| Objective:To study and observe the inhibitory effect of diosgenin on laser-induced choroidal neovascularization(CNV)in mice and to study the effects of oxidized low-density lipoprotein(Ox-LDL)after stimulating the cells of HUVECs at the macroscopic cellular level and the microscopic molecular level,respectively,and to observe the effects on oxidative damage to the endothelial mesenchymal transition(EndMT)of HUVECs and its potential mechanism,which would provide a new idea for the development of an effective traditional Chinese medicine to prevent and control AMD.Methods:This experimental subject first used laser treatment of the mice retina to establish a laser-induced mouse model and subperitoneal injections of mice using diosgeninogen with a concentration of 50 and 100 μM dissolved in dimethyl sulfoxide(DMSO)solution.Mice were examined by fundus fluorescence angiography and optical coherence tomography,followed by immunohistochemical staining of mouse RPE/choroidal membranes processed,photographed,and analyzed to evaluate the effect of diosgenin on choroidal neovascularization in mice.The expression levels of important mesenchymal marker proteins(α-SMA),endothelial marker proteins(VE-cadherin),and AKT/GSK-3β pathway-related proteins(TGF-β2,p-AKT/AKT,and p-GSK-3β/GSK-3β)in the endothelial-mesenchymal transition were quantitatively detected by western blot.HUVECs cells were divided into a normal group(normal culture),a model group(Ox-LDL action for 24 h),a drug group(treated with 10,20,and 40 μM diosgenin,respectively),and a special control group(MK-2206+Ox-LDL action for 24 h)in cell experiments.Cell counting kit(CCK-8)and flow cytometry were used to detect the survival and apoptosis of HUVECs cells,respectively,and Transwell assay was used to detect the migration rate of OxLDL-induced HUVECs cells and Matrigel matrix gel to observe the length of cell-forming tubes as a means of observing the effect of diosgenin on Ox-LDL-induced HUVECs cells on the macroscopic cellular level.The expression levels of important mesenchymal marker proteins(αSMA and Vimentin),endothelial marker proteins(VE-cadherin and ZO-1),and AKT/GSK-3βpathway-related proteins(TGF-β2,p-AKT/AKT,and p-GSK-3β/GSK-3β)in the endothelialmesenchymal transition were quantitatively detected by western blot,and the changes in the cells before and after the treatment of diosgenin were observed on the micro molecular level.Results:1.Fundus photography and fundus fluorescence angiography:Fundus photography was seen to be successful in establishing the mouse CNV model in all cases,and angiofluorography showed that in the model group,the laser injury displayed hyperfluorescence in the early stage,and the size and intensity of the laser injury increased in the late stage,and the size and intensity of the laser injury were significantly reduced after 2 weeks of diosgenin treatment with dose-dependent statistical differences.The ratios of grade 3 laser damage in the model group,Dio 50,and Dio 100 were 42%,37%,and 21%,respectively,and the percentage of grade 3 laser damage in the Dio-treated group was significantly lower than that in our model group.2.Optical coherence tomography(OCT):The model group mice showed high subretinal reflective signals with subretinal fluid,while the diosgenin-treated mice showed obviously reduced subretinal high reflective signals and no subretinal fluid.Compared with the model group,the CNV area of mice in the diosgenin treatment group was greatly reduced,with a dosedependent statistically significant difference(P<0.01).3.Immunohistochemical staining examination of RPE-choroidal-scleral pavements to detect the area of CNV foci:Compared with the model group,the area of CNV in the model mice in the diosgenin-treated group was dramatically reduced and showed a statistically significant difference in a dose-dependent manner(P<0.01).4.The expression of EndMT-related proteins in CNV model mice was detected by Western blot:Compared with the normal group,there was a statistically significant increase in the mesenchymal cell marker a-SMA and a statistically significant decrease in the endothelial cell marker VE-cadherin in the model group(P<0.01).Compared with the model group,a-SMA decreased and VE-cadherin increased in the 50 μM diosgenin element group,and the difference was statistically significant(P<0.05).Compared with the model group,α-SMA was significantly decreased and VE-cadherin was significantly increased in the 100 pM diosgenin element group,and the difference was statistically significant(P<0.01).5.Expression of AKT/GSK-3β signaling pathway-related proteins in CNV model mice detected by Western blot:Compared with the normal group,the expression of TGF-β2,p-AKT,and pGSK-3β proteins in the model group was significantly increased,and the difference was statistically significant(P<0.01).Compared with the model group,the expression of p-AKT proteins in the 10 μM Dio was significantly decreased,and the difference was statistically significant(P<0.01).Compared with the model group,the protein expression of TGF-β2,p-AKT,and p-GSK-3β was significantly reduced in 20 and 40 μM Dio,and the difference was statistically significant(P<0.01).6.Cell survival:Ox-LDL-induced HUVECs cells were treated with 0,10,20,and 40 μM diosgenin solution,and the cell survival rates of each group were 100.71± 7.104,103.33± 10.07,105.77± 9.50 and 110.33±7.02,respectively,and the differences in the survival rates of all the groups compared with the 0 μM group were not statistically significance(P>0.05).7.Apoptosis rate:The apoptosis rate in the model group was significantly higher than that in the control group(17.36 ± 0.81 vs 1.22 ± 0.23,P<0.01).Compared with the model group,treatment with different concentrations of diosgenin solution reduced the apoptosis rate of Ox-LDLinduced HUVECs cells,which were 13.66±0.80,11.35± 0.65 and 8.20±0.52,respectively,and the differences were statistically significant(P<0.01).8.Cell migration:The cell migration number in the model group was significantly higher than that in the normal group(210.00±9.94 vs 58.33± 3.22,P<0.01).Compared with the model group,different concentrations of diosgenin treatment reduced the migration number of OxLDL-induced HUVECs cells,which were 160.71± 7.77,137.32 ± 6.11,and 116.31± 8.62,respectively,and the differences were all statistically significant(P<0.01).9.Expression of EndMT-related proteins in Ox-LDL-induced HUVECs cells detected by Western blot:Compared with the normal group,there was a statistically significant increase in the mesenchymal cell markers α-SMA and Vimentin and a statistically significant decrease in the endothelial cell markers VE-cadherin and ZO-1 in the model group(P<0.01).Compared with the model group,α-SMA,and Vimentin decreased and VE-cadherin increased in 10 μM Dio,and the difference was statistically significant(P<0.05).Compared with the model group,α-SMA,and Vimentin were significantly decreased,while VE-cadherin and ZO-1 were significantly increased in 20 and 40 μM Dio,and the difference was statistically significant(P<0.01).11.Expression of EndMT-related proteins in MK-2206:Compared with the control group,αSMA,and Vimentin expression was up-regulated in the model group,and VE-cadherin and ZO-1 protein expression was down-regulated in the model group,with statistically significant differences(P<0.01).Compared with the model group,the expression of α-SMA and Vimentin was down-regulated in the MK-2206 group,and the difference was statistically significant(P<0.05).The protein expression of α-SMA and Vimentin(P<0.01)was decreased and that of VE-cadherin,ZO-1 was up-regulated in 40 μM Dio,and the difference was statistically significant(P<0.01).In the special control group,the expression of α-SMA and Vimentin proteins was dramatically decreased,and VE-cadherin and ZO-1 proteins were significantly upregulated,and the difference was statistically significant(P<0.01).Conclusions:1.Diosmetin can inhibit laser-induced CNV leakage and reduce the area of CNV in mice.EndMT is involved in CNV formation and may be inhibited by modulation of the AKT/GSK-3βsignaling pathway,thereby attenuating CNV formation.2.Diosgenin has a protective effect on Ox-LDL-induced HUVECs cells and inhibits cell migration and tube formation,which may inhibit the occurrence of EndMT in Ox-LDL-induced HUVECs cells by regulating the AKT/GSK-3β signaling pathway.3.Diosgenin may be one of the potential drug candidates for adjuvant treatment of CNV-related diseases,including nAMD. |
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