| OBJECTIVE: Vascular Dementia(VD)is a major and difficult disease that seriously affects the health of the elderly,and it is also the only dementia that can be prevented and reversible at present.Traditional Chinese medicine holds that "treating dementia without phlegm is not its cure",and modern famous Chinese medicine experts sum up their predecessors’ experience and propose that the treatment of "phlegm" can be used as one of the major therapies for vascular dementia.Based on our previous experience,we speculate that the pathogenesis of vascular dementia caused by inflammation and toxicity of excitatory a mino acids may be closely related to the pathogenesis of phlegm obstruction in traditional Chinese medicine,reducing the production of inflammatory factors and excitatory a mino acids,regulating the inflammatory response mediated by NF-κB and excitatory toxicity mediated by NMDA receptor may be the focus of Ditan Decoction in the treatment of vascular dementia.One of the important mechanisms.In this study,SD rats were used to establish VD animal model by modified 2-VO method combined with high-fat diet.The changes of rat behavior,hippocampal histopathology,blood lipid and homocysteine concentration,and the molecular biological mechanism of memory-related inflammatory factors and excitotoxicity-related receptors were observed to explore the therapeutic mechanism of Ditan Decoction on vascular dementia.METHODS: SPF male SD rats were randomly divided into six groups: blank group,model group,high-dose group of Ditan Decoction(hereinafter referred to as "Di Gao group"),middle-dose group of Ditan Decoction(hereinafter referred to as "Di Zhong group"),low-dose group of Ditan Decoction(hereinafter referred to as "Di Low group")and Western medicine group,with 12 rats in each group.Rats in the blank group were fed with common diet,while rats in the other five groups were fed with high-fat diet.After 8 weeks of feeding,the rat model of vascular dementia was established by modified 2-vessel occlusion method according to the literature.Digao group,Dizhong group,Dixiao group and Western medicine group were given orally immediately after successful modeling,and the dosage was deter mined according to the ratio of human to rat body surface area coefficient.The dosage of Ditan Decoction in high,middle and low dosage groups was 20 g kg-1,10 g kg-1 and 5 g kg-1 body weight respectively.The dosage of Ditan Decoction in western medicine group was 2 mg kg-1 body weight of Memantine Hydrochloride.The blank group and model group were given equal volume saline by gavage according to 10 ml kg-1.Once a day for 4 weeks.Morris water maze test was used to detect the learning and memory ability of rats;light microscopy was used to observe the morphological changes of hippocampus;ELISA method was used to detect serum lipid(including TC,TG,HDL,LDL)and Hcy concentration;Real-time PCR was used to detect the expression of inflammatory factors nf-κB,MMP-9 and TNF-alpha in hippocampal CA1 region of rats;enzyme-linked immunosorbent assay was used to detect the changes of serum inflammatory factors nf-κB,MMP-9 and tnf-alpha;enzyme-linked immunosorbent assay was used to detect the changes of hippocampal death factors p53,Bax and bcl-2.The changes of Glu concentration in hippocampal CA1 region of rats were detected by chemical colorimetry,and the expression and distribution of NMDAR subunits NR1,NR2 A and NR2 B in hippocampal CA1 region of rats were detected by Realtime PCR and immunohistochemistry.RESULTS:1.The results of Morris water maze test in rats:(1)Location navigation experiment: Compared with the blank group,the escape latency of the model group was significantly prolonged on the 5th day of training(P < 0.01);compared with the model group,the escape latency of the DaiGao group and the DaiZhong group was significantly shortened(P < 0.01),and the escape latency of the DaiGao group and the DaiZhong group was also shortened(P < 0.05).(2)Space exploration experiment: Compared with the blank group,the number of platform crossing in the model group was significantly reduced(P < 0.01),and the target quadrant time was significantly shortened(P < 0.01);compared with the model group,the number of platform crossing in the Ditan Decoction group and the western medicine group was significantly increased(P < 0.01),and the target quadrant time was significantly prolonged(P < 0.01).2.Histological changes of hippocampal CA1 area were observed under light microscope.HE staining results showed that the morphological structure of vertebral cells in hippocampal CA1 area of model group was incomplete and disordered,cell body was reduced,cytoplasm was turbid,nucleolus was fixed,and the number of normal cells was significantly reduced.Compared with model group,the morphological changes of vertebral cells in each group of Ditan Decoction and Western medicine group were regular,orderly and normal.The number of cells increased,the structure of cells was intact,the nuclei and nucleoli were clearly visible,the cytoplasm was abundant,and the phenomena of nuclear pyknosis and degeneration were improved.3.The results of blood lipid test in rats: Compared with the blank group,the TG,TC and LDL in the model group increased significantly(P < 0.01),while HDL decreased significantly(P < 0.01).Compared with the model group,the TG,TC and LDL in Dazhong group decreased significantly(P < 0.01),and HDL increased significantly(P < 0.01);the TG,TC and LDL in Dazhong group decreased significantly(P < 0.01),while HDL increased(P < 0.05);the TG,TC and LDL in Dazhong group decreased significantly(P < 0.05),and LDL decreased significantly(P < 0.01),HDL increased slightly(P < 0.05).Significant change(P > 0.05).4.Detection results of rat Hcy concentration: The serum Hcy concentration in model group was significantly higher than that in blank group(P < 0.01);the serum Hcy concentration in Ditan Decoction group was significantly lower than that in model group(P < 0.01).5.Detection of inflammatory and apoptotic factors in rats: RT-PCR showed that the expression of NF-κB,MMP-9 and TNF-alpha in hippocampal CA1 region of rats in model group was significantly higher than that in blank group(P < 0.01);compared with model group,the expression of NF-κB,MMP-9 and TNF-alpha in Ditan Decoction group and Western medicine group was significantly lower(P < 0.05);the expression of NF-κB,MMP-9,TNF-alpha in Ditan Decoction group was significantly lower(P < 0.05);and the expression of NF-κB,MMP-9,TNF-alpha in Di The expression of TNF-alpha was lower than that of Western medicine group(P < 0.05).The results of ELISA showed that the concentration of NF-κB,MMP-9 and TNF-alpha in serum of model group was significantly higher than that of blank group(P < 0.01);compared with model group,the concentration of NF-κB,MMP-9 and TNF-alpha in each group of Ditan decoction was significantly lower(P < 0.01);compared with western medicine group,the concentration of NF-κB in Digao group and Dizhong group was significantly lower(P < 0.01),and the concentration of MMP-9 and TNF-alpha in Digao group,Dizhong group was also somewhat lower(P < 0.01).It decreased(P < 0.05).In hippocampus of rats,the concentration of P53 and Bax in model group was higher than that in blank group(P < 0.05),and the expression of Bcl-2 was lower than that in blank group(P < 0.05).Compared with model group,the concentration of P53 and Bax in Ditan Decoction group and Western medicine group were significantly lower(P < 0.05),and the content of Bcl-2 was higher(P < 0.05);compared with western medicine group,the concentration of P53 and Bax in Ditan Decoction group and Dizhong group were lower(P < 0.05).The content of Bcl-2 in each group of Ditan Decoction increased(P < 0.05).6.The results of Glu concentration and NMDA receptor subunit expression and distribution in hippocampal CA1 region of rats:(1)The gene level of NMDAR receptor was detected by RT-PCR.The results showed that the expression of NMDAR subunits NR1,NR2 A and NR2 B in the model group was significantly higher than that in the blank group(P < 0.01);compared with the model group,the expression of NMDAR subunits NR1,NR2 A and NR2 B in the Ditan Decoction group and the western medicine group was significantly lower(P < 0.05).The expression of NR2 B in Digao group was lower than that in western medicine group(P < 0.05).(2)The positive expression and distribution of NR1,NR2 A and NR2 B were detected by immunohistochemical method.The results showed that the positive expression of NR1,NR2 A and NR2 B in CA1 area of hippocampus of rats in each group was significantly higher than that in blank group,Ditan Decoction group and Western medicine group,and the positive cells stained deeper and showed dark brown.The expression of NR1,NR2 A and NR2 B positive cells in Ditan Decoction group and Western medicine group was less than that in model group.In group B,the staining was light brown and yellow,and the expression of NR1 in group D was similar to that in group B.(3)Glu concentration test results: Glu concentration in model group was significantly higher than that in blank group(P < 0.01);Glu concentration in Ditan Decoction group and Western medicine group was lower than that in model group(P < 0.05);Glu concentration in Ditan Decoction group was also significantly lower than that in western medicine group(P < 0.01).Conclusion:1.The learning and memory abilities and spatial exploration abilities of model rats were significantly reduced,and the pathological changes of hippocampus were typical.It was a successful animal model of vascular dementia.2.The metabolism of lipid and homocysteine is closely related to cognitive ability.Regulating the level of blood lipid and homocysteine and reducing the risk of vascular dementia may be the protective mechanism of Ditan Decoction on learning and memory ability of VD rats.3.Increased expression of inflammatory factors and their activation of inflammatory chain reaction may be one of the pathogenesis of vascular dementia.Ditan Decoction can reduce the expression of NF-κB,MMP-9,TNF-alpha,P53 and Bax by inhibiting the signal pathway of NF-κB/MMP-9,and promote the expression of Bcl-2,so as to protect the integrity of blood-brain barrier,alleviate the damage of inflammatory response to the central nervous system,reduce apoptosis and improve the cognitive function of the body.4.Excitatory toxicity mediated by NMDA receptor may be one of the pathogenesis of vascular dementia.Decreasing the expression of NMDA receptor and Glu in hippocampus of VD rats and reducing the toxicity of excitatory a mino acids may be one of the important mechanisms of Ditan Decoction in protecting hippocampal neurons and improving cognitive dysfunction in VD rats. |
