<p>Ginsenoside Rg3 protects mouse leydig cells against triptolide by downregulation of miR-26a</p>

Abstract

Background Ginsenoside Rg3 has been reported to exert protection function on germ cells. However, the mechanisms by which Rg3 regulates apoptosis in mouse Leydig cells remain unclear. In addition, triptolide (TP) has been reported to induce infertility in male rats. Thus, this study aimed to investigate the protective effect of Rg3 against TP-induced toxicity in MLTC-1 cells. Methods CCK-8, immunofluorescence assay, Western blotting and flow cytometry were used to detect cell proliferation and cell apoptosis, respectively. In addition, the dual luciferase reporter system assay was used to detect the interaction between miR-26a and GSK3β in MLTC-1 cells. Results TP significantly inhibited the proliferation of MLTC-1 cells, while the inhibitory effect of TP was reversed by Rg3. In addition, TP markedly induced apoptosis in MLTC-1 cells via increasing the expressions of Bax, active caspase 3, Cyto c and active caspase 9, and decreasing the level of Bcl-2. However, Rg3 alleviated TP-induced apoptosis of MLTC-1 cells. Moreover, the level of miR-26a was obviously downregulated by Rg3 treatment. The protective effect of Rg3 against TP-induced toxicity in MLTC-1 cells was abolished by miR-26a upregulation. Meanwhile, dual-luciferase assay showed GSK3β was the direct target of miR-26a in MLTC-1 cells. Overexpression of miR-26a markedly decreased the level of GSK3β. As expected, upregulation of miR-26a could abrogate the protective effects of Rg3 against TP-induced cytotoxicity via inhibiting the expression of GSK3β. Conclusion These results indicated that Rg3 could protect MLTC-1 against TP by downregulation of miR-26a. Therefore, Rg3 might serve as a potential agent for the treatment of male hypogonadism.

Triptolide Induces Leydig Cell Apoptosis by Disrupting Mitochondrial Dynamics in Rats

“…Previous studies have shown that Leydig cells play an important role in testosterone production and spermatogenesis ( Zirkin and Papadopoulos, 2018 ), and triptolide was shown to inhibit superoxide dismutase expression, which increased intracellular ROS in rat Leydig cells, finally inducing oxidative stress damage and cell death ( Hu et al, 2015 ). In addition, triptolide increased miR-26a expression in rat Leydig cells, which decreased GSk 3β mRNA levels and inhibited testosterone production ( Liang et al, 2019 ). Another study also reported that triptolide induced Leydig cell apoptosis, and this effect might be highly associated with miR-200 ( Miao et al, 2020 ).…”

MicroRNAs as crucial mediators in the pharmacological activities of triptolide (Review)

“…In addition, triptolide exerts renal protective, cardioprotective and antiangiogenesis functions by regulating the expression of miRNAs (38)(39)(40)(41)(42). Furthermore, miRNAs are closely associated with the multiorgan toxicity of triptolide (23,43). Tables Ⅰ and Ⅱ list evidence supporting miRNAs as pivotal mediators in the pharmacological properties of triptolide.…”

“…Thus, investigating the molecular mechanisms underlying their activities is highly warranted. As a biologically active ingredient derived from the Chinese herb, Tripterygium wilfordii Hook F., recent studies have demonstrated that triptolide exerts effects on the expression of a series of endogenous miRNAs, which in turn employ several downstream cellular factors to achieve its multiple pharmacological activities, highlighting miRNAs as critical mediators for triptolide-induced effects (18,20,21,23,(34)(35)(36)(37)(38)(39)(40)(41)(42)(43). However, determining the exact role of these miRNAs is limited by the in vitro models offered by these studies.…”

“…miR-26a expression is upregulated in triptolide-induced reproductive toxicity in mouse Leydig cells ( 43 ). This results in cytotoxicity via inhibition of its downstream target, glycogen synthase kinase-3β, which possesses antiapoptotic effects ( 43 ). Collectively, these findings indicate the association between miRNAs and the multiorgan toxicity of triptolide.…”

<p>Downregulation of miR-200a Protects Mouse Leydig Cells Against Triptolide by Triggering Autophagy</p>

“…The dose of TP was calculated based on previous studies. 16,17 Flow Cytometric Analysis Apoptosis in MLTC-1 and in TM3 cells was detected by an Annexin V-FITC/PI Apoptosis Detection Kit (Sigma-Aldrich, St. Louis, MO, USA). Cells were washed three times with pre-cold PBS, and then were resuspended in 500 μL of binding buffer.…”