Exploring For The Mechanism Of Acrylamide Induced Neurotoxicity

Acrylamide(ACR)is a potential neurotoxin that commonly found in environment,as well as in hot processed foods rich in asparagine.In previous study,the mechanism of ACR-induced neurotoxicity was related to axonal degeneration of nervous system,oxidative stress,nerve cell apoptosis,and neuroinflammation.Based on the previous study,we will improve the mechanism of its neurotoxicity in U87-MG cells model,SH-SY5Y cells model and C57/BL/6j mice model,to provide new clues for a better understanding of the intervention mechanism and for exploring effective dietary constituents for toxicological intervention.Firstly,two kinds of nerve cells i.e.astrocytes U87-MG cells and the neuronal SH-SY5Y cells were selected as in vitro models to investigating the effects of ACR-induced cytotoxicity.The cells were exposed to vary concentrations of ACR,which the results showed that cytotoxicity was significantly decreased in a dose-dependent and time-dependent manner after treatment with ACR in U87-MG cells and SH-SY5Y cells.Observing the cells through a light microscope showed that U87-MG cells were shranked,and rounded after ACR treatment,suggesting apoptotsis may play a leading role in ACR-induced cytotoxicity of U87-MG.However,the SH-SY5Y cells accompanied by the production of transparent "pyrolysis vesicles" after ACR treatment.Then,related oxidative stress indicators were measured,and it was found that ACR induced the intracellular reactive oxygen and malondialdehyde levels increased significantly,and significantly reduced glutathione levels.In addition.ACR significantly increased cellular Ca2+ levels,while decreasing mitochondrial membrane potential in a dose-dependent manner.Due to the differences in the toxic phenotypes of ACR on the two types of cells,subsequent studies will be carried out separately to explore the underlay neurological mechanism.Secondly,the effects of apoptosis in ACR-induced cytotoxicity was observed by flow cytometry and Western blotting.Results showed that the cells apoptosis rate was significantly increased after cells were treated with 3.5 mM ACR for 24 h.The protein expression of p-p65 and BAX/Bcl 2 rate were increased,and then the levels of Cleaved Caspase 9,Cleaved Caspase 3 and Cleaved PARP were increased,consequentially promoted the leakage of Cyto C from mitochondria to cytoplasm,suggesting ACR induced U87-MG cells apoptosis via ROS-triggered NF-κB activation and caspase-related mitochondrial apoptosis pathway.In addition,ACR induced autophagosome accumulation in U87-MG cells.Through transfection of GFP-RFP-LC3 adenovirus and immunocolocalization technology,it was found that the autophagosome accumulation induced by ACR could probably be ascribed to blocke the autophagic flux.ACR inhibited the process of autophagosomes combining with lysosomes which was similar to chloroquine(CQ),a typical autophagy inhibitor.When CQ and ACR co-treated U87-MG cells,the apoptosis rate and apoptosis-related protein levels further increased.Therefore,the inhibition of autophagy caused by ACR further promoted the initiation of apoptosis.Thirdly,different from U87-MG cells,ACR can induce pyrolysis on SH-SY5Y cells.Inflammatory response was activated with the release of LDH,and the increased expression of down-stream cytokine TNF-α,IL-1β and IL-18 after ACR treatment.Additionally,the intracellular protein expression were measured by Western blot,and the results showed that the protein expression of NLRP3,ASC,Cleaved Caspase 1,GSDMD,and Cleaved Caspase 8,Cleaved Caspase 3,GSDME were increased after ACR treatment.Specific chemical inhibitors and siRNA of Caspase 1/3 were used to block/silence the Caspase 1 or Caspase 3 gene,and then observed the changes of pyroptosis and the related protein expression.Results showed that the Caspase 1/3 specific chemical inhibitors and caspase siRNA intervention can significantly improve the cytotoxicity which caused by ACR.The intervention effect of Caspase 1 inhibitor was slightly better than that of Caspase 3.After the combined intervention of Caspase 1 and Caspase 3,the release of cellular LDH could be blocked and further alleviated the cytotoxicity which caused by ACR.In addition,the activation sequence of the two signal pathways was investigated by Western blotting.Results showed that the NLRP3 inflammasome pathway was significantly activated when SH-SY5Y cells was exposed ACR after 6 h,and the NLRP3 inflammasome pathway and the Caspase 8/Caspase 3/GSDME pathway were significantly activated when cells were exposed ACR after 24 h.These results indicated that ACR may firstly activated the NLRP3/Caspase 1/GSDMD signaling to trigger an inflammatory response,and then activated the Caspase 8/Caspase 3/GSDME signaling,finally mediated the pyrolysis of SH-SY5Y cells.Finally,the potential mechanisms of ACR-induced neurotoxicity in C57/BL/6j mice models were evaluated.We choosed low dose(5 mg/kg/bw/d)ACR gavage for 60 d,medium-dose(25 mg/kg/bw/d)ACR gavage for 30 d,and high-dose(50 mg/kg/bw/d)ACR gavage for 15 d,to investigate the neurotoxic effect of ACR on mice.The results showed that medium and high doses of ACR can significantly reduce the weight gain of mice.Hindlimb weakness and abnormal gait were observed in the late stage of exposure.H&E staining and Nissan staining were used to investigate the pathological damage of mice brain tissue.Results showed that ACR caused neuron loss and neuron lesions in brain tissue.By measuring biochemical indicators,oxidative stress was indicated by the presence of distinct increase in cellular ROS levels,MDA and 8-OHdG contents,as well as significant decrease in GSH contents after ACR exposure.Meanwhile,ACR inhibited acetylcholine activity and dopamine activity,leading to neurotransmitter dysfunction.The protein expression of brain tissue was investigated by immunohistochemistry and Western blot.Results showed that ACR can significantly increase the protein levels of p-p65,NLRP3,ASC,Cleaved Caspase 1,IL-1β,IL-18,GSDMD,N-GSDMD and P62.The results indicated that ACR-induced neurotoxicity via oxidative stress,leading to neurotransmission barriers and autophagic flow inhibition,and aggravating neurotoxicity.