酒精对大鼠结肠内iNOS表达的上调作用及其机制研究
发布时间：2019-04-22 12:54
【摘要】： 我们最近的研究发现,酒精抑制大鼠结肠运动,该作用是由NO介导的,但其机制不清。已有报道,酒精可引起肝实质细胞和枯否细胞中iNOS mRNA表达增高,Blanco等研究者也发现,在人工培养的星形胶质细胞中酒精刺激可通过NF-κB途径使iNOS的基因表达上调。 核因子κB(nuclear factor-κB,NF-κB)作为转录因子,广泛参与肿瘤、炎症及免疫性疾病的发病过程,参与机体防御反应、组织损伤和应激、细胞分化和细胞凋亡以及肿瘤生长抑制等过程的信息转导。NF-κB包括NF-κB_1、NF-κB_2和某些癌基因蛋白(如RelA)等,通常与其抑制蛋白IκB结合,以二聚体的形式存在于胞浆中,当细胞受到外界刺激时,如病毒或细菌感染、紫外线照射、氧化应激等,IκB被迅速磷酸化,释放NF-κB使之转入胞核内,结合于特定的κB序列,启动和调节众多与免疫、炎症反应有关的基因转录。 我们推测,酒精可能通过激活NF-κB、提高iNOS表达而引起了NO释放。本课题主要是对该假设进行验证。 材料与方法 实验选用健康雄性Wistar大鼠,实验前禁食18h。快速牺牲大鼠,制备近端结肠纵形肌肌条,在灌流肌槽中孵育并记录肌条自发收缩活动。以平滑肌肌条张力变化为指标,分别观察经PDTC(NF-κB的阻断剂)和SMT(iNOS的阻断剂)预处理后,酒精对结肠纵形肌肌条运动的影响,用Western blot技术测定胞浆中iNOS和IκB表达量,以及NF-κB在胞核中的表达量,免疫组织化学的方法定位结肠内表达iNOS的细胞,NO试剂盒测定结肠组织NO释放量。 所有数据均采用均数±标准误表示,采用单因素方差分析进行统计学处理,以P＜0.05为显著性差异界值。 实验结果: 1.酒精(8.7×10~(-4)M)明显抑制离体结肠纵型肌肌条的自发收缩活动。加入酒精后,记录2～4分钟,7～9分钟,12～14分钟和17～19分钟,肌条张力的R值从1降低到0.90±0.02,0.87±0.02,0.89±0.03 and 0.87±0.01。 2.灌流液中分别加入PDTC(10~(-2)M)(NF-κB的阻断剂)或SMT(10~(-3)M)(选择性iNOS拮抗剂)后,再加入同样剂量的酒精,酒精对结肠肌条运动的抑制作用明显减弱。 3.酒精(0.17×10~(-3)M- 1.30×10~(-3)M)上调iNOS的表达,其中以8.7×10~(-4)M的酒精作用最明显。PDTC(10~(-2)M)明显抑制酒精(8.7×10~(-4)M)对iNOS的表达的上调作用。 4.酒精(8.7×10~(-4)M)增加胞核中NF-κB的表达量,但减少胞浆中IκB的表达量。PDTC逆转酒精的这种作用。 5.免疫组化发现iNOS在结肠肌间神经丛中表达。 6.正常大鼠结肠中NO含量为1.112±0.128μmol/g,加入酒精后20分钟,NO含量增加为7.194±0.497μmol/g(P＜0.05,n=6),PDTC预处理后再加入酒精,结肠中NO含量降为3.267±0.314μmol/g(P＜0.05,n=6) 结论: 酒精抑制结肠运动的作用是通过激活NF-κB,上调iNOS表达,从而增加NO释放量引起的。
[Abstract]:Our recent studies have found that alcohol inhibits colonic motility in rats, which is mediated by NO, but its mechanism is unclear. It has been reported that alcohol can increase the expression of iNOS mRNA in hepatic parenchyma cells and Kupffer cells. Blanco and other researchers have also found that alcohol stimulation can up-regulate the expression of iNOS gene through NF- 魏 B pathway in cultured astrocytes. Nuclear factor 魏 B (nuclear factor- kappa B (NF- 魏 B), as a transcription factor, is involved in the pathogenesis of tumor, inflammation and immune diseases, as well as in defense response, tissue damage and stress. NF-魏 B includes NF- kappa B _ 1, NF-魏 B _ 2 and some oncogene proteins (such as RelA), which usually bind to I 魏 B, an inhibitor of NF-魏 B, and some oncogene proteins such as NF-魏 B, NF-魏 B and some oncogene proteins, such as NF-魏 B, NF-魏 B and some oncogene proteins (such as RelA). In the form of dimer, when cells are stimulated by external stimuli, such as virus or bacterial infection, ultraviolet radiation, oxidative stress, etc., I kappa B is rapidly phosphorylated, releasing NF- 魏 B into the nucleus and binding to specific 魏 B sequences. Initiate and regulate the transcription of many genes associated with immune and inflammatory responses. We speculate that alcohol may cause NO release by activating NF- kappa B and increasing iNOS expression. The main purpose of this paper is to test the hypothesis. Materials and methods healthy male Wistar rats were selected and fasting for 18 hours before the experiment. The longitudinal muscle strips of proximal colon were prepared by rapid sacrifice of rats, and the spontaneous contraction activities of the strips were recorded and incubated in the perfusion groove. The effects of alcohol on the movement of longitudinal colonic muscle strips were observed after pretreatment with PDTC (NF- kappa B blocker) and SMT (iNOS (blocker of SMT (iNOS), and the expression of iNOS and IkB in cytoplasm were measured by Western blot technique. And the expression of NF- kappa B in nucleus, localization of iNOS-expressing cells in colon by immunohistochemical method, and determination of NO release in colon tissue by NO kit. All data were expressed by mean 卤standard error and analyzed by one-way ANOVA (P < 0.05). Experimental results: 1. Alcohol (8.7 脳 10 ~ (- 4) M) significantly inhibited the spontaneous contractile activity of isolated longitudinally isolated colonic muscle strips. After alcohol was added, the R value of muscle tension decreased from 1 to 0.90 卤0.02, 0.87 卤0.02, 0.89 卤0.03 and 0.87 卤0.01. The muscle tension was recorded for 2 minutes, 7 minutes, 9 minutes, 12 minutes, 14 minutes and 17 minutes 19 minutes after alcohol was added, and the R value decreased from 1 to 0.90 卤0.02, 0.87 卤0.02, 0.89 卤0.03 min. 2. When PDTC (10 ~ (- 2) M) (NF- kappa B blocker) or SMT (10 ~ (- 3) M) (selective iNOS antagonist) was added to the perfusate respectively, and the same dose of alcohol was added, the inhibitory effect of alcohol on the movement of colonic muscle strips was obviously weakened. 3. Alcohol (0.17 脳 10 ~ (- 3) M ~ (- 1.30) 脳 10 ~ (- 3) M) up-regulated the expression of iNOS. PDTC (10 ~ (- 2) M) significantly inhibited the up-regulation of iNOS expression by alcohol (8.7 脳 10 ~ (- 4) M). 4. Alcohol (8.7 脳 10 ~ (- 4) M) increased the expression of NF- 魏 B in nucleus, but decreased the expression of I 魏 B in cytoplasm. 5. Immunohistochemical study showed that iNOS was expressed in myenteric plexus of colon. 6. The content of NO in the colon of normal rats was 1.112 卤0.128 渭 mol / g. 20 minutes after adding alcohol, the content of NO increased to 7.194 卤0.497渭 mol / g (P < 0.05. After pretreatment with 6), PDTC, alcohol was added. The content of NO in colon decreased to 3.267 卤0.314 渭 mol / g (P < 0.05 (n = 6) conclusion: alcohol can inhibit colonic motility by activating NF- kappa B and up-regulating the expression of iNOS, thus increasing the amount of NO release.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363
[Abstract]:Our recent studies have found that alcohol inhibits colonic motility in rats, which is mediated by NO, but its mechanism is unclear. It has been reported that alcohol can increase the expression of iNOS mRNA in hepatic parenchyma cells and Kupffer cells. Blanco and other researchers have also found that alcohol stimulation can up-regulate the expression of iNOS gene through NF- 魏 B pathway in cultured astrocytes. Nuclear factor 魏 B (nuclear factor- kappa B (NF- 魏 B), as a transcription factor, is involved in the pathogenesis of tumor, inflammation and immune diseases, as well as in defense response, tissue damage and stress. NF-魏 B includes NF- kappa B _ 1, NF-魏 B _ 2 and some oncogene proteins (such as RelA), which usually bind to I 魏 B, an inhibitor of NF-魏 B, and some oncogene proteins such as NF-魏 B, NF-魏 B and some oncogene proteins, such as NF-魏 B, NF-魏 B and some oncogene proteins (such as RelA). In the form of dimer, when cells are stimulated by external stimuli, such as virus or bacterial infection, ultraviolet radiation, oxidative stress, etc., I kappa B is rapidly phosphorylated, releasing NF- 魏 B into the nucleus and binding to specific 魏 B sequences. Initiate and regulate the transcription of many genes associated with immune and inflammatory responses. We speculate that alcohol may cause NO release by activating NF- kappa B and increasing iNOS expression. The main purpose of this paper is to test the hypothesis. Materials and methods healthy male Wistar rats were selected and fasting for 18 hours before the experiment. The longitudinal muscle strips of proximal colon were prepared by rapid sacrifice of rats, and the spontaneous contraction activities of the strips were recorded and incubated in the perfusion groove. The effects of alcohol on the movement of longitudinal colonic muscle strips were observed after pretreatment with PDTC (NF- kappa B blocker) and SMT (iNOS (blocker of SMT (iNOS), and the expression of iNOS and IkB in cytoplasm were measured by Western blot technique. And the expression of NF- kappa B in nucleus, localization of iNOS-expressing cells in colon by immunohistochemical method, and determination of NO release in colon tissue by NO kit. All data were expressed by mean 卤standard error and analyzed by one-way ANOVA (P < 0.05). Experimental results: 1. Alcohol (8.7 脳 10 ~ (- 4) M) significantly inhibited the spontaneous contractile activity of isolated longitudinally isolated colonic muscle strips. After alcohol was added, the R value of muscle tension decreased from 1 to 0.90 卤0.02, 0.87 卤0.02, 0.89 卤0.03 and 0.87 卤0.01. The muscle tension was recorded for 2 minutes, 7 minutes, 9 minutes, 12 minutes, 14 minutes and 17 minutes 19 minutes after alcohol was added, and the R value decreased from 1 to 0.90 卤0.02, 0.87 卤0.02, 0.89 卤0.03 min. 2. When PDTC (10 ~ (- 2) M) (NF- kappa B blocker) or SMT (10 ~ (- 3) M) (selective iNOS antagonist) was added to the perfusate respectively, and the same dose of alcohol was added, the inhibitory effect of alcohol on the movement of colonic muscle strips was obviously weakened. 3. Alcohol (0.17 脳 10 ~ (- 3) M ~ (- 1.30) 脳 10 ~ (- 3) M) up-regulated the expression of iNOS. PDTC (10 ~ (- 2) M) significantly inhibited the up-regulation of iNOS expression by alcohol (8.7 脳 10 ~ (- 4) M). 4. Alcohol (8.7 脳 10 ~ (- 4) M) increased the expression of NF- 魏 B in nucleus, but decreased the expression of I 魏 B in cytoplasm. 5. Immunohistochemical study showed that iNOS was expressed in myenteric plexus of colon. 6. The content of NO in the colon of normal rats was 1.112 卤0.128 渭 mol / g. 20 minutes after adding alcohol, the content of NO increased to 7.194 卤0.497渭 mol / g (P < 0.05. After pretreatment with 6), PDTC, alcohol was added. The content of NO in colon decreased to 3.267 卤0.314 渭 mol / g (P < 0.05 (n = 6) conclusion: alcohol can inhibit colonic motility by activating NF- kappa B and up-regulating the expression of iNOS, thus increasing the amount of NO release.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363
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