Dongmei Zuo

Extracellular Vesicles Derived from Bone Marrow Mesenchymal Stem Cells Protect against Experimental Colitis via Attenuating Colon Inflammation, Oxidative Stress and Apoptosis

The administration of bone mesenchymal stem cells (BMSCs) could reverse experimental colitis, and the predominant mechanism in tissue repair seems to be related to their paracrine activity. BMSCs derived extracellular vesicles (BMSC-EVs), including mcirovesicles and exosomes, containing diverse proteins, mRNAs and micro-RNAs, mediating various biological functions, might be a main paracrine mechanism for stem cell to injured cell communication. We aimed to investigate the potential alleviating effects of BMSC-EVs in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis model. Intravenous injection of BMSC-EVs attenuated the severity of colitis as evidenced by decrease of disease activity index (DAI) and histological colonic damage. In inflammation response, the BMSC-EVs treatment significantly reduced both the mRNA and protein levels of nuclear factor kappaBp65 (NF-κBp65), tumor necrosis factor-alpha (TNF-α), induciblenitric oxidesynthase (iNOS) and cyclooxygenase-2 (COX-2) in injured colon. Additionally, the BMSC-EVs injection resulted in a markedly decrease in interleukin-1β (IL-1β) and an increase in interleukin-10 (IL-10) expression. Therapeutic effect of BMSC-EVs associated with suppression of oxidative perturbations was manifested by a decrease in the activity of myeloperoxidase (MPO) and Malondialdehyde (MDA), as well as an increase in superoxide dismutase (SOD) and glutathione (GSH). BMSC-EVs also suppressed the apoptosis via reducing the cleavage of caspase-3, caspase-8 and caspase-9 in colitis rats. Data obtained indicated that the beneficial effects of BMSC-EVs were due to the down regulation of pro-inflammatory cytokines levels, inhibition of NF-κBp65 signal transduction pathways, modulation of anti-oxidant/ oxidant balance, and moderation of the occurrence of apoptosis.

Over-expression of CXCR4 on mesenchymal stem cells protect against experimental colitis via immunomodulatory functions in impaired tissue

Bone marrow-derived mesenchymal stem cells (BMSCs) are attractive candidates for tissue regeneration and immunoregulation in inflammatory bowel disease. However, their in vivo reparative capability is limited owing to barren efficiency of BMSCs to injury region. Stromal cell-derived factor (SDF-1) plays an important role in chemotaxis and stem cell homing through interaction with its specific receptor CXC chemokine receptor 4 (CXCR4). The present study was designed to investigate the role of SDF-1α/CXCR4 axis in the therapeutic effects of lentivirus-preconditioned BMSCs for 2,4,6-trinitrobenzene sulfonic acid (TNBS)-colitis rats. BMSCs were isolated from female Sprague–Dawley rats and identified by flow cytometry. Lentiviral transduction was applied to over-express CXCR4/GFP (Ad-CXCR4-BMSCs) or null/GFP (Ad-GFP-BMSCs). Efficacy of engraftment was determined by the presence of enhanced green fluorescent protein (GFP) positive cells. One week after intravenous administration, Ad-GFP-BMSCs failed to colonize in the inflamed colon and had no beneficial effect in TNBS-induced colitis. Instead, Ad-CXCR4-BMSCs signally ameliorated both clinical and microanatomical severity of colitis. Immunofluorescence and western blotting showed that Ad-CXCR4-BMSCs migrated toward inflamed colon was more efficient than Ad-GFP-BMSCs. The therapeutic effect of Ad-CXCR4-BMSCs was mediated by the suppression of pro-inflammatory cytokines and STAT3 phosphorylation in injured colon. Collectively, our data indicated that over-expression CXCR4 led to enhance in vivo mobilization and engraftment of BMSCs into inflamed colon where these cells can function as an anti-inflammatory and immunomodulatory component of the immune system in TNBS-induced colitis.

miR-200b-containing microvesicles attenuate experimental colitis associated intestinal fibrosis by inhibiting epithelial-mesenchymal transition: miR-200b attenuates intestinal fibrosis

Epithelial–mesenchymal transition (EMT), characterized by the decrease of E‐cadherin (E‐Cad) and increase in vimentin and alpha‐smooth muscle actin (α‐SMA), was demonstrated to participate in inflammatory bowel disease‐related fibrosis. miR‐200b plays an anti‐fibrosis role in inhibiting EMT by targeting ZEB1 and ZEB2. But the stability of exogenous miR‐200b in blood limits its application. Microvesicles (MVs), which can transfer miRNAs among cells and prevent them from degradation, may provide an excellent transport system for the delivery of miR‐200b in the treatment of fibrosis.

Microvesicles Shuttled miR-200b attenuate Experimental Colitis associated Intestinal Fibrosis by Inhibiting the Development of EMT

Epithelial–mesenchymal transition (EMT), characterized by the decrease of E‐cadherin (E‐Cad) and increase in vimentin and alpha‐smooth muscle actin (α‐SMA), was demonstrated to participate in inflammatory bowel disease‐related fibrosis. miR‐200b plays an anti‐fibrosis role in inhibiting EMT by targeting ZEB1 and ZEB2. But the stability of exogenous miR‐200b in blood limits its application. Microvesicles (MVs), which can transfer miRNAs among cells and prevent them from degradation, may provide an excellent transport system for the delivery of miR‐200b in the treatment of fibrosis.

Adrenomedullin improves intestinal epithelial barrier function by downregulating myosin light chain phosphorylation in ulcerative colitis rats

Adrenomedullin (AM) is a pivotal endogenous vasoactive peptide, which can maintain epithelial barrier function in inflammatory bowel disease. Myosin light chain kinase (MLCK)‑dependent phosphorylated myosin light chain kinase (p‑MLC) is a key regulator of intestinal barrier function. The aim of the present study was to investigate the effect and mechanism of AM on the intestinal epithelial barrier in a rat model of ulcerative colitis (UC) induced by 2,4,6‑trinitro‑benzene‑sulfonic acid (TNBS). A total of 21 male Sprague‑Dawley rats were randomly divided into the following three groups and administered different agents for 7 days: The normal group (water and saline), model group (TNBS and saline) and the AM group (TNBS and AM; 1.0 µg). The weight of rats was recorded every day. Serum tumor necrosis factor‑α (TNF‑α) and interleukin‑6 (IL‑6) levels were detected using ELISA kits. Colon tissue was collected for the assessment of histological alterations. The protein expression of MLCK, p‑MLC and zonula occludens‑1 (ZO‑1) was examined by western blot analysis. Intestinal epithelial tight junctions were examined using transmission electron microscopy. The results demonstrated that in colitis model rats, the expression of TNF‑α, IL‑6, MLCK and p‑MLC significantly increased compared with normal rats. In addition, the expression of ZO‑1 decreased (P<0.05) and intestinal epithelial cell permeability increased. Following AM administration, TNF‑α, IL‑6, MLCK and p‑MLC expression significantly decreased compared with the model rats, the expression of ZO‑1 increased (P<0.05) and intestinal epithelial cell permeability reduced. These data indicate a protective effect of AM on intestinal epithelial barrier dysfunction via suppression of inflammatory cytokines and downregulation of MLCK‑p‑MLC in TNBS‑induced UC. In conclusion, AM/MLCK‑p‑MLC may be an important signaling pathway in the occurrence and development of UC.

Modulation of nuclear factor-κB-mediated pro-inflammatory response is associated with exogenous administration of bone marrow-derived mesenchymal stem cells for treatment of experimental colitis.

Mesenchymal stem cells (MSCs) inhibit the immune response in vitro and prevent the induction of disease in certain experimental models. As a result, MSC‑mediated therapy is a rapidly growing field of research. However, the efficacy of MSCs in the treatment of inflammatory bowel disease (IBD) has remained to be determined. In the present study, rats with 2,4,6‑trinitrobenzene sulfonic acid (TNBS)‑induced colitis were injected with prepared MSCs (1x106) into the tail vein. Two weeks following intravenous MSC administration, the concentration of tumor necrosis factor‑α (TNF‑α) in the serum was measured by an ELISA. The protein expression of nuclear factor‑κB (NF‑κBp65) in the colonic mucosa was assessed by western blot analysis. mRNA expression of TNF‑α and NF‑κBp65 was determined by reverse‑transcription quantitative polymerase chain reaction. MSCs were shown to exert an immunomodulatory effect on TNBS‑induced colitis and may be of use in the treatment of IBD. In addition, modulation of the NF‑κB‑mediated pro‑inflammatory response may contribute to the underlying mechanism by which MSCs ameliorate the clinical and histological changes associated with IBD.

The role of STAT3 and AhR in the differentiation of CD4+ T cells into Th17 and Treg cells

Background: This study aimed to investigate the role of aryl hydrocarbon receptor (AhR) and STAT3 gene during the differentiation of cluster of differentiation (CD)4+ T cells into T helper (Th)17 and T regulatory (Treg) cells. Methods: First, CD4+ T cells were isolated from the spleen of BALB/c mice. Then, stable CD4+ T cells expressing STAT3 shRNA were constructed. CD4+ T cells were assigned to one of the following treatments: Th17 group: antibodies against CD3 and CD28, 2.5 ng/mL transforming growth factor &bgr; (TGF-&bgr;)1, 30 ng/mL interleukin (IL)-6, and 30 ng/mL IL-23; 6-formylindolo[3,2-b]carbazole (FICZ) group: antibodies against CD3 and CD28, 2.5 ng/mL TGF-&bgr;1, 30 ng/mL IL-6, 30 ng/mL IL-23, and 100 nM FICZ; FICZ + STAT3 RNAi group (shSTAT3 group): antibodies against CD3 and CD28, 2.5 ng/mL TGF-&bgr;1, 30 ng/mL IL-6, 30 ng/mL IL-23, 100 nM FICZ, and STAT3 RNAi; naphthoflavone group: antibodies against CD3 and CD28, 2.5 ng/mL TGF-&bgr;1, 30 ng/mL IL-6, 30 ng/mL IL-23, and 3 &mgr;M naphthoflavone; 5) no antibodies were added in the control group. Later, the proportions of Th17 and Treg cells in each group were measured by flow cytometry; phospho-STAT3 and -STAT5 levels were measured by western blotting; and AhR, STAT3, STAT5, receptor-related orphan nuclear receptor &ggr;t (ROR&ggr;t), FOXP3, T-cell receptor (TCR), CD25, IL-6R, IL-10, and IL-17 mRNA levels were also measured by real-time PCR. Result: Th17 cells showed a rise and Treg cells showed a decrease in the FICZ group, but revised in the shSTAT3 group and the naphthoflavone group. Significant differences were observed in CD25, IL-6R, IL-10, and IL-17 mRNA levels among different groups. Conclusion: STAT3 may cooperate with AhR to regulate the differentiation of both Th17 and Treg cells.

Oxymatrine protects against DSS-induced colitis via inhibiting the PI3K/AKT signaling pathway.

Abstract Oxymatrine (OMT), an alkaloid derived from the root of the Sophora flavescens, has been reported to possess a significant effect on relieving UC owing to its anti‐inflammatory property. But the other therapeutic mechanism of OMT remains unclear. Recent studies have found, PI3K/AKT signaling pathway is involved in the pathogenesis of UC by pro‐inflammatory effects and activating T cells. Moreover, PI3K/AKT pathway is one of the most important pathways for regulating cell apoptosis. Thus, we aim to explore whether OMT protects against UC by targeting PI3K/AKT pathway. We established the UC mice models, using LY294002 (a specific inhibitor of PI3K/AKT) as a positive control, to observe the effect of low, medium and high dose of OMT on UC and its influence on PI3K/AKT signaling pathway. Our data indicated that OMT can significantly ameliorate UC through anti‐inflammatory, pro‐apoptotic, down‐regulating the differentiation of Th1 and Th17 cells via PI3K/AKT pathway. This study reveals that PI3K/AKT signaling pathway is a potential mechanism of OMT‐induced UC remission and suggests that OMT is a promising therapeutic agent for the treatment of UC. Graphical abstract Figure. No Caption available. HighlightsOxymatrine alleviates UC through regulating inflammation, apoptosis and immunity.PI3K/AKT signaling pathway mediates the pathogenesis of UC.Oxymatrine ameliorates UC by suppressing PI3K/AKT activation.

Tongxie Formula Reduces Symptoms of Irritable Bowel Syndrome

BACKGROUND & AIMS: Irritable bowel syndrome (IBS) is the most common chronic gastrointestinal disorder, yet few drugs are effective in reducing symptoms. Approximately 50% of patients with IBS attempt herbal therapy at least once. We performed a randomized controlled trial to compare the efficacy of the herb formulation tongxie vs placebo or pinaverium (an antispasmodic agent) in reducing symptoms of IBS. METHODS: We performed a trial of 1044 adult patients with IBS (based on Rome III criteria) at 5 hospitals in China, from August 2012 through January 2015. Subjects were randomly assigned (1:1:1) to groups given tongxie (a combination of A macrocephalae, P lactiflora, C reticulata, S divaricata, C pilosula, C wenyujin, C medica, and P cocos, along with other herbs, based on patient features), placebo, or pinaverium (50 mg tablets) 3 times daily for 4 weeks. Primary end points were significantly greater reductions in abdominal pain and Bristol stool score (before vs after the 4‐week study period) in patients given tongxie compared with patients given placebo or pinaverium. Secondary end points were reductions in pain and stool frequencies and abdominal discomfort and its frequency. RESULTS: Subjects given tongxie had significant reductions, before vs after the study period, in all 6 symptoms assessed, compared to patients given placebo (P < .001). A significantly higher proportion of patients given tongxie had increased stool consistency (75.6%) than patients given pinaverium (50.6%), and a significantly higher proportion of patients given tongxie had fewer daily stools (72.7%) than subjects given pinaverium (58.3%) (P < .001 for both). However, significantly higher proportions of patients given pinaverium had reduced pain (63.5%) and pain frequency (69.5%) than patients given tongxie (51.4% and 58.6%, respectively; P < .005 for both). CONCLUSIONS: In a randomized controlled trial of patients with IBS in China, we found 4 weeks of tongxie to produce significantly greater reduction in symptoms than placebo, and greater increases in stool consistency and reductions in stool frequency, than patients given pinaverium. Tongxie can therefore be considered an effective alternative therapy for patients with IBS who do not respond well to conventional therapies. Clinicaltrials.gov no: NCT01641224.

The ANXA1 released from intestinal epithelial cells alleviate DSS-induced colitis by improving NKG2A expression of Natural Killer cells

Inflammatory bowel disease (IBD) arises when intestinal immune homeostasis is broken, the maintenance of such homeostasis is principally controlled by cross talk between commensal bacteria, mucosal immune cells and intestinal epithelial cells (IECs). IECs can prevent the contact between luminal bacteria with immune cells through the formation of a physical barrier and the expression of antimicrobial peptides to maintain intestinal immune homeostasis. During Colitis the IECs can express increased ANXA1, which is important for regeneration of intestinal mucosa and function as a potent anti-inflammatory protein. Natural Killer (NK) cells can also suppress the progression of colitis. It is uncertain about the effect of the cross-talk between injured IECs and recruited NK cells during colitis. In this study, the expression of ANXA1 in IECS from DSS treated mice was increased, and more NK cells were recruited to intestinal mucosa. In addition, the expression of NKG2A was upregulated when co-cultured with NK cells. The results further proved that overexpression of NKG2A in NK cells was important for inhibiting the recruitment and activity of neutrophils to alleviate DSS-induced colitis. Here, we provide a new anti-inflammation mechanism about ANXA1 secreted from injured IECs, where ANXA1 can stimulate the expression of NKG2A in NK cells that affect the recruitment and activity of neutrophils necessary for pathology of colitis.