Naringin attenuates angiotensin II induced cardiac hypertrophy by inhibiting carbonic anhydrase II

Abstract

Nutraceuticals exert a series of health benefits, including protection against cardiovascular diseases. In this study, naringin, naringenin, and quercetin were tested for their safety and efficacy in ameliorating angiotensin (Ang) II-induced cardiac hypertrophy through carbonic anhydrase II (CA-II) inhibition. In silico molecular docking and MD simulations exhibited that naringin strongly binds CA-II with a docking score of -9.55 kcal/mol and hydrogen bonding energy of -6.07 kcal/mol. Naringin formed stable hydrogen bond interactions with Asn62, Trp5, and N-acetyl His4 via catalytic water molecule, and a continuous interaction via major water bridge with N-acetyl His4, His4, and Trp5. Moreover, naringin effectively inhibited CA-II activity with an IC₅₀ value of 82.99 ± 4.92 nM, followed by naringenin and quercetin. Of note, all the tested nutraceuticals were found to be safe as evident from the cell viability assays. Further, naringin effectively attenuated cardiac hypertrophy, as indicated by the reductions in the Ang II-induced increases in cell surface area of H9c2 cardio myoblasts (165.6 ± 1.26% Ang II vs. 109.8 ± 1.88% Ang II + naringin), followed by naringenin and quercetin. Furthermore, naringin significantly inhibited CA-II activity (191.77 ± 7.69% Ang II vs. 120.16 ± 5.52% Ang II + naringin) and suppressed Ang II-induced CA-II and Na⁺/H⁺ exchanger 1 (NHE1) protein expression. Besides, naringin suppressed Ang II-induced CA-II, NHE1, Na⁺/Ca²⁺ exchanger 1 (NCX1), and angiotensin-converting enzyme (ACE1) mRNA expression. Collectively, naringin when compared to naringenin and quercetin effectively attenuated Ang II-induced cardio myoblast hypertrophy, CA-II activity, CA-II, and NHE1 expression. The naringin-mediated attenuation of cardiac hypertrophy might be through the inhibition of CA-II enzyme activity, and the suppression of NHE1, and NCX1.

Keywords: CA-II; Cardiac hypertrophy; NCX1; NHE1; Naringin; Nutraceuticals.

Fig. 1

Molecular dynamics simulations of naringin with CA-II. (a) RMSD of naringin and CA-II complex, giving insight into its structural conformation throughout the simulation, showing fluctuation within the acceptable limit. (b) 2D geometrical interactive plot showing interactions of amino acids with naringin. (c) L-RMSF depicting the interaction of naringin atoms (given in percentage) with CA-II throughout the MDSs. (d) Summary of the interactions of CA-II-naringin complex in a normalized stacked bar chart. (e) The contact strengths between the putative binding site amino acids of CA-II with naringin throughout the MDS’s up to 200ns.

Fig. 2

Concentration-dependent inhibition of CA-II by nutraceuticals (a) naringin, (b) acetazolamide, (c) naringenin and (d) quercetin. The decreased concentration of P-nitrophenol in the test reaction compared to control and enzyme activity reaction was expressed as percentage of CA-II inhibition. Data represents mean ± SEM (n = 3).

Fig. 3

Dose-dependent cytotoxicity of nutraceuticals (a) naringin, (b) naringenin, and (c) quercetin on H9c2 cells, as determined by MTT assay. Data represents mean ± SEM (n = 3) (^* p < 0.05, ^**p < 0.01 ^***p < 0.001).

Fig. 4

Attenuation of Ang II-induced cell hypertrophy by nutraceuticals, as measured by the surface area of H9c2 cells. Cell surface area represented as mean value ± SEM (n = 3) (^# vs. control group; ^* vs. Ang II group; ^* P, 0.05, ^## and ^**P, 0.01, ^### and ^***P, 0.001).

Fig. 5

Inhibition of Ang II-induced CA-II activity by nutraceuticals in H9c2 cells (^# vs. control group; ^* vs. Ang II group; ^*P, 0.05, ^## and ^**P, 0.01, ^### and ^***P, 0.001). Data represents mean ± SEM (n = 3).

Fig. 6

Effect of nutraceuticals on CA-II and NHE1 protein expression in H9c2 cells. The β-actin served as internal control. Data represents mean ± SEM (n = 3) (^# vs. control group; ^* vs. Ang II group; ^* P, 0.05, ^## and ^**P, 0.01, ^### and ^***P, 0.001). (Original blots/gels are presented in Supplementary Figure S6-S8)

Fig. 7

Effect of nutraceuticals on (a) CA-II, (b) NHE1, (c) NCX1, and (d) ACE1 gene expression in H9c2 cells. The β-actin served as internal control. Data represents mean ± SEM (n = 3). Data represents mean ± SEM (n = 3) (^# vs. control group; ^* vs. Ang II group; ^* P, 0.05, ^## and ^**P, 0.01, ^### and ^***P, 0.001).

Fig. 8

Schema showing possible mechanisms of actions of naringin on CA-II-mediated signal transduction pathways involved in the development of Ang II-induced cardiac hypertrophy, via negative regulation of CA-II, NHE1, NCX1 and ACE1.