• <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 ?

    電針對腦缺血再灌注模型大鼠血清白介素的影響

    2015-06-04 03:50:16WangPing王平MuYanyun穆艷云ChengJie程潔ShenJie沈潔ShenMeihong沈梅紅ChenXia陳霞
    關鍵詞:白介素腦缺血

    Wang Ping (王平), Mu Yan-yun (穆艷云), Cheng Jie (程潔), Shen Jie (沈潔), Shen Mei-hong (沈梅紅), Chen Xia (陳霞),

    Li Qian (李茜)2, Sun Yong (孫永)2, Gong Mei-rong (龔美蓉)2

    1 Department of Traditional Chinese Medicine, the People’s Hospital of Jimo, Qingdao, Shandong 266200, China

    2 The Second Medical College of Nanjing University of Chinese Medicine, Jiangsu 210046, China

    Interleukin (IL) is a group of cytokines that has gained much attention for its action in ischemic cerebrovascular disease[1-2]. So far, the family members such as IL-1β[3-5], tumor necrosis factor (TNF)-α[6-8], IL-6[9-10],IL-8[11-12], and IL-10[13-15]have been involved in relevant studies. Clinical studies have found that the level of serum IL is correlated with the severity of disease and neurologic deficit. IL-6 and IL-8 are proinflammatory factors, and IL-10 is an anti-inflammatory factor; but the current laboratory researches are not completely consistent with clinical studies. Some studies have shown that IL-6 and IL-10 possibly protect brain from damage in ischemic cerebral disorders[15-16]. A large amount of clinical and experimental studies have proven the effect of acupuncture in promoting the recovery of neurological function in ischemic cerebrovascular diseases. Based on our previous works[17-20], the current study was to observe the effect of electroacupuncture (EA) on the level of serum IL-6,IL-8 and IL-10 in the rat model of cerebral ischemiareperfusion injury, and to discover the mechanism of acupuncture in the recovery of ischemic brain injury.

    1 Materials and Methods

    1.1 Animals

    Male Sprague Dawley (SD) rats of clean standard, 8 months, weighing (280±20) g, were purchased from Zhejiang Provincial Experimental Animal Center, license number: SCXK(Zhe)2008-0033. In the whole process of the experiment, the animals were treated in consistent with the requirements of the experimental animal ethics. The animals were applied to the experiment after 1 week of adaptive feeding at 25℃.

    1.2 Chemicals and equipment

    The G6805 EA apparatus (Shanghai Medical Equipment Factory, Shanghai, China); HH-4 digital thermostatic water bath (Guohua Electric Appliance Co.,Ltd., Changzhou, China); DG5033A enzyme reader(Huadong Electronics Medical Equpment Co., Ltd.,Nanjing, China); IL-6, IL-8, IL-10 Elisa kits (Jiancheng Bioengineering Company, Nanjing, China). The apparatuses were used strictly according to the instructions.

    1.3 Animal grouping

    The rats were randomized into a sham-operation (SO)group and a model control (MC) group. The rats in the SO group were then divided into a 6-hour SO group and a 24-hour SO group, 8 rats in each group. The rats that finished cerebral ischemia-reperfusion modeling process were scored for neurologic deficit according to the relevant literatures[21]. The rats scored over 2 points were randomized into a MC group and an EA group, and the two groups were also respectively subdivided into a 6-hour group and a 24-hour group, 9 in each group. The rats scored less than 2 points were sacrificed following the standard of experimental animal ethics.

    1.4 Model preparation

    The rat model of ischemia of middle cerebral artery(MCA) was developed using modified intraluminal suture occlusion method[22-23], the same way we adopted in the previous studies[16-18]. The rats were anesthetized using 10% chloral hydrate (0.35 mL/100 g)and then fixed supinely on a surgery table. After standard sterilization, the right common carotid artery(CCA), external carotid artery (ECA), and internal carotid artery (ICA) were isolated through a midline incision of about 2 cm long. A piece of suture of 0.25 mm in diameter, coated with silicon, was introduced into the ECA (0.4 cm away from the Y-section) to the opening of MCA [about (18±2) mm]. Then the incision was closed with 1 cm suture left outside. Two hours later, the suture was pulled out till the Y-section of the CCA (a slight resistance was felt), to achieve reperfusion. The neurologic deficit was scored 2 h after reperfusion.Neurologic deficit score ≥2 points indicated the success of modeling.

    The scoring criteria of neurologic deficit are as follows.

    0 point: Normal movement, no neurologic deficit.

    1 point: Homer’s sign positive on the right side.

    2 points: The tail suspended, and failure to extend left forepaw.

    3 points: Autonomic movement, circling to the left(the hemiplegia side).

    1.5 Interventions

    1.5.1 SO group

    1.5.2 MC group

    Rats in the MC group were modeled but not given EA intervention.

    1.5.3 EA group

    Two hours after modeling, rats in the EA group started to receive EA treatment.

    Acupoints: Dazhui (GV 14) and Baihui (GV 20).

    Operation: The acupoints were located following the rat’s acupoint map in the Experimental Acupuncture Science[24]. After standard sterilization, filiform needles(Huatuo Brand, Suzhou, China) of 0.25 mm in diameter and 0.25 mm in length were adopted for treatment.Baihui (GV 20) was horizontally punctured with the needle tip towards the rat’s tail, and Dazhui (GV 14) was needled by 30° obliquely, both by 0.5 cun. The needles were twirled for 30 s for stimulation and then connected to the G6805 EA apparatus, with sparseintense wave, 2 Hz/15 Hz, 1-3 mA, and 1-3 V. The stimulation should cause a slight swing of the punctured area. The intervention lasted 30 min each time.

    The EA group was subdivided into a 6-hour group and a 24-hour group. The 6-hour EA group only received one session of EA treatment, while the 24-hour EA group would receive another session before sacrifice.The two groups of rats were respectively sacrificed 6 h and 24 h after modeling.

    1.6 Detecting parameters

    1.6.1 Neurologic deficit scoring

    After modeling, the 6 h and 24 h groups were scored according to the following scoring criteria of neurologic deficit[23].

    Spontaneous activities (score 0-3): Observe the rats for 5 min to see their ability to approach the wall of cage and explore the environment.

    Balance of the four paws (score 0-3): Lift up the rat’s tail to see whether the movements of the paws are balanced.

    Balance of the two forepaws (score 0-3): Keep the rat on the edge of a table with its hind paws in the air to see the balance of the forepaws.

    Climbing (score 1-3): Place the rat on the wall of cage to observe its climbing ability, and feel its clinging ability when removing the rat from the wall.

    Proprioception (score 1-3): To observe the rat’s response to stimulation by touching the two sides of its body with a stick.

    Response to whisker touching (score 1-3): To observe the rat’s response to touching its whisker with a stick from behind.

    The result would be 3-18 points, and score 18 indicated normal activities. The lower the score, the severer the neurologic deficit.

    1.6.2 Serum levels of IL-6, IL-8 and IL-10

    When the rats were sacrificed, 5 mL blood was drawn from the abdominal aorta and placed for 10 min at room temperature. The supernatant was isolated after centrifugation at 4300 r/min and then kept in fridge at-20 ℃.

    1.7 Statistical processing

    The SPSS 16.0 version statistical software was adopted for analyses of all data. The measurement data were expressed by mean ± standard deviation (),and one-way ANOVA was chosen to analyze the variance. The least significance difference (LSD) was used for comparison of the data with equal variances;Dunnett’s T3 for the one with unequal variances.P<0.05 was considered a statistical significance.

    2 Results

    2.1 Comparison of neurologic deficit score

    Six hours after the reperfusion, the MC group scored the lowest, and the neurologic deficit score of the EA group was significantly higher than that in the MC group(P<0.01); the scores of the MC and EA groups were significantly different from the score of the SO group(both P<0.01). Twenty-four hours after the reperfusion,the scores in the MC and EA groups both increased; the MC group remained the lowest score, and it’s markedly different from the scores of SO group and EA group(both P<0.01); there was a significant difference in comparing the score between the EA group and the SO group (P<0.01), (Table 1).

    2.2 Comparison of serum IL-6 level

    Six hours after the reperfusion, the MC group had the highest level of serum IL-6, and it’s significantly different from the level in the SO group (P<0.01), while it’s insignificantly different from that in the EA group; there was no significant difference in comparing the serum IL-6 level between the SO and EA groups. Twenty-four hours after the reperfusion, every group showed a decrease in the serum IL-6 level, though the MC group remained the highest level, it’s insignificantly different from the level in the SO group but it’s significantly different from that in the EA group (P<0.05), and there was no significant difference between the SO group and the EA group. There were significant differences in comparing the serum IL-6 level between the 6-hour MC group and 24-hour MC group and between the 6-hour EA group and 24-hour EA group (both P<0.01), while there was no significant difference between the 6-hour SO group and the 24-hour SO group (Table 2).

    Table1.Comparison of neurologic deficit score (, point)

    Table1.Comparison of neurologic deficit score (, point)

    Note: Compared with the SO group, 1) P<0.01; compared with the MC group, 2) P<0.01

    Group n 6 h 24 h SO 8 17.38±0.52 17.50±0.53 MC 9 5.11±1.361) 8.56±1.671)EA 9 8.67±1.121)2) 13.11±1.051)2)

    Table2.Comparison of serum IL-6 level (, ng/L)

    Table2.Comparison of serum IL-6 level (, ng/L)

    Note: Compared with the SO group, 1) P<0.01; intra-group comparison with the 6-hour group, 2) P<0.01; compared with the MC group of the same period, 3) P<0.05

    Group n 6 h 24 h SO 8 176.79±21.83 165.67±18.95 MC 9 209.47±3.421) 180.70±14.112)EA 9 194.52±23.37 151.43±33.12)3)

    2.3 Comparison of serum IL-8 level

    Six hours after the reperfusion, the MC group had the highest level of serum IL-8, which was significantly different from the level of SO group (P<0.01) and the level of EA group (P<0.05); there was no significant difference between the SO group and the EA group.Twenty-four hours after the reperfusion, the level of serum IL-8 dropped in each group. The MC group still had the highest level, which was significantly different from that in the EA group (P<0.05), while insignificantly different from that in the SO group, and there was no significant difference between the SO and EA groups. There were significant differences in comparing the IL-8 level between the 6-hour MC group and the 24-hour MC group and between the 6-hour EA group and the 24-hour EA group (P<0.01, P<0.05).There was no significant difference in comparing the level between the 6-hour SO group and 24-hour SO group (Table 3).

    Table3.Comparison of serum IL-8 level (, ng/L)

    Table3.Comparison of serum IL-8 level (, ng/L)

    Note: Compared with the SO group, 1) P<0.05; compared with the MC group of the same period, 2) P<0.05; intra-group comparison with the 6-hour group, 3) P<0.01, 4) P<0.05

    Group n 6 h 24 h SO 8 176.06±23.64 164.88±14.13 MC 9 198.30±14.151) 177.52±13.893)EA 9 178.44±13.602) 154.63±28.052)4)

    2.4 Comparison of serum IL-10 level

    Six hours after the reperfusion, the MC group had the highest level of serum IL-10, which was significantly different from the level in the SO group (P<0.05), but not significantly different from that in the EA group;there was a significant difference between the SO and EA groups (P<0.05). Twenty-four hours after the reperfusion, the level of serum IL-10 dropped in each group, and there were no significant differences among the three groups. There were no significant differences in comparing the IL-10 level between each two subgroups (Table 4).

    Table4.Comparison of serum IL-10 level (, ng/L)

    Table4.Comparison of serum IL-10 level (, ng/L)

    Note: Compared with the SO group, 1) P<0.05

    Group n 6 h 24 h SO 8 92.31±16.06 87.22±5.13 MC 9 106.00±11.841) 98.88±15.29 EA 9 105.02±8.321) 90.76±21.30

    3 Discussion

    It’s believed that the levels of serum IL-6, IL-8 and IL-10 are correlated with the severity of cerebral ischemic injury, climbing high in the acute stage while declining with time. Therefore, the levels of serum IL-6,IL-8 and IL-10 are considered the marker for acute cerebral infarction. IL-6 and IL-8 are proinflammatory factors and IL-10 is an anti-inflammatory factor. The three cytokines can also be used for evaluating the severity and prognosis of cerebral infarction. The change of serum interleukin level suggests that cerebral ischemic injury can evoke the general immune response,which is regarded as a sub-clinical inflammatory state[25-26].

    The results showed that the EA group had a better neurologic function than the MC group[27-29], which is in consistence with the previous studies[17-19], indicating that EA intervention be effective in protecting the cerebral function.

    The levels of the three serum cytokines in the MC group were higher than that in the SO group 6 h after the reperfusion, indicating that cerebral ischemia causes the increase of the cytokine levels, and the central ischemia triggers a general reaction; the levels in the EA group were lower than that in the MC group,suggesting that early intervention of EA helps regulate the levels of the three cytokines. Twenty-four hours after the reperfusion, the levels of the three cytokines dropped in each group, and the levels of IL-6 and IL-8 in the EA group were lower than that in the MC group; the differences between the two subgroups of the MC and EA groups were statistically significant. It suggests that the intervention of EA can regulate the levels of serum IL-6, IL-8 and the general immune response;comparatively speaking, EA produces a more significant effect in inhibiting IL-6 and IL-8 than promoting IL-10.

    Although IL-6 is considered a proinflammatory factor,many experimental studies in animals have proven its action in protecting cerebral nerve cells[30-33], which doesn’t match up with the results of clinical studies. Our previous studies showed that the IL-6 level in brain tissues went down from 6 h till 24 h after the reperfusion, while the level in the EA group was higher than that in the MC group[34]. The current study showed that the serum IL-6 level also went down, but the level in the 24-hour EA group was lower than that in the MC group. The expression of IL-6 in brain tissues is not conforming to that in serum, which may be because that brain tissues are directly injured during cerebral ischemia and the reaction of brain tissues is not as same as the general reaction. Therefore, the change of cytokines in serum can’t represent the change of cytokines in brain tissues, and the serum cytokine level cannot be taken as the absolute evidence to prove the damaging effect of IL-6 to brain.

    Our past studies have discovered that EA can produce a content effect in protecting cerebral function in cerebral ischemia[17-19]. The current study revealed a more significant effect of EA in inhibiting IL-6, IL-8 and an insignificant effect in regulating IL-10. It suggests that regulation of serum interleukin level may possibly be connected with the effect of EA in protecting brain, but the details still expect further studies.

    Conflict of Interest

    The authors declared that there was no potential conflict of interest.

    This work was supported by National Natural Science Foundation of China (No. 81102633, No. 81373748).

    Statement of Informed Consent

    The treatment of animals conformed to the ethical criteria in this experiment.

    [1]Persson J, Folkersen L, Ekstrand J, Helleberg J, Gabrielsen A, Lundman P, Hedin U, Paulsson-Berne G. High plasma adiponectin concentration is associated with all-cause mortality in patients with carotid atherosclerosis.Atherosclerosis, 2012, 225(2): 491-496.

    [2]Zeng L, Wang Y, Liu J, Wang L, Weng S, Chen K, Domino EF, Yang GY. Pro-inflammatory cytokine network in peripheral inflammation response to cerebral ischemia.Neurosci Lett, 2013, 548: 4-9.

    [3]Chen XD, Zhen J, Zhao Y, Feng YL, Ma XL. Study on serum IL-6 and IL-8 in patients with cerebral infarction.Inner Mongolia Med J, 2012, 44(11): 1358-1359.

    [4]Di ZL, Wan Q, Lai HA, Wang HD. The role of IL-1β and IL-6 in inflammation of endothelial cell after global cerebral ischemia reperfusion. Xi’an Yike Daxue Xuebao,2001, 22(5): 432-434.

    [5]Guo YM, Liang XR, Du YH, Guo YT, Shi XM. The influence of brain-activating acupuncture on the IL-1β content of brain tissues and serum in rats with cerebral focal ischemia. Shanghai Zhenjiu Zazhi, 2004, 23(8):35-37.

    [6]Zhou W, Wang LP, Liu H, Bian Y. Influence of scalp acupuncture on serum tumor necrosis factor in patients with acute cerebral infarction. Shanghai Zhenjiu Zazhi,2002, 21(1): 11-12. [4]

    [7]Xu XJ, You C, Gao JJ, Liao SC. The expression and function of TNF-α in different time span of cerebral ischemia reperfusion injury. Sichuan Yixue, 2006, 27(7):670-672.

    [8]Wu J, Liu KD, Su ZQ, Rao ML, Zhang SQ. Tunor necrosis factor-α expression in ischemic neurons. Zhongfeng Yu ShenjingJibing Zazhi, 2000, 17(2): 77-78.

    [9]Ye F, Luo JQ, Chen J, Ye SH, Wu RD. Serum NSE and IL-6, IL-8 and acute cerebral infarction. Zhejiang Shiyong Yixue, 2012, 17(3): 192-193.

    [10]Qian LL, Jia K. Brain protection of Xing Nao Jing injection for patients with acute cerebral infarction and its effect on IL-6 and IL-8. Zhongchengyao, 2013, 35(8):1633-1636.

    [11]Jin ML, Yang GZ. Discovery of the correlation between plasma IL-8 and acute cerebral infarction. Zhongguo Yixue Chuangxin, 2012, 9(14): 56-57.

    [12]Cao QY, Pan XD. The change of the level of interleukin-8 in focal cerebral ischemia and ischemia-reperfusion injury in rats. Chin J Neuroimmunol & Neurol, 2001, 8(1): 20-22.

    [13]Chen BL, Li XB, Li J, Ma L. Attack of transient cerebral ischemia and IL-6, IL-8 and IL-10. Nao Yu Shenjing Jibing Zazhi, 2013, 21(4): 247-250.

    [14]Zhang CG, Qu CH, Yang H, Liu WH. Dynamic change of serum IL-17 and IL-10 in patients with acute cerebral infarction. Chin J Appl Physiol, 2014, 30(1): 36-37.

    [15]Ma J, Yan FL. Role of IL-10 in stroke-induced immunodepression syndrome. Chin J Cerebrovas Dis:Electronic Edition, 2011, 5(5): 49-54.

    [16]Zhang Q, Wang YF, Zhang YB, Li JM. Inflammatory biomarkers of stroke. Chin J Stroke, 2013, 8(4): 276-279.

    [17]Mu YY, Shen MH, Cheng J, Xia YB, Liu XH, Xiang XR.Effect of electroacupuncture on interleukin-6 expression in hippocampus of cerebral ischemia. Liaoning Zhongyi Zazhi, 2013, 40(4): 797-800.

    [18]Shen MH, Xiang XR, Li Y, Pan JL, Ma C, Li ZR. Effect of electroacupuncture on expression of γ-glutamylcysteine synthetase protein and mRNA in cerebral cortex in rats with focal cerebral inchemia-reperfusion. Zhen Ci Yan Jiu,2012, 37(1): 25-29.

    [19]Shen MH, Li C, Li ZR. Effect of electroacupuncture on the concentration of GSH and activation of GSH-Px and GR in rats with cerebral ischemia-reperfusion injury. Nanjing Zhongyiyao Daxue Xuebao, 2011, 27(2): 137-139.

    [20]Wu WZ, Li ZR, Cheng J, Shen MH, Zhou JL, Wu XL.Effect of electroacupuncture on serum iNOS and HO-1 in patients with acute cerebral infarction of wind-phlegm obstructing collaterals type. Shanghai Zhenjiu Zazhi, 2012,31(12): 858-859.

    [21]Kuluz JW, Prado RJ, Dietrich WD, Schleien CL, Watson BD. The effect of nitric oxide synthase inhibition on infarct volume after reversible focal cerebral ischemia in conscious rats. Stroke, 1993, 24(12): 2023-2029.

    [22]Longa EZ, Weinstein PR, Carlson S, Cummins R.Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke, 1989, 20(1): 84-91.

    [23]Garcia JH, Wagner S, Liu KF, Hu XJ. Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats statistical validation.Stroke, 1995, 26(4): 627-634.

    [24]Li ZR. Experimental Acupuncture Science. Beijing: China Press of Traditional Chinese Medicine, 2005: 255-257.

    [25]Senn JJ, Klover PJ, Nowak IA, Zimmers TA, Koniaris LG,Furlanetto RW, Mooney RA. Suppressor of cytokine signaling-3 (SOCS-3), a potential mediator of interleukin-6-dependentinsulin resistance in hepatocytes. J Biol Chem,2003, 278(16): 13740-13746.

    [26]Krogh-Madsen R, Plomgaard P, Keller P, Keller C,Pedersen BK. Insulin stimulates interleukin-6 and tumor necrosis factor-alpha gene expression in human subcutaneous adipose tissue. Am J Physiol Endocrinol Metab, 2004, 286(2): E234-E238.

    [27]Ge LB, Fang C, Xu MS, Xu J, Li CZ. Effects of electroacupuncture on the ability of learning and memory in rats with ischemia-reperfusion injury. J Acupunct Tuina Sci, 2009, 7(1): 3-7.

    [28]Dong ZH, Fang JQ, Shao XM. Advances in the study of mechanisms for acupuncture-moxibustion prevention and treatment of ischemia-reperfusion injury. Shanghai Zhenjiu Zazhi, 2011, 30(12): 877-880.

    [29]Liu CY, Xu MS, Ge LB. Effects of electroacupuncture plus dopamine D1 receptor antagonist on somatosensory evoked potentials and behavioral changes in rats with cerebral ischemia-reperfusion. J Acupunct Tuina Sci, 2012,10(3): 133-137.

    [30]Gertz K, Kronenberg G, K?lin RE, Baldinger T, Werner C,Balkaya M, Eom GD, Hellmann-Regen J, Kr?ber J, Miller KR, Lindauer U, Laufs U, Dirnagl U, Heppner FL, Endres M. Essential role of interleukin-6 in post-stroke angiogenesis. Brain, 2012, 135(Pt 6): 1964-1980.

    [31]Nakamachi T, Tsuchida M, Kagami N, Yofu S, Wada Y,Hori M, Tsuchikawa D, Yoshikawa A, Imai N, Nakamura K, Arata S, Shioda S. IL-6 and PACAP receptor expression and localization after global brain ischemia in mice. J Mol Neurosci, 2012, 48(3): 518-525.

    [32]Suzuki S, Tanaka K, Suzuki N. Ambivalent aspects of interleukin-6 in cerebral ischemia: inflammatory versus neurotrophic aspects. J Cereb Blood Flow Metab, 2009,29(3): 464-479.

    [33]Gravante G, Ong SL, Metcalfe MS, Sorge R, Sconocchia G,Orlando G, Lloyd DM, Dennison AR. Cytokine response to ischemia/reperfusion injury in an vivo perfused porcine liver model. Transplant Proc, 2009, 41(4): 1107-1112.

    [34]Mu YY, Shen HM, Cheng J, Xia YB, Liu XH, Xiang XR.Effect of electroacupuncture on interleukin-6 expression in hippocampus of cerebral ischemia-reperfusion rats.Liaoning Zhongyi Zazhi, 2013, 40(4): 797-799.

    猜你喜歡
    白介素腦缺血
    大黃總蒽醌提取物對腦缺血再灌注損傷的保護作用及其機制
    中成藥(2018年4期)2018-04-26 07:12:34
    原花青素對腦缺血再灌注損傷后腸道功能的保護作用
    血必凈對大鼠腦缺血再灌注損傷的保護作用及其機制
    細胞外組蛋白與腦缺血再灌注損傷關系的初探
    銀屑病患兒血清腫瘤壞死因子α、白介素6、白介素8的檢測
    哮喘患兒血清白介素與免疫球蛋白檢測的臨床意義
    分析奧拉西坦治療慢性腦缺血引起的癡呆
    白介素25 、33 在慢性蕁麻疹患者外周血中的表達及意義
    尋常性銀屑病患者外周血白介素17 、白介素23 mRNA 的表達及與病情相關性研究
    電針“內(nèi)關”、“心俞”對急性心肌缺血再灌注損傷大鼠血清白介素-1 β、白介素-10 含量及心肌組織NF-KB p65 蛋白表達的影響
    亚洲精品乱久久久久久| av天堂在线播放| 亚洲精品成人av观看孕妇| 亚洲精品av麻豆狂野| 黑人欧美特级aaaaaa片| 成人影院久久| 人妻久久中文字幕网| www.精华液| 91大片在线观看| 国产人伦9x9x在线观看| 熟女少妇亚洲综合色aaa.| 熟女少妇亚洲综合色aaa.| 欧美另类亚洲清纯唯美| 黄色丝袜av网址大全| 多毛熟女@视频| 亚洲第一欧美日韩一区二区三区 | 国产精品一区二区免费欧美| 欧美国产精品一级二级三级| 久久精品国产99精品国产亚洲性色 | 久久中文看片网| 乱人伦中国视频| 国产亚洲精品第一综合不卡| 日韩成人在线观看一区二区三区| 97在线人人人人妻| 女人高潮潮喷娇喘18禁视频| 国产一区有黄有色的免费视频| 亚洲 欧美一区二区三区| 精品熟女少妇八av免费久了| 在线观看免费午夜福利视频| bbb黄色大片| 日本撒尿小便嘘嘘汇集6| 18禁黄网站禁片午夜丰满| 日韩一卡2卡3卡4卡2021年| 夜夜夜夜夜久久久久| av又黄又爽大尺度在线免费看| 色精品久久人妻99蜜桃| 免费人妻精品一区二区三区视频| 男女免费视频国产| 亚洲七黄色美女视频| 一级,二级,三级黄色视频| 成人手机av| 免费在线观看黄色视频的| 一区二区av电影网| 免费观看a级毛片全部| 日本wwww免费看| 一区在线观看完整版| 久久精品91无色码中文字幕| 久久99热这里只频精品6学生| 精品国产国语对白av| 欧美黄色片欧美黄色片| 精品国产一区二区久久| 国产高清国产精品国产三级| cao死你这个sao货| 在线看a的网站| 免费女性裸体啪啪无遮挡网站| 大香蕉久久网| 欧美日韩亚洲综合一区二区三区_| 丝袜美腿诱惑在线| 国产精品久久久久久人妻精品电影 | 亚洲中文av在线| 亚洲av日韩精品久久久久久密| 日韩视频一区二区在线观看| 精品国产乱码久久久久久男人| 色94色欧美一区二区| 男女之事视频高清在线观看| 亚洲av第一区精品v没综合| 一级片免费观看大全| 美女主播在线视频| 两人在一起打扑克的视频| 欧美另类亚洲清纯唯美| 黄色a级毛片大全视频| xxxhd国产人妻xxx| 天天躁日日躁夜夜躁夜夜| 美女国产高潮福利片在线看| 午夜激情av网站| 999精品在线视频| 久久精品国产a三级三级三级| 精品人妻1区二区| 国产欧美日韩一区二区三| 国产精品九九99| 狠狠婷婷综合久久久久久88av| 亚洲九九香蕉| 中国美女看黄片| 精品熟女少妇八av免费久了| 99精品久久久久人妻精品| 日本vs欧美在线观看视频| 国产成人啪精品午夜网站| 国产日韩一区二区三区精品不卡| 亚洲欧美日韩高清在线视频 | 91精品三级在线观看| 国产午夜精品久久久久久| 中文字幕人妻丝袜一区二区| 国产高清激情床上av| 久久久国产欧美日韩av| 亚洲伊人色综图| 国产主播在线观看一区二区| 一本一本久久a久久精品综合妖精| 国产精品香港三级国产av潘金莲| 精品一区二区三卡| 国产免费av片在线观看野外av| 国产精品久久久人人做人人爽| 亚洲成人国产一区在线观看| 亚洲人成77777在线视频| 亚洲黑人精品在线| 波多野结衣一区麻豆| 精品欧美一区二区三区在线| 丰满迷人的少妇在线观看| 午夜福利视频精品| 精品卡一卡二卡四卡免费| 成年版毛片免费区| 精品一区二区三区视频在线观看免费 | 老司机亚洲免费影院| 久久人妻av系列| 大片电影免费在线观看免费| 亚洲欧洲日产国产| 怎么达到女性高潮| 欧美人与性动交α欧美软件| 亚洲成人免费电影在线观看| 亚洲av成人不卡在线观看播放网| 新久久久久国产一级毛片| 免费看a级黄色片| 欧美午夜高清在线| 五月开心婷婷网| 国产精品麻豆人妻色哟哟久久| 精品人妻1区二区| 国产精品久久久人人做人人爽| 国产av精品麻豆| 亚洲色图 男人天堂 中文字幕| 欧美日韩亚洲国产一区二区在线观看 | 欧美日韩黄片免| 久久久国产欧美日韩av| 99re6热这里在线精品视频| 香蕉丝袜av| 大香蕉久久成人网| 精品久久蜜臀av无| 日日摸夜夜添夜夜添小说| 国产精品98久久久久久宅男小说| 国产精品99久久99久久久不卡| 欧美成人午夜精品| 99国产极品粉嫩在线观看| 国产不卡av网站在线观看| 少妇裸体淫交视频免费看高清 | 亚洲,欧美精品.| 国产三级黄色录像| 一边摸一边抽搐一进一小说 | 别揉我奶头~嗯~啊~动态视频| 日本wwww免费看| 国产人伦9x9x在线观看| 夜夜骑夜夜射夜夜干| 欧美成人午夜精品| 少妇精品久久久久久久| 午夜福利乱码中文字幕| 91成年电影在线观看| 最新的欧美精品一区二区| 黄色片一级片一级黄色片| 国产人伦9x9x在线观看| 女性被躁到高潮视频| 国产精品亚洲一级av第二区| av线在线观看网站| 午夜福利乱码中文字幕| 久久国产精品大桥未久av| 国产成人影院久久av| 老司机午夜福利在线观看视频 | 岛国在线观看网站| 天天躁夜夜躁狠狠躁躁| 80岁老熟妇乱子伦牲交| 啦啦啦 在线观看视频| 极品教师在线免费播放| 国产色视频综合| 日韩中文字幕视频在线看片| 窝窝影院91人妻| 亚洲精品一卡2卡三卡4卡5卡| www.自偷自拍.com| 久久狼人影院| 亚洲中文av在线| 亚洲七黄色美女视频| 国产极品粉嫩免费观看在线| 免费久久久久久久精品成人欧美视频| 成人18禁高潮啪啪吃奶动态图| 99国产精品一区二区三区| 三上悠亚av全集在线观看| 久久久国产欧美日韩av| 精品人妻熟女毛片av久久网站| 女人久久www免费人成看片| 满18在线观看网站| 免费在线观看黄色视频的| 色尼玛亚洲综合影院| 欧美乱码精品一区二区三区| 国产无遮挡羞羞视频在线观看| 国产在线一区二区三区精| 国产淫语在线视频| 亚洲精品一二三| 91成年电影在线观看| 欧美精品一区二区大全| 国产欧美日韩综合在线一区二区| av一本久久久久| 成人精品一区二区免费| 热re99久久精品国产66热6| 亚洲第一av免费看| 国产精品九九99| 91国产中文字幕| www.熟女人妻精品国产| 老司机亚洲免费影院| 亚洲少妇的诱惑av| 女性生殖器流出的白浆| 麻豆av在线久日| 老司机午夜福利在线观看视频 | 久久人妻av系列| 日本撒尿小便嘘嘘汇集6| 国产精品偷伦视频观看了| 日韩成人在线观看一区二区三区| 亚洲第一青青草原| 欧美在线一区亚洲| 成年女人毛片免费观看观看9 | 免费观看av网站的网址| 嫁个100分男人电影在线观看| 久久久久久亚洲精品国产蜜桃av| 91大片在线观看| 国产精品一区二区精品视频观看| 三上悠亚av全集在线观看| av福利片在线| 欧美中文综合在线视频| 老司机亚洲免费影院| 亚洲美女黄片视频| 极品教师在线免费播放| 精品国产乱码久久久久久小说| 亚洲综合色网址| 老司机深夜福利视频在线观看| 久久久久久久国产电影| 国产一卡二卡三卡精品| 母亲3免费完整高清在线观看| 无人区码免费观看不卡 | 欧美av亚洲av综合av国产av| 狠狠婷婷综合久久久久久88av| 操出白浆在线播放| 国精品久久久久久国模美| 一二三四在线观看免费中文在| 亚洲av成人不卡在线观看播放网| 国产伦理片在线播放av一区| 男女下面插进去视频免费观看| 久久久久久亚洲精品国产蜜桃av| 精品国内亚洲2022精品成人 | 精品第一国产精品| a级毛片黄视频| 国产真人三级小视频在线观看| 亚洲av美国av| 久久精品亚洲av国产电影网| 成人18禁高潮啪啪吃奶动态图| 欧美日韩av久久| avwww免费| 老鸭窝网址在线观看| 亚洲精品国产色婷婷电影| 热99国产精品久久久久久7| 老熟妇仑乱视频hdxx| 国产在线视频一区二区| 视频在线观看一区二区三区| 多毛熟女@视频| 一本综合久久免费| 午夜福利视频在线观看免费| 免费观看av网站的网址| 操美女的视频在线观看| 成人精品一区二区免费| 国产深夜福利视频在线观看| 99精品欧美一区二区三区四区| 亚洲一码二码三码区别大吗| 欧美另类亚洲清纯唯美| 九色亚洲精品在线播放| 1024香蕉在线观看| 男女边摸边吃奶| 老司机福利观看| 亚洲精品一卡2卡三卡4卡5卡| 欧美黑人精品巨大| 日韩人妻精品一区2区三区| 一级毛片电影观看| 9热在线视频观看99| av在线播放免费不卡| 色精品久久人妻99蜜桃| 法律面前人人平等表现在哪些方面| 咕卡用的链子| 波多野结衣av一区二区av| 狠狠婷婷综合久久久久久88av| 精品久久久久久电影网| 中文字幕高清在线视频| 男女午夜视频在线观看| 国产成人精品久久二区二区91| 亚洲成国产人片在线观看| 91九色精品人成在线观看| 97在线人人人人妻| 国产欧美日韩精品亚洲av| 精品熟女少妇八av免费久了| 真人做人爱边吃奶动态| 精品亚洲成国产av| 极品教师在线免费播放| 亚洲国产欧美在线一区| 一本久久精品| 在线观看一区二区三区激情| videosex国产| 操美女的视频在线观看| 大片免费播放器 马上看| 悠悠久久av| 午夜福利免费观看在线| 成人国产av品久久久| 精品国产一区二区三区四区第35| 人人妻人人澡人人爽人人夜夜| 国产极品粉嫩免费观看在线| 精品国产国语对白av| 天天躁狠狠躁夜夜躁狠狠躁| 一级片免费观看大全| 国产精品欧美亚洲77777| 亚洲专区国产一区二区| 国产淫语在线视频| 夜夜爽天天搞| 伦理电影免费视频| 女同久久另类99精品国产91| 91精品三级在线观看| 极品人妻少妇av视频| 午夜91福利影院| 国产一区有黄有色的免费视频| 成人免费观看视频高清| 午夜老司机福利片| 亚洲午夜理论影院| 国产在线观看jvid| 久久免费观看电影| 我的亚洲天堂| 人人妻,人人澡人人爽秒播| 日本a在线网址| 久久久久视频综合| 色老头精品视频在线观看| 精品视频人人做人人爽| 成人亚洲精品一区在线观看| 桃红色精品国产亚洲av| 久久国产精品人妻蜜桃| 丝瓜视频免费看黄片| 91精品三级在线观看| 18在线观看网站| 操美女的视频在线观看| 色综合欧美亚洲国产小说| 国产精品久久久久成人av| videos熟女内射| 精品免费久久久久久久清纯 | 两性夫妻黄色片| 国产1区2区3区精品| 亚洲美女黄片视频| 免费看十八禁软件| 日日爽夜夜爽网站| 十八禁高潮呻吟视频| 久久精品国产亚洲av高清一级| 大片电影免费在线观看免费| 日日摸夜夜添夜夜添小说| 亚洲 国产 在线| 国产免费福利视频在线观看| 国产精品久久电影中文字幕 | 9色porny在线观看| 欧美日韩亚洲高清精品| 色94色欧美一区二区| 国产97色在线日韩免费| 麻豆成人av在线观看| 十八禁网站免费在线| 久久久精品国产亚洲av高清涩受| 中文字幕高清在线视频| h视频一区二区三区| 久久精品亚洲av国产电影网| 9191精品国产免费久久| 人妻 亚洲 视频| 亚洲精品自拍成人| 亚洲色图综合在线观看| 精品亚洲成a人片在线观看| 亚洲av国产av综合av卡| 99精品在免费线老司机午夜| av线在线观看网站| 亚洲av第一区精品v没综合| 在线播放国产精品三级| 99久久人妻综合| 色精品久久人妻99蜜桃| 一区二区三区国产精品乱码| 搡老岳熟女国产| 欧美日韩福利视频一区二区| 美女国产高潮福利片在线看| 99国产精品免费福利视频| 国产欧美日韩一区二区精品| 亚洲av片天天在线观看| 一区福利在线观看| 精品国产乱码久久久久久小说| 香蕉久久夜色| 99精品欧美一区二区三区四区| 日本vs欧美在线观看视频| 欧美精品亚洲一区二区| 国产在线视频一区二区| 一本色道久久久久久精品综合| 老熟妇乱子伦视频在线观看| 在线天堂中文资源库| 操美女的视频在线观看| 建设人人有责人人尽责人人享有的| 国产欧美亚洲国产| 亚洲国产欧美在线一区| 成人国产一区最新在线观看| 啪啪无遮挡十八禁网站| 丁香六月欧美| 欧美变态另类bdsm刘玥| 99热网站在线观看| 亚洲精品国产区一区二| 黄色视频,在线免费观看| 9色porny在线观看| 亚洲第一av免费看| a在线观看视频网站| 午夜91福利影院| 国产精品二区激情视频| 国产单亲对白刺激| 欧美 日韩 精品 国产| 香蕉丝袜av| 日本av手机在线免费观看| 国产xxxxx性猛交| 极品人妻少妇av视频| 国产亚洲一区二区精品| 欧美激情 高清一区二区三区| 色婷婷av一区二区三区视频| 国产精品久久久久久精品电影小说| 亚洲精品美女久久久久99蜜臀| 亚洲成a人片在线一区二区| 妹子高潮喷水视频| 国产有黄有色有爽视频| 久久影院123| 狠狠精品人妻久久久久久综合| 99国产精品一区二区蜜桃av | av一本久久久久| 嫩草影视91久久| 国产黄频视频在线观看| 黑人操中国人逼视频| 深夜精品福利| 高清av免费在线| 亚洲 国产 在线| 桃红色精品国产亚洲av| 女性被躁到高潮视频| 欧美日本中文国产一区发布| 在线播放国产精品三级| 精品亚洲成国产av| 大香蕉久久成人网| 一边摸一边抽搐一进一出视频| 亚洲精品一二三| 亚洲成a人片在线一区二区| 999久久久精品免费观看国产| 一夜夜www| 精品国产国语对白av| 中文字幕人妻丝袜一区二区| 国产精品久久电影中文字幕 | 9色porny在线观看| 波多野结衣av一区二区av| 成人手机av| 亚洲欧美精品综合一区二区三区| 国产在线免费精品| 久久久精品94久久精品| 18禁观看日本| 国产欧美日韩一区二区三| 亚洲成a人片在线一区二区| 女人被躁到高潮嗷嗷叫费观| 久久精品成人免费网站| 天天躁日日躁夜夜躁夜夜| 99在线人妻在线中文字幕 | 亚洲色图 男人天堂 中文字幕| 女人爽到高潮嗷嗷叫在线视频| 国产不卡一卡二| 99热网站在线观看| 久久久久久久大尺度免费视频| 精品福利永久在线观看| 一区二区三区国产精品乱码| 欧美黑人精品巨大| 中文字幕色久视频| 人人妻,人人澡人人爽秒播| 免费观看a级毛片全部| 亚洲欧美精品综合一区二区三区| 日韩视频一区二区在线观看| 国产成人av激情在线播放| 日韩欧美一区视频在线观看| 首页视频小说图片口味搜索| 老汉色∧v一级毛片| 丝袜人妻中文字幕| 亚洲色图av天堂| 18禁美女被吸乳视频| 最新在线观看一区二区三区| 好男人电影高清在线观看| 久久精品亚洲av国产电影网| 国产精品久久久久久精品电影小说| 国产在线视频一区二区| av网站在线播放免费| 少妇精品久久久久久久| tocl精华| 侵犯人妻中文字幕一二三四区| 丝袜美腿诱惑在线| 午夜福利视频精品| 午夜福利一区二区在线看| 精品午夜福利视频在线观看一区 | 亚洲少妇的诱惑av| 午夜精品久久久久久毛片777| av免费在线观看网站| 欧美在线一区亚洲| 两性午夜刺激爽爽歪歪视频在线观看 | 久久精品aⅴ一区二区三区四区| 性少妇av在线| 亚洲国产成人一精品久久久| a级毛片在线看网站| 一边摸一边抽搐一进一出视频| 久久中文字幕人妻熟女| 黄色毛片三级朝国网站| 日韩欧美三级三区| 中文字幕最新亚洲高清| 精品人妻在线不人妻| 18禁裸乳无遮挡动漫免费视频| 久久久久久久国产电影| 最新的欧美精品一区二区| 9热在线视频观看99| 天天操日日干夜夜撸| 欧美激情久久久久久爽电影 | 亚洲精品自拍成人| 亚洲午夜精品一区,二区,三区| 捣出白浆h1v1| 亚洲欧洲精品一区二区精品久久久| 极品少妇高潮喷水抽搐| 国产精品美女特级片免费视频播放器 | 久久精品91无色码中文字幕| 成年人午夜在线观看视频| 丝袜美腿诱惑在线| 丰满少妇做爰视频| 国产三级黄色录像| 国产成人免费无遮挡视频| 1024香蕉在线观看| www.熟女人妻精品国产| 成年动漫av网址| 亚洲综合色网址| av在线播放免费不卡| 高清av免费在线| 一个人免费看片子| 国产成人一区二区三区免费视频网站| 老汉色∧v一级毛片| 亚洲一卡2卡3卡4卡5卡精品中文| 亚洲欧美日韩另类电影网站| 久久久久国内视频| 国产视频一区二区在线看| 男女边摸边吃奶| 9热在线视频观看99| 中国美女看黄片| 人人妻人人爽人人添夜夜欢视频| 亚洲av欧美aⅴ国产| 一区二区三区乱码不卡18| 精品人妻熟女毛片av久久网站| 国产精品一区二区在线不卡| 国产xxxxx性猛交| 久久久国产一区二区| 国产一区有黄有色的免费视频| 国产区一区二久久| 女人高潮潮喷娇喘18禁视频| 少妇的丰满在线观看| 国产淫语在线视频| 操美女的视频在线观看| 涩涩av久久男人的天堂| 国产一区二区在线观看av| 97在线人人人人妻| 女人精品久久久久毛片| 免费黄频网站在线观看国产| 日本vs欧美在线观看视频| tube8黄色片| 少妇的丰满在线观看| 亚洲国产看品久久| 亚洲国产中文字幕在线视频| 亚洲精品一二三| 国产人伦9x9x在线观看| 亚洲av美国av| 久久 成人 亚洲| 美女高潮喷水抽搐中文字幕| 脱女人内裤的视频| 免费高清在线观看日韩| 亚洲av成人一区二区三| 亚洲七黄色美女视频| 一二三四在线观看免费中文在| 日韩人妻精品一区2区三区| 999久久久精品免费观看国产| 成人黄色视频免费在线看| 999精品在线视频| 日本wwww免费看| 99国产极品粉嫩在线观看| 国产成人欧美在线观看 | 日韩制服丝袜自拍偷拍| 夜夜夜夜夜久久久久| 国产日韩欧美在线精品| 免费av中文字幕在线| 国产精品国产av在线观看| 欧美精品av麻豆av| 91精品三级在线观看| 精品高清国产在线一区| 91av网站免费观看| 在线 av 中文字幕| 国产成人精品无人区| 欧美成人午夜精品| 99香蕉大伊视频| 国产野战对白在线观看| 色综合欧美亚洲国产小说| 日韩欧美一区二区三区在线观看 | 搡老岳熟女国产| 一边摸一边抽搐一进一小说 | 性高湖久久久久久久久免费观看| 18禁黄网站禁片午夜丰满| www日本在线高清视频| 国产精品国产av在线观看| 亚洲天堂av无毛| 侵犯人妻中文字幕一二三四区| 99riav亚洲国产免费| 精品一区二区三区视频在线观看免费 | 叶爱在线成人免费视频播放| 一本大道久久a久久精品| 欧美精品一区二区大全| 999精品在线视频| e午夜精品久久久久久久| h视频一区二区三区| 脱女人内裤的视频| 亚洲av日韩精品久久久久久密| 色播在线永久视频|