| Objective:Benzo(a)pyrene(BaP)is a typical polycyclic aromatic compound.At present,most of the studies on the adverse reactions of polycyclic aromatic hydrocarbons are carried out around their properties as carcinogens.There are few studies on their developmental neurotoxic substances,which may cause neurobehavioral disorders.As a fat-soluble substance,benzo(a)pyrene can cross the brain barrier.Benzo(a)pyrene can cause behavioral defects in adults.After the fetus or newborn is exposed to benzo(a)pyrene,it can cause cognitive decline,anxiety-related behaviors,and abnormal neurochemical indicators of hippocampal function and brain activity.Brain diseases(neurodegeneration)and neuronal cell death are related to various changes in mitochondrial homeostasis and function.Nerve cells depend on mitochondria for energy supply,so mitochondrial damage will induce neurotoxicity.In fact,mitochondrial dysfunction has caused some human diseases,such as neurodegenerative diseases.Resveratrol(3,5,4-trihydroxy-trans-stilbene,RSV)is an excellent polyphenolic phytotoxin,which is found in grapes,cranberries and peanuts.It is also a very good neuroprotective agent for humans and animals.In various animal models of neurodegeneration-related research,it was shown that resveratrol can inhibit neuronal apoptosis and improve learning and memory functions,which are contribute to its ability to eliminate antioxidants and reactive oxygen species.However,the regulation of mitochondrial function and its signaling pathways by resveratrol have not yet been explored too much.In this study,Wistar rats exposed to benzo(a)pyrene during embryonic and adolescent periods were used to simulate the nerve damage caused by continuous exposure of benzo(a)pyrene to humans from embryonic to adolescence.At the same time,rats were given resveratrol by gavage,which can evaluate the protective effect of resveratrol on benzo(a)pyrene-induced nerve damage in rats.This will provide new evidence to reveal the protective effect of resveratrol on mitochondrial dysfunction induced bybenzo(a)pyrene.Methods: Wistar rats were obtained from Liaoning Changsheng biotechnology.Temperature was maintained at 24 ± 1 ℃.After acclimatization to laboratory conditions for 1 week,4 female rats mated with 2 male rats(♀: ♂ = 2:1)and were randomly assigned into 7 groups: control group(corn oils),low benzo(a)pyrene exposuregroup(1mg/kg BaP),middle penzo(a)pyrene exposure group(2 mg/kg BaP),high benzo(a)pyrene exposure group(4 mg/kg BaP),resveratrol control group(30 mg/kg RSV),lower resveratrol pretreatment group(15 mg/kg RSV + 4 mg/kg BaP),and higher resveratrol pretreatment group(30 mg/kg RSV + 4 mg/kg BaP).The day when the vaginal plug was observed was the gestational day(GD)0.Pregnant rats were given BaP and/or RSV(dissolved in corn oil)daily by intragastric administration,from GD 0 to postnatal day(PND)21.The interval between BaP and RSV treatment was 4 h.Each group was culled to 10 pups on PND 21(same number of male and female rats in each group).From PND21 to PND 49,the pups were given the same treatments as described above.After the continuous exposure from GD0 to PND49,that is,from embryonic to adolescence,and the relevant indicators are tested.The neurobehavioral changes of rats were detected byopenfieldtest,watermazetestandstep-downpassiveavoidancetest;Nisslstainingand flow cytometry were used to observe brain hippocampal cell damage;transmission electron microscope and flow cytometry were used to detect membrane potential and activity oxygen and ATP detection experiments to observe mitochondrial damage;real-time PCR experiments to detect mt DNA copy number;western blot experiments to detect Beclin1,p62,LC3,Parkin,PINK1,PGC-1α,NRF1,TFAM,p-AMPK,p-PGC-1α protein levels,and observe mitochondrial autophagy and mitochondria biogenesis and phosphorylation of AMPK;the interaction between LC3 and Tom20 protein is detected by Co-IP experiment and immunofluorescenceexperiment.Results: Continuous exposure to BaP from embryonic to adolescence can induce neurotoxicity in rats.The open field test showed that the total distance,time and frequency of the central area of the rats in the high benzo(a)pyrene exposure group were significantly lower than those in the control group;the water maze test showed that the latency of the rats in the high benzo(a)pyrene exposure group was significantly higher than that of the control group,while the number of crossing times was lower than the control group,these experiments show that the rat model of continuous exposure to BaP from embryonic to adolescence is successfully established.Nissl staining experiment found that the survival rate of nerve cells in the hippocampus CA1 area of the high benzo(a)pyrene exposure group was significantly lower than that of the control group,while the survival rate of nerve cells in the high resveratrol pretreatment group was higher than that of the high benzo(a)pyrene exposure group.Flow cytometry apoptosis experiment showed that the apoptosis rate of the high benzo(a)pyrene exposure group was higher than that of the control group.Transmission electron microscopy found that the mitochondria in the high benzo(a)pyrene exposure group became swollen and vacuolated,while the mitochondria in the control group were intact,and the mitochondria in the high resveratrol pretreatment group were less swollen.Using JC-1 kit to detect mitochondrial membrane potential,the high benzo(a)pyrene group was lower than the control group,the membrane potential of the high resveratrol pretreatment group was higher than that of the high benzo(a)pyrene exposure group;and in the ATP detection experiment,the ATP concentration of the high benzo(a)pyrene exposure group was significantly lower than the control group.Western blot experiments found that the proteinexpressionlevelsof Beclin1,p62,LC3Ⅱ/LC3Ⅰ,Parkin,and PINK1 were different,and the protein expression levels of 1 mg/kg BaP group were significantly higher than those of the control group,while the 2 and 4 mg/kg BaP groups were significantly lower than 1mg/kg BaP group and the high resveratrol pretreatment group were higher than the high benzo(a)pyrene exposure group.The mt DNA copy number levels in the high benzo(a)pyrene exposure group were were significantly lower than control group,ang the high resveratrol pretreatment groupwas higher than the high benzo(a)pyrene exposure group.The protein expression levels of PGC-1α,NRF1,TFAM,p-AMPK,and p-PGC-1αin the high benzo(a)pyrene exposure group were significantly lower than those in the control group,and the 30 mg/kg RSV pretreatment group was higher than the high benzo(a)pyrene exposure group.Co-immunoprecipitation and immunofluorescence experiments showed that the interaction between LC3 and Tom20 was lower in the 4mg/kg BaP group than in the 1 mg/kg BaP group,while the 30 mg/kg RSV pretreatment group was higher than the 4 mg/kg BaP group.These indicate that BaP damages mitochondria and causes mitochondrial dysfunction,while RSV can alleviate mitochondrialdysfunction.Conclusion:Continuous exposure to benzo(a)pyrene from embryo to adolescence can cause learning and memory deficits,nerve cell and mitochondrial damage in rats.Resveratrol can activate AMPK/PGC-1α signaling pathway and has a protective effect on mitochondria biogenesis andautophagydisorderinduced bybenzo(a)pyrene. |
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