摘要
目的 探讨橄榄苦苷对创伤性关节炎大鼠的治疗作用和对OPG-RANKL-RANK信号通路的影响.方法 将大鼠随机分为假手术组、模型组、橄榄苦苷低剂量组、橄榄苦苷高剂量组、HY-N6973(RANKL-RANK信号通路抑制剂)组.改良Mankin评分评价软骨退变程度;ELISA检测大鼠关节软骨组织中TNF-α、IL-1β、IL-10、CTX-Ⅰ、CTX-Ⅱ水平;商品化试剂盒检测关节软骨组织中SOD、MDA、GSH含量;HE和番红O-固绿染色观察关节软骨组织形态学改变;qRT-PCR检测 OPG、RANKL 和 RANK 基因表达;Western Blot 检测大鼠关节软骨组织 Cleaved-caspase-3、MMP-13、OPG、RANKL、RANK蛋白表达.结果 假手术组大鼠膝关节形态、软骨组织和细胞外基质完整.与假手术组相比,模型组大鼠膝关节软骨组织严重损伤,Mankin评分、血清TNF-α、IL-1β、CTX-Ⅰ、CTX-Ⅱ、软骨组织中MDA水平、RANKL mRNA和RANK mRNA水平、Cleaved-caspase-3、MMP-13、RANKL、RANK蛋白表达显著升高,血清IL-10水平、软骨组织中SOD、GSH水平、OPG基因和蛋白表达水平显著降低(P＜0.05);与模型组相比,橄榄苦苷低、高剂量组和HY-N6973组膝关节软骨组织损伤显著减轻,Mankin 评分、血清 TNF-α、IL-1β、CTX-Ⅰ、CTX-Ⅱ、软骨组织中 MDA、RANKL mRNA 和 RANK mRNA 水平、Cleaved-caspase-3、MMP-13、RANKL、RANK蛋白表达水平显著降低,血清IL-10水平、软骨组织中SOD、GSH水平、OPG基因和蛋白表达显著升高(P＜0.05);与橄榄苦苷高剂量组相比,HY-N6973组各项检测指标差异均无统计学意义(P＞0.05).结论 橄榄苦苷可降低创伤性关节炎大鼠炎症反应和氧化应激,改善关节损伤,可能与调控OPG-RANKL-RANK信号通路有关.
Abstract
Objective To investigate therapeutic effect of oleuropein on traumatic arthritis rats and its effect on OPG-RANKL-RANK signaling pathway.Methods Rats were randomly grouped into sham surgery group,model group,low-dose oleuro-pein group,high-dose oleuropein group,and HY-N6973(RANKL-RANK signaling pathway inhibitor)group.The improved Mankin score was applied to evaluate the degree of cartilage degeneration.ELISA was applied to detect the levels of TNF-α,IL-1β,IL-10,CTX-Ⅰ,and CTX-Ⅱ in rat articular cartilage tissue.Commercial reagent kits were applied to detect the contents of SOD,MDA,and GSH in articular cartilage tissue.HE and safranine O-solid green staining were applied to observe the morphological changes of articular cartilage tissue.OPG,RANKL and RANK genes was detected by qRT-PCR.Western Blot was applied to detect the expres-sion of Cleaved-caspase-3,MMP-13,OPG,RANKL,and RANK proteins in rat articular cartilage tissue.Results The knee joint morphology,cartilage tissue,and extracellular matrix of the sham operated group rats were intact.Compared with sham surgery group,model group had severe damage to the cartilage tissue of the knee joint in rats,Mankin score,serum TNF-α,IL-1β,CTX-Ⅰ,CTX-Ⅱ,MDA in cartilage tissue,RANKL mRNA and RANK mRNA,and Cleaved-caspase-3,MMP-13,RANKL,and RANK pro-teins were greatly increased,serum IL-10,the level of SOD,GSH in cartilage tissue,andthe expression of OPG gene and protein were greatly reduced(P＜0.05).Compared with model group,the damage to the cartilage tissue of the knee joint in the low-dose and high-dose oleuropein groups and the HY-N6973 group was greatly reduced,Mankin score,serum TNF-α,IL-1 β,CTX-Ⅰ,CTX-Ⅱ,the level of MDA in cartilage tissue,RANKL mRNA and RANK mRNA,and Cleaved-caspase-3,MMP-13,RANKL,and RANK proteins were greatly reduced,serum IL-10,the level of SOD,GSH in cartilage tissue,and the expression of OPG gene and protein were greatly increased(P＜0.05).Compared with the high-dose oleuropein group,there was no statistically significant difference in all detection indicators in the HY-N6973 group(P＞0.05).Conclusion Oleuropein could reduce inflammatory re-sponse and oxidative stress in rats with traumatic arthritis and improve joint injury,which may be related to the regulation of OPG-RANKL-RANK signaling pathway.
维普
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