阳和汤含药血清对膝骨性关节炎兔膝软骨MMP-1、MMP-3和TIMP-1表达的影响

Effect of Yanghe Decoction Drug-Containing Serum on Expression of MMP-1,MMP-3 and TIMP-1 in Knee Cartilage of Rabbits with Knee Osteoarthritis

目的:阳和汤含药血清对膝骨性关节炎兔膝软骨MMP-1、MMP-3和TIMP-1表达的影响。方法:按照阳和汤药配方水煎阳和汤汤剂,将10只新西兰大白兔5只/组随机分为两组,含药血清制备组兔50ml/只,早晚各灌胃1次,连续3d;空白组灌胃等剂量蒸馏水。末次灌胃3h后,腹主动脉取血,分离血清~20℃备用。将30只白兔10只/组随机分为假手术组、模型组和阳和汤含药血清治疗组(治疗组),采用改良Hulth法制备关节炎模型,构建模型当日,治疗组兔子右膝关节腔注射1.5ml含药血清,每日1次;假手术组和模型组注射等剂量的普通血清,连续注射7d。兔处死后取右膝软骨组织通过HE染色进行病理分析和TUNEL染色进行凋亡分析,商业试剂盒检测丙二醛(MDA)、超氧化物歧化酶(SOD)和乳酸脱氢酶(LDH)水平;westernblot分析软骨组织中Bcl-2、Bad、cleaved-caspase-3、MMP-1、MMP-3和TIMP-1蛋白表达。结果:HE病理分析结果显示,与模型组相比,治疗组兔膝关节软骨损伤明显改善,Tunel显示凋亡细胞比例明显降低(P<0.05),同时软骨组织中MDA和血清中LDH的水平明显降低,SOD含量显著上升(P<0.05);Westernblot结果显示,与模型组相比,治疗组兔软骨组织中MMP-1的表达量明显降低,MMP-3和TIMP-1蛋白表达显著提高(P<0.05)。结论:阳和汤含药血清能够通过降低MMP-1的表达,上调MPP-3和TIMP-1的表达改善兔膝骨性关节炎软骨损伤。

Objective:To explore the effect of Yanghe Decoction drug-containing serum on the expression of MMP-1,MMP-3 and TIMP-1 in knee cartilage of rabbits with knee osteoarthritis.Methods:According to the formula of Yanghe Decoction,Yanghe Decoction was decocted with water,and 10 New Zealand white rabbits were randomly divided into two groups with 5 rabbits in each group.The drug-containing serum preparation group was given 50 ml for a rabbit and was intragastrically administered each once in the morning and evening consecutively for 3 days.The blank group was given an equal dose of distilled water.After 3 hours of last intragastric administration,blood was taken from the abdominal aorta,and the serum was separated at-20 °C for use.30 rabbits were randomly divided into sham operation group,model group and Yanghe Decotion drug-containing serum treatment group(treatment group),with 10 rabbits in each group.The arthritis model was prepared by modified Hulth method,and on the day of constructing model,and the treatment group was injected 1.5 ml of drug-containing serum in the joint cavity once a day.The sham operation group and the model group were injected with the same amount of normal serum for 7 days.After the rabbits were sacrificed,the right knee cartilage tissue was taken for pathological analysis by HE staining and for apoptosis analysis by TUNEL staining.Commercial kits were used to detect the levels of malondialdehyde(MDA),superoxide dismutase(SOD) and lactate dehydrogenase(LDH).Western blot was used to analyze the protein expression levels of Bcl-2,Bad,cleaved-caspase-3,MMP-1,MMP-3 and TIMP-1 in cartilage tissue.Results:The results of HE pathological analysis showed that compared with the model group,the knee joint articular cartilage injury in the treatment group was significantly improved,and Tunel showed the proportion of apoptotic cells was significantly decreased(P < 0.05),and the levels of in cartilage tissue MDA and serum LDH were significantly reduced,and the content of SOD was increased significantly(P < 0.05).The results of Western Blot showed that compared with the model group,the expression level of MMP-1 in the cartilage tissue in the treatment group was significantly decreased while the expression levels of MMP-3 and TIMP-1 proteins were significantly increased(P < 0.05).Conclusions:Yanghe Decotion drug-containing serum can improve cartilage injury of rabbits with knee osteoarthritis by decreasing the expression of MMP-1 and up-regulating the expression of MMP-3 and TIMP-1.