Effects Of Chronic Exposure To Acrylamide On The Nigrostriatal System And The Potential Mechanism Mediated By Neuroinflammation

Acrylamide(ACR)is widely present in diet as a by-product of Maillard reaction,but the relevant mechanism information of its neurotoxicity through diets is limited.This present study was used to research the effects of chronic exposure to ACR on the nigrostriatal system and the potential mechanism mediated by neuroinflammation through animal observational studies in vivo and cell intervention studies in vitro from the perspective of neuroethology,pathology and molecular biology.The purpose of this study is to further enrich and deepen the theoretical basis of ACR neurotoxicity,and provide a new direction for the prevention and treatment of ACR toxicity.Part Ⅰ Effects of chronic exposure to ACR on the nigrostriatal systemObjectives:The chronic ACR exposure model was established to investigate the impact of ACR on neurobehavior impairment,nigrostriatal neurons,glial cells and neuroinflammation in rats.Methods:36 rats were randomly divided into three groups(n=12/group)and maintained on treated drinking water providing dosages of 0,0.5,or 5 mg/kg ACR for12 months.The rats were weighed and their gait was scored weekly.The Open Field test,Balance Beam test,Rotarod test and the Landing Foot Splay test were carried out at the end of ACR exposure.All the animals were sacrificed,the organs were weighed and then calculated viscera coefficients.Oxidative stress markers and inflammatory markers in serum were determined by oxidative stress kits or ELISA,respectively.The changes of neuron structure were observed by HE and Nissl stainings.The ultrastructural changes were observed by transmission electron microscopy(TEM).The apoptosis was detected by flow cytometry.The distribution and expression of nerve injury-related and neuroinflammation-related indicators were detected using immunofluorescence staining or western blot in nigra and striatum.Results:(1)The effect of ACR on the overall toxicity and neurobehaviors of rats.(1)Overall toxicity evaluation.The rats in the low-dose group showed no obvious abnormalities,and no significant change in terms of serum oxidative stress indicators and inflammatory factors(P>0.05).The rats in the 5 mg/kg group began to show depilation,lethargy and hypokinesia symptoms from the 6th month.The levels of malondialdehyde(MDA),pro-inflammatory factors(IL-1β,IL-6,TNF-α)and anti-inflammatory factors IL-10 in serum significantly increased(P<0.01)in 5 mg/kg group rats.No statistical difference was observed between the exposure groups and the control group in terms of body weight,organ coefficients and water consumption(P>0.05).(2)Neurobehavioral changes.There was no significant changes of the exercise and balance indicators between 0.5 mg/kg group and the control group rats(P>0.05).The gait score significantly increased in 5 mg/kg ACR group rats from the 7th month.The distance of two hind limbs in the Landing Foot Splay test and the balance ability score in Balance Beam test significantly increased in 5 mg/kg ACR group rats(P<0.001).The moving distance and center distance in Open Field test,and the retention time in Rotarod test significantly shortened(P<0.001).(2)The effect of ACR on nigrostriatal neurons.There was no structural abnormality of neurons,and there was no difference in the distribution and expression of TH andα-Syn in nigra and striatum of 0.5 mg/kg group rats(P>0.05).Neu N expression significantly decreased in nigra and striatum(P<0.05).P-TH(Ser40)expression significantly decreased and MMP3 expression significantly decreased in nigra of 0.5 mg/kg group rats(P<0.01).Neuronal nuclear pycnosis was demonstrated in nigra of 5 mg/kg ACR-treated rats.The size and number of Nissl bodies as well as staining intensity decreased obviously in nigra and striatum of 5 mg/kg ACR group rats.Apoptosis and Bax/Bcl-2 value significantly increased in nigra(P<0.001).The expressions ofα-Syn and MMP3 were increased,and the expressions of TH,P-TH(Ser40),Neu N,BDNF,SYN1 and SYP were reduced in nigra and striatum of 5mg/kg ACR group rats(P<0.05).(3)The effect of ACR on glial cells in nigra and striatum.(1)Promoting proliferation of microglia and astrocytes.Compared with control,Iba-1~+cells number in nigra significantly increased in the low-dose group(P<0.05).However,there was no significant changes in the number of GFAP~+cells,Iba-1~+cells and corresponding protein expression in striatum of 0.5 mg/kg ACR group rats(P>0.05).In the 5mg/kg group,Iba-1~+cells number and Iba-1 expression significantly increased in nigra and striatum(P<0.01),and GFAP~+cells number in nigra significantly increased(P<0.05).(2)Promoting the activation of microglia.Compared with control group,there was no significant change in the proportion of branched or ameboid microglia and in the number of Iba-1~+CD68~+cells in nigra and striatum of 0.5 mg/kg group rats(P>0.05).The proportion of branched microglia significantly reduced,while the proportion of ameboid microglia and the number of Iba-1~+CD68~+cells significantly increased in nigra and striatum of 5 mg/kg group rats(P<0.001).(4)The effect of ACR on inflammatory factors and signaling pathways in nigra and striatum.(1)The effect on inflammatory factors.Compared with control group,there was no significant change in the expressions of Pro-IL-1β,IL-1β,TNF-α,IL-6 and Cox-2 in nigra and striatum of 0.5 mg/kg group(P>0.05).The expressions of the above proteins increased significantly in nigra and striatum of 5 mg/kg group(P<0.05).IL-1βin this model was mainly derived from microglia through fluorescence co-staining detection.(2)The effect on NLRP3 inflammasome and NF-κB signaling pathway.Compared with control group,there was no significant change in the fluorescence intensity of caspase-1 and in the expressions of NLRP3,ASC,Pro-caspase-1,caspase-1,P62,P-P65(Ser 536)、IκBαprotein in nigra and striatum of 0.5mg/kg ACR group rats(P<0.05).The fluorescence intensity of caspase-1 and NLRP3,caspase-1,P62 and P-P65(Ser536)expression significantly increased,and IκBαexpression significantly reduced of 5 mg/kg group rats(P<0.05).There was no significant change in the expressions of ASC and Pro-caspase-1 in 5 mg/kg ACR group rats(P>0.05).Conclusion:5 mg/kg/day ACR exposure led to motor dysfunction of rats,dopaminergic neuron damage,microglia activation,NLRP3 inflammasome and NF-κB pathway activation and inflammatory factors expression in nigra and striatum.0.5mg/kg/day ACR exposure led to neurons loss in nigra and striatum and microglia proliferation in nigra.