Effect Of ACTH On Oxidative Stress And Expression Of Glucocorticoid Downstream Genes In Weaned Sow

The hypothalamic-pituitary-adrenal cortex(HPA)axis plays an important role in animal stress response in animals,which not only regulates physiological balance,but also plays an important role in mitigating environmental stress and adaptive response.A large number of studies have reported that environmental stress is associated with animal organ oxidative stress;various stress in vivo and vitro can often lead to the increased free radicals in biological organisms tissues and cells,causing oxidative stress.Oxidative damage is considered to be one of the common ways in which various stress factors affect animal function.However,the use of HPA axis hormones to activate HPA axis and its impact on animal oxidative stress are lack of research reports.In order to study the effect of HPA axis on estrus and ovulation in weaned sows in our previous study,sows injected with ACTH for seven days after weaning,and the model of sow HPA axis activation was established successfully.In this study,the effect of ACTH on oxidative stress and expression of downstream glucocorticoid genes was explored in the HPA axis activation of sow.The main results are as follows:1.Injection of ACTH activated HPA axis in weaned sowsInstallation of jugular vein fistula in Suhuai weaning day,sows were injected with ACTH the next day after weaning,7 consecutive days and 3 times a day(interval 8 h).The result showed that cortisol level was significant increased in the plasma of weaned sow after injection with ACTH.This indicates that HPA axis can be activated ACTH injection.2.HPA axis activation induced weaning sows oxidative stressBlood samples were collected after seven days of ACTH treatment.Results showed that exogenous injection of ACTH resulted in the increased malondialdehyde(MDA)level in the blood.However,the level of glutathione(GSH)and total antioxidant capacity(T-AOC)were decreased in the blood.Furthermore,the activities of antioxidant enzymes(including SOD,GSH-Px and CAT)in blood and ovary of Suhuai sows were significantly decreased after ACTH treatment,and the mRNA expression levels of antioxidant enzyme genes(SOD,CAT and GPX)in blood and ovary were also significantly reduced.These results suggest that activation of the HPA axis can cause oxidative stress in Suhuai sows.3.HPA axis activation up-regulated the gene expression level of glucocorticoid downstream genesqRT-PCR assay showed that ACTH-activated HPA axis up-regulated mRNA expression of glucocorticoid downstream genes(CXCR4,DUSP1,IL7R,TXNIP and Fkbp5).Fkbp5 is a downstream gene of glucocorticoids,however,it binds to glucocorticoid receptors and down-regulates its sensitivity to glucocorticoids.The study found that HPA axis activation can significantly up-regulate Fkbp5 expression levels in the ovary.However,in other tissues,the expression level of Fkbp5 gene did not change significantly.Further studies have found that the protein levels of Fkbp5 in ovary and blood are also significantly up-regulated after ACTH treatment.4.HPA axis activation can induce demethylation of the Fkbp5 GRE regionThe methylation level of the GpG site around GRE region in the Fkbp5 gene were analyzed.It was observed that the methylation level of the 26th CpG site of the intron 1 was significantly reduced both in the blood and ovary;while the methylation level of 28th CpG site of the intron 1 was only significantly reduced in the blood.In intron 2,the methylation level of the 13th CpG site was significantly lower in both the blood and the ovary;while at the methylation of 1 and 4th CpG site only in the blood was significantly reduced.The levels of methylation in the blood and ovary of the 2 and 4th CpG site in the intron 6 were both significantly lower in the blood and in the ovary.In the intron 8,the methylation level of the 3 and 4th CpG site were significantly reduced in the blood.These results indicate that HPA activation induced the DNA demethylation of region of Fkbp5 gene,resulting in the up-regulation of its transcription;up-regulation of Fkbp5 may alter glucocorticoid receptor function by reducing ligand binding.In conclusion,ACTH activated HPA axis can lead to the significant increase in oxidative stress level in weaned sows and up-regulates the expression level of partial glucocorticoid downstream genes in blood.Furthermore,Fkbp5 mRNA abundance was up-regulated in the blood and ovary and the partial DNA methylation level of Fkbp5 was significantly reduced after HPA axis activation.This study clarified the effect of ACTH on oxidative stress and expression of glucocorticoid downstream genes in sows,and provided the basic data for avoiding the oxidative stress and improving the fecundity of weaned sows.