Remote limb ischemic postconditioning inhibits microglia pyroptosis by modulating HGF after acute ischemia stroke

Abstract

The repetitive inflation-deflation of a blood pressure cuff on a limb is known as remote limb ischemic postconditioning (RIPostC). It prevents brain damage induced by acute ischemia stroke (AIS). Pyroptosis, executed by the pore-forming protein gasdermin D (GSDMD), is a type of regulated cell death triggered by proinflammatory signals. It contributes to the pathogenesis of ischemic brain injury. However, the effects of RIPostC on pyroptosis following AIS remain largely unknown. In our study, linear correlation analysis confirmed that serum GSDMD levels in AIS patients upon admission were positively correlated with NIHSS scores. RIPostC treatment significantly reduced GSDMD level compared with patients without RIPostC at 3 days post-treatment. Besides, middle cerebral artery occlusion (MCAO) surgery was performed on C57BL/6 male mice and RIPostC was induced immediately after MCAO. We found that RIPostC suppressed the activation of NLRP3 inflammasome to reduce the maturation of GSDMD, leading to decreased pyroptosis in microglia after AIS. Hepatocyte growth factor (HGF) was identified using the high throughput screening. Importantly, HGF siRNA, exogenous HGF, and ISG15 siRNA were used to reveal that HGF/ISG15 is a possible mechanism of RIPostC regulation in vivo and in vitro.

Keywords: acute ischemic stroke; high throughput screening; microglial; pyroptosis; remote limb ischemic postconditioning.

FIGURE 1

RIPostC reduced infarct size and ameliorated neurological behavior after MCAO. (a) TTC‐stained coronal sections at 3 days after reperfusion from representative animals and infarct volumes and reproducible infarct volumes were observed (Student's t‐test, n = 4). (b) Representative laser speckle images showing cerebral blood flow at 72 h after reperfusion (One‐way ANOVA, n = 3). (c) The foot‐fault test, cylinder test, and mNSS test at different after reperfusion (two‐way ANOVA, n = 9). RPC is the abbreviation of RIPostC in the figure. Data presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 2

RIPostC triggered NLRP3 inflammasome activation and pyroptosis of microglia 3 days after AIS. (a) Illustration of the clinical experimental timeline. (b) The Pearson linear correlation analysis of the relevance between GSDMD protein expression and NIHSS score (r = 0.83, p < 0.001). Relative protein expression of GSDMD was assessed by ELISA in 21 AIS patients with RIPostC treatment and 21 AIS patients without RIPostC (Student's t‐test, n = 21). (c) Representative Western blot images of NLRP3, ASC, Pro‐Caspase 1, Caspase‐1, and GSDMD‐N in ischemic penumbra, as well as quantitative analysis of these proteins in determined with β‐actin for normalization (one‐way ANOVA, n = 4). (d) ELISA assays for IL‐1β and IL‐18 in the ischemic penumbra zone of brain tissues (n = 5). (e) Double immunostaining of Iba‐1 and GSDMD revealed a good co‐localization of these two makers. Treatment with RIPostC reduced GSDMD positive microglia in ischemic penumbra. bar = 50 μm (one‐way ANOVA, n = 3). Data presented as mean ± SEM. (f) Representative transmission electron microscopy images of microglia in peri‐infarct area. Magnified views of microglial cytomembrane are marked with dashed line boxes. Bar = 2 μm. Red arrowhead: membrane pores. RPC is the abbreviation of RIPostC in the figure. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 3

RIPostC increased the level of HGF after AIS. (a) Heat map of protein expression profiles in mice serum with p values <0.05 from sham, MCAO and MCAO + RIPostC group (one‐way ANOVA, n = 3). (b) Heat map of three cytokines (TREM1, HGF, P‐Cadherin) in mice serum. The analysis of HGF expression in protein chips. (c) The level of HGF in brain tissue of mice was detected by Western blot (n = 3). (d) The level of HGF in brain tissue of mice was detected by Real‐time PCR (one‐way ANOVA, n = 4). (e) HGF protein expression was assessed by ELISA in AIS patients with or without RIPostC (Student's t‐test, n = 21). (f) The ROC curve analysis of the diagnostic significance of HGF (AUC = 0.745) for RIPostC treatment (n = 21). RPC is the abbreviation of RIPostC in the figure. Data presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 4

HGF/ISG15 is necessary for RIPostC‐attenuated microglia pyroptosis after MCAO. (a) NLRP3, ASC, caspase‐1, GSDMD protein expressions in ischemic penumbra tissues were detected by western blotting 3 days in RIPostC mice with or without HGF siRNA and ISG15 siRNA treatment (one‐way ANOVA, n = 3). (b) The release of IL‐1β and IL‐18 in different treated groups measured by ELISA (n = 5). (c,d) Double immunofluorescence showed that both HGF siRNA and ISG15 siRNA treatment notably reversed the decline of GSDMD‐positive microglial cells induced by RIPostC. Bar = 50 μm (one‐way ANOVA, n = 3). (e,f) Immunopositivity particles for GSDMD were performed in different treated groups. Bar = 50 μm (one‐way ANOVA, n = 5). RPC is the abbreviation of RIPostC in the figure. Data presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 5

HGF attenuates microglia pyroptosis through ISG15 in vitro. (a) The expression levels of NLRP3, ASC, Pro‐Caspase 1, Caspase‐1, and GSDMD in microglial cells were detected by Western blotting in different treated groups (one‐way ANOVA, n = 3). (b) The release of IL‐1β and IL‐18 in different cultured groups measured by ELISA (one‐way ANOVA, n = 5). (c,d) Representative images of immunofluorescence of GSDMD acquired from different treated microglial cells (one‐way ANOVA, n = 3). All data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 6

RIPostC overcame NLRP3‐Dependent microglia pyroptosis may involve the HGF. We demonstrated that RIPostC, a noninvasive protection strategy, can inhibit microglia pyroptosis via mediating GSDMD through HGF/ISG15 axis.