Antioxidant-Rich Extract from Plantaginis Semen Ameliorates Diabetic Retinal Injury in a Streptozotocin-Induced Diabetic Rat Model

Plantaginis semen, the dried ripe seed of Plantago asiatica L. or Plantago depressa Willd. (Plantaginaceae), has been traditionally used to treat blurred vision in Asia. The aim of this work was to investigate the effect of plantaginis semen ethanol extract (PSEE) on the amelioration of diabetic retinopathy (DR) in streptozotocin (STZ)-diabetic rats. PSEE has abundant polyphenols with strong antioxidant activity. PSEE (100, 200 or 300 mg/kg) was oral administrated to the diabetic rats once daily consecutively for 8 weeks. Oral administration of PSEE resulted in significant reduction of hyperglycemia, the diameter of the retinal vessels, and retinal vascular permeability and leukostasis in diabetic rats. In addition, PSEE administration increased the activities of superoxidase dismutase (SOD) and catalase (CAT), and glutathione peroxidase (GSH) level in diabetic retinae. PSEE treatment inhibited the expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) and the phosphorylation of Akt without altering the Akt protein expression in diabetic retinae. PSEE not only down-regulated the gene expression of hypoxia-inducible factor-1α (TNF-α) and interleukin-1β (IL-1β), but also reduced ICAM-1 and VCAM-1 expression in diabetic retinae. Moreover, PSEE reduced the nuclear factor-κB (NF-κB) activation and corrected imbalance between histone deacetylases (HDAC) and histone acetyltransferases (HAT) activities in diabetic retinae. In conclusion, phenolic antioxidants extract from plantaginis semen has potential benefits in the prevention and/or progression of DR.


Introduction
Diabetes mellitus (DM) is a severe metabolic disease, and numerous complications are associated with the characteristic hypergly-cemia of this disease [1]. Of these, diabetic retinopathy (DR) is one of the major microvascular complications amongst diabetic patients, and is the primary cause of visual loss [1]. Increasing evidence indicates that the chronic uncontrolled hyperglycemic state leads to generation of reactive oxygen species (ROS), which triggers a severe inflammatory state characterized by an elevation of proinflammatory cytokines [2][3][4]. The proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) are positively correlated with blood retinal barrier (BRB) breakdown and vascular cell death [5][6][7]. Apart from this, chronic hyperglycemia stimulates synthesis and secretion of vascular endothelial growth factor (VEGF), which was transcriptional regulated by Na 2 CO 3 was mixed into each sample of 100 µL and allowed to equilibrate for 2 min before adding 50% Folin-Ciocalteu's phenol reagent (Sigma-Aldrich, Inc.). Absorbance at 750 nm was measured at room temperature. The polyphenol content of PSEE was expressed as mg of gallic acid equivalent per gram (g) of PSEE in dry weight (DW), i.e., mg gallic acid/g DW.

Total Flavonoid Content
The total flavonoid content of PSEE was determined by the aluminium chloride colorimetric method [23]. Briefly, 0.25 mL of PSEE (100 µg/mL) was added to a tube containing 1 mL of double-distilled water. Next, 0.075 mL of 5% NaNO 2 , 0.075 mL of 10% AlCl 3 , and 0.5 mL of 1 mmol/L NaOH were added sequentially at 0, 5, and 6 min. Finally, the volume of the reacting solution was adjusted to 2.5 mL with double-distilled water. The solution had an absorbance of 510 nm. The total flavonoid content was calculated from a calibration curve, and the result was expressed as mg rutin (Sigma-Aldrich, Inc.) equivalent per g dry weight, i.e., mg rutin/g DW.

Experimental Animals
All experimental methods and animal care procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Tajen University (approval number, IACUC 104-28; approval date: 12 November, 2015), in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, as well as the guidelines of the Animal Welfare Act. Male Wistar rats (8 weeks aged) weighting 200-250 g, were purchased from National Laboratory Animal Center (Taipei, Taiwan) and housed two per cage in a room under controlled temperature (20-25 • C), humidity (50% ± 5%) and lighting (12 h light/dark cycle) with food and water provided ad libitum. Rats were rendered diabetic by a single intravenous injection of 60 mg/kg streptozotocin (STZ; Sigma-Aldrich, Inc.). Eight-week age-matched control rats were injected with vehicle (sterile saline 0.9%, pH 7.4). After 1 week, rat with non-fasting blood glucose levels >350 mg/dL, polyuria, and glucosuria were defined as diabetic and used for the experiments.

Treatment Protocols
In the treatment group (n = 10 per group), STZ-diabetic rats were dosed by oral gavage once per day for 8 weeks with PSEE at dosages of 100, 200, or 300 mg/kg in a volume of 1.5 mL/kg distilled water. The dosages of PSEE were selected based on consideration of tests of Plantago extracts relating to producing preventive effects on oxidative damage in rats [25]. A vehicle-treated group (n = 10 per group). Of normal rats and STZ-diabetic rats were treated with 1.5 mL/kg distilled water only over the same treatment period. Animals had free access to standard rat diet (Harlan Teklad, Madison, WI, USA; catalogue number (Cat. No.) 2018) and water throughout the entire treatment period.
At the end of the 8-week treatment, the rats were weighed, fasted overnight and anesthetized using an intraperitoneal injection of sodium pentobarbital (60 mg/kg). While under anesthesia, they were painlessly sacrificed and blood was collected from the abdominal aorta of each animal into heparin sample bottles. Rat eyes from each group were removed and the retinae were isolated. The diagnostic kit for determination for plasma levels of glucose (Cat. No. COD12503) was purchased from BioSystem (Barcelona, Spain). Commercial enzyme-linked immunosorbent assay (ELISA) kits were used to quantify HbA 1c levels (Integrated Bio Ltd., Taipei, Taiwan; Cat. No. CSB-E08140r). All analyses were performed in accordance with the instructions provided by the manufacturers.

Fundus Photography and Vessel Diameter
Fundus photography is performed with a retina camera (Kowa Company Ltd., Tokyo, Japan). In order to accustom to the fundus photography procedure, rats were trained before start of the study. Eyes were dilated with a drop of 1% tropicamide (Synpac-Kingdom Pharmaceutical Co., Ltd., Taipei, Taiwan). Moisol eye drops were administered periodically to prevent the cornea from drying out. Fundus photography was done regularly till 8 weeks to monitor the fundus changes.
The diameter of retinal vessels was estimated by previously described method [26]. Before diameter estimation, the retinal photographs from all groups were randomized. The vessel diameter of 3 most prominent vessels was estimated at 3 sites in its widest portion at equal distance from the center. An average of 3 estimations was taken as the final retinal vessel diameter.

Quantification of Retinal Leukostasis
Quantification of leukostasis was performed at the end of the 8-week treatment by previously described method [27]. The chest cavity of each deeply anesthetized rat was carefully opened and a perfusion needle was inserted into the left ventricle. After cutting the right atrium, the animals were immediately perfused with 500 mL of PBS per kg body weight and heparin (0.1 mg/mL) to wash out nonadherent blood cells. Fluorescein isothiocyanate-coupled Concanavalin A lectin (ConA) (20 µg/mL in PBS; pH 7.4; 5 mg/kg; Vector Laboratories, Burlingame, CA, USA) was then perfused to label adherent leukocytes and vascular endothelial cells. Residual unbound ConA was flushed by PBS perfusion. Eyes were removed and fixed in 4% paraformaldehyde for 1 h. Retinas were dissected and flat mounted on a microscope slide, covered with anti-fading medium and a coverslip, and imaged via fluorescence microscopy. Only whole retinae in which the entire vascular network was stained were used for analysis. The total number of adherent leukocytes within the vessels of each retina was counted.

Retinal Permeability Assessment
Retinal vascular permeability was measured using Evans blue (EB) dye extravasation technique at the end of the 8-week treatment [28]. EB dye (Sigma-Aldrich, Inc.) was dissolved in normal saline at 45 mg/mL and was injected through the tail vein of anesthetized rats over 10 s at a dosage of 45 mg/kg. After the dye had circulated for 2 h, the rats were anesthetized with sodium pentobarbital (40 mg/kg), the chest cavity was opened, and cardiac perfusion was performed via the left ventricle with 1% paraformaldehyde in citrate buffer (0.05 mol/L, pH 3.5) under a constant pressure of 120 mmHg. Immediately after perfusion, the retinas were carefully dissected under an operating microscope. After retinas were fully dried at 4 • C, then the weights of them were measured, EB dye was extracted by incubating each sample in 150 µL formamide for 18 h at 70 • C. The extract was ultracentrifuged at a speed of 14,000 rpm for 60 min. Absorbance was measured using 100 µL of the supernatant at 620 nm and 740 nm. The concentration of EB in the extracts was calculated from a standard curve and normalized by total protein concentration in the tissue.

Assay of Retinal Antioxidant Enzymes
Retinas from right and left eyes from one rat were pooled as one sample, and then were homogenized in 10 volume of ice cold 0.1 M Tris-HCl, pH 7.4 containing 0.5% Triton X-100, 5 mmol/L β-mercaptoethanol, 0.1 mg/mL phenylmethylsulfonyl fluoride and centrifuged at 14,000× g for 5 min at 4 • C. The supernatant was collected and used for following experiments as described below. Protein concentration of the supernatant was assayed by Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). The intracellular activity of superoxide dismutase (SOD; Cat. No. ab65354), catalase (CAT; Cat. No. ab118184) and glutathione (GSH; Cat. No. ab65322) were estimated using commercially available assay kits from Abcam plc. (Cambridge, MA, USA). All assays were carried out in triplicates.

Protein Extraction and Western Blot Analyses
Retinas from right and left eyes from one rat were pooled as one sample, which were then homogenized in 1 mL of ice-cold hypotonic buffer A (10 mmol/L of HEPES, 10 mmol/L of KCl, 2 mmol/L of MgCl 2 , 1 mmol/L of dithiothreitol, 0.1 mmol/L of EDTA, and 0.1 mmol/L of phenylmethylsulfonylfluoride; pH 7.8). A solution of 80 µL of 10% Nonidet P-40 was added to the homogenates, and the mixture was centrifuged for 2 min at 14,000× g at 4 • C. Before immunoblotting, and the protein concentration of each sample was determined using a Bio-Rad protein assay kit and bovine serum albumin as a standard, to ensure equal loading among lanes.
The tissue lysates containing 40-50 mg protein were electrophoresed through 8%, 12%, and 15% sodium dodecyl sulfate-polyacrylamide gels. According to the manufacturer's instructions, separated proteins were electrophoretically transferred to a nitrocellulose membrane, blocked with 5% skim milk solution for 1 h, and incubated with primary antibodies to TNF-α (Cat , the membranes were washed three times in TBST and visualized on X-ray film. Band densities were determined using ATTO Densitograph Software (ATTO Corporation, Tokyo, Japan) and quantified as the ratio to β-actin. The mean value for samples was adjusted to a value of 1.0 from the vehicle-treated normal rats on each immunoblot, expressed in densitometry units. Then, all experimental sample values were expressed to this adjusted mean value.

Activity of NF-κB, Histone Deacetylases (HDAC) and Histone Acetyltransferases (HAT)
Nuclear extract of retina was prepared using the nuclear extract kit (Cat. No. 40410; Active Motif, Carlsbad, CA, USA) following manufacturer's protocol. Two retinas from right and left eyes from one rat were pooled as one sample. Nuclear factor-κB (NF-κB) activation was determined, TransAM ® NF-κB p65 transcription factor assay kit (Cat. No. 40596) implemented under the procedures provided by the manufacturer (Active Motif Inc., Carlsbad, CA, USA). Reaction was quantified at 450 nm. Histone deacetylases (HDAC) activity in retinal nuclear extracts was measured by HDAC activity colorimetric assay kit (Cat. No. K331-100) from BioVision Inc. (Milpitas, CA, USA). The colorimetric readings were measured at 400 nm in a spectrophotometer. Activity of histone acetyltransferases (HAT) was quantified in retinal nuclear fraction by non-radioactive indirect ELISA kit (Cat. No. K332-100) from BioVision Inc. The readings were monitored at 440 nm in a spectrophotometer.

Statistical Analysis
All statistical analyses were performed using SPSS for Windows (version 21.0; IBM Corporation, Armonk, NY, USA). The results are presented as the mean ± standard deviation (SD) for each group of animals at the number (n) indicated. The significance of differences between groups was evaluated by oneway ANOVA with Fisher's Least Significant Difference post hoc test; and p < 0.05 was considered as indicating statistically significant differences. Relationships between variables were examined using Pearson correlations.

Polyphenolic Composition and Antioxidant Activity
The content of total polyphenols and flavonoids in PSEE were 184.5 ± 4.7 mg gallic acid/g DW and 63.8 ± 0.60 mg rutin/g DW, respectively. The TEAC value of PSEE was 18.3 ± 2.6 mmol/L Trolox/100 g DW.

Effects on Body Weights and Plasma Parameters
At the end of the experimental period, plasma levels of glucose and HbA 1c in STZ-treated rats were significantly greater than those in the normal animals, while body weight of the diabetic rats was significantly less than that of the normal group. Treatment 300 mg/kg/day PSEE for 8 weeks decreased the plasma levels of glucose and HbA 1c in STZ-diabetic rats by 33.1% and 27.9%, respectively, relative to the values in vehicle-treated counterparts (Table 1). Comparison to the vehicle-treated group, the reduction in body weight was not obvious in STZ-diabetic rats receiving PSEE at the end of experimental period (Table 1).

Effects on Antioxidant Parameters in Retinae
The activities of SOD and CAT in in diabetic retinae were significantly reduced as compared to those from normal group. Enhancement of SOD and CAT activities were observed in PSEE-treated 8-week diabetic retina with a dose-dependent manner (Table 1). Retinal GSH levels were markedly lower in STZ-diabetic rats compared with normal rats. Administration of STZ-diabetic rats with PSEE for 8 weeks resulted in a dose-dependent increase of GSH levels (Table 1).

Fundus Photographs and Microvasculature Diameter
Fundus photograph from normal rat was not showing any abnormal in retinal vasculature and the optic nerve head (Figure 1) However, vascular leakage with accompanying dilatation vessels were shown on fundus photographs from STZ-diabetic rats ( Figure 1). Less vascular leakage was present from the optic disc of STZ-diabetic rats receiving 300 mg/kg/day PSEE treatment (Figure 1). In addition, the retinal arterioles and venules became mildly dilated in PSEE (300 mg/kg/day)-treated STZ-diabetic rats ( Figure 1).

Fundus Photographs and Microvasculature Diameter
Fundus photograph from normal rat was not showing any abnormal in retinal vasculature and the optic nerve head (Figure 1) However, vascular leakage with accompanying dilatation vessels were shown on fundus photographs from STZ-diabetic rats (Figure 1). Less vascular leakage was present from the optic disc of STZ-diabetic rats receiving 300 mg/kg/day PSEE treatment (Figure 1). In addition, the retinal arterioles and venules became mildly dilated in PSEE (300 mg/kg/day)-treated STZ-diabetic rats (Figure 1).

Effects on Retinal Vascular Permeability and Leukostasis
Increased leakages of EB dye were observed in the retinas of STZ-diabetic rats (Figure 2A). Treatment of STZ-diabetic rats with 100, 200 or 300 mg/kg/day PSEE for 8 weeks decreased retinal EB dye accumulation by 21.7%, 29.5% and 36.4%, respectively, when compared with the levels observed in the vehicle-paired counterparts (Figure 2A). animals. p < 0.05 and p < 0.01 compared to the values of vehicle-treated normal rats, respectively. p < 0.05 and d p < 0.01 compared to the values of vehicle-treated STZ-diabetic rats, respectively.

Effects on Retinal Vascular Permeability and Leukostasis
Increased leakages of EB dye were observed in the retinas of STZ-diabetic rats (Figure 2A). Treatment of STZ-diabetic rats with 100, 200 or 300 mg/kg/day PSEE for 8 weeks decreased retinal EB dye accumulation by 21.7%, 29.5% and 36.4%, respectively, when compared with the levels observed in the vehicle-paired counterparts (Figure 2A).
The number of adherent leukocytes in the retinal microvasculature of normal rats was negligible, while STZ-induced diabetes caused a significant increase of leukocytes adhesion to the endothelia cells ( Figure 2B). Eight-week administration of STZ-diabetic rats with 100, 200 or 300 mg/kg/day PSEE decreased retinal leukostasis by 16.4%, 34.8%, 48.4%, respectively, compared to vehicle-treated diabetic group ( Figure 2B).  . Normal or STZ-diabetic rats receiving vehicle treatment were given the same volume of vehicle (distilled water) used to prepare the test medication solutions. Retinal vascular permeability was measured with EB dye as a tracer. Evans blue was normalized by total protein concentration in the tissue. Values (mean ± SD) were obtained for each group of ten animals. a p < 0.05 and b p < 0.01 compared to the values of vehicle-treated normal rats, respectively. c p < 0.05 and d p < 0.01 compared to the values of vehicle-treated STZ-diabetic rats, respectively.
The number of adherent leukocytes in the retinal microvasculature of normal rats was negligible, while STZ-induced diabetes caused a significant increase of leukocytes adhesion to the endothelia cells ( Figure 2B). Eight-week administration of STZ-diabetic rats with 100, 200 or 300 mg/kg/day PSEE decreased retinal leukostasis by 16.4%, 34.8%, 48.4%, respectively, compared to vehicle-treated diabetic group ( Figure 2B).
STZ caused a 2.6-fold increase in retinal TNF-α mRNA, a 3.1-fold rise in retinal IL-1β mRNA, a 3.2-fold increase in retinal ICAM-1 mRNA and a 2.9-fold rise in retinal VCAM-1 mRNA compared to the levels seen in the normal group ( Figure 3B). Treatment with PSEE at the daily oral dosage of 300 mg/kg markedly suppressed the STZ-induced stimulation of retinal mRNA levels of TNF-α, IL-1β, ICAM-1 and VCAM-1 to 65.2%, 58.4%, 50.6% and 50.3%, respectively, compared to the levels seen in their vehicle-treated counterparts ( Figure 3B). A positive correlation coefficient of 0.503 (p < 0.001), 0.412 (p < 0.001), 0.529 (p < 0.001), and 0.318 (p < 0.001), were identified between mRNA and protein expression in TNF-α, IL-1β, ICAM-1 and VCAM-1, respectively. vehicle treatment were given the same volume of vehicle (distilled water) used to prepare the test medication solutions. Retinal vascular permeability was measured with EB dye as a tracer. Evans blue was normalized by total protein concentration in the tissue. Values (mean  SD) were obtained for each group of ten animals. a p < 0.05 and b p < 0.01 compared to the values of vehicle-treated normal rats, respectively. c p < 0.05 and d p < 0.01 compared to the values of vehicle-treated STZ-diabetic rats, respectively.
STZ caused a 2.6-fold increase in retinal TNF-α mRNA, a 3.1-fold rise in retinal IL-1β mRNA, a 3.2-fold increase in retinal ICAM-1 mRNA and a 2.9-fold rise in retinal VCAM-1 mRNA compared to the levels seen in the normal group ( Figure 3B). Treatment with PSEE at the daily oral dosage of 300 mg/kg markedly suppressed the STZ-induced stimulation of retinal mRNA levels of TNF-α, IL-1β, ICAM-1 and VCAM-1 to 65.2%, 58.4%, 50.6% and 50.3%, respectively, compared to the levels seen in their vehicle-treated counterparts ( Figure 3B). A positive correlation coefficient of 0.503 (p < 0.001), 0.412 (p < 0.001), 0.529 (p < 0.001), and 0.318 (p < 0.001), were identified between mRNA and protein expression in TNF-α, IL-1β, ICAM-1 and VCAM-1, respectively. Normal or STZ-diabetic rats receiving vehicle treatment were given the same volume of vehicle (distilled water) used to prepare the test medication solutions. Results in each column are mean  SD from ten rats per group. a p < 0.05 and b p < 0.01 compared to vehicle-treated normal rats, respectively. c p < 0.05 and d p < 0.01 compared to the values of vehicle-treated STZ-diabetic rats, respectively.

Effects on Retinal Angiogenic Factors Expression
Retinal protein and mRNA levels of HIF-1α in STZ-diabetic rats were clearly higher than those of the normal rats, and were down-regulated by 300 mg/kg/day PSEE treatment: decreases of 40.4% and 45.3%, respectively, when compared with the levels observed in the vehicle-treated counterparts (Figure 4). . Normal or STZ-diabetic rats receiving vehicle treatment were given the same volume of vehicle (distilled water) used to prepare the test medication solutions. Results in each column are mean ± SD from ten rats per group. a p < 0.05 and b p < 0.01 compared to vehicle-treated normal rats, respectively. c p < 0.05 and d p < 0.01 compared to the values of vehicle-treated STZ-diabetic rats, respectively.

Effects on Retinal Angiogenic Factors Expression
Retinal protein and mRNA levels of HIF-1α in STZ-diabetic rats were clearly higher than those of the normal rats, and were down-regulated by 300 mg/kg/day PSEE treatment: decreases of 40.4% and 45.3%, respectively, when compared with the levels observed in the vehicle-treated counterparts (Figure 4).
The retinal expression of VEGF was significantly increased in the STZ-diabetic rats compared with those in normal rats at both protein and mRNA levels, which were decreased by 300 mg/kg/day PSEE treatment to 45.3% and 54.3%, respectively, relative to those observed in the vehicle-treated counterparts (Figure 4). The retinal expression of VEGF was significantly increased in the STZ-diabetic rats compared with those in normal rats at both protein and mRNA levels, which were decreased by 300 mg/kg/day PSEE treatment to 45.3% and 54.3%, respectively, relative to those observed in the vehicle-treated counterparts (Figure 4). . Normal or STZ-diabetic rats receiving vehicle treatment were given the same volume of vehicle (distilled water) used to prepare the test medication solutions. Results in each column are mean  SD from ten rats per group. a p < 0.05 and b p < 0.01 compared to vehicle-treated normal rats, respectively. c p < 0.05 and d p < 0.01 compared to the values of vehicle-treated STZ-diabetic rats, respectively.

Effects on Protein Expression and Phosphorylation of Akt in Retinas
No change was observed in the protein level of Akt in the retinas of STZ-diabetic rats compared with the normal group ( Figure 5). PSEE did not affect the retinal Akt protein expression in STZdiabetic rats ( Figure 5). The immunoblot results showed that the phosphorylation of Akt on Ser 473 and Thr 308 were 2.6-and 2.2-fold greater in the retinas of STZ-diabetic rats than in the normal group, respectively ( Figure 5). These STZ-induced upregulation in Akt phosphorylation was significantly reversed in the retinas after 8-week treatment with 300 mg/kg/day PSEE (56.6% decreases in Ser 473 and 57.1% decreases in Thr 308, relative to those in vehicle-treated STZ-diabetic rats; Figure 5). The STZ markedly elevated the ratio of pAkt (Ser 473)/Akt and pAkt (Thr 308)/Akt by 2.4-and 2.2-fold relative to those in vehicle-treated STZ-diabetic rats, respectively, in the retinas of the rats ( Figure 5). Treatment of STZ-diabetic rats with 300 mg/kg/day PSEE significantly downregulated the ratios of pAkt (Ser 473)/Akt and pAkt (Thr 308)/Akt in the retinas to 1.5-and 1.1-fold relative to those in vehicle-treated STZ-diabetic rats ( Figure 5). . Normal or STZ-diabetic rats receiving vehicle treatment were given the same volume of vehicle (distilled water) used to prepare the test medication solutions. Results in each column are mean ± SD from ten rats per group. a p < 0.05 and b p < 0.01 compared to vehicle-treated normal rats, respectively. c p < 0.05 and d p < 0.01 compared to the values of vehicle-treated STZ-diabetic rats, respectively.

Effects on Protein Expression and Phosphorylation of Akt in Retinas
No change was observed in the protein level of Akt in the retinas of STZ-diabetic rats compared with the normal group ( Figure 5). PSEE did not affect the retinal Akt protein expression in STZ-diabetic rats ( Figure 5). The immunoblot results showed that the phosphorylation of Akt on Ser 473 and Thr 308 were 2.6-and 2.2-fold greater in the retinas of STZ-diabetic rats than in the normal group, respectively ( Figure 5). These STZ-induced upregulation in Akt phosphorylation was significantly reversed in the retinas after 8-week treatment with 300 mg/kg/day PSEE (56.6% decreases in Ser 473 and 57.1% decreases in Thr 308, relative to those in vehicle-treated STZ-diabetic rats; Figure 5). The STZ markedly elevated the ratio of pAkt (Ser 473)/Akt and pAkt (Thr 308)/Akt by 2.4-and 2.2-fold relative to those in vehicle-treated STZ-diabetic rats, respectively, in the retinas of the rats ( Figure 5). Treatment of STZ-diabetic rats with 300 mg/kg/day PSEE significantly downregulated the ratios of pAkt (Ser 473)/Akt and pAkt (Thr 308)/Akt in the retinas to 1.5-and 1.1-fold relative to those in vehicle-treated STZ-diabetic rats ( Figure 5).

Figure 5. Effects of treatments on protein expression and phosphorylation of Akt in retinas.
Representative immunoblots of retinal protein levels and phosphorylation degrees of Akt in rats receiving 8-week treatment. STZ-diabetic rats (STZ) were dosed by oral gavage once per day for 8 weeks with PSEE at dosages of 100 (PSEE 100), 200 (PSEE 200), or 300 mg/kg (PSEE 300). Normal or STZ-diabetic rats receiving vehicle treatment were given the same volume of vehicle (distilled water) used to prepare the test medication solutions. The pAkt/Akt ratio is expressed as the mean with mean  SD from ten rats per group. a p < 0.05 and b p < 0.01 compared to vehicle-treated normal rats, respectively. c p < 0.05 and d p < 0.01 compared to the values of vehicle-treated STZ-diabetic rats, respectively.

Effects on Activities of NF-κB, HDAC and HAT in Retinas
The activities of NF-κB and HDAC in the retinas of STZ-diabetic rats were 2.4-and 3.6-fold higher than those of the normal group, respectively ( Figure 6). These STZ-induced up-regulations in activities of NF-κB and HDAC were lowered in the retina after treatment with 300 mg/kg/day PSEE, at 58.4% and 60.8% the levels seen in the vehicle-treated STZ-diabetic rats, respectively ( Figure 6).
The retinal HAT activity was 58.6% lower in STZ-diabetic rats than in the normal group, which were enhanced by 300 mg/kg/day PSEE treatment, with a 1.6-fold elevation, respectively, when compared with the levels observed in the vehicle-treated counterparts ( Figure 6). . Normal or STZ-diabetic rats receiving vehicle treatment were given the same volume of vehicle (distilled water) used to prepare the test medication solutions. The pAkt/Akt ratio is expressed as the mean with mean ± SD from ten rats per group. a p < 0.05 and b p < 0.01 compared to vehicle-treated normal rats, respectively. c p < 0.05 and d p < 0.01 compared to the values of vehicle-treated STZ-diabetic rats, respectively.

Effects on Activities of NF-κB, HDAC and HAT in Retinas
The activities of NF-κB and HDAC in the retinas of STZ-diabetic rats were 2.4-and 3.6-fold higher than those of the normal group, respectively ( Figure 6). These STZ-induced up-regulations in activities of NF-κB and HDAC were lowered in the retina after treatment with 300 mg/kg/day PSEE, at 58.4% and 60.8% the levels seen in the vehicle-treated STZ-diabetic rats, respectively ( Figure 6).
The retinal HAT activity was 58.6% lower in STZ-diabetic rats than in the normal group, which were enhanced by 300 mg/kg/day PSEE treatment, with a 1.6-fold elevation, respectively, when compared with the levels observed in the vehicle-treated counterparts ( Figure 6).  . Normal or STZ-diabetic rats receiving vehicle treatment were given the same volume of vehicle (distilled water) used to prepare the test medication solutions. Results in each column are mean  SD from ten rats per group. a p < 0.05 and b p < 0.01 compared to vehicletreated normal rats, respectively. c p < 0.05 and d p < 0.01 compared to the values of vehicle-treated STZ-diabetic rats, respectively.

Discussion
Data from multicenter prospective studies have shown that an abnormally high glucose concentration in blood is the principal cause of microvascular and macrovascular complications [1]. Therefore, tight control of blood glucose is the key to preventing or reversing diabetic complications in diabetic patients [11]. In vitro and in vivo studies have shown that dietary polyphenols may inhibit α-amylase and α-glucosidase, inhibit glucose absorption in the intestine by sodium-dependent glucose transporter 1, stimulate insulin secretion and reduce hepatic glucose output, suggesting that polyphenols could improve glucose homeostasis through potential multiple mechanisms of action and might be one dietary therapy for the prevention and management of diabetes [13]. In the present Figure 6. Activity of nuclear factor-κB (NF-κB), histone deacetylases (HDAC) and histone acetyltransferases (HAT) in retinas of rats receiving 8-week treatment. STZ-diabetic rats (STZ) were dosed by oral gavage once per day for 8 weeks with PSEE at dosages of 100 (PSEE 100), 200 (PSEE 200), or 300 mg/kg (PSEE 300). Normal or STZ-diabetic rats receiving vehicle treatment were given the same volume of vehicle (distilled water) used to prepare the test medication solutions. Results in each column are mean ± SD from ten rats per group. a p < 0.05 and b p < 0.01 compared to vehicle-treated normal rats, respectively. c p < 0.05 and d p < 0.01 compared to the values of vehicle-treated STZ-diabetic rats, respectively.

Discussion
Data from multicenter prospective studies have shown that an abnormally high glucose concentration in blood is the principal cause of microvascular and macrovascular complications [1]. Therefore, tight control of blood glucose is the key to preventing or reversing diabetic complications in diabetic patients [11]. In vitro and in vivo studies have shown that dietary polyphenols may inhibit α-amylase and α-glucosidase, inhibit glucose absorption in the intestine by sodium-dependent glucose transporter 1, stimulate insulin secretion and reduce hepatic glucose output, suggesting that polyphenols could improve glucose homeostasis through potential multiple mechanisms of action and might be one dietary therapy for the prevention and management of diabetes [13]. In the present study, PSEE treatment showed significant and consistent reduction in fasting blood glucose levels and also improved the body weight loss in STZ-diabetic rats as compared to the vehicle treated diabetic controls, indicating its potent antidiabetic activity on an insulin deficient animal model. PSEE was rich in polyphenolic flavonoids, and its antidiabetic activity may be attributed to the presence of these.
Antioxidant nutrients and phytonutrients have been reported to inhibit the oxidation of living cells by free radicals and result in a decrease in oxidative stress [30]. In the present study, the antioxidant capacity of PSEE was measured by ABTS radical cation decolorization assay and showing promising results. It is clear that PSEE was rich in polyphenols and shown with antioxidant potential. Actually, long-term hyperglycemia could lead to an increase in ROS generation and decreased antioxidant capacity in diabetes [3]. The retina is particularly susceptible to oxidative stress because of high energy demands and exposure to light [31]. Regarding the oxidative stress affects the pathogenesis of DR, correction of oxidant-antioxidant balance may be a powerful approach for preventing vision loss associated with DR [3]. It is well known that SOD, CAT, and GSH constitute a mutually supportive team of defense against ROS [4]. In our study, decline in the activities of these enzymes in the retinal tissue of STZ-diabetic rats and attainment of near normalcy in PSEE-treated rats indicate that oxidative stress elicited in the retina of diabetic rats had been nullified due to the effect of PSEE. Thus, our results suggest that PSEE has potential to overcome the hyperglycemia-specific microvascular complications.
The growing evidence has suggested that BRB breakdown, leakage capillaries and vascular structural and functional changes are characteristic for the diabetic retina [6]. Similar with the previous study [32], we observed that the number of leukocytes adhered to the retinal vascular endothelium was increased in STZ-diabetic rats; accordingly, vascular permeability and the retinal vessels diameter were increased as well. In consistent with the attenuated leukostasis, PSEE reduced diabetic retinal vascular leakage accompanied by restrained the retinal vascular dilation in STZ-diabetic rats. Prevention of diabetes-related structural disorganization of the retina might be an important contributor of PSEE to the preventing the progression of DR.
VEGF, an endothelial angiogenic and vasopermeability factor, is known to be a key molecule leading to retinal permeability and breakdown of BRB in diabetes and other retinal diseases [33]. Regulation of VEGF expression is complex, and HIF-1α is one of the transcription factors that regulate VEGF expression under hyperglycemia [8,9]. Furthermore, Akt activation has been recognized as an upstream regulator of HIF-1α expression [34]. Therefore, promotion of the Akt-HIF-1α-VEGF signaling pathway contributes to the induction of retinal vascularization [35]. We found that the elevated contents of HIF-1α and VEGF in retinae of STZ-diabetic rats were both reduced in rats receiving PSEE treatment. In addition, the results of the present study revealed an increase in phosphorylation of Thr 308 and Ser 473 of Akt in the retinas of STZ-diabetic rats; the deficit was ameliorated by PSEE treatment. Therefore, it can be considered that PSEE rescued diabetic retinal vasculopathy by downregulation of HIF-1-mediated induction of VEGF expression via suppressing Akt activation in the retina of STZ-diabetic rats. Full activation of Akt requires phosphorylation on Thr 308 and Ser 473 by 3-phosphoinositide-dependent kinase-1 and Ser-473 kinase, respectively [36]. Thus, the role of PSEE on the alterations in Akt signaling in the development of DR will be identified in future research work.
Numerous studies show that hyperglycemia leads to oxidative stress in the diabetic retinas, which has been associated with cellular inflammation and release of inflammatory cytokines [2]. One of these mediators is TNF-α, a proinflammatory cytokine which is known as an initiator of inflammatory reactions [7]. Similarly, IL-1β can be up-regulated in the retina in diabetes [5]. In addition to increases in the above-mentioned inflammatory mediators, both molecules ICAM-1 and VCAM-1 promote chemoattraction of leukocytes into the vascular walls and their migration into retinal tissues, which accounts for the majority of diabetes-associated retinal vascular leakage [37]. Actually, plantaginis semen significantly inhibited lipopolysaccharide-induced cyclooxygenase-2 (concentration required for 50% inhibition [IC(50)] = 8.61 µg/mL, TNF-α [IC(50)] = 9.63 µg/mL, and nitric oxide [IC(50)] = 8.65 µg/mL) production in RAW 264.7 cells; anti-inflammatory activity of plantaginis semen has been reported [38]. In the present study, retinae from PSEE-treated STZ-diabetic rats showed lower levels of inflammatory cytokines and chemokines, suggesting that the extract acted against inflammatory response triggered by hyperglycemia. These results support the proposition that protection of PSEE from retinal damage in STZ-diabetic rats was mediated by blockade of diabetes-induced production of inflammatory molecules in retinal tissue, and attenuates retinal vascular leakage.
NF-κB plays a critical role in diabetes complications as it regulates transcription of a number of genes involved in inflammatory response [39]. Actually, it has been demonstrated that the subunits of the NF-κB signaling pathway, including the inhibitory transcription factor IκB, are acetylated/deacetylated by the HAT and HDAC, respectively [40]. High HDAC activity may therefore maintain deacetylated such inhibitory factor allowing for NF-κB activation [41]. Recent studies have shown that diabetes induced increase HDAC activity in the retina and kidney, that are the tissues associated with microvascular complications [42]. It has also been reported that hyperglycemia-induced superoxide overproduction activates HDAC activity and decreases HAT activity [43]. PSEE treatment results in a significant increase in HAT activity and a parallel decrease in activies of HDAC and NF-κB in retinae of STZ-diabetic rats. The effects of PSEE seems to play a role in controlling NF-κB activation and modulation of HDAC and HAT activity, consequently affecting the expression of inflammatory response genes.
Medicinal plants produced several useful biological activities; however, the inclusion of toxicological evaluation at preclinical stage will assure its safe usage in humans as a medicine [44]. Further studies are needed to clarify toxicity of PSEE to rat at the effective dosage used for treating DR. Whether PSEE is effective in human for DR improvement also need further evaluate in clinical studies.

Conclusions
The overall findings indicate that PSEE has protective effect on DR with possible mechanisms of lowering plasma glucose, rescuing oxidative stress and supression of angiogenesis via down-regulation of the HIF-1α/VEGF signaling axis accompanied by Akt inhibition. Impairment of NF-κB activation and maintain the balance between HAT and HDAC, and thereby limiting the inflammatory response has also been suggested as a possible underlying mechanism of PSEE involved in preventing the progression of diabetic retinal vascular diseases. PSEE supplementation may be considered as an alternative choice used for the prevention of retinal microvascular complications of diabetes. PSEE has abundant polyphenolic compounds with strong antioxidant activity. Polyphenols are divided into flavonoids, phenolic acids, stilbenes, and lignans [12]. Thus, the specific components of PSEE that are mainly responsible for its protective effects on retinas will be identified in future research work.