The Role And Mechanism Of Acetaldehyde Dehydrogenase 2 In Sepsis Associated Brain Injury

Background: Sepsis associated brain injury is one of the major complications of sepsis and septic shock among septic survivors.The brain is the target organ in the early stage of sepsis.The clinical symptoms can occur prior to shock,and the hippocampal CA1 sub-region is the most vulnerable area.Even if sepsis and sepsis shock has been restored,sepsis associated brain injury can still be left with long-term neurological impairment cognitive dysfunction or dyskinesia.Sepsis associated brain injury is a sterile nerve inflammation that can cause neuronal damage or brain dysfunction.Free radicals are increasingly produced in the response to inflammation.The highly instable free radicals bring about lipid peroxidation,resulting in producing excessive endogenous reactive aldehydes,and further cause organ dysfunction.Acetaldehyde dehydrogenase 2(ALDH2)is a major aldehyde metabolic enzyme,which is also widely distributed in the brain in addition to the metabolism of ethanol metabolism in the liver.Therefore,to optimize the treatment of sepsis associated brain injury and improve the quality and prognosis of patients,it is necessary to explore the potential effect and mechanism of ALDH2 in sepsis associated brain injury.It has been confirmed that the activity of ALDH2 decreased after cecal ligation and perforation(CLP)in septic mice,and phosphorylating ALDH2 might protect against sepsis.Moreover,ALDH2 protects nerve function in a variety of neuro-inflammatory processes.However,few study has evaluated whether ALDH2 protects the brain against sepsis.Objective To investigate the potential role and molecular mechanism of ALDH2 in sepsis associated brain injury.Methods Part 1 An animal model was constituted by CLP.Twenty-four rats were stochastically divided into sham group(n=6),CLP group(n=6),CLP + Alda-1 group(n=6)and CLP + Cyanamide(CYA)group(n=6).Vital signs were monitored,and arterial blood gas analysis,hippocampal histological staining and ALDH2 activity analysis were conducted.Western blot analysis and enzyme-linked immunosorbent assays(ELISA)were respectively conducted to detect the levels of NOD-like receptors family pyrin domain containing protein 3(NLRP3),caspase-1,apoptosis-associated-speck-like protein(ASC),Gasdermin D(GSDMD),interleukin-1β(IL-1β)and interleukin-1β(IL-18).Part 2 Lipopolysaccharide(LPS)-treated HT22 cells were employed as an in vitro model of sepsis associated brain injury,with and without pretreatment with Alda-1 or CYA,to further examine the potential mechanisms.Real-time quantitative polymerase chain reaction and western blot were used to determine the levels of NLRP3,caspase-1,GSDMD and ASC.ELISA were conducted to assay the levels of IL-1β and IL-18.ResultsPart 1 1.Mean arterial pressure(MAP): At 3h,6h and 12 h following CLP,the MAP of the rats in the CLP group,CLP + Alda-1 group and CLP + CYA group had decreased considerably compared with the baseline,and were significantly lower than the nonoperative rats.There was no distinct difference in MAP among the CLP groups.2.Arterial blood gas analysis index: At 6h and 12 h following CLP,the lactate levels of the operative rats elevated.There were higher lactate levels in the rats with CYA pretreatment,and there were lower lactate levels in the rats with Alda-1 pretreatment.The level of base excess(BE)in the three CLP groups was lower than that in the sham group,but there was no distinct difference in BE between the CLP groups.There was also no distinct difference in p H among the four groups.3.ALDH2 activity and pathological lesion in the hippocampal CA1 region: At 12 h following CLP,hippocampal cell injuries were found in the CLP group,with decreased ALDH2 activity.In the group pretreated with Alda-1,there were increased ALDH2 activity,and decreased hippocampal cell damage.In the group pretreated with CYA,ALDH2 activity was further declined,and the cell injury grade increased.4.Protein levels of NLRP3 inflammasome and pyroptosis-related proteins in the hippocampus: The NLRP3/caspase-1 axis were overexpressed in the CLP group.At 12 h following CLP,the protein levels of NLRP3,ASC,caspase-1 and GSDMD were further increased in the CLP + CYA group,and there were reduced protein levels of NLRP3,ASC,caspase-1 and GSDMD in the CLP + Alda-1 group.5.The levels of inflammatory cytokines: The levels of serum IL-1β and IL-18 were statistically increased following CLP.Compared with the sham group,the levels of IL-1β and IL-18 were significantly reduced in the CLP + Alda-1 group,and the levels of the two inflammatory cytokines were further increased in the CLP + CYA group.Part 2 1.Cell viability and ALDH2 activity of HT22 cells: There were decreased cell viability and ALDH2 activity in HT22 cells exposed to LPS.In the LPS + Alda-1 group,the cell viability and ALDH2 activity were increased than the LPS group.In the LPS + CYA group,cell viability and ALDH2 activity were further declined than the LPS group.2.Expression of NLRP3 inflammasome and pyroptosis-related proteins: Increased m RNA and protein levels of the NLRP3,ASC,caspase-1 and GSDMD were observed in HT22 cells exposed to LPS.Compared to the LPS group,the levels of NLRP3/caspase-1 axis were reduced in the LPS + Alda-1 group,and the levels of NLRP3/caspase-1 axis were increased in the LPS + CYA group.3.The levels of inflammatory cytokines: LPS induced significantly increased IL-1β and IL-18 levels in HT22 cells.The levels of the two inflammatory cytokines further increased in the LPS + CYA group,while the levels of the two inflammatory cytokines decreased in the LPS + Alda-1 group.Conclusions 1.The activation of ALDH2 can attenuate sepsis associated brain injury following CLP and LPS stimulation.2.The molecular mechanism may be the regulation of the NLRP3/caspase-1 axis and effect on subsequent pyroptosis.3.ALDH2 and NLRP3 inflammasome may emerge as new therapeutic targets for sepsis associated brain injury in the near future.