| Objectives: Vascular dementia is the clinical syndrome mainly are senior neurocognitive dysfunction based on damaging of brain tissue, caused by a series factors of cerebral vascular. The gradually reducing kidney essence, empty of brain, blood stasis is the basic pathogenesis of the disease, and take invigorating the kidney and promoting blood circulation as the basic treatment, thought by traditional Chinese medicine. Previous studies showed that, Bu Shen Huo Hue Fang could promote the expression of the m RNA and improve the content on the BDNF in the brain tissue, also could improve the ability of learning and memory of mice with vascular dementia, but it was still not clear that by which signal pathway mediated by BDNF can play a role through the drug. This study reveal the expression of BDNF induced by Bu Shen Huo-Xue Fang, and the mechanism of action of the neuron protected by Bu Shen-Huo Xue Fang from the angel of regulating and controlling the PI3K/AKT signal pathway by observing the effect of praxiology, hippocampal neuronal ultrastructure and expression of BDNF 、 Trk B 、 PI3 K 、 AKT coused by Bu Shen Huo Xue Fang.Methods:65 male SD rats of clean grade, about 250-300 g of weight, were provided by the Experimental animal center of Hebei Medical University. The unqualified rats were eliminated by the test of water maze, and the remaining 60 SD rats were randomly divided into the sham operation group, model group, nimodipine group, Bu Shen Huo Xue high-dose group and Bu Shen Huo Xue low-dose group, and each one takes 12 rats, after a week of adaptive feeding.SD rats were fasted for 12 hours and deprivated for water 4 hour before operation, the rats were anaesthetized by intraperitoneal injection with 10% chloral hydrate(3.5ml/kg body weight). According to the literature, the rats were treated by routine disinfection, and cutted on cervical median, and the muscle and connective tissue were bluntly separated, then bilateral common carotid artery were separated and ligated by No. 4 thread, then the muscle and skin were sutured, the skin of local wound were injected 2000 units of Gentamicin for preventing of wound infection. The bilateral common carotid artery of sham operation group were separated, but not be ligated.Each drug group were treated by medicine through weight ratio on the third day after the modeling. Bu Shen Huo Xue high-dose group were perfused the Bu Shen Huo Xue Fang solution which concentration was 1.014 g of crude drug /ml(equivalent to 10.14 g of crude drug /kg), Bu Shen Huo Xue low-dose group were perfused the Bu Shen Huo Xue Fang solution which concentration was 0.507 g of crude drug /ml(equivalent to 5.07 g of crude drug /kg), the control group were perfused Nimodiping solution which concentration was 10ml/kg(equivalent to 11.06 mg /kg). The sham operation group and model group were perfused 5% Physiological saline 10ml/kg, once a day, treating for 30 d. The dead rats were eliminated after modeling and treatment, the remaining rats of each group: 10 of sham operation group, 9 of model group, 10 of Bu Shen Huo Xue high-dose group, 9 of Bu Shen Huo Xue low-dose group, and 9 of control group.1 rats of each group were randomly selected to execute, and the brain hippocampus of rats were stripped and putted into 4% glutaraldehyde to immobilize, then the changes of neuronal ultrastructure in CA1 area of hippocampus were observed, taken photograph and recorded the microscopic picture results through H-7500 transmission electron microscope after treating 30 d. The remaining rats were tested in Morris water maze, mainly including the place navigation test and spatial probe tes. The rats of each group were decapitate to get out the brain after the praxiologyal experiment, western blot was used to test the expression level of protein on BDNF、Trk B、PI3K、AKT of rats, Real Time RT-PCR was used to test the expression of m RNA on BDNF、Trk B、PI3K、AKT of rats.The data were statisticed by using the SPSS for Windows 16.0 software processing, and the measurement data were expressed by modality of mean ± standard deviation( X ±S). The single factor analysis of variance was adoptted when comparing among many groups, then the least significant difference was adoptted to compare with each group; the lever of significant difference was standarded as 0.05 and 0.01.Results: 1 The experimental results of spatial learning and memory ability of rats in each group 1.1 Place navigation test The average time of navigation of the rats in model group was obviously prolonged compared with sham operation group, which was statistical difference(P < 0.01), The average time of navigation of the rats in each treatment group was obviously shortened after drug intervention, which was statistical difference(P<0.01 or P<0.05). Compared with Bu Shen Huo Xue high-dose group, the average time of navigation of the rats in Nimodiping group and Bu Shen Huo Xue low-dose group was not statistically significant(P>0.01). 1.2 Spatial probe test The times and staying time of throughing the original platform of rats in model group were obviously redused compared with sham operation group, which was statistical difference(P<0.01), The times and staying time of throughing the original platform of rats each treatment group were obviously increased after drug intervention, which was statistical difference(P<0.01 or P<0.05). Compared with Bu Shen Huo Xue high-dose group, the times and staying time of throughing the original platform of rats in Nimodiping group and Bu Shen Huo Xue low-dose group were obviously redused, which was statistical difference(P<0.01 or P<0.05). 2 Observation on the neuronal ultrastructure of rats in each group. In the sham operation group, The morphology of neurons were nomal, the organelle were abundant, the nucleus were big like oval, the nuclearmembrane were complete,the euchromatin were abundant, and heterochromatin were less. In the model group: the neuron were serious edema, the organelle were obviously redused, most of crista and part of membrane in Mitochondrial were fusion and blurred, mitochondrial crista had the phenomenon of fracture and losing, the grain in rough endoplasmic reticulum were fusion or degranulation. In the Bu Shen Huo Xue high-dose group: the neuron were light edema, part of crista and membrane in Mitochondrial were fusion and blurred, the grain in rough endoplasmic reticulum were fusion or degranulation. The changes of neuronal ultrastructure in Bu Shen Huo Xue lowdose and Nimodiping group were similar, all can see the edema of neuron, the number of organelle was reduced, most of crista and part of membrane in Mitochondrial were fusion and blurred, Mitochondrial crista had the phenomenon of fracture and losing, the grain in rough endoplasmic reticulum were fusion or degranulation. 3 The changes of the amount of protein expression on BDNF, Trk B, PI3 K, AKT in hippocampus of rats in each group The amount of protein expression in BDNF and Trk B in model group was obviously reduced compared with sham operation group, which was statistical difference(P<0.01 or P<0.05), the amount of protein expression in BDNF and Trk B in hippocampus of rats in each treatment group were obviously improved after drug intervention, which was statistical difference(P<0.01). Compared with Bu Shen Huo Xue high-dose group, The amount of protein expression in BDNF and Trk B in Nimodiping group and Bu Shen Huo Xue low-dose group were reduced, which was statistical difference(P<0.01 or P<0.05). The amount of protein expression in PI3 K and AKT in model group was obviously reduced compared with sham operation group, which was statistical difference(P<0.05), The amount of protein expression in PI3 K and AKT in hippocampus of rats in each treatment group were obviously improved after drug intervention, which was statistical difference(P<0.01). Compared with Bu Shen Huo Xue high-dose group, The amount of protein expression in PI3Kand AKT in Nimodiping group and Bu Shen HuoXue low-dose group were reduced, which was statistical difference(P<0.01 or P<0.05). 4 The changes of the relative content of expression on BDNFm RNA, Trk Bm RNA, PI3 Km RNA, AKTm RNA in hippocampus of rats in each group The relative content of expression on Trk Bm RNA in model group was obviously reduced compared with sham operation group, which was statistical difference(P<0.05), the relative content of expression on BDNFm RNA and Trk Bm RNA in hippocampus of rats in each treatment group were obviously improved after drug intervention, which was statistical difference(P<0.05 or P < 0.01). Compared with Bu Shen Huo Xue high-dose group, the relative content of expression on BDNFm RNA in Nimodiping group and Bu Shen Huo Xue low-dose group were reduced, which was statistical difference(P < 0.01 or P < 0.05); the relative content of expression on Trk Bm RNA in Nimodiping group were reduced, which was statistical difference(P<0.05). The relative content of expression on PI3 Km RNA and AKTm RNA in model group was obviously reduced compared with sham operation group, which was statistical difference(P<0.05 or P<0.01), the relative content of expression on PI3 Km RNA and AKTm RNA in hippocampus of rats in each treatment group were obviously improved after drug intervention, which was statistical difference(P<0.05 or P<0.01). Compared with Bu Shen Huo Xue high-dose group, the relative content of expression on PI3 Km RNA in Nimodiping group and Bu Shen Huo Xue low-dose group were reduced, which was statistical difference(P<0.05).Conclusions: An important reason which lead to the impairment of learning and memory was ischemia and hypoxia in cerebral hippocampal area is in the rats with VD. The level of BDNF and the expression of receptor Tr KB in cerebral hippocampal area was reduced, caused the obstacle of PI3K/AKT signal pathway mediated by BDNF, and aggravated the injury of hippocampal neurons, then leaded to injury of learning and memory function. This study indicated that:1 Bu Shen HuoXue Fang could reduce the injury of hippocampus neurons coused by ischemia, improve the injury of ultrastructure in neurons, and promote the ability of learning and memory in VD rats. 2 Bu Shen Huo Xue Fang could regulate the expression of BDNF and Tr KB from the level of m RNA and protein, and arouse the effect of repair and protective for neurons from the pathway of downstream. 3 Bu Shen Huo Xue Fang could regulate and control PI3K/AKT signal pathway mediated by BDNF, and reduce the ischemia injury of neurons. 4 “kidney storing essence”, vital essence could promote the growth and development of body, it was the same as the effect of promoting the maturation of the neurons by BDNF, so BDNF and the signal pathway mediated by it might be the material basis of deficiency of kidney and blood stasis syndrome in VD. |
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