Effects of total flavonoids from Semen Cuscuta on apoptosis related proteins Bax, Bcl-2 and Caspase-3 of mouse testicular Leydig cells

  Background  Studies have shown that the apoptosis of testicular Leydig cells is one of the important factors leading to male infertility, and total flavonoids from semen cuscutae (TFSC) has anti-apoptotic and anti-inflammatory activities, which has achieved a certain effect in the treatment of male infertility.

  Objective  To observe the effect of TFSC on apoptosis of mouse testicular Leydig cells (TM3) and explore its possible mechanism.

  Methods  TM3 cells were cultured in vitro and divided into control group (drug-free complete medium), model group (35 µmol/L ACR), and TFSC low (100 μg/mL), medium (200 μg/mL) and high dose (400 μg/mL) groups. The median lethal dose of Acrolein (ACR) and the range of active dose of TFSC were evaluated by CCK-8 assay. Flow cytometry was used to detect cell apoptosis, and Western blotting was used to detect the expression of apoptosis-related proteins Bcl-2, Bax and Caspase-3.

  Results  TFSC concentration of 100 µg/mL, 200 µg/mL, 400 µg/mL, 600 µg/mL significantly promoted the proliferation of TM3 cells (all P ＜ 0.05). When ACR concentration was 33.97 µmol/L, the median lethal dose was achieved. Compared with the model group, the cell survival rate of the TFSC medium and high dose groups increased significantly (all P ＜ 0.05). Flow cytometry results showed that TFSC medium and high dose groups could significantly reduce cell apoptosis compared with model group (all P ＜ 0.05). Western blotting results showed that compared with model group, the expression level of bcl-2 protein increased significantly in TFSC low-dose, medium-dose and high-dose groups (all P ＜ 0.05), while the expression levels of Bax and caspase-3 protein decreased significantly in medium-dose and high-dose groups (all P ＜ 0.05).

  Conclusion  TFSC can promote the proliferation of TM3 cells and inhibit the apoptosis of TM3 cells. The anti-apoptosis and imbalance between pro-apoptotic proteins are improved by reducing the expression levels of apoptotic proteins Bax and caspase-3 and increasing the expression level of anti-apoptotic protein Bcl-2.