The Therapeutic Effect Of Hypoxia-induced Mesenchymal Stem Cells On Radiation-induced Lung Injury And Mechanism Involved

BackgroundRadiation-induced lung injury（RILI）is a common complication occurring on nuclear war,nuclear accident and thoracic radiotherapy with high mortality.Especially in the thoracic radiotherapy,as reported,about 14.6%-37.2%of radiated patient develop to RILI,which greatly limits the radiotherapy dose and subsequently reduce the curative effect on cancer.However up to now,there are few effective means and medicines for RILI.Hence the effective and treatments for RILI are in powerful need.Generally,the pathological process of RILI includes pneumonitis in early phase and pulmonary fibrosis in the late stage.The pathological characteristics of radiation-induced pneumonitis present as destruction of pulmonary parenchyma and infiltration of immune cells.The pulmonary fibrosis is characterized as deposition of collagenous fiber and restrictive ventilatory impairment in symptom.Three theories are associated with the mechanism of RILI that are target cells theory,cytokine theory and epithelial-mesenchymal transition.According to above mechanism some clinical drugs have been developed,such as glucocorticoid against inflammation,glutathione eliminating ROS.However,there are still not perfect treatment for RILI,which are attributed to the complex process and masses of influence factors in the development of RILI.Recent studies concerning mesenchymal stem cells（MSCs）provide novel strategies and means for RILI treatment.MSC is a type of cell characterized as self-renewal and multi-directional differentiation.As their distinct advantages of hypoimmunogenicity and accessibility,MSCs have been applied in treatments for many diseases including heart failure,liver injury,skin injury and so on.In the treatment for RILI,some studies have also tried to apply MSCs and some have reported their therapeutical effect.However,as the deeply understanding in MSCs,more and more scientists think that the exogenous MSCs are limited when they are transplanted into damaged lung tissues which are induced by radiation,because the poor oxygen and high ROS environment within radiated lung tissues greatly reduce the survival of implanted MSCs and thereby lower their therapeutical effect on RILI.On the other hand,recent studies have revealed that hypoxia could enhance the MSCs’proliferation and induce their differentiation.Therefore,we speculated that hypoxia condition may promote the survival of implanted MSCs in the radiated lung tissues so that improve MSCs’therapeutical effect on RILI.Based on above hypothesis we set up this project to investigate the effect of hypoxia bone mesenchymal stem cells（BMSCs）on RILI.We will take advantage of the established mice model of RILI to test the effects of normoxia and hypoxia BMSCs on radiated lung tissues by detecting pulmonary pathology,measuring the expression of inflammatory factors and counting inflammatory cells.Next the experiments in vitro will demonstrate the cellular effect of hypoxia BMSCs.Go a step further we will deeply research the involved mechanism and investigate the role of HIF-1αin the therapeutical effect of hypoxia MSCs on RILI.Research contents1、The effect of hypoxia on BMSCs characteristic（1）The effect of various oxygen concentration on BMSCs growth and he selection of optimum oxygen concentration.（2）The influence of hypoxia on BMSCs viability,proliferation,morphology and differentiation.（3）The comparison of intracellular ROS between normoxia and hypoxia BMSCs.（4）The detection of resistance to hypoxia and high ROS condition between normoxia BMSCs and hypoxia BMSCs.2、The effect of hypoxia BMSCs on mice model of RILI（1）Homing detection of implanted hypoxia BMSCs in radiation injured lung tissues（2）Comparison of therapeutic effect on injured lung tissues between normoxia and hypoxia BMSCs（3）Measurement of inflammatory factors in lung tissues and peripheral blood3、The interacted action between hypoxia BMSCs and pulmonary parenchyma cells（1）Detection to apoptosis of radiated MLE with/without hypoxia BMSCs.（2）Influence of hypoxia BMSCs on cellular EMT induced by radiation4、The investigation to molecular mechanism（1）Expression of HIF-1α,Akt and apoptin under hypoxia condition.（2）Inhibiting HIF-1αto investigate proteic in hypoxia BMSCs（3）Test of tolerance to hypoxia and high ROS condition by inhibiting HIF-1α（4）Critical molecular screening by protein microarray（5）Verification test to confirm selected critical molecularExperimental results（1）Hypoxia could change BMSCs’characteristics and enhance their resistance to hypoxia and high ROS conditionThe BMSCs which were cultured under hypoxia condition（2.5%O₂）acquired higher viability and potential of proliferation.Hypoxia BMSCs were inclined to differentiation into osteoblast and lipoblast.Long-term hypoxia treatment could lower ROS level of BMSCs and enhance their resistance to hypoxia and high ROS environment.（2）Hypoxia BMSCs remitted RILICompared with normoxia BMSCs,implanted hypoxia BMSCs had longer survival in radiation injured lung tissues.Hypoxia BMSCs greatly reduced inflammatory exudation and inhibited the process of pulmonary fibrosis presenting as reduced positive rate of Masson andα-SMA staining in lung tissue.Besides radiated mice injected with hypoxia BMSCs had a lower level of inflammatory factors including TNF-α,TGF-β,but the level of INF-γrose at first stage and then decreased.（3）Co-cultured with hypoxia BMSCs the injury of radiated pulmonary parenchyma cells was mitigatedRLE-6TN cells were representative of pulmonary epithelial cells,and then radiated RLE-6TN were co-cultured with normoxia/hypoxia BMSCs for 24 hours.As a result,supernatant from hypoxia BMSCs reduced apoptosis rate of RLE-6TN and inhibited the development of EMT.（4）The study of molecular mechanism involved in the effect of hypoxia MSCs on RILI1)HIF-1αwas involved in the resistance of hypoxia MSCs to hypoxia stressCompared with normoxia BMSCs the hypoxia BMSCs had a higher intensity of background expression in Akt,HIF-1α.The expressions of phosphorylated Akt（p-Akt473）,HIF-1αand caspase-3 were rapidly up-regulated in normoxia BMSCs but hardly change in hypoxia BMSCs.Inhibiting the expression of HIF-1αwith 2-methoxyestradiol（2-MeOE2）could up-regulate the expression of caspase-3 in hypoxia MSCs which originally seldom to change under hypoxia condition.Above results demonstrated that HIF-1αmediated the resistant characteristic of hypoxia BMSCs to hypoxia condition.2)Cytokines screening from hypoxia BMSCs using protein microarray techniqueBy protein microarray technique,we screened critical cytokines that were secreted by hypoxia MSCs to play a vital role in RILI treatment.As set criteria:P<0.05 and fold change was greater than 1.5,we obtained 23 cytokines.That were highly especially expressed in hypoxia MSCs included GM-CSF,IL-4,IL-13,IL-17,MIP-1a,CD27,DtK,MIP-1b in which IL-13（Log2FC=2.226）,MIP-1β（Log2FC=2.071）,MIP-1α（Log2FC=1.380）presented greatest fold changes.Next above secreted cytokines were verified by EILSA test.IL-13,MIP-1βand MIP-1αmay be the critical molecular involved in the mechanism of MSCs treating RILIDiscussion and analysisDiffering from other factors induced lung injuries,the radiation induced lung injury often products extensive ROS and lead to local ischemia and hypoxia in damaged lung tissues.Therefore the effective medicines for RILI are scarce.As the development of MSCs studies they also provide new strategies for RILI treatment.However the complex and hostile environment in radiated lung tissues often limited the implanted MSCs and subsequently lower their therapeutical effect on RILI.Hence the means to enhance MSCs’survival in radiated lung tissues are in powerful need.Recently some studies demonstrated that hypoxia could promote the proliferation of MSCs and enhanced their resistance to hypoxia condition.Especially the elevated expression of HIF-1αinduced by hypoxia has verified to be the critical molecular in enhancing potential of cells’proliferation,and elevating cell viability.Thus,we speculated that hypoxia could improve the therapeutical effect of implanted BMSCs on RILI.Firstly,we established the mice model of RILI,and then test the effect by implanting normoxia/hypoxia MSCs.The results demonstrated that hypoxia BMSCs had a longer survival in radiated lung tissues than normoxia BMSCs and effectively remitted lung injury and inhibited pulmonary fibrosis.Cytological experiments demonstrated that hypoxia MSCs could reduce apoptosis induced by radiation and inhibit EMT.In the deeply study of mechanism,we investigated the role of HIF-1αinvolved and screened critical molecular by protein microarray.Finally,IL-13,MIP-1βand MIP-1αbecame candidates and need to further explore in future studies.ConclusionHypoxia BMSCs could effective remit RILI and protect pulmonary epithelial cells from radiation.The mechanism includes:one is through the hypoxia induced up-regulation of HIF-1α,thereby inhibiting the activation of the mitochondrial pathway of apoptosis pathway;on the other hand,hypoxia BMSCs secret effective cytokines to reduce radiation-induced alveolar epithelial cell injury,in which the main effector molecules are IL-13,MIP-1a and MIP-1b.