• <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 ?

    L-Arginine based polyester amide/hyaluronic acid hybrid hydrogel with dual anti-inflammation and antioxidant functions for accelerated wound healing

    2022-06-20 06:19:48TongLiuGuitingLiuJinhuZhngZhngfnDingYikeLiKrishnSigdelXioyiWngHuixuXie
    Chinese Chemical Letters 2022年4期

    Tong Liu,Guiting Liu,Jinhu Zhng,Zhngfn Ding,Yike Li,Krishn Sigdel,Xioyi Wng,*,Huixu Xie,*

    a State Key Laboratory of Oral Diseases,National Clinical Research Center for Oral Diseases,Department of Head and Neck Oncology Surgery,West China Hospital of Stomatology,Sichuan University,Chengdu 610041,China

    b The State Key Laboratory of Polymer Materials Engineering,Polymer Research Institute of Sichuan University,Chengdu 610065,China

    c College of Polymer Science and Engineering,State Key Laboratory of Polymer Materials Engineering,Sichuan University,Chengdu 610065,China

    1 These authors contributed equally to this work.

    ABSTRACT Nowadays,there are still many challenges to skin regeneration.As a new type of skin substitute,hydrogel has emerging gradually with its excellent properties.However,it is still a challenge to combine with biological active agents to facilitate skin regeneration.Under the circumstance,we synthesized argininebased poly(ester amide)(Arg-PEA)and hyaluronic acid(HA-MA),and combined them into new hybrid hydrogels via photo-crosslinking.We found that the internal structure and physicochemical properties of hybrid hydrogels were greatly improved with the increase of content of Arg-PEA.Therefore,we designed hybrid hydrogels with 5 wt% and 10 wt% of Arg-PEA content,respectively.Besides,we selected the corresponding anti-inflammatory(CRP,TNF-α)indicators to detect the anti-inflammatory properties of the hybrid hydrogels at the protein level,and the corresponding antioxidant indicators(SOD,GSH/GSSG,MDA)were selected to investigate the antioxidant properties of hybrid hydrogels at the cellular level in vitro.In addition,we also selected relevant genes to test the effect of hybrid hydrogels on fibrosis and vascularization in the process of skin wound healing in vitro and verified them in vivo with a mouse dorsum wound model.The results confirmed that Arg-PEA/HA-MA(AH)hybrid hydrogel was a prospective scaffold material for skin regeneration.

    Keywords:Hydrogel Skin wound healing Antioxidant Anti-inflammation

    As the largest organ,skin plays an important role in preventing mechanical,chemical and pathological damage as well as dehydration and infection[1–5].Once the skin is damaged,it should be very urgent to achieve wound repair,otherwise it will lead to chronic wounds and even death.In the past few decades,autologous skin grafts have always been the most frequently utilized method to repair skin defects[6–8].However,there are still some significant clinical challenges and socioeconomic burdens in autologous skin grafts,such as the limitations of the autologous tissue sources,the difficulty of skin grafts,and the low success rate of operations[9,10].Recently,tissue engineering has received widespread attention as a new method of wound repair,as it could lead to a range of new and powerful artificial skin alternatives and overcome the limitations of skin transplantations to some extent.Scaffold plays a key role in tissue engineering,it should not only provide space and place to support tissue regeneration,but also possess the properties of biocompatibility and biodegradability[11–13].At present,hydrogel has become a prospective scaffold material.It has good biocompatibility and biodegradability,and could provide a three-dimensional polymer network to facilitate cell adhesion,proliferation,differentiation,migration and transport of nutrients,which is conducive to skin regeneration.Moreover,according to the physiological characteristics of wound tissue,hydrogels can be given corresponding biological activities to accelerate wound healing.

    Generally,wound healing consists three stages:inflammatory and contractile responses,granulation tissue formation and tissue remodeling[14,15].Among them,appropriate inflammatory and contractile responses are crucial,which could play a role in hemostasis,protecting the body and preventing serious complications[15–17].However,continuous inflammatory response leads to the accumulation of a large number of reactive oxygen species(ROS).However,the antioxidant capacity of cells is limited,and a large number of ROS hinders the transition from the inflammatory stage to the proliferative stage of the wound[17–19].Many studies have shown that low levels of ROS could stimulate cell migration and angiogenesis,thus promoting wound healing,but high levels of ROS would hinder wound healing,especially in chronic wounds[20,21].Therefore,it is vital to promote effective antiinflammatory and anti-oxidation in the process of skin wound healing.L-Arginine,known as a protein amino acid and one of the most crucial nutrition supplies,is essential for wound healing[16,17].As numerous works demonstrated that L-arginine displays both anti-inflammatory and antioxidant activities,and can participate in metabolism and produce creatine,polyamines,agmatine and nitric oxide(NO),which are beneficial to tissue regeneration.However,in wound tissue,the supply of L-arginine is seriously insufficient and prolongs the inflammatory and contractile responses,hence sustained and targeted supply of L-arginine in wound site could be a potential and powerful strategy for wound repair.

    In this work,we attempted to prepare a novel L-arginine based polyester amide/hyaluronic acid(AH)hybrid hydrogel with anti-inflammation and antioxidant dual-functions for accelerated wound healing.First,Arg-PEA was prepared by chemical bonding of dip-nitrophenyl ester of dicarboxylic acids(NF)and di-p-toluene sulfonic acid salts of bis-L-arginine(or bis-DL-2-allylglycine)esters(Arg-2-S),then AH hybrid hydrogel was fabricated by chemical reaction and electrostatic interaction between Arg-PEA and methacrylic anhydride modified hyaluronic acid(HA-MA)under UV irradiation.By this design,AH hydrogel could be used for in-situ gelation,suitable for irregular wounds,and the physical property of hydrogel could be adjusted by the addition of Arg-PEA,furthermore,L-arginine could be released sustainedly and targeted by the hydrolysis reaction of Arg-PEA in wound site to achieve antiinflammatory and antioxidant dual-effects.In the present study,we aimed to characterize the composition,physicochemical properties,biological properties,anti-inflammatory and antioxidant activityin vitro,meanwhile,we verified the effect of AH hybrid hydrogels in a mouse dorsal injury modelin vivo.

    This experiment was approved by the Ethics Committee of West China Hospital of Stomatology,Sichuan University(WCHSIRBD-2017–263).In this experiment,we observed the internal microstructures of hydrogels by SEM.For the physical and chemical properties of hydrogels,we tested compressive mechanical property,swelling kinetics and degradation of hydrogels,respectively.The extracts of hydrogel samples were prepared by soaking the hydrogels in DMEM or MEM at a mass-to-volume ratio of 100 mg/mL respectively[15].Cell viability was quantitatively evaluated using CCK-8 colorimetric assay.To observe the morphology and distribution of cells under the influence of extracts of the biomaterials,the processed samples were placed under the CLSM.The protein expression of inflammatory factors(CRP,TNF-α)was measured by ELISA to detect the anti-inflammatory effect of hydrogels.To explore the anti-oxidant of HUVECs under the influence of extracts of hybrid hydrogels,we stimulated the cellular microenvironment by hydrogen peroxide(H2O2).To explore fibrosis and vascularization in skin regeneration,we tested Krt 10,Krt 14,Col 1α,Col 3α,EGF,TGF-βand VEGF by qRT-PCR,respectively.Sevenweek-old female Kunming(KM)mice weighing about 25 g were used for the experimentsin vivo.At the specific time points,the wound area and surrounding normal tissues were removed and fixed in 4% paraformaldehyde,and then these tissues were sectioned and carried out by histological and immunohistochemical staining.HE and Masson’s trichrome staining were used to assess morphology of the wound areas and collagen formation.Immunohistochemical analysis was performed with antibodies against Krt 10,Krt 14,Col 1α,Col 3α,CD 31 and CD 34 to evaluate fibrosis and vascularization of the wound areas.After all the treated sections were observed,representative locations were selected for further evaluation.Related experimental procedures are provided in Supporting information.

    The hydrogel samples were shown in Fig.1A.We found that the color of hybrid hydrogels gradually turned dark yellow with the increase of the content of Arg-PEA.The FTIR spectra of hydrogels were shown in Fig.1B.We observed that the stretching positions of amide I and amide II corresponding to C=O in Arg-PEA were 1640 cm-1and 1560 cm-1,respectively,and the stretching position of C–OH in HA-MA was 1000 cm-1.The presented FTIR indicated the successful formation of hybrid hydrogels.The microstructure of the hydrogels was shown in Fig.1C.The pore size of the HA-MA hydrogel was about 100 μm,while the pore sizes of the hybrid hydrogel became denser and decreased to about 50 μm with the increase of the content of Arg-PEA.The hybrid hydrogels have good three-dimensional porous structures,which are beneficial to cell attachment and transport of nutrients and metabolites.Under the circumstance,AH hybrid hydrogel provides a feasible choice for obtaining adequate mechanical properties of skin regeneration.

    The physical and chemical properties of hydrogels were shown in Fig.S1(Supporting information).We found that the compression modulus of HA-MA hydrogel was about 0.9 MPa.With the increase of content of Arg-PEA,the compression modulus and brittleness of hybrid hydrogels decreased.In the swelling kinetics experiment,the weight of the hydrogel samples increased rapidly in the initial 2 h,and the swelling rate tended to be stable with the passage of time.With the increase of Arg-PEA content,the equilibrium swelling ratio of hydrogels decreased from 10 to 3.In the hybrid hydrogel system,the electrostatic attraction between HAMA and Arg-PEA hinders the expansion of the network of hydrogels,which explains the low expansion rate of the hybrid hydrogel.From the perspective of molecular structure,Arg-PEA has a lot of ester bonds and amide bonds,which also means that Arg-PEA is hydrophilic and easy to hydrolysis.As a consequence,with the increase of the content of Arg-PEA,the degradation rate of the hydrogels increased gradually.At the later stage of the degradation kinetics experiment,the remaining hydrogel scaffolds were relatively stable,which also provided a basis for skin regenerationin vivo.

    In live/dead assay,the CCK-8 showed that the hydrogel samples had an effect on the proliferation ability of HUVECs(Fig.S2A in Supporting information).Induced or overexpressed arginase promotes arginine metabolism,resulting in the production of polyamines,which contribute to endothelial cell proliferation to some extent.In addition,the groups of hydrogels showed no significant cytotoxicity to HUVECs.At the same time,through CLSM(Fig.S2B in Supporting information),cells in each group showed their proper color,morphology and distribution.It is proved that AH hybrid hydrogels have a good biocompatibility with HUVECs,and the result demonstrated the bio-safety of hydrogels for skin regenerationin vitro.

    Fig.1.Characterization of the formation of obtained hybrid hydrogels:(A)An image of AH hydrogels with Arg-PEA contents of 0%,5%,10% from left to right.(B)FTIR spectra of precursors and hybrid hydrogels(a,HA-MA;b,5% AH;c,10% AH).(C)SEM images of freeze-dried pure HA hydrogel and AH hydrogels.Scale bar:100 μm.

    Fig.2.Anti-oxidant,anti-inflammation,fibrosis and vascularization properties of hybrid hydrogels.(A)Image of ROS immunofluorescence in HUVECs photographed by fluorescence microscope.(B-D)Absorbance values of GSH/GSSG,SOD and MDA under the influence of hybrid hydrogels.(E)Concentration of CRP and TNF-α.(F-H)Krt 10,Krt 14,Col 1α,Col 3α,EGF,TGF-β and VEGF mRNA expression in skin regeneration treated with hydrogels.The quantitative analysis of gene transcript level,relative to β-Actin,was determined by qRT-PCR.Error bars indicate mean ± SD,*P <0.05,**P <0.01,***P <0.001.

    Fig.3.Healing trend chart of dorsal wound of mouse model.(A)Image of dorsal wound of mouse model.(B and C)During wound healing,every skin wound area was quantitatively measured at a specific time.(B)Represents the healing tendency.Data points represent mean recovery(%)(a,Control group;b,Gauze;c,HA-MA;d,5% AH;e,10% AH).(C)Represents the statistical significance between two groups,error bars indicate mean ± SD,**P <0.01,***P <0.001.

    For anti-oxidant assay,the stained cells were observed under a fluorescence microscope and were photographed(Fig.2A).At the same time,we quantitatively detected the content of free radical(GSH/GSSG,SOD and MDA)(Figs.2B-D).SOD is one of major components of ROS.And it is the intermediate product of many biological processes and has certain cytotoxicity.Through co-cultured with hydrogels,with the increase of Arg-PEA content,stronger radical scavenging activities of superoxide were observed in HUVECs.Results showed that the generation of SOD was effectively inhibited to varying degrees by AH hybrid hydrogels.In addition,this study found that the accumulation of MDA was significantly negatively correlated with the content of Arg-PEA.These results indicated that with the increase of Arg-PEA content,the excess ROS in microenvironment could be effectively removed.In this study,electron transfer(ET)mechanism could explain the antioxidant effect of Arg-PEA[22–24].Arg-PEA could share one of its electrons to a free radical and react with it,thus terminating the chain reaction of free radicals.On this basis,high levels of Arg-PEA could be exposed to more proton/electron donors,and thus exhibiting stronger reductivity and antioxidant capacity[25,26].In addition,the electron cloud density of the functional group also has a significant effect on the activity of electron donor.High electron cloud density increases the activity of electron donor,thus increasing the ability to scavenging free radicals[27,28].Therefore,Arg-PEA promotes the availability of potential electron donors,thereby removing excess ROS in the microenvironment and ameliorating oxidative stress.Besides,an increased level of GSH was positively associated with the content of Arg-PEA.Among them,glutamate,which is contained in GSH,plays an important role in antioxidant activity.L-Arginine is a substrate for glutamate synthesis,and its supplementation may indirectly stimulate endogenous GSH synthesis.Therefore,Arg-PEA supplementation in this study could promote GSH synthesis and exert antioxidant properties.

    ELISA assay was utilized to test the effect of hydrogel samples on the expression of inflammatory factors CRP and TNF-α(Fig.2E).The expression of CRP and TNF-αdecreased in the hydrogel groups compared with control group,and the higher the Arg-PEA content,the lower the factor expression(CRP:199.7,183.4,173.4,160.1 pg/mL;TNF-α:68.49,61.95,56.07,47.65 pg/mL).Therefore,it concluded that the three hydrogels could reduce the immune stress state of the body during wound healing,and it could provide a better basis for skin wound healing.

    For the fibrosis and vascularization property of hydrogelsin vitro,we detected the expression of genes associated with skin wound healing(Krt 10,Krt 14,Col 1α,Col 3α,EGF,TGF-β,VEGF)by qRT-PCR(Figs.2F-H).The expressions of these seven genes were significantly up-regulated under the stimulation of different hydrogels(P <0.05).These results provided a basis for furtherin vivostudies.Keratin is the main skeleton protein in keratinocytes.During keratinocyte differentiation,the keratin expression profile changes due to the change of cell morphology and function.Therefore,the characterization of key keratins is the key to judge whether hydrogels could promote wound healing.As for vascular growth factors,thein vitroresults showed that the mRNA expression levels of EGF,TGF-βand VEGF were increased in different degrees in the experimental group,indicating that the hydrogel materials could promote cell proliferation during wound healing.It showed that hydrogel materials promote the synthesis of extracellular matrix,deposition of collagen and formation of granulation tissue during skin wound healing by regulating vascular growth factors,thus promoting skin wound healing.

    The dorsum wound healing of each group was shown in Fig.3A.During this experiment,mice in each group were in good physical condition.Slight redness and swelling were observed in the process of the wound healing,and no obvious signs of infection was observed.Accordingly,we performed a quantitative analysis of the percentage of defect recovery based on the collected images of skin wounds throughout the experiment(Figs.3B and C).In the experimental groups,we found that the wound diameters decreased significantly from day 0 to day 4,the reduction rates of the wound diameters slowed down from day 4 to day 8,and the wounds were gradually closed until day 14.There was no significant difference between the three experimental groups.In the empty group and gauze group,the diameters of the defects also decreased over time,but the diameters of the wounds decreased significantly from day 4 to day 8.Up to day 14,there were scabs in the middle of the defect areas indicating incomplete closure.

    By HE staining(Fig.S3A in Supporting information),the epidermal continuity of the wound area in the empty control group was interrupted in the early stage.While in the experimental group,there were more temporary matrix accumulated on the wound bed than the empty control group.As time went by,scab had formed over the wound of empty control group.Between the scab and the temporary matrix,it could be observed that the epidermal tongue gradually moved from the margin to the center of the wound.And the temporary matrix gradually covered the wound and the blue-stained collagen increased significantly.In the experimental groups,re-epithelialization of the wound bed was completed at day 8 post-surgery with a significant increase in the density of the new tissue due to collagen deposition of fibroblasts.At day 12 post-surgery,the process of re-epithelialization was still uncompleted in the empty group,while there was a more mature,layered new epidermis that was thicker than normal epidermal tissue around the wound in the experiment group.In addition,the density of collagen deposition during this time period was more uniform than before.Hybrid hydrogels facilitated the formation and maturation of new tissue in the wound.As mentioned above,the early granulation tissue formed by fibronectin and collagen,which was subsequently remodeled to the formation of the new dermis.Masson’s trichrome staining showed that in the empty control group,the collagen content in the wound was low despite an increased overall density of new tissue in the wound.While in the experimental group,collagen density significantly accumulated during wound recovery(Fig.S3B in Supporting information).

    By immunohistochemical staining(Figs.S3C and D in Supporting information),at day 4 post-surgery,in the experimental groups,the expression of Krt 10 was widely performed in new epidermal tissue around the edge of the wound.For Krt 14,it was expressed in strongly stained basal cells in the experimental group and was widely dispersed from the apical to the basal layer in the dermal tongue that migrated from the edge of the wound to the center.Staining of the experimental group at 8–12 post-surgery showed that Krt 10 was widely distributed in the new epidermis tissue and gradually migrated to the differentiated spinous layer,and the expression of Krt 14 gradually tended to be evenly dispersed in the basal layer instead of the whole layer.It indicated that keratinocytes changed from the previously dedifferentiated state to the normal differentiated state,thus further remodeling the new epidermis and promoting skin healing.In this study,the hybrid hydrogels provided a temporary matrix by direct contact with the wound and activated keratinocytes,thereby accelerating re-epithelialization.It is not difficult to find that the expressions of Krt 10,Krt 14 by hydrogelsin vivois consistent with thosein vitro.

    For vascular factors(Figs.S3E and F in Supporting information),we found that the immunofluorescence staining of CD 31 positive cells was widely distributed in the wound surfaces of each group.Moreover,the distribution of staining of CD 31 positive cells gradually spread and thickened from time goes on,indicating that CD 31 actively promoted wound healing.However,immunofluorescence staining of CD 34 positive cells was more frequent on the day 4 post-operation.As time went on,staining of CD 34 positive cells between day 8 and day 12 post-operation showed a downregulated trend,as CD 34 molecules would gradually weaken to disappear with the maturation of the cells,which was also consistent with its own function.In vivo,we focused on plateletendothelial adhesion factors(CD 31,CD 34).It could be found that although CD 31 and CD 34 play a role in wound healing process,there is no significant difference between the control group and the experimental groups.It could be seen that hybrid hydrogel materials have almost no regulated effect on CD 31 and CD 34 in the process of promoting wound healing.

    In conclusion,we successfully synthesized Arg-PEA and HAMA,and prepared AH hybrid hydrogels with different feed ratios by photo-crosslinking to explore their roles in the process of skin wound healing.The results showed that the internal morphology,swelling,mechanical and biodegradation properties of the hybrid hydrogel system could be well adjusted by changing the content of Arg-PEA.More importantly,through detecting corresponding signal molecules,hybrid hydrogels were found to have antioxidant,antiinflammatory,fibrosis and vascularization effects in the process of skin wound healing.

    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 work was supported by the National Natural Science Foundation of China(No.52103039)and Sichuan University postdoctoral interdisciplinary Innovation Fund.

    Supplementary materials

    Supplementary material associated with this article can be found,in the online version,at doi:10.1016/j.cclet.2021.10.022.

    av播播在线观看一区| 久热这里只有精品99| 黄色日韩在线| 看非洲黑人一级黄片| 欧美国产精品一级二级三级 | 少妇精品久久久久久久| 黄色日韩在线| 日韩制服骚丝袜av| 久久国产亚洲av麻豆专区| 国产乱来视频区| 夜夜爽夜夜爽视频| 国产成人精品久久久久久| 国产伦精品一区二区三区视频9| 综合色丁香网| 日韩一区二区三区影片| 亚洲人成网站在线观看播放| 午夜日本视频在线| 伦理电影免费视频| 久久精品国产亚洲网站| 亚洲国产精品999| 亚洲精品视频女| 午夜久久久在线观看| 国产精品熟女久久久久浪| 日日啪夜夜爽| 中文字幕人妻丝袜制服| 国产一区二区在线观看av| 精品久久国产蜜桃| 免费大片18禁| 麻豆乱淫一区二区| 另类亚洲欧美激情| 亚洲精品久久久久久婷婷小说| 国产男女内射视频| 水蜜桃什么品种好| 国产午夜精品一二区理论片| 欧美亚洲 丝袜 人妻 在线| 欧美少妇被猛烈插入视频| 亚洲国产色片| 久久婷婷青草| 日韩人妻高清精品专区| 久久久久人妻精品一区果冻| 在线观看www视频免费| 国产精品不卡视频一区二区| 亚洲天堂av无毛| 欧美日韩视频高清一区二区三区二| 日韩一区二区三区影片| 亚洲,欧美,日韩| 国产极品天堂在线| 成人18禁高潮啪啪吃奶动态图 | 日韩精品有码人妻一区| 成人影院久久| 久久人人爽人人片av| 秋霞伦理黄片| 久久免费观看电影| 国产成人免费观看mmmm| 色视频在线一区二区三区| 亚洲欧美一区二区三区国产| 秋霞伦理黄片| 亚洲精品aⅴ在线观看| 我的女老师完整版在线观看| 亚洲综合精品二区| 伊人久久国产一区二区| 波野结衣二区三区在线| 国产精品秋霞免费鲁丝片| 天堂8中文在线网| 久久精品夜色国产| 成年女人在线观看亚洲视频| 国产精品久久久久久久电影| 99久久中文字幕三级久久日本| 91精品国产国语对白视频| 一级黄片播放器| 日韩精品免费视频一区二区三区 | 91在线精品国自产拍蜜月| 国产日韩欧美视频二区| 国产在线男女| 亚洲精品日韩在线中文字幕| 狂野欧美激情性xxxx在线观看| 简卡轻食公司| 国产午夜精品一二区理论片| 国产精品偷伦视频观看了| 又粗又硬又长又爽又黄的视频| 精品国产国语对白av| 99热这里只有精品一区| 女人久久www免费人成看片| 日本色播在线视频| 91久久精品国产一区二区成人| 妹子高潮喷水视频| 久久精品国产亚洲av涩爱| 蜜臀久久99精品久久宅男| 欧美另类一区| 国产日韩欧美视频二区| 亚洲久久久国产精品| 欧美亚洲 丝袜 人妻 在线| 国产视频首页在线观看| 天堂俺去俺来也www色官网| 在线播放无遮挡| 国产精品国产三级专区第一集| 777米奇影视久久| 五月玫瑰六月丁香| 国产成人午夜福利电影在线观看| 超碰97精品在线观看| 一本一本综合久久| 国产在视频线精品| 少妇 在线观看| 韩国高清视频一区二区三区| 18禁动态无遮挡网站| 蜜桃久久精品国产亚洲av| 久热这里只有精品99| 国产精品99久久久久久久久| av福利片在线观看| 国内少妇人妻偷人精品xxx网站| 国产精品无大码| 一本色道久久久久久精品综合| 成人特级av手机在线观看| 在线观看免费高清a一片| 水蜜桃什么品种好| 成人综合一区亚洲| 在线观看免费日韩欧美大片 | 夜夜骑夜夜射夜夜干| 亚洲三级黄色毛片| 成人毛片60女人毛片免费| 精品一区在线观看国产| 蜜桃久久精品国产亚洲av| 夜夜看夜夜爽夜夜摸| 一边亲一边摸免费视频| 啦啦啦啦在线视频资源| 亚洲中文av在线| av免费在线看不卡| 少妇人妻精品综合一区二区| 不卡视频在线观看欧美| 日韩三级伦理在线观看| 最近2019中文字幕mv第一页| 欧美激情国产日韩精品一区| 中国三级夫妇交换| 最近2019中文字幕mv第一页| h日本视频在线播放| 各种免费的搞黄视频| 狂野欧美激情性bbbbbb| 视频中文字幕在线观看| 日韩视频在线欧美| 少妇精品久久久久久久| 高清视频免费观看一区二区| 国产成人精品久久久久久| 午夜av观看不卡| 亚洲av男天堂| 亚洲精品国产色婷婷电影| 嫩草影院入口| 国产日韩欧美视频二区| 亚洲精品乱码久久久v下载方式| 日韩一区二区视频免费看| .国产精品久久| 22中文网久久字幕| 亚洲av成人精品一二三区| 男女国产视频网站| 又爽又黄a免费视频| 亚洲av日韩在线播放| 久久久久人妻精品一区果冻| 丝袜喷水一区| 亚洲熟女精品中文字幕| 日韩亚洲欧美综合| 精品人妻熟女毛片av久久网站| 人人妻人人澡人人看| av不卡在线播放| 欧美变态另类bdsm刘玥| 九九久久精品国产亚洲av麻豆| 色婷婷久久久亚洲欧美| 国产一区二区三区综合在线观看 | 成人毛片60女人毛片免费| 久久久久人妻精品一区果冻| 国产日韩欧美亚洲二区| 插阴视频在线观看视频| 成人综合一区亚洲| 久久精品国产自在天天线| 国产精品久久久久成人av| 午夜免费观看性视频| 日本色播在线视频| 下体分泌物呈黄色| 国产精品.久久久| 在线观看三级黄色| 在线亚洲精品国产二区图片欧美 | 日韩精品有码人妻一区| 狂野欧美白嫩少妇大欣赏| 欧美激情极品国产一区二区三区 | 一区二区av电影网| 国产 精品1| 欧美日本中文国产一区发布| 国产男女内射视频| av线在线观看网站| 汤姆久久久久久久影院中文字幕| 一本久久精品| 99re6热这里在线精品视频| 桃花免费在线播放| 国产精品久久久久久精品古装| 精品国产一区二区三区久久久樱花| 欧美国产精品一级二级三级 | xxx大片免费视频| 在线观看www视频免费| 人体艺术视频欧美日本| 欧美另类一区| 涩涩av久久男人的天堂| 免费高清在线观看视频在线观看| 一本一本综合久久| 看十八女毛片水多多多| 久久久久人妻精品一区果冻| 亚洲av成人精品一区久久| 亚洲精品日韩在线中文字幕| 春色校园在线视频观看| 黄色一级大片看看| 狂野欧美激情性xxxx在线观看| 成年av动漫网址| 亚洲精品aⅴ在线观看| 国产高清有码在线观看视频| h视频一区二区三区| 久久99蜜桃精品久久| 午夜老司机福利剧场| 亚洲精华国产精华液的使用体验| 黑人高潮一二区| 免费看av在线观看网站| 男的添女的下面高潮视频| 久久青草综合色| 免费少妇av软件| 亚洲熟女精品中文字幕| 简卡轻食公司| 国产av码专区亚洲av| 亚洲欧洲日产国产| 精品亚洲成a人片在线观看| av播播在线观看一区| 国产亚洲精品久久久com| 色5月婷婷丁香| 十分钟在线观看高清视频www | 两个人的视频大全免费| 久久午夜福利片| 中国美白少妇内射xxxbb| 精品久久久久久电影网| 欧美 日韩 精品 国产| 国产女主播在线喷水免费视频网站| 久久青草综合色| 在线观看免费视频网站a站| 亚洲欧美成人综合另类久久久| 午夜激情福利司机影院| 欧美日韩一区二区视频在线观看视频在线| 91在线精品国自产拍蜜月| 久久婷婷青草| av又黄又爽大尺度在线免费看| 亚洲精品456在线播放app| 成人毛片60女人毛片免费| 国产成人a∨麻豆精品| 亚洲人成网站在线观看播放| 久久久国产一区二区| 久久久久久久国产电影| 深夜a级毛片| 一本一本综合久久| 最近中文字幕2019免费版| 国产日韩一区二区三区精品不卡 | 国产伦理片在线播放av一区| 精品午夜福利在线看| 熟妇人妻不卡中文字幕| 欧美日韩视频精品一区| 天堂俺去俺来也www色官网| 永久网站在线| av国产久精品久网站免费入址| 高清视频免费观看一区二区| 伦理电影免费视频| 一级毛片aaaaaa免费看小| 亚洲怡红院男人天堂| 日韩欧美精品免费久久| 一级爰片在线观看| 亚洲av男天堂| 亚洲欧美日韩另类电影网站| 亚洲性久久影院| 国产男女内射视频| 久久久a久久爽久久v久久| 欧美日韩精品成人综合77777| 97精品久久久久久久久久精品| 全区人妻精品视频| 国产免费一区二区三区四区乱码| 大香蕉久久网| av国产精品久久久久影院| 成人午夜精彩视频在线观看| 爱豆传媒免费全集在线观看| 久久久久久久久久久丰满| 成人黄色视频免费在线看| 国产黄色视频一区二区在线观看| 两个人的视频大全免费| 在现免费观看毛片| 亚洲欧美精品自产自拍| 精品少妇内射三级| www.av在线官网国产| 搡老乐熟女国产| 精品一区二区三卡| xxx大片免费视频| 亚洲av在线观看美女高潮| 深夜a级毛片| 一级黄片播放器| 日韩欧美 国产精品| 夜夜骑夜夜射夜夜干| h日本视频在线播放| 天美传媒精品一区二区| 亚洲人成网站在线观看播放| 欧美国产精品一级二级三级 | 天美传媒精品一区二区| 亚洲性久久影院| a级毛片免费高清观看在线播放| 久久久久久久亚洲中文字幕| 亚洲美女视频黄频| 99热这里只有精品一区| 最新中文字幕久久久久| 纯流量卡能插随身wifi吗| 免费高清在线观看视频在线观看| 99热全是精品| 日产精品乱码卡一卡2卡三| 亚洲国产色片| 天天操日日干夜夜撸| 一级毛片我不卡| 精品酒店卫生间| 天堂8中文在线网| 国产精品人妻久久久久久| 日本猛色少妇xxxxx猛交久久| 国产高清三级在线| 国产一区亚洲一区在线观看| 亚洲国产毛片av蜜桃av| 日韩大片免费观看网站| 老熟女久久久| 九九久久精品国产亚洲av麻豆| 亚洲国产成人一精品久久久| 久久精品久久久久久噜噜老黄| 另类精品久久| 亚洲熟女精品中文字幕| 欧美日韩亚洲高清精品| 观看av在线不卡| 久久精品久久久久久久性| 久久久午夜欧美精品| 水蜜桃什么品种好| 亚洲精品自拍成人| 在线免费观看不下载黄p国产| 男女免费视频国产| 国产老妇伦熟女老妇高清| 日韩不卡一区二区三区视频在线| 亚洲国产欧美日韩在线播放 | 老女人水多毛片| 视频区图区小说| 18禁动态无遮挡网站| 哪个播放器可以免费观看大片| 性色av一级| 蜜臀久久99精品久久宅男| 51国产日韩欧美| 国产永久视频网站| 亚洲精品第二区| 亚洲激情五月婷婷啪啪| 国产日韩欧美视频二区| 欧美精品一区二区免费开放| 国产精品久久久久久精品古装| 久久99蜜桃精品久久| 国产视频内射| av免费观看日本| 国产精品久久久久久精品古装| 免费人妻精品一区二区三区视频| 久久精品久久久久久久性| 自拍偷自拍亚洲精品老妇| 国产乱来视频区| 日产精品乱码卡一卡2卡三| 99热全是精品| 如日韩欧美国产精品一区二区三区 | 搡女人真爽免费视频火全软件| 久久久久久久大尺度免费视频| 欧美日韩在线观看h| 久久99热6这里只有精品| av在线观看视频网站免费| 人妻系列 视频| 午夜日本视频在线| 热re99久久精品国产66热6| 亚洲真实伦在线观看| 国产亚洲一区二区精品| 免费人妻精品一区二区三区视频| 国产精品伦人一区二区| 国产欧美日韩综合在线一区二区 | 免费人妻精品一区二区三区视频| 天堂俺去俺来也www色官网| 久久99精品国语久久久| 国产精品伦人一区二区| 亚洲欧洲国产日韩| 涩涩av久久男人的天堂| 欧美精品国产亚洲| av专区在线播放| 黑人猛操日本美女一级片| 80岁老熟妇乱子伦牲交| 成人漫画全彩无遮挡| 人人妻人人爽人人添夜夜欢视频 | 另类亚洲欧美激情| 免费大片18禁| 女人久久www免费人成看片| av卡一久久| 大陆偷拍与自拍| 春色校园在线视频观看| 中文天堂在线官网| 国产精品一二三区在线看| 亚洲欧美精品专区久久| 国产爽快片一区二区三区| 亚洲欧美成人综合另类久久久| 伊人久久国产一区二区| 777米奇影视久久| 日韩电影二区| 国产视频首页在线观看| 国产成人freesex在线| 免费观看无遮挡的男女| 精品国产露脸久久av麻豆| 2021少妇久久久久久久久久久| 美女脱内裤让男人舔精品视频| 最近手机中文字幕大全| 伊人亚洲综合成人网| 国产中年淑女户外野战色| 国产精品偷伦视频观看了| 水蜜桃什么品种好| 女性生殖器流出的白浆| 美女中出高潮动态图| 亚洲av日韩在线播放| 人人妻人人添人人爽欧美一区卜| 高清在线视频一区二区三区| 我的女老师完整版在线观看| 久久婷婷青草| 久久人人爽av亚洲精品天堂| 大香蕉久久网| 中文字幕精品免费在线观看视频 | a级一级毛片免费在线观看| 久久久久人妻精品一区果冻| 久久久久国产网址| 国产在视频线精品| a级毛片在线看网站| 麻豆精品久久久久久蜜桃| 日本av免费视频播放| 亚洲精品456在线播放app| 深夜a级毛片| 自线自在国产av| 日韩大片免费观看网站| 久久这里有精品视频免费| 成人影院久久| 国产成人精品福利久久| 夜夜骑夜夜射夜夜干| 中文天堂在线官网| 成人二区视频| 女人精品久久久久毛片| av专区在线播放| 一级二级三级毛片免费看| www.av在线官网国产| 精品久久久精品久久久| 天天躁夜夜躁狠狠久久av| 99热这里只有是精品在线观看| av福利片在线| 一级爰片在线观看| 中国国产av一级| 九草在线视频观看| 99久久精品国产国产毛片| 久久久亚洲精品成人影院| 亚洲成人一二三区av| 国产精品秋霞免费鲁丝片| 国产成人精品一,二区| 91久久精品国产一区二区三区| 亚洲在久久综合| 午夜福利视频精品| 亚洲av日韩在线播放| 久久精品国产亚洲网站| 99热国产这里只有精品6| 免费黄频网站在线观看国产| 久久久精品94久久精品| 日韩,欧美,国产一区二区三区| videossex国产| 91久久精品电影网| 久久人妻熟女aⅴ| 男人和女人高潮做爰伦理| 亚洲av中文av极速乱| 国产精品久久久久久久久免| 免费不卡的大黄色大毛片视频在线观看| av专区在线播放| 色视频在线一区二区三区| 老司机影院毛片| 亚洲国产色片| 日本午夜av视频| 涩涩av久久男人的天堂| 亚洲精品国产色婷婷电影| 国产在线免费精品| 老司机亚洲免费影院| 免费人妻精品一区二区三区视频| 精品亚洲乱码少妇综合久久| 多毛熟女@视频| 久久鲁丝午夜福利片| 日韩欧美精品免费久久| 男女啪啪激烈高潮av片| 纯流量卡能插随身wifi吗| 黑人高潮一二区| 熟女av电影| 亚洲色图综合在线观看| 亚洲欧美一区二区三区黑人 | 日日撸夜夜添| 国产成人午夜福利电影在线观看| 精品少妇内射三级| 久久久久久久久久久久大奶| 婷婷色av中文字幕| 丰满人妻一区二区三区视频av| 少妇的逼水好多| av在线播放精品| 免费观看在线日韩| 色视频www国产| 色婷婷av一区二区三区视频| 国产av一区二区精品久久| 亚洲欧美日韩卡通动漫| 永久免费av网站大全| 国产无遮挡羞羞视频在线观看| 国产成人91sexporn| 国产伦精品一区二区三区四那| 亚洲色图综合在线观看| 成人毛片60女人毛片免费| 国产精品久久久久久精品古装| 日韩大片免费观看网站| 国产伦理片在线播放av一区| 亚洲综合精品二区| 久久99蜜桃精品久久| 久久久久久久久久久丰满| 欧美国产精品一级二级三级 | 丝袜脚勾引网站| 观看av在线不卡| 日韩视频在线欧美| av在线播放精品| 成人毛片60女人毛片免费| 欧美成人精品欧美一级黄| 五月玫瑰六月丁香| 国产一区二区三区综合在线观看 | 一边亲一边摸免费视频| 免费大片黄手机在线观看| 男人和女人高潮做爰伦理| 国产一区二区三区av在线| 又黄又爽又刺激的免费视频.| 搡老乐熟女国产| 99精国产麻豆久久婷婷| 99热这里只有是精品在线观看| 日日撸夜夜添| 亚洲性久久影院| 欧美 亚洲 国产 日韩一| 2022亚洲国产成人精品| 又爽又黄a免费视频| 搡老乐熟女国产| 美女脱内裤让男人舔精品视频| a级毛色黄片| 中国三级夫妇交换| 国产精品99久久久久久久久| 色婷婷久久久亚洲欧美| 国产一区二区在线观看日韩| 一级毛片aaaaaa免费看小| 卡戴珊不雅视频在线播放| 一级,二级,三级黄色视频| 亚洲av在线观看美女高潮| 久久ye,这里只有精品| 黄色一级大片看看| 亚洲欧美一区二区三区国产| 极品人妻少妇av视频| 亚洲人成网站在线观看播放| 国模一区二区三区四区视频| 久久99精品国语久久久| 欧美精品高潮呻吟av久久| 久久青草综合色| av网站免费在线观看视频| 国产爽快片一区二区三区| 丰满乱子伦码专区| 99视频精品全部免费 在线| 人妻系列 视频| 爱豆传媒免费全集在线观看| 欧美日本中文国产一区发布| 亚洲国产最新在线播放| 亚洲电影在线观看av| 国产精品熟女久久久久浪| 国产欧美日韩精品一区二区| 亚洲精品日韩av片在线观看| 精品人妻一区二区三区麻豆| 国产熟女午夜一区二区三区 | 欧美 亚洲 国产 日韩一| 国产成人精品无人区| 亚洲欧美日韩另类电影网站| 如日韩欧美国产精品一区二区三区 | 极品人妻少妇av视频| 国产精品久久久久久久久免| 国产成人免费观看mmmm| 人人妻人人看人人澡| 男男h啪啪无遮挡| 国产成人精品久久久久久| 国产亚洲一区二区精品| 成人国产麻豆网| 亚洲av成人精品一区久久| 激情五月婷婷亚洲| 日本黄色日本黄色录像| 成人无遮挡网站| 纵有疾风起免费观看全集完整版| 青春草国产在线视频| 精华霜和精华液先用哪个| 欧美成人午夜免费资源| 久久人人爽人人爽人人片va| 久久久欧美国产精品| 国产综合精华液| 大陆偷拍与自拍| 水蜜桃什么品种好| 国产熟女午夜一区二区三区 | 另类亚洲欧美激情| 亚洲色图综合在线观看| 全区人妻精品视频| 亚洲精品成人av观看孕妇| av福利片在线| 在线观看一区二区三区激情| 亚洲精品国产成人久久av| 黄色配什么色好看| 久久国内精品自在自线图片| 精品人妻偷拍中文字幕| 国产精品伦人一区二区| 我的女老师完整版在线观看| 两个人免费观看高清视频 | 国产探花极品一区二区| 好男人视频免费观看在线| 91久久精品电影网| 91精品国产九色|