Protective Effect Of Proanthocyanidins On Hippocampal Neuron Damage Induced By Oxidative Stress Induced By Benzo[a]pyrene

Objective:1.To investigate whether the neurotoxicity of fetal rats induced by benzo[a]pyrene,B[a]P during pregnancy is related to oxidative stress,so as to provide experimental basis for the toxicity study of B[a]P.2.To investigate whether proanthidin（PC）has protective effect on hippocampal neuron damage induced by oxidative stress induced by B[a]P exposure during pregnancy,and to provide scientific basis for preventing neurotoxicity of B[a]P.Methods:Part 1:Animals are treated in two batches.The first batch:SD pregnant mice were randomly divided into 5 groups with 4 mice in each group,which were blank control group,olive oil group,B[a]P low-dose group(10 mg·kg^-1),B[a]P medium-dose group(20 mg·kg^-1)and B[a]P high-dose group(40 mg·kg^-1),once a day on the 17th day of gestation for 3 consecutive days.Intraperitoneal injection of B[a]P poisoning;The second batch:SD pregnant mice were randomly divided into 4 groups with 5 mice in each group,which were blank control group,olive oil group,B[a]P group(20 mg·kg^-1)and PC intervention group(20 mg·kg^-1 B[a]P+100 mg·kg^-1 PC).On the 17th day of gestation,the mice were intragastric injected with PC solution once a day for 3consecutive days.Intraperitoneal injection of B[a]P poisoning.Fetal rats were removed by anesthesia and laparotomy on the 20th day of gestation in both batches,and the brains of three fetal rats were taken from each group for subsequent Nith staining experiment.The remaining embryonic rats in each group were separated from the hippocampus.The protein expression levels of Nrf2,Keap1,HO-1,Bcl-2 and Bax in the hippocampus were detected by Western blot,and the m RNA expression levels of Nrf2,Keap1,HO-1,Bcl-2and Bax in the hippocampus were detected by q RT-PCR.Part Ⅱ:Hippocampal neurons were isolated and cultured in SD rats within 24 h of birth,andβ-TubulinⅢantibody was used to identify the purity of neurons.Control group,5,10,20,40,80,100μmol·L^-1 B[a]P group were set up,and cell viability was detected by CCK-8 method,and 10,20,40μmol·L^-1 B[a]P was screened for follow-up experiments.The control group,2.5,5,10,and 20μg·m L^-1 PC groups were set up,the cell viability was detected by CCK-8 method,and 2.5μg·m L^-1 PC was screened for follow-up experiments.Control group,PC group(2.5μg·m L^-1 PC),B[a]P group(20μmol·L^-1 B[a]P),B[a]P+PC group(B[a]P 20μmol·L^-1+PC 2.5μg·m L^-1),and CCK-8method were used to detect cell viability.The length and morphological changes of hippocampal neuronal processes in each group were observed by immunofluorescence method.The ROS levels of cells were determined by DCFH-DA fluorescent probe method.The protein expression levels of Nrf2,Keap1,HO-1,Bcl-2 and Bax were detected by Western blot.m RNA expression levels of Nrf2,Keap1,HO-1,Bcl-2 and Bax were detected by q RT-PCR.Results:Part Ⅰ:The results of Nishi staining showed that the neurons in the blank control group and olive oil group had a higher density,were arranged neatly and tightly,the cell morphology and structure were clear and complete,the Nishi bodies were full and abundant,and the color was deep.With the increase of B[a]P concentration,the arrangement of hippocampal neurons is gradually disordered,the pericellular space is gradually enlarged,the matrix is blurred,the cell body shrinks,and the number of nishi decreases.After PC intervention,hippocampal neurons were significantly less damaged than those in the infected group,and the neuronal arrangement was relatively neat and compact.Western blot assay results showed that compared with olive oil group,the protein expression levels of Nrf2,Keap1,HO-1 and Bcl-2 in 10,20 and 40 mg·kg^-1B[a]P groups were decreased（P<0.05 or P<0.01）,and the protein expression levels of Bax were increased（P<0.01）.Compared with B[a]P group,the protein expression levels of Nrf2,Keap1,HO-1 and Bcl-2 in B[a]P+PC group were significantly increased（P<0.01）,while the protein expression levels of Bax were significantly decreased（P<0.01）.The results of q RT-PCR showed that compared with olive oil group,the m RNA expression levels of Nrf2,Keap1,HO-1 and Bcl-2 in 10,20 and 40 mg·kg^-1 B[a]P groups were decreased（P<0.05 or P<0.01）,and the m RNA expression levels of Bax were increased（P<0.01）.Compared with B[a]P group,the m RNA expression levels of Nrf2,Keap1,HO-1 and Bcl-2 in B[a]P+PC group were significantly increased（P<0.01）,while the m RNA expression levels of Bax were significantly decreased（P<0.01）.Part Two:Compared with the control group,with the increase of B[a]P concentration,the immunofluorescence detection results showed that the neuronal protuberance gradually became curved,and then broke,the nucleus shrank,the number of cells decreased,the protuberance length gradually became shorter（P<0.01）,and the number of branches gradually decreased（P<0.01）.At the same time,CCK-8 assay showed a gradual decrease in cell viability（P<0.05）,DCFH-DA fluorescence probe assay showed a gradual increase in ROS（P<0.05）,Western blot assay showed a gradual decrease in Nrf2,Keap1,HO-1,Bcl-2 protein expression（P<0.01）.The expression of Bax protein was increased gradually（P<0.01）,the m RNA expression levels of Nrf2,Keap1,HO-1 and Bcl-2 were decreased gradually（P<0.05）,and the expression level of Bax m RNA was increased gradually（P<0.01）.Compared with B[a]P group,the immunofluorescence detection results of B[a]P+PC group showed that the length of cell process was significantly longer（P<0.01）,the number of cell branches was significantly increased（P<0.01）,and the cell number was increased.CCK-8 method showed that the cell vitality was enhanced（P<0.05）.DCFH-DA fluorescence probe showed that ROS was significantly decreased（P<0.05）,Western blot showed that the expressions of Nrf2,Keap1,HO-1 and Bcl-2 were significantly increased（P<0.05）,and the expression of Bax was significantly decreased（P<0.01）.The results of q RT-PCR showed that the m RNA expression levels of Nrf2,Keap1,HO-1 and Bcl-2 were significantly increased（P<0.01）,while the m RNA expression levels of Bax were significantly decreased（P<0.01）.Conclusions:1.Exposure to B[a]P during pregnancy can cause hippocampal neuron damage in fetal rats,which is related to oxidative stress of cells,and its mechanism may be related to Nrf2-HO-1 pathway.2.PC intervention can alleviate B[a]P-induced oxidative stress injury of fetal hippocampal neurons,and the mechanism may be related to Nrf2-HO-1 pathway.