Amelioration of cardiac function and activation of anti-inflammatory vasoactive peptides expression in the rat myocardium by low level laser therapy

Abstract

Low-level laser therapy (LLLT) has been used as an anti-inflammatory treatment in several disease conditions, even when inflammation is a secondary consequence, such as in myocardial infarction (MI). However, the mechanism by which LLLT is able to protect the remaining myocardium remains unclear. The present study tested the hypothesis that LLLT reduces inflammation after acute MI in female rats and ameliorates cardiac function. The potential participation of the Renin-Angiotensin System (RAS) and Kallikrein-Kinin System (KKS) vasoactive peptides was also evaluated. LLLT treatment effectively reduced MI size, attenuated the systolic dysfunction after MI, and decreased the myocardial mRNA expression of interleukin-1 beta and interleukin-6 in comparison to the non-irradiated rat tissue. In addition, LLLT treatment increased protein and mRNA levels of the Mas receptor, the mRNA expression of kinin B2 receptors and the circulating levels of plasma kallikrein compared to non-treated post-MI rats. On the other hand, the kinin B1 receptor mRNA expression decreased after LLLT. No significant changes were found in the expression of vascular endothelial growth factor (VEGF) in the myocardial remote area between laser-irradiated and non-irradiated post-MI rats. Capillaries density also remained similar between these two experimental groups. The mRNA expression of the inducible nitric oxide synthase (iNOS) was increased three days after MI, however, this effect was blunted by LLLT. Moreover, endothelial NOS mRNA content increased after LLLT. Plasma nitric oxide metabolites (NOx) concentration was increased three days after MI in non-treated rats and increased even further by LLLT treatment. Our data suggest that LLLT diminishes the acute inflammation in the myocardium, reduces infarct size and attenuates left ventricle dysfunction post-MI and increases vasoactive peptides expression and nitric oxide (NO) generation.

Figure 1. Myocardial infarction size after LLLT.

(A) Size of the infarct as a percentage of left ventricular (LV) perimeter in infarcted rats (MI) and infarcted rats treated with laser (MI+Laser) after three days of descending left coronary artery occlusion. (B) Frequency of large (□) or small plus mild (▪) infarctions (p value = 0.004, Chi-squared test).

Figure 2. mRNA expression of interleukins 1 beta (IL-1beta) and 6 (IL-6) on myocardial tissue after LLLT.

Myocardial infarction remote area presented an acute inflammation of the myocardium, as observed as an increased of cytokines IL-1beta and IL-6 mRNA in the left ventricles (LV) of MI rats (n = 28). A downregulation of IL-1beta (A) and IL-6 (B) mRNA expression was detected in the group MI+Laser (n = 30), suggesting an anti-inflammatory effect of LLLT. Control group was composed of 14 rats. Data are means ± S.E.M. *p<0.05 vs. Control group; ^#p<0.05 vs. MI group. P values given were determined by ANOVA.

Figure 3. Effects of LLLT on kinins receptors mRNA expression on myocardial tissue and circulating levels of plasma kallikrein.

Distinct kinin receptors mRNA modulation was observed in the remote area of MI. An increased kinin B2 receptor mRNA expression (A) and a diminished kinin B1 mRNA expression (B) was observed in the left ventricles (LV) of MI+Laser rats (n = 30). Control and MI groups were composed of 14 and 28 rats, respectively. Increased circulating levels of plasma kallikrein (C) were detected after LLLT. Data are means ± S.E.M. *p<0.05 vs. Control group; ^#p<0.05 vs. MI group. P values given were determined by ANOVA.

Figure 4. Quantification of ACE and ACE2 mRNA, and Mas receptor mRNA and protein on myocardial tissue.

An up-regulation of ACE mRNA (A) was detected after MI. This effect was blunted by LLLT. ACE2 mRNA (B) was strongly expressed in in the LV of MI+Laser (n = 30). Control and MI groups were composed of 14 and 28 rats, respectively. An up-regulation of Mas gene (C) and protein (D) expression was detected in MI+Laser rats. Data are means ± S.E.M. *p<0.05 vs. Control group; ^#p<0.05 vs. MI group. P values given were determined by ANOVA.

Figure 5. Histological changes and evaluation of capillaries density and VEGF levels of the remote myocardium after LLLT.

Representative microphotographs of the remote myocardium stained with (A) Periodic acid-Schiff for capillary counting (400× original magnification, arrows indicate some capillaries). Parameters including capillarity (B), and myocardial VEGF levels (C) were evaluated in remote myocardial samples from experimental groups. Control, MI and MI+Laser groups were composed of 7, 12 and 14 rats, respectively for each evaluation. Data are means ± SEM. *p<0.05 vs. Control group. P values given were determined by ANOVA.

Figure 6. Analyses of iNOS and eNOS mRNA content on myocardial tissue and serum total nitrites concentration.

A diminished iNOS mRNA expression (A) was observed after LLLT. Increased myocardial eNOS mRNA content (B) and plasma NO metabolites (C) were found after LLLT. Control, MI and MI+Laser groups were composed of 14, 28 and 30 rats, respectively. Data are means ± SEM. *p<0.05 vs. Control group; ^#p<0.05 vs. MI group. P values given were determined by ANOVA.