植物乳杆菌对IL-10基因敲除结肠炎小鼠黏附分子的影响

目的: 观察植物乳杆菌( Lactobacillus plantarum,LP)灌胃对IL-10基因敲除小鼠肠道炎症和淋巴细胞归巢的影响.方法: 取IL-10基因敲除(knockout,KO)小鼠和未作基因敲除的背景鼠分为4组: 对照组(野生组WT)、加植物乳杆菌组(WT+LP)、IL-10基因敲除模型组(KO)、模型加植物乳杆菌组(KO+LP).4 wk开始对照组和KO组每日予PBS灌胃,WT+LP和KO+LP组予溶于PBS的LP灌胃,持续4-8 wk结束.实验结束后取各组小鼠结肠行炎症评分和电镜亚显微结构观察,并用RT-PCR和Western blot检测归巢相关分子MAdCAM-1、ICAM-1、α4β7及CD3的表达.结果: 8 w k 后K O小鼠1 0 0%发生肠道炎症,且其CD3及黏附分子α4β7、ICAM-1和MAdCAM-1的mRNA和蛋白表达水平较WT组均明显增高(mRNA: t = 39.42,8.83,25.53,45.78,均P＜0.01;CD3、ICAM-1、MAdCAM-1蛋白: t = 19.04,29.57,12.29,均P＜0.01).予以益生菌LP灌胃后,KO+LP组小鼠CD3及黏附分子α4β7、ICAM-1和MAdCAM-1的mRNA和蛋白表达水平较KO组均明显降低(mRNA: t = 20.34;4.95;14.21;22.31,均P＜0.01;CD3、ICAM-1、MAdCAM-1蛋白: t = 6.82,14.10,7.03,均P＜0.01);WT+L P组小鼠CD3及黏附分子α4β7、ICAM-1和MAdCAM-1的mRNA较WT组均明显降低( t = 9.33,10.55,7.75,6.69,均P＜0.01),而WT+LP组小鼠CD3及黏附分子ICAM-1和MAdCAM-1的蛋白表达水平无明显降低.结论: 植物乳杆菌能下调黏附分子在IL-10基因敲除结肠炎小鼠中的高表达,这可能是其减轻炎症状态,缓解炎症性肠病的重要机制之一.     

白细胞介素-10与溃疡性结肠炎

溃疡性结肠炎是一种病因不明的慢性非特异性结肠炎症,其确切的发病机制尚不清楚,但结肠黏膜的免疫学异常尤其是细胞因子的作用已得到公认。白细胞介素(IL)-10属于抗炎性细胞因子,近年来许多研究结果表明,IL-10参与了溃疡性结肠炎的发生与发展,重组IL-10能改善其临床症状。     

双歧三联活菌胶囊对溃疡性结肠炎患者结肠黏膜白细胞介素-1β及白细胞介素-10的影响

我国溃疡性结肠炎（UC）发病率低于西方国家，但有逐年上升趋势。UC的病因及发病机制不明确。近年来，人们开始注意到肠腔内细菌在UC发病中的重要作用，提出UC是机体对肠道某些细菌不耐受而造成的免疫损伤的新观点。因此，许多学者试图利用肠道益生菌来抑制UC致病微生物的生长，从而减轻或缓解肠道炎症。因为UC存在结肠黏膜的免疫和细胞因子异常，所以我们观察了双歧三联活菌胶囊是否对黏膜白细胞介素（IL）-β和IL-10有影响，现报道如下。     

白细胞介素-23/-17轴在小鼠急性实验性结肠炎中的表达及作用

目的 研究白细胞介素(IL)-23/IL-17轴在小鼠实验性结肠炎结肠组织中的表达和作用.方法 将64只小鼠分为对照组24只、模型组24只、抗体组8只、正常血清组8只.除对照组外,其余各组建立小鼠急性实验性结肠炎模型.对照组和模型组小鼠分别于造模后24 h、48 h、7 d处死.抗体组和正常血清组小鼠分别于造模前2 h腹腔内注射多克隆大鼠抗小鼠IL-17中和抗体和正常大鼠血清,于造模48 h后处死.检测各组小鼠组织学损伤评分、肠组织髓过氧化物酶(MPO)活性;酶联免疫吸附试验检测结肠组织IL-23p19、IL-17含量;免疫组化染色检测核因子(NF)-κB p65在结肠组织中的表达;实时荧光定量(RT)PCR检测IL-23p19、IL-17、IL-12p35的mRNA表达水平.结果 模型组24 h、48 h、7 d时IL-23p19蛋白表达水平和mRNA表达水平[分别为(15.53±3.32)、(31.16±4.98)、(14.03±3.56)ng/mg蛋白和4.09±0.34、3.39±0.46、6.54±1.82]、IL-17的蛋白表达水平和mRNA表达水平[分别为(0.35±0.06)、(0.38±0.08)、(0.26±0.05)ng/mg蛋白和4.21±2.61、2.65±0.91、5.63±1.43]均显著高于正常对照组(P值均＜0.05),48 h时达高峰.IL-23与IL-17蛋白表达水平和mRNA表达水平呈正相关(r值分别为0.745和0.793,P＜0.05).抗体组IL-23p19和IL-12p35高水平表达,但NF-κB p65阳性细胞率、组织学评分及MPO活性[分别为1.86%±0.36%、0.63±0.52、(0.40±0.03)U/g]明显低于48 h模型组[分别为4.35%±0.37%、5.13±0.64、(2.29±0.40)U/g],说明中和IL-17后能明显减轻结肠炎症,抑制NF-κB活性.结论 IL-23/IL-17轴在急性实验性结肠炎早期阶段起关键作用.IL-17有望成为炎症性肠病治疗的新靶标.     

双歧杆菌对小鼠急性实验性结肠炎作用的研究

目的 研究双歧杆菌在葡聚糖硫酸钠(DSS)诱导的急性溃疡性结肠炎小鼠模型中的作用.方法 将80只BALB/C小鼠均分为8组,每组10只.除正常对照组、单纯0501菌株组、单纯c122菌株组外,其他5组动物均给予5%DSS造模7 d.在造模开始前2 d,阴性对照组用0.9%氯化钠液灌肠、阳性对照组用柳氮磺胺吡啶(SASP)20 mg/ml灌肠、DSS+0501菌株组用1×109 CFU/ml 0501菌液灌肠、DSS+c122菌株组用1×109 CFU/ml c122液菌灌肠.9 d后处死动物取结肠标本,行H-E染色,观察镜下结肠病理变化,并用免疫组化染色、RT-PCR技术分析结肠黏膜中白细胞介素(IL)-10 mRNA及蛋白表达.结果 DSS造模过程中给予双歧杆菌灌肠小鼠(0501菌株组和c122菌株组)的结肠炎性反应程度较模型组加重,结肠黏膜IL-10表达减少.而单纯给予双歧杆菌灌肠小鼠(单纯0501菌株组和单纯c122菌株组)未见结肠炎表现.结论 双歧杆菌的某些菌株在结肠黏膜屏障功能受损情况下,可加重溃疡性结肠炎小鼠的结肠黏膜损伤.     

Effect of Lactobacillus plantarum LP-Onlly on gut flora and colitis in interleukin-10 knockout mice

Background and Aim: Probiotics are used in the therapy of inflammatory bowel disease. This study aimed to determine the effects of probiotic Lactobacillus plantarum LP‐Onlly (LP) on gut flora and colitis in interleukin‐10 knockout (IL‐10^?/?) mice, a model of spontaneous colitis. Methods: IL‐10^?/? and wild‐type mice were used at 8 weeks of age and LP by gavage was administered at a dose of 10⁹ cells/day per mice for 4 weeks. Mice were maintained for another one week without LP treatment. The colonic tissues were collected for histological and ultrastructural analysis at death after 4 weeks treatment of LP, and the feces were collected at 1‐week intervals throughout the experiment for the analysis of gut flora and LP using selective culture‐based techniques. Results: Compared with control mice, IL‐10^?/? mice developed a severe intestinal inflammation and tissue damage, and had an abnormal composition of gut microflora. LP administration attenuated colitis with the decreased inflammatory scoring and histological injury in the colon of IL‐10^?/? mice. In addition, LP administration increased the numbers of beneficial total bifidobacteria and lactobacilli, and decreased the numbers of potential pathogenic enterococci and Clostridium perfringens, although the decrease of coliforms was not significant after LP treatment in IL‐10^?/? mice. Conclusions: Oral administration of LP was effective in the treatment of colitis, with the direct modification of gut microflora in IL‐10^?/? mice. This probiotic strain could be used as a potential adjuvant in the therapy of inflammatory bowel disease, although further studies are required in human.     

植物乳杆菌对2,4,6-三硝基苯磺酸诱导的小鼠结肠炎的治疗作用

目的:评估益生菌植物乳杆菌(LP)对三硝基苯磺酸(TNBS)诱导小鼠肠道炎症损伤的治疗作用,并探讨其可能的作用机制.方法:将成年♀Balb/c小鼠随机分成3组:正常对照组(Control组),TNBS灌肠诱导小鼠结肠炎组(TNBS组)和LP干预组(TNBS+LP组).TNBS诱导肠炎模型建立后,给予TNBS+LP组小鼠灌胃LP3wk,其余两组灌胃空白对照PBS液.实验结束后对大鼠一般情况、结肠大体损伤及组织学损伤进行评估,并对各组小鼠结肠组织髓过氧化物酶(MPO)活性、LTB4含量及促炎细胞因子TNF-α和IFN-γ的表达进行测定.结果:与TNBS诱导的TNBS组相比,LP明显减轻了TNBS诱导的小鼠结肠炎症,表现为疾病活动指数下降(3.37±0.36vs0.97±0.47,P<0.05),结肠大体和组织学评分显著降低(1.11±0.61vs4.62±0.40;1.48±0.40vs5.39±1.12,均P<0.05),且LP显著降低了TNBS诱导的小鼠结肠黏膜内中性粒细胞浸润,这与MPO活性降低相一致(25.14U/g±5.22U/gvs90.3U/g±7.70U/g,P<0.05).此外,LP明显降低了T...     

Lactobacillus plantarum 299V in the treatment and prevention of spontaneous colitis in interleukin-10-deficient mice

Interleukin (IL)-10-deficient (IL-10-/-) mice develop colitis under specific pathogen-free (SPF) conditions and remain disease free if kept sterile (germ free [GF]). We used four different protocols that varied the time-points of oral administration of Lactobacillus plantarum 299v (L. plantarum) relative to colonization with SPF bacteria to determine whether L. plantarum could prevent and treat colitis induced by SPF bacteria in IL-10-/- mice and evaluated the effect of this probiotic organism on mucosal immune activation. Assessment of colitis included blinded histologic scores, measurements of secreted colonic immunoglobulin isotypes, IL-12 (p40 subunit), and interferon (IFN)-gamma production by anti-CD3-stimulated mesenteric lymph node cells. Treating SPF IL-10-/- mice with L. plantarum attenuated previously established colonic inflammation as manifested by decreased mucosal IL-12, IFN-gamma, and immunoglobulin G2a levels. Colonizing GF animals with L. plantarum and SPF flora simultaneously had no protective effects. Gnotobiotic IL-10-/- mice monoassociated with L. plantarum exhibited mild immune system activation but no colitis. Pretreatment of GF mice by colonization with L. plantarum, then exposure to SPF flora and continued probiotic therapy significantly decreased histologic colitis scores. These results demonstrate that L. plantarum can attenuate immune-mediated colitis and suggest a potential therapeutic role for this agent in clinical inflammatory bowel diseases.     

敲除趋化因子受体3对延缓原发性胆汁性肝硬化模型病情进展的作用

目的 建立原发性胆汁性肝硬化(PBC)动物模型,研究趋化因子受体3(CXCR3)及其配体干扰素诱导蛋白10(CXCL10)在PBC发病中的作用.方法 6～8周雌性C57BL/6J小鼠72只,分为3组:C57BL/6J野生鼠PBC模型组、正常对照组、CXCR3基因敲除(CXCR3-/-)C57BL/6J小鼠PBC模型组,PBC模型组小鼠每次腹腔注射5 mg/kg的聚肌苷酸(聚)胞苷酸(Poly I:C),每周2次,共24周,第8、16、24周处死小鼠,收集小鼠肝脏、血清标本;酶联免疫吸附试验(ELISA)检测血清抗线粒体抗体(AMA)和CXCL10滴度;病理分析肝脏炎性细胞浸润程度.计量资料两样本间比较选用t检验.结果 第8、16、24周对照组血清中AMA-M2的平均吸光度(A)值分别为0.17±0.04、0.19±0.07、0.20±0.06;PBC野生鼠模型的AMA-M2的平均A值分别为0.70±0.41、0.48±0.35、0.69±0.44;CXCR3-/-小鼠模型的平均A值分别为0.56±0.25、0.46±0.35、0.85±0.34.第8、16、24周PBC野生鼠模型组碱性磷酸酶(ALP)平均值分别为(115±33)、(119±32)、(133±52)U/ml;PBC CXCR3-/-鼠模型组ALP平均值分别为(106±29)、(112±29)、(122±60)U/ml;对照组小鼠ALP平均值分别为(65±7)、(70±9)、(77±10)U/ml.与对照组相比,PBC模型组的AMA滴度和ALP水平明显增高(P＜0.05);CXCR3-/-小鼠和野生鼠PBC模型组之间的AMA滴度和ALP水平差异无统计学意义.PBC模型组小鼠第8周可见小胭管周围单个核细胞浸润,胆管上皮细胞变性坏死,基底膜不完整;第16周可见小叶间胆管增生,炎性细胞浸润;第24周可见小胆管损伤、闭塞,胆管内胆栓形成,周围炎性细胞浸润,肉芽肿形成,炎症从门静脉区发展到肝实质.CXCR3-/-鼠PBC模型组病变程度较同期的野生鼠PBC模型组轻,第8周可见部分小胆管周围少量炎性细胞浸润,第16周汇管区炎性细胞浸润较前增多,至第24周时无胆栓和纤维间隔形成;CXCR3-/-小鼠模型组血清CXCL10的平均滴度较对照组明显增高;ELISA检测PBC模型组血清中CXCL10的平均滴度较对照组明显升高(P＜0.05),随着病程的进展,CXCL10滴度呈逐渐上升的趋势.与野生鼠PBC模型相比,CXCR3-/-小鼠模型CXCL10的平均滴度更高,第24周时两者差异有统计学意义.结论 敲除CXCR3有助于减轻和延缓小鼠PBC模型的病情进展.

Abstract：

Objective To generate an autoimmune cholangitis animal model and investigate whether CXCR3 and its ligand were involved in the pathogenesis of primary biliary cirrhosis (PBC). Methods Female C57BL/6 wild-type (WT) mice and CXCR3 knockout (CXCR3-/-) mice were injected with 5 mg/kg of poly I:C intra-peritoneally twice a week for 24 consecutive weeks. Liver specimens were collected to evaluate the degree of cell infiltration. AMA was assayed by ELISA. Differences in pathology were compared between CXCR3-/- mice and wild-type. Student's test was used to assess the differences. Results Anti-mitochon-drial antibody (AMA) and alkaline phosphatase (ALP) level were elevated significantly in the sera of all poly Ⅰ:C injected mice compared with control mice. AMA titers in serum were increased in the poly I:C injected WT mice compared with that of the control mice at week 8, 16, and 24 respectively (0.70±0.41 vs 0.17±0.04,0.48±0.35 vs 0.19±0.07, 0.69±0.44 w 0.20±0.06, P＜0.01). There was no significant difference between AMA titers in the serum of WT PBC mice and CXCR3-/- PBC mice (0.70±0.41 vs 0.56±0.25, 0.48±0.35 vs 0.46±0.35, 0.69±0.44 vs 0.85±0.34). Serum alkaline phosphatase (ALP) levels were raised among the poly I:Cinjected WT mice compared with WT controls (115±33) vs (65±7) U/ml; (119±32) vs (70±9) U/ml; (133±52) vs (77±10) U/ml. The serum ALP levels in CXCR3-/- PBC mice were (106±29), (112±29)and (122±60) U/ml at week 8, 16 and 24 respectively. There was no significant difference in ALP level between WT and CXCR3-/- mice PBC model. Considerable numbers of infiltrating cells were detected at the portal areas 8weeks after poly I:C injection, which progressed up to 24 weeks. At week 24, the interlobular bile ducts were lost and bile-plug were evident. Compared to WT mice model, this results revealed that CXCR3-/- mice, had fewer foci of inflammation and significant reduction in total inflammatory cells in their livers than those of the WT mice at 8, 16 and 24 weeks after injection of poly I:C. At week 24, there were no cholestasis orgranulomas in CXCR3-/- mice of PBC models. Compared to the control model, CXCL10 serum level was increased in PBC model. With the disease progression, CXCL10 serum level was elevated gradually. In comparison with wild mice, CXCR3-/- mice model had a higher serum level of CXCL10. At week 24, there was significant difference of CXCL10 serum level between wild-type mice and CXCR3-/- mice model. Conclusion In conclusion, these findings indicate that, CXCR3-/- mice might have a delayed onset and ultimately could not recruit sufficient effector cells to the liver for inflammation development.     

Murine monoclonal anti-tNF antibody administration has a beneficial effect on inflammatory bowel disease that develops in IL-10 knockout mice

Although the etiology of inflammatory bowel disease (IBD) is unknown, there is increasing evidence for the pivotal role played by tumor necrosis factor- (TNF-). Recent work has shown an increased concentration of TNF- in both the bowel wall and in the stools of patients with IBD, and in children with that disease there are increased serum levels. Coincidental studies have shown that IL-10 knockout mice have increased levels of TNF- and are known to develop a syndrome of stunted growth, anemia, bloody diarrhea, and colon tumors that mimics IBD. By injecting monoclonal antibodies intraperitoneally into IL-10 knockout mice, we were able to demonstrate significant histologic improvement of inflammation that correlates well with a resolution of diarrhea and rectal bleeding. This finding is consistent with a role for anti-TNF- in the pathogenesis of IBD and suggests that this model may be of use for examining the effects of anti-TNF- antibody administration.     

鸢尾素对载脂蛋白E基因敲除小鼠动脉粥样硬化的影响

目的研究鸢尾素对载脂蛋白E基因敲除(ApoE~(-/-))小鼠动脉粥样硬化的影响。方法将ApoE~(-/-)小鼠随机分为正常对照组、动脉粥样硬化模型组、鸢尾素组,每组10只,分别给予普通饮食+生理盐水、高脂饮食+生理盐水、高脂饮食+鸢尾素,饲养至12周后处死,处死前测体重,用化学法测定总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDLC)、高密度脂蛋白胆固醇(HDLC)、血清超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量。采用HE染色和整体油红O染色分析主动脉粥样斑块面积大小,实时荧光定量(qRT-PCR)检测主动脉CD36、NF-κB p65的mRNA表达,Western blot检测主动脉CD36、NF-κB p65、SOD的蛋白表达。结果动脉粥样硬化模型组主动脉斑块面积明显高于正常对照组,内膜明显增厚,而鸢尾素组较动脉粥样硬化模型组斑块面积减少,内膜增厚程度下降。与正常对照组比较,动脉粥样硬化模型组小鼠体重、TC、TG、LDLC、MDA水平升高,NF-κB p65、CD36mRNA和蛋白表达增加,而血清SOD活性、HDLC水平减低(P0.05)。与动脉粥样硬化模型组比较,鸢尾素组小鼠体重、TC、TG、LDLC、MDA水平降低,NF-κB p65、CD36 mRNA和蛋白表达下调,而血清SOD活性、HDLC水平升高(P0.05)。结论鸢尾素能减轻ApoE~(-/-)小鼠动脉粥样硬化的发生发展,这可能与其降低小鼠体重和血脂、抑制炎症反应、抗氧化应激有关。     

益生菌治疗的实验性结肠炎中调节性T细胞的作用

目的评价不同剂量益生菌对大鼠实验性结肠炎的疗效,研究治疗后全身及肠道局部调节性T细胞(Tr)的变化情况,探讨益生菌作用机制。方法建立三硝基苯磺酸(TNBS)实验性结肠炎大鼠模型。所用益生菌为双歧三联活菌(商品名:培菲康)。设阴性对照组、泼尼松组、柳氮磺胺吡啶 (SASP)组、益生菌小、大剂量组(剂量分别为150和300 mg·kg-1·d-1)、益生菌+泼尼松或SASP(益生菌剂量为150 mg·kg-1·d-1)组。治疗2周后组织学积分评定疗效;流式细胞仪检测各组外周血、脾脏和结肠上皮内CD4+CD25+及CD8+CD28-两种Tr比例变化。采用t检验行统计学分析。结果组织学评分显示大剂量益生菌对实验性结肠炎有效(2．2±0．8比3．5±0．7,P<0．05),而小剂量益生菌单独作用无明显疗效,联合泼尼松或SASP治疗比单独应用疗效更强;大剂量益生菌治疗后CD4+ CD25+Tr比例在外周血(3．4±0．6比11．7±4．7,P<0．05)及脾脏(2．1±1.9比10．3±3．1,P<0．05) 中下降,在结肠内上升(36．6±15．0比7．9±4．7,P<0．05);CD8+CD28-Tr则相反,在外周血及脾脏中上升(91．7±4．5比59．0±4．2,97．3±0．1比88．2±6．9,P<0．05),在结肠内下降(42．2±6．0比68．5 ±8．6,P<0．05)。Tr的这种变化与泼尼松或SASP治疗存在一定差异。结论益生菌单独大剂量或小剂量联合泼尼松或SASP治疗TNBS诱导的结肠炎有效,CD4+CD25+和CD8+CD28-Tr在发挥疗效过程中可能起一定作用。     

Beneficial effect of butyrate,Lactobacillus casei and L-carnitine combination in preference to each in experimental colitis

AIM: To investigate the beneficial effect of the combination of butyrate, Lactobacillus casei, and L-carnitine in a rat colitis model.METHODS: Rats were divided into seven groups. Four groups received oral butyrate, L-carnitine, Lactobacillus casei and the combination of three agents for 10 consecutive days. The remaining groups included negative and positive controls and a sham group. Macroscopic, histopathological examinations, and biomarkers such as tumor necrosis factor-alpha (TNF-α) and interlukin-1β (IL-1β), myeloperoxidase (MPO), thiobarbituric acid reactive substances (TBARS), and ferric reduced ability of plasma (FRAP) were determined in the colon.RESULTS: The combination therapy exhibited a significant beneficial effect in alleviation of colitis compared to controls. Overall changes in reduction of TNF-α (114.66 ± 18.26 vs 171.78 ± 9.48 pg/mg protein, P < 0.05), IL-1β (24.9 ± 1.07 vs 33.06 ± 2.16 pg/mg protein, P < 0.05), TBARS (0.2 ± 0.03 vs 0.49 ± 0.04 μg/mg protein, P < 0.01), MPO (15.32 ± 0.4 vs 27.24 ± 3.84 U/mg protein, P < 0.05), and elevation of FRAP (23.46 ± 1.2 vs 15.02 ± 2.37 μmol/L, P < 0.05) support the preference of the combination therapy in comparison to controls. Although the monotherapies were also effective in improvement of colitis markers, the combination therapy was much better in improvement of colon oxidative stress markers including FRAP, TBARS, and MPO.CONCLUSION: The present combination is a suitable mixture in control of experimental colitis and should be trialed in the clinical setting.