Modulation of RAGE and the Downstream Targets of RAGE Signaling Cascades by Taurine in Lycium Barbarum (Goji Berry): Protection of uman Retinal Pigment Epithelial Barrier Function and its Potential Benefit in Diabetic Retinopathy

Background: Retinal pigment epithelial (RPE) barrier disruption is an early event in diabetic retinopathy (DR). However, the biochemical details of its pathophysiology and treatment options are unclear. Taurine is reported to be beneficial in DR and is abundant in the fruit of Lycium barbarum (Goji Berry, LB). Hence, we have investigated the effect of taurine pure compound and an extract of LB extract rich in taurine on a model of DR, the ARPE-19 cell line treated with high glucose, and whether their effects on RPE barrier disruption by glucose may contribute to protection against DR. Methods: The levels of NF-κB and ICAM-1 activity were measured by luciferase reporter gene analysis. Protein levels of RAGE and VEGF were determined by Western blot analysis. VEGF secretion levels were analysed by ELISA. The barrier function was determined by measuring TER and FITC-dextran permeability. Distribution of claudin-1 and connexin 43 proteins was examined by Western blot and immunofluorescence analysis. Results: Taurine and an extract of LB rich in taurine dose-dependently down-regulate increased levels of RAGE, NF-κB, VEGF and ICAM-1 in the ARPE-19 cell line exposed to 33.3 mM glucose. This reversal was associated with attenuation of high glucose-induced RPE barrier disruption, which was shown by increased TER, reduced FITCdextran permeability, characteristic morphological staining and dose-dependent modulation of claudin-1 and connexin 43 protein expression. Conclusions: Pure taurine and LB extract protect against barrier disruption following exposure of ARPE-19 cells to high glucose. These effects are associated with altered NF-κB, ICAM-1 activity and VEGF secretion. Taurine and LB extract decrease RAGE. As they are known to activate PPAR-γ we propose that their effects on barrier disruption may occur through their effects on RAGE and the downstream targets of RAGE signaling cascades. This pathway provides a rationale for the potential of taurine and the LB extract for protection against progression of DR. J o u r na l o f D iabetes & Mabol i s m ISSN: 2155-6156 Journal of Diabetes and Metabolism Citation: Song MK, Roufogalis BD, Huang THW (2011) Modulation of RAGE and the Downstream Targets of RAGE Signaling Cascades by Taurine in Lycium Barbarum (Goji Berry): Protection of Human Retinal Pigment Epithelial Barrier Function and its Potential Benefit in Diabetic Retinopathy. J Diabetes Metab 2:162. doi:10.4172/2155-6156.1000162


Introduction
Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes and remains a major cause of preventable blindness worldwide [18]. The retinal pigment epithelial (RPE) cells form an important permeability barrier, the blood retinal barrier (BRB), between the sensory retina and the choriocapillaris, and play a key role in the pathogenesis of DR [1,60].
Chronic hyperglycemia is believed to be the primary pathogenic factor for inducing damage to pigment epithelial cells [14]. This results in changes to the pattern of production of a number of proinflammatory mediators, followed by angiogenesis in tissue remodeling, and finally increase in retinal vasopermeability and disruption of the BRB [11,22,64]. Moreover, degradation of high glucose-induced RPE cells leads to down-regulation of the gap junction protein connexin 43 and up-regulation of the tight junction protein claudin-1, causing disruption of the epithelial barrier function [1,34].
One of the underlying pathophysological mechanisms associated with hyperglycemia-induced DR is excessive formation of advanced glycation end products (AGEs) and subsequent interaction with their receptor advanced glycation end products (RAGE) [54]. Activation of RAGE results in oxidative stress, and the subsequent activation of NF-κB transcription factor is considered to be linked to epithelial dysfunction [36]. These cascade episodes trigger serum components and inflammatory cells to enter the vitreous cavity and sub-retinal space, exposing the RPE cells to pro-inflammatory mediators, including intercellular adhesion molecule-1 (ICAM-1) and vascular endothelial growth factor (VEGF), causing BRB breakdown and vascular leakage, leading to further progression of DR [29,60].
Recent studies have shown that retinal expression of PPAR-γ was suppressed in experimental models of diabetes and in endothelial cells treated with high glucose [55]. Administration of 15-deoxy-∆ 12, 14 − prostaglandin J 2 (15d-PGJ 2 ) inhibited VEGF-stimulated angiogenesis in rat cornea [63]. Rosiglitazone has been found to inhibit retinal endothelial cell proliferation, migration, and tube formation in response to VEGF treatment [56]. A body of evidence has shown that PPAR-γ ligands inhibit the formation of AGEs [47,51]. Moreover, rosiglitazone has been shown to inhibit both retinal leukostasis and retinal leakage by the inhibition of NF-κB activation, with consequent suppression of ICAM-1 expression [42]. These findings suggest that PPAR-γ is involved in the pathogenesis of DR [52].
Fruit from Lycium barbarum L (LB) in the family Solanaceae is well-known in traditional Chinese medicine [35]. LB has been widely used in functional food products with a large variety of reported beneficial effects, such as reducing blood glucose, neuroprotection, and anti-inflammatory activity [5,45,61]. There is a growing body of evidence indicating that LB intake increases the fasting plasma zeaxathin levels, beneficial for maintaining macular pigment density in age-related macular degeneration [9]. Moreover, LB has been shown to be effective in the treatment of glaucoma and modulating immunity in retinal ganglion cells in a rat hypertension model [7,10]. However, the beneficial effect of LB and its possible mechanisms in DR has not been studied. LB contains taurine, a non-essential free amino acid, which is one of the most abundant chemical components present in this plant [6,62]. Taurine has been recommended as a complementary therapeutic agent for the prevention of diabetic complications in type II diabetes [39].
Despite the growing body of evidence both from in vitro and animal research for LB in various medical applications in recent years, there is limited research available to establish its efficacy and mode of action in modulating eye related pathology. Moreover, the taurine component of LB needs to be further investigated through molecular biology to pinpoint its therapeutic potential for the treatment of DR. Recently, we have shown that LB and, taurine, its abundant active component, may delay DR through the PPAR-γ pathway [53]. However, the beneficial effect of taurine and LB on DR by modulation of high glucose induceddisruption of RPE barrier function through the downstream targets of RAGE signaling cascades have not been extensively investigated. Therefore, the aim of this study is to investigate the relationship between the receptor function of pure taurine and an extract of LB rich in taurine in the modulation of parameters of retinal barrier permeability in a high glucose-treated retinal cell line model.

Preparation of LB extract
Dried LB was purchased in raw powder form (batch no.53101: DeDu Holdings, West Ryde, Australia). The preparation of an extract of LB was performed as previously described [21,53] with modifications. Briefly, 250 mg of fine LB powder was extracted with 2.5 ml of methanol by sonication at room temperature for 15 min followed by centrifugation for 5 min. This step was repeated four times. The solvent was then evaporated under reduced pressure below 50 o C and the remaining solid collected. The identification and quantification of taurine in the LB extract was undertaken by thin layer chromatography (TLC) analysis, as previously described [53].

Tissue culture
The human retinal epithelial cell line, ARPE-19 was a kind gift from Dr Weiyong Shen (Save Sight Institute, Sydney, Australia). The cells were cultured as previously described [16]. Briefly, the cells were cultured in a humidified incubator at 37 o C in 5% CO 2 in 10% fetal bovine serum-defined minimal essential medium (FBS-DMEM-F12 medium) containing 5.5 mM glucose, supplemented with 100 U/ml penicillin G and 100 μg/ml streptomycin. The culture medium was replaced with fresh medium every second day. Upon confluence, cultures were passaged by dissociation in 0.05% (w/v) trypsin (Gibco-Life Technologies, Roseville, MD, USA) in phosphate buffered saline (PBS) pH 7.4. Cytotoxicity of LB extract and taurine used in the experiments were examined by MTS assay (Promega, Australia) and they showed little or no effect on cell viability (>90% viability remained) (data not shown).
For high glucose-induced functional studies, cells were maintained in fresh medium containing 1% FBS for 2 h prior to use in the experiments. Cells were then treated with samples, rosiglitazone (RG), and 15d-PGJ 2 (PG) or vehicle (0.5% DMSO) for 6 h. The treatment with extract and drug samples was continued for 48 h during the incubation in normal (5.5 mM) or high (33.3 mM) D-glucose. Cells incubated in 27.5 mM mannitol (M) served as an osmotic control [67]. The cells used in these experiments were between passages 20 and 24.

Transfection and luciferase analysis of NF-κB and ICAM-1 luciferase activity
To determine NF-κB and ICAM-1 luciferase activity, the ARPE-19 cells were transiently transfected, as previously described with modifications [17,28,40]. The plasmids used for transfection were p-NF-κB-Luc (a kind gift from Dr Sheridan Henness, The University of Sydney, Australia), p-ICAM-Luc (a kind gift from Dr Young-Guen Kwon, Younsei University, Korea) and pSV-β-Galactosidase Control Vector (Promega, Australia) to normalize transfection efficiencies.
Cells were transfected with FuGENE6 transfection reagent (Roche, Australia) in accordance with the manufacturer's instructions. At 48 h before transfection, ARPE-19 cell line was seeded at 5x10 5 cells/T25 flask in 5 ml of Dulbecco's modified Eagle's medium/F-12 containing 10% FBS and supplemented with 1% penicillin and streptomycin, 1% L-glutamine, 20 mM HEPES and 5.5 mM D-glucose. After 48 h, the cells were then harvested and plated into 96-well plates at 3x10 4 cells per well in complete transfection media and allowed to attach for 12 h. The cells were then replaced with serum free media containing 1% FBS for 2 h prior to use in the experiments. Cells were treated with the PPAR-γ positive controls (RG and PG), test samples, TNF-α (a known activator of NF-κB and ICAM-1) [48,50] or vehicle (0.5% DMSO) for 6 h followed by further exposure to normal (5.5 mM) or high (33.3 mM) D-glucose. After 48 h, the cells were lysed and assayed for luciferase and β-galactosidase activities using the Bright-Glo Luciferase Assay System and Beta-Glo Assay System (Promega, Australia), respectively. There results were expressed as relative luciferase activity (fold difference compared to negative control).
proteins from the cells were prepared using the Ripa lysis buffer (25 mM Tris buffer (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS). The lysed cells were centrifuged at 12,000 rpm (Micromax RF centrifuge, Thermo IEC, MA, USA) for 10 min and supernatants resolved by SDS-PAGE, 4-12% (Invitrogen, Australia). Protein was transferred to cellulose membrane with transfer buffer (Tris base 25mM, glycine 192 mM, pH 8.3) and blocked in 5% skim milk powder (Sigma, Australia) overnight. The primary antibody were anti-RAGE mouse monoclonal (1:200 dilution), anti-VEGF mouse monoclonal (1:100 dilution) (Santa Cruz Biotechnology, USA), anticlaudin-1 mouse monoclonal (1:1000 dilution) and anti-Cx43 mouse monoclonal (1:1000 dilution) (Zymed Lab Gibco; Invitrogen, San Diego, CA, USA). After incubation with the primary antibody for 1 h at room temperature, the membrane was washed and further incubated with horseradish peroxidase conjugated anti-mouse secondary antibodies (1: 6000 dilution; Santa Cruz Biotechnology, USA). Bound antibodies were detected using enhanced chemiluminescence with Lumi-Light Western Blotting Substrate (Roche). The membranes were exposed to X-ray film (Kodak, USA) and developed using the SRX-101A X-ray developer (Konica, Taiwan). The resultant films were quantified by scanning densitometry using ImageJ (National Institutes of Health, Bethesda, MD). Protein expression was quantified by normalisation to α-tubulin. Levels in control were arbitrarily assigned a value of 1.0. The membranes were re-probed with anti-α-tubulin primary antibody (1:10,000 dilution; Santa Cruz Biotechnology, USA) after being stripped and overnight blotting with 5% skim milk. The membranes were re-incubated with horseradish peroxidase conjugated anti-mouse secondary antibody and detected using the same procedure as described above. Cell lysate protein concentrations were determined by the BCA (bicinchoninic acid) assay (Thermo, USA) according to the manufacturer's instructions.

Measurement of VEGF levels by ELISA
The levels of VEGF in cell supernatants were measured using ELISA kit (R&D Systems, Minneapolis, MN, USA), as described previously [46]. The authors used medium alone without cells incubated under the same conditions as a blank control for the ELISA. VEGF levels were normalised to total protein, as determined by the BCA assay (Thermo, USA) according to the manufacturer's instructions.

Measurement of barrier functions
Cells were seeded on 12-mm diameter, 0.4-μm pore size, polyester transwell filters (Coster, Corning Inc, NY, USA) at 1.0 x10 5 cells/well in 0.5 ml of growth medium containing 5.5 mM glucose. 1 ml of medium was added at the basal chambers to level the height of the liquid for preventing hydrostatic pressure. Transepithelial electrical resistance (TER) of cell monolayers was measured using an epithelial volt-meter (EVOM; World Precision Instruments, Sarasota, FL) according to the manufacturer's instructions. Briefly, the cells were taken from the incubator and placed at room temperature for 30 min of equilibration before the measurements. The TER (in ohms·cm 2 ) of the filter alone was measured as background and subtracted from the TERs obtained with the filters containing the ARPE-19 cells. Measurements were repeated six times for each well, and each experiment was repeated in five different wells.
The permeability of the ARPE-19 cells was determined by measuring the apical to basolateral movements of fluorescein isothiocynate (FITC)-dextran (70 kDa). The test molecule was added to the apical compartment of the cells in a concentration of 1.43 μM. Five hours later, 200 μl of sample from the basolateral chamber was collected and the fluorescence intensity was measured using a fluorometer at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. A minimum of three cultures were used for each time measurement.

Immunofluorescence detection
To study the distribution and relative amounts of claudin-1 and connexin 43, RPE monolayers maintained on glass cover slips (Livingstone International, Rosebery, Australia) were immunostained for claudin-1 and Connexin 43 proteins, as described previously [25]. Briefly, cells were washed twice in PBS for 5 min, fixed with 4% paraformaldehyde for 10 min followed by two washes with PBS. Cells were then permeabilised with 0.1% triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 10 min and blocked with 5% bovine serum albumin (BSA) for 30 min at RT. Mouse anti-claudin-1 (1: 200 dilution) and connexin 43 (1: 200 dilution) antibodies (Zymed Laboratories Inc., San Francisco, CA) were incubated overnight at 4 o C. After washing with PBS, cells were further incubated with Alexa 568 goat anti-mouse secondary antibody (Invitrogen, Australia) for 1 h at RT. After several washes with PBS, Hoechst 33342 was added for the counterstaining of the cell nuclei. Coverslips were then mounted onto glass slides using antifade reagent (Invitrogen, Australia) and examined under a fluorescence microscope (DMI3000B: Leica Microsystems Imaging Solution, Ltd.). For quantification, the fluorescence intensity (mean gray values) of images was measured using Image J software. Images were analysed from four randomly selected X63 images from the same sets of experiments [41]. In addition, we measured the florescence intensity of the background, which was later subtracted from the values obtained from the images.

Statistical analysis
All results are expressed as means ±S.E.M. Data was analysed by 1-factor analysis of variance (ANOVA). If a statistically significant effect was found, the Newman-Keuls test was performed to isolate the difference between the groups. P values less than 0.05 (P<0.05) were considered to indicate significance.

Effects of LB extract and taurine on RAGE protein expression activation in high-glucose treated ARPE-19 cells
As hyperglycemia increases expression of the receptor for advanced glycation end products (RAGEs) [66] we determined the effect of LB extract and taurine on RAGE protein expression in high glucosetreated ARPE-19 cells.

Effects of LB extract and taurine on NF-κB luciferase activation in high-glucose treated ARPE-19 cells
As hyperglycemia activates the pro-inflammatory transcriptional factor, NF-κB [31], we determined the effect of LB extract and taurine on NF-κB luciferase activity in high glucose-treated ARPE-19 cells. Incubation with high glucose for 48 h induced a significant increase in NF-κB luciferase activity (7.77-fold), compared with normal glucose culture ( Figure 2). LB extract (0.1, 0.5 and 0.75 mg/ml) dose-dependently decreased NF-κB activation by high glucose incubation (by 4.96-, 3.27-and 2.46fold, respectively). Taurine (0.001, 0.1 and 1 mM) dose-dependently decreased NF-κB activation by high glucose incubation (by 5.38-, 3.74-and 2.57-fold, respectively) ( Figure 2). The NF-κB activity in high glucose treated cells was decreased by adding the positive controls, RG (3.22-fold) and PG (3.36-fold). The osmotic control did not enhance NF-κB luciferase activity, excluding the involvement of osmotic effects of high glucose concentrations.

Effects of LB extract and taurine on ICAM-1 luciferase activation and levels of VEGF in high-glucose treated ARPE-19 cells
As activation of RAGE and activation of NF-κB transcription factor, are considered to be linked to up-regulation of the levels of ICAM-1 and VEGF [29,60], we investigated the effect of LB extract and taurine treatment on ICAM-1 activity (by luciferase assay), and levels of VEGF concentrations and protein expression.

Effect of LB extract and taurine on barrier function in high glucose-treated ARPE-19 cells
Increase in the levels of ICAM-1 and VEGF, caused by hyperglycemia, has been implicated in the disruption of RPE barrier function [2,60]. To investigate the effects of treatment with LB extract  decreased by the positive controls, RG (5.47-fold) and PG (2.80fold). The osmotic control did not enhance active claudin-1 protein level, excluding the involvement of osmotic effects of high glucose concentrations.

Effects of LB extract and taurine on connexin 43 protein expression in high-glucose treated ARPE-19 cells
Hyperglycemia also down-regulates gap junction protein connexin 43, leading to degradation of RPE cells and eventual disruption of the BRB [34]. We therefore determined the effect of LB extract and taurine on connexin 43 protein levels in high glucose-treated ARPE-19 cells.

Effects of LB extract and taurine on claudin-1 protein expression in high-glucose treated ARPE-19 cells
The up-regulation of claudin-1 occurs in the dysfunction of the RPE tight junction [1,12]. To confirm the protective effect of LB extract and taurine in barrier function in high glucose-treated ARPE-19 cells, claudin-1 protein expression, an indicator of barrier function at tight junction [26], was determined by Western blot analysis.
Incubation with high glucose for 48 h induced a significant increase in the level of claudin-1 protein expression (10.07-fold), compared with normal glucose culture ( Figure 6). positive controls, RG (0.60-fold) and PG (1.03-fold). The osmotic control did not decrease connexin 43 protein levels, excluding the involvement of osmotic effects of high glucose concentrations.

Effects of LB extract and taurine on claudin-1 and connexin 43 distribution and localisation in high-glucose treated ARPE-19 cells
To further investigate the effect of LB extract and taurine on barrier function in high glucose-treated ARPE-19 cells, we examined the morphological characteristic staining of claudin-1 (Figure 8a

Discussion
Diabetic retinopathy (DR) is a preventable microvascular diabetic complication, and hyperglycemia is considered a major factor in its development [3]. Although various hyperglycaemia-induced metabolic abnormalities are implicated in DR [30], it has been difficult to pinpoint the exact pathophysiological mechanism, thus making the rationale for a therapeutic target difficult. Numerous studies have shown that activation of RAGE and subsequent activation of NF-κB transcription factor, followed by increased production of ICAM-1 and VEGF, are one of the critical factors in the breakdown of the blood retinal barrier (BRB), which eventually leads to DR [36,52].
Retinal pigment epithelial (RPE) cells form a monolayer between the neuroretina and the choriocapillaris that are the essential components of the outer BRB and play an important role in the pathologic process of DR [4,49,60]. Moreover, hyperglycemia induces inflammation in ARPE-19 cells that causes breakdown of RPE barrier function, resulting in up-regulation of claudin-1 and down-regulation of connexin 43 [1,34,57].
PPAR-γ is heterogeneously expressed in the mammalian eye, and is prominently present in the retinal pigmented epithelium, photoreceptor outer segments and choriocapillaries [24]. A recent study has shown that retinal expression of PPAR-γ was suppressed in experimental models of diabetes and in endothelial cells treated with high glucose [55]. In previous work which formed the background leading to the present studies we determined that LB extract and pure taurine, a major component in the LB extract, activated PPAR-γ by luciferase reporter gene analysis and by mRNA and Western blotting measurement in human retinal pigment epithelial cells. At the same time both LB extract and pure taurine inhibited a variety of PPAR-γ dependent downstream effectors in the retinal cells [53]. A body of evidence has shown that PPAR-γ ligands inhibit the formation of AGEs [47,51]. PPAR-γ agonists such as RG and pioglitazone have been shown to decrease tumor necrosis factor-alpha-induced RAGE expression via suppression of NF-κB activation [37]. Moreover, telmisartan, an angiontensin II type 1 receptor block has shown to down-regulate RAGE expression and suppress its downstream signalings in various cells through its unique PPAR-γ −modulating ability [38,65,68]. PPAR-γ ligands are also potent inhibitors of corneal angiogenesis and neovascularization [42,63]. Administration of 15d-PGJ2 inhibited VEGF-stimulated angiogenesis in rat cornea [63]. RG was shown to inhibit both retinal leukostasis and retinal leakage by the inhibition of NF-κB activation, with consequent suppression of ICAM-1 expression [42]. Moreover, RG has been shown to inhibit retinal neovascularization by a mechanism downstream from VEGFinduced angiogenesis [43].
In the present study we have investigated the potential of Lycium barbarum (LB), to prevent retinal barrier disruption by high glucose exposure, and examined the protein activities associated with this protection. Lycium barbarum (LB), a traditional Chinese medicine, has been used for centuries in the East and is believed to be beneficial for treating eye related disorders [8]. Different biological activities of LB have been demonstrated, including anti-aging, anti-inflammatory activity and cytoprotection [33]. The crude polysaccharides from Lycium barbarum (LBP) have been shown to decrease the serum AGEs levels in a D-galactose induced mouse aging model, and reduce BRB disruption in retinal ischemia/reperfusion injury [15,32].
We have also investigated the effect of taurine, one of the major components in LB, in the retinal barrier permeability and its associated protein activities. Taurine is present in abundance in retina, and is essential for sustaining retinal structure and function [6]. Various studies have shown that plasma and tissue levels of taurine are reduced in diabetes [19,20,23]. Indeed, studies have shown the beneficial effect of taurine supplementation in preventing or ameliorating hyperglycemiainduced retinal defects [20,23]. Moreover, the studies have shown that high glucose-induced AGEs level and NF-κB activation are inhibited by taurine, exhibiting effective prevention against diabetic-related pathology [27,44]. In addition, taurine has been shown to protect against hyperglycemia-induced endothelial and retinal dysfunctions through the down-regulation of ICAM-1 and VEGF levels [58,69].
From previous work where we established that LB extract and the active component, taurine, delayed DR pathophysiology through PPAR-γ activation [53] we have hypothesised that taurine, may modulate high glucose-induced disruption of RPE barrier function and that this may be associated with modulation of PPAR-γ-mediated downstream targets of RAGE signaling cascades. In order to test this hypothesis, we first investigated the effect of pure taurine and an LB extract rich in taurine on levels of RAGE protein expression in high glucose-treated ARPE-19 cells. Our results demonstrated that a methanol extract of LB dose-dependently decreased levels of RAGE protein expression (Figure 1a). The extract also decreased activation of the pro-inflammatory transcriptional factor, NF-κB ( Figure 2). Since the methanol extract used here contained a high content of taurine (10.66% ) [53], we investigated the effect of pure taurine and showed that it had a similar effect in modulating levels of RAGE protein expression ( Figure 1b) and activation of NF-κB ( Figure 2). Our study also demonstrated that both LB extract and taurine dose-dependently down-regulated the increase in pro-inflammatory mediators following exposure of RPE cells with 33.3 mM glucose, as shown by the ICAM-1 luciferase activation (Figure 3), VEGF protein expression ( Figure  4a,b) and VEGF levels ( Figure 4c). We further examined the role of LB extract and taurine on barrier permeability function, and showed that they both attenuated high glucose induced-RPE barrier disruption, as measured by transepithelial electrical resistance (TER) (Figure 5a) and FITC-dextran permeability (Figure 5b). Moreover, LB extract and taurine were found to dose-dependently down-regulate the increase by high glucose exposure of claudin-1 tight junction protein (Figure 6a,b), and up-regulated the decrease in connexin 43 gap junction protein expression (Figure 7a,b). These findings were further confirmed by characteristic morphologic staining (Figure 8a-l) and densitometric analysis of immunoflorescence signals (Figure 8m,n). The results support the proposal that taurine present in the LB extract is to a large extent responsible for the modulation of high glucose induceddisruption of RPE barrier function. The range of taurine concentrations in the LB extract used in the experiments, 0.086 -0.645 mM, falls within the range of pure taurine concentrations (0.001-1.00 mM) used [53]. Hence it can be concluded that the activities of the extracts are accounted to a large extent by their taurine content.
The results of our studies have found that taurine and LB extract are PPAR-γ activators [53]  establish a direct link between taurine and LB extract activation and RAGE-dependent downstream effectors on barrier permeability. Future studies are needed to clarify the involvement of this pathway in the actions of LB and taurine. Interestingly, it has been reported in a previous study that high glucose concentration results in a reduction of permeability [59]. In contrast, a more recent study has shown that high glucose moderately increases permeability in ARPE-19 cells [57]. These differences might be explained by the different cell culture conditions. In addition, the studies have shown that the high levels of VEGF in high glucose-induced ARPE-19 cells produce a significant decrease in TER [2,34]. In the light of these conflicting findings, the exact mechanism of the changes in permeability in high glucose-treated ARPE-19 cells requires further investigation.

Control
In summary, this study has demonstrated for the first time that the traditional Chinese medicine Lycium barbarum protects against high glucose barrier disruption in retinal pigment epithelial cells. It also decreases RAGE and the downstream targets of RAGE signaling cascades, consistent with the effect being mediated through the PPAR-γ activation pathway. The effects of the extract are mimicked by taurine at concentrations identical to those present in the extracts. Though the exact mechanisms on RAGE signaling cascades has not been examined in this study, it can be speculated that taurine and, LB extract, being PPAR-γ ligands, might be modulating RAGE signaling cascades similar to other PPAR-γ agonists like RG, leading to protection against the high glucose barrier disruption. Delineation of the similar effects of LB extract and taurine provides a rationale for the therapeutic use of this valuable medicinal herb and its taurine component for the prevention of DR. However, further investigation into their exact mechanism warrants further translational studies to gather proof of efficacy and safety of LB for possible protection against DR in various preclinical and clinical settings.