硫酸镁启动自噬对兔软骨保护作用机制研究

Study on the protective mechanism of autophagy on cartilage by magnesium sulfate

目的探讨硫酸镁通过启动自噬保护兔软骨的作用机制。方法取24只成年雌性新西兰兔,采用前交叉韧带切断方法制备创伤性关节炎(post-traumatic osteoarthritis,PTOA)模型,随机分为PTOA组、蒸馏水组和硫酸镁组,每组8只。术后即刻开始,蒸馏水组及硫酸镁组分别于关节腔内注射0.5 mL蒸馏水及20 mmol/L硫酸镁溶液,每周3次,连续4周;PTOA组不作处理。术后观察动物一般情况;术后4周取材,ELISA检测关节腔积液TNF-α和Ⅱ型胶原及静脉血中Ⅱ型胶原表达量,Western blot检测股骨软骨组织中瞬时感受电位V5(transient receptor potential channel vanilloid 5,TRPV5)及微管相关蛋白1轻链3(microtubule associated protein1 light chain 3,LC3)蛋白(LC3-Ⅱ/LC3-Ⅰ)表达,实时荧光定量PCR检测滑膜组织中IL-1β、TNF-α、基质金属蛋白酶3(matrix metalloproteinases 3,MMP-3)及软骨组织中Ⅱ型胶原、蛋白聚糖(Aggrecan,AGN)、SOX9 mRNA水平,软骨组织切片行HE、Masson、阿利新蓝染色并参照改良组织学骨关节炎(osteoarthritis,OA)评分标准评分。结果各组动物均存活至实验完成。与其他两组比较,硫酸镁组关节腔积液中TNF-α以及关节腔积液及静脉血中Ⅱ型胶原表达量均降低,TRPV5蛋白表达明显降低、LC3-Ⅱ/LC3-Ⅰ比值明显升高,滑膜组织中IL-1β、TNF-α、MMP-3 mRNA表达降低,软骨组织中Ⅱ型胶原、AGN、SOX9 mRNA表达升高,OA评分亦显著降低,差异均有统计学意义(P<0.05)。PTOA组及蒸馏水组以上指标比较,差异均无统计学意义(P>0.05)。结论关节腔内注射硫酸镁可减轻兔关节内炎症,减少Ⅱ型胶原及AGN丢失,有利于软骨再生,其作用机制可能是通过抑制钙离子通道TRPV5,进而启动软骨自噬。

Objective To investigate the mechanism of magnesium sulfate in protecting rabbit cartilage by initiating autophagy. Methods Twenty-four adult female New Zealand rabbits were used to prepare post-traumatic osteoarthritis（PTOA） models by anterior cruciate ligament transection. Then, the PTOA models were randomly divided into PTOA group, distilled water group, and magnesium sulfate group, with 8 rabbits in each group. Immediately after operation, the distilled water group and the magnesium sulfate group were injected with 0.5 mL distilled water and20 mmol/L magnesium sulfate solution in the joint cavity 3 times a week for 4 weeks, respectively. The PTOA group was not treated. The general condition of the animals was observed after operation. After 4 weeks, the expressions of tumor necrosis factor α（TNF-α） and collagen typeⅡ in the joint fluid and the expression of collagen type Ⅱ in venous blood were detected by ELISA assay. The protein expressions of transient receptor potential channel vanilloid 5（TRPV5）and microtubule associated protein 1 light chain 3（LC3; LC3-Ⅱ/LC3-Ⅰ） in femoral cartilage were detected by Western blot. The mRNA expressions of interleukin 1β（IL-1β）, TNF-α, matrix metalloproteinases 3（MMP-3） in synovial tissue and collagen type Ⅱ, Aggrecan（AGN）, SOX9 in cartilage tissue were detected by real-time fluorescence quantitative PCR.Cartilage tissue sections were stained with HE staining, Masson staining, and Alcian blue staining and scored according to the modified histological osteoarthritis（OA） score. Results All animals survived until the experiment was completed.Compared with the other two groups, the expression of TNF-α in joint effusion and collagen type Ⅱ in joint effusion and venous blood were decreased in magnesium sulfate group; the protein expression of TRPV5 decreased, and the ratio of LC3-Ⅱ/LC3-Ⅰ increased significantly; the mRNA expressions of IL-1β, TNF-α, and MMP-3 in synovial tissue were decreased, and the mRNA expressions of collagen type Ⅱ, AGN, and SOX9 in cartilage tissue were increased; OA scores also decreased significantly. All differences were statistically significant（P〈0.05）. There was no significant difference in the above indicators between the PTOA group and the distilled water group（P〉0.05）. Conclusion Intra-articular injection of magnesium sulfate can reduce intra-articular inflammation, reduce the loss of collagen type Ⅱ and AGN, and is beneficial to cartilage regeneration in rabbits. The mechanism may be related to the initiation of chondroautophagy by inhibiting the calcium channel TRPV5.