Abstract：
BACKGROUND: 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.
OBJECTIVE: 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.
METHODS: 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.
CONCLUSIONS: 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.
OBJECTIVE: 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.
METHODS: 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.
CONCLUSIONS: 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.
摘要：
背景：滋肾清热利湿化瘀方（ZQLHR）是在中医理论和临床实践的基础上创建的中药配方，主要用于多囊卵巢综合征(PCOS)的治疗。然而,这一事实的潜在机制尚未明确阐明。
目的：验证ZQLHR是否通过Krüppel样因子4（KLF4）-CCATT增强子结合蛋白β（C/EBPβ）通路调节颗粒细胞（GCs）的增殖和凋亡，并为ZQLHR促进卵泡发育和治疗PCOS患者提供体外分子机制支持。
方法：基于先前的实验，我们进行了以下实验。首先,我们使用不同浓度的ZQLHR处理KGN细胞（类固醇生成的人颗粒样肿瘤细胞系）48小时，以观察各组的细胞凋亡。其次,通过定量实时PCR（q-PCR）和Westernblot检测ZQLHR给药后KGN细胞中KLF4和C/EBPβ的mRNA和蛋白表达水平。第三，在使用siRNA敲除KLF4和C/EBPβ后，检测KGN细胞中KLF4与C/EBPβ的关系。Further,细胞计数试剂盒-8测定，使用集落形成测定和流式细胞术验证ZQLHR是否通过KLF4-C/EBPβ途径促进KGN细胞的增殖和促进凋亡。最后,q-PCR和Westernblot检测ZQLHR是否介导B细胞淋巴瘤-2(Bcl-2)等增殖和凋亡相关因子,Bcl-2相关X(BAX),增殖细胞核抗原（PCNA）和caspase-3通过KLF4-C/EBPβ途径影响KGN细胞的增殖和凋亡。
结论：ZQLHR，按体积计含0.2%，给予KGN细胞导致最低的凋亡率。在ZQLHR处理后,KGN细胞中KLF4和C/EBPβ的表达水平增加。此外,ZQLHR通过KLF4-C/EBPβ途径调节增殖和凋亡相关因子，促进KGN细胞增殖，抑制凋亡。此外，我们证实KLF4和C/EBPβ在KGN细胞中相互调节。这些发现表明ZQLHR增强了GCs的增殖并抑制了它们的凋亡,这构成了治疗PCOS患者的治疗机制。
目的：验证ZQLHR是否通过Krüppel样因子4（KLF4）-CCATT增强子结合蛋白β（C/EBPβ）通路调节颗粒细胞（GCs）的增殖和凋亡，并为ZQLHR促进卵泡发育和治疗PCOS患者提供体外分子机制支持。
方法：基于先前的实验，我们进行了以下实验。首先,我们使用不同浓度的ZQLHR处理KGN细胞（类固醇生成的人颗粒样肿瘤细胞系）48小时，以观察各组的细胞凋亡。其次,通过定量实时PCR（q-PCR）和Westernblot检测ZQLHR给药后KGN细胞中KLF4和C/EBPβ的mRNA和蛋白表达水平。第三，在使用siRNA敲除KLF4和C/EBPβ后，检测KGN细胞中KLF4与C/EBPβ的关系。Further,细胞计数试剂盒-8测定，使用集落形成测定和流式细胞术验证ZQLHR是否通过KLF4-C/EBPβ途径促进KGN细胞的增殖和促进凋亡。最后,q-PCR和Westernblot检测ZQLHR是否介导B细胞淋巴瘤-2(Bcl-2)等增殖和凋亡相关因子,Bcl-2相关X(BAX),增殖细胞核抗原（PCNA）和caspase-3通过KLF4-C/EBPβ途径影响KGN细胞的增殖和凋亡。
结论：ZQLHR，按体积计含0.2%，给予KGN细胞导致最低的凋亡率。在ZQLHR处理后,KGN细胞中KLF4和C/EBPβ的表达水平增加。此外,ZQLHR通过KLF4-C/EBPβ途径调节增殖和凋亡相关因子，促进KGN细胞增殖，抑制凋亡。此外，我们证实KLF4和C/EBPβ在KGN细胞中相互调节。这些发现表明ZQLHR增强了GCs的增殖并抑制了它们的凋亡,这构成了治疗PCOS患者的治疗机制。
