Neuroprotective Effects And Mechanisms Of Lipoic Acid On Acrylamide-induced Neurophathology

With the growing concern over food quality and safety,it is urgent to minimize the damage of food-derived contaminants,and to make use of natural antioxidants as possible candidates in health-promoting.During heat processing at high temperatures,acrylamide(ACR)can be generated in the Maillard reaction in certain foods.Numerous of literatures demonstrated that long-term exposure of ACR elicited to carcinogenicity,reproduction toxicity,teratogenicity,and neurotoxicity.Lipoic acid(LA)is a naturally occurring nutraceutical synthesized in mitochondria,and can be easily found in common foods like broccoli,spinach,and animal tissues.LA can be utilized as a potential neuroprotective antioxidant due to its capability to penetrate the blood-brain barrier and to accumulate in the brain tissue.This study was designed to determine the possible protective effect of LA against ACR-induced neurotoxicity in vivo and in vitro.Main methods and results are as following:(1)To investigate the protective effect of LA against ACR-induced neurotoxicity in vivo,CD-1 male mice were administrated with dietary LA and/or ACR.Losses in body weight and organ weight of mice treated with ACR were observed in the first place,while LA supplement improved weight gaining.Behavioral assessments were performed,mice in the ACR-treated group exhibited obvious muscle weakness,gait abnormality,and significantly increased hindlimb landing foot splay distance.The footprint patterns of mice treated with ACR also indicated a deterioration of the gait.In accordance with the lack of hindlimb strength,ACR-treated mice also showed their inactivity in the locomotor activity test.Results from Y-maze,a classical behavioral test to assess basic mnemonic processing and exploratory activity,indicated neurological toxicity of ACR from the aspect of the declining mnemonic ability.However,administration of LA was found to be effective in alleviating above symptoms triggered by ACR.Mice brain tissue damages were examined,ACR treatment increased the number of fragmented,damaged or dead neurons,while LA administration significantly reduced degenerated neurons in mice cortex and hippocampus.Further examinations for neural cell degradation showed the increase of the cleaved caspase-3,cleaved PARP,as well as the expression of cytochrome c resulted from ACR exposure were restored to the control levels by LA.(2)The study investigated the underlying mechanisms of neuroprotection conferred by LA against ACR-stimulated neurotoxicity.ACR activated NF-?B pathway,elevated the secretion of inflammatory mediators such as TNF-?,IL-1? and MPO in mice serum,activated MPO m RNA and protein over-expression,depleted GSH level while elicited transcriptional upregulation of several inflammatory mediators,including Cox-2,i NOS,TNF-?,interleukin-1 beta(IL-1?),IL-6 and BACE1 in mice brain tissues.LA was shown to attenuate the ACR-induced inflammation.Down-regulation of ACR towards neurotrophic factors was reversed by LA intake.Moreover,activation of glia aroused by ACR was observed by immunostaining and immunofluorescent staining,which was suppressed by LA as well.(3)The study examined the inflammatory property of ACR and the protective effect of LA towards it in BV2 immortalized mouse microglia cell line.Mitochondrial membrane potential(??)loss as an early event that leads to alterations of mitochondrial function was found to be declined by ACR,but was reversed by LA pretreatment.The activated NF-?B pathway,intracellular ROS generation and H2O2 release,expressions of inflammatory mediators Cox-2 and i NOS,and the subsequent PGE2 and NO production stimulated by ACR exposure were also suppressed by LA,accompanied with an enhancement in GSH level brought about by LA.Mitochondrial dysfunction triggered by ACR lead to the intrinsic pathway of apoptosis,manifested as the increase of the cleaved caspase-3,cleaved PARP,Bax/Bcl-2 ratio and the expression of cytochrome c,which were down-regulated by LA as well.Enhancement of MPO expression was observed in a dose-dependent manner with ACR-intoxicate,while LA showed inhibition effect,along with the protection for BDNF reduction.With pretreatment with a representative MPO inhibitor,4-aminobenzoylhydrazide(ABAH),a similar reduction of MPO expression and recovery of BDNF can be observed.(4)Co-culture system of BV2 microglial cells and the SH-SY5 Y neuroblastoma cells was constructed in form of conditioned medium.Under the pretreatment of ABAH or LA and the incubation with ACR,Cox-2 and i NOS in microglia,cleaved caspase-3 and PARP in neuroblastoma cells were determined.Results demonstrated significant microglial inflammation and the coinstantaneous neuronal apoptosis.As in the SH-SY5 Y neuroblastoma cells,mitochondrial functions,redox status,energy metabolism,mitochondrial respiration,autophagy and cell viability were detected under the pretreatment of LA and the incubation with ACR.Results showed that ACR elicited changes in forms of GSH loss and H2O2 formation,provoked ROS and NO secretions and reduced mitochondrial membrane potential.Mitochondrial dysfunction resulted in energy metabolism disruption and the negative regulation on AMPK/GSK3? catabolic pathway.Furthermore,protein expressions of mitochondrial respiration complexes declined in cells with ACR exposure,with the reduction in Sirt1 and PGC-1? expressions.Impaired cell redox status and inflammation occurred under treatment with ACR through its regulations on redox-sensitive signaling pathways.ACR dampened the phosphorylation of Erk,while intensified the phosphorylation of JNK and p38 in MAPKs,activated NF-?B pathway,reduced Nrf2 nuclear translocation and the downstream expressions of phase II enzymes.Autophagy and mitochondrial autophagy was observed,declines in autophagy-related protein expressions brought a more compelling argument.Interplay between apoptosis and autophagy was explored under pretreatment of autophagy inhibitor(3-Methyladenine,3-MA)and exposure of ACR.Results showed that ACR exerted apoptosis in SH-SY5 Y cells could be partially reduced by inhibiting autophagy.Detection for apoptosis and cytomorphology observations revealed the toxicity of ACR on SH-SY5 Y cell viability.Nevertheless,ACR-exerted mitochondrial dysfunctions,disturbance of redox status and energy metabolism,disruption of mitochondrial respiration,autophagy and loss of cell viability could be alleviated by the pretreatment of LA.Above all,ACR-triggered acute stress in vitro was exhibited as the induction of oxidative stress,mitochondria-mediated energetic homeostasis disruption,mitochondrial dysfunction and the substantial activation of mitophagy,which as a result,lead to cell death.While LA pretreatment conferred protective effects by negatively modulating ACR-induced autophagy and apoptosis in a mitochondrial-dependent manner,and finally reversed the cytotoxicity.In mice,the positive role LA played in restoring mitochondrial functions can be partly ascribed to its regulation in cell signaling pathways and the subsequent decrease in pro-inflammation factors,which lead to the improvement in microglial cellular redox status,including the suppression of MPO over-activation and the corresponding recovery in GSH level and BDNF expression.The inhibition of microglia activation brought about by LA also reversed the neurotrophic factors impairment and nerve terminal dysfunctions,which were reflected in the mice behavioral tests.These findings provided novel insights into the neurotoxicity of ACR and the protective role of LA.