Astragalus root extract inhibits retinal cell apoptosis and repairs damaged retinal neovascularization in retinopathy of prematurity

Ethics statement

The study was approved by the Ethics Committee of Luoyang Women’s and Children’s Health Care Center. All animal experiments were in line with the Guide for the Care and Use of Laboratory Animal by International Committees. The treatment of animals in all experiments conformed to the ethic standards of experimental animals.

Astragalus root extract preparation

Astragalus was bought from Tongrentang (Beijing, China). First, the dried Astragalus was crushed and extracted by water. Next, the Astragalus was soaked in 60% ethanol for 24 h (materials vs. liquid at 1:20), then bathed in water (70°C), heated and refluxed (three times, 1.5 h/time). After that, the Astragalus was filtered and added with distilled water (dregs vs. distilled water at 1:20) for 2 h each time, hot water reflux for three times, then combined extract solution three times and concentrated, and put into the refrigerator for reserve (−20°C).

Animal grouping

One hundred and twenty SPF C57BL/6 newborn mice (aging 7 days and weighing 15–20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Chaoyang District, Beijing, China). Mice were randomly divided into six groups: normal group, OIR group, high Astragalus root extract (H-ARE) group, medium Astragalus root extract (M-ARE) group, low Astragalus root extract (L-ARE) group and saline group, 20 mice per group, male or female.

Oxygen induced retinopathy (OIR) model construction and treatment

The OIR group, the saline group, and the Astragalus root extract group (high dose group, medium dose group, low dose group) were placed in a self-sealed glass box. The glass box was: 120 cm (length) × 100 cm (width) × 80 cm (height). By adjusting the flow rate of oxygen and nitrogen in the sealed glass box, the initial concentration of oxygen was adjusted to 21%, and then medical oxygen was injected into the oxygen-permeated silica gel hose. The oxygen concentration in the glass box was maintained at 75% ± 2% (caution the oxygen concentration). Then, the OIR group, the saline group, the Astragalus root extract groups were fed for 7 d. After modeling, the mice were fed back to normal air. The saline group was fed with 20 mL/kg of saline daily by intragastric administration (ig). The mice in the Astragalus root extract groups were fed with 40, 20 and 10 mg/kg Astragalus root extract, respectively, ig daily for 4 w. The newborn mice in the normal control group were fed 5 w in the normal air. Mice in each group were fed in the standard animal room, the environment was relatively quiet, free to fresh drinking water and food, kept the indoor temperature 25 ± 2°C. In each group, 10 newborn mice were randomly perfused with Evans Blue (EB), and the rest were randomly selected for retinal preparation, electron microscopic specimen preparation, tissue sections for hematoxylin-eosin (HE) staining, the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining, oxidative stress test, immunohistochemistry detection, reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis.

EB staining

After 5 w of feeding, 10 newborn mice were randomly selected from each group, and anesthetized by intraperitoneal injection of 3% pentobarbital sodium (50 mg/kg, Sigma-Aldrich, St. Louis, MO, USA). After anesthesia, EB was injected into the tail vein of mice at a dose of 30 mg/kg by body weight. After 2 h of circulation, the sodium citrate buffer was preheated to 37°C (pH 3.5, 0.05 mol/L) to prepare 1% polyformaldehyde solution (pH 4.5) which was perfused from the left ventricle. Then, the right auricle was cut open and the perfusive pressure was maintained at 120 mmHg for 2 min. The eyeball was removed and the retina was separated. The retina with 120 µL formamide was bathed in 70°C water for 18 h. The supernatant was taken and transferred into a hypercentrifugal tube at 4°C, 12,000 r/min, and centrifuged for 45 min to obtain the supernatant (50 µL). By using a spectrophotometer, the absorbance (A) values at the wavelength of 620 nm and 740 nm were measured (average value of each sample was measured three times). The formula for calculating absorbance value = A620 – A740 nm. Retinal dry weight (mg) was used to standardize the content of EB dye (ng), and the result was expressed as ng/mg. The formula was: the content of EB in retina (ng/mg) = the concentration of EB in formamide (ng/µL) × 120 (µL)/dry weight of retina (mg).

Adenosine diphosphate (ADP) enzyme histochemical assay

The newborn mice were anesthetized by intraperitoneal injection of 3% pentobarbital sodium (50 mg/kg, Sigma-Aldrich, St. Louis, MO, USA). After anesthesia, the thoracic cavity of the newborn mice was opened and the heart was exposed. The left ventricle was perfused with a perfusion needle and the right auricle was cut open. Firstly, the right auricle was perfused with normal saline for 5 min, and then perfused with 4% polyformaldehyde solution for 5 min. Rigidity of body, limbs, and tail indicated successful perfusion. After that, the eyeballs of mice were removed and fixed in the polyformaldehyde solution (4°C, 4%) for about 12–24 h. The cornea, lens, vitreous body, sclera and choroid were cut off along the limbus of cornea. Radial incision (4°C5) was made with the optic papilla as the center. ADP staining was performed on the slides: Tris-Maleate buffer solution (4°C, 50 mmol/L, pH 7.2) was used to wash 5 times with 15 min/time. Freshly prepared Tris-Maleate (37°C, 0.2 mol/L, pH 7.2) buffer solution (containing lead nitrate 3 mmol/L, magnesium chloride 6 mmol/L, ADP 1 mg/mL) was added to incubate for 15 min. Then, the retinal blood vessels were washed 5 times with Tris-Maleat buffer (room temperature, 50 mmol/L, pH 7.2), 15 min each time, and treated with 10% ammonium sulfide (1:10) for 1 min to form dark brown precipitation. The retinal blood vessels were washed 3 times with Tris-Maleat buffer (room temperature, 50 mmol/L, pH 7.2) for 15 min. Finally, the results were observed by an optical microscope.

HE staining

The eyeballs of neonatal mice were removed and fixed for 24 h with 4% polyformaldehyde solution at 4°C, followed by conventional dehydration, clearance, and paraffin embedding. The prepared wax blocks were sliced with a Leica SM2010R paraffin microtome (Leica Microsystems, Shanghai, China) on the parallel plane of the tissue wax blocks and the sagittal axis of the optic nerve for 4 μm slices. And 10 slices were taken from each eye (the section with the optic nerve section was excluded). The pathological changes of tissues were observed by HE staining under a light microscope and images were acquired for analysis. The number of endothelial cell nuclei breaking through the inner limiting membrane of retina was counted by double-blind method, and the average number of endothelial cell nuclei breaking through the inner limiting membrane of retina was counted by every slice of each eyeball.

Preparation of electron microscopic specimens

Retinal tissues of newborn mice were taken and fixed in prepared glutaraldehyde solution (2.5%, 4°C) for 72 h after perfusion. Then, the tissues were washed in PBS, fixed by 1% osmium tetroxide, dehydrated, cleaned by acetone gradient, then embedded in Epon812 epoxy resin, and sliced into 1 μm sections for light microscopic localization, then the ultrathin sections were made. After double staining with uranium acetate and lead citrate, ultrastructural changes of the photographic retina were observed by a transmission electron microscopy (H-600, Haitachi, Tokyo, Japan).

Immunohistochemical staining

The paraffin slices were placed in xylene for 15 min and were dewaxed to water by gradient ethanol (anhydrous ethanol, 95%, 90%, 80%, 70% ethanol). Ethylene diamine tetraacetic acid (EDTA) antigen repair buffer (pH 9.0) was used for antigen repair, then the slices were immersed in PBS, and washed three times. After that, the slices were put in 3% hydrogen peroxide solution, and hatched for 25 min avoiding light. Moreover, the retina was covered by 5% bovine serum albumin (BSA) and placed at room temperature for 20 min to remove excess fluid. Then, the primary anti-vascular endothelial growth factor (VEGF) (1:1600, CST company, St. Louis, Missouri, USA), pigment epithelium-derived factor (PEDF) (1:40, CST company, Denver, Massachusetts, USA) were added at 37 for 1 h. Furthermore, the corresponding secondary antibody was added and incubated at room temperature for 50 min. Next, diaminobenzidine (DAB) solution was used until full stained. The slices were counterstained with hematoxylin, dehydrated with gradient alcohol, cleared with xylene, dried at room temperature and preserved at −20°C. The expression of VEGF and PEDF was observed, and 10 visual fields were randomly selected under a high power microscopy (× 200). The number of cells and positive cells in each high power mirror field of vision were counted.

TUNEL staining

Retinal tissues of newborn mice were fastened with paraformaldehyde (37°C, 4%) for 8 min, and the optic cup was dissected and placed in a 4% paraformaldehyde solution for 30 min. Then, the optic cup was removed and dehydrated in a gradient of sucrose solution (10%, 20%, 30%). After the dehydration completed, the optic cup was placed in Tissue-Tek OCT Compound (Sakura Fine Technical Co. Ltd., Tokyo, Japan) (no bubbles), then gently put it into liquid nitrogen until the OCT was solidified. After that, the optic cup was sliced by a CM1950 freezing slicer (Leica Microsystems, Wetzlar, Germany). The retina around the optic nerve was taken at 7 μm and adhered to the slides. After dried the slides for 30 min, the slides were rinsed with PBS 3 times with 10 min/time. After pre-cooling, the slides were immersed in freshly prepared 0.1% sodium citrate solution containing 0.1% Triton X-100, after precooling, the slides were incubated for 5 min. After that, the slides were washed in PBS 2 min. Then, TUNEL solution was added into each well, and incubated at 37°C; for 1 h. After washing and drying, the tissues were sealed by 4°C,6-diamidino-2-phenylindole 2hci (DAPI). Finally, the tissues were observed and captured by a fluorescence microscopy (Olympus IX71, Olympus Optical Co., Ltd, Tokyo, Japan).

Fluorescent probe cellROX® green oxidative stress reagent assay

Retinal tissues of newborn mice were embedded with Tissue-Tek OCT Compound (Sakura Fine Technical Co. Ltd., Tokyo, Japan). The frozen sections (7 μm) near the optic nerve were taken out. CellROX (1:500, Cat: C10444, Life Technologies, Shanghai, China) was diluted by PBS to 5 μm working fluid. Fresh slices were used to wash three times with PBS for 10 min. After dried, appropriate amount of CellROX working fluid was added and incubated at 37°C for 30 min. After that, the mounting medium containing DAPI was used for sealing. Finally, a fluorescence microscopy (Olympus IX71, Olympus Optical Co., Ltd, Tokyo, Japan) was used to observe and take photographs.

RT-qPCR

Total RNA from neonatal mice’s retinal tissue was extracted using a one-step method by Trizol (Invitrogen, Carlsbad, CA, USA). The total RNA obtained was measured for the purity of RNA (the ratio of 260/280 was between 1.8 and 2.0) using a nucleic acid detector. One μg of RNA was taken and reverse-transcribed with AMV reverse transcriptase to obtain cDNA, and glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal reference. The product was electrophoresed on an agarose gel and examined under UV light (260 nm). The threshold was selected manually at the lowest point of parallel rise of each logarithmic amplification curve, and the threshold cycle (Ct) value of each reaction tube was obtained. The data were analyzed by 2^−ΔΔCt method [Citation12]. The experiment was conducted in triplicate and the data were averaged. PCR primers were invented and synthesized by Invitrogen company (Carlsbad, California, USA) (Table 1).

Western blot analysis

The retinal tissue was homogenized with radio-immunoprecipitation assay (RIPA) lysate. The concentration of total protein in retina of neonatal mice in each group was determined by Bradford method [Citation13]. The extracted protein was added to the sample buffer and boiled at 100°C for 5 min, and centrifuged at 14,000 g for 5 min. Samples after centrifugation were taken at 20 µg per well, then 8% sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) gel was prepared. After that, the protein was transferred to the membrane and the membrane was immersed in 5% skimmed milk sealing solution and sealed at room temperature for 1 h. The primary antibody anti-VEGF (1:1000, Santa Cruz Biotechnology Company, California, USA), PEDF (1:200, Cayman Chemical Co., Ann Arbor, MI, USA), IGF-1 (1:600, CST, St. Louis, Missouri, USA), HIF-1α (1:400, San Jose Institute of Biological Sciences, California, USA), Bax (1:200, Santa Cruz Biotechnology Company, California, USA), Bcl-2 (1:100, Santa Cruz Biotechnology Company, California, USA), eNOS (1:1000; CST company, Danvers, Massachusetts, USA), nNOS (1:250, ThermoFisher Scientific Co., Ltd., Shanghai, China), and iNOS (1:800, Abcam Company, Cambridge, UK) were added and incubated at 4°C overnight. Furthermore, the retinal tissues were incubated with the secondary antibody, and incubated at room temperature for 1 h, followed by chemiluminescent reagent development, with GAPDH (1:1000, Cell Signaling Technology, Beverly, MA, USA) as the internal reference. Gel Doc EZ imager (Bio-rad, California, USA) was used for development. Image J software was used to analyze the gray value of the target band.

Statistical analysis

All data were analyzed by SPSS 21.0 software (IBM Corp., Armonk, NY, USA). The measurement data were shown as mean ± standard deviation. Comparisons between two groups were conducted by t-test, while comparisons among multiple groups were assessed by one-way analysis of variance (ANOVA) followed by Fisher’s least significant difference t-test (LSD-t). P < 0.05 was indicative of statistically significant difference, P > 0.05 equaled to no statistical significance.

Astragalus root extract protects retina blood-retinal barrier in the OIR model mice

The content of EB in the retina of newborn mice in each group was measured 2 h after EB perfusion (Figure 1(a)). EB in retina of neonatal mice in the OIR group was dramatically increased, 7.16 times higher than that of the normal group, suggested that the blood-retinal barrier was damaged. After administration of Astragalus root extract by intragastric administration, EB content in retina was reduced and the damage of blood-retinal barrier was alleviated, there was an obvious difference compared with the OIR group (P < 0.05), and the dose–effect relationship was clear (P< 0.05). In contrast to the OIR group, there was no difference in EB content of retina in the saline group (P > 0.05), indicated that Astragalus root extract protects blood-retinal barrier in OIR model mice.

Figure 1. Blood-retinal barrier in the OIR model mice was protected by Astragalus root extract. (a) Comparison of EB leakage of blood-retinal barrier in neonatal mice; (b) observation of ADP enzyme staining of retina (× 50–100); The data in the figure were measured in the form of mean ± standard deviation. One-way ANOVA was involved for comparison between multiple groups, and LSD-t-test was used for pairwise comparison after ANOVA analysis. * P < 0.05 vs. the normal group, # P < 0.05 vs. the OIR group, N = 10

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Astragalus root extract repairs the structure and morphology of retina in OIR model mice

As shown in Figure 1(b) in the normal group, retinal blood vessels were seen to be radially and evenly distributed from the optic disc. The retina was divided into two layers, deep and shallow, and the two layers of blood vessels were connected to each other by spiral blood vessels. The deep blood vessels could be seen in a polygonal mesh shape, and the shallow blood vessels could be seen in multiple radial branches. In the OIR group, a large number of vascular hyperplasia in the retina was observed, the vascular density was heightened, and non-perfusion area of varying sizes was observed. The retinal blood vessels from the optic disc were dilated and distorted with a large number of new blood vessels were formed. The blood vessels weren’t in normal radial morphology and performed polygonal reticular vascular structures. These new blood vessels were disorderly in distribution, complex in structure. The superficial vascular network and deep blood vessels were widely occluded, and a large number of non-perfused areas existed. The stretched preparation of retina in the OIR group was similar to that of the saline group. In the H-ARE, M-ARE and L-ARE groups, the retinal blood vessels were seen to be overall radially distributed from the optic disc. The retinal blood vessels were basically distributed in two layers. The deep and shallow blood vessels were connected to each other by spiral blood vessels. The deep blood vessels could be seen in a polygonal mesh shape, and multiple radial branches could be seen in the shallow blood vessels.

Astragalus root extract improves retinal vascular angiogenesis in OIR model mice

HE staining of retinal slices in neonatal mice revealed that (Figure 2(a)) the structure of the retina in the normal group was clear, the arrangement was neat, the inner limiting membrane was clear, and the chromatin was evenly distributed. Rarely to see the endothelial cell nucleus breaking through the inner membrane of the retina, no new blood vessels were found in the retina, and the edge of the tissue structure was neat and normal. In the OIR group, the whole retinal tissue was disordered, the edges of retinal tissue were irregular, and a large number of endothelial cell nuclei were broken through the inner membrane of the retina. Individual or clustered retinal endothelial vascular hyperplasia could be seen. Compared with the OIR group, the saline group showed no clear improvement, but still existed a large number of retinal vascular hyperplasia, a great number of the endothelial cell nucleus breaking through the retinal inner membrane, and retinal margins were irregular. H-ARE, M-ARE and L-ARE groups have rarely seen the endothelial cell nucleus breaking through of retinal intimal membrane. The structure of the retina was clear, and shape was regular, the edges of the structure were orderly.

Figure 2. Retinal vascular angiogenesis in the OIR model mice was improved by Astragalus root extract. (a) HE staining of retinal tissues of neonatal mice (200 ×); (b) Comparison of retinal neovascular endothelial cell nuclei in neonatal mice; the data in the figure are all measurement data, using the mean ± standard deviation. One-way ANOVA was taken for comparison among groups, and LSD-t-test was used for pairwise comparison after ANOVA analysis. * P < 0.05 vs. the normal group, # P < 0.05 vs. the OIR group, N = 10

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The number of endothelial cells breaking through the retina of neonatal mice was counted through the retina of neonatal mice. The results indicated that (Figure 2(b)) there was 1 (0.82 ± 0.88) in the normal group and 39 (38.58 ± 2.17) in the OIR group, 38 (38.03 ± 1.98) in the saline group, 26 (26.52 ± 0.49) in the H-ARE group, 29 (29.43 ± 2.32) in the M-ARE group and 34 (34.38 ± 0.74) in the L-ARE group. In contrast to the normal group, the number of neovascular endothelial cells breaking through the retinal inner membrane in the OIR group was dramatically raised (P < 0.05). No statistical significance was detected in the OIR group and the saline group (P > 0.05). The H-ARE, M-ARE and L-ARE groups obviously had fewer neovascular endothelial cells breaking through the retinal inner membrane boundary than the OIR group, and the dose–effect relationship was distinct (P < 0.05).

Astragalus root extract improves the ultrastructure of retina in OIR model mice

The ultrastructure of retinal tissues in neonatal mice in the OIR group was observed by an electron microscope (Figure 3(a)). The results presented that the endothelial cells of neonatal mice in the normal group were regular in shape, with good interconnection system between cells, and the basement membrane was continuous but not thick. The nuclei of retinal ganglion cells were mostly round or elliptic, with low electron density, uniform chromatin distribution, complete extracellular ganglion disc, clear structure, and orderly arrangement. In contrast to the normal group, the retinal endothelial cells of neonatal mice in the OIR group were markedly swollen and round with vacuoles. The interconnection system between cells was destroyed, the basement membrane became thicker, optic ganglion cells was deformed, the structure of ganglion cell layer was disordered, vacuoles were visible, and the disc of outer segments of photoreceptor cells was sparse and shorter. In relation to the neonatal mice in the OIR group, there was no major difference in the retina of the newborn mice in the saline group. In the H-ARE, M-ARE and L-ARE groups, the newborn mice showed different degrees of swelling degrade, the vacuoles were reduced, the intercellular connection system was recovered, and the basement membrane was continuous and not thick. And the structure was clear, the optic ganglion cells were round or elliptical, the ganglion cell layer structure was restored, at the same time, the vacuoles were reduced. The photoreceptor extracellular membrane disc was gradually restored to complete, also the structure was clear and the arrangement was neat.

Figure 3. The ultrastructure of retina in OIR model mice was improved by Astragalus root extract. (a) Electron microscopic observation of retinal ultrastructure in neonatal mice in each group; (b) Fluorescence probe CellROX was used to detect the expression of reactive oxygen species in the retina of each group of mice; the data in the figure are all measurement data, by using the mean ± standard deviation form, one-way ANOVA was used for multiple groups comparison, and LSD-t-test was used for the pairwise comparison after ANOVA analysis. * P < 0.05 vs. the normal group; # P < 0.05 vs. the OIR group, N = 10

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Astragalus root extract inhibits reactive oxygen species (ROS) production in retinal tissues of the OIR model mice

The detection of ROS levels in the newborn mice by CellROX® Green oxidative stress reagents (Figure 3(b)) indicated that compared with the normal group, the content of ROS increased obviously in the neonatal mice of the OIR group (P 7< 0.05). Compared with the OIR group, the newborn mice in the saline group were unable to inhibit the production of ROS after saline administration, and there was no statistical significance (P > 0.05). The Astragalus root extract in newborn mice could suppress the production of ROS and the dose–effect relationship was obvious (P< 0.05). The experimental results suggest that the Astragalus root extract inhibits the production of ROS in retinal tissues of OIR model mice.

Astragalus root extract inhibits retinal cells apoptosis in the OIR model mice

The apoptosis of retinal cells was observed by TUNEL staining of retinal tissue of newborn mice in all group (Figure 4(a)). No obvious apoptosis was observed in retinal cells of the normal group. Neonatal mice in the OIR group had different levels of apoptosis in the inner nuclear layer, outer nuclear layer, and ganglion cell layer. In contrast to the normal group, the number of TUNEL-positive cells in the retinal tissues of the OIR group increased obviously (P < 0.05). Compared with the neonatal mice in the OIR group, the number of TUNEL-positive cells in the retinal tissues of the newborn mice in the saline group did not improve dramatically, and the apoptosis was basically the same with the OIR group (P > 0.05). The number of TUNEL-positive cells in the retinal tissue of the newborn mice of the H-ARE, M-ARE and L-ARE groups was lower than that in the OIR group, and the dose–effect relationship was significant (all P < 0.05), which suggested that the Astragalus root extract inhibits the apoptosis of retinal cells in OIR model mice.

Figure 4. Bcl-2 in the OIR model mice was promoted and Bax was suppressed by Astragalus root extract. (a) TUNEL assay was used to detect the apoptosis of retina in each group; (b) The mRNA expression of Bax and Bcl-2 in the retina of newborn mice in each group detected by RT-qPCR; (c) Protein bands of Bax and Bcl-2 in the retina of newborn mice; (d) Protein expression Bax and Bcl-2 in the retina of newborn mice in each group detected by western blot analysis. The data are all measurement data, by using the mean ± standard deviation form, the comparison among multiple groups using one-way ANOVA, and LSD-t-test was used after ANOVA analysis. * P < 0.05 vs. the normal group; # P < 0.05 vs. the OIR group, N = 10

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The expression of Bax and Bcl-2 in retinal tissues of neonatal mice in each group was detected by RT-qPCR and western blot analysis. The results showed that (Figure 4(b–d)) compared with the normal group, the expression of Bax in neonatal mice of the OIR group increassed, while the expression of Bcl-2 decreased (both P < 0.05). The expression levels of each gene in the saline group were consistent with those in the OIR group, and the difference was not statistically significant (P > 0.05). In the H-ARE, M-ARE and L-ARE groups, the expression of Bax decreased and the expression of Bcl-2 increased after the treatment (both P < 0.05). The results showed that Astragalus root extract suppresses the Bax level and induces the Bcl-2 level, thus could protect the retina in the OIR model mice.

Astragalus root extract reduces VEGF expression and increases PEDF expression in retina of the OIR model mice

The expression of VEGF and PEDF in the retinal tissues of OIR model mice was observed by immunohistochemical staining. Ten fields were randomly selected under a high power microscope. The number of positive cells was counted in each high power field. The results showed (Figure 5): compared with newborn mice in the normal group, the positive expression of VEGF in the retinal tissues of the neonatal mice in the OIR group was significantly up-regulated, and the positive expression of PEDF was significantly decreased (both P< 0.05). In contrast to the OIR group, there was no significant difference in the expression of VEGF and PEDF in the retinal tissue of the newborn mice (both P > 0.05). In the H-ARE, M-ARE and L-ARE groups, the positive expression of VEGF in retinal tissues of neonatal mice was obviously decreased, and the positive expression of PEDF was increased (all P < 0.05). The results of this experiment indicated that Astragalus root extract could reduce the expression of VEGF and increase the expression of PEDF in the retinal tissues of the OIR model mice.

Figure 5. VEGF expression was reduced and PEDF expression was increased by Astragalus root extract in retina of the OIR model mice (a) immunohistochemical detection of positive expression of VEGF in retinal tissue of newborn mice (200 ×); (b) Comparison of positive expression of VEGF in retinal tissue of newborn mice in each group; (c). Immunohistochemical detection of PEDF positive expression in retina tissue of newborn mice (200 ×); (d) Comparison of positive expression of PEDF in retinal tissues of newborn mice from all group. The data in the figure are all measurement data, in the form of mean ± standard deviation, and the comparison among groups was analyzed by one-way ANOVA. The LSD-t-test was taken for pairwise comparison after ANOVA analysis; * P< 0.05 vs. the normal group; # P < 0.05 vs. the OIR group, N = 10

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Astragalus root extract inhibits VEGF and HIF-1α while promotes PEDF and IGF-1 in retinal tissues of the OIR model mice

The expression of VEGF, HIF-1α, PEDF, and IGF-1 in the retinal tissues of each group were detected by RT-qPCR and western blot analysis. The results of the experiment indicated (Figure 6(a-c)): VEGF and HIF-1α expression of neonatal mice in the OIR group increased, and PEDF and IGF-1 expression decreased compared with normal group (all P < 0.05). The expression levels of each factor in the saline group were the same with those in the OIR group (all P > 0.05). In the H-ARE, M-ARE and L-ARE groups, VEGF and HIF-1α expression decreased markedly after the treatment, and the expression of PEDF and IGF-1 increased (all P < 0.05). The results showed that Astragalus root extract inhibits the expression of VEGF and HIF-1α in the retinal tissue of the OIR model mice, and promotes PEDF and IGF-1 expression, thus inhibits the retinal vascular hyperplasia.

Figure 6. VEGF and HIF-1α were inhibited, while PEDF and IGF-1 were promoted by Astragalus root extract in retinal tissues of the OIR model mice (a) The expression of VEGF, HIF-1α, PEDF, and IGF-1 mRNA in the retina of newborn mice in each group detected by RT-qPCR; (b) The protein bands of VEGF, HIF-1α, PEDF, and IGF-1 in the retina of newborn mice; (c) Statistical analysis of protein expression in Figure b. The data are all measurement data, in the form of mean ± standard deviation, and the comparison among groups was analyzed by one-way ANOVA. The LSD-t-test was used for pairwise comparison after ANOVA analysis; * P< 0.05 vs. the normal group; # P < 0.05 vs. the OIR group, N = 10

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Astragalus root extract promotes iNOS expression and inhibits eNOS and nNOS in the retina of the OIR model mice

The expressions of eNOS, nNOS, and iNOS in the retinal tissues of newborn mice in each group were detected by RT-qPCR and western blot analysis. As shown in Figure 7(a-c), the expression of eNOS and nNOS in neonatal mice of the OIR group increased, and the expression of iNOS decreased compared with the normal group (all P < 0.05). The expression levels of each factor in the saline group were consistent with those in the OIR group, and there was no statistical significance (all P > 0.05). In the H-ARE, M-ARE and L-ARE groups, the expression of eNOS and nNOS reduced, and the expression of iNOS increased after the administration of neonatal mice (all P < 0.05). The results indicated that the extract of Astragalus promoted the expression of iNOS and inhibited the levels of eNOS and nNOS in the retinal tissue of the OIR model mice, thus protected the retina in the OIR model mice.

Figure 7. iNOS expression was promoted while eNOS and nNOS were inhibited by Astragalus root extract in the retina of the OIR model mice. (a) The mRNA expression of eNOS, nNOS and iNOS in the retina of newborn mice of each group detected by RT-qPCR; (b) Protein bands of eNOS, nNOS and iNOS in the retina of newborn mice; (c) The protein expression of eNOS, nNOS and iNOS in the retina of newborn mice of each group detected by western blot analysis. The data in the figure are all measurement data, in the form of mean ± standard deviation, and the comparison among groups was analyzed by one-way ANOVA. The LSD-t-test was used for pairwise comparison after ANOVA analysis; * P < 0.05 vs. the normal group; # P < 0.05 vs. the OIR group, N = 10

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