Study On The Mechanism Of Anxiety And Depression Phenotypes And Abnormal Hippocampal Neurogenesis In Offspring Rats Caused By High Androgen Exposure During Pregnancy

Objective: To establish an acquired pregnant hyperandrogen environment animal model with neurobehavioral abnormalities in offspring.Behavior tests were used to examine whether they have behaviors abnormalitiess and immunofluorescence was used to detect effects on neurogenesis in the hippocampal dentate gyrus(DG)of rats,and check whether lithium treatment has effort on the behavior and neurogenesis.Methods: The Sprague–Dawley rats were divided into four groups: CTL group,HA group,LICL group and HA+LICL group.The pregnant rats of HA group and HA+LICL group were treated with subcutaneous injection of a low dose of letrozole(1?g/kg/day)for 3 weeks(from G0 to G20),while control rats received only the vehicle at the same time.After delivery,the LICL group and HA+LICL group were injected lithium 2mmol/kg,and the CTL group and HA group were injected same volume saline from PND1 to PND9.Maternal serum was collected and the levels of testosterone and estradiol hormones were detected by ELISA.Between PND42 and PND56,four types of behavior tests were carried out.The forced swimming experiment was used to test depression-like behavior;the anxiety-like behavior was tested with Elevated plus maze;social interaction ability was examined by an three-chambered social interaction test;self-grooming behaviors were detected by an open-field test.At PND56,the SD rats of the four groups were received intraperitonealinjection of bromodeoxyuridinem(Brdu,100mg/kg)once a day for three days.After BrdU injection,the animals were deeply anesthetized and perfused with PBS and 4% paraformaldehyde buffer.The brain tissues were fixed in 4%paraformaldehyde for forty-eight hours.The changes of neurogenesis in the hippocampus dentate gyrus(DG)were detected by immunofluorescence assay.The positive cells of neurons and astrocytes were examined by Brdu and DCX,NeuN or GFAP antibodies.Western blot was used to detect the expression of?-catenin,DVL2,GSK-3? and TCF-4 in Wnt/?-catenin signal pathway in hippocampus.Results: Serum testosterone levels were higher in HA group and HA+LICL group than in CTL group(P<0.05).There was no significant difference in estrogen among the four groups(P>0.05).The forced swimming experiment showed that the struggling time of the HA group was significantly reduced compared with the CTL group(P<0.05),and there was no statistical difference in the immobility latency(P > 0.05).Compared with CTL group,HA+LICL group treated with chronic lithium was reduced of the open arm retention time and frequency(P<0.05),which was close to the level of CTL group.The open-field experiment showed that the self-grooming frequency of the HA group was lower than that of the CTL group(P<0.05),and the standing frequency of the HA+LiCL group was lower than that of the HA group(P<0.05).The social behavior experiment results showed no significant difference of the communication time and toy time of the four groups of rats(P> 0.05).Immunofluorescence results indicated that hyperandrogen exposure during pregnancy caused neurogenic abnormalities in the hippocampal dentategyrus of rats.Compared with the CTL group,the new DCX positive cells in the HA group were significantly decreased(P<0.05),while the lithium treatment promoted the differentiation of neural progenitor cells(NPC)(P<0.05),and there was no difference between the HA+LICL group and the CTL group(P>0.05).Hyperandrogen exposure during pregnancy also reduced the proliferation of NPC.The Brdu and NeuN labeled neurons in the HA group were significantly lower than those in the CTL group(P<0.05).Chronic lithium treatment showed that the proliferation of NPC was also increased(P<0.05),and there was no statistical difference between the neurons co-expressed by Brdu and NeuN in the HA+LICL group and the CTL group(P<0.05).However,high androgen exposure had no effect on GFAP and Brdu co-expressed astrocytes(P>0.05).Western blot analysis showed no significant difference in expression of?-catenin,DVL2,GSK-3? and TCF-4 among four groups(P > 0.05).Conclusions: Hyperandrogen exposure during pregnancy can lead to anxiety-like and depression-like behavior in offspring,resulting in neurogenic abnormalities in the hippocampal dentate gyrus of rats.The use of lithium chloride can alleviate the anxious-and depressive-like symptoms of offspring associated with high androgen exposure.Objective: To investigate the effect of hyperandrogen on proliferation,apoptosis and synaptic protein expression of neuroblastoma cells(N2a cells).Methods: Mouse N2 a cell line was cultured in vitro with letrozole treatment and lithium intervention 24 h respectively.The effects of letrozole and lithium on N2 a cell proliferation were determined by cell proliferation assay.Cell cycle and apoptosis were detected by flow cytometry;western blot and in situ hybridization were used to detect the expression of?-catenin,DVL2,GSK-3? and TCF-4 in Wnt/?-catenin signal pathway and the expression of letrozole targeting enzyme CYP19A1.The dendritic and synaptic protein markers MAP2 and Synaptophysin of N2 a neurons were detected by immunofluorescence.Results: Cell proliferation determined by CCK-8 assay showed that there were more proliferating cells in the HA group,Licl group and HA+LICL group than in the CTL group(P<0.05),and the cell proliferation was more obvious in the HA+LICL group than in the HA group(P < 0.05).Cell cycle assay results showed that the S phase in the HA group was longer than that in the CTL group(P < 0.05),and the S phase in the HA+ LICL group was shorter than that in the HA group(P < 0.05).Apoptotic cell percentages were increased in the HA group compared with the CTL group(P < 0.05),and there was no significant change in the HA+LICL group compared with the HA group(P > 0.05).In situ hybridization and Western blot analysis showed no significant difference in expression of ?-catenin,DVL2,GSK-3?,TCF-4 and CYP19A1 among four groups.Immunofluorescence revealed differences in the expression positions and amounts of MAP2 and Synaptophysin in the four groups.MAP2 and Synaptophysin were decreased in HA group compared with CTL group,and increased in HA+LICL group compared with HA group.Conclusions: Hyperandrogen could promote N2 a cell proliferation and apoptosis and affect the expression of synaptic proteins.Lithium can increase N2 a cell proliferation.