氯化钆抑制结肠粘膜炎症改善TNBS和DSS诱导的肠炎

发布时间：2018-05-17 13:20

本文选题：炎症性肠病 + 肠炎　；参考：《山东大学》2014年博士论文

【摘要】：背景炎症性肠病(inflammatory bowel disease, IBD)包括溃疡性结肠炎和克罗恩病,是一种累及回肠、直肠和结肠粘膜的慢性非特异性肠道炎症性疾病。IBD可发生于任何年龄,多见于20-49岁,亦可见于老年人或儿童。流行病学研究发现IBD发病率在世界范围内呈现逐渐增高的趋势。我国缺乏大规模流行病学调查,但亦有证据表明其呈逐年上升趋势。由于发病机制尚不明确,临床上常表现为反复发作而治愈难度大。已有的研究证实,遗传易感因素、肠道菌群、粘膜免疫异常以及环境因素共同参与了该病的发生。目前,IBD的主要治疗目的是抑制粘膜炎症,常用药物包括5-氨基水杨酸、糖皮质激素、抗生素和一些免疫抑制剂。尽管这些治疗方法在很多患者中均取得较好的治疗效果,仍有许多患者不能临床缓解,因此有待研究新的行之有效的治疗方法。结肠炎性巨噬细胞的浸润是IBD的一个重要特征,在IBD的发病中发挥着极为重要的作用。在IBD患者和实验性结肠炎模型中,外周血单核细胞可募集至结肠粘膜,分化成炎性巨噬细胞分泌包括TNFα、IL-1和IL-6在内的促炎性细胞因子。NF-κB信号通路的活化被认为是促炎性细胞因子表达和分泌的强效诱导者,且IBD患者结肠粘膜巨噬细胞中存在明显的NF-κB信号通路活化。此外,巨噬细胞促炎性细胞因子的分泌是IBD的重要发病因素之一。因此,靶向粘膜炎性巨噬细胞的治疗策略可能对研发新的治疗药物有重要的作用和价值。然而,由于缺乏有效的干预方法,针对于结肠粘膜巨噬细胞干预的研究极为有限。氯化钆(gadolinium chloride,GdCl3)已被广泛应用于实验性研究。在实验性动物中已经发现,氯化钆在肝脏中有清除巨噬细胞的作用,在肝损伤等疾病中有预防或治疗的作用。此外,实验性动物中发现氯化钆对巨噬细胞有选择性抑制作用。已经证实,氯化钆对大鼠肺组织巨噬细胞没有清除作用,但可以抑制LPS刺激后TNFα和IL-6的表达。然而,氯化钆对结肠粘膜巨噬细胞的作用知之甚少。目的 1.研究应用氯化钆后肠炎小鼠结肠粘膜炎症程度和疾病严重程度。 2.探讨氯化钆抑制肠炎小鼠粘膜炎症的机制。 3.应用RAW264.7细胞体外验证氯化钆对巨噬细胞活性的抑制作用。方法 1.动物处理体重20-25g,周龄8-10周的雄性C57BL/6J小鼠被用于该研究。静脉给予不同浓度的氯化钆,对照组给予PBS。流式细胞术检测小鼠结肠粘膜巨噬细胞的比例。 2.肠炎的诱导 (1)TNBS灌肠造肠炎模型将2mg的TNBS溶解于50%的乙醇溶液中,终体积为O.1ml。用注射器(0.1m1)吸取配置好的TNBS溶液,随后与一聚乙烯塑料管(内径为1.2mm)相连,用石蜡油充分润滑导管末端后灌肠,导管末端距小鼠的肛门约2-3cm,将注射器内的TNBS缓慢推入结肠。对照组小鼠给予同等剂量的生理盐水。 TNBS灌肠后第3天至第7天,用注射器(0.1m1)吸取配置好的氯化钆(10mg/kg体重)溶液灌肠。对照组给予等量的PBS灌肠。在TNBS灌肠后第7天或第14天,处死小鼠。 TNBS灌肠后第3天至第7天,用注射器(O.1m1)吸取配置好的氯化钆(10mg/kg体重)溶液,行尾静脉注射。对照组给予等量的PBS灌肠。在TNBS灌肠后第7天或第14天,处死小鼠。 (2)DSS诱导肠炎将DSS (40,000MW)溶于饮用水中,使其终浓度为3%(w/v),给予小鼠连续饮用7天,对照组给予正常的饮用水饮用。饮用DSS第3天,用注射器(O.1m1)吸取配置好的氯化钆(10mg/kg体重)溶液,随后与一聚乙烯塑料管(内径为1.2mmm)相连,用石蜡油充分润滑导管末端灌肠,导管末端距小鼠的肛门约2-3cm,将注射器内的氯化钆缓慢推入结肠。对照组给予等量的PBS灌肠。在DSS诱导肠炎后第7天或第14天,处死小鼠。饮用DSS诱导肠炎第3天,用注射器(O.1m1)吸取配置好的氯化钆(10mg/kg体重)溶液,行尾静脉注射。对照组给予等量的PBS灌肠。在DSS诱导肠炎后第7天或第14天,处死小鼠。 3.肠炎严重程度评估每日监测小鼠体重,肠炎程度通过疾病活动指数(DAI)进行评估。疾病活动指数评分主要评估体重减轻程度、血便情况和粪便硬度。对每组的每只老鼠进行评估,每一项的评分为0-4分,共计12分。小鼠与诱导炎症后第7天或第14天处死,取结肠标本甲醛固定,对实验设计和操作全盲的医师进行组织学炎症程度评分。 4.流式检测结肠粘膜巨噬细胞比例提取结肠粘膜细胞,孵育F4/80抗体,流式检测结肠粘膜巨噬细胞比例。 5.血清和结肠粘膜细胞因子水平检测收集小鼠外周血血清,提取结肠粘膜蛋白,ELISA检测TNFα、IL-1β和IL-6水平。 6.细胞处理 RAW264.7培养于DMEM+10%FBS的培养基中,将RAW264.7种在96孔板中,培养12h后,给予不同浓度的氯化钆分别处理不同时间点。 7.氯化钆处理后细胞活性和凋亡的检测上述RAW264.7细胞经氯化钆处理后,加入MTT检测细胞活性。此外,RAW264.7经氯化钆处理24h后,流式检测其凋亡水平。 8.LPS刺激细胞检测细胞因子分泌将RAW264.7种在24孔板中,LPS刺激后,ELISA检测TNFα、IL-1β口IL-6的分泌。 9. Western blot检测提取小鼠结肠粘膜蛋白和LPS处理的RAW2647细胞蛋白,用BCA法测定浓度,通过SDS-PAGE进行蛋白分离,然后转移到PVDF膜上。脱脂奶粉封闭后依次行一抗(NF-κB p65抗体)和辣根过氧化物酶标记二抗孵育。ECL化学发光法检测条带。结果 1.氯化钆对小鼠结肠粘膜巨噬细胞没有清除作用实验结果表明,静脉应用氯化钆后,小鼠结肠粘膜巨噬细胞比例未见明显改变。此外,血清和结肠粘膜TNFα、IL-1β和IL-6的表达水平未见升高,表明氯化钆无致炎作用。 2.氯化钆可缓解TNBS和DSS诱导的肠炎为探究氯化钆在小鼠肠炎模型中的潜在作用,我们应用TNBS和DSS诱导的两种小鼠肠炎模型进行研究。在TNBS诱导的小鼠肠炎模型中,在TNBS灌肠后第3天,给予小鼠尾静脉注射氯化钆。研究结果显示,氯化钆尾静脉注射后肠炎小鼠的死亡率、疾病严重程度、结肠缩短程度和组织学损伤程度均明显改善。TNBS灌肠诱导小鼠肠炎模型后第3天起予氯化钆灌肠至第7天。研究结果显示,氯化钆灌肠后肠炎小鼠的死亡率、疾病严重程度、结肠缩短程度和组织学损伤程度亦均明显改善。而氯化钆尾静脉注射组和氯化钆灌肠组小鼠之间无显著的统计学差异。将DSS (40,000MW)溶于饮用水中,使其终浓度为3%(w/v),给予小鼠连续饮用7天,对照组给予正常的饮用水饮用。在DSS诱导肠炎后第3天,给予小鼠尾静脉注射氯化钆。研究结果显示,氯化钆尾静脉注射亦可显著缓解DSS小鼠肠炎严重程度。DSS诱导小鼠肠炎模型后第3天起予氯化钆灌肠至第7天。研究结果显示,氯化钆灌肠后肠炎小鼠的死亡率、疾病严重程度、结肠缩短程度和组织学损伤程度亦均明显改善。而氯化钆尾静脉注射组和氯化钆灌肠组小鼠之间无显著的统计学差异。 3.氯化钆抑制TNBS和DSS诱导的TNFα、IL-1β和IL-6表达 TNBS和DSS诱导肠炎后,静脉或经肛给予氯化钆。我们发现,结肠粘膜巨噬细胞比例未见明显改变,但ELISA结果表明肠炎小鼠血清和结肠黏膜TNFα、IL-1p和IL-6的表达水平降低。 4.氯化钆降低LPS活化的RAW264.7细胞的促炎性细胞因子分泌氯化钆对RAW264.7细胞无毒性且不诱导其凋亡。给予LPS活化的RAW264.7细胞氯化钆,ELISA结果发现TNFα、IL-1β和IL-6的表达水平均明显降低。 5.氯化钆抑制肠炎小鼠结肠粘膜和LPS活化RAW2G47细胞的NF-κB活性给予肠炎小鼠氯化钆,结肠粘膜NF-κB p65的表达明显降低。此外,体外研究发现氯化钆亦能降低LPS活化的RAW264.7细胞的NF-κB p65的表达。结论本研究应用TNBS和DSS构建了两种IBD小鼠肠炎模型,首次发现氯化钆在TNBS和DSS诱导的肠炎模型中可缓解小鼠的死亡率、疾病严重程度、结肠缩短程度和组织学损伤程度,具有显著的保护作用。此外,通过相关机制的深入研究,发现氯化钆主要是通过下调NF-KB信号通路的活性抑制巨噬细胞促炎性细胞因子TNFα、IL-1β和IL-6的分泌从而抑制结肠粘膜炎症以缓解小鼠肠炎的严重程度。因此,干扰粘膜炎性巨噬细胞可能是IBD治疗的新的有前景的靶点。
[Abstract]:background
Inflammatory bowel disease (IBD), which includes ulcerative colitis and Crohn's disease, is a chronic nonspecific intestinal inflammatory disease involving the ileum, rectum and colonic mucosa,.IBD can occur at any age, more than 20-49 years old, and also in the elderly or children. Epidemiological studies have found that the incidence of IBD is in the world. There is no large-scale epidemiological survey in our country. There is a lack of large-scale epidemiological investigation in China, but there is also evidence that it is increasing year by year. Because the pathogenesis is not clear, the clinical manifestations are often repeated and it is difficult to cure. The existing research has confirmed that genetic susceptibility, intestinal flora, mucosal immune abnormalities and environmental factors are common. The main therapeutic purpose of IBD is to inhibit mucous membrane inflammation. The commonly used drugs include 5- amino salicylic acid, glucocorticoid, antibiotics and some immunosuppressants. Although these treatments have achieved good therapeutic effects in many patients, many patients are still unable to be remission, so it needs to be studied. Effective treatment.
The infiltration of colitis macrophages is an important feature of IBD, which plays an important role in the pathogenesis of IBD. In IBD patients and experimental colitis models, peripheral blood mononuclear cells can raise the colonic mucosa and differentiate into inflammatory macrophages, including TNF alpha, IL-1, and IL-6, the.NF- kappa B signal, including TNF alpha, IL-1 and IL-6 Activation of the pathway is considered to be a strong inducer of proinflammatory cytokines expression and secretion, and there is an obvious activation of NF- kappa B signaling pathway in the macrophages of IBD patients. In addition, the secretion of macrophage proinflammatory cytokines is one of the important factors of IBD. Therefore, the therapeutic strategy for targeting mucositis macrophages can be used. It has an important role and value for the development of new therapeutic drugs. However, due to the lack of effective intervention, the study of macrophage intervention in colonic mucosa is very limited.
Gadolinium chloride (GdCl3) has been widely used in experimental research. In experimental animals, gadolinium chloride has the role of removing macrophages in the liver and has the effect of prevention or treatment in liver injury. In addition, the experimental animals have found that gadolinium chloride has selective inhibition on macrophages. It is proved that gadolinium chloride has no scavenging effect on macrophages in rat lung tissue, but can inhibit the expression of TNF alpha and IL-6 after LPS stimulation. However, little is known about the effect of gadolinium chloride on macrophages in colonic mucosa.
objective
1. to study the severity and severity of colitis in mice with gadolinium chloride.
2. to explore the mechanism of gadolinium chloride inhibiting the mucositis of enteritis mice.
3. in vitro, RAW264.7 cells were used to verify the inhibitory effect of gadolinium chloride on macrophage activity.
Method
1. animal treatment
Body weight 20-25g, male C57BL/6J mice of 8-10 weeks of age were used in this study. Intravenous gadolinium chloride was given to different concentrations. The proportion of macrophages in colonic mucosa of mice was detected by PBS. flow cytometry in the control group.
Induction of 2. enteritis
(1) TNBS enterocolitis model
The TNBS of 2mg was dissolved in 50% ethanol solution. The final volume was O.1ml. using a syringe (0.1m1) to absorb the configured TNBS solution. Then it was connected to a polyethylene plastic tube (the inner diameter of 1.2mm), and the end of the catheter was fully lubricated with paraffin oil. The end of the catheter was about 2-3cm from the anus of the mouse. The TNBS in the syringe was slowly pushed into the colon. The mice were given the same dose of saline.
TNBS enema was used from third to seventh days after enema. The collocated gadolinium chloride (10mg/kg body) solution enema was absorbed with a syringe (0.1m1). The control group was given the same amount of PBS enema. The mice were killed seventh days or fourteenth days after TNBS enema.
TNBS from third to seventh days after enema was used to absorb the collocated gadolinium chloride (10mg/kg body) solution with a syringe (O.1m1). The caudal vein was injected. The control group was given the same amount of PBS enema. The mice were killed seventh days or fourteenth days after TNBS enema.
(2) DSS induced enteritis
DSS (40000MW) was dissolved in drinking water and the final concentration was 3% (w/v). Mice were given 7 days of continuous drinking, and the control group was given normal drinking water.
After drinking DSS for third days, using a syringe (O.1m1) to absorb the configured gadolinium chloride (10mg/kg body) solution, then connected to a polyethylene plastic tube (the inner diameter of 1.2mmm), the end of the catheter was fully lubricated with paraffin oil, the end of the catheter was about 2-3cm from the anus of the mouse, and the gadolinium chloride in the syringe was slowly pushed into the colon. The control group was given the equal amount of PBS irrigation. The mice were killed on the seventh or fourteenth day after DSS induced enteritis.
Drinking DSS induced enteritis for third days, using a syringe (O.1m1) to absorb the collocated gadolinium chloride (10mg/kg body) solution, injecting the tail vein. The control group was given the same amount of PBS enema. The mice were killed at seventh days or fourteenth days after DSS induced enteritis.
3. evaluation of severity of enteritis
The weight of the mice was monitored daily. The degree of enteritis was assessed by the disease activity index (DAI). The disease activity index score was mainly assessed for weight loss, blood stool and fecal hardness. Each group was evaluated with a score of 0-4, a total of 12 points. The rats were killed seventh days or fourteenth days after the induced inflammation, and the colon was taken to take the colon. Specimens were fixed with formaldehyde, and the histological degree of inflammation was scored on the experimental design and the totally blind doctors.
4. flow cytometry to detect the proportion of macrophages in colonic mucosa
Colonic mucosal cells were extracted, F4/80 antibody was incubated, and the proportion of macrophages in the colonic mucosa was detected by flow cytometry.
Detection of cytokine levels in 5. serum and colonic mucosa
The peripheral blood serum of mice was collected, and the colonic mucosal protein was extracted. The levels of TNF, IL-1 and IL-6 were detected by ELISA.
6. cell processing
RAW264.7 was cultured in DMEM+10%FBS medium, and RAW264.7 was cultured in 96 well plates. After culture 12h, different concentrations of gadolinium chloride were treated at different time points.
Detection of cell activity and apoptosis after gadolinium chloride treatment with 7. gadolinium chloride
The RAW264.7 cells were treated with gadolinium chloride and MTT was added to detect the cell viability. In addition, RAW264.7 was treated with gadolinium chloride and treated with 24h, and the apoptosis level was detected by flow cytometry.
Detection of cytokine secretion by 8.LPS stimulator cells
After stimulation of RAW264.7 seeds on 24 Kong Banzhong and LPS, ELISA was used to detect the secretion of TNF alpha and IL-1 beta IL-6.
9. Western blot detection
The protein of mouse colon mucous membrane protein and the RAW2647 cell protein treated by LPS were extracted and the concentration was measured by BCA. The protein was separated by SDS-PAGE and then transferred to the PVDF membrane. After the skimmed milk powder was closed, the first anti (NF- kappa B p65 antibody) and the horseradish peroxidase labeling two anti incubation.ECL chemiluminescence method were used to detect the strip.
Result
1. gadolinium chloride has no scavenging effect on macrophages in mouse colonic mucosa.
The results showed that the proportion of macrophages in the colon mucosa of mice was not significantly changed after intravenous gadolinium chloride. The expression level of TNF alpha, IL-1 beta and IL-6 in the serum and colonic mucosa did not increase, indicating that gadolinium chloride has no inflammatory effect.
2. gadolinium chloride can relieve TNBS and DSS induced enteritis
In order to explore the potential role of gadolinium chloride in the mouse enteritis model, we studied two mice enteritis models induced by TNBS and DSS. In the TNBS induced mouse enteritis model, the gadolinium chloride was injected into the tail vein of the mice at the third day after TNBS enema. The severity of the disease, the degree of colonic shortening and the degree of histological damage significantly improved the gadolinium enema to seventh days after the.TNBS enema induced mouse enteritis model. The results of the study showed that the mortality, the severity of the disease, the degree of colonic contraction and the degree of histological damage were also significantly improved in the mice after gadolinium chloride enema. There was no significant difference between gadolinium tail vein injection group and gadolinium chloride enema group.
DSS (40000MW) was dissolved in drinking water, the final concentration was 3% (w/v), and the mice were given a continuous drinking water for 7 days. The control group was given normal drinking water. The gadolinium chloride was injected into the tail vein of the mice for third days after DSS induced enteritis. The results showed that the gadolinium chloride tail vein injection could also significantly relieve the severity of DSS mice's enteritis to be.DSS induced to be small. The results of the study showed that the mortality, the severity of the disease, the degree of colon shortening and the degree of histological damage were also significantly improved in mice after gadolinium chloride enema, and there was no significant difference between the gadolinium chloride tail vein group and the gadolinium chloride enema group in the third days after the rat enterocolitis model.
3. gadolinium chloride inhibits TNBS and DSS induced TNF, IL-1, and IL-6 expression.
After TNBS and DSS induced enteritis, gadolinium chloride was given to the veins or through the anus. We found that the proportion of macrophages in the colonic mucosa was not significantly changed, but the ELISA results showed that the levels of TNF alpha, IL-1p and IL-6 in the serum and colonic mucosa of the colitis mice were reduced.
4. gadolinium chloride reduces proinflammatory cytokine secretion in LPS activated RAW264.7 cells
Gadolinium chloride was nontoxic to RAW264.7 cells and did not induce apoptosis. Gadolinium chloride was given to RAW264.7 cells activated by LPS. ELISA results showed that the expression level of TNF alpha, IL-1 beta and IL-6 decreased significantly.
5. gadolinium chloride inhibits NF- kappa B activity in colonic mucosa and LPS activated RAW2G47 cells of enteritis mice
The expression of NF- kappa B p65 in colonic mucosa was significantly reduced in mice treated with colitis. In addition, the in vitro study showed that gadolinium chloride could also reduce the expression of NF- kappa B p65 in LPS activated RAW264.7 cells.
conclusion
In this study, two IBD mouse enteritis models were constructed with TNBS and DSS. It was found for the first time that gadolinium chloride could alleviate the mortality of mice, the severity of the disease, the degree of colon shortening and the degree of histological damage in the model of TNBS and DSS induced enteritis. The inhibition of colonic mucositis by inhibiting the secretion of macrophage proinflammatory cytokines, TNF a, IL-1 beta and IL-6, can inhibit the severity of colitis in mice by reducing the activity of NF-KB signaling pathway. Therefore, interfering with mucositis macrophages may be a new and promising target for the treatment of IBD.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R574.62

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