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Introduction: Prostate cancer (PCa) often progresses to castration-resistant prostate cancer (CRPC), which is linked to higher treatment resistance and recurrence rates. This highlights the urgent need for new therapeutic options. Natural products, especially flavonoids, have shown promise in reducing drug resistance and possess both antioxidant and anticancer effects. Developing drugs that specifically target CRPC could offer significant therapeutic advantages. Methods: Chrysosplenetin B (CspB) was extracted and purified from the herb Laggera pterodonta (DC.) Benth. using traditional flavonoid extraction techniques, followed by high-performance liquid chromatography (HPLC) for purity assessment and nuclear magnetic resonance (NMR) for structural identification. The effect of CspB on the viability of PCa cells was evaluated using the Cell Counting Kit-8 assay. Subsequently, transcriptome analysis was conducted, and cell cycle progression was assessed through flow cytometry in conjunction with propidium iodide (PI) staining. Additionally, western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were employed to confirm the expression levels of relevant proteins and genes. Results: CspB was found to inhibit the proliferation of PC3, DU145, and LNCaP cells in a dose-dependent manner, with a stronger effect noted in PC3 and DU145 cells. Transcriptomic analysis revealed that CspB treatment led to cell cycle arrest, particularly in PC3 cells. Flow cytometry with PI staining confirmed that CspB caused G1 phase cell cycle arrest in PC3 cells. Moreover, CspB treatment significantly increased the expression of essential members of the Cip/Kip family, including CIP1/P21 and KIP1/P27, as well as CDKN2B (P15) and CDKN2D (P19) from the INK4 family. Additionally, CspB exposure notably raised the expression of the G1 phase-negative regulatory gene CDKN1C, while key cell cycle regulators like CDK6 and E2F1 were significantly downregulated at the protein level. Conclusion: Our findings indicate that CspB effectively inhibits the proliferation of CRPC cells by reducing the activity of cell cycle proteins and cyclin-dependent kinase (CDK) complexes while upregulating the expression of P21 and P27 and inducing G1 phase cell cycle arrest. These results highlight the potential of CspB as a promising candidate for developing therapeutic agents aimed at targeting CRPC.

Chrysosplenetin, a polymethoxy flavonol purified in our laboratory from the waste products generated during the industrial extraction of artemisinin, has been previously demonstrated to be a potential inhibitor of artemisinin resistance. Based on NMR-untargeted metabolomics, one of its hypothesized mechanisms of action is associated with the regulation of amino acid metabolism. In this study, we further quantified the key amino acids using LC-MS/MS targeted metabolomics and screened out the perturbed metabolic pathway network, which was characterized by tissue-specific differences. As a result, among the commonly and uniquely altered metabolites, increased levels of phenylalanine, tryptophan, and isoleucine were detected in the serum and various organs of the resistant groups. Interestingly, while the individual use of chrysosplenetin or artemisinin elevated the contents of these amino acids, their combination led to a significant down-regulation of these amino acids in the serum and intestines. Therefore, chrysosplenetin has the potential to act as a restorer of amino acid metabolism homeostasis, which is associated with artemisinin resistance in Plasmodium berghei K173.

Background Chrysosplenetin is an O-methylated flavonol compound isolated from the plant Chamomilla recutita and Laggera pterodonta . The aim of our research is to evaluate the function of Chrysosplenetin on osteogenesis of human-derived bone marrow stromal cells (hBMSCs) and inhibition of estrogen deficiency-induced osteoporosis via the Wnt/β-catenin signaling pathway. Method hBMSCs are cultured and treated by Chrysosplenetin in the absence or presence of Wnt inhibitor dickkopf-related protein 1 (DKK1) or bone morphogenetic protein 2 (BMP2) antagonist Noggin. RT-qPCR is taken to identify the genetic expression of target genes of Wnt/β-catenin pathway and osteoblast-specific markers. The situation of β-catenin is measured by western blot and immunofluorescence staining. An ovariectomized (OVX) mouse model is set up to detect the bone loss suppression by injecting Chrysosplenetin . Micro-CT and histological assay are performed to evaluate the protection of bone matrix and osteoblast number. Serum markers related with osteogenesis are detected by ELISA. Results In the present study, it is found that Chrysosplenetin time-dependently promoted proliferation and osteoblastogenesis of hBMSCs reaching its maximal effects at a concentration of 10 μM. The expressions of target genes of Wnt/β-catenin pathway and osteoblast-specific marker genes are enhanced by Chrysosplenetin treatment. Furthermore, the phosphorylation of β-catenin is decreased, and nuclear translocation of β-catenin is promoted by Chrysosplenetin . Osteogenesis effects mentioned above are founded to be blocked by DKK1 or BMP2 antagonist Noggin. In vivo study reveals that Chrysosplenetin prevents estrogen deficiency-induced bone loss in OVX mice detected by Micro-CT, histological analysis, and ELISA . Conclusions Our study demonstrates that Chrysosplenetin improves osteoblastogenesis of hBMSCs and osteogenesis in estrogen deficiency-induced bone loss by regulating Wnt/β-catenin pathway.

Background Chrysosplenetin (CHR), a polymethoxy flavonol co-occurring with artemisinin (ART) in Artemisia annua L., reverses ART resistance in Plasmodium berghei K173 potentially by downregulating intestinal P-glycoprotein (P-gp, encoded by  Mdr1a ) expression. In the present study, we further elaborated on the mechanism by comparing differences in antimalarial activity and resistance-associated molecular expression profiles between ART alone and combination therapy in blood and tissues of Mdr1a wild-type (WT) and knockout (KO) mice infected with either sensitive or resistant malarial parasites. Methods We evaluated the effects of monotherapy and combination therapy in WT and KO mice infected with sensitive and resistant P. berghei K173 strains. The mRNA expressions of multi-resistance proteins (Mrp1, 2, 4, 5) and breast cancer resistance proteins (Bcrp) were detected. Hemoglobin levels, mRNA expressions of cytokines including tumor necrosis factor-α (IFN-α), interferon-α (IFN-α), and interleukin (IL-1β) in blood and tissues, and redox balance (ROS/GSH levels), as well as gene or protein expression of signaling pathway (PI3K/AKT-mTOR and MAPK) were investigated. Results In drug-resistant mice, combination therapy maintained the highest survival (100%) and inhibition (30%) rates and the lowest parasitaemia percentage (approximately 20.0%), irrespective of Mdr1a gene status. Furthermore, combination reshaped the spatial and ART resistance-phenotypic disparities in Mrps and Bcrp mRNA expressions (with a fold change ranging from 1.35 to 38.03), ROS/GSH balance (ranging from 1.02-fold to 10.18-fold), hemoglobin levels (ranging from 1.04-fold to 1.20-fold), and cytokine profiles (ranging from 1.14-fold to 37.79-fold) induced by ART alone, which were partially dysregulated by Mdr1a deficiency. Monotherapy and combination exert oppositely regulatory effects on the PI3K/AKT-mTOR pathway in a tissue-, Mdr1a genotype-, and parasite sensitivity/resistance-dependent manner (ranging from 1.52-fold to 84.00-fold). Specifically, CHR reversed ART-induced changes via PI3K/AKT protein inhibition (ranging from 1.20-fold to 63.00-fold), which was contingent on P-gp functionality. Finally, mitogen-activated protein kinase (MAPK) pathway was involved in the antagonistic regulation between ART alone and combination therapy in a P-gp-independent manner (ranging from 1.39-fold to 16.69-fold). Conclusions The efflux pump function of P-gp is probably not a critical factor in the mechanism by which CHR reverses ART resistance. Instead, CHR acts as a homeostasis stabilizer with dual functions: it disrupts Plasmodium berghei K173 resistance to ART driven by both ABC transporters and the heme-ROS/GSH axis, in which the non-transport function of P-gp on ART is involved. Graphical Abstract

Background Artemisinin (ART) is an efficacious and safe anti-malarial drugs but has low oral bioavailability and auto-induction profiles during multiple dosing. The pharmacokinetic disadvantages have been found to partially depend on the induction of cytochrome P-450 enzymes by ART and resulted in the therapeutic failure due to insufficient drug levels. The present study, therefore, investigated the impacts of chrysosplenetin (CHR), a polymethoxylated flavonoid from Artemisia annua , on the pharmacokinetics and the anti-malarial efficacy of ART against Plasmodium berghei . The inhibition of CHR on enzymatic activity of CYP1A2, CYP2A, CYP2C19, CYP2D6, CYP2E1, and CYP3A in rat liver microsome was also investigated. IC50, Km, Ki, and inhibitory type of CHR were respectively calculated. Methods Twenty rats were randomly divided into four groups and received three-day oral doses of ART in absence or presence of CHR (in ratio of 1:0, 1:1, 1:2, and 1:4, respectively). Plasma samples were separately harvested for ART pharmacokinetics analysis using a valid liquid chromatography tandem mass spectrometric (LC–MS/MS) method. Female Kunming mice were inoculated by P. berghei K173 strain and pre-exposed to three-day oral administration of ART with or without CHR as pharmacokinetics protocol. Giemsa staining method was applied to calculate percent parasitaemia (%) and inhibition (%). In vitro rat liver microsomal model was employed to elucidate the inhibitory effect of CHR on CYP1A2, CYP2A, CYP2C19, CYP2D6, CYP2E1, and CYP3A. Results The AUC 0–t , C max , and t 1/2 of ART increased significantly ( P  < 0.05 or P  < 0.01) as well as declined CLz ( P  < 0.05 or P  < 0.01) after three-day oral doses of ART in presence of CHR (1:2) when compared with ART alone. Also, parasitaemia (%) remarkably attenuated 1.59 folds with 1.63-fold augmented inhibition (%) when the ratio between ART and CHR reached 1:2. CHR itself had no anti-malarial efficacy ( P  > 0.05). CHR inhibited in vitro activity of CYP1A2 and CYP2C19 ( P  < 0.01, IC 50  = 4.61 and 6.23 μM) in a concentration–response manner. The inhibition did not emerge on CYP2E1 and CYP3A until the CHR concentration exceeded 4.0 μM ( P  < 0.01, IC 50  = 28.17 and 3.38 µM). CHR has no impact on CYP 2A and CYP2D6 ( P  > 0.05). The inhibition types of CHR on CYP1A2 and CYP3A belonged to noncompetitive and uncompetitive, respectively. Conclusions Co-administration of ART with CHR in ratio of 1:2 achieved a synergic anti-malarial effect partly because of the noncompetitive or uncompetitive inhibition of CHR of drug-metabolism enzymes, especially CYP3A which is closely related to the auto-induction of ART.