| Objective:To establish a model of adriamycin（ADR）-induced cardiotoxicity aggravated by long-term high fat diet（HFD）in mice,we expected to verify whether the long-term HFD mice were more sensitive to ADR-induced cardiotoxicity,and whether overexpression of Sestrin 2（Sesn2）alleviated ADR-induced cardiotoxicity aggravated by obesity.Next,we further explored whether Sesn2 resisted ADR-induced cardiotoxicity aggravated by HFD through activating nuclear factor erythroid 2-related factor 2（Nrf2）,and explored the role of AKT/GSK-3β/Fyn signaling pathway in activation of Nrf2 by Sesn2 to protect against ADR-induced cardiotoxicity aggravated by obesity.Based on the above research purposes,as a medical staff,on the one hand,we can give corresponding dietary intervention and health guidance to obese cancer patients undergoing clinical chemotherapy.On the other hand,we can look for drugs or natural plant extracts that activate Nrf2,such as sulforaphane,to improve heart injury induced by chemotherapeutic drugs.Methods:Male FVB mice were randomly assigned to feed HFD for 4 months,and then treated with/without 8mg/kg ADR every other week for 1 month in the presence or absence of HFD to generate cardiotoxicity.To determine whether the long-term HFD could exacerbate cardiac dysfunction caused by ADR,echocardiography was performed as our previously reported.After the mice was anesthetized and sacrificed,H＆E staining was used to detect myocardial fibre disruption;Hypertrophic cardiomyocytes were evaluated by semi-quantitative analysis of the myocyte area with FITC-WGA staining;The collagen accumulation was dectected by Sirius red staining;The protein expression of TNF-α was examined by IHC staining;Cardiac superoxide production was detected via DHE staining;Cardiac oxidative stress was further measured by protein staining of 3-NT and 4-HNE.The related marker for cardiac hypertrophy,inflammation and fibrosis was further detected by qRT-PCR.The protein expression of Sesn2,p-AKT,T-AKT,p-GSK-3β,T-GSK-3β,Fyn and Nrf2 was detected by Western blot.Intracellular ROS production and cellular apoptosis were examined by flow cytometry in cardiomyocytes.Results:Part one.Long-term HFD aggravated ADR-induced cardiotoxicity1.Long-term HFD aggravated cardiac dysfunction induced by ADRTo determine whether long-term HFD could exacerbate cardiac dysfuntion caused by ADR,echocardiography was performed as our previously reported.The results revealed that the mice in the ADR group showed the obvious increase in systolic LVID and systolic LV VOL and decrease in EF and FS.But these cardiac function changes were seriously aggravated by ADR treatment in the HFD group.2.Long-term HFD aggravated the myocardial structural disorder induced by ADRH＆E staining showed myocardial fibre disruption in the ADR group,but not obviously in the HFD group.And the HFD mice after ADR administration showed the most serious cardiac injury compared with the HFD or ADR group.3.Long-term HFD aggravated cardiomyocyte hypertrophy induced by ADRHypertrophic cardiomyocytes were evaluated by semi-quantitative analysis of the myocyte area with FITC-WGA staining.Compared with the HFD or ADR group,cardiomyocyte hypertrophy was more evident in the HFD mice after ADR administration.Cardiomyocyte hypertrophy was further supported by the upregulation of the hypertrophy markers Myh7 and Anp for the mRNA expression.4.Long-term HFD aggravated myocardial fibrosis induced by ADRSirius red staining demonstrated significant collagen accumulation in the HFD group after ADR administration compared with the HFD or ADR group,suggesting that HFD further exacerbated ADR-induced cardiac fibrosis.Cardiac fibrosis was further detected by qRT-PCR of the pro-fibrotic mediators,Tgfb and Ctgf.These fibrotic factors were significantly increased in the HFD group after ADR administration compared with ADR group.5.Long-term HFD aggravated cardiac inflammation induced by ADRCardiac inflammation damage,defined by mRNA levels of inflammatory cytokines Tnfa and 116 and protein staining of TNF-α,was noted in ADR-treated mice and further increased in HFD mice after ADR administration.Part two.The mechanism and related molecular pathways of ADR-induced cardiotoxicity exacerbated by long-term HFD1.Long-term HFD aggravated ADR induced cardiac oxidative stressCardiac superoxide production in our mice model was detected via DHE staining.The results indicated that both the ADR and HFD group exhibited an increase in superoxide production in heart tissue,and superoxide production was significantly elevated in the HFD group after ADR administration.Cardiac oxidative stress was further measured by protein staining of 3-NT and 4-HNE.These oxidative stress markers were also increased in the ADR group,and significantly aggravated in the HFD group after ADR administration.The mRNA levels of two antioxidant genes,Cat and Sod,were also decreased in the ADR or HFD group,and significant aggravated in the HFD group after ADR administration.2.Palmitate aggravated ADR disturbed expression of Sesn2 and Nrf2 in heart tissues and cardiomyocytesOur results revealed a significant decrease in Sesn2 protein in HFD mice after ADR administration compared with HFD or ADR treatment.Additionally,the expression of Nrf2 was also analyzed by Western blot.The results indicated that a significant decrease in Nrf2 protein in HFD mice after ADR administration compared with HFD or ADR treatment.To confirm whether decreased total Nrf2 expression is accompanied by its function decline,mRNA expression of Nrf2 downstream antioxidants Ho1 and Nqo1 were also measured by qRT-PCR.The results indicated that HFD or ADR alone decreased gene expression of Ho1 and Nqo1,and HFD combined with ADR treatment further disturbed these mRNAs expressions in hearts.Part three.Sesn2 activated Nrf2 via AKT/GSK-3β/Fyn pathway1.Overexpression of Sesn2 could activate Nrf2Sesn2 was overexpressed by transfected with pCMV6-Sesn2 plasmid in H9c2 cells.Cardiomyocytes with or without Sesn2 overexpression were then subjected to the treatment of palmitate and/or ADR for 24h.Interestingly,the protein abundance of Nrf2 was obviously increased in Sesn2-transfected H9c2 cells.2.AKT/GSK-3β/Fyn pathway mediated Sesn2 induced Nrf2 activationTo find out whether the AKT/GSK-3β/Fyn pathway was crucial to beneficial effects observed in pCMV6-Sesn2-transfected cardiomyocytes,wortmannin（1μmol/L）was used to block AKT expression in our cell model.The results exhibited that the ability of Sesn2 overexpression to restore the effects of palmitate and/or ADR on AKT,and GSK-3β were inhibited by wortmannin.And wortmannin also significantly abolished Sesn2 overexpression-increased expression of Nrf2.3.Inhibition of the AKT pathway could eliminate the anti-oxidative stress and anti-apoptosis funtion of Sesn2 in cardiomyocytesCardiomyocytes were pretreated with wortmannin for 30min,followed by palmitate and/or ADR treatment for 24h.Flow cytometry was used to detect oxidative stress and apoptosis of cardiomyocytes.The results showed that wortmannin significantly abolished Sesn2 overexpression-increased expression of Nrf2,leading to a complete abolishment of Sesn2 overexpression-attenuated cellular oxidative stress and apoptosis in palmitate and ADR-treated cardiomyocytes.4.sh-Nrf2 did not alter of Sesn2 induced activation of AKT/GSK-3β/Fyn pathway.The H9c2 cells were transfected with purified sh-Nrf2 in the presence or absence of pCMV6-Sesn2 for 24h,followed by ADR and palmitate for 24h.Western blot was used to detect the protein expressions of Sesn2,p-AKT,p-GSK-3β and Nrf2.The results showed that overexpression of Sesn2 could increase phosphorylation of AKT and GSK-3β,which was not affected by Nrf2 downregulation.5.sh-Nrf2 abolished the effect of overexpression of Sesn2 on antioxidant stress and anti-apoptosis in cardiomyocytesThe H9c2 cells were transfected with purified sh-Nrf2 in the presence or absence of pCMV6-Sesn2 for 24h,followed by ADR and palmitate for 24h.Nrf2 downregulation eliminated the protective effects of overexpression of Sesn2 against intracellular ROS production examined by flow cytometry in cardiomyocytes.Similarly,cardiac Sesn2-overexpression improved the cellular apoptosis detected by flow cytometry in the sh-NC-transfected group but not in the sh-Nrf2-transfected group.Conclusion:Our study revealed for the first time that there was a close relationship between downregulation of Sesn2 and cardiac oxidative stress in the model of ADR-induced cardiotoxicity aggravated by long-term HFD（obesity）.Overexpression of Sesn2 significantly ameliorated the oxidative stress and dysfunction induced by ADR.The mechanism study found that the protective effect of Sesn2 needed the activation of Nrf2 antioxidant function,and AKT/GSK-3β/Fyn pathway mediated Sesn2 induced Nrf2 activation in ADR-induced cardiotoxicity aggragated by obesity.This study provided an important experimental and theoretical basis for further elucidating the biological function of Sesn2 and finding new targets for improving ADR-induced oxidative damage and cardiotoxicity in obesity during chemotherapy. |
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