| Aging-related nerve injury diseases cause the loss of nerve cells and nerve tissue damage due to various reasons,and it is difficult for the body to age to recover from itself,resulting in long-term motor and cognitive dysfunction.The incidence and risk factor of such diseases increase with age,which not only brings great pressure to families but also to the aging society today.Neural stem cells(NSCs)have the potential of self-renewal and multidirectional differentiation.They can differentiate into a variety of nerve cells to maintain homeostasis in the brain,so they are a good tool for nerve repair and regeneration.However,the number and proliferation capacity of NSCs are reduced by aging,and the occurrence of neurogenesis in the aging body is reduced.In modern biological studies,neither transplantation of exogenous NSCs for internal supplementation nor administration of drugs such as nutritional factors to regulate the initiation and repair of endogenous NSCs have achieved satisfactory clinical results.In traditional Chinese medicine,it is believed that the kidney is related to the brain.The kidney stores essence,essence is transformed into to make marrow,and the brain is the sea of marrow.The strength of the brain is closely related to the adequacy of kidney-essence and the filling of kidney-qi.Moreover,modern research suggests that kidney-essence and NSCs are related.NSCs can be considered as the cell form of kidney-essence in the brain.Rehmanniae Radix Praeparata(PRR)has been used in the treatment of kidney disease in traditional Chinese medicine,which can strengthen kidney-essence and supplement brain-marrow.However,the activity of PRR on NSCs lacks in-depth research,and the therapeutic material basis has not been clarified.The research results of this subject will lay the foundation for the clinical repair and regeneration of Rehmannia glutinosa and its active ingredients.In this project,the primary cultured NSCs were used as the source of the cells,and the results of CCK-8 tests were used as the proliferation judgment indicators.The activities of 11 kinds of kidney-tonifying and semen-supplementing traditional Chinese medicines were preliminarily screened.At the same time,using CCK-8 test results as the activity index,combined with a variety of chemical separation methods,the active components of Rehmannia glutinosa promoting normal NSCs proliferation were tracked to narrow the screening scope.Finally,using D-gal-induced NSCs in vitro aging model to further determine the active site,identified 50 components.Through the study on the proliferation of several characteristic chemicals in Rehmannia glutinosa,it was found that the verbasin glycosides from the active part of Rehmannia glutinosa had the strongest activity.The experimental research is divided into three chapters,which are summarized as follows:Chapter 1:The hippocampal NSCs were mechanically isolated from the brain of 15 d?17 d fetal rats,and the serum-free medium supplemented with bFGF and B27 was used for suspension culture and passage,and the purity of neural stem cells up to 95%was successfully obtained.The growth curve of NSCs was plotted with CCK-8.It was found that NSCs were in the incubation period within 24 hours after inoculation,24 h ? 96 h in the logarithmic growth phase,and entered the plateau phase after 96 h.Based on the above results,we studied 11 flavour invigorating the essence of traditional Chinese medicine(including Cornu Cervi Pantotrichum,Semen Cuscutae,Rehmanniae Radix Praeparata,Radix Achyranthis Bidentatae,Lycii Fructus,Radix Morindae Officinalis,Cistanches Herba,Rhizoma Polygonati,Epimedii Folium,Corni Fructus,Radix Polygoni multiflori)on promoting NSCs proliferation activity,found that they all have obvious promoting the activity of NSCs proliferation(P<0.01).This result provides cytological support for the "Kidney Brain Related" theory of traditional Chinese medicine,and also provides guidance for the development of Chinese medicine for nerve repair.Chapter 2:Using the chemical separation method as the separation tool and the cck-8 detection results as the activity index,the multilayer screening was conducted for the pharmacodynamic components of PRR that promoted the proliferation of NSCs.The petroleum ether fraction(SDE-Pe),ethyl acetate fraction(SDE-Ea),n-butanol fraction(SDE-nB),remaining water fraction(SDE-W)and Polysaccharide fraction(SDW-P),and SDE-Ea and SDE-nB were screened out as the effective components.Macroporous resin,ODS,and gel column chromatography were used to separate SDE-nB,and nB-CX-N3 was further selected as a medicinal component.SDE-Ea was separated by silica gel column chromatography,and Ea-GX was further selected as a medicinal component.In summary,the pharmacologically effective components of PRR for promoting the proliferation of NSCs were finally selected as nB-CX-N3 and Ea-GX.This result lays a foundation for further exploring the pharmacodynamic substance basis of PRR to promote the proliferation of NSCs.Chapter 3:By investigating the administration time(6 h,12 h,24 h,48 h,72 h,96 h)and concentration(100 mg/mL,50 mg/mL,25 mg/mL,12.50 mg/mL,6.25 mg/mL,3.13 mg/mL,1.56 mg/mL,0.78 mg/mL,0.39 mg/mL,0.20 mg/mL)of D-gal,the in vitro aging model of NSCs required by this study was successfully established,that is,12.50 mg/mL D-gal was administered for 24 h.Based on the above NSCs aging model,the proliferative activities of the screened NSCs nB-CX-N3 and Ea-GX were studied,and it was found that the optimal anti-aging efficacy group was divided into nB-CX-N3.In order to know the chemical composition of nB-CX-N3,it was analyzed and summarized by UPLC-QE-Orbitrap-MS/MS.And,50 potential anti-aging components were found,and it was speculated that phenylpropanol and its derivatives might be the structural basis of anti-aging of PRR.Finally,the representative components of various structural types in Rehmanniae Radix,such as Catalpol,Rehmannnioside D,Acteoside,Stachyose,Mannotriose and Melibiose,were studied to promote the proliferation of aging NSCs.The results showed that Catalpol,Acteoside and low concentration Stachyose had significant activities,among which Acteoside had the best effect on proliferation of D-gal aging NSCs,indirectly supporting the results of radix rehmanniae activity tracking.This study shows that Chinese medicines with the effect of invigorating the kidney and improving essence do have the effect of promoting the proliferation of NSCs,and the theory of "kidney-brain related" in Chinese medicine is verified from the cellular level.Using the cell activity tracking method,the main active parts of Rehmannia glutinosa were found and 50 of them were identified,among which the verbascoside has a stronger effect.This topic provides cytological basis for the clinical treatment of aging-related nerve injury diseases by Rehmannia glutinosa,and lays a foundation for the clinical application of Rehmannia glutinosa and its main active ingredients. |
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