• <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 ?

    Glutathione Transferase Omega 1 Promotes The Expression of MHCII via Inhibiting Its Ubiquitination*

    2022-04-24 06:15:46LIUJiWeiLIShuHangXIEDiZHANGHuiMinLIQingBAILi

    LIU Ji-Wei,LI Shu-Hang,XIE Di,ZHANG Hui-Min,LI Qing,BAI Li**

    (1)Hefei National Laboratory for Physical Sciences at Microscale,the CAS Key Laboratory of Innate Immunity and Chronic Disease,School of Life Sciences,Division of Molecular Medicine,University of Science and Technology of China,Hefei 230026,China;2)The First Affiliated Hospital of the University of Science and Technology of China,Hefei 230001,China)

    Abstract Objective We investigated the role of glutathione transferase omega 1 (GSTO1) in regulating function of bone marrow-derived dendritic cells (BMDCs). Methods The expression of MHCII on the surface of Gsto1-/- BMDCs and GSTO1 inhibitor-treated BMDCs was detected by flow cytometry and confocal microscope. To investigate the role of BMDCs, they were cocultured with CD4 T cells from OTII mice in the presence of OVA and IFN-γ production was measured. The recycling and internalization of MHCII molecule in BMDCs treated with GSTO1 inhibitor were measured by flow cytometry. The transcriptional level of MHCII molecule in BMDCs treated by GSTO1 inhibitor was detected by real-time quantitative PCR.The total protein level of MHCII molecule in BMDCs treated by GSTO1 inhibitor was detected by Western blot. The ubiquitination level of MHCII after GSTO1 inhibitor treatment was detected by co-immunoprecipitation assay. Results GSTO1 deficiency and GSTO1 inhibitor did not affect proliferation and apoptosis of BMDCs, but reduced the surface expression of antigen presentation molecule MHCII.Gsto1-/-BMDCs and BMDCs treated with GSTO1 inhibitor showed impaired CD4 T cell-activating capability.In GSTO1 inhibitortreated BMDCs, the recycling and internalization of MHCII molecule were normal. GSTO1 inhibitor showed no influence on the transcription of MHCII but reduced its total protein level. Furthermore, BMDCs treated with GSTO1 inhibitor showed higher level of ubiquitinated MHCII molecule and treatment with proteasomes inhibitor MG132 recovered the surface MHCII expression in these cells. Conclusion These data demonstrate that GSTO1 in BMDCs promotes MHCII expression and thus CD4 T cell-activating capacity by inhibiting ubiquitination of MHCII molecule.

    Key words GSTO1,MHCII,dendritic cell,ubiquitination

    Glutathione (GSH) is the main non-protein tripeptide antioxidant substance in cells, the balance between GSH and its oxidize form GSSG determines the redox state of cells. GSH binds cysteine in proteins and thereby leads to protein glutathioneylation to modulate protein function[1].Glutathionylation is a reversible post-translational modification catalyzed by different cellular oxidoreductases, which promote the formation of mixed disulfides between protein cysteine and glutathione (GSH) cysteine[2]. This process is catalyzed by glutathione transferase (GST) family,which consist of three super-families: the cytosolic GST, mitochondrial GST, and microsomal GST. The cytosolic GST family contains a variety of types,such as alpha,zeta,theta,mu,pi,sigma,and omega class[3].Glutathione transferase omega 1 (GSTO1) is a constitutively active deglutathionylating enzyme[4].As an atypical GST isoform, GSTO1 is overexpressed in several tumors and has been implicated in drug resistance[5-6]. Recent studies suggest that GSTO1 is involved in activation of NLRP3 inflammasome and inflammatory diseasesin vivo[7]. It remains unclear that whether GSTO1 regulates functions of dendritic cells(DCs)and DC-related immune responses.

    DCs are professional antigen-presenting cells(APCs), and link the innate immunity and adaptive immunity.Viaantigen presentation, DCs play essential roles in activating T cells[8-9]. Antigens presented by MHCI molecules induce activation of CD8 T cells, whereas antigens presented by MHCII molecules activate CD4 T cells. Antigenic peptide-MHCII complexes (p-MHCII) expressed on the surface of DCs are critical to antigen presentation and the initiation of CD4 T cell activation, therefore the surface expression and transport of these complexes are tightly regulated in DCs[10].The newly synthesized MHCII binds to a chaperone protein called invariant chain (li), which remains associated with MHCII while transporting to the plasma membraneviathe trans-Golgi network. Once on the plasma membrane,the MHCII-li complex is rapidly internalized by clathrin-mediated endocytosis and transported from the early endosome to the late endosome[11],where the MHCII-associated li is degraded by proteases and removed by H2-DM (a mouse MHCII-like αβ dimer which has very limited polymorphism). Then,immunogenic peptides will bind to MHCII and the newly formed p-MHCII complexes recycle back to the plasma membrane[11]. Large amounts of MHCII are stored in intracellular antigen-processing compartment and are transported to cell surface when DCs are activated[12-13].

    Ubiquitination of MHCII has been reported as an important strategy to regulate MHCII expression in APCs[14]. Oligo-ubiquitination of MHCII at a single lysine residue has been identified in the cytoplasmic tail of MHCII β-chain. It has been reported that ubiquitination of MHCII influences its internalization,transportation to antigen-processing compartments,and delivery to lysosomes for degradation[15-16].Ubiquitination of MHCII is promoted by the E3 ubiquitin ligase membrane-associated RING-CH 1(March-1), and that reduces surface expression and half life of MHCII in DCs[17]. On the other hand,activation signal blocks the ubiquitin-dependent turnover of MHCII by promoting MHCII recycling and preventing its delivery to lysosomes, thereby enhancing MHCII stability and favoring antigen presentation to CD4T cells[17].

    In this study, we investigate the role of GSTO1 in regulating functions of DCs. We show that GSTO1 activity promotes surface expression of MHCII molecule in bone marrow-derived dendritic cells(BMDCs), and thus enhances the antigen presenting function of BMDCs. Although GSTO1 does not influence the recycling or internalization or transcription of MHCII molecule, it inhibits ubiquitination of MHCII. Our results propose that GSTO1 enhances antigen presentation in BMDCs by inhibiting MHCII degradation.

    1 Materials and methods

    1.1 Mice

    Gsto1+/-andGsto1-/-mice were bred and maintained in specific pathogen-free conditions.Gsto1-/-mice were provided by Prof. XU Tao’s laboratory of Institute of Biophysics, Chinese Academy of Sciences. OTII CD45.1 mice were provided by Prof. ZHU Shu’s laboratory of University of Science and Technology of China(USTC).All animal procedures were approved by the University of Science and Technology of China(USTC) Institutional Animal Care and Use Committee. And all experiments were performed in accordance with the approved guidelines.

    1.2 Flow cytometry

    BMDCs were induced with 20μg/L GM-CSF for 7 d in complete RPMI 1640 medium[18],and then were collected and seeded in 96-well flat plate at 1×109cells/L in the presence or absence of 5 μmol/L C1-27(MCE, 568544-03-6) or 4 μmol/L ML175(AOBIOUS) for 24 h. For surface staining, cells were preincubated with anti-mouse CD16/32 antibody(BioLegend,101302)for 15 min on ice at 2 mg/L,and then were stained with monoclonal antibodies against BV421-CD11b (BioLegend, 101236), PE/cy7-CD11c(BioLegend, 117318), Percp/cy5.5-CD40 (BioLegend,124624),APC-CD80 (BioLegend, 104714),APC/cy7-CD86 (BioLegend, 105030), PE-CD1d (BioLegend,140805), FITC-MHCII (BioLegend, 107606) for 45 min on ice at 1 mg/L. For cell proliferation analysis, BMDCs were stained with PE mouse anti-Ki67 set (BD Pharmingen, 556027), according to the manufacturer’s method. For cell apoptosis analysis,BMDCs were stained with FITC Annexin V Apoptosis Detection kit (Vazyme, A211-01),according to the manufacturer’s method. For intracellular staining, cells finishing surface staining were fixed with 4% paraformaldehyde (PFA, Sigma,158127) for 15 min and then were permeabilized with 0.1% saponin (Sigma, s7900) for 30 min and blocked with 5% FBS (Biological Industries, 04-001).Antibodies against intracellular molecules were used to stain the cells for 1 h on ice. To measure the amount of intracellular MHCII, surface MHCII was blocked with purified antibody against MHCII(Abcam, ab139365), before intracellular staining with PE-conjugated antibody from same clone. Cells were washed with PBS twice and subsequently analyzed by flow cytometry.

    1.3 Cytokine analysis

    Cytokines, including IL-6 and TNF-α, in cell supernatants were analyzed with mouse Th1/Th2/Th17 CBA kit (BD Biosciences, 560485), according to the manufacturer’s instructions.

    1.4 Immunofluorescent staining

    BMDCs were collected and seeded into confocal dish at 2×109cells/L with or without C1-27(5 μmol/L)for 24 h. Then, cells were blocked with anti-mouse CD16/32 antibody for 15 min at 4℃before staining with Alexa Fluor 488 anti-mouse MHCII antibody(Santa Cruz Biotechnology, sc-23940) overnight at 4℃. Cells were washed 3 times with 0.1% Tween-20(Sigma,P1379)and sealed with DAPI Fluoromount-G(SouthernBio, 0100). Images were taken with Olympus inverted fluorescence microscope(Olympus)and analyzed with Image J software.

    1.5 RT-qPCR

    Total RNA was extracted from BMDCs with ReliaPrep RNA Cell Miniprep System (Promega,Z6011), according to the manufacturer’s protocols.cDNA was synthesized with a high-capacity cDNA reverse transcription kit (Applied Biosystems,4368814), according to the manufacturer’s instructions. Primers for qPCR were as follows:H2-Ab(forward: AAGGCATTTCGTGTACCAGTTC;reverse: CCTCCCGGTTGTAGATGTATCTG);Ciita(forward: AGACCTGGATCGTCTCGTG; reverse:AGTGCATGATTTGAGCGTCTC);β-actin(forward:GTGACGTTGACATCCGTAAAGA;reverse:GCCGGACTCATCGTACTCC). qPCR was performed with the Roche LightCycler 96 using TB Green Premix Ex Taq ?II (Takara, RR82WR). Relative expression values were normalized to a control gene(β-actin).

    1.6 MHCII recycling and internalization assay

    For recycling assay, BMDCs pre-treated with or without C1-27 were incubated with 10 μg mouse anti-MHCII antibody (Abcam, ab139365) for 30 min at 4℃to block the MHCII molecule on the surface.After washing and resuspending cells with RPMI 1640 complete medium, cells were incubated in 37℃water bath for different times and then cells were stained with FITC-conjugated anti-mouse MHCII antibody (Biolegend, 107606) for 45 min. The newly recycled MHCII stained with FITC-conjugated antimouse MHCII antibody was detected by flow cytometry. The percentage of recycled MHCII was measured using the equation (Tx-T0)/T×100, whereT0was the mean fluorescence intensity (MFI) of FITC at 0 min, andTxwas the MFI of FITC at the indicated times, andTwas the total MFI of FITC in cells that were not blocked with anti-MHCII antibody.

    For internalization assay, BMDCs pre-treated with or without C1-27 were surface-labeled with FITC-conjugated anti-mouse MHCII antibody for 45 min at 4℃.After washing with RPMI 1640 media containing 0.5% FBS, cells were incubated for different times at 37℃in complete medium to allow internalization of MHCII. Then, surface antibody was stripped with buffer containing 0.5 mol/L NaCl and 0.5% acetic acid (pH 3.0) for 10 min on ice. Cells were then fixed with 4% PFA, and the internalized MHCII was measured by flow cytometry. The percentage of internalized MHCII was measured using the equation (Tx-T0)/T×100, whereT0was the MFI of FITC at 0 min,andTxwas the MFI of FITC at the indicated times, andTwas the total MFI of FITC in cells that not treated with strip solution.

    1.7 Western blotting and coimmunoprecipitation(CoIP)

    BMDCs were treated with GSTO1 inhibitor C1-27 (10 μmol/L) for 24 h.To detect total protein of MHCII, cells were harvested and lysed with sample buffer and were boiled for 15 min. Proteins were separated by electrophoresis and were detected by western blotting. Antibodies against MHCII(Invitrogen, 14-5321-82, 1∶1 000 dilution), and β-actin (Transgen Biotech, HC201-02, 1∶1 000 dilution)were used in our experiments.

    To detect ubiquitination of MHCII, cells were harvested and washed with ice-cold PBS and then lysed with a NP-40 (Biotec, P0016F) buffer containing 1% protease inhibitor cocktail(Trans, N21024, 1∶100 dilution) and EDTA(ethylenediaminetetraacetic acid) (Trans, 10622,1∶100 dilution) on ice for 1 h.Half of cell lysates mixed with 5×lane marker loading buffer (Biotec,P0015) was heated at 101℃for 15 min and used as input sample. The rest cell lysates were incubated with 2 μg anti-MHCII antibody (Invitrogen, 14-5321-82) and 15 μl protein G beads (Invitrogen, 10004D)and rotated at 4℃ overnight. Antibody against ubiquitin was used to detect ubiquitination of MHCII in co-immunoprecipitated proteins.

    1.8 Antigen presentation and activation of CD4 T cells

    BMDCs (5×108cells/L) were loaded with or without 500 mg/L OVA (Sigma-Aldrich, A5503)overnight in the presence or absence of 5 μmol/L C1-27, and then were washed and were co-cultured with CD4 T cell enriched from splenocytes of OTII transgenic mice for 6 d in the presence of rhIL-2(1×105U/L). IFN-γ in supernatant was measured with CBA flex set(BD,558296).

    1.9 Data analysis

    Error bars represent standard error of the mean(SEM). The statistical significance of differences between two groups was determined by the unpaired two-tailed student’sttest.

    2 Results

    2.1 GSTO1 deficiency reduces surface MHCII expression in BMDCs

    Glutathionylation of protein is an important strategy to response to external signals, including sensing energy and metabolic changes, cell apoptosis,signal transduction, and protein folding[2]. GSTO1, an atypical GST, is significant in the glutathionylation cycle[19]. In order to study the functions of GSTO1 in DCs,we generatedGsto1-/-mice andGsto1+/-mice as littermate controls. The levels of surface markers,including CD80, CD86, CD40, CD1d, and MHCII, in BMDCs fromGsto1+/-andGsto1-/-mice were compared by flow cytometry. The expression of CD80, CD40, and MHCII were significantly reduced in GSTO1 deficient BMDCs, whereas CD86 and CD1d expressions were not influenced by deletion of GSTO1 (Figure 1a). To further prove the reduction of surface MHCII molecule, we stained BMDCs with Alex Fluor 488-conjugated anti-MHCII and imaged these cells with laser confocal microscopy. In comparison withGsto1+/-BMDCs,Gsto1-/-BMDCs showed lower level of surface MHCII(Figure 1b).On the other hand, GSTO1 deficiency did not influence the proliferation or apoptosis of BMDCs (Figure 1c,d). These results suggest that GSTO1 promotes the surface expression of MHCII in BMDCs.

    2.2 GSTO1 inhibitors reduce surface MHCII expression in BMDCs

    To investigate whether GSTO1 promoted surface expression of MHCIIviaits catalytic activity, we inhibited activity of GSTO1 with two inhibitors ML175 and C1-27, respectively. Both ML175 and C1-27 reduced the surface expression of CD80,CD86, and MHCII, but exhibited no influence on CD40 or CD1d in BMDCs (Figure 2a, b). Again,confocal images demonstrated reduced surface MHCII in C1-27 treated BMDCs (Figure 2c). In line with the normal proliferation and apoptosis inGsto1-/-BMDCs, GSTO1 inhibitor C1-27 showed no influence on proliferation and apoptosis of BMDCs.These results confirm that GSTO1’s catalytic activity is required for surface expression of MHCII in BMDCs.

    2.3 Deficiency of GSTO1 in BMDCs reduces the activation of CD4 T cells

    DCs promote the activation and differentiation of CD4 T cellsviapresenting antigens through MHCII molecule. Hence, we investigated whether the reduction of surface MHCII as a result of GSTO1 deficiency would impair the capability of DCs to activate CD4 T cells. We pulsedGsto1+/-BMDCs,Gsto1-/-BMDCs, and C1-27 treatedGsto1+/-BMDCs with OVA, and co-cultured them with OTII CD4 T cells, respectively. We found that presence of OVA was required for activating CD4 T cells, as indicated by the IFN-γ production in supernatants. Both deletion ofGsto1and inhibition of its activity in BMDCs reduced OVA-induced CD4 T cell activation(Figure 3).Therefore, GSTO1 promotes the capability of BMDCs to activate CD4 T cells.

    Fig.1 GSTO1 deficiency reduces the surface expression of MHCII in BMDCs

    2.4 GSTO1 promotes surface expression of MHCII via inhibiting its ubiquitination

    It is well-known that MHCII molecule undergoes internalization and recycling. Next, we investigated whether the C1-27-induced reduction of surface MHCII in BMDCs was due to alteration in MHCII internalization or recycling. We found that C1-27 displayed no influence on recycling rate or internalization rate of MHCII in BMDCs (Figure 4a).In line with the reduced surface MHCII but normal recycling and internalization rate, we found that the surface and intracellular MHCII was reduced in C1-27 treated BMDCs (Figure 4b). Besides, the protein level of total MHCII in BMDCs was also decreased in C1-27 treated BMDCs (Figure 4c). Our results indicated a decrease of total MHCII protein in GSTO1 activity deficient BMDCs.Notably,the C1-27 treatment did not influence the transcription of MHCII, as indicated by normal mRNA levels ofH2-AbandCiita, which encodes a key regulator for MHCII, in C1-27 treated BMDCs (Figure 4d).Therefore,C1-27 induced reduction of MHCII protein would likely be due to increased protein degradation.It has been reported that ubiquitination regulates the retention and degradation of MHCII in DCs[20]. In C1-27 treated BMDCs, we detected increased ubiquitination of MHCII molecule, in comparison with untreated cells (Figure 4e). Protein that has been ubiquitinated will be degraded by proteasome, and proteasome inhibitor could interfere with the protein degradation. Next, we added MG132, an inhibitor of proteasome, to C1-27 treated BMDCs, and showed that MG132 abrogated the inhibitory effect of C1-27 on MHCII surface expression (Figure 4f). Together,our results indicate that GSTO1 promotes the surface expression of MHCIIviainhibiting its ubiquitination and degradation.

    Fig.2 GSTO1 inhibitors reduce the surface expression of MHCII in BMDCs

    Fig.3 Deficiency of GSTO1 in BMDCs reduces the activation of CD4 T cells

    3 Discussion

    Fig.4 C1-27 promotes ubiqutination and degradation of MHCII

    Published studies have shown that GSTO1 plays an important role in NLRP3 inflammasome activation and inflammatory diseases[21-22]. As the link of innate immunity and adapted immunity, DCs are important in presenting antigen, and activating naive T cells. It still unclear that whether GSTO1 regulates immune responses via modulating functions of DCs. In this study, we found that the expression of CD80, CD86,and MHCII but not CD40 and CD1d were downregulated by GSTO1 inhibitor ML175 and C1-27 in BMDCs.Similar reduction of MHCII was observed in GSTO1 deficient BMDCs, although CD80 expression was not changed and CD40 expression was decreased a little in these cells. On the other hand, BMDCs proliferation and apoptosis were not influenced by GSTO1 deficiency.

    Antigen-specific CD4 T cells are activated by the antigens presented by MHCII on the surface of APCs.In line with the role of GSTO1 in promoting surface MHCII expression, GSTO1 is important for the antigen presenting function of BMDCs. Additionally,the activation of T cells also requires co-stimulation signal provided by APCs. Both MHCII and costimulation molecule play essential roles in the activation of T cells. Therefore, GSTO1 deficiency induced reduction of CD80 might also contribute to the impaired capability of BMDCs to activate CD4 T cells. Together, we propose that GSTO1 in BMDCs would influence the crosstalk between BMDCs and CD4 T cells. Thus, we investigated how GSTO1 regulated MHCII expression in DCs. We supposed that C1-27 damages the transcription of MHCII and CIITA. The results showed that MHCII expression reduction caused by C1-27 did not result from the effect of C1-27 on the transcription ofMHCIIandCiitagenes.

    It has been demonstrate that the rapid turnover of p-MHCII in immature DCs is regulated by ubiquitindependent p-MHCII degradation under the control of March-1[17]. MHCII that are transported to the cell surface are rapidly internalized and degraded in immature DCs[23-24]. It has been shown that ubiquitination of MHCII on Lys225 is responsible for the rapid turnover in immature DCs[17]. Here, our results demonstrate that GSTO1 inhibits ubiquitination of MHCII but shows no effect on internalization or recycling of MHCII. It is possible that GSTO1-regulated ubiquitination of MHCII has no influence on its internalization. Our results that GSTO1 promotes antigen presenting function of BMDCsviainhibiting MHCII ubiquitination are in line with previous findings that ubiquitination and deubiquitination are involved in regulating maturation and function of DCs[25]. However, it is still unclear how GSTO1 regulates the ubiquitination of MHCII,although our study suggests involvement of its catalytic activity. It has been reported that GSTO1 deglutathionylates proteins[4]. Whether GSTO1 inhibits ubiquitination of MHCIIviadeglutathionylating MHCII or other ubiquitinationrelated proteins remains to be explored.

    4 Conclusion

    The present study demonstrates that GSTO1 promotes the expression of MHCII in BMDCs by inhibiting its ubiquitination, and thus favors antigen presentation and CD4 T cell-activating capacity.

    AcknowledgementsWe thank Prof. XU Tao and Prof. ZHU Shu for providing withGsto1-/-mice and OTII CD45.1 mice.

    日韩av不卡免费在线播放| 99re6热这里在线精品视频| 涩涩av久久男人的天堂| 涩涩av久久男人的天堂| 丝袜喷水一区| 嫩草影视91久久| 晚上一个人看的免费电影| 高清在线视频一区二区三区| 日本欧美视频一区| 亚洲男人天堂网一区| 亚洲在久久综合| 秋霞伦理黄片| 国产淫语在线视频| 国产精品久久久人人做人人爽| 久久久欧美国产精品| 中文字幕人妻丝袜一区二区 | 精品国产一区二区久久| 国产1区2区3区精品| 超碰97精品在线观看| 在线观看免费视频网站a站| 国产成人精品在线电影| 日本wwww免费看| 啦啦啦视频在线资源免费观看| 另类精品久久| 亚洲成人国产一区在线观看 | 最近最新中文字幕免费大全7| 午夜福利视频精品| 无遮挡黄片免费观看| 久久女婷五月综合色啪小说| 性少妇av在线| 亚洲四区av| 91成人精品电影| 亚洲成人av在线免费| 亚洲欧美成人综合另类久久久| 我的亚洲天堂| 久久久久久久国产电影| 色网站视频免费| 视频区图区小说| 国产av国产精品国产| 亚洲av综合色区一区| 国产不卡av网站在线观看| 亚洲七黄色美女视频| 亚洲精品乱久久久久久| 无限看片的www在线观看| 国产在线一区二区三区精| 性高湖久久久久久久久免费观看| 99久久精品国产亚洲精品| 亚洲成人手机| 亚洲成人免费av在线播放| 成人亚洲欧美一区二区av| 精品一区二区三区四区五区乱码 | 日本欧美国产在线视频| 亚洲成人免费av在线播放| 在线天堂最新版资源| 99久久精品国产亚洲精品| av国产久精品久网站免费入址| 国产在线一区二区三区精| 久久国产精品大桥未久av| 久久久精品国产亚洲av高清涩受| 99九九在线精品视频| 国产乱人偷精品视频| 在线看a的网站| 性少妇av在线| 大片免费播放器 马上看| 久久人人97超碰香蕉20202| 黄色一级大片看看| 国产极品粉嫩免费观看在线| 欧美 亚洲 国产 日韩一| 无遮挡黄片免费观看| 制服丝袜香蕉在线| 自拍欧美九色日韩亚洲蝌蚪91| 在现免费观看毛片| 亚洲欧美成人精品一区二区| 69精品国产乱码久久久| 国产免费一区二区三区四区乱码| 亚洲成人一二三区av| 国产淫语在线视频| 男女高潮啪啪啪动态图| 亚洲av综合色区一区| 久久国产精品男人的天堂亚洲| 叶爱在线成人免费视频播放| 亚洲国产av影院在线观看| 美女扒开内裤让男人捅视频| 丰满乱子伦码专区| 亚洲综合精品二区| 18禁观看日本| 欧美变态另类bdsm刘玥| 中文天堂在线官网| 一级毛片 在线播放| 日本爱情动作片www.在线观看| 亚洲免费av在线视频| av在线观看视频网站免费| 国产极品天堂在线| 1024视频免费在线观看| 国产男女内射视频| 亚洲精品国产av成人精品| 青春草国产在线视频| 国产黄色视频一区二区在线观看| 一区二区三区激情视频| 国产精品一二三区在线看| 国产成人欧美| 欧美精品高潮呻吟av久久| 99国产精品免费福利视频| 丝袜喷水一区| 国产成人系列免费观看| 亚洲精品国产区一区二| 亚洲欧美色中文字幕在线| 99国产精品免费福利视频| 大香蕉久久网| 欧美激情高清一区二区三区 | 久久综合国产亚洲精品| 高清不卡的av网站| 日韩中文字幕欧美一区二区 | 久久天堂一区二区三区四区| 蜜桃国产av成人99| 人人妻,人人澡人人爽秒播 | 人妻 亚洲 视频| 肉色欧美久久久久久久蜜桃| 日本av免费视频播放| 精品一区二区免费观看| 成人影院久久| 久久午夜综合久久蜜桃| 卡戴珊不雅视频在线播放| 啦啦啦视频在线资源免费观看| 亚洲精品国产色婷婷电影| 女性被躁到高潮视频| 亚洲精品一区蜜桃| 欧美激情 高清一区二区三区| 成人免费观看视频高清| 欧美日本中文国产一区发布| 亚洲成人国产一区在线观看 | 国产一区二区三区综合在线观看| 免费观看av网站的网址| 欧美日韩一区二区视频在线观看视频在线| 国产熟女欧美一区二区| 丝袜美腿诱惑在线| 国产精品 国内视频| 两性夫妻黄色片| 香蕉国产在线看| 日韩av不卡免费在线播放| 国产精品秋霞免费鲁丝片| 久久精品久久精品一区二区三区| 激情五月婷婷亚洲| 精品少妇内射三级| 日韩 亚洲 欧美在线| 久久久国产欧美日韩av| 国产精品.久久久| 欧美精品一区二区免费开放| 中文字幕色久视频| 亚洲国产欧美网| 精品国产超薄肉色丝袜足j| h视频一区二区三区| 免费观看av网站的网址| 少妇人妻久久综合中文| 国产人伦9x9x在线观看| 黑人欧美特级aaaaaa片| 亚洲精品国产区一区二| 国产日韩欧美在线精品| 色94色欧美一区二区| 国产精品偷伦视频观看了| 日本vs欧美在线观看视频| 黄色毛片三级朝国网站| 日韩一区二区三区影片| 91成人精品电影| 18禁国产床啪视频网站| 最近最新中文字幕大全免费视频 | 91精品国产国语对白视频| 18禁观看日本| 亚洲第一av免费看| 1024视频免费在线观看| 哪个播放器可以免费观看大片| 亚洲精品自拍成人| 又大又爽又粗| 操美女的视频在线观看| 久久韩国三级中文字幕| av一本久久久久| 99久国产av精品国产电影| 美女中出高潮动态图| 精品人妻熟女毛片av久久网站| 99精国产麻豆久久婷婷| 别揉我奶头~嗯~啊~动态视频 | 人体艺术视频欧美日本| av有码第一页| 51午夜福利影视在线观看| 男女之事视频高清在线观看 | 男女之事视频高清在线观看 | 午夜福利,免费看| 亚洲欧美一区二区三区黑人| 最黄视频免费看| 久久精品国产亚洲av高清一级| 婷婷色麻豆天堂久久| 看非洲黑人一级黄片| 女人精品久久久久毛片| 久久热在线av| 97精品久久久久久久久久精品| av不卡在线播放| 久热爱精品视频在线9| 国产精品香港三级国产av潘金莲 | 精品人妻熟女毛片av久久网站| 不卡视频在线观看欧美| 欧美老熟妇乱子伦牲交| 久久精品aⅴ一区二区三区四区| 亚洲国产欧美一区二区综合| 欧美日韩视频高清一区二区三区二| 亚洲欧美精品综合一区二区三区| 日韩电影二区| 精品酒店卫生间| 满18在线观看网站| 99久久精品国产亚洲精品| av国产久精品久网站免费入址| 亚洲美女搞黄在线观看| 成人手机av| 亚洲欧美清纯卡通| www.av在线官网国产| 中文精品一卡2卡3卡4更新| 老汉色∧v一级毛片| 肉色欧美久久久久久久蜜桃| 精品少妇内射三级| 老汉色av国产亚洲站长工具| 久久久久人妻精品一区果冻| 日韩精品免费视频一区二区三区| 菩萨蛮人人尽说江南好唐韦庄| 最近2019中文字幕mv第一页| 叶爱在线成人免费视频播放| 中文字幕av电影在线播放| 久久久精品免费免费高清| 精品国产一区二区久久| 老司机靠b影院| 大话2 男鬼变身卡| 久久精品久久精品一区二区三区| h视频一区二区三区| 精品一区二区三区四区五区乱码 | 国产欧美日韩一区二区三区在线| 天天躁夜夜躁狠狠久久av| 男男h啪啪无遮挡| 午夜免费男女啪啪视频观看| 高清av免费在线| 天天躁夜夜躁狠狠躁躁| 国产精品一国产av| 免费黄色在线免费观看| 中文字幕人妻丝袜制服| 王馨瑶露胸无遮挡在线观看| 捣出白浆h1v1| √禁漫天堂资源中文www| 高清在线视频一区二区三区| 99热国产这里只有精品6| 国产黄色视频一区二区在线观看| 在线看a的网站| 午夜福利视频精品| 日本黄色日本黄色录像| 久久久久久久久免费视频了| 国产不卡av网站在线观看| 一本色道久久久久久精品综合| 亚洲成人一二三区av| 欧美在线一区亚洲| 不卡av一区二区三区| 九草在线视频观看| 黄色怎么调成土黄色| 久久精品熟女亚洲av麻豆精品| 久久这里只有精品19| 日韩中文字幕欧美一区二区 | 久久久久久久久久久久大奶| 在现免费观看毛片| 亚洲,欧美,日韩| av免费观看日本| 少妇 在线观看| 国产有黄有色有爽视频| 精品国产国语对白av| 亚洲,欧美,日韩| 亚洲欧洲日产国产| 日韩电影二区| 成人三级做爰电影| 免费少妇av软件| 少妇人妻 视频| 日韩中文字幕欧美一区二区 | 一级毛片电影观看| 亚洲国产看品久久| 各种免费的搞黄视频| 如何舔出高潮| 久久精品亚洲av国产电影网| www日本在线高清视频| 国产免费现黄频在线看| 91精品国产国语对白视频| 国产成人一区二区在线| 成人漫画全彩无遮挡| 国产色婷婷99| av又黄又爽大尺度在线免费看| 九九爱精品视频在线观看| 大香蕉久久成人网| 国产精品国产三级国产专区5o| 男女下面插进去视频免费观看| 欧美乱码精品一区二区三区| 色网站视频免费| 啦啦啦在线免费观看视频4| 18禁国产床啪视频网站| 成人影院久久| 国产一卡二卡三卡精品 | 亚洲国产精品国产精品| 久久精品久久久久久噜噜老黄| 久久久精品区二区三区| 在线观看免费日韩欧美大片| 一区二区三区激情视频| 大香蕉久久成人网| 久久精品久久久久久久性| 电影成人av| 免费日韩欧美在线观看| 精品免费久久久久久久清纯 | 午夜福利在线免费观看网站| 午夜免费鲁丝| 亚洲,欧美精品.| 亚洲欧洲日产国产| 精品人妻在线不人妻| 亚洲国产欧美在线一区| 天天影视国产精品| 自拍欧美九色日韩亚洲蝌蚪91| 男女午夜视频在线观看| 最近最新中文字幕免费大全7| 飞空精品影院首页| 只有这里有精品99| 亚洲精品在线美女| 午夜av观看不卡| 麻豆精品久久久久久蜜桃| 色精品久久人妻99蜜桃| 九色亚洲精品在线播放| a级毛片在线看网站| 日本欧美国产在线视频| 香蕉丝袜av| 热re99久久国产66热| 欧美激情极品国产一区二区三区| 看免费av毛片| 大香蕉久久网| 黑人欧美特级aaaaaa片| 欧美激情 高清一区二区三区| 欧美另类一区| kizo精华| 久久人人爽av亚洲精品天堂| 十八禁网站网址无遮挡| 啦啦啦中文免费视频观看日本| 欧美黑人欧美精品刺激| 综合色丁香网| 高清视频免费观看一区二区| 黑人欧美特级aaaaaa片| 91精品三级在线观看| 久久久久人妻精品一区果冻| 国产成人一区二区在线| xxxhd国产人妻xxx| 亚洲国产日韩一区二区| 国产1区2区3区精品| www.av在线官网国产| 久久99热这里只频精品6学生| 久久亚洲国产成人精品v| 男女高潮啪啪啪动态图| 宅男免费午夜| 青青草视频在线视频观看| h视频一区二区三区| 亚洲精品国产一区二区精华液| 日韩视频在线欧美| 国产亚洲欧美精品永久| 久久精品aⅴ一区二区三区四区| 亚洲精品国产一区二区精华液| 日韩视频在线欧美| 欧美国产精品一级二级三级| 国产精品久久久人人做人人爽| 一级毛片电影观看| a级片在线免费高清观看视频| 亚洲精品国产色婷婷电影| 久久毛片免费看一区二区三区| 久久久精品94久久精品| 久久天堂一区二区三区四区| 欧美日韩视频高清一区二区三区二| 亚洲,欧美,日韩| 日韩成人av中文字幕在线观看| 男女床上黄色一级片免费看| 久久青草综合色| 黄色视频不卡| 热re99久久国产66热| 人人妻人人澡人人爽人人夜夜| 大片免费播放器 马上看| 午夜福利在线免费观看网站| 欧美人与性动交α欧美软件| 国产精品久久久久久人妻精品电影 | 国产在线一区二区三区精| 精品国产一区二区久久| 丝袜喷水一区| 日本猛色少妇xxxxx猛交久久| 欧美黑人精品巨大| 成人漫画全彩无遮挡| 亚洲,欧美,日韩| 最近中文字幕高清免费大全6| 国产成人一区二区在线| 国产成人免费无遮挡视频| 女人久久www免费人成看片| 大片免费播放器 马上看| 黑丝袜美女国产一区| 天天躁日日躁夜夜躁夜夜| 国产精品av久久久久免费| 最近手机中文字幕大全| 高清黄色对白视频在线免费看| 久久精品亚洲熟妇少妇任你| 亚洲av欧美aⅴ国产| 午夜激情av网站| 午夜日本视频在线| 亚洲成人一二三区av| 别揉我奶头~嗯~啊~动态视频 | 亚洲av综合色区一区| 99香蕉大伊视频| 自线自在国产av| 亚洲av男天堂| 日本欧美视频一区| 国产视频首页在线观看| 国产一区二区三区综合在线观看| netflix在线观看网站| 免费观看av网站的网址| 最近2019中文字幕mv第一页| 制服诱惑二区| 国产在线一区二区三区精| 最近最新中文字幕免费大全7| 欧美在线黄色| 久久影院123| 91精品三级在线观看| 91精品国产国语对白视频| 99精国产麻豆久久婷婷| 一边摸一边抽搐一进一出视频| 久久免费观看电影| 国产日韩欧美视频二区| 欧美日韩福利视频一区二区| 亚洲少妇的诱惑av| 777米奇影视久久| h视频一区二区三区| 久久影院123| 19禁男女啪啪无遮挡网站| 嫩草影院入口| 永久免费av网站大全| 狂野欧美激情性bbbbbb| 香蕉国产在线看| 天天躁夜夜躁狠狠躁躁| 精品久久蜜臀av无| 亚洲欧美成人精品一区二区| 国产成人免费无遮挡视频| 叶爱在线成人免费视频播放| 水蜜桃什么品种好| 波多野结衣一区麻豆| 熟妇人妻不卡中文字幕| 自拍欧美九色日韩亚洲蝌蚪91| 国产精品久久久人人做人人爽| 观看美女的网站| 亚洲成色77777| 老司机亚洲免费影院| 国产黄色免费在线视频| 老司机靠b影院| 99久久精品国产亚洲精品| 亚洲国产看品久久| 黄片播放在线免费| 亚洲成人av在线免费| 曰老女人黄片| 亚洲美女黄色视频免费看| 999久久久国产精品视频| 一本—道久久a久久精品蜜桃钙片| 精品一品国产午夜福利视频| 久久人人爽av亚洲精品天堂| 黑丝袜美女国产一区| 曰老女人黄片| 亚洲天堂av无毛| 黄色 视频免费看| 亚洲国产av新网站| 日韩一区二区视频免费看| 精品一区二区三区av网在线观看 | 日本爱情动作片www.在线观看| 悠悠久久av| 91aial.com中文字幕在线观看| 99久久99久久久精品蜜桃| av有码第一页| 久久97久久精品| 国产精品国产av在线观看| 国产精品亚洲av一区麻豆 | 国产精品国产三级专区第一集| 亚洲五月色婷婷综合| 久久免费观看电影| 在现免费观看毛片| 老鸭窝网址在线观看| a级毛片黄视频| 国产成人精品久久久久久| 女人爽到高潮嗷嗷叫在线视频| 人人妻人人澡人人爽人人夜夜| 色播在线永久视频| 黑丝袜美女国产一区| 美国免费a级毛片| 精品国产乱码久久久久久男人| 婷婷色综合www| 国产成人精品久久久久久| a级毛片黄视频| 亚洲激情五月婷婷啪啪| 亚洲在久久综合| 精品一区在线观看国产| 一区二区日韩欧美中文字幕| 大码成人一级视频| 秋霞伦理黄片| 欧美精品av麻豆av| 久久av网站| 在线观看国产h片| 欧美黑人欧美精品刺激| 亚洲,一卡二卡三卡| 在线天堂中文资源库| 一二三四中文在线观看免费高清| 女人久久www免费人成看片| 免费观看性生交大片5| 秋霞伦理黄片| 熟女少妇亚洲综合色aaa.| 少妇被粗大的猛进出69影院| 中文精品一卡2卡3卡4更新| 在线观看免费视频网站a站| 久久ye,这里只有精品| 蜜桃在线观看..| 久久精品久久精品一区二区三区| 亚洲久久久国产精品| 日韩成人av中文字幕在线观看| 亚洲国产日韩一区二区| 国产免费一区二区三区四区乱码| 人体艺术视频欧美日本| 热99国产精品久久久久久7| a级片在线免费高清观看视频| 欧美日韩视频高清一区二区三区二| 精品人妻熟女毛片av久久网站| 精品少妇内射三级| 亚洲成人av在线免费| 精品久久久精品久久久| 亚洲成色77777| 好男人视频免费观看在线| 欧美日韩综合久久久久久| 丰满少妇做爰视频| 欧美日韩一区二区视频在线观看视频在线| 亚洲综合精品二区| 国产有黄有色有爽视频| 日本欧美国产在线视频| 亚洲国产精品一区二区三区在线| 一边摸一边抽搐一进一出视频| 电影成人av| 欧美成人午夜精品| 午夜av观看不卡| 亚洲专区中文字幕在线 | 成年人午夜在线观看视频| 午夜福利网站1000一区二区三区| 久久久久精品性色| 成人黄色视频免费在线看| 国产色婷婷99| 中文字幕av电影在线播放| 亚洲av欧美aⅴ国产| 久久人人97超碰香蕉20202| 午夜久久久在线观看| videosex国产| 国产亚洲av高清不卡| 亚洲伊人久久精品综合| kizo精华| 亚洲自偷自拍图片 自拍| 亚洲熟女精品中文字幕| 美女中出高潮动态图| 久久 成人 亚洲| av网站免费在线观看视频| 亚洲美女搞黄在线观看| 国产精品一区二区在线不卡| 国产av精品麻豆| 亚洲熟女毛片儿| 色吧在线观看| 捣出白浆h1v1| 国产 精品1| 少妇人妻 视频| 18禁国产床啪视频网站| 老司机深夜福利视频在线观看 | 又大又爽又粗| 丝袜喷水一区| 一区二区三区四区激情视频| 亚洲av欧美aⅴ国产| 久久精品国产a三级三级三级| 亚洲精品国产色婷婷电影| 国产熟女欧美一区二区| 亚洲精品国产区一区二| 美女扒开内裤让男人捅视频| 午夜福利在线免费观看网站| 国产亚洲一区二区精品| 成人三级做爰电影| videosex国产| 久久久精品区二区三区| 久久久国产一区二区| 色94色欧美一区二区| 一区二区三区激情视频| av网站在线播放免费| 90打野战视频偷拍视频| 性色av一级| 熟女少妇亚洲综合色aaa.| 波野结衣二区三区在线| 精品国产超薄肉色丝袜足j| 久久久国产一区二区| 午夜福利在线免费观看网站| 精品国产超薄肉色丝袜足j| 高清av免费在线| 国产高清不卡午夜福利| 亚洲精品国产区一区二| 欧美国产精品一级二级三级| 香蕉丝袜av| 女的被弄到高潮叫床怎么办| www.熟女人妻精品国产| 免费观看av网站的网址| 国产爽快片一区二区三区| 成人漫画全彩无遮挡| 国产女主播在线喷水免费视频网站| 国产在线一区二区三区精| 精品一区二区三区av网在线观看 | 欧美 亚洲 国产 日韩一| 久久精品久久久久久噜噜老黄| 欧美老熟妇乱子伦牲交| 国产老妇伦熟女老妇高清| 国产一区二区在线观看av| 精品少妇黑人巨大在线播放| 狂野欧美激情性bbbbbb| 各种免费的搞黄视频|