The toxicity and mechanisms of pyrotinib on male reproductive system

While Pyrotinib has proven effective in treating HER2-positive breast cancer, its impact on male patients is not well-studied. This research examines the potential toxicity of Pyrotinib on the male reproductive system using mouse Leydig cells. Our results indicate that 24 hours of Pyrotinib treatment significantly increased apoptosis and decreased cell viability. Clonogenic assays showed a marked reduction in colony formation, and flow cytometry analysis revealed that 67.2% of the treated cells were in the G1 phase, suggesting hindered progression into the S phase. Western blot analysis demonstrated an initial rise in phosphorylated AKT and ERK, which was followed by a notable decline in these phosphorylated proteins after extended treatment, indicating the involvement of specific pathways in the drug's effects. Transcriptome sequencing identified a downregulation of 197 genes due to Pyrotinib treatment, with these genes enriched in processes related to chromatin and mitosis, particularly in pathways linked to the cell cycle. These findings imply that Pyrotinib inhibits cell proliferation through various molecular mechanisms, underscoring the necessity for further research into its safety and effects on male reproductive health. TM3 cells in the logarithmic growth phase were dissociated and counted, then seeded into 6-well plates at a density of 500,000 cells per well. The treatment group received 100 nM Pyrotinib, while the control group was treated with 0.1‰ DMSO, with three replicates for each group. After 48 hours of treatment, RNA was extracted using TRIzol reagent (Santa) and sent to Beijing Novogene Co., Ltd. for transcriptome sequencing.

吡咯替尼（Pyrotinib）在治疗人类表皮生长因子受体2（HER2）阳性乳腺癌方面已被证实具有良好疗效，但其对男性患者的影响尚未得到充分研究。本研究以小鼠莱迪希细胞（Leydig cells）为模型，探究吡咯替尼对雄性生殖系统的潜在毒性。
研究结果显示，经吡咯替尼处理24小时后，细胞凋亡水平显著升高，细胞活力显著降低。克隆形成实验表明细胞集落形成能力明显下降；流式细胞术分析结果显示，经处理的细胞中有67.2%处于G1期，提示细胞向S期的进程受到阻滞。蛋白质印迹（Western Blot）分析显示，磷酸化AKT与磷酸化ERK的表达水平先出现一过性升高，在延长处理时间后则显著下调，提示该药物的作用涉及特定信号通路。转录组测序结果显示，经吡咯替尼处理后共有197个基因表达下调，这些富集基因涉及染色质调控与有丝分裂相关过程，尤其与细胞周期通路密切相关。上述结果表明，吡咯替尼可通过多种分子机制抑制细胞增殖，凸显了开展进一步研究以明确其对男性生殖健康的安全性与影响的必要性。
实验方法如下：取处于对数生长期的TM3细胞，经消化分离并计数后，以每孔5×10^5个细胞的密度接种于6孔板中。实验组加入100 nM吡咯替尼进行处理，对照组则以0.1‰二甲基亚砜（DMSO）处理，每组设置3个复孔。处理48小时后，采用TRIzol试剂（Santa）提取总RNA，并送至北京诺禾致源科技股份有限公司（Beijing Novogene Co., Ltd.）进行转录组测序。