Neurotoxicity And Neuroprotection As Well As Metabolic Differences Of Gelsemium Alkaloids

Gelsemium elegans is a species belonging to genus Gelsemium of Marchenaceae,which has anti-inflammatory,analgesic,anti-anxiety,anti-tumor and immune-enhancing effects.Although G.elegans is toxic to human beings,beneficial effects including weight gaining and growth promoting in animals like pig,cow and sheep were found.The major active ingredients of G.elegans are indole alkaloids.To date,studies showed that different alkaloids have diverse toxicity and pharmacologic effects.Most of indole alkaloids function in nervous system,yet there are few comparative studies on the neural effects of different alkaloids.Alkaloids with mild toxicity effects such as koumine and gelsemine have promising anti-anxiety functions.Long-term anxiety could be a trigger for mild cognitive impairment,however it is unclear whether alkaloids of G.elegans have cognitive-promoting effects in anti-anxiety process.This study aims to explore the cognitive-promoting effect of alkaloids,to understand protective and toxic effects of different alkaloids on diverse nerve cells,and to clarify the different metabolic processes of alkaloids on different animal species.We first studied neuroprotective and toxic effects of alkaloids on SH-SY5Y and PC12 cell lines,and then further explored the effect of alkaloids on spatial cognition,learning,memory in mice.Finally,from the perspective of HPLC-QqTOF-MS based comparative metabolism,we studied the differences of metabolic processing of alkaloids in liver microsomes of human,pig,rat and goat.The major results are as follows:1.The treatment of alkalodis on PC12 cells showed that the viabilites of poorly differentiated PC12 cells treated by koumine,gelsevirine,gelsemine and gelsenicine were all higher than 90%and that of the control,together with the IC₅₀ values all higher than 100μM.In contrast,the lowest viability of poorly differentiated PC12 cells was found in furanokoumine treatment,which was also higher than the control.After the high differentiation of PC12 was induced by NGF,furanokoumine significantly reduced the cell viability with the IC₅₀ value at the concentration of 31.6μM,and the other alkaloids had no obvious toxicity.We further used flow cytometry to detect the apoptosis effect of 25μM furanokoumine on PC12 cells.Compared with the control,25μM furanokoumine did not cause a large amount of apoptosis in PC12 well-differentiated cells.Our fluorescence quantitative PCR detecting the effect of furanokoumine on several toxicity and necrosis-related genes in PC12 cells indicated that expressions of toxic-responsive genes such as Egln3,Cd8b,Camk2a,Noslap and Bik were increased,while other toxic-responsive genes such as Notch4,SIc16a3,Tacrl and Htr3a were down-regulated.It is also showed that necrosis-related genes including Bnip3l,FUS,COMMD4,RIPK1/K3 were up-regulated,while other necrosis-related genes like Tnfrsf4,Atp6vlg2 and Foxil were down-regulated.Moreover,the gene expression results manifested that many genes related to cellular immune response,cell repair,programmed death,autophagy,mitochondrial damage were also altered during furanokoumine treatment,indicating that furanokoumine induced cytotoxicity is not only through pathways of apoptosis,but also other biological processes such as channels and neurotransmitters might be involved in.2.Furanokoumine significantly reduced SH-SY5Y cell viability at the concentration of 0.1μM,while gelsenicine displayed cytotoxicity at the concentration of 50μM.Significant cytotoxicity of koumine and gelsemine were observed at the concentration of 100μM,and similar effects were found for gelsevirine at 250μM.We further used SCOP-induced damage of SH-SY5Y cells as model to test protective effects of different alkaloids.Our results suggested that gelsemine and gelsenicine had protective effects in this model at the concentrations of 10μM and 5μM,respectively,with a significant increase in cell viability,but koumine and gelsevirine didn’t.The results of flow cytometry showed that co-treatment of cells with 50μM gelsemine and SCOP could enhance the apoptosis and necrosis induced by SCOP,similar results were also found in cases of gelsenicine and gelsevirine,but not in koumine.3.Mice were continuously injected with koumine（10 mg/kg）or gelsenicine（20μg/kg）by intraperitoneal injection for 12 days.From the 8th day on,1 hour after administration,3mg/kg scopolamine was intraperitoneally injected every day for 4 days,to construct a mouse model of mild cognitive impairment.The results of the water maze positioning navigation experiment indicated that the escape latency of the model was prolonged compared with the control,and the escape latency of the koumine treated group was shortened compared with the model,and both had significant differences;the results of space exploration showed that the times and distance of the mice in the model crossed the platform were significantly less than those in the control,while the movement distance of the koumine treated group was significantly shorter than that of the model.The comparison between HE staining for the hippocampus and the control demonstrated that the cells in the CA1 and CA3 areas of the hippocampus solidified and atrophied effects of koumine treated mice;the number of viable cells in the CA3 and PC areas decreased and necrotic cells were increased in case of koumine treated group.Taken all the results above into consideration,it is concluded that koumine could improve scopolamine-induced mild cognitive impairment and memory ability in mice,however,gelsenicine did not have protective effects on scopolamine-induced mild cognitive impairment and could damage the hippocampus of mice.4.Drug metabolism is closely associated with pharmacological and toxicological effects.This study represents the first report of the metabolic differences of koumine,gelsemine and gelsevirine in liver microsomes of different animals.Based on the technology of HPLC-QqTOF-MS,we found that the major metabolic processing of koumine in liver microsomes were oxidation,reduction,dehydrogenation and demethylation,resulting in metabolites of eight,six,six and seven koumines in mice,human,goat and pig,respectively.The major metabolic processes of gelsemine in liver microsomes were oxidation,dehydrogenation and demethylation,resulting in metabolites of three,four,three and four gelsemines in mice,human,goat and pig,respectively.The major metabolic processes of gelsevirine in liver microsomes were hydroxylation,hydrogenation,dehydrogenation,and demethylation,resulting in metabolites of three,four,three and four gelsevirines in mice,human,goat and pig,respectively.The same alkaloid had similar metabolic processing pathways among different liver microsomes,slightly different amounts of produced metabolites,but with clear different metabolisms.This study indicated that gelsenicine had no improvement on promoting cognition in animals,however it did protective effects on SCOP-induced SH-SY5Y cells,yet koumine had protective effects on SCOP-induced SH-SY5Y cells and could improve promoting cognition.Alkaloids of G.elegans in general had lower toxicity effects on tested cell lines,and there were variations in their toxicity effects and metabolic products.Different alkaloids were metabolized differently by incubating with different species of liver microsomes.This research improves the basic study of the substance action of G.elegans and provides effective support for the elucidation of the pharmacological and toxicological mechanisms of G.elegans,which is of great significance for the further clinical development and use of G.elegans drugs.