• <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 ?

    Therapeutic effect of a traditional Chinese medicine formulation on experimental choroidal neovascularization in mouse

    2021-11-08 01:45:46YuFeiZhangChuanJiangXiaoHongZhouDongYuWeiShaoHengLiPanLongManHongLiZuoMingZhangTaoChenHongJunDu
    International Journal of Ophthalmology 2021年10期

    Yu-Fei Zhang, Chuan Jiang, Xiao-Hong Zhou, Dong-Yu Wei, Shao-Heng Li, Pan Long, Man-Hong Li, Zuo-Ming Zhang, Tao Chen, Hong-Jun Du

    1Center of Clinical Aerospace Medicine, Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China

    2The Air Force Hospital from Northern Theater PLA,Shenyang 110092, Liaoning Province, China

    3Department of Aviation Medicine, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province,China

    4Ophthalmology, Children’s Hospital of Fudan University,Shanghai 201102, China

    5School of Basic Medicine, Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China

    6Department of Ophthalmology, General Hospital, Western Theater Command, Chengdu 610083, Sichuan Province, China

    7Department of Ophthalmology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China

    Abstract

    ● KEYWORDS: choroidal neovascularization; traditional Chinese medicine; mice

    INTRODUCTION

    C horoidal neovascularization (CNV)-related diseases are major eye diseases that cause blindness, especially in many developed countries[1-3]. They are characterized by pathological proliferative neovascularization and patients range from newborns to elder individuals, with a wide range of vulnerable populations. While the number of patients with of CNV-related diseases is increasing, there is currently no an ideal treatment. Symptomatic remission treatments such as anti-vascular endothelial growth factor (anti-VEGF)agents, laser photocoagulation, photodynamic therapy, and transpupillary thermotherapy are mainly used[4-7].

    CNV is the basic pathological change of many kinds of intraocular diseases and it often involves the macula and leads to serious damage of central vision[8-9]. CNV comes from choroidal capillaries and invades retinal pigment epithelium(RPE) through Bruch’s membrane. Due to the incomplete structure of CNV, the leakage fluid accumulates between RPElayer and retina, which makes photoreceptor cells separate from RPE layer and eventually leads to blindness[10]. Current studies show that the production of CNV is mainly related to VEGF, which can regulate vascular endothelial cells, promote angiogenesis and increase vascular permeability, therefore,VEGF and its receptor become the target molecules of CNV treatment[11-12]. Study has shown that Hexuemingmu (HXMM)can regulate the level of VEGF in the eyes of rats with retinal central vein occlusion[13]. With the development of traditional Chinese medicine (TCM) research, an increasing number of TCM formulations have been found to have good therapeutic effects on ophthalmic diseases that are difficult to treat conventionally[13-14]. Therefore, as a traditional ophthalmic drug, can HXMM regulate VEGF to treat CNV? This study was to explore the potential therapeutic effects of HXMM on CNV-related diseases, with the aim of providing a novel therapeutic direction for these diseases.

    Table 1 Ingredients of HXMM

    MATERIALS AND METHODS

    Ethical Approval All experiments were conducted in accordance with the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of Animals in Ophthalmic and Vision Research. All procedures were carried in accordance with the Animal Research: Reportingin vivoExperiments (ARRIVE) guidelines.

    Animals Ninety healthy adult male C57BL/6 mice (8-weekold) were obtained from the experimental animal center of the Fourth Military Medical University in Xi’an, Shaanxi Province, China (license No.SYXK 2017-0045). All animals were maintained under standard laboratory conditions, with food and water availablead libitum.

    Choroidal Neovascularization Model The mice were anesthetized by intraperitoneal injection (IP) 1 mL/kg 1%sodium pentobarbital (Sigma-Aldrich, St Louis, MO, USA;P3761). After mydriasis with compound tropicamide eye drops (Santen Pharmaceutical Co., Ltd, Japan), six laser photocoagulation spots were made around the optic disc, with 1-2 disc diameters from it by using an image-guided laser system (577 nm) with specific parameters (power, 180 mW;duration, 100ms; spot size, 100 μm)[15]. Small bubbles accompanied by slight sound and no bleeding were regarded as the success of CNV inducing. The bilateral eyes of the mice were modeled using a laser.

    Ingredients of Hexuemingmu The HXMM mainly includes the components in Table 1.

    Grouping and Intervention After modeling, the mice were randomly divided into the following groups of 18 mice each control group, low-, moderate-, and high-dose(CG, LOW, MOD, and HIGH, respectively). In addition, a sham operation (Sham) group was set up. Except for laser modeling, other procedures on the Sham group were the same as those conducted on the CG. The CG and Sham groups were administered 10 mL/kg normal saline by gavage daily,whereas the LOW, MOD, and HIGH groups were administered intragastrically with HXMM (dissolved in 10 mL/kg normal saline) at doses of 0.34, 0.68, and 1.36 g/kg, respectively.

    Electroretinography Electroretinography (ERG) was conducted 1, 4, and 8wk after drug administration. Six mice were randomly selected from each group at each time point and the ERG was performed in the right eyes of the experimental animals according to the standardized protocol of small animal ERG records established by ISCEV[16]. Briefly, before the ERG, the mice were allowed to adapt to the dark overnight and then they were anesthetized by administering conventional IP injections of 3 mL/kg 1% pentobarbital sodium and 50 μL 10% Lumianning II (Jilin Shengda Animal Pharmaceutical,Co., Ltd., Jilin Province, China).

    Compound tropicamide eye drops were used to dilate the pupils and oxybuprocaine hydrochloride eye drops (Santen Pharmaceutical Co., Ltd., Japan) were used for corneal anesthesia. The electrodes were connected correctly and the action electrode was a silver-silver chloride (Ag-AgCl)corneal ring electrode. The reference and ground electrodes were both stainless steel needle electrodes and were punctured under the cheek subcutaneous tissue and inserted into the tail subcutaneous tissue, respectively. The ERG was recorded under dim red light and at the end of the experiment,levofloxacin eye drops (Santen Pharmaceutical Co., Ltd.,Japan) were administered for infection prevention.

    Fluorescence Fundus Angiography Fluorescence fundus angiography (FFA) was performed 1, 4, and 8wk after drug administration. After the ERG, the animals were injected(IP) with 0.5 mL/kg 20% fluorescence solution (Baiyunshan Mingxing Corporation, Guangzhou, Guangdong Province,China) and FFA images were collected 1-2min later. The degree of fluorescence leakage was graded according to the method of Takehanaet al[17], and the classification criteria were as follows: no leakage, level 0; mild leakage, level 1;moderate leakage, level 2; and severe leakage level 3.

    Choroidal Flat Mount After the completion of thein vivoexperiment, the mice were anesthetized by IP injections with 6 mL/kg 1% pentobarbital, and dislocation of cervical vertebra was performed 5min later. The eyeballs of the mice were enucleated rapidly, immersed in 4% paraformaldehyde for 1h, and then the anterior segment and neuroretina were removed. After 4% paraformaldehyde fixation for 12h, four to six radial incisions were made on the choroidal complex.The rinse solution (0.2 mL Tween 20 and 0.5 g bovine serum albumin BSA dissolved in 100 mL phosphate-buffered saline,PBS, 0.1 mmol/L, pH 7.2) was used to rinse the choroidal complex 5 times for 5min.

    Then, the choroidal complex was incubated with isolectin B4-fluorescein isothiocyanate (FITC) conjugate (Sigma-Aldrich,L2895, 10 μg/mL) at 4℃ for 4h. The samples were sealed with glycerol and images of the choroidal flat mount were captured using a fluorescence microscope (BX53, Olympus, Japan).

    Western Blot Analysis The choroidal tissues of the right eyeballs of the mice were separated and homogenized on ice in radioimmunoprecipitation assay (RIPA) buffer (Beyotime Institute of Biotechnology, Jiangsu, China), and proteinase/phosphatase inhibitors were added at a 1:100 ratio. Operation of electrophoresis was according to the protocol in our laboratory[13].

    The separated proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA), which were incubated with 5% nonfat milk solution for 1h at 25℃,followed by incubation with the following antibodies:β-tubulin (1:1000, 2146s, Cell Signaling Technology), rabbit anti-hypoxia-inducible factor 1 (HIF-1) α antibody (1:250, bs-20399R; Bioss Biotechnology, Beijing, China), anti-VEGFA antibody (6 μg/mL; Ab1316, Abcam, Cambridge, MA, USA),and fibroblast growth factor (FGF)-2 antibody (1:500; sc-271847, Santa Cruz Biotechnology) overnight at 4℃.After rinsing with PBS plus tween (PBST), the PVDF membranes were incubated with anti-rabbit immunoglobulin G (IgG),horseradish peroxidase (HRP)-linked (1:5000; 7074s; Cell Signaling Technology) and goat anti-mouse IgG(H+L)HRP (1:5000; 31430; Xianfeng Biotechnology Company,Shaanxi, China) antibodies at 25℃ for 2h. After rinsing three times with PBST for 5min, an enhanced chemiluminescence solution (Millipore, USA) was used for protein visualization.The intensity of the immunoreactivity was quantified using densitometry using Quantity One software (Bio-Rad Laboratories, Inc., USA).

    Histological Staining The left eyeballs of the mice were enucleated rapidly and immersed in a fixative solution(40% formaldehyde: distilled water: 95% ethanol: glacial acetic acid, 2:2:3:2) for 48h. The eyeballs were dehydrated,embedded in paraffin wax, and 4 μm thick sections were cut.The sections containing laser spots were chosen, stained with hematoxylin and eosin (H&E), and then observed under 400×magnification.

    Immunofluorescence Staining The paraffin wax sections were dewaxed with xylene and hydrated using gradient ethanol. Sections were boiled with ethylenediaminetetraacetic acid (EDTA) antigen retrieval solution (Beyotime Institute of Biotechnology, Jiangsu, China) for 20min for antigen retrieval.The sections were washed with PBS after cooling to 25℃,blocked with 10% goat serum (Boster Biological Technology,Haimen, China) for 1h at 25℃, and then incubated with anti-VEGF antibody (1100; Ab1316, Abcam, Cambridge, MA,USA) overnight at 4℃.

    After washing with PBST, sections were incubated with Alexa Fluor 594-conjugated goat anti-mouse IgG(H+L) (1:100;ZF-0512; ZSGB-BIO Biotechnology, Beijing, China) for 1h at 25℃. Antifade mounting medium with DAPI (Beyotime Institute of Biotechnology, Jiangsu, China) was used to seal the sections. Images of the sections were obtained using a laser scanning confocal microscope (Carl Zeiss AG, Germany).

    Serum Biochemistry At 8wk after drug administration,blood samples were collected from the heart of each animal(before dislocation of cervical vertebra), centrifuged at 14 000 g for 20min at 4℃, and the supernatant was collected. The serum concentrations of aspartate transaminase (AST), alanine transaminase (ALT), uric acid (UA), blood urea nitrogen(BUN), and creatinine (CR) of the mice were detected using an automatic biochemical analyzer (Rayto Life Sciences Ltd.,Shenzhen, China).

    Figure 1 Hexuemingmu improved retinal function in mice with choroidal neovascularization A: Typical electroretinography (darkadaptation response 3.0 b-wave) performance. Amplification of b-wave (dark-adaptation 3.0 response) after HXMM administration for 1(B); 4 (C); and 8wk (D) in mice with CNV. Statistical significance was determined using one-way analysis of variance (ANOVA). Data are means±SEM. All analyses were performed in duplicate, n=6 mice per group. aP<0.05 and bP<0.01, intervention group vs control group.

    Table 2 Grade of fundus fluorescence leakage in each group of experimental animals

    Statistical Analysis The IBM statistical package for the social science (SPSS) version 23.0 software (IBM, USA) was used for the statistical analysis. The normally distributed data were expressed as mean±standard error of the mean (SEM), whereas the abnormally distributed data were expressed as medians and quartile [M (Q1, Q3)]. For normally distributed data, an analysis of variance (ANOVA), followed by Dunnett’s post hoc analysis was performed to detect significant differences among the groups. For abnormally distributed data, the Kruskal-Wallis test (Htest) and Mann-WhitneyUtest were performed to examine the significant differences among the groups. AP≤0.05 indicated statistical significance.

    RESULTS

    Effects of Hexuemingmu on Retinal Function of Choroidal Neovascularization Mice The objective examination of retinal function using ERG showed that compared with the Sham group, the retinal function of CNV mice in each experimental group was decreased. This finding was indicated by the decreased amplitude of scotopic 3.0 ERG response b-wave (Figure 1). Treatment with HXMM dose-dependently restored the amplitude of the scotopic 3.0 ERG response b-wave in each treatment group, to a certain extent compared with that of the CG (P<0.05). Furthermore, 1wk after treatment, the difference between the treatment group and CG was the highest, and showed a stable time-dependent trend.

    Reduction of Fundus Leakage by Hexuemingmu FFA was used to determine the effects of HXMM on leakage of the fundus. The FFA results showed that the CNV mice in the various groups exhibited different degrees of fluorescence leakage, and that of the HXMM-treated groups was significantly lighter than that of the CG was (Figure 2).The grading evaluation results of the analysis of fluorescence leakage intensity, showed that the CNV leakage rate and leakage intensity of the HIGH group was significantly lower than that of the CG after 1 and 8wk (Table 2,P<0.05), but not significantly at 4wk of treatment. In addition, the generation rate and leakage intensity of the MOD group was decreased after 8wk of administration. However, the generation rate and leakage intensity of the CNV mice were not significantly changed by low-dose HXMM.

    Figure 2 Typical fluorescence fundus angiography images of experimental animals White arrow: leakage point.

    Effects of Hexuemingmu on Choroidal Neovascularization Leakage in Mice As shown in Figure 3, compared with the Sham group, CNV mice in each group showed different degrees of neovascularization around the laser spot (green light fluorescence). The area of neovascularization in the HXMM intervention groups was smaller than that in the CG, mainly at 1 and 8wk after administration.

    Effects of Hexuemingmu on Pathological Changes in Choroidal Neovascularization Mice The Hematoxylin and eosin (H&E) staining (Figure 4) showed that after laserinduced CNV generation, the mouse retinas showed obvious damage, which manifested as a thinning or even disappearance of the outer nuclear layer, and the inner nuclear layer migrated to the injured area. The RPE and choroid were significantly thickened. The H&E staining also showed that the degree of choroidal thickening decreased with increasing doses of HXMM.

    Effects of Hexuemingmu on Expression of Angiogenic Proteins in Choroidal Neovascularization Mice Detection of the expression of CNV-related proteins in the mouse choroid(Figure 5) showed high-dose HXMM continuously reduced the expression of VEGFA in the choroid (P<0.05), whereas the effect of the moderate and small doses was only obvious at 1 and 4wk. In addition, the moderate- and high-dose HXMM continuously reduced the expression of FGF-2 protein in the choroid (P<0.05), thereby alleviating the generation of CNV.However, the effect of HXMM on HIF-1α expression was not obvious.

    The distribution of VEGF expression in the eyeballs of mice was observed using immunofluorescence (Figure 6). The results demonstrated that the choroidal region of the normal mice showed low fluorescent staining. In contrast the laserinduced CNV mice showed strong fluorescence staining, and the stained region decreased with increasing HXMM dose.

    Safety of Hexuemingmu The results of the serum biochemical tests (Figure 7) showed that 8wk administration of HXMM did not significantly affect the liver and kidney function of the mice, as indicated by the serum Cr, BUN, AST,ALT, and UA levels.

    DISCUSSION

    CNV is a type of abnormal vasculature originating from choroidal vessels, which is mainly caused by factors such as aging, trauma, inflammation, and tumors[18-19]. The generation of CNV increases the permeability of the vascular. This process leads to increased susceptibility to bleeding, exudation,and scar formation and eventually leads to visual impairment and even blindness[20]. The specific pathogenesis of CNV is unclear presently although numerous studies have shown that a variety of substances are closely related to its development.Among them, VEGF, FGF, epidermal growth factor (EGF),platelet-derived endothelial cell growth factor (PD-ECGF),and tumor necrosis factor (TNF) have been proved to play an important role in angiogenesis. VEGF is a soluble protein closely related to hypoxia that promotes vascular endothelial cell division, and has attracted much attention[2,7]. It is a secreted polypeptide with specific mitotic effects on vascular endothelial cells and promote the proliferation and migration of endothelial cells. Therefore, VEGF plays a central role in CNV[5-6]. FGF-2 is a multifunctional polypeptide that promotes angiogenesis, damage repair, and tissue regeneration[21-23]. In retinal tissue, FGF-2 interacts with VEGF to play a role in promoting angiogenesis, and participates in the occurrence and development of retinal vascular diseases[24].

    Figure 3 Typical choroidal stretched preparation images of mice White and red arrows indicate leakage point and optic disc, respectively.Area of choroidal neovascularization after Hexuemingmu administration for 1 (A); 4 (B); and 8wk (C) in mice with CNV. Statistical significance was determined using Kruskal-Wallis test (H test) and Mann-Whitney U test. Data are median and quartile range (M Q1, Q3); n=6 mice per group. aP<0.05, intervention groups vs control group.

    The experimental CNV model is mainly established by the thermal, photochemical, and mechanical damage effects of a laser, which raises the local tissue temperature suddenly,causing protein denaturation, tissue damage, and cell necrosis[1]. After RPE injury, local inflammatory reactions occur in the Bruch’s membrane and choroid[2]. In addition,a variety of growth factors and proteases secreted by the inflammatory cells jointly promote the generation of CNV[2].We confirmed that treatment with HXMM effectively alleviated the various manifestations of laser-induced CNV generation in an established experimental CNV animal model.Our findings showed that treatment with HXMM effectively and in a dose-dependent manner, reduced the fluorescence leakage of the fundus, improved the electrophysiological function of retinal cells, and reduced the expression of related proteins to alleviate the manifestations of CNV. The generation of CNV is affected by many factors including VEGF, FGF, and HIF-1α[25]. The results of our study showed that the expression of VEGF and FGF-2 in the choroid tissue was reduced by treatment with HXMM in the early stage of CNV, which showed a more significant effect at a higher dose. However, in the late stage of CNV, HXMM protected visual function from CNV by inhibiting expression of FGF-2. Furthermore, a longer duration of treatment was used to ensure the safety. In addition,tanshinol and baicalin, the active components of HXMM,have been shown to maintain the retinal microenvironment and improve retinal microcirculation[26-27]. Studies have shown that baicalin improved vascular endothelial function and ameliorated vascular inflammation and oxidative stress[28-30].Moreover, HXMM protected the retina and promoted the absorption of retinal edema. Therefore, it restored the electrophysiological function of the retina and reduced the degree of fundal fluorescence leakage[13].

    Figure 4 Hematoxylin and eosin images of mouse tissue samples White arrows: Thickened choroid. Outer nuclear layer thinned and the inner nuclear layer migrated to injured area. Pigment epithelium and choroid were significantly thickened and degree of choroidal thickening decreased in a Hexuemingmu (HXMM) dose-dependent manner.

    Figure 5 Hexuemingmu reduced expression of choroidal neovascularization (CNV)-related proteins Expression of CNV-related proteins after HXMM administration for 1 (A), 4 (B), and 8wk (C) in mice with CNV. Statistical significance determined using one-way analysis of variance. Data are means±SEM. All analyses were performed in duplicate, n=6 mice per group. aP<0.05 and bP<0.01, intervention groups vs control group.

    Figure 6 Vascular epidermal growth factor fluorescence staining images of mouse tissue samples Strong fluorescence signal of VEGF in injured area. Range of fluorescence staining region decreased with increasing Hexuemingmu dose.

    Figure 7 Hexuemingmu did not significantly affect liver and kidney function of mice Serum alanine transaminase (ALT; A); aspartate transaminase (AST; B); blood urea nitrogen (BUN; C); creatinine (Cr; D) and uric acid (UA; E) concentrations of mice with CNV. Statistical significance was determined using one-way analysis of variance. Data are means±SEM. All analyses were performed in duplicate, n=5 mice per group.

    In conclusion, this study confirmed that HXMM has a specific therapeutic effect on CNV in animal experiments. Its effect was mainly mediated by protecting the function of retinal cells,promoting the absorption of inflammatory exudation, and reducing the expression of related proteins. Our results provide a new reference for the treatment of CNV-related diseases.

    ACKNOWLEDGEMENTS

    The authors thank all those who have contributed to this study.

    Foundation:Supported by the Army Laboratory Animal Foundation of China (No.SYDW2016-011).

    Conflicts of Interest:Zhang YF, None; Jiang C, None; Zhou XH, None; Wei DY, None; Li SH, None; Long P, None; Li MH, None; Zhang ZM, None; Chen T, None; Du HJ, None.

    精品人妻视频免费看| 欧美成人精品欧美一级黄| 51国产日韩欧美| 国产aⅴ精品一区二区三区波| 精品一区二区免费观看| 99久久无色码亚洲精品果冻| 啦啦啦啦在线视频资源| 尾随美女入室| 日产精品乱码卡一卡2卡三| 美女黄网站色视频| 给我免费播放毛片高清在线观看| 亚洲在线自拍视频| 欧美三级亚洲精品| 色吧在线观看| 久久久色成人| 真人做人爱边吃奶动态| 一进一出抽搐gif免费好疼| 国产精品国产三级国产av玫瑰| 欧美成人a在线观看| 免费看日本二区| 日韩在线高清观看一区二区三区| 91午夜精品亚洲一区二区三区| 十八禁网站免费在线| 俺也久久电影网| 欧美国产日韩亚洲一区| 亚洲av中文字字幕乱码综合| 不卡一级毛片| 九九久久精品国产亚洲av麻豆| 亚洲av成人av| 能在线免费观看的黄片| 久久久久久久久中文| 成人毛片a级毛片在线播放| 在线免费观看的www视频| 欧美xxxx黑人xx丫x性爽| 免费黄网站久久成人精品| 免费看光身美女| 欧美日韩在线观看h| 成人漫画全彩无遮挡| 美女 人体艺术 gogo| 99精品在免费线老司机午夜| 午夜福利在线在线| 日本免费a在线| 97超级碰碰碰精品色视频在线观看| 小蜜桃在线观看免费完整版高清| av中文乱码字幕在线| or卡值多少钱| 欧美中文日本在线观看视频| 网址你懂的国产日韩在线| 亚洲精品日韩av片在线观看| 免费搜索国产男女视频| 亚洲久久久久久中文字幕| 天堂影院成人在线观看| 99在线人妻在线中文字幕| 日韩欧美精品v在线| 国产精品福利在线免费观看| 亚洲精品日韩在线中文字幕 | 赤兔流量卡办理| 国产高潮美女av| 亚洲国产欧洲综合997久久,| 亚洲人与动物交配视频| 欧美日韩国产亚洲二区| 国产不卡一卡二| 69av精品久久久久久| 欧美成人精品欧美一级黄| 少妇熟女aⅴ在线视频| 亚洲精品日韩av片在线观看| 国产精品久久久久久av不卡| 内地一区二区视频在线| 成年女人看的毛片在线观看| 亚洲精品色激情综合| 日本一二三区视频观看| 淫妇啪啪啪对白视频| 夜夜看夜夜爽夜夜摸| 国产蜜桃级精品一区二区三区| 69人妻影院| 久久精品国产99精品国产亚洲性色| 国产亚洲精品综合一区在线观看| 国产一区二区三区av在线 | 免费搜索国产男女视频| 精品99又大又爽又粗少妇毛片| 精品少妇黑人巨大在线播放 | 国产三级中文精品| 日日干狠狠操夜夜爽| 成人亚洲精品av一区二区| 一个人看的www免费观看视频| 国产伦精品一区二区三区视频9| 六月丁香七月| 国产高清三级在线| 亚洲激情五月婷婷啪啪| 在线免费观看的www视频| 偷拍熟女少妇极品色| 99久国产av精品国产电影| 99久国产av精品国产电影| 少妇裸体淫交视频免费看高清| 日本免费a在线| 观看美女的网站| 亚洲精品久久国产高清桃花| 亚洲av不卡在线观看| 亚洲经典国产精华液单| 天堂√8在线中文| 狂野欧美白嫩少妇大欣赏| 欧美高清成人免费视频www| 无遮挡黄片免费观看| 99国产精品一区二区蜜桃av| 久久精品国产鲁丝片午夜精品| 中文亚洲av片在线观看爽| 国产精品一区二区三区四区久久| 美女高潮的动态| 又黄又爽又免费观看的视频| 日韩三级伦理在线观看| 久99久视频精品免费| 国产精品电影一区二区三区| 亚洲在线自拍视频| 欧美zozozo另类| 久久韩国三级中文字幕| 日韩 亚洲 欧美在线| 成人精品一区二区免费| 久久亚洲精品不卡| 麻豆成人午夜福利视频| 不卡视频在线观看欧美| 精品一区二区免费观看| 少妇熟女欧美另类| 国产高清不卡午夜福利| 久久久久久久午夜电影| 免费黄网站久久成人精品| 全区人妻精品视频| 欧美性感艳星| 国产 一区 欧美 日韩| 一进一出抽搐gif免费好疼| a级一级毛片免费在线观看| 悠悠久久av| 最近视频中文字幕2019在线8| 国产69精品久久久久777片| 少妇高潮的动态图| 国产一区二区在线av高清观看| 国产69精品久久久久777片| 亚洲色图av天堂| 国产精品免费一区二区三区在线| 久久99热6这里只有精品| 人妻制服诱惑在线中文字幕| 国产精品一区二区三区四区久久| 国产69精品久久久久777片| 亚洲经典国产精华液单| 亚洲内射少妇av| 国产亚洲欧美98| 国产黄片美女视频| 亚州av有码| 在线免费观看不下载黄p国产| 国产精品99久久久久久久久| 97超视频在线观看视频| 国产精品伦人一区二区| 国产成人精品久久久久久| 91久久精品国产一区二区三区| 99热全是精品| 全区人妻精品视频| 亚洲av一区综合| 别揉我奶头 嗯啊视频| 国产午夜福利久久久久久| 国产一区二区亚洲精品在线观看| 成年免费大片在线观看| 日本-黄色视频高清免费观看| 丰满乱子伦码专区| 国内久久婷婷六月综合欲色啪| 不卡一级毛片| 69人妻影院| 老女人水多毛片| 久久热精品热| 国产精品久久久久久av不卡| 天堂网av新在线| 日本五十路高清| 毛片一级片免费看久久久久| 日韩一区二区视频免费看| 人人妻人人澡欧美一区二区| 亚洲va在线va天堂va国产| a级一级毛片免费在线观看| 97人妻精品一区二区三区麻豆| 午夜福利在线观看吧| 国产在线男女| 国产精品免费一区二区三区在线| 如何舔出高潮| 久久热精品热| 久久午夜福利片| 国产在视频线在精品| 色哟哟哟哟哟哟| 乱人视频在线观看| 国产精品美女特级片免费视频播放器| 最近的中文字幕免费完整| 国产私拍福利视频在线观看| av在线亚洲专区| 亚洲无线观看免费| 2021天堂中文幕一二区在线观| 欧美性猛交╳xxx乱大交人| 夜夜夜夜夜久久久久| 欧美绝顶高潮抽搐喷水| 无遮挡黄片免费观看| 天美传媒精品一区二区| 国产单亲对白刺激| 久久久久久久久久成人| 国产av在哪里看| 床上黄色一级片| 国产精品一区www在线观看| 亚洲在线自拍视频| 寂寞人妻少妇视频99o| 99热网站在线观看| 免费观看人在逋| 少妇熟女欧美另类| 波多野结衣巨乳人妻| 亚洲国产高清在线一区二区三| 日韩制服骚丝袜av| 国模一区二区三区四区视频| 国产在线精品亚洲第一网站| 自拍偷自拍亚洲精品老妇| 国产成人影院久久av| 亚洲国产日韩欧美精品在线观看| 哪里可以看免费的av片| 国内久久婷婷六月综合欲色啪| 日韩一本色道免费dvd| 99久久无色码亚洲精品果冻| 看片在线看免费视频| 男人舔女人下体高潮全视频| 久久精品国产亚洲网站| 亚洲中文字幕日韩| 偷拍熟女少妇极品色| 午夜精品在线福利| 亚洲,欧美,日韩| 男女下面进入的视频免费午夜| 综合色丁香网| 国产又黄又爽又无遮挡在线| 亚洲一区二区三区色噜噜| 久久精品综合一区二区三区| 韩国av在线不卡| 国产精品不卡视频一区二区| 两个人的视频大全免费| 久久精品久久久久久噜噜老黄 | 尾随美女入室| 久久精品国产自在天天线| 欧美成人一区二区免费高清观看| 真实男女啪啪啪动态图| 欧美日韩精品成人综合77777| 最近手机中文字幕大全| 99久久九九国产精品国产免费| 国产一区二区在线av高清观看| 亚洲av电影不卡..在线观看| 日韩人妻高清精品专区| 在线观看美女被高潮喷水网站| 可以在线观看的亚洲视频| 亚洲av中文字字幕乱码综合| 99久久久亚洲精品蜜臀av| 乱人视频在线观看| 亚洲久久久久久中文字幕| 国产探花极品一区二区| 久久精品91蜜桃| 亚洲自偷自拍三级| 久久韩国三级中文字幕| 蜜桃久久精品国产亚洲av| 国产精品国产高清国产av| 久久精品综合一区二区三区| 免费观看精品视频网站| 91麻豆精品激情在线观看国产| 久久精品国产99精品国产亚洲性色| 悠悠久久av| 国产精品久久视频播放| 天天一区二区日本电影三级| 一级毛片久久久久久久久女| 高清日韩中文字幕在线| 校园春色视频在线观看| 亚洲精品一区av在线观看| 99久久精品国产国产毛片| 亚洲最大成人中文| 少妇裸体淫交视频免费看高清| 免费观看精品视频网站| 久久久成人免费电影| 麻豆av噜噜一区二区三区| 在线观看66精品国产| 深夜精品福利| 国产美女午夜福利| 日韩欧美在线乱码| 午夜福利在线观看免费完整高清在 | 最近最新中文字幕大全电影3| 欧美三级亚洲精品| 有码 亚洲区| 久久精品国产99精品国产亚洲性色| 天天一区二区日本电影三级| 草草在线视频免费看| 亚洲精品久久国产高清桃花| 女人被狂操c到高潮| 麻豆一二三区av精品| 色在线成人网| 97超级碰碰碰精品色视频在线观看| 免费在线观看影片大全网站| 免费观看的影片在线观看| 看十八女毛片水多多多| 女人十人毛片免费观看3o分钟| 午夜免费男女啪啪视频观看 | 免费av观看视频| 国产精品一区二区三区四区久久| 国产在线精品亚洲第一网站| 久久中文看片网| 九九爱精品视频在线观看| 日韩欧美精品v在线| 亚洲人与动物交配视频| 国产乱人视频| 国产成人aa在线观看| 国产一级毛片七仙女欲春2| 欧美成人a在线观看| 伊人久久精品亚洲午夜| 嫩草影院新地址| 亚洲精华国产精华液的使用体验 | 国产成人aa在线观看| 国产午夜福利久久久久久| 久久午夜福利片| 黄色一级大片看看| 亚洲七黄色美女视频| 久久久久免费精品人妻一区二区| 乱人视频在线观看| 日韩成人伦理影院| 俄罗斯特黄特色一大片| 国产成人一区二区在线| 一级毛片久久久久久久久女| 搞女人的毛片| 亚洲最大成人av| 国产91av在线免费观看| 国产极品精品免费视频能看的| 色吧在线观看| 嫩草影院入口| 成人一区二区视频在线观看| 国产午夜精品久久久久久一区二区三区 | 亚洲欧美清纯卡通| av福利片在线观看| 在线观看午夜福利视频| 精品福利观看| videossex国产| 伦理电影大哥的女人| 搡老妇女老女人老熟妇| 国产午夜精品论理片| 在线免费观看的www视频| 美女cb高潮喷水在线观看| 五月玫瑰六月丁香| 精品一区二区三区视频在线| 国产大屁股一区二区在线视频| 色播亚洲综合网| 最近最新中文字幕大全电影3| 久久人人精品亚洲av| 超碰av人人做人人爽久久| 久久久久精品国产欧美久久久| 神马国产精品三级电影在线观看| 丝袜美腿在线中文| 天堂网av新在线| 最好的美女福利视频网| 插阴视频在线观看视频| 在线播放无遮挡| 亚洲av.av天堂| 日韩亚洲欧美综合| 内射极品少妇av片p| 免费不卡的大黄色大毛片视频在线观看 | 波多野结衣高清无吗| 人妻少妇偷人精品九色| 波多野结衣高清无吗| 午夜激情福利司机影院| 神马国产精品三级电影在线观看| 久久99热6这里只有精品| 99国产极品粉嫩在线观看| 非洲黑人性xxxx精品又粗又长| 欧美一区二区精品小视频在线| 日韩亚洲欧美综合| 在线观看av片永久免费下载| 秋霞在线观看毛片| 久久久午夜欧美精品| 欧美xxxx性猛交bbbb| ponron亚洲| 乱系列少妇在线播放| 国产爱豆传媒在线观看| 中文字幕精品亚洲无线码一区| 嫩草影视91久久| 熟女人妻精品中文字幕| 国产精品久久久久久亚洲av鲁大| 最新在线观看一区二区三区| 亚洲精品日韩av片在线观看| 欧美另类亚洲清纯唯美| 亚洲综合色惰| 精品一区二区三区av网在线观看| 长腿黑丝高跟| 18+在线观看网站| 亚洲性夜色夜夜综合| 97超碰精品成人国产| 俄罗斯特黄特色一大片| 国产成人freesex在线 | 免费在线观看影片大全网站| 亚洲人与动物交配视频| 亚洲乱码一区二区免费版| 亚洲成a人片在线一区二区| 亚洲美女黄片视频| 亚洲av电影不卡..在线观看| 久久久久久久久大av| av在线老鸭窝| 精品久久久久久久久av| 日日摸夜夜添夜夜添av毛片| 91在线精品国自产拍蜜月| 久久久久国产网址| 18禁在线无遮挡免费观看视频 | 欧美xxxx性猛交bbbb| 欧美又色又爽又黄视频| 亚洲精品456在线播放app| 婷婷色综合大香蕉| 亚洲图色成人| 国产精品一区二区三区四区久久| 国产在线男女| 搡老妇女老女人老熟妇| 人人妻人人澡人人爽人人夜夜 | 国产精品一区二区三区四区免费观看 | 亚洲中文日韩欧美视频| 欧美性感艳星| 色综合色国产| 亚洲欧美清纯卡通| 日韩人妻高清精品专区| 一级av片app| 久久精品久久久久久噜噜老黄 | 午夜精品国产一区二区电影 | 国产一区二区激情短视频| 日韩欧美在线乱码| av在线播放精品| 久久精品国产鲁丝片午夜精品| 色噜噜av男人的天堂激情| 在线免费观看不下载黄p国产| 成年女人看的毛片在线观看| 国产视频一区二区在线看| 亚洲自拍偷在线| 日韩av在线大香蕉| 亚洲,欧美,日韩| 欧美精品国产亚洲| 成人亚洲精品av一区二区| 色在线成人网| 嫩草影院入口| 中文字幕免费在线视频6| 日产精品乱码卡一卡2卡三| 99热6这里只有精品| 1000部很黄的大片| 午夜精品在线福利| 国语自产精品视频在线第100页| av在线天堂中文字幕| 亚洲在线观看片| 精品一区二区免费观看| 久久久久精品国产欧美久久久| 日本精品一区二区三区蜜桃| 人人妻人人看人人澡| 免费大片18禁| 国产白丝娇喘喷水9色精品| 国产精华一区二区三区| 日韩av不卡免费在线播放| 国产69精品久久久久777片| 少妇丰满av| av在线老鸭窝| 我要看日韩黄色一级片| 日本a在线网址| 乱码一卡2卡4卡精品| 久久国产乱子免费精品| 亚洲欧美中文字幕日韩二区| 欧美最新免费一区二区三区| 免费看美女性在线毛片视频| 亚洲丝袜综合中文字幕| 听说在线观看完整版免费高清| 伊人久久精品亚洲午夜| 亚洲精品国产av成人精品 | 午夜免费激情av| 日韩欧美 国产精品| 韩国av在线不卡| 少妇丰满av| 中文在线观看免费www的网站| 亚洲熟妇熟女久久| 中国美白少妇内射xxxbb| 欧美zozozo另类| 亚洲人成网站在线播放欧美日韩| 午夜激情欧美在线| 国产一区二区在线观看日韩| 又黄又爽又刺激的免费视频.| a级毛片免费高清观看在线播放| 免费看光身美女| 国产成人影院久久av| 日本免费一区二区三区高清不卡| 欧美成人免费av一区二区三区| 久久中文看片网| 成人精品一区二区免费| 免费人成视频x8x8入口观看| 亚洲欧美精品自产自拍| 色视频www国产| 精品一区二区免费观看| 国产在线男女| 国产成人a∨麻豆精品| av在线蜜桃| 麻豆精品久久久久久蜜桃| 久久这里只有精品中国| 国产又黄又爽又无遮挡在线| 国产精品爽爽va在线观看网站| 成人高潮视频无遮挡免费网站| 六月丁香七月| 日韩一本色道免费dvd| 成熟少妇高潮喷水视频| АⅤ资源中文在线天堂| 日本熟妇午夜| 国内精品宾馆在线| 日韩欧美精品v在线| 中文在线观看免费www的网站| 91久久精品国产一区二区三区| 伦理电影大哥的女人| 麻豆国产av国片精品| 午夜影院日韩av| 亚洲天堂国产精品一区在线| 久久久精品94久久精品| 婷婷精品国产亚洲av| 能在线免费观看的黄片| 最新中文字幕久久久久| 国产精品野战在线观看| 亚洲av熟女| 国产极品精品免费视频能看的| 成年免费大片在线观看| 国产成人91sexporn| 亚洲人成网站在线观看播放| 夜夜爽天天搞| 嫩草影视91久久| 高清毛片免费看| 国国产精品蜜臀av免费| 亚洲av五月六月丁香网| 国产成人aa在线观看| 午夜a级毛片| 欧美最黄视频在线播放免费| 欧美激情在线99| 日韩欧美 国产精品| 亚洲av不卡在线观看| АⅤ资源中文在线天堂| 亚洲国产精品合色在线| 亚洲国产色片| 国产不卡一卡二| 欧美最黄视频在线播放免费| 日日摸夜夜添夜夜爱| 人妻夜夜爽99麻豆av| 免费看日本二区| 日韩欧美三级三区| 国产免费男女视频| 亚洲欧美日韩高清专用| eeuss影院久久| 99精品在免费线老司机午夜| 免费人成视频x8x8入口观看| 又粗又爽又猛毛片免费看| 亚洲人成网站在线播| 久久久久久久午夜电影| 亚洲成人av在线免费| 在线看三级毛片| 精品一区二区免费观看| 中国美白少妇内射xxxbb| 亚洲欧美清纯卡通| 成人三级黄色视频| 99热精品在线国产| 夜夜爽天天搞| 99国产极品粉嫩在线观看| 一本精品99久久精品77| av在线天堂中文字幕| 亚洲第一区二区三区不卡| 国产精品伦人一区二区| 亚州av有码| 亚洲欧美日韩东京热| 国产欧美日韩一区二区精品| 久久精品国产自在天天线| 免费一级毛片在线播放高清视频| 免费人成视频x8x8入口观看| 日韩精品青青久久久久久| 又爽又黄a免费视频| 国产一区二区三区在线臀色熟女| 老熟妇乱子伦视频在线观看| 精品无人区乱码1区二区| 久久精品国产清高在天天线| 色5月婷婷丁香| 久久久色成人| 一区福利在线观看| 午夜视频国产福利| 俺也久久电影网| 日本色播在线视频| 可以在线观看毛片的网站| 美女内射精品一级片tv| 国产精品福利在线免费观看| 人妻丰满熟妇av一区二区三区| 免费人成视频x8x8入口观看| 波多野结衣高清无吗| 精品人妻一区二区三区麻豆 | 精品久久久久久久人妻蜜臀av| 97热精品久久久久久| 久久久午夜欧美精品| 精品久久久久久久久久免费视频| 少妇裸体淫交视频免费看高清| 两个人的视频大全免费| 一级av片app| 国国产精品蜜臀av免费| 国产精品亚洲一级av第二区| 老司机午夜福利在线观看视频| 成年女人永久免费观看视频| 亚洲人成网站在线播放欧美日韩| 国产精品久久久久久久久免| 欧美一区二区国产精品久久精品| 熟女电影av网| 午夜福利成人在线免费观看| 中文字幕熟女人妻在线| 亚洲一区二区三区色噜噜| 亚洲精品乱码久久久v下载方式| 黄色视频,在线免费观看| av在线蜜桃| 尤物成人国产欧美一区二区三区| 在线观看美女被高潮喷水网站| 国产爱豆传媒在线观看| 老女人水多毛片| 亚洲aⅴ乱码一区二区在线播放| 国产三级中文精品| 亚洲av成人精品一区久久| 夜夜爽天天搞| 国产精品无大码| 真人做人爱边吃奶动态| 久久国内精品自在自线图片|