Protective Effect And Mechanism Of Inhibition Of 12/15-LO On Viral Myocarditis

Background and Objective Viral myocarditis(VMC)is a common viral heart disease,often accompanied by myocardial necrosis,interstitial hyperplasia and other pathological changes.It’s serious harm to people’s health,especially in young adults.Coxsackievirus B3(CVB3)is identified as the most widespread causative factor of myocarditis and is responsible for more than half of the cases of acute myocarditis and one-fourth of DCM cases.Lipoxygenases(LOs),including 12-LO and 15-LO,are a family of lipid-peroxidizing enzymes.It was reported that cardiac 12/15-LO was markedly up-regulated and involved in the development of heart failure.The nuclear factor E2-related factor 2(Nrf2)is a sensitive transcription factor for oxidative stress in cells,which regulates the formation of various antioxidant proteins and plays a fundamental protective role in cardiovascular disease.In this study,we used CVB3 virus to infect mice and cardiomyocytes to establish VMC in vitro and in vivo models.The use of baicalein treatment inhibited 12/15-LO overexpression,and we intended to investigate the role of 12/15-LO in CVB3-induced myocarditis and explore the molecular mechanism.Methods 1.Four-week-old male BALB/C mice(n = 60)of 18-20 g weight in specific pathogen free(SPF)conditions were divided into three group: control group,VMC group and baicalein treatment group(n=20).Primary mouse cardiomyocytes were divided into four groups: control group,baicalein group,CVB3 group and baicalein treatment group.2.VMC group and baicalein treatment group of mice were inoculated intraperitoneally with 0.1 ml CVB3 for 3 consecutive days.Mice received another intraperitoneal injection of baicalein for 10 additional consecutive days in the treatment group.The mice intraperitoneally injected with normal saline solution were used as control.CVB3 group and baicalein treatment group of cardiomyocytes were infected at multiplicity of infection(MOI)of 10 with CVB3 for 1 h.Subsequently,the cells were treated with 25 μM baicalein for 24 h and 48 h.Cells cultured without viral infection and baicalein treatment served as the control group.3.After 10 days of treatment with baicalein,blood and heart tissue were taken under aseptic conditions.And,the cardiomyocytes were treated with baicalein for 48 hours,followed by experimental study.4.Pathological sections of heart tissue was observed,the viability of cardiomyocytes was determined and the serum level of myocardial CK-MB in heart homogenates was detected;the content of TNF-α,IL-1β,IL-6,MDA and the activities of antioxidant enzymes SOD and GSH-Px were measured,and the expression level of 12/15-LO,Nrf2,HO-1 were measured.Results 1.In the VMC mice infected with 10 days,compared with the negative control group,the serum CK-MB activity was significantly higher(P<0.05);more inflammatory lesions in the heart were observed;the concentrations of inflammatory cytokines,including TNF-α,IL-1β,IL-6 and MDAwere exceptionally higher(P<0.05);12/15-LO level was significantly elevated and the levels of Nrf2 and HO-1 were markedly reduced;the concentrations of GSH-Px and SOD were significantly lower(P<0.05).2.In the myocardial cells infected with CVB3 virus for 48 hours,compared with the control group,12/15-LO level was significantly elevated and the levels of Nrf2 and HO-1 were markedly reduced;TNF-α,IL-1β and IL-6 were exceptionally higher(P<0.05);the viability was markedly inhibited(P <0.05).3.In the VMC mice of 12/15-LO inhibition(by baicalein),compared with the model group,inflammation areas in the heart were significant decrease;the level of CK-MB was significantly reduced(P<0.05);inflammatory cytokines,including TNF-α,IL-1β and IL-6 were marked reduction(P<0.05);the expression levels of Nrf2 and HO-1 were improved to some extent.4.In the myocardial cells of 12/15-LO inhibition(by baicalein),compared with the model group,TNF-α,IL-1β and IL-6 were significant decrease(P<0.05);baicalein treatment conspicuously reversed CVB3-induced viability inhibition of cardiac myocytes(P<0.05);the expression levels of Nrf2 and HO-1 were improved to some extent.Conclusions 1.We demonstrated for the first time that inhibition of 12/15-LO attenuated the severity of myocarditis and abrogated CVB3-induced cardiac inflammation and oxidative stress.2.Our study further indicated that inhibition of 12/15-LO ameliorated VMC may by activating Nrf2,contributing to better understanding the immune-pathological mechanism in CVB3-induced myocarditis.