| Stroke,as an ischemic cerebrovascular disease,has a rapid onset and a short treatment window.It may also cause the patient's loss of behavior or even death,which seriously harms human health.Thrombolytic therapy dominates the acute phase of stroke,but with the recanalization of blood vessels,blood flow is restored,resulting in a more serious "ischemic reperfusion injury." Since cerebral ischemia-reperfusion injury is a multi-molecule,multimechanism,and interactive pathophysiological process,it is of great significance to find an active and effective treatment plan.Modern pharmacological studies have shown that the multicomponent and multi-target characteristics of traditional Chinese medicine have significant advantages in the prevention and treatment of cardiovascular and cerebrovascular diseases.Xinnaoning Capsules are made from the combination of Salvia miltiorrhiza,Ginkgo biloba,Buxus lobata,Daguomu ginger,and scallion.They have good clinical effects on cardiovascular diseases,but there are few studies in the field of cerebrovascular diseases,and the research on related mechanisms is almost blank.Therefore,to explore the protective effect of compound Chinese medicine Xinnaoning capsule on cerebral ischemia-reperfusion injury and to explore its mechanism of action have certain research significance.Objective:In this paper,Xinnaoning Capsule(XNN)is selected as the research object,the cerebral ischemia-reperfusion model was evaluated by behavioral,imaging and pathological observations to explore its protective effect on acute cerebral ischemia-reperfusion injury;Through the network pharmacology analysis of Xinnaoning Capsule treatment of cerebral ischemia-reperfusion injury and the important target and related mechanism pathway.To systematically explore the mechanism of Xinnaoning Capsule in treating cerebral ischemiareperfusion injury from the animal level to the molecular level,and to provide theoretical basis and experimental reference for the treatment of clinical ischemic stroke.Methods:In this study,male ICR mice were selected as experimental subjects,and they were treated with pre-administration before inducing ischemia-reperfusion injury.The Xinnaoning capsule group was given 3g/kg intragastric suspension of XNN content daily;the positive drug group was given daily 9mg/kg tail vein injection of edaravone(YDLF);the model group and the sham operation group were given daily gavage Normal saline for 7 days.The model of cerebral ischemia/reperfusion injury in mice was established by thread embolization of middle cerebral artery.The ischemic time was selected to be 30 min and the reperfusion time was 24 hours.That is,the detection of relevant data and the selection of tissues are all performed within 24 hours of ischemia-reperfusion.Starting from the overall animal level,the Bederson neurobehavioral score was used to evaluate the neurological function immediately after the model was awakened.Samples that did not cause ischemic damage(0 points)or were too heavy(5 points)were excluded,so that the neurological damage of the enrolled mice was at the same level as possible.The neurobehavioral scoring was performed at 24 h of ischemic perfusion to evaluate the progress of neurological damage.Subsequent scans were performed using the small animal in vivo imaging equipment Micro-CT combined with X-ray contrast agent iohexol injection,and then the threedimensional reconstruction technique was used to reproduce the brain midline of each group of mice,calculate the offset,and judge the brain edema;use TTC staining Method to evaluate the improvement of cerebral infarction area in mice.Brain tissue was taken and sectioned at 24 hours after reperfusion,TTC staining and photographing were performed,infarct area was calculated and infarct percentage was counted;H&E staining technique was used to evaluate the histopathological differences of the striatum and hippocampus in mice after ischemiareperfusion injury.After completing the pharmacodynamic evaluation,in order to explore the main mechanism of action of Xinnaoning capsule on cerebral ischemia-reperfusion injury,we used IPA analysis of network pharmacology to combine the main active components of XNN with the relevant targets of ischemic stroke.Obtain compound-target-pathway related data,analyze the most likely action pathway,and key targets on the path,and use this as an entry point to explore and verify the mechanism.We measured the expression of NF-?B p65 total protein in mouse brain tissue by immunoblotting;next we analyzed and evaluated the expression of NF-?B p65 protein in the brain nucleus of brain tissue by immunohistochemical staining;Finally we Using RT-PCR technology,the expression of IL-1?,IL-6,and TNF-? m RNAs in downstream groups of NF-?B pathway was detected.Completed the mechanism study of XNN based on inflammatory response against cerebral ischemia-reperfusion injury Finally,immunohistochemical staining was used to evaluate the effect of Xinnaoning capsule pretreatment on the expression of nuclear factor-kappa B(NF-?B)in brain tissue of mice with ischemia-reperfusion injury;Determine the expression of NF-?b protein in mouse brain tissue;use real-time quantitative PCR technology(rt-PCR)to detect the effects of downstream related IL-1,IL-6,and TNF-? m RNA expression of NF-?B pathway in each groupResults:Regarding the establishment and evaluation of the model,there were significant differences between the operation groups and the sham operation groups,and no significant differences between the operation groups,indicating that the operations were successful in inducing ischemic injury and the parallelism was good.At 24 h,the neurobehavioral scores of the XNN group and the YDLF group were significantly lower than the model group(p<0.01).Micro-CT imaging results showed that the distance of the midline offset between the Xinnaoning group and the YDLF group was significantly smaller than that of the model group(p<0.01).In terms of histopathological evaluation,the TTC staining results showed that the infarct size in the XNN group was significantly smaller than that in the model group(p<0.01),and the YDLF effect was more obvious(p<0.001);the HE results showed that the XNN group and the YDLF group compared with the model group The cell distribution and arrangement are more regular,the staining is more normal,and there is a certain interstitial edema.Network pharmacological analysis showed that XNN intervention in cerebral ischemiareperfusion injury is most likely to play a role through neuroinflammation signaling pathway,and the final core target analysis showed the highest correlation with Rel A(p65).Western blot results showed that the total protein content of NF-?B p65 in the XNN group was significantly lower than that in the model group(p<0.001);immunohistochemical staining showed that there were a large number of NF-?B p65 positive expressions in the nucleus of the model group,and positive cells in the XNN group The number is significantly different from the model group(p<0.01),but if the integrated optical density measurement method is used,the gap between the model group and the model group is more significant(p<0.001);RT-PCR results show that the XNN group NF-?B The relative expression of downstream related inflammatory factors IL-1?,IL-6,TNF-? m RNA has a certain downward trend compared with the model group,among which IL-1? is the most obvious(p<0.05).Conclusion:This study found that XNN has a protective effect on cerebral ischemia-reperfusion injury.It can improve the neurological dysfunction after MCAO model injury;it can protect the bloodbrain barrier and inhibit the progression of secondary brain tissue edema;it can save the ischemic penumbra and reduce the scope of cerebral infarction lesions;it can improve the pathological presentation Tissue and cell damage;it can inhibit the expression of NF-?B p65 after cerebral ischemia-reperfusion injury and reduce the expression of downstream related inflammatory factor m RNA.The results of related network pharmacological analysis also verified this point.From this we conclude that XNN can play a role in resisting cerebral ischemia/reperfusion injury by down-regulating the expression of NF-?B and inflammatory factors related to its pathway. |
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