| Objective To observe the neurological score,the cerebral infarction volume,the activity changes of SOD,and the expression changes of NLRP3/ASC/caspase-1/IL-1β/IL-18/MMP-9 after ischemia reperfusion injury.Based on the two aspects of the reaction of oxidative stress and inflammation studying the protection mechanism of the main active ingredients combination of Yangyin Tongnao Granules on ischemic reperfusion injury rats,and further studying the compatibility rule,clarifying the scientific of the TCM compound compatibility.Methods The male SD rats with body weight of 280±20g were randomly divided into sham operation group,model group,orthogonal compatibility group and positive control group.MCAO model was prepared by modified Longa suture method,and the suture was removed for reperfusion after occlusion at one hour.In the sham operation group,the operation was the same except for the plug.The orthogonal combination group were treated with the main active ingredients combination of Yangyin Tongnao Granules.Positive control group were treated with nimodipine.The sham operation group and model group were given the same amount of sodium carboxymethyl cellulose solution,once daily for three consecutive days.Conducting the neurological score before adopting every sample.Cerebral infarct volume was measured by TTC staining.Elisa kit was used to detect the activity of SOD.To evaluate the expression of NLRP3 protein in Ischemic brain tissue by immunohistochemistry;to detect the mRNA expression of ASC,caspase-1,IL-1,IL-18,MMP-9 in hippocampus through the method of RTPCR.Results(1)The effect of neurological deficit of the main active ingredients combination of Yangyin Tongnao Granules on ischemic reperfusion injury rats.The sham group had no neurological deficit.Compared with the sham group,model group had serious neurological deficit.And the difference was statistically significant;after given the main active ingredients of Yangyin Tongnao granule and the positive control drug,symptoms of neurological deficit reduced,compared with the model group.And the difference was statistically significant;(2)The effect of cerebral infarction volume of the main active ingredients combination of Yangyin Tongnao Granules on ischemic reperfusion injury rats.The sham operation group had no cerebral infarction,compared with the sham operation group,the model group had brain infarction.And the difference was statistically significant;after given the main active ingredients of Yangyin Tongnao granule and the positive control drug,the volume of cerebral infarction reduced,compared with the model group.And the difference was statistically significant;(3)The effect of SOD activity of the main active ingredients combination of Yangyin Tongnao Granules on ischemic reperfusion injury rats.Compared with the sham operation group,the activity of SOD of the model group decreased.And the difference was statistically significant;compared with the model group,the group of the main active ingredients combination of Yangyin Tongnao Granules and positive control drug can significantly increase the SOD value.(4)The effect of the distribution and expression of NLRP3 protein of the main active ingredients combination of Yangyin Tongnao Granules on ischemic reperfusion injury rats.Through the analysis of immunohistochemical image,we found that NLRP3 inflammasome scattered in cerebral ischemia rat spinal cord room in the sham group.And the main expression of cell were ependymal epithelial cells.And they distributed in the cytoplasm,the number of positive cells in ischemic brain significantly increased after ischemia reperfusion.And they distributed intensive in the spinal cord room of brain.after administration of the main active ingredients of Yangyin Tongnao granule and nimodipine,the number of positive cells decreased significantly.(5)The effect of the mRNA expression of ASC,caspase-1,IL-1,IL-18,MMP-9 of the main active ingredients combination of Yangyin Tongnao Granules on I/R injury rats.By real-time PCR analysis,we found that ischemia-reperfusion injury increased the mRNA expression of ASC,caspase-1,IL-1,IL-18,MMP-9 in the hippocampus of ischemic brain,after administration of the main active ingredients of Yangyin Tongnao granule and nimodipine,the mRNA expression of ASC,caspase-1,IL-1,IL-18,MMP-9 in the hippocampus of ischemic brain decreased significantly.Conclusion(1)the main active ingredients combination of Yangyin Tongnao Granules played a protective mechanism on focal cerebral ischemia reperfusion injury rats.It can play a protective role by reducing the neurological deficit,the infarction volume and the oxidative stress by increasing the value of SOD.(2)The expression of NLRP3,ASC,caspase-1,IL-1,IL-18,MMP-9 increased after cerebral ischemia reperfusion injury.The main active ingredients combination of Yangyin Tongnao Granules can reduce the cerebral ischemia reperfusion injury and the inflammatory reaction by inhibiting NLRP3 inflammasome,down-regulating the expression of ASC,caspase-1,IL-1,IL18,MMP-9 to realize the ischemia reperfusion injury neuroprotection. |
