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Research Article: High-fat diet-induced obesity impairs endothelium-dependent relaxation in rabbits: association with MLCK upregulation and partial ex vivo improvement by ML-7

Date Published: 2026-05-11

Abstract:
Obesity is an independent risk factor for cardiovascular diseases and elevated mortality, yet the underlying mechanisms remain incompletely understood. Impaired endothelium-dependent relaxation is an early manifestation of vascular injury. This study investigated the impact of high-fat diet (HFD)-induced obesity on endothelium-dependent relaxation function and explored the potential role of myosin light chain kinase (MLCK) signaling in a rabbit model. Forty-five male New Zealand White rabbits were randomized into control (normal diet), HFD, and ML-7 (HFD plus the MLCK inhibitor ML-7, 1?mg/kg/day) groups ( n =?15/group). After 8 weeks, in vivo endothelial function was assessed by flow-mediated dilation (FMD) of the iliac artery. Serum lipids and fasting glucose levels were measured. Ex vivo endothelium-dependent and endothelium-independent relaxations were evaluated in aortic rings using cumulative concentrations of acetylcholine (ACh, 0.001–10??mol/L) and sodium nitroprusside (SNP). Aortic protein expression levels of MLCK, endothelial nitric oxide synthase (eNOS), and phosphorylated myosin light chain (p-MLC) were analyzed by Western blotting. HFD-fed rabbits developed obesity (body weight +27.85% vs. control), dyslipidemia (elevated TC, LDL-C, and ox-LDL; all P <?0.001), hyperglycemia ( P <?0.05), and significant endothelial dysfunction, characterized by impaired FMD ( P <?0.05) and reduced ACh-induced relaxation of aortic rings. This was accompanied by increased aortic MLCK expression ( P <?0.001) and a higher p-MLC/MLC ratio ( P <?0.001), whereas eNOS expression and arterial NO levels remained unchanged. ML-7 treatment significantly reduced MLCK expression and MLC phosphorylation. ML-7 partially improved ACh-induced relaxation in aortic rings at higher concentrations (1 and 10??mol/L, P <?0.05 vs. HFD) but failed to enhance FMD in the iliac artery in vivo . SNP-induced, endothelium-independent relaxation was similar across all groups. Obesity-induced endothelial dysfunction is associated with visceral fat accumulation, dyslipidemia, and MLCK upregulation. MLCK inhibition with ML-7 partially restored endothelium-dependent relaxation ex vivo in a concentration-dependent manner but did not translate to improved FMD in vivo . This dissociation highlights the complex nature of vascular dysfunction and suggests that noninvasive FMD may not capture modest, pathway-specific improvements, warranting caution in its use as a sole surrogate for endothelial health in preclinical studies.

Introduction:
Obesity is a chronic disease that severely impacts physical and mental health, reduces quality of life ( 1 ), and imposes significant medical costs ( 2 ). Its prevalence has been continuously increasing across all age groups in China ( 3 ), with the prevalence of obesity among adults increasing from 3.1% in 2004 to 16.4% during 2015–2019 ( 4 ). Asian children, particularly Chinese children, exhibit higher body fat percentages and central obesity ( 5 , 6 ), which are closely associated with an increased risk of…

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