Stress Response Simulated by Continuous Injection of ACTH Attenuates Lipopolysaccharide-Induced Inflammation in Porcine Adrenal Gland.

On modern farms, animals are at high risk of bacterial invasion due to environmental stress factors. The adrenal gland is the terminal organ of the stress response. The crosstalk between adrenal endocrine stress and innate immune response is critical for the maintenance of immune homeostasis during inflammation. Thus, it's important to explore whether stresses play a pivotal role in lipopolysaccharide (LPS)-induced inflammatory response in the porcine adrenal gland. Thirty-days-old Duroc × Landrace × Large White crossbred piglets (12 ± 0.5 kg) were randomly allocated into four groups in a 2 × 2 factorial arrangement of treatments, including ACTH pretreatment (with or without ACTH injection) and LPS challenge (with or without LPS injection). Each group consisted of six male piglets. The results showed that our LPS preparation alone induced mRNA expressions of IL-1ß, IL-6, TNF-α, IL-10, COX-2, TLR2, TLR4, and GR (P < 0.05). ACTH pretreatment downregulated the TLR2 mRNA and IL-6 protein level induced by our LPS preparation significantly (P < 0.05) by one-way ANOVA analysis. Treatment with LPS alone extremely significantly decreased ssc-miR-338 levels (P < 0.01). Interaction of ACTH × LPS was significant for cNOS level (P = 0.011) and ssc-miR-338 expression (P = 0.04) by two-way ANOVA analysis. The LPS treatment significantly downregulated cNOS levels (P < 0.01), which was significantly attenuated by ACTH pretreatment (P < 0.05). Lipopolysaccharide alone did not affect ssc-miR-146b expression levels compared to that in the vehicle group. However, ACTH pretreatment in combination with LPS significantly increased this micro-RNA expression (P < 0.05). TLRs 1-10 were all expressed in adrenal tissue. The LPS challenge alone induced remarkable compensatory mitochondrial damages at the ultrastructural level, which was alleviated by ACTH pretreatment. Accordingly, ACTH pretreatment was able to block LPS-induced secretion of local adrenal cortisol (P < 0.05). Taken together, our results demonstrate that ACTH pretreatment seems to attenuate LPS-induced mitochondria damage and inflammation that decreased cNOS activity in the adrenal gland and ultimately returned local adrenal cortisol to basal levels at 6 h post LPS injection.

Sexually dimorphic effects of maternal dietary protein restriction on fetal growth and placental expression of 11ß-HSD2 in the pig.

Placental 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) inactivates glucocorticoids (GCs) to protect fetuses from over-exposure to maternal GCs, yet how maternal malnutrition affects placental 11ß-HSD2 expression is unknown. In this study, Meishan sows were fed standard-protein (SP) or low-protein (LP, 50% of SP) diets and fetuses/newborn piglets were weighed and the corresponding placenta and umbilical cord blood were collected on gestational day 70 and the day of parturition. Significant growth retardation was observed in female, but not male, fetuses (P < 0.05) and the newborns (P < 0.01) of the LP group, which was accompanied by sexually dimorphic expression of 11ß-HSD2 in placentas. Female fetuses in LP group showed significant decrease in placental 11ß-HSD2 protein content (P < 0.05) and enzyme activity (P < 0.05), whereas male fetuses demonstrated significantly enhanced placental 11ß-HSD2 activity (P < 0.05). Serum cortisol levels were significantly higher (P < 0.05) in male piglets compared to females, and the effects of maternal protein restriction on thyroid hormones (T3 and T4) in the umbilical cord blood were also sex dimorphic. Male piglets in LP group had significantly higher T3 (P < 0.01) and lower T4 (P < 0.01), whereas female piglets showed significantly lower T4 (P < 0.01) with no change in T3. As a result, male piglets in LP group exhibited significantly higher T3/T4 ratio compared to female counterparts. These results indicate that the effects of maternal protein restriction on placental 11ß-HSD2 expression are gender-dependent in the pig, and thyroid hormones may be involved in such effects.

Low amino acids affect expression of 11ß-HSD2 in BeWo cells through leptin-activated JAK-STAT and MAPK pathways.

Maternal protein restriction diminishes placental 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) activity and causes fetal growth restriction in mammals. However, it is unknown whether such effect was caused directly by nutrient deficiency, or indirectly through the mediation of maternal hormones. In the present study, a human placental cell line (BeWo) was cultured in F12K as control and F12 as low amino acids (LAA) media for 48 h to investigate the effects of amino acids deficiency on 11ß-HSD2 expression and activity. Despite a significant up-regulation of 11ß-HSD2 mRNA expression in LAA cells, 11ß-HSD2 activity and protein content were decreased by 38 and 54%, respectively (P<0.05), indicating a mechanism of post-transcriptional regulation. Among 5 miRNAs targeting 11ß-HSD2, miR-498 was expressed significantly higher in LAA cells. Leptin concentration was significantly lower (P<0.01) in LAA medium. The mRNA expression of both isoforms of leptin receptor was significantly higher in LAA cells, although no difference was detected at protein level. To further clarify whether leptin is involved in mediating the effect of LAA on 11ß-HSD2 activity, leptin was supplemented to LAA medium, whereas three specific inhibitors of leptin signaling pathways, WP1066 for JAK-STAT, PD98059 for MAPK and LY294002 for PI3K, respectively were added to control medium. Leptin restored the diminished 11ß-HSD2 activity in LAA cells, whereas WP1066 (5 nM) and PD98059 (50 nM) significantly decreased 11ß-HSD2 activity in control cells. In conclusion, the present results indicate that LAA diminishes 11ß-HSD2 expression and activity in BeWo cells through leptin-activated JAK-STAT and MAPK pathways.