Effect of HtrA serine peptidase 3 gene on choroidal neovascularization and M2 macrophage polarization / 眼科新进展

ABSTRACT

Objective To investigate the effect of the HtrA serine peptidase 3(HTRA3)gene on choroidal neovascu-larization(CNV)and M2 macrophage polarization.Methods Fasting venous blood was collected from 30 patients with wet age-related macular degeneration(wAMD group)and 30 healthy subjects(normal group).The serum HTRA3 messen-ger ribonucleic acid(mRNA)level was detected by quantitative reverse transcription polymerase chain reaction(qRT-PCR).RF/6A cells were randomly divided into the control group,NC-sh group and HTRA3-sh group.Lentiviral vectors of NC-shRNA and HTRA3-shRNA were transfected into RF/6A cells in the NC-sh group and HTRA3-sh group by Lipo-fectamine2000.HTRA3 transfection was detected by qRT-PCR and Western blot.Then,the RF/6A cells were randomly di-vided into the N group,H group,H+NC-sh group and H+HTRA3-sh group.After cell transfection,RF/6A cells in the N group were cultured in a RPMI 1640 complete medium at a normoxia state,and cells in other groups were cultured in a RP-MI 1640 medium with 200 mmol·L-1 CoCl2 at a hypoxia state.Tubule formation was measured by Matrigel.The C57BL/6J mice were divided into the control group,CNV group,CNV+NC-sh group and CNV+HTRA3-sh group,with 12 mice in each group.Mice in the control group were unmodeled mice,and mice in the other groups were laser-induced CNV model mice.NC-shRNA and HTRA3-shRNA lentiviral vectors with a titer of 1 × 1011 TU·mL-1 were administered to mice in the CNV+NC-sh group and CNV+HTRA3-sh group via intravitreal injection.Mice in the control group and CNV group were in-jected with phosphate buffered saline.After 7 days of treatment,the mice were examined by fundus fluorescein angiogra-phy,and the eyeballs received hematoxylin & eosin staining.The mRNA levels of HTRA3,chitinase-like protein 3(Ym-1),arginase 1(Arg-1),inducible nitric oxide synthase(iNOS),cyclooxygenase-2(COX-2)and vascular endothelial growth factor(VEGF)in RF/6A cells or choroidal tissues were detected by qRT-PCR.The protein expression levels of HTRA3,VEGF and nuclear factor kappa B(NF-κB)p65 in RF/6A cells or choroidal tissues were detected by Western blot.Re-sults Compared with the normal group,serum HTRA3 mRNA level of patients in the wAMD group increased(t=11.804,P＜0.001).Compared with the control group and NC-sh group,the expressions of HTRA3 mRNA and protein in RF/6A cells in the HTRA3-sh group decreased(all P＜0.05).Compared with the N group,the number of closed lumen and the mRNA and protein expressions of HTRA3 and VEGF in RF/6A cells in the H group increased(all P＜0.05).Compared with the H+NC-sh group,the number of closed lumen and the mRNA and protein expressions of HTRA3 and VEGF decreased in RF/6A cells in the H+HTRA3-sh group(all P＜0.05).Compared with the control group,the mRNA and protein expression levels of HTRA3 increased,the relative fluorescence intensity of CNV increased,the mRNA levels of Ym-1 and Arg-1 in-creased,the iNOS and COX-2 mRNA levels decreased,and the NF-κB p65 protein expression level increased in mice of the CNV group(all P＜0.05).Compared with the CNV+NC-sh group,the mRNA and protein expression levels of HTRA3 de-creased,the relative fluorescence intensity of CNV decreased,the mRNA levels of Ym-1 and Arg-1 decreased,the mRNA levels of iNOS and COX-2 increased,and the NF-κB p65 protein expression level decreased in mice of the CNV+HTRA3-sh group(all P＜0.05).Conclusion Down-regulation of HTRA3 can inhibit the formation of CNV and the polarization of M2 macrophages.HTRA3 may be an important potential target for the prevention and treatment of wAMD.