| With the aging of society,the incidence of Alzheimer's Disease(AD)is soaring year by year,among which Sporadic Alzheimer's Disease(SAD)accounts for more than 95%,and its etiology and pathogenesis are complex.There is growing evidence that patients with SAD have abnormal insulin resistance and glucose metabolism in the central nervous system,so it is also called "type ? diabetes" without elevated peripheral blood glucose.In traditional Chinese medicine,it is believed that the heart and brain are the "organs of the gods",and the representative prescription of Shenzhiling oral liquid(SZL)has the effect of improving Qi and Yang,eliminating phlegm and calming spirits,and has certain curative effect on early and middle AD.But its mechanism remains to be explored.Myelin structural abnormalities and white matter damage exist in the whole process of AD,and they preexist the early cognitive impairment caused by amyloid amyloid protein(AP)deposition and neurofibirilary tangles(NFTs).The PI3K/Akt-mTOR signaling pathway is an important pathway that regulates myelin sheath related proteins and maintains the integrity of myelin sheath.This study observed the effect of SZL,the representative prescription of treatment from heart theory on learning and memory retention in C57BL/6J mice injected with Streptozocin(STZ)in bilateral lateral ventricles(icv-STZ)which simulated the SAD.Based on PI3K/Akt-mTOR pathway and the functional brain circuits formed by myelin sheath thickness,myelin shape integrity and myelin related proteins,discuss the reversal effect of SZL on early cognitive impairment of SAD,to provide experimental basis for early intervention in AD from the perspective of traditional Chinese medicine.Methods1.A total of 30 3-month-old male C57BL/6J wild-type mice were randomly selected to receive bilateral lateral ventricle injection of artificial cerebrospinal fluid as the control group,and another 15 were randomly selected to prepare SAD model with bilateral icv-STZ.After 1,2,3 and 4 months of modeling,the step-down test was carried out to observe the changes of the learning and memory retention ability of the mice.Immunohistochemistry and Western-blot were used to observe the expression levels of A?42,p-tau and proteins related to central glucose metabolism(IR,IRS-1,GSK-3?,p-GSK-3?,GLUT1 and GLUT3)to evaluate the pathological changes related to AD.The ultrastructure of myelin sheath was observed by transmission electron microscopy,and the expression level of myelin basic protein(MBP)was observed by immunohistochemistry and Western-blot.2.90 male 3-month-old C57BL/6J mice,among which artificial cerebrospinal fluid was injected bilaterally into the lateral ventricles of 15 mice as control group,other 75 mice received bilateral icv-STZ as SAD model.After one-month adaptive feeding,the 90 mice were randomly divided into model group(distilled water),donepezil group(0.92 mg/kg/d),and SZL high,medium and low dose group(49.67g/kg/d,24.83 g/kg/d,12.42g/kg/d),15 mice in each group.All mice were given the drug according to the body weight of 0.1ml/10g mice.3.Morris water maze was used to observe the behavioral performance of each group of mice after 3-month-intervention to evaluate the effect of SZL on the learning and memory retention ability of SAD mice.4.The ultrastructure and morphology of myelin sheath were observed by Luxol Fast Blue(LFB)staining and transmission electron microscopy.Immunohistochemistry,Western blot and RT-PCR were used to observe the protein expression and gene regulation of myelin sheath specific proteins MAG,MBP,MOG and PLP,and to evaluate the myelin sheath protection effect of SZL on SAD mice.5.Immunohistochemistry,Western-blot and RT-PCR were used to observe the expression and gene regulation of related proteins in the PI3K/Akt-mTOR pathway to evaluate the role of SZL in the myelin forming pathway.Results1.AD related pathological changes in bilateral icv-STZ mice The step-down test showed that after 1,2,3 and 4 months of modeling,the average incubation period of bilateral icv-STZ mice was shorter than that in the control group,and the average errors number was increased(P<0.05,P<0.01).Immunohistochemistry and Western-blot showed that the expression of A?42 and p-tau of bilateral icv-STZ mice increased compared with that in control group(P<0.05,P<0.01).The expression of IR in bilateral icv-STZ mice was decreased compared with that in the control group(P<0.05),while the expression of IRS-1 was increased compared with that in the control group(P<0.01).The expression of GSK-3? in bilateral icv-STZ mice was increased compared with that in the control group,while the expression of p-GSK-3? was decreased compared with that in control group(P<0.05).The expression of GLUT1 and GLUT3 in bilateral icv-STZ mice decreased compared with that in control group(P<0.05,P<0.01).Transmission electron microscopy was used to observe the ultrastructure of myelin sheath in the hippocampal CA1 area of the two groups of mice.In the control group,the myelin lamellar structure was clear and complete,and the morphology of oligocytes was basically normal.In bilateral icv-STZ mice,the myelin sheath collapsed or seriously expanded,and the morphology of oligocytes was irregular.The number of myelin sheath and synapses was higher in the control group,and decreased in the model group(×2.0k).The results of immunohistochemistry and Western-blot were consistent with those of electron microscopy,and the expression of MBP in bilateral icv-STZ mice was decreased compared with that in the control group(P<0.05,P<0.01).2.Effects of SZL on behavioral behavior of SAD miceMorris water maze showed that,from day 2 to day 5,the average incubation period of mice in each group was shortened and the average swimming distance was reduced.The average incubation period in the model group was prolonged compared with that in the control group(P<0.01),and the average swimming distance was increased compared with that in the control group(P<0.01).On the 4th day,the average incubation period of donepezil group,SZL high group and SZL medium group was shorter than that of model group,and their average swimming distance was less than that of model group(P<0.05,P<0.01).On the 5th day,the average incubation period of mice in each treatment group was shorter than that in the model group(P<0.05).After withdraw,times of crossing the platform in model group was less than that of the control group(P<0.01),and its target quadrant residence time shortened(P<0.05),crossing platform number in donepezil group and SZL-treated groups increased than that of the model group(P<0.05),target quadrant residence time in donepezil group and SZL high and SZL medium group increased compared with model group(P<0.05,P<0.01).3.Effect of SZL on myelin sheath structure and g-ratio in SAD miceLFB staining showed that the myelin sheath fibers in the control group were densely distributed and showed dark blue staining.In the model group,the myelin fibers were obviously lost and stained lightly.The distribution of myelin fibers in all the treatment group was more compact than that in the model group,and the blue staining deepened.Transmission electron microscopy was used to observe the lamellar structure of the myelin sheath in the hippocampal CA1 area of each group.The myelin sheath structure in the control group was clear and complete.The myelin sheath structure in the model group was seriously disintegrated.Five fields were randomly selected to calculate the g-ratio(diameter of single axon/diameter of myelated fiber).The g-ratio of the model group increased compared with the control group(P<0.01),and the g-ratio of each treatment group(except the SZL low dose group)decreased compared with the model group(P<0.01)(×1.2k).The axonal diameter of the model group was larger than that of the control group,and the myelin sheath was thinner.The axonal diameter of the donepezil and SZL-treated groups were larger than that of the model group,and the axonal diameter of the SZL medium dose group was smaller than that of the model group,and the myelin sheath thickness was increased(×8.0k).Western-blot showed that the MAG expression of the model group was lower than that of the control group(P<0.05),and the MAG expression of all treatment groups(except the SZL low dose group)was increased than that of the model group(P<0.05).Immunohistochemical results showed that MAG positive staining was observed in all groups except the model group,in which the MAG staining was deep and dense in the control group and the SZL high dose group.MAG occasionally stained in the model group.Western-blot showed that the MBP expression in the model group was lower than that in the control group(P<0.05),and the MBP expression in the donepezil group,SZL high and medium dose groups was higher than that in the model group(P<0.01).MOG expression in the model group was lower than that in the control group(P<0.01),while MOG expression in the high and low dose groups was higher than that in the model group(P<0.05).The PLP expression in the model group was lower than that in the control group(P<0.01),and the PLP expression in all treatment groups except the SZL low dose group was higher than that in the model group(P<0.05).4.Effect of SZL on the expression of MAG,MBP,MOG and PLP mRNART-PCR showed that the expressions of MAG,MBP,MOG and PLP mRNA in the model group were lower than those in the control group(P<0.05,P<0.01),and the expressions of MAG,MBP and PLP mRNA in the donepezil and SZL high dose groups were increased than those in the model group(P<0.05,P<0.01).5.Effect of SZL on the expression of PI3K/Akt-mTOR pathway related proteinsImmunohistochemistry showed that the number of PI3K,Akt,p-Akt,mTOR and p-mTOR positive cells in the model group all decreased compared with the control group(P<0.05,P<0.01).Compared with the model group,the number of PI3K and p-Akt positive cells increased in the donepezil group and the SZL high and medium dose group(P<0.05),and the number of Akt,mTOR and p-mTOR positive cells increased in the donepezil group and the SZL high dose group(P<0.05,P<0.01).PI3K and p-PI3K Westen-blot showed that the expression of PI3K in the model group was lower than that in the control group(P<0.05),and the expression of PI3K in the SZL high and medium dose groups were higher than that in the model group(P<0.05,P<0.01).The expression of p-PI3K in the model group was lower than that in the control group(P<0.01),and the expression of p-PI3K in the donepezil and SZL high dose groups was higher than that in the model group(P<0.05).Akt and p-Akt Westen-blot showed that the expression of Akt and p-Akt in the model group was lower than that in the control group(P<0.05),while the expression of Akt and p-Akt in the donepezil and SZL high groups was higher than that in the model group(P<0.05).mTOR and p-mTOR Westen-blot showed that the expression of mTOR in the model group was lower than that in the control group(P<0.01),and the expression of mTOR in each treatment group was higher than that in the model group(P<0.05,P<0.01).The expression of p-mTOR in the model group was lower than that in the control group(P<0.05),and the expression of p-mTOR in the donepezil and SZL high dose group was higher than that in the model group(P<0.05,P<0.01).p-S6K1 Westen-blot showed that the expression of p-S6K1 in the model group was lower than that in the control group(P<0.01),and the expression of p-S6K1 in the donepezil and szl high dose group was higher than that in the model group(P<0.05).6.Effect of SZL on PI3K/Akt-mTOR pathway and downstream S6K mRNA expressionThe expression of PI3K mRNA in the model group was lower than that in the control group,with no statistical difference(P>0.05).The expression of PI3K mRNA in each treatment group increased compared with that in the model group,with no statistical difference(P>0.05).The expressions of Akt and mTOR mRNA in the model group were lower than those in the control group(P<0.05).The expressions of Akt and mTOR mRNA in donepezil group and the SZL large dose group were increased compared with the model group(P<0.05,P<0.01),and the mRNA expressions of Akt in the SZL medium dose group were increased compared with the model group(P<0.05).The expression of S6K mRNA in model group was lower than that in control group(P<0.01).The expression of S6K mRNA in donepezil and SZL high dose group increased compared with that in model group(P<0.05).Conclusion1.Bilateral icv-STZ SAD mice not only had behavioral cognitive impairment for 4 months,but also had pathological changes of AD and myelin sheath injury2.SZL improved the early cognitive impairment of SAD mice,which was related to promoting the repair after myelin sheath injury,increasing the expression of myelin sheath specific proteins,and regulating the expression of related proteins in the PI3K/Akt-mTOR pathway. |
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