Low-dose Radiation Reduces Doxorubicin-induced Cardiotoxicity Through Regulating Mitochondrial Homeostasis

Background:Doxorubicin(DOX),as a representative drug of anthracycline,has been widely used in anti-tumor therapy.However,DOX-induce cardiotoxicity severely limits its clinical application.The study on the mechanism and intervention of DOX-induced cardiotoxicity has important clinical significance.The mechanism of DOX-induced cardiotoxicity is not yet fully understood,which may involve oxidative stress,lipid peroxidation,DNA or RNA damage,mitochondrial dysfunction and apoptosis.Mitochondrial homeostasis may be the core among these factors.Mitochondrial homeostasis is the balance state of mitochondrial contents and metabolism,which mainly involves oxygen free radical system,protein and protease system,mitochondrial dynamics,etc.In recent years,low-dose radiation(LDR)has been considered harmless and may assume a protective function by inducing hormesis and adaptive response.Our previous work showed that LDR can induce adaptive response and reduce DOX-induced cardiotoxicity,which may be related to improving mitochondrial function and increasing ATP production,but the exact effect and mechanism are not clear.Based on our previous work,we postulates a hypothesis that LDR may potentially reduce DOX-induced cardiotoxicity by modulating mitochondrial homeostasis.Furthermore,subsequent investigations were conducted to elucidate the primary mechanisms underlying DOX-induced cardiotoxicity,evaluate the effect of LDR on DOX-induced cardiotoxicity,and explore the mechanism related to mitochondrial homeostasis.The study is mainly divided into the followed three parts.PART 1 Bioinformatics analysis of DOX-induced cardiotoxicityObjective:To clarify the possible mechanisms underlying the occurrence of DOX-induced cardiotoxicity.Methods:Two datasets GSE40289 and GSE59672 were selected from the high-flux Gene Expression Omnibus(GEO)database.Data grouping and gene ID conversion were performed in R studio using R language.Differential expression gene analysis was used to identify the differently expressed genes(DEGs)between normal myocardium and DOX-treated myocardium.The biological significance of differential expression gene generation was determined by Gene Ontology,Kyoto Encyclopedia of Genes and Genomes,Gene Set Enrichment Analysis,and protein-protein interaction.Results:1.We obtained 199 up-regulated genes and 235 down-regulated genes in GSE40289,91 up-regulated genes and 118 down-regulated genes in GSE59672.2.GO analysis showed that DEGs were mostly concentrated in oxidative stress,reactive oxygen species metabolism,cellular response to interleukin-γ,regulation of mitochondrion organization,regulation of mitochondrial membrane potential,mitochondrial fusion and some other biological processes.3.KEGG pathway analysis showed that DEGs were mainly related to ferroptosis,glutathione(GSH)metabolism pathway,KEAP1/NFE2L2 pathway,Nrf2 pathway,etc.It was suggested that inflammation,energy metabolism,oxidative stress and apoptosis are involved in DOX-induced cardiotoxicity mechanism,and mitochondria may be the key factor.4.In GSE40289,IFIT gene family and interferon regulatory factors(IRFs)family are considered as Hub genes,which are enriched in type I interferon signaling pathway,negative regulation of innate immune response,and cell response to interferon.Collagen(Col)family was considered to be Hub genes of GSE59672,which was enriched in collagen fiber tissue,protein digestion and absorption,collagen binding,etc.Heme oxygenase(HMOX),matrix metalloproteinases(MMPs)and Bc1211 are considered as Hub genes in the intersection dataset,which are enriched in reactive oxygen species metabolism,amino acid reaction and reaction to toxic substances.Conclusion:The process of DOX-induced cardiotoxicity may be characterized by the disruption of mitochondrial homeostasis and oxidative stress,which leads to myocardial cell injury and apoptosis.Mitochondrial structure and metabolism,along with mitochondrial function,active oxygen metabolism and inflammatory response,play pivotal roles in this process.PART 2 The inhibitory effect of LDR on DOX-induced cardiotoxicityObjective:To observe the effects of LDR on DOX-induced cardiotoxicity.Methods:Healthy female mice were randomly divided into four groups including CTR group,LDR group,DOX group and LDR+DOX group.The mice models of DOX-induced cardiotoxicity were established by intraperitoneal injection with 7.5 mg·Kg-1 DOX.LDR were performed 72 h before DOX injection.Irradiation conditions:whole-body irradiation,radiation dose of 75 mGy,dose rate of 12.5 mGy·min-1.Electrocardiogram of mice was recorded 5 days after DOX injection,then the mice were euthanized,their blood and hearts were collected.Enzyme linked immunosorbent assay(ELISA)was used to determine myocardial injury markers and inflammatory factors.Hematoxylin eosin(HE)and Masson staining was used to observe the pathological changes in the myocardial tissue of the mice.TUNEL staining were performed to detect the cell apoptosis.The expression of apoptotic proteins were detected by Western blot.Results:1.Effects of LDR on DOX-induced cardiac arrhythmia and myocardial injury markers changes.The electrocardiogram changes of mices in DOX group were obvious including arrhythmia,precontraction and ST-T changes.The LDR+DOX group showed occasional precontraction and mild ST-T changes.The level of myocardial injury markers(NTpro-BNP,CK-MB,cTnI and LDH)were significantly increased in mice of DOX group compared with that in CTR group,while these markers were significantly decreased in the LDR+DOX group(P<0.05).These results suggest that LDR has a protective effect against DOX-induced cardiotoxicity.2.Effects of LDR on DOX-induced pathological injury of cardiac tissue.We observed disordered arrangement of myocardial fibers by HE staining in the mice of DOX group,accompanied with irregular nuclei and cytoplasmic vacuolar degeneration.Compared with DOX group,the arrangement of cardiomyocyte in LDR+DOX group was slightly regular,and plasmic vacuolar degeneration was occasionally observed.Masson staining showed inflammatory cell infiltration and fibrotic lesions in mice of DOX group.While the morphology of LDR+DOX group was relatively complete,and myocardial fibrosis was alleviated than in the mice of DOX group.These results suggest that LDR intervention can reduce the pathological damage of cardiomyopathy caused by DOX.3.Effects of LDR on DOX-induced apoptosis and inflammation.After TUNEL staining,the cardiomyocyte apoptosis rate of mice in DOX group was significantly higher than that in CTR group.While the apoptosis rate of mice cardiomyocyte in LDR+DOX group significantly decreased compared with that in DOX group(P<0.05).The levels of apoptosis-related proteins were detected by Western blot.Compared with CTR group,the Bcl-2 expression of mice in DOX group was significantly decreased,while the Bax,cleaved-Caspase-3 and cleaved-Caspase-9 proteins were significantly increased(P<0.05).Compared with DOX group,the Bcl-2 protein of mice in LDR+DOX group was significantly increased,and the Bax,cleaved-Caspase-3 and cleaved-Caspase-9 expressions were significantly decreased(P<0.05).The myocardial inflammatory factors of mice in each group were detected by ELISA.The levels of inflammatory factors(TNF-α,IL-6,IL-1β,IL-18)were significantly increased in the mice of DOX group compared with the CTR group,while these inflammatory factors were significantly decreased in the LDR+DOX group compared with the DOX group(P<0.05).These results suggest that LDR may reduce DOX-induced cardiotoxicity by alleviating cardiomyocyte apoptosis and inflammatory damage.Conclusion:LDR can significantly reduce DOX-induced cardiotoxicity by alleviating cardiomyocyte apoptosis and inflammatory.PART 3 LDR reduces DOX-induced cardiotoxicity through regulating mitochondrial homeostasisObjective:To investigate the role of mitochondrial homeostasis in LDR reducing DOX-induced cardiotoxicity from oxidative stress,energy metabolism,mitochondrial structure and function.Methods:High-throughput sequencing was used to analyze the different gene expression levels of each group,and analyze the possible mechanism of LDR reduces DOX-induced cardiotoxicity.Use electron microscope to observe the ultrastructure and mitochondria of myocardial.Myocardial oxidation and antioxidant indexes(SOD,CAT,MDA,GSH)were detected by kits.The activity of myocardial mitochondrial complex I-V and ATP of different groups were measured by multiscan spectrum.The mitochondrial function proteins,mitochondrial dynamics related proteins,mitochondrial autophagy related proteins and oxidative stress related proteins of mice were detected by Western blot.The AMPK/PGC-1α pathway related proteins were detected by Western blot and immunohistochemical method.Results:1.High-throughput sequencing showed that there were 618 up-regulated genes and 235 down-regulated genes between DOX and CTR group,25 up-regulated genes and 35 down-regulated genes between LDR+DOX and DOX group.The GO and KEGG analysis suggested that the DEGs between different groups were mainly concentrated in mitochondrial complex,mitochondrial membrane structure,ATPase complex,peroxisome and other cell components.The biological processes are mainly concentrated in mitochondrial division,ATPase activity,active oxygen metabolism,apoptosis and the regulation of inflammatory reaction.The molecular functions are mainly concentrated in REDOX enzyme activity,glutathione peroxidase activity and ATPase activity.The DEGs were mainly concentrated in AMPK pathway,oxidative phosphorylation pathway,pyruvate metabolism pathway and TCA cycle pathway.These results suggest that the mitochondrial alterations and AMPK pathway are highly correlated with the attenuation of DOX-induced cardiotoxicity by LDR.2.LDR alleviates cardiomyocyte and mitochondrial injury induced by DOX.We observed disordered cardiomyocyte arrangement,mitochondrial swelling and mitochondrial ridge decreased or even disappeared in mice of DOX group,which were alleviated in LDR+DOX group.These results suggest that LDR can alleviate DOX-induced cardiomyocyte and mitochondrial injury.3.LDR reduces DOX-induced oxidative stress injury by improve myocardial antioxidant capacity.Compared with the CTR group,MDA content and CAT activity of mice in DOX group were significantly increased,while the GSH and SOD levels were significantly decreased(P<0.05).Compared with the DOX group,MDA content in LDR+DOX group was significantly decreased;while the GSH,SOD and CAT levels were significantly increased(P<0.05).The expressions of Nrf2 pathway related proteins were detected by Western blot.Compared with CTR group,the Keap1 and HO1 expressions in DOX group were significantly down-regulated,while the NQO1 and Nrf2 expressions were significantly up-regulated(P<0.05).Compared with DOX group,the HO1 expression in LDR+DOX group was significantly increased,while the expressions of Keapl and Nrf2 proteins were significantly decreased(P<0.05).These results suggest that LDR reduces DOX-induced cardiotoxi city by increasing antioxidant capacity.4.LDR alleviates DOX-induced mitochondrial energy metabolism disorders.The activity of cardiomyocyte mitochondrial respiratory chain complex Ⅰ-Ⅴ and ATP in each group of mice were detected.Compared with CTR group,the activity of mitochondrial complex Ⅰ-Ⅲ of mice in DOX group was significantly decreased,while the mitochondrial complex Ⅴ was significantly increased,and ATP content was significantly decreased(P<0.05).Compared with DOX group,the activity of mitochondrial complex Ⅰ-Ⅲ were significantly increased in LDR+DOX group,and the activity of complex Ⅴ was decreased,the ATP content was significantly increased(P<0.05).The results showed that LDR could reduce DOX-induced cardiotoxicity by regulating energy metabolism and maintaining mitochondrial metabolic balance.5.LDR resists mitochondrial function injury caused by DOX.Western blot was used to detect the expressions of mitochondrial function-related proteins in mice of each group.Compared with CTR group,the expressions of mitochondrial function related proteins(ATP5F1,CYC1 and NDUFV1)were significantly decreased in mice of DOX group(P<0.05).While the expressions of mitochondrial function related proteins were significantly increased in mice of LDR+DOX group compared with the DOX group(P<0.05).It was suggested that LDR can resist mitochondrial function impairment induced by DOX6.LDR regulates the DOX-induced mitochondrial dynamics imbalance.We used Western blot to detect mitochondrial dynamics related proteins of mice in each group.Compared with CTR group,the dynamically-related proteinl(DRP1)and P-DRP1(Ser616)expressions were significantly increased,while the expressions of OPA1,MFN1 and MFN2 were significantly decreased in DOX group(P<0.05).Compared with DOX group,the expression of DRP1 in LDR+DOX group was significantly decreased,and the expressions of OPA1,MFN1 and MFN2 were significantly increased(P<0.05).It was suggested that LDR can maintain mitochondrial homeostasis by influencing the DOX-induced mitochondrial dynamics imbalance.7.LDR alleviates the DOX-induced mitochondrial autophagy.Compared with CTR group,the autophagy assiociated proteins Parkin and PINK1 in DOX group were significantly increased(P<0.05).While the expressions of Parkin and PINK1 in LDR+DOX group were significantly decreased compared with DOX group(P<0.05).The results suggest that LDR may reduce DOX-induced cardiotoxicity by alleviating mitochondrial autophagy induced by DOX.8.LDR activated AMPK/PGC-1α pathway.Western blot and immunohistochemistry were used to detect the expression of AMPK/PGC-1α signaling pathway related proteins in each group.Compared with CTR group,the expression levels of AMPK,p-AMPK and PGC-1α of mice in DOX group were significantly down-regulated,while the expression level of PGC-1α was significantly up-regulated in mice of LDR group(P<0.05).Compared with DOX group,the expression levels of AMPK,P-AMPK and PGC-1α in LDR+DOX group were significantly increased(P<0.05).These results suggest that LDR may reduce DOX-induced cardiotoxicity by activating AMPK/PGC-1α pathway.Conclusion:1.LDR can regulate mitochondrial homeostasis and reduce DOX-induced cardiotoxicity by maintaining energy metabolism and mitochondrial dynamics balance,decreasing mitochondrial autophagy and oxidative stress injury.2.LDR may regulate mitochondrial homeostasis and reduce DOX-induced cardiotoxicity by activating AMPK/PGC-1α signaling pathway.