Xue-fu-Zhu-Yu decoction protects rats against retinal ischemia by downregulation of HIF-1α and VEGF via inhibition of RBP2 and PKM2

Retinal ischemia related diseases include central retinal artery occlusion (CRAO), central retinal vein occlusion (CRVO), branch retinal artery occlusion (BRAO), branch retinal vein occlusion (BRVO), glaucoma, and age-related macular degeneration (AMD) [1‐3]. All of these disorders can result in serious complications and thus the treatment of retinal ischemia is vital to patient outcomes.

Glutamate receptor rich retinal ganglion cells (RGCs) and amacrine cells, and their neuronal fibers are situated in the inner retina, which makes them susceptible to ischemia/reperfusion (I/R) injury [1, 3]. After I/R, enhanced vimentin immunoreactivity in Müller cells can be seen [4]. Retinal or choroidal ischemia associated angiogenesis [5] often results in subretinal fluid and bleeding, which in turn leads to visual dysfunction. It is thus important to discover novel approaches that are able to stimulate self-protective mechanisms within the eye and that help to avoid detrimental neovascularization. Elevated concentrations of the hypoxia-inducible factor-1α (HIF-1α) have been demonstrated after retinal ischemia [6]. HIF-1α interacts with hypoxia dependence components of various hypoxia related genes and up-regulates the level of vascular endothelium growth factor (VEGF) [6]. Liu et al. [7] have also shown that oxidative stress in the human retinal pigment epithelium leads to VEGF up-regulation. All the above results support the hypothesis that up-regulation of HIF-1α and VEGF in the retina is directly related to ischemic insult. The KDM5/JARID1 family of Fe(II)- and α-ketoglutarate-dependent demethylases remove methyl groups from tri- and dimethylated lysine 4 of histone H3. Figure 2c of Wang et al. [8] demonstrated that purified Jumonji demethylases representing several subfamilies exhibited distinct sensitivities to JIB-04 in vitro, with JARID1A (KDM5A, also known as retinoblastoma-binding protein 2, RBP2) being the most sensitive (IC50 = 230 nM). Other proteins include JMJD3 (KDM6B) and JMJD2C (KDM4C), which were more resistant (IC50 ~ 1 μM) to JIB-04. Presently, JIB-04, a paninhibitor of the Jumonji demethylase superfamily, was thus selected to observe the inhibition of RBP2 upregulation in retinal ischemia. Pyruvate kinase (PK), glycolytic enzyme, catalytically convert phosphoenolpyruvate into pyruvate, generating one molecule of ATP. In addition, the PKM2 inhibitor shikonin [9], and the anti-VEGF agent avastin was presently selected to observe the inhibition of PKM2 and/or VEGF overexpression after retinal ischemic insult. An investigation as to whether an overexpression of VEGF, HIF-1α, PKM2 and RBP2 coexists in the ischemic retina has also been included in the present investigation.

Xue-Fu-Zhu-Yu decoction (XFZYD) is a famous traditional Chinese medicine formula that has been used to manage with cardiovascular disorders for several centuries [10]. The study of Lee et al. [11] suggested that XFZYD increased the potential of recombinant tissue plasminogen activator (tPA) as a neuroprotection agent against rat brain ischemia. The report of Zhou et al. [12] has also indicated that XFZYD seems to be able to alleviate hypoxia and protect liver sinusoidal endothelial cell function and does this by decreasing the levels of VEGF and HIF-1α. The formulation of XFZYD consists of eleven components (with respective representative active compounds in the bracket), namely Radix Angelicae Sinensis (Dang Gui: ferulic acid) [13, 14], Radix Rehmanniae glutinosae (Di Huang: catalpol) [15], Radix Paeoniae Rubra (Chi Shao: peoniflorin) [16], Rhizoma Ligustici Chuanxiong (Chuan Xiong: ferulic acid) [13, 14], Semen Pruni Persicae (Táo Rén: amygdalin) [17], Flos Carthami Tinctorii (Hong Hua: hydroxysafflor yellow A, HSYA) [18], Radix Glycyrrhizae Uralensis (Gan Cao: glycyrrhizin) [19], Fructus Auranti (Zhi Qiao: costunolide) [20], Radix Bupleuri Chinense (Chai Hu: saikosaponin) [21], Radix Achyranthis Bidentatae (Niu Xi: Achyranthes bidentata polysaccharides) [22], and Radix Platycodi Grandiflori (Jie Geng: platycodins) [23]. The important known effects of the defined active compounds described above are as follows. Ferulic acid has been proved to scavenge hydroxyl radical and is able to provide neuroprotection against retinal ischemia [13, 14]. Catalpol and the Achyranthes bidentata polysaccharides have antioxidative potential [15, 22]. Peoniflorin [16], amygdalin [17], HSYA [18], glycyrrhizin [19], costunolide [20] and saikosaponin [21] are able to protect against brain ischemia. Furthermore, amygdalin and HSYA have been shown to act additively by decreasing plasma viscosity and platelet aggregation. Aqueous extract from Platycodon grandiflorum such as platycodins [23] has a significant hepatoprotective effect and acts by reducing nitric oxide and counteracting lipid peroxidation dose-dependently.

The purpose of the current research is to investigate if XFZYD is able to alleviate retinal ischemic insult and to explore the decoction’s influences on electroretinogram (ERG), the density of RGCs, retinal thickness, choline acetyltransferase (ChAT) immunopositive amacrine cells, and glial fibrillary acidic protein (GFAP) immunolabelling of Müller cells and vimentin immunolabelling of Müller cells. In addition, the mRNA and protein concentrations of VEGF, HIF-1α, PKM2 and RBP2 were analyzed by real-time polymerase chain reaction (rtPCR) and Western blot, respectively, in the presence/absence of XFZYD.

Ischemia is widely accepted to be involved in vision-threatening retinal disorders such as, proliferative diabetic retinopathy (PDR) [26], wet age-related macular degeneration (wAMD) [27] and central/branch retinal vein or artery occlusion [4]. Furthermore, although, anti-VEGF antibodies are clinically utilized world-wide, these are not completely effective when treating these conditions [28]. Moreover, disappointing visual results often occur in patients who have been treated with anti-VEGF drugs, even though the vitreous/subretinal blood and cystoid macular edema have been effectively treated. KDM5A (JARID1A; RBP2) was originally identified as a factor that interacts with the RB gene product [29, 30]. In mammals, there are four PK isoforms including PKM2, which has been found in the retina, an extension of the central nervous system, and the brain [31‐33]. Avastin is widely known to target all VEGF-A isoforms; isotype 165 is most abundant and biologically active in the eye. The mode of action of another recently launched anti-VEGF aflibercept is involved in VEGF-A, VEGF-B and placental growth factor. As mentioned in the Introduction, PKM2 and RBP2 co-activate HIF-1α, which further triggers VEGF secretion and induces possible subsequent angiogenesis in the ischemic/hypoxic conditions [6, 7, 34‐36]. However, there remained a need to investigate whether PKM2 might be an upstream factor that links with the activation of RBP2.

Furthermore, there is also a clinical necessity to identify anti-ischemic agents that possess novel pharmaceutical mechanisms, such as blocking RBP2 and PKM2. The enhanced mRNA/protein concentrations of RBP2 and PKM2, as well as the consequential stimulation of HIF-1α and VEGF mRNA/protein levels as measured during the present investigation are clearly associated with retinal ischemia and, possibly, wAMD. As also mentioned in the Introduction, XFZYD has been found to potentiate recombinant tPA-mediated neuroprotection against ischemic stroke in the rats [11] and act as an antioxidant. It is also well known that Radix paeoniae rubra, Rhizoma chuanxiong, Semen pruni persicae, and Carthamus tinctorius have an ability to ameliorate the blood circulation and clean up the blood clot as mentioned [11, 13, 14, 16‐18, 37]. If the present study helps to identify novel protective mechanisms, CJDHW could offer novel means of coping with the various retinal ischemic diseases mentioned earlier that threaten vision.

GFAP and Vimentin immunoreactivity were enhanced in Müller cells following retinal I/R [3], and this is supported by the current result (Figs. 5c and 6c). Dysfunctional Müller cells produce a negative influence on normal ERG b-wave [3, 38, 39], which is also found in this present results (Fig. 1). When retinas are subjected to ischemia and preadministrated with vehicle, enhanced vimentin/GFAP immunoreactivity was found to be present simultaneously with the reduction in b-wave amplitude. Notably, these ischemic changes were mitigated when there was preischemia or postischemia administration of XFZUD to the experimental rats.

After retinal I/R, glutamate receptor-containing neurons, for example RGCs and amacrines, which are present in the inner retina are widely accepted to be liable to injury [1, 3, 24, 25, 39]. In the present study, following I/R, it was confirmed that the number of RGCs was significantly decreased (Fig. 2b and f) and retinal thickness, especially inner retinal thickness, was significantly reduced (Fig. 3b and f). In addition, the result of ChAT immunohistochemistry showed that the number of the amacrine cell bodies was significantly fewer (Fig. 4c and g). Of particular clinical importance, our findings also reveal that preischemia or postischemia administration of XFZYD is able to bring about a significant counteraction of these ischemic features, namely an attenuation of the ischemia-induced decrease in the number of the RGCs (Fig. 2c~f), an alleviation of the ischemia-associated decrease in the inner/whole retina thickness (Fig. 3c~f), as well as mitigation of the ischemia-related reduction in amacrine cell bodies (Fig. 4d~g).

Previous studies have found that HIF-1α plays a vital part in the activation of hypoxia related VEGF release [6, 35, 40]. Moreover, Luo et al. [40] found that PKM2 reacts with HIF-1α in a physical and functional manner to trigger the linkage of HIF-1α with target genes, including coactivators and gene transcription. Moreover, Qi et al. [35] indicated that the RBP2 protein is able to trigger HIF-1α upregulation and, which in turn stimulates VEGF release. In the present study, it has been verified that the mRNA/protein levels of VEGF, HIF-1α, PKM2 and RBP2 (Figs 7 and 8) are increased in the ischemic retina. As shown by Fig. 8, the effect of JIB-04 on the mean protein levels of RBP2 relative to effect of XFZUD on the mean protein levels of RBP2 was 1.16 to 1.00 (p = 0.668). As discussed in the beginning, PKM2 and RBP2 co-activate HIF-1α and then stimulates VEGF release [35, 36]. Not inconsistently with the present result, preischemia or postischemia administration of XFZYD significantly counteracted these ischemia-induced increases (Fig. 8). Taken together, this implies that XFZYD might be able to prevent or protect the defined retinal ischemic changes via a synergistic inhibition of RBP2 and PKM2 as well as by the further down-regulation of HIF-1α and a subsequent reduction in VEGF-A secretion.

The Rhesus monkey studies [41, 42] have indicated that CRAO of up to 240 min leads to disastrous permanent retinal injury. BRAO eyes that have poorer presenting corrected vision are unlikely to spontaneously improve in the vision and this could result in persistent retinal injury [43, 44]. Topical brimonidine and somatostatin had been shown to act as neuroprotectants and seem to attenuate morbidity due to background diabetic retinopathy (BDR) as well as any deterioration of BDR into PDR [45]. As mentioned earlier, hypoxia, mediated by HIF-1α, both aggravates cellular injury and inflammation as well as plays an important role in angiogenesis [46]. The increased levels of HIF-1α further stimulates the overexpression of VEGF and VEGFR [47], which in turn promotes endothelium growth and new vessel formation, namely angiogenesis [11]. A recent report has indicated that XFZYD significantly reduces the level of HIF-1α and, as a consequence, VEGF [12]. In addition to the decoction’s antiangiogenic and protective effects, XFZYD would appear to have a potentially important role in preventive medicine, while at the same time providing a relevant and important pharmaceutical effect when one has a familial morbidity or have suffered from previous vision-threatening retinal ischemic disorders such as, wAMD, DR, glaucoma, CRAO, BRAO, CRVO or BRVO.

The present study has clearly demonstrated various retinal ischemic changes, namely a significant decrease in the amplitudes of ERG b-wave (indexing Müller and bipolar cells), a significant less numerous RGCs, a significantly reduced inner/whole retinal thickness, and a significant loss of the ChAT immunolabeling amacrine cells as well as an obvious enhancement in GFAP/Vimentin immunolabeling (indexing Müller cells). In addition, significant upregulation of the mRNA/protein levels of VEGF, HIF-1α, PKM2 and RBP2 were also found in the ischemic retina. Importantly, the harmful influences of these aspects of ischemia were alleviated in a dose responsive and significant manner when XFZYD was applied for seven consecutive days before or after retinal ischemia. In particular, the increases in the ischemia-associated VEGF, HIF-1α, PKM2 and RBP2 increases were blunted by XFZYD. When our findings are taken as a whole, XFZYD would seem to have a preventive or protective effect on retinal ischemia via the downregulation of HIF-1α expression and a reduction in VEGF secretion; this ocurrs via an inhibition of RBP2 and PKM2.