非甾体抗炎药相关小肠黏膜损伤发生机制及药物干预的实验研究

发布时间：2018-07-10 20:12

本文选题：共聚焦激光显微内镜 + 上皮细胞屏障　；参考：《山东大学》2014年博士论文

【摘要】：背景与目的目前非甾体抗炎药(Non-Steroid Anti-Inflammatory Drugs, NSAIDs)被广泛应用于临床,但在发挥治疗作用的同时,也可导致上消化道及肠道黏膜不同程度的损伤。60%～70%的NSAIDs相关肠病患者无特征性的症状,严重的并发症如消化道大出血、肠道狭窄、肠穿孔等少见,但一旦发生,却是致命性的。在NSAIDs相关胃黏膜损伤中,环氧合酶起着关键性作用。但研究认为环氧合酶的抑制在NSAIDs相关肠黏膜损伤过程中并不起主要作用。目前,NSAIDs目关肠黏膜损伤机制尚无定论,较为认可的是“三次打击假说”。假说提出肠黏膜上皮屏障的完整性在NSAIDs相关肠病中至关重要。肠上皮屏障由上皮细胞及细胞间紧密连接构成。上皮细胞再生的速度为脱落速度的一半,这就决定了细胞脱落后间隙的存在。脱落细胞间隙与肠黏膜屏障之间有密切的关系。已有研究证实脱落细胞间隙密度可预测炎症性肠病(Inflammatory bowel disease, IBD)的严重程度及其与复发之间的关系。本研究应用共聚焦激光显微内镜(Confocal laser endomicroscopy,CLE),观察吲哚美辛相关小肠上皮屏障损伤的脱落细胞间隙密度,并分析其发生机制,同时研究替普瑞酮和雷贝拉唑对上皮屏障的保护机制。材料及方法第一部分：NSAIDs相关小肠黏膜损伤及其药物干预的形态学研究雄性Wistar大鼠,按体重完全随机分为八组,每组6只,分别为健康对照组、模型组(采用吲哚美辛造模)、替普瑞酮预防组、雷贝拉唑预防组、治疗对照组、替普瑞酮治疗组、雷贝拉唑治疗组,以及替普瑞酮与雷贝拉唑联合治疗组(联合治疗组)。各组模型制备成功后水合氯醛腹腔注射麻醉成功后,沿十二指肠向下约10cm,取上段空肠,沿肠系膜对侧纵行剖开肠管,切口长约1-2cm,注意保护肠管的血流。暴露要观察的黏膜后心内注射荧光素钠注射液1min后用观察。将黏膜平铺开,将optiscan FIVE1探头垂直,轻柔放置于黏膜表面,观察时间控制在20min内。选择图像质量佳的5个图像,计数1000个上皮细胞中脱落细胞间隙数。观察结束后取小肠上段组织块长约1cm,冲洗掉表面血迹及粘液,立即10%中性甲醛固定12h后,沿肠系膜对侧剖开肠管,大头针固定于薄板上,尽量展平肠管,继续放入中性甲醛中固定12h。固定后的组织脱水后,蜡块包埋,切片厚度4um,苏木素-伊红(Hematoxylin and Eosin, HE)染色后观察。取空肠上段组织块,扫描电子显微镜(Scanning electron microscopy, SEM)下观察黏膜表面超微结构。所有数据应用SPSS13.0统计软件分析。数据以x±s表示,多样本间两两比较检验方差齐性,两两组间采用LSD-t检验,方差不齐时两两组间比较采用Hamhane's T2检验。P0.05为差异有统计学意义。第二部分：NSAIDs相关小肠黏膜损伤及其药物干预的分子机制研究雄性Wistar大鼠,按体重完全随机分为八组,每组6只,分别为健康对照组、模型组(采用吲哚美辛造模)、替普瑞酮预防组、雷贝拉唑预防组、治疗对照组、替普瑞酮治疗组、雷贝拉唑治疗组,以及替普瑞酮与雷贝拉唑联合治疗组(联合治疗组)。各组模型制备成功后水合氯醛腹腔注射麻醉成功后,心内穿刺抽取血,离心后取上清,按照酶联免疫吸附试验(Enzyme linked immunosorbent assay, ELISA)试剂盒指示测定血清肿瘤坏死因子-a (Tumor Necrosis Factor-α, TNF-α)浓度；沿十二指肠向下约10cm,取小肠上段组织,剥取黏膜层提取蛋白,用蛋白免疫印迹(Western Blotting, WB)方法测定组织中caspase-3及核因子-κB(nuclear factor-kappa B, NF-κB)表达及组织中闭合蛋白(occludin)、闭锁小带蛋白1(Zonula occludens protein1, ZO-1)的表达。应用SPSS13.0统计软件分析数据。数据以x±s表示,多样本间比较检验方差齐性,两两组间采用LSD-t检验,方差不齐时两两组间比较采用Hamhane'sT2检验。P0.05为差异有统计学意义。结果第一部分： 1.CLE脱落细胞间隙脱落细胞间隙呈强荧光,较吸收细胞略小,部分可见射流样征象。替普瑞酮预防组和雷贝拉唑预防组空肠组织的脱落细胞间隙密度分别为(57.43±24.55)/1000和(59.80±21.14)/1000,均低于模型组的(110.93±50.58)/1000,差异均有统计学意义(t=53.50、54.13,P均0.01)。联合治疗组的脱落细胞间隙密度为(40.53±15.39)／1000,低于治疗对照组的(93.80±40.65)/1000,差异有统计学意义(t=44.27,P0.01)。 2.HE病理学及SEM脱落细胞间隙组织固定后HE病理组织学观察,亦可见脱落或正在脱落的细胞,但脱落细胞间隙很少能观察到。扫描电镜下脱落细胞间隙清晰可见,呈空洞样,周围可见小肠上皮吸收细胞,与共聚焦激光显微内镜下表现类似。第二部分： 1.血清TNF-α浓度替普瑞酮预防组和雷贝拉唑预防组的TNF-α水平分别为(25.80±8.97)和(22.74±7.15) ng/L,均低于模型组的(44.48±7.42) ng/L,差异均有统计学意义(t=18.68、21.74,P均0.01)。联合治疗组TNF-α水平为(13.66±4.98)ng/L,低于治疗对照组的(24.67±6.70)ng/L,差异有统计学意义(t=9.02,P0.01)。 2. caspase-3及NF-κB含量替普瑞酮预防组和雷贝拉唑预防组的caspase-3水平分别为1.47±0.35和1.58±0.34,NF-κB水平分别为1.27±0.14和1.21±0.10,与模型组(2.44±0.45和1.69±0.13)相比,差异均有统计学意义(t=0.97、0.86、0.42、0.48,P均0.01)。联合治疗组的caspase-3和NF-κB水平分别为0.66±0.06和0.44±0.21,低于治疗对照组,差异均有统计学意义(t=0.34、0.56,P均0.01)。 3. occludin及ZO-1表达替普瑞酮预防组和雷贝拉唑预防组的occludin水平分别为0.69±0.16和0.74±0.11,ZO-1水平分别为0.81±0.08和0.84±0.12,与模型组(0.45±0.22和0.64±0.07)相比,差异均有统计学意义(t=0.24、0.29、0.17、0.21,P均0.01)。联合治疗组的occludin和ZO-1水平分别为2.50±0.46和1.76±0.18,高于治疗对照组,差异均有统计学意义(t=1.50、0.76,P均0.01)。结论 1.共聚焦激光显微内镜可以清晰地观察肠上皮脱落细胞间隙并进行密度测定,脱落细胞间隙密度可作为预测炎症反应程度及上皮屏障功能以及评价药物疗效的有价值指标。 2.TNF-α增加通过NF-κB-caspase-3途径导致紧密连接蛋白occludin和ZO-1表达的减少是导致吲哚美辛相关小肠黏膜损伤过程中细胞脱落加速的机制之一。 3.替普瑞酮与雷贝拉唑可以抑制TNF-α增加,通过NF-KB-caspase-3途径,改善细胞紧密连接蛋白的表达,改善肠上皮屏障的损伤,从而达到预防和治疗吲哚美辛相关小肠黏膜损伤的作用。研究意义本研究证实共聚焦激光显微内镜可以清晰得观察肠上皮脱落细胞间隙并进行脱落细胞间隙密度的测定,从而客观得评价吲哚美辛相关小肠黏膜的损伤,将来有望将其运用于临床,用于肠道炎症严重程度的评价,成为一种在体检测肠上皮细胞屏障完整性的新方法及评价药物疗效的新指标。雷贝拉唑和替普瑞酮可以抑制TNF-α增加,通过调节NF-κB-caspase-3途径,改善细胞紧密连接蛋白的表达,改善肠上皮屏障的损伤,从而达到预防和治疗吲哚美辛相关小肠黏膜损伤的作用。这为两种药物在NSAIDs所致小肠黏膜损伤治疗的临床应用提供动物实验佐证。
[Abstract]:Background and purpose
At present, Non-Steroid Anti-Inflammatory Drugs (NSAIDs) is widely used in clinical practice, but it can also lead to a different degree of damage to the upper digestive tract and intestinal mucosa in.60% to 70% of NSAIDs related bowel disease patients with no characteristic symptoms, serious complications such as massive hemorrhage of digestive tract, and intestinal stenosis. Intestinal perforation is rare, but once it occurs, it is fatal. Cyclooxygenase plays a key role in NSAIDs related gastric mucosal injury. However, the inhibition of cyclooxygenase is not a major role in NSAIDs related intestinal mucosal damage. The mechanism of NSAIDs mesenteric mucous membrane damage is not conclusive, and "three" The hypothesis suggests that the integrity of the intestinal mucosal epithelial barrier is essential in NSAIDs related bowel disease. The intestinal epithelial barrier is composed of close connections between epithelial cells and cells. The rate of epithelial cell regeneration is half of the rate of exfoliation, which determines the existence of the clearance of the cells after the exfoliation of the cells. There has been a close relationship. Studies have shown that exfoliative cell space density can predict the severity of Inflammatory bowel disease (IBD) and its relationship with recurrence. This study used confocal laser endoscopy (Confocal laser endomicroscopy, CLE) to observe the damage of indomethacin related intestinal epithelial barrier injury. The gap density was analyzed and the protective mechanism of tebuprazone and rabeprazole against epithelial barrier was investigated.
Materials and methods
Part one: morphological study of NSAIDs related intestinal mucosal injury and drug intervention
Male Wistar rats were divided into eight groups according to the total weight of body weight, with 6 rats in each group, including the healthy control group, the model group (using indomethacin model), the tipriprazole prevention group, the riprazole prevention group, the treatment control group, the tipriprone treatment group, the riprazol treatment group, and the combined treatment group of tepreone and riprazole and riprazole (combined treatment group). After successful intraperitoneal injection of chloral chloral injection after the successful preparation of the group model, the upper jejunum was taken along the duodenum, the upper jejunum was taken along the duodenum, the intestinal canal was opened along the mesenteric side, the incision was about 1-2cm, and the blood flow of the intestinal tube was protected. After the observation of the intracardiac injection of Fluorescein Sodium Injection 1min, the mucosa was observed. The mucous membrane was paved open and optiscan The FIVE1 probe is vertical and gently placed on the surface of the mucous membrane, and the observation time is controlled within the 20min. 5 images with good image quality are selected and the number of exfoliated cells in the 1000 epithelial cells is counted. After observation, the upper segment of the small intestine is about 1cm long, and the surface blood and mucus are flushed out. After the 10% neutral formaldehyde is fixed for 12h, the mesenteric side section is on the side of the mesentery. Open the intestine, the pin was fixed to the plate, and the intestine was flattened as far as possible. After the tissue dehydration was fixed in the neutral formaldehyde for 12h. fixation, the wax block was embedded, the thickness of the slice was 4um, and the hematoxylin eosin (Hematoxylin and Eosin, HE) was observed. The upper segment of the jejunum was taken and the scanning electron microscope (Scanning electron microscopy, SEM) was taken down. The ultrastructure of the mucosal surface was examined. All data were analyzed by SPSS13.0 software. The data were expressed in X + s. The 22 test variances were homogeneous in the diversity, and the 22 groups were tested by LSD-t test. The difference between the 22 groups using the Hamhane's T2 test.P0.05 was statistically significant.
The second part: NSAIDs related intestinal mucosal injury and the molecular mechanism of drug intervention.
Male Wistar rats were divided into eight groups according to the total weight of body weight, with 6 rats in each group, including the healthy control group, the model group (using indomethacin model), the tipriprazole prevention group, the riprazole prevention group, the treatment control group, the tipriprone treatment group, the riprazol treatment group, and the combined treatment group of tepreone and riprazole and riprazole (combined treatment group). After successful intraperitoneal injection of chloral chloral injection after the successful preparation of the group model, the blood was extracted from the heart, and then the supernatant was taken after centrifugation. The concentration of serum TNF -a (Tumor Necrosis Factor- alpha, TNF- alpha) was determined according to the enzyme linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA), and the concentration of the serum tumor necrosis factor -a (Tumor Necrosis Factor- a, TNF- alpha) was measured along the duodenum, and down about 10cm, The expression of Caspase-3 and nuclear factor kappa B (nuclear factor-kappa B, NF- kappa B) in tissue and the expression of closed protein (occludin) in tissue were measured by Western Blotting (WB). Analysis data. The data were expressed in X + s, and the variance was homogeneous. The 22 groups were tested by LSD-t test. When the variance was not homogeneous, the difference between the 22 groups using the Hamhane'sT2 test.P0.05 was statistically significant.
Result
Part one:
1.CLE abscission cell space
The exfoliated cell space showed strong fluorescence, slightly smaller than the absorbable cells, and some jet samples were visible. The interspace density of the exfoliated cells in the jejunum tissue of the preventative and riprazole prevention groups was (57.43 + 24.55) /1000 and (59.80 + 21.14) /1000 respectively, which were lower than that of the model group (110.93 + 50.58) /1000, and the difference was statistically significant (t=53.50,54.13 P 0.01). The interspace density of the exfoliated cells in the combined treatment group was (40.53 + 15.39) / 1000, which was lower than that of the treatment control group (93.80 + 40.65) /1000, and the difference was statistically significant (t=44.27, P0.01).
2.HE pathology and SEM exfoliative cell space
After tissue fixation, the HE histopathological observation was also observed, and the exfoliated or falling cells were also visible, but the exfoliated cell space was rarely observed. The exfoliated cell gap was clearly visible under scanning electron microscope, and the epithelial absorption cells were visible around the small intestinal epithelium, similar to the confocal laser microendoscopy.
The second part:
1. serum TNF- alpha concentration
The TNF- alpha levels of the preventonone prevention group and the riprazole prevention group were (25.80 + 8.97) and (22.74 + 7.15) ng/L respectively, which were lower than those of the model group (44.48 + 7.42) ng/L, and the difference was statistically significant (t=18.68,21.74, P 0.01). The level of TNF- alpha in the combined treatment group was (13.66 + 4.98) ng/L, which was lower than that of the treatment control group (24.67 + 6.70) ng/L. T=9.02, P0.01.
2. caspase-3 and NF- kappa B content
The caspase-3 levels of the preventonone prevention group and the riprazole prevention group were 1.47 + 0.35 and 1.58 + 0.34 respectively, and the levels of NF- kappa B were 1.27 + 0.14 and 1.21 + 0.10 respectively. Compared with the model group (2.44 + 0.45 and 1.69 + 0.13), the difference was statistically significant (t=0.97,0.86,0.42,0.48, P all 0.01). The level of Caspase-3 and NF- kappa B in the combined treatment group was 1.58, respectively. 6 + 0.06 and 0.44 + 0.21, lower than the treatment group, the difference was statistically significant (t=0.34,0.56, P all 0.01).
3. occludin and ZO-1 expression
The occludin levels of the preventonone prevention group and the riprazole prevention group were 0.69 + 0.16 and 0.74 + 0.11 respectively, and the levels of ZO-1 were 0.81 + 0.08 and 0.84 + 0.12 respectively. Compared with the model group (0.45 + 0.22 and 0.64 + 0.07), the differences were statistically significant (t=0.24,0.29,0.17,0.21, P were all 0.01). The occludin and ZO-1 levels in the combined treatment group were respectively 0.74 6 and 1.76 + 0.18 were higher than those in the treatment group, and the differences were statistically significant (t=1.50,0.76, P 0.01).
conclusion
1. confocal laser endoscopy can clearly observe the intercellular space of the intestinal epithelium and determine the density of the cells. The gap density of the exfoliated cells can be used as a valuable indicator to predict the degree of inflammation and the function of epithelial barrier and to evaluate the efficacy of the drug.
The increase of 2.TNF- alpha through the NF- kappa B-caspase-3 pathway leads to the decrease in the expression of closely connexin occludin and ZO-1, which is one of the mechanisms of cell exfoliation acceleration during indomethacin related intestinal mucosal injury.
3. tepreone and rabezole can inhibit the increase of TNF- alpha, improve the expression of close connexin by NF-KB-caspase-3 pathway, improve the damage of intestinal epithelial barrier, and thus prevent and treat indomethacin related intestinal mucosal injury.
research meaning
This study confirms that confocal laser endoscopy can clearly observe the interspace of exfoliative cells in the intestinal epithelium and determine the gap density of the exfoliated cells, thus objectively evaluating the damage of the indomethacin related small intestinal mucosa. It is expected to be used in the future for clinical evaluation of the severity of intestinal inflammation and become a kind of intestinal test. A new method for the integrity of the skin cell barrier and a new index for the evaluation of the efficacy of the drug. Rabeprazole and teprirone can inhibit the increase of TNF- alpha, improve the expression of close connexin by regulating the NF- kappa B-caspase-3 pathway, improve the damage of the intestinal epithelial barrier, and thus achieve the prevention and treatment of indomethacin related intestinal mucosal injury. This provides an animal experimental evidence for the clinical application of two drugs in the treatment of small intestinal mucosal injury caused by NSAIDs.
【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】：R96

【共引文献】