Dendrobium nobile Lindley and its bibenzyl component moscatilin are able to protect retinal cells from ischemia/hypoxia by dowregulating placental growth factor and upregulating Norrie disease protein

Defects in vasculogenesis (early retinal vessel development) seem to be mediated through the Norrin-dependent Wnt signaling pathway [1‐3]. Norrin/Frizzled-4 signaling seems to play a crucial role in vasculogenesis such as in Norrie disease and familial exudative vitreoretinopathy [1‐4], which might eventually progress into retinal ischemia and neovascularization (NV; angiogenesis). In addition to Norrie disease and familial exudative vitreoretinopathy, there were other developmental retinal vascular disorders, namely Coats disease and persistent hyperplastic primary vitreous that share similar fundus pictures, namely peripheral retinal avascularization and subretinal exudation [1‐5]. As above mentioned, these vitreoretinopathies may also cause retinal ischemia, thus giving rise to a similar threat to the patient’s vision, although they are not as common as other retinal ischemic disorders such as central/branch retinal artery occlusion (CRAO/BRAO), central/branch retinal vein occlusion (CRVO/BRVO), glaucoma, diabetic retinopathy (DR) and neovascular age related macular degeneration (nvAMD) [6]. As indicated by Beck et al. (2017) [7], persistent hypoxia has been assumed to be one of the major driving forces involved in progression of these developmental retinal vascular disorders such as Norrie disease [5, 7]. Based on the role of Norrin in vasculogenesis [1‐5, 7], we investigated here whether the level of Norrie disease protein (NDP; Norrin) might change in cells subjected to a hypoxia model system (oxygen glucose deprivation, OGD). We also investigated whether moscatilin, the bibenzyl component of Dendrobium nobile Lindley (DNL), might have the capacity to upregulate the expression level of NDP, which could potentially protect retinal cells from ischemia/hypoxia that seems to induce the progression of these developmental retinal vascular disorders.

Retinal ganglion cells (RGCs) and amacrine cells in the inner retina are susceptible to ischemia/reperfusion (I/R) [8]. Moreover, vimentin/glial fibrillary acidic protein (GFAP) immunolabelling of Müllers is elevated after ischemia [9]; this is also associated with a reduction in RGC numbers [8]. Overexpression of vascular endothelium growth factor (VEGF), of hypoxia inducible factor (HIF-1α), of pyruvate kinase M2 (PKM2) and of retinoblastoma-binding protein 2 (RBP2) are also known to occur together in the ischemic retina [8, 10‐12] and further abnormal NV (late neovessel formation) can lead to visual dysfunction due to edema and hemorrhage. Upregulation of HIF-1α and VEGF has also be observed in the Norrin depleted retina [5]. In addition to VEGF [8, 10, 12], placental growth factor (PLGF) has been reported to be increased when there are defined ischemic disorders of the retina/choroid vasculature; thus, downregulation of this factor is able to be utilized as a biomarker for visual functional outcome and treatment [13]. The present study aims to provide further confirmation regarding these ischemic alterations.

As described in “An Illustrated Chinese Materia Medica”, DNL (a member of the Orchidae family) is a “vision improving” herbal. DNL has also been used as a tonic and found to have antipyretic/anti-inflammatory effects [14] and anti-angiogenic (e.g. anti-VEGF/HIF-1α) properties [15‐17]. DNL has several active ingredients with various action mechanisms, including alkaloids (Tissue necrosis factor receptor 1 protein overexpression via inhibiting the p-p38 mitogen activated protein kinase and NF-ĸB pathway) [18], flavonal glycosides (α-glucosidase inhibitors) [19], SG-168 and polysaccharides (antioxidative effects) [20, 21]. Furthermore, an anti-angiogenic or anti-oxidative compound moscatilin is one of the active bibenzyl compounds present in DNL and this chemical seems to have a range of effects. These include an anti-VEGF/HIF-1α effect, where it acts as. OH radical scavenger, an anti-inflammation effect and an anti-apoptosis effect [15‐17, 22, 23]; there are also other unknown mechanisms of action that are different to those mentioned above. In other words, DNL could possess a number of distinct therapeutic effects that may be shared with its component moscatilin.

The aim of the present study is to examine whether DNL is able to attenuate retinal ischemic injury (see also Ischemia induction in the Methods) in the rat. Additionally, the effects of DNL and its mechanisms of action were assessed by electrophysiology, by examining the thickness of various retinal layers, by assessing RGC number, and by examining choline acetyl transferase (ChAT) immuolabeling in amacrine cells and by observing vimentin/GFAP immunoreactivity in Müller cells. Moreover, protein expression levels of HIF-1α, VEGF, PKM2 and RBP2 were analyzed. Furthermore, we for the first time investigated under hypoxia/ischemia the effect of DNL and, of novelty and significance, of its bibenzyl component moscatilin (0.1 μM; non-toxic at the concentration≤1 μM) [23] on vasculogenesis/angiogenesis in relation to the expression levels of NDP (norrin)/PLGF. As part of the NDP study, a RGC-5 cell system (retinal neuronal progenitors; see also in vitro studies in the Methods) model, namely OGD, was used to investigate the mechanisms involved in hypoxic/ischemic-like injury; this part of the study involved the assessment of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cell viability and the measurement of the expression levels of NDP by the Western blotting assay.

As mentioned in the Introduction, many of DNL’s various active components, including alkaloids, flavonal glycosides, SG-168 and polysaccharides, have known action mechanisms [18‐21]; on the other hand, moscatilin is also an active ingredient (bibenzyl) of DNL seems to have a novel mode of action. In the present study, we found that the protein levels of HIF-1α, VEGF, PKM2 and RBP2 were significantly upregulated in the ischemic retinas, which agrees with previous studies [8, 10‐12], but importantly the significant upregulation events affecting these proteins were significantly mitigated by administration of DNL; furthermore, this did not happen with vehicle alone (Fig. 8a). As shown in Fig. 8b, the ischemia-induced elevation of PLGF [13] was also significantly blunted when ischemic retinas were pre-administrated with the VEGF trap/anti-PLGF Eylea. This is similar to the effect of 1.0 g/Kg/day of DNL. This finding suggests that DNL might have a novel and clinical significant anti-angiogenesis/VEGF (PLGF) trapping effect. This is not inconsistent with previous reports where a bibenzyl component of DNL, moscatilin, has been shown to act as an anti-angiogenesis agent and inhibit HIF-1α and VEGF [15, 16, 22].

Up to the present, it has been believed that ischemia is likely to be very similar in mode of action to various diseases such as CRVO/BRVO/CRAO/BRAO, nvAMD and DR, as well as various developmental retinal vasculopathies such as familial exudative vitreoretinopathy and Norrie disease. Over the past twenty years, anti-VEGF antibodies have been used to clear ocular hemorrhage and macular edema effectively in many cases; however, disappointingly, poor visual results do occur in some patients. An increasing body of evidence supports a role of the norrin-dependent Wnt-signaling pathway in both the early normal development of retinal vessels and in the late progression of defined developmental retinal vascular diseases [1‐5, 7]. The latter condition may possibly further aggravate ischemia/hypoxia and form NV. Consistently, NDP (norrin) seems to protect the eye from abnormal angiogenesis and retinopathy and it does this by modulating the norrin-dependent Wnt signaling pathway [30]. Moreover, overexpression of NDP has been shown to protect photoreceptors and RGCs from cell death via activation of the norrin-dependent Wnt signaling pathway [31, 32]. Presently, hypoxia (OGD) is known to lead to a significant decrease in the level of NDP (Fig. 2) as well as to cause a significant decrease in the cell viability (Fig. 1). Furthermore, a significant nullification in the OGD induced reduction in the NDP level (Fig. 2) and cultured cell number (Fig. 1) in the presence of moscatilin (0.1 μM), implies that this bibenzyl ingredient of DNL is to be able to significantly alleviate hypoxic/ischemic-like (OGD) injury. As supported by the present results and by various previous reports [5, 7, 15, 16, 22, 30‐32], DNL and/or moscatillin would seem to be able to activate the NDP (norrin)-dependent Wnt signaling pathway (Graphical Abstract for DNL & moscatilin as anti-PLGF & NDP stimulator; Additional file 1) and thus provide neuroprotection against retina ischemia (Figs. 1, 3, 4, 5, 6 and 7). This presumably occurs via an inhibition of VEGF-A/PLGF (Fig. 8) and an upregulation of NDP (Fig. 2). This approach seems to be a novel promising way of protecting against retinal ischemia that should further terminate abnormal vasculogenesis, ischemia associated neovascularization (angiogenesis), and the progression of various developmental vascular disorders that are associated with persistent ischemia/hypoxia [5, 7].

After ischemic insult and the administration of vehicle, the inner retinal thickness (Fig. 4), the number of RGCs (Fig. 5) and the ChAT immunoreactivity of amacrine cells (Fig. 6) were significantly/obviously decreased, which is not inconsistent with a previous report [8]. Importantly, our findings in the present study also confirm that these ischemia induced changes were significantly or obviously blunted by pre- administration or post-administration of a high dose of DNL (at 1 g/kg/day). Moreover, in the ischemic retinas with pre−/post-administration of vehicle, vimentin/GFAP immunolabeling overexpression (Fig. 7) was found to parallel the decrease in b-wave (Fig. 3). This is of clinical importance because the present results also show that these ischemic alterations are significantly or obviously counteracted by pre-administration or post-administration of DNL at 1 g/kg/day.

The present results demonstrate that ischemia/hypoxia (ischemia-mimetic OGD), significantly or obviously affects the retina electrophysiologically (Fig. 3), morphometrically (Fig. 4), and immunohistochemically (Figs. 5, 6 and 7) as well as affect at the level of molecular biology and cellular viability (Figs. 1, 2 and 8). In terms of clinical situation, all of these changes following ischemia/OGD are effectively attenuated by pre-treatment or post-treatment with DNL or its bibenzyl component moscatilin (Figs. 1, 2, 3, 4, 5, 6, 7 and 8). This is the first study to show that the Chinese herb DNL (and its bibenzyl ingredient moscatilin) [22, 33] might be able to electrophysiologically, morphometrically, immunohistochemically and molecular biologically protect against the retinal ischemic/ischemic-like injury. These protective mechanisms are presumed to act by suppressing the upregulation of HIF-1α, VEGF-A, PKM2, RBP2, and, above all, PLGF, as well as, possibly, by upregulating the level of NDP (Figs. 2 and 8).

Taking the above findings as a whole, DNL and/or moscatilin seems to be able to protect against or even prevent defined retinal ischemic/ischemic-like alterations and this is likely to occur via the inhibition of the PLGF and also probably via the upregulation of NDP. DNL treatment (and/or moscatilin) might be an useful way of providing a complementary approach that helps to prevent and/or manage the developmental vascular disorders such as Norrie disease in patients where the diseases might progress due to the presence of persistent ischemia/hypoxia [5, 7].

The present study has that demonstrated various ischemic/hypoxic (OGD) alterations that occur in the retina or in retinal cells and these can be monitored by electroretinography, immunohistochemistry (RGCs, amacrine cells, Müller cells), histopathology (retinal thickness), cell viability and measurement of expression levels of various proteins (PLGF, HIF-1α, VEGF-A, PKM2, RBP2 and NDP). Of clinical significance and novel to this study, the protein concentration of PLGF (Fig. 8) was found to be upregulated in such circumstances and that of NDP (Fig. 2) was downregulated. Moreover, and importantly, treatment with DNL/moscatillin (DNL’s bibenzyl ingredient) significantly counteracted these alterations. Furthermore, neither moscatilin nor DMSO has an effect on cell numbers in the control groups [moscatilin (0.1 μM) + DMEM or DMSO (50 μl) + DMEM]. DNL/moscatillin might safely provide an alternative way to prevent and/or manage patients with the persistent hypoxia/ischemia associated progression that occurs in various developmental vascular disorders such as Norrie disease; this might occur via a downregulation of the level of PLGF and an upregulation of the concentration of NDP.