Heng Fan

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.

Oxymatrine improves TNBS-induced colitis in rats by inhibiting the expression of NF-κB p65

SummaryInflammatory bowel disease is thought to be regulated by the balance between Th1 and Th2 cytokines secreted by T cells, and NF-κB p65 also plays a predominant role in the intestinal inflammation. We evaluated the potency of oxymatrine, one of active components of Sophora Root, in inhibiting the immune responses and inflammation in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. The inflammation was markedly ameliorated in the oxymatrine-treated rats. The level of IL-2 was increased and that of IL-10 was decreased in colon tissue in the rat model, which was reversed by the treatment of oxymatrine. Moreover, the elevated expression of NF-κB p65 in colon tissue in the model was also improved by oxymatrine treatment. Our results suggest that oxymatrine might be beneficial for the abnormal immune responses and inflammation by regulating the unbalance of Th1 and Th2 cytokines secretion and inhibiting the expression of NF-κB p65 in colon tissue.Inflammatory bowel disease is thought to be regulated by the balance between Th1 and Th2 cytokines secreted by T cells, and NF-κB p65 also plays a predominant role in the intestinal inflammation. We evaluated the potency of oxymatrine, one of active components of Sophora Root, in inhibiting the immune responses and inflammation in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. The inflammation was markedly ameliorated in the oxymatrine-treated rats. The level of IL-2 was increased and that of IL-10 was decreased in colon tissue in the rat model, which was reversed by the treatment of oxymatrine. Moreover, the elevated expression of NF-κB p65 in colon tissue in the model was also improved by oxymatrine treatment. Our results suggest that oxymatrine might be beneficial for the abnormal immune responses and inflammation by regulating the unbalance of Th1 and Th2 cytokines secretion and inhibiting the expression of NF-κB p65 in colon tissue.

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.

Role of β2-adrenoceptor-β-arrestin2-nuclear factor-κB signal transduction pathway and intervention effects of oxymatrine in ulcerative colitis

ObjectiveTo investigate the β2-adrenoceptor (β2AR)-β-arrestin2-nuclear factor-κB (NF-κB): signal transduction pathway and the intervention effects of oxymatrine in a rat model of ulcerative colitis.MethodsForty SD rats were randomly divided into four groups, which included the normal control group, the model group, the mesalazine group and the oxymatrine treatment group, with 10 rats per group. Experimental colitis induced with trinitrobenzene sulfonic acid (TNBS) was established in each group except the normal control group. The rats in the oxymatrine treatment group were treated with intramuscular injection of oxymatrine 63 mg/(kg kg·d) for 15 days and the rats in the mesalazine group were treated with mesalazine solution 0.5 g/(kg kg·d) by gastric lavage for 15 days. The rats in the normal control group and model group were treated with 3 mL water by gastric lavage for 15 days. Diarrhea and bloody stool were carefully observed. Histological changes in colonic tissue were examined on day 7 in 2 rats per group that were randomly selected. The expression of β2AR, β-arrestin2 and NF-κB p65 in colon tissue and spleen lymphocytes were detected with immunohistochemistry and Western immunoblotting techniques on day 16 after fasting for 24 h. Six rats died of lavage with 2 each in the normal control, the model group and the mesalazine group; and were not included in the analysis.ResultsThe rats in the model group suffered from: looser stool and bloody purulent stool after modeling. But in the oxymatrine and mesalazine groups, looser stool and bloody purulent stool reduced after treatment. And the colonic wall in the model group was thickened and the colon length shortened. The colon mucosa was congested in multiple areas with edema, erosion, superficial or linear ulcer and scar formation, while the intestinal mucosa injury reduced in the mesalazine and oxymatrine groups (P<0.01). In colonic mucosa and in spleen lymphocytes, compared with the normal control group, the expression of NF-κBp65 were significantly increased (P<0.01) in the model group while the expressions of β 2AR and β-arrestin2 were significantly decreased (P<0.01). Compared with the model group, the expression of NF-κ Bp65 was significantly decreased in the mesalazine group (P<0.01) and oxymatrine treatment group (P<0.01) while the expressions of β2AR and β-arrestin2 were significantly increased (P<0.01). There were no statistically significant differences in the expression of β2AR, β-arrestin2 and NF-κBp65 between the mesalazine group and oxymatrine group (P>0.05).ConclusionsThe β2AR-β-arrestin2-NF-κB signal transduction pathway participated: in the pathologic course of ulcerative colitis. Oxymatrine attenuated ulcerative colitis through regulating the β2AR-β-arrestin2-NF-κB signal transduction pathway.

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.

Intervention effects of QRZSLXF, a Chinese medicinal herb recipe, on the DOR-β-arrestin1-Bcl2 signal transduction pathway in a rat model of ulcerative colitis.

ETHNOPHARMACOLOGICAL RELEVANCE Qingre Zaoshi Liangxue Fang (QRZSLXF) is a Chinese medicinal herb recipe that is commonly prescribed for the treatment of ulcerative colitis. It includes 5 quality assured herbs: Sophora flavescens Aiton., Baphicacanthus cusia (Nees) Bremek., Bletilla striata Rchb.f., Glycyrrhiza uralensis Fisch. and Coptis chinensis Franch. The main phytochemical ingredient of QRZSLXF includes ammothamnine, sophocarpidine, liquiritin, berberine and indirubin. QRZSLXF has been clinically proven for use in the treatment of ulcerative colitis for over twenty years. In the past ten years, research has confirmed the therapeutic effect of QRZSLXF in ulcerative colitis and partially revealed its mechanism of action. Here, we further reveal the therapeutic mechanism of QRZSLXF in ulcerative colitis. To investigate the role of the DOR-β-arrestin1-Bcl-2 signal transduction pathway in ulcerative colitis and to determine the effects of QRZSLXF on this signal transduction pathway. MATERIALS AND METHODS Eighty-four Sprague-Dawley rats were randomly divided into six groups: normal control group, model group, mesalazine group, and QRZSLXF high-dose, medium-dose group and low-dose groups (n=14). Experimental colitis was induced by trinitrobenzenesulfonic acid (TNBS) in each group, except the normal control group. After modeling, bloody stool, mental state and diarrhea were observed and recorded. Two rats were randomly selected from the model groups adfnd sacrificed on day 3 to observe pathological changes in the colon tissue by microscopy. The rats in the QRZSLXF-treated groups received intramuscular injections of different concentrations of QRZSLXF for 15 days. The rats in the mesalazine group were treated with mesalazine solution (0.5 g/kg/day) by gastric lavage for 15 days. The rats in the normal control group and the model group were treated with 3 mL water by gastric lavage for 15 days. On the 16th day, after fasting for 24 h, the remaining rats were sacrificed and their colon tissues were used to detect the mRNA and protein expressions of DOR, β-arrestin1 and Bcl-2 by Real-time PCR and immunohistochemistry, respectively. Histological changes in the colon tissues were also examined. RESULTS AND CONCLUSIONS The expressions of DOR, β-arrestin1 and Bcl-2 were significantly different among the four groups. The expressions of DOR, β-arrestin1 and Bcl-2 protein and mRNA were significantly increased in the model group compared with the other groups (P<0.05). In contrast to the model group, the expressions of DOR, β-arrestin1 and Bcl-2 were significantly decreased in the mesalazine group and the groups that received different doses of QRZSLXF (P<0.05), and there were no statistically significant differences among the mesalazine and QRZSLXF-treated groups (P>0.05). This study indicates that the DOR-beta-arrestin1-Bcl-2 signal transduction pathway may participate in the pathologic course of ulcerative colitis. Moreover, QRZSLXF could attenuate ulcerative colitis by regulating the DOR-β-arrestin1-Bcl-2 signal transduction pathway.

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.

Effect of Wumeiwan on Cytokines TNF-α, IL-6, IL-8, IL-10 and Expression of NF-κBp65 in Rats with Ulcerative Colitis

SummaryThe effects of Wumeiwan (WMW) on TNF-α, IL-6, IL-8, IL-10 and NF-κBp65 in rats with ulcerative colitis (UC) were investigated, the curative effectiveness of WMW vs salicylazosulfapyridine (SASP) was compared, and the action mechanism was analyzed. Fifty-Six Sprague-Dawley (SD) rats were randomly divided into four groups (n=14 in each group, with equal ratio of male and female): normal control group, model group, SASP group, and WMW group. Except normal control group, the rat UC models in the remaining three groups were established using the method of 2.4-dinitrochlorobenzene (DNCB) immunization and acetic acid local enema. The rats in model group, SASP group, and WMW group were treated with distilled water, SASP, and WMW respectively. The changes in the symptoms and signs were observed, and levels of IL-6, IL-8, TNF-α, IL-10 and the expression of NF-κBp65 in the colonic tissues were statistically analyzed. The results showed that the levels of IL-6, IL-8, and TNF-α were significantly increased (P<0.01), while those of IL-10 significantly reduced (P<0.01) after establishment of rat UC models as compared with normal control group. The levels of IL-6, IL-8, and TNF-α were obviously lower, but the level of IL-10 was obviously higher in WMW and SASP groups than those in model group (P<0.05). The levels of IL-6, IL-8, and TNF-α were lower, while the level of IL-10 was higher in WMW group than in SASP group. NF-κBp65 was expressed negatively or weakly in normal colonic tissues. The positive expression rate of NF-κBp65 in WMW group and SASP group was obviously lower than in model group (P<0.01), and there was significant difference between WMW group and SASP group (P<0.05). It was concluded that rat UC model was established successfully. WMW could up-regulate the expression of IL-10, down-regulate the expression of TNF-α, IL-6, IL-8, and inhibit the NF-κBp65 activity to adjust immune function, indicating WMW had better curative effects on UC in rats.The effects of Wumeiwan (WMW) on TNF-α, IL-6, IL-8, IL-10 and NF-κBp65 in rats with ulcerative colitis (UC) were investigated, the curative effectiveness of WMW vs salicylazosulfapyridine (SASP) was compared, and the action mechanism was analyzed. Fifty-Six Sprague-Dawley (SD) rats were randomly divided into four groups (n=14 in each group, with equal ratio of male and female): normal control group, model group, SASP group, and WMW group. Except normal control group, the rat UC models in the remaining three groups were established using the method of 2.4-dinitrochlorobenzene (DNCB) immunization and acetic acid local enema. The rats in model group, SASP group, and WMW group were treated with distilled water, SASP, and WMW respectively. The changes in the symptoms and signs were observed, and levels of IL-6, IL-8, TNF-α, IL-10 and the expression of NF-κBp65 in the colonic tissues were statistically analyzed. The results showed that the levels of IL-6, IL-8, and TNF-α were significantly increased (P<0.01), while those of IL-10 significantly reduced (P<0.01) after establishment of rat UC models as compared with normal control group. The levels of IL-6, IL-8, and TNF-α were obviously lower, but the level of IL-10 was obviously higher in WMW and SASP groups than those in model group (P<0.05). The levels of IL-6, IL-8, and TNF-α were lower, while the level of IL-10 was higher in WMW group than in SASP group. NF-κBp65 was expressed negatively or weakly in normal colonic tissues. The positive expression rate of NF-κBp65 in WMW group and SASP group was obviously lower than in model group (P<0.01), and there was significant difference between WMW group and SASP group (P<0.05). It was concluded that rat UC model was established successfully. WMW could up-regulate the expression of IL-10, down-regulate the expression of TNF-α, IL-6, IL-8, and inhibit the NF-κBp65 activity to adjust immune function, indicating WMW had better curative effects on UC in rats.

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.