| Objective: Based on the theory of "brain marrow",this study starts from the disease organ Suixu and Dusun of stroke disease,using methods of behavioral,pathological,molecular biology and other research methods to detect and analyze cognitive function,hippocampal pathology and ultrastructural changes in hippocampus,apoptosis protein and gene expression in hippocampal PI3K/Akt cell signal transduction pathway and other aspects of VD(Vascular Dementia)rats,to further explore the effect and mechanism of Yisui Jiedu method on cognitive function and hippocampal neuronal injury and apoptosis in VD rats,and provide new targets and experimental evidence for VD understanding and treatment of TCM.Methods: 1.The healthy SPF male SD rats were subjected to permanent ligation of bilateral common carotid arteries(2-VO),and the VD animal model was replicated.The successful model rats were randomly divided into 5 groups: vascular dementia model group(model group),the donepezil hydrochloride control group(positive control group),High,medium and low dose groups of Chinese medicine for Yisui Jiedu,15 in each group,and another 15 rats with only un-ligated common carotid artery were selected as sham operation group.The gavage treatment was started on the 2nd day after model establishment.The administration and dosage of each group were sham operation group and model group: 0.9%normal saline,positive control group: donepezil hydrochloride solution,0.052 mg/kg;Chinese medicine high and medium Low-dose group: Yishen detoxification Chinese medicine compound Nongbenfang granule solution,22.22g/kg,11/11g/kg,5.56g/kg.An equal volume(1mL/100 g)was administered to the stomach for 30 days.2.Morris water maze was used to test the behavior of rats.The test started on the first day after the end of the gavage treatment.The effects of Yisui Jiedu method on learning and memory function of VD rats were analyzed by positioning navigation experiment and space exploration experiment.3.To analyze the effect of Yisui Jiedu method on the ultrastructure of hippocampalpathological tissues and hippocampal neurons in VD rats through HE staining of rat hippocampus and observation of ultrastructure of hippocampus through TEM.4.Nissl staining and TUNEL(green fluorescence)apoptosis assay were used to analyze the effect of Yisui Jiedu method on the number of Nissl bodies and positive neurons and hippocampal tissue apoptosis in hippocampus of VD rats.5.Methods of IHC,Western-blot and RT-PCR were used to detect of PI3K/Akt signaling pathway-related factors PI3 K,Akt,p-Akt,Caspase-3,Caspase-9,NF-κB,Bad protein expression and mRNA changing,to analyze the regulation of PI3K/Akt signaling pathway in hippocampus of VD rats by Yisui Jiedu method.Results: 1.Behavioral test results: In the results of the navigation test,compared with the sham operation group,the escape latency of the VD model group was significantly prolonged(P<0.05,P<0.01),compared with the model group,the escape of the Chinese medicine group and the positive control group The latency was significantly shortened(P<0.05,P<0.01),and the difference was statistically significant.In the space exploration experiment,compared with sham operation,the time of the first arrival of the platform in the VD model group was significantly prolonged,and the platform quadrant dwell time and the number of crossing platforms were significantly reduced.The time for the Chinese medicine group to reach the platform for the first time was significantly shortened.The quadrant dwell time and the number of crossing platforms increased significantly.2.Compared with the model group,the difference was significant(P<0.05,P<0.01).Results of hippocampal pathological examination: HE staining results:The neuronal structure of hippocampal CA1 area in VD rat model group is incomplete,irregular in shape,loosely arranged and disordered.Cell interstitial edema is obvious,and nuclear pyknosis is deeply stained.The gap between the neurons and the surrounding tissues is widened,and the neurons are lost significantly.Compared with the model group,the neuronal structure of the hippocampal CA1 area of the middle dose group of Chinese medicine is relatively complete,and the morphology is relatively regular,and the level is clear.The interstitial edema is not as obvious as the model group,the nucleus pyknosis isreduced,and the gap between neurons and surrounding tissues is reduced.The neurons showed a small amount of loss.TEM results: in the model group,the hippocampal CA1 neurons had abnormal nuclear morphology,increased heterochromatin,disordered organelles,incomplete mitochondrial structure,sputum and membrane fusion,severe cavitation,and rupture and loss of sputum.Compared with the model group,the middle dose group of Chinese medicine: the structure of neurons is relatively complete,the degree of membrane dissolution is light,and the mitochondrial cavitation and expansion are significantly reduced.3.Intervention of hippocampal cell injury and apoptosis results: Nissl staining results:compared with the sham operation group,hippocampal neurons Nissl bodies of the VD model group rats lost significantly,staining became shallow,the number of positive cells decreased significantly(P <0.01),Compared with the model group,the Nissl bodies in the positive control group and the middle dose group of Chinese medicine were significantly stained with Nissl bodies,and the number of neurons positive cells increased(P<0.05,P<0.01).The difference was statistically significant.The number of apoptotic cells detected by TUNEL in the VD model group was significantly higher than that in the sham operation group,indicating that there was apoptosis in the VD process.The middle dose group of Chinese medicine could significantly reduce the number of apoptotic cells.Compared with the model group,the difference was significant(P<0.01).4.The IHC method was used to detect the related factors of PI3K/Akt signaling pathway.Compared with the sham operation group,the expression of Akt and p-Akt protein in hippocampus of VD model group was significantly decreased,and the expression of Caspase-9 and Caspase-3 protein was significantly increased(P<0.05,P<0.01),Compared with the model group,the expression of Akt and p-Akt protein was significantly increased in the positive control group and the middle dose group,and the expression of Caspase-9 and Caspase-3 protein was significantly decreased(P<0.05,P<0.01).The results of WB test showed that compared with the sham operation group,the expression of PI3 K,Akt,p-Akt and NF-κB protein in hippocampus of VD model group wassignificantly decreased,and the expressions of Caspase-9,Caspase-3and Bad protein were significantly increased(P<0.05,P<0.01).The difference between the two groups was significant and statistically significant.In the middle dose group,the expression of PI3 K,Akt,p-Akt and NF-κB protein was up-regulated,and the expression of Caspase-9,Caspase-3 and Bad protein was down-regulated.The results of Rt-PCR showed that the levels of PI3 K mRNA,Akt mRNA and NF-κB mRNAwere significantly decreased in the model group compared with the sham operation group,and the levels of Caspase-9 mRNA,Caspase-3 mRNA and Bad mRNA were significantly increased(P<0.05,P<0.01).The middle dose group of Chinese medicine can significantly up-regulate the expression of PI3 K mRNA,Akt mRNA and NF-κB mRNA and down-regulate the expression of Caspase-9 mRNA,Caspase-3 mRNA and Bad mRNA.Conclusion:1.The Yisui Jiedu method can significantly improve cognitive functions such as learning and memory of VD rats.2.Yisui Jiedu method can significantly improve the pathological structure of hippocampus in VD rats,protect the ultrastructural integrity of hippocampal neurons and repair neuronal damage.3.Yisui Jiedu method can significantly increase the number of Nissl bodies in hippocampal neurons of VD rats,repair nerve damage and fight against neuronal apoptosis.4.Yisui Jiedu method can significantly up-regulate PI3 K,Akt,p-Akt,NF-κB protein and gene expression in rat hippocampus,down-regulate Caspase-9,Caspase-3,Bad protein and gene expression,which may be one of the mechanisms due to the regulation of PI3K/Akt signaling pathway-related factors by the Yisui Jiedu method.,detoxifies the method to solve the "neurotoxicity",fight against neuronal apoptosis,repairs neuronal damage by means of the marrow,and realizes brain protection,thereby improving VD learning and cognition,playing a role in the treatment of VD. |
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