP3,ASC,Caspase-1,GSDMD,IL-1β,IL-18mRNAs and proteins and GSDMD-N protein(P<0.01).Whereas relevant to the model group,both the EA and medication groups had marked lower levels of Lequesne’s score and synovial pathological score,serum IL-1βand IL-18contents,and expression levels of NLRP3,ASC,Caspase-1,IL-1β,IL-18mRNAs and proteins,GSDMD mRNA and GSDMD-N protein(P<0.01,P<0.05).Comparison between two intervention groups showed that the contents of serum IL-1βand IL-18,and the expression levels of IL-1βmRNA and protein were significantly higher in the EA group than in the medication group(P<0.05,P<0.01).No significant differences were found between the EA and medication groups in the Lequesne’s score,synovial pathological score,NLRP3,ASC,Caspase-1and IL-18mRNAs and proteins,as well as GSDMD mRNA(P>0.05).Conclusion EA can alleviate the inflammatory response of synovial tissues of knee joints in KOA rats,which may be related to its function in down-regulating the expression levels of synovial NLRP3,ASC,Caspase-1,IL-1βand IL-18mRNAs and proteins,and GSDMD mRNA and GSDMD-N proteins,reducing the occurrence of pyroptosis.