• <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 ?

    Study on the regulatory effect of herbal cakepartitioned moxibustion on colonic CD206, AMPK and TSC2 in rats with Crohn disease

    2021-10-27 08:38:10DongXiaoqing董小慶LiXiaoying李曉瑩WangXuejun王雪君GuoXiaocong郭瀟聰LongJunyi龍俊燚LuYunqiong盧云瓊LiuLi劉力CaoyaoJiani曹姚佳妮ZhangDan張丹LuYuan陸嫄WuHuangan吳煥淦XieChen謝晨MaXiaopeng馬曉芃
    關(guān)鍵詞:張丹國家自然科學(xué)基金

    Dong Xiao-qing (董小慶), Li Xiao-ying (李曉瑩), Wang Xue-jun (王雪君), Guo Xiao-cong (郭瀟聰), Long Jun-yi(龍俊燚), Lu Yun-qiong (盧云瓊), Liu Li (劉力), Caoyao Jia-ni (曹姚佳妮), Zhang Dan (張丹), Lu Yuan (陸嫄),Wu Huan-gan (吳煥淦),, Xie Chen (謝晨), Ma Xiao-peng (馬曉芃),

    1 Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

    2 Xi’an Traditional Chinese Medicine Hospital of Encephalopathy, Shaanxi Province, Xi’an 710032, China

    3 Shanghai Research Institute of Acupuncture and Meridian, Shanghai 200030, China

    Abstract

    Keywords: Moxibustion Therapy; Indirect Moxibustion; Medicinal Cake-partitioned Moxibustion; Point, Tianshu (ST 25); Point,Qihai (CV 6); Crohn Disease; AMP-activated Protein Kinases; Tuberous Sclerosis Complex 2 Protein

    Crohn disease (CD) is a chronic inflammatory disease of the gastrointestinal tract with protracted, recurrent,and gradually progressive symptoms[1]. The lesion involves entire digestive tract, mainly the ileum and the end of colon. The typical pathological features are segmental, asymmetry, and transmural infiltrating inflammation[2]. The clinical manifestations are right lower abdomen pain, chronic diarrhea and weight loss,as well as disease-associated symptoms such as fatigue and anorexia. Patients with colon involvement often have rectal bleeding or bloody diarrhea, and half of the patients have complications such as strictures, fistulas or abscesses[3]. Epidemiological investigations have shown that CD usually occurs in people over 20 years old, and the peak happens in 50-60 years old[4]. The incidence rate is relatively high in developed countries, e.g. it is 322 per 100 000 people in European countries. In recent years, the incidence rate of CD in Asian countries has also increased, reaching 0.54 per 100 000 people[5]. The onset of CD is related to many factors, currently believed to be mainly caused by innate and adaptive immune response disorders due to genetic susceptibility and environmental factors[1]. As a key cellular component of innate immunity[6], macrophages play important roles in the pathogenesis of CD.

    Macrophage polarization refers to the phenotype and functional differentiation of mature macrophages induced by various factors[7]. According to different stimuli, surface molecular markers, and secreted cytokines and chemokines, macrophages can be divided into two polarized phenotypes: pro-inflammatory M1 type, the classically activated macrophages; antiinflammatory M2 type, the alternately activated macrophages[8]. It has confirmed that dextran sulfate sodium (DSS) induces increased M1 macrophages and decreased M2 macrophages in colitis mice[9-10].Promoting expression of colonic M2 macrophages in T cell transfer mouse model reduces intestinal inflammation[11]. AMP-activated protein kinase (AMPK)is closely related to macrophage activation. Activated AMPK directly phosphorylates tuberous sclerosis complex (TSC) 2, and competitively inhibits mammalian target of rapamycin complexes 1 (mTORC1) by promoting the formation of TSC1/2, thereby inhibiting nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome activation, promoting the polarization of macrophages to M2 phenotype to reduce inflammation[12-13]. In this study, a CD rat model was established by 2,4,6-trinitrobenzene sulfonic acid(TNBS) to observe the effect of herbal cake-partitioned moxibustion on the surface marker CD206 of colonic M2 macrophages and the upstream regulatory proteins of AMPK and TSC2; to explore the anti-inflammatory mechanism of herbal cake-partitioned moxibustion in CD treatment from the perspective of regulating colonic M2 macrophage expression.

    1 Material and Methods

    1.1 Experimental animal

    Twenty-six specific pathogen free healthy male Sprague-Dawley rats, weighing (150±20) g, were provided by Shanghai Slack Laboratory Animal Co., Ltd.[SCXK (Hu) 2013-0016]. Before experiment, the rats were adaptively reared at room temperature (20±2) ℃ and humidity of 50%-70% for 7 d. All animal experiments followed the Guiding Opinions on the Treatment of Experimental Animals[14], which was issued by the Ministry of Science and Technology, and were approved by the Experimental Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine.

    1.2 Main instruments and reagents

    Tissue dehydrator, paraffin embedding machine, slicer,spreader and slide warmer (Leica, Germany); optical microscope and analysis software (Olympus, Japan);tissue homogenizer (Fluko, Germany); multifunctional microplate reader (Bio Tek, USA); electrophoresis instrument and gel imaging system (Bio-Rad Laboratories, USA); Light Cycler 480 real-time fluorescence quantitative polymerase chain reaction (RTqPCR) instrument (Roche Diagnostic, USA).

    Moxa (Nanyang Hanyi Moxa Co., Ltd., China); Guifu No.1 prescription (Huaji Pharmaceutical Co., Ltd., China; 5%(W/V) TNBS (Sigma-Aldrich, USA); DAB chromogenic reagent, rabbit immunoglobulin G (IgG)-immunohistochemistry kit (Boster Biological Technology Co., Ltd.,China); BCA protein concentration determination kit, Cy3 labeled goat anti-rabbit IgG (H+L), SDS gel kit, HRP labeled goat anti-rabbit secondary antibody, ECL luminescent solution, Tris and TEMED (Beyotime Biotechnology Co., Ltd., China); GAPDH, AMPKα (5831),TSC2 (4308s) (Cell Signaling Technology, USA); tissue RNA extraction kit (RN001-plus, EZ Bioscience, USA); cDNA reverse transcription kit Prime Script? RT Master Mix,PCR amplification kit TB Green? Premix Ex Taq? Ⅱ(Takara Biomedical Technology, Japan); hematoxylineosin (HE) staining solution (Nanjing Jiancheng Technology Co., Ltd., China); sodium pentobarbital,absolute ethanol, neutral gum, methanol and xylene(Sinopharm Group Chemical Reagent Co., Ltd., China);trizol reagent (Thermo Fisher, USA).

    1.3 Model preparation and identification

    Twenty-six rats were randomly divided into a normal group (n=9) and a modelling group (n=17) according to the completely random segment grouping method[15].CD rat model preparation method: 5% (W/V) TNBS[100 mg/(kg·bw)] and 50% ethanol was mixed at 2:1(volume ratio) to prepare an enema solution, and enema was carried out at 3 mL/(kg·bw). Rats were fasted for 24 h to prepare intestine before enema. The enema needle was slowly inserted into rat anus by 6-8 cm in a head-down-tail-up position, the enema fluid was injected, and then the rats were kept upside down for 1 min to prevent the enema fluid from overflowing.Enema was performed once a week for 4 consecutive weeks[16]. One rat in the modelling group and one rat in the normal group were randomly selected for colonic histopathological observation to verify whether the CD model was confirmed successfully prepared. After the model was successfully prepared, group intervention started.

    1.4 Grouping and intervention

    The 16 CD rats were randomly divided into a model group and a herbal cake-partitioned moxibustion group,with 8 rats in each group. Qihai (CV 6) and bilateral Tianshu (ST 25) were selected, and the main ingredient of medicinal cake wasFu Zi(Radix Aconiti Lateralis Praeparata) powder in the herbal cake-partitioned moxibustion group. The mixture ofFu Zi(Radix Aconiti Lateralis Praeparata) powder and yellow wine was pressed into herbal cakes using a self-made special mold(0.6 cm in diameter, 0.3 cm in thickness). Moxa was pressed into cone-shaped moxa cones (0.4 cm in diameter, 0.4 cm in height, about 90 mg in weight) using a special mold. The herbal cake was first placed on the acupoints, the moxa cone was placed on the herbal cake,and then the moxa cone was lighted for moxibustion.Moxibustion was performed with 2 cones per acupoint,once a day, for 7 consecutive days. Rats in the normal group and the model group only received the same grasping and fixation as in the herbal cake-partitioned moxibustion group.

    1.5 Sample collection

    After intervention, rats were fasted for 24 h and euthanized by intraperitoneal injection of sodium pentobarbital. Rats were fixed in a supine position, the abdominal wall was cut to collect all colons from 2 cm above the anus to the lower end of the cecum, and then cut longitudinally; after rinsed with 4 ℃ pre-cooled normal saline, about 1 cm severely damaged colon determined by gross observation was selected and fixed in 4% paraformaldehyde solution. The remaining colon was placed in a cryotube and stored in -80 ℃refrigerator after quickly frozen in liquid nitrogen.

    1.6 Indicator detection

    1.6.1 Morphological observation of colon tissues

    After HE staining of colon tissues, the morphological changes were observed under an optical microscope.

    The main operation steps included: tissue dehydration, paraffin embedding, sectioning (4 μm),baking slices at 60 ℃ for 1 h, dewaxing and hydrating in xylene and gradient alcohol, staining with hematoxylin for 10 min, washing with tap water for 10 min,differentiating with 1% hydrochloric acid and alcohol for 3 s, rinsing with tap water for 5 min, staining with 0.5%eosin for 2.5 min, and then dehydrating with 70%, 80%,90% and 100% gradient alcohol in sequence. After being transparentized with xylene, the slides were mounted with neutral gum.

    1.6.2 Detection of surface marker CD206 of colonic M2 macrophages

    The CD206 expression was detected by immunohistochemistry (IHC). Paraffin sections of colon tissues were pretreated with baking slices, xylene, and gradient alcohol, then immersed in citric acid buffer and heated in microwave oven for hot antigen retrieval.Endogenous peroxidase was blocked with 3% H2O2. The sections were incubated overnight at 4 ℃ in a humidified box after blocked with BSA and dropwise added with rabbit anti-CD206 antibody (1:1 000);reheated at 37 ℃ for 45 min, washed 3 times with PBS for 3 min/time, added with secondary antibody dropwise, and incubated at 37 ℃ for 30 min; added SABC dropwise and incubated at 37 ℃ for 20 min,washed 3 times with PBS for 3 min/time; added with DAB chromogenic solution dropwise and immersed in water to stop the color development after 3 min; stained the nucleus with hematoxylin for 2 min, rinsed with tap water for 10 min, differentiated with 1% hydrochloric acid alcohol for 3 s, rinsed with tap water for 5 min to reverse blue, dehydrated with 70%, 80%, 90% and 100%gradient alcohol; mounted the slides after transparentized with xylene; observed and photographed under a light microscope. Five fields of view were randomly selected from each slice to photograph, followed by analysis with Image-Plus Pro 6.0 software. The integrated optical density (IOD) of the positive target in each image was calculated, and the average value was used as IOD value for the slice.

    1.6.3 Detection of AMPK and TSC2 protein expressions in colon

    Western blot technology was used to detect the expression of colonic AMPK protein. One milliliter of RIPA lysate, 10 μL of PMSF protease inhibitor, and 20 μL of phosphatase inhibitor were added into every 0.1 g of colon tissue. After homogenization, the supernatant was collected, and the protein concentration was determined by BCA method to prepare the loading protein with 40 μg sample. Ten percent gel was prepared for electrophoresis, blocked with 5% BSA for 1 h after transfer, incubated with AMPK primary antibody (1:1 000)overnight at 4 ℃, added with horseradish peroxidase(HRP) labeled with secondary antibody (1:1 000),incubated at room temperature for 1 h, and then analyzed with Image J software after development.

    Immunofluorescence technique was used to detect colonic TSC2 protein expression. Paraffin sections of colon tissues were baked at 60 ℃ for 1 h, dehydrated by xylene and gradient alcohol, boiled in citric acid buffer for 8 s, incubated with 3% H2O2for 25 min in dark,blocked with 5% BSA at room temperature for 25 min,and incubated with primary antibody at 4 ℃ overnight;added with fluorescent secondary antibody dropwise;mounted with DAPI-containing mounting fluid and exposed to different wavelengths. Semi-quantitative analysis was performed with Image J software to detect the average fluorescence intensity of positive expression(arbitrary units, AU).

    1.6.4 Detection of AMPK and TSC2 mRNA expressions in colon

    RT-qPCR was used to detect the mRNA expressions of AMPK and TSC2 in colon tissues. Trizol was used to extract total RNA from colon tissues, and Prime Script?RT Master Mix kit was used to generate the first cDNA strand of mRNA. The 10 μL reverse transcription reaction system was as follows: 5× Prime Script RT Master Mix 2 μL, total RNA 500 ng, and RNase free dH2O to make up to 10 μL. Reverse transcription conditions were: 37 ℃for 15 min, 85 ℃ for 5 s. RT-qPCR amplification was performed using SYBR Green dye method. The 20 μL reaction system was as follows: 10 μL of TB Green Premix Ex Taq Ⅱ, 0.8 μL of PCR forward primer (10 μmol/L),0.8 μL of PCR reverse primer (10 μmol/L), 2 μL of cDNA template, 6.4 μL of dH2O. The cyclic reaction program was: 95 ℃, 30 s, 1 cycle; 95 ℃, 5 s, 60 ℃, 30 s,40 cycles; 95 ℃, 5 s, 60 ℃, 1 min, 1 cycle; 50 ℃, 30 s,1 cycle. The 2-△△Ctmethod was used to calculate the relative expression level of mRNA[17], △Ct for each sample = Ct value of target gene - Ct value of internal reference; 2-△△Ctfor each sample = 2-△Ctfor each sample ÷ Mean value of 2-△Ctfor all samples in the normal group. The primers used in this experiment are shown in Table 1.

    Table 1. RT-qPCR primer design

    1.7 Statistical methods

    2 Results

    2.1 Morphological changes of rat colon in each group

    Observed under light microscope, the rat colonic epithelium in the normal group was intact, with uniform distribution of monolayer columnar epithelial cells,continuous intestinal mucosa, neatly arranged glands,without congestion, edema or ulcers. In the model group,the colonic mucosa was damaged, the epithelial layer was partially missing, and the glands were hyperplastic and irregularly arranged; the lamina propria and submucosa had obvious hyperemia and edema; the submucosal muscle layer was thickened; large number of monocytes and lymphocytes were infiltrated with fissure-like ulcers reaching the muscle layer. Rat mucosal epithelium structure in the herbal cake-partitioned moxibustion group was still intact, but some glands were swollen and deformed, and new mucosal epithelium appeared (as shown by the arrow in Figure 1), suggesting the formation of healed ulcers, significantly reduced submucosal hyperemia and edema, and a small amount of inflammatory cell infiltration (Figure 1).

    2.2 Expression of CD206, a surface marker of colonic M2 macrophages

    It can be seen from Figure 2 that colonic CD206 is mainly expressed in lamina propria. Compared with the normal group, the colonic CD206 expression in the model group was significantly reduced (P<0.01);compared with the model group, the CD206 expression in rat colon tissues in the herbal cake-partitioned moxibustion group was significantly increased (P<0.01),suggesting that herbal cake-partitioned moxibustion upregulated the expression of colonic CD206, promoted colonic macrophage polarization to type M2.

    2.3 Expressions of AMPK protein and mRNA in colon

    It can be seen from Figure 3 that compared with rats in the normal group, the expressions of colonic AMPK protein and mRNA in the model group were statistically significantly decreased (bothP<0.01); compared with the model group, the AMPK protein and mRNA expressions in rat colon tissues in the herbal cakepartitioned moxibustion group were statistically significantly increased (bothP<0.05).

    2.4 Expressions of TSC2 protein and mRNA in colon

    It can be seen from Figure 4 that compared with rats in the normal group, the expressions of colonic TSC2 protein and mRNA in the model group were statistically significantly decreased (bothP<0.01); compared with the model group, the TSC2 protein and mRNA expressions in rat colon tissues in the herbal cakepartitioned moxibustion group were statistically significantly increased (P<0.05,P<0.01).

    Figure 1. HE staining of rat colon tissues in each group (HE, ×100)

    Figure 2. CD206 expression in rat colon tissues in each group (immunohistochemical staining, ×100)

    Figure 3. AMPK protein and mRNA expressions in rat colon tissues in each group

    Figure 4. TSC2 protein and mRNA expressions in rat colon tissues in each group

    3 Discussion

    The pathogenesis of CD is complicated, but currently believed to be mainly caused by genetic susceptibility,environmental factors and interaction of intestinal flora,resulting in abnormal mucosal immune response and impaired epithelial barrier function[18-19]. CD is characterized by alternately clinical remission and recurrence, lingering and hard-to-heal, which brings great challenges to clinical treatment and seriously affects the quality of life in patients. At present, the basic treatment with Western medicine primarily includes induction and maintenance therapy, such as immunosuppressive agents of glucocorticoids,aminosalicylic acid, and thiopurine; biological agents such as adalimumab. Although these therapies can delay disease progression, the serious side effects such as diabetes, osteoporosis, lymphoma, and infection cannot be avoided[20-22]. Thus it is important to actively explore treatment strategies without side effects that can treat the disease, reduce complications, and control disease progression[23]. Preliminary studies of our group have confirmed that herbal cake-partitioned moxibustion can effectively alleviate CD intestinal inflammation and improve mucosal damage[24]. This study further found that herbal cake-partitioned moxibustion promoted the expression of colonic M2 macrophage marker CD206,up-regulated the expressions of colonic AMPK and TSC2 protein and mRNA in CD rats, and reduced intestinal inflammation. As innate immune cells, macrophages respond quickly to environmental signals through many receptors, maintaining a specific and optimized activation status, and are considered to be the main effector cells in many chronic inflammatory diseases[25].Macrophages are the most common mononuclear phagocytes in the lamina propria of the intestinal mucosa, which play an important role in maintaining homeostasis of intestinal immune system. Chronic intestinal inflammation can greatly change the number and sub-population diversity of macrophages[26]. Tissue microenvironment changes can induce abnormal differentiation of macrophages, resulting in two phenotypes, namely classic activation (M1 type) and alternate activation (M2 type) macrophages[27]. Different macrophage differentiation types express different surface markers. The surface of M2 type macrophages highly expresses mannose receptors of CD206 and CD163, arginase 1, anti-inflammatory factors of interleukin (IL)-4 and IL-10. It plays an important role in tissue repair, angiogenesis and metabolism[28]. CD206 is a highly specific marker for M2 macrophages. Therefore,this study used CD206 expression to characterize the expression level of M2 type macrophages. M2 type macrophages have anti-inflammatory properties, and activated M2 type macrophages and their cytokines may be immunotherapeutic targets for anti-inflammatory effects. The study of Zhu W,et al[10]showed that promoting the polarization of M2 macrophages can induce the production of IL-10 and regulatory T cells,which can effectively reduce intestinal inflammation.Yang R,et al[29]treated mice with DSS-induced colitis by intraperitoneal injection of purified M2 macrophage exosomes, and found that M2 macrophage exosomes up-regulated the expression of Treg cells and IL-4, and reduced the production of pro-inflammatory cytokines of IL-1β, IL-6 and IL-17A, effectively alleviated colon inflammation in DSS mice. In this study,immunohistochemical staining of colon tissues showed that the CD206 expression in colonic lamina propria of CD rats was significantly reduced, and herbal cakepartitioned moxibustion treatment significantly increased the colonic CD206 expression, suggesting that promoting colonic M2 macrophage expression should be one of the effective mechanisms of herbal cakepartitioned moxibustion in alleviating intestinal inflammation.

    AMPK is an important regulator of intracellular energy metabolism and widely present in gastrointestinal tissues. Phosphorylation and activation of AMPK closes some anabolic pathways to reduce the consumption of adenosine triphosphate (ATP), and to activate the catabolic pathway that produces ATP, which plays an important regulatory role in the inflammatory response[30-31]. Studies believe that AMPK activation promotes the expression of caudal type homeobox 2(CDX-2) and improve the barrier function of intestinal epithelial cells[32]; experiments have confirmed that AMPK activation reduces the levels of intestinal proinflammatory cytokines of tumor necrosis factor (TNF)-α,IL-1β and IL-6, and significantly reduces colonic inflammation[33]. Regulatory effect of AMPK on inflammation may be related to macrophage polarization. Wang Y,et al[34]found that AMPK activation promotes M2 macrophage polarization to reduce neuroinflammation. Garami A,et al[35]found that knocking down AMPK expression with siRNA promoted the polarization of M1 type macrophages, enhanced the secretion of TNF-α and IL-6, and promoted inflammation.The role of AMPK in regulating macrophage polarization may be related to the mammalian target of rapamycin(mTOR)-related signaling pathways. Research by Qing L,et al[12]confirmed that AMPK/mTOR/NLRP3 inflammasome signal pathway was involved in regulating the polarization of M2 macrophages. AMPK inhibited mTORC1 activity by phosphorylating multiple regulatory factors in mTORC1 pathway. First, activated AMPK directly phosphorylated tuberous sclerostin TSC2 to promote TSC1/2 complex formation, which directly acted on GTPase Rheb and inhibited its activity[36], and Rheb was an upstream active molecule that directly interacted with mTORC1[37], so the activity of mTORC1 was inhibited. The mTOR was also the binding partner of NLRP3[38], which inhibited the activation of NLRP3 inflammasomes by binding to NLRP3. Therefore,activated AMPK inhibited the mTORC1 expression through phosphorylation of TSC2, thereby reducing NLRP3 inflammasome activation. Reduced activation of NLRP3 inflammasomes in macrophages inhibited the polarization of M1 macrophages and the production of IL-1β[39]. At this time, the phenotype of macrophages changes towards to M2 type, which is manifested as increased M2 type macrophages and reduction of inflammation. The results of this experiment also showed that the protein and mRNA expressions of AMPK and CD206 in rat colon tissues were reduced under CD inflammation, and their expression levels were significantly increased after herbal cake-partitioned moxibustion.

    In summary, AMPK and TSC2 proteins are closely related to colonic M2 macrophages. Herbal cakepartitioned moxibustion increased the colonic CD206 expression in CD rats, up-regulated the expressions of AMPK and TSC2 proteins and mRNAs in colon of CD rats,and reduced intestinal inflammation, suggesting that herbal cake-partitioned moxibustion may increase the protein and mRNA expressions of colonic AMPK and TSC2, promote colonic macrophage polarization to type M2, and improve intestinal inflammation. However, this experiment has not been able to confirm the potential association between regulating AMPK and TSC2 with the polarization of macrophages by herbal cake-partitioned moxibustion. Therefore, further research is needed.

    Conflict of Interest

    Authors Ma Xiao-peng and Wu Huan-gan are members of the Editorial Board of Journal of Acupuncture and Tuina Science. The paper was handled by other editors and has undergone rigorous peer review process. Authors Ma Xiao-peng and Wu Huan-gan were not involved in the journal’s review or decisions related to this manuscript.

    Acknowledgments

    This work was supported by National Natural Science Foundation of China (國家自然科學(xué)基金項目, No.81674073, No. 81904303); Natural Science Foundation of Shanghai ( 上海市自然科學(xué)基金項目, No.20ZR1453000, No. 19ZR1451600).

    Statement of Human and Animal Rights

    The treatment of animals conformed to the ethical criteria in this experiment.

    Received: 15 October 2020/Accepted: 14 April 2021

    猜你喜歡
    張丹國家自然科學(xué)基金
    常見基金項目的英文名稱(一)
    常見基金項目的英文名稱(一)
    介詞的時間搭配
    常見基金項目的英文名稱(一)
    Probing Nonclassicality of Two-Mode SU(2)Generator Based on Quantum Fisher Information?
    我校喜獲五項2018年度國家自然科學(xué)基金項目立項
    Application of Communicative Approach to Junior English Teaching
    嚇人奶奶,新年快樂
    麗塔的神奇松果
    2017 年新項目
    久久青草综合色| 国产男女超爽视频在线观看| 中文欧美无线码| 亚洲成国产人片在线观看| 在线观看免费高清a一片| 一级黄片播放器| 男女无遮挡免费网站观看| 叶爱在线成人免费视频播放| 少妇人妻 视频| 日韩成人av中文字幕在线观看| av女优亚洲男人天堂| 精品亚洲成a人片在线观看| 九草在线视频观看| 日韩av在线免费看完整版不卡| 中国三级夫妇交换| 香蕉国产在线看| 欧美另类一区| 韩国av在线不卡| 欧美黑人精品巨大| 亚洲av国产av综合av卡| 国产高清国产精品国产三级| 丝袜脚勾引网站| 亚洲国产毛片av蜜桃av| 久久精品国产亚洲av高清一级| 99精品久久久久人妻精品| 久久午夜综合久久蜜桃| 妹子高潮喷水视频| 久久久久精品人妻al黑| 中文欧美无线码| 亚洲国产精品一区三区| 国产亚洲一区二区精品| 午夜免费男女啪啪视频观看| 免费人妻精品一区二区三区视频| 丰满迷人的少妇在线观看| 国产精品免费视频内射| 青春草视频在线免费观看| 欧美激情高清一区二区三区 | 国产免费又黄又爽又色| 你懂的网址亚洲精品在线观看| 亚洲欧美成人综合另类久久久| 亚洲精品久久午夜乱码| www.自偷自拍.com| 日韩熟女老妇一区二区性免费视频| 国产精品一国产av| 久久 成人 亚洲| 999精品在线视频| 午夜福利一区二区在线看| av网站免费在线观看视频| 亚洲综合色网址| 欧美人与性动交α欧美软件| 亚洲国产看品久久| 国产免费福利视频在线观看| 免费av中文字幕在线| 亚洲欧美中文字幕日韩二区| 久久毛片免费看一区二区三区| 交换朋友夫妻互换小说| 欧美精品av麻豆av| 妹子高潮喷水视频| 90打野战视频偷拍视频| 久久久久视频综合| 日韩 亚洲 欧美在线| 美女中出高潮动态图| 久久精品国产亚洲av高清一级| 精品午夜福利在线看| 欧美人与性动交α欧美软件| 久久毛片免费看一区二区三区| 视频在线观看一区二区三区| 综合色丁香网| 韩国精品一区二区三区| 男女之事视频高清在线观看 | 一级毛片电影观看| 少妇人妻精品综合一区二区| 亚洲成av片中文字幕在线观看| 一区二区三区精品91| 两个人看的免费小视频| 亚洲精品国产区一区二| 亚洲成人av在线免费| 极品人妻少妇av视频| 黄色毛片三级朝国网站| 亚洲欧美色中文字幕在线| 亚洲第一区二区三区不卡| 最近最新中文字幕免费大全7| 亚洲欧美一区二区三区久久| 久久狼人影院| 国产黄频视频在线观看| 精品少妇内射三级| 成年动漫av网址| 天天操日日干夜夜撸| 一级片'在线观看视频| a级毛片黄视频| 在线观看免费视频网站a站| 日韩av不卡免费在线播放| 精品一区在线观看国产| 亚洲国产成人一精品久久久| 肉色欧美久久久久久久蜜桃| 大片电影免费在线观看免费| 久久精品久久久久久久性| 一本一本久久a久久精品综合妖精| 国产精品偷伦视频观看了| 亚洲精品久久午夜乱码| 极品少妇高潮喷水抽搐| 中文天堂在线官网| 精品一区二区三区av网在线观看 | 伊人亚洲综合成人网| 黑人猛操日本美女一级片| 国产黄色视频一区二区在线观看| 91精品三级在线观看| 别揉我奶头~嗯~啊~动态视频 | 国产日韩一区二区三区精品不卡| 午夜精品国产一区二区电影| 久久女婷五月综合色啪小说| 一边亲一边摸免费视频| 婷婷色av中文字幕| 精品国产乱码久久久久久男人| 国产一区亚洲一区在线观看| 在线观看www视频免费| 久久久久精品人妻al黑| 国产精品人妻久久久影院| 欧美日韩av久久| 亚洲人成网站在线观看播放| 在线观看免费午夜福利视频| 国产免费一区二区三区四区乱码| 赤兔流量卡办理| 亚洲精品,欧美精品| 久久精品久久精品一区二区三区| 欧美在线黄色| 亚洲人成77777在线视频| 国产成人精品久久久久久| 又大又爽又粗| 国产极品粉嫩免费观看在线| 欧美中文综合在线视频| 精品少妇久久久久久888优播| 日韩免费高清中文字幕av| 精品少妇一区二区三区视频日本电影 | 少妇被粗大猛烈的视频| 日韩大片免费观看网站| 中文字幕最新亚洲高清| 男男h啪啪无遮挡| 最近的中文字幕免费完整| 欧美日韩亚洲高清精品| tube8黄色片| 国产免费又黄又爽又色| 蜜桃在线观看..| 国产精品嫩草影院av在线观看| 美女视频免费永久观看网站| 精品国产一区二区三区四区第35| 你懂的网址亚洲精品在线观看| 91aial.com中文字幕在线观看| 国产精品亚洲av一区麻豆 | 熟妇人妻不卡中文字幕| 久久人人97超碰香蕉20202| 天天影视国产精品| 大香蕉久久网| av.在线天堂| av网站在线播放免费| 一级a爱视频在线免费观看| 深夜精品福利| 亚洲国产欧美在线一区| 亚洲欧美清纯卡通| 国产精品久久久久久久久免| 欧美在线黄色| 国产男女超爽视频在线观看| 亚洲激情五月婷婷啪啪| 七月丁香在线播放| 天美传媒精品一区二区| 午夜91福利影院| 一级a爱视频在线免费观看| 国产亚洲av片在线观看秒播厂| 午夜免费男女啪啪视频观看| 国产av码专区亚洲av| 亚洲精华国产精华液的使用体验| 蜜桃在线观看..| 久久久久久久大尺度免费视频| h视频一区二区三区| 亚洲久久久国产精品| 狠狠婷婷综合久久久久久88av| 亚洲av欧美aⅴ国产| 99热国产这里只有精品6| 可以免费在线观看a视频的电影网站 | 国产高清国产精品国产三级| 中文乱码字字幕精品一区二区三区| 搡老岳熟女国产| 精品国产乱码久久久久久男人| 精品午夜福利在线看| 亚洲婷婷狠狠爱综合网| 成年av动漫网址| 国产一卡二卡三卡精品 | 狂野欧美激情性xxxx| 少妇猛男粗大的猛烈进出视频| 日韩中文字幕欧美一区二区 | 熟女av电影| 国产精品 国内视频| 各种免费的搞黄视频| 国产1区2区3区精品| 丰满饥渴人妻一区二区三| 熟女av电影| 丝袜人妻中文字幕| 久久久国产一区二区| 日韩欧美精品免费久久| 国产在线一区二区三区精| 美女高潮到喷水免费观看| 成人黄色视频免费在线看| 亚洲综合色网址| 国语对白做爰xxxⅹ性视频网站| 97人妻天天添夜夜摸| 久久久久久人人人人人| avwww免费| 国产免费现黄频在线看| 大话2 男鬼变身卡| 亚洲免费av在线视频| 国产1区2区3区精品| 老司机靠b影院| 欧美激情 高清一区二区三区| 一级,二级,三级黄色视频| av一本久久久久| 欧美精品亚洲一区二区| 亚洲欧美色中文字幕在线| 亚洲成av片中文字幕在线观看| 天天躁夜夜躁狠狠久久av| 国产男人的电影天堂91| 9191精品国产免费久久| 在线亚洲精品国产二区图片欧美| 亚洲,欧美,日韩| 汤姆久久久久久久影院中文字幕| 丰满乱子伦码专区| 欧美日韩av久久| 一区福利在线观看| 国产福利在线免费观看视频| 色吧在线观看| 精品第一国产精品| 亚洲,一卡二卡三卡| 婷婷成人精品国产| 80岁老熟妇乱子伦牲交| 免费女性裸体啪啪无遮挡网站| 观看av在线不卡| 高清不卡的av网站| 丝袜在线中文字幕| 国产成人精品福利久久| 国产极品粉嫩免费观看在线| 亚洲精品av麻豆狂野| 亚洲三区欧美一区| 在线观看免费视频网站a站| 亚洲成色77777| 亚洲一卡2卡3卡4卡5卡精品中文| 两个人看的免费小视频| 老司机影院成人| 在线观看一区二区三区激情| 中文字幕人妻丝袜制服| 各种免费的搞黄视频| 国产精品熟女久久久久浪| 精品一品国产午夜福利视频| 肉色欧美久久久久久久蜜桃| 99热国产这里只有精品6| 色视频在线一区二区三区| 最近的中文字幕免费完整| kizo精华| 日韩av不卡免费在线播放| 亚洲精品国产一区二区精华液| 在线 av 中文字幕| 少妇精品久久久久久久| 肉色欧美久久久久久久蜜桃| 免费久久久久久久精品成人欧美视频| 黑人欧美特级aaaaaa片| 亚洲欧美精品综合一区二区三区| 波多野结衣av一区二区av| 亚洲成av片中文字幕在线观看| 最新的欧美精品一区二区| 成人国产麻豆网| 91老司机精品| av不卡在线播放| 久久久久久人人人人人| 国产成人午夜福利电影在线观看| 男女下面插进去视频免费观看| www.精华液| 99热全是精品| 美女午夜性视频免费| 人人妻人人爽人人添夜夜欢视频| 欧美精品人与动牲交sv欧美| 亚洲伊人久久精品综合| 日本爱情动作片www.在线观看| 丝袜在线中文字幕| 成人亚洲精品一区在线观看| 亚洲av男天堂| 美女国产高潮福利片在线看| 十八禁高潮呻吟视频| 中文字幕另类日韩欧美亚洲嫩草| 久久这里只有精品19| 一级毛片我不卡| 亚洲国产看品久久| 18在线观看网站| 国产av国产精品国产| 伊人久久国产一区二区| 你懂的网址亚洲精品在线观看| 高清av免费在线| 中文字幕精品免费在线观看视频| 亚洲欧美激情在线| 亚洲在久久综合| 久久99一区二区三区| 精品午夜福利在线看| 精品少妇一区二区三区视频日本电影 | 亚洲专区中文字幕在线 | 午夜免费鲁丝| 黄色 视频免费看| 欧美黄色片欧美黄色片| 如日韩欧美国产精品一区二区三区| 日本一区二区免费在线视频| 精品卡一卡二卡四卡免费| 国产无遮挡羞羞视频在线观看| 久久精品aⅴ一区二区三区四区| 麻豆av在线久日| 亚洲色图综合在线观看| av线在线观看网站| 欧美xxⅹ黑人| 久久久久精品国产欧美久久久 | 久久久久久久久久久免费av| 青草久久国产| 久久久久人妻精品一区果冻| 蜜桃国产av成人99| 久久精品国产亚洲av涩爱| 日本av免费视频播放| 黑人猛操日本美女一级片| 国产精品99久久99久久久不卡 | 免费久久久久久久精品成人欧美视频| 亚洲三区欧美一区| 美女高潮到喷水免费观看| 中文字幕色久视频| 97人妻天天添夜夜摸| 国产成人一区二区在线| 国产 一区精品| 中文字幕av电影在线播放| 亚洲专区中文字幕在线 | 国产成人欧美在线观看 | 如日韩欧美国产精品一区二区三区| 久久久久久久久免费视频了| 熟妇人妻不卡中文字幕| 欧美日韩亚洲综合一区二区三区_| 中文精品一卡2卡3卡4更新| 韩国高清视频一区二区三区| 色网站视频免费| svipshipincom国产片| 亚洲av综合色区一区| 深夜精品福利| 最黄视频免费看| 国产成人免费无遮挡视频| 熟妇人妻不卡中文字幕| 99精国产麻豆久久婷婷| 狂野欧美激情性xxxx| 国产日韩欧美视频二区| 久久精品人人爽人人爽视色| xxx大片免费视频| 黄网站色视频无遮挡免费观看| 女人久久www免费人成看片| 亚洲免费av在线视频| 久久青草综合色| 亚洲伊人色综图| 夫妻性生交免费视频一级片| 日韩伦理黄色片| 超碰成人久久| 中文字幕人妻丝袜一区二区 | 亚洲精品在线美女| 你懂的网址亚洲精品在线观看| 在线观看国产h片| 在线观看人妻少妇| 国产成人精品无人区| 久久热在线av| 久久久久久免费高清国产稀缺| 亚洲国产日韩一区二区| 亚洲一级一片aⅴ在线观看| 老汉色∧v一级毛片| 最近中文字幕2019免费版| 国产人伦9x9x在线观看| 亚洲国产精品国产精品| 一区二区三区精品91| 亚洲四区av| 捣出白浆h1v1| 免费久久久久久久精品成人欧美视频| 婷婷色av中文字幕| 天天操日日干夜夜撸| 中文字幕av电影在线播放| 国产精品国产av在线观看| 桃花免费在线播放| 久久久久久免费高清国产稀缺| 国产免费福利视频在线观看| 久久精品久久精品一区二区三区| 国产日韩欧美亚洲二区| 这个男人来自地球电影免费观看 | 99re6热这里在线精品视频| 七月丁香在线播放| 狂野欧美激情性bbbbbb| 国产又爽黄色视频| 亚洲欧美一区二区三区黑人| 男女国产视频网站| 丝袜喷水一区| 如日韩欧美国产精品一区二区三区| 久久国产亚洲av麻豆专区| 国产野战对白在线观看| 1024视频免费在线观看| 亚洲欧美精品综合一区二区三区| 久久久亚洲精品成人影院| 最近2019中文字幕mv第一页| 18在线观看网站| 国产精品国产三级国产专区5o| 欧美亚洲 丝袜 人妻 在线| 久久久久网色| 中文字幕另类日韩欧美亚洲嫩草| 在现免费观看毛片| 最近最新中文字幕大全免费视频 | 人人妻,人人澡人人爽秒播 | 国产高清国产精品国产三级| 人妻一区二区av| 欧美久久黑人一区二区| 深夜精品福利| 日本av手机在线免费观看| 99国产精品免费福利视频| av一本久久久久| 久久毛片免费看一区二区三区| 国产人伦9x9x在线观看| 在线精品无人区一区二区三| 在线观看免费高清a一片| 黑丝袜美女国产一区| 丰满少妇做爰视频| 免费女性裸体啪啪无遮挡网站| 久久国产亚洲av麻豆专区| 80岁老熟妇乱子伦牲交| 亚洲精品自拍成人| 亚洲 欧美一区二区三区| 亚洲国产毛片av蜜桃av| 91aial.com中文字幕在线观看| 午夜日韩欧美国产| 熟妇人妻不卡中文字幕| 免费观看人在逋| 国产亚洲午夜精品一区二区久久| 97人妻天天添夜夜摸| 亚洲精品美女久久av网站| 高清av免费在线| 男女午夜视频在线观看| 亚洲精品,欧美精品| 一边摸一边抽搐一进一出视频| 女性生殖器流出的白浆| 丰满迷人的少妇在线观看| 巨乳人妻的诱惑在线观看| 成人国产麻豆网| 亚洲精品在线美女| 欧美精品人与动牲交sv欧美| 香蕉国产在线看| 一边摸一边做爽爽视频免费| 国产精品一国产av| 人人妻,人人澡人人爽秒播 | 久久午夜综合久久蜜桃| 岛国毛片在线播放| 亚洲综合精品二区| 久久久亚洲精品成人影院| 啦啦啦啦在线视频资源| 日本一区二区免费在线视频| tube8黄色片| 国产成人啪精品午夜网站| 宅男免费午夜| 亚洲色图综合在线观看| 中文字幕另类日韩欧美亚洲嫩草| 亚洲欧美精品综合一区二区三区| 青草久久国产| 精品亚洲乱码少妇综合久久| 高清av免费在线| 熟女av电影| 卡戴珊不雅视频在线播放| 一级毛片黄色毛片免费观看视频| 欧美日韩精品网址| 免费观看a级毛片全部| www.自偷自拍.com| 2018国产大陆天天弄谢| 深夜精品福利| 午夜91福利影院| 欧美在线一区亚洲| 国产福利在线免费观看视频| 少妇 在线观看| 日本午夜av视频| 精品亚洲成国产av| 国产片内射在线| 日韩,欧美,国产一区二区三区| 一区二区三区乱码不卡18| 午夜福利网站1000一区二区三区| 青春草国产在线视频| 最黄视频免费看| 丁香六月天网| 久久久精品94久久精品| 欧美激情极品国产一区二区三区| 国产精品久久久久成人av| 婷婷色麻豆天堂久久| 美女视频免费永久观看网站| 国产成人欧美在线观看 | 汤姆久久久久久久影院中文字幕| 久久久久久人妻| 成年女人毛片免费观看观看9 | 国产免费现黄频在线看| 男人爽女人下面视频在线观看| 国产黄频视频在线观看| 极品少妇高潮喷水抽搐| av一本久久久久| 黄色一级大片看看| 在现免费观看毛片| 91aial.com中文字幕在线观看| 国产在线一区二区三区精| 国产成人啪精品午夜网站| 不卡视频在线观看欧美| 又黄又粗又硬又大视频| 亚洲av电影在线进入| 欧美 亚洲 国产 日韩一| 日韩av不卡免费在线播放| 欧美 日韩 精品 国产| 亚洲av男天堂| a级片在线免费高清观看视频| 亚洲精品av麻豆狂野| 亚洲欧美精品自产自拍| 亚洲精品中文字幕在线视频| 日本vs欧美在线观看视频| 久热这里只有精品99| 中文字幕av电影在线播放| 亚洲七黄色美女视频| 性色av一级| 国产成人91sexporn| 久久久精品免费免费高清| 丁香六月欧美| 欧美在线一区亚洲| 18禁动态无遮挡网站| 午夜激情久久久久久久| 99国产精品免费福利视频| 婷婷色综合www| 久久精品亚洲av国产电影网| 丁香六月天网| 9色porny在线观看| 国产伦人伦偷精品视频| 热re99久久精品国产66热6| 成人国语在线视频| 国产成人一区二区在线| 高清欧美精品videossex| 中文欧美无线码| 91成人精品电影| 精品第一国产精品| 香蕉丝袜av| 秋霞在线观看毛片| e午夜精品久久久久久久| 考比视频在线观看| 色播在线永久视频| 亚洲av中文av极速乱| 男人舔女人的私密视频| 欧美中文综合在线视频| 人人妻人人澡人人爽人人夜夜| 美女国产高潮福利片在线看| 久久精品久久久久久噜噜老黄| 国产淫语在线视频| 国产免费视频播放在线视频| 日韩av不卡免费在线播放| 亚洲美女搞黄在线观看| 一级毛片电影观看| 亚洲成人av在线免费| 韩国av在线不卡| 中国三级夫妇交换| 91精品三级在线观看| 免费日韩欧美在线观看| 成人黄色视频免费在线看| 欧美在线黄色| 卡戴珊不雅视频在线播放| 久久免费观看电影| 久久精品国产a三级三级三级| 搡老乐熟女国产| 免费高清在线观看日韩| 国产亚洲一区二区精品| 日韩中文字幕视频在线看片| 黄色视频不卡| 亚洲欧美一区二区三区国产| 极品人妻少妇av视频| 亚洲第一青青草原| 超碰成人久久| 免费在线观看黄色视频的| 精品一品国产午夜福利视频| 色播在线永久视频| 亚洲精品,欧美精品| 欧美久久黑人一区二区| 免费观看av网站的网址| 女人被躁到高潮嗷嗷叫费观| 高清av免费在线| 人人妻人人澡人人爽人人夜夜| 麻豆乱淫一区二区| 国产97色在线日韩免费| 久久久久精品人妻al黑| 黄色视频在线播放观看不卡| 日本vs欧美在线观看视频| 亚洲男人天堂网一区| 欧美 亚洲 国产 日韩一| 69精品国产乱码久久久| 午夜福利一区二区在线看| 亚洲欧美一区二区三区久久| 成人手机av| 日韩精品免费视频一区二区三区| 精品少妇内射三级| 久久性视频一级片| 在线观看一区二区三区激情| 国产成人免费无遮挡视频| 视频在线观看一区二区三区| 少妇精品久久久久久久| 亚洲色图综合在线观看| 午夜日本视频在线| av在线app专区| 一级毛片电影观看| 99精国产麻豆久久婷婷| 婷婷成人精品国产| svipshipincom国产片| 精品国产露脸久久av麻豆| 如何舔出高潮| 黑人猛操日本美女一级片| 欧美日韩一区二区视频在线观看视频在线| 老司机深夜福利视频在线观看 | 丝袜在线中文字幕| 成人国产av品久久久| 一本一本久久a久久精品综合妖精|