糖尿病伤口是糖尿病(DM)的严重并发症,可导致持续感染、截肢甚至死亡。长期氧化应激已被广泛认为是糖尿病伤口发展的主要诱因。因此,氧化应激被认为是一个有前途的治疗靶点。在本研究中,Keap1/Nrf2 信号被证实在链脲佐菌素 (STZ) 诱导的糖尿病小鼠和甲基乙二醛 (MGO) 处理的人脐静脉内皮细胞 (HUVEC) 中被激活。通过 siRNA 敲除 Keap1,可逆转 Keap1 水平的增加,促进 Nrf2 的核转位,并增加抗氧化蛋白 HO-1 的表达。为了探索治疗递送策略,乳源性外泌体 (mEXO) 被开发为一种新型、高效且无毒的 siRNA 载体。通过超声处理将SiRNA-Keap1(siKeap1)加载到mEXO中,发现所获得的mEXOs-siKeap1可以促进HUVEC增殖和迁移,同时缓解MGO处理的HUVEC中的氧化应激。同时,在糖尿病伤口小鼠模型中,注射 mEXOs-siKeap1 显着加速糖尿病伤口愈合,增强胶原蛋白形成和新血管形成。总而言之,这些数据支持开发 Keap1 敲低作为糖尿病伤口的潜在治疗策略,并证明了 mEXO 作为可扩展、生物相容且经济高效的 siRNA 递送系统的可行性。
图形概要
评估了装载 siKeap1 的 mEXO 对糖尿病伤口愈合的治疗效果。首先,我们发现在糖尿病小鼠的伤口和用甲基乙二醛(MGO)预处理的人脐静脉内皮细胞(HUVEC)中Keap1的表达上调。接下来,我们通过差速离心从原料奶中提取外泌体,并通过超声处理将 siKeap1 加载到牛奶来源的外泌体中。体外应用合成复合物(mEXOs-siKeap1)被发现可以增加Nrf2的核定位和抗氧化蛋白HO-1的表达,从而逆转氧化应激。此外,体内 mEXOs-siKeap1 给药显着加速了糖尿病伤口的愈合速度(方案 1)。
方案1示意图。mEXOs-siKeap1 复合物的合成
。B mEXOs-siKeap1 的体外机制。C mEXOs-siKeap1对糖尿病伤口体内小鼠模型的治疗效果
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Milk-derived exosomes carrying siRNA-KEAP1 promote diabetic wound healing by improving oxidative stress
Diabetic wounds are a serious complication of diabetes mellitus (DM) that can lead to persistent infection, amputation, and even death. Prolonged oxidative stress has been widely recognized as a major instigator in the development of diabetic wounds; therefore, oxidative stress is considered a promising therapeutic target. In the present study, Keap1/Nrf2 signaling was confirmed to be activated in streptozotocin (STZ)-induced diabetic mice and methylglyoxal (MGO)-treated human umbilical vein endothelial cells (HUVECs). Knockdown of Keap1 by siRNA reversed the increase in Keap1 levels, promoted the nuclear translocation of Nrf2, and increased the expression of HO-1, an antioxidant protein. To explore therapeutic delivery strategies, milk-derived exosomes (mEXOs) were developed as a novel, efficient, and non-toxic siRNA carrier. SiRNA-Keap1 (siKeap1) was loaded into mEXOs by sonication, and the obtained mEXOs-siKeap1 were found to promote HUVEC proliferation and migration while relieving oxidative stress in MGO-treated HUVECs. Meanwhile, in a mouse model of diabetic wounds, injection of mEXOs-siKeap1 significantly accelerated diabetic wound healing with enhanced collagen formation and neovascularization. Taken together, these data support the development of Keap1 knockdown as a potential therapeutic strategy for diabetic wounds and demonstrated the feasibility of mEXOs as a scalable, biocompatible, and cost-effective siRNA delivery system.
Graphical Abstract
The therapeutic effect of siKeap1-loaded mEXOs on diabetic wound healing was assessed. First, we found that the expression of Keap1 was upregulated in the wounds of diabetic mice and in human umbilical vein endothelial cells (HUVECs) pretreated with methylglyoxal (MGO). Next, we extracted exosomes from raw milk by differential centrifugation and loaded siKeap1 into milk-derived exosomes by sonication. The in vitro application of the synthetic complex (mEXOs-siKeap1) was found to increase the nuclear localization of Nrf2 and the expression of the antioxidant protein HO-1, thus reversing oxidative stress. Furthermore, in vivo mEXOs-siKeap1 administration significantly accelerated the healing rate of diabetic wounds (Scheme 1).
Scheme 1 Schematic diagram. A
Synthesis of mEXOs-siKeap1 complex. B Mechanism of mEXOs-siKeap1 in vitro. C The treatment
effect of mEXOs-siKeap1 on an in vivo
mouse model of diabetic wounds