温阳通络胶囊干预骨关节炎大鼠软骨细胞的炎症反应和血管新生

Wenyang Tongluo capsule intervenes with inflammation and angiogenesis in rat osteoarthritic chondrocytes

背景:温阳通络胶囊在临床上治疗骨关节炎具有明显的疗效,但其作用机制不明确。目的:探讨温阳通络胶囊对骨关节炎的治疗作用,以及其对软骨细胞中miR-140/血管内皮生长因子信号的调控机制。方法:(1)动物实验:将75只SD大鼠分为5组,每组15只:正常组、模型组、药物组每天左后肢膝关节内注射miR-140-antagomir NC和siRNAVEGF NC,抑制剂组每天左后肢关节内注射mi R-140-antagomir和si RNA-VEGF NC,干扰组每天关节内注射mi R-140-antagomir和si RNA-VEGF,各组均注射2周;2周后,药物组、抑制剂组和干扰组灌胃给予温阳通络胶囊,连续灌胃4周;模型组、药物组、抑制剂组、干扰组实验的1,4,7 d关节腔内注射木瓜蛋白酶复制关节炎模型。给药结束后,进行相关检测。(2)细胞实验:将软骨细胞ATDC5分5组处理:正常组加入含mi R-140-antagomir NC和si RNA-VEGF NC的培养基;模型组加入含白细胞介素1β、mi R-140-antagomir NC和si RNA-VEGF NC的培养基;药物组加入含白细胞介素1β、mi R-140-antagomir NC、si RNA-VEGF NC及温阳通络胶囊的培养基;抑制剂组加入含白细胞介素1β、mi R-140-antagomir及温阳通络胶囊的培养基;干扰组加入含白细胞介素1β、mi R-140-antagomir、si RNA-VEGF及温阳通络胶囊的培养基,进行相关检测。结果与结论:(1)动物实验:与正常大鼠相比,骨关节炎大鼠软骨组织中miR-140 mRNA表达降低(P <0.05),血管内皮生长因子mRNA表达升高(P <0.05)。与模型组相比,药物组、抑制剂组、干扰组大鼠血清中白细胞介素6和肿瘤坏死因子α水平、软骨细胞凋亡、新生血管数量、血管内皮生长因子蛋白表达均明显减少(P <0.05),其中以药物组效果最明显。(2)细胞实验:与正常软骨细胞相比,骨关节炎软骨细胞中miR-140 mRNA表达降低(P <0.05),血管内皮生长因子mRNA表达升高(P <0.05)。与模型组相比,药物组、抑制剂组、干扰组细胞增殖活性提高(P <0.05),细胞凋亡、上清中白细胞介素6和肿瘤坏死因子α水平、新生血管数量、血管内皮生长因子基因及蛋白表达均明显减少(P <0.05),其中以药物组效果最明显。(3)温阳通络胶囊能明显抑制软骨细胞的炎性应激和血管新生,缓解关节炎大鼠软骨病理损伤,这可能与调控miR-140/血管内皮生长因子信号轴有关。

BACKGROUND:Wenyang Tongluo capsule has obvious clinical effects in the treatment of osteoarthritis,but its mechanism of action is unclear.OBJECTIVE:To investigate the therapeutic effect of Wenyong Tongluo capsule on osteoarthritis and its regulation mechanism on microRNA-140(miR-140)/vascular endothelial growth factor(VEGF) signaling in chondrocytes.METHODS:(1) Animal experiments:A total of 75 Sprague-Dawley rats were randomized into 5 groups(n=15 per group):normal group,model group,drug gro up,inhibitor group,and interference group.In the latter three groups,miR-140-antagomir NC and siRNA-VEGF NC,miR-140-antagomir and siRNA-VEGF NC,and miR-140-antagomir and siRNA-VEGF were injected into the left hindlimb joint daily,res pectively.Administration in each group lasted for 2 weeks.Afte rwards,the rats in the drug,inhibitor,and interference groups were intragastrically given Wenyong Tongluo capsule for 4 continuous wee ks.Animal models of osteoarthritis were prepared via the intraarticular injection of papain on days 1,4,and 7 in the model,drug,inhibitor,and interference groups.After the completion of drug administration,related measurements were performed.(2) Cell assays:ATDC5 chondrocytes were divided into five groups:normal group(cultured in the medium containing miR-140-antagomir NC and siRNA-VEGF NC),model group(cultured in the medium containing interleukin-1β,miR-140-antagomir NC and siRNA-VEGF NC,drug group(cultured in the medium containing interleukin-1β,miR-140-antagomir NC,siRNA-VEGF NC,and Wenyong Tongluo capsule),inhibitor group(cultured in the medium containing interleukin-1β,miR-140-antagomir and Wenyang Tongluo capsule),and interference group(cultured in the medium containing interleukin-1β,miR-140-antagomir,siRNA-VEGF and Wenyang Tongluo capsule).Relevant detections were then performed.RESULTS AND CONCLUSION:(1) Animal expe riments:Compared with normal rats,the expression of miR-140 mRNA in cartilage tissue of osteoarthritic rats was decreased(P <0.05),while the expression of vascular endothelial growth fa ctor mRNA was increased(P <0.05).Compared with the model group,interleukin-6and tumor necrosis factor-a levels,chondrocyte apoptosis,the number of new blood vessels and protein expression of vascular endothelial growth fa ctor in serum of the drug group,inhibitor group and interference group were significantly decreased(P <0.05).The drug group could most significantly interfere with the malignant progression of these indicators.(2) Cell assays:Compared with normal chondrocytes,the mRNA expression of miR-140 in osteoarthritc chondrocytes was decreased(P <0.05),while the mRNA expression of vascular endothelial growth factor was increased(P <0.05).Compared with the model group,the prolife ration activity was increased in the drug group,inhibitor group and interference group(P<0.05),while cell apoptosis,inte rleukin-6 and tumor necrosis factor-α levels in the supernatant,the number of new blood vessels,and the expression of vascular endothelial growth factor gene and protein were significantly decreased(P <0.05).The drug groups had the most obvious effects on these indicato rs.In conclusion,Wenyang Tongluo caps ule can relieve the pathological damage of ca rtilage in osteoarthritic rats by significantly inhibiting inflammatory stress and angiogenesis in chondrocytes,which may be related to the regulation of the miR-140/VEGF signal axis.