Effects Of Astragalus Polysaccharides On Aging Endothelial Cells And The Mechanisms

Background:Vascular endothelial cell aging is a risk factor for vascular aging and cardiovascular diseases.Astragalus polysaccharide(APS)is one of the main extracts of Astragalus membranaceus.Modern pharmacological studies have shown that Astragalus polysaccharide has a variety of biological functions.Methods:The rat thoracic aorta was seeded onto a 6-welled plate and removed two days after RAECs sprouting started.RAECs were assayed by immunofluorescence and flow cytometry for endothelial marker expression(CD31 and vWF),the Dil ac LDL uptake,the tube formation,the Hoechst staining apoptosis,the ?-galactosidase staining,and the CCK-8.RAECs were pre-treated with 0 ug/ml,50ug/ml,200ug/ml and 800ug/ml APS for 1h and then co-treated with ECM complete medium containing 30mmol/L glucose for 48h as HG group,HG+APS50 group,HG+APS200 group and HG+APS800 group respectively.SA-?-Gal activity was assayed,P16,P21,P53,NCLX,NLRP3,LC3B,Atg7 and p62 expression were determined by Western blotting or/and immunofluorescence staining,IL-1? was tested by ELISA.After RAECs were transfected by siNCLX,SA-?-Gal activity was assayed,P16,P21,P53,NCLX,NLRP3,LC3B,Atg7 and p62 expression were determined by Western blotting or/and immunofluorescence staining,IL-1? was tested by ELISA again.In addition,autophagosomes were observed by transmission electron microscopy.Results:1)The cultured cells expressed high levels of CD31 and vWF,94.65%of the cells were positive for CD31.Cells at passages 3 and 10 showed similar levels of CCK-8,?-galactosidase,Hoechst staining,uptake of Dil-ac-LDL,and capillary tube formation.2)APS reduced the expression of senescence-related proteins(P16,P21,P53)induced by high glucose in RAECs.3)APS enhanced the expression of mitochondrial sodium-calcium exchange protein(NCLX)in senile RAECs.4)After downregulation of NCLX,the above protective effects of APS were inhibited,and the expression of inflammatory factors(NLRP3 and IL-1)were increased.5)APS promoted the transformation from LC3BI to LC3BII,increased the aggregation of autophagy-related protein Atg7,decreased p62/SQSTM1 and promoted autophage formation.Conclusions:A novel method is developed for isolating and culturing vascular endothelial cells from rat aorta without collagenase digestion,cells at passages 3 to 10 are suitable for experiments.APS may inhibit the aging and inflammation of vascular endothelial cells induced by high glucose,which maybe depend on the regulation of NCLX.APS can promote autophagy formation through LC3-p62-Atg7 way and ensure autophagy flux,which may be one of the mechanisms of its anti-aging effect on vascular endothelial cells.