Adrenomedullin alleviates the pyroptosis of Leydig cells by promoting autophagy via the ROS–AMPK–mTOR axis

Abstract

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.

Introduction

Leydig cells, located within the interstitial compartment of the testes, mainly contribute to testosterone synthesis and secretion and play a principal role in the development of male traits, reproductive activity and male factor fertility¹. Bacterial lipopolysaccharide (LPS) can induce oxidative stress that leads to the perturbation of Leydig cell mitochondria, which may be the major influential factor involved in the steroidogenic impairment of Leydig cells^2,3,4. Therefore, understanding the cellular and molecular mechanisms underlying the recovery of steroidogenic impairment of Leydig cells has important implications.

The identification of key molecules in the recovery of impaired steroidogenic property of Leydig cells that can be targeted for therapy may help improve outcomes for patients with acute bacterial orchitis. One such potential molecule is adrenomedullin (ADM), which is a possible target for novel therapeutic intervention⁵. ADM is a 52-amino acid peptide originally discovered in human pheochromocytoma tissue and characterised by vasodilation and blood-pressure-lowering effects⁶. Given its anti-oxidant, anti-inflammatory, anti-apoptotic and proliferative properties, ADM exhibits potent protective functions under diverse pathological conditions as an endogenous peptide^{Western blot}

After Leydig cells were treated in accordance with the above-described experimental design and reached confluence, the entire cell lysates were harvested from Leydig cell monolayers. Total protein was adjusted to equal amounts, and protein mixtures were separated via SDS-PAGE and transferred to polyvinylidene difluoride membranes. After the transfer, non-specific binding sites of the membranes were blocked for 1 h at room temperature in PBS (pH 7.4) containing 5% (wt/vol) non-fat dry milk and then incubated with primary antibodies against NLRP3 (1:1000), ASC (1:1000), caspase-1 (1:200), caspase-3 (1:200), caspase-7 (1:200), IL-1β (1:400), IL-18 (1:400), GSDMD (1:1000), LC3-I/II (1:1000), Beclin-1 (1:1000), ATG5 (1:500), p62 (1:500), StAR (1:1000), P450scc (1:1000), 3β-HSD (1:500), CYP17 (1:500), p-AMPK (1:1000), AMPK (1:1000), p-mTOR (1:1000) and mTOR (1:1000) at 4 °C overnight. The membranes were probed with an anti-β-actin antibody (1:1000) to control protein loading, and then incubated for 2 h at room temperature with HRP-conjugated secondary antibodies (1:1000). The results were scanned using a gel imaging system (UVP Company, Upland, CA, USA). Densitometry measurements were performed with Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA). The band intensities were semi-quantified via densitometry analysis using Quantity-One software (Bio-Rad Laboratories, Hercules, CA, USA). Relative protein expression was normalised to β-actin and compared with the control group.

Statistical analysis

Data were expressed as mean ± standard deviation on the basis of at least five separate experiments. Data were analysed using SPSS version 19.0 (SPSS Inc., Chicago, IL, USA). Significant differences amongst the mean values of multiple groups were evaluated with one-way ANOVA followed by Student–Newman–Keuls’ method. A two-sided P value < 0.05 was considered statistically significant.

Ethical approval

This study followed the national guidelines and protocols of the National Institutes of Health and was approved by the Local Ethics Committee for the Care and Use of Laboratory Animals of the University of South China.