Anti-Adipogenic Polyphenols of Water Shield Suppress TNF-α-Induced Cell Damage and Enhance Expression of HAS2 and HAPB2 in Adiponectin-Treated Dermal Fibroblasts

Anti-Adipogenic Polyphenols of Water Shield Suppress TNF-α-Induced Cell Damage and Enhance Expression of HAS2 and HAPB2 in AdiponectinTreated Dermal Fibroblasts Hiroshi Shimoda1*, Seikou Nakamura2, Shoketsu Hitoe1, Shuko Terazawa1, Junji Tanaka1, Takahiro Matsumoto2 and Hisashi Matsuda2 1Research and Development Division, Oryza Oil & Fat Chemical Co., Ltd., 1 Numata, Kitagata-cho, Ichinomiya, Japan 2Kyoto Pharmaceutical University, 1 Shichono-cho, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan


Introduction
Water shield (Brasenia schreberi J. F. Gmel, Cabombaceae) is an aquatic plant that grows in Asia, Australia, Africa and North America. As the surfaces of its leaves and buds are covered with mucus consisting of polysaccharide [1], the texture is similar to gelatine. In Japan, water shield (junsai in Japanese) is cultivated as a vegetable and the young buds are used as a food ingredient. On the other hand, the leaves have been used as stomachic in China; however, the constituents of water shield have not been well studied. Only quercetin 7-O-glucoside and gallic acid have been reported as principal polyphenols [2]. In terms of the biological activity of the extract from water shield, anti-oxidative [2], anti-inflammatory [2] and hypolipidemic activities [3] have been reported. Considering this background, we performed isolation of the constituents of water shield leaves and identified various types of polyphenol including a new flavonol glycoside named junsainoside A.
A number of polyphenols have been reported to suppress visceral fat accumulation in adipocytes. 3T3-L1 preadipocytes [4] and primary cultured visceral adipocytes [5] have been frequently used for the assay. These results indicate the efficacy of polyphenols against obesityrelated insulin resistance, dyslipidemia and arteriosclerosis. However, from the perspective of dermatology, the effect of polyphenols on subcutaneous fat accumulation has not been well studied. Recently, the relationship between subcutaneous adipocytes and dermal fibroblasts has been focused on. Subcutaneous fat exists in the hypodermis and releases adipocytokines including tumor necrosis factor (TNF) -α and adiponectin [6]. TNF-α is also released from dermal inflammatory cells and induces fibroblast apoptosis [7]. On the other hand, adiponectin enhances hyaluronan and collagen production from fibroblasts [8]. Thus, TNF-α and adiponectin affect dermal fibroblasts with negative and positive responses and the balance of these adipocytokines is considered to be crucial to maintain stable dermal conditions. Against the above background, we investigated the effect of water shield extract (WSE) and its constituents on lipid accumulation and adipocytokine release in subcutaneous adipocytes. In addition, the effects of the constituents on fibroblasts treated with adipocytokines were evaluated.

Preparation of WSE
The leaves of water shield cultivated in Mitane town (Akita prefecture, Japan) were harvested in June 2011. The leaves were airdried and ground. The powdered leaves (200 g) were then extracted with 30 w/w% ethanol (EtOH, 6 kg) at 50°C for 2 h. The solvent was evaporated to obtain WSE (44.3 g, yield: 22.1%). The polyphenol

Abstract
Water shield (Brasenia schreberi J. F. Gmel) is an aquatic plant cultivated in Northern Japan, from which we have isolated polyphenolic compounds including a novel flavonol glycoside, junsainoside A. Recently, dermal fibroblasts have reported to be affected by adipocytokines from subcutaneous adipocytes. Hence, we evaluated the effects of polyphenolic compounds from water shield on fat accumulation, adipocytokine release and adipocytokine-induced fibroblast status. In differentiated 3T3-L1 adipocytes, ethyl gallate, caffeoyl glucose, hypolaetin 7-O-glucoside, kaempferol and junsainoside A significantly suppressed lipid accumulation at 10 µM. In addition to these constituents, ferulic acid, kaempferol 3-O-(6"-galloyl) glucoside, quercetin 3-O-(6"-galloyl) glucoside, quercetin 3-O-glucoside and gossypetin suppressed the lipid accumulation in human subcutaneous adipocytes. Ethyl gallate and junsainoside A also enhanced adiponectin production from subcutaneous adipocytes without induction of TNF-α release. On the other hand, ethyl gallate and quercetin recovered from TNF-α-induced fibroblast damage. Moreover, ferulic acid, quercetin and kaempferol enhanced the expression of hyaluronan binding protein 2 and hyaluronan synthase 2 in adiponectintreated fibroblasts. These results suggested that water shield contains various types of polyphenolic compounds with anti-adipogenic activity, some of which protect dermal fibroblasts from TNF-α damage and probably enhance hyaluronan production. It is assumed that polyphenols in water shield maybe suppress subcutaneous fat accumulation and maintain dermal fibroblast and hyaluronan-related status.

Lipid accumulation in cultured 3T3-L1 and human subcutaneous adipocytes
3T3-L1 cells (1×10 4 cells/200 µL) suspended in Dulbecco's modified essential medium (D-MEM) containing 10% fetal calf serum (FCS) were seeded onto a 48-well culture plate and cultured for 24 hr. The medium was then changed to a new one containing 10% FCS, 5 µg/mL insulin, 0.25 µM dexamethasone and 0.5 mM isobutylmethyl xanthine and the cells were cultured for 2 days to induce differentiation. The medium was then changed to a new one containing 10% FCS, 5 µg/ mL insulin and test sample dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO was 0.1%. The medium was changed to a new one every other day and the cells were cultured for a total of 6 days. The cells were stained with oil red O and observed under a microscope. The dye in cells was dissolved in isopropanol to measure the absorption at 540 nm. For the evaluation of cytotoxicities of the compounds from water shield, MTT assay was performed according to Denizot et al. [21], with modification. Briefly, cells (5×10 3 cells/100 µL) were seeded in a 96-well culture plate and treated by the same procedure as described above. Then MTT reagent was added to each well and the plate was incubated for 2-4 hr. After washing the plate with phosphate buffered saline, pH 7. formed formazan dye was dissolved in 0.04M HCl/isopropanol solvent completely and the absorbance was measured at 570 nm.
Human subcutaneous preadipocytes (2×10 4 cells/200 µL) suspended in the growth medium attached to the kit were seeded onto a 48-well culture plate and cultured for 24 hr. The medium was changed to the differentiation medium containing test sample (final concentration of DMSO: 0.1%) for 10 days. The medium was then collected and stored at -20ºC. The contents of TNF-α and adiponectin were determined using a commercial kit. The cells were stained with oil red O and absorbance was measured as described above.

Fibroblast damage induced by TNF-α
NB1RGB cells (5×10 3 cells/100 µL) suspended in D-MEM containing 10% FCS were seeded onto a 96-well culture plate and cultured for 24 hr. Then, sample (final concentration of DMSO: 0.1%) and TNF-α (10 ng/mL) were added and cultured for 20 hr. The cytotoxicity by TNF-α was evaluated by MTT assay as described above.

Evaluation of HAS2 and HABP2 expression in fibroblasts induced by adiponectin
TIG-108 cells (1×10 5 cells/2 mL) suspended in D-MEM containing 10% FCS were seeded onto a 6-well culture plate and cultured for 24 hr. The medium was changed to D-MEM containing 0.5% FCS and cultured for 24 hr. Then, sample (final concentration of DMSO: 0.1%) and adiponectin (1 ng/mL) were added and cultured for 72 hr. Cells were collected with 200 µL of RIPA buffer containing protease and phosphatase inhibitor cocktail. The protein concentration in the cell lysate was adjusted with RIPA buffer to 0.5 mg/mL. The lysate was mixed with the same volume of Laemmli sample buffer (62.5 mM Tris-HCl, 2% SDS, 5% 2-mercaptoethanol, 25% glycerol and 0.01% bromophenol blue) and heated at 95ºC for 5 min. The sample solution (15 µL) was electrophoresed on 10% SDS gel. Separated protein was then transferred to a polyvinylidene fluoride membrane. The membrane was treated with primary antibodies at the following dilution ratios: anti-HAS2 (1: 1,000), anti-HABP2 (1: 2,000) and anti-β-actin (1: 10,000) for 1 hr. HRP-conjugated anti-goat IgG (1:25,000) or anti-rabbit IgG (1: 25,000) was used as the secondary antibody. Detection was performed using by Pierce Western Blotting Substrate Plus and an imaging system (Image Quant LAS500, GE Health Care, Fairfield, CT, USA).

Statistics
The results are expressed as means and S.E. Significance of the differences was examined by one-way ANOVA followed by Dunnett's test. Differences of p<0.05 were considered significant.

Effects of WSE and the constituents of water shield on TNF-α and adiponectin production from human cultured subcutaneous adipocytes
We evaluated the effect of WSE on TNF-α and adiponectin production from subcutaneous adipocytes. TNF-α production was not suppressed by the treatment of WSE or the constituents (Table 2). On the other hand, WSE did not enhance adiponectin production; however, ethyl gallate (3) and junsainoside A (12) significantly enhanced adiponectin production at 10 µM.

Effects of WSE and the constituents of water shield on fibroblast damage induced by TNF-α
By the treatment of 10 ng/mL TNF-α, the formazan production indicating cell activity was reduced by approximately 41% compared  Asterisks denote significant differences from the control group at *: p<0.05, **: p<0.01. with that of cells without TNF-α. In WSE-treated cells, the cell activity was significantly recovered from cell damage at 1 and 10 µg/mL.

Effects of WSE and the constituents of water shield on the expression of HABP2 and HAS2 in fibroblasts treated with adiponectin
In the fibroblasts cultured with adiponectin, WSE (10 µg/mL) enhanced HABP2 and HAS2 expression ( Figure 5). Among the constituents in water shield, ferulic acid (4), quercetin (12) and kaempferol (15) enhanced the expression of HABP2 and HAS2 at 10 µM. Glycosides (3, 5, 6, 7, 8, 10, 12) and gossypetin (11) did not enhance the expression of HABP2 and HAS2. Kaempferol 3-O-glucoside (K3G) used for a reference compound of 10 also did not enhance HABP2 and HAS2 expression. On the other hand, tiliroside, a reference compound of 12, enhanced the expression of both.
To evaluate the direct effect of WSE on adipocytokine release, we measured TNF-α and adiponectin released from subcutaneous adipocytes. As WSE enhanced neither TNF-α nor adiponectin production, WSE was found not to affect to adipocytokine release directly. In general, adipocytokine production is changed toward lipid accumulation in adipocytes [22]. Thus, WSE is thought to attenuate adipocytokine release indirectly through the suppression of excessive lipid accumulation in adipocytes. However, high concentrations of 3 and 12 enhanced adiponectin release among selected compounds, showing relatively potent anti-adipogenic activity in subcutaneous adipocytes. Regarding naturally occurring compounds with similar chemical structures to 3 and 12, a green tea catechin with a galloyl moiety (epicatechin gallate) has been reported to increase serum adiponectin in hypertensive rats [23]. On the other hand, two compounds with somewhat similar structures to 12 have been reported to enhance adiponectin production. A citrus polymethoxy flavonoid, nobiletin, enhanced adiponectin secretion from ST-13 adipocytes [24]. Tiliroside, a glycosidic flavonoid with coumaric acid, also enhanced adiponectin production in obese mice [25]. The structure of tiliroside is quite similar to 12. From the activities of these known compounds, 3 and 12 may enhance adiponectin production when applied alone. Galloyl group, methoxyl group in flavonol and cinnamic acid moiety in flavonol glucoside may contribute to the adiponectin release from adipocytes. However, further studies are required to clarify the mechanism of these compounds. MTT assay (% of control)