| BackgroundAcute ischemic stroke has become an important public health problem in China because of its high incidence and disability,which has brought heavy burden to families and society.Effective treatment is crucial to alleviate or improve the symptoms of neurological deficits and reduce the burden of disease caused by stroke.Traditional Chinese medicine has a long history in the treatment for stroke.In the long-term medical practice,doctors of all dynasties have summarized rich clinical experience,especially the theory of"toxin injuring collaterals of the brain" put forward by academician Wang Yongyan,which has pushed the understanding of stroke in traditional Chinese medicine to a new level and promoted the process of stroke treatment in traditional Chinese medicine.According to the theory of "toxin injuring collaterals of the brain",the method of clearing heat detoxifying and clearing collaterals is put forward.Kudizi injection has a definite effect in the treatment of ischemic stroke,which can reduce the inflammatory reaction and improve the neurological function and clinical outcome,but the mechanism of its action is not yet clear.Recent studies have shown that Notch signaling pathway may be involved in the inflammatory response process of cerebral infarction.Therefore,this study intends to explore the mechanism of Kudizi injection in acute cerebral infarction injury and its correlation with Notch signaling pathway.ObjectiveThe purpose of this project is to observe the effects of Kudizi injection on the inflammation and Notch signaling pathway of ischemic penumbra in rats with cerebral ischemia reperfusion injury of fire-toxicity syndrome and the changes in the pathological structure of neurons and the apoptosis of neurons,and to explore the possible mechanism of Kudizi injection in the treatment of acute cerebral infarction with fire toxicity syndrome.Method128 SPF SD male rats were randomly divided into 4 groups according to the random number table method:sham operation group,model group,Notch-1 inhibition group(DAPT group)and Kudiezi group,with 32 rats in each group.In addition to the sham operation group,the other groups used Longa combined with carrageenan intraperitoneal injection to make the rat model of fire toxicity after left middle cerebral artery occlusion reperfusion.Intraperitoneal injection was performed immediately after reperfusion,in which the sham group and the model group were given equal volume of normal saline(3.6ml/kg · d),and the corresponding drugs were given intraperitoneal injection respectively for each drug administration group,in which the Kudiezi group was given Kudiezi injection 3.6ml/kg·d,and the DAPT group was given DAPT 500ug/kg·d,once a day for continuous 3 days.At 3h,24h,48h and 72h,the ischemic penumbra tissues were collected.Western blot and RT-PCR were used to detect the expressions of Notch-1,Jagged-1,Hes-1,IKK,NF-?B and caspase-3 in ischemic penumbra.The ultrastructural changes of ischemic penumbra neurons after the intervention of kudizi injection were observed by transmission electron microscopy.The morphological and structural changes of neurons were observed after HE staining.TUNEL staining is used to observe the apoptosis of neurons in ischemic penumbra.Results1.Neurological defect score of rats:intergroup comparison:neurological defect score of sham operation group was O.After reperfusion for3h,24h and 48h,DAPT group and Kudizi group showed no significant decrease in neurological function scores compared with the model group(p>0.05).After reperfusion for 72h,DAPT group and Kudizi group showed significant decrease in neurological function scores compared with the model group(p<0.05).There was no significant difference between DAPT group and Kudizi group(p>0.05).Intra-group comparison:there was no significant difference in neural function scores of the model group at each time point(p>0.05).Neurological function of DAPT group and model group presented a gradually declining trend after 24h,in which neurological function score of 72h was significantly decreased compared with 3h(p<0.05).2.The mRNA and protein levels of Notch-1,Jagged-1,Hes-1,IKK,NF-?B,caspase-3 in the model group were higher than those in the sham group in 3h(p<0.05).At the same time the expression of the above genes and proteins model group was higher than those in the sham group(p<0.05).The expressions of Notch-1,IKK and caspase-3 in the model group peaked at 3h-24h,and then decreased.The expression of NF-?B protein peaked at 24h-48h Jagged-1 and Hes-1 protein were highly expressed in 3h-48h.3.Compared with the model group,DAPT reduced the levels of Notch-1,Jagged-1,Hes-1 IKK,NF-?B,caspase-3 mRNA and protein in the ischemic penumbra,and some of the differences were statistically significant.The protein expression trend of Notch signaling pathway in DAPT group was roughly similar to that in model group.4.Compared with the model group,Kudiezi injection reduced the expression of Notch-1,Jagged-l,Hes-1,IKK,NF-icB,caspase-3 mRNA and protein in the ischemic penumbra,and some of the differences were statistically significant.The levels of Notch-1,Jagged-1,Hes-1,IKK,NF-?B,caspase-3 mRNA and protein in ischemic penumbra were lower than those in DA PT group,and some of the differences were statistically significant.Intra-group comparison:the expressions of Notch-1 and IKK protein peaked at 3h-24h,and then decreased(p<0.05).The expression of Jagged-1 and caspase-3 peaked at 24h.The expression of Hes-1 and NF-?B protein peaked at 24h?48h.5.Transmission electron microscopy observation:compared with the sham operation group,the structure of neurons in the model group showed different degrees of damage,including nuclear shrinkage,increased heterochromatin,obvious mitochondrial damage,high electron density.With the prolongation of ischemia time,the structure of neurons in the model group changed obviously.Compared with the same time of reperfusion,the structure of neuron organelle in Kudizi group was better than that in model group.The improvement of neurons in Kudiezi group was slightly better than that in DAPT group.6.HE staining:the rats in the model group were observed with destruction of infarcted lateral brain tissue,loose structure,decreased number of neurons,mostly triangular,and cytoplasmic concentration and nucleus condensation.Compared with the model group,the structure of neurons in Kudizi group was significantly improved.The structure of DAPT group was better than model group.7.TUNEL staining:the number of TUNEL positive cells in the model group was significantly higher than that in the sham group.At 48h after reperfusion,the number of apoptotic neurons in DAPT group and Kudizi group was significantly less than that in model group(p<0.05),and there was no significant difference between DAPT group and Kudizi group(p>0.05).Conclusion1.Notch signaling pathway is activated in acute cerebral infarction,and the expressions of Notch-1,Jagged-1,Hes-1,IKK,NF-?B and caspase-3 in ischemic penumbra significantly increased.2.Kudizi injection can inhibit inflammatory reaction,reduce neuronal apoptosis,alleviate cerebral ischemia reperfusion injury,and improve neurological function in rats with acute cerebral infarction of fire-toxicity syndrome.The curative effect of Kudizi injection is much better than DAPT.3.The protective effect of Kudizi injection on cerebral ischemia reperfusion injury may be related to the regulation of Notch signaling pathway.4.The Notch signaling pathway could be inhibited by Kudiezi injection after 3h,and at the same time the Notch signaling pathway activity was lower than that in the model group.Therefore,Kudizi injection should be given as early as possible in clinical treatment of acute cerebral infarction to improve neurological function. |
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