The Role And Mechanism Of Adropin In Doxorubicin Induced Cardiotoxicity

Background:Doxorubicin(DOX)is a highly effective antihunor anthracycline antibiotic.Currently,DOX-based chemotherapy remains the cornerstone of cancer treatment.However,its dose-dependent cardiataxicity greatly limits its application in clinical practice,and DOX-induced cardiotoxicity(DIG)can eventually lead to myocardial injury and heart failure.However,there are currently no good treatment options to mitigate or prevent DIG.Therefore,it is of great significance to actively explore the potential mechanism of DIG and develop safe and effective adjuvant therapy to mitigate DIC.Adropin is a highly conserved novel secretory peptide hormone composed of 76 amino acids,encoded by Energy homeostasis-associated gene(Enho),and plays an vital role in Energy homeostasis and glycolipid metabolism.More and more evidence show that Adropin is a potential cardiovascular protective factor,with specific anti-inflammatory,antioxidant stress and anti-atherosclerosis effects.Our previous studies found that the expression of Adropin was significantly decreased in the myocardial injury induced by radiotherapy,and exogenous supplemental recombinant Adropin could significantly improve the myocardial injury induced by radiotherapy by reducing oxidative stress,promoting microangiogenesis,inbibiting myocardial fibrosis and apoptosis.In addition,recent studies have shown that Adropin alleviates hepatic steatosis,inflammation,and fibrosis in the high-fat and methionine-choline deficient-induced nonalcoholic steatohepatitis(NASH)model by inhibiting NLRP3 inflammasome activation.Oxidative stress and pyroptosis mediated by NLRP3 inflammatories are important pathophysiological mechanisms of DIC.Therefore,we speculate that Adropin may play a cardioprotective role in DIC through anti-oxidative stress and anti-pyroptosis.This study aims to explore and verify the hypothesis at the molecular,cellular and animal levels,expecting to provide new perspective for the prevention and treatment of DIC.Part Ⅰ Expression changes of Adropin aud its significance in DICObjective:To explore the expression changes of Adropin and its significance in DIC.Methods:(1)Expression of Adropin in DOX myocardial injury cell modelsH9C2 cardiomyocytes were stimulated with luM DOX for 24 hours to construct the DIC cell model,and the successful construction of the DIC cell model was evaluated comprehensively through the detection of cell activity(CCK-8 experiment),LDH release level and intracellular ROS level.The expression of Adropin in mRNA and protein levels was detected by qRT-PCR and WB.(2)Expression of Adropin in DOX myocardial injury mouse modelsC57BL/6 mice were intraperitoneally injected with low dose DOX(4mg/Kg,once a week)for 4 weeks to simulate chronic DIC.During feeding,they were weighed every 1 week.At week 5(1 week after the end of drug administration),the changes of cardiac function and structural indexes of mice were evaluated by echocardiography:left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS).Serum was collected to detect the myocardial injury markers CK-MB and LDH.After that,the left ventricular tissues were taken for HE staining to evaluate the pathological morphological changes of myocardial tissue.The successful establishment of DOX myocardial injury mouse model was evaluated by appellate indexes.Finally,the serum Adropin level of mice was detected by ELISA,and the expression level of Adropin in myocardial tissue was evaluated by qRT-PCR and WB.Results:(1)DOX treatment inhibited the cell activity,increased LDH release level and intracellular ROS level in H9C2,suggesting successful construction of DIC cell model.In addition,compared with Control group,Adropin expression in DOX treatment group was significantly decreased in both mRNA and protein levels.(2)C57BL/6 mice showed decreased body weight,impaired cardiac systolic function,elevated serum markers of myocardial injury(CK-MB and LDH),and obvious myocardial cell edema and disordered nuclear arrangement after DOX treatment.These results suggest that the chronic DIC mouse model was successfully constructed.DOX treatment significantly reduced serum Adropin levels in mice.In addition,the results of qRT-PCR and WB detection further confirmed that the expression of Adropin in the myocardium of DIC model mice was significantly decreased in the mRNA and protein levels.Conclusions:During the process of DIC,Adropin expression decreased significantly,indicating that Adropin may play a crucial role in DIC,but it still needs further exploration.Part Ⅱ The role and mechanism of Adropin in DICObjective:To determine whether Adropin improves DIC through anti-oxidative stress and anti-pyroptosis and its underlying molecular mechanismMethods:(1)To investigate the effect of exogenous recombinant Adropin on DIC at the cellular levelH9C2 cardiomyocytes with good growth condition were randomly divided into four groups:①Control group:normal cell culture without drug intervention;②Adropin group:100ng/mL Adropin treatment alone for 24 h;③DOX group:1umol/L DOX treatment alone for 24 h;④DOX+Adropin group:H9C2 cells were pretreated with 100ng/mL Adropin for 30min and then co-treated with 1umol/L DOX for 24h.Cell activity,LDH release and ROS levels in each group were evaluated using CCK-8 kit,LDH test kit and Dihydroethidium(DCFH-DA)kit The intracellular MDA level and SOD activity were also detected.The cell morphology was observed by transmission electron microscopy(TEM),including cell membrane rupture,cell enlargement&deformation,organelle deformation,pyroptosis bodies.The expression of pyroptosis signaling pathway proteins(NLRP3,caspase-1,GSDMD,IL-1β,IL-18)was detected by WB.The effects of exogenous recombinant Adropin on DIC at the cellular level were comprehensively evaluated by the above indexes.(2)To investigate the effect of exogenous recombinant Adropin on DIC at the animal levelThe 8-week-old male wild-type C57BL/6 mice were randomly divided into four groups:(1)Control group:no drug treatment,normal diet,free drinking water;②Adropin group:Mice were intraperitoneally injected with 0.2mg/kg Adropin once a week for 4 weeks;③DOX group:Mice were intraperitoneally injected with 4mg/kg DOX once a week for 4 weeks;④DOX+Adropin group:Mice were intraperitoneally injected with 0.2mg/kg adropin and every other day intraperitnneally injected with 4mg/kg DOX,once a week for 4 weeks.Each group consisted of 10 animals.During the feeding process,the mice were weighed every 1 week,and their mental and activity states and fur shedding were observed.At week 5,echocardiography was used to evaluate the changes of cardiac function and structural indicators:LVEF and LVFS.Serum was collected to detect the myocardial injury markers CK-MB and LDH.After that,the left ventricular tissues were taken for HE and DHE staining to evaluate the pathological morphological changes and oxidative stress level of the myocardium.MDA levels and SOD activity in myocardial tissue were also detected.The expression of pyroptosis related signaling pathway proteins(NLRP3,caspase-1,GSDMD,IL-1β,IL-18)was detected by immunohistochemical and WB.The influence of exogenous recombinant Adropin on DIC in vivo was comprehensively evaluated by the above indexes.(3)To investigate the effect of Adropin gene knockout on DICThe 8-week-old male mice with Adropin gene knockout and wild-type C57BL/6 were randomly divided into two groups:①Control group:no drug treatment,normal diet,free drinking water;②DOX group:Mice were intraperitoneally injected with 4mg/kg DOX once a week for 4 weeks.Each group consisted of 10 animals.The detection index is the same as the above part.The efects of Adropin gene knockout on DIC in vivo were comprehensively evaluated by the above indexes.(4)To investigate whether Adropin alleviates DOX-induced myocardial injury and pyroptosis at the cellular level by inhibiting the activation of NLRP3 inflammasome.The NLRP3 inflammasome pharmacologically specific agonist nigericin was used to explore whether Adropin plays an important role in the process of DIC by mediating activation of NLRP3 inflammasome to inhibit pyroptosis.H9C2 cardiomyocytes with good growth condition were randomly divided into 5 groups:① Control group:normal cell culture without drug intervention;②DOX group:lumol/L DOX treatment alone for 24 h;③ DOX+nigericin group:H9C2 cells pretreated with 10ug/mL nigericin for 60min,and then co-treated with 1umol/L DOX for 24 hours.④DOX+Adropin group:H9C2 cells were pretreated with 100ng/mL Adropin for 30min and then co-treated with lumol/L DOX for 24h.⑤DOX+Adropin+nigericin group:H9C2 cells were pretreated by 10ug/mL nigericin for 30min,then 100ng/mL Adropin was added to treat H9C2 cells for 30min and then 1umol/L DOX was added for 24 hours.Cell activity,LDH release and ROS levels were evaluated using CCK-8,LDH and Dihydroethidium(DCFH-DA)kits,respectively.The intracellular MDA level and SOD activity were also detected.The expressions of pyroptosis related signaling pathway proteins(NLRP3,caspase-1,GSDMD,IL-1β,IL-18)were detected by WB and immunofluorescence.The above indicators were used to comprehensively evaluate whether Adropin could inhibit pyroptosis by mediating the activation of NLRP3 inflammasome,thus playing an crucial role in the process of DIC.Results:(1)In vitro,Adropin synergistic treatment effectively alleviates DOX-induced H9C2 cell damage and death,and reduces DOX-induced LDH release.In addition,DOX treatment significantly increased intracellular ROS and MDA levels,and inhibited SOD activity.Transmission electron microscopy showed that after DOX treatment,cell structure was severely abnormal,cell swelling dissolved,membrane rupture,pore formation,nucleolar chromatin concentration and agglutination,and endoplasmic reticulum was extensively and severely dilated,suggesting that pyroptosis was triggered during the process of DIC.Further WB examination confirmed that pyroptosis related proteins(NLRP3,GSDMD,caspase-1,IL-1β,and IL,18)were significantly increased after DOX treatment,and immunofluorescence further revealed increased expression of NLRP3,a pyroptosis trigger.The Adropin cooperative treatment can effectively inhibit these changes.(2)In vivo,intraperitoneal injection of recombinant Adropin can significantly reverse body weight loss,elevated serum markers of myocardial injury,cardiac systolic dysfunction,and histopathologic changes of myocardial tissue caused by DOX treatment.These improvements were accompanied by Adropin inhibition of oxidative stress damage and NLRP3 inflammasome-mediated pyroptosis,which was reflected in the following two aspects:① Intraderitoneal injection of recombinant Adropin could significantly reduce the accumulation of ROS and the increase of MAD level in myocardial tissue of DOX-induced mice,and reverse the inhibition of SOD activity by DOX treatment;② Adropin co-treatment inhibited the overexpression of DOX-induced pyroptosis related proteins(NLRP3,GSDMD,caspase-1,IL-1β,and IL-18).Similar to in vitro,immunohistochemical results also suggested that Adropin synergistic treatment inhibited DOX-induced NLRP3 overexpression.(3)In vivo,after DOX treatment,Adropin gene knockout promoted the accumulation of ROS and increased MDA levels in myocardial tissue after DOX treatment,and further inhibited the activity of SOD.WB examination suggested that Adropin gene knockout promoted the overexpression of DOX-induced pyroptosis related proteins(NLRP3,GSDMD,caspase-1,IL-1β,and IL-18).These changes were accompanied by more severe myocardial damage in mice,which was mainly reflected in that after DOX treatment,compared with WT mice,Adropin gene knockout mice showed more significant weight loss,and cardiac systolic dysfunction was further aggravated and serum markers of myocardial damage were further increased.(4)In vitro,the NLRP3 inflammasome specific pharmacological agonist nigericin blocked the inhibition of Adropin on DoX-induced NLRP3 activation and overexpression of pyroptosis proteins(GSDMD,caspase-1,IL-1β and IL-18).In addition,Nigericin has reversed the protective effect of Adropin on DIC cells,which is mainly reflected in the decreased activity of H9C2 cells and significantly increased LDH release in Adropin+DOX+Nigericin group compared with Adropin+DOX group.Conclusions:Adropin can significantly alleviate DIC through anti-oxidative stress and anti-pyroptosis,and its underlying molecular mechanism may be to inbibit pyroptosis of cardiomyocytes by inhibiting the activation of NLRP3 inflammasome.