Effects of Taxifolin in Spontaneously Hypertensive Rats with a Focus on Erythrocyte Quality

Abstract

Oxidative stress and multiple erythrocyte abnormalities have been observed in hypertension. We focused on the effects of angiotensin-converting enzyme 2 (ACE2) inhibition by MLN-4760 inhibitor on angiotensin peptides, oxidative stress parameters, and selected erythrocyte quality markers in spontaneously hypertensive rats (SHR). We also investigated the potential effects of polyphenolic antioxidant taxifolin when applied in vivo and in vitro following its incubation with erythrocytes. SHRs were divided into four groups: control, taxifolin-treated, MLN-4760-treated, and MLN-4760 with taxifolin. MLN-4760 administration increased the blood pressure rise independent of taxifolin treatment, whereas taxifolin decreased it in control SHRs. Body weight gain was also higher in ACE2-inhibited animals and normalized after taxifolin treatment. However, taxifolin did not induce any change in angiotensin peptide concentrations nor a clear antioxidant effect. We documented an increase in Na,K-ATPase enzyme activity in erythrocyte membranes of ACE2-inhibited SHRs after taxifolin treatment. In conclusion, ACE2 inhibition deteriorated some selected RBC properties in SHRs. Although taxifolin treatment did not improve oxidative stress markers, our data confirmed the blood pressure-lowering potential, anti-obesogenic effect, and some "erythroprotective" effects of this compound in both control and ACE2-inhibited SHRs. In vitro investigations documenting different effects of taxifolin on erythrocyte properties from control and ACE2-inhibited SHRs accentuated the irreplaceability of in vivo studies.

Keywords: ACE2; Na,K-ATPase; erythrocytes; hypertension; nitric oxide; osmotic resistance; oxidative stress; renin–angiotensin system; taxifolin.

Figure 1

Experimental design. Abbreviations: C—control group of spontaneously hypertensive rats (SHRs), T—taxifolin-treated SHRs, M—MLN-treated (MLN-4760, ACE2 inhibitor) SHRs, MT—MLN and taxifolin-treated SHRs, BP—systolic blood pressure, BW—body weight.

Figure 2

Erythrocyte parameters MCV (a), RDW-SD (b). Abbreviations: MCV—mean cell volume, RDW-SD—red cell distribution width, C—control group (n = 11–12), T—taxifolin-treated group (n = 8), M—MLN-treated (MLN-4760, ACE2 inhibitor) group (n = 12), MT—MLN- and taxifolin treated group (n = 12). Data are presented as mean ± standard deviations. Statistical significance: * p < 0.05.

Figure 3

Erythrocyte deformability (a), osmotic resistance (IC₅₀) (b), nitric oxide produced by erythrocytes (c), and free radical production in erythrocytes (d). Abbreviations: C—control group (n = 11–12), T—taxifolin-treated group (n = 7–8), M—MLN-treated (MLN-4760, ACE2 inhibitor) group (n = 12), MT—MLN- and taxifolin-treated group (n = 10–12), IC₅₀–NaCl concentration at which 50% hemolysis occurred, a.u.–arbitrary units. Data are presented as mean ± standard deviations. Statistical significance: ** p < 0.01.

Figure 4

Activation of the Na,K-ATPase in Na⁺ concentrations ranging from 2 to 10 mmol/L; insert—activation of the enzyme in the whole investigated concentration range of NaCl (a), kinetic parameters of Na,K-ATPase V_max (b) and K_Na (c) in erythrocyte membranes. Abbreviations: C—control group (n = 15), T—TAX-treated group (n = 9), M—MLN-treated (MLN-4760, ACE2 inhibitor) group (n = 18), MT—TAX and MLN-treated group (n = 15). V_max—maximal velocity of reaction. K_Na—NaCl concentration required for ½ maximal activation of Na,K-ATPase, data are presented as means ± standard deviations. Statistical significance: * p < 0.05 versus C group, **** p < 0.0001 versus M group.

Figure 5

Representative photographs of erythrocyte morphology. Control group (a), MLN-4760-treated group (ACE2 inhibition) (b), MLN-4760 and taxifolin-treated group (c).

Figure 6

Erythrocyte deformability (a), osmotic resistance (IC50) (b), nitric oxide produced by erythrocytes (c), and free radical production in erythrocytes (d) after in vitro taxifolin treatment. Abbreviations: iC—erythrocytes of control rats incubated with vehicle, iCT—erythrocytes of control rats incubated with taxifolin, iM—erythrocytes of MLN-treated rats (MLN-4760, ACE2 inhibitor) incubated with vehicle, iMT—erythrocytes of MLN-treated rats incubated with taxifolin, a.u.—arbitrary units. Data are presented as means ± standard deviations. Statistical significance: * p < 0.05. The count of samples per group: n = 6 for RBC deformability; n = 11–12 for osmotic resistance; iC and iCT n = 5–6, iM and iMT n = 9–10 for other parameters.