溃结安干预UC大鼠的代谢组学及人结肠癌HT-29细胞的细胞凋亡研究
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摘要
目的:1)建立大鼠溃疡性结肠炎(Ulcerative colitis,UC)动物模型并用维药西帕依溃结安干预;2)应用代谢组学技术研究溃疡性结肠炎的发病机制和维药西帕依溃结安的作用机理,以探讨代谢组学在UC的早期诊断以及维药西帕依溃结安作用机制研究中的应用;3)研究不同浓度维药西帕依溃结安对人结肠癌HT-29细胞的增殖调节作用及其对细胞凋亡的影响,以探讨维药西帕依溃结安是否有抗肿瘤作用。方法:1)采用2,4-二硝基氯苯(2,4-dinitrochlorobenzene,DNCB)复合乙酸法建立溃疡性结肠炎大鼠模型后将大鼠随机分为模型组、生理盐水(NS)阴性对照组、维药西帕依溃结安大、中、小剂量干预组,同时设正常对照组,给药治疗20天。根据大鼠体征症状变化、大便粘稠度、大体形态及组织形态学改变进行判断及疗效评价;2)给药20天后,收集各组大鼠血浆,进行1H-NMR谱测定和主成分分析;3)四甲基偶氮吐盐(MTT)比色法检测维药西帕依溃结安对人结肠癌HT-29细胞生长增殖的调节作用,Annexin V-PI双染色流式细胞术检测细胞凋亡。结果:1)组织病理学研究结果显示:正常对照组与模型组之间差异显著(P<0.05)。维药西帕依溃结安对大鼠溃疡性结肠炎治疗有效,大、中剂量治疗组间的疗效差异无统计学意义(P>0.05),大、中剂量治疗组与小剂量治疗组间疗效差异有统计学意义(P<0.05);2)代谢组学结果显示:造模后,与正常组相比,模型组中苯丙氨酸、丙酮酸、β-羟丁酸、肌酸、亮氨酸、异亮氨酸、缬氨酸含量降低,VLDL含量升高;维药西帕依溃结安小剂量给药干预后,血浆中丙氨酸、柠檬酸代谢产物含量较模型组降低;3)维药西帕依溃结安对人结肠癌HT-29细胞生长增殖具有抑制作用,并呈剂量和时间依赖性HT-29;Annexin V-PI双染色流式细胞术检测,维药西帕依溃结安诱导人结肠癌HT-29细胞凋亡,且这种诱导作用存在剂量依赖关系。结论:1)采用经典的造模方法复制了溃疡性结肠炎大鼠模型;组织病理学转归表明维药西帕依溃结安对治疗UC有效;2)基于NMR的代谢组学可以较好地用于模型组与正常对照组的区分,是寻找溃疡性结肠炎相关小分子特征标志物的有效方法;3)维药西帕依溃结安具有抗肿瘤作用。
Objective: Development of UC animal model and the intervention of Xipayi Kui Jie’an.using the metabolomics research method to study the metabonomic profile and its variation of UC and health and interfering effects of kuijiean on the metabonomic profile of UC rats plasmaits.in order to explore the feasibility of metabonomics in the application of disease diagnosis and mechanism of action of KUIJIEAN; the effect of KJA on growth of HT-29 cells was evaluated by MTT. Flow Cytometer was used to determine apoptotic rate.To investigate the anti-tumor effect and apoptosis-inducing mechanism of KJA on HT-29 cells. Methods: After preparation of the ulcerative colitis rat models were induced by“DNCB+Acetic acid”, the animals were divided into 5 groups at randomly, which was individually normal group, model group, 3 variant Xipayi Kui Jie’an treatment group included large dose group, medium and little dose group, the group was treated with physiological saline. The whole condition and pathomorphism were observed in all groups after 20 days treatment with Xipayi Kui Jie’an.after 20 days treatment with Xipayi Kui Jie’an In the end of this study, plasma of all groups of rats was collected and its1H nuclear mannetic resonance (1H-NMR) spectra were acquired, then centre its total mark value to convert with the proportion and carry on the PCA with Simca-P 10.0 software. MTT assay was used to Detect the anti-Proliferative effect of Xipayi Kui Jie’an on growth of HT-29 cells was evaluated by MTT.Flow Cytometer was used to determine apoptotic rate. Results: The results showed that, Xipayi Kui Jie’an, Uyghur traditional medicine has a therapeutic effect on UC rats, but there was no statistical significance in the therapeutic effect between the large-dosage group and the middle-dosage group (P>0.05). The statistical significance between the large-middle dosage groups and the small-dosage group (P<0.05). The plasma metabonomic approach could easily distinnuish different groups. Compared with normal control group, valine, leucine, isoleucine, pyruvate,β-hydrox- ybutyrate, creatine, phenylalanineietc.are relatively low and very low density lipoprotei, etc.are relatively high in plasma of the rat models of UC.Compared with KJA little dose group, alanine、citrate, etc.are relatively high in plasma of the rat models of UC. Xipayi Kui Jie’an could induce apoptosis in HT-29 cells in a dose-and time-dependent manner. Conclusion:‘The compound method of immune induction in combination with chemical stimulation can be used to establish ulcerative colitis in rats successfully. KJA has conspicuous treatment effect in ulcerative colitis rat.‘The compound method of immune induction in combination with chemical stimulation’, can be used to establish ulcerative colitis in rats successfully.1NMR-based metabonomies approach can be used for the distinction between Ulcerative colitis and normal control, and Uygur medicine Kuijie′an analysis, it is an effectivetive way to find characteristic biomarker and will have a broad prospects of development in the diagnosis, pathological research of diabetes in the future.Kuijie′an has the effect of Antitumor.
Objective: Development of UC animal model and the intervention of Xipayi Kui Jie’an.using the metabolomics research method to study the metabonomic profile and its variation of UC and health and interfering effects of kuijiean on the metabonomic profile of UC rats plasmaits.in order to explore the feasibility of metabonomics in the application of disease diagnosis and mechanism of action of KUIJIEAN; the effect of KJA on growth of HT-29 cells was evaluated by MTT. Flow Cytometer was used to determine apoptotic rate.To investigate the anti-tumor effect and apoptosis-inducing mechanism of KJA on HT-29 cells. Methods: After preparation of the ulcerative colitis rat models were induced by“DNCB+Acetic acid”, the animals were divided into 5 groups at randomly, which was individually normal group, model group, 3 variant Xipayi Kui Jie’an treatment group included large dose group, medium and little dose group, the group was treated with physiological saline. The whole condition and pathomorphism were observed in all groups after 20 days treatment with Xipayi Kui Jie’an.after 20 days treatment with Xipayi Kui Jie’an In the end of this study, plasma of all groups of rats was collected and its1H nuclear mannetic resonance (1H-NMR) spectra were acquired, then centre its total mark value to convert with the proportion and carry on the PCA with Simca-P 10.0 software. MTT assay was used to Detect the anti-Proliferative effect of Xipayi Kui Jie’an on growth of HT-29 cells was evaluated by MTT.Flow Cytometer was used to determine apoptotic rate. Results: The results showed that, Xipayi Kui Jie’an, Uyghur traditional medicine has a therapeutic effect on UC rats, but there was no statistical significance in the therapeutic effect between the large-dosage group and the middle-dosage group (P>0.05). The statistical significance between the large-middle dosage groups and the small-dosage group (P<0.05). The plasma metabonomic approach could easily distinnuish different groups. Compared with normal control group, valine, leucine, isoleucine, pyruvate,β-hydrox- ybutyrate, creatine, phenylalanineietc.are relatively low and very low density lipoprotei, etc.are relatively high in plasma of the rat models of UC.Compared with KJA little dose group, alanine、citrate, etc.are relatively high in plasma of the rat models of UC. Xipayi Kui Jie’an could induce apoptosis in HT-29 cells in a dose-and time-dependent manner. Conclusion:‘The compound method of immune induction in combination with chemical stimulation can be used to establish ulcerative colitis in rats successfully. KJA has conspicuous treatment effect in ulcerative colitis rat.‘The compound method of immune induction in combination with chemical stimulation’, can be used to establish ulcerative colitis in rats successfully.1NMR-based metabonomies approach can be used for the distinction between Ulcerative colitis and normal control, and Uygur medicine Kuijie′an analysis, it is an effectivetive way to find characteristic biomarker and will have a broad prospects of development in the diagnosis, pathological research of diabetes in the future.Kuijie′an has the effect of Antitumor.
引文
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