Luo Yinji, Guo Qijie, Liu Chang, Zheng Yuxuan, Wang Yichong, Wang Bin
Department of Bone Surgery, The Second Affiliated Hospital, Guangzhou Medical University, No. 250 Changgang East Road, Haizhu District, Guangzhou, 510145, Guangdong Province, China.
Acta Diabetol. 2024 Dec 16. doi: 10.1007/s00592-024-02422-x.
Diabetic foot ulcers (DFUs) cause prominent morbidity and mortality. Adipose mesenchymal stem cell (ASC)-derived extracellular vesicles (EVs) show property in facilitating diabetic wound healing, and we explored their role in DFU rats.
ASCs were cultured in vitro, passaged and then identified by flow cytometry and induction of osteogenic/adipogenic differentiation. ASC-EVs were extracted and identified. DFU rat model was treated with ASC-EVs. High glucose (HG)-induced rat dermal fibroblasts were treated with ASC-EVs or 3-MA and sh-PINK1 plasmid in vitro. Wound healing was observed. Histological changes, inflammatory cytokines (TNF-α, IL-1β), and α-SMA and p21 double-positive cell level were assessed by HE staining, ELISA, and immunofluorescence. Mitochondrial membrane potential (MMP), cell viability and senescence, and ROS production in cells were assessed by fluorescence dye JC-1, CCK-8, SA-β-gal staining, and ROS kit. p21, LC3II/I, p62, PINK1 and parkin protein levels were determined by Western blot.
DFU rats had slow wound healing and elevated levels of IL-1β, TNF-α, α-SMA and p21 double-positive cells, and SA-β-gal, while HG-induced cells had weakened viability, elevated ROS, SA-β-gal, p21 and p62 protein levels, and decreased LC3II/I, PINK1 and parkin protein levels and MMP, which were reversed by ASC-EVs. HG inhibited mitophagy by suppressing the PINK1/parkin pathway to accelerate dermal fibroblast senescence. The PINK1/parkin pathway inhibition partly mitigated the effect of ASC-EVs. ASC-EVs promoted mitophagy by activating the PINK1/parkin pathway in vivo.
ASC-EVs mediated mitophagy by activating the PINK1/parkin pathway, thereby impeding HG-induced rat dermal fibroblast senescence and promoting wound healing in DFU rats.
糖尿病足溃疡（DFUs）会导致显著的发病率和死亡率。脂肪间充质干细胞（ASC）衍生的细胞外囊泡（EVs）具有促进糖尿病伤口愈合的特性，我们探讨了它们在糖尿病足溃疡大鼠中的作用。
体外培养ASC，传代后通过流式细胞术以及成骨/成脂分化诱导进行鉴定。提取并鉴定ASC-EVs。用ASC-EVs治疗糖尿病足溃疡大鼠模型。体外将高糖（HG）诱导的大鼠真皮成纤维细胞用ASC-EVs或3-MA以及sh-PINK1质粒进行处理。观察伤口愈合情况。通过苏木精-伊红（HE）染色、酶联免疫吸附测定（ELISA）和免疫荧光评估组织学变化、炎性细胞因子（肿瘤坏死因子-α、白细胞介素-1β）以及α-平滑肌肌动蛋白（α-SMA）和p21双阳性细胞水平。通过荧光染料JC-1、细胞计数试剂盒（CCK-8）、衰老相关β-半乳糖苷酶（SA-β-gal）染色和活性氧（ROS）试剂盒评估细胞中的线粒体膜电位（MMP）、细胞活力和衰老以及ROS产生。通过蛋白质免疫印迹法测定p21、微管相关蛋白1轻链3II/微管相关蛋白1轻链3I（LC3II/I）、p62、PTEN诱导激酶1（PINK1）和帕金蛋白水平。
糖尿病足溃疡大鼠伤口愈合缓慢，白细胞介素-1β、肿瘤坏死因子-α、α-SMA和p21双阳性细胞以及SA-β-gal水平升高，而HG诱导的细胞活力减弱，ROS、SA-β-gal、p21和p62蛋白水平升高，LC3II/I、PINK1和帕金蛋白水平以及MMP降低，这些变化可被ASC-EVs逆转。HG通过抑制PINK1/帕金途径抑制线粒体自噬以加速真皮成纤维细胞衰老。PINK1/帕金途径抑制部分减轻了ASC-EVs的作用。ASC-EVs在体内通过激活PINK1/帕金途径促进线粒体自噬。
ASC-EVs通过激活PINK1/帕金途径介导线粒体自噬，从而抑制HG诱导的大鼠真皮成纤维细胞衰老并促进糖尿病足溃疡大鼠的伤口愈合。
