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, 13 (10), e0202297
eCollection

AT1-receptor Blockade: Protective Effects of Irbesartan in Cardiomyocytes Under Hypoxic Stress

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AT1-receptor Blockade: Protective Effects of Irbesartan in Cardiomyocytes Under Hypoxic Stress

Mariarosaria Boccellino et al. PLoS One.

Abstract

Hypoxia induces myocardial injury through the activation of inflammatory and oxidative processes. The pivotal role of the renin angiotensin system (RAS) in the pathogenesis of cardiovascular diseases has been firmly established in clinical trials and practice; in fact many experimental and clinical data have highlighted that its inhibition has a cardioprotective role. Activated RAS also stimulates inflammation directly inducing proinflammatory and oxidative gene expression. This study aimed to investigate the protective role of a pre-treatment (10 and 100 μM) with irbesartan on injury induced by 24 h of hypoxia in HL-1 cardiomyocytes; in particular, we have analyzed the natriuretic peptide (BNP) expression, a biomarker able to modulate inflammatory reaction to cardiac injury and some markers involved in oxidative stress and inflammation. Our results demonstrated that a pre-treatment with 100 μM irbesartan significantly increased SOD activity and catalase expression of 15 and 25%, respectively, compared to hypoxic cells (P<0.05). On the other hand, it was able to reduce the release of peroxynitrite and iNOS protein expression of 20 and 50% respectively (P<0.05). In addition irbesartan exerts an anti-inflammatory activity reducing Toll-like receptors (TLRs)-2 and -4 mRNA expression, TNF-alpha expression and activity (20%) and increasing the expression of the cytokine IL-17 (40%) (P<0.05 vs hypoxia). Our findings also showed that BNP induced by ischemia was significantly and in a concentration-dependent manner reduced by irbesartan. The findings of our study demonstrated that the AT1 receptor antagonist irbesartan exerts a protective role in an in vitro hypoxic condition reducing oxidative stress and inflammation.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Effect of irbesartan on cell viability and on oxidative stress in HL-1 hypoxic cells.
(A) Cardiomyocytes viability was determined by MTT assay in normoxia (CTRL), hypoxia alone (Hypoxia) and in combination with a pre-treatment or post-treatment with 16 h of irbesartan (Irb 10, 50, 100 μM). (B) SOD activity was determined by an ELISA assay. (C) Catalase protein expression was measured by western blot. All data are presented as mean ± S.E.M. of three independent experiments (*P<0.05 versus CTRL; #P<0.05 versus hypoxia, § P<0.05 versus irbesartan 10 μM).
Fig 2
Fig 2. Effect of irbesartan pre-treatment on oxidative stress, on cardiac marker BNP and on TLRs mRNA expression.
(A) The release of peroxynitrite was measured by Griess assay. (B) mRNA levels BNP was evaluated by RT-qPCR. (C) Western blot analysis of BNP protein, blot is representative from three independent experiments. (D) TLR2 and (E) TLR4 mRNA expressions were measured by RT-qPCR. TLR2 an TLR4 mRNA levels were normalized relative to GAPDH mRNA levels. Data represent the mean ± S.E.M. of three independent experiments (*P<0.05 versus CTRL; #P<0.05 versus hypoxia, § P<0.05 versus irbesartan 10 μM).
Fig 3
Fig 3. Role of irbesartan pre-treatment on inflammatory mediators and on cytokines activity.
(A) iNOS TNF-alpha and IL-17 proteins expressions were measured by western blot. GAPDH was used as internal standard. Representative blot from three independent experiments. Data are expressed as relative densitometric units and represent the mean ± S.E.M. of three independent experiments (*P<0.05 versus CTRL; #P<0.05 versus hypoxia, § P<0.05 versus irbesartan 10 μM). (B) TNF-alpha and (C) IL-17 activity was measured by ELISA. Final concentration of each cytokine was expressed as pg/mg and represent the mean ± S.E.M. of three independent experiments (*P<0.05 versus CTRL; #P<0.05 versus hypoxia).

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Grant support

The authors received no specific funding for this work.
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