• <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 ?

    Cellular metabolic energy modulation by tangeretin in 7,12-dimethylbenz(a) anthracene-induced breast cancer

    2016-12-13 09:27:50KuppusamyPeriyasamyVenkatachalamSivabalanKuppusamyBaskaranKannayiramKasthuriDhanapalSakthisekaran
    THE JOURNAL OF BIOMEDICAL RESEARCH 2016年2期

    Kuppusamy Periyasamy, Venkatachalam Sivabalan, Kuppusamy Baskaran, Kannayiram Kasthuri, Dhanapal Sakthisekaran

    Department of Medical Biochemistry, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai-600 113, Tamil Nadu, India.

    Cellular metabolic energy modulation by tangeretin in 7,12-dimethylbenz(a) anthracene-induced breast cancer

    Kuppusamy Periyasamy, Venkatachalam Sivabalan, Kuppusamy Baskaran, Kannayiram Kasthuri, Dhanapal Sakthisekaran?

    Department of Medical Biochemistry, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai-600 113, Tamil Nadu, India.

    Breast cancer is the leading cause of death among women worldwide. Chemoprevention and chemotherapy play beneficial roles in reducing the incidence and mortality of cancer. Epidemiological and experimental studies showed that naturally-occurring antioxidants present in the diet may act as anticancer agents. Identifying the abnormalities of cellular energy metabolism facilitates early detection and management of breast cancer. The present study evaluated the effect of tangeretin on cellular metabolic energy fluxes in 7,12-dimethylbenz(a) anthracene (DMBA)-induced proliferative breast cancer. The results showed that the activities of glycolytic enzymes significantly increased in mammary tissues of DMBA-induced breast cancer bearing rats. The gluconeogenic tricarboxylic acid (TCA) cycle and respiratory chain enzyme activities significantly decreased in breast cancer-bearing rats. In addition, proliferating cell nuclear antigen (PCNA) was highly expressed in breast cancer tissues. However, the activities of glycolytic enzymes were significantly normalized in the tangeretin pre- and post-treated rats and the TCA cycle and respiratory chain enzyme activities were significantly increased in tangeretin treated rats. Furthermore, tangeretin down-regulated PCNA expression on breast cancerbearing rats. Our study demonstrates that tangeretin specifically regulates cellular metabolic energy fluxes in DMBA-induced breast cancer-bearing rats.

    tangeretin, mitochondria, glycolysis, tricarboxylic acid cycle, proliferating cell nuclear antigen, breast cancer

    Introduction

    Breast cancer is the most common malignancy and the leading cause of death among women worldwide. Cancer cells have distinct metabolism and highly depend on glycolysis instead of mitochondrial oxidative phosphorylation. Hexokinase, phosphoglucoisomerase, aldolase and pyruvate kinase catalyze glucose through the glycolytic pathway, and have emerged as a potential regulator of the metabolic phenotype[2]. Warburg first pointed out that the metabolic disorder of cellular respiration is a common characteristic feature of cancer cells[3]. Under aerobic conditions, one glucose molecule is fully oxidized to produce CO2, H2O and 38 adenosine triphosphate (ATP). Cancer cellscan generate 2 molecules of lactic acid and 2 molecules of ATP through anaerobic respiration under hypoxic conditions. However, Warburg found that unlike normal cells, cancer cells generate energy through rapid glycolytic activity even in ample oxygen with the concomitant production of a large amount of lactic acid molecules. Oxidative phosphorylation is inhibited by the production of large amounts of lactic acid. This metabolic phenomenon is often called Warburg effect or aerobic glycolysis. Initially, Warburg thought that aerobic glycolysis was caused by the mitochondrial oxidative damages in cancer cells[4]. Other studies showed that under certain chemotherapeutical circumstances, cancer cells might have switched back to normal mitochondrial respiratory function. Chemotherapeutically-treated cancer cells grow slower than those with cancer-bearing conditions[5]. In these ways, aerobic glycolysis of cancer cells is likely to be controlled by the energy modulating effect of chemotherapeutic agents.

    Cellular metabolic enzyme is regulated by many oncogenes and it serves a dual function as the speed regulator of cancer cell metabolism[6]. The activities of glycolytic enzymes are regulated by its own regulatory genes under the active proliferative signal. Pyruvate kinase splits the product of glycolysis into two parts, one for energy requirements and another for transformation into different precursor substances. Pyruvate kinase also forms a complex with other enzymes involved glycolysis and causes glucose to be degraded into pyruvate. It is further transformed into lactic acid, and also produces an ample amount of energy molecules. These energy molecules are used for the growth of tumor cells under hypoxic and anoxic conditions. The expression and translation of hexokinase, phosphoglucoisomerase, aldolase and pyruvate kinase are controlled by many oncogenes and metabolic intermediates[7]. Cancer cells acquire the rapid growth phenotype and ultimately develop mammary carcinoma through energy-producing molecules.

    Tangeretin is a ubiquitous bioactive flavonoid in the peel of citrus fruits of the flavone family and also known to have anti-cancer, antioxidant, anti-inflammatory and hypolipidemic effects as well as antimicrobial activity. It is used in traditional Chinese medicine for treatment of various diseases including cancer[8]. In the present study, we evaluated the effect of tangeretin on cellular metabolic energy fluxes in 7,12- dimethylbenz(a) anthracene (DMBA)-induced proliferative breast cancer.

    Materials and methods

    Chemicals and reagents

    Tangeretin (4',5,6,7,8-pentamethoxyflavone) was purchased from the Indofine Chemical Co. (Hillisborough, NJ, USA) and 7,12-dimethylbenz(a) anthracene (DMBA) and other fine chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA). Primary monoclonal antibody PCNA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibodies, horseradish peroxidase (HRP) conjugated goat-antimouse IgG was obtained from Bio-Rad Laboratories (Hercules, CA, USA). The DAB kit was purchased from Genie, Bengaluru, Bangalore. Other chemicals and reagents in the study were of the highest purity and standard, which are commercially available.

    Animals

    Ninety day old healthy female Sprague-Dawley rats (Rattus norvegicus) weighing around 150 g were used in this study. The rats were housed in clean polypropylene cages, maintained in the air-conditioned animal house with a constant photoperiod of 12 hours light/ dark cycle with the light cycle from 6:00 to 18:00 and the dark cycle from 18:00 to 6:00. The rats were fed with the pellet diet and drinking water ad libitum. The study protocol was approved by the Institutional Animal Ethical Committee, University of Madras, Taramani Campus, Chennai, India (IAEC/No. 01/13/2012) and animal studies were carried out in accordance with the established institutional guidelines regarding animal care and use. Animal welfare and the experimental procedures were carried out strictly in accordance with the Guide for Care and Use of Laboratory Animals (National Research Council of USA, 1996).

    Experimental design

    The rats were divided into 5 groups, each consisting of 6 rats. Group I served as healthy control rats; Group II received a single dose of 80 mg/kg body weight of DMBA dissolved in 1 mL olive oil by gastric lavage. Breast cancer was allowed 90 days to develop and grow. Group III were pre-treated with tangeretin (50 mg/kg body weight orally) dissolved in olive oil for 30 days prior to administration of DMBA as in Group II. Group IV received DMBA as in Group II and after 90 days, were treated with tangeretin (50 mg/kg body weight) for 30 days orally. Group V rats were treated with tangeretin alone (50 mg/kg body weight) for 30 days and served as a drug control.

    At the end of the experiment, blood was collected and rats were perfused with normal saline under sodium thiopentone (40 mg/kg body weight) anesthesia. Mammary and liver tissues were dissected out immediately. Histological evaluation of the samples was prepared in 10% buffered formalin and later embedded in paraffin. The sections were used forimmunohistochemistry. Fresh tissues were used for each experiment. Homogenate of breast tissues (10%) was prepared in 0.1 mol/L, pH 7.4 Tris-Hcl buffer using a Potter-Elvejhem glass homogenizer. Dilutions were performed according to protein concentration. The protein contents were measured by the method of Lowry et al.[9].

    Isolation of mitochondria

    Mitochondria were isolated from the breast tissues by the method of Johnson and Lardy[10]. Breast tissues were homogenized (10%) in ice-cold 0.25 mol/L sucrose with 50 mmol/L phosphate buffer, pH 7.4 with a Potter-Elvehjem glass homogenizer (Belco Glass, Inc., Vineland, NJ, USA) for 30 seconds. The homogenate was then centrifuged at 500×g for 10 minutes, and the supernatant was again centrifuged at 12,000×g for 15 minutes to obtain the mitochondrial fraction. The pellet, thus obtained, was resuspended in the buffer and used for assaying mitochondrial enzymes.

    Hexokinase activity was assayed by the method of Brandstrup et al.[11]. Phosphoglucoisomerase was assayed according to Horrocks et al.[12]. Aldolase was estimated by the method of King[13]. The activities of glucose-6-phosphatase and fructose-1,6-disphosphatase were assayed by the method of Gancedo and Gancedo[14]. Pyruvate dehydrogenase activity was measured according to the method of Chretien et al.[15]. Mitochondrial isocitrate dehydrogenase (ICDH) and α-ketoglutarate dehydrogenase activities were measured according to the method of Duncan et al.[16]. Mitochondrial succinate dehydrogenase activity was measured according to the method of Veeger et al.[17]. NADH-cytochrome c oxidoreductase and cytochrome c oxidase activities were determined by the method of Goyal and Srivastava[18].

    Immunohistochemistry

    Immunohistochemical analysis of PCNA expression was performed on 5 μm thick paraffin embedded breast tissue section on poly-L-lysine coated glass slides. The tissue slides were deparaffinized by placing the slides in an oven at 60°C for 10 minutes and then rinsed twice in xylene for 5 minutes each. The slides were hydrated in a graded ethanol series for 10 minutes each. Then, the slides were washed in double distilled water for 5 minutes. The sections were incubated with 1% H2O2in double distilled water for 15 minutes at 22°C, to quench endogenous peroxidase activity. The sections were rinsed with Tris-HCl containing 150 mmol/L NaCl (pH 7.4) and blocked with blocking buffer (1×TBS, 0.05% Tween 20, 5% NFDM) for 1 hour at 22°C. After washing with 1×TBS containing 0.05% Tween 20, the sections were incubated with primary antibodies PCNA (1:200 dilution) overnight at 4°C, followed by incubation with the respective secondary antibodies IgG-HRP conjugates for 1 hour at 4°C. After washing with 1×TBS containing 0.05% Tween 20, immune-reactivity was developed with 0.01% DAB and H2O2for 1–3 minutes and the sections were observed (20×) for brown color formation under bright field in a microscope. The sections were photographed using Nikon 80i Eclipse microscope.

    Statistical analysis

    The values are expressed as mean ± SEM. The results were computed statistically (SPSS software package, Version 17) using one-way analysis of variance (ANOVA). The post-hoc testing was performed for inter-group comparison using the Tukey multiple comparison test. P < 0.05 was considered statistically significant.

    Results

    Glycolytic enzyme activities

    Glycolytic activities in mammary tumors were determined. The glycolytic enzyme activities of DMBA-induced breast cancer cells exhibited substantial increases in cancer-bearing rats. Table 1 shows the activities of glycolytic enzymes in mammary tissues of rats. The activities of hexokinase, phosphoglucoisomerase and aldolase levels significantly increased (P < 0.05 vs. control) in DMBA-induced breast cancer-bearing rats. However, tangeretin treatment for 30 days before the administration of DMBA in Group III rats and post-treatment with tangeretin in breast cancer-bearing Group IV rats significantly reduced the activities of hexokinase, phosphoglucoisomerase, and aldolase compared with tumor-bearing rats (Group II). Group V showed no effective changes (P > 0.05) compared with the control rats (Group I).

    Gluconeogenic enzymes

    The estimated gluconeogenic activities in the liver tissue of DMBA-induced breast cancer along with tangeretin pre-and post-treated rats are shown in Table 2. The activities of gluconeogenic enzymes such as fructose-1,6-bisphosphatase and glucose-6-phosphatase significantly decreased (P < 0.05) in DMBA-induced breast cancer-bearing rats when compared with the control rats (Group I). However, tangeretin pre-treated rats showed a significant increase of fructose-1,6-bisphosphataseand glucose-6-phosphatase activities (P < 0.05 vs. induced). Furthermore, post-treatment with tangeretin (50 mg/kg body weight) for 30 days significantly restored the activities of fructose-1,6-bisphosphatase and glucose-6-phosphatase in Group IV compared with cancer-bearing rats (P < 0.05). No significant enzyme activities were found in rats treated with tangeretin alone (P < 0.05 vs. control).

    Table 1 Effects of tangeretin on the glycolytic activities in the mammary tissues of rats with DMBA-induced breast cancer

    Mitochondrial TCA cycle enzymes

    Fig. 1 shows a significant decrease in the activity of pyruvate dehydrogenase (PDH), which couples glycolysis to the TCA cycle, in DMBA-induced breast cancer-bearing rats (P < 0.05 vs. control). The activities of PDH were significantly ameliorated (P < 0.05) in tangeretin pre-and post-treated against breast cancer-bearing rats when compared with Group II rats. The mitochondrial TCA cycle enzymes such as isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and succinate dehydrogenase activities were assayed according to the standard protocols. Fig. 1 shows the activities of ICDH, α-KGDH and SDH in the mammary tissue of rats. TCA cycle enzymes were significantly reduced (P < 0.05) in Group II compared with the control rats. However, tangeretin pre-treatment and post-treatment on DMBA-induced breast cancer-bearing rats showed a significant increase of ICDH, α-KGDH and SDH activities with Group II. None of the changes were observed in rats treated with tangeretin alone compared with the control rats.

    Mitochondrial respiratory chain enzymes

    The activities of mitochondrial respiratory chain enzymes like NADH-cytochrome c oxidoreductase and cytochrome c oxidase in mitochondria are shown in Fig. 2A and 2B, respectively. The mitochondrial activities of NADH cytochrome c oxidoreductase and cytochrome c oxidase were significantly decreased (P < 0.05) in the mammary tissues of rats with DMBA-induced breast cancer. However, Fig. 2C shows the activity of cytochrome c oxidase in the cytosol of DMBA-induced breast cancer cells, and there was a significant increase (P < 0.05) of cytochrome c oxidase in cytosol due to leakage from the mitochondria into the cytosol compared with the control rats. Following treatment with tangeretin in Group III and IV rats, the activities of NADH-cytochrome c oxidoreductase and cytochrome c oxidase significantly increased (P < 0.05) in the mitochondria. Conversely, the activity of cytochrome c oxidase in tangeretin treatment was significantly decreased in the cytosol. These data are presented in Fig. 2.

    PCNA

    Fig. 3 shows the immunohistochemical expression of PCNA in breast cancer tissues. PCNA was highly expressed in DMBA-induced breast cancer-bearingmammary tissues. However, PCNA expression was significantly decreased in tangeretin pre- and posttreated mammary tissues when compared with breast cancer-bearing mammary tissue. These results indicated that tangeretin may have an antiproliferative effect on DMBA-induced breast cancer.

    Fig. 1 Protective role of tangeretin on the TCA cycle activities in mammary tissues. The activities of pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and succinate dehydrogenase are depicted in the Fig. 1. Values are mean ± SEM of 6 rats in each group. The significant level is *P < 0.05.

    Discussion

    Our previous study showed that tangeretin plays a significant role against oxidative stress-induced proliferative breast cancer[8]. In the present study, we analyzed the effect of tangeretin on cellular energy metabolic variation in rats. We found thatthe cytoplasmic glycolytic enzymes accelerated high-energy production. Furthermore, there was a low-energy production through the TCA cycle as well as the mitochondrial respiratory chain system in mammary cells due to mitochondrial oxidative stress.

    Fig. 2 Effect of tangeretin on mitochondrial respiratory enzyme activities in mammary tissues. The activities of 2(a) NADH-cytochrome-c oxidoreductase, 2(b) cytochrome-c oxidase (mitochondria) and 2(c) cytochrome-c oxidase (cytosol) were determined and values are mean ± SEM of 6 rats in each group. The significant level is *P < 0.05, **P < 0.01.

    The elevated cellular metabolic energy levels account for abnormal metabolic properties of tumors. Increased rate of glycolysis in tumor cells leads to an increase in intracellular concentration of glucose-6-phosphate. It is a key precursor in de novo synthesis of nucleic acids, phospholipids and other macromolecules[20]. Increased synthesis of glycolytic components may be essential to keep rapid cell division and membrane biosynthesis during tumor growth. Thus, rapid proliferating tumor cells have an excessive energy demand for nuclear cell division[21]. Phosphoglucoisomerase acts as a catalyst in the conversion of glucose-6-phosphate to fructose-6-phosphate and it is an indicator of metastasis. Alterations in the activities of phosphoglucoisomerase might be expected by the influence of a proportion of glucose 6-phosphate, which is metabolized via the glycolytic pathway[22]. Hennipnan et al. reported increased activity of aldolase in metastatic tumor. Upon tangeretin treatment, breast cancer cells could decrease glycolytic regulatory enzyme activities, which might be due to production of high amounts of glucose-6-phosphate by repairing defective mitochondria. Previous studies reported that natural flavonoids could inhibit glycolysis in tumor cells. Tangeretin inhibits accelerated glycolytic activities in cancer and also induces the apoptotic cell death in malignant tumor cells[23].

    In the present study, we have observed that the activities of gluconeogenic enzymes were significantly lowered in the liver tissue of DMBA-induced breastcancer-bearing rats. Reduction rate of fructase-1,6-bisphosphatase might lead to a higher concentration of its substrate fructose-1,6-bisphosphate, which is an allosteric activator of pyruvate kinase. Alternatively, down regulation of glucose-6-phosphatase activity is likely to increase the concentration of its substrate glucose-6-phosphate, which might be used by pentose phosphate shunt pathway to produce ribose-5-phosphate for rapid nucleotide synthesis in cancer[24]. The diminished activities of fructose-1,6- bisphosphatase and glucose-6-phosphatase were due to higher concentration of lactic acid production in neoplastic tissues. Higher rate of glucose utilization and an increased production of lactic acid are characteristic features of neoplastic cells[25]. Gluconeogenesis from lactate on the other hand is an essential energy- requiring process as pointed out by Fenninger and Mider. Gluconeogenesis may play an important role in excessive energy expenditure of the host; thus, it is contributing to promote weight loss[26]. Tangeretin effectively acts on proliferative cancer cells by ameliorating the gluconeogenic activities.

    Mitochondria are considered as the major source of cellular ROS, which may inhibit mitochondrial Krebs cycle enzyme activities[27]. The mitochondrial TCA cycle enzyme activities were significantly lowered in cancer, which is due to membrane damages by the oxidative stress-induced free radicals. Formation of ROS disturbs the nitric oxide pathway, resulting in protein nitration, oxidative damage of DNA and lipid peroxidation. However, it is more and more evident that alterations of energy metabolism in cancer are especially due to mitochondrial dysfunction. Decreased activities of ICDH, α-KGDH and SDH could be due to the alteration in mitochondrial membranes, morphology and organelles from peroxidation of macromolecules[28]. Tangeretin plays a major role in restoring the mitochondrial function on DMBA-induced breast cancer-bearing rats.

    Inhibition of the mitochondrial TCA cycle and respiratory chain enzyme activities could lead to a leakage of electrons, which produces a reducing environment within the mitochondria, thereby generating free radicals[29]. Reduction activities of NADH cytochrome c oxidoreductase and cytochrome c oxidase were noted in DMBA-induced breast cancer. It clearly indicates that an elevated state of oxidative stress could lead to impaired regulatory functions of mitochondria. These free radicals, particularly free ?OH radical, can damage the mitochondrial membrane, which causes leakage of cytochrome c from the mitochondria into the cytosol[30]. Our current study showed that tangeretin could inhibit the highly significant release of cytochrome c in the cytosol from mitochondria. The reason for this may lie in the fact that tangeretin may be able to repair mitochondrial membrane damage. Tangeretin could increase mitochondrial enzymes, which are related to energy metabolism. Tangeretin could completely restore NADH cytochrome c oxidoreductase and cytochrome c oxidase. It indicates that tangeretin is capable of mitigating mitochondrial oxidative stress-induced proliferation of breast cancer cells.

    Cellular proliferation of the mammary tissues was observed by immunohistochemical examination of PCNA expression. PCNA was overexpressed in 80% of breast cancer according to a previous study[31]. In our study, the protein expression of PCNA significantly increased due to potential mutagenic activity of DMBA. However, tangeretin drastically decreased PCNA expression, due to the inhibition of rapid DNA synthesis. PCNA primarily expressed during the cell cycle progression and its rate of synthesis is directly correlated with proliferative cancer cells. PCNA is a cell nuclear protein, and it functions as an auxiliary protein for DNA polymerase delta. Tangeretin inhibits the cellular G1, G1/S phases in the cell cycle systemin cancer progression[32]. Juhee et al. also reported that tangeretin significantly inhibited proliferation, DNA synthesis and migration of aortic smooth muscle cells by blocking AKT activation[33]. In that way, anti-proliferative activity of tangeretin control proliferative breast cancer.

    In conclusion, pre- and post-treatment with tangeretin in breast cancer could prevent abnormal carbohydrate metabolic energy fluxes and brought back to normal the functions of mitochondria in energy metabolism.

    Acknowledgement

    The financial support from the University Grants Commission, New Delhi, in the form of UGC-BSR Research Fellowship under the UGC-SAP-DRS-III programme is gratefully acknowledged.

    References

    [1] Ferlay J, Shin HR, Bray F, et al. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008[J]. Int J Cancer, 2010,127(12):2893-2917.

    [2] Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation. Science (New York, NY), 2009,324(5930):1029-1033.

    [3] Warburg O. On respiratory impairment in cancer cells[J]. Science, 1956,124(3215):269-270.

    [4] Warburg O, Dickens F. Kaiser Wilhelm-Institut fur Biologie B. The Metabolism of Tumours: Investigations from the Kaiser-Wilhelm Institute for Biology, Berlin-Dahlem. London, UK: Constable & Co, Ltd; 1930.

    [5] Mazurek S. Pyruvate kinase type M2: a key regulator of the metabolic budget system in tumour cells[J]. Int J Biochem Cell Biol, 2011,43:969-980.

    [6] Wu S, Le H. Dual roles of PKM2 in cancer metabolism[J]. Acta Biochim Biophys Sin (Shanghai), 2013,45(1): 27-35.

    [7] Wang X, Peralta S, Moraes CT. Mitochondrial alterations during carcinogenesis: a review of metabolic transformation and targets for anticancer treatments[J]. Adv Cancer Res, 2013,119:127-160.

    [8] Periyasamy K, Baskaran K, Ilakkia A, et al. Antitumor efficacy of tangeretin by targeting the oxidative stress mediated on 7,12-dimethylbenz(a) anthracene-induced proliferative breast cancer in Sprague-Dawley rats[J]. Cancer Chemother Pharmacol, 2015,75(2):263-272.

    [9] Lowry O, Rosebrough N, Farr A, et al. Protein measurement with the Folin phenol reagent[J]. J Biol Chem, 1951,193:265-275.

    [10] Johnson, D, Lardy H. Methods in Enzymology (Estabrook RW and Pullman ME, eds. 1967,10:94-96.

    [11] Brandstrup N, Kirk JE, Bruni C. The hexokinase and phosphoglucoisomerase activities of aortic and pulmonary artery tissue in individuals of various ages[J]. J Gerontol, 1957,12(2):166-171.

    [12] Horrocks JE, Ward J, King J. A routine method for the determination of phosphoglucoseisomerase activity in body fluid[J]. J Clin Pathol, 1963,16:248-251.

    [13] King J. Practical clinical enzymology. Van Nostrand, Science 1965, p. 363.

    [14] Gancedo JM, Gancedo C. Fructose-1,6-diphosphatase, phosphofructokinase and glucose-6-phosphate dehydrogenase from fermenting and non fermenting yeasts[J]. Arch Mikrobiol, 1971,76(2):132-138.

    [15] Chretien D, Pourrier M, Bourgeron T, et al. An improved spectrophotometric assay of pyruvate dehydrogenase in lactate dehydrogenase contaminated mitochondrial preparations from human skeletal muscle[J]. Clin Chim Acta, 1995,240(2):129-136.

    [16] Duncan MJ, Fraenkel DG. Alpha-ketoglutarate dehydrogenase mutant of Rhizobium meliloti[J]. J Bacteriol, 1979,37:415-419.

    [17] Veeger C, Vartanian DV, Zeylemaker WP. Succinate dehydrogenase[J]. Meth Enzymol, 1969,13:81-90.

    [18] Goyal N, Srivastava VML. Oxidation and reduction of cytochrome c by mitochondrial enzymes of Setariacervi[J]. J Helminth, 1995,69:13-17.

    [19] Purushothaman A, Nandhakumar E Sachdanandam P. Phytochemical analysis and anticancer capacity of Shemamruthaa, a herbal formulation against DMBA-induced mammary carcinoma in rats[J]. Asian Pac J Trop Med, 2013,6(12):925-933.

    [20] Boros LG, Lee WN, Go VL. A metabolic hypothesis of cell growth and death in pancreatic cancer[J]. Pancreas, 2002,24(1):26-33.

    [21] Senthilnathan P, Padmavathi R, Magesh V, et al. Modulation of TCA cycle enzymes and electron transport chain systems in experimental lung cancer[J]. Life Sci, 2006,78(9):1010-1014.

    [22] Ebrahim GJ. Health care of adolescents[J]. J trop Pediatr, 1996,42(6):316-317.

    [23] Hennipman A, Van Oirschot BA, Smits J, et al. Glycolytic enzymes activities in breast cancer metastases[J]. Tumour Biol, 1988,9(5):241-248.

    [24] Wang B, Hsu SH, Frankel W, et al. Stat3-mediated activation of microRNA-23a suppresses gluconeogenesis in hepatocellular carcinoma by down-regulating glucose-6-phosphatase and peroxisome proliferator- activated receptor gamma, coactivator 1 alpha[J]. Hepatology, 2012,56:186-197.

    [25] Waterhouse C. Lactate Metabolism in patients with Cancer[J]. Cancer 1974,33(1):66-71.

    [26] Fenninger LD and Mider GB. Energy and Nitrogen metabolism in cancer[J]. Adv Cancer Res, 1954,2:229-253.

    [27] Fariss MW, Chan CB, Patel M, et al. Role of mitochondria intoxic oxidative stress. Mol Interv, 2005,5(2):94-111.

    [28] Dimitrova-Shumkovska J, Veenman L, Ristoski T, et al. Decreases in binding capacity of the mitochondrial 18 kdatranslocator protein accompany oxidative stress and pathological signs in rat liver after DMBA exposure[J]. Toxicol Pathol, 2010,38(6):957-968.

    [29] Martin M, Macias M, Leon J, et al. Melatonin increases the activity of the oxidative phosphorylation enzymes and the production of ATP in rat brain and liver mitochondria[J]. Int J Biochem Cell Biol, 2002,34:348-357.

    [30] Reiter RJ, Tan DX, Mancshester LC, et al. Melatonin reduces oxidant damage and promotes mitochondrial respiration: implications for aging[J]. Ann N Y Acad Sci, 2002,959:238-250.

    [31] Urruticoechea A, Smith IE, Dowsett M. Proliferation marker Ki-67 in early breast cancer[J]. J Clin Oncol, 2005,23(28):7212-7220.

    [32] Pan M, Chen W, Lin-Shiau S, et al. Tangeretin induces cell-cycle G1 arrest through inhibiting cyclin-dependent kinases 2 and 4 activities as well as elevating Cdk inhibitors p21 and p27 in human colorectal carcinoma cells[J]. Carcinogenesis, 2002,23(10):1677-1684.

    [33] Juhee S, Hyun SL, Sungwoo R, et al. Tangeretin, a citrus flavonoid, inhibits PGDF-BB-induced proliferation and migration of aortic smooth muscle cells by blocking AKT activation[J]. Eur J Pharmacol, 2011,673:56-64.

    ? Prof. D. Sakthisekaran, Former Professor and Head, UGC-BSR-Faculty Fellow, Department of Medical Biochemistry, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai-600113, Tamil Nadu, India. Tel/Fax: +91-44-24547083/+91-44-24540709,

    Email: dsakthisekaran@hotmail.com.

    04 April 2015, Revised 02 June 2015, Accepted 10 October 2015, Epub 26 February 2016

    R730.22, Document code: A

    The authors reported no conflict of interests.

    404 Not Found

    404 Not Found


    nginx
    高清视频免费观看一区二区 | av在线播放精品| 黑人高潮一二区| 美女xxoo啪啪120秒动态图| 嫩草影院精品99| 嫩草影院新地址| 搡老妇女老女人老熟妇| 淫秽高清视频在线观看| 色吧在线观看| 永久免费av网站大全| 婷婷色麻豆天堂久久 | 床上黄色一级片| 22中文网久久字幕| 久久久久免费精品人妻一区二区| videos熟女内射| 日韩一区二区视频免费看| 久久久久久久久久成人| 日韩一本色道免费dvd| 亚洲欧美精品自产自拍| 别揉我奶头 嗯啊视频| 日本午夜av视频| 国产精品久久久久久久电影| 毛片女人毛片| 日日干狠狠操夜夜爽| 91久久精品国产一区二区三区| 特级一级黄色大片| 18禁裸乳无遮挡免费网站照片| 有码 亚洲区| 欧美bdsm另类| 人人妻人人澡人人爽人人夜夜 | 国内揄拍国产精品人妻在线| 国产一区二区在线观看日韩| 亚洲成人中文字幕在线播放| 中文字幕av成人在线电影| 欧美精品一区二区大全| 欧美成人午夜免费资源| 国产乱人偷精品视频| 国产又黄又爽又无遮挡在线| 伦理电影大哥的女人| 亚洲精品自拍成人| 欧美激情久久久久久爽电影| 2021天堂中文幕一二区在线观| 高清日韩中文字幕在线| 国模一区二区三区四区视频| 久久久色成人| 久久久久性生活片| 中文字幕av在线有码专区| 精品国产一区二区三区久久久樱花 | 国产在视频线在精品| 国产成人午夜福利电影在线观看| 日本黄大片高清| 在线观看一区二区三区| 亚洲国产精品专区欧美| 亚洲婷婷狠狠爱综合网| 久久草成人影院| av专区在线播放| 国产精品1区2区在线观看.| 自拍偷自拍亚洲精品老妇| 波多野结衣高清无吗| 欧美精品一区二区大全| 日韩大片免费观看网站 | 免费观看性生交大片5| 久久韩国三级中文字幕| 最后的刺客免费高清国语| 成年免费大片在线观看| 级片在线观看| 国产精品不卡视频一区二区| 国产精品综合久久久久久久免费| 亚洲av.av天堂| 青春草亚洲视频在线观看| 性色avwww在线观看| 久久精品国产亚洲网站| 精品无人区乱码1区二区| 久久精品国产鲁丝片午夜精品| 小蜜桃在线观看免费完整版高清| 波野结衣二区三区在线| 欧美日韩在线观看h| 国产成年人精品一区二区| 97在线视频观看| 一级毛片我不卡| 欧美丝袜亚洲另类| 亚洲美女视频黄频| 亚洲国产欧洲综合997久久,| 一级毛片我不卡| 日本爱情动作片www.在线观看| 国产精品一区www在线观看| 久久精品国产鲁丝片午夜精品| 亚洲,欧美,日韩| 日韩成人伦理影院| 久久久久久九九精品二区国产| 日韩av不卡免费在线播放| 国产极品天堂在线| 欧美日韩国产亚洲二区| 日韩国内少妇激情av| 国产乱人视频| 男女国产视频网站| 亚洲av免费高清在线观看| 精品久久久久久久久久久久久| 久久久久国产网址| 97人妻精品一区二区三区麻豆| 99在线人妻在线中文字幕| 国产高清国产精品国产三级 | 亚洲精品乱码久久久v下载方式| 亚洲av中文字字幕乱码综合| 22中文网久久字幕| 午夜a级毛片| 最新中文字幕久久久久| 春色校园在线视频观看| 超碰av人人做人人爽久久| 免费大片18禁| av视频在线观看入口| 久久韩国三级中文字幕| 日韩成人av中文字幕在线观看| 亚洲欧美精品综合久久99| av黄色大香蕉| 国产精品永久免费网站| 色视频www国产| 水蜜桃什么品种好| 日韩 亚洲 欧美在线| 中文字幕免费在线视频6| 国产在线一区二区三区精 | 久久99热这里只频精品6学生 | 亚洲成av人片在线播放无| 18+在线观看网站| 精品国内亚洲2022精品成人| 免费看av在线观看网站| 三级国产精品片| 国产v大片淫在线免费观看| 如何舔出高潮| 女人被狂操c到高潮| 日韩一区二区三区影片| 欧美高清性xxxxhd video| 亚洲天堂国产精品一区在线| 国产av一区在线观看免费| a级一级毛片免费在线观看| 成人欧美大片| 国产免费又黄又爽又色| 久久精品国产亚洲av天美| 欧美丝袜亚洲另类| 一级av片app| 看片在线看免费视频| 精品不卡国产一区二区三区| 久久精品人妻少妇| 国产成人91sexporn| 美女xxoo啪啪120秒动态图| 99久久精品国产国产毛片| 国产亚洲av嫩草精品影院| 国产精品一区www在线观看| 国产单亲对白刺激| 国产在视频线在精品| .国产精品久久| 26uuu在线亚洲综合色| 成人午夜高清在线视频| 直男gayav资源| 1000部很黄的大片| 色综合站精品国产| 午夜激情福利司机影院| 91午夜精品亚洲一区二区三区| 国产黄色小视频在线观看| 日本三级黄在线观看| 少妇高潮的动态图| 亚洲精品成人久久久久久| 久久精品久久精品一区二区三区| 精品99又大又爽又粗少妇毛片| 99久久九九国产精品国产免费| www日本黄色视频网| 天美传媒精品一区二区| 中文字幕亚洲精品专区| h日本视频在线播放| 久久99热这里只有精品18| 国产精品久久久久久av不卡| 一级爰片在线观看| 久久久久久九九精品二区国产| 久久久久久国产a免费观看| 中文字幕精品亚洲无线码一区| 国产精品人妻久久久久久| 天天躁夜夜躁狠狠久久av| 一级毛片电影观看 | 观看免费一级毛片| 三级男女做爰猛烈吃奶摸视频| 精品国产三级普通话版| 男女视频在线观看网站免费| 99久久人妻综合| 欧美区成人在线视频| 久久精品夜夜夜夜夜久久蜜豆| 色播亚洲综合网| 高清在线视频一区二区三区 | 国产精品国产三级国产av玫瑰| 亚洲va在线va天堂va国产| 国产色婷婷99| 亚洲欧美精品自产自拍| 一二三四中文在线观看免费高清| 亚洲久久久久久中文字幕| av天堂中文字幕网| 欧美精品国产亚洲| 国产精品一二三区在线看| 国产伦精品一区二区三区四那| 久久久亚洲精品成人影院| 国产亚洲午夜精品一区二区久久 | 亚洲欧洲国产日韩| 2021少妇久久久久久久久久久| 乱系列少妇在线播放| 一级毛片aaaaaa免费看小| 久久欧美精品欧美久久欧美| 乱人视频在线观看| 日本三级黄在线观看| 中文亚洲av片在线观看爽| 长腿黑丝高跟| 国产中年淑女户外野战色| 亚洲性久久影院| 深夜a级毛片| 一二三四中文在线观看免费高清| 国产高清三级在线| 天天躁夜夜躁狠狠久久av| 成人漫画全彩无遮挡| 欧美3d第一页| 成人亚洲欧美一区二区av| 亚洲怡红院男人天堂| 青春草亚洲视频在线观看| 久久久午夜欧美精品| 人人妻人人澡人人爽人人夜夜 | 97人妻精品一区二区三区麻豆| 国产一区二区亚洲精品在线观看| 免费搜索国产男女视频| 中文字幕精品亚洲无线码一区| 高清在线视频一区二区三区 | 午夜激情福利司机影院| 老女人水多毛片| 日本熟妇午夜| 在线观看av片永久免费下载| 99九九线精品视频在线观看视频| 高清毛片免费看| 精品熟女少妇av免费看| 18禁在线播放成人免费| 在线播放国产精品三级| 边亲边吃奶的免费视频| 国产黄色小视频在线观看| 欧美成人午夜免费资源| 欧美一区二区精品小视频在线| 亚洲精品国产成人久久av| 女人被狂操c到高潮| 成人高潮视频无遮挡免费网站| 色噜噜av男人的天堂激情| 一区二区三区乱码不卡18| 观看免费一级毛片| 日韩一本色道免费dvd| 午夜亚洲福利在线播放| 能在线免费观看的黄片| 色5月婷婷丁香| 男女下面进入的视频免费午夜| 美女内射精品一级片tv| 日韩欧美国产在线观看| 国产精品综合久久久久久久免费| 床上黄色一级片| 人妻系列 视频| 欧美激情久久久久久爽电影| 丰满人妻一区二区三区视频av| 能在线免费观看的黄片| 麻豆成人av视频| 国内揄拍国产精品人妻在线| 亚洲四区av| 免费人成在线观看视频色| 久久精品人妻少妇| 九九热线精品视视频播放| 国产精品久久久久久久久免| 波野结衣二区三区在线| 亚洲av.av天堂| 老司机影院成人| 91狼人影院| 最新中文字幕久久久久| 男人的好看免费观看在线视频| 真实男女啪啪啪动态图| 欧美日韩综合久久久久久| 97超视频在线观看视频| 免费观看的影片在线观看| 久久精品国产99精品国产亚洲性色| 色综合色国产| 久久欧美精品欧美久久欧美| 色哟哟·www| 又爽又黄无遮挡网站| 精品人妻偷拍中文字幕| av在线播放精品| 精品人妻一区二区三区麻豆| 97超碰精品成人国产| 日产精品乱码卡一卡2卡三| 国产老妇伦熟女老妇高清| 免费av不卡在线播放| 中文字幕熟女人妻在线| 成人一区二区视频在线观看| 亚洲精品色激情综合| 亚洲图色成人| 亚洲欧美成人综合另类久久久 | 日本色播在线视频| 啦啦啦啦在线视频资源| 日本与韩国留学比较| 嫩草影院入口| 久久久久久久久久久免费av| 一夜夜www| 欧美另类亚洲清纯唯美| 99久久九九国产精品国产免费| 听说在线观看完整版免费高清| 99热全是精品| 亚洲人成网站高清观看| 国产三级在线视频| 男女视频在线观看网站免费| 国产大屁股一区二区在线视频| 纵有疾风起免费观看全集完整版 | 可以在线观看毛片的网站| av播播在线观看一区| 毛片一级片免费看久久久久| 蜜桃久久精品国产亚洲av| 久久久久久久久久久免费av| 国产精品日韩av在线免费观看| 久久久久网色| 精品酒店卫生间| АⅤ资源中文在线天堂| 国产一级毛片在线| 亚洲精品自拍成人| 插阴视频在线观看视频| 国产又色又爽无遮挡免| av在线蜜桃| 成人午夜高清在线视频| 国产免费又黄又爽又色| 亚洲av成人精品一二三区| 久久6这里有精品| 美女被艹到高潮喷水动态| 男女下面进入的视频免费午夜| av女优亚洲男人天堂| 亚洲成av人片在线播放无| 99热网站在线观看| 看十八女毛片水多多多| 日韩三级伦理在线观看| 久久精品久久久久久久性| 色网站视频免费| 中文字幕熟女人妻在线| 天堂影院成人在线观看| 国产极品天堂在线| 国产真实乱freesex| 99久久九九国产精品国产免费| 国产精品三级大全| 老司机福利观看| 寂寞人妻少妇视频99o| 麻豆国产97在线/欧美| 亚洲美女搞黄在线观看| 2022亚洲国产成人精品| 一本久久精品| 免费无遮挡裸体视频| 精品熟女少妇av免费看| 欧美zozozo另类| 国产欧美日韩精品一区二区| 中文欧美无线码| 两个人视频免费观看高清| 在线免费十八禁| 麻豆av噜噜一区二区三区| 亚洲国产精品久久男人天堂| 色5月婷婷丁香| 又粗又爽又猛毛片免费看| 国产精品av视频在线免费观看| 国产精品久久视频播放| 亚洲图色成人| 国产精品一区二区三区四区免费观看| 亚洲综合色惰| 深爱激情五月婷婷| 建设人人有责人人尽责人人享有的 | 亚洲最大成人手机在线| 夜夜爽夜夜爽视频| 日本免费在线观看一区| 精品午夜福利在线看| 插阴视频在线观看视频| 高清视频免费观看一区二区 | 国产又色又爽无遮挡免| 午夜a级毛片| 麻豆乱淫一区二区| 99视频精品全部免费 在线| 乱人视频在线观看| 看片在线看免费视频| 高清日韩中文字幕在线| 成人鲁丝片一二三区免费| 熟女电影av网| 成人漫画全彩无遮挡| 免费黄网站久久成人精品| 只有这里有精品99| 午夜精品在线福利| 久久鲁丝午夜福利片| 综合色av麻豆| 日本三级黄在线观看| 边亲边吃奶的免费视频| 美女cb高潮喷水在线观看| 国产欧美日韩精品一区二区| 免费不卡的大黄色大毛片视频在线观看 | 日韩欧美 国产精品| 最近中文字幕高清免费大全6| 久久人妻av系列| 国产一区二区三区av在线| 大香蕉97超碰在线| 人人妻人人澡人人爽人人夜夜 | 尤物成人国产欧美一区二区三区| 日韩中字成人| 国产视频内射| 人妻夜夜爽99麻豆av| 欧美成人a在线观看| 日韩欧美在线乱码| 99在线人妻在线中文字幕| 久久精品国产鲁丝片午夜精品| 免费av毛片视频| av线在线观看网站| 国产午夜精品一二区理论片| 国产男人的电影天堂91| 青春草视频在线免费观看| 国产亚洲91精品色在线| 国产午夜精品一二区理论片| 日本免费a在线| 国产精品99久久久久久久久| 日韩av在线大香蕉| 亚洲国产高清在线一区二区三| 毛片女人毛片| 又粗又爽又猛毛片免费看| 麻豆精品久久久久久蜜桃| 久久99蜜桃精品久久| 女人被狂操c到高潮| 日本色播在线视频| 别揉我奶头 嗯啊视频| 国产高清不卡午夜福利| av卡一久久| 九九在线视频观看精品| 午夜爱爱视频在线播放| 亚洲成人精品中文字幕电影| 91精品国产九色| 欧美bdsm另类| 丝袜喷水一区| 精品久久久久久久久av| 伦精品一区二区三区| 日本av手机在线免费观看| 女人久久www免费人成看片 | 国产精品一区二区性色av| 日韩 亚洲 欧美在线| 国产三级在线视频| 蜜桃久久精品国产亚洲av| 国产精品,欧美在线| 国产日韩欧美在线精品| 综合色av麻豆| www.av在线官网国产| 能在线免费看毛片的网站| 在线观看66精品国产| 亚洲欧美日韩高清专用| 久久久午夜欧美精品| 久热久热在线精品观看| 99热网站在线观看| 天堂av国产一区二区熟女人妻| 美女xxoo啪啪120秒动态图| 岛国毛片在线播放| 成人无遮挡网站| 精品国产露脸久久av麻豆 | 久热久热在线精品观看| 日韩制服骚丝袜av| 搡老妇女老女人老熟妇| 国产白丝娇喘喷水9色精品| 男插女下体视频免费在线播放| 身体一侧抽搐| 国产视频内射| 99久久精品热视频| 精品久久久噜噜| 中文字幕熟女人妻在线| 国产精品.久久久| 最后的刺客免费高清国语| 永久免费av网站大全| 建设人人有责人人尽责人人享有的 | 一区二区三区四区激情视频| 全区人妻精品视频| 狂野欧美白嫩少妇大欣赏| 美女大奶头视频| 亚洲图色成人| 亚洲国产欧美人成| 黄色配什么色好看| 最近手机中文字幕大全| 又粗又爽又猛毛片免费看| 国产精品伦人一区二区| 国产成人91sexporn| 成人一区二区视频在线观看| 久久99热这里只频精品6学生 | 亚洲欧美日韩卡通动漫| 日本-黄色视频高清免费观看| 非洲黑人性xxxx精品又粗又长| www日本黄色视频网| 国产色爽女视频免费观看| 精品久久国产蜜桃| 国产精品乱码一区二三区的特点| 国产精品久久视频播放| 午夜福利高清视频| 一卡2卡三卡四卡精品乱码亚洲| 26uuu在线亚洲综合色| 国产成人精品一,二区| 国产亚洲最大av| 黄色配什么色好看| 久久久久网色| 久久国产乱子免费精品| 99热这里只有是精品50| 国产精品三级大全| 白带黄色成豆腐渣| 麻豆成人午夜福利视频| 3wmmmm亚洲av在线观看| 日韩视频在线欧美| 久久精品久久精品一区二区三区| 午夜福利在线观看免费完整高清在| 最近的中文字幕免费完整| 男人舔女人下体高潮全视频| 成人一区二区视频在线观看| 亚洲av熟女| 国产精品一区二区性色av| 免费看av在线观看网站| 日韩欧美三级三区| 中文字幕av在线有码专区| 色播亚洲综合网| 纵有疾风起免费观看全集完整版 | 国产精品蜜桃在线观看| 男人舔女人下体高潮全视频| 直男gayav资源| 亚洲伊人久久精品综合 | 亚洲国产日韩欧美精品在线观看| 狂野欧美激情性xxxx在线观看| 成人午夜精彩视频在线观看| 成年女人永久免费观看视频| 午夜视频国产福利| 久久久久久大精品| 亚洲丝袜综合中文字幕| 色哟哟·www| 赤兔流量卡办理| 欧美一区二区国产精品久久精品| 欧美变态另类bdsm刘玥| 国产一区二区亚洲精品在线观看| 波多野结衣巨乳人妻| 男女国产视频网站| 尾随美女入室| 校园人妻丝袜中文字幕| 大香蕉久久网| 天天躁日日操中文字幕| 精品不卡国产一区二区三区| 国产亚洲精品久久久com| 亚洲av日韩在线播放| 国产真实乱freesex| 日韩精品青青久久久久久| 桃色一区二区三区在线观看| 亚洲欧美精品专区久久| 18禁动态无遮挡网站| 精品国产三级普通话版| 91精品国产九色| 久久久久久久久久黄片| 亚洲在久久综合| 1024手机看黄色片| 黄片wwwwww| 99热精品在线国产| 99国产精品一区二区蜜桃av| av在线蜜桃| 久热久热在线精品观看| 国产av不卡久久| 女人久久www免费人成看片 | 最新中文字幕久久久久| 天堂av国产一区二区熟女人妻| 男女视频在线观看网站免费| 亚洲av中文av极速乱| 亚洲天堂国产精品一区在线| 成人漫画全彩无遮挡| 国产高清不卡午夜福利| 国产成人aa在线观看| 精品一区二区免费观看| 亚洲无线观看免费| 亚洲av免费高清在线观看| 欧美日韩精品成人综合77777| 一级毛片aaaaaa免费看小| 性插视频无遮挡在线免费观看| av在线蜜桃| 久久久久久久国产电影| 国产视频首页在线观看| 全区人妻精品视频| 亚洲欧美精品综合久久99| 国国产精品蜜臀av免费| 亚洲成人av在线免费| 免费看av在线观看网站| 一边亲一边摸免费视频| 国产黄片美女视频| 建设人人有责人人尽责人人享有的 | 亚洲综合精品二区| 欧美日韩在线观看h| 国产淫语在线视频| 毛片一级片免费看久久久久| 久久99精品国语久久久| 天堂网av新在线| 男女视频在线观看网站免费| 免费看a级黄色片| 国产成人精品一,二区| 一二三四中文在线观看免费高清| 波多野结衣高清无吗| 真实男女啪啪啪动态图| av视频在线观看入口| 婷婷色麻豆天堂久久 | 一级二级三级毛片免费看| 在线a可以看的网站| 建设人人有责人人尽责人人享有的 | 久久久色成人| 久久亚洲精品不卡| 91狼人影院| 精品一区二区三区视频在线| 久久久成人免费电影| 亚洲性久久影院| 在线免费十八禁| 七月丁香在线播放| 九草在线视频观看| 久久人人爽人人片av| 日韩欧美三级三区| 精品酒店卫生间| 桃色一区二区三区在线观看| 国产国拍精品亚洲av在线观看| 波野结衣二区三区在线| 男人狂女人下面高潮的视频| 成年女人永久免费观看视频|