菟丝子总黄酮对小鼠睾丸间质细胞凋亡相关蛋白Bax、Bcl-2、Caspase-3的影响

李利娟; 刘文涛; 罗欢欢; 孟晓彤; 柴冰茹; 谷雨; 廖礼彬; 白生宾

doi:10.3969/j.issn.2095-5227.2022.02.013

菟丝子总黄酮对小鼠睾丸间质细胞凋亡相关蛋白Bax、Bcl-2、Caspase-3的影响

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

摘要

摘要:
背景研究表明睾丸间质细胞的凋亡是造成男性不育的重要因素之一，而菟丝子总黄酮(total flavonoids from semen cuscutae，TFSC)具有抗凋亡、抗炎等药理活性，并在治疗男性不育方面有一定疗效。
目的观察菟丝子总黄酮对小鼠睾丸间质细胞(TM3细胞)凋亡的影响并探究其可能作用机制。
方法体外培养TM3细胞，将其分为对照组(不含药物的完全培养基)、模型组35 µmol/L丙烯醛(acrolein，ACR)、TFSC低剂量组(100 µg/mL)、TFSC中剂量组(200 µg/mL)、TFSC高剂量组(400 µg/mL)。采用CCK-8法筛选ACR的半数致死量和TFSC的作用剂量。采用流式细胞仪检测细胞凋亡情况、Western blotting检测凋亡相关蛋白Bcl-2、Bax、Caspase-3的表达情况。
结果 TFSC浓度为100、200、400、600 µg/mL时明显促进TM3细胞增殖(P＜0.05)；ACR浓度为33.97 µmol/L时，达到细胞半数致死量。与模型组比较，TFSC中、高剂量组可提高细胞存活率(P＜0.05)。流式结果显示，与模型组相比，TFSC中、高剂量组细胞凋亡明显减少。Western blotting结果显示，与模型组比较，TFSC低、中、高剂量组Bcl-2蛋白表达明显升高(P＜0.05)，TFSC中、高剂量组Bax、Caspase-3蛋白表达明显降低(P＜0.05)。
结论 TFSC能够促进TM3细胞的增殖，抑制TM3细胞的凋亡，其机制可能是通过降低凋亡蛋白Bax、Caspase-3的表达以及升高抗凋亡的蛋白Bcl-2的表达来改善抗凋亡与促凋亡蛋白之间的失衡。

Abstract:
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.

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