The Effect And Mechanism Of Neferine On Mice Parkinson’s Disease Associated Inflammation Model

Objective:Parkinson’s Disease(PD)is a common neurodegenerative disease in neurology.The incidence of PD is increasing year by year in middle-aged and elderly people,and the main clinical manifestations are motor disorders and non-motor symptoms.Neurodegenerative changes in Parkinson’s disease are mainly neuroinflammatory responses.Microglia-induced inflammatory responses mediate dopaminergic neuron damage,and the pathogenesis of inflammation plays a dominant role in the development of Parkinson’s disease.Microglia are immune macrophages that mediate inflammatory response and play an important role in immune regulation in the central nervous system.Microglia are activated and secrete pro-inflammatory or anti-inflammatory factors at designated sites.Overactivated microglias release a lot of cellular inflammatory factors,such as IL-1β,IL-6 and TNF-α.LPS triggers TLR4-mediated signal transduction,which activates NF-κB and MAPK through different signaling bodies,ultimately inducing transcription of inflammatory proteins and cytokines.This inflammatory process also plays a damaging role in a variety of diseases.Microglial dysfunction contributes to brain-related dysfunction in PD patients,and in neurodegenerative diseases such as PD In AD,the overactivation of microglia is the main process of neuroinflammatory response,and is also the main factor for the pathological changes and neuroinflammation mediated by PD.The clinical treatment of PD still faces great challenges,and the selection of anti-inflammatory drugs targeting microglia may provide new theoretical basis for the treatment of PD.In order to better prevent and treat the neuroinflammatory reaction of PD in clinical practice,this project screened out the potential small molecule compounds of Traditional Chinese medicine that inhibit the inflammation of the nervous system through network pharmacology method,and established an in vitro model of LPS to induce the activation of mouse microglial cell line BV-2 cells,and screened the small molecule compounds of traditional Chinese medicine.The inhibitory effect of candidate small molecule compounds on the expression of inflammatory factors and the effect of small molecule compounds on cytotoxicity were evaluated.After screening,it was found that Neferine had a dose-dependent anti-inflammatory effect,and its mechanism of anti-inflammatory action was further studied.Because MPTP had obvious brain damage to DA neurons in C57BL/6J mice and could induce neuroinflammatory response,MPTP-induced mouse model had greater advantages as an inflammatory model compared with other models.Subacute PD model in mice was established by intraperitoneal injection of MPTP,and it was observed that MPTP could successfully induce PD behavior in animals,leading to the loss of DA neurons.Therefore,we studied the mechanism of Neferine inhibiting neuroinflammation in PD mouse models induced by MPTP in vivo.It can provide theoretical reference for the clinical prevention and treatment of PD.Methods:In the first part,Traditional Chinese Medicine Systems Pharmacology(TCMSP)https://tcmspw.com/)and ETCM(The Encyclopedia of Traditional Chinese Medicine,http://www.tcmip.cn/ETCM/index.php/Home/)predicted TCM small molecule compounds with potential effects against Parkinson’s disease and that can be used in the nervous system.An in vitro model of LPS-induced BV-2 cell activation was established,and multiple groups of repeated drug screening experiments were performed using the model and fluorescence quantitative PCR(q PCR)to find small molecule compounds that could inhibit microglia activation and secretion of inflammatory factors such as TNF-αIL-6 i NOS.In the second part,Annexin V/PI flow cytometry was used to determine the percentage of PI~+dead cells,cell viability was measured by CCK8 method,and the cytotoxicity of Neferine on BV-2 cells was evaluated.Enzyme-linked immunosorbent assay(ELISA)was used to detect the release of il-6 TNF-αin LIpopolysaccharide(LPS)induced BV-2 cells.Western Blot was used to detect the expression of i NOS in LIpopolysaccharide(LPS)induced BV-2 cells.In the third part,Western Blot was used to detect the pro-inflammatory factors to explore the mechanism of Neferine inhibiting BV-2 activation,and the nuclear protein extraction experiment was carried out to detect the entry of P65protein into the nucleus.ELISA was used to detect and compare the effect of Neferine and JSH23 inhibiting IL-6.In the fourth part,MPTP was used to construct PD animal models,and behavioral experiments of PD model mice were evaluated to observe the effects of screening drugs on behavioral symptoms of PD disease models.Western Blot was used to detect the expression of inflammatory factors in mouse brain tissues and the activation of relevant inflammatory signaling pathways.Results:1.Four traditional Chinese medicine ingredients that have not been reported about Parkinson’s disease were screened by network pharmacology method:Neferine,3,4-dimethoxybenzoic acid,Methoxsalen and Nuciferine;2.The results showed that Neferine inhibited the transcription of TNF-αand IL-6 in LPS-induced BV-2.Nuciferine did not significantly affect the transcription of TNF-αand IL-6 in LPS-induced BV-2,Methoxsalendid not significantly affect the transcription of TNF-αand IL-6 in LPS-induced BV-2 cells,3,4-dimethoxybenzoic acid slightly promoted the transcription of TNF-αand IL-6 in LPS-induced BV-2 cells,and Neferine significantly inhibited the inflammatory response of LPS-induced BV-2cells;3.The percentage of PI~+dead cells was determined by flow cytometry(Annexin V/PI method)and cell viability assay(CCK8 method).It was found that the effective anti-inflammatory concentration of Neferine had no cytotoxic effect;4.By ELISA,the secretion of TNF-αand IL-6 protein and the production of i NOS in LPS-stimulated BV-2 cells were inhibited by Neferine.It was found that Neferine and JSH23 had similar inhibitory effect on IL-6 production;5.Bv-2 cells were pretreated with DMSO and Neferine,then treated with LPS for 0 h,2 h and 4 h,respectively.The activation of NF-κB and MAPK signal in BV-2 cells was detected by Western Blot,and the nuclear protein was extracted and the entry of P65 protein was detected.It was found that Neferine inhibited the nucleation of P65,similar to JSH23 inhibiting IL-6production.Neferine inhibited LPS-induced BV-2 inflammatory cytokines by inhibiting the activation of NF-κB signal;6.By examining the behavioral experiments in mouse models and the expression of inflammatory factors in the substantia nigra and the activation of related inflammatory signaling pathways,it was found that Neferine can improve the movement disorders in MPTP-induced PD mice.The activation of NF-κB and the number of i NOS andα-Syn in the substania nigra of MPTP+Neferine mice were significantly decreased compared with that of MPTP alone,suggesting that Neferine improved neuroinflammation in MPTP-induced PD mice by inhibiting NF-κB signaling pathway.Conclusion:1.Neferine inhibited the activation of microglia,the secretion of TNF-αand IL-6 and the production of i NOS by inhibiting NF-κB signaling pathway;2Neferine improved motor disorders in MPTP-induced PD mouse models,and improved neuroinflammation in MPTP-induced PD mouse models;3.Neferine has the potential to become an effective drug for alleviating neurodegenerative diseases such as Parkinson’s disease.