Plant-based diets have been associated with a lower risk of developing chronic diseases such as obesity

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Abstract: It has been suggested that spinach methanolic extract (SME) inhibits the formation of advanced glycation end products (AGEs), which are increased during diabetes progression, so it is important to know if SMEs have beneficial effects on the diabetic retina. In this study, in vitro assays showed that SME inhibits glycation, carbonyl group formation, and reduced-thiol group depletion in bovine serum albumin incubated either reducing sugars or methylglyoxal. The SME effect in the retinas of streptozotocin-induced diabetic rats (STZ2) was also studied (n=10) in the normoglycemic group, STZ, STZ rats treated with SME, and STZ rats treated with aminoguanidine (anti-AGEs reference group) during 12 weeks. The retina was sectioned and immunostained for Ne-carboxymethyl lysine (CML), receptor RAGE, NADPH-Nox4, inducible nitric oxide synthase (iNOS),3-nitrotyrosine (NT), nuclear NF-kB, vascular endothelial growth factor (VEGF), glial fibrillary acidic protein(GFAP), S100B protein, and TUNEL assay. Lipid peroxidation was determined in the whole retina by malondialdehyde (MDA) levels. The results showed that in the diabetic retina, SME reduced the CML-RAGE co-localization, oxidative stress(NOX4,iNOS,NT,MDA),inflammation(NF-B,VEGF,S10B, GFAP), and apoptosis (p<0.05).Therefore, SMEs could attenuate retinal degeneration by inhibiting CML-RAGE interaction.

Keywords: spinach; diabetic retinopathy; advanced glycation end products; oxidative stress; inflammation; RAGE; carboxymethyl L-Lysine

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1. Introduction

Plant-based diets have been associated with a lower risk of developing chronic diseases such as obesity, type 2 diabetes, and coronary heart disease. Spinach (Spinacia oleracea L.)is an edible leafy vegetable consumed throughout the world due to providing an appreciable amount of fibers, vitamins, and minerals [1,2]. Several of its phytochemicals have been identified, including chlorophylls, carotenoids(lutein and zeaxanthin) [3], and phenolic compounds(flavones, flavonoids, coumarins) [4-8] (see supplement 1). It has been reported that spinach, when ingested as a food, water-soluble extract, or in a freeze-dried form, and its thylakoids have anti-cancer, anti-obesity, and hypoglycemic properties [9]. The anti-inflammatory effect of spinach has been demonstrated in animal endotoxemia models, obese animals, and healthy humans [9]. Its antioxidant effect was studied in human prostate carcinoma cells, animal models with obesity, UV-irradiated mice, and adenocarcinoma of the transgenic mouse prostate [9,10]. The anti-glycation and anti-inflammatory effects of spinach methanolic extract (SME) have been studied in the model of glucose-induced diabetes in zebrafish and isoproterenol-induced myocardial necrosis in rats, respectively [11,12]. SME diminished the glycosylated hemoglobin and fructosamine levels, including the glycated protein levels, by reducing aldose reductase activity in the lens ocular [11]. The above findings suggest that SMEs might have a preventive effect on the progression of diabetes mellitus complications such as diabetic retinopathy (DR).

DR progressively leads to loss of visual acuity or blindness that has been related to inflammation, oxidative stress, and accumulation of advanced glycation end products (AGEs)[13,14]. The AGEs induce protein crosslinking, altering structure/function and its turnover/clearance. AGEs can be produced by a non-enzyme reaction between the glucose with a free amino group of proteins, forming reversible intermediates such as Schiff's bases and Amadori products (fructosamine)[15]. Other mechanisms for AGEs formation include the "carbonyl stress" pathway, where oxidation of sugars and lipids create dicarbonyl inter-mediate compounds such as glyoxal and methylglyoxal (MGO), which lead to AGEs formation as the N:-carboxymethyl lysine(CML)[16]. Increased CML levels in serum and vitreous can be a biochemical marker for both the appearance and progression of DR [17,18]. In the retina, the activation of receptor RAGE by AGEs(AGEs-RAGE)induces the overexpression of glial fibrillary acidic protein (GFAP) and pro-inflammatory factors, regulated by the nu-clear factor kappa-light-chain-enhancer of activated B cells(NF-kB)[19]. NF-kB positively regulates RAGE expression by acting as a positive feedback mechanism [20]. what is cistanche On the other hand, high concentrations of S100B, a calcium-binding protein, may also activate NF-kB, inducing neuroinflammation and glial cell activation [21]. The overexpression of S100B in astrocytes provokes neurotoxic effects manifested by retinal astrogliosis [22].

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Strategies have been experimentally developed to prevent retinal damage. Some phytochemicals of spinach isolated from other plants have been associated with inhibition of AGEs and oxidative stress [8,23-26]. Lutein, astaxanthin, and kaempferol have protected human-derived retinal pigment epithelial cells(ARPE-19) from damage through their anti-AGEs, antioxidant, and anti-apoptosis properties [26-28]. These results suggest that the phytochemicals of spinach have a relevant role in the prevention of retinal degeneration like DR. Another strategy has been using aminoguanidine (AG) as a potent inhibitor of glycation and iNOS activity, attenuating the CML accumulation and formation of reactive di-carbonylic precursors in rats [29]. However, in a multicentric and randomized clinical trial with 690 diabetic patients, the beneficial effect was not established clearly [30].

The changes in retinal degeneration under hyperglycemic conditions have been scarcely studied. Therefore, it is interesting to know if SME protects retinal layers from damage related to its anti-AGEs, antioxidant, and anti-inflammatory properties in the retina of streptozotocin-induced diabetic rats.

2. Materials and Methods

2.1.Preparation of the Methanolic Extract of Spinach (SME)

The fresh leaves of spinach (S.oleracea L.)were harvested in the autumn-winter season in an agricultural field of the Puebla Province, Mexico, and identified by a botanist in the herbarium of The National Polytechnic Institute (IPN, Mexico City, Mexico). Voucher specimen number 4532 was deposited in the herbarium of the National School of Biological Sciences of IPN.

The leaves were dehydrated and finely ground. The dried leaves were macerated with methanol at room temperature, filtered through cellulose filters(Whatman, Maidstone, UK), and dried using a rotatory vacuum evaporator(BUCHI Rotavapor R-200, Switzerland)at 45°Cto yield a green gum(95.0g/kg dry leaves). Anti aging cistanche The SME was stored in the dark at 4°Cuntil use. For all assays, the extract was reconstituted in distilled water[11].

2.2.In Vitro Assays of SME Anti-Glycation Activity

2.2.1. Formation of Advanced Glycation End Products Derived from Bovine Serum Albumin (BSA-AGEs)

The BSA-AGEs assay was carried out, as described previously [31]. Briefly,10 mg/mL BSA(fraction V; Sigma-Aldrich, St. Louis, MO, USA)was added either in D-glucose 0.5 M, D-fructose 0.5 M, or methylglyoxal 2.5 mM, and made in a solution 0.1 M PBS (pH 7.4)and 0.02% sodium azide. SME concentrations (5, 10,25, 50, 100, and 200 mg/mL) were added to each mixture and then incubated at 37°C for 4 weeks. Afterward, the unreacted sugars were removed by dialysis against distilled water for two days at 4°C. BSA-AGE levels were determined by fluorescence spectrophotometry(Ex370/Em 440 nm; model LS 30, PerkinElmer LAS Ltd., Buckinghamshire, UK)[32]. The glycation of BSA protein by reducing sugars and MGO were a positive control (BSA glycated), while the incubation of BSA glycated with aminoguanidine(1 mg/mL)was used as the negative control (BSA glycated-AG). The assays were performed in triplicate.

2.2.2. Fructosamine Assay

The fructosamine level was determined by the nitro blue tetrazolium (NBT) assay [33], which is based on the ability of fructosamine to reduce NBT, forming formazan, a colored end-product under alkaline conditions. Ten microliters of either of the controls or glycated BSA-incubated SME concentrations （BSA-SME）were added to 90 μL of NBT at 2.5 mM， prepared in a carbonate buffer(0.1 M; pH 10.3); all were incubated at 37 ℃C for 30 min. The absorbance at 530 nm was measured. Fructosamine concentrations (nmol/mg)were calculated according to a standard curve of formazan.

2.2.3. Determination of Carbonyl Groups Formation and Thiol Groups Depletion in BSA-AGEs

Carbonyl group concentration was measured in BSA-SME and controls samples, according to Levine et al. [34]. A solution of 2,4-dinitrophenylhydrazine (DNPH;10 mM)in 400 μL of 2.5 M HCl was added to 100 μL each sample and incubated in darkness for 60 min at room temperature. cistanche benefícios Then， 500 μL of trichloroacetic acid （20%w/v）was added and kept on ice for 5 min. The samples were centrifuged at 4000 rpm for 15 min, and the protein pellet was washed three times with ethyl acetate/ethanol(1:1 v/v). Afterward, the samples were suspended in 250μL guanidine hydrochloride to 6 M. The concentration of carbonyl groups (nmol/mg protein) was calculated by spectrophotometry at 370 nm absorbance (EON, BioTek Instruments, Inc., Winooski, VT, USA) using the absorption coefficient of DNPH (22,000 M-1 cm-1).

According to Ellman's method, the determination of thiol group depletion in BSA-SME and control samples was performed. Briefly， 10 μL of 5，5'-Disulfanediylbis（2-nitrobenzoic acid）（DNTB;10 mM， prepared in PBS）with 50 μL of glycated samples was incubated for 15 min at room temperature, then the absorbance at 410 nm was measured. The level of free thiol groups was calculated using a standard curve of L-cysteine（0.4-11 μM） and expressed in nmol/mg protein [34].

2.3. Effect of SME on the Retina of Diabetic Rats 2.3.1.Diabetes Induction in the Rats

Wistar male rats weighing 250± 10 gr(N=40) fasted for 8 h were used. A dose intraperitoneal of streptozotocin(60 mg/kg) in citrate buffer(10 mM, pH 4.5)(Sigma-Aldrich, Inc., St. Louis, MO, USA)was administered [35]. Three days later, the glucose level was measured(glucometer; ACCU-CHEK active; Roche Diagnostics, GmbH, Mannheim, Germany) by collecting a drop of a blood sample from the tail. Animals with glycemic levels higher than 350mg/dL were recruited for the study. The glucose in the blood was measured weekly for 12 weeks.

2.3.2. Experimental Design

The streptozotocin-induced diabetic rats (STZ) were divided randomly into the following groups (n =10): STZ treated intragastrically with 2 mL of drinking water (STZ); STZ treated with SME at 400 mg/kg (STZ-SME); normoglycemic rats (NG); and STZ treated with50 mg/kg aminoguanidine (AG; Sigma-Aldrich, Inc.)(STZ-AG).STZ-AG was used as an anti-AGE reference group. The assigned doses were administered every 24 h (9:00 a.m.) for 12 weeks. The glycemic level in all groups was monitored weekly. The SME dosage regimen at 400 mg/kg was based on the following: 7 g of SME is obtained from 100g of fresh spinach (data not shown). This amount is equivalent to the daily consumption of an average person of 70 kg in the American diet [36], which corresponds to 100 mg of extract/kg of body weight. Cistanche Extract Anti Radiation On the other hand, due to the difference in the accelerated metabolism of rats, it is recommended to increase the consumption of any natural extract up to 6.4 times for comparison studies with humans [37]. The dose of 400 mg/kg that we used was based mainly on the results obtained in a model of myocardial necrosis induced in Wistar rats, which showed that the anti-inflammatory effect of SME is more significant at 300 mg/kg [12].

2.3.3. Histological and Immunohistochemistry Evaluations

Enucleated eyes were fixed in neutral formalin and then were dehydrated in graded alcohols and embedded in paraffin. Histological sections of 2 μm were mounted on electrocharged slides, dewaxed, and rehydrated up to antigen recovery solution (K035;10× citrate Buffer, pH6; Diagnostic BioSystems, Pleasanton, CA, USA). A polymer-based immunodetection system(PolyVuemouse/rabbit DAB detection system, Diagnostic BioSystems) was used. The following primary antibodies were used:glial fibrillary acidic protein (anti-GFAP;MAB360; Chemicon International, Inc., Temecula, CA, USA); vascular endothelial grown factor (anti-VEGF;sc-7269; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), S100 calcium-binding protein B (anti-S100B; ab52642; Abcam PLC, Cambridge, UK), and nuclear factor kappa-light-chain-enhancer of activated B cells (anti-NF-kB p65;sc-8008).All dilutions were at 1:200 and incubated overnight at 4°C. A secondary antibody (Mouse/Rabbit PolyVueTM) was added according to the supplier's instructions. cistanche herba Sections were stained with DAB plus/chromogen substrate and hematoxylin. Histological observations and the capture of images were carried out in a Carl Zeiss microscope(Carl Zeiss Microscopy GmbH, Jena, Germany).

For immunofluorescence staining, the slides were incubated overnight at 4℃C with following primary antibodies: carboxymethyl Lysine (anti-CML; ab124145; Abcam PLC, Cambridge, UK), AGE receptor (anti-RAGE; sc-365154), NADPH oxidase 4 (anti-Nox4;ab133303), 3-nitrotyrosine (anti-NT; ab110282), and nitric oxide synthase (inducible)(anti-iNOS; A00368-1;Boster Bio, Pleasanton, CA, USA). Then, the following secondary antibodies were used: for anti-RAGE, goat anti-mouse lgG(FITC)-conjugated(1:200; Santa Cruz, Biotechnology, Inc.); for anti-CML and anti-NT, mouse anti-rabbit IgG-PE (SC-3753); and for anti-iNOS and anti-Nox4, m-IgGkBP-PE (Sc-516141). All were incubated at room temperature for 60 min. Afterward, sections were mounted in a medium containing 4',6-Diamidine-2'-phenylindole dihydrochloride (DAPI). Anti-CML and anti-RAGE antibodies were co-hybridized in the same slide. FLoidTM Cell Imaging Station (Life technologies Carlsbad, San Diego, CA, USA) was used for fluorescence images.

2.3.4.Apoptosis Evaluation

The apoptosis of retinal cells was detected according to the manual described by terminal deoxynucleotidyl transferase(TdT)-mediated deoxyuridine triphosphate (dUTP)nick-end labeling (TUNEL) assay using the In Situ Cell Death Detection Kit, TMR(tetramethyl-rhodamine-5-dUTP) red, version 12 (12156792910; Roche Diagnostics). Briefly, the dewaxed slides were rehydrated and rinsed twice with PBS. Then, they were incubated with the TUNEL reaction mixture in a humidified atmosphere for 60 min at 37 C. Afterward, sections were mounted with DAPI and observed in fluorescence microscopy (FLoidTM Cell Imaging Station). IOD for TUNEL was calculated for retinal layers.

2.3.5. Lipid Peroxidation Assay

For the evaluation of malondialdehyde (MDA)levels in the retinal tissue, approximately 3 mg of fresh tissue(n = 3) were homogenated and processed as previously re-ported [38]. MDA levels were quantified following the kit supplier's instructions(OXItek-TBARS assay kit, Enzo Life Sciences, Farmingdale, NY, USA).

2.4.Statistical Analysis

For statistical analysis, all retinal micrographs were captured with a magnitude of 200× at 100 μm beyond the optic nerve region. Approximately 40 images per group of animals (n=7) were analyzed. Image analysis was performed with software Image-Pro Premier Version 9.0 (Media Cybernetics, Inc., Rockville, MD, USA). We used GraphPad Prism software (La Jolla, CA, USA; version 8.0). The figures show values graphed in box-and-whisker plots (median, first-third quartile, minimum-maximum value) for the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer(ONL). The mean and standard deviation (mean ± SD) are shown in Appendix A (Tables A1-A3). In all cases, a one-way ANOVA test followed by Tukey's test was performed. p <0.05 was considered statistically significant.