Mechanism Of Protection Of Autophagy Triggered By ERS Against LPS-induced Injury In HL-1Cardiomyocytes

Sepsis is defined as a systematic inflammation syndrome which couples with multipleorgans dysfunction and cardiovascular dysfunction. Some researches have shown thatcardiovascular dysfunction increases the mortality of sepsis. Cardiomyocyte apoptosis andmetabolic dysfunction are the major causes for cardiac dysfunction.Autophagosomes, damaged mitochondria and endoplasmic reticulum stress areobserved in cardiomyocytes during sepsis, which connect with each other tightly.Autophagy is an evolutionarily conserved catabolic process for degradation of long-livedprotein and elimination of damaged organelles to maintain cellular homeostasis，the finialdegradation products are involved in energy metabolism. Selective elimination of damagedmitochondria is benefit to maintain celluar homeostasis and cell survivial.Mitochondria are the major site for energic metabolism and involves in apopotsis.Mitochondrial quality control depends on the balance between damaged mitochondriaelimination and mitochondrial biogenesis. The effects of autophagy on mitochondiralbiogenesis during sepsis need to be further studied.Some evidence have indicated that endoplasmic reticulum stress triggers auotphagy,but the notion has not been verified in cardiomyocytes during sepsis.In current study, we investigated the mechanism of activation and cytoprotection ofautophagy in HL-1cell sepsis model in order to provide new target of therapeuticstrategies for cardiac dysfunction during sepsis. Objective: To investigate the activation and cytoprotection of autophagy against apoptosisinduced by Lipopolysaccharide（LPS）in HL-1cells. Methods: The sepsis model ofHL-1cells was formed by LPS in the final concentration of1ug/ml. The time course ofexpression of LC3II was assessed by westenblot at2、4、8、16、24h and control.Ultrastructure of autopahgosomes, green punctate dots of LC3II and expression of ATG5and ATG7mRNA were measured by Transmission elecronic microscopy (TEM)、Confocallaser scanning microscopy (CLSM) and Real-time quantitive PCR (RTq-PCR)respectively at4and24h LPS administration. Apoptosis and expression of LC3II wereobserved in the cells pretreated with or without3-methyladenine (3-MA) or Rapamycin(Rap) for48h at4、24h LPS administration and control by Flow cytometer and westenblot.Rusults: Expression of LC3II was elevated at2h LPS exposure, peaked at4h anddeclined at24h. TEM showed that double membrane autophagosomes were appeared at4h LPS exposure and declined at24h. The green punctate dots were elevated comparedwith control at4h LPS, disappeard at24h under Confocal laser scanningmicroscopy(CLSM). The expression of ATG5and ATG7mRNA were upregulated at4hLPS administration compared with control, and weaken at24h. LPS-induced apoptosis at24h LPS stimulation and was not observed at4h. The apoptosis induced by LPS24hadministration was inhibited in cells pretreated with Rapamycin, which accompanieddownregulation of LC3II expression. Pretreatment with3-MA led to apoptosis at4h LPScompanied with upregulation of LC3II protein. Conclusion: Autophagy induced by LPSis enhanced at early stage and supressed at later stage which plays a cyotprotection role inHL-1cells. Objective：To investigate the effects of autophagy on mitochondrial function andbiogenesis in HL-1cells during sepsis. Methods: The endotoxin model of HL-1cells wasformed by LPS in the final concentration of1ug/ml. Relative area and optical density,mitochondrial membrane potential and Tfam and PGC-1a mRNA of mitochondrialbiogenesis were measured in the cells at LPS24h exposure with or without3-MA orRapamycin pretreatment for48h by Transmission electronic microscopy(TEM), FlowCytometry and RTq-PCR respectively. Results: Mitochondrial relative area and opticaldensity were augmented, mitochondrial membran potential and expression of Tfam andPGC-1a mRNA were downregulated at LPS24h expousre compared with control. Theinsults induced by LPS were aggravated by3-MA pretreatment, howerver, which wereallivated by Rapamycin. Conclusion: LPS leads to mitochondrial ulrastructure damage,loss of mitochondiral membran potential and mitochondrial biogenesis dyfunction in HL-1cells. Autophagy plays a cyotprotective role against apoptosis through improvingmitochondrial function and biogenesis in HL-1cell during sepsis. Objective: To investigate wether autophagy is induced by LPS through endoplasimicreticulum stress. Methods: The sepsis model of HL-1cells was formed by LPS in thefinal concentration of1ug/ml. GRP78and IRE1a mRNA were measured at4and24h LPSexposure and control by RTq-PCR to assess the activation of endoplasmic reticulum stress. Relationship between endoplasmic reticulum stress and autophagy was observed throughexpression of GRP78and IRE1a mRNA, ATG5and ATG7mRNA, LC3II protein and greenLC3II punctate dots by RTq-PCR, westenblot and confocal microscopy in cells stimulatedby LPS with or without Tunicamycin (Tm) and Tauroursodeoxycholic acid (TUDCA).Rusults: GRP78and IRE1a mRNA were upregulated at4h LPS exposure, declined at24h.Compared with control, Tm-induced expression of GRP78、IRE1a,ATG5and ATG7mRNA,LC3II protein and green LC3II puncta dots were upregulated at4h. Compared with4h LPSadministration, pretreatment with Tm enhanced the expression of GRP78and IRE1amRNA companied with more extensive expression of ATG5and ATG7mRNA, LC3IIprotein and green LC3II punctate dots induced by4h LPS. Expression of GRP78andIRE1a mRNA induced by LPS were suppressed by TUDCA, meanwhile, activation ofautophagy was inhibited. Conclusion: LPS induces activation of endoplasmic reticulumstress and autophagy in HL-1cell. Autophagy is induced by LPS through endoplamicreticulum stress.