Ursodeoxycholyl Lysophosphatidylethanolamide Protects Against Hepatic Ischemia/Reperfusion Injury Via Mediating Mitochondrial Membrane Phospholipid

ObjectiveAim to investigate the protective role and molecular mechanism of Ursodeoxycholyl Lysophosphatidylethanolamide（UDCA-LPE）.Reveal the dynamic changes of mitochondria and lipid components in hepatic I/R,provide a theoretical basis for a novel therapeutic target for attenuating hepatic I/R.Methords1.we generate in vitro and in vivo model of hepatic I/R and construct an oxidative stress model of Hep G2 in vitro.2.Dynamic changes of oxidative stress after hepatic I/R with or without UDCA-LPE pretreatment were evaluated by MDA,SOD,GSH kit,ROS fluorescent probe,mitochondrial ROS probe,and molecular level of redox signaling pathway。3.Mitochondrial functional after hepatic I/R with or without UDCA-LPE pretreatment were evaluated by electron microscopy,ATP,mt DNA,mitochondrial membrane potential,mitochondria-mediated apoptosis signaling pathway molecular level,immunofluorescence,mitochondrial dynamic signaling pathway molecular level.4.The dynamic changes of lipid components after hepatic I/R with or without UDCA-LPE pretreatment were evaluated by chromatography-mass spectrometry.5.The content of cPLA₂ during hepatic ischemia-reperfusion with or without UDCALPE pretreatment and the binding of cPLA₂ to mitochondrial membrane phospholipids were evaluated by WB,PCR,enzyme activity kit and immunofluorescence.6.The effect of cPLA₂ on mitochondrial function was analyzed by plasmid transfection overexpression of cPLA₂.Results1.UDCA-LPE alleviated I/R-induced oxidative stressThe MDA content was significantly enhanced in the I/R group.And hepatic I/R significantly decreased the SOD and GSH levels.Pretreatment with UDCA-LPE significantly reversed elevated levels of oxidative stress induced by hepatic I/R.Hepatic I/R decreased the Thioredoxin-2（TRX2）and Thioredoxin reductase 2（TXNRD2）protein levels.UDCA-LPE treatment reversed the changes of these proteins.2.UDCA-LPE protected hepatic I/R induced mitochondria damageI/R treatment caused extensive mitochondrial cristae breakdown,mitochondrial condensation with heavy electron-dense matrix,highly swollen mitochondria and rupture of the mitochondrial membrane,whereas UDCA-LPE pretreated showed limited mitochondrial damage.Apart from the changes of mitochondrial morphology,I/R reduced cellular ATP production,mitochondrial copy number and △Ψm.UDCA-LPE inhibits I/Rinduced mitochondrial damage.I/R increased the BAX protein levels and decreased BCL2.UDCA-LPE treatment reversed the changes of these proteins And I/R promotes the release of cyto c from the mitochondria into the cytoplasm.UDCA-LPE pretreatment reduced the cyto c release into the cytosol3.UDCA-LPE inhibited I/R induced mitochondrial fissionThe confocal images revealed that although normal cells had tubular and elongated mitochondria,most mitochondria in cells treated with t-BHP had a fragmented morphology.UDCA-LPE pretreatment markedly rescued mitochondrial morphology.I/R resulted in a significant increase in DRP1 expression of dynamin-related protein 1（DRP1）and mitochondrial fission factor（MFF）,two key proteins in mitochondrial division,and UDCALPE partially reversed the increase in DRP1,and MFF.I/R significantly reduced the expression of mitochondrial fussion related proteins Optic atrophy protein 1（OPA1）,mitofusin 1（MFN1）and mitofusin 2（MFN2）,UDCA-LPE increased the content of OPA1,MFN1,and MFN2.4.UDCA-LPE alleviated liver I/R-induced phospholipid metabolism disorderCompared with the sham group,PC,PE,phosphatidylserine（PS）and phosphatidylglycerol（PG）content was markedly decreased in the I/R group.UDCA-LPE reversed this trend.The phosphatidylinositol（PI）content was almost the same across the three groups.Overall,I/R led to phospholipid metabolic disorders.Lysophosphatidylcholine（LPC）,a metabolite of PC,is elevated during I/R,but UDCA-LPE may slightly decrease LPC levels in tissues.Lysophosphatidylethanolamine（LPE）is a metabolite of PE.A upward trend occurs during the I/R process,which can be blocked by UDCA-LPE.Free fatty acids（FFAs）increase after I/R,and UDCA-LPE can block their rise.The levels of CL,a phospholipid characteristic of mitochondria,drop after I/R,and UDCA-LPE partially reverses this effect.5.UDCA-LPE inhibited cPLA₂-induced degradation of membrane phospholipidsThe m RNA and protein levels of cPLA₂ are elevated during the I/R process,while UDCA-LPE partially reverses this effect.After I/R,it showed an increase in PLA₂ activity,and UDCA-LPE pretreatment reduced the increase in liver PLA₂ activity,which was close to normal.The levels of PC and PE fall after the I/R process.The LPC/PC ratio,the LPE/PE ratio was improved after I/R exposure.UDCA-LPE inhibits this effect.cPLA₂ was diffusely distributed inside the normal cells.However,in the oxidative stress cells,the cPLA₂ fluorescence signal was significantly enhanced,and obvious dot-like enhancement occurred.cPLA₂ and the mitochondria showed obvious colocalization.UDCA-LPE reduced the fluorescence intensity of cPLA₂ and blocked the ability of cPLA₂ to bind mitochondrial membranes.6.cPLA₂-mediated degradation of membrane phospholipids leads to mitochondrial dysfunctionOverexpression of cPLA₂ significantly increased BAX expression and decreased BCL2 expression compared to the control group.MDA,GSH,SOD and ROS fluorescence results showed that Hep G2 cells overexpressing cPLA₂ had stronger oxidative stress levels than Hep G2 cells in the control group.Conclusions1.The level of oxidative stress is increased after hepatic I/R.2.There is mitochondrial damage after hepatic I/R.3.There is cPLA₂-mediated phospholipid metabolism disorder after hepatic I/R.4.cPLA₂-mediated disorder of phospholipid metabolism leads to mitochondrial damage.5.UDCA-LPE can reduce mitochondrial damage and reduce hepatic I/R injury by inhibiting cPLA₂ activity.