• <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 ?

    Effect of Drynaria total flavonoids on the expression of NMDAR1,GluR2 and CaMK Ⅱ in the brain of hydrocortisone model mice

    2022-03-16 09:31:46WANGYitongXUYanmingZHANGZhiboSUNHuifengZHANGNingYANGBo
    Journal of Hainan Medical College 2022年24期

    WANGYi-tong, XU Yan-ming, ZHANG Zhi-bo, SUN Hui-feng,, ZHANG Ning,,YANGBo?

    1.School of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin 150040, China

    2.College of Jiamusi, Heilongjiang University of Traditional Chinese Medicine, Jiamusi 154007, China

    Keywords:

    ABSTRACT Objective: To study the effect of total flavonoids of Rhizoma Drynariae on the expression of NMDAR1, GluR2 and CAMKⅡ protein in model mice of kidney deficiency induced by exogenous glucocorticoid hydrocortisone and its mechanism.Methods: Kunming (KM) mice were randomly divided into the blank group, the hydrocortisone model group, the anti-brain failure capsule group, the Drynaria total flavonoids group, the Drynaria total flavonoids+ER blocker group, with 15 animals in each group.Except for the blank group, all groups were injected intramuscularly with hydrocortisone (25 mg·kg-1·d-1) to create models.The water maze experiment, new object recognition experiment and platform jump experiment were used to conduct behavioral investigations.HE staining was used to observe the pathological changes of mouse hippocampus, and Western blotting detected the expressions of NMDR1,GluR2 and CAMKⅡ proteins in the hippocampus of mice in each group.Results: The experimental results showed that compared with the model group, the learning and memory ability of the mice in the Drynaria fortunei total flavonoids group was significantly improved,and the difference was statistically significant (P <0.01), the expression of NMDR1 and GluR2 proteins in the hippocampus of the mice was significantly increased (P<0.01), and the level of CAMKⅡ protein significantly decreased (P<0.01).Conclusion: The total flavonoids of Rhizoma Drynariae may enhance the expression of NMDAR1 and GluR2 protein in the brain of hydrocortisone model mice through ER, reduce the expression of CAMKⅡ protein, and alleviate the damage to the brain tissue of the model mice and play a neuroprotective effect.

    1.Introduction

    Alzheimer's disease (AD) is a complex and heterogeneous disease caused by a combination of factors, characterized by the entanglement of aggregated amyloid beta (Aβ) and hyperphosphorylated microtubule-associated protein (tau).knot [1].In recent years, it has been found that estrogen is closely related to learning and memory ability.If postmenopausal women do not supplement estrogen, memory and cognitive function will be significantly reduced, and the probability of AD will be greatly increased [2].However, experiments have shown that the use of hormone replacement therapy for AD has a time window, and the time window is narrow.If the time window is exceeded, there is no therapeutic effect or even serious side effects [3], so the advantages of traditional Chinese medicine phytoestrogens are highlighted.

    Rhizoma Drynariae is a kind of fern from the family Hydrangea,which has the effect of invigorating the kidney, strengthening the bone and activating blood circulation.The main chemical components in its rhizomes are flavonoids, triterpenes and lignins.Drynaria can isolate and purify more than 40 kinds of flavonoids [4],with dihydroflavonoids, catechins and their derivatives as Mainly,naringin and new erioside are both phytoestrogens, which are similar to synthetic estrogens [5], and have less toxic and side effects.Ge[6] verified the protective effect of naringin and kaempferol in total flavonoids on Aβ25-35-induced adrenal pheochromocytoma (PC12)cells in rats, suggesting that drynaria total flavonoids can improve brain function.Learning and memory effect, but its anti-AD effect is unclear and related literatures are few.In addition, studies have shown that the regulatory effect of estrogen on learning and memory is mediated through estrogen receptor (ER) [7], so it is speculated that the total flavonoids of Drynariae may be mediated through the ER pathway to treat AD, and N-methyl flavonoids-D-aspartate(NMDAR1), glutamate receptor (GluR2) and calcium/calmodulindependent protein kinase (CAMKⅡ ) protein expression levels in the brain to verify the speculation.In this study, mice with kidney deficiency induced by hydrocortisone were used as AD model mice,and the model mice were given total flavonoids of Drynariae as the treatment group.The central protective effect of total flavonoids on AD rats provides experimental support for its clinical medication.

    2.Materials and methods

    2.1 Experimental animals

    SPF grade KM mice, 75 males, 3 months old, were purchased from Changsheng Biological Company, certificate number: SCXK (Liao)2015-0001.The temperature of the rearing environment was 20~22℃, the relative humidity was 50%~70%, and water was freely fed.The experimental procedures were in compliance with the relevant regulations of Heilongjiang University of Traditional Chinese Medicine on the management of experimental animals.

    2.2 Drugs and Reagents

    Drynaria total flavonoids (self-extracted) and Kangnaoshui capsules (batch number KN15112502) were purchased from Siyao Co., Ltd.; rabbit anti-β-actin polyclonal antibody (batch number BM3873) and HRP-labeled goat anti-rabbit IgG (batch number BA1054) were purchased from Boster Company; NMDAR1 (batch number bs-23343R), GluR2 (batch number bs-9921R), CAMKⅡ(batch number bs11347R) were purchased from Bioss Company,ER blocker (model: ICI182780) was purchased from TOCRIS Company; Hydrocortisone (Lot number: B21001) was purchased from Yuanye Bio.

    2.3 Instruments

    MiniPROTEANTetra Cell electrophoresis apparatus (BIORAD company); Trans-Blot SD Cell semi-dry membrane transfer apparatus (BIO-RAD company); WD9405B horizontal shaker (Liuyi Instrument Factory); water maze equipment (Zhenghua company); New Object Recognition Equipment (Easu Company); Platform Jumping Equipment (Easu Company).

    2.4 Animal grouping and model preparation

    Seventy-five 3-month-old KM mice were randomly divided into 5 groups according to their body weight: Blank group, Model group,Kangnaoshui capsule control group (KNSC group), Drynariae total flavonoids group ( TFRD group), total flavonoids of Rhizoma Drynariae+ER blocker group (TFRD+ER group).The blank group and model group were given distilled water by gavage, Kangnaoshui capsule control group and Rhizoma Drynariae total flavonoids group were given Kangnaoshui Capsule 585 mg·kg-1·d-1and Rhizoma Drynariae total flavonoids 97.5 mg·kg-1·d-1; Rhizoma Drynariae total flavonoids + ER blocker group were given intraperitoneal injection of ER blocker IC1182780 0.072 mg·kg-1·d-1, followed by 97.5 mg·kg-1·d-1total flavonoids of Rhizoma Drynariae; continuous irrigation After 3 weeks in the stomach, the blank group was given double distilled water; the other groups were injected with hydrocortisone 25 mg·kg-1·d-1for modeling, and the drug was administered while modeling; one week later, behavioral experiments were performed.

    2.5 Behavioral experiments

    2.5.1 Novel Object Recognition Experiment (ORT)

    The new object experiment was carried out with reference to the literature method method [8].The contact time of mice to new objects and the contact time to old objects within 5 min were recorded, and the recognition index was calculated.

    2.5.2 Morris water maze test (MWM)

    The water maze experiment was carried out according to the method of literature [9].The first 4 days were the positioning cruise experiment, and the space exploration experiment was started on the 5th day.The number of times the mice crossed the platform within 90 s and the stay time in the target quadrant were recorded.In the platform jumping experiment, mice were trained for 24 h and the latency and the number of jumps were recorded under the condition of electrification.

    2.5.3 Materials

    After the behavioral study, 6 mice in each group were randomly selected for cardiac perfusion and immersed in paraformaldehyde for 48 h for HE staining experiment; the rest of the mice were quickly stripped of hippocampal tissue on ice and stored in liquid nitrogen tanks for later use.

    2.5.4 Histopathological observation

    HE staining: The left hemibrain fixed in 4% paraformaldehyde for 48 h was embedded in paraffin and sliced.After dewaxing with alcohol, they were placed in double-distilled water for dehydration;stained with hematoxylin for 5 min, washed with water, and placed in hydrochloric acid ethanol for a few seconds; immersed in eosin aqueous solution for 5 min, washed with water; dehydrated,mounted, and observed.

    2.5.5 Western blot

    The hippocampus of mice was collected on ice and treated with lysate, and the supernatant was obtained by centrifugation, and the protein concentration was determined according to the instructions.After transferring to the membrane by semi-dry transfer method and shaking and blocking for 2 h, the primary antibodies β-actin (1:5 000), NMDAR1 (1:500), GluR2 (1:500) and CAMKⅡ (1:500) were added.The mixture was placed at 4 ℃ overnight, the membrane was washed, the secondary antibody (1:5 000) was added to incubate for 2 h, the membrane was washed 4 times, and the luminescent solution was added and placed in a chemiluminescence imager for analysis.

    2.6 Statistical processing

    The data were analyzed by SPSS18.0 statistical software, and the comparison between groups was performed by one-way ANOVA.The experimental data were expressed as mean ± standard deviation(±s), and P<0.05 indicated statistical significance.

    3.results

    3.1 The effect of total flavonoids of Rhizoma Drynariae on the behavioral ability of mice in each group

    3.1.1 Experimental test results of new object recognition

    The results of the new object recognition experiment are shown in Table 1.Compared with the Blank group, the recognition index of the Model group decreased significantly (P<0.01); compared with the Model group, the recognition index of the KNSC group and TFRD group increased significantly (P<0.01); there was no significant difference between the KNSC group and the TFRD group(P>0.05); compared with the TFRD group, the recognition index of the TFRD+ER group was significantly reduced (P<0.01).

    Tab 1 Effects of total flavonoids of Drynaria fortunei on new object recognition experiment in mice (n=15, ±s)

    Tab 1 Effects of total flavonoids of Drynaria fortunei on new object recognition experiment in mice (n=15, ±s)

    Note: Compared with Blank group, **P<0.01; compared with Model group,##P<0.01; compared with TFRD group, &&P<0.01.

    Group Identification Index Blank group 0.441±0.017 Model group 0.252±0.012**KNSC group 0.385±0.015##TFRD group 0.364±0.010##TFRD+ER group 0.290±0.014&&

    3.1.2 Test results of water maze test

    In the positioning route experiment, the results are shown in Table 2.Compared with the Blank group, the latency of the Model group was significantly increased (P<0.01); compared with the Model group, the latency of the KNSC group and TFRD group was significantly decreased (P<0.01); There was no significant difference between the KNSC group and the TFRD group (P>0.05); compared with the TFRD group, the latency of the TFRD+ER group was significantly increased (P<0.01).

    Tab 2 Effects of total flavonoids of Drynaria fortunei on navigation latency of mice in each group (n=15, ±s)

    Tab 2 Effects of total flavonoids of Drynaria fortunei on navigation latency of mice in each group (n=15, ±s)

    Note: Compared with Blank group, **P<0.01; compared with Model group, ##P<0.01; compared with TFRD group, &&P<0.01.

    Group Day 1(s) Day 2(s) Day 3(s) Day 4(s)Blank group 45.953±1.702 37.899±2.596 31.674±1.380 25.152±3.509 Model group 69.262±3.301** 61.180±2.621** 55.895±3.255** 45.523±2.206**KNSC group 48.930±1.305## 41.372±1.864## 40.065±1.957## 32.352±1.383##TFRD group 49.946±2.141## 41.873±2.626## 42.582±2.658## 34.638±2.201##TFRD+ER group 58.951±1.905&& 52.858±3.346&& 49.007±2.389&& 39.656±2.157&&

    In the spatial exploration experiment, the results are shown in Table 3.Compared with the Blank group, the spatial exploration ability of the Model group decreased significantly (P<0.01); compared with the Model group, the spatial exploration ability of the KNSC group and TFRD group increased significantly (P<0.01); there was no significant difference between the KNSC group and the TFRD group(P>0.05); compared with the TFRD group, the spatial exploration ability of the TFRD+ER group was significantly reduced (P<0.01).

    In the platform jumping experiment, the results are shown in Table 4.Compared with the Blank group, the Model group's escape latency was significantly reduced, and the number of errors was significantly increased (P<0.01).Compared with the Model group,the KNSC group and TFRD group mice escaped.The latency was significantly increased, and the number of errors was significantly decreased (P<0.01); there was no significant difference between the TFRD group and the anti-KNSC group (P>0.05); compared with the TFRD group, the escape latency of the TFRD+ER group mice was significantly decreased, and the number of errors was significantly increased (P<0.01).The experimental results show that the total flavonoids of Rhizoma Drynariae can significantly improve the learning and memory ability of hydrocortisone model mice.

    Tab 3 Effects of Drynaria total flavonoids on space exploration of mice in each group(n=15, ±s)

    Tab 3 Effects of Drynaria total flavonoids on space exploration of mice in each group(n=15, ±s)

    Note: Compared with Blank group, **P<0.01; compared with Model group,##P<0.01; compared with TFRD group, &&P<0.01.

    Group Time to cross the platform (times) Stay time (s)Blank group 2.898±0.167 33.368±1.706 Model group 0.703±0.093 ** 15.654±1.419**KNSC group 2.290±0.067## 27.185±0.873##TFRD group 2.162±0.141## 26.921±1.162##TFRD+ER group 1.521±0.125&& 21.658±0.980&&

    Tab 4 Effects of Drynaria total flavonoids on error times and dark avoidance latency of mice in each group (n=15, ±s)

    Tab 4 Effects of Drynaria total flavonoids on error times and dark avoidance latency of mice in each group (n=15, ±s)

    Note: Compared with Blank group, **P<0.01; compared with Model group,##P<0.01; compared with TFRD group, &&P<0.01.

    Group Error times Dark avoidance latency(s)Blank group 1.315±0.245 86.711±5.374 Model group 8.151±0.364** 33.418±4.214**KNSC group 3.250±0.189## 65.790±3.251##TFRD group 3.854±0.124## 62.855±4.662##TFRD+ER group 5.742±0.274&& 47.120±7.514&&

    3.2 HE staining results of total flavonoids of Rhizoma Drynariae on the hippocampus of mice in each group

    The results are shown in Figure 5.The cells in the hippocampus of the mice in the Blank group are complete, tightly arranged,with a large number of neurons and clear nucleoli.The cells in the hippocampus of the Model group mice are disorderly arranged,and the number of some neurons is reduced.The phenomenon of pyknosis was obvious; the TFRD group was similar to the KNSC group, the cells in the hippocampus were more closely arranged, the number of neurons was more, and the nucleoli were clear; Normal,but clear nucleoli without obvious pyknosis.

    3.3 Effects of total flavonoids of Rhizoma Drynariae on the expressions of NMDAR1, GluR2 and CAMKⅡ proteins in the hippocampus of mice in each group

    The results are shown in Figure 6: Compared with the Blank group,the CAMKII content of the Model group increased (P<0.01), while the NMDAR1 and GluR2 contents decreased (P<0.01); compared with the Model group, the CAMKII content of the KNSC group and TFRD group decreased (P<0.01).P<0.01), the contents of NMDAR1 and GluR2 in KNSC group and TFRD group increased (P<0.01);there was no significant difference between TFRD group and KNSC group (P>0.05).Compared with TFRD group, CAMKⅡ contents in TFRD+ER group increased.(P<0.01), the content of NMDAR1 and GluR2 decreased (P<0.01).The experimental results showed that the total flavonoids of Rhizoma Drynariae could significantly increase the expression of NMDAR1 and GluR2 proteins in the hippocampus of hydrocortisone model mice, and at the same time significantly reduce the level of CAMKⅡ protein.

    Tab 5 Effects of Drynaria total flavonoids on the expression of NMDAR1, GluR2 and CAMK Ⅱ in the hippocampus of mice in each group(n=15,±s)

    Tab 5 Effects of Drynaria total flavonoids on the expression of NMDAR1, GluR2 and CAMK Ⅱ in the hippocampus of mice in each group(n=15,±s)

    Note: Compared with Blank group, **P<0.01; compared with Model group,##P<0.01; compared with TFRD group, &&P<0.01.

    Group NMDAR1 GluR2 CAMKⅡBlank group 0.618±0.021 0.785±0.012 0.872±0.021 Model group 0.474±0.011** 0.517±0.011** 2.379±0.042**KNSC group 0.542±0.015## 0.747±0.014## 1.362±0.012##TFRD group 0.508±0.012## 0.726±0.010## 1.569±0.025##TFRD+ER group 0.484±0.013&& 0.654±0.011&& 0.739±0.023&&

    Fig 2 Expression of NMDAR1, GluR2 and CAMKⅡ protein in mouse hippocampus(n=3)

    4.Discussion

    As the population ages, Alzheimer's disease is becoming a major health crisis worldwide.Although the pathogenesis of Alzheimer's disease has been greatly understood in the past few decades, there is still a lack of effective treatments in the clinic [10].Chinese medicine believes that "the kidney controls the bones, generates the marrow,and communicates with the brain", which mainly means that the kidney stores the essence, and then the essence is the marrow [11].The mouse model of Alzheimer's syndrome is a common animal model for the study of Alzheimer's disease based on the theory of traditional Chinese medicine [12].After injecting a large amount of glucocorticoid hydrocortisone into the animal, it will cause negative feedback in the body, disturb the hormone level in the body, and appear mentally depressed, Symptoms such as unresponsiveness,dry hair, and frequent nocturia can create a pathological model of kidney-yang deficiency in animals, and glucocorticoids can promote oxidative stress and cause the death of hippocampal neurons, which can acutely damage various components of working memory.[13].Therefore, this paper adopts the kidney deficiency model made by hydrocortisone to study the mechanism of the total flavonoids of Rhizoma Drynariae in improving AD-related diseases.

    Western medicine studies believe that estrogen may be related to AD treatment in some pathways through its receptors.After estrogen binds to estrogen receptors, it acts on the estrogen response element(ERE) located in the DNA of the upstream promoter region of the target gene., under the regulation of a variety of factors, enhance or inhibit the transcription of target genes [14].Although estrogen has obvious anti-AD potential, long-term use of synthetic estrogen can easily cause endometrial lesions, causing side effects such as venous thrombosis and breast cancer.Therefore, the use of natural Chinese herbal medicines and their preparations has become a hot spot in the treatment and prevention of AD.Yuanchao [15] and other studies have shown that phytoestrogens can improve the learning and memory function of ovariectomized rats.The main components of Rhizoma Drynariae, naringenin (flavonoids), quercetin (flavonols) and other compounds are all bisphenols, which are similar to synthetic estrogens and can stimulate ER and ERβ, regulate hypothalamuspituitary- Gonadal axis, and then stimulate various systems in the body to prevent AD.Estrogen exhibits multiple physiological effects in the brain, including neuronal differentiation, neurogenesis,and neuroplasticity, which are critical for inhibiting homeostasis,cognition, and retention [16].Estrogen can improve the expression of targeted proteins in AD-related hypotheses, and its role is mainly through binding to ER to induce the expression of target genes [17],mediated by second messengers, and activating multiple related pathways to exert neuroprotection.effect.ER belongs to the steroid hormone family of the supranuclear receptor family, and it was previously believed that ER is a transcription factor mainly localized in the nucleus [18].However, the ER is also present at the plasma membrane and is involved in rapid estrogen signaling.Song et al.[19]verified through experiments that phytoestrogens also play an antiosteoporosis effect through this process, and the authors speculated that phytoestrogens Drynariae also regulate the preventive effect on AD through ER mediation.

    Hyperactive glutamatergic signaling occurs in the early stages of AD pathology, which is thought to be mediated by reduced glutamate uptake [20].One mechanism that has emerged is the coupling of canonical estrogen receptors to glutamate receptors(Glu) to initiate G protein signaling cascades that ultimately affect neuronal physiology, structure, and behavior.The association of ER with different subtypes of glutamate receptors appears to drive distinct molecular outcomes that can affect processes such as cognition, motivation, movement, and pain.Glutamate receptors(Glu) are one of the most important excitatory neurotransmitter receptors in the central nervous system and are usually found in glial cells and neurons.Glutamate receptors are closely related to energy metabolism in the brain and are essential for maintaining glutamatergic neurotransmission [21].Glutamate receptors are divided into two types: ionotropic glutamate receptors and metabolic glutamate receptors.Among them, ionotropic receptor channels include N-methyl-D-aspartate (NMDA), α-aminohydroxymethyl oxazole propionic acid (AMPA) and jujube acid salt (KA) receptor channels, among which α- Aminohydroxymethyl oxazole propionic acid receptors have four subunits: GluR1, GluR2, GluR3, and GluR4, and GluR1 and GluR2 are the main ones.Metabotropic glutamate receptors are closely related to calcium ion channels on cell membranes.Studies have shown that abnormal Ca+signaling pathway may be the potential pathogenic mechanism of Alzheimer's disease due to kidney deficiency.Calmodulin (CaM/CALM) can combine with Ca+in neurons to form calcium/calmodulin-dependent protein kinase (CAMK) II initiates a variety of downstream signaling pathways, and CAMK II is activated in the hippocampus to participate in the formation of synaptic plasticity, brain learning and memory [22].N-methyl-D-aspartate (NMDAR) is the most widely studied ionotropic glutamate receptor, and it is ubiquitously present in the central nervous system and plays an important role.They are involved in excitotoxicity, which is a major factor in AD.Widely accepted causative factor for neuronal and synaptic loss [23].It has been confirmed that the pathogenesis of neurodegenerative diseases may be closely related to the dysregulation of NMDAR activity.NMDA receptors and estrogen receptors are co-expressed in the hippocampus, which are closely related to learning and memory ability, and are the main mediators of glutamatergic neuron damage in the pathogenesis of AD.The total flavonoids of Rhizoma Drynariae may rapidly activate the ERK1/2 signaling pathway through estrogen receptors, phosphorylate the NR2B subunit [24],and then activate NMDA receptors.Estrogen, estrogen receptors,and NMDA receptors all affect synapses plasticity.

    This study showed that compared with the control group, the expression of NMDAR and GluR2 proteins in the brain of the model mice was significantly reduced, and the expression of CAMKⅡ protein was significantly enhanced, the regulation of Rhizoma Drynariae on Appellate protein was reversed, and the improvement effect on learning and memory in mice basically disappeared.Combined with the contents of Appellate, it is speculated that the total flavonoids of Rhizoma Drynariae may improve the learning of hydrocortisone model mice through the ER pathway.Memory impairment, unresponsiveness and other symptoms can reduce the damage to the brain tissue of the model mice and play a neuroprotective role, thereby achieving the effect of treating AD, and providing a new direction for the development of safe and effective new drugs for the treatment of AD.

    Author's contribution:

    Wang Yi-tong: index detection, data analysis, and paper writing;Xu Yan-ming: model establishment, index detection; Zhang Zhibo: model establishment, index detection; Sun Hui-feng: model establishment and material selection; Yang Bo: research director;Zhang Ning: instructor.

    All authors declare no conflict of interest.

    91在线精品国自产拍蜜月| 看黄色毛片网站| 一个人看的www免费观看视频| 又粗又爽又猛毛片免费看| av免费在线看不卡| 俺也久久电影网| 内地一区二区视频在线| 老熟妇乱子伦视频在线观看| 3wmmmm亚洲av在线观看| 91久久精品国产一区二区成人| 人妻少妇偷人精品九色| 麻豆一二三区av精品| 日本黄色视频三级网站网址| 国产亚洲91精品色在线| 麻豆成人午夜福利视频| 青春草视频在线免费观看| 成人亚洲欧美一区二区av| 少妇丰满av| 免费大片18禁| 成人鲁丝片一二三区免费| 成人精品一区二区免费| 国产av在哪里看| 国产一区二区亚洲精品在线观看| 久久人人爽人人片av| 国产av麻豆久久久久久久| 欧美日韩综合久久久久久| 黄片wwwwww| 欧美日韩乱码在线| 亚洲久久久久久中文字幕| 久久鲁丝午夜福利片| 少妇人妻一区二区三区视频| 男女之事视频高清在线观看| 亚洲av不卡在线观看| 国产精品乱码一区二三区的特点| avwww免费| 日日干狠狠操夜夜爽| 干丝袜人妻中文字幕| 97超碰精品成人国产| 又粗又爽又猛毛片免费看| 色哟哟·www| 春色校园在线视频观看| 一本精品99久久精品77| 国产极品精品免费视频能看的| 香蕉av资源在线| 亚洲高清免费不卡视频| 亚洲人成网站高清观看| 久久热精品热| 久久久久久久午夜电影| 美女高潮的动态| 亚洲无线观看免费| 亚洲成人精品中文字幕电影| 少妇猛男粗大的猛烈进出视频 | 级片在线观看| 亚洲经典国产精华液单| 亚洲av美国av| 麻豆国产av国片精品| 啦啦啦观看免费观看视频高清| 身体一侧抽搐| 欧美性猛交黑人性爽| 国产人妻一区二区三区在| 青春草视频在线免费观看| 人妻夜夜爽99麻豆av| 亚州av有码| 99久久精品国产国产毛片| 乱码一卡2卡4卡精品| 在线观看免费视频日本深夜| 人人妻人人澡欧美一区二区| 校园人妻丝袜中文字幕| 久久精品国产鲁丝片午夜精品| 亚洲av五月六月丁香网| 久久精品国产清高在天天线| 亚洲欧美中文字幕日韩二区| 国产精品亚洲美女久久久| 久久久午夜欧美精品| 麻豆国产97在线/欧美| 国产成人aa在线观看| 国产在线男女| 欧美日韩精品成人综合77777| 91在线精品国自产拍蜜月| 成熟少妇高潮喷水视频| 在线天堂最新版资源| 久久久久久久久久黄片| 在线免费观看不下载黄p国产| a级一级毛片免费在线观看| 97碰自拍视频| 久久6这里有精品| 国产av不卡久久| 亚洲欧美清纯卡通| 啦啦啦韩国在线观看视频| 午夜福利高清视频| a级毛片a级免费在线| 国产精品一二三区在线看| 亚洲性久久影院| 亚洲精品成人久久久久久| 岛国在线免费视频观看| 中文字幕免费在线视频6| 亚洲经典国产精华液单| 欧美精品国产亚洲| 少妇熟女欧美另类| 少妇熟女aⅴ在线视频| 日韩中字成人| 嫩草影院入口| 十八禁国产超污无遮挡网站| 日韩精品青青久久久久久| 久久久久久大精品| 久久草成人影院| 日韩精品中文字幕看吧| 深夜精品福利| 久久久久性生活片| 白带黄色成豆腐渣| 亚洲一级一片aⅴ在线观看| 欧美色视频一区免费| 真人做人爱边吃奶动态| 一级a爱片免费观看的视频| 少妇猛男粗大的猛烈进出视频 | 女同久久另类99精品国产91| 国产男人的电影天堂91| 99热这里只有是精品在线观看| 搞女人的毛片| 色吧在线观看| 最近2019中文字幕mv第一页| 日韩成人伦理影院| 国产精品久久久久久精品电影| 成人无遮挡网站| 欧美一级a爱片免费观看看| 大型黄色视频在线免费观看| 免费搜索国产男女视频| 国产精品电影一区二区三区| 久久热精品热| 日日撸夜夜添| 国产美女午夜福利| 亚洲第一电影网av| 成人特级黄色片久久久久久久| 亚洲精品粉嫩美女一区| 午夜福利成人在线免费观看| 99久久久亚洲精品蜜臀av| 欧美性猛交黑人性爽| 老司机午夜福利在线观看视频| 三级经典国产精品| 久久久久久久午夜电影| 色av中文字幕| 22中文网久久字幕| 99国产精品一区二区蜜桃av| 亚洲欧美日韩高清在线视频| 97人妻精品一区二区三区麻豆| 亚洲五月天丁香| 精品一区二区三区av网在线观看| 美女免费视频网站| 久久久国产成人免费| 欧美日本视频| 美女黄网站色视频| 免费看av在线观看网站| 中文亚洲av片在线观看爽| 国产一区二区激情短视频| 美女高潮的动态| 在线观看66精品国产| 十八禁网站免费在线| 国产中年淑女户外野战色| 特级一级黄色大片| 亚洲自偷自拍三级| 晚上一个人看的免费电影| av天堂中文字幕网| 日韩强制内射视频| 国产高清三级在线| 亚洲精品日韩在线中文字幕 | 午夜福利成人在线免费观看| 1000部很黄的大片| av视频在线观看入口| 亚洲天堂国产精品一区在线| 人人妻,人人澡人人爽秒播| 免费搜索国产男女视频| 97超碰精品成人国产| 热99在线观看视频| 久久精品夜色国产| 中文亚洲av片在线观看爽| 亚洲无线在线观看| 精品人妻一区二区三区麻豆 | 性色avwww在线观看| 成人精品一区二区免费| 国产私拍福利视频在线观看| 女人被狂操c到高潮| 亚洲专区国产一区二区| 国产一区二区激情短视频| 欧美成人精品欧美一级黄| 国产亚洲精品久久久久久毛片| 国产一区二区在线av高清观看| 成人二区视频| 成人av一区二区三区在线看| 欧美性感艳星| 午夜精品一区二区三区免费看| 国产三级在线视频| av女优亚洲男人天堂| 欧美绝顶高潮抽搐喷水| 欧美精品国产亚洲| 日本在线视频免费播放| 自拍偷自拍亚洲精品老妇| 免费人成视频x8x8入口观看| 久久久久久久久中文| 国产精品av视频在线免费观看| 国产片特级美女逼逼视频| 国产精品乱码一区二三区的特点| 欧美性猛交黑人性爽| 99热这里只有精品一区| 自拍偷自拍亚洲精品老妇| 亚洲精品国产av成人精品 | 久久韩国三级中文字幕| 在线播放无遮挡| 九九久久精品国产亚洲av麻豆| 能在线免费观看的黄片| 夜夜看夜夜爽夜夜摸| 99九九线精品视频在线观看视频| 免费人成在线观看视频色| av国产免费在线观看| 麻豆国产av国片精品| 国内精品一区二区在线观看| 舔av片在线| 尤物成人国产欧美一区二区三区| 亚洲成人中文字幕在线播放| 国产中年淑女户外野战色| 日本成人三级电影网站| 俄罗斯特黄特色一大片| 精品国内亚洲2022精品成人| 久久婷婷人人爽人人干人人爱| 精品一区二区三区视频在线| 欧美日韩综合久久久久久| 中文字幕熟女人妻在线| 成人一区二区视频在线观看| 成人三级黄色视频| 久久精品国产亚洲网站| 色哟哟哟哟哟哟| 久久6这里有精品| 美女被艹到高潮喷水动态| 热99在线观看视频| 免费看日本二区| 三级国产精品欧美在线观看| 午夜老司机福利剧场| 亚洲成av人片在线播放无| 国产精品精品国产色婷婷| 精品人妻一区二区三区麻豆 | 偷拍熟女少妇极品色| 亚洲人成网站高清观看| 日韩欧美一区二区三区在线观看| 欧美色欧美亚洲另类二区| 欧美日韩一区二区视频在线观看视频在线 | 三级国产精品欧美在线观看| 一区二区三区免费毛片| 国内精品一区二区在线观看| 偷拍熟女少妇极品色| 久久久a久久爽久久v久久| 老熟妇仑乱视频hdxx| 69av精品久久久久久| 日日摸夜夜添夜夜添小说| 丰满的人妻完整版| 免费黄网站久久成人精品| 亚洲av中文av极速乱| 国产精华一区二区三区| 一个人看的www免费观看视频| 中国国产av一级| 国产高清三级在线| 国产成人91sexporn| 亚洲一区高清亚洲精品| 日韩欧美 国产精品| 少妇猛男粗大的猛烈进出视频 | 日韩欧美精品v在线| 麻豆国产av国片精品| 波野结衣二区三区在线| 狠狠狠狠99中文字幕| 日韩一区二区视频免费看| 丰满的人妻完整版| 亚洲精品乱码久久久v下载方式| av中文乱码字幕在线| 午夜精品在线福利| 你懂的网址亚洲精品在线观看 | 国产精品精品国产色婷婷| 又爽又黄a免费视频| 亚洲精品日韩av片在线观看| 97超级碰碰碰精品色视频在线观看| 国产极品精品免费视频能看的| 一个人观看的视频www高清免费观看| 国产亚洲精品综合一区在线观看| 97在线视频观看| 男女那种视频在线观看| 99久久精品热视频| 日本三级黄在线观看| 久久久午夜欧美精品| 毛片女人毛片| 在线天堂最新版资源| 女人十人毛片免费观看3o分钟| 97超碰精品成人国产| 天堂√8在线中文| 日本在线视频免费播放| 九九在线视频观看精品| 免费观看人在逋| 精品久久久久久久久久久久久| 91av网一区二区| 在线看三级毛片| 国产高清视频在线播放一区| 亚洲国产色片| 在线a可以看的网站| 男女之事视频高清在线观看| 欧美人与善性xxx| 成人特级av手机在线观看| 人妻丰满熟妇av一区二区三区| 国产成人91sexporn| 午夜福利成人在线免费观看| 黄片wwwwww| 亚洲一区高清亚洲精品| 国产精品福利在线免费观看| 国产亚洲欧美98| 在线观看av片永久免费下载| 久久久国产成人免费| 全区人妻精品视频| 禁无遮挡网站| 高清午夜精品一区二区三区 | 欧美极品一区二区三区四区| 日韩欧美精品v在线| 精品无人区乱码1区二区| 悠悠久久av| 久久国内精品自在自线图片| avwww免费| 国产黄色视频一区二区在线观看 | 亚洲无线观看免费| 国产一区二区三区在线臀色熟女| 99久久精品一区二区三区| av中文乱码字幕在线| 欧美xxxx性猛交bbbb| 免费在线观看影片大全网站| 老司机影院成人| 亚洲国产高清在线一区二区三| 午夜福利在线观看吧| 国产白丝娇喘喷水9色精品| 我要搜黄色片| 搞女人的毛片| 一本一本综合久久| 亚洲人成网站在线播放欧美日韩| 亚洲va在线va天堂va国产| 亚洲精品一卡2卡三卡4卡5卡| 亚洲欧美日韩卡通动漫| 亚洲国产精品合色在线| 岛国在线免费视频观看| 白带黄色成豆腐渣| 亚洲婷婷狠狠爱综合网| 99视频精品全部免费 在线| 美女内射精品一级片tv| 成人亚洲欧美一区二区av| av卡一久久| 老熟妇乱子伦视频在线观看| 少妇猛男粗大的猛烈进出视频 | 精品国产三级普通话版| 成年av动漫网址| 色噜噜av男人的天堂激情| 性欧美人与动物交配| 亚洲美女黄片视频| 国产69精品久久久久777片| 国产男靠女视频免费网站| 少妇熟女欧美另类| 国产精品福利在线免费观看| 一级av片app| 无遮挡黄片免费观看| 最新在线观看一区二区三区| 亚洲欧美日韩无卡精品| 午夜福利成人在线免费观看| 久久这里只有精品中国| 色尼玛亚洲综合影院| 久久婷婷人人爽人人干人人爱| 欧美高清性xxxxhd video| 国内揄拍国产精品人妻在线| 又爽又黄a免费视频| 亚洲综合色惰| 精品一区二区三区av网在线观看| 九色成人免费人妻av| 国产大屁股一区二区在线视频| 在线观看av片永久免费下载| 麻豆成人午夜福利视频| 亚洲av中文av极速乱| 精品久久久久久久末码| 欧美绝顶高潮抽搐喷水| 欧美zozozo另类| 伦理电影大哥的女人| 91久久精品国产一区二区成人| 亚洲色图av天堂| 色播亚洲综合网| 成人永久免费在线观看视频| 欧美zozozo另类| 精品福利观看| 午夜老司机福利剧场| 在线天堂最新版资源| 禁无遮挡网站| 精品人妻偷拍中文字幕| 美女高潮的动态| 久久国产乱子免费精品| 成人亚洲精品av一区二区| 最新中文字幕久久久久| 乱码一卡2卡4卡精品| 亚洲av美国av| a级一级毛片免费在线观看| 亚洲精品国产成人久久av| 久久久久免费精品人妻一区二区| 日本免费a在线| 亚洲欧美日韩东京热| av在线播放精品| 国产白丝娇喘喷水9色精品| 一区二区三区免费毛片| 日日啪夜夜撸| 成年女人毛片免费观看观看9| 3wmmmm亚洲av在线观看| 精品人妻一区二区三区麻豆 | 国产精品久久电影中文字幕| 亚洲一级一片aⅴ在线观看| 久久久精品大字幕| 久久久久久大精品| 两性午夜刺激爽爽歪歪视频在线观看| 69av精品久久久久久| av天堂中文字幕网| 欧美色视频一区免费| 国产乱人视频| 亚洲一区高清亚洲精品| 亚洲美女视频黄频| 久久久a久久爽久久v久久| 免费高清视频大片| 中文字幕av在线有码专区| 99九九线精品视频在线观看视频| 国产毛片a区久久久久| videossex国产| 永久网站在线| 亚洲人与动物交配视频| 久久国产乱子免费精品| 尤物成人国产欧美一区二区三区| 久久人人爽人人爽人人片va| 亚洲精华国产精华液的使用体验 | 老司机午夜福利在线观看视频| 色噜噜av男人的天堂激情| 尤物成人国产欧美一区二区三区| 哪里可以看免费的av片| a级一级毛片免费在线观看| 听说在线观看完整版免费高清| 国产精品亚洲美女久久久| 69av精品久久久久久| 国产视频一区二区在线看| 国产欧美日韩一区二区精品| 自拍偷自拍亚洲精品老妇| 欧美极品一区二区三区四区| 在线免费观看的www视频| 国产欧美日韩精品一区二区| 免费大片18禁| 国产高清不卡午夜福利| 国产人妻一区二区三区在| 免费看a级黄色片| 最新在线观看一区二区三区| 久久精品国产亚洲av香蕉五月| 99热这里只有是精品50| 舔av片在线| 国产毛片a区久久久久| 欧美色欧美亚洲另类二区| 婷婷精品国产亚洲av在线| 一本一本综合久久| 国产黄片美女视频| 日本欧美国产在线视频| 日韩精品青青久久久久久| av女优亚洲男人天堂| 久久精品91蜜桃| 熟女人妻精品中文字幕| 美女免费视频网站| 久久人妻av系列| 一级毛片电影观看 | 校园春色视频在线观看| 日韩欧美三级三区| 亚洲精华国产精华液的使用体验 | 亚洲av不卡在线观看| 我的女老师完整版在线观看| 成人精品一区二区免费| 91久久精品国产一区二区成人| 成人特级黄色片久久久久久久| 一进一出抽搐动态| 精品久久久久久久久亚洲| 最近最新中文字幕大全电影3| 亚洲熟妇熟女久久| 伊人久久精品亚洲午夜| 俺也久久电影网| 12—13女人毛片做爰片一| 久久人人爽人人片av| 日韩中字成人| 午夜久久久久精精品| 麻豆国产97在线/欧美| 久久精品国产亚洲av涩爱 | 国产综合懂色| 亚洲最大成人av| 免费无遮挡裸体视频| 老司机影院成人| 午夜福利18| 久久精品国产亚洲av天美| 哪里可以看免费的av片| 日韩国内少妇激情av| 春色校园在线视频观看| 日韩av不卡免费在线播放| 成熟少妇高潮喷水视频| 久久精品影院6| 内射极品少妇av片p| 亚洲va在线va天堂va国产| 日韩精品青青久久久久久| 精品99又大又爽又粗少妇毛片| 亚洲国产欧洲综合997久久,| 国产精品99久久久久久久久| 一级毛片我不卡| 男人舔奶头视频| 亚洲久久久久久中文字幕| 99在线人妻在线中文字幕| 国产探花极品一区二区| 亚洲av免费高清在线观看| 亚洲美女黄片视频| 真实男女啪啪啪动态图| 国产精品女同一区二区软件| 国产老妇女一区| 秋霞在线观看毛片| 日韩欧美一区二区三区在线观看| 久久久欧美国产精品| 亚洲高清免费不卡视频| 国产三级中文精品| 国产精品永久免费网站| 露出奶头的视频| av天堂中文字幕网| 久久人人爽人人片av| 国产一区二区三区av在线 | 国产毛片a区久久久久| 99久久精品国产国产毛片| 亚洲精品亚洲一区二区| 午夜精品国产一区二区电影 | 亚洲最大成人av| 人人妻人人澡人人爽人人夜夜 | 国产精品久久久久久久电影| 麻豆精品久久久久久蜜桃| 精品一区二区免费观看| 日韩在线高清观看一区二区三区| 免费人成视频x8x8入口观看| 亚洲中文字幕一区二区三区有码在线看| 夜夜夜夜夜久久久久| 丰满人妻一区二区三区视频av| 日韩成人伦理影院| 国产精华一区二区三区| 少妇人妻一区二区三区视频| 人妻丰满熟妇av一区二区三区| 男女之事视频高清在线观看| 人妻夜夜爽99麻豆av| 99久国产av精品| 国产av不卡久久| 国产毛片a区久久久久| 成人性生交大片免费视频hd| 亚洲美女视频黄频| 中文字幕av在线有码专区| 中国美女看黄片| 免费看a级黄色片| 男女那种视频在线观看| 日本爱情动作片www.在线观看 | 麻豆av噜噜一区二区三区| 97超视频在线观看视频| 国产精品亚洲美女久久久| 99久国产av精品国产电影| 97人妻精品一区二区三区麻豆| 国内精品久久久久精免费| 女人十人毛片免费观看3o分钟| 午夜福利高清视频| 国产av一区在线观看免费| 色哟哟·www| 国产白丝娇喘喷水9色精品| 国产麻豆成人av免费视频| 国内揄拍国产精品人妻在线| 成人欧美大片| 一区二区三区四区激情视频 | 国产男人的电影天堂91| 国产精品一区二区三区四区免费观看 | 精品福利观看| 99热6这里只有精品| 亚洲一级一片aⅴ在线观看| 两性午夜刺激爽爽歪歪视频在线观看| 亚洲国产色片| 菩萨蛮人人尽说江南好唐韦庄 | 成年女人毛片免费观看观看9| 长腿黑丝高跟| 亚洲精品一卡2卡三卡4卡5卡| 欧美色欧美亚洲另类二区| 国产高清激情床上av| 欧美高清成人免费视频www| 男插女下体视频免费在线播放| 美女免费视频网站| 真人做人爱边吃奶动态| 午夜免费激情av| 精华霜和精华液先用哪个| 天堂av国产一区二区熟女人妻| 欧美成人a在线观看| 免费看a级黄色片| 日韩大尺度精品在线看网址| 毛片一级片免费看久久久久| 亚洲精品一区av在线观看| 国产色婷婷99| 亚洲五月天丁香| 国产精品无大码| 日韩一区二区视频免费看| 日韩高清综合在线| 亚洲欧美日韩无卡精品| 国产激情偷乱视频一区二区| 久久午夜福利片| 国产 一区精品| 99精品在免费线老司机午夜| videossex国产| 久久国产乱子免费精品| 国产在线男女| 午夜福利视频1000在线观看| 一a级毛片在线观看| 日韩成人伦理影院| 精品一区二区三区视频在线| 日韩欧美一区二区三区在线观看| 国产探花极品一区二区| 久久精品夜色国产| 亚洲18禁久久av| 一本一本综合久久|