| AIMS AND SIGNIFICANCENeutron radiation can cause severe damages to the intestine which is hard to recover. Unfortunately, there is still no good cure so far. Our previous study showed that IL-11 could attenuate the intestinal injury and promote recovery of intestinal epithelium. And the protection mechanism of IL-11 was related to the up-regulations of the IL-11 receptors and Jak/STAT activation. But it's still unknown whether ERK and PI3K/Akt signaling pathways induced by IL-11 are involved in the intestinal injury and recovery after neutron irradiation. How do the ERK and PI3K/Akt signaling pathways interact in these processes? Are Bax and Bcl-2 attributed to the cytoprotection of IL-11? All the three questions remain to be answered. In addition, various genes may play a role in the regulation of radiation response of the intestine. Gene chip makes it come true to screening out differentially expressed genomics in a high-flux way and the results maybe offer helpful clues to the next exploration of IL-11 signaling. Therefore, in this study we investigated the above topics and sought to elucidate the molecular mechanism of IL-11 regulation in neutron irradiation-induced intestinal injury, which might help to find new potential therapies.MATERIAS AND METHODS168 BALB/c mice were irradiated by 3 Gy neutron or 10 Gyγirradiation and injected with rhIL-11 at a dose of 500μg/kg body weight once per day for 5 days after irradiation. The mice were sacrificed at 6 h, 1d, 2d, 3d and 5d after irradiation. IEC-6 cells were exposed to 4 Gy neutron or 10 Gyγirradiation and treated with 100ng/ml rhIL-11 12 h prior to or immediately after irradiation. 10μM U0126 or LY294002 was added 12 h prior to IL-11 treatment. Gene chip, RT-PCR, HE staining, MTT, flow cytometry, Hoechst staining, immunohistochemistry, Western Blot and image analysis were used to detect the differentially expressed genes, histopathological changes in the mouse jejunum and determine the effect of IL-11, Uo126 and LY294002 on the intestinal pathology, IEC-6 cellular biological behaviors and expressions and activities of Raf-1, MEK1/2, ERK1/2, PI3K, PDK1, PTEN and Akt.RESULTS1. Survival and intestinal histological changes in the mice: (1) All of the mice received 3 Gy neutron irradiation died in 3~5 d after irradiation; and diarrhea occurred in every irradiated mice and the body weight lost rapidly and progressively. The small intestinal mucosa of the neutron irradiated mice showed marked destruction with villus epithelium decrease, disorder and swelling; cryptal cell number decreased sharply and the crypts were destructed with rare cell number at the 2nd d. The epithelia cells showed acidophily, pyknosis and karuorrhexis. Regeneration was rare at 3d. The mice with IL-11 administration showed significant increase in survival fraction, decrease in diarrhea, more crypt regeneration and villous cells.2. Differentially expressed genes in the murine small intestine: After 6 h of 3 Gy neutron irradiation, there were 608 genes differentially expressed in the small intestine, of which 277 genes were up-regulated and 331 genes were down-regulated. The differentially expressed genes were related to cellular proliferation and apoptosis, MAPK, PI3K/Akt signaling and so on.3. Changes of IEC-6 cellular biological behaviors: (1) At 6 h after 10 Gyγirradiation, IEC-6 cells showed higher rates of apoptosis and necrosis, lower proliferating capability and cell cycle arrest. IL-11 treatment down-regulated the rate of apoptosis and necrosis. (2) At 6 h after 4 Gy neutron irradiation, IEC-6 cells became round in shape and showed higher rates of apoptosis and necrosis and lower proliferating capability; at 24 h after neutron irradiation, the rates of apoptosis and necrosis showed no marked changes but the proliferating capability was still lower than normal. IL-11-treated IEC-6 cells showed better in morphology, lower apoptosis rate and stronger proliferating capability.4. Changes of ERK pathway in IEC-6 cells and effect of U0126 on IEC-6 cellular biological behaviors: (1) The expression and activity of Raf-1 and MEK1/2 increased but ERK1/2 activation decreased in 6~24 h after 10 Gyγirradiation. IL-11 increased the activities of Raf-1 and MEK1/2 in 5~15min and inhibited ERK1/2 activity at 6 h after radiation. (2) The expression and activity of Raf-1 and MEK1/2 showed no significant changes in 6~24 h after 4 Gy neutron irradiation while the expression and activity of ERK1/2 increased. IL-11 treatment didn't change the expression of Raf-1 at 6 h and increased the activity of MEK1/2 in 6~24 h. As well as the expression and activity of ERK1/2 was concerned, IL-11 functioned as inhition at 6 h and promotion at 24 h. (3) Compared with IL-11 treated IEC-6 cells, cells with U0126 added before IL-11 showed lower proliferating capability but no obvious changed in cell death at 6 and 24 h after 4 Gy neutron irradiation.5. Changes of PI3K/Akt pathway in IEC-6 cells and effect of LY294002 on IEC-6 cellular biological behaviors: (1) At 6 h after 10 Gyγirradiation, the expression of PI3K and activation of Akt in IEC-6 cells decreased while the expression of Akt, activities of PDK1 and PTEN increased. IL-11 treatment increased the expression of PI3K and activation of Akt and decreased the expression of Akt, activities of PDK1 and PTEN. (2) After 4 Gy neutron irradiation, the expression of PI3K decreased at 6 h and recovered at 24 h. The activation of PDK1 and PTEN increased, meanwhile the activation of Akt decreased in 6~24 h. IL-11 up-regulated the expression of PI3K and activation of Akt and down-regualted the activation of PDK1 and PTEN. (3) Compared with IL-11 treated IEC-6 cells, cells with LY294002 added before IL-11 showed lower proliferating capability and higher apoptosis and necrosis rate at 6 and 24 h after 4 Gy neutron irradiation.6. Interactions between ERK and PI3K/Akt pathway: (1) Compared with IL-11 treated IEC-6 cells, U0126 brought no significant changes to the expression and activities of ERK1/2 but increased the activations of PDK1and Akt in 6~24 h after neutron irradiation. (2) Compared with IL-11 treated IEC-6 cells, LY294002 decreased the activations of both Akt and ERK1/2 in 6~24 h after neutron irradiation.8. Expressions of Bax and Bcl-2 in the murine small intestine: (1) Immunoreactivity of Bax was granulo-positive in the normal small intestinal villous cytoplasm and weakly positive in the cryptal cytoplasm. After 3 Gy neutron and 10Gyγirradiation, Bax immunoreactivity increased greatly in 6 h~3 d. IL-11 adminstration decreased the expression of Bax at 3 d after 3 Gy neutron irradiation. (2) Immunoreactivity of Bax was weakly positive in the normal small intestinal villous and cryptal cytoplasm. 3 Gy neutron brought no changes to the expression of Bcl-2 in 6 h~3 d. 10Gyγirradiation increased the expression of Bcl-2 in 6 h~3 d.CONCLUSIONS1. Neutron irradiation resulted in injuries to many molecular targets, involved multi pathway like PI3K/Akt and MAPK in the jejunum.2. In IEC-6, ERK1/2 was continuously activated by neutron irradiation. The effect of IL-11 on ERK1/2 activation fluctuated from inhibition to promotion. This suggests ERK played dual roles in different stages of neutron-induced IEC injury.3. PI3K/Akt pathway was inhibited by neutron irradiation and triggered by IL-11 to protect IEC from neutron injury.4. Neutron andγirradiation had different impacts on ERK and PI3K/Akt pathway, which might underlie their different injury response.5. There maybe a feedback loop to connect ERK and PI3K/Akt pathway when IL-11 regulated neutron induced intestinal epithelial injury: (1) Continuously activated ERK1/2 inhibited PI3K/Akt activity, while ERK inhibition synergized with IL-11 to promote PI3K/Akt activation; (2) The activity of ERK1/2 was regulated by PI3K and independent on MEK1/2.6. LY294002 was more effective than U0126 to block IL-11 protection the intestinal epithelium from neutron irradiation. This suggests that ERK and PI3K/Akt signaling played different roles in mediating the cytoprotection by IL-11: ERK was mainly associated to cellular proliferation and PI3K/Akt was involved both cellular proliferation and death.7. IL-11 decreased the ratio of Bax/Bcl-2 to confer protection on the intestine exposed to neutron irradiation.
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