Adrenomedullin (ADM) exerts anti-oxidant, anti-inflammatory and anti-apoptotic effects in Leydig cells. However, the role and mechanism of ADM in the pyroptosis of Leydig cells are poorly understood. This study first showed the protective effects of ADM on the pyroptosis and biological functions of Leydig cells exposed to lipopolysaccharide (LPS) by promoting autophagy. Primary rat Leydig cells were treated with various concentrations of LPS and ADM, together with or without N-acetyl-L-cysteine (NAC) or 3-methyladenine (3-MA). Cell proliferation was detected through CCK-8 and BrdU incorporation assays, and ROS level was measured with the DCFDA assay. Real-time PCR, western blot, immunofluorescence, transmission electron microscopy, TUNEL and flow cytometry were performed to examine ADM’s effect on the pyroptosis, autophagy and steroidogenic enzymes of Leydig cells and AMPK/mTOR signalling. Like NAC, ADM dose-dependently reduced LPS-induced cytotoxicity and ROS overproduction. ADM also dose-dependently ameliorated LPS-induced pyroptosis by reversing the increased expression of NLRP3, ASC, caspase-1, IL-1β, IL-18, GSDMD, caspase-3, caspase-7, TUNEL-positive and PI and active caspase-1 double-stained positive rate, DNA fragmentation and LDH concentration, which could be rescued via co-incubation with 3-MA. ADM dose-dependently increased autophagy in LPS-induced Leydig cells, as confirmed by the increased expression of LC3-I/II, Beclin-1 and ATG-5; decreased expression of p62 and autophagosomes formation; and increased LC3-II/LC3-I ratio. However, co-treatment with 3-MA evidently decreased autophagy. Furthermore, ADM dose-dependently rescued the expression of steroidogenic enzymes, including StAR, P450scc, 3β-HSD and CYP17, and testosterone production in LPS-induced Leydig cells. Like rapamycin, ADM dose-dependently enhanced AMPK phosphorylation but reduced mTOR phosphorylation in LPS-induced Leydig cells, which could be rescued via co-incubation with 3-MA. In addition, pyroptosis was further decreased, and autophagy was further promoted in LPS-induced Leydig cells upon co-treatment with ADM and rapamycin. ADM may protect the steroidogenic functions of Leydig cells against pyroptosis by activating autophagy via the ROS–AMPK–mTOR axis.

Context : Ursolic acid (UA; 3β-hydroxy-urs-12-en-28-oic acid), one of the pentacyclic triterpenoids found in various plants and herbs, possesses some beneficial effects under pathological conditions, including combating hepatic fibrosis. Objective : This study investigates the effects of UA on renal tubulointerstitial fibrosis in vivo and in vitro . Materials and methods : In vivo , 24 male C57BL6 mice were divided into four groups. Eighteen mice were subjected to unilateral ureteral obstruction (UUO) and the remaining six sham-operated mice served as control. UUO mice received either vehicle or UA (50 or 100 mg/kg) by gastric gavage for 6 days. In vitro , HK-2 cells were treated with 10 or 50 μM UA and 10 ng/mL recombinant human transforming growth factor-β1 (TGF-β1). The molecular mechanisms of fibrosis were investigated. Results : UUO induced marked interstitial collagen I and fibronectin deposition and epithelial-mesenchymal transition (EMT), as evidenced by increased α-smooth muscle actin (α-SMA) and decreased E-cadherin. However, UA treatment significantly reduced collagen I and fibronectin accumulation in the fibrotic kidney. UA treatment also decreased α-SMA and preserved E-cadherin in vivo . In vitro , TGF-β1-treated HK-2 cells demonstrated elevated α-SMA, snail1, slug, TGF-β1, and p-smad3, as well as diminished E-cadherin. UA pretreatment prevented E-cadherin loss and diminished α-SMA expression in HK-2 cells. UA downregulated mRNA expression of snail1 and slug. UA also lowered TGF-β1 protein expression and p-Smad3 in HK-2 cells. Conclusions : UA attenuated renal tubulointerstitial fibrosis by inhibiting EMT, and such inhibition may be achieved by decreasing profibrotic factors. UA may be a novel therapeutic agent for renal fibrosis.

To investigate whether adrenomedullin (ADM) rescues the steroidogenic functions of Leydig cells by suppressing transforming growth factor-β1 (TGF-β1) via the 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-β1 (Ad-sh-TGF-β1). Cell viability and medium concentrations of testosterone were detected. Gene expression and protein levels were detected for steroidogenic enzymes, TGF-β1, RhoA, YAP, TAZ and TEAD1. Role of Ad-ADM in the regulation of TGF-β1 promoter was confirmed by ChIP and Co-IP. Like Ad-sh-TGF-β1, Ad-ADM reversed the decreased number of Leydig cells and plasma concentrations of testosterone by rescuing the gene and protein levels of SF-1, LRH1, Nur77, StAR, P450scc, 3β-HSD, CYP17 and 17β-HSD. Similar to Ad-sh-TGF-β1, Ad-ADM inhibited the LPS-induced cytotoxicity and cell apoptosis and rescued the gene and protein levels of SF-1, LRH1, Nur77, StAR, P450scc, 3β-HSD, CYP17, 17β-HSD and the medium concentrations of testosterone in LPS-induced Leydig cells. Like Ad-sh-TGF-β1, Ad-ADM ameliorated LPS-induced TGF-β1 expression. In addition, Ad-ADM inhibited RhoA activation, enhanced the phosphorylation of YAP and TAZ, decreased TEAD1 expression which interacted with HDAC5 and then bound to TGF-β1 gene promoter in LPS-exposed Leydig cells. ADM may exert anti-apoptotic effect to rescue the steroidogenic functions of Leydig cells by suppressing TGF-β1 via the Hippo signaling.

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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 3β‐hydroxysteroid dehydrogenase (3β‐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), 3β‐HSD, cytochrome P450 17α‐hydroxylase/17, 20 lyase (CYP17) and 17β‐hydroxysteroid dehydrogenase (17β‐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, 3β‐HSD, CYP17 and 17β‐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.