Effects of Allium victorialis leaf extracts and its single compounds on aldose reductase, advanced glycation end products and TGF-β1 expression in mesangial cells

Background Accumulating evidences suggest that aldose reductase (AR) inhibitors and advanced glycation end product (AGE) formation inhibitors may prevent chronic hyperglycemia-induced long-term complication in diabetes. Transforming growth factor-beta1 (TGF-β1) plays an important role in the development of diabetic nephropathy. Allium species have been utilized in folk medicine throughout the world for the treatment of various physical disorders. However, the benefits of Allium victorialis (A. victorialis) against diabetic complications, especially nephropathy, have yet to be explored. In the present study, we investigated the protective effect of the compounds isolated from A. victorialis leaf on diabetic nephropathy. Methods In vitro AR activity, AGEs formation, and AGE-receptor for AGEs (RAGE) binding in human RAGE (hRAGE)-overexpressing cells were tested. High glucose-induced transforming growth factor-beta1 (TGF-β1) expression was also examined in mouse kidney mesangial cells (MMCs) cultured under high glucose. Results Of the isolated eight compounds from A. victorialis leaf extracts tested, quercitrin exhibited the most pronounced inhibitory effects on AR activity (IC50 value of 0.17 μM) and AGEs formation (IC50 value of 4.20 μM). Furthermore, quercitrin disrupted AGE-RAGE binding in a concentration-dependent manner in hRAGE-overexpressing cells. Additionally, of the eight compounds tested, ferulic acid significantly reduced high glucose-induced TGF-β1 expression and secretion in MMCs. Conclusions Our results suggest that active compounds isolated from A. victorialis leaf exhibit inhibitory effects on AR activity in rat lenses and AGE formation. Further, ferulic acid reduces TGF-β1 mRNA expression and secretion in MMCs under diabetic conditions. Thus, A. victorialis is a good candidate for the development of treatments for diabetic nephropathy.


Background
Chronic hyperglycemia is the most common feature of all forms of diabetes mellitus, and it accelerates the induction of aldose reductase (AR, EC 1.1.1.21) and the irreversible formation of advanced glycation end products (AGEs), which play important roles in the pathogenesis of diabetic complications [1]. Diabetic nephropathy is a major complication of diabetes mellitus, and although the mechanism of glomerulosclerosis still remains unclear, the irreversible formation of AGEs, polyol accumulation, and oxidative stress have been considered the major causes of diabetic nephropathy [2]. AR, the first rate-limiting enzyme in the polyol pathway, is present in the eyes, kidneys, and other tissues affected by diabetic complications. Increased glucose enters the polyol pathway, where it is reduced by AR to sorbitol [2,3]. AR inhibitors (ARIs), such as epalrestat, 3,3tetramethyleneglutaric acid (TMG), and fidarestat, have been developed, and some have been revealed to prevent diabetic nephropathy in animal models or patients [3][4][5][6][7]. ARIs from natural products have been found to prevent or delay the development of diabetic complications in animal models [8][9][10].
Transforming growth factor-beta 1 (TGF-β1) is a multifunctional cytokine that plays important roles in cell proliferation, wound healing, differentiation, apoptosis, and the immune response in several cells [11]. In particular, TGF-β1 is a key mediator of diabetic nephropathy that increases the levels of extracellular matrix (ECM) proteins, such as collagen I and IV, laminin, and fibronectin, in the glomeruli [11]. In addition, TGF-β has been identified as a critical regulator and mediator of pathophysiological processes of ocular tissue development or repair. TGF-β-mediated signaling is involved in the progression of diabetic nephropathy, and high levels of TGF-β are found in diabetic kidneys.
Natural products and their active constituents have been reportedly used for the treatment of diabetes and diabetic complications [10]. The genus Allium comprises more than 600 different species distributed throughout North America, North Africa, Europe, and Asia. Many Allium species have been utilized in folk medicine throughout the world for the treatment of various physical disorders such as burns, wounds, headaches, chest colds, and rheumatism [12]. Allium victorialis var. platyphyllum (Liliaceae), one of the most popular Allium species, is an edible perennial herb widely distributed on Ulleung Island and Mt. Hambeak of the Korean Peninsula. Recently, Allium victorialis (A. victorialis) has received much attention owing to its diverse and potentially significant pharmacological properties including antiarteriosclerotic, anticancer, antioxidant, antidiabetic, antiobesity, antineuroinflammatory, hepatoprotective, and nephroprotective effects [12][13][14][15][16][17][18][19][20][21].
In this paper, we examined the effects of eight compounds (1-8) isolated from A. victorialis leaf on AR activity, AGE formation, and TGF-β1 mRNA expression and protein secretion in mouse glomerular mesangial cells (MMCs) cultured under diabetic conditions. Furthermore, binding between AGE and receptor for AGE (RAGE) in human RAGE (hRAGE)-overexpressing MMCs was analyzed, and the most active compound was identified. These results show that single compounds from A. victorialis leaf extracts have preventive effects against diabetic nephropathy and may be useful as candidates for preclinical study in the treatment of diabetic nephropathy.

Rat lens AR activity
AR activity was measured as described previously [9,22]. All animal procedures were approved by the Korea Institute of Oriental Medicine Institutional Animal Care Committee on animal care at our institute and conducted according to institutional guidelines. Rat lenses were isolated from the eyes of 8-week-old Sprague-Dawley rats (Orient Co., Seongnam, Korea) and homogenized in 12 volumes of 150 mM sodium phosphate buffer (pH 6.2) and 10 mM 2mercaptoethanol. The homogenate was centrifuged at 14,000 rpm for 30 min, and the supernatant was used as crude rat lens AR. The incubation mixture contained 150 mM sodium phosphate buffer, 0.15 mM nicotinamide adenine dinucleotide phosphate (NADPH), 10 mM DL-glyceraldehyde as a substrate, and 700 μg/ml of enzyme substrate, with or without compounds or positive control, in a total volume of 1.0 ml. The reaction was initiated by the addition of NADPH at 37°C and stopped by the addition of 0.15 ml of 0.5 N HCl. Next, 0.5 ml of 6 M NaOH containing 10 mM imidazole was added, and the solution was heated at 60°C for 15 min to convert NADP to a fluorescent product. The fluorescence (ex. 360 nm/ em. 460 nm) was assayed using a spectrofluorometric detector (Synergy HT, Bio-Tek, Winooski, VT). The concentration of each test sample that inhibited activity by 50% (IC 50 ) was estimated from the least-squares regression line of the logarithmic concentration plotted against the remaining activity.

Determination of AGEs formation
AGEs formation assay was performed as previously described [23,24]. Bovine serum albumin (BSA, 10 mg/ml, Sigma-Aldrich) in 50mM phosphate buffer (pH 7.4) with containing 0.02% sodium azide to prevent bacterial growth was added to 0.2 M fructose and glucose. The reaction mixture was then mixed with compounds or aminoguanidine (AG, Sigma-Aldrich). After incubating at 37°C for 7 days, the fluorescent reaction products were assayed on a spectrofluorometric detector (BIO-TEK, Synergy HT, Ex: 350 nm/Em: 450 nm). AGEs assay was performed in quadruplicate. The concentration of each test sample giving 50% inhibition of the activities (IC 50 ) was estimated from the least-squares regression line of the logarithmic concentration plotted against the remaining activity.

Determination of secreted TGF-β1 expression in MMCs using enzyme-linked immunosorbent assay (ELISA)
The levels of TGF-β1 in the medium were determined as described previously [9]. The medium was replaced with serum-free medium containing compound under high glucose conditions for 24 h. This medium was then harvested and TGF-β1 was activated by treatment with 1 N HCl (0.1 ml/0.5 ml of conditioned media) for 10 min at room temperature, then 0.1 ml 1.2 N NaOH/0.5 M HEPES was added. Quantikine mouse TGF-β1 ELISA (R&D systems, Minneapolis, MN) was performed according to the manufacturer's protocol, and the TGF-β1 levels were normalized to those of total protein. Medium without cells that had been incubated under the same conditions was used as a control for the ELISA.
Detection of live cell-based AGE-BSA/RAGE binding AGE-BSA/RAGE binding in the cells was determined as described previously [23].

Statistical analysis
Data are expressed as mean ± S.E.M. of multiple experiments. Paired Student's t-tests were used to compare two groups, or analysis of variance with Tukey's was used for multiple comparison tests using PRISM software (Graph Pad, San Diego, CA). Values of p < 0.05 were considered statistically significant.

Rat lens AR activity, AGE formation, and AGE/RAGEbinding in hRAGE-overexpressing cells
ARIs suppressing the hyperglycemia-induced polyol pathway have been identified as potential therapeutic candidates in the treatment and prevention of diabetic complications. The IC 50 values of compounds (Table 1) in this assay were comparable to that those of known ARIs, such as TMG, which suggested that the compounds and extracts appeared to have an inhibitory effect on AR activity. Among the compounds, quercitrin (4), kaempferol (5), and quercetin (6) were significantly more potent than the previously known positive control, TMG. Previous research also demonstrated that flavonoids such as quercetin and myricitrin are effective inhibitors of lens AR [28]. We previously reported that quercitrin gallate also inhibits AR activity and xyloseinduced lens opacity and oxidation [25]. Kaempferol and its prenylated derivatives are reported to be aldolase inhibitor [32]. Kaempferol 3,4'-O-β-D-diglucopyranoside (3) (IC 50 = 9.77 ± 0.33 μM) and the A. victorialis EtOAc-soluble fraction (IC 50 = 7.53 ± 0.02 μg/ml) inhibited AR activity. Although, IC 50 level of EtOAcsoluble fraction was higher than TMG (0.94±0.01 μg/ ml), among the extracts, it has the inhibitory effects on AGEs formation (IC 50 =30.13±1.68 μg/ml; AG, IC 50 = 76.47±4.81 μg/ml). Previous research indicated that genistein has inhibitory effects of AR activity in vitro, AGEs formation, and AGE-RAGE binding in hRAGEoverexpressing cells [9,23]. Next, we examined the inhibitory effects of compounds and extracts on AGEs formation (Table 1). Quercitrin (4) (IC 50 = 4.20 ± 0.04 μM) and ferulic acid (8) (IC 50 = 7.50 ± 0.20 μM) exhibited inhibitory effects on AGEs formation. Furthermore, because of the pronounced inhibitory effect of the three compounds (4, 5, and 6) on AR and AGEs formation, AGE-RAGE binding assays were performed in hRAGEoverexpressing cells (Figure 2). Among the compounds, quercitrin (4) significantly inhibited AGE-RAGE binding in hRAGE-overexpressing cells. Although quercitrin (4) has been tested on ARI effect [28], this compound has never been examined for the AGE-RAGE binding assay in hRAGE-overexpressing cells up to data. Quercitrin has anti-inflammatory effect through the inhibition of the NF-kappa B pathway and it shows potential anticancer effect, including cell cycle regulation and tyrosine kinase inhibition [33,34].

Inhibition of high glucose-induced TGF-β1 expression and secretion in MMCs
TGF-β1 stimulates the production of ECM proteins such as fibronectin and collagen and promotes mesangial cell expansion [35,36]. In diabetic nephropathy, these changes are associated with the development of basement membrane thickening in the glomeruli [37]. Thus, TGF-β1 is considered a potential therapeutic target in diabetic nephropathy and other chronic renal diseases.
To assess which compounds from A. victorialis are involved in the regulation of both TGF-β1 mRNA and protein levels in MMCs under diabetic conditions, cells were treated with high glucose in the presence or absence of single compounds (1-8) for 48 h. As shown in Figure 3A and B, single compounds (

Conclusion
In summary, our data suggest that active compounds isolated from A. victorialis leaf exhibit inhibitory effects on AR activity and AGE formation. Further, ferulic acid reduces TGF-β1 mRNA expression and secretion in MMCs under diabetic conditions. Thus, the compounds isolated from A. victorialis leaf provide some scientific evidence to support the folk medicinal utilization of A. victorialis in the treatment of diabetic nephropathy. Furthermore, A. victorialis is a good candidate for the development of treatments for diabetic nephropathy.