The Study Of NF-κB Signaling Pathway Regulation Mechanism In Intestinal Epithelial Cells Injury Induced By Neutron Irradiation

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 METHODS1. Animal grouping and model-making method120 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 observationThe 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 assayThe 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 assayThe 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 treatmentIEC-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 assayThe 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 assayThe 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 AnalysisThe 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.RESULTS1. 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.CONCLUSIONS1. 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.