Expression And Regulation Of Genes Involved In Cortisol Action In Hippocampus Of Pigs

Stress response of animal involves the activation of hypothalamus-pituitary-adrenal axis (HPA axis) and the increase of cortisol secretion. Hippocampus can maintain organism’s homeostasis by regulating the activity of HPA axis upon receiving the feedback information of cortisol from periphery. The sensitivity of hippocampus to cortisol was determined by two types of glucocorticoid receptors (GR and MR) and two isozymes,11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) and type 2 (11β-HSD2). In our study, we first choose Erhualian (EHL) and Pietrain (PIE) as model to reveal breed difference in hippocampal expression pattern of genes involved in cortisol action in both basal and stressed conditions. In order to clarify whether the breed differences in gene expression between EHL (Ha1NN) and PIE (Ha1nn) pigs were attributed to halothane genotypes, we compared hippocampal expression pattern of genes involved in cortisol action in F2 PIEⅩEHL littermates with segregated halothane genotypes. To test the hypothesis that the breed specific pattern of hippocampal gene expression in EHL pigs is a result of natural selection under the pressure of low protein diet through generations of metabolic programming, we investigated the effect of maternal protein restriction during pregnancy and lactation on serum cortisol levels and hippocampal pattern of gene expression in offspring. To further explore whether cortisol mediates the programming of hippocampal gene expression, we conducted an in vitro study with primary cultured hippocampal neurons to examine the effect of cortisol pretreatment on cell viability and its sensitivity to cortisol. Finally, we investigated the effect of dietary supplementation of curcumin on serum cortisol concentration and hippocampal gene expression in pigs under transport stress in order to investigate stress alleviation efficacy of curcumin and its possible action pathway.1 Effect of transport stress on hippocampal expression of genes involved in cortisl action in Erhualian and Pietrain pigs In the present study,12 EHL and 12 PIE pigs at body weight of 20±2 kg were respectively allocated to 4 groups:EHL control, EHL transport, PIE control and PIE transport. Pigs in the transport groups were subjected to 2 h transport. The results showed that EHL pigs exhibited significantly lower hippocampus weight and the ratio of hippocampus weight/body weight than that of PIE pigs. Under basal condition, EHL pigs exhibited higher expression of GR, MR,11βHSD1 and ratio of 11β-HSD1/11β-HSD2, but lower 11β-HSD2, Bax-αand Bcl-xL in hippocampus compared with PIE pigs (P<0.05). Under stressed condition, EHL pigs displayed higher expression of 11β-HSD1 and ratio of 11β-HSD1/11β-HSD2, but lower expression of 11β-HSD2, Bax-α, Bcl-xL and ratios of GR/MR and Bax-α/Bcl-xL. In addition, the hippocampal expressions of GR, MR, BDNF, Bax-α/Bcl-xL in EHL pigs were significantly down-regulated (P<0.05) compared with their control counterparts, with no alteration in GR/MR ratio, whereas PIE pigs in transport group exhibited significantly up-regulated GR/MR ratio (P<0.05). Moreover, EHL pigs in transport group showed significantly elevated Bcl-xL mRNA expression, resulting in significantly lower Bax-α/Bcl-xL ratio (P<0.05), compared with the control. These results indicate that EHL pigs may be more effective in the negative feedback regulation of HPA axis activity. Furthermore, EHL pigs are more flexible and adaptative under stressed situation reflected by changes in hippocampal gene expression. All these data demonstrate significant breed-specific pattern of hippocampal expression of GR/MR, 11β-HSD1/2 and Bax-α/Bcl-xL, which may account, to some extent, for the breed-specific stress coping characteristics in the pig.2 Effect of transport stress on hippocampal expression of genes involved in cortisol action in pigs with different halothane genotypesIn order to determine the effect of halothane genes on hippocampal expression of genes involved in cortisol action in pigs, EHL sows (Ha1NN) were mated to PIE boars (Ha1nn) to produce F1 (Ha1Nn) pigs which were used for brother-sister inbreeding to produce F2 with segregated genotypes:Ha1NN, Ha1Nn, and Ha1Nn. F2 pigs were used to measure serum cortisol concentration and hippocampal expression of genes involved in cortisol action. None of parameters detected showed significant differences among different halothane genotypes, indicating that halothane genotype does not seem to be the cause for the breed-specific pattern of hippocampal expression of genes involved in cortisol action in EHL and PIE pigs. 3 Effect of maternal protein restriction on serum cortisol levels and hippocampal expression of genes involved in cortisol action in offspring pigletsTo test the hypothesis that the breed specific pattern of cortisol related gene expression in hippocampus is related to metabolic programming of protein restriction of pregnant sows, Meishan sows were divided to control and protein restriction groups. Both groups were fed with either standard protein level diet (control group) or protein restriction diet (50% of standard protein level, protein restriction group) from copulation to weaning. Serum cortisol levels of sows were detected on 60 and 90 d post copulation (dpc), as well as 2 weeks after parturition. Serum cortisol levels of offspring were detected on 0,14 and 35 d after birth. Cortisol related gene expression pattern in hippocampus of newborn offspring (0 d) and weaning piglets (35 d) were investigated. The results showed that serum cortisol level of sows was not influenced by protein restriction on 60 d of pregnancy or 2 weeks after parturition. Serum cortisol level was significantly lower in protein restriction group than control only in 14-day-old female piglets, no significant variations in serum cortisol were determined on the other age groups (0 and 35 d). Serum cortisol level showed sexual difference in an age-dependent fashion which were diminished or reversed by protein restriction. For instance, the male> female (P< 0.05) dimorphism of serum cortisol in newborn piglets was observed in control but not in protein restriction group; in contrary, the female> male (P< 0.05) dimorphism shown on 14 days of age was reversed in protein restriction group. Effect of protein restriction on gene expression in piglets’ hippocampus also showed sex- and age-dependent pattern. Significant alteration was only seen in newborn piglets, being more significant in females. In protein restriction group, female offspring expressed significantly higher (P< 0.05) GR, MR,11β-HSD1 and 11β-HSD2 mRNA compared to control, whereas in male offspring only detected a significant increase (P< 0.05) in GR transcription. In addition, male piglets exhibited higher expression of GR, MR,11β-HSD1 and 11β-HSD2 mRNA compared with females in control group (P< 0.05), but again this sexual dimorphism disappeared in protein restriction group. Furthermore, higher DNMT1 mRNA (P< 0.05) found in male piglets was also eliminated in protein restriction group. All the results above indicate that protein restriction during gestation and lactation of sow can program cortisol related gene expression in offspring, and female piglets seems to be more susceptible. Alteration of gene transcription may involve changes in DNA methylation. It supports our hypothesis to some extent that low protein diet may serve as a natural selection pressure for the formation of breed-specific pattern of cortisol related gene expression in hippocampus of EHL pigs, as the up-regulation in hippocampal expression of genes in protein restriction group resembles the pattern found in EHL pigs.4 Effect of cortisol pretreatment on viability and gene expression in porcine primary hippocampal neurons under basal and stressed situations in vitroPigs with different serum cortisol levels show different hippocampal expression pattern of genes involved cortisol action, implicating that higher cortisol exposure may influence the activity and cortisol sensitivity of hippocampal neurons. The effect of maternal protein restriction on hippocampal gene expression in offspring may also be mediated by cortisol. Therefore, in this study, primary cultured porcine hippocampal neurons were pretreated with 0 M,10-7 M and 10-6 M cortisol respectively for 6 days, then each group was subdivided to control and stress-mimic group which was challenged with 10-5M cortisol for 4 h before harvest. Neuron viability was detected with MTT, apoptosis status was evaluated by Caspase 3/7 activity and gene expression was quantitated with real time PCR. The results showed that cortisol pretreatment significantly decreased viability of neurons (P < 0.05) without remarkable alteration in caspase 3/7 activity. However, cortisol pretreatment increased neuron protection under stress. Neurons without pretreatment showed significantly increased caspase 3/7 activity when treated with 10-5 M cortisol (P< 0.05), but pretreated cells did not show such increase. Cortisol pretreatment did not change GR, MR,11β-HSD1 and 11β-HSD2 mRNA expression under basal condition, but prevented the down-regulation of GR and 11β-HSD1 expression in non-pretreated cells under stress-mimic condition. These results indicate that cortisol pretreatment is helpful to increase cytoprotection capacity of hippocampal neurons under stress, and to maintain the sensitivity of hippocampal neurons to cortisol which is essential for the effective negative feedback of cortisol on HPA axis.5 Effect of dietary curcumin supplementation on serum cortisol concentrarion and hippocampal expression of functional genes in pigs under transport stressIn order to investigate the efficacy of curcumin in alleviating transport stress in pigs and the possible action pathway, curcumin (8 mg/kg) was added in the diet of weaning piglet. The curcumin group was fed with curcumin supplemented diet for 21 days, and then loaded on a truck for 2 h transport together with control pigs. After transport, all pigs were sacrificed and blood sample and hippocampi were collected for detecting serum cortisol and hippocampal expression of genes involved in cortisol action. The results showed that 21 days of dietary curcumin treatment did not influence body weight, hippocampal or adrenal weights. Curcumin treatment alleviated increase of serum cortisol level induced by transport (P< 0.05), but did not affect hippocampal expression of GR, MR,11β-HSD1 or 11β-HSD2. However, curcumin treatment prevented attenuation of BDNF expression in hippocampus induced by transport stress (P< 0.05). These results indicate that curcumin can effectively relieve stress response and exert cytoprotection effect for hippocampus neurons possibly through BDNF-mediated pathway.