The regulation of lymphatic valve formation promotes lymphatic drainage to reduce atherosclerotic plaques: A novel mechanism of anti-atherosclerosis by Gualou-Xiebai

Ethnopharmacological relevance: Atherosclerosis (AS) is a chronic vascular disease with excessive accumulation of plaques formed by lipid accumulation and intimal thickening in the arterial wall, resulting in the lumen stenosis of vessel. Adventitial lymphatic vessels employ lymphatic valves to eliminate infiltrated harmful substances from arterial walls, thereby preserving vascular wall integrity. The traditional Chinese medicine "Gualou-Xiebai" (GLXB) has been shown to have a definite therapeutic effect on AS by inhibiting aortic plaque deposition and reducing vascular damage. However, whether the effect of GLXB on ameliorating vascular damage in AS is related to lymphatic drainage and lymphatic valves formation has not been clarified.

Aim of the study: To investigate whether GLXB alleviates the plaque lesions of AS by regulating the function of lymphatic drainage and its potential mechanism.

Materials and methods: Doppler ultrasound imaging were used to validate AS lesions to confirm successful model establishment. HE staining and morphological photography were used to evaluate the plaque area. The bilateral branches of the left common carotid artery were ligation to block the lymphatic drainage of the aortic ventricular duct in AS mice, and the mouse lymphatic vessel blockade model was established. The lymphatic vessel contraction frequency and leakage were detected by near-infrared two-zone imaging and Evans blue staining, and the lymphatic drainage function was evaluated. Lymphatic valve formation was evaluated through immunofluorescence analysis of specific biomarker expression. After lymphatic endothelial cells (LECs) were treated with serum containing GLXB or transfected with VEGFR3 in LECs, the proliferation and migration of LECs were evaluated by CCK8 method, transwell and immunofluorescent staining of Ki67. The expression of lymphatic-associated proteins was detected by Western blot, and the expression of IL-1β and IL-18 in serum was detected by ELISA kit.

Results: GLXB promotes lymphatic drainage in the adventitia and ameliorates vasculopathy in AS mice by maintaining lymphatic drainage function, which is related to the promotion of lymphatic valve formation. Meanwhile, GLXB promotes lymphatic valve formation based on the fact that GLXB promoted the proliferation and migration of LECs, while also the expression of various markers of lymphatic valve formation in LECs. Mechanistically, we demonstrated that VEGFR3 is a key target of GLXB in promoting LECs development and maintaining the function of adventiolar lymphatic drainage to improve vasculopathy in AS. Rutin, and Kaempferol-3-O-β-rutinoside are the key active components of GLXB targeting VEGFR3 to improve the proliferation and migration of LECs after injury.

Conclusion: GLXB inhibits LECs injury by targeting VEGFR3, promotes lymphatic valve formation, maintains lymphatic drainage function, and reduces AS.

Atherosclerosis; Lymphatic drainage; Lymphatic endothelial cells; Lymphatic valve formation; VEGFR3; Xiebai” herb pair; “Gualou.