Molecular Mechanism Of Plastrum Testudinis Extracts To Promote Directional Differentiation Of Neural Stem Cells By Regulating TET1

Objective:Parkinson’s Disease(PD)is an age-related neurodegenerative disease with a complex etiology and a slow course.At present,there is no radical cure for PD,which may cause a serious socioeconomic burden for the future aging society.Severe loss of substantia nigra and striatum dopamine neurons is a major pathological change in PD.Neural stem cells(NSCs)are self-renewing,can be multi-directionally differentiated,and have low immunogenicity.Under specific conditions,NSCs can be directionally differentiation into dopamine neurons to replace damaged neurons,thus acting as a therapeutic role for treatment of PD.Therefore,the use of NSCs for treatment of PD has attracted widespread attention in recent years.How to induce directionally differentiation of NSCs is a key issue in the treatment of PD by NSCs.The 10-11 translocation(TET)protein family and its mediated DNA methylation(5hmC)play an important role in the differentiation of NSCs,and 5hmC enriched in highly expressed neuron function-related genes.As a member of the TET family,TET1 is highly expressed in embryonic stem cells and the nervous system.Thus,how to regulate DNA methylation has become a key issue in the differentiation of NSCs.Previous research by our research team found that Plastrum Testudinis decoction has obvious therapeutic effect on Parkinson’s disease model rats,the number of dopamine neurons in the brain increases,and the dose-response relationship is obvious.At the same time,previous studies have found that Plastrum Testudinis extract can promote the differentiation of NSCs into dopamine neurons,but whether this effect can regulate the epigenetic mechanism of neural stem cell differentiation remains to be further studied.This study used immunohistochemical staining to observe the changes in the number of dopamine neurons and the level of DNA methylation in the substantia nigra in the PD model rats after the intervention of Plastrum Testudinis decoction,and their effects on TET1 and FOXA2 were verified.Through in vitro experiments to clarify the possible molecular mechanism of the Plastrum Testudinis extracts to promote the differentiation of neural stem cells to dopamine neurons by regulating TET1 and FOXA2.Methods1.A PD rat model was prepared,and the Plastrum Testudinis decoction was used for drug intervention treatment.The experiment was divided into:blank control group(1mg/kg/d,0.9%NaCl),sham operation group(1mg/kg/d,0.9%NaCl),PD model group(1mg/kg/d,0.9%NaCl),positive-drug group(1mg/kg/d,levodopa 10mg/kg+benserazide 2.5mg/kg),Plastrum Testudinis decoction low-dose group(1.1g/d),Plastrum Testudinis decoction high-dose group(9.9g/d).Immunohistochemical staining was used to observe the expression of Th-positive neurons in substantia nigra in brain slices of Parkinson’s disease model rats.At the same time,TET1,DNA hydroxymethylation(5hmC)levels and FOXA2 expression levels were detected by immunohistochemical staining.2.Take the embryonic fetal brain of SD female rats at E14-17,isolate and culture the cells,and use immunofluorescence staining to identify the cultured cells and use them in subsequent cell experiments.3.Using the Plastrum Testudinis extract and its effective ingredients(PTE,s6,s9)for drug intervention for 5 days,the experiment was divided into control group,PTE group(30μg/ml),s6 group(30μg/ml),s9 group(30μg/ml).Western-blot,qRT-PCR,immunofluorescence staining,Dot-blot and other methods were used to detect the effects of Plastrum Testudinis extract and its effective ingredients on Th,5hmC levels,TET1 and FOXA2 expression.4.Using the Plastrum Testudinis extract and its effective ingredients(PTE,s6,s9)for drug intervention for 5 days,the experiment was divided into control group,PTE group(30μg/ml),s6 group(30μg/ml),s9 group(30μg/ml).Through immunofluorescence co-localization and co-immunoprecipitation,to verify the interaction between TET1 and FOXA2 and the Plastrum Testudinis extract and its Effects of effective ingredients on the interaction of TET1 and FOXA2.5.Use qRT-PCR to screen out the best silent fragments of Tetl and Foxa2 genes,and separately transfect Tet1 silent fragments and Foxa2 silent fragments into differentiated neural stem cells,using Plastrum Testudinis extract and its active ingredients(PTE,s6,s9,30μg/ml)to intervene.The mRNA levels of related genes were detected by qRT-PCR,and investigation the effect correlation of Plastrum Testudinis extract and its effective ingredients with Tetl and Foxa2.Results1.In the results of immunohistochemical staining in vivo,the number of Th-positive dopaminergic neurons in the substantia nigra of PD model group was significantly reduced.Although Th-positive neurons in the positive drug group were up-regulated,the effect was not significant.The number of Th-positive neurons in the brain substantia nigra in the PD model rats of the low-dose and high-dose Plastrum Testudinis decoction groups increased significantly,with a significant dose-effect relationship.Meanwhile the level of 5hmC in the brain substantia nigra of PD rats was significantly down-regulated,and the level of 5hmC in the low-dose and high-dose groups were increased,and the upward trend was consistent with Th expression.Moreover,the Plastrum Testudinis decoction up-regulate the expression of TET1 and FOXA2 in the substantia nigra of PD model rats.2.In ex vivo experiments,Plastrum Testudinis extract and its effective ingredients(PTE,s6,s9,30μg/ml)can significantly increase the expression of TH,and the number of neural stem cells differentiated into Th-positive neurons has increased significantly.The effect of PTE is most obvious.At the same time,PTE,s6,and s9 can significantly increase the expression of TET1 and 5hmC levels during neural stem cell differentiation,and can increase the expression of FOXA2.3.Immunofluorescence co-localization and Western-blot results show that PTE,s6,and s9 can increase the expression of TET1 and FOXA2 in the nucleus;Co-IP results proved that the Plastrum Testudinis extract and its effective ingredients(PTE,s6,s9,30μg/ml)in the process of inducing neural stem cell differentiation,there is an interaction between TET1 and FOXA2,and in PTE,s6,and s9 groups the binding rate of TET1 and FOXA2 was increased.4.After silencing Tetl gene alone,the mRNA level of Th gene was significantly reduced,while the expression of Foxa2 gene was not affected.After the Tetl gene was silenced,PTE,s6,and s9 were added for drug intervention.The results showed that the mRNA level of Th gene was not significantly increased in the PTE,s6,and s9 groups.5.After silencing Foxa2 gene alone,the mRNA level of Th gene was significantly reduced,while the expression of Tetl gene was not affected.After the Foxa2 gene was silenced,PTE,s6,and s9 were added for drug intervention.The results showed that the mRNA level of Th gene was not significantly increased in the PTE,s6,and s9 groups.Conclusion1.Plastrum Testudinis decoction can increase the number of Th-positive neurons in the substantia nigra of PD model rats and change its 5hmC level.2.Plastrum Testudinis extract and its effective ingredients(PTE,s6,s9)can effectively promote the targeted differentiation of neural stem cells into Th-positive neurons.3.During the process of neural stem cell differentiation induced by Plastrum Testudinis extract and its effective ingredients(PTE,s6,s9),PTE,s6,and s9 can increase the binding rate of TET1 and FOXA2.4.Plastrum Testudinis extract and its effective ingredients(PTE,s6,s9)promote the directional differentiation of neural stem cells into dopamine neurons by regulating TET1 and FOXA2.5.Plastrum Testudinis decoction may promote the directed differentiation of endogenous NSCs into dopamine neurons through regulates the interaction between TET1 and FOXA2 in the mesencephalic substantia nigra of PD model rats,and improve PD on PD rats.