Betaine protects cerebral microvascular endothelium and ameliorates hypertension-induced cognitive dysfunction via upregulation of the endothelial nitric oxide synthase/nitric monoxide signaling pathway

Abstract

Objectives: Hypertension-induced endothelial damage in cerebral microvessels is a key factor contributing to vascular cognitive impairment (VCI). Endothelial function stabilization considerably depends on the endothelial nitric oxide synthase (eNOS)/nitrogen monoxide (NO) pathway. Furthermore, the eNOS/NO signaling pathway plays a role in stabilizing the vascular endothelium. Although betaine (bet) has been shown to improve cognitive dysfunction, its underlying mechanisms remain unclear. Therefore, this study aimed to determine whether betaine protects cognitive function by regulating eNOS/NO activity.

Methods: Male 7-month-old spontaneously hypertensive rats (SHR) were randomly assigned to four groups: SHR, Bet, Bet and N(G)-Nitroarginine methyl ester hydrochloride (L-NAME), and L-NAME groups. Male 7-month-old Wistar Kyoto rats (WKY) served as controls. All animals received treatment or saline for 4 weeks. In-vitro experiments were conducted using rat brain microvascular endothelial cells (RBMECs) treated with either homocysteine (Hcy) or betaine. Behavioral experiments, western blotting, pathological tissue staining, Doppler ultrasound technique, and ELISA were employed to assess the impact of hypertension on cognitive and endothelial functions.

Results: Hypertension led to cognitive decline in SHR by causing endothelial dysfunction, blood-brain barrier disruption, inflammation, oxidative stress, and apoptosis. Bet administration significantly improved these pathological indicators of cognitive impairment; however, the eNOS inhibitor L-NAME reversed its effects.

Conclusion: Our findings suggest that betaine protects vascular endothelium and improves VCI by modulating the eNOS/NO signaling pathway.

Keywords: betaine; endothelial dysfunction; endothelial nitric oxide synthase; hypertension; vascular cognitive impairment.

FIGURE 1

Effects of betaine on cognitive function in rats. (a–c) Morris water maze. ^#P < 0.05, ^##P < 0.01 vs. WKY group, ^∗P < 0.05, ^∗∗P < 0.01 vs. NS group, ^$P < 0.05, ^$$P < 0.01, vs. Bet group. (d) Novel object experiment. (e) Y maze. n = 6–8. ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 2

Effects of betaine on endothelial dysfunction and cerebral blood flow. (a–d) eNOS and p-eNOS protein expression in rat cortex. (e–h) eNOS and p-eNOS protein expression in RBMECs. (i,j) ELISA for rat serum and RBMEC NO content. (k–n) Changes in cerebral blood flow parameters in the middle cerebral artery of the rats. ^∗P < 0.05, ^∗∗P < 0.01, ns, not significant. Mean data ± SE. n = 4.

FIGURE 3

Effects of betaine on loss of tight junction proteins and blood-brain barrier permeability. (a–c) Changes in Occludin and Claudin-5 content in rat cortex. (d–f) Changes in Occludin and Claudin-5 content in RBMECs. (g,h) Evans Blue leakage in rat cortex and hippocampus. ^∗P < 0.05, ^∗∗P < 0.01, ns, not significant. Mean data ± SE. n = 4.

FIGURE 4

Effects of betaine on inflammation and oxidative stress levels. (a,b) Relative NF-κB levels in rat cortex. (c,d) Relative NF-κB levels in RBMECs. n = 4. (e–g) ELISA for cortex GSH, MDA, and total SOD levels in rat. n = 6. ^∗P < 0.05, ^∗∗P < 0.01, ns, not significant. Mean data ± SE.

FIGURE 5

Effect of betaine on apoptosis in Hcy-treated RBMECs. (a,b) WB for Bcl-2, Bax in RBMECs. (c,d) Effect of betaine RBMEC apoptosis rates. ^∗P < 0.05, ^∗∗P < 0.01, ns, not significant. (n = 4, Mean data ± SE.).