Ginsenoside Rh2 Inhibits Vascular Endothelial Growth Factor-induced Corneal Neovascularization

Different insults to the cornea,including ischemia and inflammation,induce ocular surface injury,leading to corneal neovascularization（CoNV）,damaging to the patients’vision.Vascular endothelial growth factor（VEGF）plays a pivotal role in the process.Hence,it is important to inhibit the expression of VEGF in controlling the disease.Ginsenoside Rh2（GRh2）is a medicinal ingredient extracted from ginseng which shows anti-tumor properties.However,the effect of GRh2 on CoNV remained unreported.Objective:The study aimed to investigate the effect of GRh2 on VEGF-induced angiogenic responses,and examine the underlying mechanisms.Methods:（1）We adopted human umbilical vein endothelial cells（HUVECs）for vitro study.HUVEC cells were treated with GRh2 for 48 hours and a（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT）assay performed.After treated with GRh2（1 and 10μM）for 30 min,the HUVECs were cultured with VEGE（10ng/mL）.MTT assay,BrdU incorporation assay,[H³]Thymidine incorporation assay and Ki-67 immunofluorescence assay were used to detect cell proliferation.Scratch wound healing assay and“Transwell”migration array were used to detect cell migration.Tube formation assay was used to detect cell tube formation.（2）HUVECs were cultured in vitro,after pre-treatment of GRh2 and VEGF,Western Blot was used to detect the expression of related proteins in VEGFR2 signaling pathway and downstream signaling pathways,including PI3K-AKT and ERK-MAPK.To examine the effect of GRh2 on the association of VEGFR2 and downstream signaling components,a co-immunoprecipitation（“Co-IP”）assay was performed.（3）The puromycin-selected stable HUVECs,expressing Gab1 shRNA（shGab1,“a/b”）or the scramble control shRNA（“shSCR”）,as well as the parental control HUVECs（“Pare”）,were treated with VEGF（10 ng/mL）for 10 min,expressions of related proteins in PI3K-AKT and ERK-MAPK signaling pathways were tested by Western Blot.The stable HUVECs,expressing Gab1 shRNA（shGab1,“b”）or the scramble control shRNA（“shSCR”）,were pretreated with GRh2（10μM）for 30 min,followed by VEGF（10 ng/mL）administration,cells were further cultured for the applied time,cell proliferation was tested by MTT assay and BrdU ELISA assay;cell migration was tested by Transwell assay.（4）Twelve-week-old male mice were received alkali burn on the central cornea,which were also treated with/out 1 mg/mL GRh2（five times a day）or PBS（vehicle control）.On day-7,CoNV was observed by slit-lamp.Hematoxylin-eosin（HE）staining was employed to detect the CoNV and inflammatory cell infiltration after alkali burn.Total mRNA was isolated from the vascularized areas of cornea with alkali burn and relative expressions of IL-1βmRNA,IL-6 mRNA,TNF-αmRNA,VEGF mRNA,MPP-9 mRNA,the human ortholog of NR₀33585,PEDF mRNA and chr8:129102060-129109035 reverse strand were tested by Real-time RT-PCR.Results:（1）GRh2 inhibited VEGF-induced HUVEC cell proliferation,migration and tube formation significantly in vitro.（2）Pre-treatment with GRh2（1 and 10μM）inhibited VEGF-induced AKT and ERK activation in HUVECs significantly.VEGF-induced Gab1 activation was largely inhibited by GRh2 pre-treatmen.VEGF-induced association between VEGFR2 and Gab1 was largely inhibited by GRh2（10μM）.（3）The knockdown of Gab1 largely inhibited VEGF-induced phosphorylation of AKT and ERK.VEGF-induced HUVEC proliferation and migration were largely inhibited by shGab1.In Gab1-silenced HUVECs,adding GRh2 was unable to further inhibit VEGF-induced cell proliferation and migration.（4）GRh2 inhibits alkali-burn-induced neovascularization and inflammatory cell infiltration in mouse cornea.The mRNA expression of IL-1β,IL-6,TNF-α,VEGF and MPP-9 as well as the expression of lncRNA the human ortholog of NR₀33585 were significantly increased in the vascularized area of alkali-burned cornea.Conversely,expression of PEDF mRNA and the lncRNA chr8:129102060-129109035 reverse strand were decreased.Conclision:In summary,GRh2 inhibits VEGF-induced angiogenic effect possibly via inhibiting VEGFR2-Gab1 signaling in vitro.It also inhibits angiogenic and inflammatory responses in alkali-treated mouse cornea.These results indicate that GRh2could be further tested as a potential supplement for the treatment for CoNV.