• <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 ?

    P-selectin glycoprotein ligand 1 deficiency prevents development of acute pancreatitis by attenuating leukocyte infiltration

    2020-12-11 03:30:24XuZhangMingZhuXiaoLiangJiangXingLiuXueLiuPanLiuXianXianWuZhiWeiYangTaoQin
    World Journal of Gastroenterology 2020年41期

    Xu Zhang, Ming Zhu, Xiao-Liang Jiang, Xing Liu, Xue Liu, Pan Liu, Xian-Xian Wu, Zhi-Wei Yang, Tao Qin

    Abstract

    Key Words: P-selectin glycoprotein ligand 1; Acute pancreatitis; Inflammation; Leukocyte adhesion; Interleukin-6

    INTRODUCTION

    Acute pancreatitis (AP) is rapid-onset pancreatic inflammation that causes local and systemic inflammatory response syndrome (SIRS) with high morbidity and mortality[1]. Although most patients with AP experience a mild and self-resolving disease course, 15%–20% of patients develop severe AP that leads to multiple organ dysfunction syndrome and poor prognosis[2]. Uncontrolled systemic inflammation in AP results in a high risk of morbidity and mortality, but no approved therapies are currently available[3]. Immune cells infiltration such as monocytes and neutrophils is the first step of inflammation and results in pancreatic injury[4]. Therefore, blocking immune cell infiltration may be a potential and promising target for AP treatment.

    P-selectin glycoprotein ligand 1 (PSGL-1), a transmembrane glycoprotein, is mainly expressed on leukocytes. PSGL-1 regulates leukocyte activation, recruitment, and infiltrationviabinding to E-selectin and P-selectin, which play an important role in initiating inflammatory responses[5]. Knockdown of PSGL-1 effectively reduces the numbers of circulating neutrophils and monocytes, improves endothelial damage, and decreases blood pressure[6]. Blocking functional PSGL-1 reduces visceral adipose inflammation and ameliorates endothelial dysfunction in atherosclerosis[7]. The contribution of PSGL-1 to the inflammatory response has been demonstrated in various inflammation-related diseases[8]. However, the role of PSGL-1 in the development of AP has not been tested.

    This study tested the hypothesis that PSGL-1 plays a significant role in the development of AP by a clinical experiment and animal experiments using caerulein induced AP models inPSGL-1-/-andPSGL-1+/+mice, which would be a new therapeutic target for the treatment of AP.

    MATERIALS AND METHODS

    Materials

    The mouse brain endothelial cell line (Bend.3) was purchased from Feng Hui Biological Technology Company Co., Ltd. (Changsha, Hunan Province, China).PSGL-1+/+andPSGL-1-/-mice were a generous gift from Dr. Xia at the Department of Molecular Biology and Biochemistry, University of Oklahoma Health Science Center. Peripheral blood mononuclear cell (PBMC) extraction kits were purchased from Millipore Sigma Co., Ltd. (St. Louis, MO, United States). Rabbit anti-mouse Interleukin-6 (IL6) and Interleukin-1beta (IL-1beta), as well as the IL-6 inhibitor galiellalactone were purchased from Santa Cruz Biotechnology Co.,Ltd (San Francisco, CA, United States). The IL-1beta inhibitor AS101 was purchased from Selleck Chemicals Co., Ltd. (Huston, TX, United States). PerCP/Cyanine5.5-conjugated antihuman CD162 antibody, APC-conjugated anti-human CD14 antibody, FITCconjugated anti-human CD33 antibody, PE-conjugated anti-human CD45 antibody, PerCP/Cyanine5.5-conjugated anti-mouse/human CD11b antibody, APC/Cyanine7-conjugated anti-mouse CD45 antibody, PE/Cyanine7-conjugated anti-mouse Ly-6G antibody, and FITC-conjugated anti-mouse Ly-6C antibody were purchased from BioLegend Co., Ltd. (San Diego, CA, United States). A PE-conjugated rat anti-Mouse CD162 was purchased from BD Bioscicences Co., Ltd. (San Jose, CA, United States). IL-1beta enzyme-linked immunosorbent assay (ELISA) kit and IL-6 ELISA kit were purchased from Shanghai Xin Fan Biological Technology Co., Ltd. (Shanghai, China). TUNEL test kits were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Caerulein and lipopolysaccharide (LPS) were purchased from Millipore Sigma Co., Ltd. (St. Louis, MO, United States).

    Clinical experiments

    Twenty-one patients with AP who were admitted from January 2018 to January 2019 were recruited. The diagnostic criteria for AP were: (1) Abdominal pain consistent with AP (acute, sudden, persistent, severe upper abdominal pain, often radiating to the back); (2) Serum amylase and/or lipase activity at least three times higher than the normal limit; and (3) Enhanced computed tomography/magnetic resonance imaging (CT/MRI) or abdominal ultrasound showing AP-related imaging changes[9]. Venous blood was collected from patients on an empty stomach in the early morning of the day after admission. Eight healthy volunteers matched by age and sex were recruited as controls. All patients and volunteers signed an informed consent form, which was approved by the hospital's ethics committee (Approval number: QT19012; Approval date: November 15, 2017).

    Animal experiments

    AP was induced in mice by intraperitoneal injection of 0.2 mg/kg caerulein (Millipore Sigma, United States) every hour for 7 h, followed by one injection of 10 mg/kg LPS (Millipore Sigma, United States). Twenty-four hours later, the animals were sacrificed by inhalation of isoflurane (1.5%-2.5%, inhaled). The animals were 6 or 8 wk old at the time of the experiments. Groups (n= 4) of age-matched wild-type and/orPSGL-1-/-mice were used. All experimental protocols were approved by the Animal Care and Use Committee at the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences and Peking Union Medical College (Approval number: YZW19007; Approval date: December 13, 2019).

    ELISA

    The levels of the inflammatory cytokines IL-1beta and IL-6 in the serum or supernatant were tested with an IL-1beta ELISA kit (Shanghai Xin Fan Biological Technology, China) and an IL-6 ELISA kit (Shanghai Xin Fan Biological Technology, China), respectively. Briefly, serum and supernatant samples were added to 96-well plate wells that were coated with IL-1beta or IL-6 mAb, incubated for 2 h at 37 °C, washed with PBS four times for 3 min each time, and then incubated with an horseradish peroxidase (HRP) linked streptavidin solution for 30 min at 37 °C in a darkroom. All samples were tested three times, and the absorbance was measured at 450 nm with a microplate reader.

    Western blot analysis

    Total protein was extracted using a Total Protein Extraction kit (Beyotime Biotechnology), and protein concentrations were determined using a Bicinchoninic Acid (BCA) Protein Assay kit (Beyotime Biotechnology), and the amount of protein

    was calculated. Twenty micrograms of protein was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 80 V for 120 min and transferred to a nitrocellulose (NC) membrane, and then the membrane was blocked with 5% skim milk powder at room temperature for 2 h and incubated with a mouse monoclonal IL-6 antibody (Santa Cruz Biotechnology) diluted 1:1000 and an IL-1beta antibody (Santa Cruz Biotechnology) diluted 1:1000, at 4 °C overnight. The next day, the membrane was incubated with an HRP-conjugated anti-rabbit antibody (Santa Cruz Biotechnology, United States) diluted 1:5000 at room temperature for 1 h, and the signals were visualized with an electrochemiluminescence kit (Millipore Sigma).

    Flow cytometry

    Blood samples were lysed with 1 × BD FACS lysing solution (BD Biosciences, United States) and stained with fluorescently labeled antibodies for 30 min in the dark on ice to identify different leukocyte cell populations. Labeled and fixed samples were analyzed by flow cytometry on a FACSCanto III system (BD Biosciences) immediately or within 24 h. Before each run, BD cytometer setup and tracking beads (BD Biosciences) were used for internal calibration. Appropriate controls were prepared for each sample to allow for compensation and detection of nonspecific binding. Cellular fluorescence was quantified as the mean fluorescence intensity or percentage of double-positive cells at each time point. All results were analyzed by using BD FACS Diva software.

    Immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTPbiotin nick end labeling (TUNEL) assay

    Paraffin-embedded tissues were sectioned, dewaxed, immersed in graded ethanol and 3% hydrogen peroxide solution (twice), washed three times with PBS solution, and subjected to high-temperature and high-pressure antigen retrieval in a weak acid solution. Blocking was performed with 10% goat serum to block nonspecific reactions. The samples were incubated with biotin-labeled solution at 37 °C for 60 min in the dark. Then, the sections were incubated with an HRP-labeled secondary antibody, stained with 3,3′-diaminobenzidine staining solution, counterstained with hematoxylin, dehydrated in ethanol dehydration, mounted with gum, and observed under a microscope. To quantify apoptosis in the tissues, a TUNEL assay was performed according to the instructions of the TUNEL kit (Beyotime Biotechnology).

    Cell culture and adhesion assay

    The pancreatic acinar cell line AR42J was purchased from American Type Culture Collection and cultivated in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C and 5% CO2. A cell model of AP was established by incubating AR42J cells with 100 nmol/L of caerulein for 24 h after the cells reached 80% confluence. The supernatants were collected and frozen at -80 °C. Bend.3 cells were cultured with DMEM containing 10% FBS, 100 μg/mL penicillin, and 10 μg/mL streptomycin in 96-well plates. Peripheral blood monouclear cells (PBMCs) were isolated from the peripheral blood ofPSGL-1+/+andPSGL-1-/-mice as previously described[10], stained with Green 5-chloromethylformacein diacetate (CMFDA) (Abcam, United Kingdom) for 30 min at 37 °C, and resuspended with normal medium or conditioned medium containing the supernatant from the cell model. Afterwards, the treated PBMCs were incubated with cultured Bend.3 cells grown in monolayers in 96-well plates for 1 h at 37 °C. The IL-1beta inhibitor AS101 (Selleck Chemicals, United States) and IL-6 inhibitor galiellalactone (Santa Cruz Biotechnology, United States) were added simultaneously according to the experimental grouping. After the nonadherent PBMCs were removed by washing with PBS, the fluorescence signals were photographed using an inverted fluorescence microscope.

    Statistical analysis

    The data are expressed as the mean ± SD of the mean. Student’st-test was used to determine significant differences between the control and experimental groups.P< 0.05 was considered statistically significant.

    RESULTS

    Increased expression of PSGL-1 on monocytes and neutrophils from patients with AP

    We found that there was a significant increase in the number of circulating monocytes and neutrophils in AP patients compared with volunteers (Figure 1A–D). The expression of PSGL-1 on monocytes and neutrophils in AP patients was significantly elevated than that in volunteers (Figure 1E and F), which suggests that PSGL-1 plays an important role in the development of AP.

    PSGL-1 deficiency alleviates caerulein-mediated inflammatory response and acinar damage

    To further examine the role of PSGL-1 in AP, an AP mouse model was induced by caerulein inPSGL-1-/-mice andPSGL-1+/+mice (Figure 2A). Hematoxylin-eosin staining showed that tissue edema and leukocyte infiltration into the pancreas inPSGL-1+/+but not inPSGL-1-/-mice (Figure 2B). The level of amylase inPSGL-1+/+AP mice was significantly higher than that in the control group, while there was no significant increase in amylase in thePSGL-1-/-AP mice (Figure 2C). The pancreases damage rate was approximately three-fold higher inPSGL-1+/+mice than inPSGL-1-/-mice (Figure 2D). The levels of the proinflammatory cytokines IL-1beta and IL-6 were not increased in the pancreatic parenchyma and serum ofPSGL-1-/-AP mice compared with those inPSGL-1+/+AP mice (Figure 2E–H). Caerulein-induced apoptosis of acinar cells quantified by TUNEL assay was blocked, at least partially, byPSGL-1gene knockout (Figure 2I and J).

    PSGL-1 deficiency attenuates caerulein-mediated leukocyte infiltration in the pancreas

    Leukocyte infiltration, especially neutrophils and macrophages, is an important process leading to the occurrence and development of AP. We detected the infiltration of leukocytes in pancreatic tissue fromPSGL-1+/+mice andPSGL-1-/-mice by immunohistochemistry. We found more pancreatic infiltration of myeloperoxidasepositive neutrophils and F4/80-positive monocytes/macrophages inPSGL-1+/+AP mice than inPSGL-1-/-AP mice (Figure 3A-D). However, infiltration of CD3-positive T cells and CD45R-positive B cells was not different (Figure 3E-H) between the two groups of mice. These results indicate that infiltration of neutrophils and macrophages, but not T cells or B cells, is the main pathological change in AP, which is consistent with the results reported in the literature[11]. Thus, PSGL-1 deficiency attenuates infiltration of neutrophils and macrophages in the pancreas.

    PSGL-1 deficiency attenuates the number of circulating leukocytes

    AP occurs early in damaged pancreatic acinar cells, resulting in a local inflammatory response[12]. We investigated the role of PSGL-1 in systemic inflammatory response caused by AP, and found that the number of neutrophils (Ly-6G; Figure 4A and C) and monocytes (Ly-6C; Figure 4B and D) in the peripheral blood was significantly increased inPSGL-1+/+AP mice, with higher expression of PSGL-1 on neutrophils and monocytes cells (Figure 4E and F), compared withPSGL-1-/-AP mice. The results are consistent with our clinical findings, and indicate that PSGL-1 deficiency significantly prevents the systemic inflammatory response caused by AP and blocks the development of AP.

    PSGL-1 deficiency alleviates caerulein-induced leukocyte-endothelial cell adhesion

    It has been reported that IL-1beta and IL-6 are important proinflammatory factors in AP[13,14]. Therefore, we detected the levels of IL-6 and IL-1beta in the supernatants of AR42J cells treated with caerulein. We found that the change in IL-6 was more obvious than that in IL-1beta (Figure 5A). The result shows that in the early stage of AP, especially within 24 h of onset, IL-6 may play the main promotion role than IL-1beta. We speculated that IL-6 recruits leukocytes and induces leukocyte adhesion and infiltration. We then detected whether PSGL-1 deficiency affects leukocyte-endothelial cell adhesion in anin vitroAP model. As shown in Figure 5B-C the number of PBMCs (green) from wild-type mice that adhered to Bend.3 cells was significantly increased when the cells were cultured with the supernatant from the AP model. Compared with the IL-1beta inhibitor, the IL-6 inhibitor significantly decreased the number of adherent cells when the cells were cultured with the supernatant from the AP model. Furthermore, the number of PBMCs (green) fromPSGL-1-/-mice that adhered to Bend.3 cells obviously decreased when the cells were cultured with the supernatant from the AP model. These results indicate that PSGL-1 plays an important role in caerulein-induced AP by regulating leukocyte-endothelial cell adhesion, which is triggered by IL-6 in the early stage of AP.

    Figure 1 Increased expression of P-selectin glycoprotein ligand 1 on neutrophils and monocytes from patients with acute pancreatitis. A: Flow cytometry for CD14-positive cells to detect monocytes in the peripheral blood of patients (n = 21) with acute pancreatitis (AP) or normal control subjects (n = 8); B: Boxplot showing quantification of monocytes; C: Flow cytometry for CD33-positive cells to detect neutrophils in the peripheral blood of patients (n = 21) with AP or normal control subjects (n = 8); D: Boxplot showing quantification of neutrophils; E: Fluorescence intensity of P-selectin glycoprotein ligand 1 (PSGL-1) on neutrophils and monocytes; F: Boxplot showing quantification of fluorescence intensity of PSGL-1 on neutrophils and monocytes. aP < 0.05, Student's t-test. PSGL-1: P-selectin glycoprotein ligand 1.

    DISCUSSION

    Severe AP is an acute abdominal disease with a strong systemic inflammatory response. Immune cell infiltration, along with chemokine and cytokine cascademediated inflammation, plays a critical role in the development of this disease[15,16]. Although increasing evidence shows that inhibiting the immune cells (neutrophils) or specific adhesion molecules (P-selectin, lymphocyte function antigen-1, and CD11a/CD18) may have a therapeutic effect on excessive inflammatory responses in AP[17-20], no drug that effectively modulates the outcome of AP has been identified[21]. It is urgent to find a novel therapeutic target for the treatment of AP.

    Figure 2 P-selectin glycoprotein ligand 1 deficiency alleviates caerulein-mediated inflammatory response and acinar damage. A: Schematic diagram of the induction of acute pancreatitis (AP) showing the frequency of caerulein and lipopolysaccharide injections as well as the sampling time points; B: Hematoxylin-eosin staining of the pancreas of wild-type (upper panels) and P-selectin glycoprotein ligand 1 (PSGL-1)-/- (lower panels) mice 0 h and 24 h after treatment with caerulein; C: Boxplots showing the expression of amylase (n = 4); D: Boxplots showing quantification of acinar cell damage (n = 4); E: Expression of IL-6 in the pancreas of wild-type and PSGL-1-/- mice; F: Expression of IL-1beta in the pancreas of wild-type and PSGL-1-/- mice; G: Expression of IL-6 in sera of wildtype and PSGL-1-/- mice (n = 4); H: Expression of IL-1beta in sera of wild-type and PSGL-1-/- mice(n = 4); I: Transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay for apoptosis in the pancreas; J: Boxplots showing quantification of the TUNEL assays (n = 4). aP < 0.05; bP < 0.001, Student's t-test. H&E: Hematoxylin-eosin; TUNEL: Transferase-mediated dUTP-biotin nick end labeling; PSGL-1: P-selectin glycoprotein ligand 1; AP: Acute pancreatitis; WT: Wild type.

    PSGL-1, the main ligand of E-selectin and P-selectin, initiates the capture and rolling steps in leukocyte-endothelial cell adhesion and extravasation into tissues, engaging multiple inflammatory signaling pathways[22-24]. Cross-linking between PSGL-1 with Pselectin regulates the rolling and tethering of neutrophils and macrophages /monocytes, plasma B cells, dendritic cells, and T cells during extravasation[25]. Evidence shows that patients with pancreatitis have more P-/E-selectin in their serum than healthy individuals, and that these factors are involved in leukocyte adhesion and inflammation[26]. However, the role of their ligand, PSGL-1, in the development of AP remains poorly understood. Herein, we explored the role of PSGL-1 in AP and found that PSGL-1 was significantly upregulated on circulating monocytes and neutrophils in patients with AP, which indicated the close relationship between PSGL-1 and AP development.

    To better understand the function of PSGL-1, we usedPSGL-1-/-mice to explore the role of PSGL-1 in the progression of AP. We found that caerulein treatment caused amylase upregulation and acinar damage inPSGL-1+/+mice, but not inPSGL-1-/-mice. Since acinar cell death is central to AP[27], we also detected whether PSGL-1 deficiency affects acinar cell death by TUNEL assay. We found that PSGL-1 deficiency significantly decreased acinar cell death, indicating the protective effect of PSGL-1 deficiency against acinar cell damage. PSGL-1, as an initiating factor of the leukocyteendothelial cell adhesion cascade, has been well described to mediate the inflammatory response in immune cells[28]. In this study, we found that PSGL-1 deficiency not only attenuated local inflammation, but also attenuated the systemic inflammation. In addition, fewer PBMCs fromPSGL-1-/-mice adhered to endothelial cells than PBMCs from wild-type micein vitro, which was in consistent with a previous study[10]and further demonstrated the protective effect of PSGL-1 deficiency on AP development by regulating leukocyte-endothelial adhesion, which might become a promising therapeutic target.

    Figure 3 P-selectin glycoprotein ligand 1 deficiency attenuates caerulein-mediated leukocyte infiltration in the pancreas. A: Immunohistochemistry of pancreatic tissue sections for F4/80 (monocytes/macrophages); B: Immunohistochemistry of pancreatic tissue sections for myeloperoxidase (MPO) (neutrophils); C: Boxplots showing quantification of F4/80-positive monocytes/macrophages in the pancreas (n = 4); D: Boxplots showing quantification of MPO-positive neutrophils in the pancreas (n = 4); E: Immunohistochemistry of pancreatic tissue sections stained for CD3 (T cells); F: Immunohistochemistry of pancreatic tissue sections stained for CD45R (B cells); G: Boxplots showing quantification of CD3-positive T cells in the pancreas (n = 4); H: Boxplots showing quantification of CD45R-positive B cells in the pancreas (n = 4); aP < 0.001, bP < 0.01, Student's t-test. PSGL-1: P-selectin glycoprotein ligand 1; MPO: Myeloperoxidase; WT: Wild type; AP: Acute pancreatitis.

    In the initial stage of AP, pancreatic acinar cells are damaged mainly due to abnormal activation of enzymes, and circulating neutrophil and monocyte recruitment[29]by cytokines and inflammation factors[30], including IL-6 and IL-1beta, thus leading to a vicious cycle[31]. In our mechanism study, the expression of IL-6 and IL-1beta was significantly increased in the pancreas of AP mouse models and in cell lines. Evidence shows that IL-6 seems to be an effective regulator during systemic inflammation in the pancreas[32].IL-6-/-mice have a lower death rate than wild-type mice with AP, while injection of IL-6 causes more lethal AP in mice[33]. IL-6 stimulates the phosphorylation of STAT3 and the production of the neutrophil attractant CXCL1 in pancreatic acinar cells[34]. Our study demonstrated that IL-6 inhibitor prevented caerulein-induced adhesion of PBMCs-endothelial cellsin vitro, but not IL-1beta inhibitor treatment. Therefore, this study implied that PSGL-1 may initiate leukocyte adhesion and infiltration and promote the development of AP when IL-6 is activated.

    CONCLUSION

    In conclusion, PSGL-1 plays an important role in the development of AP by promoting inflammatory cell infiltration initiated by leukocyte and endothelial cell adhesionviaIL-6 stimulation. PSGL-1 deficiency may become a new drug target for AP clinical therapy.

    Figure 4 P-selectin glycoprotein ligand 1 deficiency attenuates the number of circulating leukocytes. A: Flow cytometry for CD45-positive cells to detect immune cells in the serum; B: Flow cytometry for CD11b-positive cells to detect myeloid cells in the serum; C: Boxplots showing quantification of immune cells in the serum (n = 4); D: Boxplots showing quantification of myeloid cells in the serum (n = 4); E: Flow cytometry for Ly-6G-positive cells and Ly-6C-positive cells to detect neutrophils and monocytes, respectively, in the serum; F: Boxplots showing quantification of neutrophils in the serum (n = 4); G: Boxplots showing quantification of monocytes in the serum (n = 4). aP < 0.05; bP < 0.01, cP < 0.001, Student's t-test. PSGL-1: P-selectin glycoprotein ligand 1; WT: Wild type; AP: Acute pancreatitis.

    Figure 5 P-selectin glycoprotein ligand 1 deficiency alleviates caerulein-induced leukocyte-endothelial cell adhesion. A: Enzyme-linked immunosorbent assays for IL-6 and IL-1beta in the supernatants of AR42J cells cultured in caerulein-containing medium (n = 4); B: Boxplots showing the quantification of adhered peripheral blood mononuclear cells (PBMCs) (n = 4); C: The number of PBMCs that adhered to Bend.3 cells cocultured under different conditions. Bend. 3 cells were cocultured with PBMCs from wild-type or PSGL-1-/- mice and stained with Green 5-chloromethylformacein diacetate under different conditions. Fluorescent signals were captured using a confocal microscope (Leica, 400 ×). Green represents the number of adhered PBMCs. aP < 0.05; bP < 0.01, Student's t-test. PSGL-1: P-selectin glycoprotein ligand 1; WT: Wild type; PBMCs: Peripheral blood mononuclear cells; DMEM: Dulbecco's modified Eagle's medium; AP: Acute pancreatitis.

    ARTICLE HIGHLIGHTS

    Research objectives

    This research aimed to investigate the role and mechanism of PSGL-1 in the inflammatory response during the development of AP.

    Research methods

    We used flow cytometry to detect the expression of PSGL-1 in leukocytes from AP patients and a mouse model of caerulein-induced AP. Next,PSGL-1-/-mice administered caerulein were used to detect pancreatic injury, inflammatory cytokine expression, and inflammatory cell infiltration. A peripheral blood mononuclear cellendothelial cell coculture system was used to clarify the mechanism by which PSGL-1 regulates leukocyte adhesion to endothelial cells.

    Research results

    The results of this study indicated that the numbers of monocytes and neutrophils and the expression of PSGL-1 in the peripheral blood of patients were significantly increased. PSGL-1 deficiency reduced serum amylase levels, the expression of IL-1beta and IL-6 in the serum and pancreas, the number of infiltrated neutrophils and macrophages in the pancreas, and the number of peripheral circulating neutrophils and monocytes in the AP mouse model. PSGL-1 deficiency alleviated caeruleininduced leukocyte-endothelial cell adhesion.

    Research conclusions

    PSGL-1 deficiency protects against the development of AP by inducing leukocyteendothelial cell adhesion.

    Research perspectives

    Further research will explore the effect of PSGL-1 on leukocyte function and treatments for AP involving drugs targeting PSGL-1.

    ACKNOWLEDGEMENTS

    Thanks are due to Ya-Ping Zhai and Fang-Yuan Qin for assistance with the experiments.

    一级片免费观看大全| 亚洲av成人av| 国产成人精品久久二区二区91| 99在线视频只有这里精品首页| 久久伊人香网站| 国产亚洲精品综合一区在线观看 | 在线播放国产精品三级| 欧美 亚洲 国产 日韩一| 此物有八面人人有两片| 日本精品一区二区三区蜜桃| 后天国语完整版免费观看| 国产片内射在线| 午夜日韩欧美国产| 97碰自拍视频| 亚洲无线在线观看| 非洲黑人性xxxx精品又粗又长| 国内精品久久久久精免费| 一级黄色大片毛片| 国产一卡二卡三卡精品| 一边摸一边抽搐一进一小说| 亚洲天堂国产精品一区在线| 九色亚洲精品在线播放| 午夜福利一区二区在线看| 在线免费观看的www视频| 男女床上黄色一级片免费看| 19禁男女啪啪无遮挡网站| 女生性感内裤真人,穿戴方法视频| 久久中文字幕人妻熟女| 日韩三级视频一区二区三区| 久久精品影院6| 美女午夜性视频免费| 久久香蕉精品热| 看黄色毛片网站| 亚洲精品国产一区二区精华液| 在线观看午夜福利视频| 这个男人来自地球电影免费观看| 国产亚洲av嫩草精品影院| 热99re8久久精品国产| 后天国语完整版免费观看| 99香蕉大伊视频| 制服诱惑二区| 18禁黄网站禁片午夜丰满| 夜夜夜夜夜久久久久| 99久久国产精品久久久| a在线观看视频网站| 麻豆国产av国片精品| 桃色一区二区三区在线观看| 人妻久久中文字幕网| 50天的宝宝边吃奶边哭怎么回事| 美国免费a级毛片| 亚洲 欧美 日韩 在线 免费| 久久狼人影院| 久久精品人人爽人人爽视色| 亚洲人成电影观看| 亚洲欧美日韩高清在线视频| 一区二区三区高清视频在线| 一个人免费在线观看的高清视频| 亚洲成国产人片在线观看| 亚洲一区二区三区不卡视频| 亚洲欧美日韩无卡精品| 满18在线观看网站| 国产精品 欧美亚洲| 免费无遮挡裸体视频| 国产又色又爽无遮挡免费看| 国产精品久久视频播放| 午夜免费鲁丝| 成人国产一区最新在线观看| 亚洲精品美女久久av网站| 国产精品九九99| 一个人观看的视频www高清免费观看 | 可以在线观看的亚洲视频| 亚洲 欧美一区二区三区| 丝袜在线中文字幕| 啦啦啦 在线观看视频| 麻豆国产av国片精品| 成人欧美大片| 欧美日本亚洲视频在线播放| 男女之事视频高清在线观看| 在线播放国产精品三级| 99久久久亚洲精品蜜臀av| 国产午夜精品久久久久久| 人人妻人人澡人人看| 热re99久久国产66热| 人人妻,人人澡人人爽秒播| 久久久精品欧美日韩精品| av片东京热男人的天堂| 岛国在线观看网站| 91麻豆精品激情在线观看国产| 亚洲五月婷婷丁香| 成人精品一区二区免费| 波多野结衣av一区二区av| а√天堂www在线а√下载| 成人三级做爰电影| 亚洲电影在线观看av| 一二三四社区在线视频社区8| 久久久国产成人免费| 免费一级毛片在线播放高清视频 | 成人亚洲精品av一区二区| 激情在线观看视频在线高清| 99精品欧美一区二区三区四区| 国产熟女午夜一区二区三区| 国产亚洲精品一区二区www| 人人妻人人澡欧美一区二区 | 亚洲三区欧美一区| 国产午夜精品久久久久久| 老司机午夜十八禁免费视频| 激情在线观看视频在线高清| 国产精品二区激情视频| 国产精品亚洲av一区麻豆| 怎么达到女性高潮| 日本五十路高清| 香蕉丝袜av| 可以在线观看的亚洲视频| 免费在线观看日本一区| 国产男靠女视频免费网站| 黄色视频不卡| 久久久久久亚洲精品国产蜜桃av| 国语自产精品视频在线第100页| 午夜免费观看网址| 国产欧美日韩一区二区精品| 亚洲一区二区三区不卡视频| 国产三级在线视频| cao死你这个sao货| 女人被狂操c到高潮| 午夜久久久久精精品| 真人一进一出gif抽搐免费| 色av中文字幕| 国产亚洲精品久久久久久毛片| 欧美黄色片欧美黄色片| 性少妇av在线| 亚洲五月色婷婷综合| 日本精品一区二区三区蜜桃| 首页视频小说图片口味搜索| 亚洲午夜精品一区,二区,三区| 丰满的人妻完整版| 国内精品久久久久久久电影| 久久精品亚洲熟妇少妇任你| 亚洲男人天堂网一区| 十八禁人妻一区二区| 精品国产乱子伦一区二区三区| 国产精品综合久久久久久久免费 | 婷婷丁香在线五月| 精品一区二区三区视频在线观看免费| 变态另类丝袜制服| 色综合欧美亚洲国产小说| 国产成+人综合+亚洲专区| 亚洲精品国产区一区二| 亚洲九九香蕉| bbb黄色大片| 国产激情欧美一区二区| 久久久国产欧美日韩av| 高清毛片免费观看视频网站| 国产成人精品久久二区二区免费| av视频免费观看在线观看| 香蕉国产在线看| 精品国产国语对白av| 国产xxxxx性猛交| 日韩高清综合在线| 精品久久久精品久久久| 丰满的人妻完整版| 欧美日韩一级在线毛片| 国产精品国产高清国产av| 欧美黄色片欧美黄色片| 久久久国产成人精品二区| 欧美精品亚洲一区二区| 88av欧美| 操美女的视频在线观看| 国产精品亚洲一级av第二区| 国内毛片毛片毛片毛片毛片| 我的亚洲天堂| 国产一区在线观看成人免费| 别揉我奶头~嗯~啊~动态视频| 精品国产国语对白av| 欧美黑人精品巨大| 欧美成狂野欧美在线观看| 国产成人精品久久二区二区免费| 日韩精品免费视频一区二区三区| 在线观看日韩欧美| 国语自产精品视频在线第100页| 国产一区二区三区综合在线观看| 午夜福利成人在线免费观看| 男人舔女人的私密视频| 久久久久久久久久久久大奶| 热re99久久国产66热| 亚洲精品久久成人aⅴ小说| 亚洲一卡2卡3卡4卡5卡精品中文| 成年版毛片免费区| 久久久精品欧美日韩精品| 午夜成年电影在线免费观看| 男人舔女人的私密视频| 在线观看午夜福利视频| 又黄又粗又硬又大视频| 长腿黑丝高跟| 法律面前人人平等表现在哪些方面| 一区二区三区激情视频| 久久人妻熟女aⅴ| 欧美久久黑人一区二区| 国产真人三级小视频在线观看| 高清毛片免费观看视频网站| 少妇的丰满在线观看| 精品高清国产在线一区| ponron亚洲| 国产一区二区激情短视频| 别揉我奶头~嗯~啊~动态视频| 人妻丰满熟妇av一区二区三区| 黄色 视频免费看| 欧美老熟妇乱子伦牲交| 18禁裸乳无遮挡免费网站照片 | 国产色视频综合| 久久久久精品国产欧美久久久| 欧美激情久久久久久爽电影 | 在线观看日韩欧美| 国产在线精品亚洲第一网站| 黑人巨大精品欧美一区二区mp4| av在线播放免费不卡| 国产一区二区激情短视频| 最近最新免费中文字幕在线| 成人三级做爰电影| 好看av亚洲va欧美ⅴa在| 欧美精品啪啪一区二区三区| 亚洲成人免费电影在线观看| 欧美+亚洲+日韩+国产| 国产一区在线观看成人免费| 91av网站免费观看| 国产麻豆成人av免费视频| 自拍欧美九色日韩亚洲蝌蚪91| 欧美乱色亚洲激情| 国产在线观看jvid| 一个人免费在线观看的高清视频| 69精品国产乱码久久久| 成人欧美大片| 亚洲在线自拍视频| 欧美性长视频在线观看| 国产精品二区激情视频| 国产熟女午夜一区二区三区| 国产亚洲精品综合一区在线观看 | 成人亚洲精品av一区二区| 欧美精品啪啪一区二区三区| 91麻豆精品激情在线观看国产| 欧美成狂野欧美在线观看| 亚洲国产欧美网| 亚洲精品在线观看二区| 亚洲精品中文字幕一二三四区| 高清在线国产一区| 久久人人97超碰香蕉20202| 满18在线观看网站| 色av中文字幕| 国产午夜福利久久久久久| videosex国产| а√天堂www在线а√下载| 精品第一国产精品| 国产精品综合久久久久久久免费 | 欧洲精品卡2卡3卡4卡5卡区| 黄网站色视频无遮挡免费观看| 88av欧美| 香蕉国产在线看| 中文字幕av电影在线播放| 好男人电影高清在线观看| 大陆偷拍与自拍| 少妇的丰满在线观看| 国产乱人伦免费视频| 久久婷婷人人爽人人干人人爱 | 亚洲国产精品合色在线| 日韩精品青青久久久久久| 国产一区二区三区在线臀色熟女| 国产97色在线日韩免费| 久久久久国产精品人妻aⅴ院| 精品久久久久久,| 亚洲国产欧美一区二区综合| 欧洲精品卡2卡3卡4卡5卡区| 一边摸一边做爽爽视频免费| 99国产精品免费福利视频| 麻豆国产av国片精品| 咕卡用的链子| 亚洲自拍偷在线| 日韩av在线大香蕉| 国产精品一区二区在线不卡| 又紧又爽又黄一区二区| 老汉色∧v一级毛片| 91麻豆精品激情在线观看国产| 国产精品一区二区三区四区久久 | 亚洲欧美精品综合一区二区三区| 在线免费观看的www视频| 后天国语完整版免费观看| 99久久99久久久精品蜜桃| 国产av在哪里看| 亚洲av电影在线进入| 香蕉国产在线看| 夜夜躁狠狠躁天天躁| 每晚都被弄得嗷嗷叫到高潮| 欧美激情极品国产一区二区三区| 精品无人区乱码1区二区| 女人爽到高潮嗷嗷叫在线视频| 91成人精品电影| 免费搜索国产男女视频| 两个人看的免费小视频| www日本在线高清视频| 亚洲自拍偷在线| а√天堂www在线а√下载| 亚洲精品国产一区二区精华液| 一边摸一边抽搐一进一出视频| 一级a爱片免费观看的视频| 国产精品自产拍在线观看55亚洲| 少妇的丰满在线观看| 亚洲国产欧美网| 亚洲片人在线观看| 精品熟女少妇八av免费久了| 亚洲一区二区三区不卡视频| 91麻豆精品激情在线观看国产| 国产成人一区二区三区免费视频网站| 99国产精品一区二区三区| 亚洲激情在线av| 操出白浆在线播放| 咕卡用的链子| 国产av一区二区精品久久| 亚洲成av人片免费观看| 午夜免费鲁丝| 精品人妻1区二区| 男女下面插进去视频免费观看| 色尼玛亚洲综合影院| 9热在线视频观看99| 日韩精品中文字幕看吧| 一区二区日韩欧美中文字幕| 香蕉国产在线看| 老司机深夜福利视频在线观看| 99热只有精品国产| 欧美一级毛片孕妇| 日本免费一区二区三区高清不卡 | 色综合欧美亚洲国产小说| 久久久久久免费高清国产稀缺| 免费高清在线观看日韩| 黄色a级毛片大全视频| 国产xxxxx性猛交| 亚洲人成电影免费在线| 777久久人妻少妇嫩草av网站| 黄色视频,在线免费观看| 成人国语在线视频| 色老头精品视频在线观看| 一区二区日韩欧美中文字幕| 如日韩欧美国产精品一区二区三区| 在线观看舔阴道视频| 国产精品久久久久久精品电影 | 午夜久久久在线观看| 亚洲伊人色综图| 真人一进一出gif抽搐免费| 老熟妇乱子伦视频在线观看| 亚洲av电影在线进入| 制服人妻中文乱码| 人人妻人人澡欧美一区二区 | 国产伦一二天堂av在线观看| 亚洲成人国产一区在线观看| 国产亚洲欧美精品永久| 国产精品久久久久久亚洲av鲁大| 午夜福利视频1000在线观看 | 电影成人av| 好看av亚洲va欧美ⅴa在| 国产精品二区激情视频| 国产精品久久电影中文字幕| 自线自在国产av| 这个男人来自地球电影免费观看| 欧美乱码精品一区二区三区| 十八禁网站免费在线| 欧洲精品卡2卡3卡4卡5卡区| 免费看a级黄色片| 成年人黄色毛片网站| 又黄又爽又免费观看的视频| 亚洲精品美女久久av网站| 中出人妻视频一区二区| 亚洲欧美激情在线| 制服丝袜大香蕉在线| 99久久国产精品久久久| 午夜影院日韩av| 亚洲av熟女| 午夜免费鲁丝| 久久狼人影院| 亚洲人成伊人成综合网2020| 身体一侧抽搐| 日本a在线网址| 九色国产91popny在线| 丁香欧美五月| 欧美日韩福利视频一区二区| 黑人巨大精品欧美一区二区mp4| 免费看美女性在线毛片视频| 啦啦啦韩国在线观看视频| 精品人妻在线不人妻| 亚洲欧美激情综合另类| 亚洲欧美激情在线| 欧美黄色片欧美黄色片| 日本vs欧美在线观看视频| 不卡一级毛片| 国产黄a三级三级三级人| 69精品国产乱码久久久| 成人欧美大片| 好男人电影高清在线观看| 国产成人精品在线电影| 国产极品粉嫩免费观看在线| 亚洲 欧美一区二区三区| 好男人电影高清在线观看| 国产一区二区三区在线臀色熟女| 老司机午夜十八禁免费视频| 午夜福利影视在线免费观看| 国产一区在线观看成人免费| 国产精品美女特级片免费视频播放器 | 久久香蕉激情| 欧美精品啪啪一区二区三区| 亚洲av日韩精品久久久久久密| 欧美久久黑人一区二区| 日本欧美视频一区| 很黄的视频免费| 搞女人的毛片| 在线天堂中文资源库| 国产精品 欧美亚洲| 亚洲精品一区av在线观看| 香蕉国产在线看| 一级a爱视频在线免费观看| 欧美日韩亚洲综合一区二区三区_| 欧美国产日韩亚洲一区| 亚洲全国av大片| a级毛片在线看网站| 国产熟女xx| 久久久久久久午夜电影| 久久午夜综合久久蜜桃| 巨乳人妻的诱惑在线观看| 国产成人av教育| 精品国产亚洲在线| 最近最新中文字幕大全免费视频| 国内精品久久久久精免费| 欧美在线黄色| 国产精品秋霞免费鲁丝片| 黄网站色视频无遮挡免费观看| 啪啪无遮挡十八禁网站| 国产免费男女视频| 欧美激情高清一区二区三区| 人妻丰满熟妇av一区二区三区| 高清黄色对白视频在线免费看| 亚洲免费av在线视频| 午夜视频精品福利| 后天国语完整版免费观看| 精品不卡国产一区二区三区| 一个人免费在线观看的高清视频| 国产精品一区二区在线不卡| 色在线成人网| 99在线视频只有这里精品首页| 欧美国产精品va在线观看不卡| 国产三级黄色录像| 国产成人啪精品午夜网站| 久久欧美精品欧美久久欧美| 99riav亚洲国产免费| 国产亚洲精品综合一区在线观看 | 50天的宝宝边吃奶边哭怎么回事| 精品人妻在线不人妻| 精品久久蜜臀av无| 日韩欧美国产一区二区入口| 黄片大片在线免费观看| 在线观看免费视频日本深夜| 亚洲一码二码三码区别大吗| 午夜福利影视在线免费观看| 天堂√8在线中文| 亚洲男人的天堂狠狠| 嫩草影视91久久| 久久久久国内视频| 午夜日韩欧美国产| 99热只有精品国产| 国产精品永久免费网站| 亚洲第一av免费看| 99在线视频只有这里精品首页| 国产精品乱码一区二三区的特点 | 91大片在线观看| 国产真人三级小视频在线观看| av片东京热男人的天堂| 国产成人欧美| 嫁个100分男人电影在线观看| 欧美精品啪啪一区二区三区| 欧美激情极品国产一区二区三区| 国产精品 欧美亚洲| av天堂在线播放| 亚洲精品久久国产高清桃花| 精品久久久久久久久久免费视频| 亚洲国产毛片av蜜桃av| 在线观看免费日韩欧美大片| 欧美绝顶高潮抽搐喷水| 99在线人妻在线中文字幕| 黄色丝袜av网址大全| 丁香欧美五月| 一进一出抽搐动态| 777久久人妻少妇嫩草av网站| 99热只有精品国产| 国产单亲对白刺激| 亚洲五月色婷婷综合| 亚洲精品粉嫩美女一区| 婷婷丁香在线五月| 一边摸一边做爽爽视频免费| 久久久久久国产a免费观看| 国产精品亚洲av一区麻豆| 国产精品98久久久久久宅男小说| 19禁男女啪啪无遮挡网站| 国产成人欧美在线观看| 老熟妇仑乱视频hdxx| 大型av网站在线播放| 日韩大尺度精品在线看网址 | 18禁黄网站禁片午夜丰满| 少妇熟女aⅴ在线视频| 国产精品av久久久久免费| 在线播放国产精品三级| 国产欧美日韩一区二区三区在线| 国产精品 国内视频| 黄色毛片三级朝国网站| 在线观看免费视频网站a站| 18禁黄网站禁片午夜丰满| 亚洲av五月六月丁香网| 性色av乱码一区二区三区2| aaaaa片日本免费| 18禁观看日本| 久久香蕉激情| 国产高清视频在线播放一区| 后天国语完整版免费观看| 色哟哟哟哟哟哟| 青草久久国产| 亚洲电影在线观看av| 亚洲精品久久国产高清桃花| 麻豆成人av在线观看| 中国美女看黄片| av免费在线观看网站| 18美女黄网站色大片免费观看| 国产精品99久久99久久久不卡| 一个人观看的视频www高清免费观看 | 女人被狂操c到高潮| 日韩成人在线观看一区二区三区| 久久久国产成人精品二区| 国产精品免费一区二区三区在线| 好看av亚洲va欧美ⅴa在| 一本久久中文字幕| 日本五十路高清| 亚洲第一电影网av| 黑丝袜美女国产一区| 一边摸一边做爽爽视频免费| 女人精品久久久久毛片| 美女高潮喷水抽搐中文字幕| 一本综合久久免费| 欧美成狂野欧美在线观看| 日韩成人在线观看一区二区三区| 国产成人av激情在线播放| 18禁美女被吸乳视频| 不卡一级毛片| 亚洲av电影不卡..在线观看| 国产亚洲欧美在线一区二区| 一级a爱视频在线免费观看| 精品无人区乱码1区二区| 国产欧美日韩一区二区三| 看黄色毛片网站| 在线观看午夜福利视频| 午夜影院日韩av| 精品一区二区三区av网在线观看| 亚洲七黄色美女视频| 老司机靠b影院| 美女扒开内裤让男人捅视频| 成人免费观看视频高清| 麻豆成人av在线观看| 国产亚洲欧美98| 日本精品一区二区三区蜜桃| 岛国在线观看网站| 波多野结衣一区麻豆| 亚洲精品一区av在线观看| 精品第一国产精品| 日日干狠狠操夜夜爽| 午夜精品国产一区二区电影| 国内久久婷婷六月综合欲色啪| or卡值多少钱| 在线观看www视频免费| 99久久综合精品五月天人人| 国产一区二区在线av高清观看| 国产精品一区二区精品视频观看| 性少妇av在线| 亚洲久久久国产精品| 亚洲午夜精品一区,二区,三区| 国产国语露脸激情在线看| 国产欧美日韩一区二区三| 亚洲专区字幕在线| 在线观看日韩欧美| 国产蜜桃级精品一区二区三区| 搡老熟女国产l中国老女人| tocl精华| 久热这里只有精品99| 黑人操中国人逼视频| a在线观看视频网站| 国产三级在线视频| 脱女人内裤的视频| 午夜福利在线观看吧| 亚洲欧美日韩无卡精品| 成人欧美大片| 99riav亚洲国产免费| 免费少妇av软件| 黄色毛片三级朝国网站| 十八禁人妻一区二区| 丁香欧美五月| 男男h啪啪无遮挡| 午夜免费激情av| 欧美在线黄色| 99久久99久久久精品蜜桃| 香蕉国产在线看| 久久亚洲精品不卡| 午夜福利影视在线免费观看| 别揉我奶头~嗯~啊~动态视频| 日韩欧美一区二区三区在线观看| 中文字幕精品免费在线观看视频| av福利片在线| 国产91精品成人一区二区三区| 在线十欧美十亚洲十日本专区| 人人妻人人澡人人看| av在线播放免费不卡| 国内精品久久久久精免费| 欧美激情高清一区二区三区| 国产精品综合久久久久久久免费 | 欧美亚洲日本最大视频资源| 亚洲一区高清亚洲精品|