Effect and mechanism of metformin-modified bone marrow mesenchymal stem cell exosomes on regulating chondrocytes

doi:10.12307/2025.550

Abstract

Abstract: BACKGROUND: Osteoarthritis is a progressive joint condition identified by ongoing deterioration of the cartilage matrix, and there is currently no effective drug treatment plan. Metformin-modified exosomes isolated from bone marrow-derived mesenchymal stem cells can become a new method for treating osteoarthritis due to their avoidance of oral drug adverse reactions and immunogenicity.
OBJECTIVE: To study the controlling impact of exosomes from metformin-altered bone marrow-derived mesenchymal stem cells on chondrocytes.
METHODS: Rabbit bone marrow-derived mesenchymal stem cells and chondrocytes were cultured in vitro. Bone marrow-derived mesenchymal stem cells derived exosomes and metformin pretreated bone marrow-derived mesenchymal stem cells derived exosomes were collected using a high-speed centrifuge. Chondrocytes were cultured with exosome-containing culture medium for 24 hours and then treated with 100 µmol/L H2O2 for 24 hours. The capability changes of two extracellular vesicles on chondrocyte proliferation and migration were detected using CCK8 assay and scratch healing experiment, respectively. Western blot analysis and RT-qPCR were employed to examine the alterations in the expression of type II collagen, P16 protein, and their mRNA in chondrocytes. Western blot analysis was utilized to assess the changes in the expression of MKK7/JNK pathway proteins. ELISA kits were utilized to measure the activity of cell superoxide dismutase and the levels of malondialdehyde in chondrocytes.
RESULTS AND CONCLUSION: (1) In an oxidative stress environment, the proliferation and migration abilities of chondrocytes were weakened. The two types of exosomes could restore the proliferation and migration abilities of chondrocytes to a certain extent. Metformin pretreated bone marrow-derived mesenchymal stem cells derived exosomes had a significantly better improvement effect (P < 0.05). (2) Compared with normal bone marrow mesenchymal stem cell-derived exosomes, metformin pretreated bone marrow-derived mesenchymal stem cells derived exosomes could more effectively increase type II collagen expression and superoxide dismutase activity (P < 0.05), and were also more effective in reducing P16 expression and malondialdehyde levels (P < 0.05). (3) The two types of exosomes could inhibit the expression of MKK7 and p-JNK proteins to a certain extent, and the inhibitory effect of metformin pretreated bone marrow-derived mesenchymal stem cells derived exosomes was more significant (P < 0.05). The results show that in an oxidative stress environment, metformin pretreated bone marrow-derived mesenchymal stem cells derived exosomes resist chondrocyte aging and promote chondrocyte proliferation by inhibiting the MKK7/JNK pathway.

Key words: bone marrow mesenchymal stem cell, metformin, exosome, chondrocyte, osteoarthritis, oxidative stress, aging, engineered stem cell

CLC Number:

Sima Xinli, Liu Danping, Qi Hui. Effect and mechanism of metformin-modified bone marrow mesenchymal stem cell exosomes on regulating chondrocytes[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(36): 7728-7734.

Figures/Tables 3

2.1 骨髓间充质干细胞及其来源外泌体鉴定结果骨髓间充质干细胞具有成骨、成脂、成软骨分化能力[15-16]，见图1A。透射电镜观察MET-Exos与NC-Exos一样，均呈类圆形，见图1B，Western blot检测特异标志物 CD9、CD63、TSG101表达阳性，见图1C。 2.2 二甲双胍及外泌体对软骨细胞活性的影响 CCK-8结果显示，50，500 μmol/L二甲双胍作用3 d时软骨细胞活力显著提高(P < 0.05)，50 μmol/L二甲双胍提高软骨细胞活力更明显，见图2A。与NC-Exos相比，MET-Exos显著促进软骨细胞的增殖(P < 0.05)，见图2B。 2.3 软骨细胞内吞外泌体的能力共聚焦显微镜下观察，Dil标记的外泌体可以被Phalloidin标记的软骨细胞内吞，主要位于软骨细胞胞质内，分布在DAPI标记的蓝色核周围，见图3。 2.4 外泌体对软骨细胞迁移能力的影响与对照组相比，模型组划痕愈合率显著减小(P < 0.05)；与模型组和NC-Exos组相比，MET-Exos组划痕愈合率显著增大(P < 0.05)，表明MET-Exos可促进软骨细胞迁移，见图4。 2.5 外泌体对软骨细胞Ⅱ型胶原、P16和MKK7/JNK通路相关蛋白表达的影响 Western blot结果表明，与对照组相比，模型组Ⅱ型胶原蛋白表达量显著降低(P < 0.05)，P16、MKK7和p-JNK蛋白表达量显著升高(P < 0.05)；与模型组相比，外泌体治疗组P16、MKK7和p-JNK蛋白表达量显著降低(P < 0.05)，Ⅱ型胶原蛋白表达量显著升高(P < 0.05)；与NC-Exos组相比，MET-Exos组Ⅱ型胶原蛋白表达量显著升高(P < 0.05)，P16、MKK7和p-JNK蛋白表达量显著降低(P < 0.05)，见图5。RT-qPCR结果表明，与NC-Exos组相比，MET-Exos组Ⅱ型胶原mRNA表达水平显著提高，P16 mRNA表达水平显著降低(P < 0.05)，见图6，表明MET-Exos有可能通过MKK7/JNK通路发挥对抗软骨细胞衰老、促进软骨细胞增殖的作用。 "

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