The Role And Mechanism Of IDOL-LDLR Axis In Cerebral Ischemia/reperfusion Injury

Objective: To explore the role of IDOL-LDLR axis in cerebral ischemia/reperfusion injury,and clarify its mechanism.Methods: 99 C57BL/6 male mice(20-25g)were fed in the animal laboratory of University of South China,divided into the blank control group(Control),the sham operation group(Sham),the operation group(MCAO),the AAV-Gfp operation group(MCAO+AAV-Gfp),and the AAV-sh IDOL operation group(MCAO+AAV-sh IDOL).The control group was not given any treatment,and the sham group was not with nylon monofilament.Focal cerebral ischemic stroke was induced by right middle cerebral artery(MCA)occlusion with nylon monofilament of the operation group.In the AAV-Gfp operation group,mice were injected with AAV-Gfp adeno-associated virus into the lateral ventricle two weeks before modeling.In the AAV-sh IDOL operation group,mice were injected with AAV-sh IDOL adeno-associated virus into the lateral ventricle two weeks before modeling.After 24 hours the model was successfully established,the neurological deficit scores and the relative cerebral infarct area of the brain tissue of each group of mice were measured.The morphology of the brain tissue of each group was observed by HE staining,and the lipid uptake in the brain tissue of the mice was observed by oil red O staining.Immunohistochemical staining to detect the expression levels of Apo E and Caspase-3 in the brain tissue of each group of mice.Western Blot detection protein expression levels of IDOL,LDLR,Apo E and Caspase-3 in the brain tissues of mice in each group.Results:1.Construction of mouse cerebral ischemia/reperfusion injury model:the blank control group and the sham operation group did not have infarct size and no neurological deficit symptoms.The area of cerebral infarction in the operation group increased,and the neurological deficit score also increased significantly.HE staining showed that the cerebral cortex structure of the mice in the control group and the sham operation group was intact and the nerve cells were necrotic;the nerve cell nuclei of the mice in the MCAO group shrank and contained a large amount of inflammatory cell infiltration(P<0.001).2.Changes in lipid metabolism in mice with cerebral ischemia/reperfusion injury: Western Blot results showed that compared with the control group and the sham operation group,the IDOL protein expression in the MCAO group was increased(P<0.01),and the LDLR protein expression in the MCAO group was significant Decline(P<0.01).Immunohistochemistry showed that compared with the control group and the sham operation group,the number of Apo E positive cells in the MCAO group was significantly increased(P<0.001).3.Changes in cell apoptosis during cerebral ischemia/reperfusion injury in mice: Immunohistochemistry showed that the number of Caspase-3 positive cells in the MCAO group was significantly increased compared with the control group and the sham operation group(P<0.001).4.Establishment of cerebral ischemia/reperfusion injury in IDOL knockout mice: TTC staining showed that compared with the MCAO group,the infarct size and nerve damage in the MCAO+AAV-sh IDOL group were reduced(P<0.001).HE staining showed that the brain tissue of the control group and the sham operation group had no signs of necrosis,and the nerve cells in the MCAO group had a large number of necrosis.Compared with the MCAO group,the brain tissue necrosis in the MCAO+AAV-sh IDOL group was significantly reduced,the infarct size was reduced,and the cell damage and structural changes were significantly improved(P<0.001).5.Changes in lipid metabolism during cerebral ischemia/reperfusion injury in IDOL knockout mice: Western blot analysis showed that the IDOL protein expression of the IDOL knockout model(MACO+AAV-sh IDOL)was significantly decreased(P<0.001),MCAO group The expression of LDLR in MCAO+AAV-Gfp group was significantly reduced(P<0.05).The expression of Apo E protein in the MCAO+AAV-sh IDOL group was also significantly reduced(P<0.01).Immunohistochemistry showed that Apo E(P<0.001)positive cells in the MCAO+AAV-sh IDOL group were significantly reduced compared with the MCAO group.Oil red O staining results showed that no lipid particles were found in the control group,sham operation group and MCAO+AAV-sh IDOL group,while a small amount of lipid particles could be found in the MCAO group and MCAO+AAV-Gfp group.6.Changes in cell apoptosis during cerebral ischemia/reperfusion injury in IDOL knockout mice: Caspase-3 expression was up-regulated in MCAO group and MCAO+AAV-Gfp group,and Caspase-3 protein expression in MCAO+AAV-sh IDOL group was significant Decrease,the proportion of cleaved Caspase-3 decreased(P<0.05).Immunohistochemistry showed that Caspase-3(P<0.01)positive cells in the MCAO+AAV-sh IDOL group were significantly reduced compared with the MCAO group.Tunel method was used to detect cell apoptosis.The fluorescent staining results showed that the control group and sham operation group failed to find red fluorescently labeled apoptotic cells.Compared with MCAO+AAV-Gfp group and MCAO group,the MCAO+AAV-IDOL group had red fluorescence cut back.Conclusion: The IDOL-LDLR axis plays an important role in cerebral ischemia/reperfusion injury.Mice knocking out IDOL can increase the level of LDLR,inhibit Apo E and Caspase-3,and lower cholesterol during cerebral ischemia/reperfusion injury,plays a neuroprotective effect in the brain tissue.