Protective effects of the R-(+)-thioctic acid treatment: possible anti-inflammatory activity on heart of hypertensive rats

Abstract

Background: In cardiovascular disease, high blood pressure is associated with oxidative stress, promoting endothelial dysfunction, vascular remodeling, and inflammation. Clinical trials are discordant that the most effective treatment in the management of hypertension seems to be the administration of anti-hypertensive drugs with antioxidant properties. The study aims to evaluate the effects of the eutomer of thioctic acid on oxidative stress and inflammation in the heart of spontaneously hypertensive rats compared to normotensive Wistar Kyoto rats.

Methods: To study the oxidative status, the malondialdehyde and 4-hydroxynonenal concentration, protein oxidation were measured in the heart. Morphological analysis were performed. Immunohistochemistry and Western blot were done for alpha-smooth muscle actin and transforming growth factor beta to assess fibrosis; cytokines and nuclear factor kappaB to assess inflammatory processes.

Results: Spontaneously hypertensive rats were characterized by hypertension with increased malondialdehyde levels in the heart. OxyBlot in the heart of spontaneously hypertensive rats showed an increase in proteins' oxidative status. Cardiomyocyte hypertrophy and fibrosis in the ventricles were associated with an increased expression of alpha-smooth muscle actin and pro-inflammatory cytokines, reduced by the eutomer of thioctic acid supplementation.

Conclusions: Based on this evidence, eutomer of thioctic acid could represent an appropriate antioxidant molecule to reduce oxidative stress and prevent inflammatory processes on the cardiomyocytes and cardiac vascular endothelium.

Keywords: Heart; Hypertension; Inflammation; Oxidative stress; Thioctic acid.

Fig. 1

Blood pressure modulation. Systolic blood pressure values in normotensive Wistar Kyoto rats (WKY), spontaneously hypertensive rats (SHR), and SHR treated with (+)-thioctic acid lysine salt [SHR (+)-TIO]. Data, expressed in mmHg, are the mean ± S.D. (n = 8/group) *= p < 0.05 vs. WKY: #= p < 0.05 vs. SHR

Fig. 2

Parameters of oxidative stress in the heart parenchyma. Samples of the heart of normotensive Wistar Kyoto rats (WKY), spontaneously hypertensive rats (SHR), and SHR treated with (+)-thioctic acid lysine salt [SHR (+)-TIO] were immunoblotted with OxyBlot (A) and with specific anti-4-hydroxynonenal (4-HNE) antibody (B). For the OxyBlot analysis, the bar graph reports the values of optical density measured in the optical density unit (ODU). 4-HNE bar graph indicates the ratio of densitometric analysis of bands to β-actin levels used as the reference loading control. (C) Concentration of malondialdehyde (MDA) expressed in pmol/mg of tissue. Data are the mean ± S.D. = p < 0.05 vs. WKY: #= p < 0.05 vs. SHR

Fig. 3

Fibrosis in the heart parenchyma. Cardiac sub-endocardium parenchyma in heart tissue of normotensive Wistar Kyoto rats (WKY), spontaneously hypertensive rats (SHR), and SHR treated with (+)-thioctic acid lysine salt [SHR(+)-TIO] were staining with Masson’s Trichrome technique for connective tissue, with the Sirius red technique to reveal the collagen fibers and with silver impregnation histochemistry to highlighted reticulin fibers. The accumulation of connective tissue fibers was indicated with the black arrowheads. Magnification 20×. Scale bar: 50 μm

Fig. 4

Fibrosis was related to the increase of the alpha-smooth muscle actin (alpha-SMA). Lysates of the heart from normotensive Wistar Kyoto rats (WKY), spontaneously hypertensive rats (SHR), and SHR treated with (+)-thioctic acid lysine salt [SHR(+)-TIO] were immunoblotted using specific antibodies against alpha-SMA (A) and transforming growth factor-beta 1 (TGF-beta 1) (B). Values indicate the ratio of densitometric analysis of bands and β-actin levels used as the reference loading control. Data are mean ± S.D. *= p < 0.05 vs. WKY; #=p < 0.05 vs. SHR. Blots are representative of each experimental group

Fig. 5

Modulation of inflammatory markers. Lysates of the heart from normotensive Wistar Kyoto rats (WKY), spontaneously hypertensive rats (SHR), and SHR treated with (+)-thioctic acid lysine salt [SHR (+)-TIO] were immunoblotted with specific antibodies against (A) interleukin 1 beta (IL-1 beta), (B) interleukin-6 (IL-6) and (C) tumor necrosis factor-alpha (TNF-alpha). Graphs values indicate the ratio of densitometric analysis of bands to β-actin levels used as the reference loading control. Data are mean ± S.D. *= p < 0.05 vs. WKY; #=p < 0.05 vs. SHR. Blots are representative of each experimental group

Fig. 6

Up-regulation of inflammatory cytokines. Sections of the heart of normotensive Wistar Kyoto rats (WKY), spontaneously hypertensive rats (SHR), and SHR treated with thioctic acid lysine salt [SHR (+)-TIO] processed for the confocal immunofluorescence of (A) interleukin-1 beta (IL-1 beta), (B) interleukin-6 (IL-6) and (C) tumor necrosis factor-alpha (TNF-alpha). The graphs showed the values of mean fluorescence intensity (MFI). The immunoreactive cardiomyocytes are indicated with the arrowheads. Data are mean ± S.D. *= p < 0.05 vs. WKY; #=p < 0.05 vs. SHR. Magnification 40×, zoom 2. Scale bar: 10 μm. NC, negative control

Fig. 7

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) p50 modulated the inflammatory process. Lysates of the heart from normotensive Wistar Kyoto rats (WKY), spontaneously hypertensive rats (SHR), and SHR treated with thioctic acid lysine salt [SHR(+)-TIO] were immunoblotted with specific antibodies (A) NF-kB; Values indicate the ratio of densitometric analysis of bands to β actin levels used as the loading control. Data are mean ± S.D. *= p < 0.05 vs. WKY; #=p < 0.05 vs. SHR. Blots are representative of each experimental group. Sections of the heart (B) of WKY, SHR, [SHR (+)-TIO] processed for the immunohistochemistry of NF-kB p50 and quantification in optical density unit (ODU). The immunoreactive cardiomyocytes are indicated with the arrowheads. Magnification 20×. Scale bar: 50 μm