Allicin Improves Dietary Acrylamide-induced Hepatotoxicity By Regulating Oxidative Stress And Endoplasmic Reticulum Stress

This work was supported by fund from the National Natural Science Foundation of China(No.31571939).Acrylamide(AA)is an important product of Maillard reaction in the thermal processing of food.It has reproductive toxicity,neurotoxicity,liver toxicity,and potential carcinogenicity.Therefore,the safety of dietary intake of AA has caused widespread concern.Studies have found that the occurrence of AA toxicity was closely related to oxidative stress(Oxidative stress,OS)and endoplasmic reticulum stress(Endoplasmic reticulum stress,ERS),but its underlying mechanism was still in the exploratory stage.OS and ERS could be involved in regulating the activation of NLRP3(NOD-like receptor protein-3,NLRP3)inflammasome,and MAPK(Mitogen-activated protein kinase,MAPK)and NF-?B(Nuclear factor kappa-B,NF-?B)signaling pathways also played vital roles in the process of regulating inflammation.Exploring the MAPK and NF-?B signaling pathways involved in the process of AA-induced OS and ERS can not only supplement the toxic mechanism of AA,but also further improve the potential mechanism of OS-and ERS-induced life process disorders.Biologically active ingredients have been widely concerned in regulating the inflammatory response of body.As the main active substance of garlic,allicin has physiological functions such as antioxidant,antibacterial and anti-inflammatory,which shown a prominent role in AA toxicity intervention.Therefore,this study aims to explore the interaction between AA-induced activation of OS,ERS or MAPK signaling pathways and NLRP3 inflammasome activation or inflammation.And on this basis,further clarify the potential mechanism of allicin to protect the body damage caused by AA through allicin intervention.The main research contents and results are as follows:(1)The cells models were constructed using rat liver macrophages Kupffer cells and human liver cancer cells HepG2.The effects of AA on the activation of NLRP3 inflammasome in Kupffer and HepG2 cells were explored.Firstly,Kupffer and HepG2 cells were treated with 1 m M AA and the SOD(Superoxide dismutase,SOD),GSH-Px(Glutathione peroxidase,GSH-Px)enzyme activities and ROS(Reactive oxygen species,ROS)release level,as well as the ERS marker proteins GRP78(Glucose regulated protein 78 k D,GRP78)and CHOP(C/EBP homologous protein,CHOP)were detected to confirm that AA could induce OS and ERS in cells.Secondly,it was found that AA could activate the MAPK and NF-?B signaling pathways and the NLRP3 inflammasome through detecting the gene and protein levels.In addition,the ELISA method was used to detect the release levels of inflammatory factors IL-1?(Interleukin-1?,IL-1?)and IL-18(Interleukin-18,IL-18),which also proved that AA could promote the release of inflammatory factors in Kupffer and HepG2 cells and induce inflammation.On this basis,Kupffer and HepG2 cells were pretreated with ROS scavengers,endoplasmic reticulum stress inhibitors and MAPK selective inhibitors,respectively.To further clarify the effects of OS,ERS and MAPK signaling pathways in regulating AA-induced the activation of NLRP3 inflammasome in Kupffer and HepG2 cells.Studies have found that AA could activate NLRP3 inflammasome and induce inflammation through the OS/ERS-MAPK-NLRP3 signaling pathway.(2)3.75,7.5 and 15 ?M allicin was pre-treated for 1 m M AA-induced Kupffer and HepG2 cells.The effects and regulatory mechanism of allicin on AA-induced NLRP3 inflammasome activation and inflammation in Kupffer and HepG2 cells were investigated.First,SOD,GSH-Px enzyme activities and ROS fluorescent probe,as well as GRP78,CHOP mRNA and protein level were detected to prove that allicin can alleviate AA-induced OS and ERS in Kupffer and HepG2 cells.Second,the IRE1?(Inositol-requiring enzyme 1,IRE1)pathway of the unfolded protein response(UPR)branch,MAPK and NF-?B signaling pathways nodal proteins were detected,and it was found that allicin could inhibit the protein phosphorylation of IRE1?,ASK1(Apoptosis signal regulating kinase-1,ASK1),JNK(c-Jun N-terminal kinase,JNK),ERK(Extracellular regulated protein kinases,ERK),p38(p38 mitogen activited protein kinase,p38 MAPK)and nuclear transcription factor p65(p65 NF-k B)as well as the expression levels of TRAF2(TNF receptor-associated factor 2,TRAF2)and XBP-1(X-box binding protein1,XBP1)proteins,thereby blocking the signaling pathways and inhibiting the NLRP3 inflammasome activation induced by AA.Studies have found that allicin could inhibit the NLRP3 inflammasome activation in Kupffer and HepG2 cells induced by AA through the OS/ERS-MAPK-NLRP3 signaling pathway,and reduced AA induced toxic effects.(3)The SD(Sprague Dawley,SD)rats model of AA poisoning and allicin intervention was established,and the potential mechanism of AA-induced NLRP3 inflammasome activation through in vivo experiments and the regulatory effects of allicin on AA-induced inflammation were studied deeply.In vitro experiments have preliminarily clarified the potential mechanisms of AA-induced NLRP3 inflammasome activation and allicin alleviated the AA-induced inflammation.The study of allicin alleviated AA induced SD rats liver and spleen toxicity could further clarify and improve its potential mechanism.The AA(30 mg/kg)exposure and the allicin(25 mg/kg and 50 mg/kg)protection SD rats model were constructed,respectively.The liver and spleen morphological observation proved that the AA exposure SD rats model was successfully established.The OS index,ERS marker proteins and inflammation index were measured,and it was found that the levels of SOD,ROS,GRP78,CHOP,IL-1? and IL-18 were consistent with the results of in vitro experiments.In addition,the detection results of the expression levels of genes related to the MAPK and NF-?B signaling pathways showed that AA could induce the activation of NLRP3 inflammasome in the liver and spleen of rats by activating the MAPK and NF-?B signaling pathways,and further produced inflammation.Therefore,alllicin has a potential modulatory effect on this process.In summary,AA could activate the MAPK signaling pathway by inducing OS and ERS,and the activation of the MAPK signaling pathway is the key to the activation of NLRP3 inflammasome.NLRP3 inflammasome activation will further trigger the inflammation,and allicin could regulate the activation of MAPK signaling pathway by regulating OS and ERS,inhibit the production of inflammation in the body,eventually relieved the toxicity of AA.Therefore,allicin could inhibit AA-induced activation of NLRP3 inflammasome through the OS/ERS-MAPK-NLRP3 signaling pathway,and alleviated the toxic effects of AA.