Study On The Neurotoxic Material Basis Of Strychnos Alkaloids And Prophylactic Neuroprotection Of Total Glucosides Of Paeoniae Radix Alba

Semen Strychni,the mature seed from Strychnos nux-vomica L.,has a wide range of clinical application to treat rheumatism,facial paralysis,and tumors and is listed in Chinese pharmacopeia.Ancient literature records and modern clinical applications indicate that misuse or overdose of strychnine may cause severe neurotoxicity.Due to the narrow margin between effective and toxic doses,neurotoxicity induced by SAs is likely to occur when exposure to a high dose of Semen Strychni,which restricts the clinical application of Semen Strychni.Studies showed that Strychnos alkaloids(SAs)are responsible for the pharmacological and toxic properties of Semen Strychni.Total glucosides of Paeoniae Radix Alba(TGP)are a class of active compounds extracted from the roots of Paeonia lactiflora Pall.,and increasing reseaches confimed that TGP have a significant characteristics of neuroprotective effects.Therefore,aimed at the toxicity of SAs,in vivo and in vitro toxicity evaluation models were established to study the neurotoxicity of SAs and interventional effects of TGP in the present paper.The present study will provide beneficial information for the safe use of Semen Strychni and developing herbal medicines to alleviate neurotoxicity in the clinic.1.Evaluation of neurotoxicity of SAs and intervention effect of TGP based on animal toxicity modelsA toxicity model was established in SD rats to evaluate the neurotoxicity of SAs and prophylactic neuroprotection of TGP at different dose(125 mg/kg/day and 250 mg/kg/day)on the toxicity caused by SAs.Physical and behavioral testing,biochemical assay of the brain,histological examination and fluorescent staining were selected as the indicators of toxicity.Results of physical and behavioral testing showed that continuous administration of SAs(12 mg/kg/day)for 15 days could reduce body weight significantly,deteriorate rats’ appearance,decrease the amount of autonomous exploration activities,and impair the motor coordination.The behavioral results of rats in the SAs group were consistent with that in the positive control group of neurotoxicity,which confirmed the neurotoxicity of SAs preliminarily.However,TGP at a dose of 250 mg/kg/day could reduce the rats’ behavior abnormalities significantly and show neuroprotective effects against the toxicity caused by SAs.Results of biochemical assay involved in oxidative damage indicated that SAs could inhibit the activities of superoxide dismutase(SOD)and catalase(CAT),decrease the levels of glutathione(GSH)and total antioxidant capacity(T-AOC),and lead to the overload of reactive oxygen species(ROS)significantly.It suggested that the neurotoxicity of SAs may be related to oxidative stress.Pretreatment with a high dose of TGP increased the activities of SOD and CAT and the levels of GSH and T-AOC,reduced the overproduction of ROS,and relieved the oxidative stress in the brain significantly.Histopathological sections and tissue fluorescent staining results showed that SAs could cause the disorder of lipid metabolism,lead to apoptosis of tissue cells and caused brain injury.Pretreatment with a high dose of TGP decreased excessive lipid metabolism significantly,reduced apoptosis of tissue cells and result to attenuate brain injury.The neurotoxicity of SAs was confirmed on the basis of the above results and their neurotoxicity may be involved in the oxidative damage.In addition,TGP showed prophylactic neuroprotection against the neurotoxicity of SAs via anti-oxidative damage.2.Identification of SAs extract and the constituents absorbed into blood and the brain after oral administration of SAs.A Q Exactive Quadrupole-Orbitrap Mass Spectrometer high-resolution spectrometer was applied to conduct SAs extract,the constituents absorbed into blood and the brain as well as their metabolites in rat after oral administration of SAs.A total of 15 SAs were accurately identified in vitro,following relevant literature and the lysis rules of reference substances.Based on the theory of serum pharmacochemistry,the constituents absorbed into blood and the brain were identified as well as their metabolites.In comparison with blank serum and brain samples,a total of 7 alkaloids from SAs and 21 metabolites were identified in the serum.3 alkaloids,including strychnine,brucine,strychnine-N-oxide,were identified in brain tissue.No metabolites were found in the brain.This study revealed that strychnine,brucine and strychnine-N-oxide are the material basis for neurotoxicity induced by Semen Strychni and lay a foundation for further studies on the toxicity of Semen Strychni.3.Tissue time-course of strychnine and brucine following oral administration of SAsA high-performance liquid chromatography coupled with mass spectrometry method(HPLC-MS/MS)was developed to explore the tissue time-course of strychnine and brucine in brain,the effects of pretreatment with TGP on which were investigated.Chromatographic separation was carried out on a CAPCELL CORE ADME column(2.1 × 150 mm,2.7 μm,Shiseido,Japan).Acetaminophen was selected as an internal standard.The mobile phase consisted of acetonitrile and 0.1%formic acid in water.Acetonitrile precipitated protein method was used for sample preparation.For strychnine and brucine,good linearity of the method was achieved within the tested range.Selectivity,intra-and inter-day precision and accuracy,recovery,matrix effect,and stability met the accepted requirements for quantitation in biological samples.The method was applied successfully to quantitation of strychnine and strychnine in the rat brain.Results showed that strychnine and brucine were absorbed rapidly after oral administration of SAs in rats.They could cross the blood-brain barrier(BBB)and enter in the brain at 15 min and their concentrations reached the maximum at 30 min.Strychnine and brucine were eliminated at 30 min after oral administration of SAs.It suggested that the absorption and elimination behavior of strychnine and brucine was similar.The areas under the concentration-time curve of strychnine and strychnine were smaller in TGP group than those of SAs group,which indicated that pre-treatment with TGP could reduce the content of strychnine and strychnine in the brain.The above experimental results can provide useful information for clinical safty of Semen Strychni and treatment course.4.Study on the brain permeability of strychnine and brucine in Semen Strychni,Brain-to-plasma ratio is the ratio of drug concentration in the brain to drug concentration in the plasma and is used to assess the ability of the drug to cross the BBB.An HPLC-MS/MS was applied to determination of the concentration of strychnine and strychnine in rat plasma and brain.Then the brain-to-plasma ratios of strychnine and brucine were calculated and 1.40±0.13 and 0.52± 0.12,respectively.It indicated that strychnine and brucine has low brain-to-plasma ratio and their capacity to cross the BBB was limited.Strychnine has a higher brain-to-plasma ratio than brucine,which indicted that strychnine was more capable to cross the BBB.In addition,compared to the SAs group,pretreatment with TGP could reduce the brain-to-plasma ratio of strychnine and reduce its ability to pass through the BBB,and had no significant effect on the brain-to-plasma ratio of brucine.5.Study on the neurotoxicity of SAs and prophylactic neuroprotection of TGP based on animal toxicity modelsPC 12 cells were incubated in the culture medium containing SAs for 24 h.The cell morphology,cell viability,biochemical assay,and apoptosis rate were used as toxicity indicators to establish the toxic cell model to evaluate the neurotoxicity of SAs and prophylactic neuroprotection of TGP.The experimental results showed that PC12 cells became rounded,shrunk,and the cell viability was decreased significantly after 24 h incubation in the medium containing SAs;the activities of SOD and CAT were inhibited,and the levels of ROS,GSH,T-AOC,SOD and MDA were abnormal.Increased apoptosis rate indicated that SAs could cause oxidative damage to cells;pre-treatment for 24 h could significantly reduce the oxidative damage caused by SAs and showed protective effects.Western blotting and PCR analysis showed that the Nrf2 and HO-1 proteins were significantly up-regulated after SA exposure.At the same time,TGP up-regulated the expression of Nrf2 and HO-1 genes in brain tissue significantly.to counter the toxicity of SAs.The results showed that pretreatment with TGP can activate the Nrf2/ARE signaling pathway to exert its prophylactic neuroprotection against the toxicity induced by SAs.