PPARβ Mediates Mangiferin-induced Neuronal Differentiation Of Neural Stem Cells Via Regulating DNA Demethylation

Research BackgroundNeural stem cells(NSCs)are cells with neurogenesis potential.Which mainly exist in the lower granular layer of hippocampal dentate gyrus(SGZ)and the subventricular zone(SVZ).Neural stem cells can differentiate into neurons and glial cells under certain stimulus conditions,Which can repair the injury of the nervous system.The proliferation and differentiation of NSCs are regulated by internal and external factors.The internal factors include DNA methylation,and the external factors include drug effects.Mangiferin(MGF,chemical formula: C19H18O11)has some biological activities,including anti-oxidative stress,anti-tumor,anti-diabetes,anti-inflammatory,cell protection and other effects.Mangiferin can protect the central nervous system to reduce damage,in vivo and in vitro,and improve the cognitive and memory functions in mice.The effect of mangiferin on neural differentiation and its mechanism are still unclear.Peroxisome proliferation-activated receptors(PPARs)are nuclear receptors.Many studies have reported that mangiferin acts on this receptor.PPARs expressed in the brain.PPARβ agonists can reduce brain neuroinflammation in animal models.Neurodegeneration,amyloid load,and improve cognitive function.PPARs are involved in the DNA methylation process,DNA methylation is a common epigenetic modification.DNA methylation is mainly catalyzed by the DNA methyltransferase family(DNMT1,DNMT3 a,DNMT3b),and the TET protease family(TET1,TET2,TET3)is involved in the process of DNA demethylation.DNA methylation/demethylation modification plays a vital role in the development and function of the nervous system.MASH1(alternative names: ASCL1,ASH1,HASH1)is a pre-neuronal gene whose encoded protein is a transcription factor,belonging to the basic helix-loop-helix(b HLH)structure,which plays a role in the early development of specific neural lineages and participates in Initiation of neuronal differentiation.Research AimsTo investigate whether there is a causal relationship between PPARs and DNA demethylation,and if so,whether this relationship is involved in mangiferin induced the differentiation of neural stem cells into neurons.Research MethodsPart one: After culturing and treating the cells,IF and Ed U experiments were used to observe the effect of mangiferin on the differentiation and proliferation of neural stem cells.WB experiment observed the concentration and time dependence of mangiferin on the expression of PPARs protein.IF and Ed U experiments observed the effect of PPARβ lentivirus on the differentiation and proliferation of neural stem cells,and the effect of neuron differentiation-related gene Mash1.Part two: WB experiment detected the concentration and time dependence of mangiferin on TETs protein,and DB experiment verified the change of 5-hm C expression.WB detects the expression of TET1 protein after PPARβ lentivirus interference,and IF observe the effect of PPARβ lentivirus interference on 5-hm C expression.Part three: The WB experiment detected the concentration and time dependence of mangiferin on the protein expression of DNMTs,and the DB experiment verified the change of 5-m C expression.WB was used to detect DNMT3 a protein expression after PPARβ lentivirus interference,and IF was used to observe the effect of PPARβlentivirus interference on 5-m C expression.Research ResultsPart one: 1.The results of IF showed that the expression of Tuj1(neurons)in the mangiferin drug concentration gradient group gradually increased and the expression of GFAP(astrocytes)gradually decreased compared with the blank control group,in a concentration-dependent manner.Ed U results showed that the number of Ed U positive cells in the mangiferin drug group were gradually increased compared with the blank control group,which was concentration-dependent.2.WB results showed that PPARβ gradually increased in the mangiferin concentration gradient group compared with the blank control group in a concentration-dependent manner,while PPARα and PPARγ had no significant changes.Compared with the control group,the expression of PPARβ in the time gradient group of mangiferin decreased after reaching a peak at 9h,but there was no significant change in PPARα and PPARγ.3.The q PCR results showed that the expression of PPARβ m RNA in the sh RNA1 group and the sh RNA2 group was lower than that of the Vector group,and the sh RNA2 group had better knockdown efficiency than the sh RNA1 group.4.The IF results show that the Vector+MGF group has more neurons and fewer astrocytes than the Vector group,and the sh RNA group has fewer neurons and more astrocytes than the Vector group.The sh RNA+MGF group is more than Vector+MGF.The number of neurons decreased and the number of astrocytes increased.Ed U results showed that the number of positive cells in the Vector+MGF group was increased compared to the Vector group,the number of positive cells in the sh RNA group was reduced compared to the Vector group,and the number of positive cells in the sh RNA+MGF group was reduced compared to the Vector+MGF group.5.IF results showed that the expression of Mash1 in the Vector+MGF group was increased compared with the Vector group,the expression of Mash1 in the sh RNA group was decreased compared with the Vector group,and the expression of Mash1 in the sh RNA+MGF group was decreased compared with the Vector+MGF group.The results of QPCR and WB are consistent with the results of IF.Part two: 1.WB results show that TET1 in the mangiferin group was gradually increased compared with the blank control group,while TET2 and TET3 did not change significantly.Compared with the control group,the expression of TET1 in the time gradient group of mangiferin decreased after reaching a peak at 12 h,but there was no significant change in TET2 and TET3.2.The results of DB showed that5-hm C in the mangiferin concentration gradient group gradually increased compared with the blank control group,which was concentration-dependent.3.WB results showed that the expression of TET1 in the Vector+MGF group was increased compared with the Vector group,the expression of TET1 in the sh RNA group was decreased compared with the Vector group,and the expression of TET1 in the sh RNA+MGF group was decreased compared with the Vector+MGF group.There is no significant difference between them.The result of IF is consistent with the result of WB.4.The IF results showed that the expression of 5-hm C in the Vector+MGF group was increased compared to the Vector group,the expression of 5-hm C in the sh RNA group was decreased compared to the Vector group,and the expression of 5-hm C in the sh RNA+MGF group was decreased compared to the Vector+MGF group.Part three: 1.The results of WB showed that the DNMT3 a of the mangiferin concentration gradient group gradually decreased compared with the blank control group,which was concentration-dependent,while DNMT1 and DNMT3 b had no significant changes.Compared with the control group,the mangiferin time gradient group showed that the expression of DNMT3 a gradually increased after reaching the lowest value at 12 h,but there was no significant change in DNMT1 and DNMT3 b.2.The results of DB showed that the 5-m C of the mangiferin concentration gradient group was gradually reduced compared with the blank control group,which was concentration-dependent.3.WB results showed that the expression of DNMT3 a in the Vector+MGF group was reduced compared to the Vector group,the expression of DNMT3 a in the sh RNA group was increased compared to the Vector group,and the expression of DNMT3 a in the sh RNA+MGF group was increased compared with the Vector+MGF group.There is no obvious difference between DNMT1 and DNMT3 b.The result of IF is consistent with the result of WB.4.The IF results showed that the5-m C expression in the Vector+MGF group was reduced compared to the Vector group,the 5-m C expression in the sh RNA group was increased compared to the Vector group,and the expression of 5-m C in the sh RNA+MGF group was increased compared to the Vector+MGF group.Research Conclusion1)Mangiferin promotes the differentiation of neural stem cells into neurons through PPARβ.2)PPARβ regulates the expression of TET1 and participates in the differentiation of neural stem cells induced by mangiferin.3)PPARβ regulates the expression of DNMT3 a and participates in the differentiation of neural stem cells induced by mangiferin.