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Abstract：
BACKGROUND: Subarachnoid hemorrhage (SAH) is a severe stroke subtype that lacks effective treatment. Exosomes derived from human dental pulp stem cells (DPSCs) are a promising acellular therapeutic strategy for neurological diseases. However, the therapeutic effects of DPSC-derived exosomes (DPSC-Exos) on SAH remain unknown. In this study, we investigated the therapeutic effects and mechanisms of action of DPSC-Exos in SAH.
METHODS: SAH was established using 120 male Sprague-Dawley rats. One hour after SAH induction, DPSC-Exos were administered via tail vein injection. To investigate the effect of DPSC-Exos, SAH grading, short-term and long-term neurobehavioral assessments, brain water content, western blot (WB), immunofluorescence staining, Nissl staining, and HE staining were performed. The role of miR-197-3p/FOXO3 in regulating pyroptosis was demonstrated through miRNA sequencing, bioinformatics analysis, and rescue experiments. The SAH model in vitro was established by stimulating BV2 cells with hemoglobin (Hb) and the underlying mechanism of DPSC-Exos was investigated through WB and Hoechst/PI staining.
RESULTS: The expressions of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) were increased after SAH. DPSC-Exos alleviated brain edema and neuroinflammation by inhibiting the expression of FOXO3 and reducing NLRP3 inflammasome activation, leading to improved neurobehavioral functions at 24 h after SAH. In vitro, the expression of the NLRP3 inflammasome components (NLRP3 and caspase1-p20), GSDMD-N, and IL-18 was inhibited in BV2 cells pretreated with DPSC-Exos. Importantly, DPSC-Exos overexpressing miR-197-3p had a more obvious protective effect than those from NC-transfected DPSCs, while those from DPSCs transfected with the miR-197-3p inhibitor had a weaker protective effect. Functional studies indicated that miR-197-3p bound to the 3\'-untranslated region of FOXO3, inhibiting its transcription. Furthermore, the overexpression of FOXO3 reversed the protective effects of miR-197-3p.
CONCLUSIONS: DPSC-Exos inhibited activation of the NLRP3 inflammasome and related cytokine release via the miR-197-3p/FOXO3 pathway, alleviated neuroinflammation, and inhibited microglial pyroptosis. These findings suggest that using DPSC-Exos is a promising therapeutic strategy for SAH.
METHODS: SAH was established using 120 male Sprague-Dawley rats. One hour after SAH induction, DPSC-Exos were administered via tail vein injection. To investigate the effect of DPSC-Exos, SAH grading, short-term and long-term neurobehavioral assessments, brain water content, western blot (WB), immunofluorescence staining, Nissl staining, and HE staining were performed. The role of miR-197-3p/FOXO3 in regulating pyroptosis was demonstrated through miRNA sequencing, bioinformatics analysis, and rescue experiments. The SAH model in vitro was established by stimulating BV2 cells with hemoglobin (Hb) and the underlying mechanism of DPSC-Exos was investigated through WB and Hoechst/PI staining.
RESULTS: The expressions of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) were increased after SAH. DPSC-Exos alleviated brain edema and neuroinflammation by inhibiting the expression of FOXO3 and reducing NLRP3 inflammasome activation, leading to improved neurobehavioral functions at 24 h after SAH. In vitro, the expression of the NLRP3 inflammasome components (NLRP3 and caspase1-p20), GSDMD-N, and IL-18 was inhibited in BV2 cells pretreated with DPSC-Exos. Importantly, DPSC-Exos overexpressing miR-197-3p had a more obvious protective effect than those from NC-transfected DPSCs, while those from DPSCs transfected with the miR-197-3p inhibitor had a weaker protective effect. Functional studies indicated that miR-197-3p bound to the 3\'-untranslated region of FOXO3, inhibiting its transcription. Furthermore, the overexpression of FOXO3 reversed the protective effects of miR-197-3p.
CONCLUSIONS: DPSC-Exos inhibited activation of the NLRP3 inflammasome and related cytokine release via the miR-197-3p/FOXO3 pathway, alleviated neuroinflammation, and inhibited microglial pyroptosis. These findings suggest that using DPSC-Exos is a promising therapeutic strategy for SAH.
摘要：
背景：蛛网膜下腔出血（SAH）是一种严重的卒中亚型，缺乏有效的治疗方法。源自人牙髓干细胞（DPSC）的外泌体是神经系统疾病的有希望的无细胞治疗策略。然而,DPSC来源的外泌体(DPSC-Exos)对SAH的治疗效果尚不清楚.在这项研究中,我们研究了DPSC-Exos在SAH中的治疗效果和作用机制。
方法：用120只雄性SD大鼠建立SAH。SAH诱导后一小时，通过尾静脉注射施用DPSC-Exos。为了研究DPSC-Exos的效果，SAH分级,短期和长期神经行为评估，脑含水量,蛋白质印迹(WB),免疫荧光染色，尼氏染色,进行HE染色。通过miRNA测序证明miR-197-3p/FOXO3在调节焦亡中的作用,生物信息学分析，和救援实验。用血红蛋白（Hb）刺激BV2细胞建立SAH的体外模型，并通过WB和Hoechst/PI染色研究DPSC-Exos的潜在机制。
结果：促炎细胞因子（IL-1β，SAH后IL-6和TNF-α）升高。DPSC-Exos通过抑制FOXO3的表达和减少NLRP3炎性体的激活来减轻脑水肿和神经炎症,导致SAH后24小时神经行为功能改善。体外，NLRP3炎性体成分（NLRP3和caspase1-p20）的表达，GSDMD-N,在用DPSC-Exos预处理的BV2细胞中IL-18被抑制。重要的是,过表达miR-197-3p的DPSC-Exos比NC转染的DPSCs具有更明显的保护作用。而转染miR-197-3p抑制剂的DPSC的保护作用较弱。功能研究表明miR-197-3p与FOXO3的3'-非翻译区结合，抑制其转录。此外，FOXO3的过表达逆转了miR-197-3p的保护作用。
结论：DPSC-Exos通过miR-197-3p/FOXO3通路抑制NLRP3炎性体的活化和相关细胞因子的释放，缓解神经炎症，并抑制小胶质细胞焦亡。这些发现表明使用DPSC-Exos是SAH的有希望的治疗策略。
方法：用120只雄性SD大鼠建立SAH。SAH诱导后一小时，通过尾静脉注射施用DPSC-Exos。为了研究DPSC-Exos的效果，SAH分级,短期和长期神经行为评估，脑含水量,蛋白质印迹(WB),免疫荧光染色，尼氏染色,进行HE染色。通过miRNA测序证明miR-197-3p/FOXO3在调节焦亡中的作用,生物信息学分析，和救援实验。用血红蛋白（Hb）刺激BV2细胞建立SAH的体外模型，并通过WB和Hoechst/PI染色研究DPSC-Exos的潜在机制。
结果：促炎细胞因子（IL-1β，SAH后IL-6和TNF-α）升高。DPSC-Exos通过抑制FOXO3的表达和减少NLRP3炎性体的激活来减轻脑水肿和神经炎症,导致SAH后24小时神经行为功能改善。体外，NLRP3炎性体成分（NLRP3和caspase1-p20）的表达，GSDMD-N,在用DPSC-Exos预处理的BV2细胞中IL-18被抑制。重要的是,过表达miR-197-3p的DPSC-Exos比NC转染的DPSCs具有更明显的保护作用。而转染miR-197-3p抑制剂的DPSC的保护作用较弱。功能研究表明miR-197-3p与FOXO3的3'-非翻译区结合，抑制其转录。此外，FOXO3的过表达逆转了miR-197-3p的保护作用。
结论：DPSC-Exos通过miR-197-3p/FOXO3通路抑制NLRP3炎性体的活化和相关细胞因子的释放，缓解神经炎症，并抑制小胶质细胞焦亡。这些发现表明使用DPSC-Exos是SAH的有希望的治疗策略。
