Genipin Alleviates Oxo-glucose Deprivation Damage In HT22 Cells Through Mitochondrial Stress And ER Stress-regulating Mitophagy

Background:Cerebral ischemia is caused by brain flow disturbance,which will trigger continuous and complex metabolic and cell lesions,for acute ischemic stroke(Acute Ischemia Stroke,AIS)patients with intravenous thrombolysis and combined with mechanical thrombectomy,such treatment will cause cerebral ischemia reperfusion injury,analysis of its potential biological mechanism,provide a new entry point for the treatment of neuronal death.Because hippocampal neurons are more sensitive to glucose-deficient hypoxia,even a short-term lack of glucose and oxygen can cause great damage to neuronal cells,and in some cases can even cause cell death.We treated mouse hippocampal neuronal cells(HT22 cells)with glucose deficiency and hypoxia to simulate cerebral ischemic injury.Mitochondria can generate ATP to provide energy to the cells.The intramitochondrial proteases Htr A2 / OMI,LONP,CLPP,etc.,can enhance the unfolded protein reaction(UPR),remove excess proteins to restore the function of mitochondria,and play an important role in maintaining cell homeostasis.Mitochondrial dysfunction is associated with mitochondrial failure to meet cellular requirements for ATP,and enhanced reactive oxygen species generation.When the mitochondria are damaged,the internal mitochondrial protease Htr A2 /OMI is released from the mitochondria,which cleaves XIAP and promotes the development of apoptosis.Studies have shown that ER(ER)homeostasis is damaged,resulting in ER stress,which in turn initiates the unfolded protein response(UPR),an integrated intracellular signaling pathway in response to ER stress by increasing expression of ER molecular chaperones,attenuated global protein translation and degradation of unfolded proteins,and the PERK /e IF2-ATF4-ATF5-CHOP classical pathway causes ER stress when damaged.The classical pathway of mitophagy is the PINK / PARKIN pathway.When the mitochondrial membrane potential decreases,PINK is expressed on the outer mitochondrial membrane,recruiting phosphorylated PARKIN to bind ubiquitin groups,and then combining mitophagy-related proteins to cause autophagy and remove unhealthy mitochondria.Mitochondria are important organelles that play key roles in different life functions,such as energy production,Ca2 + homeostasis,carbon metabolism,production of cell growth intermediates,and induction of apoptosis.In this study,we explored the protective role of mitochondrial and mitochondrial stress in the combined induction of mitoophagy in cell injury in HT22 mice with mitochondrial stress and ER stress as the entry point.Methods:HT22 cells were deprived of hypoxia glucose(using glucose-free DMEM medium(O20.5%,5% CO25%)and Genipin into Con,OGD,OGD + Genipin.1.Cell damage was assessed by MTT,flow cytometry,nuclear morphology by Hoechst staining and protein expression levels of apoptosis-related proteins BAX,BCL-2,and ccaspase.2.The membrane potential and ROS of mitochondria were detected by flow cytometry JC-1 staining,the ROS within mitochondria was observed by fluorescence microscopy,and the mitochondrial status of seroprotease Htr A2 / OMI mitochondrial proteases LONP and CLPP in mitochondria,cytosol and total proteins was assessed by Western Blot.3.The ER stress was assessed by detecting PERK,e IF2α,p-e IF2α,ATF4,and ATF5 ER stress-related proteins by Western Blot.4.The expression of UCP2,PINK,PARKIN,LC3 II / LC3 I and P62 was detected by Western Blot;after adding autophagic flux blocker chloroquine(CQ),LC3 II / LC3 I,mitochondria by flow cytometry,mitophagic flux;IP UCP2,PARKIN and ATF4,mitochondrial stress and ER stress.Result:1.After addition of Genipin,OGD + Genipin group increased apoptosis in OGD +Genipin group;fluorescence microscopy after Hoechst staining decreased nuclear sequestration and fragmentation morphology;Western blot protein of apoptosis-related protein BAX and downstream c-caspase3,increased expression decreased in OGD + Genipin group,and anti-apoptotic protein BCL-2 in OGD + Genipin group.2.JC-1 and ROS showed that mitochondrial membrane potential increased,ROS generation and ROS decreased by fluorescence microscopy;Western Blot showed mitochondrial protein,BAX expression decreased and BCL-2 increased,while total protein and mitochondrial protein,Htr A2 / OMI,mitochondrial protease LONP,and CLPP increased in OGD group and further increased in the OGD + Genipin group.3.Western Blot detected PERK,e IF2α,p-e IF2α,ATF4,ATF5,and CHOP in the total protein,and the expression increased more significantly after the mitochondrial protein ATF5.4.Western Blot detected total and mitochondrial proteins,UCP2 increased after treatment,decreased,mitophagy-related proteins PINK,PARKIN,LC3 II / LC3 I,more significantly,P62 protein,p-UB within mitochondrial protein,and more significantly after treatment.RT-q PCR increased UCP2 expression after treatment,m RNA of PINK,more significantly,PARKIN,LC3 II,and more significantly in the additive group.After CQ addition,LC3 II /LC3 I and P62 protein expression varied significantly between con + CQ and con groups,while OGD + CQ and OGD + Genipin + CQ,the number of mitochondria increased significantly after CQ addition.Protein immunoprecipitation IP showed that less UCP2 bound more PARKIN and ATF4,while also bound PARKIN with increased ATF4 expression,and less bound UCP2.Conclusion:1.Genipin enhances the viability of HT22 cells after hypoxic glucose deprivation treatment by inhibiting apoptosis.2.Genipin enhances the mitochondrial membrane potential to increase the mitochondria and maintains mitochondrial function..3.Genipin activates the ER stress response by regulating PERK/EIF2α and promotes cell survival.4.Inhibition of UCP2 expression by Genipin enhances mitochondrial stress and ER stress,and jointly regulates mitophagy through the UCP2-ATF4-PARKIN pathway to mitigate damage in HT22 cells.