| Stroke is a frequently-occurring disease that threatens human’s life and health,metabolic disorder is one of the important pathological mechanism,thus timely regulation of glucose metabolic disorder may be an effective strategy to improve cerebral ischemia/reperfusion(I/R)injury.Celastrol is a pentacyclic-triterpene isolated from the root extracts of Tripterygium wilfordii and its metabolism-regulatory effect has attracted much attention in recent years.It has reported that HIF-1α/PDK1 pathway plays a key role in the conversion of glucose metabolism.But whether celastrol can regulate glucose metabolic-disorder through HIF-1α/PDK1 pathway to protect against cerebral I/R injury is still unknown.Part Ⅰ Observation of the protective effect of celastrol on focal cerebral ischemia/reperfusion injury in miceObjective:To observe whether celastrol has a protective effect on focal cerebral I/R injury in mice.Methods:1.Establishment of the middle cerebral artery occlusion/reperfusion(MCAO/R)model and experimental groupsSuture-occluded method was adjusted to establish MCAO/R model in mice,ischemia for 1 hour and reperfusion for 24 hours after removal of the thrombus.During the experiment,laser Doppler blood flow meter was used to monitor the changes in the cerebral blood flow of the mice in real time.Mice in the sham operation group underwent the same procedure of vascular separation without blocking the blood vessels.30 male C57 mice were randomly divided into 5 groups(18-22g): sham-operated group received equal volume 0.9% Na Cl including 1% DMSO;I/R group received equal volume 0.9% Na Cl including 1% DMSO;I/R+Celastrol group was injected with 3 mg/kg,4.5 mg/kg and 6 mg/kg celastrol respectively.The above were all intraperitoneal injected immediately at the onset of reperfusion;n=6.2.Main observation indicators and methods: TTC staining was used to observe the effect of different doses of celastrol on infarct volume;neurological function score was used to evaluate the improvement of different doses of celastrol on the neurological function in I/R mice;HE staining was adjusted to observe the effect of different doses of celastrol on the histopathological damage of cortex in I/R mice.Results1.When the middle cerebral artery was embolized,the cerebral blood flow dropped to about 40% of the baseline;the blood flow returned to the normal level after the thrombus was removed,which indicating that the MCAO/R model was successfully made.2.Compared with Sham group,the mice in I/R group exhibited severe cerebral infarct volume;compared with I/R group,3 mg/kg,4.5 mg/kg,and 6 mg/kg celastrol treatment can decrease cerebral infarct volume notably;compared with 3 mg/kg,4.5 mg/kg and 6mg/kg celastrol decreased more obvious,and there was no difference observed between4.5 mg/kg and 6 mg/kg celastrol.3.Compared with Sham group,the mice in I/R group showed severe neurological deficits;compared with I/R group,3 mg/kg celastrol reduced the neurological function scores and there was no significant difference,but 4.5 mg/kg and 6 mg/kg celastrol can significantly reduce the neurological function score and there was no statistical difference between these two groups.4.HE staining showed that the cortical neurons in I/R group had obvious nuclear pyknosis and hyperchromatic nucleus compared with Sham group;but after treatment of3 mg/kg,4.5 mg/kg,and 6 mg/kg of celastrol,nuclear pyknosis and nuclear hyperchromatism were alleviated.Compared with the 3 mg/kg,the protective effect of4.5 mg/kg and 6 mg/kg celastrol were more obvious and the effect was similar.Conclusions:1.Celastrol can protect against focal cerebral ischemia-reperfusion injury in mice.2.There was no statistical difference between the protective effect of 4.5 mg/kg and 6mg/kg celastrol.According to reports,the safety range of celastrol is narrow,we selected4.5mg/kg of celastrol for further research.Part Ⅱ Effect of celastrol on HIF-1α/PDK1 pathway and glycolysis in cortex of mice with focal cerebral ischemia/reperfusion injuryObjective:To observe whether the protective mechanism of celastrol on focal cerebral I/R injury in mice is related to the regulation of HIF-1α/PDK1 pathway mediated glycolysis.Method:(1)Experimental grouping and administration1.Effect of celastrol on glycolysis level and HIF-1α/PDK1 expression in cortex of mice with cerebral I/R injury24 male C57 mice were randomly divided into 3 groups(18-22g): sham-operated group received equal volume 0.9% Na Cl including 1% DMSO;I/R group received equal volume 0.9% Na Cl including 1% DMSO;I/R+Celastrol group was injected with 4.5mg/kg celastrol.The above were all intraperitoneal injected immediately at the onset of reperfusion,n=8.2.Effect of celastrol combined with HIF-1α agonist on cerebral I/R injury in mice40 male C57 mice(18-22g)were randomly divided into 5 groups: sham-operated group were received equal volume 0.9% Na Cl including 1% DMSO;I/R group received equal volume 0.9% Na Cl including 1% DMSO;I/R+Celastrol group were injected with 4.5mg/kg celastrol;I/R+Celastrol+ DMOG group received 4.5 mg/kg celastrol and 50mg/kg DMOG at mean time;I/R + DMOG group received 50mg/kg DMOG.The above drugs were all intraperitoneal injected immediately at the onset of reperfusion;n=8.(2)Main observation indicators and methodsTTC staining was used to observe the change in cerebral infarct volume;the neurological function score was employed to evaluate the neurological function;RT-PCR was used to detect the mRNA level of HIF-1α,PDK1 and glycolysis-related factors LDHA,GLUT1 and HK2;Western blot was adjusted to detect expressions of HIF-1α,PDK1 and the glycolysis-related factors LDHA,GLUT1 and HK2;lactic acid determination,ATP detection and glucose content determination kits were used to quantify the lactic acid accumulation,ATP production and glucose content in the cortical tissue of the infarcted side.Results:1.Effect of celastrol on glycolysis level and HIF-1α/PDK1 expression in cortex of mice with cerebral I/R injury(1)Compared with the Sham group,the mRNA and protein expressions of HIF-1αand PDK1 in I/R group increased notably.But after 4.5mg/kg celastrol treatment,the mRNA and protein expressions of HIF-1α and PDK1 significantly reduced.(2)Compared with Sham group,the mRNA and protein expressions of glycolysisrelated factors LDHA,GLUT1 and HK2 significantly increased in the I/R group,but after administration of celastrol,the mRNA and protein expressions of LDHA,GLUT1 and HK2 significantly reduced.Compared with Sham group,the lactic acid content in I/R group significantly increased,and the ATP production and glucose content significantly reduced.After the treatment of celastrol,the lactic acid content significantly reduced,accompanied by significant increase in ATP production and glucose content.2.Effect of celastrol combined with HIF-1α agonist on cerebral I/R injury in mice(1)Compared with Sham group,mice subjected to I/R group exhibited severe cerebral infarct volume and neurological deficit;compared with I/R group,cerebral infarct volume increased significantly in I/R+ DMOG group,and neurological function score increased without significant difference;compared with I/R group,celastrol treatment significantly downregulated cerebral infarct volume and neurological score;compared with I/R+Celastrol group,the cerebral infarct volume and the neurological function score significantly upregulated in I/R+Celastrol+DMOG group.(2)Compared with Sham group,the expression of HIF-1α and PDK1 in I/R group increased notably.But after 4.5mg/kg celastrol treatment,the expression of HIF-1α and PDK1 significantly reduced;compared with I/R group,the expression of HIF-1α and PDK1 in I/R+DMOG group increased;compared with I/R+Celastrol group,expressions of HIF-1α and PDK1 significantly increased in I/R+Celastrol+DMOG group.(3)Compared with Sham group,the expression of glycolysis-related factors LDHA,GLUT1 and HK2 significantly increased in I/R group,but after administration of celastrol,the expression of LDHA,GLUT1 and HK2 significantly reduced;compared with I/R group,the expression of LDHA,HK2 and GLUT1 increased in I/R+DMOG group;compared with I/R+Celastrol group,the expression of LDHA and HK2 significantly increased in I/R+Celastrol+DMOG group,GLUT1 increased but no statistically difference.(4)Compared with the Sham group,the lactic acid content in I/R group was significantly increased,and the ATP production and glucose content significantly reduced.After the treatment of celastrol,the lactic acid content significantly reduced,accompanied by significant increase of ATP production and glucose content;compared with I/R group,lactic acid content significantly increased in the I/R+DMOG group,and ATP production and glucose content were decreased;compared with I/R+Celastrol group,the lactic acid accumulation in I/R+Celastrol+DMOG group significantly increased,longing with downregulation of ATP production and glucose content.Conclusions:1.Celastrol notably inhibited glycolysis and the expression of HIF-1α/PDK1 in cortex of mice with focal cerebral I/R injury;2.Celastrol inhibited glycolysis by regulating the HIF-1α/PDK1 pathway,thereby protecting cerebral I/R injury in mice. |
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