Protective Mechanisms Of Rapamycin On Radiation-induced Intestinal Mucosal Injury In Mice

BACKGROUND It is well known that the digestive system and hematopoietic system in the body are very sensitive to radiation.In clinic,many cancer patients who receive radiation treatment are often accompanied by acute and chronic intestinal and bone marrow injury,which limits the successful treatment of cancer.In the case of nuclear accident or nuclear terrorist attack,the primarily damaged system is also the digestive system and hematopoiesis system,resulting in acute radiation syndrome.Although there are several drugs to alleviate the radiation-induced acute hematopoietic system damage,such as Amifostine and Filgrastim,which have been used in clinical practice,there is no recognized drug to effectively alleviate the radiation-induced acute intestinal damage.The main reason is that the mechanism of radiation-induced intestinal damage remains unknown.It is necessary to find new approaches through understanding potential damage mechanisms under irradiation settings.mTORC1 signaling pathway,as the hub of multiple pathways in cells,is involved in many important physiological and pathological processes,such as insulin/insulin receptor pathway,PI3K/Akt pathway and AMPK pathway.But is mTORC1 signaling pathway involved in radiation-induced intestinal damage?Whether changing the status of mTORC1 signaling pathway can protect intestine from irradiation is worthy of further investigation.In this project,the survival and pathological changes of small intestinal mucosa of mice after different doses of whole-body X-ray radiation were observed,which benefits to determine the proper radiation dose in the follow-up experiment(Part I).whole-body X-ray irradiation was utilized to understand the role of mTORC1signaling pathway in mouse small intestinal crypt cells.The autophagy,apoptosis,NF-κB signaling pathway,DNA damage and other pathophysiological changes,which are related to mTORC1 signaling pathway,were observed(Part II).Rapamycin was used to temporarily inhibit the mTORC1 signaling pathway activated by radiation to observe its effect on mouse survival and intestinal mucosal mechanical barrier.The effects of rapamycin on the intestinal stem cell injury was further confirmed the role of mTORC1 signaling pathway(Part III).To further explore the mechanisms of rapamycin inhibiting mTORC1 signaling pathway on the protection of intestinal radiation damage,autophagy,apoptosis,NF-κB signaling pathway and DNA damage repair were investigated(Part IV).Part Ⅰ Damage of intestinal mucosa in mice induced by different doses of X-ray radiationObjective:To observe irradiation-induced damage on the mechanical barrier of intestinal mucosa and intestinal stem cells in a radiation mouse model induced by whole-body X-ray irradiation.Methods:120 8-week C57BL/6 male mice were randomly divided into 4 groups:4Gy group,8 Gy group,12 Gy group and normal control group.The total body radiation was performed in Elekta Precise instrument at 4 Gy,8 Gy and 12 Gy with a radiation rate of 2.28 Gy/min.The mice in the normal control group were not radiated.40 mice were used to observe the survival of each group within 30 days after exposure.80 mice were taken after irradiation.The observation time points of each group were 6h,1d,2d,3d and 7d.The changes of intestinal permeability were observed by hematoxylin eosin(H-E)staining,ultrastructural changes of jejunal mucosal epithelial cells,distribution and changes of Olfm4~+small intestinal stem cells,Lysozyme~+Paneth cells.The distribution of ZO-1 and Occludin were observed by immunohistochemistry.The expression of ZO-1 and Occludin was detected by Western blot.Results:1.With the increase of X-ray radiation dose,the weight of mice decreased.Within 30days after radiation,all mice in 12 Gy group died within 5 days,mice in 8 Gy group died within 14 days,and mice in 4 Gy group survived.2.After different doses of X-ray radiation,the villi of jejunum and ileum showed lodging,shortening,epithelial shedding,telangiectasia and hemorrhage in the lamina propria.The ultrastructures of microvilli and mitochondria in intestinal epithelial cells were damaged post exposure.The damage under 12 Gy radiation was the most serious.With the increase of radiation dose,the pathological injury scores of jejunum and ileum mucosa increased(P<0.01).3.Compared with the normal group,the activity of DAO in serum of mice was increased significantly at the 2nd day after 8 Gy and 12 Gy of X-ray irradiation,which was dose and time-dependent(P<0.01).4.Compared with the normal group,the numbers of Olfm4~+intestinal stem cells in the crypts of jejunum and ileum were decreased at 6 hours and 1 day after radiation(P<0.01).2 and 3 days after radiation,the numbers of Olfm4~+intestinal stem cells were sharply reduced in 12 Gy group(P<0.01).The numbers of intestinal stem cells were increased on 3 days in 4 Gy and 8 Gy groups.5.In comparison to the normal group,the numbers of goblet cells in jejunal villi were significantly decreased on 2 and 3 days after 8 Gy and 12 Gy radiation(P<0.01).The numbers of goblet cells in ileal villi were significantly decreased on 2 and 3 days after8 Gy and 12 Gy radiation(P<0.01).The numbers of endocrine cells in jejunal villi were decreased on 2 and 3 days after 8 Gy and 12 Gy radiation.The numbers of Lysozyme~+cells in jejunal crypt were remarkably increased on 2 and 3 days after 8Gy radiation(P<0.01).The number of Lysozyme~+cells in jejunum and ileum were significantly decreased on 3 days after 12 Gy radiation and 7 days after 8 Gy radiation(P<0.01 or P<0.05).6.The expression of ZO-1 and Occludin in intestinal mucosa were significantly decreased with abnormal distribution after 8 Gy radiation(P<0.01).Conclusion:Different doses of X-ray radiation not only damage jejunum and ileum mucosa,the mechanical barrier of intestinal mucosa,but also reduce the numbers and differentiation of intestinal stem cells.Part Ⅱ The role of mTORC1 signaling pathway in radiation-induced intestinal mucosal injury in miceObjective: To observe the effects of radiation on mTORC1 signaling pathway,NF-κB signaling pathway and DNA damage in mouse intestinal crypt cells and explore the role of mTORC1 signaling pathway in radiation-induced intestinal mucosal damage.Methods: 85 male C57 BL / 6 mice were randomly divided into four groups: 4 Gy group,8 Gy group,12 Gy group and normal control group.The total body radiation was performed in Elekta Precise linear accelerator at 4 Gy,8 Gy and 12 Gy with a radiation rate of 2.28 Gy/min.The mice in the normal control group were not radiated.The observation time point of mice in the radiation group was 6h,1d,2d,3d and 7d,in which 8 Gy dose was added the observation time point at 12 h.The mice in each group were intraperitoneally injected with Brd U solution(100mg/kg)two hours before sacrifice,and the jejunum samples were collected after anesthesia.The expression of pS6,Brd U~+ proliferating cells,the numbers of γ-H2 AX and 53BP1 positive foci in the cell nucleus were examined by immunohistochemistry staining.The expression of pS6 in Olfm4~+ intestinal stem cells were demonstrated by double immunofluorescence staining.The changes of apoptotic cells in the intestinal crypt were observed by TUNEL staining.Western blotting was used to measure the expression of S6,pS6,mTOR,pmTOR,LC3 B,p62,Akt,pAkt,p65 and pp65.Results: 1.The integral optical density of pS6 protein in jejunal crypt was significantly higher than that in normal group at each time points from 6h to 7d after whole-body irradiation with different doses of X-ray.Western blotting also showed that the protein of pS6 and pmTOR was highly expressed in 8 Gy-irradiated crypts(P < 0.01).The positive staining of pS6 and Olfm4 was co-expressed in crypt cells(including intestinal stem cells)on 3 day after irradiation.2.Compared with the normal control group,the numbers of Brd U~+ cells in jejunum were significantly decreased after 12 Gy radiation.The numbers of Brd U~+ cells in jejunum were decreased on 1 and 2 days after 8 Gy radiation but increased on 3 days after 8 Gy radiation and 2 days after 4 Gy radiation(P < 0.01),The numbers of apoptotic cells in crypt were significantly increased after radiation.3.The expression of p62 in the intestinal crypt was higher than that in the normal group after 8 Gy radiation(P < 0.01).The ratio of LC3BII/LC3 BI was significantly lower than that in the non-radiated group(P < 0.01 or P < 0.05).Autophagy was significantly inhibited after radiation.4.Compared with the normal control group,the expression level of pAkt protein in intestinal crypt was increased at all time points after 8 Gy radiation.The expression level of pp65 protein in intestinal crypt was significantly increased after radiation(P < 0.01).5.The numbers of γ-H2 AX and 53BP1 foci in the nucleus of mouse intestinal crypt increased significantly after 8 Gy radiation(P < 0.01).Conclusion: mTORC1 signaling and NF-κB signaling pathways are over-activated after radiation.Cellular autophagy is inhibited,and DNA damage and cellular apoptosis exist in intestinal crypt cells after radiation.Part Ⅲ Protection of rapamycin on radiation-induced intestinal mucosal injury in miceObjective: To investigate the protection of rapamycin on radiation-induced intestinal mucosal damage by inhibiting mTORC1 signaling pathway in mice.Methods: 90 8-week-old C57BL/6 male mice were randomly divided into normal control group,radiation group,rapamycin treatment group,rapamycin control group.40 mice were used to observe the survival rate within 30 days after radiation.Mice were irradiated with Elekta Precise linear accelerator at 8 Gy dose.The mice in rapamycin treatment group were subcutaneously injected with rapamycin(4mg/kg)staring at 6h after whole-body radiation,and then injected once every other day.The mice in rapamycin control group were not radiated but given at the same time point.The mice in irradiation group and normal control group were not given rapamycin at the same time point.The observation time point was 1d,3d,7d after radiation.Jejunal samples were collected from mice in each group.The changes of intestinal permeability were observed by hematoxylin eosin(H-E)staining,ultrastructural changes of jejunal epithelial cells.Olfm4~+ small intestinal stem cells,Lysozyme~+ Paneth cells,pS6 and the distribution of ZO-1 and Occludin were demonstrated by immunohistochemistry.The expression of pS6 in Olfm4~+ small intestinal stem cells was demonstrated by double immunofluorescence staining.The changes of goblet and endocrine cells in intestine were assessed by PAS and argyrophil staining,respectively.The changes of ZO-1,Occludin,S6,pS6,mTOR and pmTOR were detected by immunoblotting.Results: 1.The upregulation of pS6 and pmTOR protein in intestinal crypts induced by radiation was significantly reduced after treatment with rapamycin for 3 days and 7 days(P < 0.01).Rapamycin treatment can significantly reduce the enhanced fluorescence intensity of S6 in intestinal crypt and stem cells after irradiation,indicating that rapamycin inhibits the mTORC1 signaling pathway in irradiated intestinal crypt cells including the intestinal stem cells.2.30% of the mice in rapamycin group survived in 30 days after 8 Gy dose of radiation,but all irradiated mice without rapamycin treatment died.Rapamycin treatment can improve the survival rate of the irradiated mice(P < 0.05).3.The activity of DAO in the serum of mice treated with rapamycin under radiation was significantly lower than that in the radiation group(P < 0.05).Compared with the radiation group,the pathological changes of intestinal mucosa in the rapamycin group were significantly improved.For example,the villi were increased,the damage of intestinal epithelial cells was reduced,the morphology and quantity of microvilli were improved,the tight junction structure tended to be normal,and the injury score was significantly reduced(P < 0.01).4.Compared with the radiation group,the expression of ZO-1 and Occludin protein in the intestinal mucosa of irradiated mice was increased.The distribution of ZO-1 and Occludin protein in intestinal epithelial cells was increased under rapamycin treatment after radiation.5.The numbers of Olfm4~+ cells were less in rapamycin group on day 1 and 3 than that in radiation group while the numbers of intestinal stem cells in intestinal crypt at day 7 after radiation with rapamycin were more than that in radiation group(P < 0.01).Rapamycin treatment could increase the numbers of goblet cells and Paneth cells in intestinal mucosa after radiation.Rapamycin treatment promotes the recovery of both numbers and differentiation function of intestinal stem cells after radiation.Conclusion: Rapamycin can inhibit the mTORC1 signal pathway activated by radiation,protect the intestinal mucosal barrier and promote the repairment of intestinal mucosal injury.Part Ⅳ Protective mechanism of rapamycin on radiation-induced intestinal mucosal injury in miceObjective: To explore the protective mechanism of rapamycin on intestinal mucosa in mice with whole-body X-ray radiation.Methods: 65 C57BL/6 male mice were randomly divided into normal control group,radiation group,rapamycin treatment group and rapamycin control group.The mice were irradiated with Elekta Precise Linear Accelerator at 8 Gy dose.The mice in rapamycin treatment group were subcutaneously injected with rapamycin(4mg/kg)starting at 6h after whole-body radiation,and then injected once every other day.The mice in rapamycin control group were not radiated but given rapamycin at the same time point.The observation time point was 12 h,1d,3d,7d after radiation.The mice in each group were intraperitoneally injected with Brd U(100 mg/kg)two hours before sacrifice.The numbers of Brd U~+ proliferating cells,the number of γ-H2 AX and 53BP1 positive foci in the nucleus were counted after immunohistochemistry staining.The apoptotic cells were detected by TUNEL staining.The relative m RNA expression of Puma、Bcl2、Bcl-xl、Bax 和 Bak in each group were measured by qRT-PCR The expression of LC3 B,p62,Akt,pAkt,p65 and pp65 was observed by Western blot.Results: 1.The ratio of LC3BII/LC3 BI was increased(P <0.01)in the intestinal crypt cells of rapamycin treated group when compared to the control group after radiation.The expression of p62 in the intestinal crypt cells was significantly increased after radiation,while rapamycin could significantly reduce the expression of p62 induced by radiation(P < 0.01).Rapamycin treatment significantly decreased the levels of pAkt and pp65 in crypt after radiation(P < 0.01 or P < 0.05).2.Compared with the radiation group,the numbers of Brd U~+ cells in the intestinal crypt were significantly decreased after rapamycin treatment.(P < 0.01),Rapamycin treatment significantly inhibiting over-proliferation of cells in crypt.The numbers of apoptotic cells in the intestinal crypt was decreased after rapamycin treatment(P < 0.05).Compared with the control group,rapamycin treatment significantly reduced the expression of Puma and Bak m RNA(P < 0.01 or P < 0.05).3.Compared with the radiation group,the numbers of γH2AX and 53BP1 foci in the intestinal crypt cells were significantly decreased with accelerating DNA damage repair after rapamycin treatment(P < 0.01).Conclusion: Rapamycin,an inhibitor of mTORC1 signaling pathway,can promote the repair of intestinal mucosa by promoting autophagy and inhibiting cell over-proliferation,inhibiting NF-κB signaling pathway and apoptosis,and accelerating the repair of DNA damage induced by radiation.SUMMARY According to the results from the above four parts,the summary is as below: 1.Whole body X-ray radiation can cause pathological damage of intestinal mucosa,injury of intestinal mechanical barrier,increase of permeability,decrease of numbers and differentiation ability of intestinal stem cells.2.Whole body X-ray irradiation can activate mTORC1 signaling pathway in intestinal crypt,inhibit autophagy,activate Akt and NF-κB signaling pathway,accelerate DNA damage and apoptosis to produce pathological changes.3.Rapamycin can protect the numbers and differentiation of intestinal stem cells,maintain the structural integrity of the mechanical barrier of intestinal mucosa.The survival rate of radiated mice by temporarily inhibiting mTORC1 signal pathway of intestinal crypt stem cells was increased.4.Rapamycin can promote the repair of intestinal mucosa by promoting autophagy,inhibiting NF-κB signal pathway activation and accelerating DNA damage repair.