Activation of Cholinergic Anti-Inflammatory Pathway Ameliorates Cerebral and Cardiac Dysfunction After Intracerebral Hemorrhage Through Autophagy

Abstract

Background: Intracerebral hemorrhage (ICH) is the devastating subtype of stroke with cardiovascular complications, resulting in high rates of mortality and morbidity with the release of inflammatory factors. Previous studies have demonstrated that activation of α7nAChR can reduce immune and inflammation-related diseases by triggering the cholinergic anti-inflammatory pathway (CAIP). α7nAChR mediates protection from nervous system inflammation through AMPK-mTOR-p70S6K-associated autophagy. Therefore, the purpose of this study is to explore whether the activation of α7nAChR improves cerebral and cardiac dysfunction after ICH through autophagy.

Methods: Male C57BL/6 mice were randomly divided into five groups (1): Control + saline (2), ICH+ saline (3), ICH + PNU-282987 (4), ICH+ PNU-282987 + MLA (5), ICH + PNU-282987 + 3-MA. The neurological function was evaluated at multiple time points. Brain water content was measured at 3 days after ICH to assess the severity of brain edema. PCR, immunofluorescence staining, and Western Blot were performed at 7 days after ICH to detect inflammation and autophagy. Picro-Sirius Red staining was measured at 30 days after ICH to evaluate myocardial fibrosis, echocardiography was performed at 3 and 30 days to measure cardiac function.

Results: Our results indicated that the PNU-282987 reduced inflammatory factors (MCP-1, IL-1β, MMP-9, TNF-α, HMGB1, TLR2), promoted the polarization of macrophage/microglia into anti-inflammatory subtypes(CD206), repaired blood-brain barrier injury (ZO-1, Claudin-5, Occludin), alleviated acute brain edema and then recovered neurological dysfunction. Echocardiography and PSR indicated that activation of α7nAChR ameliorated cardiac dysfunction. Western Blot showed that activation of α7nAChR increased autophagy protein (LC3, Beclin) and decreased P62. It demonstrated that the activation of α7nAChR promotes autophagy and then recovers brain and heart function after ICH.

Conclusions: In conclusion, PNU-282987 promoted the cerebral and cardiac functional outcomes after ICH in mice through activated α7nAChR, which may be attributable to promoting autophagy and then reducing inflammatory reactions after ICH.

Keywords: autophagy; inflammation; intracerebral hemorrhage; macrophage; α7nAChR.

Figure 1

Activating α7nAChR decreased brain water content 3 days after ICH and improves neurological function in ICH mice. (A) PNU-282987 significantly reduces water content in the brain in the ipsilateral hemisphere. (B) Activating α7nAChR decreases foot-fault test scores in ICH mice. (C) Activating α7nAChR decreases mNSS scores in ICH mice. n= 5/group, the results are expressed as mean ± SE, *p < 0.05, **P < 0.01, ***P < 0.0001, ns means p > 0.05.

Figure 2

Activating α7nAChR prevented ICH-induced cardiac dysfunction. (A) Representative echocardiography results at 7 days from control, ICH, ICH+PNU-282987, ICH+PNU-282987+MLA. (B) Representative echocardiography results at 30 days. (C) LVEF, LVFS dimension at 7 days and 30 days after ICH, IVS dimension at the end of diastole and systole. (D) LV volume, LVID dimension at the end of diastole and systole. n = 6 per group for echocardiography. Data are presented as mean ± SE; *p < 0.05; **P < 0.01; ***P < 0.0001; ns means p > 0.05.

Figure 3

Activating α7nAChR significantly decreased cardiac fibrosis and cardiac hypertrophy in the chronic phase of intracerebral hemorrhage. (A) Representative results for PSR immunostaining in heart tissue at 30 days. (B) Quantification of interstitial fibrosis (Picro Sirius Red). n = 6 per group. (C) The result of heart weight at 7 days. (D) The result of heart weight at 30 days. (E) The PCR results showed that treatment with PNU-282987 significantly reduced the gene expression of the ICH-caused profibrotic factor TGF-β compared with ICH mice at 30 days. (F) PNU-282987 significantly reduced the gene expression of the ICH-caused profibrotic factor MMP-9 compared with ICH mice at 30 days. n = 6 per group. Data are presented as mean ± SE; *p < 0.05; **P < 0.01; ***P < 0.0001.

Figure 4

Activating α7nAChR induced autophagy in the brain and heart of ICH mice. (A) Representative protein expression levels of LC3, Beclin, p62/SQSTM1 in brain evaluated by western blot. (B) Quantification of western blot data of LC3 in brain. (C) Quantification of western blot data of Beclin in brain. (D) Quantification of western blot data of p62/SQSTM1 in brain. (E) Representative protein expression levels of LC3, Beclin, p62/SQSTM1 in heart, as measured by western blot. (F) Quantification of western blot data of LC3 in heart. (G) Quantification of western blot data of Beclin in heart. (H) Quantification of western blot data of p62/SQSTM1 in heart. n= 6 per group. Data are presented as mean ± SE; *p < 0.05; **P < 0.01; ***P < 0.0001.

Figure 5

Activating α7nAChR induced autophagy in the brain and heart of ICH mice. (A) Immunofluorescence staining of LC3 from brain tissue of control, ICH, ICH+PNU-282987 and ICH+PNU-282987+MLA (scale bar, 100 μm). (B) The quantitation of LC3 in brain. (C) Immunofluorescence staining of LC3 from heart tissue of control, ICH, ICH+PNU-282987 and ICH+PNU-282987+MLA (scale bar, 100 μm). (D) The quantitation of LC3 in heart. n= 6 per group. Data are presented as mean ± SE; ***P < 0.0001.

Figure 6

The expression of α7nAChR was reduced in brain and heart After ICH. (A) Representative protein expression levels of α7nAChR in brain and heart, the expression of protein α7nAChR remarkably reduced at 7 days after ICH, while PNU-282987 promoted their expression. (B) Quantification of western blot data in brain. (C) Quantification of western blot data in heart. (D) ICH for 7 days significantly decreased mRNA expression of α7nAChR in brain, PNU-282987 promoted their expression. (E) ICH for 7 days significantly decreased mRNA expression of α7nAChR in heart, PNU-282987 promoted their expression. n= 6 per group. Data are presented as mean ± SE; *p < 0.05; **P < 0.01; ***P < 0.0001.

Figure 7

Activating α7nAChR mediated neuroprotection in ICH by regulating microglia/macrophage polarization toward an anti-inflammatory phenotype. (A) Typical flow cytometric graph showing the gating strategy for CD45+CD11b+F4/80+ macrophages, CD45+CD11b+Ly6G+neutrophils, CD206+ anti-inflammatory macrophages and CD86+ pro-inflammatory macrophages populations in brain. (B) Quantitative data of anti-inflammatory macrophages expression in brain at 7 days after ICH. (C) Quantitative data of pro-inflammatory macrophages expression in brain at 7 days after ICH. (D) Quantitative data of macrophages expression in brain at 7 days after ICH. n = 6 per group. (E) Quantitative data of neutrophils expression in brain at 7 days after ICH. Data are presented as mean ± SE; *p < 0.05; **P < 0.01; ***P < 0.0001.

Figure 8

The results of PCR demonstrated that PNU-282987 drastically decreased the phenotype of pro-inflammatory macrophages [CD86 (A)] and increased anti-inflammatory macrophages [CD206 (B), Arg-1 (C), YM-1 (D)] in brain. PCR results indicated that PNU-282987 significantly reduced the pro-inflammatory macrophages [CD86 (E)] and increased the phenotype of anti-inflammatory macrophages [CD206 (F), Arg-1 (G), YM-1 (H)] in heart. n= 6 per group for echocardiography. Data are presented as mean ± SE; *p < 0.05; **P < 0.01; ***P < 0.0001; ns means p > 0.05.

Figure 9

α7nAChR expression in endothelial cells promoted angiogenesis and alleviated the functional dysfunction of the blood–brain barrier after ICH. (A) PCR results indicated that PNU-282987 remarkably decreased the expression of VCAM. (B) The results of PCR demonstrated that PNU-282987 drastically increased the expression of ZO-1. (C) The results of PCR demonstrated that PNU-282987 drastically increased the expression of Claudin-5. (D) The results of PCR demonstrated that PNU-282987 drastically increased the expression of Occludin. n= 6 per group for echocardiography. Data are presented as mean ± SE; *p < 0.05; **P < 0.01; ***P < 0.0001.

Figure 10

Activating α7nAChR decreased ICH-caused oxidative stress and inflammatory responses in the brain and heart. (A–F) PCR results indicated that PNU-282987 remarkably decreased ICH-induced inflammatory factor expression as MCP-1, IL-β, MMP-9, TNF-α, TLR2 and HMGB1 compared to control mice in brain at 7 days after ICH. (G–L) PCR results indicated that PNU-282987 significantly reduced gene expression of ICH-induced inflammatory factor as TLR2, HMGB1, MCP-1, IL-β, MMP-9 and TNF-α compared to control mice in heart at 7 days after ICH. n = 6 per group for echocardiography. Data are presented as mean ± SE; *p < 0.05; **P < 0.01; ***P < 0.0001.

Figure 11

Activating α7nAChR decreased ICH-caused oxidative stress and inflammatory responses in the heart. Immunofluorescence images of inflammation factor of TGF-β (A), NOX-2 (B), MCP-1 (C) were compared in four groups in heart, (scale bar, 100 μm). Quantitative data of TGF-β (D), NOX-2 (E), MCP-1 (F) in heart. n = 6 per group. Data are presented as mean ± SE; **P < 0.01; ***P < 0.0001.