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. 2018 Oct 15;32(10):1340-1345.
doi: 10.7507/1002-1892.201804015.

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

[Article in Chinese]
Rong Chen  1 , Xue Zhou  2 , Shaomei Yin  2 , Zeyu Lu  2 , Jinpeng Nie  2 , Wencheng Zhou  2 , Xinghui Liu  3
Affiliations

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

[Article in Chinese]
Rong Chen et al. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. .

Abstract
in English, Chinese

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 and 20 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.

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 protein 1 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。.

Keywords: Post-traumatic osteoarthritis; autophagy; cartilage injury; magnesium sulfate; rabbit.

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Figures

图 1
图 1
The expressions of TNF-α and collagen type Ⅱ in each group detected by ELISA ELISA 检测各组 TNF-α 及Ⅱ型胶原表达量
图 2
图 2
The expressions of target proteins in each group　detected by Western blot Western blot 检测各组目的蛋白表达
图 3
图 3
The target gene expressions in each group detected by real-time fluorescent quantitative PCR 实时荧光定量 PCR 检测各组目的基因表达
图 4
图 4
Histological observation of each group (×100) 各组组织学观察（×100）
See this image and copyright information in PMC

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Grants and funding

湖北省教育厅指导性项目基金（B2016141）；湖北医药学院研究生启动基金（2015QDZR11）