Spontaneous colitis in IL‐10‐deficient mice was ameliorated via inhibiting glutaminase1

Immunity imbalance and barrier damage in the intestinal mucosa are the main pathogenic factors of Crohn's disease (CD). Bis‐2‐(5‐phenylacetamido‐1,2,4‐thiadiazol‐2‐yl) ethyl sulfide (BPTES) is a glutaminase 1 (Gls1) inhibitor with the dual functions of increasing glutamine levels and immune regulation. In this study, we focused on the role of BPTES in CD‐like enteritis and the possible mechanisms. We found that Gls1 expression was significantly increased in CD intestinal tissue compared with control tissue. Bis‐2‐(5‐phenylacetamido‐1,2,4‐thiadiazol‐2‐yl) ethyl sulfide treatment significantly ameliorated chronic colitis in the IL‐10^?/?, as manifested by decreased disease activity index, body weight change, histological inflammatory degree and inflammatory cytokine expression. Bis‐2‐(5‐phenylacetamido‐1,2,4‐thiadiazol‐2‐yl) ethyl sulfide treatment exerted protective effects on CD that were associated with the maintenance of intestinal barrier integrity and the Th/Treg balance. Bis‐2‐(5‐phenylacetamido‐1,2,4‐thiadiazol‐2‐yl) ethyl sulfide treatment may act in part through TCR‐mediated mammalian target of rapamycin complex 1 (mTORC1) signalling activation. In conclusion, inhibition of Gls1 expression attenuated chronic colitis by maintaining intestinal barrier integrity and the Th/Treg balance, thereby ameliorating CD‐like colitis.     

Epac‐2 ameliorates spontaneous colitis in Il‐10 −/− mice by protecting the intestinal barrier and suppressing NF‐κB/MAPK signalling

Intestinal barrier dysfunction and intestinal inflammation interact in the progression of Crohn''s disease (CD). A recent study indicated that Epac‐2 protected the intestinal barrier and had anti‐inflammatory effects. The present study examined the function of Epac‐2 in CD‐like colitis. Interleukin‐10 gene knockout (Il‐10 ^−/−) mice exhibit significant spontaneous enteritis and were used as the CD model. These mice were treated with Epac‐2 agonists (Me‐cAMP) or Epac‐2 antagonists (HJC‐0350) or were fed normally (control), and colitis and intestinal barrier structure and function were compared. A Caco‐2 and RAW 264.7 cell co‐culture system were used to analyse the effects of Epac‐2 on the cross‐talk between intestinal epithelial cells and inflammatory cells. Epac‐2 activation significantly ameliorated colitis in mice, which was indicated by reductions in the colitis inflammation score, the expression of inflammatory factors and intestinal permeability. Epac‐2 activation also decreased Caco‐2 cell permeability in an LPS‐induced cell co‐culture system. Epac‐2 activation significantly suppressed nuclear factor (NF)‐κB/mitogen‐activated protein kinase (MAPK) signalling in vivo and in vitro. Epac‐2 may be a therapeutic target for CD based on its anti‐inflammatory functions and protective effects on the intestinal barrier.     

Metformin protects against intestinal barrier dysfunction via AMPKα1‐dependent inhibition of JNK signalling activation

Disruption of the intestinal epithelial barrier, that involves the activation of C‐Jun N‐terminal kinase (JNK), contributes to initiate and accelerate inflammation in inflammatory bowel disease. Metformin has unexpected beneficial effects other than glucose‐lowering effects. Here, we provided evidence that metformin can protect against intestinal barrier dysfunction in colitis. We showed that metformin alleviated dextran sodium sulphate (DSS)‐induced decreases in transepithelial electrical resistance, FITC‐dextran hyperpermeability, loss of the tight junction (TJ) proteins occludin and ZO‐1 and bacterial translocation in Caco‐2 cell monolayers or in colitis mice models. Metformin also improved TJ proteins expression in ulcerative colitis patients with type 2 diabetes mellitus. We found that metformin ameliorated the induction of colitis and reduced the levels of pro‐inflammatory cytokines IL‐6, TNF‐a and IL‐1β. In addition, metformin suppressed DSS‐induced JNK activation, an effect dependent on AMP‐activated protein kinase α1 (AMPKα1) activation. Consistent with this finding, metformin could not maintain the barrier function of AMPKα1‐silenced cell monolayers after DSS administration. These findings highlight metformin protects against intestinal barrier dysfunction. The potential mechanism may involve in the inhibition of JNK activation via an AMPKα1‐dependent signalling pathway.     

High glucose‐induced intestinal epithelial barrier damage is aggravated by syndecan‐1 destruction and heparanase overexpression

Syndecan‐1 (Sdc1) and its endo‐beta‐d ‐glucuronidase heparanase (HPSE) are implicated in maintenance of intestinal epithelial barrier (IEB), but their alterations and roles in high‐glucose/hyperglycaemia (HG) conditions have not been fully investigated. This study aimed to determine the expression pattern, the possible regulation mechanism of Sdc1 and HPSE in HG conditions, and their potential effects on IEB. Therefore, diabetic mice/cell models were developed, and tissue/serum samples, cell lysate and culture supernatants were harvested. The expression of Sdc1 and HPSE in control, HG and designated interventions groups were detected. Phosphorylations of mitogen‐activated protein kinase signalling pathway (MAPK), the expressions of Occludin and ZO‐1, and the levels of transepithelial electrical resistance (TEER) were measured and monitored. The results showed that in HG conditions, intestinal tissue and cellular Sdc1 were significantly decreased, but the expression of HPSE, and soluble Sdc1 in serum and culture supernatants were remarkably increased. Such alterations of Sdc1 and HPSE were associated with solely p38 MAPK activation, and were correlated with the reductions of Occludin, ZO‐1 and TEER. Heparin (Sdc1 analogue) and SB203580 (a p38 MAPK inhibitor), instead of insulin, alleviated Sdc1 destruction and HPSE overexpression, and effectively prevented against the reductions of tight junctions and the abnormality of intestinal permeability in HG conditions. In conclusion, we confirm the unique alterations of Sdc1 and HPSE in HG conditions, and found their interactions with p38 MAPK activation and IEB. These indicate that Sdc1/HPSE modulation can be viewed as an important complementary treatment for relieving HG‐induced gastrointestinal damage.     

Knockdown of ghrelin‐O‐acyltransferase attenuates colitis through the modulation of inflammatory factors and tight junction proteins in the intestinal epithelium

Ghrelin‐O‐acyltransferase (GOAT) is a membrane‐bound enzyme that attaches eight‐carbon octanoate to a serine residue in ghrelin and thereby acylates inactive ghrelin to produce active ghrelin. In this study, we investigated the function of GOAT in the intestinal mucosal barrier. The intestinal mucosal barrier prevents harmful substances such as bacteria and endotoxin from entering the other tissues, organs, and blood circulation through the intestinal mucosa. Here, we established 5% dextran sodium sulfate (DSS)‐induced colitis in mice and found that the body weight and colon weight were significantly decreased in these mice. Furthermore, increased inflammation and apoptosis were observed in the tissues of DSS‐induced colitis mice, with increased expression of tumor necrosis factor‐α, interleukin‐6, phosphorylation of nuclear factor kappa B‐p65 (p‐NF‐κB‐p65), and cleaved caspase‐3, and decreased expression of tight junction (TJ) proteins such as zonula occluden‐1 and occludin. The knockdown of GOAT significantly attenuated colitis‐induced inflammation responses and apoptosis, while GOAT overexpression significantly enhanced the induction of colitis. These results suggest that knockdown of GOAT may attenuate colitis‐induced inflammation, ulcers, and fecal occult blood by decreasing the intestinal mucosal permeability via the modulation of inflammatory factors and TJ proteins.     

Expression of Toll-like receptor 2 (TLR2), TLR4, and CD14 in biopsy samples of patients with inflammatory bowel diseases: upregulated expression of TLR2 in terminal ileum of patients with ulcerative colitis.

Dysregulation of innate and adaptive intestinal immune responses to bacterial microbiota is supposed to be involved in pathogenetic mechanisms of inflammatory bowel diseases (IBDs). We investigated expression of Toll-like receptor 2 (TLR2), TLR4, and their transmembrane coreceptor CD14 in biopsy samples from patients with IBD and in non-inflamed gut mucosa from controls. Small intestine and colon samples were obtained by colonoscopy from patients with Crohn's disease (CD), ulcerative colitis (UC), and controls. Immunohistochemical analysis of cryostat sections using polyclonal and monoclonal antibodies specific for TLR2, TLR4, and CD14 showed a significant increase in TLR2 expression in the terminal ileum of patients with inactive and active UC against controls. Significant upregulation of TLR4 expression relative to controls was found in the terminal ileum and rectum of UC patients in remission and in the terminal ileum of CD patients with active disease. CD14 expression was upregulated in the terminal ileum of CD patients in remission and with active disease, in the cecum of UC patients in remission and with active disease, and in rectum of UC patients with active disease. Hence, dysregulation of TLR2, TLR4, and CD14 expression in different parts of the intestinal mucosa may be crucial in IBD pathogenesis.     

Inhibition of mitochondrial fusion by α‐synuclein is rescued by PINK1, Parkin and DJ‐1

Aggregation of α‐synuclein (αS) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of αS is largely unknown. We demonstrate with in vitro vesicle fusion experiments that αS has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, αS binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age‐dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous αS. In contrast, siRNA‐mediated downregulation of αS results in elongated mitochondria in cell culture. αS can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, αS prevents fusion of differently labelled mitochondrial populations. Thus, αS inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of αS is rescued by coexpression of PINK1, parkin or DJ‐1 but not the PD‐associated mutations PINK1 G309D and parkin Δ1–79 or by DJ‐1 C106A.     

The role of LINC00094/miR‐224‐5p (miR‐497‐5p)/Endophilin‐1 axis in Memantine mediated protective effects on blood‐brain barrier in AD microenvironment

The dysfunction of the blood‐brain barrier (BBB) is one of the main pathological features of Alzheimer's disease (AD). Memantine (MEM), an N‐methyl‐d ‐aspartate (NMDA) receptor antagonist, has been reported that been used widely for AD therapy. This study was performed to demonstrate the role of the MEM in regulating BBB permeability in AD microenvironment as well as its possible mechanisms. The present study showed that LINC00094 was dramatically increased in Abeta_1‐42‐incubated microvascular endothelial cells (ECs) of BBB model in vitro. Besides, it was decreased in MEM‐incubated ECs. Silencing LINC00094 significantly decreased BBB permeability, meanwhile up‐regulating the expression of ZO‐1, occludin and claudin‐5. Furthermore, silencing LINC00094 enhance the effect of MEM on decreasing BBB permeability in AD microenvironment. The analysis of the mechanism demonstrated that reduction of LINC00094 inhibited Endophilin‐1 expression by up‐regulating miR‐224‐4p/miR‐497‐5p, promoted the expression of ZO‐1, occludin and claudin‐5, and ultimately alleviated BBB permeability in AD microenvironment. Taken together, the present study suggests that the MEM/LINC00094/miR‐224‐5p (miR‐497‐5p)/Endophilin‐1 axis plays a crucial role in the regulation of BBB permeability in AD microenvironment. Silencing LINC00094 combined with MEM provides a novel target for the therapy of AD.     

Annexin A1 restores Aβ1‐42‐induced blood–brain barrier disruption through the inhibition of RhoA‐ROCK signaling pathway

The blood–brain barrier (BBB) is composed of brain capillary endothelial cells and has an important role in maintaining homeostasis of the brain separating the blood from the parenchyma of the central nervous system (CNS). It is widely known that disruption of the BBB occurs in various neurodegenerative diseases, including Alzheimer's disease (AD). Annexin A1 (ANXA1), an anti‐inflammatory messenger, is expressed in brain endothelial cells and regulates the BBB integrity. However, its role and mechanism for protecting BBB in AD have not been identified. We found that β‐Amyloid 1‐42 (Aβ42)‐induced BBB disruption was rescued by human recombinant ANXA1 (hrANXA1) in the murine brain endothelial cell line bEnd.3. Also, ANXA1 was decreased in the bEnd.3 cells, the capillaries of 5XFAD mice, and the human serum of patients with AD. To find out the mechanism by which ANXA1 recovers the BBB integrity in AD, the RhoA‐ROCK signaling pathway was examined in both Aβ42‐treated bEnd.3 cells and the capillaries of 5XFAD mice as RhoA was activated in both cases. RhoA inhibitors alleviated Aβ42‐induced BBB disruption and constitutively overexpressed RhoA‐GTP (active form of RhoA) attenuated the protective effect of ANXA1. When pericytes were cocultured with bEnd.3 cells, Aβ42‐induced RhoA activation of bEnd.3 cells was inhibited by the secretion of ANXA1 from pericytes. Taken together, our results suggest that ANXA1 restores Aβ42‐induced BBB disruption through inhibition of RhoA‐ROCK signaling pathway and we propose ANXA1 as a therapeutic reagent, protecting against the breakdown of the BBB in AD.     

A novel susceptibility locus in MST1 and gene‐gene interaction network for Crohn's disease in the Chinese population

The incidence of Crohn's disease is increasing in many Asian countries, but considerable differences in genetic susceptibility have been reported between Western and Asian populations. This study aimed to fine‐map 23 previously reported Crohn's disease genes and identify their interactions in the Chinese population by Illumina‐based targeted capture sequencing. Our results showed that the genetic polymorphism A>G at rs144982232 in MST1 showed the most significant association (P = 1.78 × 10^?5; odds ratio = 4.87). JAK2 rs1159782 (T>C) was also strongly associated with Crohn's disease (P = 2.34 × 10^?4; odds ratio = 3.72). Gene‐gene interaction analysis revealed significant interactions between MST1 and other susceptibility genes, including NOD2, MUC19 and ATG16L1 in contributing to Crohn's disease risk. Main genetic associations and gene‐gene interactions were verified using ImmunoChip data set. In conclusion, a novel susceptibility locus in MST1 was identified. Our analysis suggests that MST1 might interact with key susceptibility genes involved in autophagy and bacterial recognition. These findings provide insight into the genetic architecture of Crohn's disease in Chinese and may partially explain the disparity of genetic signals in Crohn's disease susceptibility across different ethnic populations by highlighting the contribution of gene‐gene interactions.     

Amyloid‐beta 1‐40 is associated with alterations in NG2+ pericyte population ex vivo and in vitro

The population of brain pericytes, a cell type important for vessel stability and blood brain barrier function, has recently been shown altered in patients with Alzheimer's disease (AD). The underlying reason for this alteration is not fully understood, but progressive accumulation of the AD characteristic peptide amyloid‐beta (Aβ) has been suggested as a potential culprit. In the current study, we show reduced number of hippocampal NG2+ pericytes and an association between NG2+ pericyte numbers and Aβ1‐40 levels in AD patients. We further demonstrate, using in vitro studies, an aggregation‐dependent impact of Aβ1‐40 on human NG2+ pericytes. Fibril‐EP Aβ1‐40 exposure reduced pericyte viability and proliferation and increased caspase 3/7 activity. Monomer Aβ1‐40 had quite the opposite effect: increased pericyte viability and proliferation and reduced caspase 3/7 activity. Oligomer‐EP Aβ1‐40 had no impact on either of the cellular events. Our findings add to the growing number of studies suggesting a significant impact on pericytes in the brains of AD patients and suggest different aggregation forms of Aβ1‐40 as potential key regulators of the brain pericyte population size.     

α‐synuclein‐induced mitochondrial dysfunction in isolated preparation and intact cells: Implications in the pathogenesis of Parkinson's disease

This study has shown that purified recombinant human α‐synuclein (20 μM) causes membrane depolarization and loss of phosphorylation capacity of isolated purified rat brain mitochondria by activating permeability transition pore complex. In intact SHSY5Y (human neuroblastoma cell line) cells, lactacystin (5 μM), a proteasomal inhibitor, causes an accumulation of α‐synuclein with concomitant mitochondrial dysfunction and cell death. The effects of lactacystin on intact SHSY5Y cells are, however, prevented by knocking down α‐synuclein expression by specific siRNA. Furthermore, in wild‐type (non‐transfected) SHSY5Y cells, the effects of lactacystin on mitochondrial function and cell viability are also prevented by cyclosporin A (1 μM) which blocks the activity of the mitochondrial permeability transition pore. Likewise, in wild‐type SHSY5Y cells, typical mitochondrial poison like antimycin A (50 nM) produces loss of cell viability comparable to that of lactacystin (5 μM). These data, in combination with those from isolated brain mitochondria, strongly suggest that intracellularly accumulated α‐synuclein can interact with mitochondria in intact SHSY5Y cells causing dysfunction of the organelle which drives the cell death under our experimental conditions. The results have clear implications in the pathogenesis of sporadic Parkinson's disease.

     

Identification of ciliary neurotrophic factor receptor α as a mediator of neurotoxicity induced by α‐synuclein

Accumulating evidence suggests that extracellular α‐synuclein (eSNCA) plays an important role in the pathogenesis of Parkinson's disease or related synucleinopathies by inducing neurotoxicity directly or indirectly via microglial or astroglial activation. However, the mechanisms by which this occurs remain to be characterized. To explore these mechanisms, we combined three biochemical techniques – stable isotope labeling of amino acid in cell cultures (SILAC), biotin labeling of plasma membrane proteins followed by affinity purification, and analysis of unique proteins binding to SNCA peptides on membrane arrays. The SILAC proteomic analysis identified 457 proteins, of which, 245 or 172 proteins belonged to membrane or membrane associated proteins, depending on the various bioinformatics tools used for interpretation. In dopamine neuronal cells treated with eSNCA, the levels of 86 membrane proteins were increased and 35 were decreased compared with untreated cells. In peptide array analysis, 127 proteins were identified as possibly interacting with eSNCA. Of those, seven proteins were overlapped with the membrane proteins that displayed alterations in relative abundance after eSNCA treatment. One was ciliary neurotrophic factor receptor, which appeared to modulate eSNCA‐mediated neurotoxicity via mechanisms related to JAK1/STAT3 signaling but independent of eSNCA endocytosis.     

Cell surface‐anchored syndecan‐1 ameliorates intestinal inflammation and neutrophil transmigration in ulcerative colitis

Syndecan‐1 (SDC1), with a variable ectodomain carrying heparan sulphate (HS) chains between different Syndecans, participates in many steps of inflammatory responses. In the process of proteolysis, the HS chains of the complete extracellular domain can be shed from the cell surface, by which they can mediate most of SDC1's function. However, the exact impact on SDC1 which anchored on the cell surface has not been clearly reported. In our study, we established the models by transfection with the cleavable resistant SDC1 mutant plasmid, in which SDC1 shedding can be suppressed during stimulation. Role of membrane SDC1 in inflammatory pathway, pro‐inflammatory cytokine secretion as well as neutrophil transmigration, and how suppressing its shedding will benefit colitis were further investigated. We found that the patients suffered ulcerative colitis had high serum SDC1 levels,presented with increased levels of P65, tumour necrosis factor alpha (TNF‐α) and IL‐1β and higher circulating neutrophils. NF‐κB pathway was activated, and secretion of TNF‐α, interleukin‐1beta (IL‐1β), IL‐6 and IL‐8 were increased upon lipopolysaccharide stimuli in intestinal epithelial cells. Syndecan‐1, via its anchored ectodomain, significantly lessened these up‐regulation extents. It also functioned in inhibiting transmigration of neutrophils by decreasing CXCL‐1 secretion. Moreover, SDC1 ameliorated colitis activity and improved histological disturbances of colon in mice. Taken together, we conclude that suppression of SDC1 shedding from intestinal epithelial cells relieves severity of intestinal inflammation and neutrophil transmigration by inactivating key inflammatory regulators NF‐κB, and down‐regulating pro‐inflammatory cytokine expressions. These indicated that compenstion and shedding suppression of cytomembrane SDC1 might be the optional therapy for intestinal inflammation.