The Mechanism Of TFEB In Myocardial Inflammatory Injury

Background: Myocardial inflammatory injury is a common complication of sepsis,with the morbidity of up to 50%,and ultimately leads to organ dysfunction.The main features of septic myocarditis are inflammatory disorder,low metabolism and immune dysfunction.Cytokine targeting therapy of septic myocarditis,inhibition of its development has become an important means of clinical treatment.Transcription factor EB(TFEB)is a major regulator of autophagy and lysosomal biogenesis,which plays an important role in promoting angiogenesis and recovery of cardiac function.In addition,TFEB is involved in the regulation of mitochondrial homeostasis and plays an important role in maintaining the balance between mitochondrial autophagy and mitochondrial biogenesis.In this study,the effect of TFEB regulation on the lipopolysaccharide(LPS)-induced inflammatory injury of human AC16 cardiomyocytes was investigated,and the effect of TFEB regulation on the LPS-induced inflammatory injury of human AC16 cardiomyocytes was further investigated.Our findings indicate a new potential therapeutic strategy for the cardiac inflammatory related diseases.Methods:(1)LPS is a useful method of inducing inflammation model in vitro,using different concentrations of LPS processing AC16 myocardial cell,quantitative real-time PCR(q PCR)was used to check the relative expression of inflammatory factors and cell adhesion molecules,DCFH-DA was applied to determine the intracellular reactive oxygen species(ROS)accumulation levels,and JC-1 was used to detect the mitochondrial membrane potential(MMP),the appropriate processing time and concentration of LPS were determined based on the above results.(2)Human AC16 cardiomyocytes were treated with the selected LPS concentration,and the endogenous TFEB protein expression was detected by western blot(WB).TFEB silencing model was established to detect the effect on LPS-induced human AC16 cardiomyocytes.(3)TFEB overexpression model was constructed to detect the effect of LPS-induced cell inflammatory injury: q PCR was used to detect the expression levels of cell inflammatory factors and autophagosomal-related genes;WB detected the expression levels of cell adhesion molecules;apoptosis and autophagy marker LC3 B protein;DCFH-DA was used to detect intracellular ROS levels.(4)Autophagy was inhibited by 3-methyladenine(3-MA)and chloroquine(CQ).WB and q PCR were used to detect the autophagy marker LC3 B,cell adhesion molecule,apoptosis protein Bax,anti-inflammatory factor IL-10 and DCFH-DA used to detect ROS expression levels.Results:(1)LPS can induce the inflammatory response,ROS accumulation and apoptosis of human AC16 cardiomyocytes.Based on the above results,the optimal treatment time and concentration of LPS induced the inflammatory injury of human AC16 cardiomyocytes is 24 h and 0.1μg/m L,respectively.(2)Compared with AC16 group,endogenous TFEB expression in human AC16 cardiomyocytes induced by LPS group was increased.The expressions of inflammatory factors,cell adhesion molecules and apoptosis were increased in the silenced TFEB group compared with the LPS group.(3)Compared with the LPS group,the expressions of inflammatory factors and cell adhesion molecules in TFEB-OE group were significantly decreased,the expression of Bax protein was decreased,and the ROS level was significantly decreased.(4)Compared with LPS+TFEB-OE group,the levels of cell adhesion molecules,ROS accumulation and apoptosis were increased in LPS+TFEB-OE+3-MA and LPS+TFEB-OE+CQ groups,while the expression of anti-inflammatory factors was decreased.Conclusions:(1)The expression of endogenous TFEB was up-regulated in the inflammatory injury of cells,but the silencing of its expression aggravated the inflammatory response and apoptosis.(2)Exogenous TFEB overexpression can inhibit LPS-induced inflammatory response,mitochondrial ROS accumulation and apoptosis in human AC16 cardiomyocytes by regulating autophagy.In conclusion,TFEB may play a protective role against myocardial inflammatory injury by activating autophagosomal pathway.Therefore,targeting TFEB and autophagy may be a viable strategy for the treatment or prevention of cardiac inflammatory injury.