The Molecular Mechanism Of Plastrum Testudinis Extracts Targets Notch Signal Pathway In Anti-Parkinson’ Disease

ObjectiveParkinson’s disease is a common neurodegenerative disease for elderly people.Its pathological features are the degeneration of dopamine neurons and the formation of Louis body in SNC,resulting in striatal dopamine deficiency and basal ganglia dysfunction.The main clinical manifestations are resting tremor,rigidity,bradykinesia and postural instability and other symptoms.At present,the treatment of Parkinson’s disease can only relieve the clinical symptoms,not cure or delay the natural development of the disease.NSCs is a kind of cell with the ability of self-renewal,and can differentiate into neuron and glial cells under certain conditions.It has been found that NSCs exist in the brain of normal adult mammals.If endogenous NSCs can be specifically induced to differentiate into dopaminergic neurons and replace the degeneration of neurons,it will bring new hope for the treatment of Parkinson’s disease.The Notch pathway can inhibit the differentiation of NSCs into neurons and promote the differentiation of NSCs into radial glial cells,indicating that the Notch pathway is an important pathway to regulate NSCs differentiation.As an important traditional Chinese Bushen medicine tortoise plastron,the decoction has been proved to have effect on the treatment of Parkinson’s disease and its extract on induced differentiation of NSCs into dopaminergic neurons play an important role.However,it is still unclear whether the regulation of NSCs differentiation is targeting the Notch pathway.Therefore,this thesis intends to explore the correlation between Decoction treatment effect on the shell of Parkinson’s disease and Notch pathway,clarify the mechanism of how PTE promote NSCs in the differentiation of dopaminergic neurons by regulating Notch pathway,and provides new ideas for the treatment of Parkinson’s disease by traditional Chinese Bushen medicine.Method1.The therapeutic effect of tortoise plastron Decoction on rat model of PD.Establishe PD rat model by 6-OHDA injection.Grouping:normal group,sham operation group,PD model group,positive drug group,tortoise plastron Decoction low dose group,tortoise plastron Decoction high dose group.HE was used to observe the pathological changes of PD rats on the morphological staining;immunohistochemistry to observe the effect of Plastrum Testudinis Decoction on rat model of PD in a-synuclein,TH and Notchl;Western-blot method was used to detect the expression levels of a-synuclein,TH and Notch l;the mRNA expression of TH is detected by qRT-PCR.2.The effect of Plastrum Testudinis extracts on expression of Notch signaling molecules during the differentiation of NSCs.Culture the primary NSCs from 14 day pregnant SD rats.Immunofluorescence staining was used to identify the differentiation ability of NSCs.First with the concentration of 30 g/ml PTE(2B),Plastrum Testudinis extracts eighteen acid ethyl ester(S6)and 4-phytosterone(S9)stimulated the differentiation of NSCs for 5 days,using immunofluorescence staining to detect the expression of tyrosine hydroxylase(TH)and Notchl.Then three different concentrations of 2B,S6 and S9 were used to stimulate the differentiation of NSCs for 7 days,and the expression of TH and Notchl were detected by Western-blot.3.Effect of Plastrum Testudinis extracts on expression of Notch signaling molecules after transfecting Hesl-siRNA.P19 cells were cultured and transfected with Hesl-siRNA.After stimulating with different concentrations of 2B,S6 and S9 for 7 days,the expression of TH,Notchl and NF200 were detected by Western-blot method.Results1.The Lewy body expression of the substantia nigra of the PD model group increases and the expression of the Turtle shell Decoction decreases,with no significant difference in striatal.The PD model group,the substantia nigra and striatum in the reduced expression of TH,increase the expression of a-synuclein and Notchl;turtle shell Decoction low dose group and high dose group TH expression increased,and the obvious dose effect relationship,reduce the expression of a-synuclein and Notchl.2.Stimulation of NSCs differentiation after 5 days,TH expression of 2B,S6 and S9 group at 30μg/ml increased,Notchl expression decreased;stimulate the differentiation of NSCs after 7 days,the TH expression of 2B,S6 and S9 in different concentration groups had different degrees of increase,all the medium dose groups(30 g/ml)are significant.The Notchl expression of 2B and S9 groups reduced,and S6 group increased.3.After transfection of Hesl-siRNA,the expression of TH and NF200 protein increased in each group of 2B,S6 and S9,in which the dose effect relationship of S6 was obvious,and the expression of TH and NF200 decreased with the increase of S9 concentrationConclusion1.The decoction of Plastrum Testudinis has therapeutic effect on PD rats.It can reduce the expression of Louis body in substantia nigra and the loss of dopamine neurons in brain tissue.The doseeffect relationship is remarkable.This may be achieved by inhibiting the expression of Notchl.2.2B,S6 and S9 might promote the differentiation of NSCs into dopaminergic neurons by inhibiting the expression of Notchl and Hes1.