Effect of recombinant Trichinella spiralis cysteine proteinase inhibitor on TNBS-induced experimental inflammatory bowel disease in mice

Inflammatory bowel disease (IBD) is a chronic autoimmune disease with a high recurrence rate. Ulcerative colitis (UC) and Crohn's disease (CD) are two types of IBD. At present, parasite-derived cysteine protease inhibitors have received extensive attention from researchers, and experiments have confirmed that these protease have an effect on certain autoimmune diseases. So we conducted experiments to investigate the effect of Trichinella spiralis cysteine protease inhibitors on TNBS-induced mouse CD models. In this experiment, 72 male BALB/c mice aged 6–8 weeks were randomly divided into two groups: prevention group and therapy group. The mice were sacrificed and harvested on the 7th day after the model was established to measure the changes of various indicators of colitis.The comparison of the TsCystatin + TNBS group with the PBS + TNBS group showed that the DAI score, MPO activity, and colonic macroscopic and microscopic damage significantly reduced, IFN-γ significantly decreased, IL-4 expression increased, and NF-κB expression decreased. The percentage of CD4+CD25+Foxp3+ Treg and CD8+CD28− Treg in spleen, and the proportion of CD4+/CD8+ Treg cells decreased. In the therapy group, we found no significant difference between the TNBS+PBS group and TNBS + TsCystatin group.Treatment with TsCystatin exerted a good intervention effect on the TNBS-induced mouse CD model. TsCystatin possibly induced a Th2-type immune response in the body, which balanced the Th1-type immune response induced by TNBS administration, thereby relieving colitis.     

The effects of Foxp3-expressing regulatory T cells expanded with CD28 superagonist antibody in DSS-induced mice colitis

Regulatory T (Treg) cells play an important role in the pathogenesis of inflammatory bowel disease (IBD). In the present study, we found that a superagonistic CD28-specific monoclonal antibody (supCD28mAb, D665) could preferentially stimulate expansion of CD4+Foxp3+ Treg cells. Foxp3(EGFP) mice were orally administrated with 3.5% DSS for 5days, and intraperitoneally injected supCD28mAb 1mg/mice in treated group. All of the mice were sacrificed on day 8, and both clinical and histological parameters showed that the severity of colitis was significantly reduced in treated group compared to controls. In treated group, the proportion of CD103, CD152 and CD62L expression on Foxp3+Treg cells in the spleen and mesenteric lymph node were higher than controls. Furthermore, qRT-PCR analysis showed that expression of anti-inflammatory cytokines such as IL-10, TGF-β was significantly increased in treated group. Taken together, our data demonstrated that supCD28mAb targets CD4+Foxp3+Treg cells expansion in vivo, maintains and enhances their regulatory functions, to reduce the damage of colon in dextran sulfate sodium (DSS)-induced mouse colitis by secreting a large amount of IL-10. It represents a major advance towards the therapeutic use of polyclonally activated Treg cells as cellular therapy for treatment of IBD.     

苦参素对溃疡性结肠黏膜细胞NF-κB表达的影响

目的探讨苦参素对溃疡性结肠黏膜细胞中NF-κB mRNA表达的影响机制。方法 SPF级SD大鼠随机分成正常对照组,UC模型组,UC+苦参素组,UC+柳氮磺胺吡啶组,实验结束时,剖取病灶结肠,RT-PCR方法检测各实验组动物结肠黏膜细胞中NF-κB mRNA表达水平。结果苦参素可显著抑制NF-κB mRNA在溃疡性结肠炎症细胞中的表达(P<0.01)。结论苦参素可干预NF-κB mRNA在炎症性结肠黏膜细胞中的表达,进而抑制溃疡性结肠炎症反应。     

Polydatin ameliorates DSS-induced colitis in mice through inhibition of nuclear factor-kappaB activation

Nuclear factor- κB (NF- κB) plays a pivotal role in the regulation of immune and inflammatory responses. The real-time expression level of NF- κB reflects the development of ulcerative colitis (UC). Polydatin has vast pharmacological activities, including inhibiting the production of inflammatory mediators, inducing the production of antioxidants, regulating immune function, etc. The purpose of this study was to investigate the potential inhibitory effects of polydatin on NF- κB pathway activation in a mouse UC model. The results showed that polydatin treatment downregulated NF- κB p65 activity and expression, blocked the expression of TNF- α, IL-6 and IL-1 β at both mRNA and protein levels, decreased myeloperoxidase (MPO) activity, and alleviated inflammatory damage of colitis in mice with UC (p < 0.05), suggesting that the anti-inflammation effects of polydatin can be attributed, at least partially, to the blocking of the NF- κB pathway.     

己酮可可碱对小鼠溃疡性结肠炎NF-κB的影响

目的:观察己酮可可碱(pentoxifylline,PTX)治疗小鼠溃疡性结肠炎(ulcerative colitis,UC)的治疗作用,探讨PTX对三硝基苯磺酸(TNBS)诱导的小鼠UC NF-κB的影响.方法:用TNBS局部灌肠法建立小鼠UC模型,随机分为正常组、模型组、PTX组、柳氮磺胺吡啶(SASP)组,评价疾病活动指数(DAI),观察结肠组织学损伤,用ELISA法测定结肠组织NF-κB的水平.结果:用TNBS灌肠可使小鼠结肠炎性改变;PTX治疗可使小鼠疾病活动指数、结肠黏膜大体形态学损伤评分及组织学评分、结肠黏膜组织NF-κB的表达明显下降,与SASP相比较差异无统计学意义(P＞0.05).结论:PTX对以TNBS诱发的小鼠UC有良好的治疗作用,其作用机制可能是通过抑制小鼠结肠黏膜组织NF-κB的表达而减轻其炎症性损害.     

Mangiferin corrects the imbalance of Th17/Treg cells in mice with TNBS-induced colitis

In the previous study, 80% ethanol extract of the rhizome mixture of Anemarrhena asphodeloides and Coptidis chinensis (AC) and its main constituent mangiferin improved TNBS-induced colitis in mice by inhibiting macrophage activation related to the innate immunity. In the preliminary study, we found that AC could inhibit Th17 cell differentiation in mice with TNBS-induced colitis. Therefore, we investigated whether AC and it main constituent mangiferin are capable of inhibiting inflammation by regulating T cell differentiation related to the adaptive immunity in vitro and in vivo. AC and mangiferin potently suppressed colon shortening and myeloperoxidase activity in mice with TNBS-induced colitis. They also suppressed TNBS-induced Th17 cell differentiation and IL-17 expression, but increased TNBS-suppressed Treg cell differentiation and IL-10 expression. Moreover, AC and mangiferin strongly inhibited the expression of TNF-α and IL-17, as well as the activation of NF-κB. Furthermore, mangiferin potently inhibited the differentiation of splenocytes into Th7 cells and increased the differentiation into Treg cells in vitro. Mangiferin also inhibited RORγt and IL-17 expression and STAT3 activation in splenocytes and induced Foxp3 and IL-10 expression and STAT5 activation. Based on these findings, mangiferin may ameliorate colitis by the restoration of disturbed Th17/Treg cells and inhibition of macrophage activation.     

Inhibitory effect of β-sitosterol on TNBS-induced colitis in mice

β-Sitosterol, a common sterol in herbal medicines, exhibits anti-inflammatory effects beneficial in the treatment of lung inflammation, asthma, and bronchospasm. To evaluate whether β-sitosterol also has anticolitic benefits, we tested the effect of β-sitosterol on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. β-Sitosterol inhibited colon shortening and led to lowered macroscopic scores and myeloperoxidase activity in TNBS-treated colitic mice. β-Sitosterol also inhibited the expression of proinflammatory cytokines TNF-α, IL-1β, and IL-6, and an inflammatory enzyme, cyclooxygenase (COX)-2, in the colons of TNBS-induced colitic mice, as well as the activation of NF-κB. Based on these findings, β-sitosterol may ameliorate colitis by inhibiting the NF-κB pathway.     

Therapeutic efficacy of carboxyamidotriazole on 2,4,6-trinitrobenzene sulfonic acid-induced colitis model is associated with the inhibition of NLRP3 inflammasome and NF-κB activation

Excess proinflammatory cytokines owing to the activation of NF-κB and NLRP3 inflammasome play the key role in inflammatory bowel disease (IBD). Previously, we reported the anti-inflammatory activity of carboxyamidotriazole (CAI) resulting from decreasing cytokines. Therefore, we investigated the therapeutic effects of CAI in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced rat colitis and the involvement of CAI action with NLRP3 inflammasome and NF-κB pathway. CAI was orally administered to TNBS-induced colitis rat. The severity of colitis was assessed, and NLRP3 inflammasome, NF-κB pathway and cytokines were determined. Our results showed that CAI significantly reduced weight loss and disease activity index (DAI) scores in colitis rats and alleviated the colonic macroscopic signs and pathological damage. In addition, the intestinal inflammatory markers and permeability index were markedly ameliorated by CAI treatment. The decreased levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, IL-18 were also detected in the colon tissues of CAI-treated colitis rats. Moreover, the activation of NLRP3 inflammasome in inflamed colon was significantly suppressed by showing an obvious reduction in the NLRP3 and activated caspase-1 levels. Furthermore, CAI reduced NF-κB p65 expression and IκBα phosphorylation and degradation in colitis rats. Therefore, CAI attenuates TNBS-induced colitis, which may be attributed to its inhibition of NLRP3 inflammasome and NF-κB activation, and down-regulation of proinflammatory cytokines. These results provide further understanding of the intestinal anti-inflammatory effect of CAI and highlight it as a potential drug for the treatment of IBD.     

Administration of geniposide ameliorates dextran sulfate sodium-induced colitis in mice via inhibition of inflammation and mucosal damage

Ulcerative colitis (UC), an idiopathic inflammatory bowel disease, not only affects millions of patients worldwide, but also increases the risk of colon cancer. Geniposide is an iridoid glycoside and has many biological activities such as anti-inflammatory and antioxidant. However, its protective efficacy and mechanism of action against UC are still unclear. In this study, we aimed to investigate the protective effects and mechanisms of geniposide on dextran sulfate sodium (DSS)-induced experimental colitis in mice. The results revealed that geniposide alleviated body weight loss, disease activity index, colon length shortening and colonic pathological damage induced by DSS. Geniposide significantly suppressed pro-inflammatory cytokines by regulating NF-κB and PPARγ pathways in vivo and in vitro. Furthermore, geniposide also significantly regulated the expressions of ZO-1 and occludin in DSS-induced experimental colitis in mice and lipopolysaccharide (LPS)-triggered inflammation in Caco-2 cells. These findings indicated that geniposide may be a new natural chemopreventive agent to combat UC.     

海南砂仁挥发油对2,4-二硝基氯苯与乙酸诱发的大鼠溃疡性结肠炎的治疗作用

目的观察海南砂仁挥发油(VOA)对溃疡性结肠炎(UC)的治疗作用及其机制。方法采用2,4-二硝基氯苯(DNCB)与乙酸复合灌肠法制备UC大鼠模型。将SD大鼠分为正常对照组、UC组、UC+VOA(0.42,0.84和1.68g·kg-1)和UC+柳氮磺吡啶(SSZ)0.52g·kg-1治疗组。正常组和UC组ig生理盐水,治疗组ig相应药物,连续给药21d后处死大鼠进行结肠大体形态和组织病理学评分。用邻苯三酚自氧化法测定结肠组织中超氧化物岐化酶(SOD)活性,比色法测定谷胱甘肽过氧化物酶(GSH-Px)活性和一氧化氮合酶(NOS)含量,SABC免疫组织化学法检测结肠组织肿瘤坏死因子α(TNF-α)和核因子-κBp65(NF-κBp65)阳性细胞百分率。结果与正常组比较,UC大鼠结肠组织学评分升高;结肠出现黏膜层缺损、淋巴组织增生、腺体排列紊乱以及以淋巴细胞为主的炎症细胞浸润和血管扩张等病理变化。结肠组织SOD和GSH-Px活性明显降低,NOS显著升高,TNF-α和NF-κBp65免疫阳性细胞百分率明显增加。与UC组比较,VOA0.84和1.68g·kg-1治疗后,明显降低UC大鼠结肠形态和组织学评分,减轻结肠病理变化,使UC结肠组织SOD和GSH-Px升高,NOS降低,结肠组织TNF-α和NF-κBp65免疫阳性细胞明显减少。结论VOA可减轻UC大鼠结肠炎症反应和黏膜损伤,作用机制可能与其抑制TNF-α和NF-κBp65表达从而抑制炎症级联反应有关。     

溃疡性结肠炎患者肠黏膜CD4CD25调节性T细胞及Foxp3表达水平的研究

目的 通过分析溃疡性结肠炎(UC)患者肠黏膜中CD4+CD25+调节性T细胞及其特异性标志物Foxp3的表达特点,探讨CD4+ CD25+调节性T细胞和Foxp3在UC发病机制中的作用.方法18例UC活动期患者和12例健康体检者作为正常对照组,分别经电子肠镜活检肠黏膜组织,以流式细胞术检测肠固有层单个核细胞( LPMC...     

Timosaponin AIII and its metabolite sarsasapogenin ameliorate colitis in mice by inhibiting NF-κB and MAPK activation and restoring Th17/Treg cell balance

The rhizome of Anemarrhena asphodeloides (AA, family Liliaceae), which contains furostanol and spirostanol saponins, is a typical herbal medicine that improves learning and memory in rats and inhibits inflammation. In a preliminary study, timosaponin AIII, one of AA main constituents, was metabolized to sarsasapogenin by gut microbiota and inhibited NF-κB activation in lipopolysaccharide (LPS)-stimulated macrophages. Here we have investigated the anti-inflammatory effects of AIII and sarsasapogenin in vitro and in vivo. Both AIII and sarsasapogenin potently inhibited NF-κB and MAPK activation, as well as IRAK1, TAK1, and IκBα phosphorylation in LPS-stimulated macrophages. Further, AIII and sarsasapogenin inhibited the binding of LPS to macrophage Toll-like receptor 4, as well as polarization of M2 to M1 macrophages. Oral administration of AIII and sarsasapogenin inhibited 2,3,4-trinitrobenzene sulfonic acid (TNBS)-induced colon shortening and myeloperoxidase activity in mice, along with reducing NF-κB activation and interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 levels, while simultaneously increasing IL-10. Both compounds inhibited Th17 cell differentiation in colonic lamina propria, but induced Treg cell differentiation. Further, AIII and sarsasapogenin inhibited the differentiation of splenic CD4⁺ T cells into Th17 cells in vitro. The vitro and in vivo anti-inflammatory effects of sarsasapogenin were more potent than AIII. These results suggest that orally administered AIII may be metabolized to sarsasapogenin by gut microbiota, which may ameliorate inflammatory diseases such as colitis by inhibiting TLR4-NF-κB/MAPK signaling pathway and restoring Th17/Treg cell balance.     

Sodium butyrate inhibits the NF-kappa B signaling pathway and histone deacetylation,and attenuates experimental colitis in an IL-10 independent manner

Butyrate is a bacterial metabolite of dietary fiber in the colon that has been used to treat inflammatory disease. However, the effect of oral supplementation with butyrate on colitis has not been fully explored. We evaluated the effects of and mechanisms underlying oral supplementation with butyrate on experimental murine colitis. In an in vitro study, we found that LPS induced the secretion of cytokines (i.e., IL-8 in COLO 205; TNF-α, IL-6, IL-12, and IL-10 in RAW 264.7; and TNF-α, IL-6 and IL-12 in peritoneal macrophages obtained from IL-10-deficient [IL-10^−/−] mice). Butyrate (100 μM and 500 μM) inhibited pro-inflammatory cytokine production (i.e., IL-8 in COLO205 and TNF-α, IL-6 and IL-12 in macrophages) but promoted anti-inflammatory cytokine (i.e., IL-10) production in RAW264.7 cells. Butyrate attenuated both the LPS-induced degradation/phosphorylation of IκBα and DNA binding of NF-κB and enhanced histone H3 acetylation. To confirm that butyrate played a protective role in colitis, an acute colitis model was induced using dextran sulfate sodium (DSS) and a chronic colitis model was induced in IL-10^−/− mice. The administration of oral butyrate (100 mg/kg) significantly improved histological scores in both colitis models, including the IL-10^−/− mice. In immunohistochemical staining, IκBα phosphorylation was attenuated, and histone H3 acetylation was reversed in the treated colons of both colitis models. Our results indicate that oral supplementation with butyrate attenuates experimental murine colitis by blocking NF-κB signaling and reverses histone acetylation. These anti-colitic effects of butyrate were IL-10-independent. Butyrate may therefore be a therapeutic agent for colitis.     

Maresin 1 alleviates dextran sulfate sodium-induced ulcerative colitis by regulating NRF2 and TLR4/NF-kB signaling pathway

ObjectiveUlcerative colitis (UC) is one of the most common gastrointestinal diseases, characterized as a chronic, relapsing inflammation that causes damage to the colonic mucosa. Maresin 1 (MaR1), a specialized proresolving mediator, has powerful anti-inflammatory activity that prevents the occurrence of various inflammatory diseases. The aim of this study was to explore the role and potential mechanism of MaR1 in DSS-induced ulcerative colitis.MethodsIn the present study, we established dextran sulfate sodium (DSS)-induced ulcerative colitis rat model in vivo. Rats with colitis received tail vein injection of MaR1, with or without intraperitoneal injection of ML385. The changes of body weight, colon length, disease activity index (DAI), colonic histopathology, inflammatory cytokines, the activity of myeloperoxidase (MPO) and reactive oxygen species (ROS), and infiltration of macrophages expressing F4/80 were analyzed for the evaluation of colitis severity. In addition, protein expressions were detected using western blot.ResultsMaR1 significantly reduced inflammatory cytokines production, and restored body weight, DAI and colonic histopathology. Besides, MaR1 improved the expression of tight junction (TJ) proteins and reduced the infiltration of neutrophil and macrophages, as well as a decreased activity of MPO and ROS. Meanwhile, MaR1 activated Nrf2 signaling and decreased toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB) activation. Furthermore, ML385, an inhibitor of Nrf2, significantly reversed the protective effect of MaR1.ConclusionMaR1 play a protective role in DSS-induced colitis by activating Nrf2 signaling and inactivating Nrf2-mediated TLR4/NF-κB signaling pathway, which mediate proinflammatory mediators and intestinal TJ proteins in rats, providing novel insights into the therapeutic strategy of colitis.     

Regulatory effect of daphnetin, a coumarin extracted from Daphne odora, on the balance of Treg and Th17 in collagen-induced arthritis

Daphnetin extracted from Daphne odora Var. marginata contains coumarin compounds, which possess properties of analgesic and anti-inflammatory effects. In this study, we investigated the therapeutic effect of daphnetin on anti-arthritis and its role on the balance of Tregs and Th17, using a collagen-induced arthritis rat model. Collagen-induced arthritis rats were treated with daphnetin for 21 days. The therapeutic effects of daphnetin were evaluated by clinical symptoms and histopathology. The levels of Th17-, Treg-, Th2-, Th1-type cytokines in serum were determined by ELISA. The expression levels of related receptors RORγt, NF-κB, Foxp3 and CD77 in joint tissues were detected by immunohistochemistry. Our results showed that administration of daphnetin significantly alleviated the severity of the arthritis, as evidenced by the reduction of arthritis scores, suppression of the infiltration of inflammatory cells and prevention of synovial hyperplasia, thereby resulting in the joint destruction in the arthritis rats. Additionally, daphnetin treatment reduced the serum level of Th17-, Th2- and Th1-type in collagen-induced arthritis rats. Correspondingly, the expression of RORγt, NF-κB and CD77 in joint tissue of collagen-induced arthritis rats was remarkably decreased, while the expression of Foxp3 and IL-10 was remarkably increased after being administered with daphnetin. Collectively, this study demonstrated that administration of daphnetin attenuated the clinical symptoms and pathological destruction of arthritis joints. The therapeutic effects were associated with the up-regulation of Tregs, down-regulation of Th17-, Th2- and Th1-type cell responses. The results provide novel evidence that daphnetin has therapeutic effects on autoimmune arthritis through modulating the balance of Tregs and Th17.     

Development of non-antibiotic macrolide that corrects inflammation-driven immune dysfunction in models of inflammatory bowel diseases and arthritis

Inflammation-driven immune dysfunction supports the development of several chronic human disorders including inflammatory bowel diseases and rheumatoid arthritis. Macrolides are effective antibiotics endowed with immunomodulatory effects. In this study we report the chemical synthesis and the pharmacological characterization of CSY0073, a non-antibiotic derivative of azithromycin. CSY0073 was tested for efficacy in two experimental models of colitis induced by administering mice with dextran sulfate (DSS) and trinitrobenzene sulphonic acid (TNBS) and in collagen induced arthritis. Like azithromycin, CSY0073 improved clinical, macroscopic and histopathological scores in mice administered DSS (12.5μmol/kg/day p.o.) and TNBS (45μmol/kg/day p.o.). When administered to TNBS-treated mice, CSY0073 effectively attenuated influx of neutrophils and macrophages into the colonic mucosa and reduced the intestinal expression pro-inflammatory cytokines TNFα, IL-2 and IFNγ. CSY0073 (0.1 to 10μM) counter-regulated TNFα, IFNγ, IL-12 and IL-23 release caused by exposure of mouse spleen monocytes and CD11b+ cells isolated from the colonic lamina propria to endotoxin. CSY0073 (25μmol/kg/day) reduced clinical scores in the collagen induced murine model of rheumatoid arthritis. In myeloid cells, CSY0073 (10μM) prevented the nuclear translocation of the p65 subunit of NF-κB and its binding to canonical NF-κB responsive elements. In summary, we report a novel class of non-antibiotic 14-member macrocycles with anti-inflammatory and immune-modulatory effects. CSY0073, the prototype of this new class of macrolides exerts counter-regulatory activity on NF-κB signaling. This study suggests the exploitation of non-antibiotic macrolides in the treatment of inflammatory disorders characterized by immune dysfunction.     

AMPK agonist downregulates innate and adaptive immune responses in TNBS-induced murine acute and relapsing colitis

AMP-activated protein kinase (AMPK), a cellular energy sensor, has been reported to participate in modulating inflammatory responses, but its role in intestinal inflammation remains unclear. IBD has been characterized by excessive innate and adaptive immune responses. Here, the roles of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an agonist of AMPK, in regulating immune responses of experimental colitis were investigated. In vitro effects of AICAR on LPS-induced macrophage activation and Th1 and Th17 differentiation, as well as in vivo effects of AICAR in mice with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis, were explored. In acute colitis, daily AICAR treatment commenced 2 days after TNBS delivery (day 1), while in relapsing colitis, AICAR treatment commenced after three weekly TNBS administrations. Colon inflammation, production of proinflammatory cytokines and NF-κB activation in colon tissues, and Th1 and Th17 cell populations in lamina propria mononuclear cells (LPMCs) and mesenteric lymph node cells (MLNs) were assayed. Results show that AICAR significantly inhibited in vitro LPS-induced macrophage activation and Th1 and Th17 cell differentiation. Administration of AICAR was therapeutically effective in ameliorating acute and relapsing experimental colitis, as shown by reduced body weight loss and significant attenuation in colon histological inflammation. Moreover, this treatment inhibited NF-κB activation in macrophages, and reduced levels of TNF, Th1- and Th17-type cytokines, and Th1 and Th17 cell populations in LPMCs and MLNs. AICAR-initiated AMPK activation may act as a central downregulator in ongoing innate and adaptive immune responses of murine colitis, providing a novel therapeutic approach in the treatment of IBD.