Hunan Provincial Key Laboratory of Pathogenic Biology Based on Integrated Chinese and Western Medicine, Hunan University of Chinese Medicine, Changsha 410208, China.
Oxid Med Cell Longev. 2021 Dec 30;2021:9925561. doi: 10.1155/2021/9925561. eCollection 2021.
As one of the fundamental components of , astragaloside IV (AST IV) exerts protective effects against cerebral ischemia-reperfusion injury (CIRI). Nevertheless, the underlying mechanisms have not yet been conclusively elucidated. To do so, here, we report on the regulatory effects of Nrf2 on NLRP3 inflammasome-mediated pyroptosis. CIRI was induced by middle cerebral artery occlusion-reperfusion (MCAO/R) in Sprague Dawley rats and modeled by oxygen and glucose deprivation/reoxygenation (OGD/R) in SH-SY5Y cells. Cerebral infarct volume and neurological deficit score served as indices to evaluate MCAO/R injury. In addition, the CCK-8 assay was used to assess cell viability, the LDH leakage rate was used as a quantitative index, and propidium iodide (PI) staining was used to visualize cells after OGD/R injury. The NLRP3/Caspase-1/GSDMD pathway, which produces the pores in the cell membrane that are central to the pyroptosis process, was assessed to investigate pyroptosis. Nrf2 activation was assessed by detecting Nrf2 protein levels and immunofluorescence analysis. We show that after MCAO/R of rats, the infarct volume and neurological deficit score of rats were strongly increased, and after OGD/R of cell cultures, cell viability was strongly decreased, and the LDH leakage rate and the proportion of PI-positive cells were strongly increased. In turn, MCAO/R and OGD/R enhanced the protein levels of NLRP3, Caspase-1, IL-1, GSDMD, and GSDMD-N. Moreover, Nrf2 protein levels increased, and Nrf2 translocation was promoted after CIRI. Interestingly, AST IV (i) reduced the cerebral infarct volume and the neurological deficit score in vivo and (ii) increased the cell viability and reduced the LDH leakage rate and the proportion of PI-positive cells in vitro. AST IV reduced the protein levels of NLRP3, Caspase-1, IL-1, GSDMD, and GSDMD-N, inhibiting NLRP3 inflammasome-mediated pyroptosis. AST IV also increased the protein levels of Nrf2 and promoted the transfer of Nrf2 to the nucleus, accelerating Nrf2 activation. Particularly revealing was that the Nrf2 inhibitor ML385 partly blocked the above effects of AST IV. Taken together, these results demonstrate that AST IV alleviates CIRI through inhibiting NLRP3 inflammasome-mediated pyroptosis via activating Nrf2.
黄芪甲苷（AST IV）作为其基本组成部分之一，对脑缺血再灌注损伤（CIRI）具有保护作用。然而，其潜在机制尚未得到明确的阐述。为了阐明这一点，我们报告了 Nrf2 对 NLRP3 炎性小体介导的细胞焦亡的调节作用。在 Sprague Dawley 大鼠中通过大脑中动脉闭塞再灌注（MCAO/R）诱导 CIRI，并通过氧葡萄糖剥夺/再氧合（OGD/R）在 SH-SY5Y 细胞中建立模型。脑梗死体积和神经功能缺损评分用于评估 MCAO/R 损伤。此外，CCK-8 测定法用于评估细胞活力，LDH 漏出率作为定量指标，碘化丙啶（PI）染色用于可视化 OGD/R 损伤后的细胞。评估 NLRP3/Caspase-1/GSDMD 通路，该通路产生细胞焦亡过程中关键的细胞膜孔，以研究细胞焦亡。通过检测 Nrf2 蛋白水平和免疫荧光分析来评估 Nrf2 激活。我们发现，大鼠 MCAO/R 后，大鼠的梗死体积和神经功能缺损评分明显增加，细胞培养物 OGD/R 后，细胞活力明显降低，LDH 漏出率和 PI 阳性细胞比例明显增加。相反，MCAO/R 和 OGD/R 增强了 NLRP3、Caspase-1、IL-1、GSDMD 和 GSDMD-N 的蛋白水平。此外，CIRI 后 Nrf2 蛋白水平增加，促进 Nrf2 易位。有趣的是，AST IV（i）降低了体内的脑梗死体积和神经功能缺损评分，（ii）增加了细胞活力，降低了体外的 LDH 漏出率和 PI 阳性细胞的比例。AST IV 降低了 NLRP3、Caspase-1、IL-1、GSDMD 和 GSDMD-N 的蛋白水平，抑制了 NLRP3 炎性小体介导的细胞焦亡。AST IV 还增加了 Nrf2 的蛋白水平，并促进了 Nrf2 向核内的转移，加速了 Nrf2 的激活。特别值得注意的是，Nrf2 抑制剂 ML385 部分阻断了 AST IV 的上述作用。总之，这些结果表明，AST IV 通过激活 Nrf2 抑制 NLRP3 炎性小体介导的细胞焦亡来减轻 CIRI。
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