Studies On The Effects Of Modified Kai-Xin-San In Alleviating The Cerebral Microangiopathy In 5xFAD Mice Model Of Alzheimer's Disease

Objective:To study the effects of Kai-Xin-San(KXS)and Modified Kai-Xin-San(MKXS)on the learning and memory behavior of 5×FAD mice;To investigate the possible mechanism of the expression of protein related to cerebrovascular disease in 5×FAD mice with KXS and MKXS;To observe the effects of KXS and MKXS on the morphology of neurons and small blood vessels in the brain area of 5 ×FAD mice.Methods:Taking wild-type mice from 3 to 4 months old as WT group(NaCl 0.1mL/lOg/d).5×FAD mice were randomly divided into 5 groups,called 5×FAD group(saline 0.1mL/10g/d),5×FAD+DP group(Donepezil 0.02mg/10g/d),5×FAD+KXS group(KXS 8.7g/kg/d),5×FAD+MKXSLD group(MKXS 15.42g/kg/d)and 5×FAD+MKXSHD group(MKXS 30.84 g/kg/d),All mice were administered drugs by gavage for 3 month,Using OFT,NOR,T/YM and MWM behavioral experimental methods to observe the memory ability of each group of mice difference.Using western blot to detect the expression of TNF-?,HIF-1?,LRP1,VEGF,IL-1?,ApoE 4 and IL-6 proteins level in hippocampus of mice.Microscopic vessels and blood vessels were observed by TEM.The morphology of endothelial cell organelles was determined by hematoxylin-eosin staining.The pathological changes of neurons in hippocampus of each group were observed after 3 months.Results:In the OFT experiment,there were no statistical differences in the total distance of spontaneous activity along all groups,nor the residence time in the central region.In the NOR behavioral experiment,the results of the recognition coefficient of the mouse on the new object indicate that,compared with the WT group,the exploration time on the new object in the 5×FAD group was significantly reduced,Compared with the KXS group and MKXS group,the exploration time on the new object increased.T/YM results showed that,the residence time of the 5×FAD group in the novel arm was significantly shortened compared with the WT group.The exploration time of the novel arm was increased in the 5×FAD+KXS group and 5xFAD+MKXS group.MWM training stage:compared with the 5xFAD group,the incubation period of escape in each group was shortened.Test phase:compared with the WT group,the number of crossing the platform and the time spent in the quadrant of the target platform was significantly reduced in the 5×FAD group.DP group and MKXS group were significantly increased.Western Blot results show that,compared with WT group,VEGF and HIF-la expression level in 5×FAD group was extraordinary increased.Compared with 5×FAD group,the expression level in each group decreased to different degrees.The expressions of KXS group and MKXS group were significantly reduced compared with 5×FAD group.Compared with the WT group,the hippocampal LRP1 level in the 5×FAD group was significantly decreased;Compared with WT group,the level of IL-1?TNF-??IL,6 in hippocampus of 5×FAD group was significantly increased,in hippocampus of 5×FAD group was significantly increased,and both KXS group and MKXS group could significantly reduce the level of protein.HE staining in mice showed that the granule cells in the hippocampus of the normal group were closely arranged and intact.In the 5×FAD group,the loss of granulosa cells in the hippocampus decreased.The morphological results of granulosa cells were similar between the two groups.The granulosa cells were closely arranged,and the structure was clear and complete.The vascular endothelial cells in the WT group were observed by TEM with close connections intact,uniform basal membrane density,smooth and clear contour,complete intracellular organelle morphology of vascular ECs,no swelling of the endoplasmic reticulum,dense granules,numerous mitochondrial cristae,and clear cristae membrane.In the 5×FAD group,the basal membrane thickened,the thickness was uneven,the endothelial nucleus contour was not smooth,the peritubular cells were swollen and vacuolated,the intracellular organelle morphology of VECs was incomplete,the endoplasmic reticulum was swollen,the texture was not clear,the granulation was removed,the mitochondrial structure was unclear,the contents were dissolved,the number of mitochondrial crista was significantly reduced,and the crista membrane was swollen.In the DP group and KXS group,the basal membrane was slightly thicker,with uneven thickness.There was swelling in the peritubular cells of the basal membrane,rough endoplasmic reticulum swelling,degranulation,increasing the number of granular ridges,and the swelling was alleviated.In the MKXS group,vascular endothelial cells were closely connected and intact,with uniform basal membrane density,smooth and clear contour,no swelling of perithelial cells,abundant andcomplete organelles,clear texture structure of endoplasmic reticulum,no degranulation,significantly increased the number of mitochondrial cristae,obvious stratification,clear crista membrane,and disappeared swelling.Conclusion:MKXS can enhance the non-spatial episodic memory ability of 5xFAD mice,improve the spatial alternation memory disorder,and have a significant effect on the spatial learning and memory ability of the mice.MKXS can significantly reduce the expression of inflammatory cytokines IL-1,TNF-?,IL-6,HIF-1? and VEGF in 5×FAD mice,and also significantly up-regulate the expression of LRP1 protein,but cannot affect the expression of ApoE 4 protein,suggesting that MKXS may improve the cerebral small blood vessel perfusion disorder,angiogenesis abnormalities,blood-brain barrier injury and intracerebral inflammatory response in 5×FAD mice.The pathomorphological results suggested that MKXS could improve the hippocampal neuron loss in 5×FAD mice at the age of 7-8 months,repair the pathological morphology of small cerebral vessels,and reduce the damage of organelles inside VECs.