Meteorin-Like (METRNL) Attenuates Myocardial Ischemia/Reperfusion Injury-Induced Cardiomyocytes Apoptosis by Alleviating Endoplasmic Reticulum Stress via Activation of AMPK-PAK2 Signaling in H9C2 Cells

Figures

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  Figure 1. The expression of METRNL was downregulated in H9C2 cells during OGD/R, and METRNL overexpression inhibited the inflammatory cytokines secretion. (A, D) The expression of METRNL in H9C2 cells was determined by western blot. (B, E) Relative expression of METRNL in H9C2 cells was evaluated by qRT-PCR. (C) The viability of H9C2 cells was analyzed by CCK8 assay. (F) The levels of inflammatory cytokines including TNF-α, IL-1β, and IL-6 were measured by corresponding ELISA kits. Error bars represent the mean±SEM from 3 independent experiments. * P < 0.05, ** P<0.01, *** P<0.001 versus Control; ## P<0.01, ### P<0.001 versus OGD/R 12 hours. METRNL – Meteorin-like; OGD/R – oxygen-glucose deprivation and reperfusion; qRT-PCR – quantitative real-time polymerase chain reaction; CCK-8 – Cell Counting Kit-8; TNF – tumor necrosis factor; IL – interleukin; ELISA – enzyme-linked immunosorbent assay; SEM – standard error of the mean.

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  Figure 2. METRNL overexpression suppressed the apoptosis of H9C2 cells induced by OGD/R. (A, B) The apoptosis rate of H9C2 cells was determined by flow cytometry. (C) The expressions of Bcl2, Bax, cleaved caspase-3, pre caspase-3, caspase-12, and PARP were evaluated by western blot. Error bars represent the mean±SEM from 3 independent experiments. *** P<0.001 versus Control; ## P<0.01, ### P<0.001 versus OGD/R 12 hours. METRNL – Meteorin-like; OGD/R – oxygen-glucose deprivation and reperfusion; PARP – poly ADP-ribose polymerase; SEM – standard error of the mean.

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  Figure 3. METRNL overexpression inhibited ER stress and promoted the PAK2 expression. (A, B) The expressions of GRP78, ATF4, ATF6, CHOP, and PAK2 were determined by western blot. Error bars represent the mean±SEM from 3 independent experiments. *** P<0.001 versus Control; ## P<0.01, ### P<0.001 versus OGD/R 12 hours. METRNL – Meteorin-like; ER – endoplasmic reticulum; PAK2 – p21-activated kinase 2; GRP78 – glucose-regulated protein 78; ATF – activating transcription factor; CHOP – C/EBP-homologus protein; SEM – standard error of the mean; OGD/R – oxygen-glucose deprivation and reperfusion.

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  Figure 4. PAK2 silencing reversed the inhibitive effect of METRNL overexpression on inflammatory response. (A) The expression of PAK2 in H9C2 cells was determined by western blot. (B) Relative expression of PAK2 in H9C2 cells was evaluated by qRT-PCR. (C) The levels of inflammatory cytokines including TNF-α, IL-1β, and IL-6 were measured by corresponding ELISA kits. Error bars represent the mean±SEM from three independent experiments. *** P<0.001 versus Control; ### P<0.001 versus OGD/R 12 hours; @@ P<0.01, @@@ P<0.001 versus OGD/R+Ov-METRNL. PAK2 – p21-activated kinase 2; METRNL – Meteorin-like; qRT-PCR – quantitative real-time polymerase chain reaction; TNF – tumor necrosis factor; IL – interleukin; ELISA – enzyme-linked immunosorbent assay; SEM – standard error of the mean; OGD/R – oxygen-glucose deprivation and reperfusion.

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  Figure 5. PAK2 silencing blocked the inhibitive effect of METRNL overexpression on cell apoptosis induced by OGD/R. (A) The apoptosis rate of H9C2 cells was analyzed by TUNEL assay (200x magnification). (B) The expressions of Bcl2, Bax, cleaved caspase-3, pre caspase-3, caspase-12, and PARP were evaluated by western blot. Error bars represent the mean±SEM from 3 independent experiments. *** P<0.001 versus Control; ## P<0.01, ### P<0.001 versus OGD/R 12 hours; @ P<0.05, @@ P<0.01 versus OGD/R+Ov-METRNL. PAK2 – p21-activated kinase 2; METRNL – Meteorin-like; OGD/R – oxygen-glucose deprivation and reperfusion; TUNEL – terminal deoxynucleotidyl transferase dUTP nick end labeling immunohistochemical; PARP – poly ADP-ribose polymerase; SEM – standard error of the mean; Ov-METRNL – overexpression-METRNL.

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  Figure 6. PAK2 silencing reversed the inhibitive effect of METRNL overexpression on c ER stress induced by OGD/R. The expressions of GRP78, ATF4, ATF6, and CHOP were determined by western blot. Error bars represent the mean±SEM from 3 independent experiments. *** P<0.001 versus Control; ## P<0.01, ### P<0.001 versus OGD/R 12 hours; @ P<0.05, @@@ P<0.001 versus OGD/R+Ov-METRNL. PAK2 – p21-activated kinase 2; METRNL – Meteorin-like; ER – endoplasmic reticulum; OGD/R – oxygen-glucose deprivation and reperfusion; GRP78 – glucose-regulated protein 78; ATF – activating transcription factor; CHOP – C/EBP-homologus protein; SEM – standard error of the mean.

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  Figure 7. (A, B) METRNL overexpression induced activation of AMPK-PAK2 signaling cascade. The expressions of p-AMPK, AMPK and PAK2 were determined by western blot. Error bars represent the mean±SEM from three independent experiments. **** P<0.001 versus Control; ## P<0.01, ### P<0.001 versus OGD/R 12 hours. METRNL – Meteorin-like; AMPK – 5′ AMP-activated protein kinase; PAK2 – p21-activated kinase 2; SEM – standard error of the mean.