The Role Of CPT1A Dysfunction-Induced Fatty Acid Metabolism Disorder In Ischemic Stroke And Drug Intervention

Objective:Fatty acid metabolism in the central nervous system is abnormal after ischemic stroke.Carnitine palmitoyltransferase 1A(CPT1A)is a key regulator and rate-limiting enzyme for fatty acid oxidative degradation that transports long-chain fatty acids into mitochondria.Inhibition of CPT1A can reduce the oxidative degradation of long fatty acids into mitochondria and cause fatty acid metabolism disorder.A previous study reported that etomoxir,a specific inhibitor of CPT1 A,aggravates transient middle cerebral artery occlusion(tMCAO)-induced ischemic brain damage,but the role of CPT1A in ischemic stroke is still unknown.Therefore,this thesis aimed to explore the role and mechanism of CPT1A in ischemic stroke and to identify therapeutic drugs targeting CPT1A.Methods:1.In vitro experiments:Primary cultured rat astrocytes and a human astrocyte cell line were used in in vitro experiments to establish an oxygen-glucose deprivation/reoxygenation(OGD/Re)model.Metabolomics was applied to detect the metabolic changes of astrocyte after OGD/Re;The etomoxir was used to pharmacologically inhibiting CPT1A;The homology modeling and molecular docking methods were used to screen CPT1A-targeted drugs;Rutin was added upon reoxygenation to observe its effect on OGD/Re-induced astrocytes damage and to explore its mechanism;The LDH analysis and PI staining were used to measure the OGD/Re-induced astrocytic damage;The formation of glial scar induced by OGD/Re was investigated with Western blotting and immunofluorescence assays;The BODIPY staining was used to detect the formation of lipid droplets in astrocytes;The level of free fatty acid was measured by using probe tools,and the level of triglyceride was detect by using GPO-PAP method;Lyso-Tracker staining and Aeridine Orange(AO)staining were used to detect the lysosomal damage;Western blotting,immunofluorescence and ELISA methods were applied to detect the expression of lysosomal cathepsin B;Immunofluorescence and Western blotting analysis were used to detect the change of Hsp70 and to observe the colocalization of Hsp70 and lysosomes in human astrocytes;the lysosomal Hsp70 levels of lysosomes fractions was measured by using Western blotting analysis;The Hsp70 palmitoylation levels was measured by using immunoprecipitation-acyl-biotin exchange method.2.In vivo experiments:Sprague-Dawley rats and C57B6 mice were used in the in vivo experiment to establish a tMCAO model.Western blotting,immunohistochemistry and enzymatic methods were used to observe the distribution of CPT1A in brain tissue,and to detect the changes of the CPT1A protein level and activity;The etomoxir was intraperitoneal injected to pharmacologically inhibit CPT1A and the specific targeting astrocytic adeno-associated virus(pAAV-GfaABC1D-CPT1A)was intraventricular injected to specifically overexpress CPT1A in astrocytes;Intraperitoneal injection of rutin immediately upon reperfusion tMCAO to observe its effect on cerebral infarction volume induced by tMCAO in the acute phase;or the rats were treated with intraperitoneal injection of rutin at day 1 after tMCAO,and then followed by daily treatment with rutin,to detect the effects of delayed administration of rutin on tMCAO-induced brain injury;The infarction volume was measured with TTC staining;The electron microscope was used to observe the formation of lipid droplets and lysosomal damage in astrocytes;The tMCAO-induced lipid droplet accumulation in astrocytes was observed by immunohistochemistry;Western blotting and immunohistochemistry were used to detect the tMCAO-induced glial scar formation.Results:Section 1 The fatty acid metabolite L-palmitoylcarnitine is decreased in astrocytes after OGD/Re.Metabolomics results showed that 97 metabolites were significantly downregulated and 149 metabolites were significantly upregulated in astrocytes after OGD/Re.Among them,the fatty acid metabolite L-palmitoylcarnitine was markedly decreased in astrocytes.Section 2 The role of CPT1A in ischemic stroke and its mechanism1.Changes in CPT1A in reactive astrocytes induced by ischemic strokeCPT1A is a key catalytic enzyme by which L-palmitoylcarnitine is produced.The results of Western blotting,immunohistochemistry and immunofluorescence analysis showed that CPT1A was mainly expressed in astrocytes and was almost not expressed in neurons;CPT1A was mainly present in the cortex,hippocampus,hypothalamus,and cerebellum and was less distributed in the striatum;the protein level and the activity of CPT1A was reduced in the rat model of tMCAO in vivo and in the OGD/Re-induced astrocyte injury model in vitro.The above results suggest that astrocytic CPT1A is functionally defective after OGD/Re.2.The role of CPT1A in ischemic strokeFurther study showed that pharmacological inhibition of CPT1A by etomoxir could increase the infarction volume induced by tMCAO in the acute stage and aggravate the astrocyte damage and glial scar formation induced by OGD/Re,while specific overexpression of astrocytic CPT1A with adeno-associated virus(AAV)transfection could reduce the infarction volume and the formation of glial scars.3.The mechanism by which CPT1A dysfunction aggravates brain damage induced by ischemic strokeAfter ischemic stroke,the dysfunction of astrocytic CPT1A induced the accumulation of lipid droplets in astrocytes.CPT1A dysfunction-induced accumulation of lipid droplets trapped Hsp70 and palmitoylated Hsp70,leading to a decrease in the level of hsp70 in lysosomes.The CPT1A inhibitor etomoxir aggravated the OGD/Re-mediated accumulation of lipid droplets in astrocytes,increased the colocalization of Hsp70 and lipid droplets,and promoted the palmitoylation of Hsp70,which led to a reduction in the level of lysosomal Hsp70,aggravating lysosomal damage,and promoting the release of cathepsin B.Section 3 Rutin reduces ischemic stroke damage by activating CPT1A1.Discovery of rutin targeting CPT1A through computer virtual drug screening technologyIn this paper,a protein structure model of CPT1A was established through homology modeling,and then the CPT1 A-targeted compound rutin was discovered through molecular docking technology.2.Rutin reduces the tMCAO-induced cerebral infarction volume in the acute phase and attenuates OGD/Re-induced astrocyte damageTTC staining results showed that rutin could significantly reduce the cerebral infarction volume induced by tMCAO in the acute phase;PI staining results showed that rutin could significantly reduce OGD/Re-induced astrocyte damage.3.Delay administration of rutin inhibits the formation of glial scars induced by ischemic strokeWestern blotting,immunohistochemistry and immunofluorescence results showed that the delayed administration of rutin could obviously reduce the formation of glial scars induced by tMCAO or OGD/Re.4.Rutin reduces astrocyte lysosomal damage mediated by lipid droplet accumulation by activating CPT1ARutin increased the expression and activity of CPT1A in the marginal zone of cortical infarction in tMCAO-treated rats and in OGD/Re-induced astrocyte injury.Rutin reduced tMCAO-and OGD/Re-induced lipid droplet formation in astrocytes.Rutin reduced the accumulation of astrocyte lipid droplets by activating CPT1A,blocked the colocalization of Hsp70 and lipid droplets,and reduced palmitoylation of Hsp70,thereby increasing the level of lysosomal Hsp70 to stabilize the lysosomal membrane,reducing lysosome damage and inhibiting the release of cathepsin B.Conclusion:1.The protein level and activity of CPT1A in astrocytes are decreased after ischemic stroke,which leads to fatty acid metabolism disorder.2.Inhibition of CPT1A aggravates ischemic brain damage and the formation of glial scars;in contrast,activation of CPT1A reduces ischemic brain damage and the formation of glial scars.3.Abnormal fatty acid metabolism mediated by CPT1A dysfunction increases the accumulation of lipid droplets in astrocytes;CPT1A dysfunction-induced accumulation of lipid droplets traps Hsp70 and palmitoylates Hsp70,which leads to downregulation of Hsp70 in lysosomes and lysosomal damage,contributing to the formation of astrogliosis and glial scars,and cell death occurs in some reactive astrocytes.4.Rutin can upregulate and activate CPT1A,reduce CPT1A dysfunction-induced lipid droplet accumulation and lysosomal damage,inhibit the formation of glial scars,and has a protective effect on ischemic stroke.