Effect And Mechanism Of Shenma Yizhi Decoction On White-matter In Rats With Vascular Dementia

Vascular dementia(VaD)is the second most common non-degenerative dementia after Alzheimer’s disease(AD).Insufficient diagnosis of VaD,lack of effective treatment options,prolonged life expectancy and VaD risk factors for hypertension,coronary heart disease,diabetes,metabolic syndrome and stroke have led to an increase in the number of patients with VaD.VaD is a type of dementia that can be prevented and treated.It is characterized by reversible and good prognosis in the early intervention.Therefore,it is urgent to develop effective diagnostic methods and drugs.Studies have shown that white matter damage caused by cerebral hypoxia is an important pathological change of VaD.Diffuse changes of white matter with loss of myelin and axons are common features of all VaD subtypes.White matter damage is closely related to the decline of cognitive function of VaD.However,its neurobiological mechanism is still unclear.Shenma Yizhi(SMYZ)decoction is the Decoction of Professor Zhou Wenquan,a famous Chinese medicine practitioner.It is commonly used in the treatment of qi deficiency and blood stasis type VaD and has shown a promising results in VaD patients.Previous studies have shown that SMYZ could inhibit calcium influx and upregulate the level of monoamine neurotransmitters,this current study investigated the effect and mechanism of Shenma Yizhi Decoction on spatial learning,and white matter damage in VaD rats.Here,we also discussed the relationship between white matter damage,BBB destruction and oxidative stress,in hope that it will be helpful to explore effective strategies against VaD in the future.Objective:To observe the effect and mechanism of Shenma Yizhi Decoction on white matter injury in VaD rats,and to explore its correlation with blood-brain barrier permeability and oxidative stress.Methods:A rat model of multiple cerebral infarction dementia was established by sodium alginate microsphere embolization.A total of 56 male Wistar rats were randomly divided into blank group,sham operation group,model group,donepezil group,and SMYZ low、medium and high dose groups.At the end of the modeling,the donepezil group was given with 0.5 mg/kg of the drug,and the low,medium and high dose groups of SMYZ group were given with 3.3 g/kg,6.6 g/kg and 16.5 g/kg of drugs respectively.The model group was given an equal amount of distilled water.2.After 4 weeks of continuous administration,Morris water maze was used to observe the escape latency,the crossing times,the original platform activity time and the distance to evaluate the spatial learning and memory ability of the rats.Hematoxylin-eosin staining(HE)and Nissl staining(Nissl)were used to evaluate the morphological changes of neurons in the hippocampal CA1 region of rats.3.Luxol fast blue(LFB)staining and Myelin basic protein(MBP)detected by Western blotting(WB)were used to assess changes in myelin loss.The protein expression of ZO-1,Occludin and CX43 in hippocampus was detected by WB to evaluate the function of BBB.The activity of CAT,GSH,SOD and the content of MDA in hippocampus were detected by colorimetry to evaluate the level of oxidative stress.Results:Effect of SMYZ Decoction on spatial learning and memory ability and pathological morphology of hippocampus in rats1.1 Effect of SMYZ Decoction on spatial learning and exploration ability in ratsIn the orientation navigation experiment of the Morris water maze,during the same day,compared with the Sham group,the escape latency of the Model group on the second day was longer(P<0.05).Compared with the Model group,donepezil group was significantly shortened on the second day(P<0.05),and the SMYZ-M and SMYZ-H groups were significantly shortened on the third day(P<0.O5).For the same group,compared with the first day,the escape latency of all groups of rats were shortened,indicating that all rats have certain learning and memory ability.The escape latency of the Control group and the Model group was significantly shortened on the second day(P<0.05),the donepezil group and the SMYZ-L,SMYZ-M,and SMYZ-H groups were significantly shortened on the third day(P<0.05).In space probe test,compared with the Control group,the crossing times,swimming distance and swimming time of the Sham group showed no significant difference(P>0.05).Compared with the Sham group,the crossing times and swimming distance in the target quadrant of the Model group decreased(P<0.05).Compared with the model group,the crossing times of the SMYZ-L and SMYZ-H groups increased significantly(P<0.05),the swimming distance in the first quadrant of the Donepezil and SMYZ-H groups showed significant difference in each group(P<0.05).1.2 The pathological morphology of rat brain tissueIn the control group,neurons in the Control and Sham group were well-aligned,with regular cell morphology,nuclei and cytoplasm were clear,and rich in Nissl bodies exhibited dark blue particles or plaques.In the model group,neurons were scattered and severe degenerated,with irregular cell morphology,nuclei and cytoplasm were not clear.The Nissl body disintegrates or loses,the boundaries were blurred and the color becomes lighter.After donepezil and SMYZ treatment,degenerative neurons were significantly reduced,the neurons in CA1 region showed different degrees of improvement neatly arranged with more cell layers and more Nissl bodies were observed in the cytoplasm with cytoplasm stained dark blue.2.Effects of SMYZ Decoction on white matter injury2.1 Myelin injury in the corpus callosumIn the control group,the myelin sheath in the corpus callosum exhibited dark blue with the myelin fibers were densely arranged.In the model group,the myelin sheath exhibited light blue with the structure of was loose and the vacuoles were observed.After Donepezil and SMYZ treatment,the staining of myelin sheath became darker and the structure began to become tighter.There was no significant difference in MBP expression between Sham group and control group(P＞0.05).Compared with the Sham group,the expression of MBP in hippocampus of model group was significantly decreased(P<0.05).Compared with the Model group,the MBP of the Donepezil group and the SMYZ-H group increased significantly(P<0.05),and the SMYZ-L and SMYZ-M groups had an improvement effect,but there was no significant difference(P>0.05).2.2 The protein expression of Occludin,ZO-1 and CX43Compared with the control group,the expression of Occludin and ZO-1 in the hippocampus of the Model group was significantly decreased,and the difference between the Model group and the Sham group was statistically significant(P<0.05).Donepezil and SMYZ Decoction intervention increased the expression of Occludin and ZO-1,and the difference between Donepezil group,SMYZ-H group and Model group was statistically significant(P<0.05).Compared with the Control group,the expression of CX43 in the Model group was significantly increased,the difference was statistically significant(P<0.05).The intervention of SMYZ-H and SMYZ-M group could significantly reduce the expression of CX43 compared with Model group(P<0.05).Donepezil treatment also reduced the expression of CX43 to a certain extent,but the difference is not statistically significant(P＞0.05).2.3 The level of oxidative stressThe colorimetric results showed that no significant difference in the activity of CAT,GSH and SOD,and MDA content were found in Sham group compared with Control group(P>0.05),Compared with the control group and the Sham group,the activity of CAT,GSH and SOD in the Model group decreased significantly(P<0.05),and the MDA content increased significantly(P<0.05).The difference between the groups was statistically significant(P<0.05).Compared with the Model group,the CAT activity in the Donepezil group and the SMYZ-H group was significantly increased,and the difference between groups was statistically significant(P<0.05).The activities of GSH and SOD in the SMYZ-M and SMYZ-H groups significantly increased compared with model group(P<0.05).The activity of CAT,GSH and SOD in SMYZ-L group was higher than that in Model group,but the difference was not statistically significant(P>0.05).Compared with the Model group,the MDA content in the Donepezil group,SMYZ-M,and SMYZ-H groups was significantly lower,and the difference between the groups was statistically significant(P<0.05).The MDA content in the SMYZ-L group was lower than that in the Model group,but the difference was not statistically significant(P>0.05).Conclusion:In all,our results show that SMYZ decoction improves the spatial learning and memory of VaD rats,alleviates morphological alternations in hippocampus,mitigates white matter damage by increasing the expression of MBP,protecting myelin sheath and reducing white matter injury.Mechanisms might be explained by(1)increasing the expressions of tight and gap junction proteins,thus enhancing BBB integrity,preventing harmful substances in plasma from leaking into the brain.Consequently,these effects restore the inner environment for the survival of neuronal cells t and retard white matter damage.(2)enhancing the clearance of free radicals and reactive oxygen species,which ameliorates neurovascular unit dysfunction,reducing the ROS-mediated neuronal apoptosis,thereby protects against cerebral multi-infarction induced white matter damage.The present study demonstrates that SMYZ might be a promising drug for the treatment of VaD,which may also provide new insight into this disease.