Protective Effect Of Apigenin On Acrylonitrile-induced Inflammatory Injury And Apoptosis In Rat Brain

Objective: To explore the mechanism of brain injury caused by acrylonitrile(ACN)as well as the interventional effect of apigenin(AP)in rats,so as to provide basis for the prevention and treatment of ACN-induced neurotoxicity and the utilization of AP.Methods: Sixty adult healthy male SD rats were randomly divided into the control group,the ACN-treated group(46.0 mg/kg ACN),the low-dose AP-intervention group(117 mg/kg AP + 46.0 mg/kg ACN),the middle-dose AP-intervention group(234 mg/kg AP + 46.0 mg/kg ACN)and the high-dose AP-intervention group(351 mg/kg AP + 46.0 mg/kg ACN).The rats were orally administered once a day,6 days a week for 4 weeks.AP was administered intragastrically 30 min before ACN gavage and the rats were sacrificed after 24 hours of the last gavage.(1)Measure the activity of rats by the open field test before and after exposure.(2)Observe the pathological changes of brain tissue via preparing HE sections.(3)The levels of malondialdehyde(MDA),glutathione(GSH),glutathione peroxidase(GSH-Px)and superoxide dismutase(SOD)were determined by spectrophotometry;enzyme-linked immunosorbent assay was used to detect the concentrations of interleukin-6(IL-6),interleukin-1?(IL-1?)and tumor necrosis factor-?(TNF-?).(4)The mRNA expressions of TLR-4,IKK-a,I?B-?,NF-?B,Bax,Bcl-2,Caspase-9 and Caspase-3 were conducted by qRT-PCR.(5)Western Blot was carried out for the protein expressions of HMGB-1,TLR-4,IKK-?,p-IKK-?,I?B-?,p-I?B-?,NF-?B p65,Cyt c,Bax,Bcl-2,Caspase-9 and Caspase-3.(6)Immunofluorescence was used for the activation and nuclear transfer of NF-?B p65.(7)The determination of the neuronal apoptosis with TUNEL assay.Results:(1)There was no significant difference in body weight changes of rats among groups during the experiment(P > 0.05).After exposure,the wet weight and organ coefficient of brain decreased in the ACN-treated group(P < 0.05);the wet weights of brain increased in the low-and high-dose AP-intervention groups,and the organ coefficients of brain raised in the low-,middle-and high-dose AP-intervention groups(P < 0.05).(2)After treatment,ACN induced the increases of large-during,large-distance and total distance of motion(P < 0.05);there were the reductions of large-during and large-distance in the middle-and high-dose AP-intervention groups,and the decrease of total distance of motion in the middle-dose AP-intervention group(P < 0.05).(3)ACN caused the pale nuclei and cytoplasm,structurally deficient of pyramidal cells with the appearance of neuronophagia in the cortex,and the reduction and derangement of nerve cell layers in hippocamcus CA1 area;there were the structure close to normal of pyramidal cells in the cortex,and the increase and neatly arrangement of nerve cell layers in hippocamcus CA1 area in the low-,middle-and high-dose AP-intervention groups,obviously in the middle-dose AP-intervention group.(4)ACN induced the increase of MDA and the decrease of GSH-Px while there were the promotion of SOD in the middle-dose AP-intervention group,and the raise of GSH and GSH-Px in the low-,middle-and high-dose AP-intervention groups(P < 0.05).(5)ACN led to the upregulated mRNA expressions of TLR-4,IKK-? and NF-?B,the increased protein expressions of HMGB-1,TLR-4,IKK-?,p-IKK-?,p-I?B-? and NF-?B p65,and the raised protein expression ratios of p-IKK-?/IKK-? and p-I?B-?/I?B-?(P < 0.05);there were the downregulated mRNA expressions of TLR-4,IKK-? and NF-?B in the middle-and high-dose AP-intervention groups,the upregulated mRNA expression of I?B-? in the low-dose AP-intervention group,the decreased protein expressions of HMGB-1,IKK-?,p-IKK-?,p-I?B-? and NF-?B p65 and the increased protein expressions of I?B-? in the middle-and high-dose AP-intervention groups,the reduced protein expression of TLR-4 in the high-dose AP-intervention group,and the lowered protein expression ratios of p-IKK-?/IKK-? and p-I?B-?/I?B-? in the low-dose AP-intervention group(P < 0.05).(6)AP inhibited the activation and nuclear transfer of NF-?B p65 induced by ACN,and the effect was more obvious with the increase of dose.(7)ACN caused the increase of IL-6 while the IL-6 and TNF-? reduced in the low-dose AP-intervention group,and the IL-6 decreased in the middle-and high-dose AP-intervention groups(P < 0.05).(8)The number of the TUNEL-positive cells in the high-dose AP-intervention group was significantly less than that in the ACN-treated group(P < 0.05).(9)ACN induced the upregulated mRNA expressions of Bax and Caspase-3,the increased protein expressions of Cyt c,Bax,Bcl-2,Caspase-9 and Caspase-3,and the decreased protein expression ratio of Bcl-2/Bax(P < 0.05);there were the reduced mRNA expressions of Bax and Caspase-9 in the high-dose AP-intervention group,the decreased mRNA expressions of Caspase-3 in the low-,middle-and high-dose AP-intervention groups,the increased mRNA expressions of Bcl-2 in the middle-and high-dose AP-intervention groups,the lessened protein expressions of Cyt c,Bax,Caspase-9 and Caspase-3 in the low-,middle-and high-dose AP-intervention groups,the promoted protein expressions of Bcl-2 in the middle-and high-dose AP-intervention groups,and the raised protein expression ratio of Bcl-2/Bax in the high-dose AP-intervention group(P < 0.05).Conclusions:(1)The activations of TLR-4/NF-?B signaling pathway and mitochondrial-mediated apoptosis pathway by oxidative stress may be one of the mechanisms of ACN-induced brain injury in rats.(2)The intervention with AP could protect the rat brain against the inflammatory injury and apoptosis caused by ACN.