Mechanism Of Swainsonine-induced Apoptosis Of Cerebral Cortex Neurons In Neonatal SD Rats

Swainsonine is the critical toxic ingredients of Astragalus and Oxytropis speciesLeguminosae plants, whose chemical name is1,2,8-trihydroxy indolizidine alkaloid. A largeamount of researches have reported that swainsonine can damage the central nervous systemof animals, inducing cellular vacuolation. In vitro, swainsonine-treatmented neurons aredecreased in size and the length of process. However, the mechanisms are not clear. Thus, inthis study, we employed the model of cerebral cortex neurons to evaluate the effects ofswainsonine on the cerebral cortex neurons from neonatal SD rats, and investigate thepossible machanisms. The following results were obtained:1. The experimental cerebral cortex neurons are obtained from neonatal SD rats in24h.To observe the morphologic changes showed that the neurons had a good refraction, and theneurites were cross-linked to form a completed neural network; The NSE/Hoechst33258fluorescein stain showed that the purity of neurons is above94%; MTT assay showed thatfrom the third day to the fifth day the viability of cerebral cortex neurons is rising, and reachto the highest on the fifth day. These results demonstrated that we were successed to establishthe modle of cerebral cortex neuron primal cultures.2. In this section, we employed the modle of cerebral cortex neuron primal cultures toevaluate the toxic effects of swainsonine. MTT assay showed that swainsonine treatmentinhibited the growth of cerebral cortex neurons, and the IC50of24h was0.886mg/mL;Treated neurons at different dose （swainsonine0.5，1.0，2.0mg/mL） for24h respectively.Inverted phase contrast microscope observation showed that cell density was decreased, andneural network was damaged seriously; Hoechst33258fluorescein stain showed thatchromatin condensation and nuclear fragmentation after swainsonine treatment; Laserscanning confocal microscope observation showed that the [Ca²⁺]ishowed a dosed-dependentincrease. These results demonstrated that swainsonine treatment concentration-dependentlyinhibited the growth of cerebral cortex neurons, and mainly induced by apoptosis.3. Treated cerebral cortex neurons at the same concentration of swainsonine asmentioned above for24h, western blot assay showed that compare with the control group,swainsonine treatment increased the expression of cleaved-caspase-8and-3（P<0.05）, whilethe expression of pro-caspase-9did not appear significant changes （P>0.05）, in the process ofswainsonine induced apoptosis. These results demonstrated that swainsonine inhibited cerebral cortex neurons growth through a caspase cascade response, which depend on theactivation of caspase-8and-3, which implied swainsonine induced cerebral cortex neuronsapoptosis in vitro through the activation of the dearh receptor pathway, but not themitochondrial pathway.4. Cerebral cortex neurons were treated with0,0.5,1.0,2.0mg/mL swainsonine for24h,respectively. Compared with the control group, western blot assay showed that swainsoninetreatment increased the expression of cleaved-caspase-12（P<0.05）. These results werepreliminarily demonstrated that swainsonine could induce endoplasmic reticulum stress tomediate cerebral cortex neurons apoptosis.This study demonstrated for the first time that swainsonine induced cerebral cortexneurons apoptosis through the death receptor pathway and endoplasmic reticulum stress invitro. And this study laide the foundation for further revealing the mechanism of swainsonineneurotoxicity.