The Effects Of Acidic Metabolic Components Of PLGA On Angiogenesis Of Vascular Endothelial Cells

ObjectiveThis study intends to use in vitro experiments and animal experiments to study the biological effects of PLGA on vascular endothelial cells under the action of acidic meTableolic components,to clarify the role and mechanism of the main products of PLGA meTableolism on angiogenesis in the material,and to explore effective targets for research and development performance.Excellent bone substitute materials provide important theoretical guidance.MethodExperiment 1 Effects of different pH values of PLGA acidic meTableolites on angiogenesis of vascular endothelial cells:Human umbilical vein endothelial cells(HUVECs)were cultured in ECM complete medium containing hydrochloric acid,lactic acid and glycolic acid at pH 7.4,6.8 and 6.2,respectively).The effects of hydrochloric acid,lactic acid and glycolic acid on cell proliferation at different pH values were detected by CCK-8 method.The effects of hydrochloric acid,lactic acid and glycolic acid on cell migration ability under different pH values were detected by scratch test.The effect of hydrochloric acid,lactic acid and glycolic acid on the tube formation ability of cells was detected by tube formation experiment.Experiment 2 The effects of different acidic components on angiogenesis of vascular endothelial cells under the condition of pH 6.8:HUVECs were cultured in ECM complete medium containing hydrochloric acid,lactic acid and glycolic acid at pH 6.8.The effects of hydrochloric acid,lactic acid and glycolic acid on cell proliferation at pH 6.8 were detected by CCK-8 method.The effects of hydrochloric acid,lactic acid and glycolic acid on cell migration ability under pH 6.8 were detected by scratch test.The effects of hydrochloric acid,lactic acid and glycolic acid on the tube-forming ability of cells were detected by tube-forming experiment under the condition of pH 6.8.The expression of VEGF,ANG-1,FGF1,Endostatin mRNA and miRNA of miR-2103p and miR-221-3p were detected by RT-PCR.The protein expressions of VEGF,ANG-1,FGF1,Endostatin and MMP12 were detected by Western Blot.Experiment 3 The effects of transfection of miR-210-3p and PI3K/AKT inhibitors on angiogenesis of vascular endothelial cells in lactic acid microenvironment:After silencing the expression of miR-210-3p by lentiviral transfection,adding PI3K/AKT inhibitor HUVECs were cultured in ECM complete medium containing lactic acid at pH 6.8 of 3-MA.The targets of lactate and miR-210-3p were predicted by bioinformatics analysis.The effect of silencing miR-210-3p on cell proliferation was detected by CCK-8 assay.The effect of silencing miR-2103p on cell migration ability was detected by scratch assay.The effect of silencing miR-210-3p on the tube formation ability of cells was detected by tube formation assay.The expression of VEGF,ANG-1,FGF1,Endostatin mRNA and miR-2103p were detected by RT-PCR.The protein expressions of VEGF,ANG-1,FGF1,Endostatin,MMP12,PI3K,p-PI3K,AKT and p-AKT were detected by Western Blot.The binding of miR-210-3p and FGFRL1 was detected by dual luciferase assay.Rapamycin-loaded material was implanted in a rabbit cranial parietal defect model,and the vascular marker Isolectin B4 immunohistochemical staining was performed at two time points of 1 week and 1 month to verify the effect of activating PI3K/AKT pathway on angiogenesis in bone repair.ResultExperiment 1:1.In the pH range of 7.4 to 6.2,the low pH environment inhibits the proliferation of cells.2.In the pH range of 7.4 to 6.2,the low pH environment inhibits the migration ability of cells.3.Within the pH range of 7.4 to 6.2,a low pH environment can enhance the tube-forming ability of cells.Experiment 2:1.The difference of cell proliferation curve appeared at 72h,the proliferation ability of NC group was greater than that of hydrogen ion group,lactic acid group and glycolic acid group,and there was no significant difference between the three acid component groups.2.The migration ability of NC group and glycolic acid group>hydrogen ion group.There was no significant difference in cell migration ability between groups.3.The tube-forming ability of lactic acid group>NC group,hydrogen ion group,glycolic acid group>NC group,there is no significant difference among other groups.4.The results of WB detection of protein expression showed that the expression of VEGF protein was in the NC group>the hydrogen ion group,the lactic acid group and the glycolic acid group.Hydrogen ion group>glycolic acid group.The expression of FGF1 protein was in lactate group>NC group.Hydrogen ion group>glycolic acid group.5.RT-PCR results showed:VEGF expression in NC group>hydrogen ion group,lactic acid group and glycolic acid group.The expression level of FGF1 in lactate group>NC groupExperiment 3:1.There are 45 targets of lactate acting on angiogenesis,and the functions are mainly enriched in the PI3K/AKT pathway.There are 28 targets of miR-2103p.Combined with the lactate target,we screened FGFRL1 as a downstream target of miR-210-3p for exploration.2.After 48 hours of cell proliferation,the silence group was greater than the non-silence group in both neutral and lactic acid environment,and the proliferation ability was enhanced after silencing micro.In the silent group,the ability of cell proliferation under neutral environment>lactate environment.3.The migration ability of cells in the silenced group was higher than that in the non-silenced group in both neutral and lactic acid environments,and the migration ability of cells was enhanced after silencing micro.In the silent group,the ability of cell proliferation under neutral environment>lactate environment.4.The tube-forming ability under the lactic acid environment of blank carrier>neutral environment.There was no difference between the remaining groups.5.RT-PCR results showed that the expression of VEGF in the silence group was greater than that in the blank group.After adding the inhibitor,the expression of VEGF decreased,and there was no significant difference.The expression of FGF1 in the blank group was greater than that in the silent group.After adding the inhibitor,FGF1 was down-regulated and recovered,and there was no significant difference between the groups.6.WB results showed the same trend as PCR.There was no significant difference in the expression of VEGF between the silent group and the blank group.After adding the inhibitor,VEGF was significantly decreased,and there was no significant difference between the two groups.The expression of FGF1 in the blank group was greater than that in the silent group.After adding the inhibitor,FGF1 was down-regulated and recovered,and there was no significant difference between the groups.Phosphorylated PI3K and AKT had no difference between the silence group and the blank group,but were significantly decreased after the addition of pathway inhibitors.7.In the dual luciferase experiment,we can see that the luciferase activity of the 210-3p overexpressing cells in the FGFRL1 wild strain is reduced,and the 210-3p overexpressing cell luciferase activity in the FGFRL1 mutant strain has no decrease.8.The blood vessel density in the drug-loaded group was significantly higher than that in the blank control group at the time points of 8,1 week and 1 month.Conclusion1.In the pH value range of 7.4-6.2,the acidic meTableolic components of PLGA increased,the pH value decreased,and the proliferation and migration ability of HUVECs cells were inhibited and the tube-forming ability was enhanced.2.Under the condition of PH value of 6.8,hydrogen ions down-regulated the expression of VEGF and inhibited the proliferation,migration and tube formation of vascular endothelial cells.Lactic acid down-regulates VEGF,up-regulates FGF1 expression,inhibits cell proliferation,and promotes cell tube formation.Glycolic acid down-regulates VEGF,up-regulates MMP12 expression,inhibits cell proliferation,and promotes cell migration and tube formation.3.The miR-210-3p-FGFRL1-PI3K/ATK signaling axis is one of the important pathways for lactate to regulate angiogenesis.Lactate regulates the expression of VEGF and FGF1 through the signaling axis,and promotes the migration ability and tube-forming ability of vascular endothelial cells.