Hemin regulates mitochondrial pathway of oxidative stress in mouse chondrocytes

doi:10.12307/2025.269

Abstract

Abstract:
BACKGROUND: Studies have shown that mitochondrial oxidative stress has an important role in the development of knee osteoarthritis, and Hemin can regulate the expression of mitochondria-related proteins.
Objective: To study the regulatory effect of Hemin on oxidative stress in mouse chondrocytes and its interventional effect and mechanism in knee osteoarthritis.
METHODS: (1) In vitro cell experiment: Primary chondrocytes from C57BL/6 mice were extracted and induced with 10 ng/mL interleukin-1β to construct an in vitro chondrocyte model of osteoarthritis. The optimal concentration of Hemin (0, 1, 10, 20, 40, 80, and 160 μmol/L) for the intervention in mouse chondrocytes was determined by cell counting kit-8 method. Chondrocytes were randomly divided into control group, model group (interleukin-1β) and Hemin group (interleukin-1β+Hemin). Reactive oxygen species, mitochondrial membrane potential and apoptosis of chondrocytes in each group were detected. (2) In vivo experiment: Adult C57BL/6 mice were randomly divided into normal group, model group (osteoarthritis) and Hemin group (osteoarthritis+Hemin), with eight mice in each group. After 4 weeks of Hemin treatment, the behavioral test and histopathological observation of the knee joint were performed in each group. Changes in extracellular matrix-related protein expression and apoptosis in chondrocytes and the expression level of Nrf2/HO-1 protein in cartilage tissue were detected.
RESULTS AND CONCLUSION: In vitro experiment: the optimal concentration of Hemin on primary chondrocytes was 40 μmol/L. Compared with the model group, the level of reactive oxygen species was significantly reduced, the mitochondrial membrane potential was significantly improved, and the apoptosis of chondrocytes was reduced in the hemin-treated interleukin-1β-induced chondrocytes. In vivo experiment: After 4 weeks of treatment, compared with the model group, the lower limb function of mice in the Hemin group was significantly improved, the histopathological score was significantly improved, and the apoptosis of knee chondrocytes was significantly reduced. All these findings indicate that Hemin can alleviate oxidative stress, restore mitochondrial function and reduce apoptosis in mouse chondrocytes induced by interleukin-1β. Hemin can improve extracellular matrix degradation, promote chondrocyte anabolism, reduce catabolism and reduce chondrocyte apoptosis in knee osteoarthritis. It may act by activating the chondrocyte Nrf2/HO-1 signaling pathway in the inflammatory environment.

Key words: knee osteoarthritis, Hemin, oxidative stress, mitochondria, apoptosis, cartilage injury

CLC Number:

He Guanghui, Yuan Jie, Ke Yanqin, Qiu Xiaoting, Zhang Xiaoling. Hemin regulates mitochondrial pathway of oxidative stress in mouse chondrocytes[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(6): 1183-1191.

Figures/Tables 9

2.1 Hemin对软骨细胞的活性影响 0，1，10，20，40 μmol/L 的Hemin(分子式见图1A)处理24 h后对小鼠软骨细胞活力没有差异，但是80，160 μmol/L 的Hemin对小鼠软骨细胞活力有显著差异，见图1B。因而后续细胞实验选择Hemin的浓度为40 μmol/L。 2.2 各组软骨细胞活性氧水平与正常组相比，模型组软骨细胞活性氧水平相对绿色荧光升高；相较于模型组，Hemin组软骨细胞活性氧水平相对绿色荧光降低，见图 2。结果表明Hemin能够降低炎症因子白细胞介素1β对小鼠软骨细胞的活性氧水平。 2.3 各组软骨细胞线粒体膜电位水平与正常相比，模型组软骨细胞线粒体膜电位水平降低；与模型组相比，Hemin组软骨细胞线粒体膜电位水平升高，见图 3。结果说明Hemin可以增加炎症因子白细胞介素1β作用的软骨细胞线粒体膜电位水平。 2.4 各组软骨细胞凋亡情况采用流式细胞术进行小鼠软骨细胞凋亡检测，结果发现，经白细胞介素1β处理后软骨细胞凋亡数量明显增加(P < 0.001)，而给予Hemin治疗后软骨细胞凋亡水平降低(P < 0.001)，表明经Hemin处理可以缓解白细胞介素1β诱导的软骨炎症，见图4。结果说明Hemin可以降低炎症因子白细胞介素1β对软骨细胞凋亡的影响。 2.5 实验动物情况分析实验选用C57BL/6J雄性小鼠24只，随机分为3组，实验过程中动物无脱失，全部纳入结果分析。 2.6 各组小鼠行为学分析治疗4周后使用CatWalk进行小鼠步态分析，小鼠自由通过固定跑道时能清晰记录四肢印记，见图5A，B。正常组小鼠节律整齐的通过固定跑道；模型组小鼠在通过跑道时节律紊乱，右后肢触地面积变少；与模型组相比，Hemin组小鼠下肢步态节律更齐，有显著的恢复效果，见图5C。进一步分析发现模型组形成后局部痛阈减小，小鼠触地面积以及摆动的时间减少，见图5D，Hemin组治疗后小鼠功能显著恢复。 2.7 各组小鼠膝关节软骨组织病理学变化见图6。 2.7.1 苏木精-伊红染色结果显示，模型组的膝关节表面非常粗糙，软骨下骨暴露于关节面，而Hemin组减少了关节表面的磨损，恢复了一定的软骨层，软骨表面相对平整光滑，软骨组织病理学表现有所改善，见图6A。根据Mankin’s评分从软骨结构、软骨细胞、软骨基质细胞染色和潮线完整性4个方面定量损伤结果，与正常组相比模型组的Mankin’s评分显著升高，而Hemin组的Mankin’s评分均显著降低，见图6C。 2.7.2 番红O-固绿染色结果显示，模型组小鼠骨关节炎有明显的软骨损伤，与正常组小鼠相比，模型组小鼠膝关节软骨退变和侵蚀、以及番红O染色强度降低；相反，Hemin治疗可以恢复番红O染色强度，改善其他病理学变化，预防软骨损伤，见图6B。使用OARSI评分从软骨基质丢失、软骨损伤面积占比、垂直裂隙延伸长度不同程度等方面的软骨退变来定量损伤后，结果发现Hemin治疗显著降低了OARSI评分，见图6D。 2.8 各组小鼠软骨组织细胞外基质相关蛋白表达相对于正常组，模型组的软骨组织中Ⅱ型胶原表达显著降低，而基质金属蛋白酶13表达则显著升高；与模型组相比，Hemin组的Ⅱ型胶原表达均显著升高，基质金属蛋白酶13表达显著降低，见图7。结果表明Hemin能够提高骨关节炎软骨的合成代谢，同时降低其分解代谢，改善骨关节炎软骨退变。 2.9 各组小鼠软骨组织细胞凋亡变化与正常组相比，模型组软骨组织中凋亡软骨细胞显著增加；与模型组相比，Hemin组的凋亡软骨细胞显著均显著下降，见图 8。结果表明Hemin可以减少骨关节炎软骨细胞的凋亡数量。 2.10 各组小鼠软骨细胞Nrf2/HO-1蛋白的表达与正常组相比，模型组软骨细胞中HO-1、Nrf2蛋白表达水平均显著下降，与模型组相比，Hemin组HO-1、Nrf2蛋白表达水平均显著上升，见图 9。结果表明Hemin可以激活炎症环境中软骨细胞的Nrf2/HO-1信号通路。"

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