Based On The Damage Of Myelin Sheath And Oligodendrocytes, The Protective Effect Of Shenzhiling Oral Liquid On AD Was Discussed

ObjectiveStarting from the theory of heart and brain in traditional Chinese medicine,we select the repre-sentative prescription shenzhi ling oral liquid of "treating from the heart" to explore the mech-anism of its intervention in Alzheimer's disease(AD)based on the myelin sheath and oligoid cell injury.AD is the most common type of dementia.Clinical manifestations can be memory,emotional and behavioral disorders,which place a heavy economic and mental burden on pa-tients' families and caregivers.Recent studies have shown that myelin damage is associated with neurodegenerative diseases such as AD,Parkinson's disease(PD),and stroke.It was found that AD is closely related to the destruction and loss of myelin sheath.The study found that myelin damage was earlier than cognitive impairment,SPs deposition and NFTs pathological changes,and myelin damage may play a key role in early AD cognitive impairment.The PI3K/Akt-mTOR pathway is a powerful driving force for myelin formation and a major driving factor for CNS myelin protein expression.Some studies have shown that abnormal and sus-tained activation of neuronal PI3K/Akt-mTOR signaling is an early feature of AD.MTOR plays a significant role in lipid synthesis,transcription factor regulation,and cytoskeleton in the oligodendrocytes constituting the myelin sheath,which is an indispensable process for oli-godendrocyte development.In addition,epigenetic block and lipid metabolism disorder are closely related to the pathology of AD.In this study,we explored whether Shenzhiling Oral Liquid can improve myelin damage through in vivo experiments.At the same time,we observed whether it can prevent early AD by protecting A?42 damaged oligodendrocytes and maintaining myelin sheath integrity.Based on PI3K/Akt-mTOR signaling pathway,epigenetic regulation and lipid metabolism,the possible mechanism was explored to provide possible evidence for Shenzhiling oral liquid to protect myelin sheath.Methods1.36 male APPswe/PS1dE9 SPF-grade double transgenic mice were randomly divided into a model group,a donepezil group,and a Shenzhiling Oral Liquid group,with 12 mice in each group.12 wild-type C57BL/6J mice of the same background and age were used as normal control group.The donepezil group was given oral administration of donepezil(0.92 mg·kg-1·d-1),and the Shenzhiling oral solution group was given oral administration of Shenzhiling oral solution(25g·kg-1 · d-1).The normal control group and the model group were perfused with 0.5%CMC.Gavage was continued for 3 months,once a day2.Using the Y-maze experiment to study the effects of Shenzhiling Oral Liquid for 3 months on the spatial recognition and memory ability of APPswe/PS1dE9 double transgenic mice.3.Transmission electron microscopy was used to study the effect of Shenzhiling Oral Liquid on the ultrastructure of amyloid plaque,myelin sheath and oligodendrocytes in the hippocam-pus of APPswe/PS1dE9 double transgenic mice after oral administration for 3 months.4.Immunohistochemistry and Western blot were used to study the effects of Shenzhiling Oral Liquid on the expression of MBP,PLP,MAG and A?42 proteins in the hippocampus of APPswe/PS1dE9SPF double transgenic mice after gavage for 3 months.5.Forty male SD adult rats(200 ± 20g)were randomly divided into the normal control group and the Shenzhiling oral liquid group,and the Shenzhiling oral liquid group was administered by gavage at twice the equivalent dose of an adult(70kg).In the normal control group,an equal volume of 0.5%CMC was administered once a day for 7 days in a row to prepare Shen Zhiling medicine-containing serum6.UHPLC-MRM-MS/MS method was used to detect the main chemical constituents of Shen-zhiling oral liquid drug-containing serum7.A?42 damages OLN-93 oligodendrocytes,and establishes a pseudo-AD cell model in vitro.8.CCK-8 method was used to screen the safe and effective doses of Shenzhiling oral liquid drug-containing serum and the positive drug donepezil to OLN-93 oligodendrocytes9.Western blot,RT-qRCR and immunofluorescence methods were used to study the effects of Shenzhiling Oral Liquid containing serum on A?42-damaged OLN-93 oligodendrocyte myelin-related protein and PI3K/Akt-mTOR signaling pathway protein and mRNA expression10.Use the PI3K/Akt-mTOR signaling pathway inhibitor LY294002(PI3K inhibitor)and Rapamycin(mTOR inhibitor)to further confirm the participation of the PI3K/Akt-mTOR signaling pathway.11.The chromatin immunoprecipitation(CHIP)experiment was used to observe the interaction between the acetylated histone and MBP gene of oligodendrocyte OLN-93 in each group12.LC-MS/MS method was used to observe the effect of Shenzhiling Oral Liquid on lipid metabolism of A?42-injured oligodendrocytes.Results1.In vivo animal experimentShenzhiling oral solution and the positive control drug donepezil for 3 months can significantly prolong the distance and duration of APPswe/PS1dE9 double transglnic mice's activity in the new allogeneic arm.Under the transmission electron microscope,the deposition of amyloid plaques near the injured oligodendrocytes was observed,but it was not observed in the normal group and the treatment group.It was also observed under transmission electron microscopy that after three months of oral administration of Shenzhiling Oral Liquid and donepezil,the structure of the myelin lamellar layer,the disintegration of myelin,and the structural destruction of oligodendrocytes were significantly improved.Based on this,immunohistochemistry and Western blot results of the hippocampus of each group of mice also showed that Shenzhiling Oral Liquid and donepezil can significantly up-regulate the myelin-related proteins MBP,PLP and Protein expression of MAG.At the same time,the APPswe/PS1dE9 double transgenic model mouse was observed to up-regulate the expression of A?42 in the hippocampus.After 3 months of oral administration of Shenzhiling Oral Liquid and donepezil,the expression of A?42 in the hippocampus of the mice could be significantly reduced.2.UHPLC-MRM-MS/MS method for detection of main chemical constituents of Shenzhiling oral solution and Shenzhiling medicated serumIn Shenzhiling oral solution,the five metabolites of paeoniflorin,cinnamic acid,glycyrrhizic acid,glycyrrhizin,and paeoniflorin can be detected.And the concentration content:glycyr-rhizic acid>paeoniflorin>paeoniflorin>cinnamic acid>glycyrrhizin.Four metabolites of cin-namic acid,glycyrrhizic acid,glycyrrhizin,and paeoniflorin could be detected in the serum of rats containing Shenzhiling oral solution.Concentration content:glycyrrhizic acid>cinnamic acid>paeoniflorin>glycyrrhizin.Although the peak of peiolide can be detected in the mass spectrum,the signal may be similar to the noise,and the quantitative detection did not detect the content.It is suggested that Shenzhiling Oral Liquid can indeed play a role by instilling blood into the stomach.The protective effect of Shenzhiling Oral Liquid on myelin sheath may be closely related to the four components of cinnamic acid,glycyrrhizic acid,glycyrrhizin and paeoniflorin.3.In vitro cell experimentsA?42 can induce OLN-93 oligodendrocytes to cause damage.Incubating cells with 100?M A?42 for 16h and incubating cells with 1?M,100?M and 100?M A?42 can significantly reduce cell viability.At the same time,1?100 ?M A?42 incubated the cells for 24 hours,and the cell damage rate increased significantly.1?100?M A?42 incubation for 24 hours can cause damage to cell morphology,cause cell shrinkage,fragmentation,and death.In addition,A?42 incubation for 24 hours can significantly increase the apoptosis level of OLN-93 cells.The CCK-8 method was used to screen the safe dose and time range of Shenzhiling oral liquid drug-containing serum and donepezil.There was no cytotoxicity to normal OLN-93 cells after incubation for 3?48h with Shenzhiling oral liquid drug-containing serum of 1%?20%concentration.The cell viability of normal OLN-93 cells could be significantly increased by incubating the Shenzhiling oral liquid drug-containing serum with 16%and 20%concentration for 24h and 48h.The safe dose and time of donepezil for normal OLN-93 cells are:3h incubation(0.01 ?M,0.1 ?M,1?M,10 ?M,100 ?M);24 h(0.01 ?M,0.1 ?M,1 ?M,10 ?M,100 ?M);Incubate for 48 h(0.01?M,0.1 ?M,1 ?M,10 ?M).CCK-8 method was used to screen the effective dose and time range of Shenzhiling oral liquid drug-containing serum and donepezil.The cells were incubated for 24h and 48h in 16%and 20%Shenzhiling oral liquid drug-containing serum.1?M,10 ?M,and 100 ?M donepezil were incubated for 3 h or 24 h;0.1 ?M,1 ?M,and 10 ?M donepezil were incubated for 48 h.A?42-damages OLN-93 oligodendrocytes,which is consistent with in vivo animal experiments AP42 significantly down-regulates the expression of oligodendrocyte myelin-related proteins MBP,PLP,MAG,and MOG,and down-regulates the mRNA expression levels of MBP and PLP.The intervention of Shenzhiling Oral Liquid containing serum and donepezil could sig-nificantly reverse the expression levels of these proteins and mRNA.On the other hand,A?42 damage to OLN-93 oligodendrocytes also appears to cause abnormalities in the PI3K/Akt-mTOR signaling pathway.A?42 can not only down-regulate the protein and mRNA expression levels of PI3K,Akt and mTOR,but also significantly down-regulate the protein expression levels of p-PI3K,p-Akt and p-mTOR.Significantly reversed the expression levels of these pro-teins and mRNA.The protective effect of Shenzhiling Oral Liquid containing medicated serum and donepezil on A?42-damaged OLN-93 oligodendrocytes can be completely reversed by inhibitors of the PI3K/Akt-mTOR signaling pathway.The specific manifestation is that whether using LY294002,Rapamycin or The combined use of LY294002 and Rapamycin can completely reverse the ability of Shenzhiling Oral Liquid containing serum and donepezil to increase MBP.CHIP experiments showed that histone H3 acetylation level increased after A?42 damaged OLN-93 oligodendrocytes.Donepezil and Shenzhiling oral liquid-containing serum down-reg-ulated histone H3 acetylation level.At the same time,after A?42 damaged OLN-93 oligoden-drocytes,histone H3 acetylation occurred in more genes MBP.Donepezil and Shenzhiling oral liquid-containing serum can down-regulate the H3 acetylated MBP gene level.The results of lipid metabolism showed that CL,PA,PC,PE,PG,PI,and PS in the normal group were significantly higher than those in the model group.Cer,DG,SM and TG in the model group were significantly higher than those in the normal group.Cerp,Hex2Cer,HexCer and Sphingosine were equal in both groups.Donepezil's Cer,CL,PA,PC,PE,PG,PI,PS were significantly higher than the model group.The SM and TG of the model group were signifi-cantly higher than those of the donepezil group.The Cer,DG,CL,PA,PC,PE,PG,PI,PS,SM of Shenzhiling oral liquid were significantly higher than the model group.The TG of the model group was significantly higher than that of Shenzhiling oral liquid group.ConclusionsShenzhiling Oral Liquid can improve the damage of myelin sheath.At the same time,Shen-zhiling Oral Liquid can protect the oligodendrocytes damaged by A?42 and maintain the integ-rity of myelin sheath to prevent early AD.The protective effect of Shenzhiling oral liquid on myelin sheath is closely related to PI3K/Akt-mTOR signaling pathway,epigenetic regulation and lipid metabolism.First,Shenzhiling Oral Liquid may increase PI3K,Akt and mTOR pro-tein expression levels by up-regulating PI3K,Akt and mTOR mRNA levels,indirectly increase the phosphorylation of PI3K,Akt and mTOR,and enhance PI3K/Akt-mTOR signaling path-way Activity,thereby exerting the protective effect of oligodendrocytes and myelin sheath.Second,Shenzhiling Oral Liquid protects oligodendrocytes by down-regulating histone H3 acetylation level and down-regulating histone H3 acetylation MBP gene level.Furthermore,Shenzhiling oral liquid protects oligodendrocytes by up-regulating the content of CL,PA,PC,PE,PG,PI,and PS,down-regulating the content of TG,and adjusting the disorder of lipid metabolism.Finally,the material basis of the myelin protection of Shenzhiling Oral Liquid may be closely related to the four components of cinnamic acid,glycyrrhizic acid,glycyrrhizin and paeoniflorin.