Pulmonary Extracellular Matrix Hydrogel On The Treatment Of Radiation-induced Lung Injury In Rats

Objective:Radiation induced lung injury（RILI）is a common complication in the treatment of thoracic tumors.The persistent inflammatory response,oxidative stress damage and post-fibrosis are the main pathophysiological features of RILI.The extracellular matrix（ECM）scaffold material is that an isolated-lung perfusion with SDS,Triton-X-100,etc.,and lysing the lipid membrane to obtain a protein complex containing a relatively complete structure and function.Which can regulate a variety of cytological behaviors.After further grinding and enzymatic digestion,the ECM scaffold material is made into a temperature-sensitive hydrogel,which can adhere to the tissue and continue to function.Previous studies have reported that ECM materials have anti-inflammatory,anti-oxidative,anti-fibrotic effects.To clarify whether lung ECM has a therapeutic effect on radiation-induced lung injury,This study established intratracheal injection of lung ECM hydrogel to treat rats with radiation-induced lung injury,on this basement,the therapeutic effect of lung ECM hydrogel on RILI rats was initially observed,and to observe the effect of lung ECM hydrogel on the process of alveolar epithelial-mesenchymal transition,explore its possible mechanism.Method and result:Part one:Preparation of rat model of radiation-induced lung injury treated by pulmonary ECM hydrogelMethods:1.Pulmonary ECM preparation and histocompatibility evaluationPreparation of lung ECM materials by decellularization,verifying the decellularization efficiency by haematoxylin and eosin staining,electron microscopy.The ECM is made into hydrogel material,and a certain dose of hydrogel material is injected into the skin of normal rats,After one week,the tissue around the injection site was excised and immunofluorescence staining was performed to observe the histocompatibility of the material.2.Evaluation of rat model of radiation-induced lung injury9 rats were divided into two groups:control group（normal rats,n=3）and irradiation group（n=6）.After anesthesia,Radiation was delivered at a single dose of 20 Gy with thorax field to prepare a model of radiation-induced lung injury in rats.Seven days after irradiation,two groups of rats were sacrificed,and the lungs were taken for H&E staining to observe the local pathological changes of the tissue.Serum was taken and the levels of TNF-αand IL-6 were detected by Elisa method.3.Screening of appropriate doses of intratracheal injection of lung ECM hydrogel in rats with radiation-induced lung injury24 RILI rats were randomly divided into 4 groups half an hour after irradiation,and the lung ECM hydrogels was injected into the trachea by0μl,300μl,500μl.800μl,the mortality of each group of rats,PaO2,and the distribution of materials in the airway were observed by immunofluorescence staining.Results:1.The lung ECM scaffold was successfully prepared by decellularization.HE staining and scanning electron microscopy showed that the nucleated cell components were completely eluted,and the three-dimensional structure of the extracellular matrix was well preserved.The lung ECM hydrogel was observed after subcutaneously for 1 week,and there was no obvious inflammatory cell infiltration around the material.Which proves that the material has good histocompatibility.2.7 days after irradiation,H&E staining of lung tissue showed that the alveolar space was thickened,the cell infiltration was increased,and some alveolar cavities showed fluid exudation and hemorrhage.The results of Elisa showed that the serum levels of TNF-α,IL-6 in the irradiated group were（98.79±12.12）pg/ml,（75.67±9.22）pg/ml,while in the control group.They were（57.11±9.11）pg/ml,（35.14±4.25）pg/ml,respectively,the irradiation group was significantly higher than the control group（P<0.05）.3.Intratracheal injection of lung ECM hydrogel 300μl or 500μl did not affect PaO₂（P>0.05）,while intratracheal injection of 800μl resulted in decreased PaO₂（P<0.05）.Under the fluorescence microscope,the hydrogel material with green fluorescence was observed on the alveolar surface in the 300μl,500μl,and 800μl groups.while some of the hydrogels in the 800μl group were retained in the airway.Part two:Therapeutic effect of lung ECM hydrogel on radiation-induced lung injury in ratsMethods:1.Effect of pulmonary ECM hydrogel on inflammatory response during radiation-induced lung injury54 rats were randomly divided into 3 groups:Control group（normal rat group,n=18）,IR+NS group（irradiation+intratracheal injection of saline,n=18）,IR+ECM group（irradiation+intratracheal injection of lung ECM hydrogel,n=18）.Six rats were randomly sacrificed at 4,8,and16 weeks after irradiation.The lung wet/dry weight ratio,H&E staining of lung tissue and pathological damage score,Masson staining and fibrosis score,and the serum levels of TNF-α,IL-6,TGF-β1.were compared at each time point.2.Effect of pulmonary ECM hydrogel on oxidative damage during radiation-induced lung injury54 rats were randomly divided into 3 groups:Control group（normal rat group,n=18）,IR+NS group（irradiation+intratracheal injection of saline,n=18）,IR+ECM group（irradiation+intratracheal injection of lung ECM hydrogel,n=18）.Six rats were randomly sacrificed at 4,8,and16 weeks after irradiation.The lung tissues were taken and the SOD activity and MDA content were measured.Results:1.Compared with IR+NS group,the levels of serum TNF-α,IL-6 and TGF-β1 in rats were decreased after intratracheal injection of lung ECM hydrogel at 4th,8th and 16th week after irradiation（P<0.05）.The wet/dry weight ratio of lung tissue was significantly decreased（P<0.05）.H&E staining showed that the pathological changes of lung tissue were alleviated and the pathological score decreased（P<0.05）.Masson staining showed that the deposition of collagen fibers in the lung tissue was reduced.The score was improved（P<0.05）.2.Compared with the IR+NS group,after intratracheal injection of lung ECM hydrogel,MDA content in lung tissue decreased at the 4th,8th,and 16th week after irradiation（P<0.05）,and SOD activity recovered significantly（P<0.05）.Part three:Effect of lung ECM hydrogel on radiation-induced alveolar epithelial-mesenchymal transitionMethods:1.Effect of pulmonary ECM hydrogel on the level of epithelial-mesenchymal transition in lung tissue36 rats were randomly divided into 3 groups:Control group（normal rats,n=12）,IR+NS group（irradiation+intratracheal injection of saline,n=12）,IR+ECM group（irradiation+intratracheal injection Lung ECM hydrogel,n=12）.Six rats were randomly sacrificed at each of the 4th and16th week after irradiation.The changes of epithelial-mesenchymal transition marker E-cadherin,vimentin andα-SMA was detected by immunohistochemical staining and western blot at different time points.2.Effect of pulmonary ECM hydrogel on hydroxyproline content in lung tissue36 rats were randomly divided into 3 groups:Control group（normal rats,n=12）,IR+NS group（irradiation+intratracheal injection of saline,n=12）,IR+ECM group（irradiation+intratracheal injection Lung ECM hydrogel,n=12）.Six rats were randomly sacrificed at the 4th and 16th week after irradiation,and lung tissues were taken.The changes of hydroxyproline content in lung tissues were detected by alkaline water method.Results:1.Compared with the IR+NS group,the expression of E-cadherin in the lung tissue of the IR+ECM group increased at 4 and 16 weeks after intratracheal injection of lung ECM hydrogel,and the expression levels of vimentin andα-SMA decreased（P<0.05）.2.Compared with the IR+NS group,the hydroxyproline content in the lung tissue of the IR+ECM group was decreased at 4 and 16 weeks after intratracheal injection of lung ECM hydrogel（P<0.05）.Conclusions:1.Intratracheal injection of 500μl of lung ECM hydrogel in rats with radiation-induced lung injury does not significantly reduce the PaO₂ or increase the mortality of rats,and the material can distribute on the alveolar surface uniformly2.Intratracheal injection of lung ECM hydrogel can reduce the systemic inflammatory response in rats with radiation-induced lung injury,reduce the local inflammatory reaction and oxidative stress level after irradiation,and reduce the formation of local fibrosis in the late stage3.Lung ECM hydrogel can inhibit the EMT process of alveolar epithelial cells in rats with radiation-induced lung injury,thereby reducing the severity of pulmonary fibrosis.This may be one of the main mechanisms for the therapeutic effect of lung ECM hydrogels.