CRF通过CRF2受体介导肠上皮细胞中TLR4的表达

目的:研究促肾上腺皮质释放因子(CRF)介导肠上皮细胞中Toll样受体4(toll-like receptor4,TLR4)的表达,并探讨其可能通过的受体途径.方法:常规培养人结肠上皮细胞株HT-29细胞,将HT-29细胞分为正常对照组(不加刺激剂),脂多糖(lipopolysaccharide,LPS)刺激组(LPS20g/L刺激24h),促肾上腺皮质释放因子(corticotrophin-releasing factor,CRF)刺激组(CRF20g/L刺激24h),CRF+LPS刺激组(预先CRF20g/L刺激12h,更换细胞液后再与LPS20g/L刺激12h),CRF+Antalarmin 组(CRF与Antalarmin20g/L共刺激24h),CRF+LPS+Antalarmin组(CRF与Antalarmin20g/L共刺激12h后再以LPS刺激12h),CRF+Astressin2B组(CRF与Astressin2B20g/L共刺激24h),CRF+LPS+Astressin2B组(CRF与Astressin2B20g/L共刺激12h后再以LPS刺激12h).刺激结束后,收取各组HT-29细胞,RT-PCR法和免疫印迹法检测各组上皮细胞中TLR4mRNA和蛋白的表达.ELISA法检测各组细胞上清液中IL-8的表达.结果:CRF可以诱导人结肠上皮细胞株HT-29细胞中TLR4表达导致IL-8分泌增多(P<0.05),C R F1受体拮抗剂不能有效地阻滞C R F对TLR4的诱导(P>0.05,CRF+LPS组vs CRF组),CRF2受体拮抗剂可阻滞CRF对TLR4的诱导(P<0.05,CRF+LPS组vs CRF组).结论:CRF通过CRF2受体通路介导肠上皮细胞中TLR4的表达.     

Epithelial restitution and wound healing in inflammatory bowel disease

Inflammatory bowel disease is characterized by a chronic inflammation of the intestinal mucosa. The mucosal epithelium of the alimentary tract constitutes a key element of the mucosal barrier to a broad spectrum of deleterious substances present within the intestinal lumen including bacterial microorganisms, various dietary factors, gastrointestinal secretory products and drugs. In addition, this mucosal barrier can be disturbed in the course of various intestinal disorders including inflammatory bowel diseases. Fortunately, the integrity of the gastrointestinal surface epithelium is rapidly reestablished even after extensive destruction. Rapid resealing of the epithelial barrier following injuries is accomplished by a process termed epithelial restitution, followed by more delayed mechanisms of epithelial wound healing including increased epithelial cell proliferation and epithelial cell differentiation. Restitution of the intestinal surface epithelium is modulated by a range of highly divergent factors among them a broad spectrum of structurally distinct regulatory peptides, variously described as growth factors or cytokines. Several regulatory peptide factors act from the basolateral site of the epithelial surface and enhance epithelial cell restitution through TGF-13-dependent pathways. In contrast, members of the trefoil factor family （TFF peptides） appear to stimulate epithelial restitution in conjunction with mucin glycoproteins through a TGF-13-independent mechanism from the apical site of the intestinal epithelium. In addition, a number of other peptide molecules like extracellular matrix factors and blood clotting factors and also non- peptide molecules including phospholipids~ short-chain fatty acids （SCFA）, adenine nucleotides, trace elements and pharmacological agents modulate intestinal epithelial repair mechanisms. Repeated damage and injury of the intestinal surface are key features of various intestinal disorders including inflammatory bowel diseases and require constant repair of the epi     

卡巴胆碱对肠上皮细胞氧化损伤的保护作用

目的:探讨卡巴胆碱对大鼠肠上皮细胞(intes- tinal epiIhelia cell,IEC)过氧化损伤的影响.方法:将H_2O_2加入IEC培养液中制成IEC过氧化损伤模型.实验设对照组、H_2O_2组(2.5 mmol/L)、卡巴胆碱组(卡巴胆碱100μmol/L).用四甲基偶氮唑盐(MTT)实验检测IEC的存活率,测定培养液中测乳酸脱氢酶(LDH)和细胞中丙二醛(MDA)含量.结果:与对照组相比,H_2O_2组LDH(7.40±2.10 vs 0.81±0.12,P<0.01)、MDA水平显著增高(P<0.0 1),细胞活力明显降低(37.25%±0.80%vs 100%±0.13%,P<0.01).卡巴胆碱组与H_2O_2组相比,LDH漏出MDA形成明显减少(4.64±1.31 vs 7.40±2.10,P<0.01),细胞的细胞活力显著增加(78.70%±2.80%vs 37.25%±0.80%,P<0.01).结论:卡巴胆碱对大鼠IEC过氧化损伤具有保护作用.     

Effects of antiadhesive agents on the healing of intestinal anastomosis

PURPOSE: The effects of antiadhesive agents on the healing of intestinal anastomosis were investigated. METHODS: Eighty rats were divided into eight groups. Colotomy and anastomosis were performed to all rats. Saline solution (control), carboxymethylcellulose, aprotinin, verapamil, tenoxicam, cyclosporine, and dextran 70 were administered intraperitoneally. Vitamin E was administered intramuscularly. The rats were killed 15 days later. Anastomotic healing was assessed by bursting pressure and the hydroxyproline content of the anastomotic tissues. The results were evaluated by Mann-WhitneyU test. RESULTS: The mean (± standard deviation) bursting pressures of carboxymethylcellulose, cyclosporine, and aprotinin groups (108 ± 6.73, 122.5 ± 14.39, and 127 ± 20.23, respectively) were significantly lower than those of the control group (234 ± 6.19). The mean level of hydroxyproline in the anastomotic tissues was significantly lower in the carboxymethylcellulose and cyclosporine groups (8.92 ± 0.6 and 8.32 ± 0.63) than that in the control group (16.33 ± 0.68). CONCLUSION: These findings indicate that carboxymethylcellulose and cyclosporine had adverse effects on intestinal anastomosis in rats.Presented at the Turkish Surgical Congress 98, zmir, Turkey, May 6 to 10, 1998.     

缺血-再灌流时卡巴胆碱对大鼠肠上皮细胞的保护作用

目的:研究缺血/再灌流(I/R)时卡巴胆碱(ca)对肠上皮细胞的保护作用及可能机制.方法:阻断Wistar大鼠SMA血量建立I/R模型,并以肠袋内注射卡巴胆碱进行处理.采用病理学方法观察不同时间肠上皮细胞损伤指数,免疫组化法检测肠上皮细胞TNF-α表达的变化;生化法检测肠组织中DAO含量(nkat/g pro)的变化.结果:I/R Ca组再灌流各时间点肠上皮细胞病理变化较I/R NS组轻(P<0.01);I/R Ca组再灌流各时间点肠袋组织DAO含量较I/R NS组显著增加(0 min:42.01±7.17 vs 18.50±7.83;30 min:44.51±8.00 vs 20.00±5.83;60 min:35.67±7.00 vs 16.34±8.17;120 min:39.00±7.33 vs 21.84±6.67;240 min:53.34±8.17 vs 45.68±6.00;均P<0.01),而I/R Ca组再灌流各时间点肠上皮细胞中呈TNF-α阳性细胞数较I/R NS组明显减少(0 min:204.4±12 vs 246.4±15.6;30 min:198.4±11.2 vs 230.4±14.4;60 min:234.4±12.1 vs 270.4±17.1;120 min:225.4±10.2 vs 260.4±18.5;240 min:196.4±10.8 vs 220.4±18.2;均P<0.01).结论:卡巴胆碱能抑制缺血-再灌流时肠上皮细胞中TNF-α表达,减轻病理损害,对肠上皮细胞具有保护作用.     

Growth hormone abolishes the negative effects of everolimus on intestinal wound healing

AIM: To investigate whether the simultaneous treatment with human growth hormone(h GH) abolishes the negative effects of everolimus on anastomotic healing.METHODS: Forty-eight male Sprague-Dawley-rats were randomized to three groups of 16 animals each(Ⅰ: vehicle; Ⅱ: everolimus 3 mg/kg po; Ⅲ: everolimus 3 mg/kg po + h GH 2.5 mg/kg sc). Animals were pretreated with h GH and/or everolimus daily for seven days. Then a standard anastomosis was created in the descending colon and treatment was continued for another seven days. The anastomosis was resected in toto and the bursting pressure was assessed as a mechanical parameter of intestinal healing. Moreover, biochemical(Hydroxyproline, PCNA, MPO, MMP-2 and MMP-9) and histological(cell density, angiogenesis, amount of granulation tissue) parameters of intestinal healing were assessed.RESULTS: Anastomotic bursting pressure was significantly reduced by everolimus and a simultaneous treatment with h GH resulted in considerably higher values(Ⅰ: 134 ± 19 mm Hg, Ⅱ: 85 ± 25 mm Hg, Ⅲ: 114 ± 25 mm Hg; P 0.05,Ⅰvs Ⅱ; P = 0.09,Ⅰvs Ⅲ and Ⅱ vs Ⅲ) Hydroxyproline concentration was significantly increased by h GH compared to everolimus alone(Ⅰ: 14.9 ± 2.5 μg/mg, Ⅱ: 8.9 ± 3.6 μg/mg, Ⅲ: 11.9 ± 2.8 μg/mg; P 0.05,?Ⅰvs Ⅱ/Ⅲ and Ⅱ vs Ⅲ). The number of MPO-positive cells was reduced significantly by h GHcompared to everolimus alone(Ⅰ: 10 ± 1 n/mm~2, Ⅱ: 15 ± 3 n/mm~2, Ⅲ: 9 ± 2 n/mm~2; P 0.05,Ⅰvs Ⅱ and Ⅱ vs Ⅲ), while the number of PCNA-positive cells were increased by h GH(Ⅰ: 28 ± 3 /mm~2, Ⅱ: 12 ± 3 /mm~2, Ⅲ: 26 ± 12 /mm~2; P 0.05,?Ⅰ?vs Ⅱ and Ⅱ vs Ⅲ). Corresponding to these biochemical findings, HEhistology revealed significantly increased amount of granulation tissue in h GH-treated animals.CONCLUSION: Inhibition of intestinal wound healing by everolimus is partially neutralized by simultaeous treatment with h GH. Both inflammation as well as collagen deposition is influenced by h GH.     

Alveolar epithelial cells: differentiation and lung injury

Abstract:   Re-epithelialization of alveolar epithelial cells is one of the important repair processes in many types of lung injury. The major functions of alveolar type II cells are synthesis and secretion of surfactant, hyperplasia in reaction to alveolar epithelial injury, and serving as progenitor cells for alveolar type I cells. The authors have examined the effects of several soluble factors on cultured alveolar type II cells in vitro , and also examined the histopathology and gene expression of surfactant proteins in the rat lungs with LPS, bleomycin and/or treated with keratinocyte growth factor. The authors next examined the effects of bone marrow stromal cells (BMSC) implanted transvenously into bleomycin-induced lungs. The authors found that keratinocyte growth factor (KGF) is a strong growth factor for alveolar type II cells, and that KGF instillation prevents bleomycin-induced lung injury. Furthermore, the authors showed the possibility of differentiation of implanted BMSC into alveolar epithelial cells. KGF and BMSC may play an important role in maintaining the alveolar epithelium and repairing the damaged epithelium after injury, and may well provide potential therapeutic alternatives.     

Probiotic modulation of dendritic cells co-cultured with intestinal epithelial cells

AIM:To investigate cytokine production and cell surface phenotypes of dendritic cells (DC) in the presence of epithelial cells stimulated by probiotics.METHODS:Mouse DC were cultured alone or together with mouse epithelial cell monolayers in normal or inverted systems and were stimulated with heat-killed probiotic bacteria,Bifidobacterium lactis AD011 (BL),Bifidobacterium bifidum BGN4 (BB),Lactobacillus casei IBS041 (LC),and Lactobacillus acidophilus AD031 (LA),for 12 h.Cytokine levels in the culture supernatants were determined by enzyme-linked immunosorbent assay and phenotypic analysis of DC was investigated by flow cytometry.RESULTS:BB and LC in single-cultured DC increased the expression of I-Ad,CD86 and CD40 (I-Ad,18.51 vs 30.88,46.11;CD86,62.74 vs 92.7,104.12;CD40,0.67 vs 6.39,3.37,P 0.05).All of the experimental probiot-ics increased the production of inflammatory cytokines,interleukin (IL)-6 and tumor necrosis factor (TNF)-α.However,in the normal co-culture systems,LC and LA decreased the expression of I-A d (39.46 vs 30.32,33.26,P 0.05),and none of the experimental probiotics increased the levels of IL-6 or TNF-α.In the inverted coculture systems,LC decreased the expression of CD40 (1.36 vs-2.27,P 0.05),and all of the experimental probiotics decreased the levels of IL-6.In addition,BL increased the production of IL-10 (103.8 vs 166.0,P 0.05) and LC and LA increased transforming growth factor-β secretion (235.9 vs 618.9,607.6,P 0.05).CONCLUSION:These results suggest that specific probiotic strains exert differential immune modulation mediated by the interaction of dendritic cells and epithelial cells in the homeostasis of gastrointestinal tract.     

Ephrin-B reverse signaling induces expression of wound healing associated genes in IEC-6 intestinal epithelial cells

AIM: Eph receptors and ephrin ligands play a pivotal role in development and tissue maintenance. Since previous data have indicated an involvement of ephrin-B2 in epithelial healing, we investigated the gene expression and downstream signaling pathways induced by ephrin-B mediated cell-cell signaling in intestinal epithelial cells. METHODS: Upon stimulation of ephrin-B pathways in IEC-6 cells with recombinant rat EphB1-Fc, gene expression was analyzed by Affymetrix rat genome 230 high density arrays at different time points. Differentially expressed genes were confirmed by real-time RT-PCR. In addition, MAP kinase pathways and focal adhesion kinase (FAK) activation downstream of ephrin-B were investigated by immunoblotting and fluorescence microscopy. RESULTS: Stimulation of the ephrin-B reverse signaling pathway in IEC-6 cells induces predominant expression of genes known to be involved into wound healing/cell migration, antiapoptotic pathways, host defense and inflammation. Cox-2, c-Fos, Egr-1, Egr-2, and MCP-1 were found among the most significantly regulated genes. Furthermore, we show that the expression of repair-related genes is also accompanied by activation of the ERK1/2 MAP kinase pathway and FAK, two key regulators of epithelial restitution. CONCLUSION: Stimulation of the ephrin-B reverse signaling pathway induces a phenotype characterized by upregulation of repair-related genes, which may partially be mediated by ERK1/2 pathways.     

Wound healing in normal and diabetic Chinese hamsters

Summary Wound healing was examined in normal and diabetic, non-ketotic Chinese hamsters by morphological and morphometric methods. Dermal, perforating wounds were made in the ears of the hamsters and the response to injury was evaluated in tissue biopsies. The response in normal hamsters was characterized by vascular and cellular migration and pronounced infiltration of polymorphonuclear leukocytes into the area closest to the wound (zone 1). The transition region (zone 2) between wounded and non-wounded tissue was infiltrated primarily by fibroblasts and capillaries. In wounds from diabetic hamsters, 8 h after injury, there was less cellular infiltration (fibroblasts 49%, polymorphonuclear leukoytes 48% of control) and vascular proliferation (47% of control). In the late phase of healing (16 h after injury) the vascular (87% of control) and polymorphonuclear leukocyte (103%) responses in diabetic wounds were not significantly different from control in zones 1 and 2. Wounds from diabetic hamsters also showed considerable oedema (143% of control) in zones 1 and 2, which was accompanied by vascular degeneration and necrosis. At 16 h the collagen content of diabetic wounds was also decreased (54% of control). Increased oedema with reduced vascular proliferation and cellular infiltration in the early healing period characterises the response to injury in the diabetic Chinese hamster.     

非侵入性方法检测肠黏膜屏障功能的研究进展

异常的肠道通透性在人类多种疾病中起着非常重要的作用,包括糖尿病、炎症性肠病、乳糜泻、多发性硬化、食物变态反应过敏症、肠易激综合征等.近年大量的研究发现:一些自身免疫性疾病伴有肠道通透性增加,这种现象发生在疾病之前,被认为与疾病的发病机制相关.研究肠黏膜屏障的功能与结构,可以提高我们对疾病的病因及病理生理认识,并且对于早期检测疾病以及对疾病进行二级预防有重要意义.目前有多种试验方法评估肠黏膜上皮细胞受损、紧密连接功能以及肠黏膜屏障的完整性.本篇综述主要探讨目前评估肠黏膜屏障功能的检测方法.     

The experimental basis of intestinal suturing

Factors that incite inflammation at the healing wound prolong the lag period of wound healing and delay the return of strength at the suture line. Inflammation activates bowel-wall collagenase, which degrades the collagen within the wound, eroding the foundation in which sutures are anchored. Experimental studies have compared the impact of various surgical techniques. Sutures placed by hand uniformly invoke an inflammatory response because dragging the thread through the bowel wall injures tissue. Single-layer anastomoses heal more rapidly than double-layer suture lines. The inner layer causes avascular necrosis of the inverted cuff. Experimental studies have not clearly shown the superiority of inverting suture lines over everting ones. Experimental studies done over the last century indicate that the single-layer inverting anastomosis recommended by Lembert and Halstead adequately compensates for enteric wound weakness during the lag period. Other techniques of sewing an anastomosis provide no clear advantage. Other factors that incite inflammation also delay enteric wound healing. Debris, necrotic tissue, or infection illicit an inflammatory response with detrimental effects on the anastomosis. Antibiotics, by assisting in the control of infection or by minimizing the size of an inoculum help speed healing. Stapling devices violate many of the doctrines of intestinal suturing. Experimental studies suggest, however, that staple lines incite a minimal inflammatory response. Consequently, wounds closed with stapling devices regain strength more rapidly than those closed with traditional surgical techniques.     

免疫血清对旋毛虫感染性幼虫侵入肠上皮细胞及其发育的影响

目的观察旋毛虫感染性幼虫排泄分泌(ES)抗原与旋毛虫感染性幼虫表面抗原免疫鼠血清对幼虫侵入HCT-8肠上皮细胞及其发育的影响。方法将旋毛虫感染性幼虫接种至半固体培养基(RPMI1640培养基+1.75%琼脂糖)+HCT-8细胞中,37℃5%CO2培养12、24、36、72和96h后镜下观察幼虫发育情况;将幼虫接种至含免疫血清的半固体培养基+HCT-8细胞中,培养15min后镜下观察幼虫形态及其对肠上皮细胞的侵入情况,36h后应用间接荧光抗体试验(IFAT)观察幼虫蜕皮,并计数Ⅰ期和Ⅱ～Ⅳ期幼虫。结果幼虫在半固体培养基培养12h可侵入HCT-8细胞单层,36～72h幼虫可蜕皮1～2次,培养96h可见早期成虫。在含ES抗原免疫血清与感染鼠血清条件下培养15min,幼虫头端可见免疫复合物形成的帽样结构,但在含表面抗原免疫血清、正常鼠血清或不含免疫血清条件下培养的幼虫头端则无帽样结构,头端带有帽样结构的幼虫不能侵入HCT-8细胞单层。在含ES抗原与表面抗原免疫血清条件下发育至Ⅱ～Ⅳ期幼虫的百分比(2.25%、2.2%)均明显低于含正常鼠血清条件下培养的幼虫(24.7%)(P0.05)。结论旋毛虫ES抗原免疫血清可阻止幼虫对肠上皮细胞的侵入,ES抗原及表面抗原免疫血清均可阻止部分幼虫的发育(蜕皮)。     

Adipose-derived mesenchymal stem cells alleviate TNBS-induced colitis in rats by influencing intestinal epithelial cell regeneration,Wnt signaling,and T cell immunity

BACKGROUND Conventional Crohn's disease(CD) treatments are supportive rather than curative and have serious side effects. Adipose-derived mesenchymal stem cells(ADSCs) have been gradually applied to treat various diseases. The therapeutic effect and underlying mechanism of ADSCs on CD are still not clear.AIM To investigate the effect of ADSC administration on CD and explore the potential mechanisms.METHODS Wistar rats were administered with 2,4,6-trinitrobenzene sulfonic acid(TNBS) to establish a rat model of CD, followed by tail injections of green fluorescent protein(GFP)-modified ADSCs. Flow cytometry, qRT-PCR, and Western blot were used to detect changes in the Wnt signaling pathway, T cell subtypes, and their related cytokines.RESULTS The isolated cells showed the characteristics of ADSCs, including spindle-shaped morphology, high expression of CD29, CD44, and CD90, low expression of CD34 and CD45, and osteogenic/adipogenic ability. ADSC therapy markedly reduced disease activity index and ameliorated colitis severity in the TNBS-induced rat model of CD. Furthermore, serum anti-sacchromyces cerevisiae antibody and panti-neutrophil cytoplasmic antibody levels were significantly reduced in ADSCtreated rats. Mechanistically, the GFP-ADSCs were colocalized with intestinal epithelial cells(IECs) in the CD rat model. GFP-ADSC delivery significantly antagonized TNBS-induced increased canonical Wnt pathway expression, decreased noncanonical Wnt signaling pathway expression, and increased apoptosis rates and protein level of cleaved caspase-3 in rats. In addition, ADSCs attenuated TNBS-induced abnormal inflammatory cytokine production, disturbed T cell subtypes, and their related markers in rats.CONCLUSION Successfully isolated ADSCs show therapeutic effects in CD by regulating IEC proliferation, the Wnt signaling pathway, and T cell immunity.     

Ephrin-B2 is differentially expressed in the intestinal epithelium in Crohn＇s disease and contributes to accelerated epithelial wound healing in vitro

AIM: Eph receptor tyrosine kinases and their membrane bound receptor-like ligands, the ephrins, represent a bi-directional cell-cell contact signaling system that directs epithelial movements in development. The meaning of this system in the adult human gut is unknown. We investigated the Eph/ephrin mRNA expression in the intestinal epithelium of healthy controls and patients with inflammatory bowel disease (IBD). METHODS: mRNA expression profiles of all Eph/ephrin family members in normal small intestine and colon were established by real-time RT-PCR. In addition, differential expression in IBD was investigated by cDNA array technology, and validated by both real-time RT-PCR and immunohistochemistry. Potential effects of enhanced EphB/ephrin-B signaling were analyzed in an in vitro IEC-6 cell scratch wound model. RESULTS: Human adult intestinal mucosa exhibits a complex pattern of Eph receptors and ephrins. Beside the known prominent co-expression of EphA2 and ephrinAl, we found abundantly co-expressed EphB2 and ephrin-B1/2. Interestingly, cDNA array data, validated by real-time PCR and immunohistochemistry, showed upregulation of ephrin-B2 in both perilesional and lesional intestinal epithelial cells of IBD patients, suggesting a role in epithelial homeostasis. Stimulation of ephrin-B signaling in ephrin- B1/2 expressing rat IEC-6-cells with recombinant EphB1-Fc resulted in a significant dose-dependent acceleration of wound closure. Furthermore, fluorescence microscopy showed that EphB1-Fc induced coordinated migration of wound edge cells is associated with enhanced formation of lamellipodial protrusions into the wound, increased actin stress fiber assembly and production of laminin at the wound edge. CONCLUSION: EphB/ephrin-B signaling might represent a novel protective mechanism that promotes intestinal epithelial wound healing, with potential impact on epithelial restitution in IBD.     

The phytoestrogen genistein promotes wound healing by multiple independent mechanisms

Genistein has been implicated in the beneficial effects of soy on human health, particularly in the context of ageing. In post-menopausal women reduced systemic estrogen leads to a range of age-associated pathologies, including delayed cutaneous wound healing. We have previously shown that this can be reversed by estrogen replacement. However, the effect of genistein on the skin is poorly understood and crucially the influence of genistein on wound healing has not been assessed. 10-week-old ovariectomised mice were systemically treated with 17β-estradiol or genistein. Genistein substantially accelerated wound repair, associated with a dampened inflammatory response. Unexpectedly, co-treatment with the ER antagonist ICI had little impact on the anti-inflammatory, healing promoting effects of genistein. Thus genistein's actions are only partially mediated via classical estrogen receptor-dependent signalling pathways. Indeed, we report that alternative (cell-type specific) signalling mechanisms are activated in the skin in response to genistein treatment.