| Cerebral ischemia-reperfusion injury(CIRI)is a condition in which tissue damage rapidly deteriorates after blood flow is restored to the ischemic part of the brain tissue.Oxidative stress and neuroinflammation are closely related to the pathological mechanism of CIRI.NADPH oxidase 2(NOX2)is one of main source of reactive oxygen species(ROS)after cerebral ischemia/reperfusion(I/R).Excessive ROS after cerebral ischemia activates inflammasomes to induce inflammatory responses.Nucleotide-binding oligomerization domain(NOD)-like receptor protein 1(NLRP1)inflammasome regulates the maturation and secretion of inflammatory factors IL-1? and IL-16 in neurons.These inflammatory factors can further induce the expression of cell adhesion molecules,cause immune cells such as neutrophils and macrophages to migrate,and mediate ischemia-reperfusion injury.Ginsenoside Rg1 is one of the important monomer components in ginseng and has anti-inflammatory and anti-oxidant effects.This study was performed to observe the change of NOX2-NLRP1 signaling pathway in cerebral ischemia-reperfusion injury and the intervention of Rg1.The study provides new targets and ideas for clinical drug treatment of cerebral ischemia.Objective To study the role of NOX2-mediated activation of NLRP1 inflammasome in cerebral ischemia-reperfusion injury,to explore the protective effect and mechanism of ginsenoside Rg1 in cerebral ischemia-reperfusion injury,and to develop ginsenoside Rg1 as a safe and effective application of neuroprotective drugs for cerebral ischemia.Methods 1.Male 3-month-old Kunming mice were randomly divided into sham operation group,I/R group,I/R + Tempol(50mg/kg)group,I/R + Apocynin(50mg/kg)group,and I/R + Rg1(5mg/kg)group,I/R + Rg1(10mg/kg)group,5 mice in each group.Bilateral common carotid artery nodule was used in the mouse cerebral ischemia model,before ischemia,after ischemia for 40 min,and after 20 minutes of reperfusion,cerebral blood flow was monitored by laser speckle hemometry.2.Male 3-month-old Kunming mice were randomly divided into sham operation group,I/R group,I/R + Tempol(50mg/kg)group,I/R + Apocynin(50mg/kg)group,and I/R + Rg1(5mg/kg)group,I/R + Rg1(10mg/kg)group,9 in each group.A model of cerebral ischemia in mice was made by ligation of common carotid arteries,and reperfusion was performed after 1 hour of ischemia.The mice were treated with drugs or distilled water for 14 days.Open field experiments were used to detect changes in mouse movement and exploratory behavior;rod climbing method was used to detect mouse neuromotor coordination ability;HE staining was used to observe the pathological and morphological changes of mouse cortex and hippocampal CA1 and CA3 neurons;Nissl staining method to detect the changes of Nissl bodies in mouse cortex and hippocampus;DHE fluorescence method was used to detect reactive oxygen species(ROS)production in mouse cortex and hippocampal CA1 and CA3 regions;Western Blotting to detect cortical and hippocampal NADPH oxidase and NLRP1 inflammasome-related proteins NOX2,p47 phox,p22phox,NLRP1,ASC,caspase-1,IL-1? expression;Western Blotting to detect the expression of Iba1 and MAP2 in the cortex and hippocampus.3.OGD/R model of PC12 cells: The cell was divided into Control group,OGD/R group, OGD/R + Tempol(50 ?mol/L)group,OGD/R + Apocynin(50 ?mol/L)group,OGD/R + Rg1(5 ?mol/L)group,OGD/R + Rg1(10 ?mol/L)group.LDH kit to detect lactate dehydrogenase content;ELISA kit to detect the expression of inflammatory factor IL-1? in cell supernatant;DCFH-DA kit to detect the expression of reactive oxygen species in PC12 cells;Annexin V-FITC/PI kit to detect apoptosis of PC12 cells;Western Blotting was used to detect the expression of NADPH oxidase and NLRP1 inflammasomerelated proteins NOX2,p47 phox,p22phox,NLRP1,ASC,caspase-1,and IL-1?.Results: 1.Cerebral blood flow results showed that compared with the sham operation group,the cerebral blood flow in the I/R model group was significantly reduced after ischemia and 20 minutes after reperfusion.Compared with the I/R model group mice,the blood flow of Tempol,Apocynin and ginsenoside Rg1(5,10mg/kg)pretreated mice was significantly increased after cerebral ischemia and 20 minutes after reperfusion,suggesting that Rg1 can improve blood flow perfusion.2.The results of the open field and pole climbing experiments showed that compared with the sham operation group,the exercise ability and limb coordination ability of the mice in the I/R group were significantly reduced.Compared with the mice in the I/R group,the exercise ability and limb coordination ability of the mice in the Tempol,Apocynin and ginsenoside Rg1(5,10 mg/kg)group were significantly improved.3.The DHE results showed that compared with the sham operation group,the cerebral tissue cortex and hippocampal CA1 and CA3 regions of the I/R model group had significantly increased ROS production.Compared with mice in the I/R model group,Tempol,Apocynin,and ginsenoside Rg1(5,10 mg/kg)treatment significantly reduced neuron ROS production,especially in Rg1(10 mg/kg)treated group.4.The HE staining results showed that compared with the sham operation group,the cerebral cortex of the I/R model mice had obvious pathological damage,and a large number of cells showed unclear nucleoli,unclear outline,nuclear shrinkage,and the phenomenon of fission vacuoles and necrosis,a small amount of nuclear shrinkage and nuclear fission in neurons in CA1 and CA3 of hippocampus.Compared with the mice in the I/R model group,the mice in the Tempol,Apocynin,and ginsenoside Rg1(5,10 mg/kg)administration group could significantly improve the pathological damage in the I/R mice.5.The results of Nissl staining showed that compared with the sham operation group,the intensity and volume of Nissl bodies in the cortical region and hippocampal CA1 and CA3 regions was reduced and the staining was pale.Compared with the mice in the I/R model group,the neuronal cell damage in the Tempol,Apocynin,and Rg1(5,10 mg/kg)pretreatment group was milder,the intensity of Nissl bodies was increased,and the staining was deeper,indicating that Rg1 treatment effectively protected the neurons.6.Western blot results showed that compared with the sham operation group,the expressions of NOX2,p22 phox,NLRP1,ASC,IL-1?,and Caspase-1 in the cortex and hippocampus of the I/R model group were significantly increased,while p47 phox expression had no significant change;compared with mice in the I/R model group,pretreatment with tempol and Apocynin had no significant effect on the expression of NOX2 and ASC,and reduced the expressions of p22 phox,p47phox,NLRP1,IL-1?,and caspase-1(P <0.05);Rg1(5,10mg/kg)pretreatment can significantly reduce the expressions of NOX2,p22 phox,p47phox,NLRP1,ASC,IL-1?,and Caspase-1 in the I/R model group.The results of this experiment indicate that Rg1 has a down-regulating effect on NADPH oxidase 2 and NLRP1 inflammasome in the brain tissue of I/R mice.7.In the cell experiment results,compared with the control group,the OGD/R group had increased cell damage,increased apoptotic cells,increased release of ROS and inflammatory factor IL-1?.The expressions of NOX2,p22 phox,p47phox,NLRP1,ASC,IL-1? and caspase-1 protein were significantly increased.Compared with the OGD/R group,pretreatment with Tempol,Apocynin,and Rg1(5,10?mol/L)significantly reduced cell damage,inhibited apoptosis and released ROS and IL-1?,down-regulated oxidative stress and inflammasome-related proteins of NOX2,p22 phox,p47phox,NLRP1,ASC,IL-1?,and Caspase-1.Conclusion Ginsenoside Rg1 can improve I/R injury in mice,reduce oxidative stress and inflammatory response caused by ischemia-reperfusion.The mechanism may be related to the inhibition of NOX2-NLRP1 signaling pathway.This study provides an experimental basis for ginsenoside Rg1 in the prevention and treatment of cerebral ischemia-reperfusion injury. |
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