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. 2017 Jul;8(4):434-445.
doi: 10.1111/jdi.12603. Epub 2017 Feb 28.

Angiotensin-(1-7) protects cardiomyocytes against high glucose-induced injuries through inhibiting reactive oxygen species-activated leptin-p38 mitogen-activated protein kinase/extracellular signal-regulated protein kinase 1/2 pathways, but not the leptin-c-Jun N-terminal kinase pathway in vitro

Yiyan Lei  1 , Qing Xu  2 , Bo Zeng  1 , Wei Zhang  2 , Yulan Zhen  3 , Yuansheng Zhai  2 , Fei Cheng  4 , Weiyi Mei  2 , Dongdan Zheng  2 , Jianqiang Feng  5 , Jun Lan  4 , Jingfu Chen  4
Affiliations

Angiotensin-(1-7) protects cardiomyocytes against high glucose-induced injuries through inhibiting reactive oxygen species-activated leptin-p38 mitogen-activated protein kinase/extracellular signal-regulated protein kinase 1/2 pathways, but not the leptin-c-Jun N-terminal kinase pathway in vitro

Yiyan Lei et al. J Diabetes Investig. 2017 Jul.

Abstract

Aims/introduction: Angiotensin-(1-7) (Ang-[1-7]), recognized as a new bioactive peptide in the renin-angiotensin system, shows biological and pharmacological properties in diabetic cardiovascular diseases. The leptin-induced p38 mitogen-activated protein kinase (MAPK) pathway has been reported to contribute to high glucose (HG)-induced injury. In the present study, we showed the mechanism of how Ang-(1-7) can protect against HG-stimulated injuries in H9c2 cells.

Materials and methods: H9c2 cells were treated with 35 mmol/L glucose (HG) for 24 h to establish a model of HG-induced damage. Apoptotic cells were observed by Hoechst 33258 staining. Cell viability was analyzed by cell counter kit-8. The expression of protein was detected by western blot. Reactive oxygen species was tested by 2',7'-dichlorodihydrofluorescein diacetate staining. Mitochondrial membrane potential was measured by 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide staining.

Results: The present results showed that treating H9c2 cells with HG obviously enhanced the expressions of both the leptin and phosphorylated (p)-MAPK pathway. However, the overexpression levels of leptin and p-p38 MAPK/p-extracellular signal-regulated protein kinase 1/2 (ERK1/2), but not p-c-Jun N-terminal kinase, were significantly suppressed by treatment of the cells with Ang-(1-7). Additionally, leptin antagonist also markedly suppressed the overexpressions of p38 and ERK1/2 induced by HG, whereas leptin antagonist had no influence on the overexpression of c-Jun N-terminal kinase. More remarkable, Ang-(1-7), leptin antagonist, SB203580 or SP600125, respectively, significantly inhibited the injuries induced by HG, such as the increased cell viability, decreased apoptotic rate, reduction of ROS production and increased mitochondrial membrane potential. Furthermore, the overexpressions of p38 MAPK, ERK1/2 and leptin were suppressed by N-actyl-L-cystine.

Conclusions: The present findings show that Ang-(1-7) protects from HG-stimulated damage as an inhibitor of the reactive oxygen species-leptin-p38 MAPK/ERK1/2 pathways, but not the leptin-c-Jun N-terminal kinase pathway in vitro.

Keywords: Angiotensin-(1-7); Cardiomyocytes; High glucose.

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Figures

Figure 1
Figure 1
Different doses of angiotensin‐(1–7) (Ang‐[1–7]) suppresse the high glucose (HG)‐induced activation of leptin and p‐38 mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated protein kinase 1/2 (ERK1/2) in H9c2 cells, but have no influence on overexpression of phosphorylated (p)‐c‐Jun N‐terminal kinase (JNK). (a–f) H9c2 cells were exposed to 35 mmol/L glucose for the indicated times (30, 60, 120, 240 and 480 min, respectively) or (3, 6, 9, 12 and 24 h, respectively). (g–l) H9c2 cells were co‐treated with 35 mmol/L glucose and indicated concentrations of Ang‐(1–7) (0.5, 1 and 2 μmol/L, respectively) for 15,240 min or 24 h. The expression levels of (a,b,g,h) p38 MAPK, (a,c,g,i) ERK1/2, (a,d,g,j) JNK and (e,f,k,l) leptin were measured by western blot assay. (b,c,d,f,h,i,j,l) Densitometric analysis of the results from (a), (e), (g) and (k), respectively. Data are presented as the mean ± standard error of the mean (n = 3). **P < 0.01 vs the control (Con) group; ‡ P < 0.01 vs the HG‐treated group. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; p‐p38, phosphorylated‐p38; t‐p38, total p38.
Figure 2
Figure 2
Angiotensin‐(1–7) (Ang‐[1–7]) suppresses the high glucose (HG)‐induced activation of leptin and p‐38 mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated protein kinase 1/2 (ERK1/2) in H9c2 cells, but has no influence on overexpression of phosphorylated (p)‐c‐Jun N‐terminal kinase (JNK). Cells were coconditioned with 1 μmol/L Ang‐(1–7) for 24 h with or without HG. (a,c,e,g) The expression of p38, ERK1/2, JNK and leptin were measured by western blot analysis. (b,d,f,h) Densitometric analysis of the related protein expression levels in (a,c,e,g), respectively. The data were quantified by densitometric analysis with IMAGEJ 1.47 i software. Data are shown as the mean ± standard error of the mean (n = 3). **P < 0.01 vs the control (Con) group; ‡ P < 0.01 vs the HG‐treated group. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; p‐p38, phosphorylatedp38; t‐p38, total p38.
Figure 3
Figure 3
Leptin antagonist (LA) decreases the high glucose (HG)‐induced activation of p‐38 mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated protein kinase 1/2 (ERK1/2) in H9c2 cells, but has no influence on overexpression of phosphorylated (p)‐c‐Jun Nterminal kinase (JNK). Cells were preconditioned with 50 ng/mL LA for 24 h with or without HG for 24 h. (a,c,e) The expression levels of p38, ERK1/2 and JNK were assessed by western blot analysis. (b,d,f) Densitometric analysis of the related protein expression levels in (a,c,e), respectively. The data was quantified by densitometric analysis with IMAGEJ 1.47 i software. Data are shown as the mean ± standard error of the mean (n = 3). **P < 0.01 vs the control (Con) group; ‡ P < 0.01 vs the HG‐treated group. p‐p38, phosphorylated‐p38; t‐p38, total p38.
Figure 4
Figure 4
Angiotensin‐(1–7) (Ang‐[1–7]), leptin antagonist (LA), SB203580 (a selective inhibitor of p38 mitogen‐activated protein kinase) and SP600152 (a selective inhibitor of extracellular signal‐regulated protein kinase 1/2) relieved cytotoxicity induced by high glucose (HG) in H9c2 cells. Cell viability was tested by using the CCK‐8 assay. Cells were treated with 1 μmol/L Ang‐(1–7), 50 ng/mL LA, 3 μmol/L SB203580 or 10 μmol/L SP600125 with or without HG. Data are shown as mean ± standard error of the mean (n = 3). **P < 0.01 vs the control (Con) group; ‡ P < 0.01 vs the HG‐treated group.
Figure 5
Figure 5
Angiotensin‐(1–7) (Ang‐[1–7]), leptin antagonist (LA), SB203580 and SP600152 decrease apoptosis induced by high glucose (HG) in H9c2 cells. (a–j) Cells were treated with different drugs. Hoechst 33258 staining followed by photofluorography was used to assess the cellular apoptosis. Data are shown as mean ± standard error of the mean (n = 3). **P < 0.01 vs the control (Con) group; ‡ P < 0.01 vs the HG‐treated group.
Figure 6
Figure 6
Angiotensin‐(1–7) (Ang‐[1–7]), leptin antagonist (LA), SB203580 and SP600152 alleviate the increased reactive oxygen species (ROS) generation induced by high glucose (HG) in H9c2 cells. (a–j) Cells were treated with different drugs, intracellular ROS level was assessed by 2′,7′‐dichlorodihydrofluorescein diacetate staining followed by photofluorography. Data are shown as mean ± standard error of the mean (n = 3). **P < 0.01 vs the control (Con) group; ‡ P < 0.01 vs the HG‐treated group.
Figure 7
Figure 7
Angiotensin‐(1–7) (Ang‐[1–7]), leptin antagonist (LA), SB203580 and SP600152 block the mitochondrial membrane potential (MMP) induced by high glucose (HG) in H9c2 cells. (a–j) Cells were treated with different drugs, and the MMP level was tested by 5,5′,6,6′‐Tetrachloro‐1,1′,3,3′‐tetraethyl‐imidacarbocyanine iodide staining followed by photofluorography. Data are shown as mean ± standard error of the mean (n = 3). **P < 0.01 vs the control (Con) group; ‡ P < 0.01 vs the HG‐treated group.
Figure 8
Figure 8
Reactive oxygen species (ROS) inhibitor suppresses the high glucose (HG)‐induced overexpressions of p38 mitogen‐activated protein kinase (MAPK), extracellular signal‐regulated protein kinase 1/2 (ERK1/2) and leptin in H9c2 cells. Cells were priority treated with 1,000 μmol/L N‐actyl‐L‐cystine for 60 min with or without HG. (a,c,e) The expressions of leptin, p38 and ERK1/2 were measured by western blot analysis. (b,d,f) Densitometric analysis of the related protein expression levels in (a,c,e), respectively. Data are shown as mean ± standard error of the mean (n = 3). **P < 0.01 vs the control (Con) group; ‡ P < 0.01 vs the HG‐treated group. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; p‐p38, phosphorylated‐p38; t‐p38, total p38.
Figure 9
Figure 9
N‐actyl‐L‐cystine alleviates HG‐induced cytotoxicity in H9c2 cells. Cells were pretreated with 1,000 μmol/L NAC for 60 min with or without high glucose (HG). Cell viability was tested by using the CCK‐8 assay. Data are shown as mean ± standard error of the mean (n = 3). **P < 0.01 vs the control (Con) group; ‡ P < 0.01 vs the HG‐treated group.
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