| Objective: To investigate whether acute aconitine poisoning results in impairment of blood-brain barrier function via an acute aconitine poisoning model in rats and further to explore its possible potential mechanism.Methods: Ninety-six healthy adult male SD rats were randomly divided into 4 groups with 24 rats in each group: control group(n=24),low dose group: AC-L group(0.5mg/kg,n=24),middle dose group: AC-M group(1.5mg/kg,n=24),high dose group: AC-H group(2.5mg/kg,n=24).According to the time of execution,each group was divided into 0.5h,2h,6h and 12 h subgroups(n = 6).Rats in the AC-L group,AC-M group and AC-H group were given aconitine solution of 0.5mg/kg,1.5mg/kg and 2.5mg/kg respectively by oral perfusion,and those in the control group were given the same amount of normal saline by oral perfusion.Three rats were injected into Evans blue(2% EB,4 ml/kg)through the caudal vein at 1h before death at each time point(0.5h were not included).After heart perfusion with normal saline and 4% paraformaldehyde,the rats were killed,and their brains were taken completely to calculate the content of EB.The remaining rats were killed at each time point to take out the brain quickly.One hemisphere was fixed with 4%paraformaldehyde for HE staining immunofluorescent staining and another for WB,ELISA detection.As for cells,AC groups were divided into control group,low concentration group(10?M),middle concentration group(20?M)and high concentration group(50?M)according to different concentration.after 12 hours of exposure to PC12 cells,cell growth inhibition rate and apoptosis rate were measured by MTT and flow cytometry to observe the effect of different concentrations of AC on the survival rate and apoptosis rate of PC12 cells.Results: HE staining of acute aconitine poisoning rats: the hippocampal formation was normal,the neurons were arranged regularly,and the pathological changes were not obvious in the control group.With the increase of the dose of aconitine,the hippocampus capillary dilated and congested,the edema on the basal part of granular cell gradually aggravated,and the number of neurons in AC-H group decreased and suffered from disorder of arrangement.With the prolongation of time,the content of EB in AC-L,AC-M and AC-H group increased gradually,and all of them increased most obviously at 12 h,which has statistical significance(P<0.05).Compared with control group,the expression of ZO-1 and claudin5 in AC-H group decreased at 0.5h,2h,6h and 12 h,The decrease of 6hours and 12 hours had statistical significance(P<0.05),The expression of ZO-1 and claudin5 protein decreased most significantly at 12h(P < 0.01),The expression of ZO-1and claudin5 protein in the AC-L,AC-M group at 12 h was significantly lower than that in the control group with statistical significance(P<0.01).Compared with the control group,the expression of XBP-1,p-IRE-1 and GRP78 in 12 h AC-L,AC-M,AC-H groups all increased,and the expression levels of AC-M and AC-H groups increased significantly(P< 0.05).Compared with the control group,the expression of GRP78 in brain tissue of the AC-L,AC-M,AC-H group at 12 h was remarkably higher than that of the control group(P<0.05).The expression of TNF-? protein in the AC-L and AC-M group was significantly higher than that of the control group with statistical significance(P<0.05),And the AC-H group was the most significant(P<0.01).It was shown that aconitine of 10?M,20?M and50?M significantly inhibited the survival rate of PC12 cells compared with the control group with statistical significance(P < 0.01),and the survival rate decreased significantly with dose increasing.The results of Annexin V-FITC/PI double labeling showed that aconitine of 10?M,20?M and 50?M significantly increased the apoptosis rate of PC12 cells compared with the control group with statistical significance(P < 0.01).Conclusion : Under this study,the content of EB in brain tissue increased and the expression of ZO-1 and claudin5 decreased after aconitine poisoning,suggesting that aconitine can cause blood-brain barrier dysfunction.The damage may be related to theup-regulation of XBP-1,p-IRE-1 and GRP78 protein expression by aconitine,which leads to over-activation of endoplasmic reticulum stress. |
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