The Studies On The Effects And Mechanisms Of Radiation-induced Dysregulated Lipid Metabolism In Regulating The Development Of Liver Damage Caused By Ionizing Radiation

Background:liver belongs to the radiosensitive organ.Radiation-induced liver damage(RILD)is the common dose-limiting toxicity and complications for the radiotherapy treatment for primary hepatocellular carcinoma(HCC),thoracic and abdominal tumors,allogeneic bone marrow or hematopoietic stem cell transplant.Until now,the underlying molecular mechanisms have not been fully elucidated.Dysregulated lipid metabolism plays an important regulatory role in a variety of physiological and pathological processes.Despite evidence which suggests that lipid metabolism dysregulated might be related to RILD,its specific molecular mechanisms in the development of liver injure induced by ionizing radiation is still unclear.Objective:The study aims to investigate the correlation between the lipid metabolic homeostasis induced by radiation and the occurrence and development of RILD,and to demonstrate the potential biological functions and the underlying molecular mechanisms.Methods:(1)The correlation between the radiation-induced dysregulated lipid metabolism and RILD.The X-RAD SmART system was used to establish a RILD mouse model,and the detection of biochemical markers of liver function(ALT,AST),hematoxylin-eosin staining,western immunoblotting,transmission electron microscopy was performed to determine the damages caused by ionizing radiation(IR)of the liver.Subsequently,the impact of radiation on liver lipid metabolism was evaluated by Oil Red O staining,intrahepatic triglycerides(TG)and free cholesterol(FC)assay kits.(2)Single-cell transcriptome sequencing technology combined with functional verification to identify the possible regulatory mechanisms.The RILD mouse model was established by X-RAD SmART system,and the 10 × Genomics single-cell transcriptome sequencing technology was used to investigate the effects of radiation on cell typing and gene expression heterogeneity.The enriched biologicalpathways of differentially expressed genes that related to lipid metabolism in different hepatic cell populations were analyzed by bioinformatics combined with quantitative real-time polymerase chain reaction(qRT-PCR)confirmation.The PPARα gene knockout mouse was constructed and the primary hepatocytes were isolated by two-step liver perfusion method followed by immunofluorescence staining identification.To test the effects of radiation on cell cycle arrest and DNA damage with the hepatocytes without PPARαgenes,the flow cytometry and immunofluorescence staining were used.A PPARαknockout mice were used for RILD model construction,and the biochemical analyzer and hematoxylin-eosin staining were used to detected the changes of biochemical indicators and pathological structurein vivo.(3)Exploration of the lipid metabolism related mechanism in regulating RILD.The MDA and Iron content in liver tissues was detected using MDA assay kit and Iron assay kit.Western blot assay was used to measure the changes in COX2 expression after IR exposure.In addition,intracellular iron deposition in the hepatocyte was visualized with Prussian blue staining.The expression levels of MDA and COX2 in PPARα deleted liver tissues were detected by MDA assay kit and western blot assay.Further,the FABP1 knockout mouse was constructed and used for primary hepatocytes isolation.According to the classical strategies of signal pathways studies,the technologies of qRT-PCR and western immunoblotting were used to detect the effects of radiation on mRNA and protein expression of key regulatoryfactors.The STRING database(https://cn.string-db.org/)was utilized to predict targeted interactions between key moleculess,followed by the dual-luciferase reporter gene assay and Coimmunoprecipitation verification.The Oil Red O staining,C11-BODIPY 581/591 prob and Prussian Blue,MDA assay kit and ROS probe(DCFH-DA)were used to investigate effects of the key molecular of PPARα-FABP1-SCD1 axis on lipid deposition,lipid peroxidation and iron deposition in vitro.(4)Preliminary study on intervention strategies for RILD by targeting the radiation-induced abnormal lipid metabolism related mechanisms.The C57BL/6J mice were adopted for RILD model construction,which were received an intraperitoneal injection of WY14643,the PPARα agonist.The expression of PPARαin liver was determined by Western blot assay.The biochemical analyzer,hematoxylin-eosin staining,MDA and Iron assay kits,Western blot and qRT-PCR assay were performed to investigate the protection of activation PPARα on the occurrence and development of RILD.Results:(1)The correlation between the radiation-induced dysregulated lipid metabolism and RILD.The RILD mice model was successfully established by using single-fraction doses of 30 Gy X-Ray.In contrast with the non-irradiation mice,the levels of serum ALT(p<0.05)and AST(p<0.05)were significantly increased 1 day post-irradiation for RILD mice.At 2 weeks after irradiation,histopathological changes,DNA double strand break and the mitochondrial membranes were fragmented,cristae rupture,indicating the radiation caused severe liver injure.In addition,the lipid droplets of RILD mice exhibited significant increase and enlargement,and the content of triglyceride(p<0.01)and cholesterol in liver tissues was higher than non-irradiation group,indicating the radiation led to the hepatic lipid metabolic disturbance and increase lipid accumulation.(2)Single-cell transcriptome sequencing technology combined with functional verification to identify the possible regulatory mechanisms.Liver tissues of C57BL/6J mice 2 weeks after irradiated with single-fraction doses of 30 Gy X-Ray were analyzed with 10× Genomics single cell transcriptome,and analysis showed radiation not affect the kind of cell population,but result in differential gene expression,which was more remarkable in hepatocyte.Total 307 differential genes were detected,of which 294 were upregulated and 13 were downregulated.These differential genes were subjected to pathway analysis using the KEGG database,and found the differential genes were mainly enriched in PPAR signal pathway,ferroptosis and fatty acid degradation,which was mainly related to lipid metabolism.Upregulation of PPARαexpression in liver after radiation,a key regulator of lipid metabolism,was confirmed by qRT-PCR.In vitro,radiation-induced DNA double strand break(p<0.05)and cell cycle G2/M phase arrest(p<0.001)in the hepatocytes without PPARα genes was significantly higher than control cells.In vivo,PPARα knockout increased the serum levels of ALT(p<0.01)and AST(p<0.05),aggravate pathological damage in mice with radiation.(3)Exploration of the lipid metabolism related mechanism in regulating RILD.Irradiation significantly increased the contents of MDA(p<0.01),Iron(p<0.0001)and COX2 in liver,and promoted iron deposition in hepatocyte.KO-PPARα caused a marked increase in MDA content(p<0.001)and COX2 expression in irradiated liver.Radiation induced both the mRNA and protein levels of FABP1 and SCD1 increased in liver,and both are regulated by PPARα.String database revealed the potential interactions among PPARα,FABP1 and SCD1.PPARα could directly bind to FABP1 promoter regions to regulated the FABP1 expression,and FABP1 affected the expression of SCD1 by proteins co-precipitation.FABP1 was not the only condition for PPARα to regulate the expression of SCD1.In vitro,inhibition of PPARα,FABP1 and SCD1 gene expression in hepatocyte resulted in the intracellular lipid deposition,the levels of lipid peroxidation and the iron content increase,demonstrated that PPARαFABP1-SCD1 axis could regulate the occurrence and progression of RILD via ferroptosis.(4)Preliminary study on intervention strategies for RILD by targeting the radiation-induced abnormal lipid metabolism related mechanisms.The C57BL/6J mice were intraperitoneally injected with a low doses WY14643(100 mg/kg/d)for 10 consecutive days,and liver PPARα expression was efficiently activated(p<0.05).Activation PPARα significantly decreased the levels of serum ALT(p<0.05),ameliorated the liver histopathologic damage induced by radiation.WY14643 could stabilize the lipid peroxidation and iron metabolism to decreased the liver ferroptosis after radiation.Specific manifestation is as follows:WY 14643 intervention resulted in significant reduction of COX2 induced by radiation(p<0.05),decreased the MDA content of liver following radiation,inhibited the radiation-induced decrease of FPN(p<0.05)and reduced iron deposition in liver(p<0.01).Conclusion:The present study identified that radiation-induced aberrant lipid metabolism was associated with the occurrence and progression of radiation induced liver damage,and the mechanism involved was related to the PPARα-FABP1-SCD1 signaling pathway regulated ferroptosis by affecting the IR-induced lipid oxidation and iron metabolism.