Nanoformulation of valsartan-loaded tablet attenuates L-NAME-induced hypertension: role of Nrf2/PPARγ/AT1 signaling pathway

Abstract

Hypertension is the most common entity globally, marked by high prevalence and heterogeneous pathophysiology. Oxidative stress is a crucial area of investigation among potential etiologies. We examined the hypothesis that blocking the angiotensin type 1 (AT1) receptor with valsartan (VST) in self-nanoemulsifying delivery systems (SNEDS) and loads in liquisolid tablets (LST-1) or valsartan and hydrochlorothiazide (VST/HCTZ) in SNEDS and loads in liquisolid tablets (LST-2) in comparison with non-SNEDS liquisolid tablets (DCT-3 and DCT-4) would lead to an improvement in hypertension management. The present study aims to explore the molecular mechanisms underlying their effect in N(G)-nitro-L-arginine methyl ester (L-NAME)-induced hypertensive rats. Male Sprague-Dawley rats were given L-NAME (40 mg/kg/day) orally for three weeks to inhibit the endogenous synthesis of nitric oxide (NO). Concurrent treatment with VST or VST/HCTZ liquisolid tablets (20 mg/kg/day for three weeks) resulted in lowering blood pressure (BP), reversing the L-NAME-induced serum NO suppression, enhancing lipid profile, and improving oxidative status. The antioxidant defense of paraoxonase was significantly increased in the LST-1- and LST-2-treated rats compared to the L-NAME-treated rats by 135% and 90%, respectively. Furthermore, SNEDS-loaded VST or SNEDS-loaded VST/HCTZ liquisolid tablets significantly lowered the elevated level of AT1 (P < 0.05), showed a marked Nrf2 expression (P < 0.01) and overexpressed PPARγ (P < 0.05), and suppressed iNOS expression (P < 0.0001). These results highlight the remarkable benefits of the novel formula, "SNEDS-loaded VST and SNEDS-loaded VST/HCTZ," as an alternative therapy in treating hypertension and its complications.

Keywords: Hypertension; L-NAME; SNEDS; Valsartan.

Fig. 1

Characterization of SNEDS-loaded VST and SNEDS-loaded VST/HCTZ: A particle size and B zeta potential

Fig. 2

SNEDS-loaded VST and SNEDS-loaded VST/HCTZ reduce blood pressure: A study design. B Bar graph represents the blood pressure. The administration of formulations LST-1, LST-2, DCT-3, or DCT-4 substantially reduces blood pressure. LST-2 reduced BP significantly compared to DCT-4. ^****P < 0.0001, ^@@@@P < 0.0001 (ANOVA)

Fig. 3

Histopathology of the heart in L-NAME-induced hypertensive rats. Microscopic examination of hearts from the control group showed normal myocardium without any detectable alterations. L-NAME-induced hypertension group revealed diffuse myocarditis manifested by mononuclear inflammatory cell infiltration (black arrow). Normal myocardium was detected in LST-1 and LST-2. Mild mononuclear inflammatory cell infiltration in between myocardial fibers and small focal aggregation of mononuclear inflammatory cells were shown in DCT-3 and DCT-4, respectively (black arrows)

Fig. 4

Cardiac RAS mRNA expressions: L-NAME upregulated the mRNA expressions of A AT1 R, B AT2 R, C ACE, and D ACE2. The administration of formulations LST-1, LST-2, DCT-3, or DCT-4 reduced the expression of AT1 R, AT2 R, ACE, and ACE2. E There are no significant differences in the Mas1l mRNA expression among groups. There are 6–8 rats per group. ^*P < 0.05, ^**P < 0.01, ^****P < 0.0001 (ANOVA)

Fig. 5

Oxidative stress in L-NAME-induced hypertensive rats after administration of SNEDS-loaded VST and SNEDS-loaded VST/HCTZ. A MDA levels in the aorta tissue homogenates were increased in the L-NAME-treated group versus the control group. LST-1 administration reduced the elevated MDA level. B The protein expression of NOX2 was upregulated in the L-NAME-treated group versus the control group and downregulated in all formulations treated rats. Signals were quantified by densitometry. ^****P < 0.0001 L-NAME-treated group versus control; ^****P < 0.0001 LST-1-, LST-2-, DCT-3-, and DCT-4-treated groups compared to the L-NAME-treated group. C NO level in aorta tissue homogenates was depleted in the L-NAME-treated group versus the control group. LST-1- and LST-2-treated groups increased NO levels compared to the L-NAME-treated group. There are no significant differences in DCT-3- and DCT-4-treated groups compared to the L-NAME-treated group. ^*P < 0.05 (ANOVA). D Paraoxonase serum level was significantly reduced in the L-NAME-treated group versus control. The LST-1-, LST-2-, and DCT-3-treated rats significantly increased paraoxonase serum levels compared to the L-NAME-treated group. ^***P < 0.001, ^****P < 0.0001, ^$P < 0.05 LST-1 versus DCT-3, ^@P < 0.05 LST-2 versus DCT-4 (ANOVA). There are 6–8 mice per group

Fig. 6

Lipid profile in L-NAME-induced hypertensive rats after SNEDS-loaded VST and SNEDS-loaded VST/HCTZ administration. A levels of triglycerides significantly decreased in LST-1-, LST-2-, DCT-3-, and DCT-4-treated groups compared to L-NAME-treated rats. ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001, ^@@P < 0.01 LST-2 versus DCT-4. B Compared to L-NAME-treated rats, total cholesterol levels significantly decreased in LST-1-, LST-2-, DCT-3-, and DCT-4-treated groups. ^****P < 0.0001, ^$P < 0.01 LST-1 versus DCT-3, ^@P < 0.01 LST-2 versus DCT-4. C HDL levels were increased in both formulations: LST-1 and LST-2. DCT-3 showed a moderate increase, and DCT-4 showed no change compared to L-NAME-treated rats. ^*P < 0.05, ^****P < 0.0001, ^$$P < 0.01 LST-1 versus DCT-3, ^@@P < 0.01 LST-2 versus DCT-4 (ANOVA). There are 6–8 mice per group

Fig. 7

Liver marker enzyme activity (ALT and AST): (A) LST-1 and LST-2 lowered the ALT significantly versus L-NAME-induced hypertensive rats. *P < 0.05, **P < 0.01 (ANOVA). Both DCT-3 and DCT-4 tended to reduce the ALT. (B) All formulations did not significantly alter the AST level compared to the L-NAME-treated group. There are 6-8 mice per group

Fig. 8

Downregulation of Nrf2 and PPARs gene expressions in L-NAME-induced hypertensive rats. A The protein expression of PPARγ was downregulated in the L-NAME-treated group versus the control group and upregulated in LST-1 and LST-2 formulations-treated rats. Signals were quantified by densitometry. ^*P < 0.05 LST-1 versus L-NAME-treated group and LST-2 versus L-NAME-treated group; ^$P < 0.05 LST-1 compared to DCT-3; ^@P < 0.05 LST-2 compared to DCT-4-treated group. B mRNA expression of Nrf2 was significantly downregulated in the L-NAME-treated group versus the control group and upregulated in LST-1 and LST-2 formulations-treated rats. ^*P < 0.05 L-NAME-treated group versus control; ^**P < 0.01 LST-1 versus L-NAME-treated group and LST-2 versus L-NAME-treated group; ^*P < 0.05 DCT-4 versus L-NAME-treated group; ^$P < 0.05 LST-1 compared to DCT-3 (ANOVA). C The protein expression of pP38 was upregulated in the L-NAME-treated group versus the control group and significantly downregulated in LST-1-, LST-2-, and DCT-3-treated rats. Signals were quantified by densitometry. ^*P < 0.05 L-NAME-treated group versus control and LST-1 versus L-NAME-treated group; ^**P < 0.01 LST-1 compared to L-NAME-treated group; ^*P < 0.05 DCT-3 compared to L-NAME-treated group. D The protein expressions of pERK (p42/44) and PPARα were upregulated in the L-NAME-treated group versus the control group and downregulated in all formulations-treated rats. Signals for pERK (p42/44) expressions were quantified by densitometry. ^**P < 0.01 L-NAME-treated group versus control and DCT-3 versus L-NAME-treated group; ^****P < 0.0001 LST-1 compared to L-NAME-treated group; ^***P < 0.001 LST-2 compared to L-NAME-treated group; ^*P < 0.05 DCT-4 compared to L-NAME-treated group. There are 6–8 mice per group

Fig. 9

Photomicrographs of the heart immunostained with iNOS (× 200). A The iNOS immunoreactivity was characteristically cytoplasmic, and the cytoplasm was stained brown; the control normotensive group had minimal immunoreactivity. L-NAME group shows a very strong immunopositive reaction. LST-1 and LST-2 show weak immunoreactivity. DCT-3 and DCT-4 groups showed moderate levels of immunoreactivity. B The bar chart represents the iNOS immunopositivity expressed as area %. Mean values with different superscripts are significantly different. ^****P < 0.0001 L-NAME-treated group versus control. ^****P < 0.0001 LST-1, LST-2, and DCT-3 versus L-NAME-treated group; ^$$$$P < 0.0001 LST-1 compared to DCT-3; ^@@@@P < 0.0001 LST-2 compared to the DCT-4-treated group; ^#P < 0.05 DCT-3 compared to the DCT-4-treated group (ANOVA)

Fig. 10

Inflammatory cytokines in L-NAME-induced hypertensive rats after SNEDS-loaded VST and SNEDS-loaded VST/HCTZ administration. A mRNA expression of NF-κB was increased in heart tissues of the L-NAME-treated group. All formulations reduced the NF-κB mRNA significantly. ^**P < 0.01 L-NAME-treated group versus control and LST-1 versus L-NAME-treated group; ^*P < 0.05 LST-2, DCT-3, and DCT-4 versus L-NAME-treated group. B Serum NF-κB level also increased in the L-NAME-treated group, and all formulations reduced its level. ^****P < 0.0001 L-NAME-treated group versus control. ^****P < 0.0001 LST-1, LST-2, DCT-3, and DCT-4 versus L-NAME-treated group; ^$P < 0.05 LST-1 compared to DCT-3; ^@P < 0.05 LST-2 compared to DCT-4-treated group. C Serum IL-6 level increased in the L-NAME-treated group, and all formulations reduced its level. ^**P < 0.01 L-NAME-treated group versus control; ^*P < 0.05 LST-1, LST-2, DCT-3, and DCT-4 compared to L-NAME-treated group. There are 6–8 mice per group

Fig. 11

Electrocardiography (ECG): the ECG of rats was recorded for 5 min using the ECG PowerLab module. VST and VST/HCTZ ameliorated the L-NAME-induced changes in ECG parameters after 21 days of treatment