Zishen Qingre Lishi Huayu Recipe May Ameliorate the Symptoms of PCOS Model Rats via Alleviating Systemic and Ovarian Inflammation.

BACKGROUND: Zishen Qingre Lishi Huayu recipe (ZQLHR) has shown significant therapeutic effects in treating sex hormone levels and follicular developmental disorders in patients with polycystic ovary syndrome (PCOS). However, little is known about the potential mechanisms of its treatment. METHODS: Dehydroepiandrosterone and a high-fat diet induced the PCOS model rat. The serum of rats was collected to detect the levels of sex hormones and inflammatory cytokines by enzyme-linked immunosorbent assay, and the ovaries were collected for ovarian histopathology and qPCR assay to detect the levels of inflammatory cytokines in ovarian tissues. Granulosa cells (GCs) were collected for western blot assay to detect of IL-1ß, IL-6R, and LOX protein expression levels. RESULTS: ZQLHR could reduce body weight, regulate estrous cycles, and improve serum sex hormone levels, follicular development, and insulin resistance (IR) in PCOS model rats. In addition, ZQLHR treatment improved the levels of inflammatory cytokines in serum and ovary, and regulated the protein expression of IL-6R, IL-1ß, and LOX in GCs of PCOS model rats. The results showed that the HOMA-IR index increased with the increasing levels of IL-6, IL-1ß, and CRP, and decreased with the increased IL-10. CONCLUSION: This study reveals that the treatment of endocrine disorders and ovulation disorders in PCOS with ZQLHR may be closely related to the improvement of systemic and ovarian inflammation in PCOS patients, as well as the inhibition of IL-6R, IL-1ß, and LOX expression in GCs, which reemphasizes the role of reducing chronic inflammatory states in the treatment of PCOS. Moreover, this study reemphasizes the correlation between multiple inflammatory mediators and IR.

Zishen Qingre Lishi Huayu recipe promotes proliferation and inhibits apoptosis of GCs of PCOS via KLF4-C/EBPß pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: 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. AIM OF THE STUDY: To verify whether ZQLHR regulates granulosa cells (GCs) proliferation and apoptosis through the Krüppel-like factor 4 (KLF4) - CCATT enhancer-binding proteinß (C/EBPß) pathway, and to provide in vitro molecular mechanism supporting for the effects of ZQLHR to enhance follicular development and treat patients with PCOS. MATERIALS AND 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/EBPß 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/EBPß using siRNAs, the relationship between KLF4 and C/EBPß 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/EBPß 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/EBPß 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/EBPß 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/EBPß pathway. Furthermore, we confirmed that KLF4 and C/EBPß 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.