Anti-apoptotic effect of adrenomedullin gene delivery on Leydig cells by suppressing TGF-ß1 via the Hippo signaling pathway.

This study aims to establish whether adrenomedullin (ADM) is capable to restore the steroidogenic functions of Leydig cells by suppressing transforming growth factor-ß1 (TGF-ß1) through Hippo signaling. Primary Leydig cells were treated with lipopolysaccharide (LPS), an adeno-associated virus vector that expressed ADM (Ad-ADM) or sh-RNA of TGF-ß1 (Ad-sh-TGF-ß1). The cell viability and medium concentrations of testosterone were detected. Gene expression and protein levels were determined for steroidogenic enzymes, TGF-ß1, RhoA, YAP, TAZ and TEAD1. The role of Ad-ADM in the regulation of TGF-ß1 promoter was confirmed by ChIP and Co-IP. Similar to Ad-sh-TGF-ß1, Ad-ADM mitigated the decline in the number of Leydig cells and plasma concentrations of testosterone by restoring the gene and protein levels of SF-1, LRH1, NUR77, StAR, P450scc, 3ß-HSD, CYP17 and 17ß-HSD. Similar to Ad-sh-TGF-ß1, Ad-ADM not only inhibited the LPS-induced cytotoxicity and cell apoptosis but also restored the gene and protein levels of SF-1, LRH1, NUR77, StAR, P450scc, 3ß-HSD, CYP17 and 17ß-HSD, along with the medium concentrations of testosterone in LPS-induced Leydig cells. Like Ad-sh-TGF-ß1, Ad-ADM improved LPS-induced TGF-ß1 expression. In addition, Ad-ADM suppressed RhoA activation, enhanced the phosphorylation of YAP and TAZ, reduced the expression of TEAD1 which interacted with HDAC5 and then bound to TGF-ß1 gene promoter in LPS-exposed Leydig cells. It is thus suspected that ADM can exert anti-apoptotic effect to restore the steroidogenic functions of Leydig cells by suppressing TGF-ß1 through Hippo signaling.

Possible Toxic Mechanisms of Deoxynivalenol (DON) Exposure to Intestinal Barrier Damage and Dysbiosis of the Gut Microbiota in Laying Hens.

Deoxynivalenol is one the of most common mycotoxins in cereals and grains and causes a serious health threat to poultry and farm animals. Our previous study found that DON decreased the production performance of laying hens. It has been reported that DON could exert significant toxic effects on the intestinal barrier and microbiota. However, whether the decline of laying performance is related to intestinal barrier damage, and the underlying mechanisms of DON induced intestine function injury remain largely unclear in laying hens. In this study, 80 Hy-line brown laying hens at 26 weeks were randomly divided into 0, 1, 5 and 10 mg/kg.bw (body weight) DON daily for 6 weeks. The morphology of the duodenum, the expression of inflammation factors and tight junction proteins, and the diversity and abundance of microbiota were analyzed in different levels of DON treated to laying hens. The results demonstrated that the mucosal detachment and reduction of the villi number were presented in different DON treated groups with a dose-effect manner. Additionally, the genes expression of pro-inflammatory factors IL-1ß, IL-8, TNF-α and anti-inflammatory factors IL-10 were increased or decreased at 5 and 10 mg/kg.bw DON groups, respectively. The levels of ZO-1 and claudin-1 expression were significantly decreased in 5 and 10 mg/kg.bw DON groups. Moreover, the alpha diversity including Chao, ACE and Shannon indices were all reduced in DON treated groups. At the phylum level, Firmicutes and Actinobacteria and Bacteroidetes, Proteobacteria, and Spirochaetes were decreased and increased in 10 mg/kg.bw DON group, respectively. At the genus levels, the relative abundance of Clostridium and Lactobacillus in 5 and 10 mg/kg.bw DON groups, and Alkanindiges and Spirochaeta in the 10 mg/kg.bw DON were significantly decreased and increased, respectively. Moreover, there were significant correlation between the expression of tight junction proteins and the relative abundance of Lactobacillus and Succinispira. These results indicated that DON exposure to the laying hens can induce the inflammation and disrupt intestinal tight junctions, suggesting that DON can directly damage barrier function, which may be closely related to the dysbiosis of intestinal microbiota.

Local adrenomedullin gene delivery inhibits Leydig cell dysfunction by rescuing steroidogenic enzymes in vivo.

Adrenomedullin (ADM) has beneficial effects on Leydig cells under pathological conditions, including lipopolysaccharide (LPS)-induced orchitis. Our previous studies demonstrated that ADM exerts a restorative effect on steroidogenesis in LPS-treated primary rat Leydig cells by attenuating oxidative stress, inflammation and apoptosis. In this study, we aim to investigate whether ADM inhibits Leydig cell dysfunction by rescuing steroidogenic enzymes in vivo. Rats were administered with LPS and injected with Ad-ADM, an adeno-associated virus vector that expressed ADM. Then, rat testes were collected for 3ß-hydroxysteroid dehydrogenase (3ß-HSD) immunofluorescence staining. Steroidogenic enzymes or steroidogenic regulatory factors or protein, including steroidogenic factor-1 (SF-1), liver receptor homologue-1 (LRH1), Nur77, steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme (P450scc), 3ß-HSD, cytochrome P450 17α-hydroxylase/17, 20 lyase (CYP17) and 17ß-hydroxysteroid dehydrogenase (17ß-HSD), were detected via gene expression profiling and western blot analysis. Plasma testosterone concentrations were measured. Results showed that ADM may inhibit Leydig cell dysfunction by rescuing steroidogenic enzymes and steroidogenic regulatory factors in vivo. The reduction in the number of Leydig cells after LPS exposure was reversed by ADM. ADM rescued the gene or protein levels of SF-1, LRH1, Nur77, StAR, P450scc, 3ß-HSD, CYP17 and 17ß-HSD and plasma testosterone concentrations. To summarize ADM could rescue some important steroidogenic enzymes, steroidogenic regulatory factors and testosterone production in Leydig cells in vivo.