| Research BackgroundDementia is a multifaceted clinical cognitive disorder that causes severe economie,social,and public health burdens.The pathogenesis of dementia is still unclear.Risk factors such as type 2 diabetes,smoking,and obesity increase the risk of dementia,and dementia caused by diabetes is receiving increasing attention.70%of patients with type 2 diabetes develop dementia,especially for women with vasculcir dementia.However,thc re re few studies on the effects of diabetes on vascular dementia in women and its mechanisms.With the advent of an aging society,it is of great praictieal signifieance to explore the mechanism of action of diabetes on vascular dementia in women and tc)develop drugs with preventive or therapeutic functions.Since the pathogenesis of dementia is unknown,there is currently no effective curative drug.Traditional Chinese medieine has rich theoretical and practical experience and potential advantages in the prevention and treatment of geriatric diseases.Angelica sinensis is scattered from Zhang Zhongjing’ s "Golden Dragonfly",which has the effect of nourishing blood and regulating the liver,strengthening the spleen and dampness.Mainly used in the treatment of gynecological pain syndrome.In the late 1980s,Japanese scholar Mizushima Shuijima first reported the clinical observation of Danggui Shaoyao San in improving dementia,and has received extensive attention from scholars at home and abroad.The pathogenesis of dementia in Chinese medicine is based on the virtual standard.The main deficiency is the loss of kidney essence and the lack of blood.The main symptoms are phlegm obstruction and qi stagnation and blood stasis.Danggui Shaoyao San can activate blood and nouri sh blood,spleen and dampness,and has the characteristics of supplementing the virtual and sputum.It not only has the functions of regulating blood lipids,anti-oxidation,scavenging free radicals,protecting vascular endothelium,but also increases the release of acetylcholine in the brain,reduces the deposition of Aβ in the brain,and reduces the damage and loss of neurons.ObjectiveThis study aims to study the pharmacological mechanism of Danggui Shaoyao San in improving the cognitive impairment of female diabetic mice in the pathogenesis of Western medicine and the pathogenesis of Chinese medicine.It provides scientific research for the development of new Chinese medicines that can delay the progression of’ diabetic dementia and improve the quality of life of patients.in accordance with.MethodsStudy on Learning,Memory and Estrogen Related Mechanism of Danggui Shaoyao San in STZ-Induced Diabetes MiceSeventy-five SPF-class C57/BL6 female mice were on average 10 weeks old.After intraperitoneal injection of streptozotocin 80 mg/kg for three consecutive days,the blood glucose level of the mice was measured using a blood glucose meter.A mouse with a blood glucose level of more than 16.7 mM in a non-fasted state is defined as a diabetic mouse.Then,according to blood sugar and body weight,they were randomly divided into three groups:the middle and lower groups of Danggui Shaoyao San and the model group,with 8 in each group.The dose of Danggui Shaoyao San in the low dose group was 1.6 g/kg·d,and the dose in the middle dose of Danggui Shaoyao San was 3.2 g/kg·d.The dose in the high dose group of Danggui Shaoyao San was 6.4 g/kg·d.The model group and the negative control group were intragastrically administrated with the same amount of distilled water.The volume of the drug solution per administration of all the experimental animals was calculated at 0.1 ml/10 g,and was administered once a day.Gastric administration continued during the animal behavior experiment.All animals were kept in the SPF laboratory of the Sanyuanli Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine.They were kept in a single cage and were free to eat.A 6-day Morris water maze test was performed 6 weeks after administration.After the water maze test was completed,the blood samples of the mice were collected by eyeball extraction,and the serum was collected after centrifugation.At the same time,the bilateral hippocampus and bilateral cortex were taken out as specimens;Western blot was used to detect the hippocampus of each group.Expression levels of O-GlcNAc glycosylation,O-linked N-acetylglucosaminyltransferase(OGT)and O-linked N-acetylglucosaminidase(OGA)in cortical tissue.The estrogen receptors ERa and ER β levels were then examined.Immunofluorescence assay was used to detect the expression of estrogen-related protein ER a and ER β protein in the brain.HE staining and β-galactosidase staining were used to detect cell senescence and pathological changes in brain tissue sections.Study on Learning,Memory and Estrogen Related Mechanism of Danggui San in Self-style Diabetic Model db MiceThirty-one SPF female db/db mice,including 23 female models of spontaneous diabetes db/db,and 8 female wild-type db/m mice of the same age.Average 12 weeks of age.Female db/db mice were divided into several experimental groups according to a simple randomization method.There were 8 blanks,9 models,8 high doses and 6 low doses.All animals were raised in the animal room of the Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine in SPF class.They were kept in single cage and fed freely.The administration was started until 10 months of age,and the administration time lasted for 12 weeks.The dose of Danggui Shaoyao San in the low dose group was 3.2 g/kg·d,and the dose in the high dose group of Danggui Shaoyao San was 6.4 g/kg·d.The model group and the negative control group were intragastrically administrated with the same amount of distilled water.The volume of thedrug solution per administration of all the experimental animals was calculated at 0.1 ml/10 g,and was administered once a day.Oral administration continued during the animal behavior experiment,and a 6-day Morris water maze test was performed 8 weeks after administration.After the water maze test was completed,the blood samples of the mice were collected by eyeball extraction,and the serum was collected after centrifugation.At the same time,the bilateral hippocampus and bilateral cortex were taken out as specimens;Western blot was used to detect the hippocampus of each group.Expression levels of O-GlcNAc glycosylation,0-linked N-acetylglucosaminyltransferase(OGT)and O-linked N-acetylglucosaminidase(OGA)in cortical tissue.The estrogen receptors ERa and ER β levels were then examined.Immunofluorescence assay was used to detect the expression of estrogen-related protein ER a and ER β protein in the brain.HE staining and β-galactosidase staining were used to detect cell senescence and pathological changes in brain tissue sections.Resu tsStudy on Learning,Memory and O-GlcNAc Related Mechanism of Danggui Shaoyao San in STZ-Induced Diabetes Mice1.The effects on the learning and memory ability of mice include two experiments:Morris water maze and new object recognition test.(1)Morris water maze behavioral resultsPositioning navigation test resultsThe swimming time and swimming distance of mice were shortened with the prolongation of time,showing a downward trend.It is proved that the process of finding the platform is a process of learning and forming memory.Compared with the normal control group,the swimming time and swimming distance of the STZ-induced diabetic model mouse model group were significantly prolonged(P<0.01),suggesting successful modeling;compared with the model group,the Danggui Shaoyao San medium and high dose group can be shortened.The mice were positioned for swimming time and swimming distance(P<0.05,P<0.01).Danggui Shaoyao San can improve the learning ability of STZ-induced diabetic model mice.Space exploration test resultsCompared with the normal group,the STZ-induced diabetes model mice had a significantly reduced quadrant dwell time in the space exploration original platform(P<0.01),and the number of crossing platforms was significantly reduced(P<0.05).Compared with the model group,the Danggui Shaoyao San medium dose group and the residence time of the quadrant in the high-dose group space exploration platform was significantly prolonged(P<0.01,P<0.05).The number of crossing platforms increased significantly(P<0.05).The above results suggest that Danggui Shaoyao San can significantly improve the learning and memory ability of STZ-induced diabetic model mice.(2)New object recognition experiment resultsIn the object recognition test,the discrimination index of new objects in STZ-induced diabetic mice(0.24±0.21)was significantly lower than that in normal mice(0.71±0.08)(P<0.01).There was also a significant difference between the dose group resolution index(0.67±0.09)and the middle dose group(0.6±0.08)and the model group(P<0.05,P<0.01).2.Effects on the oxidative stress pathway:Compared with the normal group,the SOD activity of the STZ-induced diabetic model mice was significantly decreased(p<0.05).Compared with the model group,the SOD activity of the Danggui Shaoyao San was increased(P<0.05).Compared with the normal group,the MDA content of STZ-induced diabetic model mice increased(P<0.05).Compared with the model group,the content of MDA in the middle and high dose groups of Danggui Shaoyao was decreased(P<0.05).3.Effects on cell aging:Cell senescence was detected by β-galactosidase staining.Compared with the normal group,the senescent cells of STZ-induced diabetic model mice showed dark blue color.Compared with the model group,the Danggui Shaoyao San medium and high dose group compared with the model group aging cells.There has been a noticeable improvement.4.Effects on estrogen receptor glycosylation:The results of Westernblot indicated that the expression of ER a and ER βprotein in the brain of STZ-induced diabetic mice was significantly lower than that in the normal group(P<0.01).Compared with the model group,the dose of Danggui Shaoyao San was high and high.The group can improve the expression of ERα and ER β protein in the brain of model mice(P<0.05).Compared with the normal group,the expression of OGT protein in the brain of STZ-induced diabetic mice was significantly increased,the expression of OGA protein was decreased(P<0.05),and the expression of O-GlcNAc protein was increased(P<0.05).Compared with the group,the Danggui Shaoyao San medium dose and high dose group can down-regulate the expression of OGT protein in the brain of model mice,up-regulate the expression of OGA protein(P<0.05),and decrease the expression of O-GlcNAc protein(P<0.05).The results of immunofluorescence staining indicated that compared with the normal group,the ER a and positive staining particles in the brain of STZ-induced diabetic mice were significantly reduced.Different concentrations of Danggui Shaoyao San group could improve the expression of ER a in the brain of model mice.Study on Learning,Memory and O-GlcNAc Related Mechanism of’ Danggui San in Self-style Diabetic Model db Mice1.The effects on the learning and memory ability of’ mice include two experiments:Morris water maze and new object recognition test.(1)Morris water maze behavioral resultsPositioning navigation test resultsThe swimming time and swimming distance of mice were shortened with the prolongation of time,showing a downward trend.It is proved that the process of finding the platform is a process of learning and forming memory.Compared with the normal control group db/m mice,the swimming time and swimming distance of the self-diabetic model db/db mice group were significantly prolonged(P<0.01),suggesting successful modeling;compared with the model group db/db mice The Danggui Shaoyao San medium and high-dose groups can shorten the navigation time and swimming distance of db/db mice(P<0.05,P<0.01).Danggui Shaoyao San can improve the learning ability of db/db mice with self-diabetic diabetes model.space exploration test resultsCompared with the normal group of db/m mice,the spatial retention time of the original platform of the self-diabetic model db/db mice was significantly reduced(P<0.01),and the number of crossing platforms was significantly reduced(P<0.05);Compared with the db mice,the dwell time of the original platform quadrants of the Danggui Shaoyao San middle dose group and the high dose group were significantly prolonged(P<0.01,P<0.05).The number of crossing platforms increased significantly(P<0.05).The above results suggest that Danggui Shaoyao San can significantly improve the learning and memory ability of the self-diabetic diabetes model db/db mice.(2)New object recognition experiment resultsIn the object recognition test,the db/db mouse group resolution index(0.28±0.23)was significantly lower than that of the normal group db/m mice(0.61±0.07)(P<0.05).There was also a significant difference between the index(0.65±0.12)and the model group(P<0.05).2.Effects on the oxidative stress pathway:Compared with the normal group db/m,the SOD activity of the self-diabetic model db/db mice was significantly decreased(P<0.05).Compared with the model group,the activity of SOD in the middle and high doses of Danggui Shaoyao San was increased(P<0.05).Compared with the normal group,MDA content in db/db mice increased(P<0.05);compared with db/db mice,Danggui Shaoyao San middle and high dose groups improved MDA content(P<0.05).3.HE staining results:The brain tissue of mice was detected by HE staining.Compared with the normal group,the brain tissue morphology of the self-diabetic model db/db mice showed obvious pathological changes,and the apoptosis increased significantly.Compared with the model group,In the high-dose group of Danggui Shaoyao San,the brain tissue morphology of the mice in the model group was significantly improved.4.Effects on estrogen receptor glycosylation:Western blot results showed that compared with the normal group db/m mice,the expression of ERa protein in the brain of the self-diabetic model db/db mice was significantly decreased(P<0.01);compared with the model group,the dose of Angelica sinensis and high dose group can improve the expression of ER a and ERβ protein in the brain of model mice(P<0.05).Compared with the normal group,the expression of OGT protein in the brain of the self-diabetic model db/db mice was significantly increased,the expression of OGA Protein was decreased(P<.05),and the expression of O-GlcNAc protein was increased(P<0.05).Compared with the model group,the Danggui Shaoyao San medium dose and high dose group can down-regulate the expression of OGT protein in the brain of model mice,up-regulate the expression of OGA protein(P<0.05),and decrease the expression of O-GlcNAc protein(P<0.05).Conclusion1.STZ-induced diabetes model mice and self-diabetic diabetes model db mice showed obvious insulin resistance,and the ability of learning and memory decreased significantly.The increase of senescent cell number is an ideal animal model of dementia.2.Danggui Shaoyao San medium dose and high dose groups can improve the learning and memory ability of STZ-induced diabetic model mice and self-diabetic diabetes model db mice.Low-dose Danggui Shaoyao San failed to improve the learning and memory ability of STZ-induced diabetes model mice and db mice indicates that the effect of Danggui Shaoyao San on the nervous system is positively correlated with the dose.Experimental studies have shown that Danggui Shaoyao San has different degrees of regulation on neuronal apoptosis,estrogen levels,oxidative stress damage,and abnormal O-GlcNAc modification of ERα,which can improve the pathology and delay the onset of neurodegenerative diseases.effect.Therefore,Danggui Shaoyao San has a good development prospect in the treatment of senile dementia.It can regulate multiple signaling pathways or targets related to abnormal O-GlcNAc modification of ER a,which may bring treatment for neurodegenerative diseases.new Hope. |
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