Adrenomedullin Rescues Testicular Steroidogenesis of Leydig Cells by Suppressing TGF-β1 via PI3K/Akt-Dependent Activation of GSK-3β/β-Catenin Signaling Pathway

Backgroud: Lipopolysaccharide (LPS) exposure can induce Leydig cell dysfunction. This study aims to investigate whether adrenomedullin (ADM) rescues the steroidogenic functions of Leydig cells by suppressing TGF-β1 via PI3K/Akt/ GSK-3β/β-catenin signaling. Methods: Rats were given LPS and injected with an adeno-associated virus vector that expressed ADM (Ad-ADM). Plasma concentrations of testosterone and estradiol were measured. Testes were collected and analyzed by gene expression profiling, western blot and immunofluorescence staining. Primary Leydig cells were treated with various concentrations of LPS and ADM. Cell viability and proliferation and medium concentrations of testosterone and estradiol were detected. Gene expression and protein l evels were detected for steroidogenic enzymes, TGF-β1, PI3K, Akt, GSK-3β and β-catenin. Role of ADM in the regulation of TGF-β1 promoter was confirmed by ChIP and Co-IP. Finding: ADM reversed the decreased number of Leydig cells and plasma concentrations of testosterone and estradiol by rescuing the expression of SF-1, 17β-HSD, LRH1, CYP19, StAR, P450scc, 3β-HSD and CYP17. ADM inhibited the LPS-induced cytotoxicity and reversed the cell proliferation by rescuing the expression of Ki-67. ADM also rescued the expression of SF-1, 17β-HSD, LRH1, CYP19, StAR, P450scc, 3β-HSD and CYP17 and the medium concentrations of testosterone and estradiol in LPS-induced Leydig cells. ADM ameliorated LPS-induced TGF-β1 expression in LPS-exposed Leydig cells. ADM enhanced the phosphorylation of PI3K, Akt and GSK-3β but reduced the activation of β-catenin which interacted with TCF4 and then bound to TGF-β1 gene promoter. Interpretation: ADM may rescue the steroidogenic functions of Leydig cells by suppressing TGF-β1 via the PI3K/Akt/GSK-3β/β-catenin signaling. Funding Statement: This study was supported by the National Science Foundation of China, Beijing, China (Grant Nos.: 81402100, 81401190), Natural Science Foundation of Hunan Province, China (Grant Nos.: 2019JJ40269, 2020JJ4542), Foundation for “Young Medical Talents” of Jiangsu Province, China (Grant No.: QNRC2016840), and Key Guidance Project of University of South China, China (Grant No.: USCKF201902K01). Declaration of Interests: The authors declare no competing financial interests with regard to the publication of this paper. Ethics Approval Statement: Procedures involving rats were approved by the Local Ethics Committee for the Care and Use of Laboratory Animals of University of South China and were conducted in accordance with the national guidelines and protocols of the National Institutes of Health.