• <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 ?

    Methylprednisolone promotes recovery of neurological function after spinal cord injury: association with Wnt/β-catenin signaling pathway activation

    2016-02-09 05:17:18GongbiaoLuFuwenNiuYingchunZhangLinDuZhiyuanLiangYuanGaoTingzhenYanZhikuiNieKaiGaoDepartmentofOrthopedicsJiningNoPeopleHospitalJiningShandongProvinceChinaDepartmentofInterventionalRadiologyJiningNoPeopleHospitalJi

    Gong-biao Lu, Fu-wen Niu, Ying-chun Zhang, Lin Du, Zhi-yuan Liang, Yuan Gao, Ting-zhen Yan, Zhi-kui Nie,, Kai Gao, Department of Orthopedics, Jining No. People’s Hospital, Jining, Shandong Province, China Department of Interventional Radiology, Jining No. People’s Hospital, Jining, Shandong Province, China Jining Medical University, Jining, Shandong Province, China

    Methylprednisolone promotes recovery of neurological function after spinal cord injury: association with Wnt/β-catenin signaling pathway activation

    Gong-biao Lu1, Fu-wen Niu1, Ying-chun Zhang2, Lin Du3, Zhi-yuan Liang1, Yuan Gao1, Ting-zhen Yan1, Zhi-kui Nie1,*, Kai Gao1,*
    1 Department of Orthopedics, Jining No.1 People’s Hospital, Jining, Shandong Province, China
    2 Department of Interventional Radiology, Jining No.1 People’s Hospital, Jining, Shandong Province, China
    3 Jining Medical University, Jining, Shandong Province, China

    How to cite this article:Lu GB, Niu FW, Zhang YC, Du L, Liang ZY, Gao Y, Yan TZ, Nie ZK, Gao K (2016) Methylprednisolone promotes recovery of neurological function after spinal cord injury: association with Wnt/β-catenin signaling pathway activation. Neural Regen Res 11(11):1816-1823.

    Open access statement:This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

    Funding:This work was supported by the National Natural Science Foundation of China, No. 81471854.

    Graphical Abstract

    Some studies have indicated that the Wnt/β-catenin signaling pathway is activated following spinal cord injury, and expression levels of specific proteins, including low-density lipoprotein receptor related protein-6 phosphorylation, β-catenin, and glycogen synthase kinase-3β, are significantly altered. We hypothesized that methylprednisolone treatment contributes to functional recovery after spinal cord injury by inhibiting apoptosis and activating the Wnt/β-catenin signaling pathway. In the current study, 30 mg/kg methylprednisolone was injected into rats with spinal cord injury immediately post-injury and at 1 and 2 days post-injury. Basso, Beattie, and Bresnahan scores showed that methylprednisolone treatment significantly promoted locomotor functional recovery between 2 and 6 weeks post-injury. The number of surviving motor neurons increased, whereas the lesion size significantly decreased following methylprednisolone treatment at 7 days post-injury. Additionally, caspase-3, caspase-9, and Bax protein expression levels and the number of apoptotic cells were reduced at 3 and 7 days post-injury, while Bcl-2 levels at 7 days post-injury were higher in methylprednisolone-treated rats compared with saline-treated rats. At 3 and 7 days post-injury, methylprednisolone up-regulated expression and activation of the Wnt/β-catenin signaling pathway, including low-density lipoprotein receptor related protein-6 phosphorylation, β-catenin, and glycogen synthase kinase-3β phosphorylation. These results indicate that methylprednisolone-induced neuroprotection may correlate with activation of the Wnt/β-catenin signaling pathway.

    nerve regeneration; spinal cord injury; neuroprotection; methylprednisolone; apoptosis; locomotor function; caspase-3; caspase-9; Bax; Bcl-2; neural regeneration

    Introduction

    Spinal cord injury (SCI), including primary and secondary injury, is a worldwide medical problem (Wyndaele and Wyndaele, 2006). Secondary injury plays a critical role in SCI and is induced by various factors, such as apoptosis, oxidative stress, and the inflammatory immune response. In particular, neuronal apoptosis, which also affects microglia and oligodendrocytes and inhibits recovery of white matter and neurological functions, prevents functional recovery after SCI (Yuan and Yankner, 2000; Beattie et al., 2002). Secondary damage pathways offer potential therapeutic targets for treating SCI to enable greater functional recovery (Profyris et al., 2004; Zhang et al., 2015a). Methylprednisolone (MP), a synthetic glucocorticoid used to treat inflammatory diseases, is clinically utilized to treat SCI. Several studies have shown that MP inhibits inflammation, oxidative stress, and neuronal apoptosis, contributing to the neuroprotective effect on functional recovery after SCI (Vaquero et al., 2006; Gao et al., 2014; Gocmez et al., 2015). However, some reports have started to question the neuroprotective effects of MP after SCI in recent years, stating that MP failed to exert a neuroprotective effect and additionally increased complications in SCI, such as infection, pulmonary embolism, and even death (Hall and Springer, 2004; Pereira et al., 2009; Wilson and Fehlings, 2011; Fehlings et al., 2014; Hurlbert et al., 2015). Hence, further verification of the effect and investigation of the specific molecular mechanisms of MP is of utmost importance to justify continued clinical application of MP in SCI.

    The Wnt signaling pathways, particularly the canonical Wnt/β-catenin pathway, were first identified for their role in carcinogenesis. However, accumulating evidence has shown that Wnt/β-catenin signaling plays a critical role in embryonic development. Wnt/β-catenin has been implicated in neural development, axonal guidance, neuropathic pain remission, neuronal survival, and SCI (Zhang et al., 2013b; Ziaei et al., 2015). Furthermore, in some diseases, apoptosis is induced following inhibition of the Wnt/β-catenin signaling pathway (Hsu et al., 2014; Li et al., 2015). Apoptosis of neuronal cells is an important pathological feature of several central nervous system diseases, including neurodegeneration and SCI (Yuan and Yankner, 2000; Tang et al., 2014; Zhang et al., 2015b). Neuronal apoptosis is enhanced following SCI, which leads to suppression of functional recovery, thereby providing a potential therapeutic target for SCI.

    We hypothesized that the molecular mechanism underlying the inhibitory effect of MP on apoptosis and related restoration of neurological function after SCI occursviaregulation of the Wnt/β-catenin signaling pathway. Therefore, the goal of the present study was to further confirm the neuroprotective effect of MP after SCI by examining recovery of locomotor function and the injured spinal cord, neuronal survival in the spinal cord anterior horn, and anti-apoptotic activity, as well as to correlate MP activity with alterations in the Wnt/β-catenin signaling pathway, as a potential molecular mechanism.

    Materials and Methods

    Animals

    A total of 105 adult, male, 8–12-week-old, Sprague-Dawley rats, weighing 260–300 g, were purchased from the Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China, license No. SCXK (Jing) 2012-0001) and were raised in the specific-pathogen-free Laboratory Animal Center. The rats were maintained at 23.0 ± 0.5°C with an alternating 12-hour light/dark cycle. The rats were randomly divided into the following groups: MP (n= 35; SCI + MP), saline (n= 35; SCI + saline), and sham (n= 35).

    All procedures were performed in accordance with the United States National Institutes of Health Guide for the Care and Use of Laboratory Animal (NIH Publication No. 85-23, revised 1986). The protocols were approved by the Animal Ethics Committee of Jinzhou Medical University of China. All efforts to minimize the number of animals used and their suffering were made.

    Rat SCI model and MP administration

    The rat SCI model was established as previously described (Yacoub et al., 2014). Briefly, the rats were anesthetizedviaintraperitoneal injection of 10% chloral hydrate (0.33 mL/kg), and the T9–10of the spinal cord was exposed. An impounder (diameter: 2.0 mm; weight: 10 g) was dropped from a height of 25.0 mm above the spinal cord. Congestion in the injured spinal cord was immediately observed followed by rapid withdrawal of the hind limbs. Congestion at the injury site, rapid contraction, tremor of the lower limbs, and incontinence confirmed successful model establishment. Rats with unsuccessful SCI induction were not selected for further experimentation. After SCI, the surgical wounds were cleaned with warm saline and were sutured. The bladder was massaged three times daily to improve functional recovery of automatic micturition, and penicillin was administered for 3 consecutive days.

    MP (30 mg/kg) and saline (1 mL/kg) were injected intravenouslyviathe tail immediately post-SCI and 1 and 2 days post-SCI, and then once daily for two days. The sham group underwent laminectomy only.

    Figure 1 BBB locomotor rating scale scores in a rat model of SCI.

    Analysis of locomotor activity in the SCI rat model

    Figure 2 Nissl staining of neuronal pathology in the spinal cord anterior horn in a rat SCI model.

    Figure 3 Western blot assay of expression of the canonical Wnt/β-catenin signaling pathway in spinal cord tissue in a rat model of spinal cord injury (SCI).

    Figure 4 Western blot assay of expression of activated caspase-9 and caspase-3 as markers for apoptosis in spinal cord tissue in a SCI rat model.

    The Basso, Beattie, and Bresnahan (BBB) open-field locomotorrating scale was used to evaluate locomotor function prior to injury and recovery at 1 and 3 days , as well as at 1, 2, 3, 4, 5, and 6 weeks post-SCI (Basso et al., 1995). Briefly, the BBB scores ranged from 0 (complete paralysis) to 21 (unimpaired locomotion) and were assessed by three independent examiners in a blinded fashion.

    Figure 5 Apoptosis in the spinal cord anterior horn in a rat SCI model.

    Figure 6 Immunofluorescence of Bax and Bcl-2 in the spinal cord anterior horn in a rat SCI model.

    Tissue preparation

    At 7 days post-SCI, the rats were intraperitoneally anesthetized with 10% chloral hydrate (0.3 mL/kg), and then perfused with 0.9% saline and 4% paraformaldehyde (Xue et al., 2013; Zhang et al., 2013a). The T8–12spinal cord segments (including the epicenter) were removed and immersed in 4% paraformaldehyde for 3 days, and then dehydrated in 30% sucrose. Using a cryostat microtome (Leica CM3050S; Heidelberg, Germany), 5-μm-thick cross sections (3 mm rostral to the epicenter) were prepared for terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and immunofluorescence staining, and 20-μm-thick cross sections (3 mm rostral to the epicenter) were used for Nissl staining.

    Nissl staining of spinal cord tissue following SCI

    First, 20-μm-thick coronal sections (3 mm rostral to the epicenter) were placed in mixing solution (alcohol/chloro-form, 1:1) overnight at room temperature. The following day, sections were consecutively placed in 100% alcohol, 95% alcohol, 70% alcohol, and distilled water. Subsequently, the sections were stained in 0.05% cresyl violet (pH 3.0, Sigma-Aldrich, St. Louis, MO, USA) for 10 minutes at 40°C after which sections were differentiated in 95% alcohol, dehydrated in 100% alcohol, and cleared in xylene. The images were captured by a light microscope (Leica, Heidelberg, Germany). The large and Nissl-stained anterior horn cells in the spinal cord tissue were recognized as motor neurons. Five Nissl-stained sections in every experimental rat were randomly selected for evaluating the average number of surviving neurons in the spinal cord anterior horn. The total and residual white-matter areas were measured using a BZ-Analyzer (Keyence) to measure lesion size in the spinal cord tissue in all groups (Xue et al., 2013; Zhang et al., 2013a).

    Western blot assay of spinal cord tissue following SCI

    At 3 and 7 days post-SCI, the rats were first anesthetized with 10% chloral hydrate (0.33 mL/kg)viaintraperitoneal injection and then euthanized. T9–11spinal cord tissues (3 mm cephalad and caudal from the lesion epicenter) were separated and homogenized in radioimmune precipitation assay (RIPA) lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 1 mM EDTA). Protein homogenates (40 μg) were subjected to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Subsequently, the proteins were transferred to polyvinylidene difluoride membranes and incubated with the following primary antibodies at 4°C overnight: rabbit anti-phosphorylated low-density lipoprotein receptor related protein-6 (p-LRP-6) polyclonal antibody (1:500; Cell Signaling Technology, Inc., Boston, MA, USA), rabbit anti-β-catenin polyclonal antibody (1:500; Cell Signaling Technology), rabbit anti-phosphorylated glycogen synthase kinase-3β (p-GSK-3β) polyclonal antibody (1:300; Cell Signaling Technology), rabbit anti-active-caspase-3 polyclonal antibody (1:1,000; Abcam, Cambridge, UK), rabbit anti-active-caspase-9 polyclonal antibody (1:1,000; Abcam), and rabbit anti-β-actin polyclonal antibody (1:1,000; Abcam). The following day, the membranes were incubated with secondary antibodies (goat anti-rabbit IgG; 1:2,000; Abcam) for 2 hours at room temperature and immunoreactive bands were visualized with the ChemiDoc-It?TS2 Imager (UVP LLC, Upland, CA, USA). Finally, ImageJ2x software (National Institutes of Health, Bethesda, MD, USA) was utilized to measure the relative optical density of protein bands. The optical density of protein bands was normalized to β-actin controls.

    TUNEL assay of spinal cord tissue following SCI

    TUNEL assay was utilized to detect DNA fragmentation as a measure of cell apoptosis in the spinal cord anterior horn after SCI. Briefly, the 5-μm-thick coronal sections (3 mm rostral to the epicenter) were fixed with fixation solution (4% paraformaldehyde in PBS, pH 7.4) for 30 minutes. Afterwards, the tissues were washed with PBS for 30 minutes at room temperature. The tissues were incubated in permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) at 4°C for 2 minutes and then incubated with TUNEL reaction mixture (In Situ Cell Death Detection Kit, TMR red, Roche, Mannheim, Germany) at 37°C for 1 hour. Subsequently, the tissues were subjected to 4′,6-diamidino-2-phenylindole (DAPI) (1:1,000) staining. After mounting, all samples were analyzed using fluorescence microscopy (Leica, Heidelberg, Germany). The double-stained cells (red and blue) were considered to be TUNEL-positive cells. The number of TUNEL-positive cells was quantified by randomly selecting four sections in each experimental group.

    Immunofluorescence staining of spinal cord tissue following SCI

    Briefly, coronal sections (5 μm) were placed in 0.01 M citric acid (pH 6.0) for 15 minutes for antigen retrieval. The sections were blocked with blocking buffer (5% goat serum, 0.1% Triton X-100 in PBS) for 1 hour at 4°C and incubated with the primary polyclonal antibody overnight at 4°C (rabbit anti-Bcl-2 polyclonal antibody, 1:500; rabbit anti-Bax polyclonal antibody, 1:500, Novus Biologicals, Littleton, CO, USA). The following day, the sections were incubated with fluorescein isothiocyanate-coupled secondary antibody (goat anti-rabbit IgG; 1:400, Abcam) for 2 hours at room temperature, and the nuclei were counterstained using DAPI (1:1,000). The mounted sections were imaged using fluorescence microscopy (Leica 4000B, Heidelberg, Germany). The fluorescence intensity of Bcl-2 and Bax immunoreactivity in the spinal cord anterior horn was analyzed using the ImageJ2x software.

    Statistical analysis

    All data were analyzed using GraphPad Prism version 5.0 for Windows software (GraphPad Software, La Jolla, CA, USA) and expressed as the mean ± SD. Discrepancies among multiple groups were detected using one-way analysis of variance and least significant difference test, and the BBB scores were analyzed using the Mann-WhitneyUtest.P-values < 0.05 were considered statistically significant.

    Results

    MP promoted locomotor functional recovery after SCI

    The BBB scores were utilized to determine locomotor functional recovery before surgery and at 1 and 3 days, and 1, 2, 3, 4, 5, and 6 weeks post-SCI (Figure 1). BBB scores were 21 points before injury and 0 points at 1 day post-injury in MP and saline groups. The BBB scores in the two groups gradually increased from 1 day to 2 weeks, but no statistically significant difference was detected between the MP and saline groups at 3 and 7 days (P> 0.05). However, the BBB scores were significantly higher in the MP group compared with the saline group from 2 to 6 weeks post-injury (P< 0.05). The BBB scores reached a peak between 5 and 6 weeks, indicating that the neurological function related to locomotor activity improved with MP intervention post-SCI.

    MP treatment improved pathological recovery post-SCI

    Nissl staining was performed to detect pathological morphology in the spinal cord of SCI rats (Figure 2A). The number of Nissl-positive cells in the anterior horn of the spinal cord was less in the saline group compared with the sham group.Conversely, MP intervention dramatically increased the number of large-diameter Nissl-positive neurons, morphologically appearing as alpha-motor neurons, in the spinal cord anterior horn compared with the saline group (Figure 2A). Concomitantly, the number of surviving neurons in the spinal cord anterior horn was also clearly augmented by MP treatment post-SCI (Figure 2B;P< 0.01). Furthermore, compared with the saline group, the proportion of lesion size was smaller in the MP group after treatment with MP (Figure 2C;P< 0.01). These results illustrated significantly improved pathological morphology of the spinal cord with MP treatment post-SCI.

    MP activated Wnt/β-catenin signaling pathway after SCI

    To examine the molecular mechanisms underlying the role of MP in functional recovery, western blot assay was used in spinal cord tissue from the lower thoracic region, cephalad and caudal from the lesion epicenter, to analyze alterations of the Wnt/β-catenin signal pathway, which is known to inhibit neuronal apoptosis, at 3 and 7 days post-SCI in the three groups (Figure 3A). The β-catenin protein expression levels were significantly increased in the saline group compared with the sham group, and the β-catenin expression was higher in the MP group compared with the saline group (Figure 3C) (3 days:P< 0.001; 7 days:P< 0.01). Additionally, compared with the sham group, expression levels of p-LRP-6 and GSK-3β increased after SCI, and were even higher after MP treatment (Figure 3B; 3 days:P< 0.001; 7 days:P< 0.001); (Figure 3D; 3 days:P< 0.05; 7 days:P< 0.01). These results demonstrated that the Wnt/β-catenin signaling pathway was significantly activated after SCI, potentially as part of the endogenous repair mechanism, and was further enhanced by MP treatment in the spinal cord of SCI rats.

    Cell apoptosis inhibited by MP following SCI

    Some studies have stated that neuron cell apoptosis increases from 1 to 14 days post-SCI, and the variation tendency shows more significant alterations at 3 and 7 days post-SCI (Rios et al., 2015; Yan et al., 2015). In our study, western blot analysis was used to examine the anti-apoptotic effect of MP at 3 and 7 days post-SCI (Figure 4A). Interestingly, expression of active caspase-9 and caspase-3 in the spinal cord cephalad and caudal tissue from the lesion epicenter was decreased by MP after SCI compared with elevated levels in the saline group (Figure 4B,C; 3 days:P< 0.01 orP< 0.05; 7 days:P< 0.001). Concomitantly, the number of TUNEL-positive cells was reduced by MP treatment compared with the saline group after SCI (Figure 5A,B; 7 days:P< 0.001). Furthermore, immunofluorescence analysis was utilized to detect expression of the apoptotic and anti-apoptotic proteins Bax and Bcl-2 in the spinal cord anterior horn at 7 days post-SCI (Figure 6A,C). Results showed increased Bax levels, whereas Bcl-2 levels were decreased in the saline group compared with the sham group. Conversely, MP treatment inhibited Bax expression and elevated Bcl-2 expression after SCI compared with the saline group (Figure 6B, D; 7 days:P< 0.01). Our data indicate that MP treatment inhibited neuronal apoptosis, with a potentially significant neuroprotective effect on SCI.

    Discussion

    To evaluate the effect of MP on functional recovery in a rat SCI model, BBB scores were measured to determine open-field locomotor function. MP treatment significantly promoted locomotor functional recovery, decreased lesion size, and increased the number of surviving neurons in the spinal cord anterior horn in SCI rats. The molecular and cellular mechanisms underlying these neuroprotective effects likely involved a reduction in the number of apoptotic cells, which was accompanied by reduced levels of activated caspase-3, caspase-9, and Bax protein expression, while the anti-apoptotic Bcl-2 levels were higher in the spinal cord anterior horn of MP-treated rats. Interestingly, MP further up-regulated the expression levels and activation of the Wnt/β-catenin signaling pathway, including LRP-6 phosphorylation, β-catenin, and GSK3-β phosphorylation, following SCI. Thus, the neuroprotective effect of MP treatment suppressing apoptosis may be correlated with the activation of the Wnt/β-catenin signaling pathway.

    Recent studies have tried to identify the molecular mechanisms of primary and secondary injury in SCI (Estrada and Muller, 2014). Primary injury, leading to irreparable damage, is caused by various factors, such as initial mechanical impact, compression, and contusion, resulting in damage to nerve cells, myelin, blood vessels, and supporting bone structures in SCI. Secondary injury plays a more important long-term role in SCI. The traumatic injury causes swelling and hemorrhage, which lead to increased free radicals and decreased blood flow, causing cell membrane dysfunction, oxidative stress, inflammation, and apoptosis (Kang et al., 2007; Haider et al., 2015; Saxena et al., 2015). Apoptosis is considered as a significant therapeutic target in secondary injury. The application of anti-apoptotic drugs potentially promotes functional recovery in SCI.

    The synthetic glucocorticoid MP is widely used for the clinical treatment of SCI. Initially, MP had been observed to inhibit secondary injury and improve functional recovery by intercalation into the cell membrane and inhibition of the propagation of peroxidative reactions. It was believed to inhibit posttraumatic spinal cord lipid peroxidation after SCI (Hall et al., 1987; Hall, 1993). Additionally, multiples effects of MP on spinal cord injury, such as anti-inflammatory, anti-oxidative, and autophagy-suppressive activity, have been reported (Botelho et al., 2009; Chen et al., 2012; Chengke et al., 2013; Boyaci et al., 2014). However, some studies have started to question the neuroprotective effect of MP after SCI (Liu et al., 2009; Tesiorowski et al., 2013; Harrop, 2014). Although some studies have shown that a mega dose of MP within 8 hours after SCI improves prognosis and promotes functional recovery after SCI (Bracken, 2012), other reports indicate that MP fails to exert a neuroprotective effect and actually increases complications in SCI, such as infection, pulmonary embolism, and death (Pereira et al., 2009; Hurlbert et al., 2015). In the present rat SCI model, Nissl staining results showed that MP treatment significantly decreased the proportion of lesion size after SCI and increased the number of surviving neurons in the anterior horns of the spinal cord. Notably, these Nissl-positive, large-diameter neurons inthe spinal cord anterior horn appear morphologically as alpha-motor neurons, which contribute to functional recovery after SCI (Zhang et al., 2013a). The concomitant BBB scores for functional locomotor recovery were higher in the MP group compared with the saline group after SCI. These results illustrated that MP exhibits neuroprotective propertiesin vivoleading to functional recovery after SCI.

    Some studies have demonstrated that increased apoptosis especially during a later phase, affects microglia and oligodendrocytes and inhibits white matter recovery, leading to neuronal loss and inhibition of functional recovery after SCI (Yuan and Yankner, 2000; Beattie et al., 2002). The expression levels of caspases and Bax increase, whereas anti-apoptotic Bcl-2 levels decrease after apoptosis induction. These results provide potential therapeutic targets for promoting neurological function recovery after SCI (Yuan and Yankner, 2000; Tang et al., 2014; Zhang et al., 2015b). To study apoptosis in SCI, expression levels of Bcl-2 and Bax protein were detected by immunofluorescence analysis. Western blot assay was employed to detect expression of two other apoptotic markers, activated caspase-3 and caspase-9, after SCI. Results showed that expressions of activated caspase-3, caspase-9, and Bax expression were inhibited by MP, but levels of the anti-apoptotic protein Bcl-2 were enhanced. Concomitantly, the number of TUNEL-positive cells was also reduced by MP treatment following SCI. These results indicate that neuronal apoptosis was significantly suppressed by MP treatment following SCI, and further supported the neuroprotective activity of MP and effect on functional recovery. However, the explicit molecular mechanisms underlying the neuroprotective effects of MP remain to be shown to develop novel therapeutic strategies for SCI.

    Wnts are a well-characterized family of glycoproteins and play an important role in embryonic development, influencing processes such as proliferation, composition, and survival of neurons (Hollis and Zou, 2012; Gonzalez-Fernandez et al., 2014). Wnt signaling pathways are mainly divided into three different signaling pathways: the canonical Wnt/β-catenin pathway, the non-canonical planar cell polarity (Wnt-JNK) pathway, and the Wnt-Ca2+pathway (Fernandez-Martos et al., 2011). In the canonical wnt/β-catenin signal transduction pathway, binding of the wnt protein to the receptors Frizzled and LRP-5/6 induces LRP-6 phosphorylation, which in turn induces GSK-3β phosphorylation, improves stabilization of β-catenin, and induces dissociation of β-catenin from a protein complex consisting of adenomatous polyposis coli, Axin, and GSK3-β. Subsequently, β-catenin is translocated into the nucleus where it binds to the TCF/LEF transcription factor, thereby regulating cell proliferation, cell apoptosis, or differentiation (Reya and Clevers, 2005; MacDonald et al., 2009). Recent studies have shown that the activated Wnt/β-catenin signaling pathway promotes regeneration of axons and functional recovery after SCI (Sun et al., 2013; Yang et al., 2013). Other reports demonstrate that suppression of the Wnt/β-catenin singaling pathway induces apoptosis (Wang et al., 2002; Bilir et al., 2013). However, the effect of MP on the Wnt/β-catenin signaling pathway after SCI has not been previously reported. Western blot results in the present study demonstrated enhanced phosphorylation levels of LRP-6 and GSK-3β, as well as increased β-catenin protein expression, indicating increased activation of the canonical Wnt/β-catenin signaling pathway by MP after SCI. These findings most likely indicate an increase in endogenous repair mechanisms regulated by the Wnt/β-catenin signaling following SCI. Furthermore, MP inhibited neuronal apoptosis, which may imply that the anti-apoptotic effect was mediated by further activation of the Wnt/β-catenin signaling pathway post-SCI. Thus, the neuroprotective activity of MP on SCI may be attributed to the activation of the Wnt/β-catenin signaling pathway, providing a novel molecular mechanism underlying the role of MP in SCI treatment.

    In our study, MP treatment increased the number of surviving neurons in the spinal cord anterior horn, suppressed neuronal apoptosis, and improved pathological and locomotor functional recovery following SCI in rats. In line with these results, MP treatment further activated the wnt/ β-catenin signaling pathway close to the lesion epicenter after SCI, which is known to attenuate neuronal apoptosis. Nevertheless, the effect of MP has been studied only on neurons in our study, and it will be necessary to further observe the effects of MP on the Wnt/β-catenin signaling pathway and recovery of other neural cells post-SCI throughin vivoandin vitroexperiments. Further studies are needed to verify the various effects of MP on the Wnt/β-catenin signaling pathway and neuroprotective effects on functional recovery at additional time points in SCI rats to determine the molecular mechanisms of Wnt/β-catenin signalingin vivoandin vitroto further support the potential advantages of clinical application of MP in SCI treatment.

    Author contributions:GBL, KG and ZKN designed the study and wrote the paper. FWN, YCZ and LD collected data. ZYL, YG and TZY analyzed data. All authors approved the final version of the paper.

    Conflicts of interest:None declared.

    Plagiarism check:This paper was screened twice using CrossCheck to verify originality before publication.

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

    Basso DM, Beattie MS, Bresnahan JC (1995) A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 12:1-21.

    Beattie MS, Hermann GE, Rogers RC, Bresnahan JC (2002) Cell death in models of spinal cord injury. Prog Brain Res 137:37-47.

    Bilir B, Kucuk O, Moreno CS (2013) Wnt signaling blockage inhibits cell proliferation and migration, and induces apoptosis in triple-negative breast cancer cells. J Transl Med 11:280.

    Botelho RV, Daniel JW, Boulosa JL, Colli BO, Farias Rde L, Moraes OJ, Pimenta WE Jr., Ribeiro CH, Ribeiro FR, Taricco MA, Carvalho MV, Bernardo WM (2009) Effectiveness of methylprednisolone in the acute phase of spinal cord injuries-a systematic review of randomized controlled trials. Rev Assoc Med Bras 55:729-737.

    Boyaci MG, Eser O, Kocogullari CU, Karavelioglu E, Tokyol C, Can Y (2014) Neuroprotective effect of alpha-lipoic acid and methylprednisolone on the spinal cord ischemia/reperfusion injury in rabbits. Br J Neurosurg:1-6.

    Bracken MB (2012) Steroids for acute spinal cord injury. Cochrane Database Syst Rev 1:Cd001046.

    Chen HC, Fong TH, Lee AW, Chiu WT (2012) Autophagy is activated in injured neurons and inhibited by methylprednisolone after experimental spinal cord injury. Spine 37:470-475.

    Chengke L, Weiwei L, Xiyang W, Ping W, Xiaoyang P, Zhengquan X, Hao Z, Penghui Z, Wei P (2013) Effect of infliximab combined with methylprednisolone on expressions of NF-kappaB, TRADD, and FADD in rat acute spinal cord injury. Spine 38:E861-869.

    Estrada V, Muller HW (2014) Spinal cord injury: there is not just one way of treating it. F1000Prime Rep 6:84.

    Fehlings MG, Wilson JR, Cho N (2014) Methylprednisolone for the treatment of acute spinal cord injury: counterpoint. Neurosurgery 61 Suppl 1:36-42.

    Fernandez-Martos CM, Gonzalez-Fernandez C, Gonzalez P, Maqueda A, Arenas E, Rodriguez FJ (2011) Differential expression of Wnts after spinal cord contusion injury in adult rats. PLoS One 6:e27000.

    Gao S, Ding J, Xiao HJ, Li ZQ, Chen Y, Zhou XS, Wang JE, Wu J, Shi WZ (2014) Anti-inflammatory and anti-apoptotic effect of combined treatment with methylprednisolone and amniotic membrane mesenchymal stem cells after spinal cord injury in rats. Neurochem Res 39:1544-1552.

    Gocmez C, Celik F, Kamasak K, Kaplan M, Uzar E, Arikanoglu A, Evliyaoglu O (2015) Effects of intrathecal caffeic acid phenethyl ester and methylprednisolone on oxidant/antioxidant status in traumatic spinal cord injuries. J Neurol Surg A Cent Eur Neurosurg 76:20-24.

    Gonzalez-Fernandez C, Fernandez-Martos CM, Shields SD, Arenas E, Javier Rodriguez F (2014) Wnts are expressed in the spinal cord of adult mice and are differentially induced after injury. J Neurotrauma 31:565-581.

    Haider T, Hoftberger R, Ruger B, Mildner M, Blumer R, Mitterbauer A, Buchacher T, Sherif C, Altmann P, Redl H, Gabriel C, Gyongyosi M, Fischer MB, Lubec G, Ankersmit HJ (2015) The secretome of apoptotic human peripheral blood mononuclear cells attenuates secondary damage following spinal cord injury in rats. Exp Neurol 267:230-242.

    Hall ED (1993) Neuroprotective actions of glucocorticoid and nonglucocorticoid steroids in acute neuronal injury. Cell Mol Neurobiol 13:415-432.

    Hall ED, Springer JE (2004) Neuroprotection and acute spinal cord injury: a reappraisal. NeuroRx 1:80-100.

    Hall ED, McCall JM, Chase RL, Yonkers PA, Braughler JM (1987) A nonglucocorticoid steroid analog of methylprednisolone duplicates its high-dose pharmacology in models of central nervous system trauma and neuronal membrane damage. J Pharmacol Exp Ther 242:137-142.

    Harrop JS (2014) Spinal cord injury: debating the efficacy of methylprednisolone. Neurosurgery 61 Suppl 1:30-31.

    Hollis ER, 2nd, Zou Y (2012) Expression of the Wnt signaling system in central nervous system axon guidance and regeneration. Front Mol Neurosci 5:5.

    Hsu HC, Liu YS, Tseng KC, Tan BC, Chen SJ, Chen HC (2014) LGR5 regulates survival through mitochondria-mediated apoptosis and by targeting the Wnt/beta-catenin signaling pathway in colorectal cancer cells. Cell Signal 26:2333-2342.

    Hurlbert RJ, Hadley MN, Walters BC, Aarabi B, Dhall SS, Gelb DE, Rozzelle CJ, Ryken TC, Theodore N (2015) Pharmacological therapy for acute spinal cord injury. Neurosurgery 76 Suppl 1:S71-83.

    Kang SK, Yeo JE, Kang KS, Phinney DG (2007) Cytoplasmic extracts from adipose tissue stromal cells alleviates secondary damage by modulating apoptosis and promotes functional recovery following spinal cord injury. Brain Pathol (Zurich, Switzerland) 17:263-275.

    Li B, Zeng M, He W, Huang X, Luo L, Zhang H, Deng DY (2015) Ghrelin protects alveolar macrophages against lipopolysaccharide-induced apoptosis through growth hormone secretagogue receptor 1a-dependent c-Jun N-terminal kinase and Wnt/beta-catenin signaling and suppresses lung inflammation. Endocrinology 156:203-217.

    Liu JC, Patel A, Vaccaro AR, Lammertse DP, Chen D (2009) Methylprednisolone after traumatic spinal cord injury: yes or no? PM R 1:669-673.

    MacDonald BT, Tamai K, He X (2009) Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell 17:9-26.

    Pereira JE, Costa LM, Cabrita AM, Couto PA, Filipe VM, Magalhaes LG, Fornaro M, Di Scipio F, Geuna S, Mauricio AC, Varejao AS (2009) Methylprednisolone fails to improve functional and histological outcome following spinal cord injury in rats. Exp Neurol 220:71-81.

    Profyris C, Cheema SS, Zang D, Azari MF, Boyle K, Petratos S (2004) Degenerative and regenerative mechanisms governing spinal cord injury. Neurobiol Dis 15:415-436.

    Reya T, Clevers H (2005) Wnt signalling in stem cells and cancer. Nature 434:843-850.

    Rios C, Orozco-Suarez S, Salgado-Ceballos H, Mendez-Armenta M, Nava-Ruiz C, Santander I, Baron-Flores V, Caram-Salas N, Diaz-Ruiz A (2015) Anti-apoptotic effects of dapsone after spinal cord injury in rats. Neurochem Res 40:1243-1251.

    Saxena T, Loomis KH, Pai SB, Karumbaiah L, Gaupp E, Patil K, Patkar R, Bellamkonda RV (2015) Nanocarrier-mediated inhibition of macrophage migration inhibitory factor attenuates secondary injury after spinal cord injury. ACS Nano 9:1492-1505.

    Sun L, Pan J, Peng Y, Wu Y, Li J, Liu X, Qin Y, Bauman WA, Cardozo C, Zaidi M, Qin W (2013) Anabolic steroids reduce spinal cord injury-related bone loss in rats associated with increased Wnt signaling. J Spinal Cord Med 36:616-622.

    Tang P, Hou H, Zhang L, Lan X, Mao Z, Liu D, He C, Du H, Zhang L (2014) Autophagy reduces neuronal damage and promotes locomotor recovery via inhibition of apoptosis after spinal cord injury in rats. Mol Neurobiol 49:276-287.

    Tesiorowski M, Potaczek T, Jasiewicz B, Sapa J, Zygmunt M (2013) Methylprednisolone-acute spinal cord injury, benefits or risks? Postepy Hig Med Dosw (Online) 67:601-609.

    Vaquero J, Zurita M, Oya S, Aguayo C, Bonilla C (2006) Early administration of methylprednisolone decreases apoptotic cell death after spinal cord injury. Histol Histopathol 21:1091-1102.

    Wang X, Xiao Y, Mou Y, Zhao Y, Blankesteijn WM, Hall JL (2002) A role for the beta-catenin/T-cell factor signaling cascade in vascular remodeling. Circ Res 90:340-347.

    Wilson JR, Fehlings MG (2011) Emerging approaches to the surgical management of acute traumatic spinal cord injury. Neurotherapeutics 8:187-194.

    Wyndaele M, Wyndaele JJ (2006) Incidence, prevalence and epidemiology of spinal cord injury: what learns a worldwide literature survey? Spinal Cord 44:523-529.

    Xue H, Zhang XY, Liu JM, Song Y, Liu TT, Chen D (2013) NDGA reduces secondary damage after spinal cord injury in rats via anti-inflammatory effects. Brain Res 1516:83-92.

    Yacoub A, Hajec MC, Stanger R, Wan W, Young H, Mathern BE (2014) Neuroprotective effects of perflurocarbon (oxycyte) after contusive spinal cord injury. J Neurotrauma 31:256-267.

    Yan M, Liu YW, Shao W, Mao XG, Yang M, Ye ZX, Liang W, Luo ZJ (2015) EGb761 improves histological and functional recovery in rats with acute spinal cord contusion injury. Spinal Cord 54:259-265

    Yang Z, Wu Y, Zheng L, Zhang C, Yang J, Shi M, Feng D, Wu Z, Wang YZ (2013) Conditioned medium of Wnt/beta-catenin signaling-activated olfactory ensheathing cells promotes synaptogenesis and neurite growth in vitro. Cell Mol Neurobiol 33:983-990.

    Yuan J, Yankner BA (2000) Apoptosis in the nervous system. Nature 407:802-809.

    Zhang C, Zhang G, Rong W, Wang A, Wu C, Huo X (2015a) Early applied electric field stimulation attenuates secondary apoptotic responses and exerts neuroprotective effects in acute spinal cord injury of rats. Neuroscience 291:260-271.

    Zhang HY, Wang ZG, Wu FZ, Kong XX, Yang J, Lin BB, Zhu SP, Lin L, Gan CS, Fu XB, Li XK, Xu HZ, Xiao J (2013a) Regulation of autophagy and ubiquitinated protein accumulation by bFGF promotes functional recovery and neural protection in a rat model of spinal cord injury. Mol Neurobiol 48:452-464.

    Zhang J, Cui Z, Feng G, Bao G, Xu G, Sun Y, Wang L, Chen J, Jin H, Liu J, Yang L, Li W (2015b) RBM5 and p53 expression after rat spinal cord injury: implications for neuronal apoptosis. Int J Biochem Cell Biol 60:43-52.

    Zhang YK, Huang ZJ, Liu S, Liu YP, Song AA, Song XJ (2013b) WNT signaling underlies the pathogenesis of neuropathic pain in rodents. J Clin Invest 123:2268-2286.

    Ziaei A, Ardakani MR, Hashemi MS, Peymani M, Ghaedi K, Baharvand H, Nasr-Esfahani MH (2015) Acute course of deferoxamine promoted neuronal differentiation of neural progenitor cells through suppression of Wnt/beta-catenin pathway: a novel efficient protocol for neuronal differentiation. Neurosci Lett 590:138-144.

    Copyedited by Cooper C, Robens J, Wang J, Li CH, Qiu Y, Song LP, Zhao M

    *Correspondence to: Zhi-kui Nie or Kai Gao, 1756039110@qq.com or gaohaikai88@126.com.

    orcid: 0000-0002-7158-1557 (Zhi-kui Nie) 0000-0002-0771-7755 (Kai Gao)

    10.4103/1673-5374.194753

    Accepted: 2016-10-17

    少妇熟女欧美另类| 国产爽快片一区二区三区| a级毛片免费高清观看在线播放| 免费大片黄手机在线观看| 精品99又大又爽又粗少妇毛片| 人人妻人人添人人爽欧美一区卜| 久久99精品国语久久久| 日韩,欧美,国产一区二区三区| 欧美成人午夜免费资源| 一本大道久久a久久精品| 亚洲国产欧美在线一区| 国产精品偷伦视频观看了| 久久女婷五月综合色啪小说| 少妇精品久久久久久久| 亚洲欧美日韩另类电影网站| 国产精品女同一区二区软件| 少妇丰满av| 天天操日日干夜夜撸| 内射极品少妇av片p| 国产精品麻豆人妻色哟哟久久| 亚洲欧美一区二区三区国产| 亚洲国产精品一区三区| 少妇人妻久久综合中文| 亚洲av日韩在线播放| 国产精品国产av在线观看| 极品教师在线视频| 亚洲精品国产av成人精品| 中文精品一卡2卡3卡4更新| 汤姆久久久久久久影院中文字幕| 亚洲av二区三区四区| 欧美一级a爱片免费观看看| 久久精品久久久久久久性| 美女福利国产在线| 一级a做视频免费观看| 免费播放大片免费观看视频在线观看| 午夜福利网站1000一区二区三区| 亚洲无线观看免费| 久久精品国产亚洲av天美| 欧美精品亚洲一区二区| 啦啦啦中文免费视频观看日本| 大片电影免费在线观看免费| 街头女战士在线观看网站| 91午夜精品亚洲一区二区三区| 亚洲成人av在线免费| 在线播放无遮挡| 在线观看人妻少妇| 丝瓜视频免费看黄片| 99热这里只有是精品在线观看| 99九九在线精品视频 | 一级毛片我不卡| 久久综合国产亚洲精品| 一级a做视频免费观看| 国产视频首页在线观看| 久久午夜综合久久蜜桃| 亚洲欧洲精品一区二区精品久久久 | 亚洲一区二区三区欧美精品| 全区人妻精品视频| 免费大片黄手机在线观看| 日韩精品免费视频一区二区三区 | 亚洲,欧美,日韩| 免费人成在线观看视频色| 黑人巨大精品欧美一区二区蜜桃 | 久久久久久久久久久久大奶| 亚洲av二区三区四区| 亚洲欧美成人精品一区二区| 又黄又爽又刺激的免费视频.| 欧美成人精品欧美一级黄| 少妇人妻精品综合一区二区| 春色校园在线视频观看| av在线app专区| 丰满少妇做爰视频| 99热网站在线观看| 精品久久久久久久久亚洲| 亚洲精品自拍成人| 成人亚洲欧美一区二区av| 欧美激情极品国产一区二区三区 | 日韩伦理黄色片| 午夜av观看不卡| 婷婷色麻豆天堂久久| 日本与韩国留学比较| 国产极品粉嫩免费观看在线 | 午夜精品国产一区二区电影| 性色av一级| 亚洲精品日本国产第一区| 久久午夜福利片| 嫩草影院新地址| 九色成人免费人妻av| 久久久久久伊人网av| 日本午夜av视频| 蜜臀久久99精品久久宅男| 久久免费观看电影| 国产精品一区二区在线观看99| 日韩一区二区视频免费看| 中文在线观看免费www的网站| 熟妇人妻不卡中文字幕| 超碰97精品在线观看| 最近的中文字幕免费完整| 中文字幕亚洲精品专区| 嘟嘟电影网在线观看| 纯流量卡能插随身wifi吗| 天堂俺去俺来也www色官网| 中文字幕制服av| 十八禁高潮呻吟视频 | freevideosex欧美| 街头女战士在线观看网站| 亚洲国产精品专区欧美| 一本色道久久久久久精品综合| 日本猛色少妇xxxxx猛交久久| 免费大片18禁| 国产精品久久久久久av不卡| 大香蕉97超碰在线| 欧美日韩av久久| 久久久久国产精品人妻一区二区| 一级黄片播放器| 国产亚洲一区二区精品| 国产爽快片一区二区三区| 国产乱人偷精品视频| 秋霞伦理黄片| a级一级毛片免费在线观看| 亚洲国产精品999| 国产一区二区三区综合在线观看 | 久久午夜综合久久蜜桃| 成人18禁高潮啪啪吃奶动态图 | 色94色欧美一区二区| 免费大片黄手机在线观看| 你懂的网址亚洲精品在线观看| 熟女电影av网| 在线天堂最新版资源| 久久99一区二区三区| 日韩一本色道免费dvd| 国产一区二区三区av在线| 观看av在线不卡| 欧美bdsm另类| 国产一区二区在线观看av| 亚洲精品乱久久久久久| 18禁裸乳无遮挡动漫免费视频| 中国三级夫妇交换| 夜夜看夜夜爽夜夜摸| 九九在线视频观看精品| 久久久久国产网址| 女性生殖器流出的白浆| 久久精品国产亚洲网站| 国产精品熟女久久久久浪| 日韩免费高清中文字幕av| 少妇 在线观看| 少妇熟女欧美另类| 国产精品久久久久久久久免| 欧美成人精品欧美一级黄| 晚上一个人看的免费电影| 97精品久久久久久久久久精品| 天堂俺去俺来也www色官网| 极品少妇高潮喷水抽搐| 国产精品99久久久久久久久| 国产男女超爽视频在线观看| av.在线天堂| 国产av精品麻豆| 亚洲电影在线观看av| 高清不卡的av网站| 热re99久久国产66热| 美女cb高潮喷水在线观看| 精品人妻熟女av久视频| 建设人人有责人人尽责人人享有的| 热99国产精品久久久久久7| 最近的中文字幕免费完整| 欧美3d第一页| 99久久人妻综合| 国产亚洲av片在线观看秒播厂| 国产成人精品一,二区| 中文字幕久久专区| 99re6热这里在线精品视频| 久久国产精品大桥未久av | 一级二级三级毛片免费看| 日本爱情动作片www.在线观看| 久久精品久久精品一区二区三区| 亚洲色图综合在线观看| 最后的刺客免费高清国语| 免费黄色在线免费观看| 国产美女午夜福利| 亚洲精品中文字幕在线视频 | 成年女人在线观看亚洲视频| av在线app专区| 亚洲第一av免费看| 黄色欧美视频在线观看| 欧美高清成人免费视频www| 亚洲国产日韩一区二区| 九九在线视频观看精品| 欧美三级亚洲精品| 日韩人妻高清精品专区| 亚洲真实伦在线观看| 国产熟女午夜一区二区三区 | 91午夜精品亚洲一区二区三区| 久久久午夜欧美精品| 91aial.com中文字幕在线观看| 成人黄色视频免费在线看| 少妇丰满av| 大码成人一级视频| 搡老乐熟女国产| 国产免费一区二区三区四区乱码| 青春草国产在线视频| videos熟女内射| 亚洲精品一二三| 黑丝袜美女国产一区| 一级,二级,三级黄色视频| 草草在线视频免费看| 精品久久国产蜜桃| 高清不卡的av网站| 色婷婷久久久亚洲欧美| 久久人人爽人人片av| 国产精品伦人一区二区| 亚洲精品乱码久久久v下载方式| 精品人妻一区二区三区麻豆| 久久久国产欧美日韩av| 一级毛片aaaaaa免费看小| 久久久久久久久久成人| 国内少妇人妻偷人精品xxx网站| 黄片无遮挡物在线观看| 日本vs欧美在线观看视频 | 乱人伦中国视频| 另类亚洲欧美激情| 大香蕉久久网| 全区人妻精品视频| 2022亚洲国产成人精品| 美女国产视频在线观看| 成人影院久久| 亚洲综合精品二区| 日韩免费高清中文字幕av| 婷婷色av中文字幕| 99久久精品一区二区三区| 插逼视频在线观看| 如日韩欧美国产精品一区二区三区 | 国产男女内射视频| 久久青草综合色| 美女xxoo啪啪120秒动态图| av又黄又爽大尺度在线免费看| 亚洲一级一片aⅴ在线观看| 日产精品乱码卡一卡2卡三| 亚洲av福利一区| 日日摸夜夜添夜夜爱| 国产精品嫩草影院av在线观看| 少妇熟女欧美另类| 99久久人妻综合| 尾随美女入室| 国产免费一级a男人的天堂| 深夜a级毛片| 色哟哟·www| 中文字幕精品免费在线观看视频 | 国产精品秋霞免费鲁丝片| 五月玫瑰六月丁香| 国产av国产精品国产| 亚洲综合精品二区| 国产美女午夜福利| 久久精品国产鲁丝片午夜精品| 国产又色又爽无遮挡免| 99精国产麻豆久久婷婷| 最新的欧美精品一区二区| 777米奇影视久久| 天堂8中文在线网| 一本一本综合久久| 人妻系列 视频| 国产成人aa在线观看| 欧美成人精品欧美一级黄| 国产精品不卡视频一区二区| 国产成人一区二区在线| 日韩欧美 国产精品| 亚州av有码| 成人无遮挡网站| 成人二区视频| 国产精品国产av在线观看| 高清午夜精品一区二区三区| 免费观看a级毛片全部| 夫妻午夜视频| 国产亚洲精品久久久com| 亚洲av综合色区一区| 久久婷婷青草| 你懂的网址亚洲精品在线观看| 成人黄色视频免费在线看| 偷拍熟女少妇极品色| 欧美精品亚洲一区二区| 久久这里有精品视频免费| 免费观看在线日韩| 亚洲怡红院男人天堂| 国产综合精华液| 人妻少妇偷人精品九色| av黄色大香蕉| 99re6热这里在线精品视频| 日韩欧美 国产精品| 一本—道久久a久久精品蜜桃钙片| 久久精品久久精品一区二区三区| 老司机影院成人| 乱人伦中国视频| 久久av网站| av网站免费在线观看视频| 在线观看三级黄色| 国产免费一区二区三区四区乱码| 欧美成人午夜免费资源| 国产成人freesex在线| 午夜日本视频在线| 桃花免费在线播放| 99久久综合免费| 中文字幕亚洲精品专区| 久久毛片免费看一区二区三区| 一级,二级,三级黄色视频| 亚洲一级一片aⅴ在线观看| 亚洲婷婷狠狠爱综合网| av线在线观看网站| 人妻制服诱惑在线中文字幕| 国产精品国产三级专区第一集| 精品久久久久久久久av| 久久青草综合色| 亚洲欧美日韩另类电影网站| 国产成人精品一,二区| 久久女婷五月综合色啪小说| 少妇人妻一区二区三区视频| 黑人猛操日本美女一级片| 国产片特级美女逼逼视频| 久久久a久久爽久久v久久| 亚洲国产日韩一区二区| 日韩人妻高清精品专区| 中文精品一卡2卡3卡4更新| 插阴视频在线观看视频| 国产在线一区二区三区精| 高清视频免费观看一区二区| 交换朋友夫妻互换小说| 国产欧美另类精品又又久久亚洲欧美| 六月丁香七月| 蜜桃久久精品国产亚洲av| 大陆偷拍与自拍| 视频区图区小说| 久久青草综合色| 国产无遮挡羞羞视频在线观看| 国产av一区二区精品久久| 午夜福利在线观看免费完整高清在| 最黄视频免费看| 久久ye,这里只有精品| 色婷婷久久久亚洲欧美| 少妇精品久久久久久久| 欧美三级亚洲精品| 国产一区亚洲一区在线观看| 我要看黄色一级片免费的| 新久久久久国产一级毛片| 欧美最新免费一区二区三区| 人人妻人人澡人人看| 久久 成人 亚洲| 蜜桃久久精品国产亚洲av| av有码第一页| 成人黄色视频免费在线看| 亚洲av电影在线观看一区二区三区| 夜夜骑夜夜射夜夜干| 亚洲av在线观看美女高潮| 伊人久久精品亚洲午夜| 一区二区三区精品91| 午夜久久久在线观看| 亚洲国产色片| 国产色爽女视频免费观看| 边亲边吃奶的免费视频| 欧美精品国产亚洲| 亚洲高清免费不卡视频| 九九爱精品视频在线观看| 久久久国产精品麻豆| 五月伊人婷婷丁香| 我要看黄色一级片免费的| 中文在线观看免费www的网站| 黄色日韩在线| 亚洲av男天堂| 能在线免费看毛片的网站| 精品99又大又爽又粗少妇毛片| 免费看光身美女| 26uuu在线亚洲综合色| 欧美日韩国产mv在线观看视频| 国产日韩欧美亚洲二区| 免费大片黄手机在线观看| 国产精品秋霞免费鲁丝片| 国产精品久久久久久av不卡| 久久久久久久大尺度免费视频| 内射极品少妇av片p| 街头女战士在线观看网站| 久久久久人妻精品一区果冻| 欧美丝袜亚洲另类| 久久久精品免费免费高清| 国产一区二区三区av在线| 卡戴珊不雅视频在线播放| 黑人巨大精品欧美一区二区蜜桃 | 3wmmmm亚洲av在线观看| 亚洲欧美一区二区三区黑人 | 免费看光身美女| 极品人妻少妇av视频| 亚洲综合精品二区| 久久久久久久久久成人| 久久久久久伊人网av| 色视频www国产| 高清黄色对白视频在线免费看 | 国产黄片美女视频| 有码 亚洲区| 国产精品伦人一区二区| 男的添女的下面高潮视频| 丰满少妇做爰视频| 亚洲精华国产精华液的使用体验| 国产 一区精品| 熟女人妻精品中文字幕| 中文字幕人妻丝袜制服| 大片免费播放器 马上看| 十八禁网站网址无遮挡 | 99久久精品一区二区三区| 亚洲综合色惰| 久久精品久久久久久久性| 寂寞人妻少妇视频99o| 日韩成人av中文字幕在线观看| 亚洲第一av免费看| www.色视频.com| 亚洲国产成人一精品久久久| 日韩人妻高清精品专区| 亚洲精品日本国产第一区| 欧美日韩一区二区视频在线观看视频在线| 免费观看无遮挡的男女| 精品视频人人做人人爽| 精品久久久久久久久亚洲| 欧美精品亚洲一区二区| 五月开心婷婷网| 国产综合精华液| 在线观看人妻少妇| 欧美日韩精品成人综合77777| 欧美精品亚洲一区二区| 国产熟女欧美一区二区| 亚洲精品国产成人久久av| 国产免费又黄又爽又色| 国产一区亚洲一区在线观看| 涩涩av久久男人的天堂| 亚洲美女视频黄频| 国产无遮挡羞羞视频在线观看| 亚洲激情五月婷婷啪啪| 国产在视频线精品| 五月开心婷婷网| av在线播放精品| 国产又色又爽无遮挡免| 多毛熟女@视频| 日韩视频在线欧美| 一级毛片 在线播放| 蜜桃久久精品国产亚洲av| 在线观看免费日韩欧美大片 | 最近2019中文字幕mv第一页| 欧美激情极品国产一区二区三区 | 国产永久视频网站| 亚洲av.av天堂| 51国产日韩欧美| 亚洲国产日韩一区二区| 日韩中文字幕视频在线看片| 99热全是精品| 性高湖久久久久久久久免费观看| 国产精品国产三级国产av玫瑰| 国产日韩欧美视频二区| 亚洲经典国产精华液单| 久久久久久久精品精品| 18+在线观看网站| 在线观看三级黄色| 少妇精品久久久久久久| 日本vs欧美在线观看视频 | 欧美日韩在线观看h| 一级黄片播放器| 国产av精品麻豆| 国产成人免费无遮挡视频| 亚洲丝袜综合中文字幕| 精品一区二区免费观看| 超碰97精品在线观看| 只有这里有精品99| 久久人人爽人人片av| 午夜福利在线观看免费完整高清在| 午夜91福利影院| 人人妻人人澡人人爽人人夜夜| 天堂8中文在线网| 精品一品国产午夜福利视频| 在线观看免费日韩欧美大片 | 午夜免费鲁丝| 99热这里只有是精品50| 九九久久精品国产亚洲av麻豆| 国产69精品久久久久777片| 亚洲精品视频女| 亚洲欧美日韩卡通动漫| 两个人免费观看高清视频 | 妹子高潮喷水视频| 最近中文字幕2019免费版| 日日摸夜夜添夜夜爱| 国产白丝娇喘喷水9色精品| 久久6这里有精品| 久久精品国产亚洲av涩爱| 久久综合国产亚洲精品| 午夜免费男女啪啪视频观看| 男女边吃奶边做爰视频| h视频一区二区三区| 国产成人精品婷婷| 91精品国产九色| 日韩一本色道免费dvd| 一本大道久久a久久精品| 免费大片黄手机在线观看| 亚洲av电影在线观看一区二区三区| 美女中出高潮动态图| 精品一区二区免费观看| 国产精品伦人一区二区| 国内少妇人妻偷人精品xxx网站| 国产精品熟女久久久久浪| 99精国产麻豆久久婷婷| 一区二区三区免费毛片| av有码第一页| 日本爱情动作片www.在线观看| 中国美白少妇内射xxxbb| 97超视频在线观看视频| 又爽又黄a免费视频| 五月伊人婷婷丁香| 人妻少妇偷人精品九色| 亚洲国产精品一区二区三区在线| 嫩草影院入口| av有码第一页| 国产高清有码在线观看视频| 国产伦在线观看视频一区| 精品久久国产蜜桃| 成人美女网站在线观看视频| 一级毛片 在线播放| 蜜桃久久精品国产亚洲av| 亚洲天堂av无毛| 久久久久国产精品人妻一区二区| 欧美xxⅹ黑人| 一区二区三区乱码不卡18| 国产精品.久久久| 午夜免费鲁丝| 不卡视频在线观看欧美| 日本爱情动作片www.在线观看| 精品亚洲成国产av| 亚洲国产精品专区欧美| 亚洲av在线观看美女高潮| 十分钟在线观看高清视频www | 尾随美女入室| 午夜久久久在线观看| 国产精品麻豆人妻色哟哟久久| 又大又黄又爽视频免费| 热re99久久精品国产66热6| 亚洲av日韩在线播放| 久久国产亚洲av麻豆专区| 亚洲无线观看免费| 啦啦啦在线观看免费高清www| 国产深夜福利视频在线观看| 国产精品国产三级专区第一集| 免费在线观看成人毛片| 国产黄频视频在线观看| 国产精品久久久久久精品电影小说| 亚洲成人av在线免费| 人人妻人人添人人爽欧美一区卜| h视频一区二区三区| 日韩中文字幕视频在线看片| 老女人水多毛片| 色婷婷av一区二区三区视频| 久久精品国产鲁丝片午夜精品| 少妇人妻 视频| 久久精品国产亚洲av天美| 亚洲欧洲国产日韩| 99热这里只有精品一区| 久久精品久久久久久噜噜老黄| 国产精品久久久久久久久免| 亚洲真实伦在线观看| 亚洲欧美中文字幕日韩二区| 久久久久久久久久久免费av| 日本av手机在线免费观看| 中文资源天堂在线| 色视频在线一区二区三区| 日日摸夜夜添夜夜添av毛片| 国产成人精品婷婷| 精品久久久噜噜| 久久久久久伊人网av| 久久精品国产a三级三级三级| 街头女战士在线观看网站| 免费在线观看成人毛片| 蜜臀久久99精品久久宅男| 高清欧美精品videossex| 欧美精品人与动牲交sv欧美| 亚洲人与动物交配视频| 伊人亚洲综合成人网| 三级国产精品片| 一区在线观看完整版| 亚洲精品中文字幕在线视频 | 精品久久久久久久久av| 国产精品国产av在线观看| 久久人人爽av亚洲精品天堂| 人人妻人人爽人人添夜夜欢视频 | 国产精品久久久久久久久免| 视频区图区小说| 老熟女久久久| 日韩不卡一区二区三区视频在线| 日本91视频免费播放| 亚洲欧洲日产国产| 人人妻人人爽人人添夜夜欢视频 | 日日啪夜夜爽| 亚洲综合色惰| videos熟女内射| 中文精品一卡2卡3卡4更新| 久久狼人影院| 成人特级av手机在线观看| a级片在线免费高清观看视频| 亚洲av电影在线观看一区二区三区| 尾随美女入室| av国产精品久久久久影院| 亚洲中文av在线| 午夜91福利影院| 欧美日韩一区二区视频在线观看视频在线| 五月天丁香电影| 精品熟女少妇av免费看| 日韩免费高清中文字幕av| 熟女电影av网| 亚洲国产色片| 男人舔奶头视频| 国产一级毛片在线| 亚洲天堂av无毛| 高清毛片免费看| 我的女老师完整版在线观看| 国产色婷婷99| 欧美另类一区| 久久av网站| 国产高清有码在线观看视频|