The Mechanism Of Aconitine-induced Apoptosis On HT22 Cells

Aconitum belongs to the aconitum genus and ranunculaceae family and it contains highly toxic aconitine.The characteristic symptoms of aconitum poisoning including vomiting,diarrhea,arrhythmia,vision and auditory sense weakening and muscle rigidity.Aconitine contains target organ toxicity and well known for its neurological and vascular toxicity.Studies on aconitine toxicity have mainly been focused on energy metabolism,ion channels,oxidative and damage,while neuronal apoptosis mechanism of aconitine is still unclear.Apoptosis is a process of programmed cell death.The mechanism of apoptosis mainly included the mitochondria and death receptor signaling pathways.To understand the mechanism of apoptosis in aconitine-induced neurotoxicity injury,we used normal mouse hippocampal neurons(HT22 cells)as an in vitro model to investigate the effect of aconitine on HT22 cell apoptosis.It will help understand the mechanistic details of aconitine neurotoxic damage and provides basis for prevention and treatment of animal aconitosis.The results were as follows:1.Aconitine suppressed cell proliferation and induces the generation of DA in HT22 cellsThe HT22 cells were cultured with various doses of aconitine(0,50,100,200 μg/mL)for 6,12 h.The survival rate of HT22 cells was reduced and the cellular DA contents were increased in a time-dose-dependent manner(P<0.05).The morphology of HT22 cells changed,the number of cells decreased,and obvious changes in cell damage were shown,and showing a dose-dependent manner.2.Aconitine induced apoptosis in HT22 cellsThe HT22 cells were cultured with various doses of aconitine(0,50,100,200 μg/mL)for 12 h and morphology changes were observed.The control group showed complete cell structure,large nucleus and uniform chromatin distributed in the nucleus,while aconitine treatment group showed cell membrane was fragmented,and the mitochondria were slightly swollen.Some of the mitochondrial cristae were broken and disappeared,showing a vacuole shape.The membranes were arranged to form blocks of different shapes and sizes with chromatin progressively splited into fragments.The nucleus was crescent-shaped,ring-shaped,or fragmented,formed spherical protrusions of different sizes,suggested formation of apoptotic bodies.To explore the role of apoptosis in aconitine-suppressed cell proliferation,Annexin V-FITC/PI double staining was performed.The result indicated that the early apoptosis rates were 2.47%,3.53%,5.30%,12.5%(6 h),2.6%,6.8%,6.15%,6.78%(12 h);the late apoptosis rates were 4.48%,6.43%,5.39%,5.27 %(6 h),4.67%,8.18%,9.18%,10.6%(12h);The total apoptotic rates of 0 μg/mL,50 μg/mL,100 μg/mL and 200 μg/mL were 6.95%,9.96%,10.69,17.77%(6 h),7.27%,14.98%,15.33%,17.38%(12 h),and the difference was highly significant.The early apoptotic rate decreased,the late apoptotic rate gradually increased with time,and the total apoptotic rate increased in a time-dose-dependent manner,indicating that aconitine can induce apoptosis in HT22 cells.3.Aconitine induced cell apoptosis via mitochondria and death receptor signaling pathways in hippocampus cell lineTo clarify the signaling pathway of aconitine-caused cells apoptosis,MTT,double antibody sandwich,Western blot was used to measure the survival rate,the content of Caspase3,and the expression of Caspase3 protein.After addition of Caspase8 inhibitor Z-IETD-FMK and Caspase9 inhibitor Z-LETD-FMK,the survival rate of HT22 cells was obviously raised while the activity of Caspase3 was obviously decreased.Western blot results showed that aconitine could induce apoptosis of HT22 cells through both mitochondrial pathway and death receptor pathway.The results showed that addition of Caspase8 and Caspase9 inhibitor could effectively the reverse survival rate of HT22 cells and the aconitine-induced increase of Caspase3.These results suggested that aconitine-induced apoptosis is dependent on the activation of Caspase8 and Caspase9.To determine if the aconitine-induce cell apoptosis is through the mitochondrial pathway and death receptor pathway,q RT-PCR and Western blot was used to detect the apoptosis-related gene and protein expression of HT22 cells.The results showed that the expression of apoptosis-related genes and proteins changed significantly with the increase of aconitine concentration.The gene and protein expression of Bax,Cyto C,Apaf-1,Caspase9,Caspase3,Fas,Fas L,Fadd and Caspase8 both showed a significant upward trend(P<0.05),but expression of Bcl-2 decreased significantly(P<0.05),and gene expression of Bid increased while its protein levels reversed.The above results indicate that aconitine can suppresses cell proliferation and cause dopamine release.In addition,the mitochondrial pathway and death receptor pathway are involved in aconitine-induced HT22 cell apoptosis.