| ObjectiveBy the analysis of the expression levels of mRNA and protein about the aging related genes of GnRH1?Sirt1?DNMT1?DNMT3a in hypothalamus of the aged and adult rats and the methylation of GnRH1 gene to explore the changes of the activity of DNA methyltransferase(DNMT)and the methylation of GnRH1 gene in the natural aging process of rats from the level of epigenetics;by separating the neural stem cells in hypothalamus of neonatal rats,building the aging model of neural stem cells in hypothalamus caused by D-galactose,to compare the differences of the capability of cell sphere-forming,the expression of inflammatory cytokines and NF-?B between the normal neuron stem cell group,D-galactose induced aging group and the intervention group of different concentration of Qiong Yugao,to provide experimental reference and data support for revealing the anti-aging effect on neural stem cells and its possible molecular mechanisms in Qiong Yugao.MethodsWe observed and compared the expression levels of mRNA and protein about aging related genes in hypothalamus of 6 24 month aged SD rats and 6 2 month adult SD rats:the mRNA expression levels of the gene about GnRH1?Sirt1?DNMT1?DNMT3a were detected by Real-Time PCR;the protein expression levels of above were detected by Western blot;did statistics analysis with spss19.0 software.The methylation status of GnRH1 gene was examined by means of methylation specific PCR.Separated and cultured neuron stem cells in hypothalamus of 8 neonatal SD rats(within 24 hours after birth),the expressions of NSE were detected by immunofluorescence essay in order to identify neural stem cells;the effect on neural stem cells proliferation from Qiong Yugao(OD)were tested by CCK-8 essay,calculated the value of IC50 as the basis for the group experiment with spss19.0 software.The neural stem cells were divided into 4 groups(group A,B,C,D)and cultured primarily by NSCs complete medium respectively to the eighth days,from the ninth day,group A continued to use the medium for culture;10mg/mL D-galactose has been added to group B to cause degradation according to references;group C was first added with 90?g/mL(1/2 IC50)Qiong Yugao for 6h,then added 10mg/mL D-galactose for 24h culture;group D was first added with 45?g/mL(1/4 IC50)Qiong Yugao for 6h,then added 10mg/mL D-galactose for 24h culture;The morphology and the capability of sphere-forming(SFE)of neural stem cells were observed by inverted fluorescence microscope;the expression levels of mRNA about TNF-? and IL-1? in each group were detected by Real-Time PCR;the expression levels of NF-? B was detected by Western Blot;the expression levels of the protein about TNF-? and IL-1? were detected by Elisa;did statistics analysis with spssl9.0 software.Results1.The expression levels of mRNA and protein about the genes of GnRH1?Sirt1?DNMT1 were decreased significantly in aged rats,there was a significant difference when compared with adult rats(p<0.05);the expression levels of mRNA and protein about DNMT3a gene has no obvious change in aged rats,there was no significant difference when compared with adult rats(p>0.05);GnRH1 gene in aged rats occured partly methylated but did not occur in adult rats.2.The neural stem cells in hypothalamus of neonatal rats were separated successfully,we observed cell had grown into sphere from the inverted microscope;the clear red signal fluorescent signal of NSE in cell was detected by immunofluorescence assay,it showed that NSE was positive in neural stem cells.3.The proliferation effect(OD)on neural stem cells of Qiong Yugao had a certain dependence with time and dose,the change of the concentration of Qiong Yugao in 24h was smaller;ccording to the IC50 value of 24h(185.91?g/mL),we selected 1/2 IC50(90 g/mL)and 1/4 IC50(45 g/mL)as the concentration values of Qiong Yugao in the group experiment.4.We established aging model of neural stem cell successfully,it appeared typical inflammatory changes:the capability of cell sphere-forming was decrease,the expression levels of mRNA and protein about TNF-a and IL-I10 were increased significantly,NF-?B signaling pathway was activated and the expression levels of cytoplasm P65 was increased and nuclear P65 was decreased.5.Compared with the D-galactose-induced aging model group,the ability of cell sphere-forming formation was recovered significantly(p<0.05),the expression levels of mRNA and protein about TNF-?a and IL-1? were decreased significantly and the transcriptional activity of NF-?B was inhibited in the intervention group of 90?g/mL(1/2 IC50)Qiong Yugao.Conclusion1.The expression levels of mRNA and protein about the genes of GnRHl and Sirtl had a significant difference between the aged and adult rats,it showed that the expression levels of GnRH1 and Sirtl was negatively correlated with age.2.The expression levels of mRNA and protein about DNMT1 was decreased significantly in aged rats,there was no significant difference of the expression levels of mRNA and protein about DNMT3a between the aged and adult rats,it showed that the expression levels of DNMT1 was negatively correlated with age and DNMT3a did not.3.GnRHl gene in aged rats occured partly methylated but did not-occur in adult rats,it showed that the expression levels of GnRHl gene was regulated by its gene promoter methylation,methylation of GnRHl gene promoter was associated with aging.The natural aging of rats was related to the methylation of GnRHl gene promoter and inhibited the expression of GnRH1.4.Qiong Yugao could prevent the inflammation in the D-galactose-induced aging mode of neural stem cells:recovered the ability of cell sphere-forming formation and inhibited the inflammatory index,and its effect were concentration dependent,with the concentration of Qiong Yugao from 45 g/mL to 90 g/mL,the anti-aging effect was enhanced accordingly.5.Because NF-?B has a function of negative regulation of the expression of GnRHl gene,so the possible molecular mechanisms of anti-aging in Qiong Yugao was to reduce the expression of inflammatory factors:TNF-??IL-1?in hypothalamic neuronal stem cells and the transcriptional activity of NF-?B,and then reverse the methylation of GnRH1 gene and the reduction of its expression levels of mRNA in aging process. |
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