Neuregulin‐1 alleviate oxidative stress and mitigate inflammation by suppressing NOX4 and NLRP3/caspase‐1 in myocardial ischaemia‐reperfusion injury

FIGURE 1. NRG‐1 protects against myocardial ischaemia‐reperfusion injury. A, Representative cardiac tissue sections stained with Evans blue and triphenyl tetrazolium chloride [blue, non‐ischaemic area; non‐blue, area at risk (AR); white, infarct size (IS)]. B, The in vivo experiment protocol. C, The ratio of IS/AR. (D) Serum creatinine kinase‐MB concentration. E, Representative paraffin‐embedded cardiac sections subjected to immunohistochemical staining to detect 4‐hydroxynonenal (4HNE) at the risk area. The cellular nuclei are shown in blue, whereas 4HNE‐positive areas are shown in brown. F, Representative M‐mode echocardiograms for each group, with (G) EF; H, FS quantified in bar graphs. Data are represented as mean ± standard error of mean (n = 6; *P < .05 vs. IR, # P < .05 vs. CON). CON, sham‐operated control; IR, ischaemia‐reperfusion; NRG‐1, IR + NRG‐1

FIGURE 2. NRG‐1 shows no effect on myocardial capillary density but mitigates myocardial apoptosis. A, Immunohistochemical analysis of CD31 was performed to evaluate the myocardial capillary density. Capillary density was assessed based on the capillary/myocyte nucleus (C/M) values. Magnification: 200X. The CD31+ capillary endothelial cells are shown in brown. Myocardial sections were counterstained with haematoxylin (nuclei are stained blue). B, The percentage of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)‐positive cells among the total number of cells. C, Representative myocardial sections subjected to the TUNEL assay. TUNEL‐positive cells are shown in green. The 4′,6‐diamidino‐2‐phenylindole (DAPI)‐stained nuclei are shown in blue. The cardiomyocytes stained with anti‐α‐SCA antibody are shown in red. Data are represented as mean ± standard error (n = 6; *P < .05 vs CON; ***P < .001 vs. CON; ##P < .01 vs IR). CON, sham‐operated control; IR, ischaemia‐reperfusion; NRG‐1, IR + NRG‐1

FIGURE 3. NRG‐1 down‐regulates the expression of NOX4 by activating ERK1/2. The mRNA expression levels of Nox4 (A), Gpx‐1 (B), and Cat (C) were detected using quantitative real‐time polymerase chain reaction. (D) Representative paraffin‐embedded cardiac sections subjected to immunohistochemical staining to detect NOX4 at the risk area. The cellular nuclei are shown in blue, whereas NOX4‐positive areas are shown in brown. E. Representative Western blots showing the levels of NOX4, P‐ERK1/2, T‐ERK1/2, GPX‐1 and GAPDH. Semi‐quantification of NOX4 level (F), the P‐ERK1/2 density/T‐ERK1/2 density ratio (G), and GPX‐1 level (H). The expression levels of target proteins were normalized to those of GAPDH Data are represented as mean ± standard error of mean (n = 6; *P < .05, **P < .001 vs. IR). CON, sham‐operated control; IR, ischaemia‐reperfusion; NRG‐1, IR + NRG‐1

FIGURE 4. NRG‐1 suppresses the NLRP3/caspase‐1 pathway. A. Representative Western blots showing the levels of NLRP3, caspase‐1, cleaved caspase‐1, IL‐1β and GAPDH. Semi‐quantification of NLRP3 level (B), the cleaved caspase‐1 density/caspase‐1 density ratio (C), and IL‐1β level (D). The expression levels of the target proteins were normalized to those of GAPDH. Data are represented as mean ± standard error of mean (n = 6; *P < .05,**P < .001 vs CON; #P < .05 vs. IR). CON, sham‐operated control; IR, ischaemia‐reperfusion; NRG‐1, IR + NRG‐1

FIGURE 5. PD98059 mitigates the NRG‐1‐mediated alleviation of myocardial injury. A. Triphenyl tetrazolium chloride (TTC) staining was used to detect the infarct size. Representative cardiac sections stained with TTC (red, ischaemic area; white, infarct area). B. The percentage of infarct size/left ventricle (IS/LV%). C. The Langendorff experiment protocol. D. Representative frozen cardiac sections subjected to dihydroethidium(DHE) staining to detect reactive oxygen species at the risk area. Data are represented as mean ± standard error of mean (n = 6; **P < .01 vs IR; ##P < .01 vs. NRG‐1). CON, sham‐operated control; IR, ischaemia‐reperfusion; NRG‐1, IR + NRG‐1; PD, PD98059

FIGURE 6. PD98059 increases the number of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)‐positive cells. (A) Representative myocardial sections subjected to the TUNEL assay. TUNEL‐positive cells are shown in green. The 4′,6‐diamidino‐2‐phenylindole (DAPI)‐stained nuclei are shown in blue. The α‐SCA‐positive cardiomyocytes are shown in red. (B) The percentage of TUNEL‐positive cells among the total number of cells. Data are represented as mean ± standard error of mean (n = 6; **P < .01 vs IR; #P < .05 vs. NRG‐1). CON, sham‐operated control; IR, ischaemia‐reperfusion; NRG‐1, IR + NRG‐1; PD, PD98059

FIGURE 7. PD98059 up‐regulates the expression of NOX4 and NLRP3/caspase‐1 pathway. A, Representative Western blots showing the levels of NOX4, P‐ERK1/2, T‐ERK1/2, caspase‐1, NLRP‐3, cleaved caspase‐1, IL‐1β and GAPDH. Semi‐quantification of NOX4 level (B), the P‐ERK1/2 density/T‐ERK1/2 density ratio (C), NLRP3 level (D), the cleaved caspase‐1 density/caspase‐1 density ratio (C) and IL‐1β level (E). The expression levels of target proteins were normalized to those of GAPDH. G. A proposed schematic diagram of the molecular pathway activated by NRG‐1. Data are represented as mean ± standard error of mean (n = 6; *P < .05 vs IR; ##P < .001 vs. NRG‐1). CON, sham‐operated control; IR, ischaemia‐reperfusion; NRG‐1, IR + NRG‐1; PD, PD98059