• <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.

    此物有八面人人有两片| 老司机午夜福利在线观看视频| 露出奶头的视频| 一级a爱片免费观看的视频| 亚洲成人免费电影在线观看| 成年女人毛片免费观看观看9| 两个人视频免费观看高清| 久久人人精品亚洲av| 亚洲中文日韩欧美视频| 亚洲人成伊人成综合网2020| or卡值多少钱| 91av网一区二区| 少妇高潮的动态图| 真实男女啪啪啪动态图| 国产真实乱freesex| or卡值多少钱| 欧美日韩精品成人综合77777| 麻豆成人av在线观看| 欧美国产日韩亚洲一区| 毛片一级片免费看久久久久 | 国产一级毛片七仙女欲春2| 国产免费男女视频| 亚洲avbb在线观看| 久久草成人影院| 1024手机看黄色片| 亚洲av日韩精品久久久久久密| 亚洲一区二区三区色噜噜| 亚洲在线自拍视频| 中亚洲国语对白在线视频| 免费电影在线观看免费观看| 欧美日韩精品成人综合77777| 窝窝影院91人妻| 免费搜索国产男女视频| 人人妻人人澡欧美一区二区| 中文字幕久久专区| 乱人视频在线观看| 亚洲熟妇中文字幕五十中出| 国产国拍精品亚洲av在线观看| 身体一侧抽搐| 国产成人a区在线观看| 欧美色视频一区免费| 久久久久久久午夜电影| 深夜a级毛片| 欧美日韩瑟瑟在线播放| 中文字幕免费在线视频6| 观看美女的网站| 色在线成人网| 色综合婷婷激情| xxxwww97欧美| 亚洲国产欧洲综合997久久,| av福利片在线观看| 亚洲精品乱码久久久v下载方式| 麻豆国产97在线/欧美| 免费av观看视频| 亚洲精品国产成人久久av| 欧美极品一区二区三区四区| 久久久午夜欧美精品| 99久久精品国产国产毛片| 日韩,欧美,国产一区二区三区 | 国产高清激情床上av| 久99久视频精品免费| 日韩,欧美,国产一区二区三区 | 亚洲精品国产成人久久av| 色5月婷婷丁香| 非洲黑人性xxxx精品又粗又长| 88av欧美| 国产爱豆传媒在线观看| 国产精品人妻久久久久久| 日韩 亚洲 欧美在线| 亚洲七黄色美女视频| 午夜福利在线在线| 国产亚洲精品av在线| 久久久国产成人精品二区| 日本a在线网址| 国产精品人妻久久久影院| 男人舔奶头视频| 三级男女做爰猛烈吃奶摸视频| 国产 一区 欧美 日韩| 美女cb高潮喷水在线观看| 国产男人的电影天堂91| 国产爱豆传媒在线观看| 午夜免费男女啪啪视频观看 | 亚洲七黄色美女视频| avwww免费| 一个人看的www免费观看视频| 高清在线国产一区| 国产淫片久久久久久久久| 欧美日韩乱码在线| 男女做爰动态图高潮gif福利片| 亚洲成人久久性| 亚洲av中文字字幕乱码综合| 欧美xxxx性猛交bbbb| 亚洲人成伊人成综合网2020| 日本黄色片子视频| 国产成人福利小说| 搡老熟女国产l中国老女人| 亚洲性夜色夜夜综合| 成年版毛片免费区| 久久精品综合一区二区三区| av中文乱码字幕在线| 91在线观看av| 一个人观看的视频www高清免费观看| 嫩草影院入口| 日韩精品中文字幕看吧| 亚洲图色成人| 精品人妻熟女av久视频| 桃红色精品国产亚洲av| 亚洲av一区综合| 最近视频中文字幕2019在线8| 在线免费观看的www视频| 国产男靠女视频免费网站| 亚洲专区国产一区二区| 亚洲国产日韩欧美精品在线观看| 精品日产1卡2卡| 中文字幕精品亚洲无线码一区| 亚洲精华国产精华精| 亚洲性夜色夜夜综合| 亚洲熟妇熟女久久| 亚洲精品日韩av片在线观看| 小说图片视频综合网站| 搞女人的毛片| 日日摸夜夜添夜夜添小说| 午夜久久久久精精品| 蜜桃久久精品国产亚洲av| 中文字幕精品亚洲无线码一区| 日韩欧美精品免费久久| 国产伦人伦偷精品视频| 听说在线观看完整版免费高清| 国产黄a三级三级三级人| 欧美bdsm另类| 大型黄色视频在线免费观看| 身体一侧抽搐| 三级男女做爰猛烈吃奶摸视频| 国产伦在线观看视频一区| 一本精品99久久精品77| 99热6这里只有精品| 天美传媒精品一区二区| 亚洲18禁久久av| 深夜a级毛片| 一区二区三区激情视频| 韩国av一区二区三区四区| 国产单亲对白刺激| 亚洲黑人精品在线| 国产成人福利小说| 亚洲av.av天堂| 成人国产麻豆网| av国产免费在线观看| 精品一区二区三区人妻视频| 亚洲av二区三区四区| 成人永久免费在线观看视频| 国产在视频线在精品| 干丝袜人妻中文字幕| av女优亚洲男人天堂| 毛片女人毛片| 99久久精品国产国产毛片| 女人十人毛片免费观看3o分钟| a级毛片免费高清观看在线播放| 色综合亚洲欧美另类图片| 一级a爱片免费观看的视频| 午夜福利18| 亚洲成人精品中文字幕电影| 欧美三级亚洲精品| 99视频精品全部免费 在线| 免费无遮挡裸体视频| 天天一区二区日本电影三级| 成人欧美大片| 久久人妻av系列| 人妻久久中文字幕网| 性欧美人与动物交配| 热99re8久久精品国产| 国产淫片久久久久久久久| 好男人在线观看高清免费视频| 亚洲黑人精品在线| 少妇裸体淫交视频免费看高清| 国产精品日韩av在线免费观看| 在线免费观看的www视频| 一进一出好大好爽视频| 3wmmmm亚洲av在线观看| 18禁黄网站禁片免费观看直播| 少妇的逼好多水| 日本免费a在线| 久久久精品欧美日韩精品| 国产男人的电影天堂91| 日日撸夜夜添| 露出奶头的视频| 琪琪午夜伦伦电影理论片6080| 天堂动漫精品| 少妇丰满av| 婷婷六月久久综合丁香| 欧美日韩精品成人综合77777| 深爱激情五月婷婷| 丰满的人妻完整版| 91av网一区二区| 午夜免费成人在线视频| 亚洲成人久久性| 成人毛片a级毛片在线播放| .国产精品久久| 亚洲中文字幕日韩| 熟女电影av网| 波野结衣二区三区在线| 欧美日韩瑟瑟在线播放| 亚洲最大成人中文| 一个人看的www免费观看视频| 国产精品亚洲美女久久久| 熟妇人妻久久中文字幕3abv| 特级一级黄色大片| 熟妇人妻久久中文字幕3abv| 久久久久国产精品人妻aⅴ院| 亚洲精品一卡2卡三卡4卡5卡| 国产精品亚洲一级av第二区| 不卡一级毛片| 老熟妇仑乱视频hdxx| 神马国产精品三级电影在线观看| 午夜a级毛片| 日韩欧美三级三区| 十八禁网站免费在线| 国产一区二区在线av高清观看| 欧美日韩中文字幕国产精品一区二区三区| 国产精品自产拍在线观看55亚洲| 一级毛片久久久久久久久女| 99国产精品一区二区蜜桃av| 我要看日韩黄色一级片| 成人国产一区最新在线观看| 18+在线观看网站| 日本黄色片子视频| 欧美在线一区亚洲| www.色视频.com| 男女啪啪激烈高潮av片| 老司机深夜福利视频在线观看| 九九爱精品视频在线观看| 好男人在线观看高清免费视频| 舔av片在线| 人人妻人人澡欧美一区二区| 小说图片视频综合网站| 日日摸夜夜添夜夜添av毛片 | 免费在线观看日本一区| 老司机午夜福利在线观看视频| 搡老妇女老女人老熟妇| 99久久精品热视频| 精品午夜福利在线看| 久久人人爽人人爽人人片va| 一个人免费在线观看电影| 欧美性猛交╳xxx乱大交人| 校园人妻丝袜中文字幕| 国产精品久久久久久av不卡| 午夜精品在线福利| 久久精品国产99精品国产亚洲性色| 亚洲久久久久久中文字幕| 国产探花在线观看一区二区| av在线亚洲专区| 国产老妇女一区| 日韩精品青青久久久久久| 男人舔女人下体高潮全视频| 十八禁网站免费在线| av在线天堂中文字幕| 欧美xxxx黑人xx丫x性爽| 久99久视频精品免费| 免费黄网站久久成人精品| 黄色配什么色好看| 男人舔女人下体高潮全视频| 最后的刺客免费高清国语| 国产精品一区www在线观看 | 欧美三级亚洲精品| 亚洲美女黄片视频| 99热只有精品国产| 日本免费a在线| 美女cb高潮喷水在线观看| 欧美xxxx性猛交bbbb| 中文在线观看免费www的网站| 日日摸夜夜添夜夜添av毛片 | 久久久久久久午夜电影| 夜夜爽天天搞| 美女 人体艺术 gogo| 亚洲第一电影网av| 国产精品1区2区在线观看.| 人人妻,人人澡人人爽秒播| 丰满的人妻完整版| 在线观看美女被高潮喷水网站| 干丝袜人妻中文字幕| 免费看光身美女| eeuss影院久久| 国产精品日韩av在线免费观看| 免费人成视频x8x8入口观看| 熟女人妻精品中文字幕| 亚洲第一电影网av| 91狼人影院| 欧美一区二区精品小视频在线| 亚洲一区二区三区色噜噜| 此物有八面人人有两片| 99热网站在线观看| 制服丝袜大香蕉在线| 久久热精品热| netflix在线观看网站| 亚洲专区国产一区二区| 午夜影院日韩av| 有码 亚洲区| 欧美xxxx黑人xx丫x性爽| 午夜激情欧美在线| 直男gayav资源| 亚洲三级黄色毛片| 国语自产精品视频在线第100页| 91午夜精品亚洲一区二区三区 | 日本撒尿小便嘘嘘汇集6| 久久久久久久亚洲中文字幕| 国产亚洲精品综合一区在线观看| 夜夜看夜夜爽夜夜摸| 色在线成人网| 男女边吃奶边做爰视频| 18禁裸乳无遮挡免费网站照片| 日本一本二区三区精品| 国产乱人伦免费视频| 亚洲国产精品sss在线观看| 亚洲国产欧美人成| 麻豆成人午夜福利视频| 十八禁国产超污无遮挡网站| 国产精品1区2区在线观看.| 自拍偷自拍亚洲精品老妇| 有码 亚洲区| 麻豆成人午夜福利视频| 成人美女网站在线观看视频| 亚洲专区国产一区二区| 最新中文字幕久久久久| 少妇裸体淫交视频免费看高清| 国内精品久久久久久久电影| 欧美最黄视频在线播放免费| 亚洲电影在线观看av| 久久精品国产清高在天天线| 亚洲七黄色美女视频| xxxwww97欧美| 日韩在线高清观看一区二区三区 | 草草在线视频免费看| 欧美性感艳星| 最新在线观看一区二区三区| 精品久久久久久久人妻蜜臀av| 一进一出抽搐gif免费好疼| 久久中文看片网| 禁无遮挡网站| 国产精品一区二区免费欧美| 亚洲中文字幕一区二区三区有码在线看| 久久久久久伊人网av| 久久天躁狠狠躁夜夜2o2o| 日韩欧美在线二视频| 不卡视频在线观看欧美| 亚洲av中文av极速乱 | 亚洲av免费高清在线观看| 欧美黑人欧美精品刺激| 国产av一区在线观看免费| 国产高潮美女av| 天堂√8在线中文| 国产不卡一卡二| 波多野结衣高清作品| 观看美女的网站| 三级毛片av免费| 少妇的逼水好多| 欧美+亚洲+日韩+国产| 狂野欧美激情性xxxx在线观看| 亚洲最大成人手机在线| 老女人水多毛片| 国产精品亚洲一级av第二区| www.色视频.com| 嫩草影院入口| 九九爱精品视频在线观看| 成人高潮视频无遮挡免费网站| 日韩欧美精品v在线| 极品教师在线免费播放| 日本撒尿小便嘘嘘汇集6| 91在线观看av| 国内少妇人妻偷人精品xxx网站| 少妇的逼好多水| 国产精品嫩草影院av在线观看 | 久久久久久久久久成人| 1000部很黄的大片| 亚洲成人精品中文字幕电影| 91av网一区二区| 久久精品夜夜夜夜夜久久蜜豆| 97超视频在线观看视频| 国产亚洲91精品色在线| 中文字幕熟女人妻在线| 免费人成视频x8x8入口观看| 一本一本综合久久| 人妻少妇偷人精品九色| 亚洲精品456在线播放app | 国产熟女欧美一区二区| 成人国产一区最新在线观看| 国产三级在线视频| 少妇人妻一区二区三区视频| 一进一出好大好爽视频| 亚洲国产欧洲综合997久久,| 亚洲av日韩精品久久久久久密| 久久天躁狠狠躁夜夜2o2o| aaaaa片日本免费| 天堂av国产一区二区熟女人妻| 国产乱人视频| 国产精品自产拍在线观看55亚洲| 麻豆精品久久久久久蜜桃| 国产成人一区二区在线| 国产男靠女视频免费网站| 日本免费a在线| 男人狂女人下面高潮的视频| 少妇熟女aⅴ在线视频| 成人国产麻豆网| 男人的好看免费观看在线视频| 国产又黄又爽又无遮挡在线| 国产一级毛片七仙女欲春2| 国产男人的电影天堂91| 国内精品一区二区在线观看| 美女大奶头视频| 亚洲黑人精品在线| 国产aⅴ精品一区二区三区波| 三级国产精品欧美在线观看| 久久久久久国产a免费观看| 国产视频内射| 亚洲va日本ⅴa欧美va伊人久久| 男女下面进入的视频免费午夜| 性插视频无遮挡在线免费观看| 乱码一卡2卡4卡精品| av.在线天堂| 天堂动漫精品| 好男人在线观看高清免费视频| 麻豆一二三区av精品| 精品人妻1区二区| 久久久久九九精品影院| 成人综合一区亚洲| 亚洲乱码一区二区免费版| 一个人观看的视频www高清免费观看| 日韩欧美在线二视频| 国产一级毛片七仙女欲春2| 十八禁网站免费在线| 97超级碰碰碰精品色视频在线观看| 亚洲欧美日韩东京热| 香蕉av资源在线| 热99re8久久精品国产| 黄色配什么色好看| 久久久久久伊人网av| 欧美xxxx性猛交bbbb| 欧美日韩瑟瑟在线播放| 精品乱码久久久久久99久播| 美女 人体艺术 gogo| 日本熟妇午夜| 少妇丰满av| 久久国内精品自在自线图片| netflix在线观看网站| 久久这里只有精品中国| 日韩亚洲欧美综合| 日日摸夜夜添夜夜添av毛片 | xxxwww97欧美| 亚洲精品一卡2卡三卡4卡5卡| 国产一区二区激情短视频| 亚洲一区高清亚洲精品| 一个人看的www免费观看视频| 国产成人福利小说| 婷婷丁香在线五月| 动漫黄色视频在线观看| 国内毛片毛片毛片毛片毛片| 级片在线观看| 精品人妻1区二区| 嫩草影院入口| 国产乱人视频| 久久久久久伊人网av| 韩国av在线不卡| 99视频精品全部免费 在线| 欧美一区二区国产精品久久精品| 老司机福利观看| 国产一级毛片七仙女欲春2| 俄罗斯特黄特色一大片| 观看免费一级毛片| 少妇人妻一区二区三区视频| 久久午夜亚洲精品久久| 内射极品少妇av片p| 在线免费观看的www视频| 99热这里只有是精品在线观看| 小说图片视频综合网站| 综合色av麻豆| 老女人水多毛片| 午夜影院日韩av| 国产亚洲精品久久久com| 伊人久久精品亚洲午夜| 国产成人福利小说| 日本三级黄在线观看| 久久精品人妻少妇| 一区二区三区免费毛片| 国产精品不卡视频一区二区| 99热只有精品国产| 91精品国产九色| 12—13女人毛片做爰片一| 欧美在线一区亚洲| 亚洲午夜理论影院| 亚洲熟妇熟女久久| 亚洲国产精品久久男人天堂| 婷婷精品国产亚洲av在线| 一本久久中文字幕| 欧美绝顶高潮抽搐喷水| 国产av不卡久久| 直男gayav资源| 久久国产乱子免费精品| 一进一出抽搐动态| 在线观看免费视频日本深夜| 嫩草影院新地址| 亚洲av中文字字幕乱码综合| 日韩欧美一区二区三区在线观看| 亚洲一区高清亚洲精品| 1000部很黄的大片| 夜夜爽天天搞| 国产精品亚洲美女久久久| 亚洲内射少妇av| 热99re8久久精品国产| 搞女人的毛片| 久久精品综合一区二区三区| 在线免费观看不下载黄p国产 | 欧美性猛交黑人性爽| 99热这里只有是精品50| 国产精品av视频在线免费观看| 两人在一起打扑克的视频| 1000部很黄的大片| 在线观看午夜福利视频| 日韩,欧美,国产一区二区三区 | 国产精品精品国产色婷婷| 国产高清三级在线| 婷婷六月久久综合丁香| 99久国产av精品| 国产av在哪里看| 国产成人aa在线观看| 大型黄色视频在线免费观看| 麻豆国产av国片精品| 久久久久国产精品人妻aⅴ院| 舔av片在线| 免费人成在线观看视频色| 在线观看一区二区三区| 干丝袜人妻中文字幕| 男人舔奶头视频| 在现免费观看毛片| 日本成人三级电影网站| 99国产精品一区二区蜜桃av| 国产精品不卡视频一区二区| 国产精品一区二区三区四区久久| 久久人妻av系列| 99热网站在线观看| 别揉我奶头 嗯啊视频| 午夜福利欧美成人| 婷婷丁香在线五月| 少妇人妻一区二区三区视频| xxxwww97欧美| 午夜福利在线观看免费完整高清在 | 亚洲av日韩精品久久久久久密| 老师上课跳d突然被开到最大视频| 久久精品夜夜夜夜夜久久蜜豆| 国产日本99.免费观看| 大型黄色视频在线免费观看| 麻豆国产av国片精品| 国产高清不卡午夜福利| 夜夜爽天天搞| 日韩精品中文字幕看吧| 搡老妇女老女人老熟妇| 成人永久免费在线观看视频| 欧美性猛交黑人性爽| a级一级毛片免费在线观看| 国产欧美日韩精品亚洲av| 十八禁国产超污无遮挡网站| 日日撸夜夜添| 国产综合懂色| 国产精品久久久久久精品电影| 日本三级黄在线观看| 久久精品久久久久久噜噜老黄 | 如何舔出高潮| 日本色播在线视频| 成年女人毛片免费观看观看9| 精品人妻1区二区| 99热6这里只有精品| 波多野结衣高清无吗| 狂野欧美白嫩少妇大欣赏| 日韩一本色道免费dvd| 人妻少妇偷人精品九色| 久久久久久九九精品二区国产| 久9热在线精品视频| 国内精品美女久久久久久| 精品一区二区三区视频在线| 欧美区成人在线视频| 国产精品久久视频播放| 变态另类成人亚洲欧美熟女| 亚洲精品久久国产高清桃花| 三级男女做爰猛烈吃奶摸视频| 男女视频在线观看网站免费| 国产黄片美女视频| 夜夜爽天天搞| 又紧又爽又黄一区二区| 性插视频无遮挡在线免费观看| 婷婷亚洲欧美| 欧美激情在线99| 亚洲国产精品合色在线| 久久6这里有精品| 黄色配什么色好看| 国产伦人伦偷精品视频| 人人妻人人看人人澡| 91久久精品国产一区二区三区| a级一级毛片免费在线观看| 亚洲无线在线观看| 中国美白少妇内射xxxbb| 一级毛片久久久久久久久女| 日韩国内少妇激情av| 女人十人毛片免费观看3o分钟| 日韩精品中文字幕看吧| 日日干狠狠操夜夜爽| 国产精品国产高清国产av| 亚洲五月天丁香| av.在线天堂| 午夜精品久久久久久毛片777| 久久久久久九九精品二区国产| 国产美女午夜福利| av天堂在线播放| 免费看日本二区| 在线观看一区二区三区| 99久久成人亚洲精品观看| 亚洲一级一片aⅴ在线观看| 啦啦啦观看免费观看视频高清| 亚洲av成人av|