Effect of Proanthocyanidin Rich Grape Seed Extract

Introduction

The reactive oxygen species (ROS) play an important role on pathological processes in the gastro intestinal tract (GI tract) including Crohn’s disease, ulcerative colitis and colon cancer. The prolonged exposure of intestine to ROS leads to oxidative stress which induces lipid peroxidation, protein degradations and damages essential cellular macro molecules which progress to metabolic syndromes like hyperglycemia, insulin resistance, hypertension, dyslipidemia, and central obesity (Almenier, Al Menshawy, Maher, & Al Gamal, 2012; Yang et al., 2014). Currently most of the scientific studies evidence the role of dietary polyphenols which potentially prevents the pathological process linked with oxidative stress generated by ROS.

Dietary polyphenols which included in our daily life through fruits, vegetables, cereals, tea, coffee and wine were large heterogeneous group of compounds characterized by hydroxylated phenyl moieties which is classified into flavonoids and non-flavonoids (Puupponen-Pimiä et al., 2002). Flavonoids has a very good antioxidant properties which scavenge the ROS, chelates the metal ion and terminate the reaction of free radicals (Crozier, Jaganath, & Clifford, 2009). Grapes one of the polyphenol rich fruit which has been consumed as fresh or dried fruit, juice and wine. Grape polyphenols (GP) has many reports and reviews stating that, it reduces obesity, atherosclerosis, changes in gut microbiota and improves endothelial dysfunctions . However, the poor bioavailability of polyphenols has been reported, recent studies has been showed that the dietary polyphenols are also able to induce cellular antioxidant defense by modulating the protein and gene expressions (). In the present study we hypothesized the protective effect of grape seed derived polyphenols against lipopolysaccharide induced oxidative stress and inflammation in CaCO2 colon model.

Materials and Methods

Grape seed extraction and biochemical characterization

Grounded grape seed powder was added to 25% ethanol in a ratio of 1:5 (grams: milliliters) and mixed thoroughly. The mixture was sonicated for 25 minutes at room temperature and then centrifuged at 4000 rpm for 10 minutes. The supernatant collected were decanted through miracloth. The filtered extract was used for the following experiment.

The biochemical characterization grape seed extract (GSE) were done with LC/MS system consisting of Dionex UltiMate 3000 UPLC including Dionex HPG-RS pump, RS autosampler, RS column compartment, and Dionex UltiMate photodiode array detector (PDA). After PDA the sample flow was guided to a Thermo Scientific Q Exactive Plus orbitrap high resolution, high mass accuracy mass spectrometer using the methodology Zhang et al., 2018.

Cell culture and maintenance

Human colorectal adenocarcinoma cell line Caco-2 were grown in culture flasks with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% fetal bovine serum (FBS), L-glutamine, and antibiotic solution (Penicillin and Streptomycin) at 37C in a humidified 5% CO2, 95% atmosphere incubator. The medium was replaced every alternate day and 80% confluent cells were used for all assays.

Cell Viability Assay

Cell viability was measured by the MTT method (Slinkard & Singleton, 1977). Briefly, the cells were seeded into 96-well cell culture plates, with the concentration of 5×104 cells/well. Cells, which were treated with GSE in different dilution for 24 h. Subsequently, cells were incubated with MTT (0.5 mg/mL) for 4 h, and the generated formazan precipitate was dissolved with 50 ?L of DMSO. Finally, the absorbance was measured at 490 nm using a Synergy HT plate reader (Biotek, Winooski, VT).

Determination of Intracellular Reactive Oxygen Species

The intracellular ROS was determined by fluorescence of 2’, 7’-dichlorofluorescein (DCFH2-DA) (Chen et al., 2008). Caco-2 cells were seeded into 24-well cell culture plates at a concentration of 5 ×104 cells/well and cultured for 24 h. After that, cells were pretreated with GSE along with/without lipopolysaccharide (LPS) (25 ?g/mL) for 24 h. Subsequently, cells were washed with PBS incubated with 10?M DCFH-DA at 37C for 30 min, and then washed with PBS again and visualized with a fluorescence microscope. Mean fluorescence intensity was quantified using ImageJ software after background staining correction. Cellular oxidant levels were expressed as the mean DCF fluorescence intensity.

Determination of Mitochondrial Superoxide

The mitochondrial superoxide level were determined by fluorescence of MitoSOX Red fluorescence stain (Wolfe & Liu, 2007). Caco-2 cells were seeded into 24-well cell culture plates at a concentration of 5 ×104 cells/well and cultured for 24 h. After that, cells were pretreated with GSE along with/without LPS (25 ?g/mL) for 24 h. Subsequently, cells were washed with PBS and incubated with 5?M MitoSOX Red for 30 min, and then were washed twice with PBS. Fluorescent images were captured using a fluorescence microscope. The results were expressed as mean fluorescence intensity, quantified using ImageJ software after background staining correction

Detection of Mitochondrial Membrane Potential

Mitochondrial membrane potential (MMP) were determined by using the fluorescent dye Rh-123 staining method as reported earlier (Ji, Qu, & Zou, 2011; Mániková et al., 2014). Caco-2 cells were seeded into 24-well cell culture plates at a concentration of 5 ×104 cells/well and cultured for 24 h. After that, cells were pretreated with GSE along with/without LPS (25 ?g/mL) for 24 h. Cells were washed with PBS and stained with 10 ?M of Rh-123 at 37°C for 30 min in the dark. After washing with PBS, fluorescence intensity of cells was visualized under a fluorescence microscope. The results were expressed as mean fluorescence intensity, quantified using ImageJ software after background staining correction.

Transepithelial Electrical Resistance (TEER) Assay in Caco-2 Cell Monolayers

Caco-2 cells were grown in transwell polycarbonate filters (diameter, 12 mm; pore size, 0.4 ?m (Costar 3460, Corning Inc.) in DMEM with 10% fetal calf serum and antibiotics and incubated at 37 °C with 5% CO2. The basolateral and apical compartments were filled with 1.5 and 0.5 mL of culture medium, respectively. The culture medium was changed three times per week, and cells became confluent after 21 days culture. Transepithelial resistance (TEER) was measured as described by Li et al., 2010 using an ohm/voltmeter (EVOM, WPI). According to preliminary tests, cells were considered as confluent when basal TEER was higher than 600 ?. The apical medium was then removed and added with GSE along with/without LPS (25 ?g/mL) contain medium for 24 h and TEER was measured for each treatment. Resistance values were calculated in ?·cm2 by multiplying the resistance values by the filter surface area.

Detection of interleukins by Enzyme Linked Immunosorbent Assay (ELISA)

Caco-2 cells were seeded into 24-well cell culture plates at a concentration of 5 ×104 cells/well and cultured for 24 h. After that, cells were pretreated with GSE along with/without LPS (25 ?g/mL) for 24 h. The supernatant from each treatment were used to determine the level of interleukin 8 (IL8) and interleukin 6 (IL6) released by the cells by using commercial Human DuoSet ELISA kit (R&D Systems, Minneapolis, MN).

Immunofluorescence microscopy

CaCO2 cells monolayer were grown on 22-mm glass coverslips in a CO2-humidified incubator. After that, cells monolayer was pretreated with GSE along with/without LPS (25 ?g/mL) for 24 h and fixed. The tight junction protein zona occluding 1 (ZO1), occludin, claudin was immunolabelled with fluorescence polyclonal antibody and the images were acquired with confocal microscopy. Relative fluorescence intensity was measured using imageJ.

Statistical analyses

All values are expressed as means ± S.E.M (n = 3). Data were analyzed by using a one-way ANOVA and the two-tailed Student’s t test using the GraphPad prism software and the differences between the means were assessed post-hoc using Tukey’s test. P

Results

Proanthocyanidin rich GSE

The GSE was characterized with LC-MS, in which the proanthocyanidin (PAC) monomers (catechin, epicatechin), Dimers (procyanidin B1, B2, B3, B4, B5, B7 etc.), trimers, tetramers, pentamers and corresponding gallates were found (Figure 1). The presence of multiple derivatives of PAC showed that the GSE are rich in PAC polyphenols.

Cytotoxic effect of GSE

The cytotoxic effect of GSE on CaCO2 cell was determined using MTT assay, for that different concentration of GSE starting from higher of 50 µg/mL to lower of 3.13 µg/mL were used (Figure 2). No cytotoxic effect was observed after 24 h of the treatment (p > 0.05).

Effect of GSE on oxidative stress

The failure of antioxidant defense and mitochondrial dysfunction cause increase in free radicals which leads to oxidative stress. We have examined the effect of GSE in CaCO2 cells on mitochondrial dysfunction, intracellular ROS and mitochondrial superoxide induced with LPS (Figure 3 and 4). GSE recover the mitochondrial damage and significantly reduces the intracellular ROS and mitochondrial superoxide affected by LPS. We have also examined the effect of GSE on expression of selected antioxidant enzymes such as Glutathione reductase (Gsr), superoxide dismutase 1 (Sod1), superoxide dismutase 2 (Sod2) and Glutathione peroxidase (Gpx) in the transcriptional level (Figure 5). The result shows that the antioxidant enzyme gene expression suppressed by LPS and GSE significantly elevate the expression. Overall result showed that GSE reduce the oxidative stress by recovering mitochondrial damage and by balancing free radicals with the antioxidant defense mechanism by significantly increasing the expression of antioxidant enzymes.

Effect of GSE on Tight junction barrier

The oxidative stress also causes the tight junction leakage/damage by affecting the tight junction protein, we have examined the effect GSE on tight junction leakage induced by LPS with TEER resistance across the CaCO2 cell monolayer. The resistance value was decreased because of damage caused by LPS whereas the GSE protect the tight junction leakage which leads to increase in TEER value (Figure 6). We also examined the expression of transmembrane protein such as zona occluding 1 (ZO1), occludin and claudin 1 in CaCO2 monolayer with immunocytochemistry (Figure 7). We found that LPS suppress the expression of transmembrane protein which leads to less integrity of tight junction barrier and GSE protect the integrity by increasing the expression of transmembrane protein significantly. We have also examined the gene expression for the tight junction protein (Figure 8), which is correspondence/ proof the above result we obtained.

Effect of GSE on inflammation

Cytokines plays an important role in inflammatory response, we have examined anti-inflammatory effect of GSE induced by LPS in CaCO2 cells by measuring the proinflammatory cytokines IL8, IL6 level in the supernatant with ELISA method (Figure 9) and also by gene expression of some inflammation markers like IL1a, IL6, TNFa, IL10 and Tgfb1 (Figure 10). The result showed that the pro-inflammatory cytokines like IL1a, IL6, IL8 and TNFa was induced by LPS which was significantly reduced by the GSE and the anti-inflammatory cytokines like IL10 and Tgfb1 were induced by GSE.

Discussion

Grapes, one of the polyphenol rich fruit which has been consumed as fresh or dried fruit, juice and wine. Grape polyphenols (GP) has many reports and reviews stating that, it reduces obesity, atherosclerosis, changes in gut microbiota and improves endothelial dysfunctions (Sun et al., 2002; Visoili and Devalos 2011; Zhang et al., 2018). Dietary factors play an important role in oxidative stress and inflammation therapy (Fiorella et al., 2013), particularly in the GI tract. High fat diet cause degradation of GI barrier function may lead to leakage of toxic luminal substances into the mucosa which may cause inflammatory processes and mucosal injury (Lambert 2009; Fiorella et al., 2013; Landy et al., 2016). So, here we focus on the effect of proanthocyanidin rich GSE on oxidative stress, gut barrier dysfunction and inflammation, for that we have used colon CaCO2 cell monolayer which was widely used invitro model for gut barrier (Manna et al., 1997; Rao et al., 2002). The oxidative stress disturbs the gut barrier and leads to microbial translocation which cause inflammatory bowel diseases, here LPS from gram negative bacteria play an important role (Wells et al., 1993; Kallapura et al., 2014). In the present study we used LPS to induce oxidative stress and inflammation. We also evidence the capacity proanthocyanidin rich GSE to significantly reduce the oxidative stress, inflammation, regulation of gene, expression of tight junction protein and increase in gut barrier integrity.

The imbalance in redox potential leads to increase in free radicals which regulates various physiological functions such as cell growth, differentiation, aging, senescence and apoptosis, especially by superoxide anion (Chiste et al., 2015). LPS increases the production of ROS leads to inflammatory signaling pathway as well as mitochondrial membrane integrity (Hsu and Wen 2002; Bognar et al., 2013). Our result shows that GSE reduces ROS and mitochondrial super oxide level by up regulating the antioxidant ezyme gene expression and recover the mitochondrial dysfunction (Figures 3, 4 and 5).

The tight junction permeability is associated with expression of transmembrane protein and it plays important role in cell to cell contact, maintaining the structure and barrier function (Van Itallie 2009). Our study shows that GSE increase the expression of transmembrane protein like ZO1, occludin and claudin 1 expression which was suppressed by LPS. Some of the proinflammatory cytokines (TNF?, IL1 and IFN?) increased the permeability by diminishing the tight junction proteins (Nunes et al., 2019). Our result showed that LPS increase the pro inflammatory cytokines TNF? and IL1? which may cause the suppression of tight junction proteins and GSE increase the expression of tight junction protein and decreases the expression of TNF? and IL1? (Figures 8 and 10). Normally the inflammation induced by pro-inflammatory cytokines will be regulated by anti-inflammatory cytokines (Coussens and Werb, 2002), in our study the anti-inflammatory cytokines IL10 and Transforming growth factor beta 1(Tgfb1) was suppressed by LPS which may cause increase in the pro inflammatory cytokines IL6, IL8, TNF? and IL1?, but with GSE change it into vice versa (Figures 9 and 10). Tgfb1 is a multifunctional cytokine which enhances epithelial barrier by up-regulating the expression of tight junction proteins (Planchon et al., 1999; Howe et al., 2005), our results show that the GSE up regulate the expression of Tgfb1 and tight junction protein as well.

In conclusion, our result showed that proanthocyanidin rich GSE was significantly reducing the free radicals like ROS and superoxide and balancing the redox potential by regulating the antioxidant enzyme genes. GSE also regenerate the mitochondrial function by increasing the mitochondrial membrane potential. In addition, GSE upregulate the tight junction protein and anti-inflammatory cytokine expression and down regulate the pro-inflammatory cytokine expression. GSE was increasing the epithelial barrier integrity and reducing the inflammation. However, our result show GSE reduces the oxidative stress and inflammation, further studies are needed to answer many unanswered questions like, bioavailability, microbiota degradation and mechanism of action in several physiological conditions.

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