• <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 ?

    The protective effect of icariin on glucocorticoid-damaged BMECs explored by microfluidic organ chip

    2022-07-11 03:39:34TngqiLiQingyuZhangFuqiangGaoYaiLiuWiSunaYiyangDong
    Chinese Chemical Letters 2022年6期

    Tngqi Li,Qingyu Zhang,Fuqiang Gao,Yai Liu,Wi Suna,,?,Yiyang Dong

    a Department of Orthopedics,Peking University China-Japan Friendship School of Clinical Medicine,Beijing 100029,China

    b Department of Orthopedics,Peking University Shougang Hospital,Beijing 100144,China

    c Department of Orthopedics,Shandong Provincial Hospital Affiliated to Shandong First Medical University,Ji’nan 250021,China

    d Department of Orthopedics,China-Japan Friendship Hospital,Beijing 100029,China

    e Office of Academic Affairs,Beijing University of Chemical Technology,Beijing 100029,China

    f Department of Pharmaceutical Analysis,College of Life Science and Technology,Beijing University of Chemical Technology,Beijing 100029,China

    Keywords:Osteonecrosis of the femoral head Bone microvascular endothelial cells Glucocorticoid Microfluidic chip Bioinformatic analysis

    ABSTRACT Osteonecrosis of the femoral head (ONFH) is a devastating musculoskeletal disease characterized by the impaired circulation of bone.The purpose of this study was to explore the underlying mechanisms of the protective effect of icariin on the glucocorticoid-induced injury of bone microvascular endothelial cells(BMECs).Normal BMECs were extracted from the femoral heads by enzymatic isolation and magneticactivated cell sorting methods.Dexamethasone and icariin were used to intervene BMECs in microfluidic organ chips,and phalloidin staining was conducted to observe the cell morphology and viability.Then next-generation transcriptome sequencing and real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were performed to identify the differentially expressed genes (DEGs) in different groups.Through the microfluidic organ chip,it can be observed that after dexamethasone intervention,the filamentous structure in cell fibers disappeared and the cell morphology changed from spindle to round until death.Icariin could relieve these changes and showed a protective effect on glucocorticoiddamaged BMECs.In addition,201 DEGs were detected between the icariin protection group and the dexamethasone group,which were significantly enriched in 17 signaling pathways.8 of the top ten selected hub genes (IL6,PTGS2,VEGFA, etc.) were confirmed by qRT-PCR.Transcription factors (TFs)-gene network showed 63 connections between 18 TFs and 12 DEGs.For instance,GATA2 could regulate 5 DEGs.The associations between 92 miRNA and 12 DEGs were visualized in a miRNA-gene network.The hub miRNA,has-mir-335–5p was predicted to interact with 8 DEGs (PTGS2,VEGFA, etc.).Microfluidic organ chips could provide excellent morphological results for cell experiments,by which it could be observed that icariin showed a protective effect on the glucocorticoid-induced injury of BMECs.Beside,these DEGs,possible regulatory TF (GATA2,FOXC1, etc.) and miRNA (has-mir-335–5p) might be dysregulated in the initiation of ONFH and have prospective importance in ONFH diagnosis and therapy.

    Osteonecrosis of the femoral head (ONFH) is a common musculoskeletal disease with rapid progression and a high disability rate [1].It imposes a heavy economic burden on individual families and society.As its pathogenesis remains unclear,treatment choice is challenging to be exact [2].In the early stage of ONFH,patients normally have no obvious clinical symptoms,and when there are pain and mobility problems,the course of the disease is often severe,and the femoral head often has collapsed [3,4].At present,more and more scholars believe that the disruption of blood supply around the femoral head is closely related to the occurrence of ONFH (Fig.S1 in Supporting information) [5,6].Therefore,the study of bone microvascular endothelial cells (BMECs) that constitute bone microvessels may help to further reveal the pathogenesis of ONFH and provide alternatives for treatment.Icariin is the main active ingredient of epimedium brevicornum,a traditional Chinese herb known for “strengthening bone and tonifying kidney”[7].Icariin could decrease the risk of glucocorticoid-induced ONFH by promoting the proliferation and mineralization of osteoblasts and decreasing the activity of osteoblasts but efforts to understand the underlying mechanisms are still ongoing [7,8].

    In recent years,multidisciplinary and cross-professional collaboration has become the trend of clinical investigation.The continuous development of microfluidic technology is bound to drive the growth of biological medicine and other industries.Many sophisticated microfluidic devices can be used to simulate tissues and organs,and provide physiological microenvironments for cells [9–11].More advanced microfluidic models have been used for diseases investigation and drug screening [12–14].Sinhaet al.[15]reported a single-cell analysis using microfluidic technology,which provided development platforms for novel therapies and rapid diagnostics.Mature organ chips can even replace animal experiments in biomedical,pharmaceutical,and toxicology applications,showing excellent development potential.Jo and Lee [16]used microfluidic technology to produce polymeric microparticles (MPs) with designed structure and composition by precise control of multiphase flow on a micro-scale.These MPs can be used in curative effect observation,3D cell culture,and biomolecular sensing.The application prospects of microfluidics technology,from simple cell culture chip to organ chip,are unlimited.

    The dysregulation of various RNAs,such as circRNA,miRNA,and mRNA,has been used to study the pathogenesis of orthopedic disorders such as osteoarthritis and osteonecrosis [17,18].Xianget al.[19]extracted synovial samples of knee osteoarthritis and compared them with controls.They found that 122 circRNAs were differentially expressed between two groups,suggesting that these circRNAs might be related to the pathogenesis of osteoarthritis.Messenger RNA (mRNA) is a type of single-stranded ribonucleic acid transcribed from a strand of DNA and detecting the change of mRNA expression level can help to understand the change of cell protein synthesis.Leijtenet al.[20]found some mRNAs such as GREM1,FRZB and DKK1 in articular cartilage were inversely associated with osteoarthritis disease.Udomsinprasertet al.[21]demonstrated that the level of interleukin-34 mRNA expression could predict the severity of synovitis in knee osteoarthritis.It can be seen that in the pathogenesis of many diseases,changes at the cellular level,such as various protein indicators and different RNA expression levels,tend to appear earlier in comparison with clinical symptoms,and identification of these differences may facilitate the early detection and diagnosis of these diseases.

    In the present study,to explore the pathogenesis of glucocorticoid-induced ONFH and the protective effect of icariin,BMECs were extracted and inoculated in microfluidic organ chips,and treated with glucocorticoids and icariin to observe the changes in cell morphology.Next,the mRNA differential expression of cells after different interventions was detected by next-generation transcriptome sequencing technology.The differentially expressed genes (DEGs) were further explored to predict the hub genes by using bioinformatics analysis.Finally,the screened hub genes were verified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR).We hope these findings will help us to further explore the pathogenesis of ONFH and guide a more accurate early diagnosis and treatment.

    As a result,a live view and schematic diagram of the chip are shown in Fig.1.There are four channels with precisely the same parameters.Fig.2 shows the isolated cells were almost 100% positive in CD31 and vWF and 100% negative in CD133,indicating that these cells were BMECs.Under the transmission electron microscope,these cells showed polygonal shape with large ovular nuclei and the cell surface was covered with microvilli formed by cytoplasm bulge.

    Fig.2.(A) Immunofluorescence staining of endothelial cell markers.(B) Observation under transmission electron microscope observation.

    Through image acquisition and data analysis (Fig.3),we can find that the number of surviving cells in group C (34.7±10.3) is significantly less than that in group A (73.3±6.7,P=0.015).The cytoskeleton area coverage percentage in group B (26.3% ± 3.9%,P=0.003) and C (18.4% ± 4.2%,P<0.001) is significantly less than that in group A (43.5% ± 3.1%).In addition,the comparison of fiber length showed that group B (6906.62±1721.75,P=0.001) and C(7961.69±1891.98,P=0.001) is significantly shorter than the control group (16,708.15±1181.78).As shown in group C (Fig.3A),the skeleton fiber length of individual cells recovered somewhat.However,due to the small number of cells,the overall fiber length showed no significant difference between groups B and C(P=0.72).In addition to data analysis,we can also see from morphology that the normal morphology of cells disappeared after dexamethasone (DXMS) injury,and the cells were deformed from spindle to ellipse or even round.When these connections between cells were broken,blood vessel structures were damaged.After icariin intervention,the morphology of single cells recovered to some extent,but there was still a difference compared with the blank group.

    Fig.3.Microfluidic experiment part results.(A) The left is confocal microscopy image of BMECs in the three groups (40-fold oil lens;scale bars,100 μm).The right is diagram of fiber length analysis.(B) Data statistics of all the chips conducted by ImageJ software.(C) Comparison of statistical differences between the three groups.?P < 0.05 vs.group A.Group A,0.0 mg/mL dexamethasone;Group B,0.6 mg/mL dexamethasone;Group C,0.6 mg/mL dexamethasone+icariin.

    Unlike former literature,in this study,the protective drug icariin was used for post-intervention rather than pre-protection.The primary purpose of this study was to investigate whether icariin could still protect BMECs 24 h after glucocorticoid injury.Microfluidics chip experiments showed that after icariin intervention,the morphology of BMECs was improved,and the filamentous structure of some cells was restored,but the morphology and length of BMECs were still different compared with the blank group.In addition,the number of cells was also significantly reduced,indicating that after the intervention of a high concentration of glucocorticoid,the cells would gradually undergo morphological changes and develop to cell death.Although the highconcentration glucocorticoid intervenes stopped after 24 h and some cell morphology recovered after icariin protection,the fact that a large number of cells died could not be changed.

    Data normalization was performed to confirm the crosscomparability of biological variability between different samples(Fig.S2A in Supporting information).In total,143 DEGs were identified between the DXMS group and the control group,including 102 upregulated and 41 downregulated genes listed in Table S2(Supporting information).Meanwhile,201 DEGs were detected between the icariin protection group and the DXMS group,including 143 upregulated and 58 downregulated genes (Table S3 in Supporting information).In addition,a volcano plot and a heatmap of all DEGs between three groups were generated using the R ggplot2 package (Figs.S2B and S3 in Supporting information).

    Baiet al.[22]found that in patients with ONFH,the alteration of bone trabecular tissue structure was preceded by discovering alteration of mRNA profile through gene sequencing.Liet al.[23]studied the difference of serum miRNAs in patients with both systemic lupus erythematosus (SLE) and ONFH,and those with SLE alone.They found 42 differentially abundant miRNAs (14 upregulated and 28 downregulated) between the two groups.These could also be targets for future treatments.Beside,the mRNA of cells also changed considerably after icariin treatment.These upregulated or downregulated genes provide a reference for the study of its mechanism.Yuet al.[24]conducted a similar experiment using glucocorticoid intervention with BMECs and finally found 239 differentially expressed long noncoding RNAs.Xieet al.[25]established the model of rabbit ONFH by methylprednisolone.After the intervention with icariin,it was found that the repairability of bone defect in the icariin group was significantly higher than that in the control group,and the expression of VEGF was also considerably elevated.Yuet al.[26]found that icariin could significantly promote the migration of BMECs and the expression of factors related to tubular and angiogenesis.However,no one has previously studied the molecular mechanism by which icariin affects BMECs,and our study provides an essential reference for the study of its molecular mechanism.

    Gene ontology (GO) enrichment analysis of 143 DEGs between the DXMS group and the control group was performed to identify the most relevant biological processes (BPs),molecular functions (MFs),and cellular components (CCs).The top ten enriched terms in BP,CC,and MF were presented in Figs.S4A–C (Supporting information).Meanwhile,based on the KEGG pathway analysis,the DEGs were significantly enriched in 8 signaling pathways,such as cytokine-cytokine receptor interaction (7 DEGs) and PI3KAkt signaling pathway (7 DEGs) (Fig.S4D in Supporting information).There is also extensive literature investigating the functions of these pathways,such as inhibition of glycogen synthesis and promotion of glycolysis [27].Inhibition of PI3K/Akt/mTOR signaling pathway could promote autophagy in mouse articular cartilage[28].

    Meanwhile,GO enrichment analysis of 201 DEGs between the icariin protection group and the DXMS group was performed and the top ten enriched terms in BP,CC,and MF are presented in Fig.S5A–C (Supporting information).Meanwhile,based on KEGG pathway analysis,the DEGs were significantly enriched in 17 signaling pathways,such as tumor necrosis factor (TNF) signaling pathway (12 DEGs),PI3K-Akt signaling pathway (12 DEGs) and pathways in cancer (11 DEGs) (Fig.S5D in Supporting information).This suggested that the PI3K-Akt signaling pathway plays an important role in both the deleterious effect of DMXS and the protective effect of icariin on BMECs.The top 20 terms associated with 201 DEGs,enriched in BP,CC and MF as well as various KEGG pathways are presented in Table S4 (Supporting information).

    The interactions between the proteins expressed from DEGs between the icariin protection group and the DXMS group consisted of 139 nodes and 404 edges (Fig.S6A in Supporting information).In addition,11 of the DEGs revealed connectivity degrees>15:IL6 (Degree=47),VEGFA (Degree=38),FOS (Degree=30),PTGS2(Degree=26),ATF4 (Degree=22),CXCR4 (Degree=17),ATF3 (Degree=17),ICAM1 (Degree=17),DDIT3 (Degree=17),CXCL12 (Degree=16),and NOS3 (Degree=16).In addition to connectivity degrees,top 10 hub genes were also selected by CytoHubba based on the maximal clique centrality (MCC) and maximum neighborhood component (MNC) classification methods (Table S5,Fig.S7 in Supporting information),showing overlapping genes with different priority.Seven significant modules (Fig.S6B in Supporting information) were obtained by module analysis using MCODE from Cytoscape.

    The top ten selected hub genes by MCC were further determined by qRT-PCR analysis,the expression of eight genes (IL6,PTGS2,VEGFA,CXCR4,ICAM1,CXCL12,MMP3,and FOS) between the icariin protection group and the DXMS group showed a statistical difference (P <0.05,Fig.4).IL6 can promote the development of blood cells [29];the expression of IL6 was significantly decreased in the DXMS group,and significantly increased in the icariin protection group.COX-2 encoded by PTGS2 can promote cell proliferation,inhibit cell apoptosis,and promote angiogenesis [30].CXCR4,ICAM1,and other genes could also regulate angiogenesis[31,32].Compared with the DXMS group,the expression of these genes was significantly increased in the icariin protection group.These results indicated that icariin can effectively reduce the damage of BMECs induced by DXMS.This is similar to the research conclusions in many related orthopedic diseases.For example,icariin can relieve knee osteoarthritis in mice [33]and effectively prevent postmenopausal osteoporosis in women through inhibiting NLRP3 mediated pyroptosis [34].

    According to the binding site and genetic coordinate position provided on ENCODE,the potential TFs targeting DEGs between the icariin protection group and the DXMS group were predicted.Eventually,a total of 63 associations between 18 TFs and 12 DEGs were obtained (Fig.S8 in Supporting information).The miRNAgene pairs were identified through network analysis of 201 DEGs between the icariin protection group and the DXMS group using the TarBase and miRTarBase databases.Finally,a total of 128 associations between 92 microRNAs and 12 DEGs were identified (Fig.S9 in Supporting information).Therefore,the TF-gene-miRNA interaction involved 12 genes,18 TFs,and 92 microRNAs (Fig.5,Table S6 in Supporting information).For instance,GATA2 regulated 5 DEGs (e.g.,VEGFA and CXCR4),IRF1 regulated 5 DEGs (e.g.,ICAM1 and VEGFA) and TFDP1 regulated 5 DEGs (e.g.,ICAM1 and VEGFA).VEGFA was regulated by 50 interacting microRNAs,while a hub microRNA,hsa-mir-335–5p,was predicted to interact with 8 genes(PTGS2,VEGFA,ICAM1,NOS3,KLF4,CXCL2,IL6,and FOS).Has-miR-335–5p,as an essential regulator of bone homeostasis,has also been demonstrated in other orthopedic diseases.Tornero-Estebanet al.[35]found that the expression of ha-miR-335–5p was significantly down-regulated during induced osteogenesis of bone marrow mesenchymal stem cells.In the pathogenesis of ONFH,we believe that has-miR-335–5p also plays a particular regulatory role,which needs to be further confirmed in the future.

    Different from traditional culture methods,microfluidic chips provide a new environment for cells to live in.The main feature of microfluidic platform is fluidity.Cells can be fused with rat tail collagen,suspended in it,and can move around slightly.Meanwhile,cell mobility can be increased by installing microfluidic pumps.In this way,different from ordinary Petri dishes,cells are no longer limited by plant growth and can grow to the surrounding space,which increases the chance of contact between cells and makes it easier to form tubular and vascular structures.Another feature of this platform is the independent operation.Four channels can be operated independently.Affected by liquid surface tension,the material exchange can only be realized when liquid exists in adjacent channels simultaneously,so the four channels are both independent and interconnected.Our previous research [36]has confirmed that microfluidic chips can provide high-definition morphological results for cell experiments,which is significantly better than the images collected in ordinary Petri dishes.As a cell culture device,this microfluidic chip can also be used to culture other vascular endothelial cells,such as human umbilical venous endothelial cells.We will further verify the possibility for culturing non-vascular cell culture in this chip.However,the feature of disposability should not be neglected.The matrix cured in channels cannot be removed,and therefore after cell intervention and dyeing,the chip will be scrapped.

    Through a microfluidic organ chip investigation,we intuitively saw the damage of dexamethasone to BMECs and the protective effect of icariin.Beside,for the first time we summarized the effect of dexamethasone and icariin on mRNA expression of BMECs and the RNA network map was constructed by bioinformatic analysis.The GO and KEGG pathways analysis revealed that PI3K-Akt signaling pathway was one of the enrichment pathways of DEGs that may be involved in the intervention of dexamethasone and icariin on BMECs.These identified DEGs,possible regulatory TF (GATA2,FOXC1,etc.) and miRNA (has-mir-335–5p) might be dysregulated in the initiation of ONFH and have prospective importance in ONFH diagnosis and therapy.

    Fig.4.Validation of the top ten differentially expressed mRNAs between the icariin protection group and the DXMS group by qRT-PCR.(A) IL6,(B) PTGS2,(C) VEGFA,(D)CXCR4,(E) ICAM1,(F) CXCL2,(G) CXCL12,(H) MMP3,(I) FOS,(J) CX3CL1.?P < 0.05,??P < 0.01.

    Fig.5.The TF-gene-microRNA interaction network between the icariin protection group and the DXMS group.

    Declaration of competing interest

    The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

    Acknowledgments

    This study was supported by the Beijing Natural Science Foundation (No.7182146),the Biomedical Translational Engineering Research Center of BUCT-CJFH (No.RZ2020–02),the National Natural Science Foundation of China (No.82072524),the Young Taishan Scholars Program of Shandong Province (No.tsqn201909183),the Academic promotion program of Shandong First Medical University(No.2020RC008) and the Natural Science Foundation of Shandong Province (No.ZR201911090016).

    Supplementary materials

    Supplementary material associated with this article can be found,in the online version,at doi:10.1016/j.cclet.2021.11.093.

    97碰自拍视频| 午夜福利一区二区在线看| 亚洲人成77777在线视频| 一区二区三区精品91| 如日韩欧美国产精品一区二区三区| 男女之事视频高清在线观看| 麻豆一二三区av精品| 久久久久久久久中文| 窝窝影院91人妻| 一级毛片精品| av国产精品久久久久影院| 丁香六月欧美| 免费在线观看日本一区| 成人精品一区二区免费| 99国产极品粉嫩在线观看| 国产精品九九99| 18禁裸乳无遮挡免费网站照片 | 91成人精品电影| 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| 搡老乐熟女国产| a级毛片黄视频| 天天影视国产精品| 亚洲中文av在线| 国产91精品成人一区二区三区| 激情在线观看视频在线高清| a级毛片黄视频| 成年版毛片免费区| 黑人巨大精品欧美一区二区mp4| 夜夜看夜夜爽夜夜摸 | 久久人人精品亚洲av| 亚洲国产精品sss在线观看 | 麻豆av在线久日| 亚洲五月婷婷丁香| 国产精品偷伦视频观看了| 久久精品亚洲av国产电影网| 国产欧美日韩一区二区精品| 免费在线观看亚洲国产| 亚洲成国产人片在线观看| 免费在线观看亚洲国产| 成人国语在线视频| 国产97色在线日韩免费| 麻豆成人av在线观看| 亚洲国产精品sss在线观看 | 老司机深夜福利视频在线观看| 久久人人97超碰香蕉20202| 在线天堂中文资源库| 丝袜人妻中文字幕| 我的亚洲天堂| 动漫黄色视频在线观看| 亚洲成人精品中文字幕电影 | av片东京热男人的天堂| 热re99久久国产66热| 身体一侧抽搐| 亚洲精品国产精品久久久不卡| 国产熟女午夜一区二区三区| 黑人操中国人逼视频| 水蜜桃什么品种好| 久久精品成人免费网站| 国产黄a三级三级三级人| 宅男免费午夜| www.999成人在线观看| 精品人妻在线不人妻| 亚洲第一av免费看| 在线观看免费视频网站a站| 男女床上黄色一级片免费看| 亚洲人成77777在线视频| 性少妇av在线| 老熟妇乱子伦视频在线观看| 国产精品国产av在线观看| 国产精品久久视频播放| 最近最新中文字幕大全电影3 | 久久久久久亚洲精品国产蜜桃av| 99国产精品一区二区三区| 无遮挡黄片免费观看| 性色av乱码一区二区三区2| 少妇的丰满在线观看| 国产精品久久久久成人av| 久久国产精品男人的天堂亚洲| 亚洲国产精品一区二区三区在线| 91大片在线观看| 女同久久另类99精品国产91| 无遮挡黄片免费观看| 亚洲成av片中文字幕在线观看| 久久人妻熟女aⅴ| 男人的好看免费观看在线视频 | www.精华液| 亚洲一区二区三区不卡视频| 久久国产精品男人的天堂亚洲| 交换朋友夫妻互换小说| 最新美女视频免费是黄的| 亚洲熟女毛片儿| 精品乱码久久久久久99久播| 热99re8久久精品国产| 99久久99久久久精品蜜桃| 男女午夜视频在线观看| 啪啪无遮挡十八禁网站| 黄色a级毛片大全视频| 在线观看www视频免费| 老汉色av国产亚洲站长工具| 欧美日韩福利视频一区二区| 免费在线观看视频国产中文字幕亚洲| 男女做爰动态图高潮gif福利片 | 久久精品国产99精品国产亚洲性色 | 90打野战视频偷拍视频| 中文字幕色久视频| 亚洲人成网站在线播放欧美日韩| 国产精品九九99| 亚洲成a人片在线一区二区| 久久精品国产综合久久久| 亚洲国产精品合色在线| 亚洲国产欧美一区二区综合| 在线看a的网站| 一级作爱视频免费观看| 美女扒开内裤让男人捅视频| 精品高清国产在线一区| 少妇 在线观看| 亚洲国产欧美日韩在线播放| 亚洲国产毛片av蜜桃av| 国产亚洲欧美在线一区二区| 久久中文字幕一级| 啪啪无遮挡十八禁网站| 国产成人欧美在线观看| 丰满迷人的少妇在线观看| 亚洲va日本ⅴa欧美va伊人久久| 麻豆久久精品国产亚洲av | 亚洲黑人精品在线| 男人操女人黄网站| 夫妻午夜视频| 淫妇啪啪啪对白视频| 欧美久久黑人一区二区| 看黄色毛片网站| 老司机亚洲免费影院| 老熟妇乱子伦视频在线观看| 日韩精品青青久久久久久| 黄片小视频在线播放| 人成视频在线观看免费观看| 亚洲免费av在线视频| 久久人妻av系列| 精品卡一卡二卡四卡免费| 日本黄色视频三级网站网址| 一级a爱片免费观看的视频| 国产欧美日韩精品亚洲av| 欧美激情高清一区二区三区| 欧美成人性av电影在线观看| 又大又爽又粗| 欧美人与性动交α欧美精品济南到| 高清av免费在线| 久久精品国产综合久久久| 一边摸一边做爽爽视频免费| 国产亚洲精品第一综合不卡| 亚洲男人天堂网一区| 国产精品爽爽va在线观看网站 | 精品久久久久久电影网| 淫妇啪啪啪对白视频| 欧美日韩av久久| 日韩免费av在线播放| 亚洲片人在线观看| 日韩欧美免费精品| 精品国产一区二区久久| 伦理电影免费视频| 午夜91福利影院| 国产精品亚洲一级av第二区| 天天躁狠狠躁夜夜躁狠狠躁| 欧美黑人欧美精品刺激| 免费搜索国产男女视频| 欧美色视频一区免费| 中文字幕色久视频| 看黄色毛片网站| 麻豆久久精品国产亚洲av | 久久久久久亚洲精品国产蜜桃av| 亚洲 欧美一区二区三区| 国产精品99久久99久久久不卡| 日韩成人在线观看一区二区三区| 久久香蕉国产精品| 香蕉国产在线看| 免费久久久久久久精品成人欧美视频| 精品国产国语对白av| 神马国产精品三级电影在线观看 | 99国产精品免费福利视频| 淫秽高清视频在线观看| 男人操女人黄网站| 成人三级做爰电影| 中文字幕高清在线视频| 女同久久另类99精品国产91| 午夜影院日韩av| 亚洲久久久国产精品| 色精品久久人妻99蜜桃| 欧美成人免费av一区二区三区| 国产成人免费无遮挡视频| 成人特级黄色片久久久久久久| 在线观看一区二区三区| 午夜亚洲福利在线播放| 狂野欧美激情性xxxx| 不卡av一区二区三区| 免费看a级黄色片| 成人免费观看视频高清| cao死你这个sao货| 久久九九热精品免费| 桃色一区二区三区在线观看| 国产1区2区3区精品| 亚洲精品在线观看二区| 成人亚洲精品一区在线观看| 制服诱惑二区| 黄色a级毛片大全视频| 又紧又爽又黄一区二区| 午夜a级毛片| 大陆偷拍与自拍| 波多野结衣一区麻豆| 久久国产精品人妻蜜桃| 又紧又爽又黄一区二区| 亚洲自拍偷在线| 久久精品人人爽人人爽视色| 欧美日韩视频精品一区| 日本三级黄在线观看| 又黄又粗又硬又大视频| 天堂俺去俺来也www色官网| 亚洲欧美日韩无卡精品| 成在线人永久免费视频| 亚洲人成伊人成综合网2020| 怎么达到女性高潮| 在线十欧美十亚洲十日本专区| 不卡一级毛片| 国产精品免费一区二区三区在线| 精品福利永久在线观看| svipshipincom国产片| 精品福利观看| 在线视频色国产色| 精品国产乱码久久久久久男人| 久久这里只有精品19| 伦理电影免费视频| 免费在线观看黄色视频的| 国产aⅴ精品一区二区三区波| 老熟妇乱子伦视频在线观看| 99久久人妻综合| 国产成年人精品一区二区 | 多毛熟女@视频| 不卡av一区二区三区| 精品一区二区三卡| 少妇裸体淫交视频免费看高清 | 在线看a的网站| 欧美另类亚洲清纯唯美| 国产伦一二天堂av在线观看| 一进一出抽搐gif免费好疼 | aaaaa片日本免费| 久久国产精品人妻蜜桃| 丝袜美足系列| 999久久久精品免费观看国产| 国产精品香港三级国产av潘金莲| 国产成人精品无人区| 国产国语露脸激情在线看| 可以在线观看毛片的网站| 黑人巨大精品欧美一区二区蜜桃| 极品教师在线免费播放| √禁漫天堂资源中文www| 亚洲自偷自拍图片 自拍| 亚洲全国av大片| 亚洲性夜色夜夜综合| 国产成人一区二区三区免费视频网站| 精品国产国语对白av| 成人三级黄色视频| 亚洲九九香蕉| 精品无人区乱码1区二区| 韩国av一区二区三区四区| 波多野结衣高清无吗| 天天添夜夜摸| 亚洲国产精品sss在线观看 | 99久久精品国产亚洲精品| 一区二区三区激情视频| 久久精品91无色码中文字幕| 老司机深夜福利视频在线观看| 国产精品九九99| 国产高清国产精品国产三级| 亚洲精品在线观看二区| 母亲3免费完整高清在线观看| netflix在线观看网站| 国产真人三级小视频在线观看| 男女下面插进去视频免费观看| 99久久99久久久精品蜜桃| 亚洲熟妇中文字幕五十中出 | 欧美激情极品国产一区二区三区| 久久久久久久久免费视频了| 国产精品久久久人人做人人爽| 少妇的丰满在线观看| 好男人电影高清在线观看| 久久九九热精品免费| 国产精品1区2区在线观看.| 日本免费a在线| 亚洲色图综合在线观看| 天堂√8在线中文| 国产片内射在线| 美女午夜性视频免费| 久久这里只有精品19| 麻豆久久精品国产亚洲av | 97碰自拍视频| 黄色片一级片一级黄色片| 成人特级黄色片久久久久久久| 免费人成视频x8x8入口观看| a级毛片黄视频| 久久人妻福利社区极品人妻图片| 在线观看舔阴道视频| 欧美激情极品国产一区二区三区| 免费在线观看日本一区| 美女高潮喷水抽搐中文字幕| 制服人妻中文乱码| 久久精品国产清高在天天线| 中文字幕最新亚洲高清| 一区二区日韩欧美中文字幕| 午夜久久久在线观看| 国产成人系列免费观看| 中文字幕色久视频| 亚洲第一av免费看| 色综合欧美亚洲国产小说| 国产三级在线视频| e午夜精品久久久久久久| 999久久久国产精品视频| 如日韩欧美国产精品一区二区三区| 一级黄色大片毛片| 18禁裸乳无遮挡免费网站照片 | 久久久久精品国产欧美久久久| 天天影视国产精品| 欧美成人免费av一区二区三区| 国产亚洲精品第一综合不卡| 国产97色在线日韩免费| 无遮挡黄片免费观看| 免费在线观看亚洲国产| 亚洲少妇的诱惑av| 亚洲欧美激情综合另类| 欧美+亚洲+日韩+国产| 麻豆国产av国片精品| 亚洲成人免费电影在线观看| 大香蕉久久成人网| 黄网站色视频无遮挡免费观看| 中国美女看黄片| 亚洲色图av天堂| 精品免费久久久久久久清纯| 99国产精品一区二区三区| 叶爱在线成人免费视频播放| 又黄又粗又硬又大视频| а√天堂www在线а√下载| 韩国av一区二区三区四区| 午夜福利免费观看在线| 91在线观看av| 日韩欧美免费精品| 国产精品乱码一区二三区的特点 | 女人被狂操c到高潮| 国产av在哪里看| 国产精品秋霞免费鲁丝片| 12—13女人毛片做爰片一| 久久九九热精品免费| 日韩中文字幕欧美一区二区| 国产精品偷伦视频观看了| 一边摸一边做爽爽视频免费| 中文欧美无线码| 激情视频va一区二区三区| 亚洲一区二区三区欧美精品| 午夜两性在线视频| 国产精华一区二区三区| 亚洲精品在线美女| 男人操女人黄网站| 精品电影一区二区在线| 老司机午夜十八禁免费视频| 无遮挡黄片免费观看| 国产97色在线日韩免费| 巨乳人妻的诱惑在线观看| 久久久国产成人精品二区 | 正在播放国产对白刺激| 岛国视频午夜一区免费看| 最新美女视频免费是黄的| 亚洲成人国产一区在线观看| 亚洲欧美日韩高清在线视频| 国产一区二区激情短视频| 免费观看精品视频网站| 国产精品九九99| 看片在线看免费视频| 久久久久久久午夜电影 | 交换朋友夫妻互换小说| 色婷婷久久久亚洲欧美| 精品福利永久在线观看| 亚洲国产精品合色在线| 国产精品爽爽va在线观看网站 | 亚洲人成77777在线视频| 国产片内射在线| 久久国产精品男人的天堂亚洲| 两人在一起打扑克的视频| 好男人电影高清在线观看| 麻豆国产av国片精品| 精品国产亚洲在线| 18禁美女被吸乳视频| 中文字幕高清在线视频| 美女高潮喷水抽搐中文字幕| 色综合欧美亚洲国产小说| 精品久久久久久,| av片东京热男人的天堂| 日本免费一区二区三区高清不卡 | e午夜精品久久久久久久| 男人操女人黄网站| 国产片内射在线| 日本 av在线| 亚洲人成伊人成综合网2020| 三上悠亚av全集在线观看| www.www免费av| 97人妻天天添夜夜摸| 露出奶头的视频| 一级毛片女人18水好多| 一区二区日韩欧美中文字幕| 十分钟在线观看高清视频www| 大型黄色视频在线免费观看| tocl精华| 成人特级黄色片久久久久久久| 母亲3免费完整高清在线观看| 欧美国产精品va在线观看不卡| 99精品欧美一区二区三区四区| 亚洲精品国产一区二区精华液| 99久久人妻综合| 91国产中文字幕| 色婷婷久久久亚洲欧美| 久久中文看片网| 精品日产1卡2卡| 国产精品影院久久| 麻豆国产av国片精品| 一级a爱视频在线免费观看| 午夜亚洲福利在线播放| 99国产精品99久久久久| 亚洲欧美日韩无卡精品| 亚洲精品国产一区二区精华液| 日韩欧美一区二区三区在线观看| 日韩大尺度精品在线看网址 | 欧美成狂野欧美在线观看| 悠悠久久av| 人人妻人人澡人人看| 女人高潮潮喷娇喘18禁视频| 夜夜躁狠狠躁天天躁| 日本精品一区二区三区蜜桃| 欧洲精品卡2卡3卡4卡5卡区| 一级a爱片免费观看的视频| 久久99一区二区三区| 亚洲性夜色夜夜综合| 亚洲avbb在线观看| 18禁观看日本| 精品国产乱码久久久久久男人| 国产精品 欧美亚洲| 国产av在哪里看| 如日韩欧美国产精品一区二区三区| 国产成+人综合+亚洲专区| 久久久水蜜桃国产精品网| av天堂久久9| 精品国产一区二区三区四区第35| 18禁美女被吸乳视频| 亚洲精品在线观看二区| 亚洲专区字幕在线| 国产精品国产av在线观看| 日韩有码中文字幕| 国产精品免费视频内射| 日韩欧美免费精品| 亚洲色图综合在线观看| 亚洲激情在线av| 亚洲第一欧美日韩一区二区三区| 亚洲全国av大片| 国产欧美日韩一区二区精品| 在线十欧美十亚洲十日本专区| 久久久精品欧美日韩精品| 久久久久久久久免费视频了| 国产一区在线观看成人免费| 中文字幕另类日韩欧美亚洲嫩草| 一级毛片女人18水好多| 另类亚洲欧美激情| 精品久久蜜臀av无| 嫩草影视91久久| 这个男人来自地球电影免费观看| 无限看片的www在线观看| 亚洲精品美女久久av网站| 搡老乐熟女国产| 亚洲精品久久成人aⅴ小说| 大码成人一级视频| 最好的美女福利视频网| 99热国产这里只有精品6| 天堂俺去俺来也www色官网| 久久精品成人免费网站| 一边摸一边抽搐一进一出视频| 精品国产乱码久久久久久男人| 黑人操中国人逼视频| 久久精品人人爽人人爽视色| 啦啦啦在线免费观看视频4| www.999成人在线观看| 国产在线精品亚洲第一网站| 在线永久观看黄色视频| 亚洲avbb在线观看| 精品一区二区三卡| 中出人妻视频一区二区| 一级a爱片免费观看的视频| 精品日产1卡2卡| 午夜福利,免费看| 丝袜美足系列| 一级片免费观看大全| 免费在线观看影片大全网站| 在线av久久热| 午夜免费观看网址| 9191精品国产免费久久| 又大又爽又粗| 亚洲激情在线av| 日本wwww免费看| 亚洲 欧美 日韩 在线 免费| 亚洲av熟女| 丝袜美足系列| 欧美黑人欧美精品刺激| 啦啦啦免费观看视频1| 国产欧美日韩综合在线一区二区| 999精品在线视频| 久久精品亚洲av国产电影网| 在线观看66精品国产| 大型av网站在线播放| 欧美 亚洲 国产 日韩一| 露出奶头的视频| 看片在线看免费视频| 国产一区二区三区综合在线观看| 国产亚洲欧美精品永久| 亚洲人成网站在线播放欧美日韩| 免费人成视频x8x8入口观看| 精品久久久精品久久久| 免费av毛片视频| 成人亚洲精品av一区二区 | 满18在线观看网站| 亚洲午夜精品一区,二区,三区| 最近最新中文字幕大全免费视频| 久久精品亚洲熟妇少妇任你| 在线看a的网站| 午夜日韩欧美国产| 亚洲精品国产精品久久久不卡| 天堂影院成人在线观看| 91成人精品电影| 真人一进一出gif抽搐免费| 一边摸一边抽搐一进一出视频| 国内毛片毛片毛片毛片毛片| 午夜福利影视在线免费观看| 国产精品av久久久久免费| 午夜91福利影院| 琪琪午夜伦伦电影理论片6080| 久久青草综合色| 91精品三级在线观看| 可以在线观看毛片的网站| videosex国产| 99久久99久久久精品蜜桃| 久久中文字幕人妻熟女| 国产精品乱码一区二三区的特点 | 亚洲一码二码三码区别大吗| 超碰成人久久| 狠狠狠狠99中文字幕| 中文字幕高清在线视频| 日本黄色日本黄色录像| 国产高清国产精品国产三级| 欧美在线黄色| 久久久国产成人精品二区 | 免费不卡黄色视频| 亚洲国产欧美日韩在线播放| 两性夫妻黄色片| 亚洲人成网站在线播放欧美日韩| 丝袜人妻中文字幕| 黄网站色视频无遮挡免费观看| 欧美精品一区二区免费开放| 成人av一区二区三区在线看| 国产国语露脸激情在线看| 91成人精品电影| 一级片'在线观看视频| 两个人免费观看高清视频| 国产在线精品亚洲第一网站| 亚洲精品粉嫩美女一区| 丝袜美足系列| 午夜亚洲福利在线播放| 曰老女人黄片| 久久精品91无色码中文字幕| 女同久久另类99精品国产91| 一边摸一边抽搐一进一小说| 午夜激情av网站| 一个人观看的视频www高清免费观看 | 亚洲七黄色美女视频| 亚洲国产精品合色在线| 色精品久久人妻99蜜桃| 国产无遮挡羞羞视频在线观看| 69精品国产乱码久久久| 亚洲专区中文字幕在线| 十分钟在线观看高清视频www| 久久狼人影院| 国产日韩一区二区三区精品不卡| 啦啦啦 在线观看视频| 9191精品国产免费久久| 国产精品av久久久久免费| 国产熟女xx| 国产伦人伦偷精品视频| 一本综合久久免费| 国产亚洲精品久久久久5区| a级毛片黄视频| 18美女黄网站色大片免费观看| 亚洲欧美日韩另类电影网站| 日韩高清综合在线| 999精品在线视频| 国产麻豆69| 免费少妇av软件| 麻豆一二三区av精品| 久久99一区二区三区| 黄网站色视频无遮挡免费观看| 免费在线观看黄色视频的| 国产亚洲精品第一综合不卡| 国产亚洲欧美98| 99re在线观看精品视频| 久久伊人香网站| 日韩欧美一区二区三区在线观看| 国产日韩一区二区三区精品不卡| 亚洲男人天堂网一区| av国产精品久久久久影院| 久久中文字幕人妻熟女| 午夜激情av网站| 午夜精品在线福利| 国产欧美日韩一区二区三区在线| aaaaa片日本免费|