• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Roots extracts of Adenophora triphylla var. japonica improve obesity in 3T3-L1 adipocytes and high-fat diet-induced obese mice

    2015-10-31 02:18:27DongRyungLeeYoungSilLeeBongKeunChoiHaeJinLeeSungBumParkTackManKimHanJinOhSeungHwanYangJooWonSuh

    Dong-Ryung Lee, Young-Sil Lee, Bong-Keun Choi, Hae Jin Lee, Sung-Bum Park,Tack-Man Kim, Han Jin Oh, Seung Hwan Yang*, Joo-Won Suh

    1NutraPham Tech, Giheung-gu, Yongin, Gyeonggi, Korea

    2Center for Nutraceutical and Pharmaceutical Materials, Myongji University, Yongin, Gyeonggi, Korea

    3Interdisciplinary Program of Biomodulation, Myongji University, Yongin, Gyeonggi, Korea

    4DONG IL Pharmtec, Gangnam-gu, Seoul, Korea

    5Department.of Family Medicine, VIEVIS NAMUH Hospital, Seoul, Korea

    Roots extracts of Adenophora triphylla var. japonica improve obesity in 3T3-L1 adipocytes and high-fat diet-induced obese mice

    Dong-Ryung Lee1#, Young-Sil Lee2#, Bong-Keun Choi1,2, Hae Jin Lee3, Sung-Bum Park3,Tack-Man Kim4, Han Jin Oh5, Seung Hwan Yang2,3*, Joo-Won Suh2

    1NutraPham Tech, Giheung-gu, Yongin, Gyeonggi, Korea

    2Center for Nutraceutical and Pharmaceutical Materials, Myongji University, Yongin, Gyeonggi, Korea

    3Interdisciplinary Program of Biomodulation, Myongji University, Yongin, Gyeonggi, Korea

    4DONG IL Pharmtec, Gangnam-gu, Seoul, Korea

    5Department.of Family Medicine, VIEVIS NAMUH Hospital, Seoul, Korea

    ARTICLE INFO

    Article history:

    in revised form 20 September 2015

    Accepted 15 October 2015

    Available online 20 November 2015

    Roots of Adenophora triphylla var. japonica extract

    High-fat diet-induced obese mice

    Adipocytes

    Anti-obesity

    Lipogenesis

    Objective: To investigate the anti-obesity activity and the action mechanism of the roots of Adenophora triphylla var. japonica extract (ATE) in high-fat diet (HFD)—induced obese mice and 3T3-L1 adipocytes. Methods: The roots of Adenophora triphylla were extracted with 70% ethanol. To demonstrate the compounds, linoleic acid was analyzed by using gas chromatography; and the anti-obesity effects and possible mechanisms of ATE were examined in 3T3-L1 adipocytes and HFD-induced obese mice. Results: Treatment with ATE inhibited the lipid accumulation without cytotoxicity in 3T3-L1 adipocytes. Furthermore,200 and 400 mg/kg ATE treatment significantly decreased the body weight gain, white adipose tissues (WATs) weight and plasma triglyceride level, while 100 and 200 mg/kg ATE treatment increased the plasma high-density lipoprotein cholesterol level in the HFD-induced obese mice, as compared with the HFD group. Treatment with 200 and 400 mg/kg ATE also lowered the size of adipocytes in adipose tissue and reduced the lipid accumulation in liver. ATE treatment showed significantly lower expression level of adipogenesis-related proteins,such as peroxisome proliferator-activated receptorγ, fatty acid binding protein (aP2), fatty acid synthase in 3T3-L1 adipocytes; and furthermore, decreased peroxisome proliferatoractivated receptorγ, aP2, fatty acid synthase, sterol regulatory element binding protein-1c, and lipoprotein lipase mRNA expression levels in WAT of the HFD-induced obese mice. Conclusions: These results suggested that the ATE has an anti-obesity effect, which may be elicited by regulating the expression of adipogenesis and lipogenesis-related genes and proteins in adipocytes and WAT of the HFD-induced obese mice.

    Document heading doi: 10.1016/j.apjtm.2015.10.011

    1. Introduction

    Adenophora triphylla (A. triphylla) var. japonica (Korean name: Jandae, Japanese name: lady bell, English name: Three-leaf ladybell)belongs to the Adenophora species (Campanulaceae), which has been used as an oriental medicinal plant in Korea, China and Japan for anti-inflammation, anti-tussive[1] and hepatoprotective effects. Alkaloids, triterpenoids and essential oil components such as pyrrolidine, piperidine, triphyllol, nonacosane, heptacosane,lupenone and saponine have been identified from A. triphylla. These chemicals have anti-inflammatory and anti-oxidant properties, and are used to prevent obesity in Korea.

    Obesity has become a significant clinical problem in recent decades since it could contribute to life-threatening diseases such as type 2 diabetes, hypertension, hyperlipidemia, and cardiovascular disease[2]. It is characterized by increased fat mass, which isassociated with increased cell number (hyperplasia) and sizes(hypertrophy)[3,4]. Adipocyte hypertrophy is caused by excessive accumulation of lipid such as triglyceride (TG) formed from energy intake[5]. Adipocytes hyperplasia results from a complex interaction between proliferation and differentiation in preadipocytes that is involved in the adipogenic process where undifferentiated preadipocytes are converted to differentiated adipocytes[6,7]. Therefore, adipogenesis and TG accumulation play an important role in fat mass increase. Accordingly, controlling adipogenesis and TG accumulation in adipocytes could help prevent obesity and its associated diseases.

    There are many different pharmacological approaches to prevent and treat obesity, but they reportedly have a number of limitations such as undesirable side effects and adverse effects[8,9]. Recently,there is increasing public attention on using natural products to prevent and treat obesity with various plants such as fruits,vegetables, and herbs that have beneficial health effects and minimal side effects[10].

    A. triphylla root extracts have anti-obesity and hypolipidemia effects[11,12], however, A. triphylla var. japonica have not yet been studied in adipocytes and high-fat diet (HFD)-induced obese mice related adipogenesis and lipogenesis mechanisms.

    In the present study, we examined the effect of A. triphylla var. japonica extracts (ATE) on obesity and the underlying lipid mechanisms, lipid deposition and lipogenesis, in adipocytes and HFD-induced obese mice.

    2. Materials and methods

    2.1. Chemicals and reagents

    5'-diallyl-2, 2'-biphenyldiol, 3-isobutylmethylxanthine insulin, and dexamethasone were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Oil-Red O, isopropanol, and TRI reagent were purchased from Sigma-Aldrich (MO, USA). Polyclonal antibodies against peroxisome proliferator-activated receptor γ(PPARγ), aP2,and α-tubulin were purchased from Santa Cruz Biotechnology(CA, USA). Horseradish peroxidase-linked anti rabbit IgG and HRP-linked anti-mouse IgG were purchased from Bio-Rad (CA, USA). SYBR Green reaction buffer was purchased from Takara (Shiga,Japan).

    2.2. Preparation and component analysis of ATE

    A. triphylla var. japonica was provided from DONG IL Pharmtec(Seoul, Korea). The dried A. triphylla var. japonica was extracted with 70% aqueous ethyl alcohol and subjected to vacuum evaporation. Gas chromatography was carried out with an Agilent 7890A GC-FID instrument for the analysis of linoleic acid as a reference compound. GC conditions were equipped on HP-INNOWAX (30 m ×0.25 mm × 0.25 μm) column. The GC oven temperature was maintained at 80 ℃for 3 min and increased to 260 ℃ with helium as a carrier gas at a flow rate of 1.0 mL/min, and . Both injector and detector were set at 260 ℃ and 290 ℃. The total analysis time was 25 min.

    2.3. Animal studies

    Male C57BL/6J mice were purchased from Samtaco (Gyeonggido, Korea) at 4 wk of age. The mice were housed individually in stainless steel cages and maintained under a temperature of(23 ± 3) ℃ in a humidity-controlled room with a 12 h-light/dark cycle. Mice were given free access to water and food (Certified irradiated global 18% protein diet, 2918C, Harlan Laboratories,Inc., Indianapolis, Indiana, USA). After acclimatization for 1 wk,they were fed with either the normal-fat diet (NFD, n = 8, Rodent diet with 10% kcal fat, D12450B) or HFD, n = 32, Rodent diet with 60% kcal fat, D12492) purchased from Research Diet Inc., (New Brunswick, NJ, USA) for 4 wk to induce obesity. After obesity induction, the mice were divided into 4 subgroups (n = 8/group)that were matched with body weight. The following 4 groups were studied for 6 wk: HFD; HFD with oral administration of ATE at 100 mg/kg of body weight (HFD + ATE 100); HFD with ATE at 200 mg/ kg of body weight (HFD + ATE 200); and HFD with ATE at 400 mg/ kg of body weight (HFD + ATE 400). The body weight of the mice was measured once wk for 6 wk. Food intake was measured every 5 d on a per-cage basis throughout the study. Food intake (g/mouse/ day) was determined by subtracting the remaining food weight from the initial food weight of the previous feeding day, and dividing by the number of mice housed in the cage. At the end of the experiment,the mice were sacrificed, and the liver, kidney, and white adipose tissue (WAT) were excised immediately. The parts were then rinsed,weighed, frozen in liquid N2, and stored at —80 ℃ until analysis. The experimental design was approved by The Animal Experiment Committee of GyeongGi Bio Center, and the mice were maintained in accordance with their guidelines.

    2.4. Plasma biochemical analysis

    After 6 wk of feeding, 4 h-fasted mice were anesthetized using ether and blood was collected. Plasma was obtained from the blood by centrifugation at 800 g for 10 min at 4 ℃ for biochemical analyses of plasma parameters. The separated plasma was stored at — 80 ℃until analysis. Plasma total cholesterol (T-CHO), TG, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)—cholesterol, and glucose levels were determined enzymatically using commercial assay kits (Asan Chemical, Seoul, Korea). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using commercial assay kits (Sigma-Aldrich, MO, USA).

    2.5. Histological analysis

    Histological examination of liver was conducted, and epididymal adipose tissue were dissected and fixed in 10% neutral formalin solution and embedded in paraffin. The tissues were studied under hematoxylin and eosin staining. The sections were viewed with alight microscope (Olympus, Tokyo, Japan) and photographed at × 200 magnification.

    2.6. Cell culture

    Mouse 3T3-L1 preadipocytes obtained from the Dr. Rhee, Sang Dal(Korea Research Institute of Chemical Technology, Daejeon, Korea),were cultured in Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% fetal bovine serum (FBS) and antibiotics[100 U/mL penicillin and 100 g/mL streptomycin (Gibco, Carlsbad,CA, USA)] at 37 ℃ under a humidified 5% CO2atmosphere. For differentiation of 3T3-L1 preadipocytes to mature adipocytes,preconfluent cells (defined as day 0) were cultured in differentiation medium containing 0.5 mM 3-isobutylmethylxanthine, 5 μg/mL insulin, and 1 μM dexamethasone in DMEM supplemented with 10% FBS (MDI differentiation medium). After 3 d, the cell culture medium was changed to DMEM containing 5 μg/mL insulin and 10% FBS. The medium was replaced again with DMEM containing 10% FBS after another 2 d. To determine the effect of ATE on adipogenesis, differentiated preadipocytes were treated with presence or absence of ATE for 5 d.

    2.7. Oil-red O staining

    After differentiation, the cells were fixed with 4% formaldehyde solution in phosphate-buffered saline for 1 h, and were subsequently washed thrice with water. The cells were stained with oil-red O(0.5% in 60% isopropanol) for 1 h and washed thrice with distilled water. Stained cells were photographed under an optical microscope(Olympus, Tokyo, Japan). Stained lipid droplets were extracted with isopropanol and the absorbance was measured at 520 nm with a microplate reader (Tecan, Mannedorf, Switzerland).

    2.8. Cell cytotoxicity assay

    For cell cytotoxicity, the cell were treated for 24 h with various concentrations (0, 100, 300, and 500 μg/mL) of ATE extracts in DMEM containing 10% FBS. Filtered MTT reagent (5 mg/mL) was added to the cells to reach a final concentration of 0.5 mg MTT/ mL, and the cells were further incubated for 4 h at 37 ℃. The violet formazan crystals were dissolved in dimethyl sulfoxide, and the absorbance was measured at 570 nm by using a microplate reader. The cell viability (%) was calculated by comparing the absorbance of the samples and the control (untreated cell).

    2.9. Protein extraction and Western blot analysis

    Cold phosphate-buffered saline washed 3T3-L1 cells and epididymal adipose tissue were homogenized in RIPA buffer containing 0.1 mM phenylmethylsulfonyl fluoride and 1% of protease inhibitor cocktail (Sigma-Aldrich, MO, USA). The homogenates were centrifuged at 12 000 rpm for 15 min at 4 ℃, and supernatant was collected. The protein concentrations were calculated by Bio-Rad protein assay reagent (Bio-Rad Laboratories,Hercules, CA, USA). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories,Hercules, CA, USA). The membranes were blocked with 5% of nonfat dried skim milk and then incubated with appropriate antibodies. The membranes were washed with wash buffer and incubated with anti-mouse or anti-rabbit horseradish peroxidaseconjugated secondary antibodies for 1 h. The immunoreactive bands were enhanced by chemiluminescence reagents and detected by chemi-luminometer (CLINX Science Instruments Co. Ltd.,Shanghai, China).

    2.10. Isolation of total RNA and quantitative real-time PCR(qRT-PCR)

    Total RNA of epididymal adipose tissue was isolated by TRI reagent (Sigma-Aldrich, MO, USA) according to the manufacturer'sprotocols. Total RNA was reverse transcribed to cDNA by using random primers and a reverse transcription system (Promega, Piscataway, NJ) according to manufacturer's recommendations. The mRNA expression levels of adipogenesisrelated genes were analyzed by real-time PCR iQTM5 system (Bio-Rad Laboratories, Hercules, CA, USA) using a SYBR Green Master PCR Kit (Takara, Shiga, Japan) according to the manufacturer's protocols. The real-time PCR cycling parameters were as follows:2 min at 95 ℃; 45 cycles of 20 s at 95 ℃, 20 s at 60 ℃, 40 s at 72 ℃, and 30 s at 72 ℃; and a final extension for 5 min at 72 ℃,followed by a melting curve analysis. All samples were normalized to the expression level of β-actin, and the results were expressed as the fold changes relative to the HFD group. The primers used in the experiments were listed in Table 1.

    2.11. Statistical analysis

    Statistical evaluation of the data was expressed as the mean ± standard error (SEM). The statistical significance of differences between the mean values for the treatment groups was analyzed by Student's t-tests and one-way analysis of variance (ANOVA) using the software Origin 7 (Microcal Software, USA). Values of P<0.05 were considered as statistical significance.

    3. Results

    3.1. Linoleic acid content of A. triphylla

    The linoleic acid content in A. triphylla was elucidated by comparing the chromatographic profile of standard on gas chromatography analysis. We confirmed that linoleic acid content was approximately 2.0% (Figure 1), and the optimized A. triphylla was used in all subsequent experiments.

    Table 1 Primer sequences used for quantitative real-time PCR.

    3.2. Effects of ATE on adipogenesis of 3T3-L1 preadipocytes

    To determine the effect of ATE on lipid accumulation during differentiation, 3T3-L1 preadipocytes were induced to differentiate into adipocytes in the presence of ATE for 5 d. Accumulated lipid droplets were measured by Oil-Red O staining. ATE inhibited the lipid accumulation in 3T3-L1 adipocytes (Figure 2A); especially,500 μg/mL ATE treatment significantly reduced the lipid content by 49.6% ,as compared to the control (P<0.05) (Figure 2B). The cytotoxic effect of ATE on 3T3-L1 adipocytes was evaluated by the MTT assay. ATE had no cytotoxic activity at various concentrations ie., 100-500 μg/mL for 24 h (Figure 2C). To investigate how ATE inhibits lipid accumulation, we measured the expression levels of adipogenesis-related proteins using Western blotting. As shown in Figure 2D, PPARγ protein expression level decreased in 300 and 500 μg/mL of ATE treatment, as compared to the control. Target proteins ie.,aP2 and FAS protein expression level also decreased as compared to the control.

    3.3. Effects of ATE on body weight and food intake

    ATE inhibited lipid accumulation in 3T3-L1 cells. Based on these results, we investigated the effect of ATE on anti-obesity in HFD-induced obese mice. The body weight gain and food intakes were shown in Figure 3. The body weight gain of the HFD group was significantly increased, as compared to the NFD group after 6 wk of feeding; whereas administration of ATE at 100, 200, and 400 mg/kg showed a tendency to decrease body weight gain than the HFD group (Figure 3A). Food intake did not display significant group wise differences (Figure 3B).

    Table 2 Effects of ATE on plasma biochemical parameters in HFD-induced obese mice

    3.4. Effects of ATE on organs weight

    The relative weight of liver, epididymal, perirenal, and mesenteric WAT were compared to those in the HFD group (Figure 4). Supplementation with ATE did not show significant differences in kidney weight in mice fed with HFD (Figure 4A). However, the HFD plus ATE presented a decreased liver weight when compared to the mice fed HFD (Figure 4B). Epididymal, perirenal, mesenteric,and total WAT weights in the HFD group were higher than those of the NFD group (Figure 4C). ATE significantly decreased epididymal,perirenal, mesentric, and total WAT weights in HFD plus ATE 200 and 400 mg/kg fed mice, respectively.

    3.5. Effects of ATE on the plasma biochemical levels

    The plasma biochemical parameters were compared to the HFD fed group. As shown in Table 2, glucose, T-CHO, and TG levels in the HFD group were significantly increased, as compared to the ND fed mice group. In HFD plus ATE groups, decreased HFD-induced plasma glucose, T-CHO; and TG levels showed a propensity to decrease in a dose-dependent manner, although not significantly, as compare to the HFD group. However, TG level in the HFD plus ATE 200 and 400 groups were significantly decreased, as compared to the HFD group. HDL-cholesterol levels in HFD fed with ATE groups were significantly increased, when compared to the HFD fed group. There were no significant differences in the LDL cholesterol level, among the HFD and ATE-treated groups, but the ATE fed groups presented a tendency towards decreased levels in. ALT level in the HFD fed group was more significantly increased than that of the ND fed group. The HFD + ATE 200 and 400 groups exhibited tendency of decreased ALT, although there were no statistically significant differences, as compared to HFD fed group. AST level in the NFD and HFD + ATE 100 group did not differ, as compared to the HFD group, but showed a significant reduction in HFD fed with 200 and 400 mg/kg of ATE groups.

    3.6. Effects of ATE on liver and epididymal adipose tissue morphology

    Histological observations of liver and epididymal adipose tissue were conducted through 5 groups as follows: NFD; HFD; HFD + ATE 100, 200, and 400 groups (Figure 5). Sections of liver and epididymal adipose tissue were stained with hematoxylin and eosin to examine the lipid accumulations and cellular morphology. In WAT, adipocytes size of the HFD group showed an increase, as compared to the NFD group; however, adipocytes size of the HFD + ATE 200 and 400 groups were reduced, as compared to the HFD group. In the liver, lipid droplet sizes of the HFD group were larger than that of the NFD group; however, ATE treatment decreased the lipid droplet size, in a dose-dependent manner.

    3.7. Effects of ATE on expression levels of adipogenesisrelated genes and proteins

    Of the adipogenesis-related protein expression levels in ATE-treated 3T3-L1 adipocytes, we found that PPARγ, aP2, and FAS protein expression levels were decreased in a dose-dependent manner. We determined whether ATE affects the protein expression of adipogenesis-related genes and protein in epididymal adipose tissues of HFD-induced obese mice, using the quantitative real-time PCR and Western blot. As shown in Figure 6A, PPARγ, aP2, FAS,sterol regulatory element binding protein-1c (SREBP-1c), and LPL mRNA expression levels in the HFD fed group were increased than those of the NFD fed group, but the difference was not statistically significant. However, the mRNA expression level was significantly decreased in the HFD plus ATE groups, as compared to the HFD group. SREBP-1c and FAS mRNA expression level in the HFD group was significantly increased, as compared to the NFD group,whereas SREBP-1c mRNA expression level significantly decreased in 3 ATE-treated groups. FAS mRNA expression level showed a tendency of decrease, and was significantly decreased in the HFD plus 400 mg/kg ATE group, as compared to the HFD group. Additionally, ATE treatments increased LPL mRNA level, in a dose dependent manner. Consistently, PPARγ protein expression level in the HFD plus ATE groups was decreased in a dose-dependent manner, and it especially showed statically significant difference in the HFD + ATE 200 and 400 groups (Figure 6B).

    4. Discussion

    Long-chain polyunsaturated fatty acid, linoleic acid, has less known effects when derived from plants, especially from roots of A. triphylla[14]. Polyunsaturated fatty acid is a potent compound with biological responses that are required for health. In the present study, we demonstrated the 18-carbon polyunsaturated fatty acid,linoleic acid (LA, 18:2n-6, the precursor of arachidonic acid) as a reference compound by gas chromatography analysis with 2 % from ATE. Linoleic acid is an essential fatty acid that is synthesized in the human body as an unsaturated fatty acid for membrane phospholipids. Recent publications have shown that linoleic acid has anti-bacterial[15], anti-oxidative[16], anti-inflammatory[17] activities and attenuates the development of obesity—related pathogenesis,such as insulin resistance[18,19].

    Recently, several studies showed that A. triphylla has compounds including saponine, β-sitosterol, cycloartenyl acetate, and taraxenone[11,13]. It has been reported that ATE and isolated compounds show anti-inflammatory, hepatoprotective, and antitumor effects. Based on these results, the present study confirmed an anti-obesity effect including lipid synthesis of ATE and elucidated the mechanisms underlying such effects in 3T3-L1 adipocytes and HFD-induced obese mice.

    Our results demonstrated that ATE treatment dose-dependently inhibited the differentiation and lipid accumulation in 3T3-L1 adipocytes. This result was further supported by in vivo experiment,demonstrating that ATE treatment for 6 wk significantly reduced the body weight gain in HFD-induced obese mice. These results are in accordance with previous reports that A. triphylla root ethanol extract has anti-obesity effects[11,12]. Additionally, ATE treatment did not affect the food intake in HFD-induced obese mice. These results suggested that ATE treatment has anti-obesity effects, and reduction in weight gain induced by ATE treatment is related to decreasing food efficiency and not decreasing food intake.

    Obesity is characterized by adipose tissue remodeling associated with hyperplasia and hypertrophy[20,21]. Adipocytes hypertrophy is caused by an excessive accumulation of TG from the intakeof excess energy, and it results from an energy imbalance. Since adipocytes hyperplasia results from a complex interaction between proliferation and differentiation in preadipocytes[22]. It is examined by 3T3-L1 adipocytes differentiation system. To differentiate from preadipocytes into adipocytes, proliferation of preadipocytes is a prerequisite[23]. Adipogenic signals MDI differentiation medium,initiating the differentiation after mitotic clonal expansion[24]. It has been suggested that inhibition of proliferation may be the potential mechanism for reducing the adipocytes number[25]. We confirmed that ATE treatment inhibited lipid accumulation during differentiation in 3T3-L1 adipocytes without cytotoxicity. Also,ATE with HFD fed mice exhibited the tendency to decrease the epididymal, perirenal, and total WAT weights in HFD-induced obese mice. Moreover, histological data showed a decrease in the adipocytes size of adipose tissue in ATE-treated mice. These results indicated that ATE treatment may affect both the cell number and sizes of adipocytes in adipose tissue, which is likely to be associated with reduced WATs weight. However, cell proliferation and cell cycle need to be further investigated.

    Fatty liver results from increased free fatty acid uptake and endogenous hepatic fatty acid synthesis[26]. Histological data of liver revealed that ATE treatment reduced lipid droplet size and number,indicating that ATE treatment was responsible for the inhibition of lipid accumulation in liver. In our study, ATE treatment lowered plasma TG level, while 100 and 200 mg/kg ATE treatment increased plasma HDL-cholesterol level in HFD-induced obese mice, as shown in previous reports[11,12]. These results collectively suggest that ATE treatment reduces the accumulation in liver, resulting in the improved hyperlipidemia in HFD-induced obese mice.

    Excessive fat intake can cause the oxidative stress, which induces dysfunction and fatty degeneration in the liver through abnormal activation of AST and ALT. Previous reports have demonstrated that various extracts of A. triphylla have an antioxidant effect that reduces plasma ALT and AST levels in HFD-induced obese mice[11,12]. In our study, 200 and 400 mg/kg ATE treatment showed a tendency to decrease plasma ALT and AST levels in HFD-induced obese mice,as compared to the HFD group. These results showed that ATE treatment may improve the liver function.

    To examine the underlying mechanism, we measured the expression levels of adipogenesis-related genes and proteins in 3T3-L1 adipocytes and WAT in HFD-induced obese mice. Adipogenesis is closely associated with etiologies of obesity, in which the preadipocytes are differentiated into mature adipocytes. Adipogenesis is regulated by sequential expression of adipogenic and lipogenic genes[27]. PPARγ is a transcription factor that regulates adipocytes differentiation and adipogenesis. C/EBP promotes adipocytes differentiation through cooperation with PPAR γ, which increases the expression of adipocytes-specific genes such as aP2[28,29]. SREBP-1c is also a transcription factor that plays key roles in lipid metabolism, by increasing the expressions of several lipogenic genes such as FAS in adipose tissue and liver. It activates PPARγ through regulation of its expression and production of an endogenous PPARγ ligand[30,31]. The lipoprotein lipase is a key regulator of blood TG levels via decomposition of lipoprotein triglyceride. It plays a critical roles in transporting the fats and hydrolysis fat-carried lipoproteins[32]. Combined action of these transcription factors causes expression of target genes and proteins that are responsible for TG synthesis and storage, which leads to accelerated adipogenesis. We found that the high concentration of ATE treatment reduced the PAPRγ, C/EBP , aP2, and FAS protein expression level in 3T3-L1adipocytes. Furthermore, PAPRγ and SREBP-1c mRNA expression level were significantly decreased in epididymal adipose tissue of 100, 200, and 400 mg/kg ATE-treated mice. Additionally, aP2 and FAS mRNA expression level was decreased in 400 mg/kg ATE-treated mice. PPARγ protein expression levels tended to be reduced specifically in adipose tissues of HFD fed with ATE groups, and significant down-regulation was observed in the 200 and 400 mg/kg ATE treatment groups. On the other hand, the reduction in body weight gain and total WATs weight were unfortunately not observed in the 100 mg/kg of ATE treatment group, despite the down-regulation of adipogenesis-related genes. There is a possibility that there are differences in PPARγ protein expression levels in ATE-treated groups. However, more study is needed on the expression levels of other genes related to adipogenesis in adipose and other tissues. Based on our results, ATE treatment affects the expression levels of adipogenesis-related genes and proteins. Furthermore, it is likely that the anti-obesity effects of ATE treatment are caused by suppression of gene expressions and proteins involved in adipogenesis.

    In conclusion, our results showed that ATE treatment inhibited the lipid accumulation in 3T3-L1 adipocytes and reduced the body weight gain, WATs weight, adipocytes sizes, and plasma TG levels, without any toxic effects in HFD-induced obese mice. These effects were at least partially accompanied by the regulation of gene expressions and proteins associated with adipogenesis/lipogenesis,in 3T3-L1 adipocytes and HFD-induced obese mice. These findings indicated that ATE prevents the development of obesity and hyperlipidemia in 3T3-L1 adipocytes and HFD-induced obese mice,which suggest the possibility of its use for the prevention of obesity and obesity-related diseases.

    Conflict of interest statement

    We declare that we have no conflict of interest.

    Acknowledgments

    This work was carried out with the support of ‘Cooperative Research Program for Agriculture Science &Technology Development (Project No.PJ01134801)' Rural DevelopmentAdministration, Republic of Korea.

    [1] Konno C, Saito T, Oshima H, Hikino C. Kabuto: Structure of methyl adenophorate and triphyllol, triterpenoids of Adenophora triphylla var. japonica roots. Planta Medica 1981; 42(7): 268-274.

    [2] World Health Statistics. Geneva: World Health Organization; 2012.

    [3] Spiegelman MB, Flier JS. Adipogenesis and obesity: rounding out the big picture. Cell 1996; 87(3): 377-389.

    [4] Fujioka K. Management of obesity as a chronic disease:nonpharmacologic, pharmacologic, and surgical options. Obes Res 2002;10: 116S-123S.

    [5] Gregoire FM. Adipocytes differentiation: from fibroblast to endocrine cell. Exp Bio Med 2001; 226(11): 997-1002.

    [6] Nawrocki AR, Scherer PE. Keynote review: the adipocyte as a drug discovery target. Drug Discov Today 2005; 10(18): 1219-1230.

    [7] S. H. Kim SH, Park HS, Lee MS, Cho YJ, Kim YS, Hwang JT, et al. Vitisin A inhibits adipocyte differentiation through cell cycle arrest 3T3-L1 cells. Biochem Biophys Res Commun 2008; 372(1): 108-113.

    [8] Derosa G, Maffioli P. Anti-obesity drugs: A review about their effects and their safety. Expert Opin Drug Saf 2012; 11(3): 459-471.

    [9] Mayer MA, Hocht CL, Puyo A, Taira CA. Recent advances in obesity pharmacotheraphy. Curr Clin Pharmaco 2009; 4(1): 53-61.

    [10] Yun JW. Possible anti-obesity therapeutics from nature-A review. Phytochemistry 2010; 71(14-15): 1625-1641.

    [11] Choi HJ, Chung MJ, Ham SS. Antiobese and hypocholesterolaemic effects of an Adenophora triphylla extract in HepG2 cells and high fat diet-induced obese mice. Food Chem 2010; 119(2): 437-444.

    [12] Lee SE, Lee HE, Lee TJ, Kim SW, Kim BH. Anti-obesity effects and action mechanism of Adenophora triphylla root ethanol extract in C57BL/6 obese mice fed a high fat diet. Biosci Biotechnol Biochem 2013;77(3): 544-550.

    [13] Ahn EK, Oh JS. Lupenone isolated from Adenophora triphylla var. japonica extract inhibits adipogenic differentiation through the downregulation of PPARγ in 3T3-L1 cells. Phytother Res 2013; 27(5):761-766.

    [14] Hung PF, Wu BT, Chen HC, Chen CL, Wu MH, Liu HC, et al. Antimitogenic effect of green tea (-) epigallocatechingallate on 3T3-L1 preadipocytes depends on the ERK and Cdk2 pathways. AM J Physiol Cell Physiol 2005; 288(5): C1094-C1108.

    [15] Dilika F, Bremner PD, Meyer JJ. Antibacterial activity of linoleic acid and oleic acids isolated from Helichrysum pedunculatum: a plant used during circumcision rites. Fitoterapia 2000; 71(4): 450-452.

    [16] Duh PD, Tu YY, Yen GC. Antioxidant activity of water extract of HarngJyur (Chrysantemum morifolium Ramat). LWT Food Sci Tech 1999;32(5): 269-277.

    [17] Chen PY, Wang J, Lin YC, Li CC, Tsai CW, Liu TC, et al. 18-carbon polyunsaturated fatty acids ameliorate palmitate-induced inflammation and insulin resistance in mouse C2C12 myotubes. J NutrBiochem 2015. DOI: http://dx.doi.org/10.1016/j.jnutbio.2014.12.007

    [18] Matravadia S, Herbst EA, Jain SS, Mutch DM, Holloway GP. Both linoleic acid -linoleica acid prevent insulin resistance but have divergent inpacts on skeletal muscle mitochondrial bioenergetics in obese Zucker rats. Am J Physiol Endocrinol Metab 2014; 307(1): 102-114.

    [19] Cranmer-Byng MM, Liddle DM, De Boer AA, Monk JM, Robinson LE. Proinflammatory effects of arachidonic acid in a lipopolysaccharide induceud inflammatory microenvironment in 3T3-L1 adipocytes in vitro. Appl Physiol Nutr Metab 2015; 40(2): 142-154.

    [20] Rosenbaum M, Leibel RL, Hirsch J. Obesity. N Engl J Med 1997; 337(6):396-407.

    [21] Ducharme NA, Bicke PE. Lipid droplets in lipogenesis and lipolysis. Endocrinology 2008; 149(3): 942-949.

    [22] Hsieh YH, Wang SY. Lucidone from Linera erythrocarpa Makino fruits suppresses adipogenesis in 3T3-L1 cells and attenuates obesity and consequent metabolic disorders in high-fat diet C57BL/6 mice. Phytomedicine 2013; 20(5): 394-400.

    [23] Zhang JW, Tang QQ, Vinson C, Lane MD. Dominant-negative C/ EBP disrupts mitotic clonal expansion and differentiation of 3T3-L1 adipocytes. Proc Natl Acad Sci USA 2004; 101(1): 43-47.

    [24] TangQQ, Otto TC, Lane MD. Mitotic clonal expansion: a synchronous process required for adipogenesis. Proc Natl Acad Sci USA 2003; 100(1):44-49.

    [25] Kim HK, Kim NJ, Han SN, Nam JH, Na HN, Ha TJ. Black soybean anthocyanins inhibit adipocytes differentiation in 3T3-L1 cells. Nutr Res 2012; 32(10): 770-777.

    [26] Browning JD, Horton JD. Molecular mediators of hepatic steatosis and liver injury. J Clin Invest 2004; 114(2): 147-152.

    [27] Spiegelman BM, Choy L, Hotiamisligil GS, Graves RA, Tontonoz P:Regulation of adipocyte gene expression in differentiation and syndromes of obesity/diabetes. J BiolChem 1993; 268(10): 6823-6826.

    [28] Kim JB, Spiegelman BM. ADD/SREBP1 promotes adipocytes differentiation and gene expression linked to fatty acid metabolism. Genes Dev 1996; 10(9): 1096-1107.

    [29] Farmer SR. Regulation of PPARγ activity during adipogenesis. Int J Obes 2005; 1: 13-16.

    [30] Rosen ED, Puigserver CJP, Spiegelman BM. Transcriptional regulation of adipogenesis. Genes & Dev 2000; 14(11): 1293-1307.

    [31] Park J, Rho HK, Kim KH, Choe SS, Lee YS, Kim JB. Overexpression of glucose-6-phosphate dehydrogenase is associated with lipid dysregulation and insulin resistance in obesity. Mol Cell Biol 2005; 25(12): 5146-5157.

    [32] Peterson J, Bihain BE, Bengtsson-Olivecrona G, Deckelbaum RJ,Carpentier YA, Olivecrona T. Fatty acid control of lipoprotein lipase: a link between energy metabolism and lipid transport. Proc Natl Acad Sci USA 1990; 87(3): 909-913.

    15 August 2015

    #These authors contributed equally to this work.

    SH Yang. PhD, Professor, JW Suh, PhD, Professor, Center for Nutraceutical and Pharmaceutical Materials, Myongji University, Cheoin-gu,Yongin, Korea.

    Tel: +82-31-330-6881 Fax: +82-31-336-0870

    E-mail: ymichigan@mju.ac.kr, jwsuh@mju.ac.kr

    Foundation project: This work was carried out with the support of ‘Cooperative Research Program for Agriculture Science &Technology Development (Project No.PJ01134801)' Rural Development Administration, Republic of Korea.

    淫秽高清视频在线观看| 国产不卡一卡二| 色视频www国产| 成人漫画全彩无遮挡| 日韩欧美 国产精品| 日本一本二区三区精品| 少妇熟女欧美另类| 禁无遮挡网站| 色av中文字幕| 在线播放无遮挡| 亚洲人成网站在线播放欧美日韩| 波多野结衣高清无吗| 寂寞人妻少妇视频99o| 狂野欧美激情性xxxx在线观看| 精品福利观看| 欧美日本视频| 老司机午夜福利在线观看视频| 久久精品影院6| 色在线成人网| 日韩成人伦理影院| 国产单亲对白刺激| 欧美3d第一页| 亚洲国产色片| 麻豆成人午夜福利视频| 免费观看人在逋| 一进一出好大好爽视频| 日韩欧美 国产精品| 亚洲精品亚洲一区二区| 插逼视频在线观看| 国产在视频线在精品| 日本三级黄在线观看| 一级毛片电影观看 | 国产熟女欧美一区二区| 秋霞在线观看毛片| 校园春色视频在线观看| 久久久久久久久久成人| 亚洲av中文av极速乱| 99久久中文字幕三级久久日本| 国产一区二区三区在线臀色熟女| 俄罗斯特黄特色一大片| 99九九线精品视频在线观看视频| 最近手机中文字幕大全| 国产精品久久久久久久久免| 色5月婷婷丁香| 三级经典国产精品| 97碰自拍视频| 国产精品久久久久久精品电影| 国产黄色小视频在线观看| 国产精品嫩草影院av在线观看| 可以在线观看的亚洲视频| 久久人人爽人人片av| 精品无人区乱码1区二区| 久久精品国产亚洲av天美| 日韩成人伦理影院| 人妻夜夜爽99麻豆av| 自拍偷自拍亚洲精品老妇| 99久国产av精品国产电影| 婷婷亚洲欧美| 亚洲av中文字字幕乱码综合| 天天躁日日操中文字幕| 十八禁网站免费在线| 人妻制服诱惑在线中文字幕| 中文在线观看免费www的网站| av黄色大香蕉| 亚洲av美国av| 国产精品野战在线观看| 在线免费十八禁| 国产成人福利小说| 国产 一区精品| 亚洲av五月六月丁香网| 18+在线观看网站| 亚洲av熟女| 色噜噜av男人的天堂激情| 国产真实伦视频高清在线观看| 免费在线观看影片大全网站| 午夜视频国产福利| 精品久久久久久成人av| 亚洲av免费高清在线观看| 免费不卡的大黄色大毛片视频在线观看 | 观看美女的网站| 国产精品亚洲美女久久久| 老司机福利观看| 99riav亚洲国产免费| 欧美xxxx黑人xx丫x性爽| 国产一区二区三区在线臀色熟女| av免费在线看不卡| 亚洲精品色激情综合| av国产免费在线观看| 嫩草影视91久久| 搡老岳熟女国产| 婷婷六月久久综合丁香| a级毛片a级免费在线| 欧美性感艳星| 可以在线观看的亚洲视频| 国产在线男女| 久久欧美精品欧美久久欧美| 亚洲av一区综合| 国产精品一区www在线观看| 人妻丰满熟妇av一区二区三区| 麻豆一二三区av精品| 亚洲人成网站在线观看播放| 综合色av麻豆| 三级国产精品欧美在线观看| 欧美最新免费一区二区三区| 在线a可以看的网站| 欧美不卡视频在线免费观看| 中文资源天堂在线| 国产成人影院久久av| 成熟少妇高潮喷水视频| 国产午夜精品论理片| 国产精品综合久久久久久久免费| 亚洲av成人av| 亚洲最大成人手机在线| 夜夜夜夜夜久久久久| 欧美潮喷喷水| 国产极品精品免费视频能看的| 国内揄拍国产精品人妻在线| 日日摸夜夜添夜夜爱| 欧洲精品卡2卡3卡4卡5卡区| 国产一区二区在线av高清观看| 欧美性猛交╳xxx乱大交人| 最近的中文字幕免费完整| 中文字幕av在线有码专区| 最好的美女福利视频网| av视频在线观看入口| 国产av一区在线观看免费| 亚洲第一区二区三区不卡| 国产亚洲91精品色在线| 精品无人区乱码1区二区| 一个人看视频在线观看www免费| 看非洲黑人一级黄片| 久久韩国三级中文字幕| 老女人水多毛片| 亚洲一区二区三区色噜噜| 国产成年人精品一区二区| 99九九线精品视频在线观看视频| 中文字幕人妻熟人妻熟丝袜美| 国内精品美女久久久久久| 久久久久久国产a免费观看| 欧美3d第一页| 精品熟女少妇av免费看| 嫩草影院精品99| 又爽又黄无遮挡网站| 黄色一级大片看看| 久久精品影院6| 夜夜夜夜夜久久久久| 免费无遮挡裸体视频| 我的女老师完整版在线观看| 99在线视频只有这里精品首页| 人人妻人人看人人澡| 嫩草影视91久久| 成人午夜高清在线视频| 一本精品99久久精品77| 婷婷精品国产亚洲av在线| 99国产精品一区二区蜜桃av| 99热精品在线国产| 久久精品国产自在天天线| 男插女下体视频免费在线播放| 欧美潮喷喷水| 久久99热这里只有精品18| 亚洲欧美日韩高清专用| 免费看光身美女| 国产精品国产三级国产av玫瑰| 午夜激情福利司机影院| 亚洲经典国产精华液单| or卡值多少钱| 久久久a久久爽久久v久久| 男女啪啪激烈高潮av片| 欧美成人一区二区免费高清观看| 啦啦啦观看免费观看视频高清| 日日啪夜夜撸| 一边摸一边抽搐一进一小说| 国产亚洲精品av在线| 少妇丰满av| 国产亚洲精品久久久com| 国产亚洲精品综合一区在线观看| 久久久久久久久大av| 久99久视频精品免费| 一个人看视频在线观看www免费| 国产私拍福利视频在线观看| 久久精品国产亚洲网站| 国产亚洲精品久久久久久毛片| 亚洲精品国产成人久久av| 一级av片app| 一个人看的www免费观看视频| 国产精品,欧美在线| 99热全是精品| 国产精品久久久久久久电影| 中国国产av一级| 日本在线视频免费播放| 久久久午夜欧美精品| 卡戴珊不雅视频在线播放| 久久人妻av系列| 91午夜精品亚洲一区二区三区| 日本爱情动作片www.在线观看 | 最好的美女福利视频网| 最近在线观看免费完整版| 国产午夜精品论理片| 高清日韩中文字幕在线| 精品日产1卡2卡| 国产一级毛片七仙女欲春2| 中国国产av一级| 国产乱人偷精品视频| 插逼视频在线观看| 日日摸夜夜添夜夜爱| 欧美潮喷喷水| eeuss影院久久| 给我免费播放毛片高清在线观看| 黄色一级大片看看| 国产一区二区激情短视频| 晚上一个人看的免费电影| 国产极品精品免费视频能看的| 午夜亚洲福利在线播放| 国产在线精品亚洲第一网站| 亚洲欧美成人综合另类久久久 | 亚洲婷婷狠狠爱综合网| 久久久久久久久中文| www.色视频.com| 午夜精品在线福利| 国产av麻豆久久久久久久| 亚洲成人久久爱视频| 干丝袜人妻中文字幕| av专区在线播放| 在线观看66精品国产| www日本黄色视频网| 久久久久久久午夜电影| 91久久精品国产一区二区成人| 又爽又黄无遮挡网站| 91狼人影院| 又粗又爽又猛毛片免费看| 国内揄拍国产精品人妻在线| 午夜亚洲福利在线播放| 国产69精品久久久久777片| 亚洲精华国产精华液的使用体验 | 99视频精品全部免费 在线| 日本 av在线| 欧美zozozo另类| 国产精品国产高清国产av| 少妇熟女欧美另类| 男女那种视频在线观看| 免费观看在线日韩| 亚洲一级一片aⅴ在线观看| 最后的刺客免费高清国语| 天堂影院成人在线观看| 搡老熟女国产l中国老女人| av在线观看视频网站免费| 日韩成人av中文字幕在线观看 | 九九在线视频观看精品| 精品乱码久久久久久99久播| 国产精华一区二区三区| 99热全是精品| 欧美日本视频| 精品一区二区三区视频在线观看免费| 色综合亚洲欧美另类图片| 秋霞在线观看毛片| 在线天堂最新版资源| 日本a在线网址| 亚洲丝袜综合中文字幕| 亚洲自偷自拍三级| 最近最新中文字幕大全电影3| av国产免费在线观看| 国产精品美女特级片免费视频播放器| 久久精品91蜜桃| 亚洲aⅴ乱码一区二区在线播放| 国产激情偷乱视频一区二区| 久久精品影院6| 成年av动漫网址| 亚洲精品亚洲一区二区| 久99久视频精品免费| 女同久久另类99精品国产91| 亚洲高清免费不卡视频| 日本熟妇午夜| 搡老妇女老女人老熟妇| 伦精品一区二区三区| 床上黄色一级片| 国产精品永久免费网站| 久久国产乱子免费精品| 免费看av在线观看网站| 日产精品乱码卡一卡2卡三| 99在线视频只有这里精品首页| 国产精品1区2区在线观看.| 国产一区二区三区在线臀色熟女| 真人做人爱边吃奶动态| 天堂√8在线中文| 午夜久久久久精精品| 少妇人妻一区二区三区视频| aaaaa片日本免费| 12—13女人毛片做爰片一| 国产成人影院久久av| 国产精品国产高清国产av| 中国国产av一级| 一级毛片aaaaaa免费看小| 国产蜜桃级精品一区二区三区| 国产精品久久久久久久久免| 日韩精品青青久久久久久| 岛国在线免费视频观看| 色综合亚洲欧美另类图片| 校园人妻丝袜中文字幕| 如何舔出高潮| 国产精品国产高清国产av| www日本黄色视频网| 久久综合国产亚洲精品| 午夜久久久久精精品| 美女大奶头视频| 欧美xxxx性猛交bbbb| 免费av不卡在线播放| 91在线精品国自产拍蜜月| 欧美日本视频| 久久精品综合一区二区三区| 成人精品一区二区免费| 深夜精品福利| 成人av在线播放网站| 免费av观看视频| 无遮挡黄片免费观看| 欧美一区二区国产精品久久精品| 久99久视频精品免费| 久久久久国产精品人妻aⅴ院| 男女边吃奶边做爰视频| 天堂av国产一区二区熟女人妻| 久久婷婷人人爽人人干人人爱| 晚上一个人看的免费电影| 亚洲激情五月婷婷啪啪| av女优亚洲男人天堂| 99视频精品全部免费 在线| 一个人观看的视频www高清免费观看| 搡老熟女国产l中国老女人| 黄片wwwwww| 久久久精品欧美日韩精品| 性欧美人与动物交配| 国产在线精品亚洲第一网站| 亚洲精品一卡2卡三卡4卡5卡| 国产一区二区在线观看日韩| 观看美女的网站| 亚洲第一电影网av| 两个人的视频大全免费| 我要搜黄色片| 国产在线精品亚洲第一网站| 搡老岳熟女国产| 禁无遮挡网站| av在线亚洲专区| avwww免费| 午夜福利在线观看吧| 亚洲欧美日韩高清在线视频| 一区二区三区高清视频在线| 色综合站精品国产| 国产精品一区www在线观看| 国产精品不卡视频一区二区| 在线观看av片永久免费下载| 色综合站精品国产| 亚洲av不卡在线观看| 不卡一级毛片| 嫩草影院新地址| 真实男女啪啪啪动态图| 亚洲真实伦在线观看| 99在线视频只有这里精品首页| 亚洲电影在线观看av| 在线观看午夜福利视频| 国产熟女欧美一区二区| 在现免费观看毛片| 12—13女人毛片做爰片一| 最近在线观看免费完整版| 精品一区二区三区人妻视频| 欧美色视频一区免费| 欧美日韩一区二区视频在线观看视频在线 | 97超碰精品成人国产| 久久久久久久午夜电影| 91在线观看av| 欧美性猛交╳xxx乱大交人| 97超碰精品成人国产| 久久久精品欧美日韩精品| 免费av观看视频| 乱人视频在线观看| 国产成人一区二区在线| 综合色丁香网| 黄色一级大片看看| 九色成人免费人妻av| 成人性生交大片免费视频hd| 亚洲四区av| 亚洲av中文字字幕乱码综合| 欧美日本亚洲视频在线播放| 久久天躁狠狠躁夜夜2o2o| 国产亚洲精品av在线| 99热网站在线观看| 桃色一区二区三区在线观看| 伦理电影大哥的女人| 变态另类丝袜制服| 亚洲av中文字字幕乱码综合| 欧美一区二区亚洲| 国内少妇人妻偷人精品xxx网站| 国产国拍精品亚洲av在线观看| 别揉我奶头 嗯啊视频| 久久欧美精品欧美久久欧美| 禁无遮挡网站| 精品福利观看| 日本a在线网址| 国产成人一区二区在线| 淫秽高清视频在线观看| 亚洲在线自拍视频| 99久久精品热视频| 欧美中文日本在线观看视频| 三级国产精品欧美在线观看| 久久6这里有精品| 啦啦啦观看免费观看视频高清| 男人舔女人下体高潮全视频| 最近的中文字幕免费完整| 欧美区成人在线视频| 综合色丁香网| 国产探花极品一区二区| 久久天躁狠狠躁夜夜2o2o| 亚洲中文字幕日韩| 好男人在线观看高清免费视频| av在线蜜桃| 最近在线观看免费完整版| 寂寞人妻少妇视频99o| 午夜爱爱视频在线播放| 波多野结衣高清无吗| 免费看av在线观看网站| 婷婷六月久久综合丁香| 午夜精品一区二区三区免费看| 欧美精品国产亚洲| 亚洲人成网站在线观看播放| 精品免费久久久久久久清纯| 亚洲精品一卡2卡三卡4卡5卡| 俺也久久电影网| 色哟哟哟哟哟哟| 51国产日韩欧美| 日本五十路高清| 女人被狂操c到高潮| 男人舔女人下体高潮全视频| 国产在视频线在精品| 亚洲熟妇中文字幕五十中出| 亚洲精华国产精华液的使用体验 | 国内精品宾馆在线| 12—13女人毛片做爰片一| 99热6这里只有精品| 人人妻人人澡欧美一区二区| 亚洲久久久久久中文字幕| 久久99热这里只有精品18| 精品一区二区三区av网在线观看| 亚洲无线在线观看| 久久久久国产精品人妻aⅴ院| 成人无遮挡网站| 国产免费一级a男人的天堂| 国产美女午夜福利| 国产午夜精品久久久久久一区二区三区 | 欧美国产日韩亚洲一区| 99久久中文字幕三级久久日本| 99热这里只有是精品50| 久久人人爽人人爽人人片va| 中国美白少妇内射xxxbb| 69av精品久久久久久| 全区人妻精品视频| 一本一本综合久久| 高清午夜精品一区二区三区 | 精品99又大又爽又粗少妇毛片| 男女做爰动态图高潮gif福利片| 久久久久精品国产欧美久久久| 亚洲电影在线观看av| 亚洲欧美日韩高清专用| 欧美日本亚洲视频在线播放| 别揉我奶头 嗯啊视频| 天堂动漫精品| 亚洲性久久影院| 欧美zozozo另类| 亚洲电影在线观看av| 51国产日韩欧美| 22中文网久久字幕| 欧美成人免费av一区二区三区| 久久精品夜夜夜夜夜久久蜜豆| 夜夜夜夜夜久久久久| 无遮挡黄片免费观看| 欧美高清性xxxxhd video| 国产一区亚洲一区在线观看| 中文资源天堂在线| 欧美成人一区二区免费高清观看| 22中文网久久字幕| 久久久国产成人精品二区| 亚洲成人中文字幕在线播放| 中出人妻视频一区二区| 国产一区二区在线观看日韩| 国产成人a区在线观看| 久久久久久久午夜电影| 久久精品国产亚洲av涩爱 | 国产 一区 欧美 日韩| 亚洲自偷自拍三级| 18+在线观看网站| 免费人成视频x8x8入口观看| 午夜福利在线观看免费完整高清在 | 国产精品一区www在线观看| 日韩欧美精品v在线| 精品人妻一区二区三区麻豆 | 中文字幕人妻熟人妻熟丝袜美| 国产成人福利小说| 亚洲国产精品合色在线| 在现免费观看毛片| 又粗又爽又猛毛片免费看| 午夜福利成人在线免费观看| 97超视频在线观看视频| 亚洲人成网站在线观看播放| 国产精品久久久久久av不卡| 久久热精品热| 国产精品久久电影中文字幕| 在线免费观看的www视频| 97超碰精品成人国产| 校园人妻丝袜中文字幕| 国产极品精品免费视频能看的| 韩国av在线不卡| 草草在线视频免费看| 国产av麻豆久久久久久久| 国产高清有码在线观看视频| 永久网站在线| 久久草成人影院| 亚州av有码| 97超碰精品成人国产| 国产精品久久视频播放| 亚洲精品日韩在线中文字幕 | 日本成人三级电影网站| 一级黄片播放器| 日本 av在线| 日日摸夜夜添夜夜添小说| 校园人妻丝袜中文字幕| 国产成人精品久久久久久| 又爽又黄无遮挡网站| 亚州av有码| 国产精品亚洲美女久久久| 亚洲va在线va天堂va国产| 日韩精品青青久久久久久| 欧洲精品卡2卡3卡4卡5卡区| 一级毛片电影观看 | 天堂影院成人在线观看| 中国国产av一级| 国产视频一区二区在线看| 麻豆乱淫一区二区| 免费电影在线观看免费观看| 91狼人影院| 禁无遮挡网站| 少妇人妻一区二区三区视频| 黄色欧美视频在线观看| 女人十人毛片免费观看3o分钟| 不卡一级毛片| 色av中文字幕| 久久精品综合一区二区三区| 免费av不卡在线播放| 中文字幕精品亚洲无线码一区| 99久久精品热视频| 亚洲国产日韩欧美精品在线观看| 亚洲欧美清纯卡通| 国产 一区精品| 国产色婷婷99| 91在线精品国自产拍蜜月| 两个人的视频大全免费| 亚洲av电影不卡..在线观看| 国产一区二区亚洲精品在线观看| 国产精品1区2区在线观看.| 中文字幕精品亚洲无线码一区| www.色视频.com| 日韩成人伦理影院| 亚洲四区av| eeuss影院久久| 国产激情偷乱视频一区二区| 欧美xxxx黑人xx丫x性爽| 婷婷精品国产亚洲av在线| 国产综合懂色| 日本a在线网址| 午夜福利高清视频| 日本三级黄在线观看| 久久久精品欧美日韩精品| 夜夜看夜夜爽夜夜摸| 亚洲人与动物交配视频| 欧美日本亚洲视频在线播放| 校园春色视频在线观看| 久久久久久伊人网av| 国产综合懂色| 哪里可以看免费的av片| 亚洲人成网站在线观看播放| 观看美女的网站| 老熟妇乱子伦视频在线观看| 欧美成人一区二区免费高清观看| 日本黄色视频三级网站网址| 国产片特级美女逼逼视频| 又粗又爽又猛毛片免费看| 久久99热6这里只有精品| 少妇被粗大猛烈的视频| 亚洲天堂国产精品一区在线| 亚洲一级一片aⅴ在线观看| 可以在线观看毛片的网站| 欧美性猛交╳xxx乱大交人| 日韩精品中文字幕看吧| 嫩草影院入口| 波野结衣二区三区在线| 午夜精品在线福利| 日产精品乱码卡一卡2卡三| 国产精品伦人一区二区| 高清日韩中文字幕在线| 不卡视频在线观看欧美| 最近2019中文字幕mv第一页| 99久久中文字幕三级久久日本| 国产精品乱码一区二三区的特点| 别揉我奶头~嗯~啊~动态视频| 国产伦精品一区二区三区视频9| 97超碰精品成人国产| 一本一本综合久久| 搡女人真爽免费视频火全软件 | 在线免费观看的www视频| 又黄又爽又免费观看的视频| 少妇人妻精品综合一区二区 | 国产精品爽爽va在线观看网站| av.在线天堂| 蜜桃久久精品国产亚洲av| a级毛片a级免费在线| 国产午夜福利久久久久久| 午夜精品国产一区二区电影 | videossex国产| 美女大奶头视频| 在线国产一区二区在线|