Abstract：
BACKGROUND: Polycystic ovary syndrome (PCOS) is a common and complex endocrine disease in women, with a prevalence of 5% to 18% worldwide. HeQi San (HQS) is a Chinese medicine compound prescription, which has been applied to treat various endocrine and metabolic diseases.
OBJECTIVE: The study was intended to investigate the effect of HQS on PCOS, and clarify the potential mechanism via in vivo and in vitro experiments.
METHODS: The PCOS mouse model was established by injecting the dehydroepiandrosterone (DHEA) subcutaneously and fading high-fat diet for 3 weeks. After making model, PCOS mice were treated with HQS (8.75 g/kg and 17.5 g/kg, ig.) for 4 weeks. Firstly, we assessed the histopathological changes in ovary tissues and detected the hormone level. Subsequently, the study evaluated the capability of anti-inflammatory and regulating macrophage polarization of HQS in vivo and in vitro. The secretion of inflammation indicators was measured with Elisa kits, and the expression level of phosphorylated nuclear factor kappa-B (P-NFκB) and B-lymphocyte activation antigen B7 (CD80) was measured by immunofluorescence and Western blot. Meanwhile, the apoptosis of ovarian granulosa cells was detected via tunel staining and Western blot. The co-culture model in vitro was utilized to assess the effect between macrophage polarization and human ovarian granulosa cells (KGN cells) apoptosis. Furthermore, 16S rDNA sequencing was utilized to elevate gut microbiota change in PCOS mice.
RESULTS: HQS reversed the abnormal hormone increase, ameliorated insulin resistance, and improved histopathological changes of the ovary tissue to exert the therapeutic effect. HQS inhibited the expression of P-NF-κB and decreased the production of interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) to further prohibit the macrophage M1 polarization in ovary tissues and macrophages. The apoptosis-positive cells, Bcl-2 Assaciated X protein (BAX), and cleaved-caspase 3 expression were also decreased in the treatment group. The B-cell lymphoma-2 (Bcl2) expression was enhanced after HQS treatment in vivo. The co-culture experiments also verified that HQS could prevent the apoptosis of KGN cells. Furthermore, HQS mediated the abundance of gut flora. The abundance of bifldobacterium and parasutterella was increased and the abundance of lachnoclostridium was decreased.
CONCLUSIONS: The study verified that HQS has the effect of anti-inflammation and inhibits macrophage M1 polarization. Besides, HQS could mediate the abundance of gut microbiota in mice with PCOS. Thus, this study would provide more reasonable basis of HQS for clinical use. In conclusion, HQS might be a potential candidate for PCOS treatment.
OBJECTIVE: The study was intended to investigate the effect of HQS on PCOS, and clarify the potential mechanism via in vivo and in vitro experiments.
METHODS: The PCOS mouse model was established by injecting the dehydroepiandrosterone (DHEA) subcutaneously and fading high-fat diet for 3 weeks. After making model, PCOS mice were treated with HQS (8.75 g/kg and 17.5 g/kg, ig.) for 4 weeks. Firstly, we assessed the histopathological changes in ovary tissues and detected the hormone level. Subsequently, the study evaluated the capability of anti-inflammatory and regulating macrophage polarization of HQS in vivo and in vitro. The secretion of inflammation indicators was measured with Elisa kits, and the expression level of phosphorylated nuclear factor kappa-B (P-NFκB) and B-lymphocyte activation antigen B7 (CD80) was measured by immunofluorescence and Western blot. Meanwhile, the apoptosis of ovarian granulosa cells was detected via tunel staining and Western blot. The co-culture model in vitro was utilized to assess the effect between macrophage polarization and human ovarian granulosa cells (KGN cells) apoptosis. Furthermore, 16S rDNA sequencing was utilized to elevate gut microbiota change in PCOS mice.
RESULTS: HQS reversed the abnormal hormone increase, ameliorated insulin resistance, and improved histopathological changes of the ovary tissue to exert the therapeutic effect. HQS inhibited the expression of P-NF-κB and decreased the production of interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) to further prohibit the macrophage M1 polarization in ovary tissues and macrophages. The apoptosis-positive cells, Bcl-2 Assaciated X protein (BAX), and cleaved-caspase 3 expression were also decreased in the treatment group. The B-cell lymphoma-2 (Bcl2) expression was enhanced after HQS treatment in vivo. The co-culture experiments also verified that HQS could prevent the apoptosis of KGN cells. Furthermore, HQS mediated the abundance of gut flora. The abundance of bifldobacterium and parasutterella was increased and the abundance of lachnoclostridium was decreased.
CONCLUSIONS: The study verified that HQS has the effect of anti-inflammation and inhibits macrophage M1 polarization. Besides, HQS could mediate the abundance of gut microbiota in mice with PCOS. Thus, this study would provide more reasonable basis of HQS for clinical use. In conclusion, HQS might be a potential candidate for PCOS treatment.
摘要：
背景：多囊卵巢综合征（PCOS）是女性常见且复杂的内分泌疾病，全球患病率为5％至18％。何芪散（HQS）是一种中药复方，已用于治疗各种内分泌和代谢疾病。
目的：本研究旨在研究HQS对PCOS的影响，并通过体内外实验阐明潜在的机制。
方法：皮下注射脱氢表雄酮（DHEA），淡化高脂饮食3周，建立PCOS小鼠模型。制作模型后，PCOS小鼠用HQS治疗（8.75g/kg和17.5g/kg，伊格.）4周。首先,我们评估了卵巢组织的组织病理学变化并检测了激素水平。随后，该研究评估了体内和体外HQS的抗炎和调节巨噬细胞极化的能力。用Elisa试剂盒测定炎症指标的分泌,免疫荧光和Westernblot检测磷酸化核因子κB（P-NFκB）和B淋巴细胞活化抗原B7（CD80）的表达水平。同时,通过tunel染色和Westernblot检测卵巢颗粒细胞的凋亡。体外共培养模型用于评估巨噬细胞极化与人卵巢颗粒细胞（KGN细胞）凋亡之间的作用。此外，16SrDNA测序用于提高PCOS小鼠的肠道微生物群变化。
结果：HQS逆转了激素异常增加，改善胰岛素抵抗，改善卵巢组织病理学改变,发挥治疗作用。HQS抑制了P-NF-κB的表达，减少了白细胞介素6（IL-6）和肿瘤坏死因子-α（TNF-α）的产生，从而进一步抑制了卵巢组织和巨噬细胞的巨噬细胞M1极化。凋亡阳性细胞，Bcl-2转运X蛋白(BAX),在治疗组中，cleaved-caspase3的表达也降低。体内HQS治疗后，B细胞淋巴瘤2（Bcl2）的表达增强。共培养实验也证实了HQS可以防止KGN细胞凋亡。此外，HQS介导了肠道菌群的丰度。双歧杆菌和副叶杆菌的丰度增加，而衣原体的丰度降低。
结论：本研究证实HQS具有抗炎作用，抑制巨噬细胞M1极化。此外,HQS可以介导PCOS小鼠肠道菌群的丰度。因此，本研究将为HQS的临床应用提供更合理的依据。总之,HQS可能是PCOS治疗的潜在候选者。
目的：本研究旨在研究HQS对PCOS的影响，并通过体内外实验阐明潜在的机制。
方法：皮下注射脱氢表雄酮（DHEA），淡化高脂饮食3周，建立PCOS小鼠模型。制作模型后，PCOS小鼠用HQS治疗（8.75g/kg和17.5g/kg，伊格.）4周。首先,我们评估了卵巢组织的组织病理学变化并检测了激素水平。随后，该研究评估了体内和体外HQS的抗炎和调节巨噬细胞极化的能力。用Elisa试剂盒测定炎症指标的分泌,免疫荧光和Westernblot检测磷酸化核因子κB（P-NFκB）和B淋巴细胞活化抗原B7（CD80）的表达水平。同时,通过tunel染色和Westernblot检测卵巢颗粒细胞的凋亡。体外共培养模型用于评估巨噬细胞极化与人卵巢颗粒细胞（KGN细胞）凋亡之间的作用。此外，16SrDNA测序用于提高PCOS小鼠的肠道微生物群变化。
结果：HQS逆转了激素异常增加，改善胰岛素抵抗，改善卵巢组织病理学改变,发挥治疗作用。HQS抑制了P-NF-κB的表达，减少了白细胞介素6（IL-6）和肿瘤坏死因子-α（TNF-α）的产生，从而进一步抑制了卵巢组织和巨噬细胞的巨噬细胞M1极化。凋亡阳性细胞，Bcl-2转运X蛋白(BAX),在治疗组中，cleaved-caspase3的表达也降低。体内HQS治疗后，B细胞淋巴瘤2（Bcl2）的表达增强。共培养实验也证实了HQS可以防止KGN细胞凋亡。此外，HQS介导了肠道菌群的丰度。双歧杆菌和副叶杆菌的丰度增加，而衣原体的丰度降低。
结论：本研究证实HQS具有抗炎作用，抑制巨噬细胞M1极化。此外,HQS可以介导PCOS小鼠肠道菌群的丰度。因此，本研究将为HQS的临床应用提供更合理的依据。总之,HQS可能是PCOS治疗的潜在候选者。
