Effect of Bletilla Striata Polysaccharide on human umbilical vein endothelial cells in high-fat and high-sugar environment based on Wnt/β-catenin signaling pathway
Objective It is to explore the effect of Bletilla Striata Polysaccharide(BSPS)on human umbilical vein en-dothelial cells(HUVECs)in high-fat and high-sugar environment,and to reveal its molecular mechanism in promoting the early healing of diabetic foot ulcers(DFU).Methods The HUVECs were cultured with palmitate acid(PA)and glucose(Glc)in vitro to mimic the models of vascular endothelial injury induced by high-fat and high sugar in vivo.The optimal action concentrations of PA,BSPS,pathway inhibitor ICG-001 and cytotoxicity of BSPS were detected by CCK-8 assay,the cell growth statuses after the intervention of the optimal concentration of Glc,PA,BSPS,ICG-001 were observed by micro-scope,the cell growth was detected by trans-endothelial electrical resistance assay,the nuclear translocation of β-catenin in cells was detected by immunofluorescence,the protein expressions of β-catenin,glycogen synthase kinase 3β(GSK-3β)and c-Myc were detected by Western blot,and the mRNA expressions of vascular endothelial growth factor(VEGF)and basic fi-broblast growth factor(bFGF)in cells were detected by RT-PCR.Results The optimal concentration of Glc was 25 mmol/L,PA was 300 pmol/L,BSPS was 300 μg/mL,and ICG-001 was 10 μmol/L,and the models of vascular endothelial injury were established by the optimal concentrations of Glc and PA.The growth of the cells in the Glc+PA 300 μmol/L group and Glc+PA 300 pmol/L+ICG-001 group were inhibited,and their adhesion and viability were significantly reduced;the number of cell fragments and morphologically abnormal cells were significantly reduced in the Glc+PA 300 μmol/L+BSPS group and Glc+PA 300 μmol/L+ICG-001+BSPS group,their adhesion was strengthened and viability was significantly increased.The resistive TEER value,the relative expression of β-catenin protein in cytoplasm and nucleus,the relative ex-pression of c-Myc protein in nucleus,and the relative expressions of VEGF and bFGF mRNA in the cells were significantly lower than those in the normal control group(all P＜0.05),and the relative expression of GSK-3β in cytoplasm was signif-icantly higher than that in the normal control group(P＜0.05),the fluorescence intensity of β-catenin expression in cyto-plasm and nucleus was lower than that in the normal control group(P＜0.05),and the above indexes were reversed in the Glc+PA 300 μmol/L+BSPS group.The resistive TEER value,the relative expression of β-catenin protein in cytoplasm and nucleus,the relative expression of c-Myc protein in nucleus,and the relative expressions of bFGF mRNA in the cells in the Glc+PA+ICG-001 group were significantly lower than those in the Glc+PA 300 µmol/L group(all P＜0.05),the fluorescence intensity of β-catenin expression in cytoplasm and nucleus was lower than that in the Glc+PA 300 μmol/L group(P＜0.05),and the above indexes were reversed in the Glc+PA 300 μmol/L+ICG-001+BSPS group.Conclu-sion BSPS could promote angiogenesis by activating Wnt/β-catenin pathway,so as to repair diabetic wounds.
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