Using STZ To Damage OLN93 Cells As An AD Model, Explore The Protective Mechanism Of Shenzhiling Myelin Sheath From PI3K/AKT-mTOR Pathway

ObjectiveAD is the most common type of dementia.With the acceleration of aging,the number of AD patients worldwide will reach 80 million.The main clinical manifestations of AD are memory loss,cognitive and behavioral disorders,learning and thinking disorders,and personality changes.The neuropathological features of AD are mainly characterized by neurofibrillary tangles formed by hyperphosphorylated Tau in neocortex and gray matter.Senile plaques(SPs)formed by abnormal deposition of amyloid-beta(Aβ).The researchers believe that in addition to the classic nerve cell damage,white matter degeneration and demyelination are also patho-logical manifestations of AD.The differentiation of oligodendrocytes,the process of mye-lination and remyelination are strictly regulated by a number of coordinated signal transduc-tion pathways,and the PI3K/Akt-mTOR pathway is one of them.Shenzhiling Oral Liquid(SZL)is the first Chinese medicine compound medicine approved by the State Food and Drug Administration.It is a theoretical cube of Chinese medicine ’Kai Xin San’ in Sun simiao’s Bei Ji Qian Jin Yao Fang.Derived on the basis of ginseng,medlar,polygala and calamus,it has a positive effect on AD,but the effect of SZL on the structure and function of early oligoden-drocytes and myelin in AD is poorly understood.The aim of this study was to use Streptozoto-cin(STZ)to damage rat oligodendrocyte OLN-93 cells and establish a model of in vitro spo-radic AD cells with PI3K/Akt-mTOR signaling pathway as a target.To study the protective effect of SZL on oligodendrocytes and myelin,and provide further evidence for the clinical application of SZL oral liquid in the treatment of AD.Methods1.40 male SD adult rats(200±20g)were randomly divided into normal group(n=10)and Shenzhiyu oral liquid group(n=30),and Shenzhiyu oral liquid group was equivalent to adult(70kg).The dose was 2 times the amount of the stomach,the normal group was given the same volume of double distilled water,the stomach was intragastric once a day at 8:00,con-tinuous gavage for 7 days,2 hours after the last administration,intraperitoneal injection of 4%chloral hydrate anesthesia Blood was taken from the abdominal aorta of mice to prepare se-rum containing Shenzhiyu oral solution.2.UHPLC-MRM-MS/MS method for the control of paeoniflorin,cinnamic acid,paeoni-florin,glycyrrhizin,glycyrrhizic acid Five indicator components were quantitatively analyzed.3.The in vitro AD model was established by STZ-induced oligodendrocyte OLN-93cells.The model was evaluated by HE staining,CCK-8/LDH leakage rate detection,RT-PCR and flow cytometry.4.The cell viability was determined by CCK-8 method,and the optimal dose of the drug-containing serum and the donepezil was determined.5.OLN-93 cells were divided into 4 groups:(1)normal control group:serum-free DMEM+15%normal rat serum;(2)model group:serum-free DMEM+15%normal rat serum;(3)SZL drug-containing serum Group:serum-free DMEM+15%SZL oral liquid containing drug serum15%;(4)donepezil group:serum-free DMEM+500 nM donepezil.6.Western blot and RT-PCR were used to detect the effect of SZLmedicated serum on PI3K/Akt-mTOR pathway and myelin-associated protein in STZ-induced OLN-93 cells.7.PI3K/Akt signaling pathway specific inhibitor LY294002 and mTOR specific inhibitor Rapamycin,Western blot and RT-PCR were used to detect PI3K/Akt-mTOR after PI3K/Akt-mTOR pathway blockade.Pathway and the effects of myelin-associated proteins.8.Chromatin immunoprecipitation to study the effect of histone deacetylation on the ap-parent regulation of oligodendrocyte genes.9.Liquid chromatography-mass spectrometry coupled with chromatographic detection of total flavonoids in OLN-93cells with STZ injury.Results1.UHPLC-MRM-MS/MS method was used to detect SZL drug-containing serum.It was found that the extracted SZL drug-containing serum contained ce rtain content of paeoniflorin,cinnamic acid,glycyrrhizin and glycyrrhizic acid,indicating that the above drugs were indeed filled.The stomach enters the gastrointestinal tract of the rat,enters the bloodstream through the liver and intestine,and metabolizes,thereby exerting a certain therapeutic effect.2.The in vitro AD cell model was established by injecting OLN-93 cells with 1 mM STZ for 3 h.Compared with the normal group,the CK value of the model group was significantly lower than that of the normal group(P<0.01);the LDH leakage rate of the LDH leakage rate test model group was significantly increased(P<0.01);the model group was detected by RT-PCR.The transcription levels of IR and IRS1 mRNA were significantly decreased(P<0.01).The expression of ROS in the model group was significantly increased by flow cy?tometry(P<0.01).3.CCK-8 results showed that20%and 15%SZL drug-containing serum could increase cell viability after 3h of 1mMSTZ injury(P<0.05);10%SZL drug-containing serum group could increase in 16h-24h The cell viability(P<0.05)was not statistically significant after 48h(P>0.05).The cell viability of 5%SZL drug-containing serum group was not statistically sig-nificant(P>0.05).The cell viability was not statistically significant when treated with 1μM donepezil for 16h(P>0.05).When the effect was 24-48h,the cell viability was significantly increased(P<0.01).500nM donepezil could significantly increase the cell viability(P<0.01);250nM group The OD value of donepezil was significantly increased at 24h(P<0.01)·4.Western blot results showed that the expression of PI3K,Akt,p-Akt,mTOR and p-mTOR protein in the model group was significantly lower than that in the normal group(P<0.01).Compared with the model group,500nM of donepezil and15%of SZL-containing serum significantly increased the protein expression of PI3K,Akt,and p-Akt(P<0.01).500 nM ofdonepezil increased the expression of mTOR and p-mTOR protein(P<0.05);15%of SZL-containing serum significantly increased mTOR protein expression(P<0.01)and in-creased p-mTOR protein expression(P<0.05).At the same time,the expression of MBP,MOG and PLP protein in the model group was significantly decreased(P<0.01).Compared with the model group,15%of SZL-containing serum significantly increased MBP and MOG protein expression(P<0.01)and increased PLP protein expression(P<0.05).500nM of donepezil increased MBP protein expression(P<0.05)and MOG protein expression(P<0.01),and increased PLP protein expression,but there was no statistical significance(P>0.05).5.RT-PCR results showed that the transcription of Akt,p-Akt and mTOR mRNA in the model group was significantly decreased(P<0.01).Compared with the model group,15%of SZL-containing serum can increase the transcription level of Akt and P-Akt mRNA(P<0.05);15%of SZL-containing serum and 500nM of donepezil can significantly increase mTOR mRNA transcr:iption level(P<0.01);500nM donepezil significantly increased the transcrip-tion level of Akt and p-Akt mRNA(P<0.01).At the same time,the normal group was able to normally transcribe MBP mRNA,and the MBP mRNA transcription was significantly re-duced in the model group compared with the normal group(P<0.01).Compared with the model group,15%of SZL-containing serum and 500nM of donepezil significantly increased MBP mRNA transcription levels(P<0.01).6.After using PI3K/Akt signaling pathway specific inhibitor LY294002 and mTOR specific inhibitor Rapamycin,the results of Western blot showed that the expression of p-Akt and MBP protein in the model group was significantly decreased(P<0.01);compared with the model group the expression of p-Akt and MBP in the model+LY294002 group was signifi-cantly decreased(P<0.01).Compared with the model group+LY294002 group,15%of SZL drug-containing serum+LY294002 and 500nM donepezil+LY294002 were able to be in the PI3K/Akt pathway.In the background of blocking,the expression levels of p-Akt and MBP protein were significantly increased(P<0.01 or P<0.05).At the same time,compared with the normal group,the expression of p-mTOR and MBP protein in the model group was sig-nificantly decreased(P<0.01).Compared with the model group,the expression of p-mTOR and MBP in the model+Rapamycin group was significantly decreased(P<0.01).Compared with the model+Rapamycin group,15%of SZL-containing serum+Rapamycin group and 500nM donepezil+Rapamycin group could increase p-mTOR and MBP protein expression levels in the context of mTOR pathway blockade(P<0.05).7.After applying PI3K/Akt signaling pathway specific inhibitor LY294002 and mTOR spe-cific inhibitor Rapamycin,the results of RT-PCR showed that the MBP mRNA transcription in the model group was significantly reduced(P<0.01);compared with the model group,the model+MBP mRNA transcription was significantly reduced in the LY294002 group and the model+Rapamycin group(P<0.01).Compared with the model+LY294002group,15%of SZL drug-containing serum+LY294002group and 500nM donepezil+LY294002 group could significantly increase MBP mRNA transcription level in the background of LY294002 block-ing PI3K/Akt pathway(P<0.01).Compared with the model+Rapamycin group,15%of SZL-containing serum+Rapamycin group and 500nM donepezil+Rapamycin group could significantly increase the transcription level of MBP mRNA in the background of mTOR pathway block(P<0.01).8.ChIP assay showed that the deacetylation level of HDAC2 was significantly increased in the model OLN-93 cell olig2 promoter sequence(P<0.01).15%SZL drug-containing serum and 500nM donepezil significantly reduced HDAC2 deacetylation level(P<0.01).9.Liquid chromatography-mass spectrometry showed that the saturated fatty acids lauric acid,myristic acid,palmitic acid,stearic acid,γ-linolenic acid,a-linolenic acid,and 19 in the model group were compared with the normal group.The contents of acid,trans-eicosaenoic acid,docosaenoic acid,arachidonic acid,behenic acid,ebutyric acid,docosahexaenoic acid,lauric acid,and nervonic acid are all significantly reduced(P<0.01);palmitoleic acid,cis-9-oleic acid,isooleic acid,cis-9,12-linoleic acid,cis-11,14-eicosadienoic acid,cis-11 The contents of14,17-eicosatrienoic acid,eicosapentaenoic acid and sinapic acid were signifi-cantly increased(P<0.01);the contents of other fatty acids were not statistically significant(P>0.05);In contrast,15%of the medicated serum of ginseng can increase lauric acid,stearic acid,α-linolenic acid,trans-eicosaenoic acid,docosaenoic acid,arachidonic acid,and ebutyric acid.Content(P<0.01 or P<0.05);lower palmitoleic acid,isooleic acid,cis-9,12-linoleic acid,nonadecanic acid,cis-11,14,17-eicosatriene Acid,eicosapentaenoic acid content(P<0.01 or P<0.05);500nM donepezil can increase the content of myristic acid,palmitic acid,pal-mitoleic acid,stearic acid,cis-9-oleic acid,docosaenoic acid,arachidonic acid and ebutyricacid(P<0.01 or P<0.05);reduce the content of cis-9,12-linoleic acid,isooleic acid,lauric ac-id,eicosapentaenoic acid(P<0.01 or P<0.05).ConclusionsSZL can stimulate Akt phosphorylation,increase PI3K/Akt protein and gene expression,reg-ulate downstream mTOR protein and gene expression,play a role in myelin protection,and possibly improve fatty acids by improving the activity of PI3K/Akt-mTOR signaling pathway.At the same time,we hypothesized that SZL can also enhance the expression of olig2 gene by reducing the level of deacetylation of HDAC2,and exert its myelin protection in epigenetics.