Effects Of Triptolide And Tripchlorolide On The Expression Of Neuroligin-1 In Hippocampus Of APP/PS1 Transgenic Mice And Their Epigenetic Regulatory Mechanisms

Object:Alzheimer’s disease(AD)is a degenerative disorder of the central nervous system characterized by progressive cognitive impairment and behavioral impairment,and is the most common type of dementia in the elderly.The typical histopathological changes of AD were neuritic plaques,neurofibrillary tangles,synaptic loss and gliosis.The main component of neuritic plaque is β-amyloid protein(Aβ),which activates microglia in the brain to secrete and release various immunoinflammatory mediators,leading to synaptic structure and function damaged.The specific mechanism of this role is still unclear.The study found that Aβ-induced neuroinflammation can reduce the expression of NLGN1 by affecting the epigenetic modification of NLGN1,and ultimately damage the structure and function of synapses.NLGN1 is a cell-adhesion molecule located on the excitatory postsynaptic membrane and plays a crucial role in the regulation of synaptic formation,development,maturation and synaptic plasticity.Dysfuntion of NLGN1 is closely related to the pathogenesis of AD.Epigenetics refers to stable and heritable changes in gene expression without altering the nucleotide sequence.Its major regulatory mechanisms include DNA methylation,histone modification,and microRNA interference.Triptolide(T10)is a diterpene triepoxide isolated from the traditional Chinese herb Tripterygium Wilfordii Hook F and has potent anti-inflammatory and immunosuppressive effects,and also has epigenetic modification.Tripchlorolide(T4)is a derivative of T10 and has stronger anti-inflammatory and immunosuppressive effects than T10,but its toxicity is lower than T10.Evidences have shown that T10 and T4 can inhibit neuroinflammation in the brain of AD model animals,improve the learning and memory ability of AD model animals,and have a clear synaptic protective effect.However,the mechanisms by which T10 and T4 play their role in synaptic protection remain unclear.This study investigated the synaptic protection of T10 and T4 against AD from the perspective of the epigenetic regulation of NLGN1 in the AD model.Methods:1.This study used APP/PS1 double transgenic AD model mice as an animal model of AD.The double transgenic AD model mice were randomly divided into three groups: AD model group,T10 treatment group,and T4 treatment group,with 20 rats in each group.Twenty wild-type female mice of the same type were used as a control group.2.T10 treatment group was intraperitoneally injected with T10 0.1 mg/kg(T10 dissolved in 1% DMSO saline),and T4 treatment group was intraperitoneally injected with T4 25 μg/kg(T4 dissolved in 1% DMSO saline).A total of 60 days were administered.3.Immunofluorescence staining,western blot,real-time,qPCR were used to detect the expression of NLGN1 in hippocampal neurons of each group of experimental animals.To observe the effect of T10 and its derivative T4 on the expression of NLGN1 in hippocampal neurons of AD animal models.4.The expressions of HDAC2 and MeCP2 in hippocampal neurons of each group were detected by western blot and qPCR.The effects of T10 and its derivative T4 on the expression of HDAC2 and MeCP2 in hippocampal neurons of AD animal model were observed.5.The interaction between HDAC2 and MeCP2 in hippocampal neurons of experimental animals was detected by CoIP.The effects of T10 and its derivative T4 on the interaction of HDAC2 and MeCP2 in hippocampal neurons of AD animal model were observed.6.MeDIP was used to detect the expression of MeCP2 in the NLGN1 promoter region of hippocampal neurons in each group of experimental animals,and the effect of T10 and its derivative T4 on methylation of NLGN1 promoter region in hippocampal neurons of AD animal model was observed.7.ChIP was used to detect the expression of HDAC2 and histone acetyltransferase 3 in the NLGN1 promoter region of hippocampal neurons in each group,and the effect of T10 and its derivative T4 on acetylation of NLGN1 promoter region in hippocampal neurons of AD animal model was observed.Results:1.The protein expression of NLGN1 in hippocampus of AD model mice was significantly decreased compared with control mice.T10 and T4 up-regulated the expression of NLGN1 in AD model mice.2.Compared with the control mice,the protein expression of HDAC2 in the hippocampus of AD model mice was significantly increased.T10 and T4 inhibited the expression of HDAC2 in the APP/PS1 double transgenic AD model mouse.3.The protein expression of MeCP2 in hippocampus of AD model mice was significantly higher than that of the control mice.T10 and T4 inhibited the expression of MeCP2 in the APP/PS1 double transgenic AD model mouse.4.Compared with the control mice,the formation of HDAC2-MeCP2 complex in the hippocampus of AD model mice was significantly increased,while T10 and T4 inhibited the formation of HDAC2-MeCP2 complex in APP/PS1 double transgenic AD model mice.5.Compared with control mice,AD model mice hippocampal NLGN1 promoter region MeCP2 and the expression of HDAC2 was significantly increased,while T10 and T4 inhibited the expression of MeCP2 and HDAC2 in the NLGN1 promoter region of the APP/PS1 double transgenic AD model mouse.6.Compared with the control mice,the histone H3 acetylation in the NLGN1 promoter region of the hippocampus of AD model mice was significantly decreased,while the T10 and T4 enhanced the expression of histone H3 acetylation in the NLGN1 promoter region of APP/PS1 double transgenic AD model mice.7.Compared with the control mice,cytosine methylation in the NLGN1 promoter region of the hippocampus of AD model mice was significantly increased,while T10 and T4 decreased cytosine methylation in the NLGN1 promoter region of APP/PS1 double transgenic AD model mice.Conclusions:1.T10 and T4 have the effect of increasing the expression of NLGN1 in hippocampal neurons of AD model mice.2.T10 and T4 have the effect of inhibiting the expression of HDAC2 and MeCP2 in hippocampal neurons of AD model mice.3.T10 and T4 have the effect of inhibiting the binding of MeCP2 and HDAC2 in hippocampal neurons of AD model mice.4.T10 and T4 have the effect of inhibiting the expression of MeCP2 and HDAC2 in the NLGN1 promoter region of hippocampal neurons in AD model mice.5.T10 and T4 have the effect of increasing the expression of histone H3 acetylation in the NLGN1 promoter region of hippocampal neurons in AD model mice.6.T10 and T4 have the effect of inhibiting cytosine methylation in the NLGN1 promoter region of hippocampal neurons in AD model mice.