Jiangxi University of Chinese Medicine, Nanchang, 330000, China.
Jiangxi University of Chinese Medicine, Nanchang, 330000, China; Institute of Obstetrics and Gynecology, Jiangxi University of Chinese Medicine, Nanchang, 330000, China.
J Ethnopharmacol. 2024 Jun 28;328:118027. doi: 10.1016/j.jep.2024.118027. Epub 2024 Mar 25.
Zishen Qingre Lishi Huayu recipe (ZQLHR) is a herbal recipe created on the basis on the theory of traditional Chinese medicine and clinical practice, and is mainly used in the treatment of polycystic ovary syndrome (PCOS). However, the underlying mechanism for this fact has not been clearly elucidated.
To verify whether ZQLHR regulates granulosa cells (GCs) proliferation and apoptosis through the Krüppel-like factor 4 (KLF4) - CCATT enhancer-binding proteinβ (C/EBPβ) pathway, and to provide in vitro molecular mechanism supporting for the effects of ZQLHR to enhance follicular development and treat patients with PCOS.
Based on previous experiments, we performed the following experiments. Firstly, we treated KGN cells (a steroidogenic human granulosa-like tumor cell line) for 48 h using different concentrations of ZQLHR in order to observe apoptosis in each group. Secondly, the mRNA and protein expression levels of KLF4 and C/EBPβ in KGN cells after administrated with ZQLHR were examined by quantitative real-time PCR(q-PCR) and Western blot assay. Thirdly, after knocking down KLF4 and C/EBPβ using siRNAs, the relationship between KLF4 and C/EBPβ in KGN cells was detected. Further, cell counting kit-8 assay, colony formation assay and flow cytometry were used to verify whether ZQLHR promotes proliferation and facilitates apoptosis in KGN cells through the KLF4-C/EBPβ pathway. Finally, q-PCR and Western blot were used to test whether ZQLHR mediated proliferation and apoptosis-related factors such as B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (BAX), proliferating cell nuclear antigen (PCNA) and cleaved caspase-3 to affect the proliferation and apoptosis of KGN cells through the KLF4-C/EBPβ pathway.
ZQLHR, containing 0.2% by volume, administered to KGN cells resulted in the lowest rate of apoptosis. The expression levels of KLF4 and C/EBPβ were increased in KGN cells following ZQLHR treatment. Additionally, ZQLHR promoted proliferation and inhibited apoptosis of KGN cells by modulating proliferation and apoptosis-related factors via the KLF4-C/EBPβ pathway. Furthermore, we confirmed that KLF4 and C/EBPβ regulate each other in KGN cells. These findings indicate that ZQLHR enhances the proliferation of GCs and suppresses their apoptosis, which constitutes a therapeutic mechanism for treating patients with PCOS.
滋肾清热利湿化瘀方（ZQLHR）是基于中医理论和临床实践而创立的一种中药方剂，主要用于治疗多囊卵巢综合征（PCOS）。然而，其作用机制尚不清楚。
验证 ZQLHR 是否通过 Krüppel 样因子 4（KLF4）-CCAAT 增强子结合蛋白β（C/EBPβ）通路调节颗粒细胞（GCs）增殖和凋亡，并为 ZQLHR 增强卵泡发育和治疗 PCOS 患者提供体外分子机制支持。
基于先前的实验，我们进行了以下实验。首先，我们用不同浓度的 ZQLHR 处理 KGN 细胞（一种类固醇生成的人颗粒状样肿瘤细胞系）48 小时，以观察每组的细胞凋亡情况。其次，用 ZQLHR 处理 KGN 细胞后，通过实时定量 PCR（q-PCR）和 Western blot 检测 KLF4 和 C/EBPβ的 mRNA 和蛋白表达水平。然后，用 siRNA 敲低 KLF4 和 C/EBPβ后，检测 KGN 细胞中 KLF4 和 C/EBPβ之间的关系。进一步，用细胞计数试剂盒-8 检测、集落形成检测和流式细胞术检测 ZQLHR 是否通过 KLF4-C/EBPβ 通路促进 KGN 细胞增殖和诱导凋亡。最后，用 q-PCR 和 Western blot 检测 ZQLHR 是否通过 KLF4-C/EBPβ 通路调节增殖和凋亡相关因子，如 B 细胞淋巴瘤-2（Bcl-2）、Bcl-2 相关 X（BAX）、增殖细胞核抗原（PCNA）和半胱天冬酶-3 切割，从而影响 KGN 细胞的增殖和凋亡。
ZQLHR 以 0.2%体积分数作用于 KGN 细胞，导致细胞凋亡率最低。ZQLHR 处理后，KGN 细胞中 KLF4 和 C/EBPβ 的表达水平增加。此外，ZQLHR 通过调节增殖和凋亡相关因子，通过 KLF4-C/EBPβ 通路促进 KGN 细胞增殖，抑制其凋亡。进一步证实 KLF4 和 C/EBPβ 相互调节 KGN 细胞。这些发现表明，ZQLHR 增强 GCs 的增殖并抑制其凋亡，这构成了治疗 PCOS 患者的治疗机制。
