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

Sepsis was definited as a systematic inflammation syndrome which companied with multiple organs dysfunction and cardiovascular dysfunction. Some studies showed that cardiovascular dysfunction increased the mortality of sepsis. Cardiomyocytes apoptosis and metabolic dysfunction were the major causes for cardiac dysfunction.Autophagosomes, damaged mitochondria and endoplasmic reticulum stress were observed in cardiomyocytes during sepsis, which connected with each other tightly. Autophagy is an evolutionarily conserved catabolic process for degradation of long-lived protein and elimination of damaged organelles to maintain cellular homeostasis, the finial degradation product were involed in energy metabolism. Selective elimination of damaged mitochondria was benefit to maintain celluar homeostasis and cell survivial.Mitochondria is the major site for energic metabolism and involves in apopotsis. Mitochondrial quality control depend on the balance between damaged mitochondria elimination and mitochondrial biogenesis. The effects of autophagy on mitochondiral biogenesis during sepsis need to be further studied.Some evidence showed that endoplasmic reticulum stress could trigger auotphagy, but the notion has not been verifed in cardiomyocytes during sepsis.In current study, we investigate the mechanism of activation and cytoprotection of autophagy in HL-1cell sepsis model in order to provide new targets of therapeutic strategies for cardiac dysfunction during sepsis. Part â…  The cytoprotective role of autophagy against apoptosis induced by LPS in HL-1cardiomyocytesObjective:To investigate the activation and cytoprotection of autophagy against apoptosis induced by Lipopolysaccharide(LPS)in HL-1cells. Methods:The sepsis model of HL-1cells was formed by LPS in the final concentration of1ug/ml. The time course of expression of LC3II was assessed by westenblot at2,4,8,16,24h and control. Ultrastructure of autopahgosomes, green puncta dots of LC3II and expression of ATG5and ATG7mRNA were measured by Transmission elecronic microscopy (TEM),confocal laser scanning microscopy (CLSM) and Real-time PCR (RTq-PCR) respectly at4and24h LPS adminstration. Apoptosis and expression of LC3II were observed in the cells pretreated with or without3-methyladenine (3-MA) or Rapamycin (Rap) for48h at4,24h LPS adminstration and control by Flow cytometer and westenblot. Rusults:Expression of LC3II was elevated at2h LPS exposure, peaked at4h and declined at24h. TEM showed that double membrane autophagosomes were appeared at4h LPS exposure and declined at24h. The green puncta dots were elevated compared with control at4h LPS, disappeard at24h under Confocal laser scanning microscopy(CLSM). The expression of ATG5and ATG7mRNA were upregulated at4h LPS administration compared with control, and weaken at24h. LPS-induced apoptosis at24h LPS stimulation and was not observed at4h. The apoptosis induced by LPS24h administration was inhibited in the cells with downreguation of LC3II expression pretreated with Rapamycin. Pretreatment with3-MA led to apoptosis at4h LPS companied with upgreguation of LC3II protein. Conclusion:Autophagy was induced by LPS was enhanced at early stage and supressed at later stage which plays a cyotprotection role in HL-1cells. Part â…¡ Effects of autophagy on mitochondrial function in HL-1cardiomyocytes during sepsisObjective:To investigate the effects of autophagy on mitochondrial function and biogenesis in HL-1cells during sepsis. Methods:The endotoxin model of HL-1cells was formed by LPS in the final concentration of1ug/ml. Relative area and optical density, mitochondrial membrane potential and Tfam and PGC-1a mRNA of mitochondrial biogenesis were measured in the cells at LPS24h exposure with or without3-MA or Rapamycin pretreatment for48h by Transmission electronic microscopy(TEM), Flow Cytometry and RTq-PCR respectively. Results:Mitochondrial relative area and optical density were augmented, mitochondrial membran potential and expression of Tfam and PGC-1a mRNA were downregulated at LPS24h expousre compared with control. The insults induced by LPS were aggravated by3-MA pretreatment, howerver, which were allivated by Rapamycin. Conclusion:LPS leads to mitochondrial ulrastructure damage, loss of mitochondiral membran potential and mitochondrial biogenesis dyfunction in HL-1cells. Autophagy play a cyotprotective role against apoptosis through improving mitochondrial function and biogenesis in HL-1cell during sepsis. Part III The mechanism of autophagy induced by LPS in HL-1cardiomyocytesObjective:To investigate wether autophagy is induced by LPS through endoplasimic reticulum stress. Methods:The sepsis model of HL-1cells was formed by LPS in the final concentration of lug/ml. GRP78and IRE la mRNA were measured at4and24h LPS exposure and control by RTq-PCR to assess the activation of endoplasmic reticulum stress. Relationship between endoplasmic reticulum stress and autophagy was observed throught expression of GRP78and IREla mRNA, ATG5and ATG7mRNA, LC3II protein and green LC3II puncta dots by RTq-PCR, westenblot and confocal microscopy in cells stimulated by 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 at4h. Compared with4h LPS administration, pretreatment with Tm enhanced the expression of GRP78and IRE1a mRNA companied with more extensive expression of ATG5and ATG7mRNA, LC3II protein and green LC3II puncta dots induced by4h LPS. Expression of GRP78and IREla mRNA induced by LPS were suppressed by TUDCA, meanwhile, activation of autophagy was inhibited. Conclusion:LPS induced activation of endoplasmic reticulum stress and autophagy in HL-1cell.Autophagy was induced by LPS through endoplamic reticulum stress.