Inhibition of Hypoxia-Induced Retinal Angiogenesis by Specnuezhenide, an Effective Constituent of Ligustrum lucidum Ait., through Suppression of the HIF-1α/VEGF Signaling Pathway.

Jianming Wu,Xiao Ke,Na Ma,Wei Fu,Manxi Zhao,Xiaoping Gao,Zhirong Zhang,Xiaofeng Hao,Hongcheng Zhang,Wei Wang

doi:10.3390/molecules21121756

Abstract

Specnuezhenide (SPN), one of the main ingredients of Chinese medicine “Nü-zhen-zi”, has anti-angiogenic and vision improvement effects. However, studies of its effect on retinal neovascularization are limited so far. In the present study, we established a vascular endothelial growth factor A (VEGFA) secretion model of human acute retinal pigment epithelial-19 (ARPE-19) cells by exposure of 150 μM CoCl2 to the cells and determined the VEGFA concentrations, the mRNA expressions of VEGFA, hypoxia inducible factor-1α (HIF-1α) & prolyl hydroxylases 2 (PHD-2), and the protein expressions of HIF-1α and PHD-2 after treatment of 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1, 1.0 μg/mL) or SPN (0.2, 1.0 and 5.0 μg/mL). Furthermore, rat pups with retinopathy were treated with SPN (5.0 and 10.0 mg/kg) in an 80% oxygen atmosphere and the retinal avascular areas were assessed through visualization using infusion of ADPase and H&E stains. The results showed that SPN inhibited VEGFA secretion by ARPE-19 cells under hypoxia condition, down-regulated the mRNA expressions of VEGFA and PHD-2 slightly, and the protein expressions of VEGFA, HIF-1α and PHD-2 significantly in vitro. SPN also prevented hypoxia-induced retinal neovascularization in a rat model of oxygen-induced retinopathy in vivo. These results indicate that SPN ameliorates retinal neovascularization through inhibition of HIF-1α/VEGF signaling pathway. Therefore, SPN has the potential to be developed as an agent for the prevention and treatment of diabetic retinopathy.

Highlights

Beta-actin was was used used as with as the the loading loading control. These results indicate that the inhibition of SPN on vascular endothelial growth factor A (VEGFA) secretion of acute retinal pigment epithelial-19 (ARPE-19) cells under hypoxia
After we demonstrated the inhibitory effect of SPN on VEGFA secretion in ARPE-19 cells vitro, we further investigated the preventive effect of SPN on the hypoxia-induced retinal in vitro, we further investigated the preventive effect of SPN on the hypoxia-induced retinal neovascularization with superficial vascular plexuses using ADPase staining in a rat pup model
We found the mRNA expressions of VEGFA and prolyl hydroxylases 2 (PHD-2) and the protein expressions of VEGFA, hypoxia inducible factor-1α (HIF-1α) and prolyl neovascularization hydroxylases (PHDs)-2 were induced in hypoxia-mimicking condition by CoCl2

Summary

Introduction

The fruit of Ligustrum lucidum Ait. (L. lucidum), known as “Nü-zhen-zi” in Chinese, is a famous traditional Chinese medicine with liver-, kidney- and vision-improving functions [1,2].As a well-known Chinese traditional medicine, the use of L. lucidum for its immunomodulation [3], antioxidant [4,5,6], anti-cancer [7], hypolipidemia [8], neuroprotection [9], hypoglycemia [10,11], Molecules 2016, 21, 1756; doi:10.3390/molecules21121756 www.mdpi.com/journal/molecules anti-atherosclerosis [12], periodontal pathogen inhibition [13], hepatoprotection [14], antiviral [15], anti-osteoporosis [16], anti-obesity [17], anti-hepatitis C virus [18]and other activities has been reported.[12], periodontal pathogen inhibition [13], hepatoprotection [14], antiviral [15], anti-osteoporosis [16], phytochemical studies have[18]revealed the activities main chemical components of L. lucidum anti-obesityC virus and other has been reported. As a well-known Chinese traditional medicine, the use of L. lucidum for its immunomodulation [3], antioxidant [4,5,6], anti-cancer [7], hypolipidemia [8], neuroprotection [9], hypoglycemia [10,11], Molecules 2016, 21, 1756; doi:10.3390/molecules21121756 www.mdpi.com/journal/molecules anti-atherosclerosis [12], periodontal pathogen inhibition [13], hepatoprotection [14], antiviral [15], anti-osteoporosis [16], anti-obesity [17], anti-hepatitis C virus [18]. Revealed the activities main chemical components of L. lucidum anti-obesity. To include terpenoids, iridoid glycosides, flavonoids, phenethyl alcohol glycosides, phytochemical studies have revealed the main chemical components of L. lucidum to volatile include oils, phospholipids, polysaccharides, fatty acids, amino acids and so on [19]. Methyl terpenoids, iridoid glycosides, flavonoids, phenethyl alcohol glycosides, volatile oils, phospholipids, polysaccharides, fatty acids, amino acids and so on [19]. It Recently, it has been reported that some preparations mainly composed of L. lucidum, such as has been reported that some preparations mainly composed of L. lucidum, such as Tangmuning

Methods

Discussion

Conclusion