ETHNOPHARMACOLOGICAL RELEVANCE:Leonurus japonicus Houtt (L. japonicus, Chinese motherwort), known as Yi Mu Cao which means "good for women", has long been widely used in China and other Asian countries to alleviate gynecological disorders, often characterized by estrogen dysregulation. It has been used for the treatment of polycystic ovary syndrome (PCOS), a common endocrine disorder in women but the underlying mechanism remains unknown.AIM OF THE STUDY:The present study was designed to investigate the effect and mechanism of flavonoid luteolin and its analog luteolin-7-methylether contained in L. japonicus on aromatase, a rate-limiting enzyme that catalyzes the conversion of androgens to estrogens and a drug target to induce ovulation in PCOS patients.MATERIALS AND METHODS:Estrogen biosynthesis in human ovarian granulosa cells was examined using ELISA. Western blots were used to explore the signaling pathways in the regulation of aromatase expression. Transcriptomic analysis was conducted to elucidate the potential mechanisms of action of compounds. Finally, animal models were used to assess the therapeutic potential of these compounds in PCOS.RESULTS:Luteolin potently inhibited estrogen biosynthesis in human ovarian granulosa cells stimulated by follicle-stimulating hormone. This effect was achieved by decreasing cAMP response element-binding protein (CREB)-mediated expression of aromatase. Mechanistically, luteolin and luteolin-7-methylether targeted tumor progression locus 2 (TPL2) to suppress mitogen-activated protein kinase 3/6 (MKK3/6)-p38 MAPK-CREB pathway signaling. Transcriptional analysis showed that these compounds regulated the expression of different genes, with the MAPK signaling pathway being the most significantly affected. Furthermore, luteolin and luteolin-7-methylether effectively alleviated the symptoms of PCOS in mice.CONCLUSIONS:This study demonstrates a previously unrecognized role of TPL2 in estrogen biosynthesis and suggests that luteolin and luteolin-7-methylether have potential as novel therapeutic agents for the treatment of PCOS. The results provide a foundation for further development of these compounds as effective and safe therapies for women with PCOS.

Caspase-1 plays a central role in innate immunity, as its activation by inflammasomes induces the production of proinflammatory cytokines and pyroptosis. However, specific inhibition of the enzymatic activity of this protease is not effective in suppressing inflammation, owing to its enzyme-independent function. Herein, we identified a cyclohexenyl isothiocyanate compound (CIB-1476) that potently inhibited caspase-1 activity and suppressed the assembly and activation of the NLRP3 inflammasome and gasdermin-D-mediated pyroptosis. Mechanistically, CIB-1476 directly targeted pro-caspase-1 as an irreversible covalent inhibitor by binding to Cys285 and Cys397, resulting in more durable anti-inflammatory effects in the suppression of enzyme-dependent IL-1β production and enzyme-independent nuclear factor κB activation. Chemoproteomic profiling demonstrated the engagement of CIB-1476 with caspase-1. CIB-1476 showed potent therapeutic effects by suppressing inflammasome activation in mice, which was abolished in Casp1 –/– mice. These results warrant further development of CIB-1476 along with its analogues as a novel strategy for caspase-1 inhibitors.

目的:探讨羟基γ-异山椒素抑制人体卵巢颗粒细胞雌激素合成的相关机制.方法:利用CCK-8检测4种山椒素对人卵巢颗粒细胞系(KGN细胞株)增殖的影响;Elisa法检测4种山椒素对KGN细胞雌二醇合成的影响;RT-qPCR检测KGN细胞芳香化酶基因;Western blot法检测芳香化酶蛋白及相关通路蛋白.结果:羟基γ-异山椒素抑制雌二醇生物合成效果最佳[半抑制浓度(IC50)=(16.63±0.39)μmol·L-1],并且在该浓度下对KGN细胞的增殖无明显抑制作用.在羟基γ-异山椒素干预后,芳香化酶基因和蛋白表达水平显著下降(P<0.05),且芳香化酶的活性未受到明显影响;同时,羟基γ-异山椒素能显著降低P-CERB,P-AKT,P-p38蛋白的表达(P<0.05).结论:羟基γ-异山椒素可能通过抑制p38/MAPK和PI3K/AKT通路的激活,降低CREB的磷酸化,抑制芳香化酶转录,最终抑制KGN细胞雌激素生物合成.

Three new compounds (1, 11, and 12), together with 32 known ones, were isolated from the root bark of Morus alba L. using various chromatographic methods. The structures of the undescribed compounds were elucidated based on 1D, 2D NMR, and HRESIMS dataanalysis, while the known ones were identified by comparison of their spectroscopic data with those reported in the literature. All the isolates were evaluated for their cytotoxic activities against human gastric cancer HGC27 cells by CCK-8 assay. Among them, compounds 5, 8, 10, and 30 exhibited cytotoxic activities on HGC27 cells with IC50 values of 33.76 ± 2.64 μM, 28.94 ± 0.72 μM, 6.08 ± 0.34 μM, and 10.24 ± 0.89 μM, respectively. Furthermore, compound 10 was confirmed to reduce proliferation ability, increase apoptosis rate, and inhibit cell migration pathway by annexin V/PI double staining experiment, transwell experiment, and Western blot analysis.

Caspase-1 is an integral regulator of innate immunity, which plays a key role in inflammasome activation and the release of pro-inflammatory cytokines. The development of novel non-peptidic small molecule caspase-1 inhibitors is an important strategy for antagonizing excessively activated caspase-1 induced by inflammatory diseases, including gouty arthritis. In the present study, we identified 63 caspase-1 inhibitors, with different structures and potencies, from bioactive compound libraries. Among them, NSC697923 potently inhibited the enzymatic activity of caspase-1, with an IC50 value of 1.737 μM. This compound adopted a favorable conformation in the active pocket of caspase-1. Furthermore, NSC697923 potently decreased mature interleukin (IL)-1β secretion in macrophages stimulated by lipopolysaccharide plus nigericin, ATP, and monosodium urate crystal. NSC697923 also inhibited NLRP3 protein expression by suppressing the NF-κB signaling pathway and the interaction between receptor interacting protein-2 (RIP2) and pro-caspase-1, thereby blocking the priming of the NLRP3 inflammasome. In addition, NSC697923 significantly inhibited caspase-1 mediated gasdermin D cleavage and pyroptosis in macrophages. In an animal model of gouty arthritis, NSC697923 effectively inhibited joint swelling, IL-1β release, and NLRP3 inflammasome activation. Our results indicate that NSC697923 can effectively suppress NLRP3 inflammasome activation by inhibiting caspase-1, thus warranting further investigation as a potential therapeutic for treating NLRP3 inflammasome-related diseases.

Mechanical stimulation induces bone growth and remodeling by the secondary messenger, cyclic guanosine 3’, 5’-monophosphate (cGMP), in osteoblasts. However, the role of cGMP in the regulation of estrogen biosynthesis, whose deficiency is a major cause of osteoporosis, remains unclear. Here, we found that the prenylated flavonoids, 3-O-methoxymethyl-7-O-benzylicaritin (13), 7-O-benzylicaritin (14), and 4'-O-methyl-8-isopentylkaempferol (15), which were synthesized using icariin analogs, promoted estrogen biosynthesis in osteoblastic UMR106 cells, with calculated EC50 values of 1.53, 3.45, and 10.57 µM, respectively. 14 and 15 increased the expression level of the bone specific promoter I.4-driven aromatase, the only enzyme that catalyzes estrogen formation by using androgens as substrates, in osteoblastic cells. 14 inhibited phosphodiesterase 5 (PDE5), stimulated intracellular cGMP level and promoted osteoblast cell differentiation. Inhibition of cGMP dependent-protein kinase G (PKG) abolished the stimulatory effect of 14 on estrogen biosynthesis and osteoblast cell differentiation. Further, PKG activation by 14 stimulated the activity of SHP2 (Src homology 2 domain-containing tyrosine phosphatase 2), thereby activating Src and ERK (extracellular signal-regulated kinase) signaling and increasing ERK-dependent aromatase expression in osteoblasts. Our findings reveal a previously unknown role of cGMP in the regulation of estrogen biosynthesis in the bone. These results support the further development of 14 as a PKG-activating drug to mimic the anabolic effects of mechanical stimulation of bone in the treatment of osteoporosis.

腺苷酸环化酶(adenylate cyclase,AC)通过调节环磷酸腺苷(cyclic adenosine cyclophosphate,cAMP)的合成从而调控细胞相关功能.在卵巢中,cAMP调控卵母细胞的发育及卵子的成熟,cAMP含量变化影响下游芳香化酶的表达,进而影响雌激素的生物合成.为开发靶向AC的候选药物,根据AC1、AC2两个活性区域,利用大肠杆菌系统克隆表达可溶ⅠC1-ⅡC2重组融合蛋白,使用ATP检测试剂盒,检测ⅠC1-ⅡC2的催化活性,从而建立有效的ⅠC1-ⅡC2蛋白体外筛选抑制剂模型.利用该模型筛选了由2 861个化合物组成的文库.化合物来那替尼(neratinib)可明显抑制ⅠC1-ⅡC2蛋白活性,且呈浓度依赖性,其ⅠC50为10.2±0.152 2 μmol/L.进一步研究发现,来那替尼可显著降低人卵巢颗粒细胞KGN细胞中芳香化酶的表达,并且抑制雌激素的生物合成.本研究通过建立蛋白体外筛选模型发现来那替尼具有抑制AC的活性,结果可为开发新的靶向芳香化酶治疗多囊卵巢综合征等疾病的候选药物提供参考.