Osmotin Protects H9c2 Cells from Simulated Ischemia-Reperfusion Injury through AdipoR1/PI3K/AKT Signaling Pathway

Abstract

Objective: This study aimed to investigate the effect of osmotin on myocardial ischemia/reperfusion (I/R), as well as the underlying mechanisms. Methods:In vitro I/R injury model was established on rat cardiac myoblast H9c2 cells by oxygen and glucose deprivation followed by reperfusion (OGD/R). Cells were administrated with osmotin, and transfected with small interfering RNAs (siRNAs) which specifically target adiponectin receptor 1 or 2 (AdipoR1/2). Besides, the cells were incubated with or without LY294002 as inhibitor of phosphatidylinositol 3-kinase (PI3K) under OGD/R condition. Cell viability, apoptosis, expressions of apoptosis-related proteins and inflammatory factors were analyzed. Results: The results showed that osmotin significantly increased H9c2 cells viability compared with the cells treated with vehicle (P < 0.05), and decreased H9c2 cells apoptosis by regulating expressions of apoptosis-related proteins. Moreover, we observed that osmotin statistically reduced the release of proinflammatory factors and increased the release of anti-inflammatory factors in H9c2 cells (P < 0.05). However, these effects were markedly reversed by AdipoR1 silence but not AdipoR2. Furthermore, osmotin dramatically upregulated the phosphorylation levels of PI3K, AKT, ERK, and downregulated the phosphorylation level of NF-κB (P < 0.05). While administration of LY294002 reduced cell viability, increased cell apoptosis, and aggravated inflammatory response (P < 0.05). Conclusion: Our results suggested that the protective effect of osmotin on the simulated OGD/R injured H9c2 cells might be associated with AdipoR1/PI3K/AKT signaling pathway.

Keywords: AKT signaling pathway; AdipoR1/PI3K signaling pathway; acute myocardial infarction; ischemia-reperfusion injury; osmotin.

Figure 1

The time axis of OGD/R exposure and osmotin administration with or without LY294002 treatment. OGD/R, oxygen and glucose deprivation/reperfusion; AdipoR, adiponectin receptor; siRNA, small interfering RNA; semi-qRT-PCR, Semi-quantitative real-time reverse transcriptase polymerase chain reaction; LDH, lactate dehydrogenase; ROS, reactive oxidative stress; MTT, 3-(4, 5-dimethylthiazol-yl)-2, 5-diphenyl-2-H-tetrazolium bromide.

Figure 2

Effects of osmotin combine with AdipoRs silence on H9c2 cell viability. H9c2 cells were pre-treated with OGD/R, and then been administrated with osmotin and/or transfection with specifically siRNAs for AdipoR1 or AdipoR2. (A) Effects of osmotin on the cell viability (n = 5); (B–D). Transfection efficiency of siRNAs for AdipoR1 and AdipoR2 were measured by semi-qRT-PCR (n = 5), and western blot assay. GAPDH acted as internal control; (E) Effects of osmotin and/or combine with AdipoR1 or AdipoR2 siRNAs transfection on cell viability (n = 5). (F) Relative LDH release of H9c2 cells after corresponding administrations (n = 5). ^*P < 0.05 compare to corresponding control; ns, no significant compared to the osmotin group. OGD/R, oxygen and glucose deprivation/reperfusion; AdipoR, adiponectin receptor; NC, negative control; siRNA, small interfering RNA; semi-qRT-PCR, semi-quantitative Real-time reverse transcriptase polymerase chain reaction; LDH, lactate dehydrogenase.

Figure 3

Effects of osmotin and silencing of AdipoRs on apoptosis of H9c2 cells. H9c2 cells were pre-treated with OGD/R, and then been administrated with osmotin and/or transfection with specifically siRNAs for AdipoR1 or AdipoR2. (A) Effects of osmotin and silencing of AdipoRs on the percentages of apoptotic cells (n = 5); (B) The expressions of apoptosis-related proteins were measured by Wearer blot. (C–E) Relative protein expression levels of apoptotic related proteins (n = 5). (F) Effects of osmotin and silencing of AdipoRs on the number of ROS positive cells (n = 5). ^*P < 0.05 compare to corresponding control; ns, no significant compared to the osmotin group. OGD/R, oxygen and glucose deprivation/reperfusion; AdipoR, adiponectin receptor; Bcl, B-cell lymphoma; siRNA, small interfering RNA; ROS, reactive oxidative stress; ns, non-significant.

Figure 4

Effects of osmotin and silencing of AdipoRs on expressions of proinflammatory factors in H9c2 cells. H9c2 cells were pre-treated with OGD/R, and then been administrated with osmotin and/or transfection with specifically siRNAs for AdipoR1 or AdipoR2. (A) Expressions of proinflammatory factors including IL-1β, IL-6, IL-8, and TNF-αmeasured by western blot. (B–E), Relative protein expression levels of proinflammatory factors IL-1β, IL-6, IL-8, and TNF-α(n = 5). ^*P < 0.05 compare to corresponding control; ns, no significant compared to the osmotin group. OGD/R, oxygen and glucose deprivation/reperfusion; AdipoR, adiponectin receptor; IL, interleukin; TNF, tumor necrosis factor; NF-κB, nuclear factor-kappa B; siRNA, small interfering RNA; ns, non-significant.

Figure 5

Effects of osmotin and silencing of AdipoRs on inflammatory factors in H9c2 cells. H9c2 cells were pre-treated with OGD/R, and then been administrated with osmotin and/or transfection with specifically siRNAs for AdipoR1 or AdipoR2. (A–D). The relative contents of proinflammatory factors IL-1β, IL-6, IL-8, and TNF-α were measured by ELISA assay (n = 5). (E) Protein expressions of anti-inflammatory factors IL-4, IL-10 and IL-13 were measured by western blot. ^*P < 0.05 compare to corresponding control; ns, no significant compared to the osmotin group. OGD/R, oxygen and glucose deprivation/reperfusion; AdipoR, adiponectin receptor; IL, interleukin; TNF, tumor necrosis factor; siRNA, small interfering RNA; ns, non-significant.

Figure 6

Effects of osmotin and silencing of AdipoRs on the expressions of PI3K, AKT, NF-κB, and ERK in H9c2 cells. H9c2 cells were pre-treated with OGD/R, and then been administrated with osmotin and/or transfection with specifically siRNAs for AdipoR1 or AdipoR2. (A) Expressions of p/t-PI3K and p/t-AKT in H9c2 cells measured by western blot after corresponding administrations. (B) The ratio of p/t PI3K (n = 5). (C) The ratio of p/t AKT (n = 5). (D) Expressions of p/t-NF-κB and p/t-ERK1/2 in H9c2 cells measured by western blot after corresponding administrations. (E) The ratio of p/t NF-κB. (F) The ratio of p/t ERK1/2 ^*P < 0.05 compare to corresponding control; ns, no significant compared to the osmotin group. OGD/R, oxygen and glucose deprivation/reperfusion; AdipoR, adiponectin receptor; PI3K, phosphatidylinositol 3-kinase; NF-κB, nuclear factor kappa B; siRNA, small interfering RNA; ns, non-significant.

Figure 7

Effects of osmotin and administration of LY294002 on H9c2 cell viability and apoptosis. H9c2 cells were pre-treated with OGD/R, and then been administrated with osmotin and/or LY294002 as inhibitor of PI3K. (A) Effects of osmotin and/or LY294002 administration on cell viability (n = 5); (B) Relative LDH release of administrated H9c2 cells (n = 5). (C) Effects of osmotin and administration of LY294002 on cell apoptosis (n = 5). (D) Expressions of apoptotic related factors measured by western bolt. GAPDH acted as internal control. (E–G) Relative expression levels of apoptotic related factors (n = 5). (H) Effects of osmotin and/or LY294002 administration on the number of ROS positive cells (n = 5). ^*P < 0.05 compare to corresponding control. OGD/R, oxygen and glucose deprivation/reperfusion; LY294002, inhibitor of phosphatidylinositol 3-kinase (PI3K); LDH, lactate dehydrogenase; ROS, reactive oxidative stress; LY294002, inhibitor of phosphatidylinositol 3-kinase (PI3K).

Figure 8

Effects of osmotin and administration of LY294002 on the proinflammatory factors expressions in H9c2 cells. H9c2 cells were pre-treated with OGD/R, and then been administrated with osmotin and/or LY294002 as inhibitor of PI3K. (A) Expressions of IL-1β, IL-6, IL-8, and TNF-α in H9c2 cells after administration were measured by western blot. (B–E) Relative expression levels of IL-1β, IL-6, IL-8, and TNF-α(n = 5). ^*P < 0.05 compare to corresponding control. OGD/R, oxygen and glucose deprivation/reperfusion; LY294002, inhibitor of phosphatidylinositol 3-kinase (PI3K); IL, interleukin; TNF, tumor necrosis factor.

Figure 9

Effects of osmotin and administration of LY294002 on the inflammatory factors contents in H9c2 cells. H9c2 cells were pre-treated with OGD/R, and then been administrated with osmotin and/or LY294002 as inhibitor of PI3K. (A–D) Results of ELISA assay about the contents of proinflammatory factors IL-1β, IL-6, IL-8, and TNF-α(n = 5). (E) Protein expressions of anti-inflammatory factors IL-4, IL-10 and IL-13 were detected by western blot ^*P < 0.05 compare to corresponding control. OGD/R, oxygen and glucose deprivation/reperfusion; AdipoR, adiponectin receptor; LY294002, inhibitor of phosphatidylinositol 3-kinase (PI3K); IL, interleukin; TNF, tumor necrosis factor.