Fushiming Capsule Attenuates Diabetic Rat Retina Damage via Antioxidation and Anti-Inflammation

Aims Diabetic retinopathy (DR) remains one of the leading causes of acquired blindness. Fushiming capsule (FSM), a compound traditional Chinese medicine, is clinically used for DR treatment in China. The present study was to investigate the effect of FSM on retinal alterations, inflammatory response, and oxidative stress triggered by diabetes. Main Methods Diabetic rat model was induced by 6-week high-fat and high-sugar diet combined with 35 mg/kg streptozotocin (STZ). 30 days after successful establishment of diabetic rat model, full field electroretinography (ffERG) and optical coherence tomography (OCT) were performed to detect retinal pathological alterations. Then, FSM was administered to diabetic rats at different dosages for 42-day treatment and diabetic rats treated with Calcium dobesilate (CaD) capsule served as the positive group. Retinal function and structure were observed, and retinal vascular endothelial growth factor-α (VEGF-α), glial fibrillary acidic (GFAP), and vascular cell adhesion protein-1 (VCAM-1) expressions were measured both on mRNA and protein levels, and a series of blood metabolic indicators were also assessed. Key Findings In DR rats, FSM (1.0 g/kg and 0.5 g/kg) treatment significantly restored retinal function (a higher amplitude of b-wave in dark-adaptation 3.0 and OPs2 wave) and prevented the decrease of retinal thickness including inner nuclear layer (INL), outer nuclear layer (ONL), and entire retina. Additionally, FSM dramatically decreased VEGF-α, GFAP, and VCAM-1 expressions in retinal tissues. Moreover, FSM notably improved serum antioxidative enzymes glutathione peroxidase, superoxide dismutase, and catalase activities, whereas it reduced serum advanced glycation end products, methane dicarboxylic aldehyde, nitric oxide, and total cholesterol and triglycerides levels. Significance FSM could ameliorate diabetic rat retina damage possibly via inhibiting inflammation and improving antioxidation.


Introduction
Diabetic retinopathy (DR) is a kind of neurovascular complication of diabetes [1]. Despite the availability of effective treatment, DR remains one of the most common causes of blindness among working age adults [2]. It is expected that the global number of diabetes patients will double by 2030 comparing with about 400 million in 2017 [3,4]. Furthermore, more than a third of people with diabetes have symptoms of DR [5]. Currently, the main effective methods for DR treatment include laser photocoagulation, vitrectomy, and intravitreal injections of corticosteroids or anti-VEGF agents [2,6]. These mentioned methods exhibit modest clinical effects, but none is yet to fully deaden clinical progression or reverse retinal damage [7]. Additionally, the frequent intraocular injections probably cause side effects involving corneal scarring and intraocular infection, not to mention the expenses related to frequent ophthalmology clinician visits. The alternative solution is limited for patients, which makes it an urgency to find new medications.
It is well known that Calcium dobesilate (calcium 2,5dihydroxybenzenesulfonic acid, CaD) has been prescribed for decades to some diabetic patients in the early stage of DR to stave off its progression [31]. According to the previous study, this synthetic compound exhibited systemic antiinflammatory and antioxidative properties in the treatment of microcirculatory disorders [32]. Therefore, in this work, CaD was administered in the positive group, for 42 days, to diabetic animals, after 30 days of diabetes.
Among the factors implicated in the pathogenesis of DR, hyperglycemia-mediated inflammation and oxidative stress are considered as major relevant instigators of DR [33]. Inflammatory process, such as cytokine network and leukocyte adhesion (e.g., vascular cell adhesion protein-1 (VCAM-1)), induces vascular hyperpermeability and blood-retinal barrier breakdown [34]. Oxidative stress can directly or indirectly stimulate the release of proinflammatory cytokines, vascular endothelial growth factor-(VEGF-), and nitric oxide (NO), which results in retinal cell damage, pathological neovascularization, and the succeeding development of DR pathogenesis. Additionally, inflammation responses and low activities of antioxidative enzymes such as glutathione peroxidase (GSH-Px), Catalase (CAT), and superoxide dismutase (SOD) contribute to further increasing accumulation of reactive oxygen species (ROS) [35,36]. This results in enhanced oxidative stress and aggravated retinal neuron damage and cell apoptosis. Moreover, hyperglycemia also causes Müller cells to produce proinflammatory cytokines to restore the retinal homeostasis [37,38]. This is characterized by upregulation of glial fibrillary acidic (GFAP) and VEGF, which leads to a glial reaction and blood barrier hyperpermeability [39]. Based on the above observations, the present study  was designed to test whether Fushiming capsule, a specific  compound traditional Chinese medicine, could alleviate  diabetic retina damage induced by high-fat and high-sugar  diet combined with a low dose STZ injection and to determine how Fushiming capsule modulates oxidative stress and  inflammatory

Diabetic Retinopathy Rat Model and Drug Administration.
SD rats were fed with high-fat and high-sugar diet (breeding rodent material 54.6%, lard 16.9%, sucrose 14%, casein 10.2%, premix 2.1%, and maltodextrin 2.2%) (Slac Laboratory Animal, Shanghai, China) for 6 weeks after the rats were allowed to acclimate for 3 days. Then, the diabetic rat model was induced by a single intraperitoneal injection of streptozotocin (35 mg/kg body weight [40]) (Sigma, USA) after 6-week high-fat and high-sugar diet. Seventy-two hours after STZ injection, blood glucose level was measured by ACCU-CHEK Performa (Roche, Germany) and rats showing a blood glucose level above 16.7 mmol/L were considered as diabetes mellitus and selected for the study feeding with high-fat and high-sugar diet. After 30 days, ERG and OCT were performed for observing pathological changes in diabetic retinas. Then, according to the amplitude of the electroretinogram OPs2 wave, 40 successfully constructed diabetic retinopathy (DR) model rats were selected and randomly divided into 5 groups (n=8): untreated diabetic model group (normal saline, equal volume), positive group (calcium dobesilate (CaD) capsule, 0.2 g/kg, Guizhou Tian'an Pharmaceutical Co., Ltd., China) (CaD group), FSM high-dose group (FSM, 1.0 g/kg, Xi'an Lejian Biological Technology Co., Ltd., China), FSM medium-dose group (FSM, 0.5 g/kg), and FSM low-dose group (FSM, 0.25 g/kg), and different treatments were given for 42 consecutive days. In addition, 8 normal rats with matched ages were selected as the normal group (normal saline, equal volume) and were fed with ordinary diet. The method of administration is gavage, once a day. The rats were monitored throughout the study for body weight and blood glucose and were maintained on their respective diets throughout the treatment period.

Full Field Electroretinography (ffERG).
Full field electroretinography (ffERG) measurements were obtained at 30 d after diabetic rat model established and 42 d posttreatment (72 d of total duration of diabetes). ERG operation procedures were performed like the method described previously [41,42] which was in compliance with the ISCEV guidelines. The ffERG recording items covered dark-adapted 0.01 response, dark-adapted 3.0 response, dark-adapted oscillatory potential response, light-adapted 3.0 response, and lightadapted flicker response. Specifically, animals were placed in a dark environment for dark adaption overnight (>12 h) and prepared for recording under a dim red-light condition. Anesthesia was applied with intraperitoneal (IP) injection of 3 mL/kg 1% sodium pentobarbital (Sigma, USA) and 50 L sumianxin II (Jilin Shengda Animal Pharmaceutical Co., Ltd., China). The pupils were dilated with 0.5% tropicamide solution (Shenyang Xingji Corporation, China). FfERG was recorded using the full-field (Ganzfeld) stimulation and a computer system (RETI port, Roland Consult GmbH, Germany) with custom-made silver chloride electrodes. The active electrode was a ring electrode placed at the center of the cornea. Stainless steel needle electrodes were placed in the cheek and tail to serve as the reference and ground leads, respectively. Levofloxacin eye drops (Suzhou, China) were used three times a day after ERG testing to avoid infection.

Optical Coherence Tomography (OCT).
Optical coherence tomography (OCT) images were taken to observe structural changes of the retina at 30 d after diabetic rat model established and 42 d posttreatment (72 d of total duration of diabetes). OCT scans were performed with a Micron IV fundus camera and an OCT Scan Head equipped with a mouse objective lens. Linear OCT scans consisted of a series of 1024 single point A-Scans. Rat eyes had previously been dilated with 0.5% tropicamide. And hydroxyl ethyl cellulose l (Bausch & Lomb Freda, China) was used as a coupling gel. Fundus and OCT images were captured from 20 positions for each eye using a Retinal Imaging System (OPTO-RIS, OptoProbe, Canada) and 4D-ISOCT Microscope Imaging System (ISOCT, OptoProbe, Canada).

2.5.
Determination of the Retinal VEGF-, GFAP, and VCAM-1 mRNA Expression. After blood samples were taken, the left eyes of rats were removed and then the retina tissues were collected at 42 d posttreatment (72 d of total duration of diabetes). Total RNA was extracted from the retinal tissues using Trizol Reagent (Servicebio, G3013, Wuhan, China). RNA concentration was detected by Nanodrop 2000 (Thermo, New York, USA). cDNA was synthesized according to the manufacturer's instructions of RevertAid M-MuLV cDNA synthesis kit (Thermo, #k1622, USA). The fluorescent quantitative polymerase chain reaction (FQ-PCR) was performed using ABI Stepone plus real-time PCR device (New York, USA) according to the protocol of FastStart Universal SYBR-Green Master kit (Roche, Mannheim, Germany). Cycling conditions were as follows: 95 ∘ C, 10 min, and 40 cycles of 95 ∘ C, 15 s, 60 ∘ C, 60 s. The primers of VEGF-were GAGCAGAAAGCCCATGAAGTG (forward) and ACTCCAGGGCTTCATCATTGC (reverse). The primers of GFAP were AGTCGGCGAGTTACCAGGAG (forward) and TTAATGACCTCGCCATCCCG (reverse). The primers of VCAM-1 were TGAACCCAAACAAAG-GCAGAGTA (forward) and TTGGGAGTTGGAAAAC-CATCAC (reverse). The primers of -actin were TGCTAT-GTTGCCCTAGACTTCG (forward) and GTTGGCATA-GAGGTCTTTACGG (reverse). Quantification of VEGF-, GFAP, and VCAM-1 mRNA was normalized to -actin mRNA, and analysis was performed using 2−ΔΔCt method. The final results were expressed as percentage relative to the normal group.

Measurement of Blood Metabolic Indicators.
After drug had been administered for 42 days (72 d of total duration of diabetes), fasted (12 h) rats were weighed and anesthetized (1% sodium pentobarbital (Sigma, USA), 3 mL/kg, i.p.). Blood specimens were collected from the vena cava. Serum samples were isolated after centrifugation of whole blood at 3500 r/min at 4 ∘ C for 15 min and were stored at −80 ∘ C. Enzyme activities of serum antioxidant parameters glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT), and levels of serum oxidative stress parameter methane dicarboxylic aldehyde (MDA) and proinflammatory indicator nitric oxide (NO) were assessed using commercially available ultraviolet spectrometry method kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), according to the manufacturer's instructions. The levels of serum glucose (GLU), triglycerides (TG), and total cholesterol (TC) were estimated using commercially available kits from Huili Biotech (Changchun, China), according to the manufacturer's instructions. Additionally, advanced glycation end-product formations (AGEs) level was detected by commercially available enzyme-linked immunosorbent assay (ELISA) kits (Cloud-Clone Corp., Wuhan, China), according to the manufacturer's instruction.

Statistical
Analysis. All data were expressed as mean ± standard deviation. All statistical analyses were conducted using the SPSS version 19.0 software package (IBM, Chicago, IL, USA). One-way analysis of variance (ANOVA) test was performed, and post hoc multiple comparisons were conducted with LSD. P < 0.05 was regarded to be significant. was found that OPs2 wave decreased to 50-60% of normal eyes (all p < 0.01) and b (d3.0) wave decreased to 70-80% of normal eyes (all p < 0.01). According to the amplitude of OPs2 wave, diabetic rats were randomly divided into 5 groups as previously described, and the age matching normal SD rats were selected as normal group. The final results were expressed as percentage relative to the normal group.
The final results were expressed as percentage relative to the normal group.

OCT Analysis
3.2.1. OCT Analysis before the Treatment. OCT was applied to evaluate retinal morphometric structure. As shown in Figure 3, the total retinal thickness decreased significantly at 30 d after diabetic rat model established. Particularly, ganglion cell layer (GCL) and inner plexiform layer (IPL), outer plexiform layer (OPL), and entire retina thickness became thinner (all p < 0.01). Thus, diabetic rats exhibited a thinner retinal thickness, which indicated to some extent that the retinal structure alterations occurred.

OCT Analysis after the Treatment.
OCT was examined to observe retinal structural alterations after 42-d treatment (72 d of total duration of diabetes). As shown in Figure 4, the retina thickness of diabetic rats, especially, ganglion cell layer and inner plexiform layer (GCL and IPL), inner nuclear layer (INL), outer nuclear layer (ONL), and entire retinal thickness were declining compared with normal group (all p < 0.05). Additionally, CaD group, FSM high-dose, FSM medium-dose, and FSM low-dose group showed a lower decrease in INL, ONL, and total retinal thickness (all p < 0.01) compared with untreated diabetic rats. Moreover, CaD group and FSM high-dose group had thicker GCL and IPL thickness than untreated diabetic rats (all p < 0.01). Besides, CaD group, FSM high-dose, and FSM medium-dose group showed thicker external limiting membrane (ELM) and photoreceptor inner/outer segment (IS/OS) thickness than untreated diabetic rats (all p < 0.05). FSM and CaD both showed protective effect on retinal structure integrity. As for the entire thickness of internal limiting membrane (ILM) and nerve fiber layer (NFL), there existed no significant difference among different groups (all p > 0.05).

VEGF-, GFAP, and VCAM-1 mRNA Expressions.
In order to evaluate the potential anti-inflammatory properties and effect on glial cell reactivity, VEGF-, GFAP, and VCAM-1 mRNA levels were analyzed by FQ-PCR after 42-d treatment (72 d of total duration of diabetes). As is shown in Figure 5, the expressions of VEGF-, GFAP, and VCAM-1 in all diabetic groups, excepting the expression of GFAP in FSM high-dose group, were incredibly more than those in normal group (all p < 0.05). Nevertheless, the expression of GFAP in CaD group, FSM high-dose, FSM medium-dose, and FSM lowdose group was decreasing compared with that in untreated diabetic model group (all p < 0.05). Moreover, the expressions of VEGF-and VCAM-1 in CaD group, FSM high-dose, and FSM medium-dose group were less than those in untreated diabetic model group (all p < 0.05). shown in Figure 6, the expressions of VEGF-, GFAP, and VCAM-1 in all diabetic groups, excepting the expression of GFAP in FSM high-dose group, were notably more than those in normal group, in agreement with the results obtained by FQ-PCR. However, the expressions of VEGF-and GFAP in CaD group, FSM high-dose, FSM medium-dose, and FSM low-dose group were less than those in untreated diabetic model group (all p < 0.05). Additionally, the expression of VCAM-1 in CaD group and FSM high-dose and FSM medium-dose groups was less than that in untreated diabetic model group (all p < 0.05), in consistence with the results obtained by FQ-PCR.

Blood Metabolic Indicators
3.5.1. The Level of GLU, TC, and TG. As compared with normal rats, serum levels of GLU, TC, and TG in diabetic rats were markedly increased (all p < 0.05) (Figure 7). There is no significant difference in GLU among diabetic groups (all p > 0.05). After 42-d treatment (72 d of total duration of diabetes), CaD group, FSM high-dose group, and FSM medium-dose group notably decreased TC level (all p < 0.01 versus the untreated diabetic model group). Additionally, CaD group and FSM medium-dose group significantly decreased TG level (all p < 0. 05 versus the untreated diabetic model group).

The
Level of Detrimental Accumulation of AGEs, MDA, and NO. Serum AGEs, MDA, and NO levels were measured to assess the degree of oxidative stress and inflammatory condition/nitrosative stress in rats after 42-d treatment (72 d of total duration of diabetes). As compared with normal rats, serum levels of AGEs, MDA, and NO in diabetic rats, excepting the levels of MDA in FSM high-dose and mediumdose group, were markedly increased (all < 0.05) ( Figure 8).
As compared with untreated diabetic model group, CaD group, FSM high-dose group, and FSM medium-dose group notably decreased AGEs and NO levels (all < 0.05).
Additionally, CaD group, FSM high-dose, FSM mediumdose, and FSM low-dose group notably decreased MDA level (all < 0.01 versus the untreated diabetic model group).

The Activity of Antioxidant Enzymes GSH-Px, SOD, and CAT.
Considering the antioxidant stress property of FSM, the effects of this compound were evaluated on serum GSH-Px, SOD, and CAT activities after 42-d treatment (72 d of total duration of diabetes). As compared with normal rats, serum levels of GSH-Px, SOD, and CAT in diabetic rats, excepting the levels of SOD and CAT in FSM high-dose group, were markedly decreased (all < 0.05) (Figure 9). However, CaD group, FSM high-dose, and FSM medium-dose group notably increased GSH-Px level (all < 0.05 versus the untreated diabetic model group). FSM high-dose and FSM mediumdose group significantly increased SOD level (all < 0.05 versus the untreated diabetic model group). Additionally, CaD group and FSM high-dose group significantly decreased CAT level (all < 0.05 versus the untreated diabetic model group).

Discussion
Diabetic retinopathy (DR) is the leading cause of visual impairment and preventable blindness [1], which represents a significant socioeconomic burden for healthcare systems worldwide [7]. In the study, we evaluated the effect of compound traditional Chinese medicine FSM on DR treatment. A better retinal function and structure were found in FSM treatment groups because of an enhanced ability of antioxidation and anti-inflammation.
In the present study, diabetes was established by 6week high-fat and high-sugar diet combined with 35 mg/kg streptozotocin (STZ) injection. We found that, at 30 days post diabetic rat model established, retinal function in the diabetic rats was decreased, both b (d3.0) wave and OPs2 wave compared with normal rats. Furthermore, diabetic rats were randomly grouped by OPs2 indicator. It is reported that oscillatory potentials (OPs) are considered the most relevant ERG test for DR diagnosis and progression [43]. Clinical research found that a frequent reduction or absence of OPs was shown in diabetic patients even in a preclinical stage of retinopathy, and the lower the amplitude of OPs2 is, the severer the DR is [44]. Otherwise, the reduction of retina thickness showed in OCT affirmed the retina morphological alterations. After 42-day treatment, retinal function in the FSM treated groups was restored in both b wave and OPs Our results suggested a vascular endothelial dysfunction in diabetic rat retina, as estimated by assays of VCAM-1 protein and VCAM-1 mRNA expression. It was found that the upregulation of VCAM-1, a cell adhesion molecule, associated to increased inflammation-related genes transcription such as tumor necrosis factor-alpha (TNF-), interleukin-1 (IL-1), and nuclear factor-kappa B (NF-B) [46]. Thus, we speculated that the enhanced endothelial activation reflected the inflammatory nature in this hyperglycemic state [47]. Interestingly, hyperglycemia also caused the stress condition of astrocytes and Müller cells, which was suggested by the increased expression of retinal GFAP in DR rats and these findings were in agreement with previous studies [38,48]. The induction of GFAP expression usually occurred after retinal damage and indicated that the retina was undergoing degeneration [49]. It was worth mentioning that the upregulation of GFAP also initiated proinflammatory cytokines release [50]. Our findings suggested that FSM and CaD could relieve the inflammation by inhibiting the increase of VCAM-1 and GFAP expression. Furthermore, FSM and CaD reduced the upregulated expression of retinal VEGF-, a major angiogenic factor, in DR rats. VEGF-was considered to associate with oxidative stress and inflammatory response and induce retinal neovascularization, vascular leakage, and macular edema [51][52][53]. Additionally, the effect of CaD on VEGF-was confirmed by other researches [54,55], but FSM probably revealed better effects, at least according to the PCR and western blot results in our research. Our results presented that the compound traditional Chinese medicine FSM could downregulate the expression of VEGFto depress angiogenesis of DR, which may provide a new option or supplement for patients.
It was noteworthy that diabetic rats showed not only upregulation of inflammatory mediators and endothelial activation in the retina, but also a systemic inflammatory response and oxidative stress in this model. It was reported that high serum NO production would increase systemic oxidative damage and nitrosative stress, which was related to increased severity of DR [18,56,57]. Moreover, upregulation of ROS could lead to enhanced activation of NF-kB, which, in turn, increased the release of proinflammatory cytokines and NO. Therefore, oxidative stress might indirectly contribute to DR through stimulating inflammation or directly contribute to the DR via oxidative damage to cells. Additionally, MDA, one of the breakdown products of peroxidized polyunsaturated fatty acids, was considered to be sensitive and reliable for the assessment of oxidative stress [58,59]. AGEs accumulated under hyperglycemic conditions were reported to increase proinflammatory mediators, stimulate ROS production, cause retinal microvascular endothelial cells dysfunction, and enhance NO and VEGF secretion. They all occurred against a background of the various metabolic derangements that were inherent to diabetes [60]. Our results revealed that, compared with untreated diabetes, serum NO, MDA, and AGEs concentrations were downregulated and antioxidative SOD, GSH-Px, and CAT activities were upregulated by FSM, which indicated that FSM could protect the retinal structure and function via regulating nitrogen species homeostasis, reducing oxidative stress, and ameliorating inflammatory response.
Moreover, dyslipidemia was found in DR rats. Longterm effects of dyslipidemia might be of importance for progression of DR and it was associated with retinal hard exudate and visual loss [61]. Reducing the levels of serum triglycerides showed an effect on the progression of DR, as reported in several observation studies [1]. In consistence with those reports, our data supported the fact that TC and TG levels were increased in DR rats. Furthermore, FSM depressed serum TC and TG level, and this might be related to the hypolipidemic activity of puerarin, the main compound constituting FSM [8], which could rapidly be absorbed in the small intestines [62] and increase the fecal excretion of TG, promote the oxidation of fatty acids, and suppress the synthesis of fats [63].
Our findings not only revealed the effects of FSM on retinal damage but also determined the pharmacological effect of CaD that was confirmed by other studies, and our results were consistent with those previous reports about CaD. In this regard, FSM showed satisfying benefits on protecting retinal function and structure and blood homeostasis, which resembled or rivaled CaD. However, lack of research on further mechanism of the major compounds of FSM may be the limitation in our study. In the next step, we would like to thoroughly evaluate the accurate mechanisms of the components of FSM on DR.

Conclusion
In summary, FSM exerted a multifaceted action on protecting the diabetic rat retinal function and morphological structure integrity. The potential mechanism of FSM protective role would be associated with antioxidant effects directly by scavenging free radicals or indirectly by increasing the antioxidant enzymes, regulating nitrogen species homeostasis, and ameliorating inflammatory response. Therefore, FSM could be contemplated as an alternative candidate for treatment of DR.

Data Availability
The original data used to support the findings of this study are included within the article.