实验性结肠炎大鼠Toll样受体2和4表达及益生菌治疗的作用

目的 探讨和分析实验性结肠炎大鼠结肠Toll样受体2(TLR2)和Toll样受体4(TLR4)的表达及意义,并分析益生菌治疗的作用.方法 30只雄性Wistar大鼠均分为正常对照组(NC组)、模型对照组(UC组)和益生菌治疗组(PC组).UC和PC组建立2,4,6-三硝基苯磺酸(TNBS)实验性结肠炎大鼠模型.PC组大鼠给予双歧三联活菌悬液(2.2×109 CFU/只)治疗,1次/d,共4周.光镜下观察肠黏膜炎性反应并评分.Western印迹法和实时荧光定量PCR法检测大鼠结肠TLR2和TLR4蛋白及mRNA表达,分析其变化及益生菌治疗的作用.结果 NC、PC和UC组炎性反应评分分别为4.35±0.88、10.25±1.36和7.94±0.85.PC组炎性反应评分较UC组明显改善(P＜0.05),但高于NC组水平(P＜0.01).NC、UC、PC组TLR2和TLR4蛋白表达水平分别为10.26±4.24、47.20±4.62、36.64±3.67和8.16±4.50、25.84±4.46、19.71±3.28.二者在UC组的表达明显高于NC组(P＜0.01)和PC组(P＜0.05),PC组高于NC组(P＜0.01).NC、UC、PC组TLR2和TLR4 mRNA表达水平分别为240±140、10 570±2690、6640±1420和210±110、8580±2710、5290±1540.二者在UC组的表达明显高于PC组(P＜0.05)和NC组(P＜0.01),PC组高于NC组(P＜0.01).结论 实验性结肠炎大鼠结肠TLR2和TLR4表达明显增高,提示肠腔内抗原摄取增多.双歧三联活菌可明显降低二者的表达水平,提示该益生菌可能通过降低TLR2和TLR4表达减少肠腔抗原的摄取.     

Toll样受体4的信号转导与溃疡性结肠炎的研究进展

溃疡性结肠炎(ulcerative colitis, UC)的发病机制尚不明确,目前研究表明,Toll样受体4(Toll-like receptor 4,TLR4)与UC的发病密切相关,TLR4通过髓样分化因子88 (myeloid differentiation factor 88, MyD88)依赖性和非依赖性等途径激活下游信号分子,引发肠黏膜释放TNF-α、IL-1、IL-6等炎症介质,最终导致UC的发生,同时对此信号转导通路的阻断研究为临床治疗UC带来了新的契机.     

清肠栓对葡聚糖硫酸钠诱导溃疡性结肠炎大鼠Toll样受体表达的影响

目的探讨清肠栓对葡聚糖硫酸钠诱导溃疡性结肠炎大鼠Toll样受体(TLRs)表达的影响。方法选取大鼠60只,根据随机数字表法分为对照组、模型组、清肠栓组,每组20只,然后对大鼠进行模型建造,造模时对照组给予蒸馏水,其余组均给予5%的葡聚糖硫酸钠。模型建立后清肠栓组给予清肠栓灌肠,比较大鼠疾病活动指数(DAI)、结肠指数以及TLR1、TLR2、TLR3以及TLR4表达。结果模型组DAI评分显著高于对照组,清肠栓组显著低于模型组(P0.05);清肠栓组结肠指数显著低于模型组(P0.05),与对照组比较无统计学意义(P0.05);模型组TLR2和TLR4表达显著高于对照组,清肠栓组TLR2和TLR4表达显著低于模型组(P0.05),各组TLR1和TLR3比较无统计学意义(P0.05)。结论清肠栓对葡聚糖硫酸钠诱导溃疡性结肠炎疗效显著,能显著降低大鼠TLR2和TLR4表达水平。     

Toll样受体4单克隆抗体干预小鼠实验性溃疡性结肠炎对信号转导通路的影响

目的 探讨Toll样受体4单克隆抗体(Toll like receptor 4 monoclonal antibodies,TLR4mAb)干预葡聚糖硫酸钠(DSS)诱导溃疡性结肠炎(UC)小鼠肠黏膜中TLR4-P38丝裂原活化蛋白激酶(MAPK)-c-fos/c-jun信号转导通路的影响情况.方法 30只BALB/C小鼠分为对照组、模型组以及低、中、高剂量干预组.对照组小鼠饮用蒸馏水7 d;其他四组小鼠饮用5%DSS溶液7 d建立急性期UC模型.造模同时,干预组小鼠分别以低(2 μg)、中(10 μg)、高剂量(20 μg)TLR4mAb腹腔注射,共4次,7 d后处死小鼠,观察疾病活动指数(DAI)、结肠组织病理学评分(HPS).RT-PCR法检测各组肠黏膜TLR4的mRNA表达,Western印迹法测定各组肠黏膜P38MAPK、磷酸化P38MAPK、c-fos、c-jun的蛋白表达.结果 模型组TLR4 mRNA表达明显高于对照组(P＜0.01).与模型组相比,低、中、高剂量干预组DAI指标和HPS指标均有不同程度缓解.P38MAPK及c-jun蛋白表达在低、中、高剂量干预组呈逐步递减,且均较模型组有明显下降(P＜0.01).与模型组相比,低、中、高剂量干预组小鼠肠黏膜磷酸化P38MAPK蛋白及c-fos蛋白表达差异均无统计学意义(P＞0.05).结论 早期应用TLR4mAb对DSS诱导小鼠急性期UC有干预作用,其作用机制可能与抑制TLR4--P38MAPK--c-jun信息转导通路有关.     

溃疡性结肠炎中Toll样受体的变化

溃疡性结肠炎(ulcerative colitis,UC)是一种反复发作的非特异性肠道黏膜慢性炎症性疾病.其病变主要累及直肠和结肠黏膜层,以溃疡为主.溃疡性结肠炎近年来在我国的病例报道有快速增加趋势[1],已成为临床常见的难治性疾病.其发病机制复杂,目前认为UC可能与一系列的遗传易感基因、环境因素及免疫系统的相互作用有关[2].肠道黏膜免疫系统异常反应导致的炎性反应在炎症性肠病的发病中起重要作用已被广大学者所公认.     

益生菌对溃疡性结肠炎模型大鼠肠黏膜Toll样受体表达的影响及其意义

目的探讨益生菌对葡聚糖硫酸钠诱导的溃疡性结肠炎模型大鼠肠黏膜Toll样受体(TLR)的影响及其临床意义。方法 40只SD大鼠随机分为正常对照组、模型组、益生菌组和美沙拉嗪组。对照组正常饲养,不予任何处理;模型组、益生菌组和美沙拉嗪组给予含5%葡聚糖硫酸钠饮用水10 d建立溃疡性结肠炎模型,益生菌组和美沙拉嗪组于第11天分别给予酪酸梭菌活菌和美沙拉嗪灌胃治疗14 d。14 d后处死所有大鼠,观察疾病活动指数,进行组织学评分,RT-PCR法检测TLR2和TLR4的表达,免疫组化方法检测转转录因子(NF)-κB的活性。结果模型组的疾病活动指数和组织学评分显著高于对照组,益生菌组和美沙拉嗪组低于模型组(P<0.05)。模型组TLR2和TLR4的表达显著高于对照组,益生菌组和美沙拉嗪组高于对照组且低于模型组(P<0.05),而益生菌组和美沙拉嗪组间差异不显著(P>0.05)。模型组NF-κB的活性显著高于对照组,益生菌组和美沙拉嗪组高于对照组且低于模型组(P<0.05),而益生菌组和美沙拉嗪组间差异不显著(P>0.05)。结论溃疡性结肠炎模型大鼠中TLR表达异常,NF-κB的活性增加,益生菌及美沙拉嗪制剂可降低溃疡性结肠炎模型大鼠TLR表达和NF-κB的活性,是治疗溃疡性结肠炎的有效方法。     

溃疡性结肠炎患者外周血单核细胞中Toll样受体4、NF-κB的表达及其临床意义

目的 分析溃疡性结肠炎(UC)患者外周血单核细胞中Toll样受体4(TLR4)、核因子-κ,B(NF-κB)水平的变化,探讨两者在UC发病中作用.方法 根据结肠镜检和病理活检结果,106例UC患者分为活动期组(57例)和缓解期组(49例),另同期健康体检患者50例作为对照组,采用实时荧光定量聚合酶链反应(RT-PCR)技术,测定所有对象外周血单核细胞中TLR4和NF-κB的表达情况.结果 与对照组比较,UC患者外周血单核细胞TLR4、NF-κBmRNA表达水平显著升高,且活动期组与缓解期组间比较差异有统计学意义(P＜0.05):按病理分级与Ⅰ级比较,Ⅱ级、Ⅲ级患者外周血单核细胞TLR4、NF-κB mRNA表达水平显著升高,且随着病理分级升高,TLR4 、NF-κB mRNA表达依次增加(P ＜0.05).UC患者TLR4与NF-κB mRNA表达均呈线性正相关,相关系数r=0.753(P ＜005).结论 UC患者外周血单核细胞中TLR4及NF-κB呈高表达状态,且TLR4的表达与NF-κB的表达密切相关.     

TNF-α对肥大细胞Toll样受体4表达的调节

目的探讨肿瘤坏死因子-α（TNF-α）对肥大细胞Toll样受体4（TLR4）表达的调节。方法不同浓度TNF-α与鼠肥大细胞P815作用后,用流式细胞技术、实时定量逆转录聚合酶链式反应（real-time polymerase chain reaction,RT-PCR）,在蛋白水平、mRNA水平检测肥大细胞的TLR4表达。结果 TNF-α上调肥大细胞P815的TLR4表达,TNF-α抗体的应用可阻断此上调作用。结论 TNF-α能够上调肥大细胞P815表面TLR4的表达,为进一步探讨肥大细胞TLR4参与过敏反应提供了实验依据。     

Toll样受体4与肝损伤

Toll样受体是近几年发现的天然免疫受体，主要参与病原微生物产物的识别及炎症信号传导，是天然免疫与获得性免疫的连接点。Toll样受体4介导内毒素引起的感染性免疫反应，参与了肝脏内的损伤、纤维化、再生与修复等炎症反应过程。     

辛伐他汀对糖尿病大鼠肾组织Toll样受体4表达的影响

用免疫组化和RT-PCR方法检测糖尿病大鼠肾组织中Toll样受体4(TLR4)的表达.结果显示糖尿病组TLR4表达较正常对照组升高(P＜0.05),而辛伐他汀干预组较糖尿病组低(P＜0.05),提示TLR4可能参与了糖尿病肾病的发生及发展.辛伐他汀可能通过降低TLR4的表达保护肾组织.     

复方青黛颗粒对溃疡性结肠炎模型大鼠结肠toll样受体2,4基因表达的影响

[目的]观察复方青黛颗粒对溃疡性结肠炎（UC）模型大鼠结肠toll样受体2,4（TLR2,TLR4）基因表达的影响,探讨其治疗UC的可能作用机制。[方法]用三硝基苯磺酸（TNBS）制备UC大鼠模型,将52只SD实验大鼠随机分为正常对照（空白）组、模型组、柳氮磺胺吡啶（SASP）组（500 mg/kg）,复方青黛颗粒低（600 mg/kg）、中（900 mg/kg）、高（1 200 mg/kg）剂量组,从造模后第3天开始分别每天灌胃给药1次至实验结束,第14 d（灌胃10 d后）,用逆转录聚合酶链反应（RT-PCR）法检测TLR2、TLR4的基因表达水平。[结果]模型组TLR2、TLR4基因相对表达量均明显高于空白组（P〈0.01）;复方青黛颗粒中、高剂量组TLR2相对表达量及高剂量组TLR4相对表达量与SASP组比较差异均无统计学意义,但均明显低于模型组（均P〈0.05）。[结论]复方青黛颗粒能有效治疗TNBS诱导的UC模型大鼠,可能与复方青黛颗粒抑制TLR2、TLR4基因表达有关。     

内毒素性肝衰竭大鼠TLR4mRNA表达、血清肿瘤坏死因子-α及肝细胞凋亡的动态变化

目的 观察急性内毒素性肝衰竭大鼠肝组织中TLR4mRNA表达变化规律及其与血清肿瘤坏死因子-α水平、肝细胞凋亡的关系。方法雌性Wistar大鼠给予D氨基半乳糖／脂多糖同时腹腔注射，计算动物死亡率及生存时间，动态观察给药后4、8、12h肝功能、血清肿瘤坏死因子-α、肝组织TLR4mRNA表达及病理变化，以TUNEL法检测原位细胞凋亡，计算凋亡指数。结果80％大鼠死于急性肝衰竭，平均生存时间15．6h±1．8h，病理表现为肝脏大块或亚大块坏死。给药后4、8、12h血清TNF—α增高含量及肝细胞凋亡均增加，血清TNF—α变化早于肝细胞凋亡指数的增加，肝组织TLR4mRNA的表达与血清TNF—α含量呈正相关（r=0．709，P=0．000）。结论 内毒素通过单核吞噬系统TLR4介导TNF—α大量产生，激活炎症级联反应并诱导肝细胞凋亡是内毒素性肝衰竭的重要病理机制之一，阻断肝内外单核吞噬系统TLR4介导的的生理学作用，可能会对内毒素性肝衰竭起到一定防治作用。     

VEGF在溃疡性结肠炎患者中的表达水平及其临床意义

目的研究血管内皮生长因子(VEGF)在溃疡性结肠炎患者组织中的表达情况,探讨其在溃疡性结肠炎的发病机制和疾病发展中的作用及意义。方法选取溃疡性结肠炎患者30例,其中轻度8例,中度10例,重度12例;肠易激综合征组20例。采用免疫组化法检测各组中VEGF的表达情况,判断不同组间其表达水平是否存在差异。结果溃疡性结肠炎患者VEGF阳性细胞数为64.50±6.15,高于正常对照组VEGF阳性细胞数的27.05±4.10(P0.01)。轻度、中度、重度的活动期溃疡性结肠炎病人的组织中,VEGF的阳性细胞数三者之间无显著差异性(P0.05)。结论VEGF在溃疡性结肠炎患者的黏膜表达显著升高。VEGF在轻度、中度、重度的溃疡性结肠炎患者的黏膜表达无显著差异性。     

罗格列酮对大鼠溃疡性结肠炎的疗效及其机制

目的观察罗格列酮对大鼠溃疡性结肠炎的疗效并探讨其可能存在的机制。方法应用三硝基苯磺酸(TNB)/乙醇灌肠制备大鼠溃疡性结肠炎模型。实验设正常对照组、模型对照组、阳性药物组(柳氮磺胺吡啶组,100 mg/kg)、罗格列酮给药组(2 mg、4mg、8 mg/kg),每天灌胃给药1次,给药时间从造模后第2天开始至实验结束共8 d,观察大鼠疾病活动指数(DAI)、结肠黏膜损伤指数(CMDI)及组织学评分(HS)。生化法检测大鼠结肠组织髓过氧化物酶(MPO)、超氧化物歧化酶(SOD)活性及丙二醛(MDA)的含量。结果与正常组相比,模型组大鼠DAI、CMDI、HS明显增加(P<0.01),结肠组织MPO活性、MDA水平显著升高(P<0.01),SOD活性下降(P<0.01)。罗格列酮4 mg、8 mg/kg和SASP可不同程度改善DAI、CMDI和HS(P<0.05,P<0.01),降低MPO活性和MDA水平(P<0.01),增加SOD活性(P<0.01)。结论罗格列酮对大鼠溃疡性结肠炎有保护作用,其机制可能与减少脂质氧化,增加清除氧自由基的能力有关。     

TLR3与TLR9在溃疡性结肠炎中表达状态及临床意义的研究

目的观察TLR3和TLR9在溃疡性结肠炎(UC)患者病变组织和正常大肠组织中的表达情况,探讨其在UC发病机制中的作用。方法收集UC病例及正常对照结肠镜活检标本各30例。采用免疫组化和实时荧光定量PCR技术,检测UC患者及正常对照组肠黏膜中TLR3、TLR9的表达情况。结果 UC病变组织、正常结肠组织中均有TLR3 mRNA及TLR9 mRNA的表达,但UC组织中TLR9 mRNA表达显著高于正常结肠组织,而TLR3 mRNA与正常结肠组织相比差异无统计学意义。在免疫组化染色图片上,发现TLR3、TLR9主要在细胞的胞浆中表达,在UC病变组织中TLR9阳性表达率明显高于正常结肠组织(P0.05)。结论TLR9在UC患者中表达高度上调,推测它可能参与了UC的发病过程。     

Effects of Proteoglycan on Dextran Sulfate Sodium-Induced Experimental Colitis in Rats

Proteoglycans (PG) are macromolecules composed of glycosaminoglycan chains covalently attached to a protein core. In this study, we examined the effects of PG on dextran sulfate sodium (DSS)-induced experimental colitis in rats. First, to examine whether PG may ameliorate acute established DSS colitis, PG was administered orally for 5 days to the model animals. We evaluated the effects of PG on the basis of clinical symptoms, hematological analysis, macroscopic observation, and microscopic examination. We then examined whether PG administered orally to rats was detectable in their colonic lumen. After administration of PG, the colonic contents were collected, and the molecular weight of PG in the sample was analyzed by gel filtration high-performance liquid chromatography. Furthermore, we examined whether orally administered PG affected the concentrations of short-chain fatty acids (SCFAs) in the colonic feces. Orally administered PG ameliorated the clinical symptoms of bloody stools and diarrhea, and attenuated the increase in the white blood cell count in rats with established DSS colitis. Histologically, orally administered PG reduced the degree of mucosal erosion and inflammatory cell infiltration into the erosive area induced by DSS. Orally administered PG was detected in rat colon, although its molecular weight was slightly decreased. Orally administered PG significantly increased the concentration of total SCFAs and n-butyrate in rat colonic feces. This is the first study to indicate that exogenous PG ameliorates experimental colitis, suggesting the potential usefulness of PG for clinical treatment of colitis.     

Effects of Exopolysaccharide-Producing Probiotic Strains on Experimental Colitis in Rats

Purpose Inflammatory bowel disease is suggested to result from a dysregulated immune response toward intestinal microflora, which may be restored by probiotic therapy based on the concept of healthy microflora. Ideal probiotic bacteria may be beneficial in inflammatory bowel disease; however, the mechanism of action and the clinical efficacy of probiotic usage are still unclear. In the present study, the effect of exopolysaccharide producing probiotics was evaluated on an experimental colitis model in rats. Methods Colitis was induced by intracolonic administration of acetic acid. Then, rats were treated daily with two probiotic strains, Lactobacillus delbrueckii subsp. bulgaricus B3 strain (exopolysaccharide of 211 mg/l: high-EPS group) or Lactobacillus delbrueckii subsp. bulgaricus A13 strain (EPS of 27 mg/l: low-EPS group), which were given into the stomach. The non-colitis–fed control group was only treated with high-exopolysaccharide strain. The model-control and control groups were treated only with tap water. Rats were killed after a seven-day treatment period. Disease activity was quantified by use of histologic scores and colonic myeloperoxidase activity, which is a marker of neutrophil infiltration during inflammation. Results The enhanced inflammatory response was accompanied by a higher level of myeloperoxidase activity in the colitis group. Histologic scores of colonic damage and myeloperoxidase activity were lower in both probiotic-treated groups compared with those of the colitis control group (P < 0.001), although the mentioned scores improved significantly more in the high-EPS group than in the low-EPS group (P < 0.001). Conclusions Exopolysaccharide-producing probiotics significantly attenuate experimental colitis, which may be mediated by exopolysaccharide in a dose-dependent manner. Therefore, exopolysaccharide-producing probiotics may be a promising therapeutic role in inflammatory bowel disease. Supported in part by the Scientific and Technological Research council of Turkey (TUBITAK), TBAG-2090 (101T129) (the probiotic properties of experimental studies and the isolations of the strains used in this study).     

Expression and effect of TLR4 in rats with diabetic nephropathy

ObjectiveTo observe the expression of TLR4 in kidney tissue of rats with diabetic nephropathy and discuss the role of TLR4 in the occurrence and development of the diabetic nephropathy.MethodsA total of 60 clean male SD rats were selected and randomly divided into the modeling group and control group after 1 week of breeding, including 30 rats in each group. Biochemical indices as well as the protein expression of TLR4 were observed and compared between two groups at 2 w, 4 w, 6 w, 8 w and 12 w after the modeling, and the correlation between TLR4 and each biochemical indexes was analyzed.ResultsRats in the modeling group had higher levels of blood glucose, 24-hour urine protein and blood urea nitrogen after the modeling, and showed the increase in the serum creatinine, kidney/body weight ratio, CRP and serum TNF-α at 4w after the modeling, with the significant difference compared to results of the control group (P<0.05). The cross-section area and mean volume of glomerulus in the modeling group at 4 w, 6 w, 8 w and 12 w were significantly higher than those in the control group, with the statistically significant difference (P<0.05). The expression of TLR4 at each time point in the control group was relatively low. Rats in the modeling group had the high expression of TLR4 in kidney's glomerular basement membrane, proximal convoluted tubule and renal interstitial area since 2 w, with the significant difference compared to the control group (P<0.05). The expression in rats of the modeling group was higher than the one of the control group since the 2nd week. As the time flied, its expression increased, with the statistically significant difference between two groups (P<0.05). There was certain correlation between the protein expression of TLR4 and the increased serum titer of 24-hour urine protein excretion, serum creatinine, CRP and TNF-α.ConclusionsTLR4 may activate the immuno-inflammatory reactions to play a role in the occurrence and development of the diabetic nephropathy.