Inhibitory effects of safranal on laser-induced choroidal neovascularization and human choroidal microvascular endothelial cells and related pathways analyzed with transcriptome sequencing
Authors:   Department of Ophthalmology, Affiliated Hospital of Inner Mongolia University for Nationalities, Tongliao 028007, Inner Mongolia Autonomous Region, China, Qin-Xiao Qin-Xiao , Lu Zhan-Jun ,   Department of Ophthalmology, Affiliated Hospital of Inner Mongolia University for Nationalities, Tongliao 028007, Inner Mongolia Autonomous Region, China, Zhang Tian-Zi ,   Department of Ophthalmology, Affiliated Hospital of Inner Mongolia University for Nationalities, Tongliao 028007, Inner Mongolia Autonomous Region, China, Li Shan-Shan ,   Department of Ophthalmology, Affiliated Hospital of Inner Mongolia University for Nationalities, Tongliao 028007, Inner Mongolia Autonomous Region, China, Hua Ting ,   College of Mongolian Medicine, Inner Mongolia University for Nationalities, Tongliao 028000, Inner Mongolia Autonomous Region, China, Suriguga Suriguga ,   College of Mongolian Medicine, Inner Mongolia University for Nationalities, Tongliao 028000, Inner Mongolia Autonomous Region, China, Chen Wen-Lin ,   College of Mongolian Medicine, Inner Mongolia University for Nationalities, Tongliao 028000, Inner Mongolia Autonomous Region, China, Ran Lin-Lin ,   College of Mongolian Medicine, Inner Mongolia University for Nationalities, Tongliao 028000, Inner Mongolia Autonomous Region, China, Yu Wen-Zhen ,   Department of Ophthalmology, Peking University People&#x2019;s Hospital, Beijing100044, China, Yang Fei ,   Department of Ophthalmology, Peking University International Hospital, Beijing 100026, China, Burenbatu Burenbatu ,   Department of Hematology, Affiliated Hospital of Inner Mongolia University for Nationalities, Tongliao 028007, Inner Mongolia Autonomous Region, China
Published: June 2021

AIM: To determine the effects of safranal on choroidal neovascularization (CNV) and oxidative stress damage of human choroidal microvascular endothelial cells (HCVECs) and its possible mechanisms.
METHODS: Forty-five rats were used as a laser-induced CNV model for testing the efficacy and safety of safranal (0.5 mg/kg·d, intraperitoneally) on CNV. CNV leakage on fluorescein angiography (FA) and CNV thickness on histology was compared. HCVECs were used for a H2O2-induced oxidative stress model to test the effect of safranal in vitro. MTT essay was carried to test the inhibition rate of safranal on cell viability at different concentrations. Tube formation was used to test protective effect of safranal on angiogenesis at different concentrations. mRNA transcriptome sequencing was performed to find the possible signal pathway. The expressions of different molecules and their phosphorylation level were validated by Western blotting.
RESULTS: On FA, the average CNV leakage area was 0.73±0.49 and 0.31±0.11 mm2 (P?=?0.012) in the control and safranal-treated group respectively. The average CNV thickness was 127.4±18.75 and 100.6±17.34 μm (P=0.001) in control and safranal-treated group. Under the condition of oxidative stress, cell proliferation was inhibited by safranal and inhibition rates were 7.4%-35.4% at the different concentrations. For tube formation study, the number of new branches was 364 in control group and 35, 42, and 17 in 20, 40, and 80 μg/mL safranal groups respectively (P<0.01). From the KEGG pathway bubble graph, the PI3K-AKT signaling pathway showed a high gene ratio. The protein expression was elevated of insulin receptor substrate (IRS) and the phosphorylation level of PI3K, phosphoinositide-dependent protein kinase 1/2 (PDK1/2), AKT and Bcl-2 associated death promoter (BAD) was also elevated under oxidative stress condition but inhibited by safranal.
CONCLUSION: Safranal can inhibit CNV both in vivo and in vitro, and the IRS-PI3K-PDK1/2-AKT-BAD signaling pathway is involved in the pathogenesis of CNV.