The abnormal expression of kisspeptin regulates pro-inflammatory cytokines, cell viability and apoptosis of macrophages in hyperandrogenism induced by testosterone

Abstract

Background

Increasing evidences have proposed that kisspeptins may be involved in polycystic ovary syndrome (PCOS) including hyperandrogenism. This work aimed to investigate the effect of kisspeptin in hyperandrogenism induced by testosterone.

Methods

The most suitable concentration of testosterone to induce hyperandrogenism was determined by detecting the mRNA changes of kisspeptin and macrophages pro-inflammatory cytokines. The role of kisspeptin in hyperandrogenism was investigated by RT-PCR of kisspeptin and pro-inflammatory cytokines, by CCK-8 of cell viability, by Annexin V-FITC/PI staining followed by flow cytometry of apoptosis, by ELISA of pro-inflammatory cytokines and by Western blot of kisspeptin and antiapoptotic Bcl-2 and proapoptotic Bax.

Results

We found that testosterone elevated kisspeptin, pro-inflammatory cytokines expressions and nitrite release in excessive androgen stimulated macrophages and further inhibited the macrophages cell viability and increased apoptosis. Kisspeptin knockdown reversed the tendency caused by testosterone and decreased apoptosis in macrophages treated with testosterone. Moreover, mRNA and protein expression levels of Bcl-2 and Bax were assessed. We showed a reduction in Bcl-2 mRNA and protein expression levels and an overexpression of Bax mRNA and protein expression levels. Kiss1 silencing reversed Bcl-2 and Bax expressions.

Conclusion

Kisspeptin inactivation confers resistance in hyperandrogenism by inhibiting pro-inflammatory cytokines expressions and apoptosis. Our results may help to comprehend the role of kisspeptin in the mechanisms of hyperandrogenism.

摘要

背景：越来越多的证据表明kisspeptin可能与包括高雄激素血症在内的多囊卵巢综合征(PCOS)有关。本研究旨在探讨kisspeptin在睾酮诱导的高雄激素血症中的作用。

方法：通过检测kisspeptin和巨噬细胞促炎细胞因子的mRNA变化, 确定最适合导致高雄激素血症的最适睾酮浓度。通过以下手段分析kisspeptin在高雄激素血症中的作用：kisspeptin和促炎细胞因子的RT-PCR检测, 检测细胞活力的CCK-8, 膜联蛋白V-FITC / PI染色后流式细胞术检测细胞凋亡, 促炎细胞因子酶联免疫吸附试验, Kisspeptin和抗凋亡Bcl-2和促凋亡Bax的蛋白质印迹分析。

结果：我们发现睾酮引起的高雄激素刺激巨噬细胞导致了kisepteptin和促炎性细胞因子的表达的增加和亚硝酸盐释放的增加, 并进一步抑制了巨噬细胞的细胞活力和增加了细胞凋亡。敲除Kisspeptin基因后由睾酮引起的以上趋势发生了逆转, 并降低了经睾酮处理后的巨噬细胞凋亡。此外, 还对 Bcl-2和Bax的mRNA和蛋白的表达水平进行了评估。我们发现Bcl-2 的mRNA和蛋白表达降低, Bax的 mRNA和蛋白呈过表达。抑制Kiss1可逆转Bcl-2和Bax的表达。

结论：Kisspeptin失活导致促炎细胞因子的表达和细胞凋亡被抑制从而降低高雄激素血症。我们的结果可能有助于理解Kispeptin在高雄激素血症发生机制中的作用。。

Keywords:

• Kisspeptin
• hyperandrogenism
• testosterone

Disclosure statement

The authors report no conflicts of interest.

Additional information

Funding

This study was supported by Basic Research Program of Yunnan Province (No.: 2018FE001(-117)), Open Program of Yunnan Clinical Medicine Center (No.: 2019LCZXKF-SZ03), and The Talent Development Program of Yunnan Province (No.: D-2018054).