| ObjectiveTo expLore the pathogenesis of PI3K/AKT/GSK3βsignaL transduction pathway in AD with kidney essence deficiency syndrome,phLegm turbidity obstructing orifice syndrome and bLood stasis obstructing coLLateraLs syndrome.Methods1.Construction of animal models（1）Reproduction of AD disease model:LocaLized injection of Aβ1-42-42 toxic fragment 5mul into Lateral ventricle of rats on the 34th day of the experiment.（2）AD kidney essence deficiency syndrome model replication:rats were intraperitoneally injected with D-gal,50 mg/kg daiLy for 48 days;at the same time,the toxic fragment of Aβ1-42-42 was injected into the lateraL ventricle of rats on the 34th day of the experiment.（3）AD phlegm turbidity obstruction orifice syndrome model duplication:rats were intraperitoneally injected with D-gal 50 mg/kg daily for 48 days,and fed with high-fat diet;on the 34th day of the experiment,rats were injected with Aβ1-42-42 toxic fragment 5 mL Locally in the lateral ventricle.（4）Combination of disease and syndrome in the bLood stasis group:rats were intraperitoneaLLy injected with d-gaL for 48 days at a dose of 50mg/kg per day,and were frozen in the refrigerator from the fourth week.Toxic fragments of Aβ1-42-42 were injected into the lateral ventricles of the rats one week before the behavioraL test.2.Verification of the model of disease-syndrome combination.Morris water maze and hippocampal tissue Nissl staining were used to validate AD disease model from behavioral and histomorphological aspects;to detect the changes of antioxidant capacity reLated indicators of rats in each group to validate the syndrome model of kidney essence deficiency;to detect the changes of blood lipid level to validate the syndrome model of phLegm turbidity obstruction;to detect the changes of hemorheology to validate the syndrome model of blood stasis obstruction collaterals.3.Western blot（WB）and immunohistochemistry were used to detect the difference of PI3K/AKT/GSK3beta signaling pathway expression in the three typical syndromes of AD.Results1.Model validation（1）AD disease model:Morris water maze behavior showed that the learning and memory ability of rats in both the sham operation group decreased.The degree of apoptosis of neurons in the hippocampus was also different,indicating that the ability to Learn and remember was impaired after injection of A into the lateral ventricle.（2）Deficiency of kidney essence:the antioxidant capacity of cortex and hippocampus of rats in the kidney deficiency group,phlegm turbidity group,blood stasis group was decreased,and lipid peroxidation products was increased,which proved that the syndrome model had been successfully constructed after the injection of d-gal.（3）Syndrome of phlegm turbidity obstruction:the content of TC,TG in rats in this group increased,indicating that the syndrome model of phlegm turbidity obstruction obstruction has been successfully constructed.（4）AD model of blood stasis and collateraL obstruction syndrome:Compared with sham operation group,the plasma viscosity,hematocrit and fibrinogen content in the model group increased（p<0.05）,and the whole blood low shear reduction viscosity decreased（p<0.01）.2.Validation of disease models:Morris water maze test showed that the learning and memory abilities of AD group and different syndromes groups were decreased,and neurons in hippocampus were apoptotic to varying degrees.3.WB test results:（1）The phosphorylation LeveLs of tau protein phosphorylation sites Thr181 and Thr231 increased in AD disease model,AD kidney essence deficiency model,AD phlegm turbidity obstruction syndrome model and AD blood stasis obstruction syndrome model（p<0.05）,and the increase of AD phlegm turbidity obstruction syndrome model was the most significant.（2）AD kidney essence deficiency syndrome model:Compared with sham operation group,AD kidney essence deficiency syndrome model,AD phlegm turbidity obstruction syndrome model,AD blood stasis obstruction syndrome model rat cortex,hippocampus antioxidant capacity decreased（p<0.05）,malondialdehyde content increased（p<0.05）.（3）The expression of p-AKT in AD disease model,AD kidney essence deficiency syndrome model,AD phlegm turbidity obstruction orifice syndrome model and AD blood stasis obstruction collateral syndrome model decreased（p<0.05）,and AD phlegm turbidity obstruction orifice syndrome model increased most significantly.（4）The expression of PI3K in AD disease model,AD kidney essence deficiency syndrome model,AD phlegm turbidity obstruction orifice syndrome modeL and AD blood stasis obstruction collateraL syndrome model decreased（p<0.05）,and AD phlegm turbidity obstruction orifice syndrome model increased most significantly.Conclusion1.The three typical syndromes of AD have common pathogenesis in PI3K/AKT/GSK3beta signaling pathway,that is,up-regulation of PI3K/AKT may inhibit it,and activation of GSK3βmay promote it.2.PI3K/AKT/GSK3βsignaling pathway regulates the three typical syndromes differently.That is,up-reguLation of PI3K/AKT has the most significant inhibitory effect on AD model of phlegm turbidity obstructing orifices,followed by AD model of kidney essence deficiency,and AD model of blood stasis obstructing collaterals is the smallest.Activating GSK3beta has the most significant effect on promoting AD model of phlegm turbidity obstructing orifices,followed by AD model of kidney essence deficiency,and AD model of blood stasis obstructing collaterals is the smallest. |
