Protective effect of angiotensin-(1-7) against hyperglycaemia-induced injury in H9c2 cardiomyoblast cells via the PI3KAkt signaling pathway

Figures

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  Figure 1. Ang-(1-7) protects against HG-triggered decrease in viability of H9c2 cells. Cell viability was detected using the Cell Counting Kit-8. (A) H9c2 cells were treated with 35 mM HG for 24 h in the absence or presence of the indicated concentrations of Ang-(1-7). (B) H9c2 cells were treated with HG for the indicated times in the absence or presence of 1 μM Ang-(1-7). Data are presented as mean ± standard error of the mean (n=3). **P<0.01 compared with the control group; ##P<0.01 vs. the HG-treated group. Ang-(1-7), angiotensin-(1-7); HG, high glucose (35 mM); LY, LY294002.

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  Figure 2. Ang-(1-7) mitigates HG-induced dephosphorylation of phosphoinositide 3-kinase and protein kinase B (PI3K/Akt) in H9c2 cells. H9c2 cells were treated for 24 h with 35 mM glucose alone, or co-treated with 1 μM Ang-(1-7) and 35 mM glucose, or co-treated with 35 mM glucose, 1 μM Ang-(1-7) and 1 μM A-779, or treated with 1 μM Ang-(1-7) alone, or 1 μM A-779 alone. The expression of PI3K and Akt were detected by western blot analysis. (A and C) Variations in the expression levels of (A) PI3K and (C) Akt in the indicated groups. (B and D) Densitometric analysis for the results in (A) and (C), respectively. Data are presented as mean ± standard error of the mean (n=3). **P<0.01 compared with the control group; ##P<0.01 compared with the HG-treated group; ΔΔP<0.01 vs. the group co-treated with Ang-(1-7) and HG. Ang-(1-7), angiotensin-(1-7); HG, high glucose (35 mM); LY, LY294002.

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  Figure 3. Activation of phosphoinositide 3-kinase and protein kinase B (PI3K/Akt) participates in the protective effect of Ang-(1-7) against HG-induced decreased viability of H9c2 cells. H9c2 cells were treated for 24 h with 35 mM glucose alone, or co-treated with 1 μM Ang-(1-7) and 35 mM glucose, or co-treated with 35 mM glucose, 1 μM Ang-(1-7) and 1 μM A-779, or co-treated with 35 mM glucose, 1 μM Ang-(1-7) and 10 μM LY294002, or treated with 1 μM Ang-(1-7) alone, or 1 μM A-779 alone, or 10 μM LY294002 alone. Cell viability was detected using the Cell Counting Kit-8. Data are presented as mean ± standard error of the mean (n=3). **P<0.01 compared with the control group; ##P<0.01 compared with HG-treated group; ΔΔP<0.01 vs. the group co-treated with Ang-(1-7) and HG. Ang-(1-7), angiotensin-(1-7); HG, high glucose (35 mM); LY, LY294002.

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  Figure 4. Activation of phosphoinositide 3-kinase and protein kinase B (PI3K/Akt) is involved in the protective effect of Ang-(1-7) against HG-induced apoptosis in H9c2 cells. (A-H) After the indicated treatments, cellular apoptosis was assessed by Hoechst 33258 staining followed by photofluorography. (A) Control group. (B) H9c2 cells were exposed to 35 mM glucose for 24 h. (C) H9c2 cells were co-treated with 1 μM Ang-(1-7) and 35 mM glucose for 24 h. (D) H9c2 cells were co-treated with 1 μM Ang-(1-7) and 35 mM glucose in the presence of 1 μM A-779 for 24 h. (E) H9c2 cells were co-treated with 1 μM Ang-(1-7) and 35 mM glucose in the presence of 10 μM LY294002 for 24 h. (F) H9c2 cells were treated with 1 μM Ang-(1-7) for 24 h followed by 24-h culture. (G) H9c2 cells were treated with 1 μM A-779 for 24 h followed by 24-h culture. (H) H9c2 cells were treated with 10 μM LY294002 for 24 h followed by 24-h culture. (I) The apoptosis rate was analysed with a Cell Counting Kit-8 and ImageJ 1.41o software. Data are presented as mean ± standard error of the mean (n=3). **P<0.01 compared with the control group; ##P<0.01 compared with the HG-treated group; ΔΔP<0.01 vs. the group co-treated with…

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  Figure 5. Activation of phosphoinositide 3-kinase and protein kinase B (PI3K/Akt) is implicated in the protective effect of Ang-(1-7) against HG-induced reactive oxygen species (ROS) production in H9c2 cells. (A-H) Intracellular ROS level was observed with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining followed by photofluorography. (A) Control group. (B) H9c2 cells were exposed to 35 mM glucose for 24 h. (C) H9c2 cells were co-treated with 1 μM Ang-(1-7) and 35 mM glucose for 24 h. (D) H9c2 cells were co-treated with 1 μM Ang-(1-7) and 35 mM glucose in the presence of 1 μM A-779 for 24 h. (E) H9c2 cells were co-treated with 1 μM Ang-(1-7) and 35 mM glucose in the presence of 10 μM LY294002 for 24 h. (F) H9c2 cells were treated with 1 μM Ang-(1-7) for 24 h followed by 24-h culture. (G) H9c2 cells were treated with 1 μM A-779 for 24 h followed by 24-h culture. (H) H9c2 cells were treated with 10 μM LY294002 for 24 h followed by 24-h culture. (I) Quantitative analysis of the mean fluorescence intensity (MFI) of DCFH-DA in (A-H) with ImageJ 1.41o software. Data are presented as means ± standard error of the mean (n=3). **P<0.01 compared with the control group; ##P<0.01 compared…

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  Figure 6. Activation of phosphoinositide 3-kinase and protein kinase B (PI3K/Akt) contributes to the protective effect of Ang-(1-7) against HG-induced reduction of mitochondrial membrane potential (MMP). (A-H) MMP was detected with rhodamine 123 (Rh123) staining followed by photofluorography. (A) Control group. (B) H9c2 cells were exposed to 35 mM glucose for 24 h. (C) H9c2 cells were co-treated with 1 μM Ang-(1-7) and 35 mM glucose for 24 h. (D) H9c2 cells were co-treated with 1 μM Ang-(1-7) and 35 mM glucose in the presence of 1 μM A-779 for 24 h. (E) H9c2 cells were co-treated with 1 μM Ang-(1-7) and 35 mM glucose in the presence of 10 μM LY294002 for 24 h. (F) H9c2 cells were treated with 1 μM Ang-(1-7) for 24 h followed by 24-h culture. (G) H9c2 cells were treated with 1 μM A-779 for 24 h followed by 24-h culture. (H) H9c2 cells were treated with 10 μM LY294002 for 24 h followed by 24-h culture. (I) Quantitative analysis of the mean fluorescence intensity (MFI) of Rh123 in (A-H) using ImageJ 1.41o software. Data are shown as means ± standard error of the mean (n=3). **P<0.01 compared with the control group; ##P<0.01 compared with the HG-treated group; ΔΔP<0.01 vs. the group co-treated…

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  Figure 7. Ang-(1-7) protects against HG-triggered inflammation in H9c2 cells. H9c2 cells were treated for 24 h with 35 mM glucose alone, or co-treated with 1 μM Ang-(1-7) and 35 mM glucose, or co-treated with 35 mM glucose, 1 μM Ang-(1-7) and 1 μM A-779, or co-treated with 35 mM glucose, 1 μM Ang-(1-7) and 10 μM LY294002, or treated with 1 μM Ang-(1-7) alone, 1 μM A-779 alone, or 10 μM LY294002 alone. After the indicated treatments, the release of (A) IL-1β, (B) IL-6 and (C) TNF-α was assessed via ELISA. Data are presented as mean ± standard error of the mean (n=3). **P<0.01 compared with the control group; ##P<0.01 compared with the HG-treated group; ΔΔP<0.01 vs. the group co-treated with Ang-(1-7) and HG. Ang-(1-7), angiotensin-(1-7); HG, high glucose (35 mM); LY, LY294002; IL, interleukin; TNF, tumor necrocis factor.

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  Figure 8. Ang-(1-7) reduced HG-induced increased expression levels of cleaved caspase-1, -3 and -12 in H9c2 cells. H9c2 cells were treated for 24 h with 35 mM glucose alone, or co-treated with 1 μM Ang-(1-7) and 35 mM glucose, or co-treated with 35 mM glucose, 1 μM Ang-(1-7) and 1 μM A-779, or co-treated with 35 mM glucose, 1 μM Ang-(1-7) and 10 μM LY294002, or treated with 1 μM Ang-(1-7) alone, or 1 μM A-779 alone, or 10 μM LY294002 alone. The expression of cleaved caspase-1, -3 and -12 was detected by western blot analysis. (A) Changes in the expression levels of cleaved caspase-1, -3 and -12 in the indicated groups. (B-D) Densitometric analysis of the results in (A). Data are presented as mean ± standard error of the mean (n=3). **P<0.01 compared with the control group; ##P<0.01 compared with the HG-treated group; ΔΔP<0.01 vs. the group co-treated with Ang-(1-7) and HG. Ang-(1-7), angiotensin-(1-7); HG, high glucose (35 mM); LY, LY294002.