Neuregulin 4 Alleviates Atherosclerosis In Mice And Its Possible Mechanism

Background and objectiveAtherosclerotic heart disease is the most disabling and fatal disease in the world.The ways to alleviate the occurrence and development of atherosclerosis are urgent to be explored.Recent research has identified that atherosclerosis is a chronic inflammatory disease,and neuregulin 4(Nrg4),which is enriched in brown adipose tissue,has shown its anti-inflammation effects in non-alcoholic steatohepatitis.Therefore,we investigated whether brown adipose tissue-derived Nrg4 could inhibit the inflammation response in aortic endothelial cells,thus protecting arteries from atherosclerotic damage.Methods1.Nrg4 knockout mice(KO mice)and nrg4-/-apoe-/-double knockout mice(DKO mice)were used for loss-of-function experiments.Adeno-associated virus(AAV)-Nrg4 which was injected into the brown adipose tissue in the interscapular region thus overexpressing Nrg4 protein was used for gain-of-function experiments.2.Western diet(WD)feeding was performed to promote endothelial damage in mice.Acetylcholine-induced relaxation of the arterial ring showed endothelial function.PCR measured the inflammation and adhesion response of endothelium.In situ TUNEL staining was used to detect apoptosis of endothelial cells in aortic tissue,and electron microscopy observed the integrity of the endothelium.3.Apoe-/-mice(AKO)were fed with WD to establish the animal model of atherosclerosis.The atherosclerotic lesions were detected by oil red O staining,and the plaque components were analyzed by immunohistochemistry staining of CD3,CD68,α-SMA and collagen.4.Gain-of-function cell experiments were conducted by exogenous supplementation of recombinant Nrg4 protein(rNrg4)and co-culture with brown adipocytes in the transwell chamber.The macrophage migration experiment was performed in a transwell chamber.The blocking experiments were conducted by Si-RNA for ErbB4.After blocking,Sc79 was used for the rescue experiments.5.The concentrations of inflammation and adhesion molecules were detected by Elisa assays.Apoptosis was measured by flow cytometry using Annexin V/PI staining.The protein expression level was detected by Western Bolt.The nuclear translocation of p65 was detected by immunofluorescence.Results1.Nrg4 can alleviate the inflammation and adhesion response of MAECs in vivo and in vitro,reducing endothelial cell apoptosis,and protecting the integrity of the endothelium.2.Nrg4 can inhibit endothelial inflammation and adhesion response,thus improving endothelial function,alleviating endothelial cell apoptosis,inhibiting macrophage migration,and last alleviating the occurrence and development of atherosclerosis.3.Mechanistically,the ErBb4-Akt-Nf-κB signaling pathway is involved in the antiinflammatory and anti-apoptotic effects of Nrg4 on MAECs.ConclusionBrown adipose tissue-derived Nrg4 can participate in the regulation of the ErBb4Akt-Nf-κB signaling pathway in MAECs,therefore inhibiting inflammation and adhesion response,alleviating MAECs apoptosis,protecting endothelial integrity and function,reducing the migration of mononuclear macrophages,and last alleviating the occurrence and development of atherosclerosis.