Protection Of Silymarin Against Acrylamide-induced Oxidative Damage In PC12 Cells

Acrylamide(AA)is mainly used in industry and causes neurotoxicity,reproductive toxicity,genotoxicity and carcinogenicity in humans and animals.Recently,it has been reported that AA can be formed during the high-temperature(?120 ?)processiong of food containing starch via the Maillard reaction.Therefore,it is necessary to prevent and inhibit the AA toxicity in human.The phytochemical,which has strong biological activity and good medicina,has been widely focused.Silymarin(SM)is a well known polyphenolic flavanoid purified from the seeds of mike thistle(Silybum marianum).SM has function of anti-inflammatory and antioxidant and inhibition of enzyme activity involved in AA metabolize.In this study,we used PC12 cell line as model of nervous system and oxidative stress,in order to explore the mechanism of SM against AA neurotoxicity in vitro.The consequences of this study provide new ideas for reducing AA toxicity in food to human health.The details were as follows:(1)The effect of AA cytotoxicity in PC12 cells were detected by MTT reduction assay.PC12 cells were treated with various concentrations of AA(0,1.25,2.5,5.0,10 and 20 mM)for 12 h,24 h and 48 h,and the viability was reduced by AA in dose-dependent and time-dependent manner.The viability was inhibited approximately 61 % in the presence of 5 mM AA for 24 h,and we selected 5 mM AA for 24 h in the following experiments.(2)The effect of SM on the PC12 cells proliferation and the protective effect of SM against AA induced toxicity in PC12 cells were detected by MTT reduction assay.Cell viability was analysed after treatment with different concentrations of SM(0,12,24,48,96 and 192 ?g/mL).The results indicated that 12 h of SM treatment was not cytotoxic to PC12 cells(P>0.05).Then,the cells were treated with 5 mM AA for 24 h after pretreatment with 0,12,24,48,96 and 192 ?g/m L SM for 1.5 h,3 h and 6 h.Cell viability results showed that SM had a cytoprotective effect against AA in PC12 cells in a dose-dependent manner and that SM treatment was significantly protective at 3 h,and we selected SM for 24 h in the following experiments.(3)The protective effects of SM against AA induced toxicity in PC12 cells were observed by inverted microscope analysis.The results showed that SM effectively protected PC12 cells against AA-induced injury.(4)The effects of SM on oxidative damage in AA-induced PC12 cells were systematical evaluated.Cells were treated with different concentrations of SM(0,12,24,48,96 and 192 ?g/mL)for 3 h and then exposed to 5 mM AA for 24 h.The data revealed that SM could reduce reactiveoxygen species(ROS)and malondialdehyde(MDA)levels and increase intracellular glutathione(GSH)levels in AA-induced PC12 cells in a dose-dependent manner.(5)The effects of SM on the m RNA expression of E2-related factor 2(Nrf2),glutathione peroxidase(Gpx),glutamate cysteine ligase catalytic subunit(GCLC)and glutamate cysteine ligase modifier subunit(GCLM)in AA-induced PC12 cells were determined by quantitative real-time PCR(q RT-PCR).Cells were treated with different concentrations of SM(0,12,24,48,96 and 192?g/mL)for 3 h and then exposed to 5 mM AA for 24 h.The data revealed that SM treatment significantly induced the mRNA expression of Nrf2,Gpx,GCLC and GCLM in a dose-dependent manner in AA-induced PC12 cells.(6)The effects of SM on the protein of the nuclear and cytosolic fractions Nrf2,Gpx,GCLC and GCLM in AA-induced PC12 cells were determined by Western bolt.PC12 cells were treated with various concentrations of SM(0,12,24,48,96 and 192 ?g/mL)for 3 h and then exposed to 5mM AA for 24 h.As a result,SM significantly enhanced accumulation of nuclear Nrf2,Gpx,GCLC and GCLM protein and reduced cytoplasmic Nrf2 protein levels in AA-exposed PC12 cells in a dose-dependent manner.The data suggested that SM can protect PC12 cells against AA induced toxicity by improving Nrf2 translocation.In summary,SM has a effective protection on AA-induced PC12 cells toxicity via a activation of the defence mechanism of Nrf2 signalling pathways involved in GSH metabolism,increasement of GSH levels and reduction of ROS and MDA levels.The consequences of this study provide a support in vitro for SM neuroprotection and a new pathway for restrain of AA toxicity in vitro.