Xin Ma, Junxia Jiao, Mayila Aierken, Hong Sun, Li Chen
{"title":"缺氧诱导因子-1α通过ROS/NLRP3途径调控急性缺血性卒中小胶质细胞灼伤机制","authors":"Xin Ma, Junxia Jiao, Mayila Aierken, Hong Sun, Li Chen","doi":"10.2147/BTT.S444714","DOIUrl":null,"url":null,"abstract":"<p><strong>Purpose: </strong>In vitro experiments explored how the hypoxia-induced factor-1α (HIF-1α) regulates the regulation of pyroptosis in microglial cells (BV 2) in acute ischemic stroke through ROS/NLRP3 pathway.</p><p><strong>Methods: </strong>The microglia acute phase oxygen-glucose deprivation/reoxygenation (OGD/R) was established, CCK-8 was applied to determine the optimal timing of intervention modeling. HIF-1α was overexpressed with stabilizer GF-4592 and HIF-1α small molecule interfering RNA (HIF-1α-siRNA), which was divided into group A (blank group), group B (OGD/R model group), group C (model+FG-4592 intervention group), group D (model+siRNA negative control group) and group E (model+HIF-1α-siRNA group). Cell proliferation of different groups was measured by CCK-8 assay. Pyroptosis and intracellular ROS levels were measured by flow cell technology. IL-18, IL-1β levels were measured by ELISA. HIF-1α, GSDMD-D, GSDMD-N, clean-Caspase-1 and NLRP3 protein expression levels were measured by Western blot. On the above experiments, ROS and NLRP3 response experiments were performed to explore how HIF-1α regulates pyroptosis through ROS/NLRP3 pathway.</p><p><strong>Results: </strong>Hypoxia for 6 h then reoxygenation for 12 h was the optimal intervention time. Compared with groups B and D, cell proliferation in group C was significantly enhanced, pyroptosis, intracellular levels of ROS, IL-18, IL-1β and the expression of GSDMD-D, GSDMD-N, clean-Caspase-1, and NLRP3 proteins were significantly decreased in group C (<i>P</i> < 0.05). However, in group E, the performance of these test indicators were exactly the opposite, and the difference was statistically significant (<i>P</i> < 0.05). Through ROS and NLRP3 response experiments, it was found that HIF-1α Inhibition of Pyroptosis by inhibiting ROS/NLRP3 pathway.</p><p><strong>Conclusion: </strong>Overexpression of HIF-1α factor can inhibit microglia pyroptosis. HIF-1α factor has an inhibitory effect on the ROS/NLRP 3 pathway, which can inhibit the pyroptotic process in microglia.</p>","PeriodicalId":9025,"journal":{"name":"Biologics : Targets & Therapy","volume":"17 ","pages":"167-180"},"PeriodicalIF":5.3000,"publicationDate":"2023-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10748736/pdf/","citationCount":"0","resultStr":"{\"title\":\"Hypoxia Inducible Factor-1α Through ROS/NLRP3 Pathway Regulates the Mechanism of Acute Ischemic Stroke Microglia Scorching Mechanism.\",\"authors\":\"Xin Ma, Junxia Jiao, Mayila Aierken, Hong Sun, Li Chen\",\"doi\":\"10.2147/BTT.S444714\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Purpose: </strong>In vitro experiments explored how the hypoxia-induced factor-1α (HIF-1α) regulates the regulation of pyroptosis in microglial cells (BV 2) in acute ischemic stroke through ROS/NLRP3 pathway.</p><p><strong>Methods: </strong>The microglia acute phase oxygen-glucose deprivation/reoxygenation (OGD/R) was established, CCK-8 was applied to determine the optimal timing of intervention modeling. HIF-1α was overexpressed with stabilizer GF-4592 and HIF-1α small molecule interfering RNA (HIF-1α-siRNA), which was divided into group A (blank group), group B (OGD/R model group), group C (model+FG-4592 intervention group), group D (model+siRNA negative control group) and group E (model+HIF-1α-siRNA group). Cell proliferation of different groups was measured by CCK-8 assay. Pyroptosis and intracellular ROS levels were measured by flow cell technology. IL-18, IL-1β levels were measured by ELISA. HIF-1α, GSDMD-D, GSDMD-N, clean-Caspase-1 and NLRP3 protein expression levels were measured by Western blot. On the above experiments, ROS and NLRP3 response experiments were performed to explore how HIF-1α regulates pyroptosis through ROS/NLRP3 pathway.</p><p><strong>Results: </strong>Hypoxia for 6 h then reoxygenation for 12 h was the optimal intervention time. Compared with groups B and D, cell proliferation in group C was significantly enhanced, pyroptosis, intracellular levels of ROS, IL-18, IL-1β and the expression of GSDMD-D, GSDMD-N, clean-Caspase-1, and NLRP3 proteins were significantly decreased in group C (<i>P</i> < 0.05). However, in group E, the performance of these test indicators were exactly the opposite, and the difference was statistically significant (<i>P</i> < 0.05). Through ROS and NLRP3 response experiments, it was found that HIF-1α Inhibition of Pyroptosis by inhibiting ROS/NLRP3 pathway.</p><p><strong>Conclusion: </strong>Overexpression of HIF-1α factor can inhibit microglia pyroptosis. HIF-1α factor has an inhibitory effect on the ROS/NLRP 3 pathway, which can inhibit the pyroptotic process in microglia.</p>\",\"PeriodicalId\":9025,\"journal\":{\"name\":\"Biologics : Targets & Therapy\",\"volume\":\"17 \",\"pages\":\"167-180\"},\"PeriodicalIF\":5.3000,\"publicationDate\":\"2023-12-19\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10748736/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biologics : Targets & Therapy\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.2147/BTT.S444714\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2023/1/1 0:00:00\",\"PubModel\":\"eCollection\",\"JCR\":\"Q1\",\"JCRName\":\"MEDICINE, RESEARCH & EXPERIMENTAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biologics : Targets & Therapy","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.2147/BTT.S444714","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2023/1/1 0:00:00","PubModel":"eCollection","JCR":"Q1","JCRName":"MEDICINE, RESEARCH & EXPERIMENTAL","Score":null,"Total":0}
引用次数: 0
摘要
目的：体外实验探讨缺氧诱导因子-1α（HIF-1α）如何通过ROS/NLRP3通路调控急性缺血性脑卒中小胶质细胞（BV 2）的热凋亡：方法：建立小胶质细胞急性期氧-葡萄糖剥夺/复氧（OGD/R）模型，应用CCK-8确定干预模型的最佳时机。用稳定剂GF-4592和HIF-1α小分子干扰RNA（HIF-1α-siRNA）过表达HIF-1α，分为A组（空白组）、B组（OGD/R模型组）、C组（模型+FG-4592干预组）、D组（模型+siRNA阴性对照组）和E组（模型+HIF-1α-siRNA组）。不同组的细胞增殖情况通过 CCK-8 检测法进行测定。流式细胞技术检测细胞的嗜热和细胞内 ROS 水平。IL-18和IL-1β水平通过ELISA检测。通过 Western blot 检测 HIF-1α、GSDMD-D、GSDMD-N、clean-Caspase-1 和 NLRP3 蛋白表达水平。在上述实验的基础上，进行了ROS和NLRP3反应实验，以探讨HIF-1α如何通过ROS/NLRP3途径调控热凋亡：结果：缺氧 6 小时然后复氧 12 小时是最佳干预时间。与 B 组和 D 组相比，C 组细胞增殖明显增强，细胞内的 ROS、IL-18、IL-1β 和 GSDMD-D、GSDMD-N、clean-Caspase-1 和 NLRP3 蛋白的表达明显降低（P < 0.05）。但在 E 组，这些检测指标的表现正好相反，差异有统计学意义（P < 0.05）。通过ROS和NLRP3反应实验发现，HIF-1α通过抑制ROS/NLRP3通路抑制脓毒症的发生：结论：HIF-1α因子的过表达可抑制小胶质细胞的脓毒症。HIF-1α因子对ROS/NLRP 3通路具有抑制作用，可抑制小胶质细胞的嗜热过程。
Hypoxia Inducible Factor-1α Through ROS/NLRP3 Pathway Regulates the Mechanism of Acute Ischemic Stroke Microglia Scorching Mechanism.
Purpose: In vitro experiments explored how the hypoxia-induced factor-1α (HIF-1α) regulates the regulation of pyroptosis in microglial cells (BV 2) in acute ischemic stroke through ROS/NLRP3 pathway.
Methods: The microglia acute phase oxygen-glucose deprivation/reoxygenation (OGD/R) was established, CCK-8 was applied to determine the optimal timing of intervention modeling. HIF-1α was overexpressed with stabilizer GF-4592 and HIF-1α small molecule interfering RNA (HIF-1α-siRNA), which was divided into group A (blank group), group B (OGD/R model group), group C (model+FG-4592 intervention group), group D (model+siRNA negative control group) and group E (model+HIF-1α-siRNA group). Cell proliferation of different groups was measured by CCK-8 assay. Pyroptosis and intracellular ROS levels were measured by flow cell technology. IL-18, IL-1β levels were measured by ELISA. HIF-1α, GSDMD-D, GSDMD-N, clean-Caspase-1 and NLRP3 protein expression levels were measured by Western blot. On the above experiments, ROS and NLRP3 response experiments were performed to explore how HIF-1α regulates pyroptosis through ROS/NLRP3 pathway.
Results: Hypoxia for 6 h then reoxygenation for 12 h was the optimal intervention time. Compared with groups B and D, cell proliferation in group C was significantly enhanced, pyroptosis, intracellular levels of ROS, IL-18, IL-1β and the expression of GSDMD-D, GSDMD-N, clean-Caspase-1, and NLRP3 proteins were significantly decreased in group C (P < 0.05). However, in group E, the performance of these test indicators were exactly the opposite, and the difference was statistically significant (P < 0.05). Through ROS and NLRP3 response experiments, it was found that HIF-1α Inhibition of Pyroptosis by inhibiting ROS/NLRP3 pathway.
Conclusion: Overexpression of HIF-1α factor can inhibit microglia pyroptosis. HIF-1α factor has an inhibitory effect on the ROS/NLRP 3 pathway, which can inhibit the pyroptotic process in microglia.
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