| Part 1 The effect of chronic acrylamide exposure on rat neurobehavioral functions and cerebellar injury.Objective: To investigate the effect of chronic acrylamide(ACR)exposure at low dose on rat neurobehavioral functions and cerebellar injury,and to research the correlated mechanism of ACR neurotoxicity at low dose by apoptosis,endoplasmic reticulum stress(ERS)and autophagy.Methods: 36 healthy male SPF Sprague-Dawley rats,weighing 80~100g,were distributed randomly in three groups,12 rats per group,involving control group,low-dose group(0.5mg/kg),high-dose group(5mg/kg).The rats of different groups had drunk water with ACR at different concentrations for 12 month,weight and gait scores of rats were recorded every week.3 days before the last ACR treatment,the hindlimb landing foot splay measurement,balance beam tests(2.5cm,5cm,7cm)and rotorod tests were conducted,after 24 h of the last treatment,4 rats in every group were fixed with 4% formaldehyde via heart perfusions,the cerebellums were separated for general pathological examination,and two cerebellums out of the four were used to do transmission electron microscope examination.The remaining rats were sacrificed and collected the brain,heart,liver,spleen,kidney and testicles and weighted them.Then separated cerebellum,used left part of cerebellum to measure the apoptosis rate via flow cytometry,remaining cerebellar tissue-80?.Used right part of cerebellums out of 7 randomly to test protein expressions of apoptosis-,ERS-and autophagy-related proteins,such as PERK,GRP78,ATF4,CHOP,Beclin1,LC3,Bax,Bcl-2,Caspase-7.Results: 1.General toxicity: during the 12-month exposure time,the weight of rats of each group all gradual increased with time,and there was no signific ant difference among these groups(P > 0.05).The organ coefficient of every organ in ACR.2.Alternation in motor function:(1)after the observation of gait,no gait abnormality was found in rats of the control group and the 0.5mg/kg group,the score of 5mg/kg group was significantly higher than control group from the 6th month to 12 th month(P < 0.05).(2)The results of hindlimb spray distance measurement,balance beam test and rotorod test all show that there was no obvious difference between the outcomes of each behavior test of 0.5mg/kg group and the control group(P > 0.05),while the rats of 5mg/kg group showed evident difference compared with control group(P < 0.05).3.The pathological alternations of cerebellar tissue:(1)the results of HE staining and Nissl's staining showed that all three layers of cells were normal with a great number of Nissl body,no or few abnormal Purkinje cell was observed in the control group and the 0.5mg/kg group,while in 5mg/kg group,many Purkinje cells were abnormal in morphology,loose and irregular arrangement with decreased number of Nissl body.(2)The structures of organelles were basically normal in the control group and 0.5mg/kg group under the transmission electron microscope(TEM),and there was no or few autophagosome,but many autophagosomes,swollen mitochondria and dilated endoplasmic reticulum were observed under TEM in rats of 5mg/kg group.4.Mechanism research:(1)effect of ACR on cell apoptosis: the results of FCM and Western blot showed that compared with control group,the apoptotic rate and the expression of pro-apoptotic proteins(Bax,Caspase-7)of 5mg/kg group were increased signficantly(P < 0.05),while there was no significant difference between the 0.5 mg/kg group and the control group(P > 0.05),the expression of Bcl-2 of three groups had no significant difference(P > 0.05).(2)Effect of ACR on endoplasmic reticulum stress(ERS): Western blot results showed that the ratio of p-PERK/t-PERK and the protein expressions of GRP78,ATF4 and CHOP in the 5mg/kg group were evidently incr eased compared with the control group(P < 0.05),while there was no significant difference between the 0.5mg/kg group and the control group(P > 0.05).(3)Effect of ACR on autophagy: Western blot results showed that the ratio of LC3-II/LC3-I and the protein expressions of Beclin-1 in the 5mg/kg group were distinctly increased compared with the control group(P < 0.05).Conclusions: Rats were treated with ACR(5mg/kg)by drinking water for 12 months could cause abnormal gait,imbalance and decreasing of motor coordination,pathologically,the Purkinje cells are abnormal in morphology,loose and irregular arrangement,and the number of Nissl body was significantly decreased.The apoptosis,ERS and autophagy contributed to neurotoxicity caused by ACR.Part 2 The role of ERS in apoptosis and autophagy in SH-SY5 Y cells caused by ACR.Objective: Using PERK inhibitor to research the role of ERS in ACR-induced apoptosis and autophagy in SH-SY5 Y cells,and to provide experimental basis for exploring potential molecular targets for the prevention and control of ACR toxicity.Methods: There were five groups in the general toxicity test,including control group,0.63mmol/L ACR group,1.25 mmol/L ACR group,2.5mmol/L ACR group,5mmol/L ACR group,treated cell for 24 h,and tested the cell viability and apoptotic rate in each group by using CCK8 and FCM.ERS inhibition test was divided into two groups: ACR(2.5mmol/L)group and GSK2606414(0.5?mol/L)+ACR(2.5mmol/L)group.In the last one group,the cells had to be treated with PERK inhibitor for 2h before exposing to ACR for 24 h.Measured the apoptotic rate by FCM,and test the protein expressions of ERS-,apoptosis-and autophagy-related proteins in these two groups.Results: 1.General toxicity: the results of CCK8 and FCM sho wed that,compared with control group,the cell viability of SH-SY5 Y was evident decreased when the dose is 1.25 m M~5m M(P < 0.01),the apoptotic rate of SH-SY5 Y cells were elevated significantly when the dose is 2.5m M~5m M(P < 0.05).2.Mechanism research:(1)the effect of PERK inhibitor: compared with the ACR alone group,the ratio of p-PERK/t-PERK and expressions of ATF4,CHOP all decreased in the ACR+PERK inhibitor group(P < 0.05).(2)The influence of ERS inhibition on apoptosis: the outcomes of FCM indicated compared with the ACR alone group,the apoptosis rates of ACR+PERK inhibitor group obviously decreased(P < 0.05),the results of Western blot also showed that the expressions of Bax,Caspase-7 in the ACR+PERK inhibitor group significantly declined,meanwhile,the expression of Bcl-2 increased(P < 0.05).(3)The influence of ERS inhibition on autophagy: the results of WB showed the expression of Beclin-1 and the ratio of LC3-II/LC3-I decreased in the ACR+PERK inhibitor group(P < 0.05).Conclusions: The inhibition of ERS by PERK specific inhibitor could efficiently reverse the autophagy and apoptosis caused by ACR,indicating ERS mediated ACR-induced apoptosis and autophagy. |
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