• <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 ?

    GPP (composition of Ganoderma lucidum polysaccharides and Polyporus umbellatus polysaccharides) protects against DSS-induced murine colitis by enhancing immune function and regulating intestinal flora

    2022-06-20 08:31:18LiyunLiYnnnGuoQingHungXiojinShiQingqingLiuFngWngQingfeiLiuKngYuZhoWng
    食品科學與人類健康(英文) 2022年4期

    Liyun Li, Ynnn Guo, Qing Hung, Xiojin Shi, Qingqing Liu,Fng Wng, Qingfei Liu, Kng Yu, Zho Wng,*

    a MOE Key Laboratory of Protein Science, School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China

    b Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China

    c Tianjin Research Institute for Advanced Equipment, Tianjin 300300, China

    d Department of Clinical Nutrition and Department of Health Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China

    Keywords:

    Ganoderma lucidum polysaccharides

    Polyporus umbellatus polysaccharides

    Immune function

    Intestinal flora

    Colitis

    A B S T R A C T

    Previous study have demonstrated that a compound composed of water-soluable Ganoderma lucidum polysaccharides (GLP) and Polyporus umbellatus polysaccharides (PUP) in a ratio of 3 : 1 named GPP enhances innate immune function in mice through enhancing the function of macrophage cells and activity of natural killer (NK) cells. Here in our research, we further investigated the effect of GPP on the diversity and composition of intestinal flora, and explored its effect on colitis model mice. The immunoregulatory verification experiments of GPP were conducted in both normal and DSS-induced mice model. Our research showed that GPP increased the diversity of intestinal microorganisms in mice with the extension of administration time. Daily GPP intake attenuated DSS-induced colon injury, protected the splenic lymphocyte proliferation ability, enhanced the serum hemolysin synthesis, and increased peripheral phagocytes and NK cell activity in model mice. Comparisons of the predominant gene pathways of the bacterial microbiota showed that DNA repair and recombination, base mismatch repair pathways was stronger in GPP-treatment group than in control group, indicating the possible molecular mechanisms of immune function regulation.Our study showed that GPP regulated immune function in both health and colitis model, and had a positive effect on maintaining intestinal flora homeostasis.

    1. Introduction

    Ganoderma lucidumandPolyporus umbellatusare rare edible and medicinal fungi that have been used to treat diseases and regulate body balance in China for more than 2 000 years [1-3].According to the theory of traditional Chinese medicine, the combination ofG. lucidumandP. umbellatusare of great significance since they had the characteristic of supplement without causing stagnation.G. lucidumhad the activity to strengthen body resistance and consolidate the constitution of patients (in traditional Chinese called Fuzheng Guben) [4], whileP. umbellatuscould induce diuresis and excrete dampness [5].G. lucidumandP. umbellatuscontains a variety of active components, among which researchers most interested in the polysaccharides. Polysaccharides from edible mushrooms (EMPs) are a class of natural macromolecule polymers that have a wide range of biological activities and relatively low toxicity [6,7]. The different biological functions of EMPs include anti-tumor, anti-oxidant, regulating gut microbiota and immunoregulatory activities [8]. BothG. lucidumpolysaccharides(GLP) andP. umbellatuspolysaccharides (PUP) exhibited immunoregulation effect, which regulated the function of a variety of immune cells, such as macrophages, T cells, B cells, dendritic cells and NK cells [3,9-11]. In combination with chemotherapy drugs, both GLP and PUP can enhance drug efficacy and reduce toxic side effect [12,13].

    Previous studies showed that GPP, a combination of GLP and PUP in a ratio of 3 : 1, enhanced the function of macrophages by enhancing the phagocytic ability, the production of nitric oxide (NO),the mRNA expression level of inducible nitric oxide synthase (iNOS)and tumor necrosis factor-α (TNF-α). GPP was slightly better than GLP but was significantly better than PUP in a certain concentration range in enhancing macrophage functions, indicating that the two polysaccharides GLP and PUP can synergistically enhance the phagocytosis function of macrophages in a certain ratio and concentration. Moreover, GPP significantly improved macrophage phagocytic function and natural killer (NK) cells activity after being administered to mice at a dose of 0, 36, 120, 360 mg/kg BW orally for 30 days, demonstrating that GPP is capable of enhancing innate immune function in mice [14]. The study demonstrated the potential of GPP develop into immunomodulation dietary supplement.However, as edible and medicinal fungus, the molecular mechanism about how GPP affect the immune system and immune cells, and whether GPP has immune modulating function on disease model remains unknown. Gut microbiota is an important constituent part of intestinal microecosystem, it is extremely complicated and dynamic.More than 1011CFU/g of microbes were found in colon, where they complete their colonization. The host provides nutrients for gut microbiota, and gut microbiota in return maintains the intestinal endothelial barrier, immune homeostasis, and protects the host from the threat of pathogenic bacteria [15,16]. By metabolizing nutrients in the diet, gut microbiota provides large quantities of metabolites to human body. As a result, diet are considered to be critical in the regulation of gut microbiota and host health factors [17].

    Previous studies showed that certain polysaccharides from traditional Chinese medicine can affect the ecological structure and metabolism of gut microbiota, thus improve the health status of the host [18]. GLP can reduce the ratio of Firmicutes to Bacteroidetes in the gut microbiota of mice fed with high fat diet, maintain the integrity of intestinal barrier, reduce metabolic endotoxemia, and thus reduce the weight and fat accumulation in mice [19]. In a chronic pancreatitis mouse model induced by diethyldithiocarbamate (DDC),supplementation of GLP improved the symptoms of inflammation,altered the composition and diversity of intestinal microbiota,decreased the relative abundance of phylum Bacteroidetes and increased that of phylum Firmictutes. At the genus level, GLP increased the relative abundance of the beneficial bacteria such as Lactobacillales, Roseburia and Lachnospiraceae [20]. Furthermore,GLP can regulate the gut microbiota of normal rats, improve the intestinal barrier function, regulate the intestinal immune function and gut microbiota abundance in rats [21].

    Ulcerative colitis (UC) is a type of inflammatory bowel disease(IBD), mainly characterized by chronic intestinal mucosal damage caused by recurrent IBD in susceptible populations [22,23]. A widely accepted view of the etiology and pathogenesis of UC is that the development of the disease is accompanied by the change or disorder in the function of immune response, mucosal barriers, and metabolic balance in intestinal epithelium [24]. DSS-induced colitis is a wellestablished animal model for the study of colon inflammation, which has similar clinical symptoms and histological changes to IBD patients [25,26].

    In this study, we mainly investigated the effect of GPP on immune system and the structure of gut microbiota in both normal and DSS-induced colitis model mice, and the possible molecular mechanism behind them. Our research found that GPP increased the diversity of intestinal microorganisms in normal mice.The administration time of GPP has significant influence on the species distribution of gut microbiota compared with the dosage of administration in normal mice.

    In DSS-induced mice model, daily GPP intake attenuated DSS-induced colon injury, including decreasing the disease activity index(DAI) score and reducing the histological injury of colon. GPP treatment in DSS-induced mice also protected the splenic lymphocyte proliferation ability, enhanced the serum hemolysin synthesis,increased peripheral phagocytes and NK cell activity in colitis model mice, indicating that GPP promotes immune function in both normal and colitis model mice. Comparisons of the predominant gene pathways of the bacterial microbiota in different groups showed that DNA repair and recombination, base mismatch repair pathways is stronger in GPP treatment group, suggesting its protective function.Taken together, these findings revealed that GPP regulates immune function in both normal and colitis model and has a positive effect on maintaining intestinal flora homeostasis.

    2. Materials and methods

    2.1 Materials

    GLP and PUP sheep were purchased from Yuanye Biotechnology(Shanghai, China). Lipopolysaccharide (LPS), NO assay kits, Neutral Red Cell Proliferation, Cytotoxicology Assay Kits were obtained from Beyotime Biotechnology (Shanghai, China).

    2.2 Animals and induction of colitis

    Six to eight-week-old ICR mice weighing about 17–22 g were purchased from the laboratory animal center of Tsinghua University(Beijing, China) and were housed in cages with free access to food(Keao Xieli Feed Co., Ltd, Beijing) and water in a room with an ambient temperature of (22 ± 2) °C and a 12 h light/dark cycle.All animal experiments were conducted according to the relevant guidelines and regulations and with the approval of the Institutional Ethical Committee of China. All the animal experiments are followed by the ARRIVE reporting guidelines [27].

    The immunoregulatory verification experiments of GPP were divided into two parts, in normal animals and in DSS-induced mice model. In the normal animal treatment part, a total of 192 experimental animals were randomly divided into 4 batches of 48 each. The animals were divided into solvent control group, low dose group (treated with 0.036 g/kg BW GPP per day), medium dose group(treated with 0.12 g/kg BW GPP per day) and high dose group (treated with 0.36 g/kg BW GPP per day), with 12 animals in each group.In the DSS-induced mice model parts, a total of 240 experimental animals were randomly divided into 4 batches of 60 each. The animals were divided into solvent control group, DSS group as positive control, DSS + low dose group, DSS + medium dose group and DSS + high dose group (the GPP concentrations were the same as described above), and also with 12 animals in each group. GPP of different concentrations was given by oral administration to animals at a fixed time from day 1 to day 30 (control groups with water),separately.

    In the DSS-induced mice model parts, animals started to receive DSS from day 23 by drinking 5% (m/V) DSS (relative molecular weight 36 000–50 000; MP Bi medicals) or regular water in control group for 7 days to induce acute colitis. All mice were carefully monitored daily for signs of disease, including weight loss, stool consistency and rectal bleeding. On day 31, mice were humanely sacrificed and colon and feces were harvested. Fresh faces samples of each group of different time point were collected and stored at ?80 °C immediately, the samples were subsequently sent to analyze intestinal flora (Novogene, China).

    2.3 16S rRNA analysis

    The total genome DNA of feces samples was extracted and 16S rRNA of 16S V4 regions were amplified used specific primer 16S V4: 515F – 806R with the barcode. PCR reactions were set up with 15 μL of Phusion? High-Fidelity PCR Master Mix (New England Biolabs); 2 μmol/L of forward and reverse primers, and about 10 ng of template DNA. Thermal cycling consisted of initial denaturation at 98 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, and elongation at 72 °C for 30 s. Finally 72 °C for 5 min. After amplification generation, PCR products were quantitated by 2% agarose gel electrophoresis. The sequencing libraries were generated using TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, USA) following manufacturer’s recommendations and index codes. The library quality was then assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on an Illumina NovaSeq platform and 250 bp paired-end reads were generated. Data were split and assembled by FLASH (V1.2.7) [28].Data filtration was performed with QIIME (V1.9.1) [29]. We used UCHIME algorithm [30]to detect and remove chimera sequences to obtain the effective tags, and adopted Uparse software [31]to perform sequence analysis. Sequences with more than 97% similarity were regarded as the same OTUs. The taxonomic information of OTUs was then annotated with Silva Database [32]based on Mothur algorithm. The phylogenetic relationship of different OTUs was conducted by MUSCLE software (V3.8.31) [33].OTUs abundance information were normalized based on the least sequences sample as a standard of sequence number. Alpha diversity, beta diversity and further analysis were all performed basing on this normalized abundance data.

    2.4 Measurement of immune organ indexes

    On day 31, mice were weighed and sacrificed. The spleen and thymus were immediately removed and weighed. The spleen or thymus index was calculated according to the following formula:

    2.5 Macrophage phagocytosis assay

    Chicken blood was placed in a conical flask with glass beads,shaken fully in one direction, washed for 3 times with normal saline,centrifuged (2 000 r/min, 10 min) and prepared into chicken red blood cell suspension of 2% (V/V) with normal saline. Each mouse was intraperitoneally injected with 1% chicken red blood cell suspension.After 30 min, the mice were sacrificed by cervical dislocation. Inject 2 mL normal saline intraperitoneally, and gently rub the abdomen of the mice for 20 seconds. Put the mouse into enamel box with wet sand cloth and transfer it to 37 °C incubation box for 30 min. Rinse with normal saline to remove uncoated cells. Dry and fix the cells with 1 : 1 acetone methanol solution, and dye with 4% (V/V) Giemsaphosphoric acid buffer for 3 min, then rinse with distilled water and dry them, and were observed and counted under the microscope.

    2.6 The determination of NK cell activity

    The splenic cell suspension (effector cells) was prepared at a final concentration of 5 × 106cells/mL with complete culture medium of RPML1640. For NK cell activity detection, both 100 μL target cells(YAC-1 cells) and 100 μL effector cells (with a ratio of 50 : 1) were seeded to U-shaped 96-well culture plate. The cells were cultured at cell incubator of 37 °C with 5% CO2for 4 h. The plate was then centrifuged at 1 500 r/min for 5 min, and 100 μL supernatant was pipetted from each well to a new 96-well culture plate and added with LDH matrix solution. After reaction for 5 min, 30 μL of 1 mol/L HCl was added to each well, and the optical density value at 490 nm were measured.

    2.7 Tlymphocyte proliferation assay

    The spleen cell suspension was prepared by filtering with 200-mesh sieve and washed with Hanks solution, and adjusted to a final concentration of 3 × 106cells/ml. For the lymphocyte proliferation reaction, each splenic cell suspension was divided into two wells, one well was added with 50 μL ConA solution (equivalent to 5.0 g/mL)and the other well was used as control. The cells were cultured at cell incubator of 37 °C with 5% CO2for 72 h. Before the end of culture, 0.7 mL of supernatant was gently pipetted from each well and 0.7 mL of RPMI1640 without calf serum was added for 4 h. 50 μL MTT(5 mg/mL) was subsequently added to each well for further culture for 4 h. Discard the supernatant, add 1 mL acidic isopropanol into each well, blow and mix well to completely dissolve the purple crystal.The samples were pipetted into 96-well culture plates with 3 parallel wells. The OD value was determined by a microplate reader at a wave length of 570 nm.

    2.8 Other immune function test

    Other immune function test like the determination of the hemolytic value (HC50), detection of antibody generating cells, induction of DTH in mice by Sheep Red Blood cell (SRBC), carbon clearance test were also conducted. All the experiments were operated according to the criteria for the enhancement of immune function in the“Technical Specification for Inspection and Evaluation of Health Food”(2003 edition) issued by the Ministry of Public Health of China.

    2.9 PAS staining

    Paraffin slides were washed by pure xylene, pure ethanol and 75% ethanol, and were kept in tap water. For PAS staining, sections were stained with PAS dye solution B for 10–15 min, PAS A for 25–30 min in the dark, and then were stained with PAS C for 30 s(Wuhan Servicebio technology Co., Ltd, China). Finally, the slides were dehydrated by pure ethanol and pure xylene, and covered with neutral resin. All slices were captured by automatic slides scanner machine (Panoramic SCAN, 3DHISTECH, Hungary).

    2.10 Histological analysis

    The histological score of each mouse was calculated according to epithelium damage and cell inflltration [34]. The slices stained with PAS and Alcian Blue were analyzed and the scores were con firmed as follows: 0, no significant change; 1, low level of inflammation with scattered inflammatory cells; 2, moderate inflammation with multiple foci; 3, high level of inflammation with marked wall thickening;4, maximal severity of inflammation with transmural leukocyte inflltration and loss of goblet cells [35]. The neutrophil inflltration in colon was also analyzed according to the characteristic of neutrophil,and the number of neutrophil aggregation was also recorded [35].

    2.11 Goblet cells quantification assay

    The quantification of goblet cells was based on Alcian Blue staining slice scan image. Following the method described by Montrose et al. [36], Fiji/ImageJ (NIH) software was used to perform color deconvolution and Gaussian blur. After color deconvolution,the total mucosal area was selected by Gaussian blur in sigma 20 and threshold auto-selection method “Huang”. The selected area was masked, measured and labeled as “mucosal area”. Similar to the selection of total mucosal area, total goblet cells area was selected by Gaussian blur in sigma 2 and threshold auto-selection method“Default”. Then the selected area was masked, measured and labeled as “goblet cell area”. The ratio of goblet cell area and total mucosal area was regarded as the proportion of mucosal surface occupied by goblet cell as an index to quantify goblet cells.

    2.12 Statistical analysis

    Results were expressed as mean ± SEM. Comparison of more than two groups was made with one-way analysis of variance (ANOVA).P< 0.05 was considered as statistically significant differences. The histogram was made by GraphPad Prism 6.0 software.

    3. Results

    3.1 Effect of GPP on intestinal bacteria community in normal mice

    In order to investigate whether GPP had an effect on intestinal bacteria community, Chao1 index of intestinal flora in mice was calculated to reflect the abundance and diversity of microbial community among different samples. The GPP was administrated to normal mice for 30 days with different doses, during the GPP treatment, mice feces of different time point were collected for analysis. On day 31, the animals were sacrificed for subsequent experiment (Fig. 1A). As shown in Fig. 1B, with the increase of GPP dose, the species richness of intestinal flora in mice gradually increased, indicating that GPP increased the diversity of intestinal microorganisms in mice. In Fig. 1C, the species richness of intestinal flora in mice increased with the extension of GPP administration time, the treatment of GPP for 15 days significantly enhanced the intestinal flora species diversity. Next, unweighted principal components analysis (PCA) dimensionality reduction analysis was performed for each sample according to GPP doses. PCA with a twodimensional principal component cumulative variance of 29.28% showed that GPP doses had less influence on the species distribution of intestinal flora in mice (Fig. 1D). However, the unweighted PCA on different time showed that GPP administration time can affect the species distribution of intestinal flora in mice. The 15-days group and 30-days group consisted of two apart sections and were significantly distinguished from each other (Fig. 1E). The results suggested that the GPP improved the richness of intestinal microbiota and that GPP doses had less influence on the species distribution compared with the administration time.

    Fig. 1 The alpha and beta diversity index of intestinal flora in mice. (A) Protocol for GPP treatment in normal mice. (B) With the increase of GPP dose, the species richness of intestinal flora in mice gradually increased. (C) The microbial diversity in the intestinal tract of mice increased with the extension of GPP administration time. (D) Unweighted PCA of samples collected with different doses of GPP. (E) Unweighted PCA of samples collected with different time point of GPP treatment (n = 4).

    3.2 Effect of GPP on the composition of the intestinal micro flora in normal mice

    To investigate whether the dominant microflora and their abundance changes with the different doses of GPP treatment, the top 20 microflora at phylum level and top 10 microflora at genera level were plotted (Figs. 2A, 2B), even though the proportion of each intestinal microflora has changed after GPP treatment of different doses, the dominant flora were consistent on phylum level.In the phylum level, the the dominant flora were Bacteroidetes and Firmicutes (Fig. 2A). In the genera level, the dominant flora wereLactobacillus,Clostridium, Lachnospiraceae, etc.The results showed that GPP did not affect the whole intestinal microorganism abundance and diversity, indicating that GPP may have influence on the balance of intestinal flora.

    Fig. 2 (A) The effect of GPP on bacterial phylum alternation during the 15-days and 30-days treatment. (B) Effect of GPP on bacterial genera alternation during the 15-days and 30-days treatment. (C) Comparisons of the predominant gene pathways of the bacterial microbiota in different groups. Orange stands for control group and blue stands for GPP medium treatment group (n = 4).

    16S rRNA marker gene sequences were used to predict the functional profiling of microbial communities. After treated by medium concentration of GPP for 15 days, DNA repair and recombination related proteins and base mismatch repair pathways were up-regulated, which also indicate the protect role of GPP on maintaining the homeostasis of intestinal microenvironment (Fig. 2C).

    3.3 Daily GPP intake attenuated DSS-induced colon injury

    Since GPP can both improve immune function and maintain the homeostasis of intestinal flora, we hope to explore whether GPP has identical biological effect on disease model. A classical colitismouse-model induced by DSS was established to investigate the effect of GPP on immune function and the composition of intestinal flora (Fig. 3A). The DAI score of each group were calculated and the scores in DSS-induced groups significantly increased compared with control group, and the treatment of GPP significantly decreased the score of DAI (Fig. 3B), indicating that GPP protected the colon from the damage of DSS. In the control group, goblet cells were evenly distributed, the structure of mucous layer was integrated,and there were no obvious inflammatory cells (Fig. 3C). In the DSS treated group, severe hyperplasia of goblet cells was observed,along with increased secretion of mucin, which eventually formed the vacuolated structure (Fig. 3D). The submucosa became swelled and widened, and some part of which showed necrosis and exfoliation (Fig. 3D). The number of monocytes in the columnar epithelium was increased, and the inflammatory response was obviously observed (Fig. 3E). After DSS induction, the colon structure of the GPP treatment group was more integrated than that of the DSS treated group, no necrosis or exfoliation was observed in submucosa. Goblet cells formed vacuole structure can only be found in low dose of GPP administration group, but the volume of vacuole was significantly smaller than that of DSS positive control group(Fig. 3F-3H). The degree of mononuclear cell infiltration is also lowered in GPP administration group compared with DSS group.These results demonstrated that GPP intake can attenuate colon injury induced by DSS.

    Fig. 3 Pathological changes in colitis mouse model induced by DSS. (A) Protocol for GPP treatment. (B) Change of DAI score in each group. (C–H) Colon tissues from mice were stained with PAS for histological analysis. (C) Control; (D–E) DSS (D-Ctrl); (F) DSS + 36 mg/kg BM of GPP (D-Low); (G) DSS + 120 mg/kg BM of GPP (D-Med); (H) DSS + 360 mg/kg BM of GPP (D-High). (I) The quantification of goblet cells. Data were expressed as mean ± SEM (n = 12).*P < 0.05,**P < 0.01, ***P < 0.001 compared with control group. #P < 0.05, ##P < 0.01, ###P < 0.001 compared with D-Ctrl group.

    3.4 GPP treatment in DSS-induced mice affected the spleen index and protected the splenic lymphocyte proliferation ability

    Spleen and thymus are both important immune organs, the organ index of which is often used as a preliminary indicator to evaluate the immunopharmacology of drugs [37]. As a result, both the spleen index and thymus index of mice were detected after being treated with different doses of GPP for 30 days, which is also the last day of DSS treatment.

    The results showed that the spleen index is significantly increased compared with control group, indicating an increased inflammatory response after DSS induction. However, the treatment of 120 mg/kg BW GPP reversed the upregulation, suggesting that GPP may have protection function for the inflammatory response induced by colitis(Fig. 4A). At the same time, the thymus index in each dose group did not changed compared with the control group, indicating that GPP does not affect the size of thymus (Fig. 4B).

    Fig. 4 The effects of GPP on immune functions of DSS-induced model mice. The effect of GPP on spleen index (A) and thymus index (B) after treatment with 36, 120, 360 mg/g BW. GPP for 30 days in the 5 groups (n = 12). (C) The effect of GPP on lymphocyte proliferation and (D) hemolytic value. (E) GPP increased peripheral phagocytes activity in mice. The uptaking capacity of peripheral phagocytes was detected by mouse peritoneal macrophage phagocyte chicken erythrocytes test, and the phagocytic index represented the phagocytosis of macrophages. (F) LDH releasing assay was used to detect the NK cell activity of the spleen. Data were expressed as mean ± SEM (n = 12). * P < 0.05, ** P < 0.01, *** P < 0.001.

    Lymphocyte are active immune cells, the proliferation and differentiation of lymphocytes are important phases during the immune response. As a result, the detection of lymphocytes proliferation can be used to evaluate the lymphocyte function in cellular immunity. As shown in Fig. 1C, DSS treatment significantly reduced the lymphocyte proliferation ability, but low-dose and medium-dose of GPP treatment group significantly increased the lymphocyte proliferation ability compared with DSS group(Fig. 4C), illustrating a protective effect of GPP on splenic lymphocytes. However, high-dose of GPP treatment does not enhance the lymphocyte proliferation in both normal and DSS treatment group(Fig. 4C, Fig. S1B), indicating that GPP can enhance the lymphocyte proliferation only within a certain concentration range.

    3.5 GPP enhanced the serum hemolysin synthesis

    Normal cells can produce hemolysin (anti-erythrocyte antibody)after being immunized with sheep erythrocyte, which can dissolve red blood cells (RBCs) and release hemoglobin when incubatedin vitrowith sheep blood cell complement. The RBC hemolysis is related to the amount of hemolysin, which can be regarded as an indicator to evaluate the humoral immune function. In normal mice treatment groups, the HC50 level does not change with the administration of GPP (Fig. S1C). However, in DSS-induced groups, medium-dose of GPP treatment significantly enhanced the HC50 level compared with DSS-treatment group (Fig. 4D), indicating that GPP may enhance humoral immune function in colitis model mice.

    3.6 GPP increased peripheral phagocytes and NK cell activity

    In order to investigate the role of GPP on the innate immune system of DSS-treated mice, the phagocytosis of peritoneal macrophages and the NK cell activity of spleen were detected after the 30-day GPP treatment.

    Both the phagocytic rate and phagocytic index significantly enhanced in low, medium and high dose groups compared with the control group (Fig. 4E), which was the same as in normal mice GPP treatment groups (Fig. S1A). The results suggested that DSS treatment have less effect on the phagocytic rate and phagocytic index, while GPP can enhance the phagocytosis of peritoneal macrophages in both normal and DSS-treated mice (Fig. 4E, Fig. S1A). Moreover,the cytotoxic activity of NK cells against YAC-1 tumor cells was significantly increased in the high dose GPP group compared with the DSS-control group (Fig. 4F), indicating that GPP protected the NK cell activity in DSS-treatment group. The above experimental results suggested that GPP was involved in the regulation of innate immunity in mice.

    3.7 Daily GPP intake changed the composition of the intestinal micro flora in colitis model mice.

    To investigate whether GPP had an effect on intestinal bacteria community in colitis model mice, Chao1 index and Shannon value of intestinal flora in mice was calculated. As shown in Figs. 5A and 5B, with the increase of GPP doses, the species richness of intestinal flora does not show significant change. However, the Chao1 value of high dose group and the Shannon value of medium and high dose group were similar as control group, indicating that GPP dose might affect the diversity of intestinal microorganisms in model mice by certain concentrations. Next, unweighted PCA dimensionality reduction analysis was also performed for each sample according to GPP doses. The result showed that the intestinal microflora significantly changed under DSS treatment (Fig. 5C).GPP treatment of medium dose were consisted of different sections and were significantly distinguished from DSS-control group, which was consistent with Shannon result. The results showed that GPP treatment had changed the species distribution of intestinal flora caused by DSS.

    Fig. 5 (A-B) The α-diversity index and the Shannon value of intestinal flora in mice. (C) Unweighted PCA of sample collected with DSS and different dose of GPP. (D) Effect of GPP on bacterial phylum alternation during the 7-days DSS treatment. (E) Effect of GPP on bacterial genera alternation during the 7-days DSS treatment (n = 3).

    The composition of the intestinal micro flora also had undergone changes. In the phylum level, Campilobacterota was decreased after DSS stimulation, and the level of Bacteroidota undergone slightly increase but decreased after medium dose of GPP treatment(Fig. 5D). In the genera level, the content ofLactobacillusin the intestinal tract was significantly reduced compared with control group after DSS stimulation, while the content ofLactobacillusin the medium dose group was significantly increased after the administration of GPP (Fig. 5E).Clinical reports have shown that the increase ofLactobacilluscontent can enhance the immune ability and resist intestinal disorders [38].In addition, the content ofDesulfovibrioandAlistipes_ finegoldiialso increased after DSS stimulation but were significantly decreased after GPP treatment (Fig. 5E). Previous studies have shown thatDesulfovibriois closely related with ulcerative enteritis [39], andAlistipes_finegoldiiis related with inflammation and cancer [40].These results indicates the protective role of GPP on intestinal microorganism.

    4. Discussion

    Gut microbiota is a complicated ecological environment that has numerous essential roles in human physiology and immune system,and is related to many chronic diseases [16,41]. Previous study of acute oral toxicity test demonstrated that GPP belonged to the non-toxic grade [14]. In our research, orally intake GPP of different doses for 30 days does not change the dominant micro flora in normal mice, the top micro flora at phylum level and top micro flora at genera level did not show much difference compared with control group (Fig. 2), this relative stable status indicated that GPP does not have deleterious effect on micro flora, which was consistent with the oral toxicity test result [14]. Moreover, after treated by medium concentration of GPP for 15 days, the comparisons of the predominant gene pathways of the bacterial microbiota showed that DNA repair and recombination related proteins and base mismatch repair pathways were up-regulated in GPP treatment group (Fig. 2C), indicating the protective role of GPP on maintaining the homeostasis of intestinal microenvironment in normal mice.

    By the metabolism of gut microbes, polysaccharide went through degradation into simple sugars and short chain fatty acids (SCFAs),which improved the efficiency of energy utilization. SCFAs are the energy source material of the epithelial cells of colon and small intestine, which are able to maintain intestinal morphology and function. Previous studies have highlighted the importance of SCFAs such as acetate, propionate and butyrate in amelioration of chronic inflammatory diseases and promotion of colonocyte health [19,42]. For example, SCFAs were reported to ameliorate colitis by suppressing the production of pro-inflammatory cytokines,enhancing interleukin-10 (IL-10) expression and activating Treg cells [19,43]. In our research, the comparisons of the predominant gene pathways of the bacterial microbiota showed that the purine and propionic acid metabolic pathways were up-regulated in GPP treatment group in normal mice, indicating an upregulated metabolism of SCFAs (Fig. 2C). These results indicated that GPP might maintain the homeostasis of intestinal microenvironment through a SCFAs dependent way.

    It has been reported widely that decreased richness or diversity of bacterial species were found both in fecal samples of human patients with ulcerative colitis and Crohn’s disease, and in fecal samples of rats with dextran sulfate sodium DSS-induced colitis [21,44,45].In our research, both Chao1 and unweighted PCA showed that the microbial diversity in the intestinal tract of mice increased with GPP doses and the extension of GPP administration time (Fig. 1). The improved richness of intestinal microbiota is consistent with the enhanced immune function in GPP treatment groups [14].

    The mononuclear macrophage system is an important part of innate immunity, which is composed of mononuclear cells,macrophages and their precursor cells in bone marrow [46]. The NK cell is an important innate immune cell, which has a strong cytolytic function to tumor cells, virus infected cells and other physiological stress cells [47]. In our last research works, GPP increased bothin vitroandin vivophagocytosis, and enhanced the activity of NK cells,indicating a specific influence of GPP on innate immune cells [14].

    In DSS-induced mice model, daily GPP intake attenuated DSS-induced colon injury, including the reduction of DAI score and histological injury (Fig. 3). DSS induction did not change the phagocytic index or NK cell viability compared with control group.GPP treatment of different doses significantly increased peripheral phagocytes activity in DSS-induced mice (Fig. 4E), which was consistent with what has been observed in normal mice (Fig. S1A).The NK cell viability was also increased in high-dose of GPP treatment in DSS-induced mice (Fig. 4F), which was also consistent with what has been observed in normal mice [13]. These results demonstrated that GPP can enhance innate immune function of both normal and colitis mice, and the function of GPP was not affected by DSS. The mechanism how GPP influence the innate immune still needs further investigation.

    GPP treatment in DSS-induced mice also protected the splenic lymphocyte proliferation ability (Fig. 4C) and enhanced the serum hemolysin synthesis (Fig. 4D), which was different from what we have observed in normal mice GPP treatment groups [14], indicating a more significant immune protective effect of GPP in DSS-induced model mice. After DSS-induction, the spleen index has increased (Fig. 4A),and the splenic lymphocyte proliferation ability has decreased significantly (Fig. 4C), which may be due to the inflammatory reaction in spleen after DSS inducement. GPP-treatment has reversed the phenomenon (Fig. 4C), indicating that GPP have protective function for the spleen and somehow reduce the inflammatory accumulation in spleen. The HC50 level is commonly regarded as an index for humoral immunity, different from what we have observed in normal mice(Fig. S1C), medium-dose of GPP treatment significantly enhanced the HC50 level compared with DSS-treatment group (Fig. 4D),indicating that GPP may also enhance humoral immune function.

    SCFAs, as mentioned above, were reported to ameliorate colitis by suppressing the production of pro-inflammatory cytokines. They are produced mainly from fermentation of polysaccharides by gut bacteria, such asBacteroides[19], which is a dominated intestinal microbiota in both normal and GPP treatment mice. Previous studies also showed that polysaccharides derived fromG. lucidumincreased the production of TNF-α, IL-2 and interferon-γ (IFN-γ) in primary cultures of human peripheral blood mononuclear cells [48,49]and improved the levels of serum IL-2, IL-4 in rats [21,50], which are key cytokines against pathogen infections in innate and adaptive immunity.G. lucidumis the main component of GPP, comparisons of the predominant gene pathways of the bacterial microbiota in different groups illustrated that DNA repair and recombination, base mismatch repair pathways is stronger, and the purine and propionic acid metabolic pathways were up-regulated in GPP treatment group,representing a higher DNA repair ability and upregulated metabolism of SCFAs in GPP treatment group. However, further exploration is needed to figure out how the GPP derived SCFAs regulate splenic lymphocyte proliferation ability and the synthesis of serum hemolysin.

    In our research work, GPP also changed the intestinal bacteria community in colitis model mice. According to the PCA dimensionality reduction analysis, the species distribution of intestinal flora significantly changed under DSS treatment (Fig. 5C). GPP treatment groups, especially the medium dose of GPP treatment group, are consisted of different sections and were distinguished from DSS-control group, indicating a significant change in the gut microbiota of GPP treatment group.

    The increase ofLactobacilluscontent can enhance the immune ability, prevent the production of inflammatory cytokines and resist intestinal disorders [38,51]. In the genus level, the content ofLactobacillusin the intestinal tract was significantly reduced compared with control group after DSS stimulation, while the content ofLactobacillusin the medium dose group was significantly increased after the administration of GPP (Fig. 5E).Desulfovibriois predominant member of sulphate-reducing bacteria inhuman gut microbiota [52], it is closely related with the pathopoiesis of ulcerative enteritis [39], patients withDesulfovibrioinfections are usually elderly men with abdominal disorders, especially hepatobiliary diseases [53].Alistipes_finegoldiiis regarded as a novel microbial driver of colitis and tumorigenesis, which is related with inflammation and cancer [40,54]. In our research, the content ofDesulfovibrioandAlistipes_ finegoldiihas increased after DSS stimulation but was significantly decreased after GPP treatment (Fig. 5E), indicating the protective role of GPP on intestinal microorganism.

    Taken together, the results indicated the protective role of GPP in regulating the homeostasis of intestinal flora in DSS-induced colitis mice model. These findings suggested that GPP regulated immune function in both health and colitis model and has a positive effect on maintaining intestinal flora homeostasis. A schematic diagram was made to represent the effect of GPP on the immune function and intestinal flora of colitis model mice (Fig. 6).

    Fig. 6 The schematic diagram represents the effect of GPP on the immune function and intestinal flora of colitis model mice.

    5. Conclusions

    In summary, our research showed that GPP increased the diversity of intestinal microorganisms without influencing the dominant intestinal microbiota constitution in normal mice, and attenuated DSS-induced colon injury, protected the splenic lymphocyte proliferation ability, enhanced the serum hemolysin synthesis, and increased peripheral phagocytes and NK cell activity in DSS-induced model mice. Our findings revealed that GPP regulated immune function in both normal and colitis model, and had a positive effect on maintaining intestinal flora homeostasis. Our research will provide theoretical basis for the development of immune-enhancing healthy food and contribute to the further utilization of GLP and PUP in enhancing immune function.

    Conflicts of interest

    The authors declare no conflict of interest.

    Acknowledgments

    This study was financially supported by grants from the National Key R&D Program of China (2018YFD0400204), the National Natural Science Foundation of China (81974503, 81871095),and the Key International S&T Cooperation Program of China(2016YFE113700).

    Appendix A. Supplementary data

    Supplementary data associated with this article can be found, in the online version, at http://doi.org/10.1016/j.fshw.2022.03.010.

    亚洲在线观看片| 又黄又爽又刺激的免费视频.| 欧美在线一区亚洲| 色哟哟·www| 免费高清视频大片| 淫妇啪啪啪对白视频| 亚洲精品久久国产高清桃花| 国产高清激情床上av| 欧美激情久久久久久爽电影| aaaaa片日本免费| 好男人在线观看高清免费视频| 一进一出好大好爽视频| ponron亚洲| 日日摸夜夜添夜夜添小说| 极品教师在线免费播放| 婷婷色综合大香蕉| 一区二区三区高清视频在线| 不卡视频在线观看欧美| 亚洲av中文字字幕乱码综合| 免费观看精品视频网站| av在线老鸭窝| 亚洲在线自拍视频| 别揉我奶头 嗯啊视频| 男人和女人高潮做爰伦理| 欧美+亚洲+日韩+国产| 毛片女人毛片| 国产私拍福利视频在线观看| 午夜免费男女啪啪视频观看 | 特级一级黄色大片| 欧美高清性xxxxhd video| 国产真实乱freesex| 欧美黑人欧美精品刺激| 午夜影院日韩av| 1024手机看黄色片| 婷婷六月久久综合丁香| 在线观看午夜福利视频| 99热网站在线观看| 在线天堂最新版资源| 网址你懂的国产日韩在线| 伊人久久精品亚洲午夜| 色综合婷婷激情| 韩国av一区二区三区四区| 日日摸夜夜添夜夜添小说| 欧美高清成人免费视频www| 精品一区二区三区视频在线| 五月玫瑰六月丁香| 精品久久久久久久久亚洲 | 日本免费a在线| 露出奶头的视频| 男女下面进入的视频免费午夜| 亚洲精品在线观看二区| 色在线成人网| 午夜福利高清视频| 日韩欧美 国产精品| 波野结衣二区三区在线| 亚洲国产日韩欧美精品在线观看| 国产伦一二天堂av在线观看| 亚洲va日本ⅴa欧美va伊人久久| 国产精品乱码一区二三区的特点| 亚洲va日本ⅴa欧美va伊人久久| 国内精品一区二区在线观看| 亚洲av二区三区四区| 亚洲av免费高清在线观看| 他把我摸到了高潮在线观看| 国产精品一区二区性色av| 99精品在免费线老司机午夜| 久久久国产成人免费| 天堂动漫精品| 日本黄大片高清| 伊人久久精品亚洲午夜| a级一级毛片免费在线观看| 国产色婷婷99| 亚洲无线观看免费| 成人高潮视频无遮挡免费网站| 亚洲欧美日韩东京热| 国内精品一区二区在线观看| 国产精品一区二区三区四区免费观看 | 国产爱豆传媒在线观看| 在线播放无遮挡| 亚洲四区av| 18禁黄网站禁片免费观看直播| 亚洲欧美日韩高清在线视频| 精品一区二区三区视频在线| 国国产精品蜜臀av免费| 国产精品av视频在线免费观看| 精品人妻视频免费看| 久久久午夜欧美精品| 亚洲中文字幕一区二区三区有码在线看| 亚洲性久久影院| 久久久久久久久中文| 又黄又爽又刺激的免费视频.| 尤物成人国产欧美一区二区三区| 男插女下体视频免费在线播放| 国产精品国产三级国产av玫瑰| 久久天躁狠狠躁夜夜2o2o| 亚洲成av人片在线播放无| 麻豆一二三区av精品| 尾随美女入室| 欧美最新免费一区二区三区| 国产精品亚洲一级av第二区| 日韩人妻高清精品专区| 国产精品无大码| 国产精品人妻久久久影院| 成人特级av手机在线观看| 香蕉av资源在线| 欧美一区二区精品小视频在线| 精品不卡国产一区二区三区| 欧美日韩黄片免| 欧美日本亚洲视频在线播放| 成人国产麻豆网| 亚洲精品在线观看二区| 一进一出抽搐gif免费好疼| 又粗又爽又猛毛片免费看| 亚洲精品日韩av片在线观看| 日日撸夜夜添| 舔av片在线| 丝袜美腿在线中文| 日本欧美国产在线视频| 桃色一区二区三区在线观看| 欧洲精品卡2卡3卡4卡5卡区| 中文字幕久久专区| 亚洲,欧美,日韩| 欧美日韩瑟瑟在线播放| 三级男女做爰猛烈吃奶摸视频| 校园春色视频在线观看| 欧美黑人巨大hd| 男女之事视频高清在线观看| 3wmmmm亚洲av在线观看| 黄色欧美视频在线观看| 九九爱精品视频在线观看| 一级黄片播放器| 欧美激情在线99| 亚洲最大成人av| 狠狠狠狠99中文字幕| 中亚洲国语对白在线视频| 国产真实乱freesex| 免费看日本二区| 中文在线观看免费www的网站| 看片在线看免费视频| 少妇的逼水好多| 国产白丝娇喘喷水9色精品| 国产精品综合久久久久久久免费| 午夜福利18| 久久婷婷人人爽人人干人人爱| 此物有八面人人有两片| 国产精品亚洲美女久久久| 日本在线视频免费播放| 美女cb高潮喷水在线观看| 免费看美女性在线毛片视频| 亚洲狠狠婷婷综合久久图片| 综合色av麻豆| 日韩强制内射视频| 露出奶头的视频| 国产精品女同一区二区软件 | 精品久久久久久成人av| 日本三级黄在线观看| 中文在线观看免费www的网站| 久久久久精品国产欧美久久久| 在线播放国产精品三级| 中文在线观看免费www的网站| 日日夜夜操网爽| 中文在线观看免费www的网站| 在线看三级毛片| 中出人妻视频一区二区| 啦啦啦观看免费观看视频高清| 99热精品在线国产| 亚洲av二区三区四区| 日韩欧美国产一区二区入口| 高清日韩中文字幕在线| 免费观看人在逋| 在线免费十八禁| 久久精品国产99精品国产亚洲性色| 亚洲四区av| 国产亚洲av嫩草精品影院| 精品一区二区三区视频在线| 人妻少妇偷人精品九色| 成年免费大片在线观看| 别揉我奶头 嗯啊视频| 精品午夜福利视频在线观看一区| 国产精品伦人一区二区| 99热精品在线国产| 婷婷亚洲欧美| 男人舔奶头视频| 色吧在线观看| 国产精品爽爽va在线观看网站| 夜夜爽天天搞| 成年免费大片在线观看| 日本一本二区三区精品| 尾随美女入室| 99久久久亚洲精品蜜臀av| 成人国产麻豆网| 国产高清视频在线观看网站| 村上凉子中文字幕在线| 国产精品av视频在线免费观看| 成年女人永久免费观看视频| 男人的好看免费观看在线视频| 日本黄色片子视频| 久99久视频精品免费| 午夜免费激情av| 亚洲精品影视一区二区三区av| 免费人成视频x8x8入口观看| 色综合站精品国产| 69人妻影院| 亚洲欧美清纯卡通| 久久99热6这里只有精品| 真实男女啪啪啪动态图| 欧美精品啪啪一区二区三区| 欧美又色又爽又黄视频| 欧美黑人欧美精品刺激| 性色avwww在线观看| 成人亚洲精品av一区二区| 亚洲中文日韩欧美视频| 国产一区二区三区视频了| 国产 一区精品| 久久人人爽人人爽人人片va| 亚洲内射少妇av| 亚洲国产欧美人成| 国产高清视频在线观看网站| 亚洲国产欧洲综合997久久,| 99在线人妻在线中文字幕| 中文字幕熟女人妻在线| 精品久久国产蜜桃| av视频在线观看入口| 男人的好看免费观看在线视频| 久99久视频精品免费| 男人狂女人下面高潮的视频| 国产 一区 欧美 日韩| 中国美白少妇内射xxxbb| 少妇高潮的动态图| 国产高清视频在线播放一区| 欧美日韩精品成人综合77777| 亚洲av美国av| 男女边吃奶边做爰视频| 久久久久久久午夜电影| 日本a在线网址| 丰满乱子伦码专区| 日本爱情动作片www.在线观看 | 2021天堂中文幕一二区在线观| 国产精品福利在线免费观看| 女生性感内裤真人,穿戴方法视频| 国产精华一区二区三区| 蜜桃久久精品国产亚洲av| 亚洲欧美日韩东京热| 日韩欧美精品v在线| 亚洲狠狠婷婷综合久久图片| 欧美色欧美亚洲另类二区| 啦啦啦观看免费观看视频高清| 中国美白少妇内射xxxbb| 两性午夜刺激爽爽歪歪视频在线观看| 夜夜看夜夜爽夜夜摸| 老熟妇乱子伦视频在线观看| av在线蜜桃| 美女黄网站色视频| 久久久久久大精品| а√天堂www在线а√下载| 18禁黄网站禁片午夜丰满| 黄色欧美视频在线观看| 亚洲中文字幕日韩| 日韩欧美一区二区三区在线观看| 欧美激情在线99| 亚洲一区高清亚洲精品| 国产伦精品一区二区三区四那| 1000部很黄的大片| 亚洲自偷自拍三级| 亚洲精品久久国产高清桃花| 蜜桃久久精品国产亚洲av| 亚洲欧美日韩东京热| av中文乱码字幕在线| 少妇被粗大猛烈的视频| 久久国产乱子免费精品| 精品久久久久久久久亚洲 | 最近最新免费中文字幕在线| 日本撒尿小便嘘嘘汇集6| 欧美3d第一页| 亚洲avbb在线观看| 国产精品久久电影中文字幕| 蜜桃久久精品国产亚洲av| av在线亚洲专区| 亚洲成av人片在线播放无| 国产精品98久久久久久宅男小说| 91av网一区二区| 亚洲aⅴ乱码一区二区在线播放| 成年免费大片在线观看| 亚洲午夜理论影院| 婷婷精品国产亚洲av在线| 久久久久久久久大av| 男插女下体视频免费在线播放| 免费黄网站久久成人精品| 日韩国内少妇激情av| 少妇被粗大猛烈的视频| 精品一区二区免费观看| 一本久久中文字幕| 久久精品综合一区二区三区| 99久久中文字幕三级久久日本| 男女边吃奶边做爰视频| 狂野欧美激情性xxxx在线观看| 欧美性感艳星| 国产美女午夜福利| 一本精品99久久精品77| 亚洲性久久影院| 久久精品人妻少妇| 久久久久久久久久黄片| 丝袜美腿在线中文| 一级av片app| 免费无遮挡裸体视频| 亚洲av成人av| 久9热在线精品视频| 亚洲成av人片在线播放无| 精品久久久久久久久久免费视频| 国语自产精品视频在线第100页| 午夜福利18| 日韩强制内射视频| 国产成人影院久久av| 日本黄色片子视频| 中文资源天堂在线| 精品不卡国产一区二区三区| 国产不卡一卡二| 亚洲av免费在线观看| 18禁在线播放成人免费| 国内精品宾馆在线| 一卡2卡三卡四卡精品乱码亚洲| 亚洲va日本ⅴa欧美va伊人久久| 日韩中字成人| 97热精品久久久久久| 啦啦啦韩国在线观看视频| 日韩精品青青久久久久久| 少妇裸体淫交视频免费看高清| 老司机福利观看| 国产精品1区2区在线观看.| 久久久久久伊人网av| 夜夜爽天天搞| 日韩 亚洲 欧美在线| 成人美女网站在线观看视频| 国产精品一区二区三区四区免费观看 | 一本久久中文字幕| 黄色欧美视频在线观看| 一个人看的www免费观看视频| 美女免费视频网站| 日本a在线网址| 91精品国产九色| 成年版毛片免费区| 亚州av有码| 日韩亚洲欧美综合| 国产亚洲精品综合一区在线观看| 国产av在哪里看| av视频在线观看入口| 色尼玛亚洲综合影院| 精品一区二区三区视频在线观看免费| 国产精品人妻久久久影院| 国产麻豆成人av免费视频| 欧美激情久久久久久爽电影| 午夜a级毛片| 男人的好看免费观看在线视频| 国产美女午夜福利| 色尼玛亚洲综合影院| 在线观看舔阴道视频| av.在线天堂| 午夜福利18| 欧美性猛交╳xxx乱大交人| 久久久久国内视频| 又爽又黄a免费视频| 91狼人影院| 无遮挡黄片免费观看| 长腿黑丝高跟| 欧美色视频一区免费| 亚洲av电影不卡..在线观看| 色5月婷婷丁香| 97人妻精品一区二区三区麻豆| 亚洲国产精品成人综合色| 久久久久久久亚洲中文字幕| 久久热精品热| 亚洲狠狠婷婷综合久久图片| 欧美另类亚洲清纯唯美| 老熟妇乱子伦视频在线观看| 国产男人的电影天堂91| 午夜激情欧美在线| 白带黄色成豆腐渣| 日本精品一区二区三区蜜桃| 亚洲欧美激情综合另类| 国产亚洲精品av在线| 嫁个100分男人电影在线观看| 久久国内精品自在自线图片| 亚洲在线观看片| 22中文网久久字幕| 亚洲av日韩精品久久久久久密| 美女高潮的动态| 欧美+日韩+精品| 91av网一区二区| 欧美成人一区二区免费高清观看| 99久久成人亚洲精品观看| 亚洲专区国产一区二区| 99热网站在线观看| 99久久无色码亚洲精品果冻| 久久久久精品国产欧美久久久| 成人特级黄色片久久久久久久| 男女边吃奶边做爰视频| 人妻久久中文字幕网| 国内精品美女久久久久久| 国产精品自产拍在线观看55亚洲| 国产久久久一区二区三区| 日本黄大片高清| 国产精品女同一区二区软件 | 国产女主播在线喷水免费视频网站 | 午夜亚洲福利在线播放| 国产中年淑女户外野战色| 国产精品国产高清国产av| 淫秽高清视频在线观看| 亚洲男人的天堂狠狠| 欧美一区二区精品小视频在线| 嫩草影院新地址| 99久久成人亚洲精品观看| 日本黄色视频三级网站网址| 国产国拍精品亚洲av在线观看| 成年女人毛片免费观看观看9| 亚洲中文字幕一区二区三区有码在线看| 成人特级av手机在线观看| 亚洲久久久久久中文字幕| 亚洲成人久久性| 亚洲七黄色美女视频| 天堂网av新在线| 美女免费视频网站| 午夜爱爱视频在线播放| 日韩,欧美,国产一区二区三区 | 一进一出抽搐动态| 日韩在线高清观看一区二区三区 | 国内揄拍国产精品人妻在线| 日韩高清综合在线| 欧洲精品卡2卡3卡4卡5卡区| 成人国产综合亚洲| 欧美bdsm另类| 在现免费观看毛片| 国内精品久久久久精免费| 欧美一区二区精品小视频在线| 国产亚洲91精品色在线| 麻豆成人午夜福利视频| 免费人成在线观看视频色| 成人av一区二区三区在线看| 国产男人的电影天堂91| av女优亚洲男人天堂| 欧美潮喷喷水| 国产av在哪里看| a在线观看视频网站| 日韩大尺度精品在线看网址| 91av网一区二区| 久久久国产成人免费| 精品福利观看| 最近视频中文字幕2019在线8| 黄色丝袜av网址大全| av在线观看视频网站免费| 18禁裸乳无遮挡免费网站照片| 亚洲午夜理论影院| 深夜精品福利| 99久国产av精品| 麻豆久久精品国产亚洲av| 91在线精品国自产拍蜜月| 国产av麻豆久久久久久久| 在现免费观看毛片| 又爽又黄a免费视频| 亚洲精品在线观看二区| 国产精品久久久久久av不卡| 精品国内亚洲2022精品成人| 99热这里只有是精品在线观看| 欧美人与善性xxx| 日韩欧美三级三区| 国产69精品久久久久777片| 欧美日韩综合久久久久久 | 校园人妻丝袜中文字幕| 九九爱精品视频在线观看| 中文字幕av成人在线电影| 女人十人毛片免费观看3o分钟| 99精品在免费线老司机午夜| 91av网一区二区| 成人性生交大片免费视频hd| 国产精品亚洲一级av第二区| 18禁在线播放成人免费| 国产成人a区在线观看| 亚洲乱码一区二区免费版| 亚洲最大成人手机在线| av福利片在线观看| 国内毛片毛片毛片毛片毛片| 午夜视频国产福利| 亚洲精品粉嫩美女一区| 国产aⅴ精品一区二区三区波| 少妇被粗大猛烈的视频| 99热这里只有是精品在线观看| 神马国产精品三级电影在线观看| 真人一进一出gif抽搐免费| 一个人看的www免费观看视频| av在线天堂中文字幕| 亚洲av中文字字幕乱码综合| 日本精品一区二区三区蜜桃| 日本在线视频免费播放| 美女大奶头视频| 色视频www国产| 欧美三级亚洲精品| 听说在线观看完整版免费高清| 如何舔出高潮| 国产成人一区二区在线| 99热网站在线观看| 免费看av在线观看网站| 日本黄大片高清| 嫩草影院入口| 日本一本二区三区精品| 成人国产一区最新在线观看| 久久久久久久精品吃奶| 内地一区二区视频在线| av在线蜜桃| 亚洲精品久久国产高清桃花| 国内精品一区二区在线观看| 亚洲最大成人av| 美女被艹到高潮喷水动态| 国产精品,欧美在线| 精品午夜福利视频在线观看一区| 日韩精品青青久久久久久| 国内精品宾馆在线| 变态另类成人亚洲欧美熟女| 欧美zozozo另类| 国产伦在线观看视频一区| 国产精品日韩av在线免费观看| 亚洲在线观看片| www.色视频.com| 性欧美人与动物交配| 日韩人妻高清精品专区| 好男人在线观看高清免费视频| 亚洲国产高清在线一区二区三| 少妇裸体淫交视频免费看高清| 高清在线国产一区| 亚洲精品日韩av片在线观看| 免费观看的影片在线观看| av在线亚洲专区| 亚洲熟妇中文字幕五十中出| 极品教师在线视频| 国产高潮美女av| 夜夜爽天天搞| 看黄色毛片网站| 免费高清视频大片| 亚洲av不卡在线观看| 欧美一区二区精品小视频在线| 久久亚洲真实| 在线国产一区二区在线| 变态另类丝袜制服| 久久人妻av系列| 男女做爰动态图高潮gif福利片| 中出人妻视频一区二区| 99国产极品粉嫩在线观看| 18禁黄网站禁片午夜丰满| 免费在线观看影片大全网站| 桃红色精品国产亚洲av| 国产老妇女一区| 性色avwww在线观看| 欧美最新免费一区二区三区| 国产精品久久久久久av不卡| 又黄又爽又免费观看的视频| 国产精品女同一区二区软件 | 亚洲av免费在线观看| 老师上课跳d突然被开到最大视频| 婷婷六月久久综合丁香| 免费黄网站久久成人精品| 婷婷六月久久综合丁香| 九九爱精品视频在线观看| 3wmmmm亚洲av在线观看| 如何舔出高潮| 国产精品无大码| 亚洲成a人片在线一区二区| 又粗又爽又猛毛片免费看| 好男人在线观看高清免费视频| 成人特级黄色片久久久久久久| 国产成人影院久久av| 日本成人三级电影网站| 99国产精品一区二区蜜桃av| 国内少妇人妻偷人精品xxx网站| 亚洲真实伦在线观看| 午夜福利视频1000在线观看| 变态另类成人亚洲欧美熟女| 欧美黑人巨大hd| 亚洲国产精品合色在线| 男女之事视频高清在线观看| 伦精品一区二区三区| 在线观看美女被高潮喷水网站| 久久久久免费精品人妻一区二区| 亚洲无线在线观看| 午夜日韩欧美国产| 噜噜噜噜噜久久久久久91| 欧美最黄视频在线播放免费| 天堂动漫精品| 免费观看精品视频网站| 国产av不卡久久| 日韩一区二区视频免费看| 国产精品日韩av在线免费观看| 亚洲av中文字字幕乱码综合| 麻豆国产97在线/欧美| 精品久久久久久久人妻蜜臀av| 男女边吃奶边做爰视频| 99热精品在线国产| 深夜精品福利| 亚洲在线观看片| 免费高清视频大片| 久久久久久久亚洲中文字幕| 国产精品,欧美在线| 国产在线精品亚洲第一网站| 日本精品一区二区三区蜜桃| 亚洲在线观看片| 国产欧美日韩精品亚洲av| 精品一区二区三区av网在线观看| 美女高潮喷水抽搐中文字幕| 99久久精品一区二区三区| 中文字幕熟女人妻在线| 亚洲最大成人中文| 性插视频无遮挡在线免费观看| 亚洲第一区二区三区不卡| 国产淫片久久久久久久久| 99国产极品粉嫩在线观看| 亚洲欧美精品综合久久99| 久久久久久伊人网av| 久久久精品大字幕|