Effects Of Er Stress On Apoptosis Of Rat Cerebral Cortical Neurons Induced By Cd And Protective Effect Of ?-LA

Cadmium(Cd)is a common heavy metal pollutant in the environment.A large number of studies have shown that Cd damage on organism was multi-systematic.The brain is one of the target organs of Cd damage.Cd can induce neuronal apoptosis.The pathways of apoptosis include death receptor pathway,mitochondrial pathway and endoplasmic reticulum(ER)stress.It has been found that Cd could induce apoptosis of rat cerebral cortical neurons by mitochondrial pathway and death receptor pathway in the previous investigation.However,the effect of ER stress on the apoptosis of rat cerebral cortical neurons induced by Cd and the protective mechanism of a-lipoic acid(a-LA)has not been elucidated.In this study,sprague dawley(SD)rats were used as experimental animals to explore the role of ER stress in apoptosis of rat cerebral cortical neurons induced by Cd and the protective effect of a-LA in vitro and in vivo.1.In vitro studiesThe cerebral cortical neurons of SD rats at 18?19 days of gestation were used as cell models.? The rat cerebral cortical neurons were treated with different concentrations(0,5,10,20 ?mol/L)of Cd at different times(0,6,12,24 h).The western blot was used to measure the expression of ER stress related protein,BiP,p-eIF2a,ATF4,CHOP and Cleaved caspase-12,Cleaved caspase-3.The results showed that the expression of BiP,p-eIF2a,ATF4,CHOP and Cleaved caspase-12,Cleaved caspase-3 were significantly or significantly increased in Cd group cpmpared with the control group(p<0.05 or p<0.01),Cd and ER stress-related protein expression have relation of dose-and time-effect.It indicated that Cd can cause ER stress in rat cerebral cortical neurons.?The rat cerebral cortical neurons were treated with 10 ?mol/L Cd and 5?mol/L Salubrinal(eIF2a specific inhibitor)or 20?mol/L Z-ATAD-FMK(Caspase-12 specific inhibitor).The western blot was used to measure the expression of p-eIF2a,CHOP,Cleaved caspase-12,Cleaved caspase-3 and Bax,Bcl-2.DAPI staining was used to observe the nuclear morphological changes of rat cerebral cortical neurons.The results showed that Salubrinal can significantly inhibit the increase of p-eIF2a,CHOP protein and Bax/Bcl-2 ratio induced by Cd(p<0.01),and Z-ATAD-FMK can significantly inhibit Cd-induced increase of Cleaved caspase-12 and Cleaved caspase-3(p<0.01).Salubrinal and Z-ATAD-FMK can inhibit the Cd-induced nuclear condensation and fragmentation.Therefore,it indicated that ER stress plays an important role in the apoptosis of rat cerebral cortical neurons induced by Cd.? The rat cerebral cortical neurons were treated with 100?mol/L a-LA and 10?mol/L Cd alone or in combination for 12 h.The western blot was used to measure the expression of BiP,p-eIF2a,ATF4,CHOP,Cleaved caspase-12,Cleaved caspase-3 and Bax,Bcl-2.DAPI staining was used to observe the nuclear morphological changes of rat cerebral cortical neurons.The results showed that a-LA can significantly inhibit the increase of BiP,p-eIF2a,ATF4,CHOP,Cleaved caspase-12,Cleaved caspase-3 and Bax/Bcl-2 ratio induced by Cd(p<0.01),a-LA can inhibit Cd-induced nuclear condensation,fragmentation and other changes in cell morphology.Therefore,it indicated that a-LA have protective effect on Cd-induced ER stress and apoptosis in rat cerebral cortical neurons.2.In vivo studiesTwenty-four female SD rats,aged 21 days,were randomly divided into four groups.There are control group,a-LA group(50 mg/kg.bw),Cd group(50 mg/L),a-LA and Cd co-treated group(50 mg/kg.bw +50 mg/L).The control group rats were free to drink ultrapure water,Cd group rats consumed the Cd(50 mg/L)as drinking water,a-LA group rats were taken a-LA(50 mg/kg.bw)by intragastic administration,a-LA and and Cd co-treated group rats consumed the Cd(50 mg/L)as drinking water and the rats also taken a-LA(50 mg/kg.bw)by intragastic administration.The rat cerebral cortex was taken after 12 weeks of continuous treatment.?Determination of Cd in cerebral cortex was measured by Atomic Absorption Spectrophotometer.The results showed that the Cd content in the cortex of the Cd group was significantly higher than that of the control group(p<0.01).Compared with Cd group,the content of Cd in the cerebral cortex of a-LA and Cd co-treated group was significantly decreased(p<0.05).Hence,it indicated that Cd can accumulate in the rat cerebral cortex,and a-LA can reduce Cd accumulation.? The ultrastructural variations of Cerebral Cortex was observed by Transmission Electron Microscopy.The results showed that compared with the control group,Cd group cerebral cortex cell nuclear shrinkage,chromatin distribution is uneven,mitochondrial ridge decreased or disappeared.Furthermore,compared with the Cd group,the nucleus and mitochondrial damage of the cerebral cortex was relatively small in a-LA and Cd co-treated group.Therefore,it indicated that Cd can cause rat cerebral cortex damage,and a-LA can relieve the damage caused by Cd in rat cerebral cortex.? The western blot was used to measure the expression of BiP,p-eIF2a,ATF4,CHOP,Cleaved caspase-12,Cleaved caspase-3 and Bax,Bcl-2.The results showed that the expression of BiP,p-eIF2a,ATF4,CHOP,Cleaved caspase-12,Cleaved caspase-3 and Bax/Bcl-2 ratio in Cd group were significantly higher than those in control group(p<0.01).Besides,compared with Cd group,the expression of BiP,p-eIF2a,ATF4,CHOP,Cleaved caspase-12,Cleaved caspase-3 and Bax/Bcl-2 ratio were significantly decreased in a-LA and Cd co-treated group(p<0.01).Accordingly,it indicated that Cd can damage rat cerebral cortex and induce ER stress,a-LA has a protective effect on ER stress and damage induced by Cd in rat cerebral certex.