• <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 ?

    Sodium glucose co-transporter 2 inhibition reduces succinate levels in diabetic mice

    2020-07-10 07:10:12LakshiniHeratNatalieWardAaronMagnoElizabethRakoczyMarcioKiuchiMarkusSchlaichVanceMatthews
    World Journal of Gastroenterology 2020年23期

    Lakshini Y Herat, Natalie C Ward, Aaron L Magno, Elizabeth P Rakoczy, Marcio G Kiuchi, Markus P Schlaich,Vance B Matthews

    Abstract

    Key words: Sodium glucose co-transporter inhibitors; Sodium glucose co-transporter 2;Diabetes; Diabetic retinopathy; Mouse; Gut microbiota; Empagliflozin; Dapagliflozin;Succinate; Akimba

    INTRODUCTION

    The gut microbiome plays an important role in physiology, the immune system development and digestion[1]. Microbial richness and the metabolic capacity of the microbiome is an indicator of health status. Most of the microbiota reside in the gut.This accounts for > 5 million genes and > 1000 species, which consists of a small number of phyla[1,2]. In addition, the large diversity and complexity of the microbiota can be divided into clusters of living organisms based on their ecosystem[1,3].

    In recent years, the area of gut microbiome research has significantly increased in both human and murine studies. These studies highlight that changes in the gut microbiome and gut dysbiosis play a critical role in susceptibility to numerous diseases[4]. Although, it is widely accepted that diabetes and its complications are multifactorial, recent studies emphasise the importance of perturbations in the gut microbiota as a factor in the pathogenesis of diabetes[5-9]and its complications[10,11]. The mechanisms leading to an altered gut microbiota and gut dysbiosis during diabetes is associated with altered glycaemic control, obesity and insulin resistance, increased inflammation, oxidative stress and vascular permeability[12]. Interestingly, more studies are now focusing on the gut microbiome as a potential source of biomarkers of diabetes and its complications[13].

    Sodium glucose co-transporter 2 inhibitors (SGLT2) are oral medications for the treatment of type 2 diabetes (T2D). This drug class blocks SGLT2 in the renal proximal tubules, thereby facilitating glucosuria and subsequently reduced plasma glucose levels and improved glycaemic parameters. The SGLT2 inhibitors canagliflozin,empagliflozin and dapagliflozin have been shown to decrease glycosylated haemoglobin levels, fasting glucose levels, body weight and blood pressure[14-17].Furthermore, SGLT2 inhibitors target two of the main problems of diabetes which are cardiovascular disease (CVD) and renal disease[15,17]. SGLT2 inhibitor related studies are currently underway in the field of type 1 diabetes (T1D)[18-20]. Given the limited understanding of SGLT2 inhibition and its beneficial effects on T1D, further preclinical studies are urgently needed. The objective of our investigation was to examine the effects of two independent SGLT2 inhibitors on gut health of a highly relevant mouse model of T1D and its complications.

    MATERIALS AND METHODS

    Animals

    Specific pathogen free 10-wk-old male C57BL6/J, Kimba, Akita and Akimba mice[21-25]were obtained from the Animal Resources Centre (ARC, Perth, Australia). All experimental and animal handing activities were performed at the Harry Perkins Institute for Medical Research animal holding facility (Perth, Western Australia)according to the guidelines of the Institutional Animal Care and Use Committee.Animal ethics approval (AE141/2019) was received from the Harry Perkins Institute for Medical Research Animal Ethics Committee.

    All mice were acclimatised for seven days. Mice were housed under a 12 h light/dark cycle, at 21 ± 2°C and were given a standard chow diet (Specialty Feeds,Glen Forrest, WA, Australia) with free access to food and drinking water containing the SGLT2 inhibitor (dapagliflozin or empagliflozin; Ark Pharma Scientific Limited,China; 25 mg/kg) or vehicle dimethylsulfoxide. Mice underwent treatment for a period of 8 wk. Drinking water containing the SGLT2 inhibitor or vehicle was freshly prepared and replaced on a weekly basis. The specific number of animals per treatment group is as follows: (1) C57BL6/J:n= 7 for vehicle;n= 5 for dapagliflozin;n= 4 for empagliflozin; (2) Kimba:n= 5-6 for vehicle;n= 6 for dapagliflozin;n= 7 for empagliflozin; (3) Akita:n= 7 for vehicle;n= 6 for dapagliflozin;n= 3 for empagliflozin; and (4) Akimba:n= 2-3 for vehicle;n= 3 for dapagliflozin;n= 3 for empagliflozin.

    Tissue and serum sample collection

    At the end of the experiment, mice were deeply anesthetised using isoflurane inhalation. Blood samples were collected using cardiac puncture and placed on ice immediately. Samples were centrifuged, serum was collected and stored at -80 °C for subsequent experiments. Mice were sacrificed, the cecum content and kidneys were collected, snap frozen and stored at -80°C for subsequent experiments. Pancreatic tissue was collected and fixed for 24 h in 10% buffered formalin for histology.

    Succinate and short chain fatty acid analysis

    The concentration of succinate and the short chain fatty acid (SCFA) butyric acid was measured in serum using gas chromatography-mass spectrometry as previously described[26,27]. Data were calculated in μmol/L units.

    Pancreatic tissue histology and digital imaging

    Upon fixation and processing in 70% ethanol overnight, pancreatic tissue was paraffin-embedded and sectioned at 5 μm thickness using a Leica semi-automated RM2245 microtome (Leica Biosystems, Sydney, Australia). Sections were stained with Gill’s hematoxylin (Sigma-Aldrich, Sydney, Australia) and alcoholic eosin (Sigma-Aldrich New South Wales, Australia) for the analysis of pancreatic histology. Tissue sections were visualised and imaged using the inverted Nikon Eclipse Ti microscopic system (Nikon, Tokyo, Japan) and a CoolSNAP HQ2 digital camera (Photometrics,Tucson, AZ, United States). Image analysis was conducted using NIS-Elements Advanced Research software (Nikon, Tokyo, Japan).

    Insulin enzyme-linked immunosorbent assay

    Serum insulin was determined using a rat/mouse Insulin enzyme-linked immunosorbent assay (ELISA) (Millipore, Australia; EZRMI-13K) according to the manufacturer instructions.

    ELISA for leptin

    Mouse serum was diluted 1 in 20 and leptin concentrations were determined by Mouse/rat leptin ELISA kit following manufacturer instructions (Quantikine ELISA;Mouse/rat leptin immunoassay; R&D Systems; Minneapolis, MN, United Sates;MOBOO).

    ELISA for norepinephrine

    Frozen mouse kidney tissue was homogenized on ice in phosphate buffered saline containing cOmplete?, ethylenediaminetetraacetic acid-free protease inhibitor cocktail (Merck, Victoria, Australia) at 10 μL per 1 mg of tissue. Debri free supernatant was analysed for norepinephrine content according to manufacturer instructions(Mouse Norepinephrine NA ELISA Kit; Cusabio, China; CSB-E07870m).

    Statistical analysis

    The number of mice required was determined with the assistance of Mrs Sally Burrows (Biostatistician, Royal Perth Hospital). Data was analysed using a two-tailed Student'sttest. Quantitative data is presented as mean ± SE. Data was deemed significant whenP< 0.05. Graphs were produced with GraphPad Prism 8 (GraphPad Software Inc., CA, United States).

    RESULTS

    Type 1 diabetes promotes hypoinsulinemia and pancreatitis in Akita and Akimba mice

    In our studies, we used 4 strains of mice. We used C57BL/6J mice as our wild-type strain. The Kimba (VEGF+/+) mouse model possesses transient overexpression of human vascular endothelial growth factor (hVEGF) in photoreceptors and hence,displays changes associated with diabetic retinopathy (DR) but lacks the hyperglycaemic background. The Akita (Ins2Akita) mouse is a T1D model that possesses a dominant mutation in the Mody4 locus in theinsulin 2gene. Male heterozygous mice possess hyperglycaemia and features of DR. The insulin protein is incorrectly folded in Akita mice and this results in β-cell toxicity. By crossing Akita and Kimba mice, the Akimba (Ins2AkitaVEGF+/-) mouse model is generated where the interplay between high blood glucose levels and VEGF-induced pathology can be investigated in diabetic complications such as DR. Insulin destruction and a concomitant reduction in insulin production is a hallmark of T1D. As predicted, the Akita allele in the Akita and Akimba mice conferred hypoinsulinemia compared to C57BL/6J and Kimba mice (Figure 1). In agreement with the higher insulin levels,islets were frequently observed in pancreata from C57BL/6J (Figure 2A) and Kimba mice (Figure 2B). However, islets were observed less frequently in Akita (Figure 2C)and Akimba mice (Figure 2D). In addition, apoptotic bodies were observed in the islets of Akita mice (Figure 2C). The acinar cells of Akita (Figure 2C) and Akimba(Figure 2D) mice appeared swollen which is indicative of acute injury or pancreatitis.These cells possessed high levels of eosin staining.

    SGLT2 inhibition influences the main products and intermediate metabolites of gut metabolism in Akita and Akimba mice

    Succinate is an intermediate metabolite of gut metabolism and has been shown to be a pathogenic factor in DR[28-30]. We hypothesised that SGLT2 inhibition may reduce the levels of this pathogenic factor in Akimba mice which develop DR. We did indeed demonstrate that dapagliflozin significantly reduced succinate levels (Figure 3A) and empagliflozin suppressed succinate levels (Figure 3B).

    The main products of gut metabolism are SCFAs. We examined levels of the beneficial SCFA butyric acid and observed that levels were significantly increased in Akita mice treated with dapagliflozin and mildly elevated with empagliflozin therapy(Figure 3C).

    Figure 1 lnsulin dysregulation prevails in mice with type 1 diabetes. Serum insulin levels are reduced in Akimba and Akita mice. n = 3-8 mice/group. All data represented as mean ± SE.

    SGLT2 inhibition with dapagliflozin and empagliflozin reduces activation of the sympathetic nervous system in type 1 diabetic mice

    Our previously published findings have uncovered that one of the mechanisms by which SGLT2 inhibition imparts metabolic benefits is by reduced activation of the sympathetic nervous system (SNS)[31,32]. Levels of the major neurotransmitter of the sympathetic system, norepinephrine, were measured in the kidneys of our T1D mice.Consistent with our previously published work, SNS activation as evidenced by norepinephrine content, was significantly reduced with both dapagliflozin and empagliflozin therapy in Akita mice (Figure 4A). Norepinephrine content was also significantly reduced after empagliflozin treatment of Akimba mice (Figure 4B).

    SGLT2 inhibition promotes hypoleptinemia in non-diabetic C57BL/6J and Kimba mice

    As leptin is a metabolically beneficial adipokine, we examined the impact of SGLT2 inhibition on serum leptin levels. Previous studies have shown that SGLT2 inhibition may reduce serum leptin levels[33]. Interestingly, C57BL/6J mice also displayed significantly reduced serum leptin after empagliflozin treatment and reduced leptin after dapagliflozin therapy (Figure 5A). Kimba mice also possessed significantly reduced serum leptin after dapagliflozin treatment (Figure 5B) and suppressed leptin levels after empagliflozin therapy (Figure 5C). The inhibition of SGLT2 failed to influence leptin levels in Akita and Akimba mice.

    DISCUSSION

    Inhibition of SGLT2 in the kidney is a therapeutic approach to improve glucose homeostasis in diabetic patients. The clinical trial known as the EMPA-REG OUTCOME study has shown that the SGLT2 inhibitor, empagliflozin, increased glycaemic control and significantly reduced the progression of renal disease[15]. In addition, empagliflozin promoted a substantial 38% relative risk reduction in death from cardiovascular causes[17]. To date, the number of studies assessing the impact of SGLT2 inhibition on T1D and its complications including DR in preclinical and human cohorts is limited[34]. In our current study, we aimed to assess whether SCFAs and intermediate metabolites of gut metabolism may be influenced by SGLT2 inhibition in a mouse model of T1D and its complications. Furthermore, we attempted to uncover mechanisms by which SGLT2 inhibitors may impact the progression of disease in our mouse models.

    Figure 2 Acute pancreatic injury in Akita and Akimba mice. A: Representative pancreatic images of C57BL/6J mice; B: Representative pancreatic images of Kimba mice; C: Representative pancreatic images of Akita mice; D: Representative pancreatic images of Akimba mice. Black arrows indicate islets; Yellow arrows and dotted outline indicates swollen acinar cells and orange arrows indicate apoptotic bodies in islets; Scale bar = 100 μm.

    There is great interest in the role the gut microbiome plays in the metabolic syndrome and T2D[27]. Many chronic diseases including diabetes demonstrate altered bacterial composition[35], which may influence glucose metabolism and development of diabetic complications such as retinopathy[10,36]. SCFAs are a product of the gut microbiota. These products include acetate and butyric acid and contribute to immune and inflammatory responses, as well as control of lipid and glucose homeostasis. Studies from our group and others indicate that numerous drug classes alter the gut microbiome[26,37]. Additionally, a reduction in SCFA production is characteristic of metabolic dysfunction. In our previous studies, we analysed caecal SCFAs from dapagliflozin and vehicle treated hypertensive mice and demonstrated that SGLT2 inhibition results in an increase in the levels of acetate and butyric acid in hypertensive Schlager mice[31]. In a chronic kidney disease mouse model, Mishimaet al[38]also demonstrated that the SGLT2 inhibitor canangliflozin significantly increased the cecal SCFA acetate, propionate and butyrate. Our previous data highlighted a potentially advantageous impact of SGLT2 inhibition on the microbiome.Additionally, as our results were produced only after 2 wk of therapy, future studies should allow a more thorough examination of the status of the microbiome to determine whether a longer duration of therapy confers further benefits on the microbiome. In our current study, our T1D and DR mice were treated with SGLT2 inhibitors for 8 wk. We found that SGLT2 inhibition promoted beneficial changes in the metabolite succinate (Figure 3A and 3B) and the SCFA butyrate (Figure 3C).Recent findings have uncovered that dapagliflozin treatment of T2D Db/Db mice promotes increases in the beneficial bacterial speciesAkkermansia muciniphilaand reduces the Firmicutes : Bacteroidetes (F : B) ratio which is associated with a lean phenotype[39]. Our future studies will plan to assess the levels ofAkkermansia muciniphilaand the F:B ratio in our T1D and DR mouse models before and after SGLT2 inhibition.

    Succinate is an intermediate metabolite of gut metabolism that has been correlated with a worsened DR phenotype. It signals through the succinate specific receptor known as GPR91 and it has been shown that succinate may promote the angiogenic factor VEGF in retinal ganglion cells and this may promote the pathogenesis of DR[40].Matsumoto and colleagues[29]demonstrated that vitreous levels of succinate were elevated in human subjects with active proliferative DR. In our novel Akimba mouse model which reproducibly presents with DR[21], SGLT2 inhibition with both dapagliflozin (Figure 3A) and empagliflozin (Figure 3B) lowered serum succinate levels. It would be interesting to measure vitreous succinate and VEGF levels in our mice in future studies. In addition, it would be mechanistically insightful to assess GPR91 expression in the eyes of all mouse strains before and after SGLT2 inhibition as GPR91 and SGLT2 (unpublished data) are both expressed in retinal ganglion cells.

    Figure 3 The diabetic retinopathy pathogenic metabolite, succinate, is downregulated with sodium glucose co-transporter 2 inhibition in Akimba mice. A:Serum succinate levels in Akimba mice treated with dapapagliflozin; B: Serum succinate levels in Akimba mice treated with empagliflozin; C: Serum levels of the beneficial short chain fatty acid, butyric acid, are elevated in diabetic Akita mice with sodium glucose co-transporter 2 inhibition. n = 3-7 mice/group for all groups except for control Akimba (Figure 3B) where n = 2; aP < 0.03; Data represented as mean ± SE.

    Hyperactivation of the SNS is characteristic of many metabolic diseases such as obesity and T2D[41,42]. Our team previously studied the impact of SGLT2 inhibition on SNS activation[31,32]. We were the first to demonstrate in human proximal tubular cells and mouse models of obesity and neurogenic hypertension that: (1) Activation of the SNSviaincreased secretion of norepinephrine up-regulates SGLT2 protein levels and promotes SGLT2 translocation to the cell membrane; (2) Treatment of mice with a high fat diet and the SGLT2 inhibitor dapagliflozin promotes glucosuria, decreased weight gain and facilitated better glucose control; and (3) SGLT2 inhibition reduced SNS innervation in the kidney and the heart. Our data suggests that SGLT2 expression is upregulated by the major neurotransmitter of the SNS and that SGLT2 inhibition promotes sympatho-inhibition. Excitingly, we also now show that SGLT2 inhibition is once again sympathoinhibitory in our T1D and DR mouse models (Figure 4).

    It appears that dapagliflozin was more bioactive than empagliflozin when it comes to the effects on succinate levels (Figure 3A and B). It has been shown in mouse models that dapagliflozin is superior to empagliflozin as it has an increased half-life,longer duration of action, higher distribution and long retention period in the kidney[43]. Therefore, this could be the likely reasoning for the dapagliflozin being more bioactive in relation to succinate levels.

    Interestingly, empagliflozin was able to reduce norepinephrine levels to a greater degree compared to dapagliflozin in diabetic Akita mice (Figure 4A). This is interesting in the context of the EMPA-REG study[17]. In this study, factors driven by an increased SNS such as mortality from CVD were reduced to a markedly greater degree compared to the dapagliflozin DECLARE-TIMI 58 clinical trial[44]. Hence,regulation of the SNS may be one factor where empagliflozin may be specifically more effective than dapagliflozin.

    The activation status of the SNS in T1D is currently quite controversial. It has been shown that there was a reduction in islet selective sympathetic nerves in human T1D pancreata isolated during autopsy compared to non-diabetic controls[45]. On the other hand, it has been demonstrated that children with T1D have a higher incidence of postural orthostatic tachycardia syndrome which is associated with sympathetic overactivity[46,47]. Our study conclusively shows that SGLT2 inhibition reduced levels of norepinephrine in our T1D and DR mouse models.

    We assessed serum levels of the adipokine leptin in our mice treated with SGLT2 inhibitors or not. It was clear that leptin levels were reduced with SGLT2 inhibition with dapagliflozin and empagliflozin, particularly in C57BL/6J and Kimba mice(Figure 5A-C). This appears to be a common phenomenon that has been observed in response to a range of SGLT2 inhibitors which include ipragliflozin and canagliflozin[33,48]. We can conclude that the reductions in leptin were not due to reductions in body weight after SGLT2 inhibition in C57BL/6J and Kimba mice (data not shown). Reductions in leptin may also be metabolically beneficial as leptin is known to activate the SNS and may impair pancreatic β-cell function[49,50]. In future studies, we will also assess the effect of SGLT2 inhibition on other adipokines such as TNF-α and IL-6.

    We are the first study to compare and contrast the effects of SGLT2 inhibition on the main products and intermediate metabolites of gut metabolism in Akita and Akimba mice. Excitingly, our results highlighted that after SGLT2 therapy, the pathogenic biomarker succinate is reduced in our Akimba DR mouse model. Our next task is to ascertain whether our finding may be reproduced in a human DR cohort treated with the SGLT2 inhibitor empagliflozin.

    Figure 4 lnhibition of sodium glucose cotransporter 2 promotes sympathoinhibition in the kidney of diabetic Akita and Akimba mice. A: Renalnorepinephrine levels in Akita; B: Renal norepinephrine levels in Akimba mice. n = 3-7 mice/group; aP < 0.05; bP < 0.005; All data represented as mean ± SE.

    Figure 5 lnhibition of sodium glucose cotransporter 2 reduces serum leptin levels. A: Serum leptin levels in C57BL/6J. B: Serum leptin levels in Kimba mice treated with dapagliflozin. C: Serum leptin levels in Kimba mice treated with empagliflozin. n = 4-7 mice/group; aP < 0.05; All data represented as mean ± SE.

    ARTICLE HIGHLIGHTS

    In comparison to C57BL/6J and Kimba mice, both Akita and Akimba mice showed reduced levels of insulin production due to the presence of the Akita allele. In line with this, Akita mice also showed the presence of apoptotic bodies within the pancreatic islets and the acinar cells of both the Akita and Akimba mice displayed swelling which is suggestive of acute injury or pancreatitis. In Akimba mice, SGLT2 inhibition with dapagliflozin for 8 wk significantly reduced succinate levels when compared to vehicle treated mice. Furthermore, succinate levels in Akimba mice treated with the SGLT2 inhibitor empagliflozin showed a similar trend. In diabetic Akita mice, the beneficial SCFA butyric acid was significantly increased after dapagliflozin treatment when compared to vehicle. There was a significant reduction in the kidney norepinephrine content in both dapagliflozin and empagliflozin treated Akita mice.Furthermore, the diabetic Akimba mice also showed a significant reduction in kidney norepinephrine content when treated with empagliflozin. Lastly, both non-diabetic C57BL/6J and Kimba mice showed significantly reduced serum leptin levels after dapagliflozin therapy.

    Research conclusions

    Our novel study compares and contrasts the effects of SGLT2 inhibition on the main products and intermediate metabolites of gut metabolism particularly in Akita and Akimba mice. We conducted studies using two independent SGLT2 inhibitors and showed that both inhibitors reduced the pathogenic biomarker succinate in our novel T1D Akimba mouse model of retinopathy. However, in relation to succinate levels in Akimba mice, dapagliflozin was more bioactive than empagliflozin, potentially due to factors such as increased half-life, longer duration of action, higher distribution and long retention period in the kidney. Furthermore, we demonstrate for the first time that SGLT2 inhibition is sympathoinhibitory in a T1D mouse model.

    Research perspectives

    In line with our findings, it would be mechanistically insightful in the future to assess the expression of the succinate specific receptor GPR91 in ocular tissue before and after SGLT2 inhibition as SGLT2 is expressed in the eye. Furthermore, it is important to determine whether our findings can be reproduced in patients with T1D and its complications who are treated with SGLT2 inhibitors.

    ACKNOWLEDGEMENTS

    The authors acknowledge Wei Ern Ong (School of Biomedical Sciences, University of Western Australia, Perth, Western Australia).

    亚洲精品美女久久久久99蜜臀| 在线观看66精品国产| 免费人成视频x8x8入口观看| 欧美国产精品va在线观看不卡| 久久久久九九精品影院| 纯流量卡能插随身wifi吗| 久久久久久亚洲精品国产蜜桃av| 亚洲片人在线观看| 一级毛片女人18水好多| 91av网站免费观看| 欧美一区二区精品小视频在线| 一级毛片女人18水好多| 又大又爽又粗| 国产一区在线观看成人免费| 一边摸一边抽搐一进一出视频| 一卡2卡三卡四卡精品乱码亚洲| a级毛片在线看网站| 国产xxxxx性猛交| 黑人巨大精品欧美一区二区mp4| 美女扒开内裤让男人捅视频| 电影成人av| 国产高清激情床上av| 精品福利观看| 成人国语在线视频| 免费高清视频大片| 亚洲专区中文字幕在线| 亚洲中文日韩欧美视频| 97超级碰碰碰精品色视频在线观看| 久久久久久人人人人人| 在线国产一区二区在线| 美女高潮喷水抽搐中文字幕| 欧美日本视频| 18禁黄网站禁片午夜丰满| 久久香蕉国产精品| 不卡一级毛片| 91九色精品人成在线观看| 黄色视频不卡| 黄网站色视频无遮挡免费观看| 欧美日韩亚洲国产一区二区在线观看| 精品国产美女av久久久久小说| 亚洲 欧美一区二区三区| 久久精品国产亚洲av高清一级| 久久久国产精品麻豆| 自线自在国产av| 国产亚洲精品一区二区www| 夜夜夜夜夜久久久久| 香蕉丝袜av| 国产男靠女视频免费网站| 日日摸夜夜添夜夜添小说| 国产精品永久免费网站| 欧美色欧美亚洲另类二区 | 侵犯人妻中文字幕一二三四区| 欧美中文日本在线观看视频| www.999成人在线观看| 亚洲国产精品合色在线| 午夜久久久久精精品| 日本撒尿小便嘘嘘汇集6| 国产精品自产拍在线观看55亚洲| 亚洲成人国产一区在线观看| av天堂在线播放| 久久伊人香网站| 制服丝袜大香蕉在线| 亚洲精品在线美女| 午夜日韩欧美国产| 级片在线观看| 一个人观看的视频www高清免费观看 | 波多野结衣av一区二区av| 亚洲专区国产一区二区| 精品久久久久久久久久免费视频| 国内精品久久久久久久电影| 亚洲一码二码三码区别大吗| 国产男靠女视频免费网站| 激情在线观看视频在线高清| 亚洲人成电影免费在线| 大型av网站在线播放| 夜夜躁狠狠躁天天躁| 在线天堂中文资源库| 精品国产超薄肉色丝袜足j| 亚洲精品av麻豆狂野| aaaaa片日本免费| 欧美激情高清一区二区三区| 国产xxxxx性猛交| 亚洲熟女毛片儿| 丰满人妻熟妇乱又伦精品不卡| 久久香蕉激情| av福利片在线| 久久香蕉激情| 国产精品秋霞免费鲁丝片| 在线观看日韩欧美| 久久性视频一级片| 真人一进一出gif抽搐免费| 精品欧美国产一区二区三| 18禁黄网站禁片午夜丰满| 久久九九热精品免费| 亚洲五月天丁香| 桃红色精品国产亚洲av| 国产亚洲欧美在线一区二区| 精品人妻1区二区| 国产亚洲av高清不卡| 中文字幕高清在线视频| 伦理电影免费视频| 校园春色视频在线观看| 国产成年人精品一区二区| 老汉色∧v一级毛片| 国产成人免费无遮挡视频| 久久草成人影院| 99香蕉大伊视频| 日日干狠狠操夜夜爽| 免费高清视频大片| 国产又色又爽无遮挡免费看| 女人被躁到高潮嗷嗷叫费观| 在线十欧美十亚洲十日本专区| 级片在线观看| 一区在线观看完整版| 国产精品爽爽va在线观看网站 | 搡老妇女老女人老熟妇| 亚洲精品一区av在线观看| 欧美 亚洲 国产 日韩一| 可以在线观看毛片的网站| 国产成人av激情在线播放| 国产av又大| 亚洲人成77777在线视频| 纯流量卡能插随身wifi吗| 久久午夜综合久久蜜桃| 在线观看免费午夜福利视频| 欧美日本中文国产一区发布| 久久久久久人人人人人| av免费在线观看网站| 国产精品永久免费网站| 美女扒开内裤让男人捅视频| 波多野结衣一区麻豆| 老汉色av国产亚洲站长工具| 在线观看免费视频网站a站| 最近最新中文字幕大全电影3 | 国产一级毛片七仙女欲春2 | 欧美绝顶高潮抽搐喷水| 熟女少妇亚洲综合色aaa.| 叶爱在线成人免费视频播放| 精品一区二区三区视频在线观看免费| 1024视频免费在线观看| av视频免费观看在线观看| 国产日韩一区二区三区精品不卡| 一级,二级,三级黄色视频| 天堂√8在线中文| 波多野结衣av一区二区av| 女性被躁到高潮视频| 美女扒开内裤让男人捅视频| 午夜老司机福利片| 久久性视频一级片| 午夜免费激情av| 韩国精品一区二区三区| 波多野结衣巨乳人妻| 精品久久久久久成人av| 美女 人体艺术 gogo| 制服诱惑二区| 18禁裸乳无遮挡免费网站照片 | 国产人伦9x9x在线观看| x7x7x7水蜜桃| 久久久国产成人精品二区| 波多野结衣巨乳人妻| 亚洲九九香蕉| 日韩中文字幕欧美一区二区| 亚洲精品中文字幕在线视频| av电影中文网址| 亚洲最大成人中文| 麻豆久久精品国产亚洲av| 欧美日韩黄片免| 国产一区在线观看成人免费| 变态另类丝袜制服| 国产成人影院久久av| x7x7x7水蜜桃| 免费看十八禁软件| 国产一区二区在线av高清观看| 国产精品香港三级国产av潘金莲| 色婷婷久久久亚洲欧美| 一级毛片女人18水好多| 亚洲国产精品久久男人天堂| 久久久精品国产亚洲av高清涩受| 满18在线观看网站| 一区二区三区高清视频在线| 又黄又爽又免费观看的视频| 91精品国产国语对白视频| 黄色丝袜av网址大全| 免费观看精品视频网站| 亚洲国产欧美网| 波多野结衣巨乳人妻| 色播在线永久视频| 99久久99久久久精品蜜桃| 99精品在免费线老司机午夜| 9色porny在线观看| 国产激情欧美一区二区| 50天的宝宝边吃奶边哭怎么回事| 色尼玛亚洲综合影院| 亚洲天堂国产精品一区在线| 欧美中文综合在线视频| 亚洲avbb在线观看| 亚洲国产精品久久男人天堂| 级片在线观看| 丰满人妻熟妇乱又伦精品不卡| 色尼玛亚洲综合影院| 欧美国产日韩亚洲一区| 欧美日韩亚洲国产一区二区在线观看| 桃色一区二区三区在线观看| 亚洲一区高清亚洲精品| 老司机午夜十八禁免费视频| 很黄的视频免费| 久久久国产成人精品二区| 亚洲精品国产一区二区精华液| 精品日产1卡2卡| 国产精品久久电影中文字幕| 国产激情久久老熟女| 老司机午夜福利在线观看视频| 精品无人区乱码1区二区| 国产极品粉嫩免费观看在线| 欧美成狂野欧美在线观看| 丝袜人妻中文字幕| 男女下面插进去视频免费观看| 高清黄色对白视频在线免费看| av视频在线观看入口| 亚洲五月天丁香| 91国产中文字幕| 好男人在线观看高清免费视频 | 69av精品久久久久久| 亚洲国产欧美一区二区综合| 亚洲aⅴ乱码一区二区在线播放 | 精品日产1卡2卡| 国产激情久久老熟女| 欧美久久黑人一区二区| 黄片播放在线免费| 成人精品一区二区免费| 欧美绝顶高潮抽搐喷水| 曰老女人黄片| 亚洲成人免费电影在线观看| 12—13女人毛片做爰片一| 神马国产精品三级电影在线观看 | 97超级碰碰碰精品色视频在线观看| e午夜精品久久久久久久| 成年女人毛片免费观看观看9| 精品高清国产在线一区| 久久久久久久久中文| 亚洲熟女毛片儿| 两个人免费观看高清视频| 搡老妇女老女人老熟妇| 三级毛片av免费| 日韩大尺度精品在线看网址 | 天天躁狠狠躁夜夜躁狠狠躁| 日韩精品免费视频一区二区三区| 国产91精品成人一区二区三区| 国产一区二区三区综合在线观看| 成在线人永久免费视频| 大型av网站在线播放| 男人的好看免费观看在线视频 | 欧美不卡视频在线免费观看 | 黄频高清免费视频| 国产精品 国内视频| 欧美日韩福利视频一区二区| 亚洲一区高清亚洲精品| 久久伊人香网站| 亚洲在线自拍视频| 热re99久久国产66热| 不卡av一区二区三区| 精品国产亚洲在线| 伊人久久大香线蕉亚洲五| 免费少妇av软件| 18禁国产床啪视频网站| 久久久久久大精品| cao死你这个sao货| 91国产中文字幕| 99久久精品国产亚洲精品| 欧美中文综合在线视频| 国产在线精品亚洲第一网站| 淫秽高清视频在线观看| 欧美在线黄色| 他把我摸到了高潮在线观看| 亚洲精品美女久久久久99蜜臀| 99国产综合亚洲精品| 日韩av在线大香蕉| 久9热在线精品视频| 两人在一起打扑克的视频| 亚洲精品国产色婷婷电影| 热re99久久国产66热| 侵犯人妻中文字幕一二三四区| 999精品在线视频| 91成人精品电影| 亚洲久久久国产精品| 国产伦人伦偷精品视频| 给我免费播放毛片高清在线观看| 午夜久久久在线观看| 日日爽夜夜爽网站| 超碰成人久久| 亚洲一区二区三区色噜噜| 午夜影院日韩av| 免费在线观看完整版高清| 丝袜美腿诱惑在线| 大型av网站在线播放| 中文字幕人妻熟女乱码| 亚洲国产精品久久男人天堂| 麻豆一二三区av精品| 一级毛片高清免费大全| 女性被躁到高潮视频| 纯流量卡能插随身wifi吗| 亚洲精品久久成人aⅴ小说| 国产精品爽爽va在线观看网站 | 国产xxxxx性猛交| 国产一区二区在线av高清观看| 一级,二级,三级黄色视频| 变态另类丝袜制服| 亚洲国产欧美一区二区综合| 少妇被粗大的猛进出69影院| 一个人免费在线观看的高清视频| 这个男人来自地球电影免费观看| 亚洲成a人片在线一区二区| 天堂影院成人在线观看| 色综合婷婷激情| 欧美日韩精品网址| 久久久久亚洲av毛片大全| 亚洲成人国产一区在线观看| av视频在线观看入口| bbb黄色大片| 国产成人啪精品午夜网站| 777久久人妻少妇嫩草av网站| 两个人免费观看高清视频| tocl精华| av电影中文网址| 波多野结衣av一区二区av| av视频在线观看入口| 性少妇av在线| 日日爽夜夜爽网站| 亚洲成人免费电影在线观看| 国产精品香港三级国产av潘金莲| 国产精品精品国产色婷婷| tocl精华| 久久伊人香网站| 欧美 亚洲 国产 日韩一| 精品电影一区二区在线| 国产精品久久久久久精品电影 | 一a级毛片在线观看| 久久久久久久午夜电影| 自拍欧美九色日韩亚洲蝌蚪91| 国产亚洲精品一区二区www| 精品国产亚洲在线| 黄色女人牲交| 满18在线观看网站| 十分钟在线观看高清视频www| 窝窝影院91人妻| 精品一区二区三区视频在线观看免费| 国产不卡一卡二| 日本免费a在线| 久久这里只有精品19| 怎么达到女性高潮| 精品国产亚洲在线| 丰满的人妻完整版| 母亲3免费完整高清在线观看| 国产精品亚洲av一区麻豆| 久久国产亚洲av麻豆专区| 亚洲专区国产一区二区| 国产成人系列免费观看| or卡值多少钱| 人人妻人人澡人人看| 国产av一区在线观看免费| 757午夜福利合集在线观看| 免费看a级黄色片| 天天添夜夜摸| 日本一区二区免费在线视频| 日日夜夜操网爽| 妹子高潮喷水视频| 欧美午夜高清在线| 精品国产乱码久久久久久男人| 日韩有码中文字幕| 色尼玛亚洲综合影院| 国产乱人伦免费视频| 国产又爽黄色视频| 少妇裸体淫交视频免费看高清 | 香蕉国产在线看| 久久人妻av系列| 在线观看免费视频日本深夜| 一区福利在线观看| 精品国产乱码久久久久久男人| 免费在线观看亚洲国产| 男人舔女人下体高潮全视频| 久久香蕉国产精品| 亚洲欧美激情综合另类| 亚洲色图综合在线观看| 国产亚洲av高清不卡| 亚洲成人国产一区在线观看| 国产成人影院久久av| 91大片在线观看| 动漫黄色视频在线观看| 黄片小视频在线播放| 18禁裸乳无遮挡免费网站照片 | 99国产精品一区二区蜜桃av| www国产在线视频色| 日韩国内少妇激情av| 天天躁狠狠躁夜夜躁狠狠躁| 男女之事视频高清在线观看| 狂野欧美激情性xxxx| 美国免费a级毛片| 日本五十路高清| 国产精品九九99| 国产亚洲av嫩草精品影院| 亚洲欧美日韩高清在线视频| 国产精品亚洲一级av第二区| 久久亚洲精品不卡| 国产精品久久视频播放| 色综合欧美亚洲国产小说| 精品久久久精品久久久| 国产成人系列免费观看| 满18在线观看网站| 最新在线观看一区二区三区| 女人爽到高潮嗷嗷叫在线视频| 色在线成人网| 俄罗斯特黄特色一大片| 国产精品秋霞免费鲁丝片| 久久久久久久久免费视频了| 国产精品亚洲一级av第二区| 黄片小视频在线播放| 亚洲五月婷婷丁香| 真人做人爱边吃奶动态| 免费少妇av软件| 黄片大片在线免费观看| 人妻丰满熟妇av一区二区三区| 一进一出抽搐gif免费好疼| 男女之事视频高清在线观看| 校园春色视频在线观看| 国产精品1区2区在线观看.| 午夜免费观看网址| 99在线视频只有这里精品首页| 91精品国产国语对白视频| 欧美日本亚洲视频在线播放| 69av精品久久久久久| 制服人妻中文乱码| 午夜精品国产一区二区电影| 大陆偷拍与自拍| 欧美成人免费av一区二区三区| 手机成人av网站| 欧美老熟妇乱子伦牲交| 在线观看免费午夜福利视频| 精品卡一卡二卡四卡免费| 97人妻精品一区二区三区麻豆 | 乱人伦中国视频| av网站免费在线观看视频| 黑人欧美特级aaaaaa片| 精品电影一区二区在线| 少妇裸体淫交视频免费看高清 | 99久久99久久久精品蜜桃| 巨乳人妻的诱惑在线观看| 亚洲aⅴ乱码一区二区在线播放 | 在线视频色国产色| 日日摸夜夜添夜夜添小说| 日本黄色视频三级网站网址| 深夜精品福利| av片东京热男人的天堂| 国产精品一区二区精品视频观看| 岛国视频午夜一区免费看| 国产精华一区二区三区| 在线国产一区二区在线| 中文字幕人成人乱码亚洲影| 18美女黄网站色大片免费观看| 最新美女视频免费是黄的| 一进一出抽搐gif免费好疼| 1024香蕉在线观看| 亚洲人成伊人成综合网2020| 久久久久国产精品人妻aⅴ院| 久久狼人影院| 1024视频免费在线观看| 久久热在线av| 99久久久亚洲精品蜜臀av| 亚洲av日韩精品久久久久久密| 禁无遮挡网站| 国产精品野战在线观看| 午夜影院日韩av| 久久精品成人免费网站| 50天的宝宝边吃奶边哭怎么回事| 亚洲伊人色综图| 欧美日本中文国产一区发布| 很黄的视频免费| 国产精品亚洲美女久久久| 男人舔女人下体高潮全视频| 麻豆国产av国片精品| 欧美乱码精品一区二区三区| 国产又色又爽无遮挡免费看| 69精品国产乱码久久久| 不卡av一区二区三区| 乱人伦中国视频| 午夜福利在线观看吧| 亚洲国产欧美网| 黄色女人牲交| 精品一区二区三区四区五区乱码| 黄色成人免费大全| 久久久久久久午夜电影| 欧美另类亚洲清纯唯美| 午夜视频精品福利| 久久久久久久久久久久大奶| 国产成人av激情在线播放| 亚洲自拍偷在线| 香蕉国产在线看| 无限看片的www在线观看| 99热只有精品国产| 99久久精品国产亚洲精品| 18美女黄网站色大片免费观看| 男男h啪啪无遮挡| 国产精品久久久人人做人人爽| 可以在线观看的亚洲视频| 免费在线观看日本一区| 亚洲国产精品合色在线| 亚洲精品美女久久久久99蜜臀| 可以免费在线观看a视频的电影网站| 激情视频va一区二区三区| 国产三级黄色录像| 国产片内射在线| 国产在线观看jvid| 国产单亲对白刺激| 大型黄色视频在线免费观看| 麻豆av在线久日| 亚洲一区二区三区色噜噜| av超薄肉色丝袜交足视频| 亚洲自偷自拍图片 自拍| 欧美激情高清一区二区三区| 国产亚洲精品av在线| 琪琪午夜伦伦电影理论片6080| 熟女少妇亚洲综合色aaa.| 天天躁夜夜躁狠狠躁躁| 亚洲五月天丁香| 美国免费a级毛片| 天堂√8在线中文| 午夜成年电影在线免费观看| 涩涩av久久男人的天堂| 黄片大片在线免费观看| 欧美国产精品va在线观看不卡| 国产精品九九99| 中文字幕最新亚洲高清| 午夜免费成人在线视频| 久9热在线精品视频| 97人妻天天添夜夜摸| 国产精品,欧美在线| 久久九九热精品免费| 波多野结衣巨乳人妻| 美女午夜性视频免费| 在线观看日韩欧美| 黄色成人免费大全| 日日干狠狠操夜夜爽| 脱女人内裤的视频| 一区二区日韩欧美中文字幕| 麻豆国产av国片精品| 亚洲五月色婷婷综合| 99国产精品一区二区蜜桃av| 一级毛片精品| 久久婷婷人人爽人人干人人爱 | 女同久久另类99精品国产91| 成在线人永久免费视频| 高清在线国产一区| av在线播放免费不卡| 精品国产一区二区久久| 成人特级黄色片久久久久久久| 国产伦人伦偷精品视频| 欧美最黄视频在线播放免费| 亚洲第一青青草原| 老司机福利观看| 亚洲伊人色综图| 国产av又大| 在线观看66精品国产| 日韩av在线大香蕉| 无限看片的www在线观看| 国产午夜精品久久久久久| 久久久久精品国产欧美久久久| 欧美av亚洲av综合av国产av| 国产极品粉嫩免费观看在线| 亚洲欧美激情在线| 免费看十八禁软件| 色婷婷久久久亚洲欧美| 亚洲av电影在线进入| 一级毛片女人18水好多| 日本 av在线| 国产亚洲欧美精品永久| 国产野战对白在线观看| 在线观看免费午夜福利视频| 亚洲欧美激情在线| 夜夜爽天天搞| 欧美国产精品va在线观看不卡| 国产精品av久久久久免费| 国产精品电影一区二区三区| 宅男免费午夜| 成熟少妇高潮喷水视频| 午夜福利18| 国产97色在线日韩免费| 好男人电影高清在线观看| 波多野结衣一区麻豆| 国产午夜福利久久久久久| 亚洲精华国产精华精| 一边摸一边抽搐一进一出视频| 51午夜福利影视在线观看| 韩国av一区二区三区四区| 久久久久久久午夜电影| 9色porny在线观看| 国产精品,欧美在线| 久久影院123| 亚洲精品久久成人aⅴ小说| 可以免费在线观看a视频的电影网站| 好男人电影高清在线观看| 亚洲人成电影免费在线| 每晚都被弄得嗷嗷叫到高潮| 精品欧美国产一区二区三| 每晚都被弄得嗷嗷叫到高潮| 两性夫妻黄色片| 欧美成人午夜精品| 又大又爽又粗| 亚洲中文字幕日韩| 高清毛片免费观看视频网站| 亚洲国产欧美网| av视频免费观看在线观看| 久久久久国产一级毛片高清牌| 一区二区三区激情视频| 99国产综合亚洲精品| 成人永久免费在线观看视频| 国产av精品麻豆|