Study On The Key Targets And Mechanism Of Curcumin Inhibition Abnormal Neurogenesis In Epilepsy

BackgroundThe causes of epilepsy are complex and numerous,but all are closely related to transient dysfunction triggered by abnormal neuronal discharges in the brain.Currently,there are more than 70 million patients with epilepsy worldwide,and the treatment plan focuses on controlling seizures and reducing neurological damage as soon as possible,but there are problems such as side effects and easy drug resistance,and about 20-25%of patients eventually develop refractory epilepsy due to drug resistance.Recurrent seizures can exacerbate neuronal damage and cause excessive neural stem cell repair,as well as lead to ectopic nerve repair and create abnormal neural loops,which can lead to focal epilepsy or generalized seizures.The application of TCM discriminatory treatment or combined with Western medicine has sufficient evidence of efficacy and low side effects,but often attributes its mechanism of action to multiple components and multiple targets.Due to the lack of identification methods for core targets of action,it is difficult to determine the link of onset of action and elucidate its exact molecular mechanism,which affects the scientific evaluation and development and utilization of TCM.Target discovery and identification are key to constructing correlations between chemical components and pharmacological effects.Traditional target screening methods are limited by the complexity of the pharmacodynamic components of Chinese medicine,and target discovery has become a bottleneck in the study of the mechanism of action of Chinese medicine.In the preliminary data mining,it was found that phlegm-transforming drugs such as E zhu,Yujin,and Shi changpu,which are frequently used in the treatment of epilepsy,all contain curcumin,which is now widely reported to have better efficacy in epilepsy.Curcumin is a polyphenolic compound that has been shown in several clinical studies to have a high safety profile and has shown good efficacy in the treatment of inflammatory diseases,tumors,neurological disorders,and acute and chronic epilepsy,but the targets and exact mechanisms of its efficacy are not yet clear.To investigate the molecular mechanism of curcumin inhibition of abnormal neurogenesis,based on the public database data acquisition method,we combined Affinity Chromatography and Drug Affinity Responsive Target Stability(DARTS),We also analyzed the key pathways using bioinformatics to explore the role of curcumin in the early stages of epilepsy and the possible targets and pathways.PurposeUsing DARTS and affinity chromatography,we analyzed the key targets and possible pathways of curcumin to inhibit abnormal neurogenesis of seizures in the epileptic pathological process,established the screening method for the key targets of curcumin action in epilepsy,and explored the possible molecular mechanisms.Methods1.Mouse strains were selected to establish epileptic mouse models.To simulate the pathological state of recurrent seizures,the susceptibility of mice to epilepsy was evaluated by comparing the epileptogenic mechanisms of pentylenetetrazol and pilocarpine,and the final modeling conditions were selected as follows:ICR mice were given an intraperitoneal injection of scopolamine(1.5 mg/kg)30 min before intraperitoneal injection of pilocarpine(290 mg/kg).Mice in status epilepticus(SE)for 2 h were given an intraperitoneal injection of diazepam(10 mg/kg)to terminate the seizures.2.The herbal formulas were divided and reduced,and the different formulas were used to treat mice with epilepsy,and the seizure level was evaluated by observing the degree of seizures in mice.The efficacy between the different formulae was evaluated by immunofluorescence staining using transcardial perfusion.3.Public databases were used to obtain and screen disease targets of epilepsy and drug targets of curcumin,and the data were preprocessed to analyze and construct curcumin-epilepsy disease-target-core pathway network relationships.4.The target protein of curcumin was captured by affinity chromatography by coupling curcumin to epoxy-activated agarose gel 6B and using it as a stationary phase.The protein samples eluted by the gradient were detected by silver staining and identified by mass spectrometry after successful sample preparation.5.The DARTS technique was used to screen the differential proteins by Kaumas Brilliant Blue staining and cut gels for mass spectrometry analysis,using the principle that the binding of small molecules and proteins increases the stability of protein folding and has resistance to protease hydrolysis.6.The obtained targets were integrated and analyzed by proteomics and bioinformatics to obtain the core targets of curcumin action in epilepsy.And the above methods were compared.Result1.It was clarified that curcumin,as the active component of Yujin,is the main component of the formula of Longmu baijin to exert its medicinal effect.To identify the key effective drugs in the LMBJ formula,we disassembled it,which was specifically divided into LMBJ formula:composed of sheng long chi,sheng mu li,sheng bai fan,yu jin.LMB formula:composed of sheng long chi,sheng mu li,sheng bai fan.BJ formula:composed of sheng bai fan,yu jin.B formula:composed of sheng bai fan.And nano-Curcumin.After staining and comparison,it was found that LMB formula without turmeric had no therapeutic effect as B formula and epilepsy group.LMBJ group,BJ group and Nano-curcumin group can inhibit epileptic neurogenesis,but BJ group may have some side effects,and the survival rate of mice was significantly lower than other groups.Therefore,as an effective substance of turmeric,curcumin is the main component of the prescription of dragon peony platinum.2.Using the public database data acquisition method(hereafter referred to as network pharmacology),440 potential targets of action were predicted,51 core targets,involving 494 GO biological processes and 133 KEGG signaling pathways.The analysis of GO and KEGG results mainly focused on excitatory and inhibitory neurogenic,mostly around synaptic signaling(Figure 3.1.3).The analysis may be explained by the fact that the data were obtained from publicly published data in databases,which tends to reflect the fact that most scholars’ hotspots of epilepsy disease research stay in the hypothesis of excitatory and inhibitory neuronal imbalance.The disadvantage of network pharmacology based on public data is that the experimental results are concentrated in one research direction,making it difficult to propose innovative ideas and discover new mechanisms.3.Seventy-three core targets were screened using affinity chromatography,involving 191 GO biological processes and 50 KEGG signaling pathways.In order to investigate the new targets and pathways of curcumin in epilepsy treatment,this study obtained 164 proteins in the control group and 116 proteins in the curcumin group by mass spectrometry using affinity chromatography,and 55 proteins were shared between the two groups.Among the shared proteins,12 proteins with PSMs values greater than 1.5 times that of the control group and 61 proteins unique to the curcumin group(73 in total)were enriched to obtain 35 annotations of cell composition(CC),25 annotations of molecular function(MF),131 annotations of biological processes(BP),and a total of 50 annotations of the KEGG signaling pathway that may be involved in curcumin treatment of epilepsy(Figure 3.1).A total of 50 annotations were obtained from the KEGG signaling pathway enrichment analysis(Figure 3.2.3).The results of the analysis were more diverse than those of the network pharmacology,except for the imbalance of synaptic signaling,mainly involving Neurotrophin Signaling Pathway,Pyruvate Metabolic Process,Scaffold Protein Binding,etc.4.212 core targets were screened using DARTS,involving 214 GO biological processes and 46 KEGG signaling pathways.In order to obtain relatively real,fair and reliable experimental data and to compensate for some shortcomings of the affinity chromatography experiments,in this study,717 proteins in the control group and 700 proteins in the curcumin group were obtained by mass spectrometry analysis using the DARTS technique,with a total of 618 proteins in both groups,and we enriched 130 proteins with MS/MS ratios greater than 1.5 times the control group from the total proteins in both groups and The results of GO and KEGG results analysis were mainly focused on the roles of cell motility and adhesion related to cellular energy metabolism(Figure 3.3.3).5 Protein-protein interaction analysis was performed on the enriched proteins under the key pathways obtained by both affinity chromatography and DARTS technology,and a comprehensive study was conducted in combination with expert workshops to obtain Laminin,Mapk3,Pik3cb,Pdpkl,Pik3c3,Crk,Mapk10,Acan,Ncan,Haplnl,Rps6ka2,Pebp1,Ppp2r1 aA key targets 14,and then deep literature study and analysis,identified Laminin as one of the key characterization proteins of the extracellular matrix,and further experimental study using immunofluorescence staining to verify its role in neurogenesis.Conclusion1.The study established an epileptogenic mouse model and conducted an in-depth study on the role of epileptogenic formula and curcumin,the main component of Yujin,in abnormal neurogenesis of hippocampal neurons in epileptic mice,and found that curcumin is an important core pharmacological component of epileptogenic formula with inhibitory effect on abnormal neurogenesis in post-epileptic brain.2.Experiments were conducted to identify the core targets of curcumin’s action in inhibiting abnormal neurogenesis after epilepsy using DARTS coupled with affinity chromatography.The obtained data were resolved by GO and KEGG enrichment analysis,protein-protein interaction network(PPI),and verified by immunofluorescence staining experiments,which revealed the extracellular matrix protein complex as the core target of curcumin action.