Changes in the Microbiota and their Roles in Patients with Type 2 Diabetes Mellitus.

An association between type 2 diabetes mellitus (T2DM) and gut microbiota is well established, but the results of related studies are inconsistent. The purpose of this investigation is to elucidate the characteristics of the gut microbiota in T2DM and non-diabetic subjects. Forty-five subjects were recruited for this study, including 29 T2DM patients and 16 non-diabetic subjects. Biochemical parameters, including body mass index (BMI), fasting plasma glucose (FPG), serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), and hemoglobin A1c (HbA1c), were analyzed and correlated with the gut microbiota. Bacterial community composition and diversity were detected in fecal samples using direct smear, sequencing, and real-time polymerase chain reaction (PCR). In this study, it was observed that indicators such as BMI, FPG, HbA1c, TC, and TG in T2DM patients were on the rise, concurrent with dysbiosis of the microbiota. We observed an increase in Enterococci and a decrease in Bacteroides, Bifidobacteria, and Lactobacilli in patients with T2DM. Meanwhile, total short-chain fatty acids (SCFAs) and D-lactate concentrations were decreased in the T2DM group. In addition, FPG was positively correlated with Enterococcus and negatively correlated with Bifidobacteria, Bacteroides, and Lactobacilli. This study reveals that microbiota dysbiosis is associated with disease severity in patients with T2DM. The limitation of this study is that only common bacteria were noted in this study, and more in-depth related studies are urgently needed.

Intestinal flora alterations in patients with ulcerative colitis and their association with inflammation.

Ulcerative colitis (UC), which is a type of inflammatory bowel disease, is a chronic intestinal disorder of multifactorial etiology. Numerous studies have indicated an association between UC and intestinal bacteria. However, a limited number of studies regarding the expression of interleukin-17 (IL-17) and interleukin-23 (IL-23) in association with intestinal bacteria have been performed. The aim of the current study was to investigate the gut microbiota alterations in patients with UC, at a number of taxonomic levels, and their relationship with intestinal inflammation by analyzing the protein expression of IL-17 and IL-23. Specimens were collected from 10 healthy controls and 16 patients with UC. A histological examination was performed in colonic tissues, IL-17 and IL-23 protein expression was detected by immunohistochemistry, fecal samples were sequenced using 16S rDNA sequencing and bioinformatics analysis was performed. The UC group exhibited an increased histological score (P<0.01) and upregulated IL-17 and IL-23 expression (P<0.01). At the order level, the bacterial diversity of the UC group was decreased. ß-diversity analyses, including principal component analysis, principal coordinate analysis and non-metric multidimensional scaling, demonstrated that the two groups of samples were separated into two taxonomic categories, as distinct variations were observed in the analysis of group differences (P=0.001). Regarding the differences in species composition between the groups, Enterococcus was indicated to be the species with the greatest difference in abundance compared with the healthy control group (P<0.01), followed by Lactobacillus (P<0.05), Escherichia-Shigella (P<0.05), Bifidobacterium and Bacteroides. In addition, the average optical density of IL-17 was positively correlated with the histological score (ρ=0.669; P=0.035), Enterococcus (r=0.843; P<0.001), Lactobacillus (r=0.737; P=0.001), Bifidobacterium (r=0.773; P<0.001) and Escherichia-Shigella (r=0.663; P=0.005), and the average optical density of IL-23 was positively correlated with the histological score (ρ=0.733; P=0.016), Enterococcus (r=0.771; P<0.001), Lactobacillus (r=0.566; P=0.022), Bifidobacterium (r=0.517; P=0.041) and Escherichia-Shigella (r=0.613; P=0.012). The results of the present study indicated that the intestinal microbiota of patients with UC differed from that of healthy controls at multiple taxonomic levels. The alterations of the intestinal microflora were closely associated with the degree of inflammation. The IL-23/IL-17 axis, as a key factor in the development of UC, maybe associated with the alterations of intestinal microflora. The interaction between intestinal microflora and the IL-23/IL-17 axis may serve an important role in the pathogenesis of UC.

Irisin attenuates inflammation in a mouse model of ulcerative colitis by altering the intestinal microbiota.

Evidence has demonstrated that the gut microbiota, which consists of probiotics and pathogenic microorganisms, is involved in the initiation of ulcerative colitis (UC) via the dysregulation of intestinal microflora and normal immune interactions, which ultimately leads to intestinal mucosal dysfunction. Irisin is released from muscle cells and displays anti-inflammatory effects; however, the mechanisms underlying irisin-mediated anti-inflammatory effects in UC have not been previously reported. In the present study, mice were divided into the following four groups: i) Control; ii) irisin; iii) dextran sulfate sodium (DSS) salt; and iv) DSS + irisin. Subsequently, the effects of irisin were investigated by observing alterations in intestinal microbes. Irisin significantly reduced the degree of inflammation in UC by reversing alterations to the macroscopic score, histological score, number of CD64+ cells and inflammatory cytokine alterations (P<0.05). Analysis of the microbial diversity in the stools of mice with active UC indicated that the five bacteria that displayed the greatest alterations in relative abundance were Alloprevotella, Bacteroides, Lachnospiraceae-UCG-001, Prebotellaceae-UCG-001 and Rikenellaceae-RCB-gut-group. Furthermore, Bactoroides were positively correlated with the histopathological score (P=0.001; R=0.977) and interleukin (IL)-23 levels (P=0.008; R=0.924). Alloprevotella (P=0.001; R=-0.943), Lachnospiraceae-UCG-001 (P=0.000; R=-0.973) and Rikenollaceae-RC8-gut-group (P=0.001; R=-0.971) were negatively correlated with the histopathological score. Furthermore, Lachnospiraceae-UCG-001 (P=0.01; R=-0.873) and Rikenollaceae-RC8-gut-group (P=0.049; R=-0.814) were negatively correlated with IL-23 levels. In summary, the results of the present study suggested that irisin improved inflammation in a UC mouse model potentially via altering the gut microbiota.

Identification of genetic variants in a pedigree associated with epilepsy by using whole exome sequencing and whole genome sequencing.

Epilepsy is a common heterogeneous group of neurological disorders including electroencephalographic and brain imaging. We used whole exome sequencing and whole genome sequencing to identify variants in a pedigree associated with epilepsy. Cranium CT scan showed that the lateral right parietal lobe was hyperdense, and there were no clear boundaries with brain tissue in affected cases. Using WES, one exclusive nonsynonymous mutant in gene TSC2 (Chr16:2138307; c.5240 T > G; p.Ile1747Ser) was involved in this disease. Further analysis showed that de novo variant in TSC2 was high conserved across different species. Moreover, the two affected sisters and their father had the same compound heterozygous variants in TSC2, while the father had no epilepsy but depigmentation. These variants demonstrated that variant in TSC2 may result in epilepsy with incomplete penetrance in humans, and the CNV and SV variants we identified probably be involved in this disease.

Immunoglobulin D-λ/λ biclonal multiple myeloma: A case report.

BACKGROUND: Immunoglobulin D (IgD) multiple myeloma (MM) is a rare subtype of MM and commonly occurs in younger subjects but at a later stage of the International Staging System (ISS) when admitted. As a special type of IgD myeloma, IgD-λ/λ biclonal MM is rarer. Its serum protein electrophoresis and serum immuno-fixation electrophoresis (IFE) might find no anomalies even if the bone marrow (BM) examination is performed. Thus, it is easy to miss the diagnosis. CASE SUMMARY: A 62-year-old man diagnosed as IgD-λ/λ myeloma (ISS stage III) was admitted with fatigue and weight loss. The physical examination suggested an anemic face, a few moist rales at the left lung base, and mild concave edema in both lower extremities. Laboratory examinations showed the elevated creatinine levels, ß2-microglobulin, lactic dehydrogenase, and erythrocyte sedimentation rate, while the decreased neutrophils, granulocytes, and hemoglobin. In the serum protein electrophoresis, there appeared two inconspicuous M-spikes. Serum IFE indicated an over-representation of lambda light chain and yielded two monoclonal bands in λ region, but only one corresponding heavy chain band in the antisera to IgD region. The BM histology and BM cytology both supported the diagnosis of IgD-λ/λ myeloma. CONCLUSION: This case highlights the differential clinical manifestations and laboratory findings of IgD-λ/λ myeloma to help minimize the chance of misdiagnosis.

Primary pulmonary plasmacytoma accompanied by overlap syndrome: A case report and review of the literature.

BACKGROUND: Extramedullary plasmacytoma (EMP) is a rare kind of soft tissue plasma cell neoplasm without bone marrow involvement; this type of plasma cell neoplasm involves a lack of other systemic characteristics of multiple myeloma. Primary pulmonary plasmacytoma (PPP), with no specific clinical manifestations, is an exceedingly rare type of EMP. Because of its complexity, PPP is often difficult to diagnose, and there is no report in the literature on cases accompanied by overlap syndrome (OS). CASE SUMMARY: A 61-year-old woman without a familial lung cancer history was admitted to our hospital in 2018, for intermittent cough, expectoration, and a stuffy feeling in the chest for 50 years; these symptoms appeared intermittently, especially occurred after being cold, and had been aggravated for the last 10 d. She was diagnosed with pulmonary fibrosis and emphysema, bronchiectasis, OS, and autoimmune hepatic cirrhosis in 2017. A pulmonary examination revealed rough breath sounds in both lungs; other physical examinations found no obvious abnormalities. A routine laboratory work-up showed decreased haemoglobin, increased ESR, and abnormal GGT, ALT, IgG, γ-globulin, κ-light chain, λ-light chain, rheumatoid factor, and autoimmune antibodies. Emission computed tomography demonstrated abnormally concentrated 99mTc-MDP. Chest computed tomography revealed a soft tissue mass in the middle and lower lobes of the right lung. After right middle and inferior lobe resection with complete mediastinal lymph node dissection, immunohistochemical analysis revealed an isolated pulmonary plasmacytoma. The patient received chemotherapy for more than 1.5 years and remains in good general condition. CONCLUSION: PPP is a type of EMP, and we report an exceedingly rare presentation of PPP accompanied by OS.

IgG plasma cells initiate changes in the protein C system in mouse ulcerative colitis through CD14+CD64+ macrophage activation.

BACKGROUND: Inhibition of the protein C system (PCS) might be one of the mechanisms of ulcerative colitis (UC). OBJECTIVES: The aim of the study was to explore the role of IgG plasma cells in changes in the PCS in UC. MATERIAL AND METHODS: Dextran sulfate sodium (DSS) was chosen to induce mouse UC. Inflammation was assessed using hematoxylin & eosin (H&E) staining and immunofluorescence. The profiling of colonic plasma cells and macrophages from colitis mice was analyzed with flow cytometry. After stimulation of macrophages with IgG type immune complex (IgG-IC), western blot was used to determine tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) protein levels. After co-incubation of colonic mucosa microvascular endothelial cells (MVECs) with TNF-α or IL-6, mitogen-activated protein kinase (MAPK) expression was detected. RESULTS: The DSS-colitis mice showed higher inflammatory indexes (p < 0.05 or p < 0.01), accompanied by greater infiltration of CD38+IgG+ plasma cells (p < 0.01), CD14+CD64+ macrophages (p < 0.01) and IgG-IC than healthy mice. Enhancement of TNF-α and IL-6 protein expression was demonstrated in this subset of macrophages when stimulated by IgG-IC (p < 0.01). After MVECs were incubated with TNF-α or IL-6, the expression of ß-arrestin1, pP38 MAPK and pJNK MAPK exhibited an increase (p < 0.05 or p < 0.01), but downregulation of endothelial protein C receptor (EPCR) expression was observed (p < 0.05 or p < 0.01); this inhibition of EPCR expression was reversed by SB203580, SP600125 or U0126 (p < 0.05 or p < 0.01). In addition, changes in activated protein C (APC) presented results similar to those for EPCR expression (p < 0.05 or p < 0.01). CONCLUSIONS: These results reveal that the PCS is inhibited during UC processing. There is a possibility that the interaction between IgG plasma cells and CD14+CD64+ macrophages, as well as further secretion of cytokines from CD14+CD64+ macrophages by the formation and stimulation of IgG-IC, subsequently influence MVECs through the ß-arrestin-MAPK pathway. Enhancement of PCS activity may represent a novel approach for treating UC.

Short-term efficacy of transvaginal specimen extraction for right colon cancer based on propensity score matching: A retrospective cohort study.

BACKGROUND: Recently, the incidence of colorectal cancer has increased each year. Natural orifice specimen extraction surgery (NOSES) removes the specimen from a natural cavity of the human body (anal or vaginal) and completes reconstruction of the digestive tract. There are only a few trocar scars in the abdomen after surgery. Transvaginal specimen extraction for right-sided colon cancer is one of the classic NOSES surgeries. As NOSES is accepted by increasing numbers of colorectal surgeons, NOSES technology is becoming increasingly widely used in China and abroad. Studies have confirmed the feasibility and safety of NOSES. Therefore, it is necessary to conduct further clinical studies to evaluate the short-term efficacy of the NOSES procedure. OBJECTIVE: To investigate the short-term efficacy of transvaginal specimens for laparoscopic right colon cancer (NOSES). METHODS: We conducted a retrospective analysis of 90 cases of laparoscopic right colon cancer radical surgery performed continuously in the anorectal surgery of our Hospital from June 2015 to December 2018. Thirty-two patients underwent complete laparoscopic anastomosis and transvaginal specimen removal (NOSES group), and 58 patients underwent conventional abdominal wall removal specimen surgery (LAP group). The general data of the patients were matched by the propensity score matching (PSM) method 1:1. Thirty-one pairs of cases were successfully matched, and the intraoperative and postoperative data were analysed. RESULTS: After PSM, the baseline data were balanced between the two groups. A total of 62 patients in the two groups were successfully operated without conversion. There were no significant differences in intraoperative blood loss, lymph node dissection, sputum tumour cell positive rate, bacterial culture positive rate, postoperative follow-up and postoperative pelvic floor function evaluation (P > 0.05). Neither tumour cells nor bacteria were detected in the rinse solution at the start of the operation. Compared with the LAP group, the incidence of postoperative complications was lower in the NOSES group (6.4% vs. 29.0%, P = 0.006), and the gastrointestinal function recovery time was shorter (2.58 ±â€¯0.92 vs. 3.42 ±â€¯0.92, P = 0.001), postoperative hospital stay was shorter (6.68 ±â€¯1.47 vs. 9.58 ±â€¯2.22, P < 0.001), postoperative pain score was lower (postoperative day 1: 2.35 ±â€¯1.52 vs. 4.87 ±â€¯1.50; postoperative day 3: 1.81 ±â€¯1.11 vs. 4.00 ±â€¯1.18; postoperative day 5: 1.45 ±â€¯1.00 vs. 2.97 ±â€¯1.17; P < 0.001), additional analgesic drug use rate was lower (12.9% vs. 61.3%, P < 0.001), and patients were more satisfied with the appearance of the abdominal wall after surgery (100% vs. 23.6%, P < 0.001). CONCLUSION: This study used PSM to remove confounding factors and retrospectively analysed the short-term efficacy of transvaginal specimens for laparoscopic right colon cancer radical resection. The results showed that the laparoscopic right colon cancer radical resection was satisfactory, ensuring sterility. At the same time, there is a clear advantage in reducing postoperative pain, shortening postoperative hospital stays, reducing the incidence of postoperative complications, and improving the appearance of the abdominal wall.

Short-term efficacy of natural orifice specimen extraction surgery for low rectal cancer.

BACKGROUND: This case-control study compared the short-term clinical efficacy of natural orifice specimen extraction surgery (NOSES) using a prolapsing technique and the conventional laparoscopic-assisted approach for low rectal cancer. AIM: To further explore the application value of the transanal placement of the anvil and to evaluate the short-term efficacy of NOSES for resecting specimens of low rectal cancer, as well as to provide a theoretical basis for its extensive clinical application. METHODS: From June 2015 to June 2018, 108 consecutive laparoscopic-assisted low rectal cancer resections were performed at our center. Among them, 26 specimens were resected transanally using a prolapsing technique (NOSES), and 82 specimens were resected through a conventional abdominal wall small incision (LAP). A propensity score matching method was used to select 26 pairs of matched patients, and their perioperative data were analyzed. RESULTS: The baseline data were comparable between the two matched groups. All 52 patients underwent the surgery successfully. The operative time, blood loss, number of harvested lymph nodes, postoperative complication rate, circumferential margin involvement, postoperative follow-up data, and postoperative anal function were not statistically significant. The NOSES group had shorter time to gastrointestinal function recovery (2.6 ± 1.0 d vs 3.4 ± 0.9 d, P = 0.006), shorter postoperative hospital stay (7.1 ± 1.7 d vs 8.3 ± 1.1 d, P = 0.003), lower pain score (day 1: 2.7 ± 1.8 vs 4.6 ± 1.9, day 3: 2.0 ± 1.1 vs 4.1 ± 1.2, day 5: 1.7 ± 0.9 vs 3.3 ± 1.0, P < 0.001), a lower rate of additional analgesic use (11.5% vs 61.5%, P = 0.001), and a higher satisfaction rate in terms of the aesthetic appearance of the abdominal wall after surgery (100% vs 23.1%, P < 0.001). CONCLUSION: NOSES for low rectal cancer can achieve satisfactory short-term efficacy and has advantages in reducing postoperative pain, shortening the length of postoperative hospital stay, and improving patients' satisfaction in terms of a more aesthetic appearance of the abdominal wall.

Authenticity of pulmonary Lophomonas blattarum infection: A case report.

Pulmonary protozoal infections are rare. A 28-year-old woman was admitted to hospital with chief complains of cough, sputum, and dyspnea. The clinical laboratory tests for blood revealed an increased eosinophil percentage of 31.3% and significantly elevated total IgE. The chest computed tomography scan revealed that bilateral bronchial walls were thickening, accompanied with patchy spots scattered throughout bilateral lungs. A suspected multiflagellated protozoan was observed under a light microscope. But some different features were observed by electron microscopy, such as the orientation of flagella and nucleus. Besides, both bronchoalveolar lavage fluid and bronchoscopic brush smears underwent Gram staining and Pap staining, which revealed that numerous respiratory ciliated cells were scattered or accumulated in the sample. Finally, she was diagnosed with eosinophil pneumonia. Metronidazole, bronchodilators, and mucolytics were taken for 5 d and symptoms and pulmonary ventilation function improved. We herein report a case of chronic eosinophilic pneumonia, which was misdiagnosed as multiflagellated protozoan infection, and it is suggested that reliable diagnosis approaches are necessary, rather than clinical symptoms and morphological features.

[Role of IgG plasma cells in the change of protein C system in ulcerative colitis].

The present study is designed to explore the role of plasma cells in the change of protein C system (PCS) in ulcerative colitis (UC). Dextran sulfate sodium (DSS, 4% in concentration) was used to induce mouse UC model. The plasma cells and the type of immune complex in colon were observed by immunofluorescence. The amount and type of plasma cells separated from colonic mucosal lamina propria were detected by flow cytometry using anti-CD54+CD38+ and IgA/M/G antibodies, respectively. After stimulation of macrophages by IgG type immune complex, TNF-α and IL-6 levels were evaluated by ELISA. After co-incubation of microvascular endothelial cells with TNF-α or IL-6, the expressions of endothelial protein C receptor (EPCR) and thrombomodulin (TM), and the activity of activated protein C (APC) were examined. As the results showed, the IgG type plasma cells infiltration and the quantity of IgG type immune complex were increased in DSS group in comparison with control group. After incubation with IgG type immune complex, the levels of TNF-α and IL-6 in the supernatant of macrophages were increased (P < 0.01) in a concentration-dependent manner. Meanwhile, after incubation with TNF-α or IL-6, the expressions of EPCR and TM in the microvascular endothelial cells were decreased (P < 0.05 or P < 0.01), while the activity of APC was reduced (P < 0.05 or P < 0.01). These results suggested that the quantity of IgG type plasma cells increases in UC and forms immune complexes, which affect the secretion of cytokines from macrophage, thereby affecting the function of endothelial cells and finally inhibiting PCS in UC. Therefore, plasma cell may be a novel target for the treatment of UC.

[Emodin ameliorates the peritoneal dialysis-related peritoneal fibrosis via inhibiting the activation of Notch pathway].

Long term peritoneal dialysis (PD) is often associated with peritoneal fibrosis. The aim of this study was to explore the effect of emodin on PD-related peritoneal fibrosis and its related cellular and molecular mechanism. PD-related peritoneal fibrosis rats and cultured rat peritoneal mesothelial cells were recruited in the experiment. PD-related peritoneal fibrosis was induced by intraperitoneal injection of lactate-buffered solution containing 4.25% glucose. The peritoneal equilibrium test (PET) was performed at the end of 2 weeks, 4 weeks, and 6 weeks, respectively. HE staining and Masson staining were used for histopathological evaluation. Enzyme linked immunosorbent assay (ELISA) was used to measure the plasma N-terminal procollagen III propeptide (PIIINP) level. Real-time PCR technique was used to detect the mRNA levels of Notch1, Jagged-1, and Hes-1 in peritoneal tissue. Western blot was applied to identify the protein levels of Notch1, Jagged-1, Hes-1, and Notch intracellular domain (NICD). In vitro, Notch1 overexpressing or knockdown rat peritoneal mesothelial cells were established and Western blot was used to examine the effect of emodin on the expressions of Hes-1 and Hey. Compared with the control group, HE staining revealed that PD rats suffered from decreasing in mesothelial cells, or detaching from surface of parietal peritoneum, accompanied by infiltration of inflammatory cells; Masson staining result showed thickened peritonea (P < 0.01), and the collagen deposition in the parietal peritoneum was increased; also, PIIINP level in plasma was elevated (P < 0.01). Treatment of the PD rats with emodin increased mesothelial cells in peritoneal tissue, and decreased the peritoneal thickness (P < 0.01), collagen depositions, as well as the plasma PIIINP level (P < 0.05). The expressions of Notch1, Jagged-1, Hes-1 and NICD in peritoneal tissue were also attenuated (P < 0.05 or P < 0.01). In cultured rat peritoneal mesothelial cells, compared with emodin group, emodin further inhibited the expressions of Hes-1 and Hey induced by Notch1-overexpression (P < 0.05), but not the expressions of Hes-1 and Hey induced by Notch1-knockdown (P > 0.05). Therefore, the activation of Notch pathway may be involved in the pathological process of PD-induced peritoneal fibrosis. Emodin may ameliorate the PD-related peritoneal fibrosis through inhibiting the activation of Notch pathway.

Anti-inflammatory effect of cannabinoid agonist WIN55, 212 on mouse experimental colitis is related to inhibition of p38MAPK.

AIM: To investigate the anti-inflammatory effect and the possible mechanisms of an agonist of cannabinoid (CB) receptors, WIN55-212-2 (WIN55), in mice with experimental colitis, so as to supply experimental evidence for its clinical use in future. METHODS: We established the colitis model in C57BL/6 mice by replacing the animals' water supply with 4% dextran sulfate sodium (DSS) for 7 consecutive days. A colitis scoring system was used to evaluate the severity of colon local lesion. The plasma levels of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), and the myeloperoxidase (MPO) activity in colon tissue were measured. The expressions of cannabinoid receptors, claudin-1 protein, p38 mitogen-activated protein kinase (p38MAPK) and its phosphorylated form (p-p38) in colon tissue were determined by immunohistochemistry and Western blot. In addition, the effect of SB203580 (SB), an inhibitor of p38, was investigated in parallel experiments, and the data were compared with those from intervention groups of WIN55 and SB alone or used together. RESULTS: The results demonstrated that WIN55 or SB treatment alone or together improved the pathological changes in mice with DSS colitis, decreased the plasma levels of TNF-α, and IL-6, and MPO activity in colon. The enhanced expression of claudin-1 and the inhibited expression of p-p38 in colon tissues were found in the WIN55-treated group. Besides, the expression of CB1 and CB2 receptors was enhanced in the colon after the induction of DSS colitis, but reduced when p38MAPK was inhibited. CONCLUSION: These results confirmed the anti-inflammatory effect and protective role of WIN55 on the mice with experimental colitis, and revealed that this agent exercises its action at least partially by inhibiting p38MAPK. Furthermore, the results showed that SB203580, affected the expression of CB1 and CB2 receptors in the mouse colon, suggesting a close linkage and cross-talk between the p38MAPK signaling pathway and the endogenous CB system.

[Study of the change and role of protein C system in ulcerate colitis].

Hypercoagulable state and thrombosis are major lethal causes of ulcerate colitis (UC). The aim of the present study is to explore the change and role of protein C (PC) system in UC thrombosis. 4% dextran sulfate sodium (DSS) was used to induce the UC model, and the body weight, the length of colon, and the weight of spleen were measured after intake of DSS as drinking water for 1 week. The macroscore and microscore were examined. The quantity of macrophage in colon smooth muscle was observed by immunofluorescence, and TNF-α and IL-6 levels in plasma were evaluated by ELISA. Intravital microscopy was applied to observe colonic mucosal microvascular circulation, activities of PC and protein S (PS) were determined by immunoturbidimetry, endothelial cell protein C receptor (EPCR) and thrombomodulin (TM) expressions were detected by immunohistochemistry. In vitro, TNF-α and IL-6 levels were tested in supernatant of macrophage separated from colonic tissue. After stimulation of mouse colonic mucosa microvascular endothelial cells by TNF-α and IL-6 respectively, the activities of PC, PS, activated protein C (APC) were evaluated, and the expressions of EPCR and TM were detected by Western blotting. The results revealed that compared with control, the DSS mouse showed weight loss (P < 0.05), a shortened colon (P < 0.05), and swelled spleen (P < 0.05), accompanied by higher histological score (P < 0.05), as well as infiltration of macrophages, elevated TNF-α and IL-6 levels in plasma (P < 0.01). The intravital microscopy results revealed that compared with control, DSS mice showed significantly enhanced adhesion of leukocytes and colonic mucosal microvascular endothelial cells (P < 0.01), meanwhile, decreased activity of PC and PS in plasma (P < 0.01 or P < 0.05), and down-regulated expression of EPCR (P < 0.01). The degree of inflammation was negatively correlated with the PC activity. In vitro, TNF-α and IL-6 levels were increased in the supernatant of macrophages from DSS mice colonic tissue (P < 0.05), and after incubation of TNF-α or IL-6 with colonic mucosal microvascular endothelial cells, the APC activity was decreased (P < 0.05 or P < 0.01), and expression of EPCR was down regulated (P < 0.05). These results suggest that PC system is inhibited in UC mouse. Presumably, the mechanism may be due to the secretion of cytokines from macrophages and subsequential influence on the function of endothelia cells. Furthermore, enhancement of PC system activity may serve as a new strategy for the treatment of UC.

[Apocynin relieves inflammation in dextran sulfate sodium-induced ulcerative colitis mice: the role of NOXs-ROS-p38MAPK pathway].

The study is aimed to explore the molecular mechanism of the treatment of apocynin in dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) mice. 5% DSS was used to mimic the UC model, and 2% apocynin was applied to treat the UC mice. HE staining was used for histopathological evaluation. Chemiluminescence technique was used to measure reactive oxygen species (ROS) production, and the rate of consumption of NADPH inhibited by DPI was detected to determine the NADPH oxidases (NOXs) activity. Western blot was applied to identify the level of p38MAPK phosphorylation, Griess reaction assay to analyze NO production, immunoenzymatic method to determine prostaglandin E2 (PGE2) production, real time RT-PCR and Western blot to identify the expression of iNOS and COX2, and enzyme linked immunosorbent assay to detect inflammatory cytokines TNF-α, IL-6, IFN-γ, IL-1ß. Rat neutrophils were separated, and then ROS production, NOXs activity, NO and PGE2 production, NOX1 and p-p38MAPK expression were detected. Compared with the UC group, apocynin decreased ROS over-production and NOXs activity (P < 0.01), reduced p38MAPK phosphorylation, inhibited NO, PGE2 and cytokines production (P < 0.01). Apocynin also decreased NOXs activity and ROS over-production (P < 0.01), inhibited p38MAPK phosphorylation and NOX1 expression, and reduced NO and PGE2 production (P < 0.01) in separated neutrophils from UC mice. Therefore, apocynin could relieve inflammation in DSS-induced UC mice through inhibiting NOXs-ROS-p38MAPK signal pathway, and neutrophils play an important role.

[Role of orphan G protein-coupled receptor 55 in diabetic gastroparesis in mice].

The aim of the present study was to explore the role of orphan G protein-coupled receptor 55 (GPR55) in diabetic gastroparesis (DG). Streptozotocin (STZ) was used to mimic the DG model, and the body weight and blood glucose concentration were tested 4 weeks after STZ injection (i.p.). Electrogastrogram and phenolsulfonphthalein test were used for detecting gastric emptying. Motilin (MTL), gastrin (GAS), vasoactive intestinal peptide (VIP), and somatostatin (SS) levels in plasma were determined using radioimmunology. Real-time PCR and Western blot were applied to identify the expression of GPR55 in gastric tissue, and immunohistochemistry was used to detect the distribution. The effect of lysophosphatidylinositol (LPI), an agonist of GPR55, was observed. STZ mice showed increased blood glucose concentration, lower body weight, decreased amplitude of slow wave, and delayed gastric emptying. LPI antagonized these effects of STZ. Compared to the control group, STZ caused significant decreases of MTL and GAS levels (P < 0.01), as well as increases of SS and VIP levels (P < 0.01). The changes of these hormones induced by STZ were counteracted when using LPI. GPR55 located in mice stomach, and it was up-regulated in DG. Although LPI showed no effects on the distribution and expression of GPR55 in normal mice, it could inhibit STZ-induced GPR55 up-regulation. These results suggest GPR55 is involved in the regulation of gastric movement of DG, and may serve as a new target of DG treatment. LPI, an agonist of GPR55, can protect against STZ-induced DG, and the mechanism may involve the change of GPR55 expression and modification of gastrointestinal movement regulating hormones.

Role of endogenous cannabinoid system in the gut.

The plant Cannabis has been used in clinic for centuries, and has been known to be beneficial in a variety of gastrointestinal diseases, such as emesis, diarrhea, inflammatory bowel disease and intestinal pain. In this text, we'll review the components of the endogenous cannabinoid system as well as its role in the regulation of gastrointestinal activities, thus providing relative information for further study. Moreover, modulation of the endogenous cannabinoid system in gastrointestinal tract may provide a useful therapeutic target for gastrointestinal disorders.

Anti-inflammatory role of cannabidiol and O-1602 in cerulein-induced acute pancreatitis in mice.

OBJECTIVES: The anti-inflammatory effects of O-1602 and cannabidiol (CBD), the ligands of G protein-coupled receptor 55 (GPR55), on experimental acute pancreatitis (AP) were investigated. METHODS: Acute pancreatitis was induced in C57BL mice by intraperitoneal injection of 50 µg/kg cerulein hourly, with a total of 6 times. Drugs (O-1602, 10 mg/kg, or CBD, 0.5 mg/kg) were given by intraperitoneal injection 2 times at 30 minutes before the first injection and immediately before the fifth cerulein injection. At 3 hours after the last injection, the blood, the lungs, and the pancreas were harvested for the pancreatic enzyme activity, myeloperoxidase activity, and pro-inflammatory cytokines measurement; and the expressions of GPR55 mRNA and protein in the pancreas were detected. RESULTS: Cannabidiol or O-1602 treatment significantly improved the pathological changes of mice with AP and decreased the enzyme activities, IL-6 and tumor necrosis factor α; levels, and the myeloperoxidase activities in plasma and in the organ tissues. G protein-coupled receptor 55 mRNA and protein expressed in the pancreatic tissue, and the expressions were decreased in the mice with AP, and either CBD or O-1602 attenuated these changes to a certain extent. CONCLUSION: Cannabidiol and O-1602 showed anti-inflammatory effects in mice with AP and improved the expression of GPR55 in the pancreatic tissue as well.

Cannabinoid HU210 protects isolated rat stomach against impairment caused by serum of rats with experimental acute pancreatitis.

Acute pancreatitis (AP), especially severe acute pancreatitis often causes extra-pancreatic complications, such as acute gastrointestinal mucosal lesion (AGML) which is accompanied by a considerably high mortality, yet the pathogenesis of AP-induced AGML is still not fully understood. In this report, we investigated the alterations of serum components and gastric endocrine and exocrine functions in rats with experimental acute pancreatitis, and studied the possible contributions of these alterations in the pathogenesis of AGML. In addition, we explored the intervention effects of cannabinoid receptor agonist HU210 and antagonist AM251 on isolated and serum-perfused rat stomach. Our results showed that the AGML occurred after 5 h of AP replication, and the body homeostasis was disturbed in AP rat, with increased levels of pancreatic enzymes, lipopolysaccharide (LPS), proinflammtory cytokines and chemokines in the blood, and an imbalance of the gastric secretion function. Perfusing the isolated rat stomach with the AP rat serum caused morphological changes in the stomach, accompanied with a significant increment of pepsin and [H+] release, and increased gastrin and decreased somatostatin secretion. HU210 reversed the AP-serum-induced rat pathological alterations, including the reversal of transformation of the gastric morphology to certain degree. The results from this study prove that the inflammatory responses and the imbalance of the gastric secretion during the development of AP are responsible for the pathogenesis of AGML, and suggest the therapeutic potential of HU210 for AGML associated with acute pancreatitis.