NF-κB信号通路在中子辐射致肠上皮细胞损伤中的调控机制研究
发布时间：2018-07-25 12:26
【摘要】：中子辐射较γ射线损伤重,肠道是中子辐射高度敏感的靶器官,中子辐射肠道损伤重、难恢复。目前中子辐射肠道损伤的病理特点已基本明确,然而其损伤机制仍未完全阐明,尚缺乏有效的治疗措施。为此,本课题在既往研究的基础上,复制中子照射肠上皮细胞损伤整体和离体模型,以NF-κB信号通路在中子辐射肠道损伤中的作用为切入点,开展NF-κB信号通路在中子辐射致肠上皮细胞损伤中的调控作用研究,为阐明中子辐射肠上皮细胞损伤的分子机制、寻找新的防治措施和发现新的治疗靶点提供依据。 材料与方法 一、实验动物分组与模型制作方法 选取二级雄性BALB/c小鼠120只,随机分为对照组(C组,n=30)、3Gy中子照射组(N组,n=60)和3Gy中子照射+姜黄素组(Cur组,n=30);采用3Gy中子全身均匀照射N组和Cur组小鼠;照射后即刻给予Cur组小鼠腹腔注射姜黄素混悬液1次,剂量为200 mg·kg-1·d-1,此后连续给药5d(1次/d),N组和C组小鼠腹腔注射同体积的生理盐水。 二、小鼠整体行为和空肠组织结构光镜和电镜观察 照射后每天观察并记录动物整体行为、体重变化、腹泻及死亡情况;分别于照射后6h、1d、3d和5d活杀C组和N组动物,Cur组于照射后3d和5d活杀,留取各组各时间点小鼠空肠组织;通过光镜和电镜观察3Gy中子照射及应用Cur后小鼠空肠组织结构以及超微结构的变化。 三、小鼠空肠上皮细胞增殖与死亡的检测 采用AgNOR和Feulgen染色方法检测中子照射及应用姜黄素后小鼠空肠上皮细胞的嗜银蛋白及DNA含量变化;采用TUNEL方法检测小鼠空肠上皮细胞凋亡改变。 四、小鼠空肠组织NF-κB通路中信号分子检测 通过IHC和EMSA方法检测中子照射及应用姜黄素后小鼠空肠上皮细胞NF-κB的表达及活性变化;采用WB检测小鼠空肠上皮细胞NF-κB、IKKβ、IκBα、PI3K和Akt的表达变化;通过Co-IP技术检测小鼠空肠组织中Akt和IKKβ相互作用;通过Real-time PCR方法检测IKKβmRNA的变化。 五、IEC-6细胞培养、分组、照射及处理 IEC-6细胞传代培养后,随机分为5组:对照组(C)、4Gy中子照射组(N)、4Gy+LY294002组(LY)、10Gyγ射线照射组(R)和10Gyγ射线+LY294002组(LY2);分别采用4Gy中子和10Gyγ射线对IEC-6细胞进行均匀照射;LY294002处理方法:于中子和γ射线照射前24h,LY组和LY2组细胞更换为含LY294002终浓度为10μmol/L的培养液。 六、IEC-6细胞增殖与死亡检测 于中子和γ射线照射后6h和24h,通过IPCM观察IEC-6细胞的形态变化;于中子照射后6h和24h收集细胞留取样品,γ射线照射后15min、30min、1h、6h和24h收集细胞留取样品;分别采用MTT方法和FCM检测IEC-6细胞的增殖活力以及凋亡与坏死率改变。 七、IEC-6细胞NF-κB通路中信号分子检测 通过WB方法检测中子和γ射线照射后IEC-6细胞中NF-κB、IKKα/β和IκBα的表达及其磷酸化水平改变。 八、图像分析和定量方法 (一)小鼠空肠细胞AgNOR、DNA含量及NF-κB免疫组化结果,采用CMIAS-Ⅱ系列多功能真彩色病理图像分析系统,在光镜10×40倍视野下,每组切片采集10个视野,检测其MOD和IOD。 (二)WB结果采用Image Pro 5.0软件进行图像分析,测定电泳条带的IOD,将各目的蛋白条带与相应内参GAPDH的IOD比值进行定量分析。 (三)IKKβ和相应GAPDH mRNA应用7300 system SDS软件进行定量分析。 (四)Co-IP结果采用CMIAS-Ⅱ图像分析系统进行IOD测定和定量分析。 九、统计学处理 文中数据以均数和标准差(?X±s)表示;采用SPSS 13.0统计软件进行一元方差分析Excel软件作图;vs对照组,*示P0.05,**示P0.01;vs照射组,#示P0.05,##示P0.01。 实验结果 一、小鼠整体行为、空肠组织学及超微结构改变 (一)小鼠整体行为的改变:3Gy中子照射及应用姜黄素后,小鼠精神状态逐渐变差,不进食水,体重进行性下降,并出现严重腹泻症状;于照射后3~5d内全部死亡,出现了肠型放射病典型的“三天半效应”。 (二)小鼠空肠组织学改变:3Gy中子照射后,光镜下可见肠黏膜大面积坏死脱落,绒毛上皮细胞稀疏、排列较乱,细胞肿胀,隐窝细胞见核固缩、碎裂,其数量进行性减少;照射后3~5d可见隐窝细胞再生,但隐窝中细胞数目较少;姜黄素组在治疗3d和5d时隐窝细胞再生较明显,且可见丰富的绒毛深入肠腔。 (三)小鼠空肠超微结构改变:3Gy中子照射后3d,电镜下,小鼠空肠隐窝细胞减少,可见胞核碎裂,核染色质浓缩边移,隐窝细胞呈空泡状,出现坏死和凋亡的细胞,以坏死为主,照射后5d可见小肠绒毛有轻度增生;Cur组于3Gy中子照射后3~5d,空肠组织中可见有新生微绒毛及隐窝,隐窝内细胞数目较照射组略有增加。 二、小鼠空肠上皮细胞嗜银蛋白及凋亡改变 (一)小鼠空肠上皮细胞嗜银蛋白和DNA含量:3Gy中子照射照射后6h~5d,N组和Cur组小鼠空肠上皮细胞嗜银蛋白和DNA含量进行性下降(P0.01),照射后3~5d,Cur组小鼠空肠上皮细胞嗜银蛋白和DNA含量均较照射组增加(P0.05)。 (二)小鼠空肠上皮细胞凋亡TUNEL检测结果:3Gy中子照射照射后6h~1d,N组小鼠空肠上皮可见大量凋亡细胞;照射后3d,N组和Cur组小鼠空肠上皮细胞偶见凋亡细胞。 三、小鼠空肠上皮细胞NF-κB及通路中信号分子的变化 (一)小鼠空肠上皮细胞NF-κB的表达变化:NF-κB在正常肠绒毛及隐窝上皮细胞胞浆内呈弱阳性,3Gy中子照射后6h~5d,NF-κB于上皮细胞核呈阳性,并于照射后5d达到高峰(P0.01),Cur组在照射后3d和5d,NF-κB在绒毛及隐窝上皮细胞核及浆内表达呈阳性,强度弱于照射组(P0.05)。 (二)小鼠空肠组织NF-κB的DNA结合活性变化:3Gy中子照射后6h~3d,N组小鼠空肠组织中NF-κB与DNA结合活性明显增强;Cur组在照射后3d,小鼠空肠组织中NF-κB与DNA结合活性较N组明显降低。 (三)小鼠空肠组织NF-κB通路中信号分子的变化:(1)小鼠空肠组织NF-κB和IKKβ蛋白于3Gy中子照射后6h和1d表达上调(P0.05),照射后3d和5d表达明显上调(P0.01);Cur组在照射后3d和5d,NF-κB和IKKβ表达下调(P0.05)。(2)小鼠空肠组织IκBα蛋白于3Gy中子照射后6h和1d表达下调(P0.05),照射后3d和5d表达明显下调(P0.01);Cur组在照射后3d和5d,IκBα表达上调(P0.05)。(3)3Gy中子照射后6h小鼠空肠组织IKKβmRNA表达增加(P0.05),至照射后24h表达明显增加(P0.01);Cur组在照射后6h和24h,IKKβmRNA表达水平较N组明显降低(P0.05)。 四、小鼠空肠上皮细胞PI3K、Akt的表达及Akt和IKKβ相互作用变化 (一)小鼠空肠组织PI3K和Akt的表达变化:小鼠空肠组织PI3K和Akt蛋白于3Gy中子照射后6h和1d表达上调(P0.05),照射后3d和5d表达明显上调(P0.01);Cur组在照射后3d和5d,PI3K和Akt表达下调(P0.05)。 (二)小鼠空肠组织Akt和IKKβ的相互作用:3Gy中子照射后6h和1d,小鼠空肠组织中Akt和IKKβ相互作用明显增强(P0.01),照射后3d和5d,Akt和IKKβ的相互作用减弱(P0.05);Cur组在照射后3d和5d,Akt和IKKβ相互作用较照射组减弱(P0.05)。 五、IEC-6细胞形态、增殖活力、凋亡及坏死率改变 (一)IEC-6细胞形态的变化:4Gy中子和10Gyγ射线照射后6h和24h,细胞肿胀,形态变圆,折光性增强;应用LY294002处理后,培养液漂浮大量死细胞。 (二)IEC-6细胞增殖活力、凋亡及坏死率改变:4Gy中子和10Gyγ射线照射后,细胞增殖活力较对照组明显下降(P0.01),细胞凋亡与坏死率均显著增加(P0.01),辐射后6h凋亡达高峰,24h以坏死为主;应用LY294002处理后,细胞的增殖活力较照射组明显降低(P0.05),凋亡和坏死率增加(P0.05)。 六、IEC-6细胞NF-κB通路中信号分子变化及蛋白磷酸化改变 (一)IEC-6细胞NF-κB通路中信号分子变化:(1)IEC-6细胞内NF-κB、IKKα和IKKβ蛋白于4Gy中子照射后6h表达上调(P0.05),24h表达明显上调(P0.01);应用LY294002处理组,于照射后6h和24h,上述蛋白表达均下调(P0.05)。(2)IκBα于4Gy中子照射后6h表达下调(P0.05),24h表达明显下调(P0.01);应用LY294002处理组,于照射后6h和24h,IκBα表达上调(P0.05)。 (二)IEC-6细胞NF-κB信号通路中蛋白磷酸化改变:(1)10Gyγ射线照射后15~30min,IEC-6细胞内磷酸化NF-κB和磷酸化IKKα/β蛋白表达上调(P0.05),照射后1h表达明显上调(P0.01);应用LY294002处理组,三种磷酸化蛋白的表达均下调(P0.05)。(2)IEC-6细胞内磷酸化IκBα蛋白在10Gyγ射线照射后15~30min未见表达,1h见明显表达(P0.01);应用LY294002处理组,于照射后1h磷酸化IκBα的表达较照射组明显上调(P0.01)。 结论 一、3Gy中子照射可引起小鼠轻度肠型放射病,肠上皮严重损伤,上皮细胞凋亡与坏死并存,细胞增殖能力降低;表明3Gy中子照射成功建立了中子辐射小鼠空肠损伤的动物模型。 二、姜黄素治疗可减轻中子辐射肠道损伤的程度,促进空肠粘膜上皮再生修复,对中子辐射肠上皮损伤具有保护作用。 三、3Gy中子照射可使小鼠空肠NF-κB信号通路活化,通路中关键信号分子IKKβ表达上调,NF-κB表达上调且发生核转位,进而可能通过调控多种参与炎症反应的靶基因,促进炎症因子的表达,导致中子照射后肠道炎症发生。 四、姜黄素可抑制中子照射后小鼠空肠NF-κB信号通路活化,下调NF-κB和IKKβ的表达水平,这可能是其发挥保护作用的机制之一。 五、3Gy中子照射后,小鼠空肠Akt和NF-κB信号通路中关键激酶IKKβ存在相互作用,表明中子照射后NF-κB信号通路受到PI3K/Akt信号通路的调控。 六、4Gy中子和10Gyγ射线照射均可使IEC-6细胞增殖活力降低,凋亡与坏死率增加,应用LY294002可以进一步降低照射后IEC-6细胞增殖活力,增加凋亡和坏死率。 七、4Gy中子和10Gyγ射线照射均可使IEC-6细胞NF-κB信号通路活化,应用PI3K的抑制剂LY294002可相应抑制NF-κB信号通路的活化,表明NF-κB信号通路活化可对IEC-6细胞损伤发挥保护作用。
[Abstract]:The radiation of neutron radiation is heavier than that of gamma ray, and the intestinal tract is a highly sensitive target organ of neutron radiation. The intestinal damage of neutron radiation is heavy and difficult to recover. The pathological characteristics of the intestinal damage of neutron radiation are basically clear, but the damage mechanism is still not fully elucidated, and the effective treatment measures are still lacking. Therefore, this subject has been replicated on the basis of previous research. The effects of NF- kappa B signaling pathway on the intestinal damage of neutron radiation are the breakthrough point, and the regulation of the NF- kappa B signal pathway in the intestinal epithelial cell damage caused by neutron radiation is studied. The molecular mechanism of the neutron radiation of intestinal epithelial cell damage is clarified to find new preventive measures. And the discovery of new therapeutic targets provides a basis.
Materials and methods
First, group and model making method of experimental animals
120 male BALB/c mice were randomly divided into control group (group C, n=30), 3Gy neutron irradiation group (N group, n=60) and 3Gy neutron irradiation + curcumin group (Cur group, n=30), N group and Cur group were irradiated with 3Gy neutrons all over the whole body. After irradiation, the intraperitoneal injection of curcumin suspension was given 1 times and the dose was 200. The mice in N and C groups were intraperitoneally injected with the same volume of normal saline for 5 days (once a day).
2. Observation of whole behavior and jejunum structure in mice by light and electron microscopy
The overall behavior, weight change, diarrhea and death of the animals were observed and recorded every day after irradiation. After irradiation, 6h, 1D, 3D and 5D were killed in C and N groups. The Cur group killed the 3D and 5D after irradiation and left the jejunum tissues of each time point in each group. The tissue structure of the jejunum in the mice after the 3Gy neutron irradiation and the Cur after the use of Cur was observed by light and electron microscopy. Changes in the ultrastructure.
3. Detection of proliferation and death of mouse jejunal epithelial cells
AgNOR and Feulgen staining were used to detect the changes of eosinophils and DNA in the jejunum epithelial cells after neutron irradiation and the use of curcumin. The apoptosis of mouse jejunum epithelial cells was detected by TUNEL method.
4. Detection of signal molecules in NF-kappa B pathway in mouse jejunum
The expression and activity changes of NF- kappa B in jejunum epithelial cells after neutron irradiation and curcumin were detected by IHC and EMSA methods. The expression of NF- kappa B, IKK beta, I kappa B alpha, PI3K and Akt were detected by WB. Changes in IKK beta mRNA.
Five, IEC-6 cell culture, grouping, irradiation and treatment
After subculture, IEC-6 cells were randomly divided into 5 groups: the control group (C), the 4Gy neutron irradiation group (N), the 4Gy+LY294002 group (LY), the 10Gy gamma ray irradiation group (R) and the 10Gy gamma ray +LY294002 group (LY2). The culture solution containing LY294002 at the final concentration of 10 micromol/L was replaced.
Six, IEC-6 cell proliferation and death detection
After irradiation of neutron and gamma rays 6h and 24h, the morphological changes of IEC-6 cells were observed by IPCM. After the neutron irradiation, 6h and 24h collected the cells to collect the samples. After gamma ray irradiation, the samples were collected by 15min, 30min, 1H, 6h and 24h, and the proliferation activity and the apoptosis and necrosis rate of the cells were detected by MTT methods.
Seven, detection of signal molecules in the NF- kappa B pathway of IEC-6 cells
WB method was used to detect the expression of NF- kappa B, IKK alpha / beta and I kappa B alpha and their phosphorylation levels in IEC-6 cells after neutron and gamma irradiation.
Eight, image analysis and quantitative method
(I) the AgNOR, DNA content and the immunohistochemical results of NF- kappa B in mouse jejunum cells, using CMIAS- II Series multi-function true color pathological image analysis system. Under the 10 x 40 fold visual field of light microscope, 10 fields of vision were collected from each group, and their MOD and IOD. were detected.
(two) WB results were analyzed by Image Pro 5 software, and the IOD of the electrophoresis strip was measured. The ratio of each target protein strip to the IOD ratio of the corresponding internal reference GAPDH was quantitatively analyzed.
(three) IKK beta and corresponding GAPDH mRNA were quantitatively analyzed by 7300 system SDS software.
(four) Co-IP results were analyzed by IOD and quantitative analysis using CMIAS- II image analysis system.
Nine, statistical treatment
The data in this paper are represented by mean and standard deviation (? X + s); Excel software is made by using SPSS 13 statistical software for the analysis of one variance analysis software; vs control group, * P0.05, vs irradiation group, vs exposure group, P0.01. P0.01., P0.01. P0.01..
experimental result
(1) the whole behavior of mice, the histological and ultrastructural changes of jejunum.
(1) changes in the overall behavior of mice: after 3Gy neutron irradiation and curcumin application, the mental state of the mice gradually became worse, no water was eaten, the weight of the mice decreased and the symptoms of severe diarrhea occurred. After the irradiation, all the mice died in 3~5d, and the typical "three half day and half effect" of intestinal type radiation disease was found.
(two) histological changes in the jejunum of mice: after 3Gy neutron irradiation, large area necrosis and shedding of intestinal mucosa were observed under light microscope, and the villous epithelial cells were sparsely scattered, the cells were swollen, the recess cells were reformed, the number of the recess cells were reduced, and the recess cells regenerated after irradiation, but the number of the recess cells was less; the curcumin group was in the treatment of 3D. The regeneration of crypt cells was more obvious when compared with 5D, and abundant villus were seen in the intestinal cavity.
(three) the ultrastructural changes in the jejunum of mice: 3Gy neutron irradiated 3D, under electron microscope, the cells of the jejunum recess decreased, the nuclei of the nucleus were broken, the nucleus chromatin was concentrated, the recess cells were vacuoled, necrotic and apoptotic cells appeared, the necrotic cells were necrotic, and the 5D of the small intestine had mild hyperplasia after irradiation; group Cur was irradiated with 3Gy neutrons, 3~5d, jejunum after irradiation. Microvilli and crypt were seen in the tissue. The number of cells in the crypt increased slightly compared with that in the irradiated group.
Two, the changes of Ag protein and apoptosis in mouse jejunal epithelial cells.
(1) the content of Ag and DNA in the mouse jejunum epithelial cells: after irradiation of 3Gy neutron irradiation, 6h~5d, N and Cur mice were decreased (P0.01) in the jejunum epithelial cells (P0.01). The contents of eosinophils and DNA in the jejunum epithelial cells of the mice were increased after irradiation (P0.05).
(two) TUNEL detection results of apoptosis in mouse jejunum epithelial cells: a large number of apoptotic cells were found in the jejunum epithelium of group N after irradiation of 3Gy neutron irradiation, and apoptotic cells were found in 3D, N group and Cur group of jejunum epithelial cells.
Three, the changes of NF- kappa B and signal molecules in the pathway of mouse jejunal epithelial cells.
(1) the expression of NF- kappa B in the mouse jejunum epithelial cells: NF- kappa B was weakly positive in the cytoplasm of normal intestinal villi and recess epithelial cells, 6h~5d after 3Gy neutron irradiation, NF- kappa B in epithelial cell nuclei and 5D reached its peak after irradiation (P0.01). Cur group was expressed in the nucleus and pulp of the villus and recess epithelium after irradiation. The intensity is weaker than that of the irradiated group (P0.05).
(two) the DNA binding activity of NF- kappa B in the jejunum of mice: 3Gy neutron irradiated 6h~3d, and the binding activity of NF- kappa B and DNA in the jejunum tissue of group N mice increased obviously; the Cur group was 3D after irradiation, and the binding activity of NF- kappa and kappa was significantly lower in the jejunum tissue of mice than that in the mice.
(three) the changes in the signal molecules in the NF- kappa B pathway in the jejunum tissue of mice: (1) the expression of 6h and 1D in the jejunum tissue of mice was up to up (P0.05) after 3Gy neutron irradiation, and the expression of 3D and 5D after irradiation was obviously up (P0.01). After irradiation, the expression of 6h and 1D was down (P0.05), and the expression of 3D and 5D was obviously down (P0.01). The expression of 3D and 5D, I kappa B alpha was up (P0.05) in Cur group after irradiation. (3) after irradiation, the expression of the mouse jejunum tissue was increased, and the expression was obviously increased after irradiation. Decrease (P0.05).
Four, the expression of PI3K, Akt and the interaction of Akt and IKK beta in mouse jejunal epithelial cells.
(1) the expression of PI3K and Akt in the jejunum tissue of mice: the expression of PI3K and Akt protein in the jejunum tissue of mice was up regulated by 6h and 1D after 3Gy neutron irradiation (P0.05), and the expression of 3D and 5D increased obviously after irradiation (P0.01).
(two) the interaction of Akt and IKK beta in the jejunum tissue of mice: 6h and 1D after 3Gy neutron irradiation, the interaction of Akt and IKK beta in the jejunum tissue of mice increased significantly (P0.01). The interaction between 3D and 5D, Akt and IKK beta was weakened after irradiation.
Five, IEC-6 cell morphology, proliferation activity, apoptosis and necrosis rate change.
(1) changes in IEC-6 cell morphology: 6h and 24h after irradiation of 4Gy neutrons and 10Gy gamma rays, the cells swelled, the morphology became round, and the refraction was enhanced; after the LY294002 treatment, the culture fluid was floating a large number of dead cells.
(two) the proliferation activity, apoptosis and necrosis rate of IEC-6 cells were changed: after 4Gy neutron and 10Gy gamma ray irradiation, the cell proliferation activity was significantly lower than that of the control group (P0.01), the apoptosis and necrosis rate increased significantly (P0.01), the apoptosis reached the peak of 6h after radiation, and the 24h was mainly necrotic, and the proliferation activity of the cells was significantly lower than that of the irradiated group after the use of LY294002. Low (P0.05), the increase of apoptosis and necrosis (P0.05).
Six, changes in signal molecules and protein phosphorylation in NF- kappa B pathway in IEC-6 cells.
(1) the change of signal molecules in the NF- kappa B pathway of IEC-6 cells: (1) NF- kappa B in IEC-6 cells, IKK A and IKK beta protein expression up up (P0.05) after 4Gy neutron irradiation, and the expression of 24h expression was obviously up-regulated. The expression of h was significantly down regulated (P0.01), and the expression of 6h and 24h, I kappa B was increased (P0.05) in LY294002 treated group.
(two) the changes in protein phosphorylation in the NF- kappa B signaling pathway in IEC-6 cells: (1) the expression of phosphorylated NF- kappa B and phosphorylated IKK alpha / beta protein expression in IEC-6 cells increased after 10Gy gamma ray irradiation (P0.05), and the 1H expression was obviously up (P0.01) after irradiation. The expression of three phosphorylated proteins decreased (2) phosphoric acid within the cells. The expression of I kappa B alpha protein was not expressed in 15~30min after 10Gy gamma ray irradiation, and the expression of 1H was obviously expressed (P0.01). The expression of I kappa B alpha in 1H phosphorylated after irradiation was significantly up (P0.01) in the LY294002 treatment group than in the irradiated group.
conclusion
First, 3Gy neutron irradiation can cause mild intestinal radiation disease in mice, the intestinal epithelium is seriously damaged, the apoptosis and necrosis of epithelial cells coexist, and the cell proliferation ability is reduced, which indicates that 3Gy neutron irradiation has successfully established the animal model of the jejunum damage in mice irradiated by neutron radiation.
Two, curcumin treatment can reduce the degree of intestinal injury induced by neutron irradiation, promote the regeneration and repair of jejunal epithelium, and protect the intestinal epithelium from neutron irradiation.
Three, 3Gy neutron irradiation can activate the NF- kappa B signal pathway of jejunum in mice. The expression of key signal molecule IKK beta in the pathway is up-regulated and the expression of NF- kappa B is up-regulated and has nuclear transposition, which may promote the expression of inflammatory factors by regulating various target genes involved in the inflammatory response, leading to the occurrence of intestinal inflammation after the irradiation of the middle children.
Four, curcumin can inhibit the activation of NF- kappa B signal pathway in jejunum of mice after neutron irradiation, and down regulate the expression level of NF- kappa B and IKK beta, which may be one of its protective mechanisms.
Five, after 3Gy neutron irradiation, the key kinase IKK beta in the Akt and NF- kappa B signaling pathway in the mouse jejunum was interacted, indicating that the NF- kappa B signal pathway was regulated by the PI3K/Akt signaling pathway after the neutron irradiation.
Six, 4Gy neutrons and 10Gy gamma rays can reduce the proliferation of IEC-6 cells and increase the apoptosis and necrosis rate. LY294002 can further reduce the proliferation activity of IEC-6 cells after irradiation and increase the rate of apoptosis and necrosis.
Seven, both 4Gy neutrons and 10Gy gamma rays can activate the NF- kappa B signaling pathway in IEC-6 cells. The activation of NF- kappa B signaling pathway can be inhibited by PI3K inhibitor LY294002, indicating that NF- kappa B signaling pathway can be activated for IEC-6 cells.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363
本文编号：2143814
[Abstract]:The radiation of neutron radiation is heavier than that of gamma ray, and the intestinal tract is a highly sensitive target organ of neutron radiation. The intestinal damage of neutron radiation is heavy and difficult to recover. The pathological characteristics of the intestinal damage of neutron radiation are basically clear, but the damage mechanism is still not fully elucidated, and the effective treatment measures are still lacking. Therefore, this subject has been replicated on the basis of previous research. The effects of NF- kappa B signaling pathway on the intestinal damage of neutron radiation are the breakthrough point, and the regulation of the NF- kappa B signal pathway in the intestinal epithelial cell damage caused by neutron radiation is studied. The molecular mechanism of the neutron radiation of intestinal epithelial cell damage is clarified to find new preventive measures. And the discovery of new therapeutic targets provides a basis.
Materials and methods
First, group and model making method of experimental animals
120 male BALB/c mice were randomly divided into control group (group C, n=30), 3Gy neutron irradiation group (N group, n=60) and 3Gy neutron irradiation + curcumin group (Cur group, n=30), N group and Cur group were irradiated with 3Gy neutrons all over the whole body. After irradiation, the intraperitoneal injection of curcumin suspension was given 1 times and the dose was 200. The mice in N and C groups were intraperitoneally injected with the same volume of normal saline for 5 days (once a day).
2. Observation of whole behavior and jejunum structure in mice by light and electron microscopy
The overall behavior, weight change, diarrhea and death of the animals were observed and recorded every day after irradiation. After irradiation, 6h, 1D, 3D and 5D were killed in C and N groups. The Cur group killed the 3D and 5D after irradiation and left the jejunum tissues of each time point in each group. The tissue structure of the jejunum in the mice after the 3Gy neutron irradiation and the Cur after the use of Cur was observed by light and electron microscopy. Changes in the ultrastructure.
3. Detection of proliferation and death of mouse jejunal epithelial cells
AgNOR and Feulgen staining were used to detect the changes of eosinophils and DNA in the jejunum epithelial cells after neutron irradiation and the use of curcumin. The apoptosis of mouse jejunum epithelial cells was detected by TUNEL method.
4. Detection of signal molecules in NF-kappa B pathway in mouse jejunum
The expression and activity changes of NF- kappa B in jejunum epithelial cells after neutron irradiation and curcumin were detected by IHC and EMSA methods. The expression of NF- kappa B, IKK beta, I kappa B alpha, PI3K and Akt were detected by WB. Changes in IKK beta mRNA.
Five, IEC-6 cell culture, grouping, irradiation and treatment
After subculture, IEC-6 cells were randomly divided into 5 groups: the control group (C), the 4Gy neutron irradiation group (N), the 4Gy+LY294002 group (LY), the 10Gy gamma ray irradiation group (R) and the 10Gy gamma ray +LY294002 group (LY2). The culture solution containing LY294002 at the final concentration of 10 micromol/L was replaced.
Six, IEC-6 cell proliferation and death detection
After irradiation of neutron and gamma rays 6h and 24h, the morphological changes of IEC-6 cells were observed by IPCM. After the neutron irradiation, 6h and 24h collected the cells to collect the samples. After gamma ray irradiation, the samples were collected by 15min, 30min, 1H, 6h and 24h, and the proliferation activity and the apoptosis and necrosis rate of the cells were detected by MTT methods.
Seven, detection of signal molecules in the NF- kappa B pathway of IEC-6 cells
WB method was used to detect the expression of NF- kappa B, IKK alpha / beta and I kappa B alpha and their phosphorylation levels in IEC-6 cells after neutron and gamma irradiation.
Eight, image analysis and quantitative method
(I) the AgNOR, DNA content and the immunohistochemical results of NF- kappa B in mouse jejunum cells, using CMIAS- II Series multi-function true color pathological image analysis system. Under the 10 x 40 fold visual field of light microscope, 10 fields of vision were collected from each group, and their MOD and IOD. were detected.
(two) WB results were analyzed by Image Pro 5 software, and the IOD of the electrophoresis strip was measured. The ratio of each target protein strip to the IOD ratio of the corresponding internal reference GAPDH was quantitatively analyzed.
(three) IKK beta and corresponding GAPDH mRNA were quantitatively analyzed by 7300 system SDS software.
(four) Co-IP results were analyzed by IOD and quantitative analysis using CMIAS- II image analysis system.
Nine, statistical treatment
The data in this paper are represented by mean and standard deviation (? X + s); Excel software is made by using SPSS 13 statistical software for the analysis of one variance analysis software; vs control group, * P0.05, vs irradiation group, vs exposure group, P0.01. P0.01., P0.01. P0.01..
experimental result
(1) the whole behavior of mice, the histological and ultrastructural changes of jejunum.
(1) changes in the overall behavior of mice: after 3Gy neutron irradiation and curcumin application, the mental state of the mice gradually became worse, no water was eaten, the weight of the mice decreased and the symptoms of severe diarrhea occurred. After the irradiation, all the mice died in 3~5d, and the typical "three half day and half effect" of intestinal type radiation disease was found.
(two) histological changes in the jejunum of mice: after 3Gy neutron irradiation, large area necrosis and shedding of intestinal mucosa were observed under light microscope, and the villous epithelial cells were sparsely scattered, the cells were swollen, the recess cells were reformed, the number of the recess cells were reduced, and the recess cells regenerated after irradiation, but the number of the recess cells was less; the curcumin group was in the treatment of 3D. The regeneration of crypt cells was more obvious when compared with 5D, and abundant villus were seen in the intestinal cavity.
(three) the ultrastructural changes in the jejunum of mice: 3Gy neutron irradiated 3D, under electron microscope, the cells of the jejunum recess decreased, the nuclei of the nucleus were broken, the nucleus chromatin was concentrated, the recess cells were vacuoled, necrotic and apoptotic cells appeared, the necrotic cells were necrotic, and the 5D of the small intestine had mild hyperplasia after irradiation; group Cur was irradiated with 3Gy neutrons, 3~5d, jejunum after irradiation. Microvilli and crypt were seen in the tissue. The number of cells in the crypt increased slightly compared with that in the irradiated group.
Two, the changes of Ag protein and apoptosis in mouse jejunal epithelial cells.
(1) the content of Ag and DNA in the mouse jejunum epithelial cells: after irradiation of 3Gy neutron irradiation, 6h~5d, N and Cur mice were decreased (P0.01) in the jejunum epithelial cells (P0.01). The contents of eosinophils and DNA in the jejunum epithelial cells of the mice were increased after irradiation (P0.05).
(two) TUNEL detection results of apoptosis in mouse jejunum epithelial cells: a large number of apoptotic cells were found in the jejunum epithelium of group N after irradiation of 3Gy neutron irradiation, and apoptotic cells were found in 3D, N group and Cur group of jejunum epithelial cells.
Three, the changes of NF- kappa B and signal molecules in the pathway of mouse jejunal epithelial cells.
(1) the expression of NF- kappa B in the mouse jejunum epithelial cells: NF- kappa B was weakly positive in the cytoplasm of normal intestinal villi and recess epithelial cells, 6h~5d after 3Gy neutron irradiation, NF- kappa B in epithelial cell nuclei and 5D reached its peak after irradiation (P0.01). Cur group was expressed in the nucleus and pulp of the villus and recess epithelium after irradiation. The intensity is weaker than that of the irradiated group (P0.05).
(two) the DNA binding activity of NF- kappa B in the jejunum of mice: 3Gy neutron irradiated 6h~3d, and the binding activity of NF- kappa B and DNA in the jejunum tissue of group N mice increased obviously; the Cur group was 3D after irradiation, and the binding activity of NF- kappa and kappa was significantly lower in the jejunum tissue of mice than that in the mice.
(three) the changes in the signal molecules in the NF- kappa B pathway in the jejunum tissue of mice: (1) the expression of 6h and 1D in the jejunum tissue of mice was up to up (P0.05) after 3Gy neutron irradiation, and the expression of 3D and 5D after irradiation was obviously up (P0.01). After irradiation, the expression of 6h and 1D was down (P0.05), and the expression of 3D and 5D was obviously down (P0.01). The expression of 3D and 5D, I kappa B alpha was up (P0.05) in Cur group after irradiation. (3) after irradiation, the expression of the mouse jejunum tissue was increased, and the expression was obviously increased after irradiation. Decrease (P0.05).
Four, the expression of PI3K, Akt and the interaction of Akt and IKK beta in mouse jejunal epithelial cells.
(1) the expression of PI3K and Akt in the jejunum tissue of mice: the expression of PI3K and Akt protein in the jejunum tissue of mice was up regulated by 6h and 1D after 3Gy neutron irradiation (P0.05), and the expression of 3D and 5D increased obviously after irradiation (P0.01).
(two) the interaction of Akt and IKK beta in the jejunum tissue of mice: 6h and 1D after 3Gy neutron irradiation, the interaction of Akt and IKK beta in the jejunum tissue of mice increased significantly (P0.01). The interaction between 3D and 5D, Akt and IKK beta was weakened after irradiation.
Five, IEC-6 cell morphology, proliferation activity, apoptosis and necrosis rate change.
(1) changes in IEC-6 cell morphology: 6h and 24h after irradiation of 4Gy neutrons and 10Gy gamma rays, the cells swelled, the morphology became round, and the refraction was enhanced; after the LY294002 treatment, the culture fluid was floating a large number of dead cells.
(two) the proliferation activity, apoptosis and necrosis rate of IEC-6 cells were changed: after 4Gy neutron and 10Gy gamma ray irradiation, the cell proliferation activity was significantly lower than that of the control group (P0.01), the apoptosis and necrosis rate increased significantly (P0.01), the apoptosis reached the peak of 6h after radiation, and the 24h was mainly necrotic, and the proliferation activity of the cells was significantly lower than that of the irradiated group after the use of LY294002. Low (P0.05), the increase of apoptosis and necrosis (P0.05).
Six, changes in signal molecules and protein phosphorylation in NF- kappa B pathway in IEC-6 cells.
(1) the change of signal molecules in the NF- kappa B pathway of IEC-6 cells: (1) NF- kappa B in IEC-6 cells, IKK A and IKK beta protein expression up up (P0.05) after 4Gy neutron irradiation, and the expression of 24h expression was obviously up-regulated. The expression of h was significantly down regulated (P0.01), and the expression of 6h and 24h, I kappa B was increased (P0.05) in LY294002 treated group.
(two) the changes in protein phosphorylation in the NF- kappa B signaling pathway in IEC-6 cells: (1) the expression of phosphorylated NF- kappa B and phosphorylated IKK alpha / beta protein expression in IEC-6 cells increased after 10Gy gamma ray irradiation (P0.05), and the 1H expression was obviously up (P0.01) after irradiation. The expression of three phosphorylated proteins decreased (2) phosphoric acid within the cells. The expression of I kappa B alpha protein was not expressed in 15~30min after 10Gy gamma ray irradiation, and the expression of 1H was obviously expressed (P0.01). The expression of I kappa B alpha in 1H phosphorylated after irradiation was significantly up (P0.01) in the LY294002 treatment group than in the irradiated group.
conclusion
First, 3Gy neutron irradiation can cause mild intestinal radiation disease in mice, the intestinal epithelium is seriously damaged, the apoptosis and necrosis of epithelial cells coexist, and the cell proliferation ability is reduced, which indicates that 3Gy neutron irradiation has successfully established the animal model of the jejunum damage in mice irradiated by neutron radiation.
Two, curcumin treatment can reduce the degree of intestinal injury induced by neutron irradiation, promote the regeneration and repair of jejunal epithelium, and protect the intestinal epithelium from neutron irradiation.
Three, 3Gy neutron irradiation can activate the NF- kappa B signal pathway of jejunum in mice. The expression of key signal molecule IKK beta in the pathway is up-regulated and the expression of NF- kappa B is up-regulated and has nuclear transposition, which may promote the expression of inflammatory factors by regulating various target genes involved in the inflammatory response, leading to the occurrence of intestinal inflammation after the irradiation of the middle children.
Four, curcumin can inhibit the activation of NF- kappa B signal pathway in jejunum of mice after neutron irradiation, and down regulate the expression level of NF- kappa B and IKK beta, which may be one of its protective mechanisms.
Five, after 3Gy neutron irradiation, the key kinase IKK beta in the Akt and NF- kappa B signaling pathway in the mouse jejunum was interacted, indicating that the NF- kappa B signal pathway was regulated by the PI3K/Akt signaling pathway after the neutron irradiation.
Six, 4Gy neutrons and 10Gy gamma rays can reduce the proliferation of IEC-6 cells and increase the apoptosis and necrosis rate. LY294002 can further reduce the proliferation activity of IEC-6 cells after irradiation and increase the rate of apoptosis and necrosis.
Seven, both 4Gy neutrons and 10Gy gamma rays can activate the NF- kappa B signaling pathway in IEC-6 cells. The activation of NF- kappa B signaling pathway can be inhibited by PI3K inhibitor LY294002, indicating that NF- kappa B signaling pathway can be activated for IEC-6 cells.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363
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