Wang Jia, Tang Hao, Tian Jianan, Xie Yibo, Wu Yun
Department of Neurology, The Second Affiliated Hospital of Harbin Medical University, Harbin 150001, China.
Department of Neurology, The Second Affiliated Hospital of Harbin Medical University, Harbin 150001, China.
Int Immunopharmacol. 2025 Jan 27;146:113881. doi: 10.1016/j.intimp.2024.113881. Epub 2024 Dec 24.
Mounting data indicates that extracellular vesicles (EVs) have the potential to improve the injury after a stroke. Pyroptosis is a recently identified kind of programmed cell death that initiates an inflammatory reaction. We aimed to ascertain the therapeutic implications and possible molecular processes of EVs obtained from adipose-derived stem cells (ADSCs) in inhibiting pyroptosis in ischemic stroke.
The investigation employed transient middle cerebral artery occlusion (tMCAO) rat model and a BV2 of oxygen-glucose deprivation/reoxygenation (OGD/R) to ascertain ADSCs-EVs implications on inflammation and pyroptosis as assessed by neurological deficit scores, TTC staining, IHC, HE, CCK8, WB, ELISA, and immunofluorescence. RNA-Seq was performed on BV2 cells in the control, OGD/R, and OGD/R + ADSCs-EVs groups. Using sequencing data analysis, in the OGD/R group, we screened the upregulated genes regulated by EVs, overlapped with 74 pyroptosis-related genes, and identified Guanylate-binding protein 2 (Gbp2) and Guanylate-binding protein 3 (Gbp3) as key genes. Following the validation of the sequencing results in vivo and in vitro, Gbp3 was selected for further study. To test its regulatory effects on inflammation and pyroptosis, Gbp3 was knocked down and overexpressed in vitro.
The administration of ADSCs-EVs resulted in a significant reduction in neurological involvement scores and reduced infarct volume in rats with tMCAO. They were also protective against BV-2 cells after OGD/R. In vivo and in vitro, ADSCs-EVs inhibited inflammatory response and pyroptosis after stroke. The outcomes of the RNA-Seq data analysis manifested that the protective implications of EVs after stroke are mediated by the modulation of inflammation-related mechanisms. Moreover, treatment with EVs led to a significant reduction in Gbp3 expression in post-ischemic brain tissue and cells. When Gbp3 was knocked down, the expression of inflammatory molecules and proteins linked to pyroptosis had a significant decline. When Gbp3 was overexpressed, the opposite results were obtained.
ADSCs-EVs modulate the NLRP3/GSDMD signaling pathway via Gbp3 to attenuate the inflammatory response and reduce pyroptosis that occurs after stroke.
越来越多的数据表明，细胞外囊泡（EVs）有改善中风后损伤的潜力。焦亡是最近发现的一种程序性细胞死亡，可引发炎症反应。我们旨在确定从脂肪来源干细胞（ADSCs）获得的细胞外囊泡在抑制缺血性中风焦亡中的治疗意义及可能的分子机制。
本研究采用短暂性大脑中动脉闭塞（tMCAO）大鼠模型和氧糖剥夺/复氧（OGD/R）的BV2细胞，通过神经功能缺损评分、TTC染色、免疫组化、苏木精-伊红染色、CCK8、蛋白质免疫印迹法（WB）、酶联免疫吸附测定（ELISA）和免疫荧光来确定ADSCs-EVs对炎症和焦亡的影响。对对照组、OGD/R组和OGD/R + ADSCs-EVs组的BV2细胞进行RNA测序。通过测序数据分析，在OGD/R组中，我们筛选出由细胞外囊泡上调的基因，与74个焦亡相关基因重叠，并确定鸟苷酸结合蛋白2（Gbp2）和鸟苷酸结合蛋白3（Gbp3）为关键基因。在体内和体外验证测序结果后，选择Gbp3进行进一步研究。为了测试其对炎症和焦亡的调节作用，在体外敲低和过表达Gbp3。
给予ADSCs-EVs可显著降低tMCAO大鼠的神经功能缺损评分并减少梗死体积。它们对OGD/R后的BV-2细胞也有保护作用。在体内和体外，ADSCs-EVs均可抑制中风后的炎症反应和焦亡。RNA测序数据分析结果表明，中风后细胞外囊泡的保护作用是通过调节炎症相关机制介导的。此外，细胞外囊泡治疗可使缺血后脑组织和细胞中Gbp3的表达显著降低。当敲低Gbp3时，与焦亡相关的炎症分子和蛋白质的表达显著下降。当过表达Gbp3时，则得到相反的结果。
ADSCs-EVs通过Gbp3调节NLRP3/GSDMD信号通路，以减轻炎症反应并减少中风后发生的焦亡。
