Menstrual blood-derived mesenchymal stem cells attenuate inflammation and improve the mortality of acute liver failure combining with A2AR agonist in mice.

BACKGROUND AND AIM: Acute liver failure (ALF) poses a serious public health issue. The menstrual blood-derived mesenchymal stem cells (MenSCs) have been applied to cure various liver-related diseases. However, the efficacy and mechanism are far from clear. This study aims to explore the efficacy and potential mechanism of MenSCs to cure ALF. METHODS: We investigate the potential mechanism of MenSCs on the ALF in vitro and in vivo. A2A adenosine receptor (A2AR) activation was investigated as the potential reinforcer for MenSCs treatment. Lipid polysaccharide/d-galactosamine (d-GalN) was employed to induce ALF. Diverse techniques were used to measure the inflammatory cytokines and key signaling molecules. Hematoxylin-eosin stain and aminotransaminases were applied to evaluate the liver injury. Flow cytometry was employed to assess the T cells. RESULTS: The MenSCs can decrease the lipid polysaccharide-induced inflammatory cytokine elevation and related signaling molecules in ALF, including TLR4, phosphorylated-NF-kBp65 (p-NF-kBp65), PI3K, and p-AKT, p-mTOR and p-IKK in vitro. Moreover, MenSCs also can significantly reverse the liver injury, inflammatory cytokines elevation and related signaling molecules increase, and Treg/Th17 ratio decrease in vivo. In addition, MenSCs plus A2AR agonist can enhance the above changes. CONCLUSIONS: The MenSCs can attenuate the ALF-induced liver injury via inhibition of TLR4-mediated PI3K/Akt/mTOR/IKK signaling. Then, this inhibits the p-NF-κBp65 translocate into nuclear, which causes a decrease of inflammatory cytokines release. Moreover, A2AR agonist can play a synergic role with MenSCs and enhance the above-mentioned effects.

Protective effect of Saccharomyces boulardii on intestinal mucosal barrier of dextran sodium sulfate-induced colitis in mice.

BACKGROUND: The effect and mechanism of Saccharomyces boulardii (Sb) in inflammatory bowel disease are unclear. The objective of the study was to evaluate the impact of Sb on intestinal mucosal barrier and intestinal flora in a colitis mouse model. METHODS: Forty C57BL/6J male mice were randomly assigned to five groups: normal control group (A), pathologic control group (B), Sb treatment group (C), mesalazine treatment group (D), and Sb combined with mesalazine treatment group (E). Colitis was induced by the addition of 2.5% (wt/vol) dextran sodium sulfate (DSS) in the drinking water ad libitum for 7 days. The general condition, weight change, stool property, and bloody stool level of mice were observed to evaluate the disease activity index. The expression of zona occludens-1 (ZO-1) and occludin in intestinal tissue were measured by immunohistochemistry. The level of tumor necrosis factor-α (TNF-α) and interleukin (IL)-8 in plasma was measured by enzyme linked immunosorbent assay. Inter-cellular tight junctions were observed by transmission electron microscopy. The feces and intestinal contents were collected sterilely, and intestinal flora was analyzed by 16S rRNA sequencing. RESULTS: Compared with group B, Sb reduced the disease activity index and histological score of group C (disease activity index: group B 2.708 ±â€Š0.628, group C 1.542 ±â€Š0.616, PBC = 0.005; histological score: group B 9.875 ±â€Š3.271, group C 4.750 ±â€Š1.832, PBC = 0.005) in DSS-induced colitis in mice. Sb exerted a protect effect on the expression of ZO-1 (group B 2.075 ±â€Š1.176, group C 4.225 ±â€Š1.316, PBC = 0.019) and occludin (group B 2.200 ±â€Š0.968, group C 3.525 ±â€Š1.047, PBC = 0.023). Compared with group B, Sb decreased the level of TNF-α and IL-8 of group C (TNF-α: group B 716.323 ±â€Š44.691 ng/L, group C 521.740 ±â€Š90.121 ng/L, PBC = 0.001; IL-8: group B 128.992 ±â€Š11.475 pg/mL, group C 106.283 ±â€Š15.906 pg/mL, PBC = 0.012). Treatment with Sb preserved the tight junctions and ameliorated microvilli and inter-cellular space. Treatment with Sb also showed its own characteristics: a higher percentage of Bacteroidetes and a lower percentage of Firmicutes, with significant differences or a significant trend. The proportion of the S24-7 family was increased significantly in the Sb treatment group. CONCLUSIONS: Sb shows an anti-inflammatory effect and has a protective effect on the intestinal mucosal mechanical barrier. Sb may up-regulate the abundance of family S24-7 specifically, and maybe a mechanism underlying its function.

High Expression and Clinical Significance of Elafin in Colorectal Cancer.

OBJECTIVE: The decrease of Elafin is associated with several inflammatory diseases. Exogenous Elafin may be a treatment for IBD. Little data has shown the expression of Elafin in patients of colorectal cancer. Here, we tried to explore Elafin expression in human tissues of colorectal cancer. METHODS: We examined the protein expression of Elafin in human tissues of adjacent nontumor and colorectal tumor by immunohistochemistry (IHC) or quantitative real-time polymerase chain reaction (qRT-PCR), then analyzed the clinical and RNA-seq data presented in The Cancer Genome Atlas (TCGA) database to confirm the relationship between Elafin levels and colorectal tumor. RESULTS: Of the 88 paired samples, 68 colorectal cancer tissues indicated a high expression of Elafin compared with 52 matched adjacent noncancerous tissues. And the mRNA levels of Elafin in 35 paired tissues showed a similar trend. The RNA-seq and clinical data were available in 438 colorectal cancer tissues and 41 normal tissues in TCGA database. The RNA-seq data showed that Elafin mRNA was upregulated about twofold in colorectal cancer samples as compared to adjacent noncancerous samples (176.42 ± 402.13 vs. 96.75 ± 150.07; P = 0.208). No statistically significant correlation was found between the Elafin expression and the age, gender, tumor invasive stage, lymph node metastasis, and distant metastasis both at the protein and mRNA levels. However, the Elafin expression was correlated with clinical stage based on the AJCC guidelines at protein levels but not mRNA levels. CONCLUSIONS: Elafin was upregulated in patients of colorectal cancer, resulting to potential limitations for exogenous Elafin treatment.

Imbalanced mucosal microcirculation in the remission stage of ulcerative colitis using probe-based confocal laser endomicroscopy.

BACKGROUND: Microcirculatory disturbance is an important factor in the pathogenesis of Inflammatory Bowel Disease (IBD) but there have been few studies in this field. Confocal Laser Endomicroscopy (CLE) has been used over the last 10 years and has made it possible to explore the changes in microcirculation of the colonic mucosa. METHODS: We retrospectively selected patients who underwent probe-based Confocal Laser Endomicroscopy (pCLE) between 2014 and 2016. There were 7 patients with ulcerative colitis (UC) in clinical remission and 7 healthy subjects included in this study; all the UC patients' medical data were reviewed. For each patient, three segments of the colon were examined using pCLE including the ascending, transverse/descending and sigmoid colon. In each segment, the representative pCLE images of the three sites were selected for analysis. Four indicators, including Mean Vessel Diameter (MVD), Diameter Standard Deviation (DSD), Functional Capillary Density-long (FCDL) and Functional Capillary Density-area (FCDA), were measured with a specially designed detection software algorithm. The four indicators were compared between UC patients and healthy subjects. According to the different blood flow patterns, three types of distribution were established: the Around (A), Cobweb (C) and Deficiency (D) type. The relationships between the recurrence and blood flow patterns of UC patients were analyzed. RESULTS: MVD, DSD, FCDL and FCDA were 10.62 ± 0.56 µm, 2.23 ± 0.26, 0.030 ± 0.019 µm and 0.289 ± 0.030 for the healthy subjects and 11.06 ± 1.10 µm, 2.68 ± 0.29, 0.026 ± 0.005 µm and 0.272 ± 0.034 for the UC patients, respectively. Compared with healthy subjects, DSD was significantly increased and FCDA was significantly decreased (P < 0.01 for both). There was no difference in MVD and FCDL between UC patients and healthy subjects. The type A and type C blood flows were observed in healthy subjects (66.67 and 33.33%, respectively) while type C appears more in UC patients (71.3%) and type D blood flow could only be found in UC patients (14.29%) P < 0.01. UC patients who showed Type D blood flow had a shorter recurrence interval. CONCLUSIONS: Some local mucosal capillary density in UC patients was decreased, particularly in the inflammation-affected segment. The three mucosal blood flow patterns can be used as an indicator of mucosal healing.

Helicobacter pylori induces caudal-type homeobox protein 2 and cyclooxygenase 2 expression by modulating microRNAs in esophageal epithelial cells.

Dysregulation of microRNAs (miRNAs) has been linked to virulence factors of Helicobacter pylori. The role of H. pylori in esophageal disease has not been clearly defined. We previously reported that H. pylori esophageal colonization promotes the incidence of Barrett's esophagus and esophageal adenocarcinoma in vivo. Here, we studied the direct effects of H. pylori on the transformation of esophageal epithelial cells, with particular focus on whether H. pylori exerts its effects by modulating miRNAs and their downstream target genes. The normal human esophageal cell line HET-1A was chronically exposed to H. pylori extract and/or acidified deoxycholic acid for up to 36 weeks. The miRNA profiles of the esophageal epithelial cells associated with H. pylori infection were determined by microarray analysis. We found that chronic H. pylori exposure promoted acidified deoxycholic acid-induced morphological changes in HET-1A cells, along with aberrant overexpression of intestinal metaplasia markers and tumorigenic factors, including caudal-type homeobox protein 2 (CDX2), mucin 2, and cyclooxygenase 2 (COX2). Helicobacter pylori modified the miRNA profiles of esophageal epithelial cells, particularly aberrant silencing of miR-212-3p and miR-361-3p. Moreover, in biopsies from Barrett's esophagus patients, esophageal H. pylori colonization was associated with a significant decrease in miR-212-3p and miR-361-3p expression. Furthermore, we identified COX2 as a target of miR-212-3p, and CDX2 as a target of miR-361-3p. Helicobacter pylori infection of esophageal epithelial cells was associated with miRNA-mediated upregulation of oncoprotein CDX2 and COX2. Our observations provide new evidence about the molecular mechanisms underlying the association between H. pylori infection and esophageal carcinogenesis.

Expression and Clinical Significance of Elafin in Inflammatory Bowel Disease.

BACKGROUND: The expression of elafin in inflammatory bowel disease (IBD) is controversial. Here, we detected the expression of elafin in the peripheral blood and colonic mucosa of patient with IBD and then explored its role and value in assessing the activity and severity of IBD. MATERIALS AND METHODS: Sixty-eight patients with IBD were selected as an experimental group. The control group included 38 healthy individuals. The expression of elafin mRNA in peripheral blood leukocytes and in serum was detected by qRT-PCR and enzyme-linked immunosorbent assay, respectively. The inflamed and noninflamed tissues were collected by colonoscopy. The expression of elafin in the intestinal mucosa was determined by immunohistochemistry staining and qRT-PCR. The expression of elafin between groups and among each stage of IBD was compared. The correlations of elafin expression with erythrocyte sedimentation rate and C-reactive protein were determined by Spearman's correlation analysis and with clinical disease activity indices (Best Crohn's Disease Activity Index and modified Mayo scores) by Pearson's correlation analysis. RESULTS: Elafin mRNA levels decreased significantly in active ulcerative colitis (UC) but increased in remission UC. However, in Crohn's disease (CD), we did not detect the aforementioned significant differences. Although serum IL-8 levels increased, serum elafin concentrations decreased both in UC and in CD, but the differences among stages were not significant. The expression of elafin in the inflamed colonic mucosa in both CD and UC was lower than that in the normal mucosa in controls and lower than that in the noninflamed mucosa in IBD. Moreover, the relative expression of elafin mRNA in peripheral blood leukocytes in UC was negatively correlated with erythrocyte sedimentation rate, C-reactive protein, and modified Mayo scores, and in CD, it was negatively correlated with Best Crohn's Disease Activity Index scores. CONCLUSIONS: Elafin decreased in active patients with IBD and was negatively correlated with disease activity, suggesting that elafin may play a protective role and could be used as an index to evaluate disease activity in IBD.

Characteristics of Good's Syndrome in China: A Systematic Review.

BACKGROUND: Good's syndrome (GS) is a rare disease characterized by thymoma, hypogammaglobulinemia, low or absent B-cells, decreased T-cells, an inverted CD4+/CD8+ T-cell ratio and reduced T-cell mitogen proliferative responses. GS is difficult to diagnose preoperatively due to its rarity and lack of typical symptoms, the characteristics of Chinese GS patients are still lacking. This study aimed to systematically review all the clinical, laboratory, and immunologic findings of reported cases of Chinese patients with GS. METHODS: We searched for case reports and articles up to January 2017 using PubMed, China National Knowledge Infrastructure, Wangfang database and China Science and Technology Journal Database with the following words in combinations as key words: "thymoma," "hypogammaglobulinemia," and "Good's syndrome." The text words and MeSH terms were entered depending on the databases characteristics. The reference lists from retrieved articles were also screened for additional applicable studies. The authors were restricted to Chinese. There was no language restriction. RESULTS: Forty-seven patients were reported in 27 studies. We found that GS has a nationwide distribution and that most cases (83%) have been described on the mainland of China. The initial clinical presentation is varied, ranging from symptoms related to the thymoma to infections resulting from immunodeficiency. Type AB (50%) is the most common histologic type of thymomas in Chinese GS patients according to the World Health Organization classification of thymomas. With respect to infection, sinopulmonary infection (74%) is the most common type, followed by skin infection (10%) and intestinal tract infection (10%). Diarrhea was presented in 36% of patients, and autoimmune manifestations were presented in 36% of patients. CONCLUSIONS: GS is a rare association of thymoma and immunodeficiency with a poor prognosis. Astute clinical acumen and increased awareness of the clinical and immunological profile of GS are needed to increase early diagnosis, that would benefit improved therapeutic effects.

Macrophage targeting contributes to the inhibitory effects of embelin on colitis-associated cancer.

Macrophages are a major component of inflammatory and tumor microenvironment. We previously reported that embelin suppresses colitis-associated tumorigenesis. Here, the role of macrophage targeting in the anti-inflammatory and anti-tumor properties of embelin was investigated. By using colitis-associated cancer (CAC) model, we demonstrated that embelin significantly depleted colon macrophages by blocking their recruitment. Moreover, embelin attenuated M2-like polarization of macrophages within the tumor microenvironment and eliminated their tumor-promoting functions during the development of CAC. Embelin potently inhibited NF-κB signaling in macrophages and decreased the production of key pro-inflammatory cytokines and tumorigenic factors involved in CAC, such as TNFα, IL-6 and COX-2. In addition, embelin directly reduced the polarization of M2 macrophages in vitro even in the presence of Th2 cytokines. These results suggested that targeting macrophages is, at least in part, responsible for the anti-tumor activity of embelin in CAC. Our observations strengthen the rationale for future validation of embelin in the prevention and treatment of CAC.

Esophageal Helicobacter pylori colonization aggravates esophageal injury caused by reflux.

AIM: To investigate esophageal Helicobacter pylori (H. pylori) colonization on esophageal injury caused by reflux and the related mechanisms. METHODS: An esophagitis model, with acid and bile reflux, was surgically produced in male rats. The rats were randomly divided into either: (1) an esophagogastroduodenal anastomosis (EGDA) group; (2) an EGDA with H. pylori infection group; (3) a pseudo-operation with H. pylori infection group; or (4) a pseudo-operation group. All rats were kept for 36 wk. Based on the location of H. pylori colonization, the EGDA rats with H. pylori infection were subdivided into those with concomitant esophageal H. pylori colonization or those with only gastric H. pylori colonization. The esophageal injuries were evaluated grossly and microscopically. The expressions of CDX2 and MUC2 were determined by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry. Ki-67 antigen expression was determined by immunohistochemistry. The mRNA levels of cyclin D1, c-Myc, Bax and Bcl-2 were determined by RT-PCR. Cell apoptosis was evaluated using the TdT-mediated dUTP nick-end labeling method. RESULTS: Esophagitis, Barrett's esophagus (BE), and esophageal adenocarcinoma (EAC) developed in rats that underwent EGDA. When comparing rats with EGDA and concomitant esophageal H. pylori colonization to EGDA-only rats, the severity of injury (87.9 ± 5.2 vs 77.2 ± 8.6, macroscopically, 92.5 ± 8.0 vs 83.8 ± 5.5, microscopically, both P < 0.05) and the incidences of BE (80.0% vs 33.3%, P = 0.055) and EAC (60.0% vs 11.1%, P < 0.05) were increased. These increases were associated with upregulation of CDX2 and MUC2 mRNA (10.1 ± 5.4 vs 3.0 ± 2.9, 8.4 ± 4.6 vs 2.0 ± 3.2, respectively, Ps < 0.01) and protein (8.1 ± 2.3 vs 3.3 ± 3.1, 7.3 ± 4.0 vs 1.8 ± 2.7, respectively, all P < 0.05). The expression of Ki-67 (8.9 ± 0.7 vs 6.0 ± 1.7, P < 0.01) and the presence of apoptotic cells (8.3 ± 1.1 vs 5.3 ± 1.7, P < 0.01) were also increased significantly in rats with EGDA and concomitant esophageal H. pylori colonization compared with rats with EGDA only. The mRNA levels of cyclin D1 (5.8 ± 1.9 vs 3.4 ± 1.3, P < 0.01), c-Myc (6.4 ± 1.7 vs 3.7 ± 1.2, P < 0.01), and Bax (8.6 ± 1.6 vs 5.1 ± 1.3, P < 0.01) were significantly increased, whereas the mRNA level of Bcl-2 (0.6 ± 0.3 vs 0.8 ± 0.3, P < 0.01) was significantly reduced in rats with EGDA and concomitant esophageal H. pylori colonization compared with rats with EGDA only. CONCLUSION: Esophageal H. pylori colonization increases esophagitis severity, and facilitates the development of BE and EAC with the augmentation of cell proliferation and apoptosis in esophageal mucosa.

SchA-p85-FAK complex dictates isoform-specific activation of Akt2 and subsequent PCBP1-mediated post-transcriptional regulation of TGFß-mediated epithelial to mesenchymal transition in human lung cancer cell line A549.

A post-transcriptional pathway by which TGF-ß modulates expression of specific proteins, Disabled-2 (Dab2) and Interleukin-like EMT Inducer (ILEI), inherent to epithelial to mesenchymal transition (EMT) in murine epithelial cells through Akt2-mediated phosphorylation of poly r(C) binding protein (PCBP1), has been previously elucidated. The aims of the current study were to determine if the same mechanism is operative in the non-small cell lung cancer (NSCLC) cell line, A549, and to delineate the underlying mechanism. Steady-state transcript and protein expression levels of Dab2 and ILEI were examined in A549 cells treated with TGF-ß for up to 48 h. Induction of translational de-repression in this model was quantified by polysomal fractionation followed by qRT-PCR. The underlying mechanism of isoform-specific activation of Akt2 was elucidated through a combination of co-immunoprecipitation studies. TGF-ß induced EMT in A549 cells concomitant with translational upregulation of Dab2 and ILEI proteins through isoform-specific activation of Akt2 followed by phosphorylation of PCBP1 at serine-43. Our experiments further elucidated that the adaptor protein SchA is phosphorylated at tyrosine residues following TGF-ß treatment, which initiated a signaling cascade resulting in the sequential recruitment of p85 subunit of PI3K and focal adhesion kinase (FAK). The SchA-FAK-p85 complex subsequently selectively recruited and activated Akt2, not Akt1. Inhibition of the p85 subunit through phosphorylated 1257 peptide completely attenuated EMT in these cells. We have defined the underlying mechanism responsible for isoform-specific recruitment and activation of Akt2, not Akt1, during TGF-ß-mediated EMT in A549 cells. Inhibition of the formation of this complex thus represents an important and novel therapeutic target in metastatic lung carcinoma.

Embelin reduces colitis-associated tumorigenesis through limiting IL-6/STAT3 signaling.

The interleukin-6 (IL-6)/STAT3 signaling regulates survival and proliferation of intestinal epithelial cells and plays an important role in the pathogenesis of inflammatory bowel disease and colorectal cancer. Embelin is a small molecule inhibitor of X-linked inhibitor of apoptosis protein (XIAP), with antioxidant, anti-inflammatory, and antitumor activities. We previously showed that embelin inhibits the growth of colon cancer cells in vitro, and effectively suppresses 1,2-dimethylhydrazine dihydrochloride-induced colon carcinogenesis in mice. Here, we explored the antitumor effects and mechanisms of embelin on colitis-associated cancer (CAC) using the azoxymethane/dextran sulfate sodium (AOM/DSS) model, with a particular focus on whether embelin exerts its effect through the IL-6/STAT3 pathway. We found that embelin significantly reduced incidence and tumor size in CAC-bearing mice. In addition to inhibiting proliferation of tumor epithelial cells, embelin suppressed colonic IL-6 expression and secretion, and subsequently STAT3 activation in vivo. Importantly, in vitro studies have revealed that in colon cancer cells, embelin diminished both the constitutive and IL-6-induced STAT3 activation by stimulating Src homology domain 2-containing protein tyrosine phosphatase (SHP2) activity. Moreover, embelin protected mice from AOM/DSS-induced colitis before tumor development. Embelin decreased IL-1ß, IL-17a, and IL-23a expression as well as the number of CD4(+) T cells and macrophages infiltrating the colonic tissues. Thus, our findings demonstrated that embelin suppresses CAC tumorigenesis, and its antitumor effect is partly mediated by limiting IL-6/STAT3 activation and Th17 immune response. Embelin may be a potential agent in the prevention and treatment of CAC.

Let-7b is involved in the inflammation and immune responses associated with Helicobacter pylori infection by targeting Toll-like receptor 4.

OBJECTIVES: Toll-like receptors (TLRs) are important initiators in native immune responses to microbial infections. TLR4 is up-regulated in response to H.pylori infection in gastric epithelial cells. However, the regulatory mechanisms for the expression of TLR4 in H.pylori infection have not been clearly defined. The aims of this study are to present the evidence that microRNA let-7b directly regulates TLR4 expression in human gastric epithelial cells, and subsequently influences the activation of NF-κB and the expression of the downstream genes in H.pylori infection. METHODS: The expression of let-7b was determined in gastric mucosa specimens and in two gastric epithelial cell lines using quantitative RT-PCR. The expression of TLR4 was determined by immunohistochemistry staining and RT-PCR. The potential target of let-7b was identified by luciferase reporter assay and Western blot. Let-7b mimics and inhibitors were used to examine the effects of let-7b on NF-κB activity. The expression of the downstream genes of NF-κB was also determined in cells infected with H.pylori 26695. RESULTS: Let-7b was significantly decreased in gastric mucosa specimens and in gastric epithelial cell lines (AGS, GES-1) infected with H.pylori 26695 (cagA+). Let-7b was complementary to the 3'-UTR of TLR4 mRNA and regulated TLR4 expression via post-transcriptional suppression in gastric epithelium. Infection of H.pylori induced the expression of TLR4 and activated NF-κB in AGS and GES-1 cells. Overexpression of let-7b by mimics downregulated TLR4, and subsequently attenuated NF-κB, MyD88, NF-κB1/p50, RelA/p65. The expression of IL-8, COX-2 and CyclinD1 was inhibited in H.pylori infected cells with let-7b overexpression. Both TAK-242 (TLR4 inhibitor) and SN50 (NF-κB inhibitor) significantly inhibited the H.pylori induced downregulation of let-7b. CONCLUSIONS: Let-7b targets at TLR4 mRNA, and regulates the activation of NF-κB and the expression of the downstream genes related to the inflammation and immune responses in H.pylori infection.