Abstract：
BACKGROUND: Exosomes generated from adipose-derived mesenchymal stem cells (Exos), and in particular hypoxia-pretreated ADSCs (HExos), possess therapeutic properties that promote spinal cord repair following spinal cord injury (SCI). Nevertheless, the regulatory mechanisms through which HExos exert their effects remain unclear.
METHODS: Here, next-generation sequencing (NGS) was utilized to examine abnormal circRNA expression comparing HExos to Exos. Bioinformatics analysis and RNA pulldown assays together with luciferase reporter assays were applied to determine interactions among miRNAs, mRNAs and circRNAs. ELISA and immunofluorescence staining were used to examine inflammatory cytokine levels, apoptosis and ROS deposition in LPS-treated HT-22 cells, respectively. The therapeutic effects of Exos and HExos on a mouse model of SCI were analyzed by immunohistochemistry and immunofluorescence staining.
RESULTS: Our findings confirmed that HExos have more significant therapeutic influences on decreasing ROS and inflammatory cytokine levels post-SCI than Exos. NGS revealed that circ-Wdfy3 expression levels were significantly higher in HExos than Exos. Downregulation of circ-Wdfy3 led to a decrease in HExo-induced therapeutic effects on spinal cord repair post-SCI, indicating that circ-Wdfy3 has a critical role in the regulation of HExo-mediated protection against SCI. Our bioinformatics, RNA pulldown and luciferase reporter data demonstrated that GPX4 and miR-423-3p were downstream targets of circ-Wdfy3. GPX4 downregulation or miR-423-3p overexpression reversed the protective effects of circ-Wdfy3 on LPS-treated HT-22 cells. Furthermore, overexpression of circ-Wdfy3 led to an in increase in the Exo-induced therapeutic effects on spinal cord repair post-SCI through the inhibition of ferroptosis.
CONCLUSIONS: circ-WDfy3-overexpressing Exos promote spinal cord repair post-SCI through mediation of ferroptosis via the miR-138-5p/GPX4 pathway.
METHODS: Here, next-generation sequencing (NGS) was utilized to examine abnormal circRNA expression comparing HExos to Exos. Bioinformatics analysis and RNA pulldown assays together with luciferase reporter assays were applied to determine interactions among miRNAs, mRNAs and circRNAs. ELISA and immunofluorescence staining were used to examine inflammatory cytokine levels, apoptosis and ROS deposition in LPS-treated HT-22 cells, respectively. The therapeutic effects of Exos and HExos on a mouse model of SCI were analyzed by immunohistochemistry and immunofluorescence staining.
RESULTS: Our findings confirmed that HExos have more significant therapeutic influences on decreasing ROS and inflammatory cytokine levels post-SCI than Exos. NGS revealed that circ-Wdfy3 expression levels were significantly higher in HExos than Exos. Downregulation of circ-Wdfy3 led to a decrease in HExo-induced therapeutic effects on spinal cord repair post-SCI, indicating that circ-Wdfy3 has a critical role in the regulation of HExo-mediated protection against SCI. Our bioinformatics, RNA pulldown and luciferase reporter data demonstrated that GPX4 and miR-423-3p were downstream targets of circ-Wdfy3. GPX4 downregulation or miR-423-3p overexpression reversed the protective effects of circ-Wdfy3 on LPS-treated HT-22 cells. Furthermore, overexpression of circ-Wdfy3 led to an in increase in the Exo-induced therapeutic effects on spinal cord repair post-SCI through the inhibition of ferroptosis.
CONCLUSIONS: circ-WDfy3-overexpressing Exos promote spinal cord repair post-SCI through mediation of ferroptosis via the miR-138-5p/GPX4 pathway.
摘要：
背景：由脂肪间充质干细胞（Exos）产生的外泌体，特别是缺氧预处理的ADSC（HExos），具有促进脊髓损伤（SCI）后脊髓修复的治疗特性。然而，HExos发挥作用的调节机制尚不清楚。
方法：这里，我们利用下一代测序(NGS)来检测异常circRNA表达,并将HExos与Exos进行比较.应用生物信息学分析和RNA下拉测定以及荧光素酶报告基因测定来确定miRNA之间的相互作用。mRNAs和circRNAs。ELISA和免疫荧光染色检测炎性细胞因子水平,LPS处理的HT-22细胞中的凋亡和ROS沉积，分别。通过免疫组织化学和免疫荧光染色分析Exos和HExos对SCI小鼠模型的治疗作用。
结果：我们的发现证实，与Exos相比，HExos对SCI后降低ROS和炎性细胞因子水平具有更显著的治疗作用。NGS显示circ-Wdfy3表达水平在HExos中显著高于Exos。circ-Wdfy3的下调导致HExo诱导的对SCI后脊髓修复的治疗作用降低，表明circ-Wdfy3在调节HExo介导的针对SCI的保护中具有关键作用。我们的生物信息学，RNA下拉和荧光素酶报告数据表明GPX4和miR-423-3p是circ-Wdfy3的下游靶标。GPX4下调或miR-423-3p过表达逆转了circ-Wdfy3对LPS处理的HT-22细胞的保护作用。此外，circ-Wdfy3的过表达通过抑制铁性凋亡导致Exo诱导的对SCI后脊髓修复的治疗作用增加。
结论：circ-WDfy3过表达Exos通过miR-138-5p/GPX4通路介导铁性凋亡促进SCI后脊髓修复。
方法：这里，我们利用下一代测序(NGS)来检测异常circRNA表达,并将HExos与Exos进行比较.应用生物信息学分析和RNA下拉测定以及荧光素酶报告基因测定来确定miRNA之间的相互作用。mRNAs和circRNAs。ELISA和免疫荧光染色检测炎性细胞因子水平,LPS处理的HT-22细胞中的凋亡和ROS沉积，分别。通过免疫组织化学和免疫荧光染色分析Exos和HExos对SCI小鼠模型的治疗作用。
结果：我们的发现证实，与Exos相比，HExos对SCI后降低ROS和炎性细胞因子水平具有更显著的治疗作用。NGS显示circ-Wdfy3表达水平在HExos中显著高于Exos。circ-Wdfy3的下调导致HExo诱导的对SCI后脊髓修复的治疗作用降低，表明circ-Wdfy3在调节HExo介导的针对SCI的保护中具有关键作用。我们的生物信息学，RNA下拉和荧光素酶报告数据表明GPX4和miR-423-3p是circ-Wdfy3的下游靶标。GPX4下调或miR-423-3p过表达逆转了circ-Wdfy3对LPS处理的HT-22细胞的保护作用。此外，circ-Wdfy3的过表达通过抑制铁性凋亡导致Exo诱导的对SCI后脊髓修复的治疗作用增加。
结论：circ-WDfy3过表达Exos通过miR-138-5p/GPX4通路介导铁性凋亡促进SCI后脊髓修复。
