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ObjectiveTo investigate the protective effect of Liuwei Dihuangwan on neurovascular injury in SAMP8 mice. MethodThe Alzheimer's disease （AD） model with insufficiency of kidney essence was induced in 75 SAMP8 mice aging 6 months. The model mice were divided into model group， positive control group （donepezil hydrochloride， 0.747 mg·kg-1·d-1）， and high-， medium-， and low-dose Liuwei Dihuangwan groups （2.700， 1.350， 0.675 g·kg-1·d-1）， with 15 mice in each group. Fifteen SAMR1 mice were assigned to a normal control group. All mice were administered continuously for 2 months. The spatial memory of mice was tested by the Morris water maze. Hematoxylin-eosin （HE） staining was used to observe the pathological changes in the hippocampus and cortex of brain tissues. The immunohistochemical method （IHC） was used to detect the deposition of amyloid β-protein （Aβ） and the expression of von Willebrand factor （vWF） and CD34 in the hippocampus and cortex of brain tissues. Electron microscopy was used to observe the ultrastructural changes in cerebral microvessels. Western blot was used to detect the protein expression levels of the receptor of advanced glycation endproduct （RAGE）， low-density lipoprotein receptor-related protein 1 （LRP1）， vascular endothelial growth factor A （VEGF-A）， and P-selection in the hippocampus and cortex of brain tissues. ResultCompared with the normal control group， the model group showed prolonged escape latency and swimming distance （P<0.01）， increased number of glial cells， decreased number of nerve cells， blurred tight junctions or enlarged gap of the brain microvascular endothelial cells， severely injured membrane structure， swollen mitochondria of endothelial cells， ruptured membrane， massive dissolution in cristae， increased protein expression of Aβ and vWF in the hippocampus and cortex （P<0.01）， reduced protein expression of CD34 （P<0.05）， elevated protein expression of RAGE and P-selection in the cortex （P<0.01）， and decreased protein expression level of LRP1 and VEGF-A （P<0.01）. Compared with the model group， the Liuwei Dihuangwan groups showed shortened escape latency and swimming distance （P<0.05）， reduced number of glial cells in the cortex and hippocampus， increased number of microvessels in the cortex， clear double-layer membrane structure in tight junctions between the microvascular endothelial cells， increased number of mitochondria with intact membrane and recovered mitochondrial cristae， decreased protein expression of Aβ， vWF， RAGE， and P-selection in the hippocampus and cortex （P<0.05）， and increased protein expression of CD34， LRP1， and VEGF-A （P<0.05）. ConclusionLiuwei Dihuangwan can regulate Aβ metabolism through the RAGE/LRP1 receptor system and promote cerebral microvascular angiogenesis by inhibiting vWF expression and increasing VEGF-A and CD34， thereby improving cerebral microvascular injury in SAMP8 mice.
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ObjectiveTo investigate the effect of Liuwei Dihuangwan on memory function of senescence-accelerated mouse prone 8 （SAMP8） mice by regulating autophagy through the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/forkhead box O3a （FoxO3a） pathway. MethodSix male senescence-accelerated mouse resistant 1 （SAMR1） mice of SPF grade aging 6 months were assigned to a normal group， and 24 male SAMP8 mice of SPF grade aging 6 months were randomly divided into a model group， a donepezil group （0.747 mg·kg-1）， and high- and low-dose Liuwei Dihuangwan groups （2.700 and 1.350 g·kg-1）， with 6 mice in each group. The mice were treated with drugs by gavage for 2 months. Morris water maze was used to detect the learning and memory abilities of mice in each group. Nissl staining was used to observe the neurons in the cortex and hippocampus. The positive expression of microtubule-associated protein 1 light chain 3B （LC3B） in the cortex and hippocampus was detected by immunohistochemistry （IHC）. Western blot was used to detect the protein expression of the mammalian ortholog of yeast ATG6 （Beclin-1）， B cell lymphoma-2 （Bcl-2）， autophagy-related gene 5 （ATG5）， cysteinyl aspartate-specific protease 3 （Caspase-3）， Caspase-9， Akt， p-Akt， FoxO3a， and p-FoxO3a. ResultCompared with the normal group， the model group showed prolonged escape latency （P<0.05，P<0.01）， reduced number of platform crossings and the residence time in the target quadrant （P<0.01）， decreased neurons with reduced volume and dispersed distribution in the cortex and hippocampus， increased positive expression of LC3B （P<0.01）， elevated expression of Beclin-1 and ATG5 in the cortex （P<0.01）， declined Bcl-2 expression （P<0.01）， up-regulated Caspase-3 and Caspase-9 expression （P<0.01）， and decreased expression levels of p-Akt/Akt and p-FoxO3a/FoxO3a （P<0.01）. Compared with the model group， the donepezil group and the Liuwei Dihuangwan groups showed shortened 3 d escape latency （P<0.05，P<0.01）， increased number of platform crossings （P<0.01）， and prolonged residence time in the target quadrant （P<0.01）. In the donepezil group， the number of neurons in the cortex and hippocampus was increased. In the Liuwei Dihuangwan groups， the number of neurons and Nissl bodies increased with denser distribution and lower degree of cell damage. The positive expression of LC3B in the cortex and hippocampus was decreased in the donepezil group and Liuwei Dihuangwan groups （P<0.01）. The expression of Beclin-1 was decreased in the Liuwei Dihuangwan groups （P<0.01）. The expression of ATG5 was decreased in the donepezil group and the low-dose Liuwei Dihuangwan group （P<0.01）. The donepezil group and the Liuwei Dihuangwan groups showed the increased expression level of Bcl-2 in the cortex （P<0.01）， decreased expression level of Caspase-3 （P<0.01）， reduced expression level of Caspase-9 （P<0.05，P<0.01）， and elevated expression levels of p-Akt/Akt and p-FoxO3a/FoxO3a （P<0.01）. ConclusionLiuwei Dihuangwan can effectively improve the learning and memory abilities of the SAMP8 mice and protect neurons. Its mechanism may be related to the regulation of the PI3K/Akt/FoxO3a signaling pathway， down-regulation of the expression of ATG5， Beclin-1， and LC3B， and the inhibition of apoptosis.
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BACKGROUND:Cerebral ischemia often occurs in underlying pathological conditions, such as hypertension, hyperlipidemia and diabetes. Therefore, it is of great significance to construct a cerebral ischemia rat model with hyperlipidemia and to study the effect of basic pathological changes on the cerebral ischemia. OBJECTIVE:To observe the brain tissue pathological changes of rat models with coexistence of hyperlipidemia and cerebral ischemia, and the effect of hyperlipidmia on cerebral ischemia. METHODS:The rats were fed with high-fat diet to establish the hyperlipidmia models, and then focal cerebral ischemia models were prepared with suture method. At 3 and 7 days after modeling, the 2,3,5-triphenyltetrazolium chloride staining was used to observe the volume of brain tissue ischemia, and hematoxylin-eosin staining was performed to observe pathological change of the margin of the brain tissue ischemia zone. RESULTS AND CONCLUSION:2,3,5-Triphenyltetrazolium chloride staining staining results showed that the volume of cerebral ischemia was significantly reduced in the hyperlipidemia+cerebral ischemia 7 day group. Hematoxylin-eosin staining results showed there was typical ischemic changes in al the cerebral ischemia models, and the number of microglial cel s after cerebral ischemia for 7 days was significantly smal er than that after cerebral ischemia for 3 days, and the changes were more obvious in the hyperlipidemia+7-day cerebral ischemia group when compared with the hyperlipidemia+3-day cerebral ischemia group. Ultrastructure showed there were neuronal and glial nuclear membrane shrinkage in al the cerebral ischemia models, mitochondria cristae was disappeared completely, endothelial cel mitochondria was decreased, most of the synaptic vesicles of nerve synapse were dissolved;the damages above were improved after ischemia for 7 days, especial y hyperlipidemia+cerebral ischemia for 7 days, the neuronal degeneration and necrosis were reduced, the mitochondrial damage was repaired, the number of mitochondrial cristae was increased significantly, and the synaptic vesicles of nerve synapse were recovered significantly. The results indicate that hyperlipidemia can promote the recovery of cerebral ischemic injury, probably because the hyperlipidemia factors can activate the protection mechanism.