Berberine alters the gut microbiota metabolism and impairs spermatogenesis

Abstract

Berberine (BBR) is used to treat diarrhea clinically. However, its reproductive toxicity is unclear. This study aims to investigate the impact of BBR on the male reproductive system. Intragastric BBR administration for 14 consecutive days results in a significant decrease in the serum testosterone concentration, epididymal sperm concentration, mating rate and fecundity of male mice. Testicular treatment with testosterone propionate (TP) partially reverses the damage caused by BBR to the male reproductive system. Mechanistically, the decrease in Muribaculaceae abundance in the gut microbiota of mice is the principal cause of the BBR-induced decrease in the sperm concentration. Both fecal microbiota transplantation (FMT) and polyethylene glycol (PEG) treatment demonstrate that Muribaculaceae is necessary for spermatogenesis. The intragastric administration of Muribaculaceae intestinale to BBR-treated mice restores the sperm concentration and testosterone levels. Metabolomic analysis reveals that BBR affects arginine and proline metabolism, of which ornithine level is downregulated. Combined analysis via 16S rRNA metagenomics sequencing and metabolomics shows that Muribaculaceae regulates ornithine level. The transcriptomic results of the testes indicate that the expressions of genes related to the low-density lipoprotein receptor (LDLR)-mediated testosterone synthesis pathway decrease after BBR administration. The transcriptional activity of the Ldlr gene in TM3 cells is increased with increased ornithine supplementation in the culture media, leading to increased testosterone synthesis. Overall, this study reveals an association between a BBR-induced decrease in Muribaculaceae abundance and defective spermatogenesis, providing a prospective therapeutic approach for addressing infertility-related decreases in serum testosterone triggered by changes in the gut microbiota composition.

1. Introduction

The infertility rate in couples of reproductive age is as high as 15%, of which male infertility accounts for approximately 50% [1]. Over the past 40 years, the seminal sperm concentration of adult males has decreased by more than 50% in Western countries due to multiple factors, such as unhealthy diet and lifestyle, exposure to toxic chemicals, and the abuse of medications [2]. For example, accumulating evidence shows that traditional Chinese medicine (TCM) might cause reproductive dysfunction. Triptonide, a TCM-derived drug for the treatment of immune-related diseases, was reported to induce sperm malformation and decrease motility [3]. Gossypol, a toxic crystalline compound present in cotton seed oil, has also been shown to repress intercellular communication via gap junctions among Sertoli cells and steroidogenesis of Leydig cells in mouse testes [4,5]. In addition, gossypol has been reported to disrupt spermatogenesis by inhibiting testis-specific lactate dehydrogenase C4 in mice [6]. Aristolochic acid induces testicular injury by inhibiting amino acid metabolism, glucose metabolism, β-oxidation of fatty acids and the tricarboxylic acid cycle in male mice [7]. Recently, berberine (BBR) has been used to treat diarrhea [8], inflammatory diseases [9], and colon cancer [10]. The daily oral administration of 0.9 g of BBR to patients receiving chemotherapy relieves chemotherapy-induced radiation intestinal syndrome [11]. A daily dose of 0.6 g BBR is effective in reducing the risk of recurrence of colorectal adenoma [12]. However, the effect of BBR on the male reproductive system remains to be elucidated.

The gut microbiota is involved in many important physiological processes of the host, including the regulation of immunity and maintaining metabolic homeostasis and cognitive functions [13,14]. Many intestinal diseases can cause imbalances in the gut microbiota, including inflammatory bowel disease, irritable bowel syndrome, chronic constipation and osmotic diarrhea [15]. Diarrhea is often accompanied by changes in the osmotic pressure of the gut, leading to changes in the abundance of osmolality-sensitive microbiota. It has been reported that the osmotic laxative PEG changes the composition of the gut microbiota, resulting in a significant decrease in Muribaculaceae abundance [16,17]. Numerous herbal ingredients have low bioavailability and have been shown to function in regulating the gut microbiota. Saponins were reported to facilitate the growth of beneficial bacteria and suppress cachexia-like symptoms in mice [18]. Rhein, one of the main components of rhubarb, increases the abundance of Lactobacillus and