Effect And Mechanism Of TLR4 Gene Knockout On Myocardial Injury In Sepsis Mice

BackgroundSepsis Induced Myocardial Injury(SIMI)is featured by severe myocardial dysfunction and remains one of the lethal complications in clinical sepsis.Toll-like receptor 4(TLR4)signaling is known as a classical innate pathway in heart diseases,whereas the precise underlying mechanism of TLR4 in SIMI remains elusive.In recent years,numerous studies have confirmed a role for TLR4/MyD88/NF-κB signaling in various cardiovascular pathologies including heart failure,coronary microembolization,myocardial infarction,cardiac dysfunction in post-traumatic stress disorder.However,little evidence has depicted a role for TLR4/MyD88/NF-κB signaling cascade in the regulation of inflammatory cascade and cardiomyocyte apoptosis under SIMI.Thus,this study was designed to explore the effect of TLR4 gene knockout on inflammation level,apoptosis level and TLR4/MyD88/NF-κB signaling pathway in mice with sepsis,providing a theoretical basis for the molecular mechanism of SIMI and cardiac dysfunction,and further enrich the signaling mechanism of myocardial damage in sepsis based on TLR4/MyD88/NF-κB signaling pathway.MethodsTLR4 deficiency(TLR4-/-,n=32)mice and wild type(WT,n=3 2)littermates were subjected to lipopolysaccharide(LPS,4 mg/kg,6 h)to establish SIMI.The mice were divided into Control group,LPS group,TLR4-/-group and TLR4-/-+LPS group.Protein expression of TLR4 signaling molecules(TLR4,MyD88,and NF-κB),proinflammatory cytokines(TNF-α,IL-1 β,and IL-6),and apoptotic markers(Bax,Bcl2)were assessed using ELISA and Western blot.In vitro study,H9c2 cardiomyocytes were challenged with LPS(1 μg/ml,24 h)in the presence or absence of TAK-242(TLR4 inhibitor,30 μM,24 h)prior to the assessment of biochemical indices.They were divided into Control group,LPS group,TAK-242 group and TAK-242+LPS group.Myocardial injury markers(CK-MB,cTnT)were detected by ELISA.The mRNA levels of inflammatory mediators and TLR4related pathway molecules were detected by RT-PCR.The expression of TLR4 signaling pathway proteins(TLR4,MyD88,p-NF-κB,NF-κB)and apoptosis-related proteins(Bax,Bcl-2)were detected by Western blot.Cell apoptosis was detected by flow cytometry.Results1.The expression levels of inflammatory cytokines(TNF-α,IL-1 β and IL-6)and pro-apoptotic protein Bax were significantly decreased in TLR4-/-+LPS group compared with Control group,while the expression levels of anti-apoptotic protein Bcl2 were significantly increased in TLR4-/-+LPS group.2.The inflammation level and apoptosis level of H9c2 myocardial cells in LPS group were significantly increased,while the inflammation le vel and apoptosis level in TAK-242+LPS group were significantly decreased compared with LPS group,At the same time,the expression levels of TLR4,MyD88 and p-NF-κB/NF-κB in TAK-242+LPS group were significantly lower than those in LPS group.3.TLR4 gene knockout or inhibition can inhibit the activation of TLR4/MyD88/NF-κB signaling pathway.ConclusionTaken together,our results demonstrated a role for the TLR4/MyD88/NF-κB signaling in the activation of proinflammatory responses and apoptosis in SIMI.TLR4 deficiency or inhibition ameliorated myocardial damage by inhibiting TLR4/MyD88/NF-κB signaling and reduced pro-inflammatory cytokines and apoptosis in SIMI.