Buyang Huanwu Decoction ameliorates atherosclerosis by regulating TGF-β/Smad2 pathway to promote the differentiation of regulatory T cells

Ethnopharmacological relevance: As a classic prescription of Traditional Chinese Medicine in Correction on the Errors of Medical Works, Buyang Huanwu Decoction (BYHWD) has a good curative effect on prevention of atherosclerosis (AS). Aim of the study: This study aims to elucidate the anti-atherosclerosis mechanism of BYHWD, which may promote the differentiation of regulatory T cells by regulating the TGF-beta/Smad2 pathway. Materials and methods: ApoE-/- mice were fed a high-fat diet for 12 weeks, then drugs group were given BYHWD with intragastric administration once a day for 4 weeks. The effect of BYHWD on lipid content in peripheral blood and plaque was evaluated by blood lipid test and oil red O staining. The number of Tregs in peripheral blood was tested by flow cytometry, and that in the spleen was evaluated by immunohistochemistry methods. Gene and protein expression relating with Tregs differentiation pathway in mice were checked by RT-PCR and Western blot experiments. CD4(+)T cells were isolated and interfered by BYHWD drug-loaded serum. The proportion of Tregs was evaluated by flow cytometry. The chemical compositions of BYHWD and rat drug-loaded serum were analyzed by ultra-high performance liquid chromatograph and liquid chromatography-tandem mass spectrometry. Results: BYHWD significantly reduced plaque area and cholesterol accumulation, increased the number of Tregs in spleen and peripheral blood of ApoE-/- AS mice, raised the proportion of Tregs in CD4(+)T cells, and regulated the levels of inflammatory factors. It also increased the TGF-beta and Smad2 mRNA and protein levels relating with Tregs differentiation pathway in vivo. The mRNA levels of Foxp3/TGF-beta/Smad2 were enhanced via BYHWD in vitro. Conclusions: BYHWD regulates TGF-beta/Smad2 signaling pathway to promotes the peripheral differentiation of Tregs, increases the number of Tregs, restores the immune balance between CD4(+)T cells, regulates lipid metabolism, inhibits inflammatory reaction and possesses the potential of enhancing plaque stability.