“杜仲-当归”调控NLPR3炎症小体对骨关节炎大鼠改善作用的研究

Study of the improvement effect of"DuZhong-DangGui"on osteoarthritis rats via regulating NLPR3 inflammasome

目的:探究药对“杜仲-当归”对骨关节炎(osteoarthritis,OA)大鼠膝组织的改善效果及对NLPR3炎症小体的调控。方法:24只雄性SD大鼠随机挑选18只采用前交叉韧带切断术(ACLT)进行OA造模,随后将大鼠分为正常组、OA模型组、杜仲-当归组和阳性药物组,每组6只,后两组分别给与“杜仲-当归”或西乐葆和奥泰灵混合液治疗8周。收集大鼠外周血,ELISA测定IL-1β、IL-6、IL-18、TNF-α水平;另取大鼠膝关节软骨组织,HE染色观察病理改变,番红-固绿染色观察软骨钙化情况并行OARSI评分,免疫组化和TUNEL染色分别检测膝关节软骨组织Caspase1、CollagenⅡ表达和软骨细胞凋亡情况,Western blot检测基质金属蛋白酶-13(MMP-13)、基质金属蛋白酶抑制剂-1(TIMP-1)、p53、p21、NLPR3、凋亡相关斑点样蛋白(ASC)、Pro-Caspase-1蛋白表达。结果:与正常组相比,OA模型组大鼠血清IL-1β、IL-6、IL-18、TNF-α水平均显著上升(P<0.01),膝关节损伤明显,OARSI评分显著升高(P<0.01),软骨细胞凋亡显著增加(P<0.01),组织Caspase1含量、MMP-13、p53、p21、NLPR3、ASC、Pro-Caspase-1蛋白表达均显著升高(P<0.01),而CollagenⅡ含量和TIMP-1蛋白表达显著降低(P<0.01)。与OA模型组相比,杜仲-当归组和阳性药物组大鼠血清IL-1β、IL-6、IL-18、TNF-α水平显著下降(P<0.05),膝关节损伤改善,OARSI评分降低(P<0.01),软骨细胞凋亡显著减少(P<0.01),组织Caspase1含量,MMP-13、p53、NLPR3、Pro-Caspase-1蛋白表达显著降低(P<0.05)、CollagenⅡ含量和TIMP-1蛋白表达显著增加(P<0.05),杜仲-当归组大鼠p21、ASC蛋白表达显著降低(P<0.01)。结论:“杜仲-当归”可改善OA大鼠软骨病变,其作用可能与介导NLPR3/ASC/Pro-Caspase-1炎性体通路,下调IL-1β及相关炎性因子水平、抑制软骨细胞凋亡有关。

AIM:To investigate the protective effect of"DuZhong-DangGui"(DZ-DG)on the knee tissue of rats with osteoarthritis(OA)and its regulation role on NLPR3 inflammasome.METHODS:Twenty-four SPF male SD rats,eighteen rats were randomly selected to establish OA model by anterior cruciate ligament amputation(ACLT)method,and rats were divided into control group,OA model group,DZ-DG group and positive drug group,6 in each group,treatment for 8 weeks.The peripheral blood were collected,ELISA was used to detect the levels of IL-1β,IL-6,IL-18,and TNF-α;cartilage tissue of knee joint were collected,HE staining was used to observe pathology changes,OARSI staining was used to observe cartilage calcification and preform quantitative OARSI scoring;immunohistochemistry and TUNEL staining were used to detect the contents of Caspase1 and Collagen II and the number of apoptosis in the tissue,respectively,and western blot was used to detect the protein expressions of matrix metalloproteinase-13(MMP-13),tissue inhibitor of metalloproteinase-1(TIMP-1),p53,p21,NLPR3,apoptosis-associated speck-like protein containing a CARD(ASC)and Pro-Caspase-1.RESULTS:Compared to control group,OA model group rats serum levels of IL-1β,IL-6,IL-18,and TNF-αsignificantly increased(P<0.01),OARSI scores significantly increased(P<0.01),chondrocyte apoptosis was increased(P<0.01),Caspase-1 content and MMP-13,p53,p21,NLPR3,ASC,Pro-Caspase-1 protein expressions significantly increased(P<0.01),while Collagen II content and TIMP-1 protein expression significantly decreased(P<0.01).Compared with the OA model group,DZ-DG group and positive drug group rats serum levels of IL-1β,IL-6,IL-18 and TNF-αsignificantly decreased(P<0.05),chondrocyte apoptosis were significantly decreased(P<0.01),Caspase1 content,MMP-13,p53,NLPR3,Pro-Caspase-1 significantly decreased(P<0.05),CollagenⅡcontent and TIMP-1 protein expression(P<0.01);DZ-DG group rats protein expression of p21 and ASC were decreased(P<0.01).CONCLUSION:The DZ-DG have protection role on cartilage of OA rats,its effect may related to mediation of NLPR3/ASC/Pro-Caspase-1 pathway,to decrease of IL-1β,and inhibition of cell apoptosis.