Beneficial In Vitro Effects of a Low Myo-Inositol Dose in the Regulation of Vascular Resistance and Protein Peroxidation under Inflammatory Conditions

Abstract

Oxidative stress induces functional changes in arteries. Therefore, the effect of myo-inositol, a possible anti-inflammatory/antioxidant agent was studied on human plasma and rat thoracic arteries. Aortic rings from male Wistar rats (3 months of age) were incubated with myo-inositol (1, 10 and 100 μM, 120 min) and analyzed using the gas chromatography (GC) method. In another experiment, aortic rings were protected first with myo-inositol (1 µM, 60 min) and then subjected to a thromboxane receptor agonist (U-46619, 0.1 nM, 60 min). Therefore, these four groups under the following conditions were studied: (i) the control in the vehicle; (ii) myo-inositol; (iii) the vehicle plus U-46619; (iv) myo-inositol plus U-46619. The hemostatic parameters of human plasma and an H2O2/Fe2+ challenge for lipid and protein peroxidation were also performed. Myo-inositol was not absorbed into the pre-incubated aortic rings as measured by the GC method (0.040 µg/mg, p ≥ 0.8688). The effect of myo-inositol was more significant in the impaired arteries due to U-46619 incubation, which resulted in an improved response to acetylcholine (% Emax: 58.47 vs. 86.69), sodium nitroprusside (logEC50: −7.478 vs. −8.076), CORM-2 (% Emax: 44.08 vs. 83.29), pinacidil (logEC50: −6.489 vs. −6.988) and noradrenaline (logEC50: −7.264 vs. −6.525). This was most likely a possible response to increased nitric oxide release (×2.6-fold, p < 0001), and decreased hydrogen peroxide production (×0.7-fold, p = 0.0012). KCl-induced membrane depolarization was not modified (p ≥ 0.4768). Both the plasma protein carbonylation (×0.7-fold, p = 0.0006), and the level of thiol groups (×3.2-fold, p = 0.0462) were also improved, which was not significant for TBARS (×0.8-fold, p = 0.0872). The hemostatic parameters were also not modified (p ≥ 0.8171). A protective effect of myo-inositol was demonstrated against prooxidant damage to human plasma and rat thoracic arteries, suggesting a strong role of this nutraceutical agent on vasculature which may be of benefit against harmful environmental effects.

Keywords: U-46619; acetylcholine; hydrogen peroxide; myo-inositol; nitric oxide; pinacidil; reactive oxygen species; thromboxane A2.

Figure 1

Presence of myo-inositol (0.040 ± 0.002 µg/mg) in aortic rings (12.55 ± 2.065 mg) analyzed with a gas chromatograph with flame-ionization detector. Control—black line; samples after pre-incubation in myo-inositol solutions (120 min): 1 μM—red line, 10 μM—blue line, 100 μM—green line.

Figure 2

The cumulative concentration-response curves to acetylcholine (A), sodium nitroprusside (B), carbon monoxide releasing molecule (C), and pinacidil (D) in the isolated thoracic arteries of the control (a), myo-inositol only (b), vehicle plus U-46619 (c), myo-inositol plus U-46619 (d). Aortic rings were pre-incubated with either myo-inositol (1 µM, 60 min) for groups b and d or vehicle (KHS)—groups a and c. Next, aortic rings were subjected to U-46619 (0.1 nM, 60 min)—groups c and d. Results (means ± SEM, n = 6) are expressed as a percentage of inhibition of the contraction induced by noradrenaline (0.1 μM), p < 0.05, two-way ANOVA with Tukey’s multiple comparisons test. Abbreviations: ACh, acetylcholine; SNP, sodium nitroprusside; CORM-2, carbon monoxide releasing molecule.

Figure 3

The cumulative concentration-response curves to noradrenaline (A,B) and 75 mM KCl (C) in the isolated thoracic arteries of the control (a), myo-inositol alone (b), vehicle plus U-46619 (c), myo-inositol plus U-46619 (d). Aortic rings were pre-incubated with either myo-inositol (1 µM, 60 min)—groups b and d or vehicle (KHS)—groups a and c. Next, aortic rings were subjected to U-46619 (0.1 nM, 60 min)—groups c and d. Contraction to KCl was performed before and after the 2-h pre-incubation period, (1°) or (2°), respectively. Results (means ± SEM, n = 6) are expressed as a percentage of the contraction induced by KCl (75 mM) and as mg of developed tension, p < 0.05, two-way ANOVA with Tukey’s multiple comparisons test.

Figure 4

The effect of pre-incubation with myo-inositol (1.0 µM) on the basal release of NO in control arteries and arteries subjected to U-46619. Results (means ± SD) are expressed as arbitrary units of fluorescence (AU) per milligram of tissue. Number of animals: n = 6–8.

Figure 5

The effect of pre-incubation with myo-inositol (1.0 µM) on the production of hydrogen peroxide (H₂O₂) in control arteries and arteries subjected to U-46619. Results (means ± SD) are expressed as nanomoles of H₂O₂ per microgram of protein. Number of animals: n = 6–8.

Figure 6

The effect of myo-inositol (1.0 µM, pre-incubation time—5 min, 37 °C) on the lipid peroxidation (A), and the oxidative damages of blood plasma protein measured as protein carbonylation (B), and the level of thiol groups (C) treated with H₂O₂/Fe²⁺ (incubation time—25 min, 37 °C). Results are given as means ± SD (n = 9). Control negative refers to the plasma not treated with H₂O₂/Fe²⁺, whereas control positive refers to the plasma treated with H₂O₂/Fe²⁺. One-way ANOVA followed by a Dunnett’s multiple comparisons test.

Figure 7

The effect of myo-inositol (1.0 µM, incubation time—30 min, 37 °C) on the hemostatic parameters: the prothrombin time (A), the thrombin time (B), and the activated partial thromboplastin time (C) of human plasma. Data are expressed as means ± SD (n = 6). Unpaired t test for TT and APTT, and Mann-Whitney test for PT. Abbreviations: APTT, activated partial thromboplastin time; PT, prothrombin time; TT, thrombin time.