Radiation-induced Mitochondrial DNA Release Continuously Activates The CGAS-STING-NFκB-TGFβ1 Pathway In Vitro And In Clinical Study

Part1 Establish the cell line model of EA.Hy926 which secreted TGF-β1 induced by X-ray and the changes in mitochondrial function.Objective Radiation-induced fibrosis is the most common problem following radiotherapy and TGF-β1 has a fundamental role in fibrosis.However,the cause of the increase of TGF-β1 after radiation was inconclusive.We established the radiationinduced secretion of TGF-β1 in EA.Hy926 cell line.We investigated the connection between mitochondrial function and TGFβ1 secretion.Methods Human umbilical vein fusion cell line EA.Hy926 was irradiated by 2Gy split dose of 6MV-X-ray(2Gy/F,5F/W,Total 40Gy).Following X-ray exposure,TGF-β1 expression was analyzed and we successfully established EA.Hy926-R cell.The m RNA expression and protein were measured through PCR and WB.Mitochondrial complex I activity was detected by microplate reader.The MMP and ROS were assessed by JC-1 and DCFH-DA probe.FISH and immunofluorescence and colocalization experiments were utilized to detect mt DNA release.WB and the confocal fluorescence microscope were applied for observing mitochondrial dynamics changes.Transmission Electron Microscopy was applied for micro-structure.Results TGF-β1 m RNA levels were assessed in EA.Hy926-R cells 1,2,3,4,5 months after radiation.The content of TGF-β1 m RNA(p=0.0004)and protein(p=0.0165)in EA.Hy926-Rcells were increased.Mitochondrial complex I activity(p=0.0045)and the levels of mitochondrial complex I subunits were reduced(p<0.05).MMP(p<0.0001)and ROS(p<0.0001)were lessened.Mt DNA contents had no significant changes(p=0.5249).The release of mt DNA into the cytoplasm were observed under a confocal fluorescence microscope and the increased mitochondrial fusion and mitochondrial autophagy was activated.TEM revealed marked alterations in mitochondrial morphology.Mitochondrial morphology was damaged,mitochondrial membranes were broken,cristae structures were disordered or invisible.Conclusion EA.Hy926-R cells which secreted TGF-β1 after radiation were established.The function of the mitochondria of the model cells was destroyed,and the dysfunction of the mitochondria caused the translocation of mt DNA to the cytoplasm.Part2 The mechanisms of continuous TGF-β1 secretion and pharmacological interventions in EA.Hy926-R cells.Objective To research the association between TGF-β1 and mitochondrial dysfunction in EA.Hy926-R cells and potential signaling machinery of TGF-β1 secretion.To explore the use of pharmacological agents for interventions and possible targets for improving fibrosis by radiotherapy.Methods WB was used to disclose the content of c GAS,STING and NFκB.Mt DNA released into the cytoplasm was observed by CSLM.TDP-43 content in mitochondria were detected by WB.Western blotting method was used to detect c GAS-STING-NFκB pathway and the changes in the content of TGF-β1 in various interventions.Results The protein levels of double-strand DNA sensor c GAS,stimulator of interferon gene STING and transcription factor NFκB were increased in EA.Hy926-R cells(p<0.05)which meant that the c GAS-STING-NFκB signaling pathway was activated.The expression of TGF-β1 was decreased after inhibition of c GAS-STINGNFκB signaling pathway(p<0.05).The mitochondrial and cytoplasmic TDP-43 accumulated in EA.Hy926-R cells(p<0.05).After the suppression of human TDP-43 expression,mt DNA spillover was reduced and TGF-β1 was efficiently inhibited(p<0.05)through the c GAS-STING-NFκB signaling pathway(p<0.05).The protein level verified that the content of overexpression cell line STING increased(p=0.0268).No alterations in TGF-β1 expression occurred after inhibiting the expression of TDP-43 in cells transduced with STING-expressing lentivirus(p>0.05)which further illustrated TDP-43 stimulated TGF-β1 expression by promoting c GAS-STING-NFκB pathway.The MMP was disrupted in EA.Hy926 cells after addition of CCCP(p<0.05),mt DNA was released into the cytoplasm,c GAS-STING-NFκB pathway was activated(p<0.05).Aspirin and captopril inhibited the c GAS-STING-NFκB pathway(p<0.05),and in turn led to reduce expression of TGF-β1(p<0.05).Conclusion TDP-43 was increased and aberrantly localized to the mitochondria in EA.Hy926-R cells.It resulted in mt DNA released into the cytoplasm in a membranepotential-dependent manner.c GAS-STING-NFκB pathway was activated and triggered TGF-β1 expression.Aspirin and captopril suppressed the c GAS‐STING-NFκB and TGF-β1 expression.Therefore,our findings provide effective molecular targets for the prevention and treatment of radiation fibrosis.Part3 Validation of specimens and further clinical trials for TGF-β1 secretion after X-ray exposureObjective To verify the effect of c GAS-STING-NFκB-TGFβ1 pathway in the radiation field undergoing X-ray radiotherapy and the blocking effect of drug intervention on the increase of peripheral blood TGF-β1 after radiotherapy.It provides the basis and preliminary data for follow-up experiments and clinical applications.Methods The clinical data and pathological database were collected from Zhong nan Hospital.The TDP-43、STING and TGF-β1 expressions were detected in tissue in radiation field by immunohistochemistry.Peripheral blood was drawn from patients before and after radiotherapy and TGF-β1 concentration were determined by ELISA.Experimental group given captopril 12.5mg bid and control group with placebo.Peripheral blood was drawn from patients and TGF-β1 concentration were determined by ELISA.Results The contents of TDP-43、STING and TGF-β1 were increased in radiation field tumor tissues of patients received neoadjuvant radiotherapy,respectively(p<0.05);Peripheral blood TGF-β1 content were measures in patient before radiotherapy and 1、2、3、4 months after radiotherapy by ELISA.The TGF-β1 levels were increased at first and then decreased and finally increased after X-ray irradiation.Compared with the control group,the patients taking captopril have suppressed the increase of TGF-β1 in peripheral blood caused by radiotherapy.(p<0.05).Conclusion The contents of TDP-43、STING and TGF-β1 were increased in irradiation field of patients underwent X-ray,which agreed with experiment.The TGF-β1 levels were increased at first and then decreased and finally increased after X-ray irradiation.The TGFβ1 contents variation in blood of therapy group were declined.The above results provide a theoretical and clinical basis for further clinical studies in radiological response of normal tissues.