Institute of Burns, Wuhan Third Hospital (Tongren Hospital of WuHan University), Wuhan 430060, China.
School of Medicine, Jianghan University, Wuhan, 430056, China.
Biochem Biophys Res Commun. 2024 Aug 30;722:150149. doi: 10.1016/j.bbrc.2024.150149. Epub 2024 May 21.
The objective of this study was to examine the potential of USP7 as a target for senolytic therapy and to investigate the molecular mechanism by which its inhibitor selectively induced apoptosis in senescent HDF and enhanced DFU wound healing.
Clinical samples of DFU were collected to detect the expression of USP7 and aging-related proteins using immunohistochemistry and Western blot. In addition, β-galactosidase staining, qPCR, flow cytometry, ROS and MMP kits, and Western blot were used to analyze the biological functions of P5091 on senescence, cycle, and apoptosis. RNAseq was employed to further analyze the molecular mechanism of P5091. Finally, the DFU rat model was established to evaluate the effect of P5091 on wound healing.
The expression of USP7 and p21 were increased in DFU clinical samples. After treatment with d-glucose (30 mM, 7 days), β-galactosidase staining was deepened, proliferation rate decreased. USP7 inhibitors (P5091) could reduce the release of SASP factors, activate the production of ROS, and reduce MMP. In addition, it induced apoptosis and selectively clears senescent cells through the p53 signaling pathway. Finally, P5091 can improve diabetic wound healing in rats.
This study clarified the molecular mechanism of USP7 inhibitor (P5091) selectively inducing apoptosis of high glucose senescent HDF cells. This provides a new senolytics target and experimental basis for promoting DFU wound healing.
本研究旨在探讨 USP7 作为衰老细胞溶解疗法靶点的潜力，并研究其抑制剂选择性诱导衰老 HDF 细胞凋亡和增强糖尿病足溃疡（DFU）愈合的分子机制。
收集 DFU 的临床样本，通过免疫组化和 Western blot 检测 USP7 和衰老相关蛋白的表达。此外，使用β-半乳糖苷酶染色、qPCR、流式细胞术、ROS 和 MMP 试剂盒以及 Western blot 分析 P5091 对衰老、细胞周期和凋亡的生物学功能。采用 RNAseq 进一步分析 P5091 的分子机制。最后，建立 DFU 大鼠模型以评估 P5091 对伤口愈合的影响。
DFU 临床样本中 USP7 和 p21 的表达增加。用 d-葡萄糖（30mM，7 天）处理后，β-半乳糖苷酶染色加深，增殖率降低。USP7 抑制剂（P5091）可减少 SASP 因子的释放，激活 ROS 的产生，并降低 MMP。此外，它通过 p53 信号通路诱导凋亡并选择性清除衰老细胞。最后，P5091 可改善糖尿病大鼠的伤口愈合。
本研究阐明了 USP7 抑制剂（P5091）选择性诱导高糖衰老 HDF 细胞凋亡的分子机制。这为促进 DFU 伤口愈合提供了新的衰老细胞溶解治疗靶点和实验依据。
