The Effect of Aqueous Lessertia frutescens Extract on TM3 Leydig Cells Exposed to TNF- in vitro

Background: Extractions of Lessertia frutescens (Lf) are shown to have immune modulation, anti-inflammatory and antioxidant properties. However, Lf is also cytotoxic, antiproliferative, and pro-apoptotic in vitro. Furthermore, Lf extractions may influence steroidogenesis. Nevertheless, the impact on Leydig cell function has not previously been investigated. As tumor necrosis factor-alpha (TNF-alpha) is known to cause Leydig cell dysfunction under inflammatory conditions, it is further proposed that Lf extracts may protect against the negative impact of TNF-alpha on Leydig cells. The aim of the study was to investigate the effect of an aqueous Lessertia frutescens extract (LFE) on Leydig cells exposed to TNF-alpha in vitro. Methods: Human chorionic gonadotrophin-stimulated TM3 Leydig cells were exposed for 24 h to (a) TNF-alpha (0.1, 1, 10, 100 ng/mL), (b) LFE (0.01, 0.1, 1, 10, 100 ng/mL), and (c) co-exposure to 10 ng/mL TNF-alpha and LFE (0.01, 0.1, 1, 10, 100 ng/mL). We analyzed cell viability, cytotoxicity, caspase 3/7 activation, testosterone concentration, and intracellular superoxide.Results: TNF-alpha exposure decreased cell viability, increased cytotoxicity, and caspase 3/7, with no significant effect on intracellular superoxide in TM3 Leydig cells. When LFE concentrations of 0.01-10 ng/mL were tested, we observed improved vitality and reduced levels of caspase 3/7. At 100 ng/mL, LFE decreased viability and increased cytotoxicity and caspase 3/7. However, LFE did not affect intracellular superoxide. Furthermore, LFE protected against 10 ng/mL TNF-alpha-induced cytotoxicity and apoptosis, except at the highest concentration. LFE alone and in co-culture with 10 ng/mL TNF-alpha increased testosterone at high concentrations.Conclusions: In our TM3 Leydig cell model, LFE protected against TNF-alpha-induced cytotoxicity and early apoptosis, except at the highest experimental concentrations, where it was cytotoxic. These effects were not mediated through a change in intracellular superoxide. Although further investigations are warranted, aqueous LFE may protect against TNF-alpha-induced Leydig cell dysfunction.