This study evaluated the effects of genistein on the ERK signaling pathway and neovascularization in retinal tissue of ovariectomized diabetic rats. We showed that the ERK phosphorylation, VEGF, MMP2, and Nf-kB proteins level and inflammatory factors were significantly higher in the OVX.D group than in the sham group at four weeks after OVX surgery and STZ administration. Genistein reversed these effects in OVX.D.G groups in comparison to OVX.D groups. Also, glutathione decreased, and MDA increased in OVX.D, but genistein reversed these effects in OVX.D.G groups.
A causal association between diabetic abnormal high glucose level and the progress of retinal vascular dysfunction has been recognized, such that glucose regulation in diabetic patients decreases the development of the disease (Control and Group, 1993). Therefore, hyperglycemia has been associated with the activation of more critical signaling pathways, including nitrative and oxidative stress, which contribute to retinal neovascularization and diabetic retinopathies (Huang and Sheibani, 2008). Hypoxia/reoxygenation conditions in diabetes or any pathological conditions are the most important causes of ERK, MMP2, and VEGF expression, which are implicated in several neovascularization (Zhang and Tao, 2017). Molecular studies have been shown that ERK expression is related to vascular endothelial cell proliferation, thereby contributing to angiogenesis. Activated ERK was detected in accelerated neovascularization during wound healing and ischemia (Elsherif et al., 2014). Also, it has been shown that the expression of IL1β, ERK, and caspase 3 increases in the lungs of the OVX.D group, which genistein decreased them in lung tissue of ovariectomized diabetic rats (Daghigh et al., 2017). These results indicate the role of ERK in tissue damage. While genistein improves these effects. One of the present study findings was an increase of MMP2 in the OVX and ovariectomized diabetic group, which was decreased by genistein. Similar to this study, it has been shown that MMP proteins decrease in retinal pigment epithelium choroid complex when treated with soy isoflavone genistein after choroidal neovascularization induction (Kinoshita et al., 2014).
In the neovascularization process, angiogenic factors increased endothelial cell's basement membrane degradation, migration, and proliferation, causing new capillary tube formation (Hanahan and Folkman, 1996). The endothelial cell's basement membrane is disrupted by proteolytic enzymes such as MMPs (Vu and Werb, 2000). MMP-2 and MMP-9 play an essential role in the hydrolyzation of endothelial cell's basement membrane components (Kinoshita et al., 2014, Hiraoka et al., 1998). Also, our results showed that the NF-kB expression markedly increased in the ovariectomized diabetic group than in the sham control groups. This shows that MMP-2 was upregulated via the increased activity of the NF-kB. This result is consistent with the previous investigations, which showed that the NF-kB activation is increased proliferation, angiogenesis, and apoptosis in endometriosis(Kunicka et al., 2019, Calibasi-Kocal et al., 2019). It has been shown that NF-βB acts via regulation of the MMPs expression in the adhesion and invasion of endometriosis cells on the peritoneal surface (Lee et al., 2010, Gottschal et al., 2002). A previous study has shown that MMP-2 is expressed in the endometrial graft in the peritoneum (Ueda et al., 2002).
Sex hormone estrogen increases transcription factors that modify the activity of chief proinflammatory cytokines (Corcoran et al., 2010). The sex hormone estrogen receptors in the retina suggest that estrogen has many functions in this tissue. Additionally, the estrogen has potent anti-oxidation properties, which may cause neuroprotection without including a receptor-mediated process (Azcoitia et al., 2011).
Oxidative stress and neovascularization lead to diabetic retinopathy, an essential complication of diabetes mellitus (DM) and the leading causes of blindness and visual complication in diabetic populations (Varma et al., 2007). Previous studies have shown that hyperglycemia can increase mitochondrial reactive oxygen species (ROS), stimulates many signaling pathways causing tissue injury (Wu et al., 2014). Retinal ROS increasing stabilizes hypoxia-inducible factor-1𝛼 and causes the upregulation of VEGF and other angiogenic factors (Masuda et al., 2017).
VEGF is distributed in animal and human organs such as the brain, kidney, liver, eye, and other tissues. Under normal conditions, there is low content of VEGF in the retinal cells (Sant et al., 2018). Under physiological conditions, the low concentration of VEGF in the eyes is essential to preserve the integrity of the ocular blood vessels. However, overexpression of this angiogenic factor in ocular tissue will cause neovascularization (Du et al., 2018). VEGF plays a vital role in the pathogenesis of diabetic retinopathy, which is produced from the retinal ganglion cells, pigment epithelium cells, smooth muscle cells, and Muller cells in the human choroids and retinas, and its expression is regulated mainly by tissue oxygen levels (Ishida et al., 2003). In the present study, we showed VEGF overexpression in the ovariectomized diabetic group.
Similar to our investigation, in the previous studies, it has been shown that VEGF expression increases in the retina of STZ-induced diabetic rats. These results suggest that diabetes induces neovascularization through increasing VEGF expression in the retinas. Our results showed that genistein decreases the VEGF levels in the OVX.D.G group.
On the other hand, estrogen insufficiency by menopause or ovariectomy causes inflammation and stress oxidation by increasing the TNF-α and MDA levels, respectively, which can cause eye complications in these patients (Daghigh et al., 2017). In this study, we showed that oxidative factors increased and anti-oxidants decreased in OVX.D. However, genistein reversed these effects in OVX.D.G groups; reducing oxidative stress may lead to a decrease in neovascularization in the retina by reducing VEGF and inflammatory factors.
Moreover, blockade of ERK signaling pathway decreases VEGF induced proliferation of endothelial cell in a placental artery (Liao et al., 2009). MMP2 is another factor involved in angiogenesis. The processing of angiogenesis is related to vascular basement membrane degradation, necessary for migration of endothelial cells and proliferation (Song et al., 2015).
VEGF expression is increased by hypoxia and hyperglycemia, two major causes of retinal neovascularization. Besides, VEGF is regulated by ERK signaling cascade (Hashimoto et al., 2006). In the present study, we showed that ERK phosphorylation, VEGF, MMP2, and Nf-kB proteins level were significantly higher in the OVX.D group that it can induce retinal neovascularization and injuries. Nevertheless, genistein reversed these effects in the retina of OVX.D.G.