| [Objective]:To study the role of Ca2+-calpain/p35-cdk5/p25pathway in neurotoxicity induced by BDE-153in vivo and in vitro.[Methods]:Method in vivo:Male puppy rats at postnatal day10(PND10) were randomly divided into four groups by littersand weights,10rats per each group, and administrated with different concentrtions of BDE-153solution (olive oil,1mg/kg,5mg/kg and10mg/kg BDE-153) by intraperitoneal injection oncebased on10ml/kg body weight. After1and2months, Morris water maze, step-down test,step-through tast were used to detect rats’neurobehavioral functions, open-field test was used totest rats’ spontaneous behavior. Two months later, hippocampus mophorlogy and ultrastructurewere observed under optical and electronic microscope, Nerve cell apoptosis rate and Ca2+concentration were assayed by TUNEL staining and flow cytometry, and lactate dehydrogenaseleakage rate was detected by spectrophotometry. Calpain, CDK5and p25and p35gene androtein expression was detected by RT-QPCR and Western blotting, and calpain, cdk5/p25andcdk5/p35kinase activities were detected by ELISA.Method in vitro:Primary cultured neurons in a logarithmic phase, was randomely categorized into5groups,blank group, DMSO control group,10μmol/L,20μmol/L,40μmol/L, was observed themorphological change under an optical microscope after treated for48h. Cell vitality wasdetected by CCK-8kit, neuron damage was observed by Hochest33258dying, AO/EB doublefluorescent staining methods. Cell apoptosis rate and the Ca2+concentration were detected byflow cytometry, LDH leakage rate detected with LDH kit. The levels of mRNA expression andprotein expression of the Calpain, cdk5and p35were detected by RT-QPCR andimmunofluorescence technique. Calpain, cdk5/p25and cdk5/p35kinase activities weredetermined with ELISA kits. After calpain inhibition by PD150606, we detected neuron vitalityand calpain kinase activity, cdk5/p25and cdk5/p35kinase activity. [Results]Results in vivo:1. Neurobehavioral function results: water maze experiment results showed that, with theincrease of BDE-153dose, rats’ spatial learning and memory abilities were significantlydecreased compared with the control group, swimming time and distance in the target quadrantwere significantly reduced in the5,10mg/kg BDE-153groupS (P <0.05). Step-down testresult showed that rats’ short-term learning and memory abilites were reduced, the latency timewas significantly shortened in the treated groups compared to the controls,(P <0.05).2. The pathomorphogy observation in HE staining showed that the control rat’s hippocampalnerve cells were aligned and uniform in size in CA3region,but disarranged, shrunk and reduced,the chromatin was condensed and margined to the nuclear membrane after deal with BDE-153;Ultrastructure results: control group nerve cells in the hippocampus in morphosis were normal,with large and round nucleus and nuclear membrane integrity, chromatin evenly, have abundantorganellesin intercellular substance;With the increase of infected dose, shrinking nucleus,chromatin concentrated together, some nucleolus disintegration, split, organelles occurredsignificantly reduce the number and degeneration and even disappear.3. The neural cell apoptosis rate, intercellular calcium ion concentration and LDH leakage rate:TUNEL staining results showed that the number of positive cells in hippocampus graduallyincreased with the increase of treated dose, compared to control,1,5and10mg/kg dose groupof IHS score is significantly higher than the control (P <0.05). Compared to the controls,nerve cell apoptosis rate were significantly increased in5mg/kg group and10mg/kg group (P <0.05). LDH leakage rate was significantly increased in the10mg/kg BDE-153groupcompared to the controls and1mg/kg BDE-153group (P <0.01); With the increase ofBDE-153dose, compared with the controls, intercellular Ca2+were significantly decreased inthe5mg/kg and10mg/kg group (P <0.01).4. Changes of CDK5, calpain1, calpain2and p35gene expression in rats’ hippocampus:Compared to the controls, CDK5gene expression were significantly increased in the3treatedgroups (P <0.05); Calpain2gene expression was gradually increased with the dose ofBDE-153,5mg/kg and10mg/kg group was significantly higher than that in the controls (P <0.05). However, calpain1gene expression had no obvious difference among the groups.;compared to the controls, p35gene expression was increased by7.06,7.44and11.36times,respectively.5. Changes of CDK5, calpain2and p35, p25protein expression in rats’ hippocampus:Compared with control groups, cdk5protein expression was significantly increased in the5and10mg/kg group (P <0.01); Calpain2protein expression significantly higher in1mg/kg,5mg/kgand10mg/kg groups than the control group (P <0.05); P35protein expression was reduced with the dose of BDE-153, which was lower in control group than that in5and10mg/kggroup,lower in the1mg/kg group than in5mg/kg group (P <0.01); P25protein expression of5and10mg/kg group was significantly higher than that in control group and1mg/kg group,10mg/kg group was significantly higher than that in5mg/kg group, differences werestatistically significant (P <0.01).6. Calpain, cdk5/p25and cdk5/p35kinase activity: calpain kinase activity was increaseddependently on the dose. Compared with the control group, rats’ calpain kinase activity wassignificantly increased in1,5and10mg/kg group (P <0.01). cdk5/p25kinase activity increasedwith the dose of BDE-153while cdk5/p35kinase activity was reduced. Compared to the control,cdk5/p25kinase activity increased significantly in the1,5and10mg/kg group (P <0.01),cdk5/p35kinase activity was significantly decreased in the1,5and10mg/kg group (P <0.01).Results in vitro:1. The toxicity test results: BDE-153infected48hours later, with the increaseing of the dose,neurons and cell-connections became reduced; axons and dendrites were decreased andshortened. cell activity decreased (P <0.001), with the BDE-153dose in Hoechst33258andAO/EB staining, and the neuron apoptosis rates were significantly increased compared to theblank control and DMSO group.With the increase of exposed, nerve cells apoptosis rateincreased significantly, compared with control,20and40μmol/L increased significantly, thedifference was statistically significant (P <0.05),40μmol/L group compared with10μmol/L,the difference was statistically significant (P <0.05). Moreover, the LDH activities wasstatistically increased in the10,20,40μmol/L BDE-153groups compared with the blankcontrols (P <0.05).2.changes of intercellular calcium ion concentration, calpain kinase, cdk5, calpain2and p35gene and protein expression: compared to the control group, neuron intracellular calcium ionconcentration were significantly increased with the dose of BDE-153in, the20μmol/L and40μmol/L BDE-153groups (P <0.05); Compared to blank group and DMSO solvent group,cdk5gene expression were statistically increased in the treated groups (P <0.05); Calpain2gene expression were also significantly increased in the20μmol/L and40μmol/L BDE-153groups compared to the blank group (P <0.05), and were statistically higher in the20μmol/Land40μmol/L BDE-153groups than that in the DMSO solvent group (P <0.05). CDK5proteinexpression was increased with BDE-153dose increasing, was significantly higher in the40μmol/L BDE-153group than in the blank group and the solvents (P <0.05). Calpain2expression was significantly in the treated groups compared to the blank group and solventgroup (P <0.05). 3. Calpain, cdk5/p25and cdk5/p35kinase activity changes: Compared with blank control groupand DMSO solvent group, calpain2kinase activity were statistically increased in the10,20and40μmol/L BDE-153groups,20and40μmol/L groups were higher than in the10μmol/Lgroup (P <0.01). And cdk5/p25kinase activities were statistically increased in the3treatedgroups compared to the the blank control (P <0.01). While cdk5/p35kinase activities weredecreased in the10,20and40μmol/L BDE-153treated group compared with the blank controlgroup and DMSO group (P <0.01).4. Compared to the20μM BDE-153group, the cell viability were prevented to reduce afterpre-treated by calpain inhibitor PD150606, enhanced-calpain kinase activity was drop down bythe calpain inhibitor, and cdk5/p25kinase activity was decreased significantly whereascdk5/p35kinase activity was increased statistically; Generally, the changes depended on thedose of PD150606, and which is better in the20μM PD150606pre-treated group than in the5μM PD150606(P <0.05).[Conclusion]:1. BDE-153have neurotoxic effects,it damaged rats’ learning and memory abilities, disruptedrats’spontaneous behavior, and induced neuron cell apoptosis.2. The neurotoxicity and neuros infected of BDE-153have a significant dose-dependent mannerwith BDE-153.3. BDE-153affected intracellular calcium ion concentration and activatedcalpain kinase which degraded p35into p25and formed cdk5/p25complex,and bring forthneurotoxicity. |
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