• <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 ?

    Qingjie Fuzheng granules inhibit colorectal cancer cell growth by the PI3K/AKT and ERK pathways

    2019-06-11 07:30:20HongYangJianXinLiuHaiXiaShangShanLinJinYanZhaoJiuMaoLin
    關(guān)鍵詞:歧路建設(shè)者共同體

    Hong Yang, Jian-Xin Liu, Hai-Xia Shang, Shan Lin, Jin-Yan Zhao, Jiu-Mao Lin

    Hong Yang, Jian-Xin Liu, Hai-Xia Shang, Shan Lin, Jin-Yan Zhao, Jiu-Mao Lin, Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122,Fujian Province, China

    Hong Yang, Jian-Xin Liu, Hai-Xia Shang, Shan Lin, Jin-Yan Zhao, Jiu-Mao Lin, Fujian Key Laboratory of Integrative Medicine on Geriatrics, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, Fujian Province, China

    Abstract

    Key words:Qingjie Fuzheng granules; Colorectal cancer; Proliferation; Apoptosis;PI3K/AKT; ERK

    INTRODUCTION

    As a result of changing lifestyles and aging populations, the prevalence of colorectal cancer (CRC) remains high and accounts for approximately 25% of the world’s cancerrelated deaths[1-3]. Although surgical resection and chemoradiotherapy are the most commonly used clinical options, surgery is not an option for all patients, and longterm chemoradiotherapy can cause adverse side effects, such as drug resistance,recurrence and metastasis[4-7]. Therefore, the search for novel therapies has attracted worldwide attention.

    Natural products, which often have fewer side effects than synthetic drugs, are important in the treatment of many diseases and have a long history of use in China.Therefore, natural products have been studied by many researchers to find better antitumor drugs[8-11]. Qingjie Fuzheng granules (QFGs) is a traditional Chinese medicine (TCM) formula (Table 1) that consists of a mixture of four herbs (Scutellaria barbataD. Don, malt,Hedyotis diffusaWilld, and Astragalus) that together confer properties of anti-inflammation, antioxidative, antibacterial, immunity enhancement and digestion promotion. QFGs have been widely used and found to be clinically effective in various cancer treatments, including CRC, and have few side effects.However, the precise mechanisms and molecular signaling pathways involved in the activity of QFGs’ anticancer effect have not been reported in the literature.

    CRC develops because of a cell growth imbalance caused by excessive proliferation or lack of apoptosis. Eukaryotic cell proliferation is controlled by the cell cycle, which consists of the G0, G1, S, G2 and M phases. In the detection of cell cycle progression,the G1/S transition is one of the main checkpoints[12]. The main regulatory factors in G1/S progression are cyclin D1 and cyclin-dependent kinase 4 (CDK4), which can form complexes to regulate this progress[13-15]. A CDK inhibitor, p21, can change the function of CDK-cyclin complexes by binding to them and then suppressing cell proliferation[16]. Normal cell apoptosis can eliminate surplus, redundant, and aberrant cells in animals, so it is essential for normal tissue maintenance. Disorders in this process trigger many diseases, including CRC[17-19]. The pathways involved in the apoptotic process are the mitochondria-dependent pathway, also called the intrinsic apoptosis pathway, and the death receptor-mediated apoptosis pathway[20]. The former is modulated by the Bax (proapoptotic) and Bcl-2 (anti-apoptotic) family proteins[21], which control the release of apoptotic correlation factors, such as cytochrome C (Cyt C)[22]. When intracellular damage occurs, mitochondria-dependent apoptosis is triggered. Then, Cyt C, together with Apaf-1 and caspase-9, cleaves caspase-3[23]. Receptor-mediated apoptosis originates from outside the cell, with the binding of the Fas ligand (termed FasL or CD95L) to the Fas receptor (termed CD95).Once the death receptor pathway is successfully activated, the Fas-associated death domain and caspase-8 will accumulate, and caspase-8 will be cleaved. Then, caspase-8 cleaves caspase-3, which generates the activated form of caspase-3 that serves as the ultimate activator of apoptosis[24]. Therefore, one of the key approaches in the development of antitumor drugs is to promote apoptosis and inhibit tumor cell proliferation, two processes that typically promote cancer growth. There are multiple signaling pathways that regulate cancer growth, including the PI3K/AKT and ERK signaling pathways, and abnormal activation of these signaling pathways can lead to irregular expression of these factors.

    The aim of this study is to better understand the mechanism underlying the potential anticancer effect of QFGs by investigating their biological function using the human CRC cell variants HCT-116 and HCT-8. Our results showed that QFGs inhibit proliferation and increase apoptosis in HCT-116 and HCT-8 cells by inactivating the PI3K/AKT and ERK pathways.

    MATERIALS AND METHODS

    Cell culture

    The human colon carcinoma HCT-8 and HCT-116 cell lines were purchased from the American Type Culture Collection. The two cell lines were cultured in Roswell Park Memorial Institute-1640 medium (C11875500BT; Life Technologies Corp. Grand Island, United States) containing 10% fetal bovine serum, 1% penicillin, and 1%streptomycin, and were grown at 37 °C in 5% CO2.

    Preparation of QFGs and caspase inhibitors

    QFGs were obtained and prepared as previously described[11]. Briefly, QFGs powder was dissolved in 1× PBS (store concentration of QFGs is 200 mg/mL) and stored at 20°C. Inhibitors of caspase-3/-8/-9 (Z-DEVE-FMK, ab120488; Z-IETD-FMK, ab141382;Z-LEHD-FMK, ab142026, Abcam, CA, United States) were dissolved in DMSO to a concentration of 10 mM and stored at -20 °C.

    MTT assays

    HCT-8 and HCT-116 cells were placed into 96-well plates (1 × 105cell/well). After 12 h, the cells were treated with different doses of QFGs (0.5, 1 and 2 mg/mL) and grown for 24 h, or the cells were treated with a designated dose of QFGs (2 mg/mL)in combination with inhibitors of caspase-3 (Z-DEVE-FMK), caspase-8 (Z-IETD-FMK),and caspase-9 (Z-LEHD-FMK) at a concentration of 10 μmol/L each and then incubated for 24 h. Then, MTT (0.5 mg/mL) was added to each well (100 μL/well)and incubated for 4 h. Subsequently, all wells were treated with DMSO (100 μL/well).Absorbance at 570 nm in each well was measured by using an ELISA reader (Infinite M200 PRO; Tecan Austria GmbH, Austria).

    Lactate dehydrogenase assays

    Cells were seeded into 12-well plates (1 × 105cell/well), treated with different dose of QFGs (0.5, 1 and 2 mg/mL) and grown for 24 h. Then, a lactate dehydrogenase (LDH)release assay kit (Beyotime, Shanghai, China) was used to determine the LDH activity according to the kit’s manual.

    Table 1 Composition of Qingjie Fuzheng granules

    Colony formation assays

    After treatment with different concentrations of QFGs (0.5, 1, and 2 mg/mL) for 24 h,a colony formation assay was performed as described previously[11].

    Hoechst 33258 staining

    QFGs and 10 μmol/L caspase inhibitors (Z-DEVE-FMK, Z-IETD-FMK, Z-LEHD-FMK)were added to the cells and grown for 24 h. Subsequently, 4% paraformaldehyde was used to fix the cells for 15 min. Then, 4% paraformaldehyde was removed and 1× PBS was used to rinse the cells three times. Then, Hoechst 33258 (c0003; Beyotime,Shanghai, China) (100 μL/well) was added to all wells in the dark for 15 min. The Hoechst 33258 solution was then removed, and 1× PBS was used to rinse the stained cells three times, followed by 1× addition of fresh PBS. An inverted fluorescence microscope (Leica DMI4000B; Leica Camera AG, Solms, Germany) was used to observe and photograph the cells.

    Cell cycle assays

    Cell cycle was measured in the HCT-8 and HCT-116 cells after treatment with the indicated concentrations of QFGs (0.5, 1 and 2 mg/mL). Cell cycle progression was estimated by using a propidium iodide (PI) kit (KGA512; KeyGen Biotech, Nanjing,China) and fluorescence activated cell sorting according to the manufacturer’s instructions.

    Annexin V-FITC/PI staining flow cytometry assays

    Cells were seeded into 6-well plates (1.5 × 105cell/well), treated with different doses of QFGs (05, 1 and 2 mg/mL) and grown for 24 h. Then, cells were stained by using an Annexin V/PI kit (KGA108; KeyGen BioTech, Nanjing, China) according to the kit’s manual. Annexin V-positivity and PI-negativity (lower-right quadrant)represented early apoptotic cells, whereas Annexin V-positivity and PI-positivity(upper-right quadrant) represented late apoptotic cells.

    Caspase activity assays

    A caspase activity assay kit (caspase-3, KGA204; caspase-8, KGA304; caspase-9,KGA404; KeyGen BioTech, Nanjing, China) was used to detect the activity of caspases according to the manufacturer’s instructions. In brief, cell lysates were prepared after the addition of the indicated reaction buffer (provided in the kit) at 37 °C for 4 h in the dark. Absorbance at 405 nm was measured by using an ELISA reader.

    Western blot analysis

    When CRC cells were treated with different doses of QFGs (05, 1 and 2 mg/mL) for 24 h, a cell lysis buffer containing a cocktail to lyse the cells was added. The bicinchoninic acid assay was used to detect the total protein concentrations, and 50 μg of total protein was used for electroblotting. Five percent skim milk was used to block the NC membranes, and then primary antibodies against Fas, FasL, p-PI3K and p-AKT (ab-110021, ab-15285, ab182651, ab38449; 1:1000, Abcam, CA, United States), p-ERK (sc-16982; 1:1000, Santa Cruz Biotechnology, CA, United States), cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9, β-actin, Bcl-2 and Bax (#9662, #4790, #9508,#4967, #4223, #5023; 1:1000, Cell Signaling, Beverly, MA, United States), PI3K, AKT and ERK (13329-1-AP, 10176-2-AP, 16443-1-AP; 1:2000, Proteintech, United States)were added at 4 °C overnight. On the second day, the appropriate HRP-conjugated secondary antibodies (goat anti-mouse IgG secondary antibody, #L3032; goat antirabbit IgG secondary, #L3012; 1:5000, Signalway Antibody, PA, United States) were added and the SuperSignal West Pico Chemiluminescent Substrate was used to detect the signal.

    Statistical analysis

    One-way ANOVA and SPSS software (version 18.0) were used to analyze all of the data in this study. The data are expressed as the mean ± standard deviation.P< 0.05indicated statistical significance.

    信息素養(yǎng)主要指個(gè)體利用信息技術(shù)及技能搜索、獲取、鑒別、分析、利用信息的能力,是當(dāng)今信息社會(huì)必備的技能之一?!耙粠б宦贰敝铝τ诖蛟烊祟惷\(yùn)共同體,但各國文化、政治、法律等差異較大,要求參與建設(shè)者必須具備一定的信息素養(yǎng)能力,了解、熟悉、掌握相關(guān)的經(jīng)濟(jì)、政治、文化信息,才能在項(xiàng)目、活動(dòng)開展過程中不走歧路、合作共贏。

    RESULTS

    QFGs decreased cell viability and increased cytotoxicity in HCT-116 and HCT-8 cells

    The MTT assays and LDH activity assays were used to evaluate the effect of QFGs on the growth of the two cell types. QFGs inhibited cell viability in a dose-dependent manner (Figure 1A, B), showing cytotoxicity at 0.5-2.0 mg/mL (Figure 1C, D). In Figure 1A and B, cell viability after treatment with QFGs (0.5-2.0 mg/mL, 24 h)decreased by (6.90% ± 1.03%)-(59.70% ± 1.51%) (HCT-116) and (5.56% ± 4.52%)-(49.44% ± 2.47%) (HCT-8) relative to the viability in control cells (P< 0.05). In Figure 1C and D, treatment with QFGs (0.5-2.0 mg/mL, 24 h) increased the LDH activity rate of the cells from 0.52 ± 0.023 to 0.77 ± 0.002 (HCT-116) and from 0.56 ± 0.054 to 0.81 ±0.044 (HCT-8) relative to that in control cells (P< 0.01). These results proved that QFGs treatment reduced cell viability and increased cytotoxicity in both cell types.

    QFGs inhibited the proliferation of HCT-116 and HCT-8 cells by arresting the cell cycle

    Cell colony formation assays were used to evaluate the changes in cell growth after treatment with QFGs. As shown in Figure 2A, QFGs dose-dependency inhibited colony formation in HCT-116 and HCT-8 cells. Subsequently, cell cycle assays were used to verify the proliferation-inhibiting effects of QFGs. As shown in Figure 2B-E,the percentages of S phase in HCT-116 cells after treatment with 0, 0.5, 1, and 2 mg/mL QFGs were 44.7% ± 2.77%, 33.45% ± 3.30%, 16.50% ± 2.12%, and 12.86% ±2.51%, respectively (P< 0.01), and the percentages of S phase in HCT-8 cells after treatment with 0, 0.5, 1, and 2 mg/mL QFGs were 44.55% ± 3.32%, 26.71% ± 2.17%,25.60% ± 2.19%, and 21.99% ± 3.30%, respectively (P< 0.01). These results suggested that QFGs can inhibit proliferation of both cell types by arresting the cell cycle.

    QFGs induced apoptosis of HCT-116 and HCT-8 cells via the mitochondriadependent and death receptor pathways

    Hoechst 33258 staining assays were used to evaluate the changes in cell nuclear morphology after treatment with QFGs (Figure 3A). The degree of staining was low in untreated cells, but it gradually increased in the other three groups, which indicated a gradual increase in apoptosis. Subsequently, Annexin V-FITC/PI assays were used to verify the apoptosis-inducing effect of QFGs. As shown in Figure 3B, apoptosis percentages in HCT-116 cells after treatment with 0, 0.5, 1, and 2 mg/mL QFGs were 4.07% ± 0.48%, 11.87% ± 0.5%, 12.77% ± 0.67%, and 31.13% ± 0.73%, respectively (P<0.01), and apoptosis percentages in HCT-8 cells after treatment with 0, 0.5, 1, and 2 mg/mL QFGs were 2.23% ± 0.50%, 9.34% ± 0.69%, 17.19% ± 0.55%, and 33.93% ±0.93%, respectively (P< 0.01). We also found that QFGs upregulated the expression of cleaved-caspase-3/-8/-9 in both cell types (Figure 3C, D;P< 0.01). At the same time,the caspase activity was measured using a commercial caspase activity assay kit.Identical to the western blot results, the activities of caspase-3/-8/-9 were significantly enhanced by QFGs treatment in both cell types (Figure 3E, F;P< 0.01).These results indicated that QFGs induced apoptosis in the two cell types, and suggested that apoptosis occurredviaboth the mitochondria-dependent and death receptor-mediated pathways.

    To further confirm these findings, various specific caspase inhibitors were used. As shown in Figure 4A, Z-DEVD-FMK, Z-IETD-FMK, and Z-LEHD-FMK markedly inhibited the inhibitory effect of QFGs on cell viability in both cell types (P< 0.05 and 0.01, respectively). In addition, we used the Hoechst 33258 staining assay to detect nuclear morphological changes, and all three caspase inhibitors clearly inhibited the apoptosis-induced effect of QFGs (2 mg/mL) in both cell types (Figure 4B). These findings proved that QFGs induced apoptosisviathe mitochondria-dependent and death receptor-mediated pathways in both HCT-116 and HCT-8 cells.

    QFGs regulated the expressions of survivin, cyclin D1, CDK4, p21, Bax, Bcl-2, Fas,and FasL in HCT-116 and HCT-8 cells

    Figure 1 Effect of QFGs on the viability and cytotoxicity of HCT-116 and HCT-8 cells.

    During cell cycle regulation, survivin is a key protein that indicates cell cycle progression, and the complex of cyclin D1 and CDK4 directly regulates cell cycle progression[13]. In a previous study, overexpression of this complex has been found to induce cell proliferation, whereas p21 inhibited the effect of the cyclin D1/CDK4 complex[14]. During the regulation of cell apoptosis, Bax and Bcl-2 regulate the mitochondria-dependent pathway[21], while Fas and FasL activate the death receptormediated pathway[24]. We found that QFGs can inhibit proliferationviaarrest of the cell cycle in HCT-116 and HCT-8 cells. We used western blotting to test the protein expression levels of survivin, cyclin D1, CDK4, and p21 after the cells were treated with QFGs. As shown in Figure 5A and B, we found that QFGs downregulated the expression of survivin, cyclin D1 and CDK4, but upregulated the level of p21 in both cell types (P< 0.05 and 0.01, respectively). Since we found that QFGs induced apoptosisviathe mitochondria-dependent and death receptor-mediated pathways in HCT-116 and HCT-8 cells, we used western blotting to test the protein expression levels of Bcl-2, Bax, Fas, and FasL after the cells were treated with QFGs. As shown in Figure 5C and D, we found that QFGs decreased the expression of Bcl-2 but promoted the expression of Bax, Fas, and FasL in both cell types (P< 0.05 and 0.01, respectively).Briefly, these findings suggest that QFGs inhibit proliferationviacell cycle arrest and induce apoptosisviathe mitochondria-dependent and death receptor-mediated pathways in HCT-116 and HCT-8 cells.

    QFGs suppressed the PI3K/AKT and ERK signaling pathways in HCT-116 and HCT-8 cells

    Cancer occurrence and progression are highly associated with the regulation of multiple signaling pathways, including PI3K/AKT and ERK[25,26]. To further explore the potential mechanisms underlying the observed anticancer effects of QFGs, we examined the expression of major regulation factors involved in the PI3K/AKT and ERK signaling pathways. As shown in Figure 6A and B, after treatment with QFGs,the ratios of the phosphorylation expression level to the total expression level were significantly downregulated in PI3K, AKT and ERK in both cell types (P< 0.05 and 0.01, respectively), which suggested that the anticancer effect of QFGs on CRC cells occursviasuppression of the PI3K/AKT and ERK signaling pathways.

    DISCUSSION

    CRC is a deadly disease, primarily due to its high rate of metastasis and recurrence in patients. Multidrug combination therapy and surgical treatment are the main therapeutic methods that can significantly improve patients survival. However, there are many patients with cancer that is drug resistant and recurrent after traditional clinical treatment[27,28]. Therefore, there is a need to discover new kinds of anticancer drugs, such as herbal products. In recent years, TCM has attracted increasing attention in the field of oncology because of its relative security and long history of application[29-32]. QFGs are a four-herb TCM formula that consists ofScutellaria barbataD. Don, malt,Hedyotis diffusaWilld, and Astragalus. In the past few years, somestudies have proven thatHedyotis diffusaWilld andScutellaria barbataD. Don are capable of promoting apoptosis and inhibiting growth and angiogenesis in many types of cancer cells, including CRC[33,34]. Malt could boost the movement of Qi and improve food digestion, and Astragalus is a vital component in many TCM formulas that have been used in clinics to cure many cancer patients, reduce the incidence of complications, and improve the quality of life of cancer patients[35]. However, there are no reports on the underlying signaling pathways and mechanisms involved in QFGs anticancer activity.

    Figure 2 Effect of QFGs on the proliferation of HCT-116 and HCT-8 cells.

    Figure 3 Effect of QFGs on HCT-116 and HCT-8 cells apoptosis.

    In this study, we found that QFGs exhibited significant anticancer effects on HCT-116 and HCT-8 cells. The anticancer effect of QFGs is mainly through the inhibition of proliferation and induction of apoptosis, which are mechanisms commonly exploited in tumor therapy. As demonstrated in the current study, QFGs reduced cell viability and increased cytotoxicity in HCT-116 and HCT-8 cells in a dose-dependent manner.Furthermore, we used cell colony formation, nuclear staining, and flow cytometry assays to demonstrate that these effects in HCT-116 and HCT-8 cells resulted from the inhibition of proliferation and induction of apoptosis by QFGs.

    Cell proliferation is regulated by the cell cycle, which consists of the G0, G1, S, G2 and M phases. DNA synthesis is completed in S phase, which is responsible for the initiation and completion of DNA replication[20]. Therefore, the G1/S transition is one of the two main checkpoints in the cell cycle. Using a cell cycle assay, we observed that the inhibitory effects of QFGs on HCT-116 and HCT-8 cell proliferation were associated with blocking the G1 to S phase transition. The G1/S process is highly regulated by cyclin D1, which forms complexes with CDK4[21,22]. Overexpression of the cyclin D1/CDK4 complex can enhance cell proliferation, whereas p21 can bind to this complex and inhibit its activity[24]. Therefore, the expression of CDK4, cyclin D1 and p21 indicate the proliferation state of HCT-116 and HCT-8 cells to some extent. This study proved that QFGs administration upregulates p21 protein expression while downregulating cyclin D1 and CDK4 protein expression. These results showed that QFGs inhibit HCT-116 and HCT-8 cell proliferation.

    Cell apoptosis or programmed death, which is an essential process in a healthy organism, removes surplus and damaged cells[36-38]. Failure to execute the apoptosis process may lead to various diseases, such as cancer[39]and autoimmune diseases[40],whereas too much apoptosis may lead to neurodegenerative diseases[41]. There are two pathways involved in apoptosis:the mitochondria-dependent and death receptormediated pathways. The mitochondria-dependent pathway is initiated by caspase-9,and the death receptor pathway is initiated by caspase-8. Both pathways ultimately rely on the activation of caspase-3[17-19].

    Figure 4 Effect of QFGs on the mitochondria-dependent and death receptor pathways in HCT-116 and HCT-8 cells.

    Figure 5 Effects of QFGs on the expression of survivin, cyclin D1, CDK4, p21, Bax, Bcl-2, Fas, and FasL in HCT-116 and HCT-8 cells.

    Figure 6 Effect of QFGs on the regulation of PI3K/AKT and ERK signaling pathways in HCT-116 and HCT-8 cells.

    In the mitochondria-dependent apoptosis pathway, mitochondrial dysfunction directly leads to the occurrence of apoptosis and is central to the apoptotic pathway[42].Mitochondrial outer membrane permeabilization (MOMP), an essential event in the mitochondria-mediated apoptosis pathway, causes the transfer of Cyt C and other apoptotic proteins from the mitochondria into the cytosol, which leads to caspase activation and apoptosis[43]. Caspases are the key proteins in the regulation of cell apoptosis. During mitochondria-mediated apoptosis, caspase-3 is an important activator that can be cleaved by its upstream initiators, such as caspase-9. The present study showed that QFGs promoted the activation of both caspase-9 and caspase-3 in HCT-116 and HCT-8 cells. In addition, the process of HCT-116 and HCT-8 cell death induced by QFGs was followed by an increase in the cleavage of caspases-9 and caspase-3, which then promotes the molecular cascade leading to apoptosis. The Bcl-2 protein is the key regulatory protein in mitochondria-dependent apoptosis. Some studies have reported that MOMP occurs when proapoptotic Bax-like proteins form pores in the mitochondria; however, the effect of anti-apoptotic Bcl-2-like members on MOMP is opposite to that of proapoptotic Bax-like proteins on MOMP. Therefore, the ratio of Bax to Bcl-2 is the key to determining cell survival[44,45]. Our study proved that QFGs administration upregulated Bax protein expression and downregulated Bcl-2 protein expression. These results showed that QFGs induce HCT-116 and HCT-8 cell apoptosisviathe mitochondria-dependent pathway.

    During the death receptor apoptosis pathway, caspase-3 is also the ultimate activator of apoptosis. Caspase-3 is cleaved by its downstream initiators, such as caspase-8. In this study, we discovered that both caspase-8 and caspase-3 can be cleaved by QFGs in HCT-116 and HCT-8 cells. In addition, as noted earlier, HCT-116 and HCT-8 cell death induced by QFGs was followed by increased cleavage of caspase-8 and caspase-3, which accelerates apoptosis. In this pathway, death signals are transmittedviacell surface receptors that communicate with the FasL/Fas signaling pathway, which is part of the death receptor pathway. After binding to FasL, Fas trimerizes and interacts with Fas-associated protein with a death domain,which contributes to the cleavage of caspase-8 and caspase-10 and leads to activationof downstream effector caspases, including caspase-3, caspase-6 and caspase-7,ultimately causing apoptosis[24]. In this study, we demonstrated that QFGs treatment upregulated FasL and Fas protein expression. These results showed that QFGs induce HCT-116 and HCT-8 cell apoptosis through the extrinsic apoptosis pathway.

    To determine if the two classic apoptosis pathways were both involved in this study, we added caspase-3/-8/-9 inhibitors and performed the MTT and Hoechst 33258 staining assays to test cell viability and cell apoptosis once again. We found that all three inhibitors markedly inhibited the inhibitory effect of QFGs on cell viability in CRC cells, and inhibited the apoptosis induced by QFGs. This result verified that QFGs induced apoptosis via the mitochondria-dependent and death receptor apoptosis pathways in HCT-116 and HCT-8 cells, which directly revealed the multitarget inhibitory effects of QFGs on CRC cells.

    The pathogenic mechanisms underlying the development of cancer, including CRC,are heterogeneous and regulated by multiple signaling pathways, including PI3K/AKT and ERK[25,26]. Previous studies have reported that the PI3K/AKT and ERK signaling pathways regulate cell growth, apoptosis and metastasis[46]. As one of the important intracellular signal transduction pathways, PI3K/AKT signaling has been reported to play important roles in cell survival, apoptosis and metastasis[47]. In previous studies, activated AKT existed in CRC tumors, which has been shown to be a poor prognostic factor for CRC patients[48]. Overexpression of downstream factors of AKT may result in the activation of the PI3K signaling pathway[46]. ERK signaling is also an important pathway that highly regulates cell proliferation and apoptosis. ERK is a mitogen-activated protein kinase (MAPK), which can be activated by MAPK kinase kinase (e.g., Raf), MAPK kinase (e.g., MEK), and MAPK (e.g., ERK)[49].Activation of the ERK pathway regulates the expression of various genes and proteins that mediate cell proliferation and apoptosis. The present study demonstrated that QFGs suppressed the activations of PI3K, AKT and ERK, which showed that the anticancer effect of QFGs acts on CRC cells via the PI3K/AKT and ERK signaling pathways.

    ARTICLE HIGHLIGHTS

    Research background

    Colorectal cancer (CRC) is a major public health problem, representing the third cause of cancer deaths worldwide. Surgery and adjuvant chemotherapy are the main treatment for CRC.However, 40-50% of patients still die due to recurrence, metastases and drug resistance. In addition, severe side effects caused by chemotherapy agents lead to the deterioration of patient quality-of-life and therapeutic application. Therefore, the search for novel therapies has attracted worldwide attention. Qingjie Fuzheng granules (QFGs) is a traditional Chinese medicine formula with properties of anti-inflammation, antioxidative, antibacterial, immunity enhancement, and digestion promotion. QFGs has been widely used and found to be clinically effective in various cancer treatments, including CRC, and has few side effects. However, the precise mechanisms and molecular signaling pathways involved in the activity of QFGs’anticancer effects have not been reported in the literature. In this study, we hypothesized that QFGs can inhibit the growth of CRC cells, and that its mechanism is closely related to one or more intracellular signal transduction pathways.

    Research motivation

    To better understand the mechanism underlying the potential anti-cancer effect of QFGs on the human CRC cell variants HCT-116 and HCT-8.

    Research objectives

    To elucidate the effect of QFGs on the biological function of CRC cells, and to investigate this biological function to explore the exact mechanism of QFGs effects on CRC cells.

    Research methods

    First, cell viability and cytotoxicity were measured by performing MTT and LDH assays. We evaluated the role of QFGs in cell proliferation and apoptosis by assessing colony formation using Hoechst 33258. Second, cell cycle and apoptosis levels were measured by fluorescenceactivated cell sorting. The expression levels of survivin, cyclin D1, CDK4, p21, Bax, Bcl-2, Fas,FasL, and cleaved-caspase-3/-8/-9 were measured by performing western blotting and caspase activity assays. Furthermore, inhibitors of caspase-3/-8/-9 were also used to elucidate the exact apoptosis pathway induced by QFGs in cancer cells. Finally, activation of the PI3K/AKT and ERK signaling pathways was examined using the western blot assay to investigate the possible mechanism.

    Research results

    MTT and LDH assays revealed that after 0.5-2.0 mg/mL of QFGs treatment, cell viability was reduced by (6.90% ± 1.03%)-(59.70% ± 1.51%) (HCT-116; P < 0.05) and (5.56% ± 4.52%)-(49.44% ±2.47%) (HCT-8; P < 0.05). Cytotoxicity was increased from 0.52 ± 0.023 to 0.77±0.002 (HCT-116; P< 0.01) and from 0.56 ± 0.054 to 0.81 ± 0.044 (HCT-8;P< 0.01) compared with non-QFGs treatment groups. Additionally, colony formation and Hoechst 33258 staining assays showed that QFGs inhibited proliferation and induced apoptosis in CRC cells. QFGs also increased the expression levels of Bax, Fas, and FasL, decreased the level of Bcl-2, and stimulated the activation of caspase-3/-8/-9, which were revealed by western blot and caspase activity assays. In contrast,upon adding the three caspase inhibitors, the suppression effect of QFGs on cell viability and apoptosis were markedly inhibited. Moreover, QFGs suppressed the phosphorylation levels of PI3K, AKT and ERK.

    Research conclusions

    These results demonstrated that QFGs inhibit CRC cell proliferation and induce apoptosis by suppressing the PI3K/AKT and ERK signaling pathways. This indicated that QFGs are a potential new therapeutic treatment for CRC and other cancers.

    Research perspectives

    Traditional Chinese Medicine (TCM) is important for the treatment of many cancers and has a long history of clinical use. If the effects and mechanisms of TCM are further elucidated, it may provide a more effective treatment for many cancer types.

    猜你喜歡
    歧路建設(shè)者共同體
    建設(shè)者
    《金憶元街的建設(shè)者》
    火花(2022年5期)2022-06-16 11:03:18
    愛的共同體
    共建人與自然生命共同體
    建設(shè)者
    歧路亡①羊
    構(gòu)建和諧共同體 齊抓共管成合力
    甘肅教育(2020年17期)2020-10-28 09:01:36
    共同體的戰(zhàn)斗
    無題(5)
    夜路吟
    在线观看免费视频日本深夜| 亚洲一区二区三区不卡视频| 久久久久免费精品人妻一区二区| 少妇熟女aⅴ在线视频| 精品久久久久久久人妻蜜臀av| 国产精品亚洲一级av第二区| 国产私拍福利视频在线观看| 精品乱码久久久久久99久播| a在线观看视频网站| 亚洲avbb在线观看| 欧美精品亚洲一区二区| 色综合亚洲欧美另类图片| 国产高清有码在线观看视频 | 超碰成人久久| 国内久久婷婷六月综合欲色啪| 精品一区二区三区av网在线观看| 黑人欧美特级aaaaaa片| 动漫黄色视频在线观看| 中文字幕人妻丝袜一区二区| 嫩草影院精品99| 成人av在线播放网站| 亚洲人成电影免费在线| 亚洲av中文字字幕乱码综合| 久久久久久人人人人人| 久久久久久国产a免费观看| 黄片大片在线免费观看| 在线观看免费午夜福利视频| 啦啦啦观看免费观看视频高清| 中文亚洲av片在线观看爽| 老汉色∧v一级毛片| 亚洲激情在线av| a级毛片在线看网站| 老司机福利观看| 欧美乱色亚洲激情| av在线播放免费不卡| 精品国产乱码久久久久久男人| 午夜激情福利司机影院| 欧美最黄视频在线播放免费| 国产精品久久久久久亚洲av鲁大| 国产午夜精品久久久久久| 制服诱惑二区| 精品久久久久久久人妻蜜臀av| 精品福利观看| 999久久久国产精品视频| 国产免费av片在线观看野外av| 丰满人妻一区二区三区视频av | 久久婷婷人人爽人人干人人爱| 成人午夜高清在线视频| 色尼玛亚洲综合影院| 99国产精品一区二区三区| 亚洲自拍偷在线| 听说在线观看完整版免费高清| 99久久国产精品久久久| 国产午夜精品论理片| 男女午夜视频在线观看| 黄色视频,在线免费观看| 国产成人av教育| 一级毛片精品| 国产野战对白在线观看| av在线天堂中文字幕| 在线看三级毛片| 日韩欧美在线乱码| 亚洲国产精品999在线| 俄罗斯特黄特色一大片| 国产激情久久老熟女| www日本在线高清视频| 美女大奶头视频| 日韩欧美免费精品| 欧美中文综合在线视频| 国产99白浆流出| 亚洲18禁久久av| 91国产中文字幕| 精品第一国产精品| 久久香蕉精品热| 日本精品一区二区三区蜜桃| 在线观看日韩欧美| 亚洲专区字幕在线| 无遮挡黄片免费观看| 一个人免费在线观看电影 | 一本大道久久a久久精品| 亚洲欧美日韩无卡精品| 亚洲国产欧美一区二区综合| 人妻丰满熟妇av一区二区三区| 国产一区二区三区视频了| 天堂动漫精品| 久久久精品国产亚洲av高清涩受| 老司机深夜福利视频在线观看| 亚洲成人精品中文字幕电影| 精品久久久久久久末码| www.熟女人妻精品国产| 麻豆久久精品国产亚洲av| 久久精品影院6| 操出白浆在线播放| 搞女人的毛片| 少妇被粗大的猛进出69影院| 麻豆国产av国片精品| 国产探花在线观看一区二区| 9191精品国产免费久久| 免费看a级黄色片| 男女之事视频高清在线观看| 观看免费一级毛片| ponron亚洲| 九九热线精品视视频播放| 国产视频内射| 亚洲va日本ⅴa欧美va伊人久久| 国产精品久久久av美女十八| 麻豆av在线久日| 观看免费一级毛片| 天天躁夜夜躁狠狠躁躁| 欧美性猛交╳xxx乱大交人| 成人国语在线视频| 精品不卡国产一区二区三区| 午夜福利免费观看在线| 特大巨黑吊av在线直播| 国产黄片美女视频| 变态另类丝袜制服| 精品久久蜜臀av无| 特级一级黄色大片| 国产精品亚洲一级av第二区| 国内揄拍国产精品人妻在线| 91国产中文字幕| 可以在线观看毛片的网站| 国产精品影院久久| 亚洲aⅴ乱码一区二区在线播放 | 久久久精品欧美日韩精品| 最新在线观看一区二区三区| 小说图片视频综合网站| 九九热线精品视视频播放| 久久久久久亚洲精品国产蜜桃av| 成人高潮视频无遮挡免费网站| 婷婷六月久久综合丁香| 日韩有码中文字幕| 亚洲国产精品合色在线| 亚洲av成人av| 成熟少妇高潮喷水视频| 一进一出抽搐gif免费好疼| 90打野战视频偷拍视频| 国产精品久久电影中文字幕| 午夜视频精品福利| 成人午夜高清在线视频| 国产aⅴ精品一区二区三区波| 久久婷婷成人综合色麻豆| 欧美日韩中文字幕国产精品一区二区三区| 色av中文字幕| 久久久久久九九精品二区国产 | 亚洲欧美精品综合一区二区三区| 国产精品九九99| 成年免费大片在线观看| 又紧又爽又黄一区二区| 亚洲精品粉嫩美女一区| 国产久久久一区二区三区| 午夜两性在线视频| 性色av乱码一区二区三区2| 亚洲国产精品久久男人天堂| 男人舔女人的私密视频| 男女视频在线观看网站免费 | 亚洲人成77777在线视频| 色综合欧美亚洲国产小说| 小说图片视频综合网站| 一个人免费在线观看的高清视频| 国产片内射在线| 悠悠久久av| 久久中文字幕人妻熟女| 亚洲人成网站高清观看| 久久精品人妻少妇| 亚洲精华国产精华精| 黄色a级毛片大全视频| 亚洲av成人不卡在线观看播放网| 麻豆av在线久日| 黄色毛片三级朝国网站| 久久婷婷人人爽人人干人人爱| 麻豆国产av国片精品| 国产午夜精品论理片| 国产精品久久久人人做人人爽| tocl精华| 曰老女人黄片| 国产精品九九99| 欧美一级毛片孕妇| 999久久久精品免费观看国产| 九九热线精品视视频播放| 亚洲av电影不卡..在线观看| 嫩草影视91久久| 人成视频在线观看免费观看| 亚洲第一电影网av| 欧美 亚洲 国产 日韩一| 国产又色又爽无遮挡免费看| 嫁个100分男人电影在线观看| 欧美成人免费av一区二区三区| 一边摸一边做爽爽视频免费| 少妇裸体淫交视频免费看高清 | 国产一区二区在线观看日韩 | 亚洲熟妇中文字幕五十中出| 午夜成年电影在线免费观看| 亚洲乱码一区二区免费版| 色精品久久人妻99蜜桃| 国产激情欧美一区二区| 国产又色又爽无遮挡免费看| 操出白浆在线播放| 国产精品久久久久久久电影 | 国产高清视频在线观看网站| 亚洲国产中文字幕在线视频| 欧美一区二区精品小视频在线| 久久精品国产亚洲av高清一级| 最近最新中文字幕大全电影3| 97碰自拍视频| tocl精华| 精品少妇一区二区三区视频日本电影| 叶爱在线成人免费视频播放| 伦理电影免费视频| 搡老岳熟女国产| 真人一进一出gif抽搐免费| 国产精品亚洲美女久久久| 香蕉av资源在线| 在线看三级毛片| 久久香蕉精品热| 叶爱在线成人免费视频播放| 丁香六月欧美| 午夜免费观看网址| 老司机福利观看| 国产精品综合久久久久久久免费| 俺也久久电影网| 国产亚洲精品一区二区www| 国产精品一及| 女人被狂操c到高潮| 精品国产超薄肉色丝袜足j| 午夜亚洲福利在线播放| 国产成人av激情在线播放| 亚洲国产精品sss在线观看| 一本一本综合久久| 在线观看免费视频日本深夜| 午夜亚洲福利在线播放| 午夜日韩欧美国产| www.999成人在线观看| 久久草成人影院| 三级国产精品欧美在线观看 | 国产单亲对白刺激| 男人舔女人的私密视频| 一本一本综合久久| 久热爱精品视频在线9| 久久久久久大精品| 人妻丰满熟妇av一区二区三区| www日本黄色视频网| 欧美色视频一区免费| 757午夜福利合集在线观看| 麻豆国产97在线/欧美 | 国产精品av视频在线免费观看| 欧美色欧美亚洲另类二区| 欧美成人午夜精品| 两个人视频免费观看高清| 国产精品野战在线观看| 免费在线观看完整版高清| 欧美黄色淫秽网站| 男人舔奶头视频| 国产精华一区二区三区| 欧美精品亚洲一区二区| 18禁美女被吸乳视频| 国产精品电影一区二区三区| 久久精品国产亚洲av香蕉五月| 中文字幕精品亚洲无线码一区| 精品少妇一区二区三区视频日本电影| 欧美zozozo另类| 精品久久久久久久人妻蜜臀av| 一进一出好大好爽视频| 亚洲av电影不卡..在线观看| 国产成人系列免费观看| 精品无人区乱码1区二区| 全区人妻精品视频| av超薄肉色丝袜交足视频| 国产亚洲精品综合一区在线观看 | 亚洲午夜理论影院| 国产一区二区激情短视频| 色尼玛亚洲综合影院| 久久久国产欧美日韩av| 国产麻豆成人av免费视频| 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| 国产精品乱码一区二三区的特点| 丝袜美腿诱惑在线| 亚洲国产日韩欧美精品在线观看 | 国产av一区二区精品久久| 国产69精品久久久久777片 | 香蕉丝袜av| 可以在线观看毛片的网站| 久久天堂一区二区三区四区| 国产激情久久老熟女| 欧美日本亚洲视频在线播放| 国内精品久久久久久久电影| www.自偷自拍.com| 99精品久久久久人妻精品| 精品少妇一区二区三区视频日本电影| 欧美黑人巨大hd| 欧美乱码精品一区二区三区| 九色成人免费人妻av| 国产精品 欧美亚洲| 天天躁狠狠躁夜夜躁狠狠躁| 美女 人体艺术 gogo| 亚洲精品中文字幕在线视频| 国产精华一区二区三区| 夜夜看夜夜爽夜夜摸| 亚洲黑人精品在线| 手机成人av网站| 极品教师在线免费播放| 99久久99久久久精品蜜桃| 亚洲熟妇中文字幕五十中出| 91老司机精品| 99精品欧美一区二区三区四区| 日本三级黄在线观看| 美女大奶头视频| 国内精品久久久久久久电影| 国产精品久久久久久亚洲av鲁大| 午夜老司机福利片| 国内少妇人妻偷人精品xxx网站 | 黄色毛片三级朝国网站| 长腿黑丝高跟| 看免费av毛片| 亚洲熟妇中文字幕五十中出| 怎么达到女性高潮| 国产精品一区二区三区四区久久| 精品一区二区三区av网在线观看| 五月玫瑰六月丁香| 黑人欧美特级aaaaaa片| 亚洲精品色激情综合| 国产精品永久免费网站| 在线永久观看黄色视频| 亚洲欧美精品综合久久99| 久久欧美精品欧美久久欧美| 久久这里只有精品19| 国产爱豆传媒在线观看 | 色在线成人网| 欧美中文日本在线观看视频| 成人三级黄色视频| 91麻豆av在线| 午夜福利欧美成人| 人妻久久中文字幕网| 亚洲国产精品sss在线观看| 99久久精品国产亚洲精品| 大型黄色视频在线免费观看| 三级毛片av免费| 老司机靠b影院| 午夜免费激情av| 在线a可以看的网站| 亚洲aⅴ乱码一区二区在线播放 | 精品熟女少妇八av免费久了| 久久久久久人人人人人| 麻豆成人午夜福利视频| 免费在线观看影片大全网站| 麻豆国产av国片精品| 亚洲精品在线美女| 欧美色视频一区免费| 亚洲 欧美 日韩 在线 免费| 在线观看免费日韩欧美大片| 日本黄色视频三级网站网址| 嫁个100分男人电影在线观看| 在线a可以看的网站| 色精品久久人妻99蜜桃| 熟女电影av网| 久久性视频一级片| 夜夜看夜夜爽夜夜摸| 俺也久久电影网| 又紧又爽又黄一区二区| 日本一二三区视频观看| 久久婷婷成人综合色麻豆| 制服诱惑二区| 久久久久久九九精品二区国产 | 搡老岳熟女国产| 又爽又黄无遮挡网站| 亚洲美女黄片视频| 欧美成人免费av一区二区三区| 啦啦啦免费观看视频1| 性欧美人与动物交配| 日本五十路高清| 精品一区二区三区四区五区乱码| 国产亚洲精品第一综合不卡| 91麻豆av在线| 久久99热这里只有精品18| 亚洲国产精品久久男人天堂| 亚洲成人国产一区在线观看| 怎么达到女性高潮| 欧美一区二区国产精品久久精品 | 88av欧美| 欧美最黄视频在线播放免费| 九九热线精品视视频播放| 久久久久久久久中文| or卡值多少钱| 国产伦一二天堂av在线观看| 亚洲一码二码三码区别大吗| 国语自产精品视频在线第100页| 久久亚洲真实| 国产精品1区2区在线观看.| 啪啪无遮挡十八禁网站| 精品不卡国产一区二区三区| tocl精华| 久久欧美精品欧美久久欧美| e午夜精品久久久久久久| 人人妻人人澡欧美一区二区| 久久精品国产99精品国产亚洲性色| 国产在线观看jvid| svipshipincom国产片| 99久久精品热视频| xxx96com| 国产亚洲欧美在线一区二区| 国产亚洲精品一区二区www| 制服丝袜大香蕉在线| 亚洲欧美日韩东京热| 黄频高清免费视频| 国产精品1区2区在线观看.| 亚洲精品久久国产高清桃花| 妹子高潮喷水视频| 国产成人av教育| 亚洲真实伦在线观看| 中文字幕精品亚洲无线码一区| 人人妻人人澡欧美一区二区| 校园春色视频在线观看| 精品国产美女av久久久久小说| 精品第一国产精品| av有码第一页| 熟妇人妻久久中文字幕3abv| 久久久久久久精品吃奶| 日本成人三级电影网站| 成年人黄色毛片网站| 少妇粗大呻吟视频| 免费在线观看成人毛片| 99re在线观看精品视频| 色尼玛亚洲综合影院| 国产精品免费一区二区三区在线| 精品国内亚洲2022精品成人| a级毛片a级免费在线| 国产亚洲精品一区二区www| 狠狠狠狠99中文字幕| 国产精品98久久久久久宅男小说| 久久性视频一级片| 此物有八面人人有两片| 老司机福利观看| 日韩欧美一区二区三区在线观看| 成人国语在线视频| 亚洲成人中文字幕在线播放| 日韩欧美国产在线观看| 日本精品一区二区三区蜜桃| 精品久久久久久久人妻蜜臀av| or卡值多少钱| 亚洲av五月六月丁香网| 中亚洲国语对白在线视频| 亚洲国产高清在线一区二区三| a在线观看视频网站| 成人av一区二区三区在线看| 免费在线观看成人毛片| 国产精品香港三级国产av潘金莲| 国产精品一区二区免费欧美| 中文字幕人妻丝袜一区二区| 精品高清国产在线一区| 叶爱在线成人免费视频播放| 亚洲一区中文字幕在线| 这个男人来自地球电影免费观看| 国产高清视频在线播放一区| 夜夜夜夜夜久久久久| 亚洲国产看品久久| 国产又黄又爽又无遮挡在线| 国产一区二区在线av高清观看| 国产亚洲精品av在线| 亚洲欧美一区二区三区黑人| 日本成人三级电影网站| 久99久视频精品免费| 日本五十路高清| 久久草成人影院| 一进一出好大好爽视频| 免费一级毛片在线播放高清视频| 亚洲性夜色夜夜综合| 亚洲av五月六月丁香网| 好男人在线观看高清免费视频| 久久精品人妻少妇| 精品欧美一区二区三区在线| 在线免费观看的www视频| 一级毛片高清免费大全| av视频在线观看入口| 日韩中文字幕欧美一区二区| 99精品久久久久人妻精品| 人人妻,人人澡人人爽秒播| 欧美日韩亚洲国产一区二区在线观看| 欧美黑人欧美精品刺激| 国产熟女xx| 亚洲七黄色美女视频| 中文亚洲av片在线观看爽| 亚洲人与动物交配视频| av在线播放免费不卡| 99热这里只有是精品50| 亚洲人成77777在线视频| 天天一区二区日本电影三级| 丝袜人妻中文字幕| 久久欧美精品欧美久久欧美| 成人亚洲精品av一区二区| 色综合亚洲欧美另类图片| 精品国产乱子伦一区二区三区| or卡值多少钱| 亚洲专区国产一区二区| 99精品久久久久人妻精品| 精品久久久久久,| 亚洲天堂国产精品一区在线| 久久久久久久午夜电影| 亚洲一区中文字幕在线| 最近在线观看免费完整版| 亚洲成人精品中文字幕电影| 久久伊人香网站| 日韩三级视频一区二区三区| 搡老熟女国产l中国老女人| 一个人观看的视频www高清免费观看 | 久久久久久亚洲精品国产蜜桃av| 欧美黑人精品巨大| 国产精品野战在线观看| 日日干狠狠操夜夜爽| 久久精品91蜜桃| e午夜精品久久久久久久| 丰满的人妻完整版| 国产亚洲精品综合一区在线观看 | 久久婷婷成人综合色麻豆| 国产精品亚洲av一区麻豆| 国产蜜桃级精品一区二区三区| 国产精品野战在线观看| 啪啪无遮挡十八禁网站| 亚洲专区中文字幕在线| 法律面前人人平等表现在哪些方面| 日本精品一区二区三区蜜桃| 欧美绝顶高潮抽搐喷水| 桃红色精品国产亚洲av| 成人国产一区最新在线观看| 人妻久久中文字幕网| 哪里可以看免费的av片| 在线十欧美十亚洲十日本专区| 午夜精品在线福利| 美女大奶头视频| 黄色a级毛片大全视频| 一个人免费在线观看电影 | 国产高清激情床上av| 国产1区2区3区精品| 一夜夜www| 老汉色∧v一级毛片| 久久久久国产一级毛片高清牌| 日本精品一区二区三区蜜桃| 国产伦在线观看视频一区| 十八禁人妻一区二区| av天堂在线播放| 丰满人妻熟妇乱又伦精品不卡| 亚洲国产欧美人成| 欧美不卡视频在线免费观看 | 国产不卡一卡二| 啦啦啦观看免费观看视频高清| 欧美性猛交╳xxx乱大交人| 国产精品一区二区精品视频观看| 精品熟女少妇八av免费久了| 亚洲美女视频黄频| 99国产精品99久久久久| 欧美成人一区二区免费高清观看 | 成在线人永久免费视频| 午夜两性在线视频| 国产单亲对白刺激| 亚洲精品美女久久av网站| 激情在线观看视频在线高清| 欧美日韩一级在线毛片| 啦啦啦免费观看视频1| 丰满人妻熟妇乱又伦精品不卡| 美女黄网站色视频| 国产av麻豆久久久久久久| 母亲3免费完整高清在线观看| 精品国产超薄肉色丝袜足j| 亚洲成人久久爱视频| 亚洲中文字幕一区二区三区有码在线看 | 欧美日韩国产亚洲二区| av有码第一页| 精品高清国产在线一区| 露出奶头的视频| 又黄又粗又硬又大视频| 国产精品 国内视频| 日本黄色视频三级网站网址| 久久久国产成人免费| 999久久久国产精品视频| av片东京热男人的天堂| 亚洲国产看品久久| 国产人伦9x9x在线观看| 国产av不卡久久| 亚洲中文日韩欧美视频| 欧美日韩乱码在线| 国产乱人伦免费视频| 国产亚洲av嫩草精品影院| 好男人电影高清在线观看| 又爽又黄无遮挡网站| 天天躁狠狠躁夜夜躁狠狠躁| 亚洲欧美激情综合另类| 色噜噜av男人的天堂激情| 欧美大码av| 国产一区二区激情短视频| 亚洲国产欧美网| 51午夜福利影视在线观看| 精品少妇一区二区三区视频日本电影| 久久午夜亚洲精品久久| 亚洲精品在线观看二区| 午夜福利成人在线免费观看| 久久中文看片网| 成人精品一区二区免费| 免费看美女性在线毛片视频| 国产精品一区二区精品视频观看| 制服诱惑二区| 亚洲真实伦在线观看| 国产成人啪精品午夜网站| 国产成年人精品一区二区| netflix在线观看网站| 亚洲一码二码三码区别大吗| 亚洲熟女毛片儿| 午夜老司机福利片| 国产精品香港三级国产av潘金莲| 淫妇啪啪啪对白视频| 午夜福利免费观看在线| 成人国产综合亚洲| 亚洲美女视频黄频| 色综合欧美亚洲国产小说| 国产精品一及| 91在线观看av|