生姜泻心汤预防伊立替康所致迟发性腹泻的机理研究

发布时间：2018-05-08 01:13

本文选题：生姜泻心汤 + 伊立替康　；参考：《北京中医药大学》2017年博士论文

【摘要】：目的1.在伊立替康迟发性腹泻大鼠模型上,研究探讨中药生姜泻心汤对肠粘膜细胞凋亡、细胞增殖和干细胞标志物表达、肝脏UGT1A1、肠道β-葡萄糖醛酸酶活性的影响,揭示该方在预防伊立替康肠毒性的作用靶点和作用机制,为中药预防化疗性腹泻提供科学依据。2.为临床应用生姜泻心汤预防伊立替康肠毒性提供实验依据。方法中药的作用具有多靶点的特点,探索中药生姜泻心汤在不同靶点抑制化疗性肠毒性的作用,是阐明对肠毒性预防机理的关键。根据伊立替康的代谢及其肠毒性的发生机制,利用伊立替康腹泻大鼠模型进行体内试验,从调节肝脏UGT1A1酶、肠道β-葡萄糖醛酸酶以及促进肠粘膜自身修复机能三个环节进行研究。1.建立伊立替康腹泻模型大鼠;2.通过观察实验过程中各组大鼠体重、进食量的变化,验证生姜泻心汤对伊立替康腹泻模型大鼠一般状况的影响;并根据文献记载腹泻等级和评分标准,对各组大鼠在伊立替康注射后进行腹泻评估,验证生姜泻心汤对伊立替康腹泻模型大鼠腹泻症状的影响;3.通过HE染色技术,观察伊立替康对腹泻模型大鼠空肠、回肠、盲肠、结肠肠粘膜的损伤作用,并对比各组大鼠间不同肠段肠粘膜在镜下病理上的差异,验证生姜泻心汤对伊立替康腹泻模型大鼠肠粘膜损伤的影响;4.通过TUNEL染色和caspase-3酶活性检测技术,分别定性和定量的检测各组大鼠空肠肠粘膜细胞的凋亡状况,验证生姜泻心汤对伊立替康腹泻模型大鼠肠粘膜细胞凋亡的影响;5.通过免疫组化染色技术,采用增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)和细胞黏附分子(homing cell adhesion molecule,CD44)抗体分别染色标记空肠中增殖的肠粘膜细胞和肠隐窝处的肠干细胞,观察各组大鼠空肠粘膜上皮细胞和肠干细胞在镜下的染色情况,并通过Image-Pro Plus(IPP)软件测定所拍摄的图像中染色部分的整体光密度(Integrated option density,IOD)值,并计数每个视野下PCNA染色阳性细胞数目,定量地分析各组大鼠空肠粘膜上皮细胞和肠干细胞表达的差异性,验证生姜泻心汤对伊立替康腹泻模型大鼠肠粘膜细胞增殖的影响;6.通过实时定量PCR技术,检测各组大鼠空肠组织肠干细胞标志物Lgr5 mRNA表达情况,从mRNA水平验证生姜泻心汤对伊立替康腹泻模型大鼠肠干细胞表达的影响;7.通过酶活性检测技术,测定各组大鼠肠道粪便中β-葡萄糖醛酸酶活性,验证生姜泻心汤对伊立替康腹泻模型大鼠肠道内环境的影响;8.通过实时定量PCR技术,检测各组大鼠肝脏组织UGT1A1 mRNA表达情况,验证生姜泻心汤对伊立替康腹泻模型大鼠肝脏代谢酶的调节作用。结果1.通过对伊立替康腹泻模型大鼠一般情况、腹泻症状的观察,及对其肠粘膜病理、免疫组化的检测,均证实伊立替康腹泻模型大鼠造模成功;2.对各组大鼠一般情况的观察结果显示,从实验第7天(末次注射伊立替康后48h)起,中、高剂量中药组大鼠的累积体重增加值显著高于模型对照组(p0.05,p0.01),但不同剂量中药组间的差异不具有统计学意义,直到第9天,与低剂量中药组相比,高剂量中药组大鼠才显示出更高的累积体重增加值(p0.05)。从实验第4天起,与空白对照组相比,模型对照组和低、中、高剂量中药组大鼠进食量显著减少(p0.01),而各剂量中药组大鼠进食量恢复明显快于模型对照组;至实验第9天,模型对照组大鼠的进食量仍显著低于空白对照组(p0.01),各剂量中药组大鼠的进食量则显著高于模型对照组(p0.01),而与空白对照组无差异,但不同剂量中药组间的差异不具有统计学意义。对各组大鼠腹泻症状的观察结果显示,末次伊立替康注射24h后,模型对照组和各剂量中药组大鼠的腹泻评分均值开始升高,其中模型对照组大鼠腹泻评分升高幅度明显高于各剂量中药组,在末次用药60h和70h后,与模型对照组相比,中剂量和高剂量中药组大鼠的腹泻等级分布均具有明显差异(p0.05,p0.01)3.肠粘膜病理镜下观察结果显示,模型对照组大鼠肠粘膜可观察到伊立替康引起的损伤,包括:肠上皮大体结构不完整、上皮细胞排列紊乱、肠隐窝和肠绒毛减少、变短等,其中,以空肠、盲肠黏膜的损伤最为严重;而低、中、高剂量中药组大鼠肠黏膜结构更加完整,肠隐窝和肠绒毛得到了更好的保存。4.TUNEL染色和caspase-3酶活性检测结果显示,中药组镜下空肠黏膜阳性凋亡细胞明显少于模型对照组,且低、中、高剂量中药组肠道caspase-3酶活性(U)分别为0.66±0.10、0.48±0.11、0.68±0.19,均明显低于模型对照组(vs1.00±0.17,p0.01);5.PCNA和CD44染色标记免疫组化检测结果显示,高剂量中药组PCNA阳性细胞数明显高于模型对照组(531.20±101.64 vs 312.40±43.23,p0.01);高剂量中药组PCNA 的 IOD 值明显高于模型对照组(2494.93±615.00 vs 1026.26±339.00,p0.05);中、高剂量中药组CD44的IOD值分别为511.35±83.18、613.04±102.42,均明显高于模型对照组(vs 360.99± 156.87,p0.05,p0.01);6.实时定量PCR技术结果显示,与模型对照组相比,各剂量中药组大鼠肠道Lgr5 mRNA表达均有所增多,但只有高剂量中药组Lgr5 mRNA表达显著增多,具有统计学意义(1.75±0.53 vs 0.56±0.54,p0.01);7.β 葡萄糖醛酸酶活性检测结果显示,在伊立替康注射前,与空白对照组、模型对照组相比,低、中剂量中药组大鼠肠道粪便β-葡萄糖醛酸酶活性无明显差异;而高剂量中药组大鼠肠道粪便β-葡萄糖醛酸酶活性明显低于模型对照组(3.88±1.78 vs 5.92±1.47,p0.05);在注射伊立替康后,低、中、高剂量中药组β-葡萄糖醛酸酶活性分别为12.56±1.81、13.55±1.17、11.62±1.78,均明显低于模型对照组(vs 16.09± 1.40,p0.05,p0.01),而各剂量中药组间的差异并不显著;8.实时定量PCR技术结果显示,与空白对照组相比,模型对照组和低、中、高剂量中药组大鼠肝脏UGT1A1酶mRNA表达显著降低;而与模型对照组相比,各剂量中药组大鼠肝脏UGT1A1酶mRNA的表达均未表现出明显差异。结论1.生姜泻心汤可有效减轻伊立替康所引起的体重下降,促进注射伊立替康后大鼠的进食;2.生姜泻心汤可以降低伊立替康所引起的腹泻级别和评分;3.生姜泻心汤可以减轻伊立替康引起的肠粘膜病理性损伤;4.生姜泻心汤可减少伊立替康所导致的肠道细胞凋亡;5.生姜泻心汤可促进肠黏膜细胞的增殖;6.生姜泻心汤可促进肠干细胞的表达;7.生姜泻心汤可降低大鼠注射伊立替康后肠道粪便中β-葡萄糖醛酸酶活性;8.生姜泻心汤对肝脏UGT1A1酶mRNA的表达无明显影响。
[Abstract]:Objective 1. to investigate the effect of ginger Xiexin Decoction on intestinal mucosal cell apoptosis, cell proliferation and expression of stem cell markers, liver UGT1A1 and intestinal beta glucuronoate activity in the rat model of irinotecan delayed onset diarrhea, and to reveal the target and mechanism of this prescription in preventing the toxicity of irinotecan. Providing scientific basis for the treatment of diarrhoea,.2. provides experimental basis for the clinical application of ginger Xiexin Decoction in the prevention of irinotecan enterotoxicity. Methods the effect of Chinese medicine has the characteristics of multiple targets. The effect of ginger Xiexin Decoction on the inhibition of chemotherapeutic intestinal toxicity at different targets is explored, and the key to the mechanism of intestinal toxicity prevention is clarified. According to erinoteca's generation A rat model of irinotecan diarrhea rat model was carried out in vivo by using the three links of regulating the liver UGT1A1 enzyme, intestinal beta glucuronoenzyme and promoting the self repair function of the intestinal mucosa to establish the rat model of erineecan diarrhea model by.1., and 2. by observing the weight of each group of rats during the experiment. To verify the effect of ginger Xiexin Decoction on the general condition of the rat model of irinotecan diarrhea model, and to evaluate the diarrhoea in the rats after injection of irinotecan according to the rank of diarrhoea and scoring in the literature, and to verify the effect of ginger Xiexin Decoction on diarrhea in the rat model of irinotecan diarrhea model; 3. The effects of irinotecan on jejunum, ileum, cecum and colon mucosa of rats with diarrhea model, and the pathological changes of intestinal mucosa in different intestinal segments in each group were compared, and the effect of ginger Xiexin Decoction on intestinal mucosal injury in the rat model of irinotecan diarrhea model was verified. 4. by TUNEL staining and caspase-3 enzyme activity detection techniques, respectively The effect of ginger Xiexin Decoction on the apoptosis of intestinal mucosa in the rat model of irinotecan diarrhea model was tested by qualitative and quantitative analysis of the apoptosis of intestinal intestinal mucosa cells in each group. 5. by immunohistochemical staining, proliferating cell nuclear antigen (PCNA) and cell adhesion molecules (homing cell adhesi) were used. On molecule, CD44) antibodies were stained respectively to mark the intestinal stem cells of intestinal mucosa cells and intestinal crypts in the jejunum, and observe the staining of the intestinal mucosal epithelial cells and intestinal stem cells under the microscope in each group, and determine the overall light density (Integrated option de) of the stained parts in the images taken by the Image-Pro Plus (IPP) software. Nsity, IOD) value, and count the number of PCNA staining positive cells in each field of vision, quantitative analysis of the difference in the expression of intestinal mucosal epithelial cells and intestinal stem cells in each group, and verify the effect of ginger Xiexin soup on the proliferation of intestinal mucosa cells in the rat model of irinotecan diarrhea model; 6. through real-time quantitative PCR technique to detect the jejunum tissue in each group The expression of Lgr5 mRNA in the intestinal stem cell marker, the effect of ginger Xiexin Decoction on the expression of intestinal stem cells in the rat model of irinotecan diarrhea model from mRNA level; 7. the activity of beta glucuronuronase in intestinal feces of rats was measured by enzyme activity detection technique, and the internal environment of the rat model of yinecan diarrhea model was verified by the ginger Xiexin soup. 8. through real-time quantitative PCR technique, the expression of UGT1A1 mRNA in rat liver tissues was detected, and the regulation of ginger Xiexin Decoction on liver metabolic enzymes in the rat model of irinotecan diarrhea model was verified. Results 1. through the observation of the general condition of the rat model of the irinotecan diarrhea model, the observation of the diarrhea symptoms, and the immunohistochemistry of the intestinal mucosa and immunohistochemistry. The experimental results of irinotecan diarrhea model rats were confirmed successfully. 2. the general situation of the rats in each group showed that the cumulative weight increase value of the rats in the high dose group of Chinese medicine group was significantly higher than that of the model control group (P0.05, P0.01), but the difference between the different doses of Chinese medicine group was no longer than that of the model control group (48h after the last injection of irinotecan). It was statistically significant, until ninth days, compared with the low dose Chinese medicine group, the high dose group of Chinese medicine group showed higher cumulative weight gain (P0.05). From the fourth day of the experiment, the intake of the rats in the model control group and the low, middle and high dose group of Chinese medicine group decreased significantly (P0.01), and the intake of the rats in each dose group was compared with the blank control group. The recovery of the model control group was significantly faster than that of the model control group. After ninth days of the experiment, the intake of the rats in the model control group was still significantly lower than that in the blank control group (P0.01). The intake of the rats in each dose group was significantly higher than that of the model control group (P0.01), but there was no difference between the control group and the blank control group, but the difference between the different doses of the Chinese medicine group was not statistically significant. The observation of diarrhea symptoms in rats showed that after the injection of 24h, the mean value of diarrhoea score in the model control group and the Chinese medicine group began to rise, and the increase of the diarrhea score in the model control group was significantly higher than that of the traditional Chinese medicine group. The middle dose and the high dose of the model control group were compared with the model control group after the last dose of 60H and 70h. The diarrhea grade distribution in the dose group of the Chinese medicine group had obvious difference (P0.05, P0.01). The observation of 3. intestinal mucosa pathological mirror showed that the intestinal mucosa of the model control group could observe the injury caused by irinotecan, including the incomplete general structure of the intestinal epithelium, the disorder of the epithelial cells, the decrease of the intestinal recess and the intestinal villi, and so on. The mucosal structure of the jejunum and the cecum was the most serious, but the intestinal mucosal structure of the rats in the low, middle and high doses of the Chinese medicine group was more complete. The intestinal recess and intestinal villi were better preserved by.4.TUNEL staining and the detection of Caspase-3 enzyme activity. The positive apoptotic cells of the jejunum mucosa under the traditional Chinese medicine group were significantly lower than those in the model control group, and the middle and high doses were low in the middle and high doses. The intestinal caspase-3 enzyme activity (U) in the drug group was 0.66 + 0.10,0.48 + 0.11,0.68 + 0.19, respectively, which were significantly lower than that in the model control group (vs1.00 0.17, P0.01). The results of 5.PCNA and CD44 staining markers showed that the number of PCNA positive cells in the high dose Chinese medicine group was significantly higher than that in the model control group (531.20 + 101.64 vs 312.40 + 43.23, P0.01). The IOD value of PCNA in the drug group was significantly higher than that in the model control group (2494.93 + 615 vs 1026.26 + 339, P0.05), and the IOD value of CD44 in the high dose Chinese medicine group was 511.35 + 83.18613.04 + 102.42, respectively higher than that of the model control group (vs 360.99 + 156.87, P0.05, P0.01), and 6. real-time quantitative PCR technique results showed that compared with the model control group, each dose was compared with the model control group. The expression of Lgr5 mRNA in the intestinal tract of the rats in the traditional Chinese medicine group increased, but the expression of Lgr5 mRNA in the high dose group was significantly increased, with statistical significance (1.75 + 0.53 vs 0.56 + 0.54, P0.01); 7. beta glucuronide activity detection results showed that before the injection of irinotecan, compared with the empty white control group, the low, medium dose group of Chinese medicine group was compared with the model control group. The activity of beta glucuronate in intestinal feces of rats had no significant difference, but the activity of beta glucuronate in intestinal feces of rats in high dose group was significantly lower than that of model control group (3.88 + 1.78 vs 5.92 + 1.47, P0.05). After injection of irinotecan, the activity of beta glucuronase in low, medium and high dose group was 12.56 + 1.81,13.55 + 1.17,1, respectively 1.62 + 1.78, significantly lower than the model control group (vs 16.09 + 1.40, P0.05, P0.01), but the difference between different doses of traditional Chinese medicine group was not significant; 8. real-time quantitative PCR technique results showed that compared with the blank control group, the expression of UGT1A1 enzyme mRNA in the liver of the model control group and the low, middle and high dose group of Chinese medicine group decreased significantly; and compared with the model control group, each group was compared with the model control group. There was no obvious difference in the expression of UGT1A1 enzyme mRNA in the liver of the rats in the dose group. Conclusion 1. ginger Xiexin Decoction can effectively reduce the weight loss caused by erinotecan and promote the feeding of rats after injection of irinotecan; 2. ginger Xiexin soup can reduce the grade and score of diarrhea caused by erinotecan; 3. ginger diarrhea heart soup can be reduced. 4. ginger Xiexin Decoction can reduce the apoptosis of intestinal cells caused by erinotecan, 5. ginger Xiexin soup can promote the proliferation of intestinal mucosa cells, 6. ginger Xiexin soup can promote the expression of intestinal stem cells, and 7. ginger Xiexin Decoction can reduce the beta glucosaldehyde in intestinal feces after injection of irinotecan in rats The activity of UGT1A1 enzyme mRNA was not significantly affected by ginger 8. Xiexin Decoction.

【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R730.5

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