独一味环烯醚萜苷对大鼠炎症性肠病的治疗作用及其机制研究

发布时间：2018-06-05 18:58

本文选题：炎症性肠病 + 独一味　；参考：《重庆医科大学》2015年硕士论文

【摘要】：实验目的：研究独一味环烯醚萜苷(IGLR)对大鼠急性IBD模型的治疗作用；为将IGLR开发成为治疗IBD的有效新药提供实验依据和理论基础。实验方法：1分组：将90只Wistar大鼠随机分成9组：正常对照组(C)、TNBS模型对照组(M1)、TNBS模型的IGLR高、中、低(300mg/kg、 150mg/kg、75mg/kg)三种剂量治疗组(T1h、T1m、T1l)、柳氮磺吡啶(SASP)阳性对照组(S1), DSS模型对照组(M2)、DSS模型的IGLR 300mg/kg治疗组(T2)、SASP阳性对照组(S2)。2大鼠IBD模型制作：用TNBS-无水乙醇(1：1)溶液一次性灌肠诱导大鼠TNBS性IBD模型；给予6% DSS溶液自由饮用8天诱导大鼠DSS性IBD模型；C组以等量蒸馏水灌肠或饮用。3给药和结果观察：造模完成后次日开始灌胃给药,各对照组给等容积蒸馏水,期间观察大鼠活动、大便性状及隐血检测；连续处理7天后处死大鼠,取结肠组织标本,观察记录各组大鼠结肠病变；HE染色光镜下观察各组组织学改变并进行组织学评分；取结肠标本做分子生物学机制研究。4分子生物学相关指标检测：采用IHC法检测结肠组织MPO及EPO的表达；采用ELISA法检测血清标本中INF-γ、IL-4、IL-10及IL-17的表达；采用Western Blot法及RT-PCR法检测结肠组织中GATA-3、T-bet、Foxp3、ROR-yt蛋白及其mRNA的表达。实验结果：1一般状况：M1,T11组大鼠实验期间期间毛发竖立,进食、饮水及活动显著减少,体重明显下降,腹胀,无排便,DAI呈较恒定高水平,与M1相比,S1、T1m及T1h大鼠在给药第2-4天起开始排便,体重回升,一般状况明显好转,给药两天后起,DAI呈明显下降趋势；M2组大鼠在实验期间一般状况差,出现大量稀便及脓血便,体重明显降低,DAI呈持续高水平,无明显下降,与M2相比S2、T2组于治疗开始后2-3天基本无肉眼血便,6-7天时大便呈梭形固体颗粒,隐血(-),体重逐渐恢复,但未达C组水平,给药后3天起DAI下降趋势明显。2结肠组织大体形态及组织学改变：结肠组织肉眼观察表明,M1组存在大片肠壁溃疡,部分节段肠壁僵硬,完全失去活性,组织学评分与C组差异显著(p0.05),T11组有一定缓解,但不明显,S1、T1h及Tim溃疡面明显缩小,仍存在部分小片及点状溃疡,评分与M1存在显著差异(p0.05)；M2组大鼠结肠壁溃疡、水肿、充血严重,有明显出血灶及血丝,评分与C组相较差异显著(p0.05),S2及T2组结肠壁溃疡、水肿、充血、出血明显减少,存在少量出血点,与M2相比评分下降具有统计学意义(p0.05)。HE染色后光镜下可见M1组肠壁组织破坏深达肌层,大量组织坏死及炎症细胞浸润；与M1组相较,S1、Ti1、T1h及Tim组有不同程度缓解,呈剂量依赖性；M2组可见肠黏膜细胞肿胀坏死,但损伤未达肌层,S2及T2可见炎症修复和及再生的腺体及杯状细胞。与C组相比,M1、M2组织学评分显著升高(p0.05),而各治疗组均呈显著下降(p0.05)。3 HIC检测MPO及EPO：与C组对照,M1及M2组大鼠结肠溃疡组织处MPO及EPO均表达增强,呈棕黄色深染,其主要分布于胞浆内,炎症致细胞破坏后,MPO及EPO大量溢出,检测评分结果升高显著(p0.05)。经IGLR和SASP处理7天后,与M1或M2组相比,各给药组大鼠结肠病变区MPO及EPO均表达均呈现不同程度的降低(p0.05)。4 ELISA检测血清蛋白：检测结果显示,与C组相比,INF-γ及IL-17在M1、M2组中均有显著升高,IL-10则显著下降,IL-4仅在M2中出现明显下降(p0.05)；治疗后,各治疗组INF-γ均出现显著下降(p0.05), IL-10均出现显著上升(p0.05), IL-17在S1、T1m、T1h、S2及T2组中出现明显下降(p0.05),在TNBS诱导的炎症性肠病模型中IL-4无显著变化,而在DSS模型的治疗组S2、T2中有明显回升(p0.05)。5结肠溃疡组织蛋白质及mRNA表达：检测结果提示,与C组对照,Ml及M2组在溃疡组织处T-bet和ROR-yt的蛋白及mRNA表达均显著提升(p0.05),经过治疗,T2、S2、Tim及T1h均出现显著下降(p0.05); GATA-3蛋白及mRNA在M1组中表达量改变不显著,而在M2组中明显上升(p0.05),治疗后T2、S2及T1h出现表达量下降,其该变量具有统计学意义(p0.05); Foxp3的表达在M1及M2组中均显著降低(p0.05),而与之相比,所有治疗组表达均出现明显升高(p0.05)。实验结论：1 TNBS诱导的大鼠IBD模型主要表现为腹胀、便秘及体重急剧下降,严重的结肠炎性反应,大片结肠壁溃疡,部分节段肠壁僵硬,溃疡深达肌层,其免疫病理学改变以Thl及Th17细胞因子作用为主导；DSS诱导的大鼠IBD模型以腹泻、脓血便及体重急剧下降为主要表现,结肠炎性反应严重,溃疡主要损伤结肠黏膜层,以水肿、出血为主,镜下可见腺窝缺损和杯状细胞破坏,其免疫病理学改变可能包括Th1、Th2及Th17细胞因子等的共同作用。2 IGLR对TNBS及DSS诱导的两类大鼠IBD模型均有显著疗效,可有效抑制IBD的炎症反应和结肠损伤。3 IGLR对大鼠1BD治疗作用的机制可能是通过调节Th1、Th2及Th17细胞因子及其转录因子T-bet、GATA-3及RORyt的表达,抑制其炎症反应及结肠损伤而产生。4 IGLR还通过促进Treg细胞因子及其转录因子Foxp3的表达,产生其抗炎及治疗IBD的作用。
[Abstract]:Objective: To study the therapeutic effect of cyclo iridoid glycoside (IGLR) on acute IBD model in rats; to provide experimental basis and theoretical basis for developing IGLR as an effective new drug for the treatment of IBD. Experimental methods: 1 groups: 90 Wistar rats were randomly divided into 9 groups: normal control group (C), TNBS model control group (M1), TNBS model IGLR Three doses of 300mg/kg, 150mg/kg, 75mg/kg, T1h, T1m, T1l, SASP positive control group (S1), DSS model control group (M2), DSS model IGLR treatment group. The model of DSS induced rat IBD was induced free drinking of 6% DSS solution for 8 days. Group C was treated with equal amount of distilled water enema or drinking.3, and the results were observed: after the model was completed the next day, the rats were given the medicine, and the control groups were given equal volume distilled water. During the period, the rats' activity, stool character and occult blood test were observed, and the rats were killed after 7 days of treatment. Colonic tissue specimens were taken to observe the colonic lesions of the rats in each group. The histological changes were observed and the histological scores were observed under the HE staining light microscope. The molecular biological mechanism of the colon specimens was examined for the detection of the.4 molecular biology related indexes: the expression of MPO and EPO in the colon tissue was detected by the IHC method; the serum specimens were detected by ELISA method. The expression of INF- gamma, IL-4, IL-10 and IL-17, Western Blot and RT-PCR methods were used to detect the expression of GATA-3, T-bet, Foxp3, ROR-yt protein and mRNA in colon tissue. Experimental results: 1 general conditions: during the period of experiment, the hair was erected, eating, drinking and activity decreased significantly, weight decreased obviously, abdominal distention and defecation were presented. Compared with the constant high level, compared with M1, S1, T1m and T1h rats began to defecate on the 2-4 day of administration, the body weight recovered and the general condition obviously improved. After two days of administration, DAI showed a significant decline trend. In the M2 group, the general condition of the rats was poor during the experiment, a large number of dilute stool and pus and blood were found, the body weight was significantly reduced, the DAI showed a sustained high level, and no significant decline. Compared with M2, S2, group T2 had no naked eye blood stool at 2-3 days after treatment, and the stool was spindle solid particles, occult blood (-) and body weight gradually recovered on the 6-7 day, but not in group C. The trend of DAI descending after 3 days was obviously.2 colonic tissue general morphology and histological changes: intestinal tissue naked eye observation showed that there were large bowel wall ulcers in group M1. The intestinal wall of the segment was stiff and completely lost the activity. The histological score was significantly different from the C group (P0.05), and the T11 group had some relief, but the S1, T1h and Tim ulcer surfaces were obviously narrowed, there were still some small fragments and punctate ulcers, and there were significant differences between the scores and M1 (P0.05), and the colon wall ulcers, edema, severe congestion, obvious hemorrhagic foci and blood in the group M2 rats. The score was significantly different from that of the C group (P0.05). The colonic wall ulcers in S2 and T2 groups were sored, edema, hyperemia, bleeding obviously decreased, and there was a small number of bleeding points. Compared with M2, the score decreased significantly (P0.05).HE staining showed that the intestinal wall tissue of M1 group destroyed the deep myometrium, a large number of tissue necrosis and inflammatory cell infiltration, compared with the M1 group, S1, T. The I1, T1h and Tim groups were in a dose-dependent manner. In the M2 group, the intestinal mucosa cells were swollen and necrotic, but the damage did not reach the myometrium, and the S2 and T2 showed the glandular and goblet cells of the inflammatory repair and regeneration. Compared with the C group, the histological score of M1 and M2 increased significantly (P0.05). Compared with group C, the expression of MPO and EPO in the tissues of colon ulcers in M1 and M2 rats was enhanced and stained with brown yellow, which was mainly distributed in the cytoplasm. After the destruction of the cells, a large number of MPO and EPO spillovers, and the results of the detection score increased significantly (P0.05). After 7 days of IGLR and SASP treatment, the colon lesion areas of the rats were compared with M1 or M2 groups. All the expression showed different degrees of decrease (P0.05).4 ELISA detection of serum protein: the results showed that compared with the C group, INF- and IL-17 were significantly increased in M1, M2 group, IL-10 decreased significantly, IL-4 only decreased in M2 (P0.05). The increase (P0.05), IL-17 in the S1, T1m, T1h, S2 and T2 groups decreased significantly (P0.05), and there was no significant change in the TNBS induced inflammatory bowel disease model. The protein and mRNA expression of ET and ROR-yt increased significantly (P0.05). After treatment, T2, S2, Tim and T1h decreased significantly (P0.05), and the expression of GATA-3 protein and mRNA in the M1 group was not significant. The expression in M1 and M2 groups was significantly decreased (P0.05), and the expression of all the treatment groups increased significantly (P0.05). Experimental conclusion: 1 TNBS induced rat IBD models were mainly abdominal distention, constipation and weight decline, severe colitis, large colonic wall ulcers, partial segmental stiffness of the bowel wall, ulcers reaching the myometrium, The immunhistopathological changes were dominated by the action of Thl and Th17 cytokine, and DSS induced rat IBD model was characterized by diarrhea, pus and blood stool and body weight drastic decline. The colitis was seriously damaged, the ulcers mainly damaged the colonic mucosa layer, with edema and hemorrhage, and the pathological changes of the gland fossa and goblet cells could be seen under the microscope. The interaction of Th1, Th2 and Th17 cytokines, such as.2 IGLR, has a significant effect on TNBS and DSS induced IBD models of two types of rats. It can effectively inhibit the inflammatory reaction of IBD and the mechanism of colon injury.3 IGLR on rat 1BD treatment. The expression of YT, the inhibition of its inflammatory response and colon injury, and the production of.4 IGLR by promoting the expression of Treg cytokine and its transcription factor Foxp3, and producing its anti-inflammatory and therapeutic effect on IBD.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R574.62

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