丹皮酚对溃疡性结肠炎的治疗效果及机制研究

发布时间：2018-06-04 23:48

本文选题：丹皮酚 + 溃疡性结肠炎　；参考：《郑州大学》2017年博士论文

【摘要】：目的:溃疡性结肠炎是一类临床常见疾病,目前缺乏较好的治疗方法,本研究在临床实践中发现了丹皮酚(Pae)注射液治疗溃疡性结肠炎的疗效显著。本文观察其临床效果,并对其作用机制进行研究,为溃疡性结肠炎的临床治疗提供可参考的新方向。方法:采用临床实验结合动物实验,通过丹皮酚注射液对溃疡性结肠炎患者的临床治疗,观察其临床治疗效果;通过动物实验,探究其可能存在的作用机理。全文实验共分为五部分:(1)丹皮酚对溃疡性结肠炎的临床疗效观察。选取2015年1月-2016年9月郑州大学第二附属医院消化内科收治的80例溃疡性结肠炎患者作为研究对象,分为对照组(40例)和治疗组(40例)。对照组采用常规药物(美沙拉嗪或泼尼松片)治疗;治疗组在常规药物治疗的基础上给予肌内注射丹皮酚注射液。治疗周期14天,观察指标包括血小板水平、血红蛋白水平、白蛋白水平、C反应蛋白水平、红细胞沉降速率、机体氧化应激水平、炎性因子含量变化情况及ICAM-1、MMP-9蛋白表达水平,评价疗效;(2)丹皮酚对葡聚糖硫酸钠(DSS)诱导溃疡性结肠炎大鼠模型的保护作用。利用DSS诱导制备溃疡性结肠炎大鼠模型,随机分为对照组、模型组、丹皮酚(低、高)剂量组及阳性对照组,观察大鼠一般状态、体重变化情况及疾病活动指数(DAI),ELISA法检测血清炎性因子水平,血浆MDA和SOD水平,RT-PCR法检测结肠组织炎性因子mRNA表达水平,Western Blot法检测结肠粘膜组织ICAM-1、MMP-9蛋白表达水平,并评价组织损伤程度;(3)丹皮酚对DSS诱导所致肠炎的抗炎效果及机制研究。利用DSS诱导制备溃疡性结肠炎大鼠模型,随机分为对照组、模型组、丹皮酚(低、中、高剂量)组及阳性对照组,ELISA法测定大鼠血清炎性因子水平,RT-PCR法测定大鼠血清炎性因子mRNA表达水平,Western Blot法测定NF-κB和MAPK信号通路蛋白表达水平;(4)丹皮酚对大鼠CD4+T细胞亚群及其相关细胞因子表达的影响及机制研究。利用DSS诱导制备溃疡性结肠炎大鼠模型,随机分为正常对照组、模型组、丹皮酚治疗组,流式细胞仪检测CD4+T细胞亚群Thl、Th2、Thl7和Treg细胞的比例,RT-PCR法检测CD4+T细胞各亚型转录因子的表达,抗体芯片检测CD4+T各细胞因子的表达,免疫组织化学染色法检测大鼠结肠粘膜组织Thl、Th2、Thl7和Treg细胞的表达,Western Blot法测定IL-23R/Jak2/Stat3信号通路蛋白表达水平;(5)丹皮酚对模型大鼠肠黏膜相关生长因子的影响。利用DSS诱导制备溃疡性结肠炎大鼠模型,随机分为对照组、模型组、丹皮酚(低、高剂量)组及阳性对照组,SABC免疫组化染色法观察大鼠肠黏膜平均血管密度,MASSON染色法观察肠黏膜血管微血栓形成,ELISA法检测血浆及结肠黏膜血管内皮生长因子(VEGF)、成纤维细胞生长因子(bFGF)、血小板衍生因子(PDGF)水平,放射免疫分析法测定血浆及结肠一氧化氮(NO)水平,Western Blot法测定血浆及结肠缺氧诱导因子(HIF-1)水平。结果:(1)丹皮酚可有效改善患者的临床症状,改善肠黏膜病变,消除并发症,降低血小板、C反应蛋白浓度,降低红细胞沉降速率,降低ICAM-1、MMP-9蛋白表达水平,提高血红蛋白、白蛋白水平,同时不会引起严重的不良反应;(2)丹皮酚注射液可有效抑制大鼠体重下降趋势,改善模型大鼠DAI指数,降低体内IL-1、IL-4、INF-γ、TNF-α及氧化应激水平,提高IL-10水平,同时对炎性因子mRNA表达具有调节作用,降低ICAM-1、MMP-9蛋白的表达,改善大鼠肠黏膜平均血管密度,抑制微血栓形成;(3)丹皮酚可以显著抑制DSS诱导的实验性肠炎大鼠促炎症因子IL-1β、IL-4、IL-6、IFN-γ、TNF-α和MCP-1及其mRNA的表达,免疫印迹结果显示,丹皮酚可抑制NF-κB通路p-IκB、p-p65、p-IKKα/β水平和MAPK通路中p-Erk1/2、p-JNK、p-p38水平,从而抑制NF-κB通路和MAPK通路活性;(4)丹皮酚可提高CD4+T细胞亚型Treg细胞的比例,下调Thl、Th2和Thl7细胞的比例,提高FoxP3的表达水平,下调GATA-3、RORc及T-bet表达水平,降低细胞因子IL-2、IL-4、IL-21和IL-17表达水平。抑制IL-23R和JAK-2蛋白的表达,并抑制stat3磷酸化,促进smad磷酸化,由此抑制IL-23R/Jak2/Stat3信号通路活性;(5)丹皮酚注射液可有效改善模型大鼠肠黏膜平均血管密度及肠黏膜血管微血栓形成个数,降低血浆及结肠组织VEGF、NO、bFGF及HIF-1水平。结论:(1)丹皮酚注射液对溃疡性结肠炎具有较好的治疗效果,治疗机制可能与减低体内氧化应激、炎性因子水平及血管细胞因子的表达有关;(2)丹皮酚对模型大鼠的保护作用可能是通过降低氧化应激水平及炎性因子mRNA的表达,消除炎症反应,抑制ICAM-1、MMP-9蛋白的表达,改善肠道及血管功能,从而起到对模型大鼠的保护作用;(3)丹皮酚可降低肠炎模型大鼠炎性水平,其作用机制可能与抑制NF-κB通路和MAPK通路活性有关;(4)丹皮酚通过抑制CD+T细胞各亚型失衡对慢性溃疡性结肠炎产生治疗效果,该治疗效果与丹皮酚可以抑制IL-23R/Jak2/Stat3信号通路活性有关;(5)丹皮酚对溃疡性结肠炎大鼠肠黏膜的保护作用机制可能为改变肠黏膜相关生长因子的水平,影响相关的代谢通路。
[Abstract]:Objective: ulcerative colitis is a kind of common clinical disease and lack of good treatment. This study found the curative effect of Pae injection in the treatment of ulcerative colitis in clinical practice. The clinical effect of this study was observed and the mechanism of action was studied to provide reference for the clinical treatment of ulcerative colitis. New directions. Methods: the clinical treatment of ulcerative colitis was treated by clinical experiment combined with animal experiment, and the clinical therapeutic effect of ulcerative colitis was observed by Paeonol Injection. The possible mechanism of action was explored through animal experiments. The full text was divided into five parts: (1) the clinical effect of Paeonol on ulcerative colitis. 80 cases of ulcerative colitis were treated in the digestive department of the Second Affiliated Hospital of Zhengzhou University, January 2015 -2016 in September. The patients were divided into control group (40 cases) and treatment group (40 cases). The control group was treated with conventional drugs (mesalazine or prednisone), and the treatment group was given intramuscular injection of Paeonol on the basis of conventional drug treatment. The treatment period was 14 days. The indexes included platelet level, hemoglobin level, albumin level, C reaction protein level, erythrocyte sedimentation rate, oxidative stress level, inflammatory factor content change and ICAM-1, MMP-9 protein expression level, evaluation of therapeutic effect, and (2) paeonol induced ulcerative junction with dextran sodium sulfate (DSS). The rat model of ulcerative colitis was induced by DSS. The rat model of ulcerative colitis was induced and divided into control group, model group, paeonol (low, high) dose group and positive control group. The general state, body weight change and disease activity index (DAI) were observed in rats. The level of serum inflammatory factors was detected by ELISA method, plasma MDA and SOD levels, RT-P CR assay was used to detect the expression level of inflammatory factor mRNA in colon tissue, Western Blot method was used to detect the expression of ICAM-1 and MMP-9 protein in colonic mucosa, and the degree of tissue damage was evaluated. (3) the anti-inflammatory effect and mechanism of Paeonol on DSS induced enteritis. The rat model of ulcers induced colitis was induced by DSS, and the model was randomly divided into the control group and the model was randomly divided into the control group. Group, paeonol (low, medium, high dose) group and positive control group, ELISA assay was used to determine the level of inflammatory factors in rat serum, RT-PCR assay was used to determine the expression level of inflammatory factor mRNA in rat serum. Western Blot method was used to determine the expression level of NF- kappa B and MAPK signaling pathway protein; (4) the effect of Paeonol on the expression of CD4+T lymphocyte subsets and related cytokines in rats The rat model of ulcerative colitis was prepared by DSS. The model was randomly divided into normal control group, model group, paeonol treatment group, flow cytometry to detect the proportion of Thl, Th2, Thl7 and Treg cells in CD4+T cell subsets. RT-PCR method was used to detect the expression of transcription factors of each subtype of CD4+T cells, and the antibody chip was used to detect the various cytokines of CD4+T. The expression of Thl, Th2, Thl7 and Treg cells in the colon mucosa of rats was detected by immunohistochemical staining. The expression level of IL-23R/Jak2/Stat3 signaling protein was measured by Western Blot method; (5) the effect of Paeonol on the intestinal mucosa related growth factors in the model rats. The rat model of ulcerative colitis was prepared by DSS inducement, and was randomly divided into control. Group, model group, paeonol (low, high dose) group and positive control group, SABC immunohistochemical staining method was used to observe the average vascular density of intestinal mucosa of rats, MASSON staining method was used to observe the formation of vascular microthrombus in intestinal mucosa, and ELISA method was used to detect vascular endothelial growth factor (VEGF), fibroblast growth factor (bFGF) and platelet derived factor (bFGF). PDGF) level, radioimmunoassay to determine plasma and colon nitric oxide (NO) levels, Western Blot method for the determination of plasma and colon hypoxia inducible factor (HIF-1). Results: (1) paeonol can effectively improve the clinical symptoms of patients, improve intestinal mucosal lesions, eliminate complications, reduce platelet, C reaction protein concentration, and decrease the rate of erythrocyte sedimentation Rate, decrease ICAM-1, MMP-9 protein expression level, improve hemoglobin and albumin level, and not cause serious adverse reactions. (2) Paeonol Injection can effectively inhibit the trend of weight loss in rats, improve the DAI index of model rats, decrease the level of IL-1, IL-4, INF- gamma, TNF- A and oxidative stress in the body, improve the level of IL-10, and also cause inflammatory causes. The expression of submRNA can reduce the expression of ICAM-1 and MMP-9 protein, improve the mean blood vessel density and inhibit the formation of microthrombus in the intestinal mucosa of rats. (3) paeonol can significantly inhibit the expression of IL-1 beta, IL-4, IL-6, IFN- gamma, TNF- A and MCP-1 and its mRNA in the DSS induced experimental enteritis rats, and the results of Western blot show that the Western blot results show paeonol. Inhibition of NF- kappa B pathway p-I kappa B, p-p65, p-IKK alpha / beta level and MAPK pathway p-Erk1/2, p-JNK, p-p38 levels, thus inhibiting the NF- B pathway and the activity of the pathway; (4) paeonol can improve the proportion of subtype cells. Low cytokine IL-2, IL-4, IL-21 and IL-17 expression levels, inhibit the expression of IL-23R and JAK-2 protein, inhibit STAT3 phosphorylation, promote Smad phosphorylation, and inhibit the activity of IL-23R/Jak2/Stat3 signaling pathway. (5) Paeonol Injection can effectively improve the average vascular density of intestinal mucosa and the number of intestinal mucosal microthrombus formation in the model rats, and reduce the number of intestinal mucosal microthrombus formation. Low plasma and colonic tissue VEGF, NO, bFGF and HIF-1 levels. Conclusions: (1) Paeonol Injection has a good therapeutic effect on ulcerative colitis. The mechanism of treatment may be related to reducing oxidative stress in the body, the level of inflammatory factors and the expression of vascular cell factors; (2) the protective effect of Dan paeonol on model rats may be by reducing oxidation. Stress level and inflammatory factor mRNA expression, eliminate inflammatory reaction, inhibit the expression of ICAM-1, MMP-9 protein, improve intestinal and vascular function, and thus protect the model rats; (3) paeonol can reduce the inflammatory level of rats with enteritis model, its mechanism can be related to the inhibition of NF- kappa B pathway and MAPK pathway activity; (4) paeonol The effect of inhibition of CD+T cell subtypes on chronic ulcerative colitis, the effect of the treatment and paeonol can inhibit the activity of IL-23R/Jak2/Stat3 signaling pathway; (5) the protective mechanism of Paeonol on the intestinal mucosa of ulcerative colitis rats may be related to the level of altered intestinal mucosa related growth factors. Metabolic pathways.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R574.62

【参考文献】