The Regulation Of Autophagy On Hydrogen Alleviated The Organ Injury Induced By Sepsis

Objective:Sepsis is defined as a life?threatening organ dysfunction that is caused by a dysregulated host response to infection.The pathogenesis of sepsis is complex.Multiple organ failure is one of the main factors of early death in sepsis and septic shock.Inflammatory dysfunction of invasive infections is still a trigger event of sepsis,which is closely related to persistent excessive inflammation,immunosuppression and abnormal homeostasis.Autophagy is a highly conservative homeostasis process,which is involved in the occurrence and development of sepsis.In recent years,more and more scholars believe that hydrogen could be used as a new medical gas molecule to protect sepsis.In this study,we observed the protective effect of H₂ on sepsis and related organ damage in sepsis in vitro and in vivo and explore the specific role of autophagy regulation in this treatment.Methods:Part ?:The role of PINK1/Parkin mediated mitochondrial autophagy in H2alleviated the sepsis-induced-organ damageIn vitro experiment:Raw 264.7,a mouse macrophage line,was cultured in DMEM medium containing 10%fetal bovine serum and was seeded on 6 or 96 well,3 mL/well or 200?L/well,with a cell density of 2*10⁶/mL.Firstly,the cells were treated by LPS and hydrogen for 6 and 24 hours.Membrane potential was detected,autophagy was observed by electron microscopy,PINK1,Parkin and LC3 protein expression were detected by Western blot,and PINK1 and LC3 were localized by immunofluorescence.Secondly,after cells were treated with LPS and hydrogen-rich medium for 6 hours,cell activity,LDH release,membrane potential and ROS release were detected.The expression of PINK1,Parkin,LC3,P62,Beclin1 and VDAC were detected by Western blot,and the expression of PINK1 and LC3 was detected by immunofluorescence.Thirdly,transfected by PINK1 shRNA,cells were treated by LPS and hydrogen-rich medium,autophagy was observed by electron microscopy,cell activity,membrane potential and ROS release were detected,the expression of TNF-?and HMGB1 was detected by ELISA,the expression of PINK1,Parkin,LC3,P62 and VDAC was detected by Western blot,and the expression of PINK1 and LC3was detected by immunofluorescence.In vivo experiments:adult male C57BL/6 mice aged 6-8 weeks,weighing 20-25g,were induced by cecal ligation and puncture.The sepsis mice were injected by intraperitoneally 5 ml/kg hydrogen-rich saline at 1 h and 6 h after CLP.PINK1shRNA was transfected through tail vein mice before operation.At 24 hours after operation,lung W/D,PaO₂/FiO₂,MPO activity,mitochondrial ROS release and lung tissue staining and score were measured,autophagy was observed by electron microscopy,the expression of PINK1,Parkin,LC3,P62 and VDAC were detected by western blot.Part ?:H₂ alleviates mitochondrial dysfunction by autophagy mediated inflammasome NLRP3 inactivating in sepsis.In vitro experiment:primary mouse alveolar macrophages were extracted and cultured in DMEM medium containing 10%fetal bovine serum.The cells were seeded on 6 or 96 well cell culture plates,3 mL/well or 200 mL/well with 2*10⁶/mL.Firstly,after the cells were treated with LPS and ATP for 6 hours,the expressions of IL-1?,IL-18,TNF-?and IL-6 were detected by ELISA and Caspase-1,NLRP3 and ASC were detected by western blot.the MMP,RCR,ATP and mtDNA were detected.Secondly,cells were treated with LPS,ATP and hydrogen-rich medium.The expression of PINK1,Parkin,LC3,Beclin1 and VDAC were detected by western blot.Finally,LPS and hydrogen-rich saline were given to cells with the administration of autophagy induced Rap and inhibition 3-MA,and the expressions of IL-1?,IL-18,TNF-?and IL-6 were detected by ELISA.The expressions of Caspase-1,NLRP3 and ASC were detected by Western blot.The MMP,RCR,ATP and mtDNA were detected.In vivo experiments:adult male C57BL/6 mice aged 6-8 weeks,weighing 20-25g,were induced by cecal ligation and puncture.The sepsis mice were injected by intraperitoneally 5 ml/kg hydrogen-rich saline at 1 h and 6 h after CLP.Autophagic agonist Rap and inhibitor 3-MA were given by intraperitoneal injection before operation.At 24 hours after operation,lung W/D,MPO activity,BAL total protein,HE pathological examination and score of lung,liver and kidney tissues were detected,blood were collected to test serum biochemical indexes such as ALT,AST,Cr and BUN,7 days survival rate were observed,expression of IL-1?,IL-18 and TNF-?were detected by ELISA,and expression of Caspase-1,NLRP 3 and ASC in lung,liver and kidney tissues were detected by western blot.Part ?:H₂ alleviated organ damage and dysfunction:the role of crosstalk between autophagy and ERS.Adult male C57BL/6 mice aged 6-8 weeks,weighing 20-25 g,were induced by cecal ligation and puncture.The sepsis mice were injected by intraperitoneally 5ml/kg hydrogen-rich saline at 1 h and 6 h after CLP.First,lung tissues were collected at different time points.The expressions of PERK and eIF2?,p-PERK,p-eIF2?and IRE1?of lung tissues were detected by western blot.Secondly,septic mice was treated by 4-PBA and TM before operation,followed by CLP and saturated hydrogen-rich saline.The expression of PERK,eIF2?,p-PERK,p-eIF2?,CHOP,ATF,IRE1?and XBP1 of lung tissue were detected by western blot.The expression of LC3,Beclin1,PINK 1 and Parkin of lung,liver and kidney tissues were detected by western blot,autophagosomes of liver were observed by transmission electron microscopy,HE staining of lung,liver and kidney tissues and histopathological scores were detected.The MPO,BAL total protein,W/D of lung,ALT,AST,Cr and BUN of serum biochemical indicators and 7 days survival rate in mice were detected.Thirdly,septic mice were treated by Rap and 3-MA before operation,followed by CLP and saturated hydrogen-rich saline.The expression of p-PERK,p-eIF2?,CHOP,ATF,IRE1?and XBP1 of lung,liver and kidney tissues were detected by western blot.Fourthly,septic mice were treated by TM and 3-MA before operation,followed by CLP and saturated hydrogen-rich saline.the expression of p-PERK,p-eIF2?,IRE1?,XBP1,LC3,Beclin1 and nuclear protein NF-?B were detected by western blot.The expression of TNF-?,IL-1?,IL-6 and HMGB1 were detected by ELISA.Lung,liver and kidney tissues were collected and to observe and calculated by HE staining.The expression of MPO of lung,BAL total protein,W/D in lung tissue,ALT,AST,Cr and BUN in serum biochemical indexes were detected,and 7 days survival rates were measured.Results:Part ?:The role of PINK1/Parkin mediated mitochondrial autophagy in H2alleviated the sepsis-induced-organ damageLPS resulted in membrane potential decline,autophagosome and related protein PINK1,Parkin and LC3 expression and co-localized expression of PINK1 and LC3increasing,cell activity decreasing and LDH and ROS release increasing.Compared with LPS group,the cell activity was increased,the release of LDH and ROS was decreased,the membrane potential was elevated,the expression of autophagy related proteins PINK1,Parkin,LC3,P62,Beclin1 and VDAC were further increased,and the co-localized expression of PINK1 and LC3 were further increased in LPS+H2group.Compared with LPS+H2 group,the autophagosome was decreased,the membrane potential was decreased,the release of LDH and ROS was increased,the expression of cytokines TNF-?and HMGB1 were increased,the expression of autophagy related proteins PINK1,Parkin,LC3,P62 and VDAC were decreased and the co-localization expression of PINK1 and LC3 was decreased in LPS+H2+shRNA group.Compared with CLP group,the lung injury score was decreased,W/D,PaO₂/FiO₂,MPO activity and mitochondrial ROS release were decreased,autophagosome was increased,and the expressions of autophagy related proteins PINK1,Parkin,LC3,P62 and VDAC were increased.Compared with CLP+H2group,the above indicators were aggravated in LPS+H2+shRNA group.Part ?:H₂ alleviates mitochondrial dysfunction by autophagy mediated inflammasome NLRP3 inactivating in sepsis.LPS and ATP increased the expression of inflammatory mediators IL-1?,IL-18,TNF-?,IL-6 and Caspase-1 P-10,NLRP3 and ASC,autophagy related proteins PINK1,Parkin,LC3,Beclin1 and VDAC,decreased the MMP,RCT,ATP and increased the mtDNA.H₂ decreased the expression of IL-1?,IL-18,TNF-?,IL-6 and Caspase-1,NLRP3 and ASC,and increased the expression of autophagy related proteins PINK1,Parkin,LC3,Beclin1 and VDAC,increased MMP,RCR and ATP and decreased the mtDNA.Compared with LPS+H2 group,the expression of IL-1?,IL-18,TNF-?,IL-6,Caspase-1 and NLRP 3 and ASC were decreased,MMP,RCT and ATP were increased,mtDNA were decreased in LPS+H2+Rap group;the expression of IL-1?,IL-18,TNF-?,IL-6,Caspase-1 and NLRP 3 and ASC were increased,MMP,RCT and ATP were decreased,mtDNA were increased in LPS+H2+3-MA group.Compared with CLP group,the histopathological scores of lung,liver and kidney were reduced,the dysfunction of lung,liver and kidney were alleviated,the survival rate of mice was increased,and the expression of IL-1?,IL-18,TNF-?and Caspase-1,NLRP3 and ASC were increased in lung,liver and kidney tissues in CLP+H2 group.Compared with CLP+H2 group,the histopathological scores of lung,liver and kidney were further reduced,the dysfunction of lung,liver and kidney were alleviated,the survival rate of mice were increased,the expressions of IL-1?,IL-18,TNF-?and Caspase-1,NLRP 3 and ASC were alleviated in lung,liver and kidney tissues in CLP+H2+Rap group;the histopathological scores of lung,liver and kidney were increased,the dysfunction of lung,liver and kidney were aggravated,the survival rate of mice were decreased,the expressions of IL-1?,IL-18,TNF-?and Caspase-1,NLRP 3 and ASC were increased in lung,liver and kidney tissues in CLP+H2+3-MA group.Part ?:H₂ alleviated organ damage and dysfunction:the role of crosstalk between autophagy and ERS.Sepsis induced by CLP increased the expression of p-PERK,p-eIF2?and IRE1?in lung tissue.TM treatment increased the expression of p-PERK,p-eIF2?,IRE1?,CHOP,ATF and XBP-1 and inhibited the expression of LC3,Beclin1,PINK1 and Parkin,reduced the number of autophagosome,aggravated the pathological scores and organ dysfunction of lung,liver and kidney tissues,reduced survival rate;4-PBA treatment in sepsis mice improved the variation of the above indicators.3-MA treatment increased the expression of p-PERK,p-eIF2?,IRE1?,CHOP,ATF and XBP-1 in lung,liver and kidney tissues of sepsis mice,while Rap treatment reduced the changes of the above indicators.H2 treatment reduced the expression of p-PERK,p-eIF2?,IRE1?,XBP-1,NF-?b,TNF-?,IL-1?,IL-6 and HMGB1 in sepsis mice,increased the expression of LC3 and Beclin1,reduced the histopathological scores and dysfunction of lung,liver and kidney and increased the survival rate of mice.TM or 3-MA treatment reversed the protection of H2 on the above indicators.Conclusion:H2 protects macrophages from mitochondrial dysfunction induced by LPS through PINK1/Parkin mediated mitophagy.H2 protects mitochondrial dysfunction and lung injury in sepsis through PINK1/Parkin mediated mitophagy.H2 improves mitochondrial dysfunction induced by LPS in macrophages through autophagy-mediated inflammasome NLRP3 pathway.H2 alleviated damage and dysfunction of lung,liver and kidney and improves the survival rate via autophagy mediated inflammasome NLRP3 pathway in sepsis mice.Sepsis can induce endoplasmic reticulum stress and autophagic activity.H2 can alleviate ERS in sepsis mice and increase the activity of autophagy.H2 can improve the inflammatory reaction,pathological damage,organ dysfunction and improve the survival rate through the crosstalk of ERS inactivation and autophagy.