藤黄健骨胶囊调控AMPK/PGC-1α信号通路改善膝骨关节炎大鼠基质代谢失衡和线粒体生物发生

Tenghuangjiangu Capsule Improves Imbalance of Matrix Metabolism and Mitochondrial Biogenesis in Knee Osteoarthritis Rats by Regulating the AMPK/PGC-1α Signaling Pathway

关键词:  骨藤黄健骨胶囊  膝骨关节炎  AMPK/PGC-1α信号通路  基质代谢  线粒体生物发生

Key words:  tenghuangjiangu capsule  knee osteoarthritis  AMPK/PGC-1α signal pathway  matrix metalloproteinases  mitochondrial biogenesis

摘要:

目的 探讨藤黄健骨胶囊对膝骨关节炎大鼠基质代谢的影响及分子机制。方法 双60只SD大鼠随机分为假手术组、模型组、西药组（硫酸氨基葡糖0.17g·kg?1）及藤黄健骨胶囊高、中、低剂量治疗组（藤黄健骨胶囊0.36、0.18、0.09g·kg?1），每组10只。除假手术组外其他各组采用改良Hulth法制备KOA大鼠模型，建模6周后分别灌胃给予硫酸氨基葡糖及相应剂量的藤黄健骨胶囊，假手术组和模型组灌胃给予0.9%生理盐水。上述药物连续灌胃2个月后采用Lequesne膝关节级别评分法评估大鼠右膝对疼痛的刺激反应步态改变、关节活动度和关节的肿胀程度，番红固绿O染色法观察关节软骨病理形态并进行OARSI评分；ELISA法检测血清中核心结合因子β亚基（core-binding factor subunit beta，Cbf-β）、基质金属蛋白酶13(matrixmetalloproteinase13，MMP-13)、组织金属蛋白酶抑制剂2（tissue inhibitor of metalloproteinase 2，TIMP-2）、Ⅱ型胶原羧基末端肽（Type II collagen carboxy terminal peptide，CTX-Ⅱ）、血小板反应蛋白解整合素金属肽酶4（ADAMTS-4）和血小板反应蛋白解整合素金属肽酶5（ADAMTS-5）浓度；RT-PCR法和Western-blot法观察关节软骨组织中AMPK介导的AMPK/PGC-1α信号通路关键分子单磷酸腺苷激活的蛋白激酶（AMP- activated protein kinase，AMPK）、过氧化物酶增殖激活受体γ共激活子-1α ( peroxisome proliferator-activated receptor γ coactivator-1α，PGC-1α)、MMP-13 mRNA表达和蛋白表达。结果 藤与假手术组比较，模型组Lequesne MG评分呈现增加趋势，血清Cbf-β、MMP-13、CTX-Ⅱ、ADAMTS-4、ADAMTS-5浓度呈现上升趋势，MMP-13 mRNA表达和蛋白表达则呈现上升趋势，模型组和藤黄健骨低剂量组大鼠OARSI评分也呈现上升趋势，而血清TIMP2浓度呈现下降趋势，右膝关节软骨组织PGC-1α、AMPK mRNA表达也呈现下降趋势（P<0.01）。与模型组比较，藤黄健骨胶囊高剂量组大鼠血清ADAMTS-5浓度呈现下降趋势、TIMP-2浓度呈现上升趋势（P<0.01），藤黄健骨胶囊中、高剂量组Lequesne MG评分、OARSI评分、血清Cbf-β、CTX-Ⅱ和ADAMTS-4含量均呈现下降趋势，右膝关节软骨组织PGC-1α蛋白表达及蛋白表达灰度值呈现增强趋势（P<0.05或P<0.01），藤黄健骨胶囊低、中、高剂量组大鼠血清MMP-13含量、MMP-13 mRNA表达、蛋白表达及蛋白表达灰度值呈现下降趋势，右膝关节软骨组织AMPK、PGC-1α mRNA表达呈现上升趋势，AMPK蛋白表达及蛋白表达灰度值也呈现增（P<0.05或P<0.01）。结论 藤黄健骨胶囊延缓KOA模型大鼠的关节软骨退变可能与维持其关节软骨组织基质代谢稳态和逆转线粒体生物发生损伤有关，其中AMPK/PGC-1α信号通路激活可能发挥着关键调控作用。

Abstract:

OBJECTIVE This study aimed to investigate the effect of the tenghuangjiangu capsule on matrix metabolism in rats with knee osteoarthritis and its molecular mechanism. METHODS Sixty SD rats were randomly divided into the sham operation group, model group, western medicine group (glucosamine sulfate: 0.17g·kg?1), and high-, middle-, and low- dose treatment groups (tenghuangjiangu capsule: 0.36, 0.18, 0.09 g·kg?1), with 10 rats in each group. KOA rat models were made using a modified version of the Hulth method, excluding the sham operation group. After six weeks of modelling, glucosamine sulfate and the corresponding dosage of the tenghuangjiangu capsule were administered through the stomach, and 0.9% normal saline was administered through the stomach in the sham operation group and the model group. After two months of continuous intragastric administration of the above drugs, the knee joint grade of rats was observed using the Lequesne knee joint grade score; the gait change, joint range of motion, and joint swelling degree of rats were evaluated with the Lequesne knee joint grade score, the pathological morphology of the joint cartilage was observed through saffron solid green O staining, and preform quantitative OARSI scoring. Serum concentrations of core-binding factor subunit beta (Cbf-β), matrix metalloproteinase 13 (MMP-13), tissue inhibitor of metalloproteinase 2 (TIMP-2), type II collagen terminal peptide (CTX-II), platelet reactive protein disintegrin metallopeptidase 4 (ADAMTS-4), and platelet reactive protein disintegrin metallopeptidase 5 (ADAMTS-5) were detected by ELISA. The expression of AMPK-activated protein kinase (AMPK), peroxidase proliferator-activated receptor γ coactivator 1α (PGC-1α), MMP-13 mRNA and protein in the articular cartilage were observed with RT-PCR and Western blotting.. RESULTS Compared with the sham-operated group, the serum Cbf-β, MMP-13, CTX-II, ADAMTS-4, and ADAMTS-5 concentrations, and the MMP-13 mRNA and protein expression levels increased in the model group, the Lequesne MG score and the OARSI score also increased in the model group and low-dose groups, but serum TIMP-2 concentration decreased in the model group. The expression levels of PGC-1α and AMPK mRNA in the cartilage of the right knee joint also exhibited a downward trend (P<0.01). Compared with the model group, the concentration of ADAMTS-5 and TIMP-2 in the high-dose group exhibited a downward trend, while the TIMP-2 concentration exhibited an upward trend (P<0.01). The Lequesne MG score, OARSI score, and Cbf-β, CTX-Ⅱ, and ADAMTS-4 levels in the middle- and high-dose groups all showed a downward trend. The expression of the PGC-1α protein and its grey value in the cartilage tissue of the right knee joint exhibited an increasing trend (P<0.05 or P<0.01). The content of MMP-13 and MMP-13 mRNA, and protein expression in the low-, middle-, and high-dose groups exhibited a downward trend, and the expression levels of AMPK and the PGC-1α mRNA in the cartilage tissue of the right knee joint also increased, the expression of the AMPK protein and its grey value in the cartilage tissue of the right knee joint exhibited an increasing trend(P<0.05 or P<0.01). CONCLUSION Tenghuangjiangu capsule delays the degeneration of the articular cartilage in KOA model rats, which may be related to maintaining the homeostasis of the articular cartilage matrix and reversing the damage of mitochondrial biogenesis, and the activation of the AMPK/PGC-1α signal pathway may play a key regulatory role.