Premature ovarian insufficiency (POI) affects up to 1 % of the female population worldwide. Icariin (ICA) has shown promising potential for the alleviation of POI. However, the molecular mechanisms underlying the protective effects of ICA warrant further exploration. In this study, we aimed to elucidate the mechanisms by which ICA improves ovarian function via antioxidant effects mediated by the ER(3/SIRT3 pathway. Cisplatin-induced murine models of POI were used to systematically investigate the cytoprotective efficacy of ICA by comprehensive ovarian histopathological analysis, follicular quantification, estrous cycle monitoring, serum hormonal profiling, and the evaluation of biomarkers of ovarian oxidative stress and apoptosis. Then, an integrated methodological approach encompassing molecular docking, dual-luciferase reporter gene assay, and chromatin immunoprecipitation, was implemented to analyze the mechanism of the ER(3/SIRT3 pathway in conferring antioxidant protection in vitro against ICA. In cisplatin-induced murine models of POI, the administration of ICA significantly elevated follicle counts, restored normal estrous cyclicity, and normalized serum hormone levels (p < 0.01). Furthermore, ICA activated the ER(3/SIRT3 pathway, thus ameliorating cisplatin-induced ovarian oxidative damage. In vitro, ICA conferred protection against H2O2-induced injury in KGN cells. Mechanistically, ICA enhanced the binding of ER(3 to the promoter of the SIRT3 gene, thereby promoting the activation of SIRT3. Critically, the protective effects of ICA were abolished following the pharmacological inhibition of ER(3 or SIRT3. ICA ameliorated cisplatin-induced ovarian oxidative damage by activating the ER(3/SIRT3 pathway, providing scientific evidence for its potential as a therapeutic agent for POI. This finding facilitates further optimization of drug design and could enhance the quality-of-life for patients with POI.

This study investigated the molecular mechanism by which icariin regulates mitophagy via the estrogen receptor α(ERα)-mediated silencing of the regulatory protein 1(SIRT1)/forkhead box protein O3a(FOXO3a) pathway and its downstream PTEN-induced kinase 1(PINK1)/Parkin signaling axis, thereby influencing bone metabolism and osteoblast differentiation to ameliorate postmenopausal osteoporosis(PMOP). Network pharmacology and molecular docking were initially employed to identify key targets of icariin and potential signaling pathways related to PMOP, followed by validation of icariin's binding affinity to these targets. For in vivo experiments, 36 female C57BL/6J mice were randomly divided into six groups: sham-operation, model, estradiol(E_2)-treated, and low-, medium-and high-dose icariin-treated groups, respectively. In the sham operation group, some fat pads around the ovaries were removed from the mice, while the PMOP models were established through castration surgery in other groups. The drug administration groups were respectively given estradiol or icariin for continuous intervention for 8 weeks. Estrus cycle changes were monitored, while serum hormone and bone metabolism levels were measured by ELISA. Meanwhile, the femoral microstructure was evaluated using Micro-CT and HE staining, and bone anabolism was assessed by Western blot, Masson staining, and Goldner staining, respectively. In addition, mitophagy and expression of related proteins were examined by Western blot and transmission electron microscopy, while SIRT1/FOXO3a pathway proteins were analyzed by Western blot. For in vitro experiments, MC3T3-E1 cells were divided into control(osteogenic induction) and icariin-treated groups(low, medium, and high doses), with additional ERα antagonist and SIRT1 inhibitor interventions. Osteogenic differentiation and extracellular matrix mineralization were evaluated using ALP staining, alizarin red staining, and Western blot. Mitophagy and expression of related proteins were examined by Western blot and immunofluorescence detection, while the expression of proteins related to SIRT1/FOXO3a pathway was detected by Western blot. The results of network pharmacology analysis showed that SIRT1/FOXO3a was identified as a critical pathway to regulate PMOP, with icariin exhibiting high binding affinity to ERα. The results of in vivo experiments showed that compared to the sham operation group, the model group exhibited disrupted estrous cycles along with significantly decreased serum E_2 and procollagen type Ⅰ N-terminal propeptide(P1NP) levels, while follicle-stimulating hormone(FSH), luteinizing hormone(LH), and C-terminal telopeptide of type Ⅰ collagen(CTX-1) levels were markedly elevated, indicating successful establishment of the PMOP model. Following icariin intervention, the treatment group showed significantly increased serum P1NP levels and decreased CTX-1 levels compared to the model group. Meanwhile, icariin improved femoral microstructure, increased the areas of collagen deposition and mineralized bone matrix, and upregulated osteogenic-specific proteins, such as osteopontin(OPN) and osteoprotegerin(OPG). Icariin promoted the formation of autophagolysosome-like structures in osteoblasts. It inhibited the expression of autophagy-related protein P62 while upregulating mitophagy-related proteins PINK1, Parkin, microtubule-associated protein 1A/1B light chain 3A(LC3Ⅱ/LC3Ⅰ) and Beclin1, as well as the key pathway proteins SIRT1 and FOXO3a. The results of in vitro experiments demonstrated that icariin upregulated the expression of OPN and OPG, while promoting osteogenic differentiation and extracellular matrix mineralization. It downregulated P62 protein expression while enhancing the expression of PINK1, Parkin, LC3Ⅱ/LC3Ⅰ and Beclin1. Additionally, icariin increased the co-localization fluorescence intensity of LC3 with MitoTracker. Upon the addition of the ERα antagonist, the expression levels of SIRT1, FOXO3a, PINK1, and Parkin were significantly reduced, accompanied by weakened co-localization fluorescence intensity. When the SIRT1 inhibitor was introduced, the expression of acetylated FOXO3a increased, while the expression of PINK1, Parkin, and FOXO3a markedly decreased, along with diminished co-localization fluorescence intensity. In summary, icariin ameliorates PMOP by enhancing PINK1/Parkin-dependent mitophagy via the ERα-SIRT1/FOXO3a pathway, thereby regulating bone metabolism and promoting bone remodeling.

Objective: To explore the pharmacological effects and molecular mechanisms of a Jiao'ai decoction (JAD) in treating premature ovarian failure. Specifically, we evaluated the receptor for advanced glycation end (RAGE) products pathway, metabolic disorders, and intestinal flora dysbiosis. Methods: Forty female rats with normal estrous cycles were randomly divided into five groups: control, model, estradiol, low-dose JAD, and high-dose JAD, with 8 rats in each. Except for the control group, the rats in other groups were injected intraperitoneally with cisplatin for 8 days (1.5 mg/kg) to establish a premature ovarian failure model. Starting on the fifth day of cisplatin injections, the estradiol, low-dose JAD, and high-dose JAD groups were administered corresponding drugs for 21 days. Sex hormone levels and pathological changes in the ovaries were measured. Key proteins in the RAGE pathway related to apoptosis, aging, and inflammation, were tested using Western blot. A 16S rRNA analysis of feces and non-targeted metabolism in serum was performed to determine the effects of JAD on intestinal flora and metabolism. Results: Body weight, ovarian index, and the number of follicles at all levels increased in the JAD group. Regarding serum hormones, estradiol, anti-mullerian hormone (AMH) and P levels increased, whereas follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels decreased in the JAD group. The levels of phosphorylated Akt protein (P-Akt), receptor for advanced glycation end products (RAGE), tumor protein p53 (P53), C-reactive protein (CRP), apoptosis regulator BAX (BAX) and Caspase3 were downregulated by JAD, whereas B cell leukemia/lymphoma 2 (Bcl-2) and Endothelial nitric oxidase synthase (eNOS) were upregulated. JAD was also found to play an important role in the regulation of metabolic disorders and intestinal ecological imbalances by adjusting species composition and diversity. Conclusion: JAD can protect ovaries by exerting anti-inflammatory and anti-apoptotic effects via inhibition of the RAGE pathway. JAD can also regulate metabolic disorders and maintain the dynamic balance of intestinal flora, thereby contributing to the improvement of the ovarian reserve function.

Premature ovarian insufficiency (POI) affects up to 1 % of the female population worldwide. Icariin (ICA) has shown promising potential for the alleviation of POI. However, the molecular mechanisms underlying the protective effects of ICA warrant further exploration. In this study, we aimed to elucidate the mechanisms by which ICA improves ovarian function via antioxidant effects mediated by the ERβ/SIRT3 pathway. Cisplatin-induced murine models of POI were used to systematically investigate the cytoprotective efficacy of ICA by comprehensive ovarian histopathological analysis, follicular quantification, estrous cycle monitoring, serum hormonal profiling, and the evaluation of biomarkers of ovarian oxidative stress and apoptosis. Then, an integrated methodological approach encompassing molecular docking, dual-luciferase reporter gene assay, and chromatin immunoprecipitation, was implemented to analyze the mechanism of the ERβ/SIRT3 pathway in conferring antioxidant protection in vitro against ICA. In cisplatin-induced murine models of POI, the administration of ICA significantly elevated follicle counts, restored normal estrous cyclicity, and normalized serum hormone levels (p < 0.01). Furthermore, ICA activated the ERβ/SIRT3 pathway, thus ameliorating cisplatin-induced ovarian oxidative damage. In vitro, ICA conferred protection against H2O2-induced injury in KGN cells. Mechanistically, ICA enhanced the binding of ERβ to the promoter of the SIRT3 gene, thereby promoting the activation of SIRT3. Critically, the protective effects of ICA were abolished following the pharmacological inhibition of ERβ or SIRT3. ICA ameliorated cisplatin-induced ovarian oxidative damage by activating the ERβ/SIRT3 pathway, providing scientific evidence for its potential as a therapeutic agent for POI. This finding facilitates further optimization of drug design and could enhance the quality-of-life for patients with POI.

ETHNOPHARMACOLOGICAL RELEVANCE:Erxian decoction (EXD) is widely used in the treatment of various gynecological conditions, including menopausal syndrome, ovarian insufficiency, and premature ovarian failure (POF). As society ages, the prevalence of postmenopausal osteoporosis (PMOP) rises annually, accompanied by a progressive increase in the incidence of sarcopenia, a skeletal muscle disorder associated with osteoporosis. Recent studies have revealed that the diverse pharmacological agents and active compounds in EXD have been utilized in clinical and experimental contexts for sarcopenia; nevertheless, the mechanism by which EXD addresses postmenopausal sarcopenia remains unclear. AIM OF THE STUDY:This study aims to elucidate the molecular mechanisms and active constituents of EXD in alleviating postmenopausal sarcopenia through a combination of network pharmacology and experimental validation both in vivo and in vitro. MATERIALS AND METHODS:The potential anti-sarcopenic mechanisms of EXD were initially explored using network pharmacology. In vivo, successful construction of mouse model of ovarian aging. while forelimb grip strength and inverted hanging tests assessed muscle function. Levels of sex hormone secretion, muscle injury, and inflammatory markers were evaluated using ELISA, HE staining, Masson trichrome staining, and ATPase staining. IHC, RT-qPCR and Western blot analyses were conducted to quantify the expression of genes and proteins related to the estrogen signaling pathway and muscle atrophy. In vitro studies also utilized a C2C12 myotube atrophy model induced by Cisplatin. Cell proliferation was evaluated using the CCK-8 assay, apoptosis was assessed via flow cytometry, and expression levels of muscle-related proteins were determined through Western blot analysis. RESULTS:Network pharmacology analysis suggested that the estrogen signaling pathway may play a key role in the mechanism by which EXD alleviates sarcopenia associated with ovarian aging. In vivo, EXD improved muscle strength in mice with ovarian aging, restored ovarian function, and prevented myofibrillar atrophy. In vitro, quercetin significantly reduced the apoptosis rate of C2C12 myotube cells and promoted cell proliferation, indicating its protective effect against muscle atrophy. CONCLUSIONS:EXD can influence phytoestrogenic effects by regulating the estrogen signaling pathway to protect skeletal muscle in mice in ovarian aging.

In this study, we explored the pharmacological effects of Siwu Decoction in treating premature ovarian insufficiency(POI) and its molecular mechanism based on the mitophagy pathway modulated and mediated by estrogen receptor(ER) subtypes. Female Balb/c mice were divided into a control group, model group, as well as high-dose and low-dose groups of Siwu Decoction. The POI mice model was constructed by intraperitoneal injection of cisplatin. The high-dose and low-dose groups of Siwu Decoction were administered intragastrically with Siwu Decoction each day for 14 days. During this period, we monitored the estrous cycle and body weight of the mice and calculated the ovarian index. The morphology of the ovaries was detected by hematoxylin-eosin(HE) staining, and the number of primordial follicles was counted. The apoptosis of the ovarian tissue was detected by TUNEL staining. The expression levels of anti-Müllerian hormone(AMH), apoptosis-associated and mitophagy-associated proteins, ER subtypes, and the expression levels of key proteins of its mediated molecular pathways were detected by Western blot and immunohistochemistry. KGN cells were divided into a control group, model group, Siwu Decoction group, and gene silencing group. The apoptosis model was induced by H_2O_2, and PTEN-induced putative kinase 1(PINK1) gene silencing was induced by siRNA transfection. The Siwu Decoction group and gene silencing group were added to the medium containing Siwu Decoction. Cell viability was detected by CCK-8 assay. Cell senescence was detected by senescence-associated-β-galactosidase. The expression levels of apoptosis-associated and mitophagy-associated proteins were detected by Western blot. The results of in vivo experiments showed that compared with the model group, the mice in the high-dose and low-dose groups of Siwu Decoction significantly recovered the rhythm of the estrous cycle, and the levels of ovarian index, number of primordial follicles, and expression of AMH, representative indexes of ovarian function, were significantly higher, suggesting that the level of ovarian function was significantly improved. The expression levels of the apoptosis-related proteins, cytochrome C(Cyt C), cysteinyl aspartate specific proteinase 3(caspase 3), B-cell lymphoma-2(Bcl-2)-associated X(Bax), and mitophagy-associated indicator(Beclin 1) were significantly decreased, and the expression levels of Bcl-2 was significantly elevated. The positive area of TUNEL was significantly reduced, suggesting that the apoptosis level of the ovaries was significantly reduced. The expression levels of PINK1, Parkin, and sequestosome 1(p62) were significantly reduced, suggesting that the level of ovarian mitophagy was significantly down-regulated. The expression levels of ERα and ERβ were significantly elevated, and the ratio of ERα/ERβ was significantly reduced. The expression levels of key proteins in the pathway, phosphoinositide 3-kinase(PI3K) and protein kinase B(Akt), were significantly reduced, suggesting that the regulation of ER subtypes and the mediation of PI3K/Akt pathway were the key mechanisms. In vitro experiments showed that compared with the model group, the proportion of senescent cells in the Siwu Decoction group was significantly reduced. Cyt C, caspase 3, Beclin 1, Parkin, and p62 were significantly reduced, which was in line with in vivo experimental results. The proportion of senescent cells and the expression level of the above proteins were further significantly reduced after PINK1 silencing. It can be seen that Siwu Decoction can regulate the expression level and proportion of ER subtypes in KGN cells, then mediate the PI3K/Akt pathway to inhibit excessive mitophagy and apoptosis, and exert therapeutic effects of POI.

Ethnopharmacological relevance: Siwu decoction (SWD) is widely used in gynecological diseases, such as peripheral menopause syndrome, premature ovarian failure, and menstrual disorder. However, the mechanism of SWD on postmenopausal osteoporosis (PMOP) remains unclear. Aim of the study: To discover the phytoestrogenic osteoprotective effect of SWD on PMOP. Materials and methods: The potential mechanism of SWD on PMOP was filtered through network pharmacology research. The potential mechanism was verified in MC3T3-E1 cell lines in vitro. CCK8 assay was conducted to assess cell proliferation and the expressions of ER/PI3K/AKT pathway were analyzed using Western blot. Female F-344 rats were chosen to set up the PMOP model. The osteoprotective effect of SWD in vivo was evaluated using Hematoxylin-eosin staining, TRAP staining, Goldner staining and DXA. The potential mechanism was verified in vivo through Western blot and immunohistochemistry. RT-qPCR was conducted to unveil the expressions of osteogenesis genes. Results: Network pharmacology research showed that ER/PI3K/AKT pathway may be the potential mechanism of SWD on PMOP. SWD promoted the proliferation of osteoblasts and regulated the protein expressions of ER/PI3K/ AKT pathway in vitro. SWD improved the morphological structure, bone mineralization and bone mineral density of femurs and suppressed osteoclastogenesis in PMOP rat model via ER/PI3K/AKT pathway in vivo. In addition, SWD regulated the mRNA expressions of osteogenesis-related genes. Conclusions: SWD exerts a phytoestrogenic osteoprotective on PMOP by regulating ER/PI3K/AKT pathway, which marks it as a valuable medicine or supplement of PMOP.

Liver fibrosis is a dynamic wound-healing response characterized by the agglutination of the extracellular matrix (ECM). Si-Wu-Tang (SWT), a traditional Chinese medicine (TCM) formula, is known for treating gynecological diseases and liver fibrosis. Our previous studies demonstrated that long non-coding RNA H19 (H19) was markedly upregulated in fibrotic livers while its deficiency markedly reversed fibrogenesis. However, the mechanisms by which SWT influences H19 remain unclear. Thus, we established a bile duct ligation (BDL)-induced liver fibrosis model to evaluate the hepatoprotective effects of SWT on various cells in the liver. Our results showed that SWT markedly improved ECM deposition and bile duct reactions in the liver. Notably, SWT relieved liver fibrosis by regulating the transcription of genes involved in the cytoskeleton remodeling, primarily in hepatic stellate cells (HSCs), and influencing cytoskeleton-related angiogenesis and hepatocellular injury. This modulation collectively led to reduced ECM deposition. Through extensive bioinformatics analyses, we determined that H19 acted as a miRNA sponge and mainly inhibited miR-200, miR-211, and let7b, thereby regulating the above cellular regulatory pathways. Meanwhile, SWT reversed H19-related miRNAs and signaling pathways, diminishing ECM deposition and liver fibrosis. However, these protective effects of SWT were diminished with the overexpression of H19 in vivo. In conclusion, our study elucidates the underlying mechanisms of SWT from the perspective of H19-related signal networks and proposes a potential SWT-based therapeutic strategy for the treatment of liver fibrosis.

Ethnopharmacological relevance: Diminished ovarian reserve (DOR) was considered a refractory reproductive endocrine condition that negatively affected female reproductivity. Yangjing Zhongyu Decoction (YJZYD) had effects on treating infertility. However, there were few studies on the mechanisms of YJZYD preserving ovarian reserve.Aim of the study: To explore the possible mechanisms of YJZYD against DOR by UPLC-ESI-MS/MS, network pharmacology, and experimental validation.Methods: The chemicals of YJZYD were measured by UPLC-ESI-MS/MS. The correlating targets of YJZYD and DOR were identified by the ETCM database, GeneCards database, and PubMed database. The common targets were employed with the DAVID database and visualized with the PPI network. GO and KEGG enrichment analyses were carried out to explore biological progression and pathways. In vivo experiments, energy production was assessed by ATP, and apoptosis rate was analyzed by TUNEL. The serum FSH, AMH, and E2 levels were evaluated by ELISA. Western blotting and immunohistochemistry were used to measure the expression of SIRT1, PGC1 & alpha;, NRF1, COX IV, FSHR, CYP19A1, PI3K, p-Akt, Akt, Bcl-2, and Bax.Results: 132 components in YJZYD were identified by UPLC-ESI-MS/MS. 149 overlapped targets were extracted from YJZYD and DOR, and the top 20 common targets included AKT1 and CYP19A1. ATP binding was involved in GO analysis. In the KEGG enrichment analysis, the metabolic pathway was the top, and the PI3K-Akt signaling pathway was included. In vivo experiments, YJZYD improved ovarian index and histomorphology. After YJZYD treatment, serum FSH, E2, and AMH were well-modulated, and the content of ATP was up-regulated. Besides, the expression of Bax was suppressed in ovarian tissue, while the expressions of SIRT1, PGC1 & alpha;, NRF1, COX IV, FSHR, CYP19A1, PI3K, Bcl-2, and p-Akt/Akt were enhanced.Conclusion: YJZYD could attenuate reproductive endocrine disturbance and ovarian lesions in vivo by mediating steroidogenesis, energy metabolism, and cell apoptosis. This study uncovered the mechanisms of YJZYD against DOR, providing a theoretical basis for further study.

The targets and mechanisms of Si-Wu-Tang (SWT) against (Breast cancer) BRCA were identified and a survival model and nomogram was construted by network pharmacology, bioinformatic analysis and in vitro experiments. A total of 72 anti-breast cancer SWT targets were selected, among which eleven genes (MAOA、SQLE、CACNA2D1、GLI1、RORB、ITGB3、TACR1、NR3C2、CA3、RBP4 and PTK6) were used to construct a novel prognostic model of breast cancer. The anti-breast cancer activity of SWT was related to the modulation of the receptor tyrosine kinases signaling pathways. Moreover, two compounds, mairin and senkyunone were found to bind directly to ITGB3 and RORB proteins. Finally, mRNA and protein expression of ITGB3 and RORB was observed to be significantly down-regulated after incubation of MCF-7 cells with SWT. Overall, our results indicated that mairin and senkyunone were the key ingredients present in SWT, and ITGB3 as well as RORB proteins were the major targets affected by SWT. The prognostic model can be used to predict the outcome of BRCA patients.

Due to the lack of classic estrogen receptors, there has been a shortage of targeted therapy for triple-negative breast cancer (TNBC), resulting in a poor prognosis. However, the newly discovered G protein-coupled estrogen receptor (GPER) has been found to be expressed in TNBC cells. Salvia miltiorrhiza (Danshen) is an essential Chinese medicine for gynecological disorders, and its component tanshinone IIA (Tan IIA) exerts an anticancer effect. Therefore, this study attempted to investigate whether GPER is involved in the inhibitory effect of Tan IIA on TNBC. We applied various databases and GO pathway analysis to predict the possible mechanism of Tan IIA. We identified 39 overlapping targets, including c-Jun, c-Fos, and caspase-3, and enriched cell cycle-related pathways. Next, we demonstrated the strong binding ability of Tan IIA to GPER by molecular docking assay. In the subsequent validation tests, Cell Counting Kit-8 (CCK8) assay showed that Tan IIA inhibited proliferation of MDA-MB-231 cells time and dose dependently without affecting normal cells. Using Transwell plate, flow cytometry, and Western blot assays, we showed that Tan IIA inhibited migration and induced apoptosis of MDA-MB-231 dose dependently. Importantly, protein expressions of GPER, epidermal growth factor receptor (EGFR), extracellular regulated protein kinases (ERK), c-Fos, and c-Jun were all decreased by Tan IIA dose dependently. Administration of GPER inhibitor partly abolished these effects. Furthermore, nuclear translocation of c-Fos and c-Jun as well as cell cycle-related proteins was downregulated by Tan IIA dose dependently. In summary, Tan IIA could inhibit the proliferation and migration of MDA-MB-231 cells and induce apoptosis, and the possible mechanism may be the regulation of GPER-mediated pathways, suggesting that GPER could be a therapeutic target for TNBC.

乳腺癌的转移是其临床治疗面临的重点和难点,其侵袭性的高低受周围基质的影响很大.癌症相关成纤维细胞(CAFs)是肿瘤基质的重要成分,同时也是新型膜雌激素受体——G蛋白耦联雌激素受体(GPER)阳性细胞.研究表明,GPER可能是作用于癌细胞与肿瘤微环境(TM)的多种重要传导因子之间的关键交叉点,GPER在CAFs中活化对乳腺癌发生、发展和转移过程中发挥着重要的作用.多种中药单体的抗乳腺癌作用也与其靶向激活CAFs中的GPER通路是相关的.现就GPER在CAFs中的活化机制及其对乳腺癌转移的作用、中药单体对该过程的干预研究进行综述,以期为寻找乳腺癌治疗的新靶点、拓展乳腺癌症治疗策略提供思路.

目的:通过观察小檗碱(BBR)对卵巢颗粒细胞衰老的影响,探究其保护作用及调节机制.方法:应用H2O2诱导建立人卵巢颗粒样肿瘤(KGN)细胞衰老模型;设置空白组、模型组、BBR高剂量(1 μmol·L-1)组和BBR低剂量(0.5 μmol·L-1)组,模型组与BBR组加入浓度为10 μmol·L-1H2O2,孵育40 min.通过细胞增殖与活性检测(CCK-8)分析检测BBR对KGN细胞增殖的影响;通过β-半乳糖苷酶染色检测BBR对KGN细胞衰老状态的影响;应用流式细胞术检测BBR对KGN细胞凋亡和ROS含量的影响;实时荧光定量聚合酶链式反应(Real-time PCR)检测BBR对KGN细胞抗凋亡蛋白B细胞淋巴瘤-2(Bcl-2)/促凋亡蛋白Bcl-2相关X蛋白(Bax)比值、胱天蛋白酶-3(Caspase-3)、叉头框转录因子O1(FoxO1)及过氧化氢酶(CAT)mRNA表达的影响;蛋白免疫印迹法(Western blot)检测BBR对KGN细胞沉默信息调节因子1(SIRT1)、超氧化物歧化酶2(SOD2)、c-Jun氨基末端激酶(JNK)、FoxO1、自噬相关蛋白微管相关蛋白轻链3Ⅱ(LC3BⅡ)、自噬关键分子酵母Atg6(Beclin-1)及泛素结合蛋白p62蛋白表达的影响.结果:H2O2诱导40 min后,与空白组比较,模型组细胞增殖率显著下降(P＜0.01);与模型组比较,BBR干预组细胞增殖率明显上升(P＜0.05);β-半乳糖苷酶染色结果显示,与空白组比较,模型组细胞呈现明显的衰老状态(P＜0.01),BBR干预组细胞衰老情况较模型组显著降低(P＜0.01);流式细胞术检测显示,与空白组比较,模型组细胞凋亡率显著上升(P＜0.01),BBR干预组细胞凋亡率较模型组明显降低(P＜0.05);同时,与空白组比较,模型组ROS含量显著增加(P＜0.01);与模型组比较,BBR干预组细胞ROS含量显著降低(P＜0.01);Real-time PCR结果显示,与空白组比较,模型组KGN细胞CAT、Bcl-2/Bax mRNA表达明显降低,Caspase-3与FoxO1 mRNA表达明显增加(P＜0.05);与模型组比较,BBR干预后KGN细胞CAT与Bcl-2/Bax mRNA表达明显增加(P＜0.05),Caspase-3与FoxO 1 mRNA表达较模型组明显降低(P＜0.05).Western blot结果显示,与空白组比较,模型组SIRT1、SOD2及p62蛋白水平显著降低(P＜0.01),JNK、FoxO1、LC3B Ⅱ与Beclin-1蛋白水平明显升高(P＜0.05);BBR干预后,SIRT1、SOD2及p62蛋白水平较模型组显著增加(P＜0.01),JNK、FoxO1、LC3BⅡ与Beclin-1蛋白水平较模型组明显降低(P＜0.05).结论:BBR具有抑制卵巢颗粒细胞衰老效应,其机制与通过SIRT1/FoxO1通路介导抑制细胞凋亡与自噬有关.

Ethnopharmacological relevant: Erxian Decoction (EXD) has been used empirically for more than 70 years to treat premature ovarian failure (POF), but more research is needed to understand how it works.Aim of the research: The study aims to ascertain both in vivo and in vitro rewards of EXD.Materials and methods: EXD is composed of Curculiginis Rhizoma, Epimedii Folium, Morindae Officinalis, Angelicae Sinensis, Anemarrhenae Rhizoma, and Phellodendri Chinensis Cortex. UPLC/MS analysis was used to investigate the components of EXD. Using a POF model created by administering cisplatin to rats intraperitoneally, the pharmacodynamic effects of EXD were investigated. Three dose groups of EXD were garaged into rats: high (15.6 g/kg), medium (7.8 g/kg), and low (3.9 g/kg). By using a vaginal smear, the impact of EXD on the rat estrous cycle was evaluated. An ELISA test was used to measure the anti-Mullerian hormone (AMH), estradiol (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels in the serum of rats. By using HE stains, pathological alterations in the ovaries may be seen. MDA and SOD levels in ovarian samples were used to measure the degree of ovarian oxidation. TUNEL labeling of ovarian sections was used to find apoptosis levels. By using ATP, energy production was evaluated. The relative expression of proteins connected to aging and the RAGE pathway was assessed using Western blot. Then, using H2O2, a model of senescent human ovarian granulosa cells (KGN) was created in vitro. The impact of EXD and H2O2 on cellular senescence was discovered using-galactosidase staining. Cell apoptosis levels were found using PI/Hoechest33342. By using DCFH-DA, intracellular ROS was examined. MDA and SOD concentrations were used to measure the degree of cellular oxidation. RAGE-related mRNA and protein expression were evaluated using RT-qPCR and western blotting.Results: Using UPLC/MS analysis, 39 chemicals in EXD were found. Rats' estrous cycles were enhanced by EXD, which increased ovarian index and follicle count and reduced the proportion of atretic follicles in the rats. EXD reduced LH and FSH output while restoring AMH and E2 secretion. In ovarian tissues, EXD reduced the amount of apoptosis and MDA while raising SOD activity and ATP levels. The protein levels of p16, p21, p53, and Lamin A/C were among the senescence-related proteins that EXD lowered, along with the levels of RAGE, PI3K, BAX, and CASPASE 3. Anti-apoptotic protein BCL-2 was also raised in the RAGE pathway. Senescence, apoptosis, ROS, and MDA levels in the KGN cells were lowered in vitro by EXD. Additionally, EXD increased the anti-apoptotic potential by changing the expression of CAT, SOD2, and SIRT1. RAGE, BAX, BCL-2, CASPASE 3, and p38 expression levels were altered by EXD, enhancing its anti-apoptotic capability.Conclusion: EXD boosted the ovary's antioxidant and anti-apoptotic capabilities while enhancing the estrous cycle and hormone output. These findings strongly suggested that EXD may contribute to the alleviation of POF and ovarian granulosa cells senescence.

目的:探讨四物汤通过胰岛素样生长因子-1(Insulin-ike growth factors-1,IGF-1)/磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/雷帕霉素靶蛋白(Mammalian target of rapamycin,mTOR)mTOR信号通路发挥对4-乙烯基环己烯二环氧化合物(4-Vinylcyclohexene Diepoxide,VCD)诱导的卵巢衰老大鼠骨骼肌的保护作用及其分子机制.方法:选用28日龄雌性F-344大鼠,随机选取6只作为空白对照组,剩余大鼠连续腹腔注射VCD溶液(160mg/kg/d)20天,每天阴道涂片检测动情周期,连续观察12 d无角化细胞或仅有少量角化细胞即符合"卵巢衰老"表现,经动情周期筛选出24只造模成功的大鼠,分为模型组(Model)、阳性(戊酸雌二醇,E2)对照组、四物汤高剂量(SWT-H)组和四物汤低剂量(SWT-L)组,每组6只,灌胃持续3周后取材.取大鼠骨骼肌组织进行HE染色观察病理组织结构;MASSON染色骨骼肌纤维形态变化;RT-PCR检测骨骼肌组织中各基因的mRNA水平.结果:与空白组比较,模型组骨骼肌肌纤维排列疏松,分布不均且有断点,胞浆不均,细胞核增多,出现增生的结缔组织,胶原纤维逐渐增多且肌纤维间距变宽;与模型组相比,E2组、SWT-H组与SWT-L组肌纤维排列相对整齐,胞浆较为均匀.肌肉形态较规则,纤维间距缩短,胶原纤维减少.RT-PCR结果显示,与空白组相比,模型组IGF-1、PI3K、AKT、mTOR基因的mRNA表达量明显下调,E2组、SWT-H组与SWT-L组的IGF-1、PI3K、mTOR的表达明显上升,SWT-H组AKT表达无明显变化,无统计学意义,SWT-L组AKT表达相对降低.与模型组相比,E2组、SWT-H组与SWT-L组的IGF-1、AKT、mTOR的表达明显增加,且具有剂量依赖性,PI3K表达增加,但无明显剂量依赖性.结论:四物汤可以明显改善VCD诱导的卵巢衰老大鼠的肌肉减少情况,其机制可能是通过IGF-1-PI3K-AKT-mTOR信号通路来发挥其抑制肌肉流失的作用.

Danshen Si Wu is a Traditional Chinese Medicine used for menopausal complains. Beside tanshinone IIA (Tan IIA), Danshen also contains tanshinone I (Tan I), cryptotanshinone (CT) and dihydrotanshinone (DT). The aim of this study was to compare the biological activity of these tanshinones and to determine their cytotoxicity and genotoxicity. Purities and stabilities of the substances were analyzed by LC-DAD and LC-MS analyses. DT and CT concentrations decreased rapidly in dimethylsulfoxide and were converted to Tan I and Tan IIA, respectively. In aqueous solution concentration of all tanshinones decreased after 24 h. Tan I and Tan IIA showed dose-dependent bioactivity mediated by ERα and ERβ. No cytotoxic and genotoxic effects for Tan I and Tan IIA were detected. In a yeast transactivation assay Tan I and Tan IIA showed antiandrogenic activity. A significant anabolic activity in C2C12 cells could be detected for Tan I and Tan IIA. In conclusion our data provide evidence that Tan I and Tan IIA are the most relevant bioactive tanshinones in Danshen. Our finding that all tanshinones display a certain instability in aqueous solutions is relevant when discussing their potential therapeutic benefits in humans.

目的:基于体外细胞实验,探索丹参酮ⅡA对三阴性乳腺癌细胞MDA-MB-231迁移的抑制作用及其分子机制.方法:选取三阴性乳腺癌细胞MDA-MB-231,利用细胞增殖实验检测丹参酮Ⅱ A对MDA-MB-231细胞增殖的作用,并筛选适宜的药物浓度;应用划痕实验检测丹参酮ⅡA对MDA-MB-231细胞迁移率的影响;Western Blot法检测丹参酮Ⅱ A对G蛋白偶联雌激素受体(G protein-coupled estrogen receptor,GPER)及基质金属蛋白酶 9(Matrix metalloprotein-9,MMP-9)表达的影响.结果:细胞增殖实验结果显示,丹参酮ⅡA可以抑制MDA-MB-231细胞增殖,且呈剂量依赖性(P＜0.05);划痕实验结果显示,丹参酮Ⅱ A可以抑制MDA-MB-231细胞迁移,且呈剂量依赖性(P＜0.01),加入GPER特异性抑制剂G15后迁移率有所上升(P＜0.01).Western Blot结果显示,丹参酮ⅡA可以显著下调GPER和MMP-9蛋白的表达水平并呈剂量依赖性(P＜0.05),加入GPER特异性抑制剂G15后,MMP-9表达有所上升(P＜0.01).结论:丹参酮Ⅱ A可以抑制三阴性乳腺癌细胞MDA-MB-231迁移,其机制可能与抑制GPER介导的MMP-9表达相关.

This study explored the phytoestrogen-like effect of Siwu Decoction(SWD) and the estrogen receptor(ER)-mediated molecular mechanism based on network pharmacology and in vivo experiment. The active components and targets of SWD were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), and related targets of "estrogen" from GeneCards and Online Mendelian Inheritance in Man(OMIM). Cytoscape and STRING were employed to construct the protein-protein interaction(PPI) network and "chemical component-target-disease" network and core targets were identified, followed by Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment of the core targets by R software. For the in vivo experiment, the 22-day-old SD female rats were treated(ig) with SWD for 4 days. Via hematoxylin-eosin(HE) staining, the morphological changes of rat uterus were observed. Reverse transcriptase-polymerase chain reaction(RT-PCR) was performed to detect mRNA expression of ER subtypes, estrogen-related targets, and the main regulatory factors in the estrogen signaling pathway. The results indicated 74 targets of SWD exerted phytoestrogen-like effect. KEGG pathway enrichment result suggested that estrogen signaling pathway was closely related to the phytoestrogen-like effect of SWD. Rats in SWD group demonstrated significantly thickened endometrium and significantly decreased expression of ERα, ERβ, and G protein-coupled estrogen receptor(GPER) mRNA in ovarian tissue. In addition, significant lowering of ERα and ERβ mRNA expression and significant rise of GPER mRNA expression in uterine tissue were observed in the SWD group. The expression of mitogen-activated protein kinase(MAPK) p38, MEK1/2 and extracellular signal-regulated kinase(ERK)1/2 mRNA was significantly low while that of epidermal growth factor receptor(EGFR) mRNA was significantly high in both ovarian and uterine tissues of SWD group compared with those in the control group. In conclusion, the phytoestrogen-like effect of SWD is closely related to the estrogen signaling pathway. The result lays a basis for revealing molecular mechanism of SWD in the treatment of gynecological diseases.

目的 探究人参皂苷Rg1对顺铂损伤大鼠卵巢颗粒细胞的保护作用及分子机制.方法 选取22～24 d SD的大鼠提取卵巢颗粒细胞进行原代培养,应用顺铂诱导建立卵巢早衰模型.设置正常组,模型组,人参皂苷Rg1低浓度组、中浓度组、高浓度组和雌二醇(E2)组.HE染色及免疫细胞化学法进行颗粒细胞鉴定;CCK8法检测人参皂苷Rg1作用24 h、48 h对顺铂损伤颗粒细胞的保护作用;Hoechst 33258染色检测细胞凋亡;Western blot法检测FSHR、PI3K、p-AKT、AKT、Bcl-2和Bax的蛋白表达.结果 细胞形态观察和胞质FSHR表达检测结果均表明所提取细胞为卵巢颗粒细胞;顺铂处理12h后,颗粒细胞增殖率呈剂量依赖性下降,而给予人参皂苷Rg1后,细胞增殖率下降被显著抑制,且呈剂量和时间依赖性;与模型组比较,人参皂苷Rg1和E2处理后卵巢颗粒细胞中FSHR、PI3K、p-AKT/AKT及Bcl-2/Bax蛋白表达水平显著上升,而PI3K抑制剂干预后,Bcl-2蛋白表达显著下降.结论 人参皂苷Rg1对顺铂损伤的卵巢颗粒细胞具有保护作用,该保护作用可能通过激活FSHR/PI3K/AKT通路,抑制卵巢颗粒细胞凋亡实现.