Study Of The Special Tau Protein Hyper-Phosphorylated Sites In Different Syndrome Types Of Alzheimer's Disease

Objective:Study of the special tau hyperphosphorylated sites in different syndrome types of Alzheimer's disease,contains deficiency of kidney essence,phlegm blocking brain collaterals,and blood-stasis blocking collaterals.Methods:1.Build AD three typical mouse models combined disease and syndrome,AD disease model and sham model.(1)AD mouse model of essence deficiency of kidney:mouse in this group were dealt with D-gal(hypodermic injection)for 48 days,to build subacute aging model,simulate the kidney essence deficiency syndrome.A week prior to the behavior detection,microinjected A?25-35 into both side of CA1 section in hippocampus.In order to build up compound model of AD with kidney essence deficiency syndrome.(2)AD mouse model of phlegm blocking brain collaterals:mouse in this group were dealt with D-gal(hypodermic injection)for 48 days,to build subacute aging model,simulate the kidney essence deficiency syndrome.Meanwhile,give a high-fat diet.A week prior to the behavior detection,microinjected A?25-35 into both side of CA1 section in hippocampus,to build up compound model of AD with phlegm blocking brain collaterals.(3)AD mouse model of blood-stasis blocking collaterals:mouse in this group were dealt with D-gal(hypodermic injection)for 48 days,to build subacute aging model,simulate the kidney essence deficiency syndrome.Meanwhile,giving ice-cold water bathing for 28 days.A week prior to the behavior detection,microinjected A?25-35 into both side of CA1 section in hippocampus,to build up compound model of AD with blood-stasis blocking collaterals.(4)AD disease model:a week before the behavior detection,mouse in this group were microinjected AP25-35 into both side of CA1 section in hippocampus,to build up model of AD.(5)Sham model:mouse in this group were dealt with 0.9%saline(hypodermic injection)for 48 days,and a week before behavior detection,they were microinjected 0.9%saline into both side of CA1 section in hippocampus,to build up model of sham.In this experiment,mouse were raised by ordinary feed except the group of phlegm blocking brain collaterals.2.Validation of disease and syndrome models.Capacity of antioxidant were tested in order to examine model of essence deficiency of kidney;the change of serum lipid level were used to examine model of phlegm blocking brain collaterals;changes of hemorheology were used to check model of blood-stasis blocking collaterals.Morris water maze(MWM)test and Nissl's stain were validated AD model through ethology and histomorphometry.3.Test of special tau hyperphosphorylated sites in different syndrome types of Alzheimer's disease.Western blot,immunohistochemistry,immunofluorescence were used to evaluate changes between AD's different syndromes.These sites contains Thr181?Thr231?Ser409?Thr212?Ser396?Ser214?Thr205?Ser199?Ser404.4.Test of protein kinases which related to tau protein hyperphosphoryaltion.Using WB detected the expression of cyclin dependence kinase 5 and glycogen synthesis kinase 3?.Results:1.Validation of syndrome model:(1)Model of essence deficiency of kidney:mouse in groups of different syndromes had a low level of antioxidant capacity,serum NOS and MDA increased,proved model of deficiency of kidney essence established.(2)Model of phlegm blocking brain collaterals:total cholesterol,triglyceride,and low density lipoprotein in serum were increased obviously in the hyperlipemia diet group,proved the model of phlegm blocking brain collaterals established.(3)Model of blood-stasis blocking collaterals:in this group,who were treated with ice-cold bath,showed highly whole blood and plasma viscosity,risen hematokrit and fibrinogen.Demonstrated the model of blood-stasis blocking collaterals established.2.Validation of disease model:Those mouse who treated with A?25-35 showed significant impairments of spatial memory and cell apoptosis in different degree,suggested the model of Alzheimer's disease established.3.Results of Western blot:(1)In the group of deficiency of kidney essence,tau sites including Thr181?Thr231?Ser409?Thr212?Ser396 were hyperphosphorylated,but there were no special sites.(2)In the group of syndrome of phlegm blocking brain collaterals,tau hyperphosphorylated in Thr 181?Thr231?Ser409 sites.(3)In the group of blood-stasis blocking collaterals,Thr212 and Ser396 were hyperphosphorylated.(4)The content of two kinases,GSK3? and CDK5,were both increased in AD group and other three syndromes groups.Conclusion:1.Continuously hypodermic injected D-gal in order to build up kidney essence deficiency model;high-fat diet to build up phlegm blocking brain collaterals mouse model;ice-cold water bathing to build up blood-stasis blocking collaterals mouse model;combined with microinjected A?25-35 into both side of CA1 section in hippocampus,to build up compound model of AD with kidney essence deficiency,phlegm blocking brain collaterals,and blood-stasis blocking collaterals.These methods accord with traditional Chinese medicine etiology and pathogenesis theory.The changes of capacity of antioxidant,level of serum lipid,hemorheology,MWM,nissle's stain could be used to validate whether models were build.2.Different sites were exist in three syndrome models of Alzheimer's disease.In the syndrome of deficiency of kidney essence,phosphorylation of Thr181?Thr231?Ser409?Thr212?Ser396 were all increased,but no specific.Thr181?Thr231?Ser409 were more related with the syndrome of phlegm blocking brain collaterals;Thr212?Ser396 were more related with the syndrome of blood-stasis blocking collaterals.3.GSK3? was increased in three syndrome models of Alzheimer's disease,indicated that this protein kinase might participate in the progress of tau hyperphosphorlation in related sites.4.These study want to prove the traditional.Chinese medicine theory about the same disease with different syndromes through check different phosphorylated sites in tau.At the same time,these results explained the Chinese medicine pathology of Alzheimer's disease also,that deficiency of kidney essence is the root,while phlegm and blood-stasis are branches.