Liang Haiyan, Zhang Suwei, Li Zhiling
Reproductive Center, The First Affiliated Hospital of Shantou University Medical College, Shantou University, Shantou, Guangdong 515031, People's Republic of China.
Department of Clinical Laboratory Medicine, Shantou Central Hospital, Shantou, Guangdong 515031, People's Republic of China.
Drug Des Devel Ther. 2019 Jun 24;13:2057-2066. doi: 10.2147/DDDT.S208328. eCollection 2019.
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.
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.
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.
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.
据报道人参皂苷Rg3对生殖细胞具有保护作用。然而，Rg3调节小鼠睾丸间质细胞凋亡的机制仍不清楚。此外，雷公藤内酯醇（TP）已被报道可导致雄性大鼠不育。因此，本研究旨在探讨Rg3对TP诱导的MLTC-1细胞毒性的保护作用。
分别采用CCK-8法、免疫荧光法、蛋白质印迹法和流式细胞术检测细胞增殖和细胞凋亡。此外，采用双荧光素酶报告系统检测MLTC-1细胞中miR-26a与糖原合成酶激酶3β（GSK3β）之间的相互作用。
TP显著抑制MLTC-1细胞的增殖，而Rg3可逆转TP的抑制作用。此外，TP通过增加Bax、活化的半胱天冬酶3、细胞色素c和活化的半胱天冬酶9的表达以及降低Bcl-2水平，显著诱导MLTC-1细胞凋亡。然而，Rg3减轻了TP诱导的MLTC-1细胞凋亡。此外，Rg3处理明显下调了miR-26a的水平。上调miR-26a消除了Rg3对TP诱导的MLTC-1细胞毒性的保护作用。同时，双荧光素酶检测显示GSK3β是MLTC-1细胞中miR-26a的直接靶点。miR-26a的过表达显著降低了GSK3β的水平。正如预期的那样，上调miR-26a可通过抑制GSK3β的表达消除Rg3对TP诱导的细胞毒性的保护作用。
这些结果表明，Rg3可通过下调miR-26a保护MLTC-1细胞免受TP损伤。因此，Rg3可能是治疗男性性腺功能减退的潜在药物。
