| Stroke is a disease in which a blood vessel in the brain suddenly bursts or blocks,causing circulation problems and damaging brain tissue.Ischemic Stroke(IS)accounts for more than 80% of the total number of strokes.Its complex pathological processes mainly include disturbance of energy metabolism,excitatory amino acid toxicity,oxidative stress,autophagy,pyrodeath and iron death,etc.,which are followed by mutual influence and causation.Therefore,it is necessary to further study the pathophysiological mechanism of ischemic brain injury in order to elucidate the fate and regulation mechanism of damaged neurons in ischemic region.Energy metabolism disorder is one of the early pathological reactions and important mechanisms of ischemic stroke,involving energy hunger,substrate utilization changes,mitochondrial dysfunction,etc.When IS occurs,the level of oxidative phosphorylation decreases due to ischemia and hypoxia,and the increase of anaerobic glycolysis leads to the accumulation of lactic acid and pyruvate,which leads to the sharp decline of ATP synthesized by mitochondria.At the same time,the respiratory chain is damaged,and the activity of complex I-ⅳ is reduced,which further blocks ATP synthesis and aggravates mitochondrial damage,thus forming a vicious cycle.And that eventually leads to neuron damage.In conclusion,energy metabolism disorder runs through the whole process of ischemic stroke,and is one of the important factors in its occurrence,development and exacerbation.Restoring energy metabolism is an important link to improve mitochondrial function and reduce cerebral ischemia reperfusion injury.Traditional Chinese medicine has been used to treat stroke for thousands of years.According to the medication patterns of ancient prescriptions,Ligusticum chuanxiong hort was the most common in the frequency of single drug use or combination use.The volatile oil is the effective part of Ligustilide Ligustilide,but the composition is complex and the mechanism of action is unclear.Studies have shown that ligustilide(LIG)is a high active component in the volatile oil of Ligustilide Ligustilide and has good biological activity.It has anti-inflammatory,anti-oxidative stress,enhance learning and memory,blood-brain barrier protection and neuronal protection.Previous studies have shown that ligustilide can cross the blood-brain barrier,inhibit the reduction of mitochondrial membrane potential after OGD/R injury,and reduce the release of cytochrome C from mitochondria to cytoplasm,suggesting that ligustilide can improve mitochondrial function,and mitochondria are the main site of energy metabolism of cells.The regulatory effects and specific mechanisms of ligustilide on post-IS energy metabolism are not yet clear.Therefore,this study took mitochondrial energy metabolism as a breakthrough point to explore the improvement of ligustilide on neuronal function after ischemic stroke and the specific mechanism of action.purposeIn this study,based on the role of AMPK/PGC-1α signaling pathway in regulating energy metabolism,the effects of ligustilide on the improvement of mitochondrial function and neuronal injury were investigated,which provided certain experimental basis for the basic research of ligustilide in reducing ischemic brain injury.method1.Protective effect of LIG on brain tissue of MCAO/R rats(1)Middle Cerebral Artery Occlusion and Reperfusion,(1)Middle cerebral artery occlusion and reperfusion,MCAO/R)Cerebral Ischemia Reperfusion Injury(CIRI)model was established.(2)The changes of neurobehavioral scores in MCAO/R rats were measured by Longa5-point scale;(3)The brain index of MCAO/R rats was measured by wet weight method.(4)The cerebral infarction volume of MCAO/R rats was measured by triphenyltetrazolium chloride staining(TTC)(2,3,5-triphenyltetrazolium chloride);(5)Hematoxylin-eosin(HE)staining was used to observe the morphologic changes of brain tissue in MCAO/R rats.2.Protective effects of LIG on mitochondrial function and energy metabolism in MCAO/R rats(1)The rat CIRI model was established by MCAO/R;(2)Tetramethylrhodamine Ethyl Ester(TMRE)was used to detect mitochondrial membrane potential in MCAO/R rats.(3)Mitochondrial ROS(mt ROS)of MCAO/R rats were measured by Mito SOX Red(Mitochondrial Superoxide Indicator).(4)The expression of mitochondrial/cytoplasmic Cyt-C protein in MCAO/R rats was detected by Western Blot.(5)Mitochondrial ATP content in MCAO/R rats was determined by colorimetry.(6)The expressions of mitochondrial respiratory strepase complex Ⅰ-Ⅳ and GLUT4 in MCAO/R rats were detected by Western Blot.(7)The content of lactic acid(LA)in MCAO/R rats was determined by colorimetry;3.Molecular docking technology was used to verify the combination of LIG and AMPK(1)Molecular docking of LIG with AMPK;(2)DARTS experiment verified the combination of LIG and AMPK;(3)The combination of LIG and AMPK was verified by CETSA experiment.4.Protective effect of LIG on HT22 cells damaged by OGD/R(1)Oxygen-Glucose Deprivation and Reperfusion(OGD/R)were used to establish CIRI model of HT22 cells in vitro.(2)Drug concentration screening by CCK8 method;(3)The proliferation of HT22 cells damaged by OGD/R was detected by CCK8 method.(4)Morphological changes of HT22 cells injured by OGD/R were observed under inverted microscope.5.To explore the protective effect of LIG on mitochondrial function and energy metabolism of HT22 cells damaged by OGD/R based on AMPK/PGC-1α signaling pathway(1)Oxygen-Glucose Deprivation and Reperfusion(OGD/R)were used to establish CIRI model of HT22 cells in vitro.(2)The mitochondrial membrane potential of HT22 cells injured by OGD/R was detected by JC-1 method.(3)ATP content in HT22 cells damaged by OGD/R was detected by colorimetry.(4)The expression of AMPK,p-AMPK and PGC-1α in HT22 cells damaged by OGD/R was detected by Western Blot.(5)GLUT4 expression in HT22 cells after OGD/R injury was detected by Western Blot.(6)The expression of mitochondrial respiratory chain complex Ⅰ-Ⅳ protein in HT22 cells damaged by OGD/R was detected by Western Blot.(7)The proliferation level of HT22 cells damaged by OGD/R was detected by CCK8 method.result1.Protective effect of LIG on brain tissue of MCAO/R ratsTTC staining,brain index and neurobehavioral score showed that compared with model group,the cerebral infarction volume,brain index and neurobehavioral score of rats in LIG administration group were significantly decreased.HE staining showed that the morphology of cells in the brain of rats was improved and tended to be normal after the intervention of LIG.2.Protective effects of LIG on mitochondrial function and energy metabolism in MCAO/R ratsFlow cytometry showed that mitochondrial membrane potential increased and mt ROS content decreased significantly after LIG intervention.The results of WB showed that LIG intervention inhibited the release of Cyt-C from mitochondria to cytoplasm,and significantly increased the activity of GLUT4 and respiratory chain complex I,II,III,IV.Mitochondrial ATP and lactate contents were detected.The results showed that LIG and ALC increased ATP expression and decreased lactate content.3.Molecular docking results of LIG and AMPKMolecular docking results showed that LIG could theoretically bind to AMPK.DARTS and CETSA experiments confirmed that LIG could combine with AMPK.4.Protective effect of LIG on HT22 cells damaged by OGD/RThe safe concentration of LIG on HT22 cells damaged by OGD/R was screened by CCK8 method,and 5μM,10μM,20μM were selected as the low,medium and high dose groups of LIG.Compared with the model group,LIG increased the proliferation rate of HT22 cells damaged by OGD/R,protected the damaged cell morphology,and had neuroprotective effects.5.To explore the protective effect of LIG on mitochondrial function and energy metabolism of HT22 cells damaged by OGD/R based on AMPK/PGC-1α signaling pathwayATP content in HT22 cells after OGD/R injury was significantly increased after LIG intervention.The increase of mitochondrial membrane potential was observed,which was consistent with the results in vivo.The results of WB showed that LIG increased the expression of p-AMPK,AMPK and PGC-1α in HT22 cells damaged by OGD/R,and Compound C could counteract the effect.Compound C also offset the effect of LIG on the proliferation of HT22 cells,which increased the expression of GLUT4 protein and mitochondrial respiratory chain complex I-ⅳ.Molecular docking results showed that LIG could theoretically bind to AMPK.DARTS and CETSA experiments confirmed that LIG could combine with AMPK.Conclusion1.Ligustilide had a protective effect on cerebral ischemia reperfusion injury and was related to improving mitochondrial function.2.The regulation of ligustilide on energy metabolism may be the main reason for improving mitochondrial function and alleviating nerve injury after ischemia.3.Ligustilide activated AMPK and induced the activation of its downstream protein PGC-1α,which may be the main target of ligustilide in regulating energy metabolism. |
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