Study On The Mechanism Of Qingda Granules Inhibiting Local Inflammatory Response In Rats With Cerebral Ischemia-reperfusion Based On NF-?B Signaling Pathway

Obejective:In this paper,the effect of QingDa Granule(QDG)on local inflammatory response after cerebral ischemia reperfusion injury(CIRI)in rats was studied by constructing middle cerebral artery occlusion(MCAO)model in rats to simulate ischemic stroke.The protective mechanism of QDG on CIRI injury was discussed from NF-?B signal pathway.With a view to further elucidating the mechanism of QDG in treating ischemic stroke,it will provide further experimental basis for the clinical application of QDG in the treatment of ischemic stroke.MethodsTake male SD rats weighing 250~280 g,and the right middle cerebral artery ischemia(MCAO)model of rats was prepared by using silicone thread plugs.Reperfusion was performed after 1.5 hours of ischemia,and no thread plug was inserted in the SHAM operation group(SHAM).Three rats were randomly selected at 24 h after surgery to collect fresh tissues,and the tissues were stained and observed with TTC staining.At the same time,the remaining postoperative rats were examined by MRI to observe whether the model was successfully constructed.Then,the successful models were randomly divided into model group,QDG 0.8 g / kg.d group,and QDG 1.6 g / kg.d group,respectively marked as MCAO group,MCAO + QDG-L group,MCAO + QDG-H group.Except for the QDG intervention group,the other groups were given the same amount of normal saline,which were all given by intragastric route.During the intervention period,the weight and neurobehavioral scores of rats in each group were measured daily.After 7 days of intervention,brain tissue was collected after perfusing through the left ventricle with cold saline and 4%paraformaldehyde.The HE staining method was used to observe the pathological changes of the cortical area in the affected side of each group of rats.The GFAP labeled astrocyte method was used to observe the expression of astrocytes in the cortical area of the affected side of each group of rats.IHC was used to detected the expression of local inflammatory factors IL-1?,IL-6,TNF-? and the expression of I?B and IKK in the affected cortical area of each group of rats.Use fluorescence dual-label co-localization method to confirm the translocation of NF-?B in the cerebral cortex infarcted area of each group.Results1.The weight measurement results showed that compared with SHAM group,the weight of rats in MCAO group and MCAO + QDG groups decreased significantly at 24 hours after operation.From the 4th day after operation,the weight loss of QDG intervention group began to slow down,until the 7th day after operation,the weight loss of the QDG intervention group was significantly reduced compared with the MCAO group.2.Neurobehavioral scores showed that at 24 hours after cerebral ischemia(before intervention),the neurobehavioral scores of the surgical group were significantly increased compared with SHAM group.After 7 days of intervention by QDG,the neurobehavioral scores of rats decline.3.The HE staining results showed that the brain tissue of the SHAM operation group was edema-free,the nerve cells were arranged neatly,the shape was complete,the cytoplasm was stained uniformly,and there was almost no cell necrosis.The MCAO group haddisordered cell arrangement,interstitial edema,and interstitial looseness The number of neurons decreased,the nucleus contracted,fragmented or eosinophilic,and a large number of inflammatory cells were infiltrated.After QDG intervented,the pathological changes in the ischemic cerebral cortex infarction area of rats were improved.4.The result of labeling astrocytes with GFAP showed that the GFAP in the affected cortical area of the MCAO group was significantly activated and increased in number compared with the SHAM group.Howerver,The number of astrocytes decreased significantly after the intervention of QDG.What is more,the data showed that the number of GFAP in the MCAO + QDG-H group was even less than that in the MCAO+QDG-L group,which was a significant difference.5.The IHC results showed that QDG down-regulated the expressions of IL-1?,IL-6 and TNF-? in the cerebral infarcted area of CIRI rats,which were inflammatory factors.6.The IHC results showed that compared with the SHAM group,the expression of phosphorylated IKK and I?B in the cortical area of the affected side of the MCAO group increased.After QDG intervention,the expression of phosphorylated IKK and I?B in the affected side cortical area decreased with a obvious difference.Besides,there was no significant difference in total I?B expression when the four groups compared with each other.7.The IF results revealed that compared with the SHAM group,NF-?B p65 in the cortical area of the MCAO group was activated,and the number of NF-?B p65 in the nuclei was significantly increased.After QDG intervention,the activation of NF-?B p65 was suppressed,and the number of NF-?B p65 in the nuclei was significantly reduced,besides,the inhibitory effect in QDG-H group was more obvious than that in QDG-L group.Conclusion1.QDG can effectively improve neurological damage after CIRI.2.QDG can prevent weight loss after CIRI and promote weight recovery.3.QDG can improve the pathological damage of cortical infarcted area after CIRI.4.QDG can reduce inflammation damage by down-regulating the expression of IL-1?,IL-6,TNF-? in the cortical infarcted area after CIRI.5.QDG can inhibit the transilocation of NF-?B by inhibiting the phosphorylation of I?B and IKK in the NF-?B signaling pathway,and finally reduce inflammation damage after CIRI.