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
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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P<0.05,**示P<0.01;vs照射组,#示P<0.05,##示P<0.01。
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(P<0.01),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(P<0.05)。
(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(P<0.01),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(P<0.05)。
(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(P<0.05),照射后3d和5d表达明显上调(P<0.01);Cur组在照射后3d和5d,NF-κB和IKKβ表达下调(P<0.05)。(2)小鼠空肠组织IκBα蛋白于3Gy中子照射后6h和1d表达下调(P<0.05),照射后3d和5d表达明显下调(P<0.01);Cur组在照射后3d和5d,IκBα表达上调(P<0.05)。(3)3Gy中子照射后6h小鼠空肠组织IKKβmRNA表达增加(P<0.05),至照射后24h表达明显增加(P<0.01);Cur组在照射后6h和24h,IKKβmRNA表达水平较N组明显降低(P<0.05)。
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(P<0.05),照射后3d和5d表达明显上调(P<0.01);Cur组在照射后3d和5d,PI3K和Akt表达下调(P<0.05)。
(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(P<0.01),照射后3d和5d,Akt和IKKβ的相互作用减弱(P<0.05);Cur组在照射后3d和5d,Akt和IKKβ相互作用较照射组减弱(P<0.05)。
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(P<0.01),细胞凋亡与坏死率均显著增加(P<0.01),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(P<0.05),凋亡和坏死率增加(P<0.05)。
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(P<0.05),24h表达明显上调(P<0.01);应用LY294002处理组,于照射后6h和24h,上述蛋白表达均下调(P<0.05)。(2)IκBα于4Gy中子照射后6h表达下调(P<0.05),24h表达明显下调(P<0.01);应用LY294002处理组,于照射后6h和24h,IκBα表达上调(P<0.05)。
(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(P<0.05),照射后1h表达明显上调(P<0.01);应用LY294002处理组,三种磷酸化蛋白的表达均下调(P<0.05)。(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(P<0.01);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(P<0.01)。
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。
AIMS AND SIGNIFICANCE Neutron irradiation can cause bodies more severe damages thanγrays. Intestine is highly sensitive to neutron radiation. Intestine is severely injuried by neutron irradiation and is hard to recovers. As we all know, the pathological change of intestine induced by neutron irradiation is on the whole elucidated, while the mechanisms of the injury were not elucidated completely. Unfortunately, there is still no good cure so far. Therefore, this study was based on the previous studies. Intestinal epithelial cell(IEC) model in vitro and vivo injuried by neutron irradiation are made. In this study, we investigated NF-κB signaling pathway in the regulation of intestinal epithelial injury induced by neutron irradiation. This can be sought to elucidate the molecular mechanism of neutron irradiation-induced intestinal injury, which might help to find new potential therapies.
MATERIAS AND METHODS
1. Animal grouping and model-making method
120 BALB/c mice were randomly divided into four groups: 30 of control group(C), 60 of neutron irradiation dose of 3Gy group(N), 30 of curcumin treatment group(Cur). The mice of N and Cur group were wholly irradiated by neutron irradiation of 3Gy. The mice of Cur group were injected through enterocoelia with curcumin at a dose of 200 mg·kg-1·d-1 body weight once per day for 5 days after irradiation. The same volume sodium chloride was injected through enterocoelia into the mice of C and N group.
2. The overall behaviors of the mice, histology and ultrastructure of the mice jejunums observation
The overall behaviors, body weight changes, diarrhea and mortality rates of the mice were observed after exposure to neutron irradiation. The mice of C and N group were sacrificed at 6 h, 1d, 3d and 5d after neutron irradiation. The mice of Cur group were sacrificed at 3d and 5d after neutron irradiation. The jejunums of the mice were stored in the -80℃refrigerator. The histology and ultrastructure of the mice jejunums were observed by means of light microscope and transmission electron microscope after neutron irradiation and therapied of curcumin.
3. Proliferation and death of the jejunum epithelial cells of the mice assay
The contents of argyrophilic of nucleaolar organizer regions(AgNOR) and DNA in the intestinal epithelial after neutron irradiation and therapied of curcumin were detected by means of AgNOR staining and Feulgen staining. The apoptosis of the jejunum epithelial cells were assayed through terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling(TUNEL) after neutron irradiation and therapied of curcumin.
4. NF-κB signaling pathway molecules in the jejunum tissues of the mice assay
The expression and activity of NF-κB in the jejunum tissues of the mice were observed by means of immunohistochemistry(IHC) staining and electrophoretic mobility shift assay(EMSA). The expressiones of NF-κB, IKKβ, IκBα, PI3K and Akt in the jejunum tissues were assayed by western blot. The mRNA expression of IKKβin the jejunum tissues were detected by means of Real-time PCR. The interaction between Akt and IKKβin the jejunum tissues were assayed through co-immunoprecipitation(Co-IP).
5. IEC-6 cells culture, group, irradiation and treatment
IEC-6 cells were subcultured and randomly divided into five groups: control group(C), 4Gy neutron irradiation group(N), 4Gy neutron irradiation + LY294002 treatment group(LY), 10Gyγray irradiation group(R), 10Gyγray irradiation + LY294002 treatment group(LY2). The IEC-6 cells of N and LY group were exposed to neutron irradiation of 4Gy. The IEC-6 cells of R and LY2 group were radiated byγray irradiation of 10Gy. The IEC-6 cells of LY and LY2 group were treated by 10μmol/L LY294002 at 24 hours before neutron andγray irradiation.
6. Proliferation and death of the IEC-6 cells assay
The morphologic changes of the IEC-6 cells were observed by inverted phase contrast microscope (IPCM) at 6h and 24h after neutron andγray irradiation. The proliferation activity, apoptosis and necrosis of the IEC-6 cells were detected by MTT colorimetry and flow cytometry(FCM) at 6h and 24h after neutron irradiation of 4Gy. These datas were assayed by MTT colorimetry and FCM at 15min, 30min, 1h, 6h and 24h afterγray irradiation of 10Gy.
7. NF-κB signaling pathway molecules in the IEC-6 cells assay
The expressiones of NF-κB and phos-NF-κB, IKKα/βand phos-IKKα/β, IκBαand phos-IκBαin the IEC-6 cells were assayed by means of western blot after neutron andγray irradiation.
8. Image analysis and quantitative methods
(1) The MOD and IOD of AgNOR and DNA content, expression of NF-κB in in the jejunum tissues of the mice were analyzed by CMIAS-Ⅱmultifunctional true color pathological image analysis system.
(2) The results of western blot were analyzed by means of Image Pro 5.0 soft. The IOD ratio of the electrophoretic bands of interest proteins and GAPDH were quantitatively analyzed.
(3) The mRNA expressiones of IKKβand GAPDH in the jejunum tissues were analyzed by 7300 system SDS soft.
(4) The results of Co-IP were analyzed by CMIAS-Ⅱpathological image analysis system.
9. Statistical Analysis
The datas were described using of mean and standard deviation (?X±s) which were analyzed significant differences by means of the SPSS13.0 software one-way ANOVA test. vs control group, * is P<0.05, ** is P<0.01; vs irradiation group, # is P<0.05, ## is P<0.01. Values of P<0.05 were considered statistically significant.
RESULTS
1. The changes of the mice overall behaviors, histology and ultrastructure of the jejunums
(1) The changes of the mice overall behaviors: The mice mental state became worse and worse, and ate or drunk nothing, and them bodies weight decreased obviously, and all of the mice had severe diarrhea symptom after neutron irradiation of 3Gy and therapied of curcumin. All of the mice died at 3~5 day after neutron irradiation of 3Gy. It was the feature of intestinal radiation sickness which named "three days and a half effect".
(2) The changes of the mice jejunums histology: After neutron irradiation of 3Gy, the small intestinal mucosa of the neutron irradiated mice showed marked destruction with villus epithelium decrease, disorder and swelling; cryptal cells number decreased sharply and the crypts were destructed with rare cell number. The epithelia and cryptal cells showed acidophily, pyknosis and karyorrhexis. Regeneration was rare at 3d and 5d. The curcumin treatment group cryptal regenerated obviously at 3d and 5d and there were lots of intestine villis in the enteric cavity.
(3) The changes of the mice jejunums ultrastructure: The number of the intestine crypts decreased obviously. The epithelia cells showed karyorrhexis and chromatin condensation edging. The cryptal cells showed vacuolization. Apoptosisi and necrosis cells were found and necrosis cells were more than apoptosis cells. The intestine villis regenerated gently at 5d after neutron irradiation of 3Gy. Microvillus, novo-crypts and more Pents particles were found in the curcumin treatment group.
2. The changes of the mice jejunums proliferation and death
(1) The changes of the AgNOR and DNA content in the mice jejunums: The contents of AgNOR and DNA in the mice jejunums were decreased progressively at 6h~5d after neutron irradiation of 3Gy (P<0.01). Those of the curcumin treatment group were more higher than the irradiation group at 3~5d after neutron irradiation of 3Gy (P<0.05).
(2) The changes of apoptosis in the mice jejunums epithelium: Lots of apoptosis cells were found in the mice jejunums epithelium of irradiation group at 6h~1d after neutron irradiation. There were scarcely apoptosis cells at 3d and 5d after neutron irradiation of 3Gy.
3. The changes of NF-κB signaling pathway molecules in the jejunum tissues
(1) The changes of the expression of NF-κB in the intestinal epithelial cells: NF-κB was weakly positive expression in the normal intestinal villi and crypt epithelial cells intracytoplasm, and positive expressiones in the intestinal villi epithelial cells nucleus and the value showed peak at 5d (P<0.01) after neutron irradiation of 3Gy. The expressions of NF-κB in the curcumin treatment group were weaker than those of the irradiation group (P<0.05).
(2) The changes of NF-κB and DNA binding activity in the mice jejunum tissues: NF-κB and DNA binding activity in the mice jejunum tissues was increased obviously at 6h~1d after neutron irradiation of 3Gy. The data in the curcumin treatment group were fewer than those of the irradiation group.
(3) The changes of NF-κB signaling pathway molecules in the jejunum tissues:①The expressiones of NF-κB and IKKβwere increased at 6h and 1d (P<0.05), and incresed obviously at 3d and 5d (P<0.01) after neutron irradiation of 3Gy. The expressiones of them in the curcumin treatment group were decreased at 3d and 5d (P<0.05) after neutron irradiation of 3Gy.②The expression of IκBαwas decreased at 6h and 1d (P<0.05), decresed obviously at 3d and 5d (P<0.01) and increased at 3d and 5d (P<0.05) in the curcumin treatment group after neutron irradiation of 3Gy.③The mRNA expression level of IKKβwas increased at 6h (P<0.05) and incresed obviously at 24h (P<0.01) after neutron irradiation of 3Gy. The mRNA expression level of IKKβin the curcumin treatment group were lower than those of the irradiation group (P<0.05) after neutron irradiation of 3Gy.
4. The changes of PI3K and Akt in the mice jejunum tissues and the interaction between Akt and IKKβ
(1) The changes of PI3K and Akt in the mice jejunum tissues: The expressiones of PI3K and Akt were increased at 6h and 1d (P<0.05), incresed obviously at 3d and 5d (P<0.01) and decreased at 3d and 5d in the curcumin treatment group (P<0.05) after neutron irradiation of 3Gy.
(2) The interaction between Akt and IKKβin the mice jejunum tissues: The interaction between Akt and IKKβwas increased obviously at 6h and 1d (P<0.01), decresed at 3d and 5d (P<0.05) and decreased at 3d and 5d in the curcumin treatment group (P<0.05) after neutron irradiation of 3Gy.
5. The changes of the IEC-6 cells morphology, proliferation and death
(1) The changes of the IEC-6 cells morphology: The IEC-6 cells swelled and beame rounded and their refraction ability enhanced after neutron andγray irradiation. Those features in the LY294002 treatment groups were worse than those in the irradiation groups. Lots of dead cells were found floating in the culture solution.
(2) The changes of the IEC-6 cells proliferation and death: The IEC-6 cells proliferation activity was decreased obviously at after neutron andγray irradiation (P<0.01). The IEC-6 cells apoptosis and necrosis rate was increased obviously after irradiation (P<0.01). The IEC-6 cells showed apoptosis peak at 6h and necrosis was chief at 24h after irradiation. The IEC-6 cells proliferation activity in the LY294002 treatment groups was lower than those in the irradiation groups (P<0.05). Their apoptosis and necrosis rate in the LY294002 treatment groups was higher than those in the irradiation groups (P<0.05).
6. The changes of the IEC-6 cells NF-κB signaling pathway molecules
(1) The changes of the IEC-6 cells NF-κB signaling pathway molecules:①The expressiones of NF-κB, IKKαand IKKβwere increased at 6h (P<0.05), incresed obviously at 24h (P<0.01) and decreased at 6h and 24h in the LY294002 treatment group (P<0.05) after neutron irradiation of 3Gy.②The expressiones of IκBαwere decreased at 6h (P<0.05), decresed obviously at 24h (P<0.01) and increased at 6h and 24h in the LY294002 treatment group (P<0.05) after neutron irradiation of 3Gy.
(2) The changes of the proteins phosphorylation in the IEC-6 cells NF-κB signaling pathway:①The expressiones of phos-NF-κB and phos-IKKα/βwere increased at 15~30min (P<0.05), incresed obviously at 1h (P<0.01) and decreased in the LY294002 treatment group (P<0.05) afterγirradiation of 10Gy.②The expressiones of phos-IκBαwere not found at 15~30min, incresed at 1h (P<0.01) and incresed obviously in the LY294002 treatment group (P<0.01) afterγirradiation of 10Gy.
CONCLUSIONS
1. Neutron irradiation of 3Gy caused the mice gently intestinal type of radiation sickness. The intestinal epitheliums were injuried seriously. The intestinal epithelium cells showed apoptosis and necrosis and their proliferation ability decreased. The intestinal damage animal model were made successfully by neutron irradiation of 3Gy.
2. Curcumin can relieve the degree of intestine damage caused by neutron irradiation. It can promote regeneration and repair of intestinal mucosa and protect the intestinal epitheliums injuried by neutron irradiation.
3. The NF-κB signaling pathway in the mice jejunums were activied by neutron irradiation. Upregulated NF-κB showed nuclear translocation. It maybe regulate the inflammation factors which led to intestinal inflammation caused by neutron irradiation.
4. Curcumin can inhibit the activition of NF-κB signaling pathway in the mice jejunums caused by neutron irradiation and decrease the expression of NF-κB and IKKβ, which maybe one of the protetive mechanisms.
5. The interaction between Akt and IKKβwas found in the mice jejunums after neutron irradiation of 3Gy, which indicates NF-κB signaling pathway is regulated by PI3K/Akt signaling pathway.
6. The IEC-6 cells proliferation activity was decreased after neutron irradiation of 4Gy andγray irradiation of 10Gy, while the apoptosis and necrosis rate of the IEC-6 cells increased obviously after neutron andγray irradiation. LY294002 can aggravate the IEC-6 cells damage caused by neutron andγray irradiation.
7. NF-κB signaling pathway in the IEC-6 cells can be activited by neutron irradiation of 4Gy andγray irradiation of 10Gy. LY294002, inhibitor of PI3K, can inhibite the activition of NF-κB signaling pathway caused by neutron andγray irradiation. It indicates that the activition of NF-κB signaling pathway can protect IEC-6 cells injuried by neutron andγray irradiation.
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P<0.05,**示P<0.01;vs照射组,#示P<0.05,##示P<0.01。
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(P<0.01),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(P<0.05)。
(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(P<0.01),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(P<0.05)。
(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(P<0.05),照射后3d和5d表达明显上调(P<0.01);Cur组在照射后3d和5d,NF-κB和IKKβ表达下调(P<0.05)。(2)小鼠空肠组织IκBα蛋白于3Gy中子照射后6h和1d表达下调(P<0.05),照射后3d和5d表达明显下调(P<0.01);Cur组在照射后3d和5d,IκBα表达上调(P<0.05)。(3)3Gy中子照射后6h小鼠空肠组织IKKβmRNA表达增加(P<0.05),至照射后24h表达明显增加(P<0.01);Cur组在照射后6h和24h,IKKβmRNA表达水平较N组明显降低(P<0.05)。
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(P<0.05),照射后3d和5d表达明显上调(P<0.01);Cur组在照射后3d和5d,PI3K和Akt表达下调(P<0.05)。
(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(P<0.01),照射后3d和5d,Akt和IKKβ的相互作用减弱(P<0.05);Cur组在照射后3d和5d,Akt和IKKβ相互作用较照射组减弱(P<0.05)。
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(P<0.01),细胞凋亡与坏死率均显著增加(P<0.01),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(P<0.05),凋亡和坏死率增加(P<0.05)。
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(P<0.05),24h表达明显上调(P<0.01);应用LY294002处理组,于照射后6h和24h,上述蛋白表达均下调(P<0.05)。(2)IκBα于4Gy中子照射后6h表达下调(P<0.05),24h表达明显下调(P<0.01);应用LY294002处理组,于照射后6h和24h,IκBα表达上调(P<0.05)。
(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(P<0.05),照射后1h表达明显上调(P<0.01);应用LY294002处理组,三种磷酸化蛋白的表达均下调(P<0.05)。(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(P<0.01);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(P<0.01)。
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。
AIMS AND SIGNIFICANCE Neutron irradiation can cause bodies more severe damages thanγrays. Intestine is highly sensitive to neutron radiation. Intestine is severely injuried by neutron irradiation and is hard to recovers. As we all know, the pathological change of intestine induced by neutron irradiation is on the whole elucidated, while the mechanisms of the injury were not elucidated completely. Unfortunately, there is still no good cure so far. Therefore, this study was based on the previous studies. Intestinal epithelial cell(IEC) model in vitro and vivo injuried by neutron irradiation are made. In this study, we investigated NF-κB signaling pathway in the regulation of intestinal epithelial injury induced by neutron irradiation. This can be sought to elucidate the molecular mechanism of neutron irradiation-induced intestinal injury, which might help to find new potential therapies.
MATERIAS AND METHODS
1. Animal grouping and model-making method
120 BALB/c mice were randomly divided into four groups: 30 of control group(C), 60 of neutron irradiation dose of 3Gy group(N), 30 of curcumin treatment group(Cur). The mice of N and Cur group were wholly irradiated by neutron irradiation of 3Gy. The mice of Cur group were injected through enterocoelia with curcumin at a dose of 200 mg·kg-1·d-1 body weight once per day for 5 days after irradiation. The same volume sodium chloride was injected through enterocoelia into the mice of C and N group.
2. The overall behaviors of the mice, histology and ultrastructure of the mice jejunums observation
The overall behaviors, body weight changes, diarrhea and mortality rates of the mice were observed after exposure to neutron irradiation. The mice of C and N group were sacrificed at 6 h, 1d, 3d and 5d after neutron irradiation. The mice of Cur group were sacrificed at 3d and 5d after neutron irradiation. The jejunums of the mice were stored in the -80℃refrigerator. The histology and ultrastructure of the mice jejunums were observed by means of light microscope and transmission electron microscope after neutron irradiation and therapied of curcumin.
3. Proliferation and death of the jejunum epithelial cells of the mice assay
The contents of argyrophilic of nucleaolar organizer regions(AgNOR) and DNA in the intestinal epithelial after neutron irradiation and therapied of curcumin were detected by means of AgNOR staining and Feulgen staining. The apoptosis of the jejunum epithelial cells were assayed through terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling(TUNEL) after neutron irradiation and therapied of curcumin.
4. NF-κB signaling pathway molecules in the jejunum tissues of the mice assay
The expression and activity of NF-κB in the jejunum tissues of the mice were observed by means of immunohistochemistry(IHC) staining and electrophoretic mobility shift assay(EMSA). The expressiones of NF-κB, IKKβ, IκBα, PI3K and Akt in the jejunum tissues were assayed by western blot. The mRNA expression of IKKβin the jejunum tissues were detected by means of Real-time PCR. The interaction between Akt and IKKβin the jejunum tissues were assayed through co-immunoprecipitation(Co-IP).
5. IEC-6 cells culture, group, irradiation and treatment
IEC-6 cells were subcultured and randomly divided into five groups: control group(C), 4Gy neutron irradiation group(N), 4Gy neutron irradiation + LY294002 treatment group(LY), 10Gyγray irradiation group(R), 10Gyγray irradiation + LY294002 treatment group(LY2). The IEC-6 cells of N and LY group were exposed to neutron irradiation of 4Gy. The IEC-6 cells of R and LY2 group were radiated byγray irradiation of 10Gy. The IEC-6 cells of LY and LY2 group were treated by 10μmol/L LY294002 at 24 hours before neutron andγray irradiation.
6. Proliferation and death of the IEC-6 cells assay
The morphologic changes of the IEC-6 cells were observed by inverted phase contrast microscope (IPCM) at 6h and 24h after neutron andγray irradiation. The proliferation activity, apoptosis and necrosis of the IEC-6 cells were detected by MTT colorimetry and flow cytometry(FCM) at 6h and 24h after neutron irradiation of 4Gy. These datas were assayed by MTT colorimetry and FCM at 15min, 30min, 1h, 6h and 24h afterγray irradiation of 10Gy.
7. NF-κB signaling pathway molecules in the IEC-6 cells assay
The expressiones of NF-κB and phos-NF-κB, IKKα/βand phos-IKKα/β, IκBαand phos-IκBαin the IEC-6 cells were assayed by means of western blot after neutron andγray irradiation.
8. Image analysis and quantitative methods
(1) The MOD and IOD of AgNOR and DNA content, expression of NF-κB in in the jejunum tissues of the mice were analyzed by CMIAS-Ⅱmultifunctional true color pathological image analysis system.
(2) The results of western blot were analyzed by means of Image Pro 5.0 soft. The IOD ratio of the electrophoretic bands of interest proteins and GAPDH were quantitatively analyzed.
(3) The mRNA expressiones of IKKβand GAPDH in the jejunum tissues were analyzed by 7300 system SDS soft.
(4) The results of Co-IP were analyzed by CMIAS-Ⅱpathological image analysis system.
9. Statistical Analysis
The datas were described using of mean and standard deviation (?X±s) which were analyzed significant differences by means of the SPSS13.0 software one-way ANOVA test. vs control group, * is P<0.05, ** is P<0.01; vs irradiation group, # is P<0.05, ## is P<0.01. Values of P<0.05 were considered statistically significant.
RESULTS
1. The changes of the mice overall behaviors, histology and ultrastructure of the jejunums
(1) The changes of the mice overall behaviors: The mice mental state became worse and worse, and ate or drunk nothing, and them bodies weight decreased obviously, and all of the mice had severe diarrhea symptom after neutron irradiation of 3Gy and therapied of curcumin. All of the mice died at 3~5 day after neutron irradiation of 3Gy. It was the feature of intestinal radiation sickness which named "three days and a half effect".
(2) The changes of the mice jejunums histology: After neutron irradiation of 3Gy, the small intestinal mucosa of the neutron irradiated mice showed marked destruction with villus epithelium decrease, disorder and swelling; cryptal cells number decreased sharply and the crypts were destructed with rare cell number. The epithelia and cryptal cells showed acidophily, pyknosis and karyorrhexis. Regeneration was rare at 3d and 5d. The curcumin treatment group cryptal regenerated obviously at 3d and 5d and there were lots of intestine villis in the enteric cavity.
(3) The changes of the mice jejunums ultrastructure: The number of the intestine crypts decreased obviously. The epithelia cells showed karyorrhexis and chromatin condensation edging. The cryptal cells showed vacuolization. Apoptosisi and necrosis cells were found and necrosis cells were more than apoptosis cells. The intestine villis regenerated gently at 5d after neutron irradiation of 3Gy. Microvillus, novo-crypts and more Pents particles were found in the curcumin treatment group.
2. The changes of the mice jejunums proliferation and death
(1) The changes of the AgNOR and DNA content in the mice jejunums: The contents of AgNOR and DNA in the mice jejunums were decreased progressively at 6h~5d after neutron irradiation of 3Gy (P<0.01). Those of the curcumin treatment group were more higher than the irradiation group at 3~5d after neutron irradiation of 3Gy (P<0.05).
(2) The changes of apoptosis in the mice jejunums epithelium: Lots of apoptosis cells were found in the mice jejunums epithelium of irradiation group at 6h~1d after neutron irradiation. There were scarcely apoptosis cells at 3d and 5d after neutron irradiation of 3Gy.
3. The changes of NF-κB signaling pathway molecules in the jejunum tissues
(1) The changes of the expression of NF-κB in the intestinal epithelial cells: NF-κB was weakly positive expression in the normal intestinal villi and crypt epithelial cells intracytoplasm, and positive expressiones in the intestinal villi epithelial cells nucleus and the value showed peak at 5d (P<0.01) after neutron irradiation of 3Gy. The expressions of NF-κB in the curcumin treatment group were weaker than those of the irradiation group (P<0.05).
(2) The changes of NF-κB and DNA binding activity in the mice jejunum tissues: NF-κB and DNA binding activity in the mice jejunum tissues was increased obviously at 6h~1d after neutron irradiation of 3Gy. The data in the curcumin treatment group were fewer than those of the irradiation group.
(3) The changes of NF-κB signaling pathway molecules in the jejunum tissues:①The expressiones of NF-κB and IKKβwere increased at 6h and 1d (P<0.05), and incresed obviously at 3d and 5d (P<0.01) after neutron irradiation of 3Gy. The expressiones of them in the curcumin treatment group were decreased at 3d and 5d (P<0.05) after neutron irradiation of 3Gy.②The expression of IκBαwas decreased at 6h and 1d (P<0.05), decresed obviously at 3d and 5d (P<0.01) and increased at 3d and 5d (P<0.05) in the curcumin treatment group after neutron irradiation of 3Gy.③The mRNA expression level of IKKβwas increased at 6h (P<0.05) and incresed obviously at 24h (P<0.01) after neutron irradiation of 3Gy. The mRNA expression level of IKKβin the curcumin treatment group were lower than those of the irradiation group (P<0.05) after neutron irradiation of 3Gy.
4. The changes of PI3K and Akt in the mice jejunum tissues and the interaction between Akt and IKKβ
(1) The changes of PI3K and Akt in the mice jejunum tissues: The expressiones of PI3K and Akt were increased at 6h and 1d (P<0.05), incresed obviously at 3d and 5d (P<0.01) and decreased at 3d and 5d in the curcumin treatment group (P<0.05) after neutron irradiation of 3Gy.
(2) The interaction between Akt and IKKβin the mice jejunum tissues: The interaction between Akt and IKKβwas increased obviously at 6h and 1d (P<0.01), decresed at 3d and 5d (P<0.05) and decreased at 3d and 5d in the curcumin treatment group (P<0.05) after neutron irradiation of 3Gy.
5. The changes of the IEC-6 cells morphology, proliferation and death
(1) The changes of the IEC-6 cells morphology: The IEC-6 cells swelled and beame rounded and their refraction ability enhanced after neutron andγray irradiation. Those features in the LY294002 treatment groups were worse than those in the irradiation groups. Lots of dead cells were found floating in the culture solution.
(2) The changes of the IEC-6 cells proliferation and death: The IEC-6 cells proliferation activity was decreased obviously at after neutron andγray irradiation (P<0.01). The IEC-6 cells apoptosis and necrosis rate was increased obviously after irradiation (P<0.01). The IEC-6 cells showed apoptosis peak at 6h and necrosis was chief at 24h after irradiation. The IEC-6 cells proliferation activity in the LY294002 treatment groups was lower than those in the irradiation groups (P<0.05). Their apoptosis and necrosis rate in the LY294002 treatment groups was higher than those in the irradiation groups (P<0.05).
6. The changes of the IEC-6 cells NF-κB signaling pathway molecules
(1) The changes of the IEC-6 cells NF-κB signaling pathway molecules:①The expressiones of NF-κB, IKKαand IKKβwere increased at 6h (P<0.05), incresed obviously at 24h (P<0.01) and decreased at 6h and 24h in the LY294002 treatment group (P<0.05) after neutron irradiation of 3Gy.②The expressiones of IκBαwere decreased at 6h (P<0.05), decresed obviously at 24h (P<0.01) and increased at 6h and 24h in the LY294002 treatment group (P<0.05) after neutron irradiation of 3Gy.
(2) The changes of the proteins phosphorylation in the IEC-6 cells NF-κB signaling pathway:①The expressiones of phos-NF-κB and phos-IKKα/βwere increased at 15~30min (P<0.05), incresed obviously at 1h (P<0.01) and decreased in the LY294002 treatment group (P<0.05) afterγirradiation of 10Gy.②The expressiones of phos-IκBαwere not found at 15~30min, incresed at 1h (P<0.01) and incresed obviously in the LY294002 treatment group (P<0.01) afterγirradiation of 10Gy.
CONCLUSIONS
1. Neutron irradiation of 3Gy caused the mice gently intestinal type of radiation sickness. The intestinal epitheliums were injuried seriously. The intestinal epithelium cells showed apoptosis and necrosis and their proliferation ability decreased. The intestinal damage animal model were made successfully by neutron irradiation of 3Gy.
2. Curcumin can relieve the degree of intestine damage caused by neutron irradiation. It can promote regeneration and repair of intestinal mucosa and protect the intestinal epitheliums injuried by neutron irradiation.
3. The NF-κB signaling pathway in the mice jejunums were activied by neutron irradiation. Upregulated NF-κB showed nuclear translocation. It maybe regulate the inflammation factors which led to intestinal inflammation caused by neutron irradiation.
4. Curcumin can inhibit the activition of NF-κB signaling pathway in the mice jejunums caused by neutron irradiation and decrease the expression of NF-κB and IKKβ, which maybe one of the protetive mechanisms.
5. The interaction between Akt and IKKβwas found in the mice jejunums after neutron irradiation of 3Gy, which indicates NF-κB signaling pathway is regulated by PI3K/Akt signaling pathway.
6. The IEC-6 cells proliferation activity was decreased after neutron irradiation of 4Gy andγray irradiation of 10Gy, while the apoptosis and necrosis rate of the IEC-6 cells increased obviously after neutron andγray irradiation. LY294002 can aggravate the IEC-6 cells damage caused by neutron andγray irradiation.
7. NF-κB signaling pathway in the IEC-6 cells can be activited by neutron irradiation of 4Gy andγray irradiation of 10Gy. LY294002, inhibitor of PI3K, can inhibite the activition of NF-κB signaling pathway caused by neutron andγray irradiation. It indicates that the activition of NF-κB signaling pathway can protect IEC-6 cells injuried by neutron andγray irradiation.
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