• <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 ?

    Ginsenoside Rg1 protects against neurodegeneration by inducing neurite outgrowth in cultured hippocampal neurons

    2016-12-02 02:30:09LiangHuangLifengLiuJuanLiuLingDouGeyingWangXiaoqingLiuQionglanYuan
    關(guān)鍵詞:苗齡北方地區(qū)種皮

    Liang Huang, Li-feng Liu, Juan Liu, Ling Dou, Ge-ying Wang, Xiao-qing Liu, Qiong-lan Yuan

    Department of Neurology, Shanghai Tongji Hospital, Tongji University School of Medicine, Shanghai, China

    RESEARCH

    Ginsenoside Rg1 protects against neurodegeneration by inducing neurite outgrowth in cultured hippocampal neurons

    Liang Huang#, Li-feng Liu#, Juan Liu, Ling Dou, Ge-ying Wang, Xiao-qing Liu, Qiong-lan Yuan*

    Department of Neurology, Shanghai Tongji Hospital, Tongji University School of Medicine, Shanghai, China

    Graphical Abstract

    # These authors contributed equally to this work.

    orcid: 0000-0001-6165-2107 (Qiong-lan Yuan)

    Ginsenoside Rg1 (Rg1) has anti-aging and anti-neurodegenerative effects. However, the mechanisms underlying these actions remain unclear. The aim of the present study was to determine whether Rg1 affects hippocampal survival and neurite outgrowth in vitro after exposure to amyloid-beta peptide fragment 25-35 (Aβ25-35), and to explore whether the extracellular signal-regulated kinase (ERK) and Akt signaling pathways are involved in these biological processes. We cultured hippocampal neurons from newborn rats for 24 hours, then added Rg1 to the medium for another 24 hours, with or without pharmacological inhibitors of the mitogen-activated protein kinase (MAPK) family or Akt signaling pathways for a further 24 hours. We then immunostained the neurons for growth associated protein-43, and measured neurite length. In a separate experiment, we exposed cultured hippocampal neurons to Aβ25-35for 30 minutes, before adding Rg1 for 48 hours, with or without Akt or MAPK inhibitors, and assessed neuronal survival using Hoechst 33258 staining, and phosphorylation of ERK1/2 and Akt by western blot analysis. Rg1 induced neurite outgrowth, and this effect was blocked by API-2 (Akt inhibitor) and PD98059 (MAPK/ERK kinase inhibitor), but not by SP600125 or SB203580 (inhibitors of c-Jun N-terminal kinase and p38 MAPK, respectively). Consistent with this effect, Rg1 upregulated the phosphorylation of Akt and ERK1/2; these effects were reversed by API-2 and PD98059, respectively. In addition, Rg1 significantly reversed Aβ25-35-induced apoptosis; this effect was blocked by API-2 and PD98059, but not by SP600125 or SB203580. Finally, Rg1 significantly reversed the Aβ25-35-induced decrease in Akt and ERK1/2 phosphorylation, but API-2 prevented this reversal. Our results indicate that Rg1 enhances neurite outgrowth and protects against Aβ25-35-induced damage, and that its mechanism may involve the activation of Akt and ERK1/2 signaling.

    nerve regeneration; ginsenoside Rg1; neurite outgrowth; Aβ25-35; hippocampal neurons;Akt; MAPK; apoptosis; growth associated protein-43; Hoechst 33258 staining; PD98059; API-2; neural regeneration

    Introduction

    The prevalence of neurodegenerative diseases such as Alzheimer’s disease (AD) is increasing, owing to an aging world population. However, despite considerable research efforts, the pathogenic mechanisms of AD remain poorly understood, and the effectiveness of currently available clinical treatments is limited. Ginsenoside Rg1 (Rg1), the major pharmacologically active ingredient of ginseng, crosses the blood-brain barrier and has anti-aging and anti-neurodegenerative effects (Cheng et al., 2005). Amyloid beta (Aβ), a 39-43 amino acid β-sheet peptide, is a key constituent of amyloid plaques and contributes to cognitive, neuronal and synaptic malfunctioning in AD. Notably, Rg1 reduces the level of Aβ in the brains of aged and transgenic AD mice, as well as in PC12 cells in vitro (Chen et al., 2006; Wang and Du, 2009; Shi et al., 2010). These studies indicate that Rg1 modulates the generation of Aβ, which may contribute to its enhancement of cognitive performance in vivo. Aβ25-35is a short Aβ fragment with large β-sheet fibrils, which possesses the same neurotoxicity as the full-length peptide (Iverson et al., 1995). It was recently reported that Rg1 prevents Aβ25-35-induced apoptosis in cultured hippocampal neurons by upregulating the ratio of the apoptotic regulators Bcl-2/Bax (Gong et al., 2011). However, its mechanism of action needs further exploration.

    Neurite growth is an important process in neuronal development, synapse formation, and regeneration. There is great clinical interest in neurite growth, and the process can be used to assess neurotrophic properties of pharmaceuticals (Mitchell et al., 2007). It has been reported that Rg1 promotes neurite outgrowth in PC12 cells (Rudakewich et al., 2001). In contrast, Radad et al (2004a) showed that Rg1 did not promote neurite outgrowth in mesencephalic dopaminergic cells. Furthermore, the same group reported that Rg1 reversed the reduction in neurite length and number induced by glutamate or 1-methyl-4-phenylpyridinium (MPP+) (Radad et al., 2004a, b). Neurite outgrowth provides the morphological basis for synaptogenesis, the foundation of learning and memory. Whether Rg1 has the ability to affect neurite growth in neurons in the hippocampus, the learning and memory center of the brain, has not yet been investigated. Previous studies have shown that Rg1 increases the number of synapses and the density of synaptophysin, which provides the morphological basis for Rg1-induced facilitation of learning and memory (Mook-Jung et al., 2001).

    Mitogen-activated protein kinases (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling are involved in many physiological and pathological processes. The MAPK family includes extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 MAPK (Roux et al., 2004), and is phosphorylated by MAPK/ERK kinase (MEK). The Akt signaling pathway is well-known for its role in cell survival and anti-apoptosis (Kennedy et al., 1999; Brunet et al., 2001). ERK/MAPK signaling has been implicated in hippocampal synaptic plasticity and hippocampus-dependent memory formation (Sweatt et al., 2004). Conversely, JNK and p38 MAPK are activated by a variety of stress signals and are implicated in the induction of apoptotic cell death (Mielke et al., 2000).

    Here, we investigated the effect of Rg1 on neurite outgrowth and survival of hippocampal neurons after exposure to Aβ25-35in vitro, and explored Akt and MAPK signaling in these biological processes.

    Materials and Methods

    Chemicals and reagents

    Rg1 (purity 98%, lot No. 110703-200424) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Neurobasal Medium, B27 supplements and GlutaMAX were from Gibco BRL (Gaithersburg, MD, USA). Bovine serum albumin (BSA), HEPES, poly-L-lysine solution, DNase I, soybean trypsin inhibitor, and bovine trypsin were all from Sigma-Aldrich (St. Louis, MO, USA). Mouse anti-rat growth associated protein-43 (GAP-43) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Peroxidase-conjugated rabbit anti-mouse IgG was from Beyotime Institute of Biotechnology (Haimen, China). Rabbit anti-rat ERK1/2 and rabbit anti-rat phospho-ERK1/2 (Thr202/Tyr204), rat anti-human Akt (Ser473), rat anti-human phospho-Akt (Ser473), and horseradish peroxidase-conjugated goat anti-rabbit or anti-rat IgG were purchased from Cell Signaling Technology (Beverly, MA, USA). API-2, PD98059, SP600125 and SB203580 were purchased from Tocris Cookson, Inc. (Ballwin, MO, USA). BCA protein assay and enhanced chemiluminescence (ECL) kits were from Beyotime. Synthetic Aβ25-35and insulin-like growth factor-1 (IGF-1) were obtained from AnaSpec (Fremont, CA, USA) and ProSpec (Rehovot, Israel), respectively.

    Rg1 was dissolved in 0.9% NaCl to 2 mM and stored as a stock solution at -80° C. PD98059, API-2, SP600125 and SB203580 were dissolved in dimethyl sulfoxide to 25 mM and stored at -80°C. IGF-1 was dissolved in sterile ionized water to 100 μg/mL, or as a 3 mM stock solution, and stored at -20°C. Aβ25-35was dissolved in distilled water to make a 2 mM stock solution; aliquots were stored at -20°C for more than 24 hours and thawed at 37°C for 3 days to induce aggregation prior to use.

    Animals

    Ninety newborn Sprague-Dawley rats, 45 females and 45 males, aged less than 24 hours and weighing approximately 2 g, were purchased from the Shanghai Institute of the Chinese Academy of Sciences (license No. SCXK (Hu) 2007-0003). All animal procedures were performed under a protocol reviewed and approved by the Institutional Ethical Committee of Tongji University School of Medicine, China.

    Primary culture of hippocampal neurons

    Primary cultured hippocampal neurons were prepared from the rat neonates as described previously (Banker et al., 1977). Briefly, the hippocampi were dissected out and trypsinized (0.25% bovine trypsin) for 9-10 minutes at 37°C. The cells were plated in NeurobasalTMMedium with 2% B27 supplement, 10 μL/ mL penicillin-streptomycin, 1% GlutaMAX, 0.4% BSA and 20 mM HEPES at 37°C in a humidified 5% CO2incubator.Neurons were characterized using phase contrast microscopy (Nikon Corporation, Tokyo, Japan) and immunocytochemistry for GAP-43. Neuronal cultures were over 90% pure.

    Figure 1 Ginsenoside Rg1 (Rg1) promoted neurite outgrowth of cultured hippocampal neurons via ERK1/2 and PI3K/Akt signaling.

    Figure 2 Rg1 increased phosphorylation of Akt and ERK in cultured hippocampal neurons.

    Neurite outgrowth of hippocampal neurons in vitro

    Primary hippocampal neurons were plated at a density of 1 × 104/cm2, onto coverslips pre-coated with 10 mg/mL poly-L-lysine, in 24-well plates for 24 hours for GAP-43 immunostaining. The medium was then refreshed and 50 μM Rg1 was added, with or without the following drugs: 10 μM API-2; 10 μM PD98059; 5, 10 or 15 μM SP600125; 5, 10 or 15 μM SB203580. After a 24 hour incubation, the cells were fixed in 4% paraformaldehyde for 30 minutes at room temperature, and incubated with blocking solution (5% BSA) for 30 minutes at 37°C in a moisture chamber to block nonspecific binding. The cells were then incubated overnight at 4°C with mouse anti-rat GAP-43 antibody (dilution 1:1,000), followed by peroxidase-conjugated rabbit anti-mouse IgG (dilution 1:100) at 37°C for 2 hours. Finally, the cells were incubated with avidin-biotin complex (dilution 1:100) at 37 ° C for 1.5 hours. Diaminobenzidine(Sigma, St. Louis, MO, USA) was used as a chromogen for light microscopy. A negative control was carried out using the same procedures without primary antibody.

    Figure 3 Rg1 post-treatment protected against Aβ25-35-induced neurotoxicity via ERK1/2 and Akt signaling in hippocampal neurons.

    Figure 4 Rg1 post-treatment reversed Aβ25-35-induced reduction of ERK1/2 and Akt phosphorylation in hippocampal neurons.

    An investigator blinded to the cell treatment analyzed neurite outgrowth in cell cultures by counting the number of neuronal cell bodies (n = 100 per well) and measuring neurite length. Digitized images of cells were taken at 20× magnification using a digital camera connected to a microscope (Nikon, NY, USA). The ratio between neurite length and number of cell bodies was used to calculate the average neurite length, using Image Tool software (Pro Plus V 6.0) (Media Cybernetics, Inc, Silver Spring, MD, USA). The experiment was replicated fivetimes.

    For western blot analysis, cells were seeded at a density of 2 × 105/cm2in 60 mm diameter flasks pre-coated with 10 mg/ mL poly-L-lysine. After 24 hours in culture, the cell pellets were collected for western blot analysis. This experiment was performed in triplicate.

    Hippocampal neurons exposed to Aβ25-35in vitro

    After 24 hours in culture, the cells were divided into groups: (1) Normal control group: cells cultured for a further 48 hours with no treatment; (2) Aβ group: cells exposed to 20 μM Aβ25-35for 48 hours; (3) Aβ + treatment group: cells exposed to 20 μM Aβ25-35for 48 hours, with the following reagents and inhibitors added after 30 minutes: IGF-1 (50 ng/mL; positive control); Rg1 (50 μM); or Rg1 (50 μM) with either PD98059 (10 μM), API-2 (10 μM), SB203580 (5, 10 or 15 μM) or SP600125 (5, 10 or 15 μM). The inhibitors were added immediately after Rg1 treatment. All cells were cultured for 48 hours.

    For western blot analysis, cells were plated at a density of 2 × 105/cm2in 60 mm diameter flasks. After 24 hours, the cells were exposed to Aβ25-35, Rg1, and inhibitors as described above. After 48 hours, cell precipitations were collected for western blot analysis. The experiment was performed in triplicate.

    Hoechst 33258 staining

    After 48 hours in culture, the cells were fixed in 4% paraformaldehyde for 30 minutes at room temperature, and Hoechst 33258 was added to the medium for 15 minutes at 37°C. Images were obtained using an inverted fluorescence microscope (Nikon). Viable cells were identified by round nuclei with pale blue fluorescence, and apoptotic cells were characterized by condensation and fragmentation of nuclei. A researcher blinded to the cell treatment counted apoptotic and viable cells (n = 200 per well) and calculated the percentage of apoptotic cells (number of apoptotic neurons/[number of surviving + apoptotic neurons]). The experiment was replicated five times.

    Western blot analysis

    The cells were washed in ice-cold phosphate buffered saline and lysed in radioimmunoprecipitation assay buffer for 30 minutes at 4°C. Cell lysates were centrifuged at 18,514 × g (reactive centrifugal force) for 30 minutes at 4°C, and protein concentration was determined using a BCA protein assay kit. Total protein (40 μg) was dissolved in sample buffer and boiled for 5 minutes prior to loading onto polyacrylamide gels. The concentrations of the separation and stacking gels were 12% and 5%, respectively. Proteins were then transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween-20, and then incubated with primary antibodies against phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-Akt (Ser473) and Akt (all 1:1,000 dilution) overnight at 4°C. Finally, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-rat IgG (dilution 1:3,000) for 1 hour at room temperature and visualized using an ECL kit. Proteins were quantified by densitometric analysis of the bands.

    Statistical analysis

    Data are expressed as the mean ± SEM and were analyzed using SPSS 18.0 software (SPSS, Chicago, IL, USA). One-way analysis of variance followed by Newman-Keuls post hoc tests was carried out to assess the differences between the relevant control and each experimental condition. Statistical significance was set at P < 0.05.

    Results

    Rg1 promoted neurite outgrowth of cultured hippocampal neurons, and was inhibited by PD98059 and API-2 but not SP600125 or SB203580

    We assessed the neurotrophic effects of Rg1 on hippocampal neurons in vitro by measuring neurite outgrowth. Intact cell bodies and moderate neurite outgrowth were observed in control cells (Figure 1A). Cells incubated with Rg1 for 24 hours showed larger cell bodies with more numerous and longer neurites. Cells treated with Rg1 had significantly greater neurite outgrowth than control cells (Figure 1B).

    北方地區(qū),早春及秋末氣候較為冷涼,一般3月末至4有初在設(shè)施內(nèi)育苗,終霜后定植于露地,6~9月份收獲。因苦瓜種子種皮較厚,播前要進(jìn)行浸種催芽。先用50~60℃的溫水浸種,水冷后繼續(xù)浸泡36~48小時,使其充分吸水膨脹。浸種后用紗布包好,放置于30℃左右的保溫環(huán)境下催芽。將出芽后的種子直接播于營養(yǎng)缽內(nèi)進(jìn)行護(hù)根育苗。苗齡30~40天,2~3片真葉時移植。

    To further explore the intracellular signaling mechanisms underlying Rg1-promoted neurite outgrowth, MAPK and Akt signaling pathway inhibitors were applied. Incubation with Rg1 plus the MEK inhibitor PD98059 (10 μM) resulted in malnourished cells with small cell bodies and rough membrane surfaces compared with Rg1 alone (Figure 1A; Rg1 + PD98059). Rg1 with the Akt inhibitor API-2 (10 μM) resulted in fewer viable cells, with shorter and fewer neurites, than Rg1 alone (Figure 1A; Rg1+API-2). In contrast, addition of different concentrations of SP600125 (p38 MAPK inhibitor) or SB203580 (JNK inhibitor) to Rg1-treated cells did not affect the survival or morphology of the cells compared with Rg1 alone. Rg1-induced neurite outgrowth was significantly reversed by PD98059 and API-2, but was not affected by SP600125 or SB203580. These results indicate that the ERK1/2 and Akt signaling pathways, but not the JNK or p38 MAPK pathways, are involved in Rg1-mediated neurotrophic effects in cultured hippocampal neurons.

    Rg1 activated ERK1/2 and Akt phosphorylation in cultured hippocampal neurons

    Phosphorylation levels of ERK1/2 and Akt were determined by western blot analysis to further investigate the role of ERK1/2 and Akt signaling in Rg1-mediated neurotrophic effects. Cells treated with Rg1 for 24 hours showed significantly greater phosphorylation of Akt (Figure 2A, C) and ERK1/2 (Figure 2B, D) than untreated cells. These effects were blocked by PD98059 and API-2. Together with the effects of PD98059 and API-2 described above (Figure 1A-D), these results strongly indicate that ERK1/2 and Akt signaling is involved in Rg1-induced hippocampal neurite outgrowth.

    Rg1 protected against Aβ25-35-induced neurotoxicity, and was inhibited by PD98059 and API-2, but not SP600125 or SB203580

    After 24 hours in culture, cells were exposed to Aβ25-35, followed by Rg1 in the presence or absence of Akt, ERK, JNK and p38 MAPK inhibitors for a further 48 hours. In untreatedcontrol cells, long and numerous neurites were observed under an inverted phase contrast microscope. Conversely, cells exposed to Aβ25-35for 48 hours were fewer in number and showed fragmented and floating cell debris. Hoechst 33258 staining was used to classify viable and apoptotic cells. Viable cells were identified by regular, round nuclei with pale blue fluorescence, and apoptotic cells were condensed and fragmented (Figure 3A). Aβ25-35-exposed cells showed a significantly greater degree of apoptosis than untreated cells. The positive control, IGF-1, significantly reduced Aβ25-35-induced apoptosis (Figure 3B), as did post-treatment with Rg1, and this effect was blocked by co-incubation with API-2 and with PD98059 (Figure 3B). In contrast, SP600125 and SB203580 did not significantly alter the anti-apoptotic effect of Rg1 (Figure 3C, D). These results indicate that Rg1 treatment after Aβ25-35exposure prevents apoptosis in hippocampal neurons, and the underlying mechanisms may involve the Akt and ERK1/2 pathways, but not JNK or p38 MAPK.

    Rg1 treatment reversed Aβ25-35-induced reduction of ERK1/2 and Akt phosphorylation

    To further investigate the possibility that the Akt and ERK1/2 signaling pathways are involved in the neuroprotective effects of Rg1 in hippocampal neurons after Aβ25-35exposure, phosphorylation levels of Akt and ERK1/2 were measured by western blot analysis (Figure 4). Aβ25-35exposure for 48 hours significantly inhibited phosphorylation of Akt and ERK1/2 compared with that observed in untreated control cells. IGF-1 significantly reversed Aβ25-35-induced reduction of Akt and ERK1/2 phosphorylation to levels observed in untreated cells, and cells treated with Rg1 also showed greater levels of Akt and ERK1/2 phosphorylation after Aβ25-35exposure than cells exposed to Aβ25-35alone. In contrast, API-2 blocked Rg1-induced Akt and ERK1/2 phosphorylation. These results strongly suggest that Aβ25-35exposure inhibits phosphorylation of Akt and ERK1/2 in hippocampal neurons, resulting in neurotoxicity, and the protective effects of Rg1 against this neurotoxicity may be mediated by the ERK1/2 and Akt signaling pathways.

    Discussion

    In the present study, we have demonstrated that Rg1 promotes neurite outgrowth in cultured hippocampal neurons and protects against Aβ25-35-induced damage, and that these effects are mediated by the Akt and ERK1/2 signaling pathways.

    When we exposed cells to API-2 with Rg1, a large amount of degeneration was observed, and the cells remaining were smaller and had shorter neurites than those incubated with Rg1 alone. However, compared with another active ingredient of ginseng, Rb1, which, when combined with API-2, resulted in a large number of fragmented cells, Rg1 may provide a greater level of resistance against Akt signaling inhibition. These results are in agreement with the widely accepted view that Akt signaling is related to neuronal survival (Kennedy et al., 1999; Brunet et al., 2001). In contrast, PD98059 blocked Rg1-induced neurite outgrowth, but did not significantly affect the number of cells in the medium compared to Rg1 alone. Interestingly, neither SP600125 nor SB203580 affected neurite outgrowth. These results suggest that signaling by ERK1/2, but not p38 MAPK or JNK, is involved in Rg1-induced neurite outgrowth. In addition, western blot analysis further demonstrated that Rg1 treatment upregulated phosphorylation of ERK1/2 and Akt compared to that in normal untreated cells, and these effects were reversed by PD98059 and API-2. Taken together, these results confirm that Akt and ERK1/2 signaling may be involved in Rg1-induced neurite outgrowth in cultured hippocampal neurons. However, how Rg1 affects Akt and ERK1/2 signaling is uncertain. Rg1 is a functional ligand of the glucocorticoid receptor, and exerts steroid hormone-like activity (Lee et al., 1997). Leung et al. (2007) reported that the neuroprotective effects of Rg1 on primary nigral neurons against rotenone toxicity could be abolished by RU486, an antagonist at both the glucocorticoid (GR) and progesterone receptors, and suggested that Rg1 exerted neuroprotective effects via a GR-dependent mechanism. Recently, Rg1 has been reported to promote non-amyloidogenic cleavage of APP via estrogen receptor signaling and to ameliorate Aβ25-35-induced cortical neuronal apoptosis at least in part by two complementary estrogen receptor α- and GR-dependent downstream pathways (Shi et al., 2012; Wu et al., 2012). Further investigations are necessary to determine whether Rg1 crosses the cellular membrane and binds to cytoplasmic molecules, or acts on membrane receptors that trigger a signaling cascade.

    Previous reports have shown that Rg1 pretreatment improves the viability of cells injured by Aβ, reduces the levels of intracellular Aβ1-42, and attenuates the activation of caspase-3 and apoptosis in vitro (Ji et al., 2006; Wei et al., 2008; Choi et al., 2010). Those studies focused mainly on Rg1 pretreatment and used cortical neurons, suggesting its potential role for preventing AD. However, post-treatment is more clinically relevant, and the hippocampus is a key structure for learning and memory. It is also the site where senile plaques are formed in AD. Here, we have demonstrated that Aβ25-35exposure for 48 hours induces apoptosis of hippocampal neurons, and that Rg1 post-treatment for 48 hours significantly reverses this. In vivo studies have also confirmed that administration of ginsenosides including Rg1 can reduce cerebral Aβ generation in transgenic AD mice (Chen et al., 2006) as well as aging mice (Shi et al., 2010). Taken together, these data highlight the potential of Rg1 as a new therapeutic drug for AD. Our results show that the neuroprotective effect of Rg1 against Aβ25-35insult is blocked by API-2 and PD98058, but not by SP600125 or SB203580, suggesting that signaling involving ERK1/2 andAkt, but not JNK or p38 MAPK, may be involved in the action of Rg1. It was also previously demonstrated that no significant change in p38 or JNK phosphorylation was observed after Aβ25-35exposure (Wu et al., 2012). These results imply that the Akt and ERK1/2 signaling pathways are involved in the neuroprotective effect of Rg1 against damage induced by Aβ25-35. Additionally, western blot analysis of ERK1/2 and Akt phosphorylation showed that Rg1 post-treatment reversed the Aβ25-35-induced inactivation of ERK1/2, supporting previous studies. Wu et al. (2012) found that U0126, another specific inhibitor of MEK, inhibited Rg1-induced Erk phosphorylation. In addition, we confirmed that Rg1 post-treatment also reversed inactivation of Akt induced by Aβ25-35, and that API-2 counteracted this effect. Together, these results provide evidence that the mechanisms underlying the neuroprotective effect of Rg1 against Aβ25-35insult involve Akt and ERK signaling.

    In conclusion, the results from the present study demonstrate that Rg1 induces neurite outgrowth in cultured hippocampal neurons through Akt and ERK signaling. Moreover, Rg1 post-treatment prevents Aβ25-35-induced apoptosis via Akt and ERK signaling. Rg1 activation of Akt and ERK signaling needs to be explored further. These observations suggest that ginsenoside has potential as a treatment for progressive neurodegenerative diseases such as AD.

    Acknowledgments: We thank Ryan Splittgerbe (Ph.D., University of Nebraska Medical Center, USA) for corrections of the paper.

    Author contributions: LH, QLY and XQL designed the study. LH, SA, JL and LD performed experiments. SA, JL, LD, LFL and GYW performed the statistical analysis. QLY and LH wrote the paper. All authors approved the final version of the paper. Conflicts of interest: None declared.

    Plagiarism check: This paper was screened twice using Cross-Check to verify originality before publication.

    Peer review: This paper was double-blinded and stringently reviewed by international expert reviewers.

    Banker GA, Cowan WM (1977) Rat hippocampal neurons in dispersed cell culture. Brain Res 126:397-342.

    Brunet A, Datta SR, Greenberg ME (2001) Transcription-dependent and -independent control of neuronal survival by the PI3K-Akt signaling pathway. Curr Opin Neurobiol 11:297-305.

    Chen F, Eckman EA, Eckman CB (2006) Reductions in levels of the Alzheimer’s amyloid beta peptide after oral administration of ginsenosides. FASEB J 20:1269-1271.

    Cheng Y, Shen LH, Zhang JT (2005) Anti-amnestic and anti-aging effects of ginsenoside Rg1 and Rb1 and its mechanism of action. Acta Pharmacol Sin 26:143-149.

    Choi RC, Zhu JT, Leung KW, Chu GK, Xie HQ, Chen VP, Zheng KY, Lau DT, Dong TT, Chow PC, Han YF, Wang ZT, Tsim KW (2010) A flavonol glycoside, isolated from roots of panax notoginseng, reduces amyloid-beta-induced neurotoxicity in cultured neurons: signaling transduction and drug development for Alzheimer’s disease. J Alzheimers Dis 19:795-811.

    Gong L, Li SL, Li H, Zhang L (2011) Ginsenoside Rg1 protects primary cultured rat hippocampal neurons from cell apoptosis induced by β-amyloid protein. Pharm Biol 49:501-507.

    Iverson LL, Mortishire-Smith RJ, Pollack SJ, Shearman MS (1995) The toxicity in vitro of beta-amyloid protein. Biochem J 311:1-16.

    Ji ZN, Dong TT, Ye WC, Choi RC, Lo CK, Tsim KW (2006) Ginsenoside Re attenuate beta-amyloid and serum-free induced neurotoxicity in PC12 cells. J Ethnopharmacol 107:48-52.

    Kennedy SG, Kandel ES, Cross TK, Hay N (1999) Akt/protein kinase B inhibits cell death by preventing the release of cytochrome c from mitochondria. Mol Cell Biol 19:5800-5810.

    Lee YJ, Chung E, Lee KY, Lee YH, Huh B, Lee SK (1997) Ginsenoside-Rg1, one of the major active molecules from Panax ginseng, is a functional ligand of glucocorticoid receptor. Mol Cell Endocrinol 133:135-140.

    Leung KW, Yung KK, Mak NK, Chan YS, Fan TP, Wong RN (2007) Neuroprotective effects of ginsenoside-Rg1 in primary nigral neurons against rotenone toxicity. Neuropharmacology 52:827-835.

    Mielke K, Herdegen T (2000) JNK and p38 stresskinases--degenerative effectors of signal-transduction-cascades in the nervous system. Prog Neurobiol 61:45-60.

    Mitchell PJ, Hanson JC, Quets-Nguyen AT, Bergeron M, Smith RC (2007) A quantitative method for analysis of in vitro neurite outgrowth. J Neurosci Methods 164:350-360

    Mook-Jung I, Hong H, Boo JH, Lee KH, Yun SH, Cheong MY, Joo I, Huh K, Jung MW (2001) Ginsenoside Rb1 and Rg1 improve spatial learning and increase hippocampal synaptophysin level in mice. J Neurosci Res 63:509-515.

    Radad K, Gille G, Moldzio R, Saito H, Rausch WD (2004a) Ginsenosides Rb1 and Rg1 effects on mesencephalic dopaminergic cells stressed with glutamate. Brain Res 1021:41-53.

    Radad K, Gille G, Moldzio R, Saito H, Ishige K, Rausch WD (2004b) Ginsenosides Rb1 and Rg1 effects on survival and neurite growth of MPP+-affected mesencephalic dopaminergic cells. J Neural Transm 111:37-45.

    Rausch WD, Liu S, Gille G, Radad K (2006) Neuroprotective effects of ginsenosides. Acta Neurobiol Exp (Wars) 66:369-375.

    Roux PP, Blenis J (2004) ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol Mol Biol Rev 68:320-344.

    Rudakewich M, Ba F, Benishin CG (2001) Neurotrophic and neuroprotective actions of ginsenosides Rb1 and Rg1. Planta Med 67:533-537.

    Shi C, Zheng DD, Fang L, Wu F, Kwong WH, Xu J (2012) Ginsenoside Rg1 promotes nonamyloidgenic cleavage of APP via estrogen receptor signaling to MAPK/ERK and PI3K/Akt. Biochim Biophys Acta 1820:453-460.

    Shi YQ, Huang TW, Chen LM, Pan XD, Zhang J, Zhu YG, Chen XC (2010) Ginsenoside Rg1 attenuates amyloid-beta content, regulates PKA/CREB activity, and improves cognitive performance in SAMP8 mice. J Alzheimers Dis 19:977-989.

    Sweatt JD (2004) Mitogen-activated protein kinases in synaptic plasticity and memory. Curr Opin Neurobiol 14:311e317.

    Wang YH, Du GH (2009) Ginsenoside Rg1 inhibits beta-secretase activity in vitro and protects against Abeta-induced cytotoxicity in PC12 cells. J Asian Nat Prod Res 11:604-612.

    Wei C, Jia J, Liang P, Guan Y (2008) Ginsenoside Rg1 attenuates beta-amyloid-induced apoptosis in mutant PS1 M146L cells. Neurosci Lett 443:145-149.

    Wu J, Pan Z, Wang Z, Zhu W, Shen Y, Cui R, Lin J, Yu H, Wang Q, Qian J, Yu Y, Zhu D, Lou Y (2012) Ginsenoside Rg1 protection against β-amyloid peptide-induced neuronal apoptosis via estrogen receptor α and glucocorticoid receptor-dependent anti-protein nitration pathway. Neuropharmacology 63:349-361.

    Copyedited by Slone-Murphy J, Robens J, Li CH, Song LP, Zhao M

    10.4103/1673-5374.177741 http://www.nrronline.org/

    How to cite this article: Huang L, Liu LF, Liu J, Dou L, Wang GY, Liu XQ, Yuan QL (2016) Ginsenoside Rg1 protects against neurodegeneration by inducing neurite outgrowth in cultured hippocampal neurons. Neural Regen Res 11(2):319-325.

    Funding: This study was financially supported by the National Program on Key Basic Research Project of China (973 Program), No. 2010CB945600, 2011CB965100; the National Natural Science Foundation of China, No. 81070987, 30971531, 81371213; and a grant from the International Science & Technology Collaboration Program, No. 2011DF30010.

    Accepted: 2015-03-10

    *Correspondence to: Qiong-lan Yuan, Ph.D., yqiongl@#edu.cn.

    猜你喜歡
    苗齡北方地區(qū)種皮
    東北地理所發(fā)現(xiàn)PG 031基因具有改良大豆種皮吸水性的應(yīng)用潛力
    北方地區(qū)湖泊河蟹養(yǎng)殖技術(shù)探討
    不同苗齡大花序桉造林效果分析
    不同接穗苗齡對小果型西瓜嫁接育苗效果的影響
    北方地區(qū)大規(guī)格梅花觀賞樹的嫁接和接后管理
    閩產(chǎn)薏苡種皮油的提取工藝研究
    苗齡、光照強度和施氮量對水稻營養(yǎng)物質(zhì)的影響及其與抗褐飛虱的關(guān)系
    應(yīng)用種皮葉綠素?zé)晒庵甘緹煵莘N子成熟度的研究
    北方地區(qū)羔羊傳染性膿皰病的防治措施
    北方地區(qū)養(yǎng)殖魚類肝膽綜合癥成因及治療
    狠狠精品人妻久久久久久综合| 日本av手机在线免费观看| 日韩av在线免费看完整版不卡| 飞空精品影院首页| 欧美 亚洲 国产 日韩一| 男男h啪啪无遮挡| 中文字幕人妻丝袜制服| 天堂8中文在线网| av免费观看日本| 老司机影院成人| 九色亚洲精品在线播放| 女的被弄到高潮叫床怎么办| 成人免费观看视频高清| 秋霞伦理黄片| 午夜免费观看性视频| 欧美在线黄色| av在线老鸭窝| 老熟女久久久| 18禁动态无遮挡网站| 久久久精品区二区三区| 久久久久国产精品人妻一区二区| 曰老女人黄片| 一边摸一边抽搐一进一出视频| 热99国产精品久久久久久7| 五月天丁香电影| 在线 av 中文字幕| 最近的中文字幕免费完整| 黄网站色视频无遮挡免费观看| 丰满饥渴人妻一区二区三| 国产无遮挡羞羞视频在线观看| 免费黄色在线免费观看| 亚洲熟女毛片儿| 老司机靠b影院| 亚洲欧美激情在线| 亚洲精品第二区| 丝袜在线中文字幕| 亚洲国产成人一精品久久久| 成年av动漫网址| 亚洲成人免费av在线播放| 一个人免费看片子| 日韩精品免费视频一区二区三区| h视频一区二区三区| 蜜桃在线观看..| 欧美日本中文国产一区发布| 五月开心婷婷网| www.av在线官网国产| 国产成人精品在线电影| 精品国产露脸久久av麻豆| www.av在线官网国产| 国产熟女欧美一区二区| 亚洲国产精品一区二区三区在线| 亚洲欧美一区二区三区黑人| 亚洲av欧美aⅴ国产| 如日韩欧美国产精品一区二区三区| 亚洲天堂av无毛| 日韩不卡一区二区三区视频在线| 亚洲av福利一区| 中文字幕色久视频| 久久久国产一区二区| 综合色丁香网| 亚洲婷婷狠狠爱综合网| 亚洲国产最新在线播放| 女性被躁到高潮视频| 成年人午夜在线观看视频| 久久久久国产精品人妻一区二区| 妹子高潮喷水视频| 黑人巨大精品欧美一区二区蜜桃| tube8黄色片| 久久av网站| 美女高潮到喷水免费观看| 亚洲欧洲日产国产| 国产高清国产精品国产三级| 黄色怎么调成土黄色| 一本久久精品| 侵犯人妻中文字幕一二三四区| 婷婷色av中文字幕| 精品亚洲成国产av| kizo精华| 亚洲人成77777在线视频| 欧美精品一区二区大全| 亚洲色图 男人天堂 中文字幕| 国产成人免费观看mmmm| 综合色丁香网| 一本大道久久a久久精品| 精品少妇一区二区三区视频日本电影 | 99热全是精品| 熟妇人妻不卡中文字幕| avwww免费| 亚洲国产日韩一区二区| 肉色欧美久久久久久久蜜桃| 免费女性裸体啪啪无遮挡网站| 久久免费观看电影| 亚洲国产av影院在线观看| 又粗又硬又长又爽又黄的视频| 成人影院久久| 亚洲精品av麻豆狂野| 不卡av一区二区三区| 精品国产超薄肉色丝袜足j| 久久精品国产a三级三级三级| 男人添女人高潮全过程视频| 啦啦啦在线观看免费高清www| 高清欧美精品videossex| a 毛片基地| 国产欧美日韩一区二区三区在线| 国产色婷婷99| 捣出白浆h1v1| av网站免费在线观看视频| bbb黄色大片| 国产精品欧美亚洲77777| 丁香六月欧美| 2021少妇久久久久久久久久久| 国产精品一二三区在线看| 天天躁狠狠躁夜夜躁狠狠躁| 热99久久久久精品小说推荐| 国产免费现黄频在线看| 久久天躁狠狠躁夜夜2o2o | 午夜老司机福利片| 久久久久国产一级毛片高清牌| 国产片内射在线| 99九九在线精品视频| 欧美精品亚洲一区二区| 熟妇人妻不卡中文字幕| 国产伦理片在线播放av一区| 久久国产精品大桥未久av| 久久免费观看电影| 免费在线观看黄色视频的| 午夜久久久在线观看| 亚洲色图综合在线观看| 亚洲精品国产av成人精品| 一边摸一边做爽爽视频免费| 亚洲国产精品国产精品| 1024香蕉在线观看| 久久精品国产亚洲av高清一级| 波多野结衣一区麻豆| av网站在线播放免费| 国产成人免费无遮挡视频| 777久久人妻少妇嫩草av网站| 久久久久久人人人人人| 久久精品aⅴ一区二区三区四区| 女人久久www免费人成看片| av网站免费在线观看视频| 99久久精品国产亚洲精品| 精品少妇久久久久久888优播| 免费在线观看视频国产中文字幕亚洲 | 国产精品.久久久| 国产精品 国内视频| 99久久精品国产亚洲精品| 午夜免费鲁丝| 国产精品免费视频内射| 久久精品国产a三级三级三级| 久久久欧美国产精品| 51午夜福利影视在线观看| 这个男人来自地球电影免费观看 | 亚洲国产毛片av蜜桃av| 岛国毛片在线播放| 国产精品成人在线| 欧美国产精品一级二级三级| 大片免费播放器 马上看| 国产亚洲一区二区精品| 欧美97在线视频| 午夜福利在线免费观看网站| 在线观看www视频免费| 国产精品女同一区二区软件| 嫩草影院入口| 亚洲熟女毛片儿| 日本欧美视频一区| 精品人妻熟女毛片av久久网站| 免费观看av网站的网址| 日日撸夜夜添| 精品少妇久久久久久888优播| 欧美日韩av久久| 免费少妇av软件| 欧美激情极品国产一区二区三区| 欧美精品人与动牲交sv欧美| 日本vs欧美在线观看视频| 精品人妻在线不人妻| 精品福利永久在线观看| 精品人妻熟女毛片av久久网站| 18禁观看日本| 午夜精品国产一区二区电影| 日韩欧美一区视频在线观看| 美女午夜性视频免费| 免费看不卡的av| 精品国产露脸久久av麻豆| 免费在线观看黄色视频的| 中国三级夫妇交换| 欧美日韩亚洲国产一区二区在线观看 | 国产成人欧美| 国产精品熟女久久久久浪| 天堂8中文在线网| netflix在线观看网站| 国产成人啪精品午夜网站| 色94色欧美一区二区| 高清视频免费观看一区二区| 精品酒店卫生间| www.av在线官网国产| 精品少妇黑人巨大在线播放| 欧美激情极品国产一区二区三区| 国产精品二区激情视频| 欧美黑人精品巨大| 80岁老熟妇乱子伦牲交| 国产探花极品一区二区| 两个人免费观看高清视频| 欧美黑人欧美精品刺激| 国产一区二区三区综合在线观看| 欧美97在线视频| 中文字幕人妻丝袜一区二区 | 看免费av毛片| 黑人欧美特级aaaaaa片| 日韩欧美精品免费久久| 亚洲欧美清纯卡通| 一级毛片我不卡| 国产精品嫩草影院av在线观看| 亚洲精品aⅴ在线观看| 国产日韩一区二区三区精品不卡| 欧美中文综合在线视频| 大片电影免费在线观看免费| 久久青草综合色| 水蜜桃什么品种好| 性色av一级| 老司机靠b影院| 丁香六月天网| 免费黄频网站在线观看国产| 日日啪夜夜爽| 日日啪夜夜爽| 少妇被粗大的猛进出69影院| 久久av网站| av视频免费观看在线观看| 日韩熟女老妇一区二区性免费视频| 国产不卡av网站在线观看| 日本午夜av视频| 精品久久久精品久久久| 欧美黑人欧美精品刺激| 免费久久久久久久精品成人欧美视频| 18禁观看日本| 在线观看免费日韩欧美大片| 不卡av一区二区三区| 亚洲成人国产一区在线观看 | 国产精品人妻久久久影院| 欧美成人午夜精品| 黄频高清免费视频| 高清不卡的av网站| 考比视频在线观看| 色吧在线观看| 国产成人欧美| 麻豆av在线久日| 久久韩国三级中文字幕| 国产成人午夜福利电影在线观看| 十分钟在线观看高清视频www| 国产 精品1| 国产在线免费精品| 精品第一国产精品| 美女中出高潮动态图| 制服诱惑二区| 水蜜桃什么品种好| 亚洲第一区二区三区不卡| 亚洲情色 制服丝袜| 九九爱精品视频在线观看| 国产成人精品在线电影| 大香蕉久久成人网| av在线老鸭窝| av在线播放精品| 亚洲成人手机| 国产精品女同一区二区软件| 日日摸夜夜添夜夜爱| 高清欧美精品videossex| av女优亚洲男人天堂| 美女视频免费永久观看网站| 国产一区有黄有色的免费视频| 国产在线视频一区二区| 十八禁人妻一区二区| 久久久久视频综合| 精品卡一卡二卡四卡免费| av电影中文网址| 女的被弄到高潮叫床怎么办| 国产免费福利视频在线观看| 大片免费播放器 马上看| 国产精品国产av在线观看| 十分钟在线观看高清视频www| 麻豆精品久久久久久蜜桃| 成人亚洲欧美一区二区av| 国产日韩一区二区三区精品不卡| 久久精品aⅴ一区二区三区四区| 又大又黄又爽视频免费| 老汉色∧v一级毛片| 一级片免费观看大全| 亚洲成人手机| 大香蕉久久网| 久久久久久久久免费视频了| 80岁老熟妇乱子伦牲交| 欧美日韩亚洲国产一区二区在线观看 | 久久人人爽av亚洲精品天堂| 日韩一区二区视频免费看| 少妇人妻 视频| 久久久精品区二区三区| 麻豆av在线久日| 亚洲四区av| 岛国毛片在线播放| 国产一区二区 视频在线| 亚洲国产看品久久| 亚洲国产av新网站| 18禁观看日本| 中文字幕人妻熟女乱码| 天堂俺去俺来也www色官网| 亚洲七黄色美女视频| 亚洲精品久久午夜乱码| 欧美老熟妇乱子伦牲交| 多毛熟女@视频| 新久久久久国产一级毛片| 观看av在线不卡| 亚洲熟女毛片儿| 亚洲七黄色美女视频| 精品卡一卡二卡四卡免费| 久久精品国产亚洲av涩爱| 午夜久久久在线观看| 免费黄频网站在线观看国产| 99热网站在线观看| 九九爱精品视频在线观看| 日本av免费视频播放| 欧美日韩精品网址| 大话2 男鬼变身卡| 又黄又粗又硬又大视频| 你懂的网址亚洲精品在线观看| 最近中文字幕高清免费大全6| 久热这里只有精品99| 色吧在线观看| 无遮挡黄片免费观看| 久久99一区二区三区| 大片电影免费在线观看免费| bbb黄色大片| 男女边吃奶边做爰视频| 美女国产高潮福利片在线看| 天天影视国产精品| 啦啦啦 在线观看视频| 亚洲免费av在线视频| 青春草国产在线视频| 狠狠婷婷综合久久久久久88av| 亚洲精品国产色婷婷电影| 国产欧美日韩一区二区三区在线| 久久精品国产亚洲av高清一级| 国产日韩一区二区三区精品不卡| 亚洲美女搞黄在线观看| 欧美xxⅹ黑人| 成人18禁高潮啪啪吃奶动态图| 国产爽快片一区二区三区| 男的添女的下面高潮视频| 久久97久久精品| 亚洲精品久久成人aⅴ小说| 男人操女人黄网站| 女性被躁到高潮视频| 熟女av电影| 成人国语在线视频| 一边摸一边抽搐一进一出视频| 肉色欧美久久久久久久蜜桃| 十分钟在线观看高清视频www| 日韩免费高清中文字幕av| 亚洲精品国产av蜜桃| 国产在线免费精品| 18禁裸乳无遮挡动漫免费视频| av电影中文网址| 日本91视频免费播放| 日韩人妻精品一区2区三区| 国产亚洲午夜精品一区二区久久| 日韩一区二区三区影片| 国产一卡二卡三卡精品 | 欧美精品一区二区大全| 国产亚洲最大av| 日韩一区二区三区影片| 亚洲精华国产精华液的使用体验| 国产精品偷伦视频观看了| av国产精品久久久久影院| 99久久人妻综合| 两性夫妻黄色片| 国产亚洲av高清不卡| 纵有疾风起免费观看全集完整版| av女优亚洲男人天堂| 午夜福利网站1000一区二区三区| 曰老女人黄片| 欧美最新免费一区二区三区| 国产一区二区在线观看av| 18在线观看网站| 国产成人啪精品午夜网站| 亚洲av综合色区一区| 一级毛片我不卡| 亚洲色图综合在线观看| 久久久久久人妻| 国产熟女欧美一区二区| 日韩大片免费观看网站| 亚洲国产精品成人久久小说| 国产免费现黄频在线看| 日韩精品有码人妻一区| 高清欧美精品videossex| 尾随美女入室| av免费观看日本| 久久 成人 亚洲| 极品少妇高潮喷水抽搐| 精品亚洲成国产av| 午夜精品国产一区二区电影| 老司机影院毛片| 多毛熟女@视频| 国产深夜福利视频在线观看| 天天操日日干夜夜撸| 国精品久久久久久国模美| 国产免费又黄又爽又色| 香蕉国产在线看| 我要看黄色一级片免费的| 赤兔流量卡办理| 人人妻人人澡人人看| 婷婷色综合www| 午夜免费男女啪啪视频观看| 国产一区二区在线观看av| 美女国产高潮福利片在线看| 欧美日本中文国产一区发布| 777米奇影视久久| 中文字幕亚洲精品专区| 欧美精品人与动牲交sv欧美| av卡一久久| 欧美黑人精品巨大| 国产成人免费观看mmmm| 欧美亚洲日本最大视频资源| 亚洲一码二码三码区别大吗| 少妇猛男粗大的猛烈进出视频| 久久久精品免费免费高清| 日本vs欧美在线观看视频| 久久久久久免费高清国产稀缺| 国产女主播在线喷水免费视频网站| 美女福利国产在线| 一本一本久久a久久精品综合妖精| 午夜激情久久久久久久| 十八禁高潮呻吟视频| 黄片无遮挡物在线观看| 晚上一个人看的免费电影| av福利片在线| 伊人久久国产一区二区| 日韩中文字幕视频在线看片| 久久毛片免费看一区二区三区| 久久久久人妻精品一区果冻| 国产成人精品福利久久| 午夜91福利影院| 欧美激情高清一区二区三区 | 成人免费观看视频高清| 一级毛片我不卡| 男女免费视频国产| 久热爱精品视频在线9| 18在线观看网站| 丝袜在线中文字幕| 国产成人免费观看mmmm| 秋霞伦理黄片| 久久综合国产亚洲精品| 国产精品一区二区在线观看99| 中文字幕精品免费在线观看视频| 中文天堂在线官网| 亚洲三区欧美一区| 欧美日韩一级在线毛片| 高清在线视频一区二区三区| 亚洲av福利一区| 免费看不卡的av| av在线播放精品| 欧美日韩综合久久久久久| 欧美 亚洲 国产 日韩一| 日韩人妻精品一区2区三区| 亚洲精品在线美女| 成人国产麻豆网| 国产成人系列免费观看| 天天躁夜夜躁狠狠躁躁| 免费不卡黄色视频| 美女午夜性视频免费| av天堂久久9| 亚洲久久久国产精品| xxxhd国产人妻xxx| 成人亚洲欧美一区二区av| 国产老妇伦熟女老妇高清| 中文字幕人妻丝袜制服| 王馨瑶露胸无遮挡在线观看| 亚洲成色77777| 女性被躁到高潮视频| 超色免费av| 熟妇人妻不卡中文字幕| av又黄又爽大尺度在线免费看| 国产成人午夜福利电影在线观看| 在线观看免费高清a一片| 欧美日韩亚洲综合一区二区三区_| 亚洲精品日本国产第一区| 国产黄色免费在线视频| 中文字幕亚洲精品专区| 国产男人的电影天堂91| 男女床上黄色一级片免费看| 一级毛片我不卡| 国产精品 国内视频| 老司机靠b影院| 成人国产av品久久久| 视频区图区小说| 亚洲成人免费av在线播放| 国产男女内射视频| 久久鲁丝午夜福利片| 人妻人人澡人人爽人人| 日韩大码丰满熟妇| 久久久久久人妻| 欧美日韩亚洲国产一区二区在线观看 | 下体分泌物呈黄色| 69精品国产乱码久久久| 日本欧美视频一区| 中国三级夫妇交换| 成年动漫av网址| xxxhd国产人妻xxx| 亚洲欧美一区二区三区黑人| 最近的中文字幕免费完整| 亚洲熟女精品中文字幕| 午夜影院在线不卡| 丰满迷人的少妇在线观看| 黄色一级大片看看| 曰老女人黄片| 日韩伦理黄色片| 国产日韩欧美在线精品| 久久ye,这里只有精品| 久久精品人人爽人人爽视色| 久久久久网色| 男人操女人黄网站| 日本欧美视频一区| 久久久久久久精品精品| 中文字幕人妻丝袜制服| 在线观看www视频免费| 国产精品秋霞免费鲁丝片| 午夜免费观看性视频| 日本爱情动作片www.在线观看| 丝袜美腿诱惑在线| 少妇人妻精品综合一区二区| 成人国产麻豆网| 日韩一区二区视频免费看| 日日爽夜夜爽网站| 欧美精品人与动牲交sv欧美| 天天躁夜夜躁狠狠躁躁| 国产精品欧美亚洲77777| 久久鲁丝午夜福利片| 五月天丁香电影| 免费观看a级毛片全部| 一个人免费看片子| 国产成人av激情在线播放| 国产av精品麻豆| 久久久久国产精品人妻一区二区| 亚洲国产av新网站| 亚洲欧美成人精品一区二区| 人妻人人澡人人爽人人| 国产黄频视频在线观看| 国产一区有黄有色的免费视频| 久久久久久久久免费视频了| 男女之事视频高清在线观看 | 国产深夜福利视频在线观看| 男女国产视频网站| 看免费成人av毛片| 午夜激情av网站| 久久久精品国产亚洲av高清涩受| 日韩一区二区视频免费看| 久久韩国三级中文字幕| 亚洲一区二区三区欧美精品| 亚洲图色成人| 国产精品人妻久久久影院| 在线观看免费视频网站a站| 国产视频首页在线观看| 国产免费视频播放在线视频| 考比视频在线观看| 中国国产av一级| 亚洲成人国产一区在线观看 | 女人精品久久久久毛片| 国产精品久久久av美女十八| 中文字幕色久视频| 国产一区有黄有色的免费视频| 色综合欧美亚洲国产小说| h视频一区二区三区| 日韩av免费高清视频| 一区福利在线观看| 成人影院久久| 国产成人一区二区在线| 啦啦啦视频在线资源免费观看| 亚洲成色77777| 午夜福利影视在线免费观看| 国产成人精品无人区| 精品少妇一区二区三区视频日本电影 | 人成视频在线观看免费观看| a 毛片基地| 这个男人来自地球电影免费观看 | 91国产中文字幕| 美女视频免费永久观看网站| 日韩制服骚丝袜av| 日本91视频免费播放| 国产亚洲午夜精品一区二区久久| 人妻一区二区av| 亚洲精品av麻豆狂野| 香蕉丝袜av| 丝袜脚勾引网站| 在线观看免费日韩欧美大片| 成年av动漫网址| 中国三级夫妇交换| 国产精品国产av在线观看| 国产1区2区3区精品| 肉色欧美久久久久久久蜜桃| a 毛片基地| 精品国产一区二区久久| 国产伦理片在线播放av一区| 老司机亚洲免费影院| 人人妻人人澡人人看| 亚洲,欧美精品.| 超碰成人久久| 又大又黄又爽视频免费| 亚洲欧美成人精品一区二区| www日本在线高清视频| 一区二区av电影网| 国产一卡二卡三卡精品 | 亚洲人成77777在线视频| 国产精品国产三级国产专区5o| xxx大片免费视频| 国产一区二区激情短视频 | 成年人午夜在线观看视频| 亚洲国产av影院在线观看| a级片在线免费高清观看视频|