Interleukin-9 Aggravates Doxorubicin-Induced Cardiotoxicity By Promoting Inflammation And Apoptosis In Mice

Objective:Interleukin（IL）9 is a pleiotropic cytokine,and recent studies have demonstrated that IL-9 is associated with several cardiovascular diseases,via regulation of the inflammatory response.Doxorubicin（DOX）is known to induce severe cardiac injury and dysfunction by enhancing inflammation.This study aimed to investigate the role of IL-9 in DOX-induced cardiotoxicity.Methods:DOX was used to induce cardiac dysfunction and the expression of IL-9 in the murine cardiac tissues was measured by western blot and immunofluorescence.And immunofluorescence was performed to seek the source of IL-9 in the heart tissues.The mice were intraperitoneally injected with recombinant mouse IL-9（rm IL-9）or anti-IL-9 neutralizing antibody（IL-9n Ab）for investigating the effect of IL-9 on DOX-induced cardiac injury and dysfunction.8-week-old mice were randomly divided into the following 5 groups（n=10 mice/group）:the saline group,the DOX group,the DOX+IL-9n Ab group,the DOX+IL-9 isotype antibody（Ig G）group and the DOX+rm IL-9 group.The general condition and body weight of mice in each group was recorded regularly every day for five consecutive days.On the fifth day,echocardiography and hemodynamic experiments were performed,and the body weight,heart weight and tibia length of mice were measured.Lactate dehydrogenase（LDH）and creatine kinase isoenzyme（CK-MB）detecting kits were used to detect LDH and CK-MB expression in the left ventricular tissues and serum of mice.The messenger ribonucleic acid（m RNA）expression levels of the pro-inflammatory cytokines were determined in each group by quantitative real-time polymerase chain reaction（RT-q PCR）.Meanwhile,the effect of different concentrations（0,0.25,0.5,1,2,4,6,8μmol/L）of DOX stimulation was evaluated by CCK-8 assay in 12h on the viability of H9C2 cells and the half effective inhibition concentration（IC50）was determined.The effect of rm IL-9 or IL-9n Ab on DOX-induced apoptosis was determined both in vivo and vitro.Results:IL-9 levels significantly increased in the heart following DOX injection,and IL-9was mainly derived from CD4+Th cells.Cardiac injury and dysfunction were induced by DOX,and treatment with IL-9n Ab significantly alleviated DOX-induced injury,whereas rm IL-9 administration aggravated the cardiac damage.IL-9n Ab decreased the expression of pro-inflammatory cytokines in the DOX-treated mice,while rm IL-9 administration increased the levels of pro-inflammatory cytokines.IL-9n Ab reduced DOX-induced myocardial apoptosis,whereas rm IL-9 administration produced the opposite results.The results of CCK-8 showed that the viability of H9C2 cells decreased gradually with the increase of DOX concentration,and the IC₅₀of 12h was 1μmol/L.Additionally,IL-9n Ab mitigated the DOX-induced apoptosis in H9C2 cells,while administration of rm IL-9 produced the opposite effect.Conclusions:Our results demonstrated that IL-9 aggravated DOX-induced cardiac injury and dysfunction by promoting the inflammatory response and cardiomyocyte apoptosis.