BACKGROUND:Toosendanin(TSN),a triterpenoid derivative extracted from the bark of Melia toosendanin Sieb et Zucc,has been demonstrated to be an effective drug for treatment of experimental botulism.OBJECTIVE:To explore its antibotulismic mechanism by observing the effect of toosendanin on association of botulinum toxin(BoTx)with rat brain synaptosomes under different conditions.DESIGN:A randomized controlled experiment.SETTING:Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences.MATERIALS:Sprague-Dawley rats,weighing(220±12)g,were involved.TSN was a sample recrystallized in ethanol with a purity＞98%.BoTx/A and BoTx/C of 500 ku(1.8x107mouse LD50/mg)were purchased from Wako(Japan),Horse antitoxins to BoTx/A and/C(pdmary antibodies)were purchased from Lanzhou Institute of Biological Products(China).METHODS:Major experiments were finished between March 2005 and October 2005 in key laboratory of neurobiology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences.①Preparation of synaptosome of rats:one aliquot of synaptosome was pre-incubated with TSN(17μmol/L)for 20 minutes at 4℃or 37℃(another aliquot of synaptosome which was untouched was used as control).Then,BoTx was respectively added for another 20-minute incubation.②A homochronous experiment was still Derformed at 37℃.Differently,high level of K+ was used to stimulate synaptosome for 25 minutee.Two aliquots of synaptosomes with or without TSN(17 μmol/L)were preincubated for 15 minutes.Then,30 mmol/L KCl was separately added in two aliquots,5 minutes later,13 nmol/L BoTx/C was separately added followed by 20-minute incubation.[3]The effect of TSN on BoTx binding was observed by West blot and synchronization method.③ttest was used for comparing the difference of measurement data.MAIN OUTCOME MEASURES:The gray value of BoTx bands from westem blot was used to estimate the bound amount of BoTx.RESULTS:①Preincubation of synaptosomes with TSN(17 μmol/L)at 4℃ decreased the association of BoTx type A(BoTx/A)by(11.4±8.7)%(P＜0.05,n=8)and that of BoTx type C(BoTx/C)by(9.2±2.8)%(P＜0.05,n=8).②When incubetion temperture was set at 37℃,preincubation of synaptosomes with TSN (17μmol/L)decreased the association of BoTx/C by(18.0±6.7)%(n=5,vs.the control without TSN at 37℃,P＜0.05),but there was no significant change observed when decreasing TSN to a concentration of 0.17 μmol/L.③When synaptosomes preincubated with TSN in high level of K+ at 37℃,the inhibition of TSN on BoTx/C association further increased to(32.6±6.1)%(n=4,vs the control without TSN at 37℃ with high level of K+,P＜0.05).CONCLUSION:These results indicate that TSN inhibits the binding step of BoTx intoxication,and probably its subsequent temperature-and depolarization-dependent intalization of toxin.