• <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 ?

    Granulocyte-macrophage colony-stimulating factor protects mice against hepatocellular carcinoma by ameliorating intestinal dysbiosis and attenuating inflammation

    2020-10-22 04:31:38YongNaWuLeiZhangTuoChenXunLiLiHongHeGuangXiuLiu
    World Journal of Gastroenterology 2020年36期

    Yong-Na Wu, Lei Zhang, Tuo Chen, Xun Li, Li-Hong He, Guang-Xiu Liu

    Abstract

    Key Words: Granulocyte-macrophage colony-stimulating factor; Microbiome; Inflammation; Hepatocellular carcinoma

    INTRODUCTION

    Hepatocellular carcinoma (HCC) is currently the third leading cause of cancer mortality[1]. There are approximately 466000 new cases of HCC in China each year, accounting for approximately 55% of the total cases worldwide. HCC has become an extremely serious public health problem in China due to its high incidence, poor prognosis, and high rate of postsurgical recurrence[2,3]. Unfortunately, lack of effective treatment for liver cancer further increases the overall burden on the patients. There is currently substantial research on the characterization of the immunology and biochemistry of the gut, which scarcely involve metabolic organs. Recent interest has been focused on important physiological functions of the gut, such as maintaining energy homeostasis[4].

    In recent decades, researchers have focused on the important contributions of the gut microbiota to the key aspects of human health, notably metabolism and immunity[5,6]. The liver is the largest solid organ in our body; it processes 80% of the blood derived from the gastrointestinal tractviaoutflow from the portal vein[7]. A large number of resident immune cells within the liver modulate immune responses, promote liver repair and detoxification, and resist invasion of pathogens[8,9]. The gut microbiome is a critical factor that regulates liver immunity and maintains liver homeostasisviaa variety of mechanisms, including those involving endotoxin, Tolllike receptors (TLR), and bile acid metabolism[10-15]. Of particular interest, extensive studies have confirmed specific differences in the intestinal flora between patients with HCC and healthy control subjects. However, the specific mechanisms underlying these findings remain unclear[16,17].

    Granulocyte–macrophage colony-stimulating factor (GM-CSF) is a host cytokine that stimulates bone marrow precursors to generate monocytes and macrophages. Furthermore, it enhances monocyte antibacterial and antitumor activities[18]. Microbiota can promote the release of GM-CSF; this process is of great importance in maintaining intestinal immune homeostasis. Interestingly, macrophage-mediated sensing of microbial signals controls GM-CSF levels[19]. Serum and tissue levels of GMCSF were decreased in patients with HCC. Conversely, we found that GM-CSF overexpression inhibited the proliferation, invasion, and migration of liver cancer cells and promoted their apoptosis (unpublished data). We hypothesize that GM-CSF plays a protective role against HCC by regulating intestinal microecology. In this study, we examined the effects of GM-CSF overexpression on the intestinal microecology and metabolism of HCC. Our findings demonstrated that GM-CSF overexpression protects mice against HCC by ameliorating intestinal dysbiosis, stabilizing the intestinal barrier, attenuating inflammation, and reducing serum endotoxin levels. These findings may have profound implications for the development of therapies for HCC.

    MATERIALS AND METHODS

    Ethics statement

    This study was approved by the Ethics Committee of the First Hospital of Lanzhou University (No. LLYYLL-2017-18) and was performed in accordance with the Guidelines for Experimental Animals of the Ministry of Science and Technology (Beijing, China).

    Animals and treatments

    Seven-week-old male BALB/c nude mice were housed in a specific pathogen-free environment with temperature (23 ± 2°C) and humidity controls (approximately 50%) and a daily cycle of 12-h light/12-h dark together withad libitumaccess to food and drinking water. After 1 wk of acclimatization, the mice were randomly divided into three groups: Control (n= 10), HCC (n= 13), and HCC + GM-CSF (GM-CSF overexpression,n= 13) groups. In the HCC and HCC + GM-CSF groups, subcutaneous tumors were implanted in the early stage. Mouse orthotopic transplantation tumor models of HCC were established after subcutaneous tumors were successfully implanted. The specific operation method is as follows. The human GM-CSF sequence was transfected using lentivirus into the human HCC cell line HCCLM3. The cells growing in the log phase were injected into the armpit of nude mice under sterile conditions (1 × 107/mL). Then, the tumors were subcutaneously inoculated with cells for approximately 4 wks (approximately 1 cm in diameter, no rupture on the surface). The tumors were trimmed into a tumor mass of 1.5 mm × 2 mm × 1 mm, the abdominal wall was cut, and the right lobe of the liver was exposed and incised. The aforementioned spare tumor mass was implanted, followed by suturing and closing of the abdomen. After the effects of anesthesia wore off, the mice were returned to the cage for feeding. They were kept in this environment for 4 wk. The construction method of the HCC orthotopic tumor model was as described above.

    Sample collection

    Fecal samples were collected from all mice and stored in a -80°C freezer. Blood samples were collected and centrifuged at 3000 r/min for 15 min; all serum aliquots were stored at -80°C. Liver and colon samples were divided into halves; tissues from one part were fixed in 4% neutral buffered formaldehyde for paraffin embedding, whereas the other part was frozen in liquid nitrogen and stored at -80°C for subsequent RNA extraction.

    Histological evaluation of liver and colon tissues

    Paraffin-embedded liver and colon tissue sections were stained with hematoxylin and eosin (H&E) to observe the morphology of these tissues and to detect liver injury. Images were scanned using a NanoZoomer digital pathology system (Hamamatsu Photonics, K.K., Japan), which digitally scans the sections into a specific image format for further evaluation.

    Serum parameter analysis

    Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using the ROCHE CABAS C311 analyzer (Roche, Germany). Lipopolysaccharide (LPS) concentrations as an indirect measure of endotoxin levels were measured with ELISA according to the manufacturer’s instructions (Guduo, Shanghai, China). Immune inflammatory factors were measured using commercial ELISA kits (Abbkine Scientific Co, United States).

    Immunohistochemical staining

    Liver and colon tissue sections were stained using an immunohistochemistry kit (DAKO, Denmark) as previously described[20]. The NanoZoomer digital pathology scanner was used to scan the stained sections into a format suitable for analysis. Each section was randomly selected at 200× and 400× magnifications for analysis. Image-Pro Plus software was used to measure the average optical density of tight junction protein-1 (ZO-1).

    RNA extraction and real-time PCR analysis

    Total RNA was extracted from liver and colon tissues using an RNA kit (Qiagen, Germantown, MD, United States) according to the manufacturer’s protocol. Real-time PCR was performed with the Applied Biosystems 7500 system using the one-step SYBR PrimeScript plus RT-PCR kit (Takara, Japan). The PCR primer sequences used in this study are listed in Supplementary Table S1. The expression data of the samples were normalized to those of the internal control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and were calculated according to the ΔΔCt method.

    Analysis of microbial communities in fecal samples

    The QIAamp Rapid DNA Kit (Qiagen) was used to extract DNA from mouse stool samples. The DNA concentration and integrity were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Hudson, NH, United States) and agarose gel electrophoresis, respectively. Suitable DNA samples were transported to Genesky Biotechnologies Inc., Shanghai, China, for absolute quantification and 16S rRNA gene amplicon sequencing. The spike-in sequences contained conserved regions identical to those of natural 16S rRNA genes and artificial variable regions and were distinct from those found in nucleotide sequences in public databases. These served as internal standards and facilitated absolute quantification across samples. Appropriate mixtures with known copy numbers of spike-in sequences were added to the sample DNAs. The V3-V4 regions of the 16S rRNA genes and spike-in sequences were PCRamplified and sequenced using Illumina HiSeq.

    Metabolomic profiling of fecal samples

    Metabolomics analysis was performed using an Agilent 7890 gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). ChromaTOF software was used to analyze the mass spectrum data for peak extraction, baseline correction, deconvolution, peak integration, and peak alignment[21]. For specific identification of biomolecules, the LECO-Fiehn Rtx5 database was used to match mass spectra and the retention time index. Finally, the peaks with a detection rate below 50% or relative standard deviation (RSD) > 30% in the quality control (QC) samples were removed[22]. Metabolites were identified from the US National Institutes of Standards and Technology (NIST) and Fiehn databases. Orthogonal partial least squares-discriminant analysis (OPLS-DA) was performed to visualize metabolic differences between experimental groups. Differential detection of metabolites was based on the statistical significance (Pvalue) of the two-tailed Student’stteston the normalized peak areas, and metabolites withvariable importance in the projection values > 1 andPvalues < 0.05 were included.

    Statistical analysis

    The data are presented as the means ± SE. For most data, one-way ANOVA with Tukey’s post hoc test was used to determine statistically significant differences between the groups. The Wilcoxon rank-sum test was performed to evaluate alpha diversity and principal coordinates between the different cohorts in absolute quantificative 16S rRNA amplicon sequencing analysis. PERMANOVA (Adonis) was used to test microbial community clustering using both weighted and unweighted UniFrac and Bray-Curtis distance matrices. Correlations between the variables were determined using Spearman’s rank correlation;Pvalues < 0.05 were considered significant. The data were analyzed using SPSS version 19.0 for Windows (SPSS Inc., United States).

    RESULTS

    GM-CSF overexpression protected against hepatic injury, normalized liver function, stabilized the intestinal barrier, and reduced serum endotoxin levels secondary to HCC

    To assess the effects of GM-CSF on hepatic injury, liver function, intestinal barrier function, and serum endotoxin levels, we established an orthotopic transplantation tumor model of HCC by transecting GM-CSF-encoded lentivirus into an HCC cell line. The morphology of the livers in the HCC + GM-CSF group improved compared with that in the mice with HCC alone; tumor tissue infiltration was also reduced in the HCC + GM-CSF group (Figure 1A). The normal structure of the liver of the mice in the HCC group was extensively destroyed by tumor tissue infiltration, and there was considerable accumulation of vesicular fat, which is related to mixed inflammatory infiltration. In contrast, GM-CSF showed no impact on colon morphology (Figure 1C).

    The mice in the HCC group had significantly higher serum ALT and AST levels than the controls. However, GM-CSF overexpression in HCC cells suppressed serum ALT and AST levels (Figure 1E). Zhuet al[23]reported that damage to intestinal tight junctions was associated with loss of intestinal barrier function. To explore this possibility as well as to examine the distribution of ZO-1 in the liver and colon[24], we first confirmed the increase in the levels of ZO-1 protein in the HCC + GM-CSF groupviaimmunohistochemistry staining. These findings suggest that GM-CSF overexpression reversed intestinal barrier dysfunction observed in response to HCC (Figure 1B and D). In parallel, the expression of ZO-1 transcripts in the liver and colon was upregulated among mice in the HCC + GM-CSF group (Figure 1F).

    Endotoxin activates specific Toll-like receptors[25]; serum LPS levels were significantly reduced in the mice in the HCC + GM-CSF group compared with those in the HCC alone group (Figure 1G). Taken together, these results suggest that GM-CSF overexpression alleviated hepatic injury, normalized liver function tests, stabilized the intestinal barrier, and reduced serum endotoxin levels observed in response to HCC.

    GM-CSF overexpression resulted in normalized cytokine profiles and Toll-like receptor expression

    Current research indicates that cytokines play an important role in the progression of HCC[26]. GM-CSF promoted significant downregulation of proinflammatory cytokines [interleukin (IL)-1β and IL-2] and increased the levels of anti-inflammatory cytokines IL-4 and IL-10, with no effects on tumor necrosis factor levels. To explore and extend these findings with respect to our mouse model, we measured the expression of cytokine transcripts in the liver tissue. GM-CSF overexpression resulted in a significant decrease in the expression of IL-1β and TGFβ; however, no changes were observed with respect to IL-4 expression (Figure 2B). Furthermore, we found that GMCSF overexpression resulted in increased IL-4 expression in the colon, whereas there were no changes in IL-1β expression (Figure 2C). Toll-like receptor (TLR)-4 and its coreceptor CD14 are central to LPS recognition[27]. Endotoxin-associated increases in both TLR4 and CD14 expression in the liver and colon were reversed by GM-CSF. These results suggest the direct effects of GM-CSF on modulating the expression of both TLR4 and CD14.

    GM-CSF ameliorated intestinal dysbiosis induced by HCC

    To obtain a greater understanding of the protective effects of GM-CSF, we examined the intestinal microbiome of the three study groups using the absolute quantificative 16S rRNA amplicon sequencing technology with an artificially synthesized DNA internal standard; use of this standard permits us to simultaneously measure the absolute and relative abundance of specific fecal bacterial communities. While previous studies have reported the relative abundance of microorganisms in specific fecal samples, increasing concern has been focused on determining the absolute abundance of targeted micro-organisms in different samples.

    Figure 1 Granulocyte-macrophage colony-stimulating factor alleviated hepatic injury, normalized liver function, improved intestinal barrier function, and reduced serum endotoxin levels detected in response to hepatocellular carcinoma. A: Liver histology was assessed by hematoxylin and eosin (H&E) staining, scale bar: 200 μm (200 ×); B: Liver histology was assessed via ZO-1 immunohistochemical staining (scale bar, 200 μm); C: Colon histology was assessed via H&E staining, scale bar: 200 μm (400 ×); D: Colon histology was assessed via ZO-1 immunohistochemical staining (scale bar, 200 μm); E: Serum alanine aminotransferase and aspartate aminotransferase (U/L) levels; F: Expression of the intestinal barrier function marker ZO-1 in the liver and colon; G: Serum lipopolysaccharide levels (μmol/L). Data are presented as the means ± SE, n = 11–13 mice per group detected via one-way ANOVA with post hoc Tukey’s test. aP < 0.05, bP < 0.01, and eP < 0.001, HCC vs control; cP < 0.05, dP < 0.01, and fP < 0.001, HCC + GM-CSF vs HCC. HCC: Hepatocellular carcinoma; GM-CSF: Granulocyte-macrophage colony-stimulating factor; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; LPS: Lipopolysaccharide; H&E: Hematoxylin and eosin.

    Alpha diversities were calculated using diversity indexes. Our results showed that the Chao index and number of operational taxonomic units were significantly increased in the fecal samples of HCC + GM-CSF mice; however, there were no significant differences in the Shannon index (Supplementary Figure 1A). Βeta diversity values were calculated by weighted UniFrac principal coordinates analysis to assess the phylogenetic relationship between microbial communities. The results from the control, HCC, and HCC + GM-CSF groups can be clearly separated into different clusters (Supplementary Figure 1C). Similarly, the structure of the intestinal flora of the HCC and HCC + GM-CSF groups was clearly divided into distinct clusters.

    The relative abundance and absolute copy number of each bacterial group at the phylum level are shown in Figure 3A. The microbial composition of the HCC + GMCSF group was significantly different from that of the HCC alone group. Of note, the absolute abundance of the micro-organisms in the HCC group was reduced by 12% compared with the control group. Furthermore, the absolute abundance of intestinal microorganisms among the fecal samples from the HCC + GM-CSF group was 1.35 times higher than among those in the HCC group; these findings suggest that GM-CSF limits HCC-induced intestinal dysbiosis. The intestinal microbiota identified in all three groups primarily included bacteria within the phyla Firmicutes, Bacteroidetes, Deferribacteres, Tenericutes, Proteobacteria, Actinobacteria, and Cyanobacteria (Figure 3A). Among them, the abundance of Tenericutes, which is related to hepatopancreatic necrosis, was 2.45 times higher in the samples from the HCC groups than those from the control. GM-CSF overexpression resulted in a 63% decrease in the absolute abundance of Tenericutes compared with that observed in samples from mice with HCC. At the genus level (Figure 3B), the intestinal flora mainly includes

    Parabacteroides,Prevotella,Anaerobacterium,Eubacterium,Clostridium_lV,Anaerotruncus,Mucispirillum,Rosecusburia, andButyricicoccus. We analyzed the absolute abundance at the genus level as shown in Figure 4.

    To evaluate the differences in the intestinal microbiome at the bacterial genus level, we performed statistical evaluation focusing on the most prominent differences among the three groups. GM-CSF overexpression contributed to the high abundance of protective genera (e.g.,Roseburia,Blautia, andButyricimonass), along with a significant reduction inPrevotella,Parabacteroides,Anaerotruncu,Streptococcus,Clostridium, and

    Mucispirillum. Particularly notable was the genusPrevotella, which was reduced 10 000-fold in response to GM-CSF overexpression. To identify additional differences in the intestinal flora among the different groups, we performed the linear discriminant analysis effect size (LEfse) analysis based on the ribosomal database project classification data (Supplementary Figures 2C and D) and identified the most important differences between the HCC and HCC + GM-CSF groups, including those identified among thePrevotella,Clostridium, andAnaerotruncusgenera.

    GM-CSF normalization of the fecal metabolomic profile

    We further elucidated the intestinal dysfunction secondary to HCC through a nontargeted metabolomics study. A total of 305 metabolites included in the following Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways were identified: Biotin (vitamin) anabolic, sphingolipid, steroid anabolic, pyrimidine, citric acid cycle (TCA cycle), purine, and primary bile acid metabolism (Figure 5C). A corresponding OPLS-DA analysis was performed. As shown in Figure 5A, the score graph reveals that the metabolomic profiles from each group are clearly clustered. We further established the OPLS-DA model corresponding to the HCC and HCC + GMCSF groups; the results demonstrated that the HCC and HCC + GM-CSF groups could be clearly divided (Figure 5B). A total of 69 metabolites were screened; we identified significantly higher concentrations of biotin and oleic acid in the HCC + GM-CSF group. These two metabolites were involved in biotin and lipid metabolism, respectively. Likewise, succinic acid, fumaric acid, adenosine, and maleic acid were detected at significantly reduced concentrations in samples from the HCC + GM-CSF group (Figure 5D).

    GM-CSF remodeling of intestinal microecology and function

    Figure 2 Granulocyte-macrophage colony-stimulating factor normalized cytokine profiles and regulated Toll-like receptor expression. A: Cytokine levels in serum; B and C: Cytokine gene expression in the liver and colon. Data are presented as the means ± SE and analyzed via one-way ANOVA with post hoc Tukey’s test. n = 11-13 mice per group. aP < 0.05, bP < 0.01, and eP < 0.001 hepatocellular carcinoma (HCC) vs control; cP < 0.05, dP < 0.01, and fP < 0.001 HCC + GM-CSF vs HCC. IL-1β: Interleukin-1β; TGFβ: Transforming growth factor β; HCC: Hepatocellular carcinoma; GM-CSF: Granulocyte-macrophage colonystimulating factor.

    To determine the beneficial impact of GM-CSF on intestinal microecology, we conducted a correlation analysis of the interaction matrix. As part of the analysis, we defined the following: Representative microbial genera with significant alterations in abundance in the HCC and HCC + GM-CSF groups, metabolites detected at significantly different concentrations in the aforementioned two groups, and other representative parameters related to liver and intestine function (Figure 6). GM-CSF overexpression can promote remodeling of the intestinal microecology and function resulting from the development of HCC. A significant reduction in the abundance ofPrevotellain the HCC + GM-CSF group positively correlated with serum ALT and LPS levels and inversely correlated with serum IL-10 and GM-CSF levels. Additionally, the abundance of bacteria of thePrevotellagenus positively correlated with succinic acid levels, with the latter being significantly higher in fecal samples from the HCC group. Similarly, the abundance of the bacterial genusAnaerotruncus, which may be involved in the inflammatory response, was strongly positively correlated with serum ALT levels and inversely correlated with ZO-1 levels and IL-4 expression. Biotin levels, which were high in samples in response to GM-CSF overexpression, inversely correlated with IL-2 expression. Likewise, succinic acid, which was detected at relatively low levels in the fecal samples of mice in the HCC + GM-CSF group, positively correlated with ALT, AST, and LPS levels and inversely correlated with IL-4 and IL-10 expression. Taken together, these data provide evidence that GM-CSF plays a dominant role in remodeling the aberrant intestinal microecology and function that develop in response to HCC.

    Figure 3 Relative and absolute abundances of the major bacteria at phylum (A) and genus (B) levels.

    DISCUSSION

    This study demonstrated that GM-CSF overexpression can repair intestinal dysbiosis, liver damage, inflammation, and damage to liver function induced by HCC. Specifically, several complementary GM-CSF-mediated mechanisms have been revealed, including a role in (1) protecting the intestinal barrier; (2) regulating biotin and lipid metabolism; (3) attenuating inflammation; and (4) reducing endotoxin levels.

    Figure 4 Profiles of the gut microbes among the hepatocellular carcinoma + granulocyte-macrophage colony-stimulating factor, hepatocellular carcinoma and control groups. The absolute abundance of each genus is indicated on the y-axis. The ordinate represents the abundance value among the three groups. The Kruskal-Wallis rank-sum test was used to analyze the different species. The figure shows all the significantly different genera in the diversity indexes among the groups, and a P value < 0.05 was considered significantly different. HCC: Hepatocellular carcinoma; GM-CSF: Granulocytemacrophage colony-stimulating factor.

    Enterohepatic recycling depends heavily on the nature and function of the intestinal micro-organisms; the intestinal flora also plays a critical role in protecting against infections[28]. TLR signaling triggers the initial inflammatory response by releasing cytokines, which may promote intestinal dysbiosis along with secondary TLR activation and signaling[29]. In this study, we found that GM-CSF overexpression resulted in significant downregulation of TLR4 expression in both liver and colon tissues. These findings suggest that GM-CSF provides protection from HCC-mediated destruction of the intestinal barrier and limits the inflammatory response secondary to LPS-mediated TLR4 signaling. The gut microbiota and metabolic products are translocated to the liverviathe portal circulation, consequently activating TLR4 in the immune cells localized in the hepatic tissue. Emerging studies have shown that intestinal mucosal damage and epithelial barrier disruption result in bacterial translocation and overgrowth; as such, endotoxins can accumulate in the liver. As the liver loses its toxin clearing ability, endotoxins will find their way into the systemic circulation[30]. This scenario has been reported in patients with alcoholic fatty liver in which significant elevations in systemic LPS have been detected[13]. In this study, we found that LPS levels were significantly elevated in the serum of mice with HCC; these levels were markedly reduced in mice with HCC and GM-CSF overexpression. Recent studies have shown that elevated LPS levels in patients with liver injury are mainly due to impaired intestinal barrier function and intestinal dysbiosis[31]. LPS is a pathogen-associated molecular pattern molecule that can trigger severe inflammatory responses both directly and indirectly[14]. Taken together, our data suggest that GMCSF may limit LPS-mediated activation of the TLR4 signaling pathway, thereby protecting against the inflammatory mic-roenvironment generated in response to HCC.

    Figure 5 Granulocyte-macrophage colony-stimulating factor normalized the fecal metabolomic profile detected in response to hepatocellular carcinoma. A: OPLS-DA score scatter plot comparing the control (purple), hepatocellular carcinoma (HCC) (red), and HCC + granulocytemacrophage colony-stimulating factor (GM-CSF) (blue) groups; B: OPLS-DA score scatter plot comparing the HCC (red) and HCC + GM-CSF (blue) groups; C: Differential involvement of the metabolic pathways between the three groups; D: Heat map detailing the differential detection of metabolites comparing the HCC and HCC + GM-CSF groups. HCC: Hepatocellular carcinoma; GM-CSF: Granulocyte-macrophage colony-stimulating factor.

    Figure 6 Correlation analysis of the representative microbial genera, characterized metabolites, and environmental factors (parameters indicating liver and gut impairment) among the control, hepatocellular carcinoma, and hepatocellular carcinoma + granulocytemacrophage colony-stimulating factor groups. Spearman’s rank correlation was used, and significant associations with P < 0.05 and r > 0.5 were found. Blue nodes, representative injury parameters; green nodes, differentially distributed genera; yellow nodes, metabolites with a high discriminative power between the groups. Green lines between nodes indicate positive linkages, and orange lines represent negative relationships. The thickness of the connection represents the correlation coefficient, with thicker lines indicating higher r values. ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; LPS: Lipopolysaccharide; IL-2: Interleukin-2; GM-CSF: Granulocyte-macrophage colony-stimulating factor.

    Previous studies have revealed thatBacillus subtilispromotes the expression of the tight junction protein ZO-1, alters intestinal immune activity, and affects the integrity of the intestinal barrier[32,33]. In this study, we detected ZO-1 expressionviaimmunohistochemistry and gene transcription analysis, and the results revealed that mice with HCC experienced intestinal barrier dysfunction, as indicated by a significant decrease in ZO-1 expression. Conversely, GM-CSF resulted in an increase in ZO-1 expression. Overall, our results indicate that GM-CSF overexpression protects against HCC-mediated disruption of intestinal barrier function.

    Tumor growth typically takes place in a complex environment with cytokines, chemokines, and extracellular matrix components, which are collectively considered to be the “tumor microenvironment”[34]. HCC is a typical inflammation-related tumor; its initiation and development are closely associated with a specific tumor microenvironment[35]. Pollardet al[36]reported that macrophages in the tumor microenvironment are polarized into M1 and M2 phenotypes in response to GM-CSF. Macrophages can be recruited to tumor tissues where they can modulate and regulate the local immune response[37]. In this study, we found that liver and systemic inflammatory responses were ameliorated in response to GM-CSF overexpression; these observations may be directly related to the impact of GM-CSF on macrophage activation. In addition, we found that serum levels of the proinflammatory cytokines IL-1β and IL-2 and the liver enzymes ALT and AST were significantly downregulated in response to GM-CSF overexpression, whereas the expression of the antiinflammatory cytokines IL-4 and IL-10 was increased. These findings suggest that liver function was protected by GM-CSF overexpression and that liver cell damage and the associated inflammation were reduced.

    A previous study has shown that translocation of intestinal flora results in chronic inflammation in the liver, thereby exacerbating HCC[38]. To generate an accurate evaluation of the structural characteristics of the intestinal flora, we determined the absolute copy number of the 16S rRNA genes in fecal samples from control mice and mice with HCC with or without GM-CSF overexpression. We identified an increase in the overall abundance of Tenericutes associated with hepatopancreatic necrosis disease in mice with HCC. In contrast, GM-CSF overexpression in association with HCC led to a 63% reduction in the overall abundance of bacteria of this phylum. It is also worth noting that GM-CSF overexpression in association with HCC resulted in a reduction in the overall abundance ofParabacteroide, Prevotella,Streptococcus, andClostridiumspp. and increased the overall abundance of anti-inflammatory bacterial species, includingBlautia,Butyricimonass, andRoseburia[39-41].

    Recent research findings have suggested that patients with type 2 diabetes mellitus, inflammatory bowel diseases, colorectal adenocarcinoma, and liver cancer present with moderate dysbiosis, including a net decrease in the anti-inflammatory bacterial genera and an increase in various potential pathogens[42-45]. These results parallel our findings on the beneficial effects of GM-CSF overexpression in association with HCC.

    In our previous research, we found an increase in the abundance ofPrevotellain the feces of patients with HCC compared with healthy volunteers[17].Prevotellais a conditional pathogen that was originally identified in the oral cavity and vagina;Prevotellaspp. is associated with periodontal disease. The identification of a large number of these bacteria in the feces of patients with HCC suggests translocation of oral bacteria to the gastrointestinal tract. The bacterial genomes ofPrevotellaspp. encode superoxide reductase, which facilitates resistance to host-derived reactive oxygen species and promotes intestinal inflammation by generating thioredoxin[46]. In this study, we found thatPrevotellapositively correlated with ALT, IL-2, and LPS levels and inversely correlated with IL-10 and GM-CSF; this finding is consistent with the conclusions from the aforementioned previous studies. Absolute numbers of bacteria from the phylumAnaerotruncuswere also present in significantly reduced numbers in the fecal samples of mice overexpressing GM-CSF; these bacteria may promote both intestinal and hepatic inflammatory responses.Anaerotruncuslevels strongly positively correlated with serum ALT levels and inversely correlated with the expression of the intestinal barrier function protein ZO-1 and the anti-inflammatory factor IL-4. Overall, these results imply that bacterial populations responding significantly to GM-CSF overexpression may be those regulating inflammation, immunity, and barrier functions.

    Microbes are connected to each otherviacomplex metabolic chains. Studying microbial metabolism in the intestines may help in determining the interactions among the members of the microbial community. Therefore, to achieve a better understanding of the protective effects of GM-CSF against HCC, we integrated metabolomics into this study. We found that GM-CSF overexpression was associated with a significant increase in biotin and oleic acid levels and a concomitant decrease in succinic acid, adenosine, fumaric acid, lipoic acid, and maleic acid levels. As a group, these metabolites are closely related to biotin and lipid metabolism pathways. Biotin is a vitamin synthesized by intestinal microorganisms[47]that enhanced expression of glucokinase in rat liver[48]. When biotin levels are low, protein synthesis in the liver and intestinal mucosa is inhibited; protein synthesis is restored after biotin supplementation[49]. Likewise, succinic acid levels were diminished in the fecal samples of GM-CSF-overexpressing mice. Earlier studies have reported that succinic acid is a toxic factor directly associated with inflammation and disease[50]. However, no changes in bile acids were detected in our study. This may be because our research used a GCMS detection platform to collect data and the Fiehn and National Institute of Standards and Technology (NIST) databases to determine the nature of the substances. Because the database of the GC platform itself contains relatively few bile acids, there are no qualitative bile acids available. In the future, the LC platform will be used to detect related bile acid substances, and relevant research will be conducted. We found that succinic acid levels positively correlated with serum ALT, AST, and LPS levels and inversely correlated with IL-4 and IL-10 levels; these findings are consistent with previous results. Interestingly,Prevotellaare succinic acid-producing bacteria, explaining the positive correlation between succinic acid andPrevotella. GM-CSF overexpression resulted in the reduced abundance ofPrevotella[51], possibly constituting an effective feedback loop in the intestinal microecosystem, serving to magnify the protective effects of GM-CSF overexpression.

    CONCLUSION

    In summary, we have demonstrated that GM-CSF overexpression can ameliorate intestinal dysbiosis, attenuate inflammation, and reduce endotoxin levels associated with HCC pathogenesis. In light of our findings, a more comprehensive understanding of the impact of GM-CSF in this setting might lead to new insights into the treatment for HCC.

    ARTICLE HIGHLIGHTS

    Research background

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays a contributing role in the pathogenesis of hepatocellular carcinoma (HCC) progression, and GM-CSF is a critical factor in promoting intestinal immunity.

    Research motivation

    GM-CSF may protect against the development of HCC by regulating immunity as well as the intestinal microecology.

    Research objectives

    To investigate the impact of GM-CSF on the gut microbiome and metabolic characteristics of HCC.

    Research methods

    We utilized established mouse models of HCC and overexpressed GM-CSF in HCC. Liver injury, intestinal barrier function, immune inflammation and the fecal microbiome and metabolome were studied.

    Research results

    Overexpression of GM-CSF had a significant impact on the gut microbiome of mice with HCC and resulted in a high abundance of organisms from the generaRoseburia,BlautiaandButyricimonass, along with a significant reduction inPrevotella,Parabacteroides,Anaerotruncus,Streptococcus,Clostridium, andMucispirillum. GM-CSF overexpression resulted in a substantial increase in fecal biotin and oleic acid, along with a prominent decrease in the fecal levels of succinic acid, adenosine, fumaric acid, lipoic acid, and maleic acid. The intestinal microbiota and fecal metabolites induced by GM-CSF are primarily involved in the pathways related to the reduction in the inflammatory response, biotin metabolism and intestinal barrier dysfunction.

    Research conclusions

    GM-CSF protects mice against HCC by ameliorating intestinal dysbiosis and attenuating inflammation.

    Research perspectives

    Our findings indicate that GM-CSF can protect against the development of HCC by regulating immunity as well as by modulating the abundance of specific intestinal micro-organisms and their metabolites. This study provides new insights into the treatment of HCC.

    国语自产精品视频在线第100页| 久久久精品94久久精品| 在线a可以看的网站| 麻豆av噜噜一区二区三区| 男女国产视频网站| 亚洲精品久久久久久婷婷小说 | 能在线免费观看的黄片| 亚洲国产欧美人成| 黄片wwwwww| 人妻制服诱惑在线中文字幕| 99热全是精品| 一级毛片久久久久久久久女| 久久国产乱子免费精品| 成人亚洲欧美一区二区av| 国产欧美日韩精品一区二区| 女人被狂操c到高潮| 亚洲va在线va天堂va国产| 成人午夜高清在线视频| 色噜噜av男人的天堂激情| 日本免费在线观看一区| 小蜜桃在线观看免费完整版高清| 久久99热6这里只有精品| 丝袜美腿在线中文| 亚洲18禁久久av| 欧美区成人在线视频| 亚洲色图av天堂| 青春草亚洲视频在线观看| 春色校园在线视频观看| av国产免费在线观看| 亚洲中文字幕日韩| 国产精品一区二区三区四区免费观看| 国产又色又爽无遮挡免| 最近最新中文字幕大全电影3| 色视频www国产| 欧美色视频一区免费| 99久久无色码亚洲精品果冻| 乱人视频在线观看| 欧美精品国产亚洲| 国产私拍福利视频在线观看| 3wmmmm亚洲av在线观看| 国产一级毛片七仙女欲春2| 亚洲欧美日韩东京热| 久久这里有精品视频免费| 女的被弄到高潮叫床怎么办| 免费在线观看成人毛片| 精品欧美国产一区二区三| 女人十人毛片免费观看3o分钟| 欧美97在线视频| 99久久人妻综合| 成年免费大片在线观看| 国产老妇伦熟女老妇高清| 性插视频无遮挡在线免费观看| 美女xxoo啪啪120秒动态图| 国产精品一及| 欧美日韩在线观看h| 久久热精品热| 色综合色国产| 午夜精品一区二区三区免费看| 久久精品夜色国产| videossex国产| 乱人视频在线观看| 美女国产视频在线观看| 寂寞人妻少妇视频99o| 欧美潮喷喷水| 蜜桃亚洲精品一区二区三区| 国语对白做爰xxxⅹ性视频网站| 日本猛色少妇xxxxx猛交久久| 日韩欧美精品v在线| 小说图片视频综合网站| 国产淫语在线视频| 直男gayav资源| 麻豆乱淫一区二区| 97热精品久久久久久| 欧美性感艳星| 国产成人91sexporn| 亚洲av日韩在线播放| 国产伦一二天堂av在线观看| 精品人妻一区二区三区麻豆| 久久精品夜色国产| 免费观看的影片在线观看| 舔av片在线| 亚洲18禁久久av| 日韩制服骚丝袜av| 亚洲真实伦在线观看| 白带黄色成豆腐渣| 免费观看精品视频网站| 高清毛片免费看| 麻豆乱淫一区二区| 精品一区二区三区视频在线| 日日摸夜夜添夜夜爱| 色吧在线观看| 日本熟妇午夜| 国产片特级美女逼逼视频| 日韩精品有码人妻一区| 免费人成在线观看视频色| 日本黄色视频三级网站网址| 六月丁香七月| 精品人妻偷拍中文字幕| 大又大粗又爽又黄少妇毛片口| 美女内射精品一级片tv| 大话2 男鬼变身卡| 人妻系列 视频| 精品一区二区免费观看| 免费看光身美女| 国产亚洲精品久久久com| 亚洲精品日韩av片在线观看| 乱码一卡2卡4卡精品| 亚洲av熟女| av在线天堂中文字幕| 少妇的逼好多水| 我的女老师完整版在线观看| 男女下面进入的视频免费午夜| 又粗又爽又猛毛片免费看| 日本黄色视频三级网站网址| 黄色一级大片看看| 欧美日本视频| av专区在线播放| 精品久久久久久久久久久久久| 欧美3d第一页| 亚洲国产色片| 美女cb高潮喷水在线观看| 久久精品久久精品一区二区三区| 亚洲欧洲国产日韩| 黄片无遮挡物在线观看| 国产精品综合久久久久久久免费| 美女内射精品一级片tv| 最近视频中文字幕2019在线8| 欧美一区二区国产精品久久精品| 在线免费观看的www视频| 3wmmmm亚洲av在线观看| 色噜噜av男人的天堂激情| 婷婷色麻豆天堂久久 | 七月丁香在线播放| 国产午夜精品久久久久久一区二区三区| 国产精品一区二区性色av| 精品国产一区二区三区久久久樱花 | 欧美xxxx黑人xx丫x性爽| 欧美精品国产亚洲| 七月丁香在线播放| 看非洲黑人一级黄片| 亚洲精品久久久久久婷婷小说 | 老司机影院毛片| 免费人成在线观看视频色| 亚洲av不卡在线观看| 久久精品夜色国产| 中文乱码字字幕精品一区二区三区 | 久久这里有精品视频免费| 精品一区二区三区视频在线| 欧美色视频一区免费| 精品欧美国产一区二区三| 中文乱码字字幕精品一区二区三区 | 又粗又爽又猛毛片免费看| 久久欧美精品欧美久久欧美| 国产三级中文精品| 亚洲乱码一区二区免费版| 亚洲自拍偷在线| 亚洲乱码一区二区免费版| 97人妻精品一区二区三区麻豆| 97人妻精品一区二区三区麻豆| 男女下面进入的视频免费午夜| 亚洲av成人精品一二三区| 国产成人91sexporn| 高清日韩中文字幕在线| 亚洲熟妇中文字幕五十中出| 中文精品一卡2卡3卡4更新| 国产成人福利小说| 成人特级av手机在线观看| 深爱激情五月婷婷| 日本av手机在线免费观看| av视频在线观看入口| 一区二区三区免费毛片| 毛片女人毛片| 日韩一区二区三区影片| 免费看av在线观看网站| 91精品国产九色| 91精品国产九色| 99久久成人亚洲精品观看| 中文字幕人妻熟人妻熟丝袜美| 久久久a久久爽久久v久久| 亚洲在线观看片| 亚洲人成网站在线观看播放| 欧美人与善性xxx| 看免费成人av毛片| 2021天堂中文幕一二区在线观| 九九久久精品国产亚洲av麻豆| 九九久久精品国产亚洲av麻豆| 一本—道久久a久久精品蜜桃钙片 精品乱码久久久久久99久播 | av免费在线看不卡| 黑人高潮一二区| 成人特级av手机在线观看| 午夜亚洲福利在线播放| 亚洲五月天丁香| 99久国产av精品国产电影| 精品人妻熟女av久视频| 啦啦啦啦在线视频资源| 国产精品久久电影中文字幕| 国产又黄又爽又无遮挡在线| 国产欧美日韩精品一区二区| 国产伦在线观看视频一区| 网址你懂的国产日韩在线| 精品熟女少妇av免费看| 少妇人妻一区二区三区视频| 亚洲国产精品专区欧美| 精品久久国产蜜桃| 午夜亚洲福利在线播放| 国产在视频线精品| 午夜激情欧美在线| 国产一级毛片在线| 天堂影院成人在线观看| 夜夜爽夜夜爽视频| 亚洲国产日韩欧美精品在线观看| 伦理电影大哥的女人| 国产极品精品免费视频能看的| 婷婷色麻豆天堂久久 | 中文字幕精品亚洲无线码一区| 国产精品蜜桃在线观看| 色尼玛亚洲综合影院| 国产日韩欧美在线精品| 欧美日韩国产亚洲二区| 人人妻人人澡人人爽人人夜夜 | 日本一本二区三区精品| 欧美高清性xxxxhd video| 国产又黄又爽又无遮挡在线| 尾随美女入室| 国产高清不卡午夜福利| 国产三级中文精品| 99久久精品热视频| 国国产精品蜜臀av免费| av在线播放精品| 性色avwww在线观看| 免费大片18禁| 黄色欧美视频在线观看| 老司机福利观看| 国产麻豆成人av免费视频| 内射极品少妇av片p| 网址你懂的国产日韩在线| 22中文网久久字幕| 九九久久精品国产亚洲av麻豆| 亚洲精品国产成人久久av| videossex国产| 免费在线观看成人毛片| 国产 一区 欧美 日韩| 色5月婷婷丁香| 国产午夜福利久久久久久| av免费观看日本| 国产一区二区亚洲精品在线观看| 国产成人a∨麻豆精品| 日韩 亚洲 欧美在线| 国产一区有黄有色的免费视频 | 青春草国产在线视频| 老女人水多毛片| 黄色一级大片看看| 日韩精品青青久久久久久| 婷婷六月久久综合丁香| 视频中文字幕在线观看| 国产精品嫩草影院av在线观看| 中文字幕亚洲精品专区| 久久这里有精品视频免费| 欧美区成人在线视频| 日本色播在线视频| 麻豆国产97在线/欧美| 性色avwww在线观看| 欧美成人免费av一区二区三区| 波多野结衣巨乳人妻| 国产高潮美女av| 亚洲伊人久久精品综合 | 午夜爱爱视频在线播放| 婷婷色av中文字幕| 国产淫语在线视频| 亚洲成人av在线免费| 国产午夜精品久久久久久一区二区三区| 爱豆传媒免费全集在线观看| 亚洲av熟女| 一级黄片播放器| 亚洲天堂国产精品一区在线| 亚洲国产日韩欧美精品在线观看| 国产极品精品免费视频能看的| 少妇被粗大猛烈的视频| 国产精品爽爽va在线观看网站| 插阴视频在线观看视频| 特大巨黑吊av在线直播| 午夜免费男女啪啪视频观看| 人体艺术视频欧美日本| 汤姆久久久久久久影院中文字幕 | 插逼视频在线观看| 亚洲精品自拍成人| 美女国产视频在线观看| 精品免费久久久久久久清纯| 狂野欧美白嫩少妇大欣赏| 国产成人a区在线观看| 久久鲁丝午夜福利片| 91午夜精品亚洲一区二区三区| 在线观看一区二区三区| 久久精品影院6| 97热精品久久久久久| 免费看日本二区| 中文字幕av在线有码专区| 麻豆久久精品国产亚洲av| 国产高清国产精品国产三级 | 高清av免费在线| 久久韩国三级中文字幕| 麻豆av噜噜一区二区三区| 国产伦精品一区二区三区四那| 精品不卡国产一区二区三区| 少妇被粗大猛烈的视频| 嫩草影院精品99| 国产精品久久久久久精品电影小说 | 日韩精品有码人妻一区| 国产亚洲午夜精品一区二区久久 | 美女被艹到高潮喷水动态| 国产精品乱码一区二三区的特点| 亚洲精品亚洲一区二区| 日韩三级伦理在线观看| 少妇猛男粗大的猛烈进出视频 | 三级经典国产精品| 中文天堂在线官网| 欧美一区二区精品小视频在线| ponron亚洲| 亚州av有码| 青青草视频在线视频观看| 久久99热这里只频精品6学生 | 好男人视频免费观看在线| 啦啦啦啦在线视频资源| 波多野结衣巨乳人妻| 国产精品综合久久久久久久免费| 97超视频在线观看视频| av卡一久久| 蜜桃亚洲精品一区二区三区| kizo精华| 日韩一本色道免费dvd| 爱豆传媒免费全集在线观看| 天天躁日日操中文字幕| 国产v大片淫在线免费观看| 中文资源天堂在线| 国产精品国产三级专区第一集| 中文字幕熟女人妻在线| 一本一本综合久久| 如何舔出高潮| 久久久欧美国产精品| 国产高潮美女av| 国产精品人妻久久久影院| 春色校园在线视频观看| av黄色大香蕉| 一级黄色大片毛片| 3wmmmm亚洲av在线观看| 日日干狠狠操夜夜爽| 国产精品伦人一区二区| 国产亚洲精品av在线| 日韩一本色道免费dvd| 国产av不卡久久| 国产亚洲av嫩草精品影院| 日本三级黄在线观看| 国语自产精品视频在线第100页| 少妇丰满av| 久久精品久久精品一区二区三区| 成人鲁丝片一二三区免费| 91狼人影院| 自拍偷自拍亚洲精品老妇| av在线天堂中文字幕| 免费黄色在线免费观看| 看黄色毛片网站| 亚洲精品亚洲一区二区| 久久久久久大精品| 久久人人爽人人片av| 亚洲精华国产精华液的使用体验| 精品人妻偷拍中文字幕| 国产精品三级大全| 欧美xxxx性猛交bbbb| 精品久久久久久久人妻蜜臀av| 91久久精品电影网| 国产亚洲午夜精品一区二区久久 | av天堂中文字幕网| 亚洲美女视频黄频| 国产精品久久视频播放| 精品久久久久久久久久久久久| 成年版毛片免费区| 国产伦精品一区二区三区视频9| 毛片一级片免费看久久久久| 成人亚洲精品av一区二区| 精品酒店卫生间| 男人舔女人下体高潮全视频| 欧美成人a在线观看| 又爽又黄a免费视频| 我的老师免费观看完整版| 九色成人免费人妻av| 老司机影院成人| 国产淫语在线视频| 中文字幕av成人在线电影| 国产亚洲午夜精品一区二区久久 | 高清午夜精品一区二区三区| 国产精品爽爽va在线观看网站| 午夜精品国产一区二区电影 | 中文天堂在线官网| 国国产精品蜜臀av免费| 三级毛片av免费| 在线播放无遮挡| 欧美日韩在线观看h| 久久韩国三级中文字幕| 精品久久久久久久久久久久久| 精品不卡国产一区二区三区| 99久久中文字幕三级久久日本| 禁无遮挡网站| 久久这里有精品视频免费| 2022亚洲国产成人精品| 免费大片18禁| 欧美极品一区二区三区四区| .国产精品久久| 亚洲精品色激情综合| 国产片特级美女逼逼视频| 看黄色毛片网站| av免费观看日本| 成人无遮挡网站| 天堂网av新在线| 91午夜精品亚洲一区二区三区| 噜噜噜噜噜久久久久久91| 一级av片app| 一级黄色大片毛片| 亚洲国产欧美在线一区| 熟女人妻精品中文字幕| 亚洲性久久影院| 国产不卡一卡二| 一区二区三区免费毛片| 成人美女网站在线观看视频| 精品人妻视频免费看| 六月丁香七月| 啦啦啦韩国在线观看视频| 黄片无遮挡物在线观看| 毛片一级片免费看久久久久| 日韩视频在线欧美| 两个人的视频大全免费| 青春草亚洲视频在线观看| 九九爱精品视频在线观看| 中文亚洲av片在线观看爽| 国国产精品蜜臀av免费| 亚洲精品456在线播放app| 国产高清不卡午夜福利| 有码 亚洲区| 又爽又黄无遮挡网站| 国产av码专区亚洲av| 国产 一区 欧美 日韩| 日本免费在线观看一区| 成人特级av手机在线观看| 51国产日韩欧美| 岛国毛片在线播放| 日韩大片免费观看网站 | 极品教师在线视频| 欧美最新免费一区二区三区| 亚洲熟妇中文字幕五十中出| 乱系列少妇在线播放| 日韩,欧美,国产一区二区三区 | 中文字幕免费在线视频6| 日本免费在线观看一区| 久久久精品欧美日韩精品| 亚洲国产精品国产精品| 日本熟妇午夜| 爱豆传媒免费全集在线观看| 久久人人爽人人片av| 国产精品1区2区在线观看.| 亚洲内射少妇av| 中文字幕制服av| 欧美日韩一区二区视频在线观看视频在线 | 黄片wwwwww| 中文乱码字字幕精品一区二区三区 | 99久久精品国产国产毛片| 欧美变态另类bdsm刘玥| www日本黄色视频网| 亚洲欧美清纯卡通| 久久久久免费精品人妻一区二区| 婷婷六月久久综合丁香| 亚洲国产精品成人久久小说| 人妻制服诱惑在线中文字幕| 在线免费观看的www视频| 非洲黑人性xxxx精品又粗又长| 不卡视频在线观看欧美| 亚洲自偷自拍三级| 麻豆av噜噜一区二区三区| 欧美日韩一区二区视频在线观看视频在线 | 在线观看一区二区三区| 午夜免费激情av| 直男gayav资源| 高清毛片免费看| 国产精品不卡视频一区二区| 亚洲成人av在线免费| 国产一区二区三区av在线| 免费电影在线观看免费观看| 99久久中文字幕三级久久日本| 男女国产视频网站| 中文字幕制服av| 91久久精品电影网| 好男人视频免费观看在线| 欧美日韩国产亚洲二区| 亚洲精品色激情综合| 特大巨黑吊av在线直播| 又粗又爽又猛毛片免费看| av国产久精品久网站免费入址| 国产成人午夜福利电影在线观看| 久久久久久久久大av| 女人被狂操c到高潮| 成人毛片60女人毛片免费| 国产精品综合久久久久久久免费| 久久久久精品久久久久真实原创| 中文字幕精品亚洲无线码一区| 国产熟女欧美一区二区| 边亲边吃奶的免费视频| 久久久久久久久久黄片| 久热久热在线精品观看| 日韩一本色道免费dvd| 午夜福利视频1000在线观看| 精品酒店卫生间| 偷拍熟女少妇极品色| 美女被艹到高潮喷水动态| av又黄又爽大尺度在线免费看 | 一级爰片在线观看| 免费观看a级毛片全部| 最近2019中文字幕mv第一页| 久久久久久久久久久免费av| av在线天堂中文字幕| 啦啦啦观看免费观看视频高清| 久久这里只有精品中国| 国产69精品久久久久777片| 国内精品美女久久久久久| 一个人观看的视频www高清免费观看| 午夜视频国产福利| 日韩欧美三级三区| 女的被弄到高潮叫床怎么办| 亚洲精品aⅴ在线观看| 一区二区三区免费毛片| 国产成人freesex在线| 久久精品综合一区二区三区| 中文字幕人妻熟人妻熟丝袜美| 国产真实伦视频高清在线观看| 简卡轻食公司| 夜夜看夜夜爽夜夜摸| 国产精品久久久久久av不卡| 欧美日韩国产亚洲二区| 欧美一区二区国产精品久久精品| 免费在线观看成人毛片| 国产精品一区二区性色av| 一区二区三区免费毛片| 搞女人的毛片| 舔av片在线| 日韩一区二区视频免费看| 爱豆传媒免费全集在线观看| 亚洲精品乱码久久久v下载方式| 国产三级在线视频| 狂野欧美白嫩少妇大欣赏| 日本黄大片高清| 亚洲国产精品成人综合色| 日日摸夜夜添夜夜添av毛片| 亚洲精品成人久久久久久| 亚洲国产最新在线播放| 国产探花在线观看一区二区| 91午夜精品亚洲一区二区三区| 超碰av人人做人人爽久久| 成年av动漫网址| 日本-黄色视频高清免费观看| 亚州av有码| 少妇熟女欧美另类| av在线亚洲专区| 久久久久久久久大av| 在线免费十八禁| 国产精品99久久久久久久久| www.色视频.com| 亚洲欧美一区二区三区国产| 免费看光身美女| 亚洲av福利一区| 超碰97精品在线观看| 精品国产一区二区三区久久久樱花 | 免费av不卡在线播放| 又粗又硬又长又爽又黄的视频| 麻豆精品久久久久久蜜桃| 国产探花极品一区二区| 色综合亚洲欧美另类图片| 亚洲国产色片| 亚洲最大成人中文| 97超视频在线观看视频| 日韩一区二区三区影片| 国产白丝娇喘喷水9色精品| 国产亚洲91精品色在线| 成年免费大片在线观看| 夫妻性生交免费视频一级片| 日本猛色少妇xxxxx猛交久久| 大又大粗又爽又黄少妇毛片口| 最近最新中文字幕免费大全7| 欧美成人午夜免费资源| 日韩中字成人| 亚洲国产成人一精品久久久| 精华霜和精华液先用哪个| 免费看日本二区| av播播在线观看一区| 欧美丝袜亚洲另类| 午夜福利在线观看免费完整高清在| 久久久久九九精品影院| 插逼视频在线观看| 亚洲va在线va天堂va国产| 你懂的网址亚洲精品在线观看 | 秋霞伦理黄片| 国产一区二区在线av高清观看| 国产欧美日韩精品一区二区| 国产黄a三级三级三级人| 国产伦精品一区二区三区四那| 久久精品综合一区二区三区| 国产午夜精品一二区理论片| 亚洲精品国产成人久久av| 国产伦在线观看视频一区| 在线观看美女被高潮喷水网站| 熟妇人妻久久中文字幕3abv| 国产黄片视频在线免费观看| 国内精品一区二区在线观看| 少妇猛男粗大的猛烈进出视频 | 日韩av在线大香蕉| 网址你懂的国产日韩在线| videossex国产| 精品国产露脸久久av麻豆 | 久久久久久伊人网av|