Dimethyl Fumarate Attenuates LPS-induced Mitochondrial Injury In Cardiomyocytes

Background Sepsis is a life-threatening disorder of systemic inflammatory response syndrome,and it can develop into septic shock and multiple organ dysfunction syndrome.Studies have shown that mortality rate of sepsis patients with cardiac dysfunction is about 70%-90%,which is significantly higher than those without cardiac dysfunction(20%).Amout of evidence has proven that oxidative stress,abnormal mitochondrial morphology,and mitochondrial dysfunction might be the main pathophysiological mechanisms of sepsisinduced cardiac injury.However,effective therapeutic strategies to prevent sepsisinduced cardiac dysfunction are still not yet established.Dimethyl fumarate(DMF)is one of the derivations of fumaric acid.DMF and its metabolite monomethyl fumarate have been reported to exert anti-inflammatory and antioxidative properties in many tissues such as nerve system,liver,and heart.So,we hypothesized that it might also protect against sepsis-induced myocardial dysfunction.Nuclear factor E2-related factor 2(Nrf2)is an important anti-oxidative protein,which is ubiquitously expressed in heart,lung and liver.During inactive conditions,Nrf2 is mainly contained in cytoplasm by Kelch-like ECH-associated protein 1(Keap1).After stimulated,Nrf2 is released from Keap1,and translocates into nuclear.In nuclear,Nrf2 binds with antioxidant response element(ARE),and induces expression of antioxidants.Studies have also shown that neuroprotective effect of DMF is mediated by a Nrf2-dependent mechanism.Whether Nrf2 is also involved in the cardioprotection of DMF against sepsisinduced cardiac damage is still unclear.Objectives The aims of present study were to investigate whether DMF can prevent lipopolysaccharide(LPS)-induced cardiomyocyte injury,and to explore whether activation of Nrf2 pathway is involved in the cardioprotection of DMF.Methods 1)H9C2 cells were treated with 1?g/m L LPS to establish cardiomyocyte injury model.2)H9C2 cells were divided into following groups: control group,LPS group,DMF group,LPS+DMF group,Nrf2-si RNA group,LPS+Nrf2-si RNA group,LPS+DMF +Nrf2-si RNA group,LPS+DMF+PD98059 group,PD98059 group.3)Survival rate of H9C2 cells was measured by using CCK-8 kit.Apoptosis of cells was detected by using Annexin V/PI apoptosis kit and evaluated by a flow cytometry.4)Expression of Nrf2,HO-1,p-ERK1/2,ERK1/2,Lamin A/C and GAPDH proteins were analyzed by using Western blotting.5)Lactate dehydrogenase(LDH)leakage,malondialdehyde(MDA)content,glutathione(GSH)content,superoxide dismutase(SOD)activity,and glutathione peroxidase(GSH-Px)activity were evaluated by five commercial kits.6)ELSIA kits were used to measure IL-1? and IL-18 levels.7)Location of Nrf2 protein and mitochondrial morphology were analyzed using immunofluorescence technique.8)Mitochondria Membrane potential was evaluated by using JC-1 fluorescent probe.9)Mitochondrial superoxide production was analyzed by using Mito SOX Red probe.10)Mitochondrial respiratory function was analyzed by Seahorse bioanalyzer.Results1)In LPS-treated H9C2 cells,cell viability was significantly reduced,the amount of LDH leakage and apoptosis were increased.Compared with LPS group,pretreatment with DMF(10,20,or 40?M)generated a higher cell viability,a lower LDH leakage and apoptosis(P <0.01).2)LPS caused decreased expression of total Nrf2 protein and nuclear Nrf2 level in H9C2 cells.DMF pretreatment could not only increase the expression of total Nrf2 and nuclear Nrf2,but also enhance HO-1 protein expression when compared with LPS(P<0.05).3)Nrf2-si RNA inhibited the DMF-indued overexpression of Nrf2 and HO-1.It could also prevented DMF-induced increase of cell viability,and DMF-induced decrease of LDH release,apoptosis,inflammatory factors,MDA and GSH content,SOD and GSH-Px activities(P<0.01).4)DMF induced enhancement of p-ERK1/2 ratio in LPS-challenged cells.ERK1/2 inhibitor PD98059 could not only prevent DMF-induced increase of nuclear Nrf2 level and HO-1 protein,but also inhibit DMF-induced increase of cell viability.5)Compared with LPS group,DMF pretreatment reduced mitochondrial superoxide production,attenuated mitochondrial fragmentation,increased mitochondrial membrane potential and mitochondrial respiration function.The above protective effect of DMF could be eliminated by Nrf2-si RNA(P <0.01).Conclusion In conclusion,DMF could protect H9C2 cells aganist LPS-induced injury.ERK1/2-dependent activation of Nrf2/HO-1 pathway is responsible for DMF-induced cardioprotection via reduction of oxidative stress,improvement of mitochondrial morphology and energy metabolism.