Abstract:

Objective To investigate the ameliorative effect of phloretin on pulmonary vascular remodeling in the rats with monocrotaline （MCT）-induced pulmonary arterial hypertension （PAH） and its effects on the proliferation and migration of pulmonary artery smooth muscle cells （PASMCs）， and to provide evidence for targeted therapy of PAH. Methods For in vivo experiments， twenty male Sprague-Dawley rats were randomly divided into control group， MCT group （60 mg·kg^-1）， low dose of phloretin group （10 mg·kg^-1）， and high dose of phloretin group （20 mg·kg^-1）. Echocardiography method was used to detect the pulmonary artery acceleration time （PAT） and mean pulmonary artery pressure （mPAP） in the rats in various groups. HE staining method was used to detect the percentages of pulmonary artery wall thickness （WT%） and percentages of pulmonary artery wall area （WA%） in the rats in various groups. Immunofluorescence staining method was used to detect the expression levels of proliferating cell nuclear antigen （PCNA） protein in the lung tissue of the rats in various groups. Western blotting method was used to detect the expression level of osteopontin （OPN） in the lung tissue of the rats in various groups. For in vitro experiments， the primary PASMCs were extracted from the rats using enzymatic digestion method. The cells were divided into control group， epidermal growth factor （EGF） group （20 ng·L^-1）， EGF+10 μmol·L^-1 phloretin group， and EGF+20 μmol·L^-1 phloretin group. Cell counting kit-8 （CCK-8） method was used to detect the activities of the cells in various groups. Wound healing assay and Transwell chamber assay were used to detect the wound healing rates and the numbers of migration cells of PASMCs in cells in various groups. Western blotting method was used to detect the expression levels of PCNA protein in PASMCs in the cells in various groups. Results The in vivo experiments results showed that compared with control group， PAT in the rats in MCT group was significantly shortened （P<0.05）， mPAP was significantly increased （P<0.05）， the WT% and WA% of the pulmonary artery were significantly increased （P<0.05 or P<0.01）， the expression level of PCNA protein in the lung tissue was significantly increased （P<0.05）， and the expression level of OPN protein was significantly increased （P<0.05）. Compared with MCT group， PATs in the rats in low dose of phloretin group and high dose of phloretin group were significantly prolonged （P<0.05 or P<0.01）， mPAPs were significantly decreased （P<0.05 or P<0.01）， the WT% and WA% of the pulmonary artery of the rats were significantly decreased （P<0.05 or P<0.01）， and the expression levels of PCNA protein in the lung tissue were significantly decreased （P<0.05）； the expression level of OPN protein in the lung tissue of the rats in high dose of phloretin group was significantly decreased （P<0.05）. The in vitro results showed that compared with EGF group， the cell activity of PASMCs in EGF+10 μmol·L^-1 phloretin group was significantly decreased （P<0.05）， the number of migration cells was significantly decreased （P<0.05）， the expression level of PCNA protein in the cells was significantly decreased （P<0.05）， while no significant difference was observed in the wound healing rate（P>0.05）； compared with EGF group， the cell activity of PASMCs in EGF+20 μmol·L^-1 phloretin group was significantly decreased （P<0.05）， the wound healing rate was significantly decreased （P<0.01）， the number of migration cells was significantly decreased （P<0.01）， and the expression level of PCNA protein was significantly decreased （P<0.01）. Conclusion Phloretin not only effectively ameliorates MCT-induced pulmonary vascular remodeling in the MCT-induced PAH rats， but also inhibits EGF-induced proliferation and migration of PASMCs， suggesting that phloretin may serve as a potential candidate drug for the treatment of PAH.

Key words: Phloretin, Pulmonary arterial hypertension, Vascular remodeling, Monocrotaline, Pulmonary arterial smooth muscle cells, Cell proliferation, Cell migration