Research On The Effect And Mechanism Of Osthole In The Treatment Of AD With NT-3-BM-NSCs Transplantation

Purpose:This work was supported by the National Natural Science Foundation of China“Study on the mechanism of isolated coumarin combined with transgenic bone marrow-derived neural stem cells in the treatment of Alzheimer's disease”?grant No.81173580?.Material and method:1.Mice were divided into three groups:normal group?5-month-old C57BL/6 mice?,model group and osthole group?all use 5-month-old APP/PS1 double transgenic AD mice?.Osthole group was given 20mg/kg Osthole twice daily for 6 weeks.Normal and model groups were given normal saline with CMC-Na.The mice were sacrificed 1 h after the last administration,and the whole brain was taken.The brain tissue samples were analysised by UPLC-MS and metabolomics methods.By searching the databases such as HMDB and KEGG and searching the literatures,we can find the different metabolites that the osthole may regulate and analyze the related metabolic pathways,and then get the signaling pathways that osthole may affect.2.BM-NSCs were generated from BM mesenchymal cells of adult?5-to 8-week-old?C57BL/6 mice.Cells from bone marrow at the 5th to 10th passages were used in the following experiments.Immunofluorescence cytochemistry method was used to identify BM-NSCs.BM-NSCs were pre-treated with different doses of Ost and treated with H₂O₂.The cell counting kit-8?CCK-8?method and lactate dehydrogenase?LDH?leakage assay were used to determine cell viability.TUNEL assay was used to evaluated the effect of Ost on cell apoptosis.RT-PCR analysis was used to determine the expression of the anti-apoptosic gene Bcl-2 and the pro-apoptosis gene Bax.To further study the protective mechanism of Ost against BM-NSC oxidative damage,we analyzed the expression of PI3K,Akt and p-Akt proteins by western blot.ImageJ software was used to analyze the protein expression with?-actin as the internal control for total protein level in each sample.3.L.v.-NT-3-GFP and L.v.-GFP were transfected into BM-NSCs.Three days after transfection,immunocytochemistry and western blotting were used to detect the expression of NT-3.The proliferation potential of transfected-BM-NSCs was determined by BrdU labeling.To detect the effect of NT-3 on differentiation of BM-NSCs into cholinergic neurons in vitro,we analyzed the expression of ChAT by immunocytochemical staining.Consistent with the results of immunocytochemistry,we detected the expression of ChAT mRNA by RT-PCR analysis.Next,we assayed the level of Ach released by BM-NSCs.To investigate whether the molecular mechanisms underlying NT-3-enhanced proliferation and differentiation of BM-NSCs into cholinergic neurons are dependent on the Notch signaling pathway,RT-PCR and WB analyses were used.4.Mice were anesthetized and placed in a stereotaxic frame,about 1?10⁵NT-3-BM-NSCs in 2?l PBS were injected into each side of the hippocampus in 5 min.The cannula was left in situ for 5 min to allow diffusion into the surrounding tissue before being slowly withdrawn.Morris Water Maze Test was used to evaluate learning and memory capacity of the mice.Immunohistochemistry method was use to determine the expression of ChAT in the brain.The expression of inflammatory cytokines was detected by ELISA.To further study the mechanism of Ost to promote the survival of transplanted NT-3-BM-NSCs,we analyzed the expression of PI3K,Akt and p-Akt proteins by western blot.Results:1.PLS-DA results showed that the normal group,the model group and the osthol group could be distinguished.There were some differences in the metabolism level between the three groups.After the administration of osthole,APP/PS1 double transgenic AD mice were able to recover in the direction of normal mice.The differences in metabolites were adenosine,xanthine 5-triphosphate,5-aminoimidazole ribonucleotides,hypoxanthine,adenine,Nicotinic acid,nicotinic acid amide,phosphatidylcholine,phosphatidylethanolamine and1-acyl-sn-glycero-3-phosphocholine.Metabolic pathway analysis showed that the main metabolic pathways regulated by osthole were purine metabolism,nicotinic acid and nicotinamide metabolism,and glycerol phospholipid metabolism.By querying KEGG and other databases and literature search,we found that the main signal pathways regulated by osthole are PI3K,MAPK,Jak-SAKT,TGF-?signaling pathways.2.After 3 to 5 weeks in culture,a number of individual BM-NSCs proliferated and formed distinct neurospheres,which expanded in the subsequent cultures.These BM-derived spheres stained positive for the NSC marker Nestin and SOX2.After 14 days of culture in differentiation medium,BM-NSCs changed morphology and developed into neurons?NeuN⁺?,oligodendrocytes?NG2⁺?,and astrocytes?GFAP⁺?as verified by immunostaining,indicating that the BM-NSCs were multipotent.Compared with the control group,the survival rate of BM-NSCs in the model group decreased,but the release of LDH in the supernatant increasedsignificantly.The neuroprotective effect of Ost reached its peak at the concentration of 100?M.The concentration of 50?M Ost was used in the following experiments.The results of TUNEL assay showed that the percentage of TUNEL-positive cells decreased in the Ost group compared to the model group?p<0.05?.The results indicated that Ost inhibited H2O2-induced apoptosis.The results of RT-PCR analysis in this study showed that oxidative stress induced by H₂O₂ stimulation increased the Bax level and decreased the Bcl-2expression level.Ost pretreatment attenuated the H2O2-induced Bax level in BM-NSCs and reversed the decreased level of Bcl-2 significantly?p<0.01?.Therefore,Ost lessened the ratio of Bax/Bcl-2 mRNA,which may contribute to the increased cell viability?p<0.01?.Western blot results indicated that the expression of PI3K and p-Akt protein were significantly decreased after the addition of H₂O_2.Ost pre-treatment increased the protein levels of PI3K and p-Akt in BM-NSCs compared to the H2O2 group.There was no significant difference in the protein expression of Akt.In the presence of LY294002,Ost-mediated neuroprotection of BM-NSCs from apoptosis induced by H₂O₂ was abolished.These results indicated that Ost prevented BM-NSC apoptosis mediated by oxidative stress through PI3K/Akt-1 signaling pathways.3.Three days after transfection,NT-3 expression was detected and it was steadily expressed by these BM-NSCs up to the 10th passage.NT-3 transduction can promote the proliferation and differentiation of BM-NSCs into cholinergic neurons and elevate the levels of acetylcholine?ACh?in the supernatant.Furthermore,NT-3 gene overexpression increase the expression of Hes1,decreased the expression of Mash1 and Ngn1 during proliferation of BM-NSCs.Whereas,the expression of Hes1 was down-regulated,and Mash1 and Ngn1expression were up-regulated during differentiation of BM-NSCs.4.Ost combined with NT-3-BM-NSCs transplantation enhances the ability of learning and memory in mice,increases the expression of ChAT in the brain and decreases the level of inflammatory cytokines.At last,Ost combined with NT-3-BM-NSCs transplantation increased the protein levels of PI3K and p-Akt.Conclusion:1.The mechanism of Ost against AD may be related to participation in the purine metabolism,nicotinic acid and nicotinamide metabolism,glycerophospholipid metabolism;the relative signaling pathway maybe including PI3K,MAPK,Jak-SAKT,TGF-?,et al.2.Ost protects BM-NSCs against oxidative stress injury,and Ost might act through the activation of the PI3K/Akt-1 signaling pathway,thereby reducing the apoptosis of BM-NSCs.3.NT-3 gene transfection can not only promote the in vitro proliferation of BM-NSCs,by also promote the differentiation of BM-NSCs into cholinergic neurons.The molecular mechanisms are most likely by affecting the Notch signaling pathway.4.Osthole can reduce brain inflammation and promote the survival of NT-3-BM-NSCs and differentiate into cholinergic neurons.The mechanism may be related to the activation of PI3K/Akt-1 signaling pathway.