| Part I Apatinib inhibits mice model of laser-induced choroidal neovascularizationObjective:To observe the effect of apatinib on inhibition of choroidal neovascularization induced by laser in mice model.Methods:Sixty healthy male 6-8 weeks C57BL/6 mice were randomly devided into four groups(each number=5),normal control group and experiment groups(normal saline group,low-doage apatinib group,high-dosage apatinib group).After the choroidal neovascularization models were successfully induced by laser,experiment groups(normal saline group,low-doage apatinib group,high-dosage apatinib group)were administated with corresponding treatments for seven days.FFA,histological observation and flat mounts of choroidal neovascularization were carried out to assess the areas of neovascularization area.Results:(1)The results of FFA:compared with normal control group,large choroidal neovascularization could be seen in normal saline group,which mean the models of choroidal neovascularization were successfully induced.The fluorescence leakage score in each group showed that the differences were significant between groups(F=51.76;P=0.000).The fluorescence leakage score in normal saline group was(8.43 ± 0.50);in low dosage of apatinib group was(7.84 ±0.50),in high dosage of apatinib group was(6.13 ± 0.90);Compared with normal saline group,the fluorescence leakage score decreased both in low dosage of apatinib group significantly(P=0.020)and in high dosage of apatinib group(P=0.000).The fluorescence leakage score in high dosage of apatinib group was also lower than that in low dosage of apatinib group(P=0.000).(2)The results of flat mounts of choroidal neovascularization indicated that retinal pigment epithelium in normal control group was regular hexagonal structure;However,the area of IB4 labled radial neovascularization in normal saline group was(100.0 ± 6.0)μm2;There was significant difference among the between the groups(F=143.72;P=0.001);Compared with normal saline group,the area of IB4 labled neovascularization in high dosage of apatinib group was(28.00 ± 4.00)μm2,and(66.88 ± 8.41)μm2 in low dosage of apatinib group,both of them were less than that of normal saline group,the difference was statistically significant(P=0.001),the area of IB4 labled neovascularization in high dosage of apatinib group was less than that in low dosage of apatinib group(P=0.000).(3)The results of HE staining in neovascularization region indicated that there was significant difference among the groups(F=1899;P=0.000);compared with that of normal control group,the normal saline group had larger and longer neovascularization region,the length of neovascularization was(392.86 ± 3.54)μm and thickness was(96.05 ± 6.58)pm(P=0.000);both the length(164.99 ± 8.24)pm and thickness(62.68 ± 3.81)μm of neovascularization in apatinib groups decreased significantly comparing with those in normal saline group(P=0.000),and also lower than those in low dosage of apatinib group(P=0.000):the length was(81.16 ± 6.53)μm and the thickness(25.58 ± 4.02)μm.Conclusion:Apatinib is effective in preventing mice model of choroidal neovascularization induced by laser.Part II Apatinib modulates the proliferation,migration,lumen formation and the expression of related proteins of the human retinal microvascular endothelial cellsObjective:To observe the influence and molecular mechanism of the proliferation,migration and the lumen formation of HRMEC by apatinib.Methods:Cultured HRMEC,add the apatinib for reagent,compare the cell proliferation,migration and lumen formation in apatinib group and control group;Collect cellular proteins,western blotting verify the effect of apatinib on the expression of VEGFR2,p-VEGFR2,ZO-1 and Occludin.Results:(1)Apatinib inhibited the proliferation viability of HRMEC:the results of CCK-8 indicated that with the increasing of the dosage of apatinib,the proliferation viability of HRMEC decreased in a dose-dependent manner,the viability of HRMEC in each group was 0.05μM(96.8%),0.1μM(84.5%),0.2μM(74.2%),0.4μM(63.5%),0.8μM(56.0%),1.6μM(45.5%),3.2μM(42.0%),6.4μM(35.6%),respectively;Compared with control group,the differences were significant(P<0.05).(2)Apatinib inhibited the migration of HRMEC:the results of migration test indicated that there was significant difference between the groups(F=119.30;P=0.000);Compared with the number of migration cells,(64.00 ± 5.57)per well in control group,the number of migration cells(97.00 ± 4.23)per well in rhVEGF group increased significantly(P=0.000);When stimulating HRMEC with rhVEGF + 5uM or 1μM apatinib,the number of migration cells was(32.00 ± 4.00)per well in rhVEGF + 5uM apatinib group,and(44.67 ± 3.51)per well in 1μM apatinib group.Compared with rhVEGF group,apatinib treated groups could significantly inhibit the migration of HRMEC(P=0.000);and the number of migration cells in rhVEGF and 5uM apatinib group was lower than that in rhVEGF + 1uM apatinib group(P=0.035).(3)Apatinib inhibited the formation of closed lumen of HRMEC:the results of closed lumen indicated that there was significant difference between the groups(F=112.10;P=0.000);The number of closed tubes in control group was(10.53 ± 0.58)per well,and(17.00 ± 1.00)per well in rhVEGF group,compared with control group,rhVEGF group significantly increased the number of closed lumen(P=0.000);When stimulate HRMEC with rhVEGF and 5uM or 1μM apatinib,the number of closed lumen was(4.0 ± 1.0)per well in rhVEGF + 5uM apatinib group,and(7.0 ± 1.0)in rhVEGF + 1μM apatinib group.Compared with rhVEGF group,apatinib treated groups can significantly inhibit the formation of closed lumen(P=0.000);and the number of closed lumen in rhVEGF + 5uM apatinib group was less than that in rhVEGF + 1uM apatinib group(P=0.016).(4)Apatinib inhibited the expression of VEGFR2 and p-VEGFR2 but promoted the expression of ZO-1 and Occludin.The results of western blot indicated the relative grey value of VEGFR2,p-VEGFR2,ZO-1 and Occludin in control group was 0.29±0.02,0.28±0.02,0.29±0.02 and 0.23 ±0.01,respectively;the relative grey value of VEGFR2,p-VEGFR2,ZO-1 and Occludin in rhVEGF treated group was 0.52±0.04,0.56±0.04,0.11 ±0.003 and 0.08±0.002,respectively;the relative grey value of VEGFR2,p-VEGFR2,ZO-1 and Occludin in rhVEGF+1μM apatinib was 0.26±0.02,0.20±0.002,0.54±0.02 and 0.41 ±0.02,respectively;the relative grey value of VEGFR2,p-VEGFR2,ZO-1 and Occludin in rhVEGF+ 5μM apatinib was 0.17 ±0.01,0.06 ±0.004,0.54 ±0.02 and 0.61 ±0.04,respectively;There was significant difference between the groups,the VEGFR2(F=110.35;P=0.001),p-VEGFR2(F=274.98;P=0.000),ZO-1(F=556.74;P=0.000)and Occludin(F=338.49;P=0.000);Compared with rhVEGF treated group,the expression levels of VEGFR2(P=0.001)and p-VEGFR2(P=0.000)in rhVEGF+ 5μM or 1μM apatinib decreased significantly,the expression levels of VEGFR2 and p-VEGFR2 in rhVEGF+ 5μM apatinib were less than those of rhVEGF+ 1μM apatinib group(P=0.000);However,the expression levels of ZO-1(P=0.000)and Occludin(P=0.000)in rhVEGF+ 5μM or 1μM apatinib increased comparing with those in rhVEGF group(P=0.000);and the expression levels of ZO-1(P=0.000)and Occludin(P=0.000)in rhVEGF+ 5μM apatinib group increased(P=0.000)comparing with that in rhVEGF+ 1μM apatinib group.Conclusion:Apatinib is effective in preventing the ability of the proliferation,migration and lumen formation of human retinal microvascular endothelial cells,and this effect may be associated with inhibiting the expression of VEGFR2/p-VEGFR2 but promoting the expression of ZO-1 and Occludin. |
PDF Full Text Request