Investigation In The Role Of 33-oxoLCA And Its Underlying Mechanism In Cerebral Is Chemic Injury

Objective: To investigate the effect of 3-oxo-lithocholic acid(3-oxoLCA)on ischemic injury model of middle cerebral artery occlusion(MCAO)in rats and its neuroprotective mechanism,and to propose transcranial direct current stimulation(t DCS)for treatment.Methods: The effects of 3-oxoLCA on neurons after stroke were studied in the MCAO model in vitro and oxygen and glucose deprivation(OGD)model in vivo.Western blot was used to detect the expression of PLOD2 and PTEN in cortical neurons after cerebral ischemia-reperfusion injury.The area of cerebral infarction after ischemia-reperfusion injury in rats was detected by TTC staining,and the survival rate of primary neurons was detected by CCK-8 cell survival assay and LDH release assay.The levels of PLOD2 and PTEN were suppressed and overexpressed using inhibitors and lentivirus transfection methods.Finally,the effects of t DCS on 3-oxoLCA-PLOD2-PTEN signal transduction and nerve cell survival were tested after cerebral ischemia-reperfusion injury.Results: We found that increased 3-oxoLCA in neurons promoted ischemia-induced neuronal death after cerebral ischemia-reperfusion(I/R)injury in rats.TTC showed that3-oxoLCA supplementation increased cerebral infarction volume,while decreased3-oxoLCA level had a neuroprotective effect.CCK-8 and LDH release assay showed increased neuronal survival.Our results showed that the expression of PLOD2(Procollagen-Lysine,2-oxoglutarate 5-Dioxygenases 2)was decreased after ischemia-reperfusion in rats,and it was consistent that the expression of PLOD2 was decreased in neurons after OGD injury.We also overexpressed PLOD2 by transfection of PLOD2 inhibitor minoxidil and lentivirus,and PTEN inhibitor Bp V(PIC)by lentivirus transfection and overexpression of PTEN,proving that PLOD2 plays a role in upstream of PTEN phosphatase and downstream of 3-oxoLCA,mediating OGD-induced neuronal injury.Interestingly,we demonstrate that direct current stimulation(DCS)reduces membranous apical sodium-dependent bile acid transporter(ASBT)and intraconal3-oxoLCA in OGD-injured neurons,and that transcranial direct current stimulation(t DCS)reduces infarct volume in MCAO rats by inhibiting ASBT.These data suggest that neuronal death in I/R injury is driven by increased ASBT expression in the3-oxoLCA-PLOD2-PTEN signaling pathway.t DCS therapy is a potential means of inhibiting ischemia-induced,thereby conferring neuroprotection after brain I/R injury.Conclusion: Our experimental study found the mechanism of action of 3-oxoLCA in ischemia-reperfusion injury.The increase of 3-oxoLCA increased ischemia-reperfusion injury,and studied the mechanism of action of 3-oxoLCA through ASBT-PLOD2-PTEN signaling pathway.t DCS successfully inhibited 3-oxol CA from entering neurons after ischemia-reperfusion injury,increased the protein level of PLOD2,inhibited the expression of PTEN,alleviated ischemia-reperfusion injury,and protected the survival of neurons.