温针灸对兔膝骨关节炎软骨组织中NLRP3、IL-1β和IL-18表达的影响

Study on the Effect of Warm Acupuncture and Moxibustion on the Expression of NLRP3,IL-1βand IL-18 in Cartilage of Rabbits Knee Osteoarthritis

目的观察温针灸对兔膝骨关节炎(knee osteoarthritis,KOA)软骨组织中nod样受体蛋白3(nod-like receptor protein 3,NLRP3)、白细胞介素(interleukin,IL)-1β和IL-18表达的影响,阐明温针灸治疗KOA的效应机制。方法选取6月龄新西兰雄兔40只,随机分为空白组、模型组、温针灸组、NLRP3抑制剂组,每组10只,除空白组外其余3组均采用右后肢伸直位石膏管型固定法固定4周建立损伤差异无统计学意义的兔KOA模型,分别予以相应的干预手段治疗2周,采用Lequesne MG评分进行行为学评估,HE染色观察兔KOA软骨组织形态学结构并进行病理学Mankin’s评分,免疫组化法检测软骨组织中NLRP3、IL-1β、IL-18的表达。结果造模前Lequesne MG评分各组差异无统计学意义(P>0.05);造模结束后,与空白组比较,其余组Lequesne MG评分均升高(P均<0.05);治疗结束后,与模型组比较,温针灸组、NLRP3抑制剂组评分均降低(P均<0.05)。HE染色结果显示:空白组软骨表面平整光滑,软骨细胞排列整齐,软骨层结构完整清晰;模型组软骨结构完整性被破坏,软骨表层粗糙、缺损,细胞形态不规整,排列紊乱;温针灸组软骨表面基本光滑完整,软骨细胞排列规则,分布均匀,软骨层结构较完整;NLRP3抑制剂组软骨表面相对完整,软骨细胞分布较均匀,软骨层结构相对清晰。Mankin’s评分:治疗结束后,与空白组比较,其余组兔膝关节病理学改良Mankin’s评分均升高(P均<0.05);与模型组比较,温针灸组和NLRP3抑制剂组Mankin’s评分均降低(P均<0.05)。免疫组化:与空白组比较,模型组、温针灸组和NLRP3抑制剂组中NLRP3表达均升高(P均<0.05),与模型组比较,温针灸组与NLRP3抑制剂组表达水平均降低(P均<0.05);与空白组比较,模型组、温针灸组和NLRP3抑制剂组中IL-1β表达均升高(P均<0.05),与模型组比较,温针灸组与NLRP3抑制剂组表达水平均降低(P均<0.05);与空白组比较,模型组、温针灸组和NLRP3抑制剂组中IL-18表达均升高(P均<0.05),与模型组比较,温针灸组与NLRP3抑制剂组表达水平均降低(P均<0.05)。结论温针灸可有效抑制NLRP3炎性因子的表达,进而降低炎性级联放大反应,保护关节软骨,达到防治KOA的目的。

Objective To observe the effects of warm acupuncture and moxibustion on the expressions of nod-like receptor protein 3(NLRP3),interleukin-1β(IL-1β)and interleukin-18(IL-18)in cartilage tissue of rabbit knee osteoarthritis(KOA),and to clarify the effect mechanism of warm acupuncture and moxibustion on KOA.Methods A total of 40 New Zealand male rabbits aged 6 months,which were randomly divided into blank group,model group,warm acupuncture group and NLRP3 inhibitor group,with 10 rabbits in each group.Except blank group,the other three groups were fixed with right hindlimb extension plaster tube fixation for 4 weeks to establish the rabbit KOA model with no difference in injury,and were treated with corresponding intervention methods for 2 weeks respectively.After the experiment,Lequesne MG was used to mark behavior assessment;HE staining was used to observe the morphology and structure of cartilage,pathological Mankin's score was performed,and immunohistochemistry was used to detect the expression of NLRP3,IL-1βand IL-18 protein in cartilage.Results Lequesne MG score showed that there was no significant difference among all groups before modeling.After modeling,compared with blank group,the Lequesne MG score of the other three groups increased(P all<0.05);After treatment,compared with model group,the scores of warm acupuncture group and NLRP3 inhibitor group were significantly lower(P all<0.05).The HE staining results showed that in blank group,the cartilage surface was smooth,chondrocytes were arranged neatly,and the structure of cartilage layer was complete and clear.In the model group,the structural integrity of cartilage was destroyed,the surface of cartilage was rough and defective,and the cell morphology was irregular and disordered.In warm acupuncture group,the cartilage surface was basically smooth and complete,the chondrocytes were arranged regularly and distributed evenly,and the cartilage layer structure was relatively complete.In NLRP3 inhibitor group,the cartilage surface was relatively intact,chondrocytes were evenly distributed,and the cartilage layer structure was relatively clear.Mankin's score:After treatment,compared with the blank group,the modified Mankin's score of knee pathology in the other three groups were significantly increased(P all<0.05);Compared with the model group,the Mankin's score of warm acupuncture group and inhibitor group were significantly decreased(P all<0.05).The Immunohistochemistry results showed that the expression of NLRP3 in blank group was higher than that in model group,warm acupuncture group and NLRP3 inhibitor group(P all<0.05),and the expression of NLRP3 in warm acupuncture group and inhibitor group was lower than that in model group(P all<0.05).The expression of IL-1βin blank group was higher than that in model group,warm acupuncture group and NLRP3 inhibitor group(P all<0.05),and the expression of IL-1βin warm acupuncture group and inhibitor group was lower than that in model group(P all<0.05).The expression of IL-18 in blank group was higher than that in model group,warm acupuncture group and NLRP3 inhibitor group(P all<0.05),and the expression of IL-18 in warm acupuncture group and inhibitor group was lower than that in model group(P all<0.05).Conclusion Warm acupuncture and moxibustion can effectively inhibit the expression of NLRP3 inflammatory factor,reduce inflammatory cascade amplification reaction,protect articular cartilage,and achieve the purpose of prevention and treatment of KOA.