Treatment And Mechanism Of Radiation-induced Intestinal Mucosal Injury With Keratinocyte Growth Factor 2 Mutant

Basic science promotes the development of applied science.Since Wilhelm Conrad Roentgen observed and documented X-rays in 1895 for first time,radiation related technologies,such as nuclear weapons,nuclear power plant,clinical radiotherapy and so on,are now flourishing.However,radiation injury is a common occurrence,and the injury of intestinal mucosa is often accompanied with hematopoietic system damage in severe cases,for which there is no effective treatment at present.Keratinocyte growth factor(KGF)had been found to reduce the incidence and duration of severe mucositis induced by high dose chemotherapy/ radiotherapy,and was approved by FDA in 2004.Keratinocyte growth factor-2(KGF-2),as a member of the same family of KGF,is expected to play a similar role in the treatment of mucosal injury.Our team developed a KGF-2 mutant with higher receptor affinity and proliferation activity,which significantly improved the survival rate after low dose ?-ray irradiation in mice.On this basis,we carry out this study to provide a new strategy for the treatment of radiation-induced intestinal mucosal injury.[Objective]To investigate the therapeutic effect and molecular mechanism of KGF-2 mutant on radiation-induced intestinal mucosal injury.[Content]We will carry out the research at animal,tissue and cell level.We will optimize the administration dosage,interval of KGF-2 mutant in the treatment of radiation-induced intestinal mucosal injury,and the possible mechanism will be investigated as well.We will also study the effect of KGF-2 mutant on autophagy and apoptosis.[Methods and Results]To study the relation between therapeutic effect and KGF-2 mutant administration dosage,interval,and the number of transplanting bone marrow cells,6 specific pathogen free(SPF)C57BL/6J mice were randomly divided,and radiated with one-time whole body ?-ray exposure of 12 Gy.The therapeutic effect of KGF-2 mutant was analyzed by survival rate,average body weight within 30 days after radiation,changes of blood cells and inflammatory factors,the pathological status,and the apoptosis as well as autophagy status of small intestine at 72 hours after exposure.The effect of KGF-2 mutant on radioprotection in Ha Ca T cell line was also studied.In the dose-effect study we found that mice of untreated group died within 10 days and mice administered with KGF-2 mutant(6mg/kg or 12mg/kg)and bone marrow cell transplantation had a higher survival rate than untreated group at 30 th day after irradiation.In the time-effect study we found that mice administered with KGF-2 mutant at two days before irradiation and transplantation had a higher survival rate than those administered at three days before irradiation and transplantation.Then,we adjusted the number of bone marrow cells for transplantation(2.0×106/each mouse),and found that the mice treated with KGF-2 mutants(6mg/kg,-2d)combined with bone marrow transplantation showed a 100% survival rate at 30 th day,which is significantly higher than the untreated group(0)and the bone marrow transplantation only group(60%).The average weight data at 7th day after irradiation are as follows: the untreated group,14.26±0.48g;group treated with KGF-2 mutant,15.11±0.90g;group treated with KGF-2 mutant combined with bone marrow transplantation,16.24±0.98g;group treated with bone marrow transplantation,15.39±1.08 g.The mice in KGF-2 mutant combined with bone marrow transplantation group had a slower weight loss,earlier recovery,more stable average weight in the 30 day's observation period than that in other groups.After ?-ray exposure,pathological status of the intestinal specimens showed that mice in untreated group got a serious intestinal damage,an increased autophagy and apoptosis level at intestinal epithelial cells;mice in KGF-2 mutant combined with bone marrow transplantation group had a more intact structure,a lower apoptosis level,and a higher autophagy level at intestinal epithelial cells,as analysed by HE staining and immunohistochemistry.Compared with the normal mice,blood cells of mice in each experimental group were decreased significantly after exposure.And administration with KGF-2 mutant and/or bone marrow transplantation couldn't 7 recover the WBC decrease,while the recovery of RBC and Hb in KGF-2 mutant(-2d)combined bone marrow transplantation group was much better,compared with the untreated group.ELISA showed the serum IL-6 level decreased in KGF-2 mutant(-2d)combined bone marrow transplantation group compared with the untreated group.At the cellular level,we investigated the molecular mechanism of KGF-2 mutant on radioprotect in the human keratinocyte Ha Ca T cell line.After ?-ray exposure,Western blot analysis showed that Cleaved Caspase-3 and LC3 level were increased,and the autophagy was take place earlier than apoptosis.We also found that the KGF-2 mutant was able to activate the NF-?B,PI3K-AKT and MAPK-ERK pathways,which was speculated that the KGF-2 mutant could play an important role in the regulation of autophagy and apoptosis in cells.Then we investigated the effects of KGF-2 mutants on autophagy and apoptosis in starvation model.Cells were subjected to a serum free starvation in the presence or absence of KGF-2 mutant,Western blot analysis showed that Cleaved Caspase-3 and Cleaved PARP level were increased in long time serum free culture,while stimulation of KGF-2 mutant could reduce the expression of Cleaved Caspase-3 and Cleaved PARP,indicating that KGF-2 mutant can inhibit cell apoptosis.Cells were starved by Earle's Balanced Salt Solution,and the levels of LC3 were analyzed by western blot.A significant increase of LC3 levels was clearly evident after nutrition deficiency,and administration of KGF-2 mutant could abolish this increase.LC3 level which was increased in cells by Chloroquine(CQ)treatment alone was decreased in cells treated with CQ and KGF-2 mutant.Then the Ha Ca T cells were transfected with p EGFP-C3-MAP1LC3 B plasmid,and the size and shape of GFP-LC3 particles were observed by confocal laser scanning microscope.Starvation or CQ treatment increased the number of EGFP-LC3-positive dots per cell,while KGF-2 mutant decreased it.These results indicated that KGF-2 mutant can inhibit the cell autophagic process.[Conclusion]At the whole animal level,we confirmed that KGF-2 mutant combined with bone marrow transplantation had a significant therapeutic effect on radiation-induced intestinal mucosal injury,which could improve the survival rate and reduce the level of inflammation of irradiated mice.At tissue level,we found that KGF-2 mutant combined with bone marrow transplantation could promote the mucositis healing,impact the autophagy and apoptosis status of intestinal crypt cells.At the cellular level we showed that ?-ray exposure could increase the level of autophagy and apoptosis in cell,and autophagy was occurred earlier than apoptosis.KGF-2 mutant could activate the NF-?B,PI3K-AKT and MAPK-ERK pathways,also inhibit cell apoptosis and autophagy induced by long-term nutrition deficiency.In summary,this study demonstrated that KGF-2 mutant combined with bone marrow transplantation has a definite therapeutic effect on radiation-induced intestinal mucosal injury,and KGF-2 mutant can inhibit autophagy and apoptosis in human keratinocyte.