糖尿病是一种世界范围内的流行疾病，可导致多种并发症，包括肾衰竭、失明和截肢。糖尿病足溃疡具有慢性伤口的特征，是糖尿病进展的破坏性组成部分，可导致下肢截肢。在这项研究中，我们着手研究糖尿病足溃疡伤口愈合的机制。采用qRT-PCR检测糖尿病足溃疡患者皮肤组织和经晚期糖基化末端（AGE）产物处理的HSF2人皮肤成纤维细胞中USP30的表达，并采用CCK-8、细胞划痕和ELISA法检测细胞活力Col I、Col III、MMP-2、MMP-9、IL-1β 和 IL-18 的迁移和水平。 USP30 和 NLRP3 之间的相互作用通过免疫共沉淀和泛素化测定进行了验证。 Western blot检测USP30、NLRP3和caspase-1 p20的表达。 USP30抑制剂MF-094用于治疗链脲佐菌素（STZ）建立的糖尿病大鼠模型。我们发现，与正常皮肤组织相比，糖尿病足溃疡患者皮肤组织中的去泛素酶 USP30 表达上调。在体外，我们发现用晚期糖基化终末 (AGE) 产品（已知会导致糖尿病并发症）处理 HSF2 人类皮肤成纤维细胞，会导致 HSF2 细胞的活力和迁移受到抑制，并导致 USP30 mRNA 和蛋白质水平增加。从功能上讲，通过 shRNA 介导的敲低或 USP30 抑制剂 MF-094 处理来下调 USP30，可恢复 AGE 处理的 HSF2 细胞的活力和迁移。我们确定 NLRP3 炎症小体是 USP30 在 AGE 诱导功能中的关键靶标。 从机制上讲，我们证明 USP30 通过去泛素化 NLRP3 来激活 NLRP3 炎症小体。最后，我们表明，通过 MF-094 治疗抑制 USP30 可促进糖尿病大鼠的伤口愈合，并导致 NLRP3 及其下游靶标 caspase-1 p20 的蛋白水平降低，从而确立了已识别的 USP30-NLRP3 联系的生理重要性。总之，我们的研究结果表明 USP30 对糖尿病足溃疡具有治疗潜力。
"点击查看英文标题和摘要"
MF-094, a potent and selective USP30 inhibitor, accelerates diabetic wound healing by inhibiting the NLRP3 inflammasome
Diabetes is a prevalent disease worldwide that can result in several complications, including renal failure, blindness, and amputation. Diabetic foot ulcers, which have the characteristics of chronic wounds, are a devastating component of diabetes progression that can lead to lower extremity amputation. In this study, we set out to investigate the mechanisms involved in wound healing of diabetic foot ulcers. The expression of USP30 in skin tissues of patients with diabetic foot ulcers and HSF2 human skin fibroblasts treated with advanced glycation end (AGE) products was detected by qRT-PCR, and CCK-8, cell scratch and ELISA assay were used to detect cell viability, migration and levels of Col I, Col III, MMP-2, MMP-9, IL-1β and IL-18. The interaction between USP30 and NLRP3 was verified by co-immunoprecipitation and ubiquitination assays. The expression of USP30, NLRP3 and caspase-1 p20 was detected by Western blot. USP30 inhibitor MF-094 was used to treat diabetic rat model established by streptozotocin (STZ). We found that USP30, a deubiquitinase, was upregulated in skin tissues of patients with diabetic foot ulcers compared with normal skin tissues. In vitro, we found that treatment of HSF2 human skin fibroblasts with advanced glycation end (AGE) products, known to contribute to diabetic complications, resulted in suppressed viability and migration of HSF2 cells, as well as increased levels of USP30 mRNA and protein. Functionally, downregulation of USP30 via shRNA-mediated knockdown or treatment with the USP30 inhibitor MF-094, restored viability and migration of AGE-treated HSF2 cells. We identified the NLRP3 inflammasome as a critical target of USP30 in AGE-induced functions. Mechanistically, we demonstrate that USP30 activates the NLRP3 inflammasome by deubiquitinating NLRP3. Finally, we show that inhibition of USP30 via MF-094 treatment facilitated wound healing in diabetic rats and resulted in decreased protein levels of NLRP3 and its downstream target caspase-1 p20, thus establishing the physiological importance of the identified USP30-NLRP3 link. Together, our findings suggest a therapeutic potential for USP30 in diabetic foot ulcers.
京公网安备 11010802027423号