Boosting Akt Pathway by Rupatadine Modulates Th17/Tregs Balance for Attenuation of Isoproterenol-Induced Heart Failure in Rats

Abstract

Disruption of Th17/Tregs homeostasis plays a crucial role in governing the immune response during myocardial fibrosis and its progression to heart failure. The present study aimed to assess for the first time the possible protection afforded by rupatadine against isoproterenol-induced heart failure in rats. It also explored the role of PI3k/Akt as a possible mechanistic pathway, through which rupatadine could modulate Th17/Tregs balance to display its effect. Isoproterenol (85 and 170 mg/kg/day) was injected subcutaneously for 2 successive days, respectively and rupatadine (4 mg/kg/day) was then given orally for 14 days with or without wortmannin (PI3K/Akt inhibitor). Rupatadine succeeded to completely ameliorate isoproterenol-induced cardiac dysfunction as demonstrated by improvements of electrocardiographic and echocardiographic measurements. Moreover, rupatadine prevented the marked elevation of PAF and oxidative stress in addition to Th17 promoting cytokines (IL-6, IL-23, and TGF-β). Accordingly, rupatadine prevented Th17 stimulation or expansion as indicated by increased Foxp3/RORγt ratio and decreased production of its pro-inflammatory cytokine (IL-17). Rupatadine treatment mitigated isoproterenol-induced activation of STAT-3 signaling and the imbalance in p-Akt/total Akt ratio affording marked decrease in atrogin-1 and apoptotic biomarkers. Finally, this therapy was effective in averting cardiac troponin loss and reverting the histological alterations as assessed by myocardial fibrosis and hypertrophy grading. Contrariwise, co-administration of wortmannin mostly attenuated the protective effects of rupatadine affording more or less similar results to that of isoproterenol-untreated rats. In conclusion, rupatadine could be an effective therapy against the development of isoproterenol-induced heart failure where PI3K/Akt pathway seems to play a crucial role in its protective effect.

Keywords: Akt; T helper 17; myocardial fibrosis; platelet activating factor; rupatadine.

FIGURE 1

Effect of RUP with or without wortmannin on ISO-induced changes in myocardial contents of (A) PAF (B) IL-6 (C) IL-23, and (D) TGF-β. Each value represents the mean of six experiments and error bars represent SD. Statistical analysis was done using One way ANOVA followed by Tukey’s post-hoc test where a p < 0.05 vs. normal, b p < 0.05 vs. ISO, c p < 0.05 vs. RUP.

FIGURE 2

Effect of RUP with or without wortmannin on ISO-induced changes in myocardial contents of (A) TBARS (B) GSH (C) SOD and (D) catalase. Each value represents the mean of six experiments and error bars represent SD. Statistical analysis was done using One way ANOVA followed by Tukey’s post-hoc test where a p < 0.05 vs. normal, b p < 0.05 vs. ISO, c p < 0.05 vs. RUP.

FIGURE 3

Effect of RUP with or without wortmannin on ISO-induced changes in protein expression of (A) Foxp3 and (B) RORγt in addition to (C) Foxp3/RORγt ratio and myocardial content of (D) IL-17. Each value represents the mean of six experiments and error bars represent SD. Statistical analysis was done using One way ANOVA followed by Tukey’s post-hoc test where a p < 0.05 vs. normal, b p < 0.05 vs. ISO, c p < 0.05 vs. RUP.

FIGURE 4

Effect of RUP with or without wortmannin on ISO-induced changes in myocardial contents of (A) p-STAT 3 and (B) pAkt/total Akt ratio. Each value represents the mean of six experiments and error bars represent SD. Statistical analysis was done using One way ANOVA followed by Tukey’s post-hoc test where a p < 0.05 vs. normal, b p < 0.05 vs. ISO, c p < 0.05 vs. RUP.

FIGURE 5

Effect of RUP with or without wortmannin on ISO-induced changes in myocardial content of (A) atrogin-1 as well as the protein expression of (B) troponin I and (C) troponin T. Each value represents the mean of six experiments and error bars represent SD. Statistical analysis was done using One way ANOVA followed by Tukey’s post-hoc test where a p <0.05 vs. normal, b p <0.05 vs. ISO, c p <0.05 vs. RUP.

FIGURE 6

Effect of RUP with or without wortmannin on ISO-induced changes in myocardial contents of (A) Bax and (B) Bcl2 in addition to (C) Bax/Bcl2 ratio and (D) caspase-3 activity. Each value represents the mean of six experiments and error bars represent SD. Statistical analysis was done using One way ANOVA followed by Tukey’s post-hoc test where a p < 0.05 vs. normal, b p < 0.05 vs. ISO, c p < 0.05 vs. RUP.

FIGURE 7

Effect of RUP with or without wortmannin on ISO-induced histological changes. Normal group (A,F,K), ISO group (B,G,L), RUP group (C,H,M) and RUP + Wort group (D,I,N). (A–D) Specimens stained with Masson’s trichrome for estimation of myocardial fibrosis (blue color). Specimens stained with hematoxylin and eosin for estimation of degree of myocardial damage (F–I) and hypertrophy (K–N). Percentage of fibrosis (E) and left ventricular (LV) thickness (O). Each value represents the mean of 5 experiments and error bars represent SD. Statistical analysis was done using One way ANOVA followed by Tukey’s post-hoc test. Histopathological scores (J) are expressed as the median of 5 animals. Statistical analysis was done using Kruskal-Wallis test followed by Dunn’s test where a p < 0.05 vs. normal, b p < 0.05 vs. ISO, c p < 0.05 vs. RUP.