Shikonin, a natural naphthoquinone phytochemical, exerts anti-leukemia effects in human CBF-AML cell lines and zebrafish xenograft models.

Core binding factor acute myeloid leukemia (CBF-AML) stands out as the most common type of adult AML, characterized by specific chromosomal rearrangements involving CBF genes, particularly t(8;21). Shikonin (SHK), a naphthoquinone phytochemical widely employed as a food colorant and traditional Chinese herbal medicine, exhibits antioxidant, anti-inflammatory, and anti-cancer activities. In this study, we aim to investigate the antileukemic effects of SHK and its underlying mechanisms in human CBF-AML cells and zebrafish xenograft models. Our study revealed that SHK reduced the viability of CBF-AML cells. SHK induced cell cycle arrest, promoted cell apoptosis, and induced differentiation in Kasumi-1 cells. Additionally, SHK downregulated the gene expression of AML1-ETO and c-KIT in Kasumi-1 cells. In animal studies, SHK showed no toxic effects in zebrafish and markedly inhibited the growth of leukemia cells in zebrafish xenografts. Transcriptomic analysis showed that differentially expressed genes (DEGs) altered by SHK are linked to key biological processes like DNA repair, replication, cell cycle regulation, apoptosis, and division. Furthermore, KEGG pathways associated with cell growth, such as the cell cycle and p53 signaling pathway, were significantly enriched by DEGs. Analysis of AML-associated genes in response to SHK treatment using DisGeNET and the STRING database indicated that SHK downregulates the expression of cell division regulators regarding AML progression. Finally, we found that SHK combined with cytarabine synergistically reduced the viability of Kasumi-1 cells. In conclusion, our findings provide novel insights into the mechanisms of SHK in suppressing leukemia cell growth, suggesting its potential as a chemotherapeutic agent for human CBF-AML.

Dual folate/biotin-decorated liposomes mediated delivery of methylnaphthazarin for anti-cancer activity.

Chemotherapy is an effective strategy for mitigating the global challenge of cancer treatment, which often encounters drug resistance and negative side effects. Methylnaphthazarin (MNZ), a natural compound with promising anti-cancer properties, has been underexplored due to its poor aqueous solubility and low selectivity. This study introduces a novel approach to overcome these limitations by developing MNZ-encapsulating liposomes decorated with folate and biotin (F/B-LP-MNZ). This dual-targeting strategy aims to enhance the anti-cancer efficacy and specificity of MNZ delivery. Our innovative F/B-LP-MNZ formulation demonstrated excellent physicochemical properties, stability, and controlled drug release profiles. In vitro studies revealed that MNZ-loaded liposomes attenuate the toxicity associated with free MNZ while F/B-LP-MNZ significantly increased cytotoxicity against HeLa cells, which express high levels of folate and biotin receptors, compared to non-targeted liposomes. Enhanced cellular uptake and improved dynamic flow attachment further confirmed the superior specificity of F/B-LP in targeting cancer cells. Additionally, our results revealed that F/B-LP-MNZ effectively inhibits HeLa cell migration and adhesion through EMT suppression and apoptotic induction, indicating its potential to prevent cancer metastasis. These findings highlight the potential of dual folate and biotin receptors-targeting liposomes as an effective delivery system for MNZ, offering a promising new avenue for targeted cancer therapy.

Analysis of action of 1,4-naphthoquinone scaffold-derived compounds against acute myeloid leukemia based on network pharmacology, molecular docking and molecular dynamics simulation.

1,4-Naphthoquinone scaffold-derived compounds has shown considerable pharmacological properties against cancer, including acute myeloid leukemia (AML) However, its impact and mechanisms in AML are uncertain. In this study, the mechanisms of 1,4-naphthoquinone scaffold-derived compounds against AML were investigated via network pharmacology, molecular docking and molecular dynamics simulation. ASINEX database was used to collect the 1,4-naphthoquinone scaffold-derived compounds, and compounds were extracted from the software to evaluate their drug similarity and toxicity. The potential targets of compounds were retrieved from the SwissTargetPrediction Database and the Similarity Ensemble Approach Database, while the potential targets of AML were obtained from the GeneCards databases and Gene Expression Omnibus. The STRING database was used to construct a protein-protein interaction (PPI) network, topologically and Cyto Hubb plugin of Cytoscape screen the central targets. After selecting the potential key targets, the gene ontology (GO) function annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed for the intersection targets, and a network map of "compounds-potential targets-pathway-disease" were constructed. Molecular docking of the compounds with the core target was performed, and core target with the strongest binding force and 1,4-naphthoquinone scaffold-derived compounds was selected for further molecular dynamics simulation and further molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) approach verification. In addition, the Bloodspot database was applied to perform the overall survival of core targets. A total of 19 1,4-naphthoquinone scaffold-derived compounds were chosen out, and then 836 targets of compounds, 96 intersection targets of AML were screened. Core targets include STAT3, TLR4, HSP90AA1, JUN, MMP9, PTPRC, JAK2, PTGS2, KIT and CSF1R. GO functional enrichment analysis revealed that 90 biological processes, 10 cell components and 12 molecular functions were enriched while KEGG pathway enrichment analysis revealed 34 enriched signaling pathways. Analysis of KEGG enrichment hinted that these 10 core genes were located in the pathways in cancer, suggesting that 1,4-naphthoquinone scaffold-derived compounds had potential activity against AML. Molecular docking analysis revealed that the binding energies between 1,4-naphthoquinone scaffold-derived compounds and the core proteins were all higher than - 6 kcal/mol, indicating that the 10 core targets all had strong binding ability with compounds. Moreover, a good binding capacity was inferred from molecular dynamics simulations between compound 7 and MMP9. The total binding free energy calculated using the MM/GBSA approach revealed values of - 6356.865 kcal/mol for the MMP9-7 complex. In addition, Bloodspot database results exhibited that HSP90AA1, MMP9 and PTPRC were associated with overall survival. The findings provide foundations for future studies into the interaction underlying the anti-AML potential of compounds with 1,4-naphthoquinone-based scaffold structures. Compounds with 1,4-naphthoquinone-based scaffold structures exhibits considerable potential in mitigating and treating AML through multiple targets and pathways.

Pharmacological Features and Therapeutic Implications of Plumbagin in Cancer and Metabolic Disorders: A Narrative Review.

Plumbagin (PLB) is a naphthoquinone extracted from Plumbago indica. In recent times, there has been a growing body of evidence suggesting the potential importance of naphthoquinones, both natural and artificial, in the pharmacological world. Numerous studies have indicated that PLB plays a vital role in combating cancers and other disorders. There is substantial evidence indicating that PLB may have a significant role in the treatment of breast cancer, brain tumours, lung cancer, hepatocellular carcinoma, and other conditions. Moreover, its potent anti-oxidant and anti-inflammatory properties offer promising avenues for the treatment of neurodegenerative and cardiovascular diseases. A number of studies have identified various pathways that may be responsible for the therapeutic efficacy of PLB. These include cell cycle regulation, apoptotic pathways, ROS induction pathways, inflammatory pathways, and signal transduction pathways such as PI3K/AKT/mTOR, STAT3/PLK1/AKT, and others. This review aims to provide a comprehensive analysis of the diverse pharmacological roles of PLB, examining the mechanisms through which it operates and exploring its potential applications in various medical conditions. In addition, we have conducted a review of the various formulations that have been reported in the literature with the objective of enhancing the efficacy of the compound. However, the majority of the reviewed data are based on in vitro and in vivo studies. To gain a comprehensive understanding of the safety and efficacy of PLB in humans and to ascertain its potential integration into therapeutic regimens for cancer and chronic diseases, rigorous clinical trials are essential. Finally, by synthesizing current research and identifying gaps in knowledge, this review seeks to enhance our understanding of PLB and its therapeutic prospects, paving the way for future studies and clinical applications.

Expression profile of microRNAs in bovine lymphocytes infected with Theileria annulata and treated with buparvaquone.

Several miRNA-based studies on Theileria-transformed bovine cells have been conducted; however, the mechanism by which transformed cells exhibit uncontrolled proliferation is not yet fully understood. Therefore, it is necessary to screen more microRNAs that may play a role in the transformation process of host cells infected with Theileria annulata to better understand the transformation mechanisms of Theileria-infected cells. RNA sequencing was used to analyze miRNAs expression in the host bovine lymphocytes infected with T. annulata at different time points after buparvaquone (BW720) treatment and DMSO treatment (control groups). Differential miRNAs related to cell proliferation and apoptosis were identified through comparison with gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and a regulatory network of miRNA-mRNA was constructed. In total, 272 differentially expressed miRNAs were found at 36, 60 and 72 h. The miRNAs change of bta-miR-2285t, novel-miR-622, bta-miR-2478, and novel-miR-584 were significant. Analysis of 27 of these co-differential expressed miRNAs revealed that 15 miRNAs were down-regulated and 12 miRNAs were up-regulated. A further analysis of the changes in the expression of each of these 27 miRNAs in the three datasets suggested that bta-miR-2285t, bta-miR-345-5p, bta-miR-34a, bta-miR-150, and the novel-miR-1372 had significantly changed. Predicted target genes for these 27 miRNAs were analyzed by KEGG and the results demonstrated that EZR, RASSF, SOCS1 were mainly enriched in the signaling pathway microRNAs in cancer. MAPKAPK2, RELB, FLT3LG, and GADD45B were mainly enriched in the MAPK signaling pathway, and some genes were enriched in Axon guidance. This study has provided valuable information to further the understanding of the regulatory function of miRNAs in the host microenvironment and host-parasite interaction mechanisms.

Natural Product-Inspired Discovery of Naphthoquinone-Furo-Piperidine Derivatives as Novel STAT3 Inhibitors for the Treatment of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and STAT3 has emerged as an effective drug target for TNBC treatment. Herein, we employed a scaffold-hopping strategy of natural products to develop a series of naphthoquinone-furopiperidine derivatives as novel STAT3 inhibitors. The in vitro assay showed that compound 10g possessed higher antiproliferative activity than Cryptotanshinone and Napabucasin against TNBC cell lines, along with lower toxicity and potent antitumor activity in a TNBC xenograft model. Mechanistically, 10g could inhibit the phosphorylation of STAT3 and the binding affinity was determined by the SPR assay (KD = 8.30 µM). Molecule docking studies suggested a plausible binding mode between 10g and the SH2 domain, in which the piperidine fragment and the terminal hydroxy group of 10g played an important role in demonstrating the success of this evolution strategy. These findings provide a natural product-inspired novel STAT3 inhibitor for TNBC treatment.

ß,ß-Dimethylacrylalkannin, a key component of Zicao, induces cell cycle arrest and necrosis in hepatocellular carcinoma cells.

BACKGROUND: ß,ß-Dimethylacrylalkannin (DMAKN), a natural naphthoquinone found in Zicao, a traditional Chinese medicine (TCM), serves as the designated quantitative marker in the Chinese Pharmacopoeia. Despite its established role in assessing Zicao quality, DMAKN's biological potential remains underexplored in research. METHODS: We investigated DMAKN's involvement in Zicao's anti-hepatocellular carcinoma (HCC) properties using a combination of HPLC content analysis and comprehensive bioinformatics. Subsequently, both in vitro and in vivo experiments were conducted to evaluate DMAKN's efficacy against HCC. Mechanistic investigations focused on elucidating DMAKN's impact on cell cycle regulation and induction of cell death. RESULTS: Integrated HPLC analysis and bioinformatics identified DMAKN as the primary active compound responsible for Zicao's anti-HCC activity. In vitro and in vivo studies confirmed DMAKN's potent efficacy against HCC. Notably, DMAKN demonstrated dual effects on HCC cells: inhibiting proliferation at lower doses and inducing rapid cell death at higher doses. Mechanistic insights revealed that low-dose DMAKN induced G2/M phase cell cycle arrest through modulation of CDK1 and Cdc25C phosphorylation, while high-dose DMAKN triggered necrosis. Importantly, high-dose DMAKN caused a sharp increase in intracellular ROS levels in a short time, while low-dose DMAKN gradually increased ROS levels over a long period. Additionally, low-dose DMAKN-induced ROS activated the JNK pathway, crucial for cell cycle arrest, whereas high-dose DMAKN-induced necrosis was ROS-dependent but JNK-independent. CONCLUSION: This study underscores DMAKN's pivotal role as the principal anti-HCC compound in Zicao, delineating its differential effects and underlying mechanisms. These results demonstrate the potential of DMAKN as a therapeutic agent for the treatment of HCC, providing important information for further study and advancement in cancer therapy.

Juglone suppresses vasculogenic mimicry in glioma through inhibition of HuR-mediated VEGF-A expression.

Vasculogenic mimicry (VM) serves as a vascular-like channel that provides important substances for tumor growth and is a primary factor in glioblastoma (GBM) drug resistance. Human Antigen R (HuR)-an mRNA-binding protein-is highly expressed in GBM, closely related to tumor progression, and deemed a potential drug target. Although some small-molecule compounds have been identified to disrupt HuR binding to target mRNA, they remain in the preclinical research stage, suggesting the need for further validation and development of HuR inhibitors. In our study, we aim to screen for potential HuR inhibitors and investigate their efficacy and molecular mechanisms in GBM. We employed the fluorescence polarization method to identify HuR inhibitors from a natural compound library, confirming the efficacy of juglone in effectively inhibiting the binding of HuR to AREVegf-a. Further validation of the binding of juglone to HuR at the protein level was conducted through electrophoretic mobility shift analysis, surface plasmon resonance, and molecular docking. Furthermore, juglone demonstrated inhibitory effects on glioma growth and VM formation in vitro and in vivo. Moreover, it was observed that juglone reversed epithelial-mesenchymal transition by inhibiting the VEGF-A/VEGFR2/AKT/SNAIL signaling pathway. Finally, we established the capability of juglone to target HuR in U251 cells through HuR knockdown, mRNA stability, and cell thermal shift assays. Therefore, this study identifies juglone as a novel HuR inhibitor, potentially offering promise as a lead compound for anti-VM therapy in GBM by targeting HuR. Abbreviations: AKT, protein kinase B; ARE, adenine-and uridine-rich elements; CETSA, cellular thermal shift assay; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme linked immune sorbent assay; EMSA, electrophoretic mobility shift assay; EMT, epithelial mesenchymal transition; FP, fluorescence polarization; GBM, glioblastoma; HTS, high-throughput screening; HuR, human antigen R; IF, Immunofluorescence; PAS, periodic acid-Schiff; PI3K, phosphoinositide-3 kinase; qRT-PCR, quantitative real-time PCR; RRMs, RNA recognition motifs; SPR, surface plasmon resonance. TMZ, temozolomide; VM, vasculogenic mimicry; VEGF-A, Vascular endothelial growth factor-A; VEGFR2, Vascular endothelial growth factor receptor-2.

Impact of Juglone, a PIN1 Inhibitor, on Oral Carcinogenesis Induced by 4-Nitroquinoline-1-Oxide (4NQO) in Rat Model.

Background and Objectives: PIN1 is overexpressed in several human cancers, including prostate cancer, breast cancer, and oral squamous carcinomas. Juglone (J), derived from walnut, was reported to selectively inhibit PIN1 by modifying its sulfhydryl groups. In this study, the potential effects of juglone, also known as PIN1 inhibitor, on oral cancer and carcinogenesis were investigated at the molecular level. Materials and Methods: 4-Nitroquinoline N-oxide (4-NQO) was used to create an oral cancer model in animals. Wistar rats were divided into five groups: Control, NQO, Juglone, NQO+J, and NQO+J*. The control group received the basal diet and tap water throughout the experiment. The NQO group received 4-NQO for 8 weeks in drinking water only. The Juglone group was administered intraperitoneally in a juglone solution for 10 weeks (1 mg/kg/day). The NQO+J group received 4-NQO in drinking water for 8 weeks, starting 1 week after the cessation of 4-NQO treatment. They were then administered intraperitoneally in a juglone solution for 10 weeks. (1 mg/kg/day). NQO+J* group: received 4 NQO for 8 weeks in drinking water and administered intraperitoneally in a juglone solution for 10 weeks (1 mg/kg/day). They were sacrificed at the end of the 22-week experimental period. The tongue tissues of the rats were isolated after the experiment, morphological changes were investigated by histological examinations, and the molecular apoptotic process was investigated by rt-qPCR and western blot. Results: Histological results indicate that tumors are formed in the tongue tissue with 4-NQO, and juglone treatment largely corrects the epithelial changes that developed with 4-NQO. It has been determined that apoptotic factors p53, Bax, and caspases are induced by the effect of juglone, while antiapoptotic factors such as Bcl-2 are suppressed. However, it was observed that the positive effects were more pronounced in rats given juglone together with 4-NQO. Conclusions: The use of PIN1 inhibitors such as juglone in place of existing therapeutic approaches might be a promising and novel approach to the preservation and treatment of oral cancer and carcinogenesis. However, further research is required to investigate the practical application of such inhibitors.

ß,ß-Dimethylacrylalkannin, a Natural Naphthoquinone, Inhibits the Growth of Hepatocellular Carcinoma Cells by Modulating Tumor-Associated Macrophages.

Tumor-associated macrophages (TAMs) are pivotal in the tumor microenvironment (TME) of hepatocellular carcinoma (HCC), influencing various stages from initiation to metastasis. Understanding the role of TAMs in HCC is crucial for developing novel therapeutic strategies. Macrophages exhibit plasticity, resulting in M1 and M2 phenotypes, with M1 macrophages displaying antitumor properties and M2 macrophages promoting tumor progression. Targeting TAMs to alter their polarization could offer new avenues for HCC treatment. ß,ß-dimethylacrylalkannin (DMAKN), a natural naphthoquinone, has gained attention for its antitumor properties. However, its impact on TAMs modulation remains unclear. This study investigates DMAKN's modulation of TAMs and its anti-HCC activity. Using an in vitro model with THP-1 cells, we induced M1 macrophages with LPS/IFN-γ and M2 macrophages with IL-4/IL-13, confirming polarization with specific markers. Co-culturing these macrophages with HCC cells showed that M1 cells inhibited HCC growth, while M2 cells promoted it. Screening for non-toxic DMAKN concentrations revealed its ability to induce M1 polarization and enhance LPS/IFN-γ-induced M1 macrophages, both showing anti-HCC effects. Conversely, DMAKN suppressed IL-4/IL-13-induced M2 polarization, inhibiting M2 macrophages' promotion of HCC cell viability. In summary, DMAKN induces and enhances M1 polarization while inhibiting M2 polarization of macrophages, thereby inhibiting HCC cell growth. These findings suggest that DMAKN has the potential to regulate TAMs in HCC, offering promise for future therapeutic development.

Plumbagin's Antiproliferative Mechanism in Human Cancer Cells: A Copper-Dependent Cytotoxic Approach.

Cancer is a serious global health problem, causing the loss of millions of lives each year. Plumbagin, a compound derived from the medicinal plant Plumbago zeylanica, has shown promise in stopping the growth of tumor cells both in laboratory settings and in living organisms. Many plant-based compounds exert their effects through copper's ability to produce reactive oxygen species (ROS). This study aimed to understand how plumbagin, dependent on copper, induces cell death (apoptosis) in human cancer cells through various experiments. The results demonstrate that plumbagin hinders the growth of pancreatic cancer cells PNAC-1 and MIA PaCa-2 by utilizing the copper naturally present in the cells. Unlike metal chelators that remove iron and zinc (desferrioxamine mesylate and histidine), a specific copper chelator called neocuproine lessens the cell death caused by plumbagin. When ROS scavengers are used, plumbagin-induced apoptosis is inhibited, indicating that ROS plays a role in initiating cell death. The study also proves that plumbagin prevents copper from leaving cancer cells by suppressing the expression of specific genes (CTR1 and ATP7A). It is confirmed that plumbagin targets the nuclear copper, leading to signals that promote oxidative stress and, ultimately, cell death. These findings provide valuable insights into the potential of plumbagin as a substance to combat cancer, highlighting the importance of understanding how copper behaves within cancer cells.

Exploring naphthoquinone and anthraquinone derivatives as antibiotic adjuvants against Staphylococcus aureus biofilms: Synergistic effects of menadione.

Given the ability of Staphylococcus aureus to form biofilms and produce persister cells, making infections difficult to treat with antibiotics alone, there is a pressing need for an effective antibiotic adjuvant to address this public health threat. In this study, a series of quinone derivatives were evaluated for their antimicrobial and antibiofilm activities against methicillin-susceptible and methicillin-resistant S. aureus reference strains. Following analyses using broth microdilution, growth curve analysis, checkerboard assay, time-kill experiments, and confocal laser scanning microscopy, menadione was identified as a hit compound. Menadione exhibited a notable antibacterial profile (minimum inhibitory concentration, MIC = 4-16 µg/ml; minimum bactericidal concentration, MBC = 256 µg/ml) against planktonic S. aureus and its biofilms (minimum biofilm inhibitory concentration, MBIC50 = 0.0625-0.25 µg/ml). When combined with oxacillin, erythromycin, and vancomycin, menadione exhibited a synergistic or additive effect against planktonic cells and biofilms of two S. aureus reference strains and six clinical isolates, highlighting its potential as a suitable adjuvant for further development against S. aureus biofilm-associated infections.

Genome Mining and Genetic Manipulation Reveal New Isofuranonaphthoquinones in Nocardia Species.

The identification of specialized metabolites isolated from microorganisms is urgently needed to determine their roles in treating cancer and controlling multidrug-resistant pathogens. Naphthoquinones act as anticancer agents in various types of cancers, but some toxicity indicators have been limited in their appropriate application. In this context, new isofuranonaphthoquinones (ifnq) that are less toxic to humans could be promising lead compounds for developing anticancer drugs. The aim of this study is to identify and characterize novel furanonaphthoquinones (fnqs) from Nocardia sp. CS682 and to evaluate their potential therapeutic applications. Analysis of the genome of Nocardia sp. CS682 revealed the presence of a furanonaphthoquinone (fnq) gene cluster, which displays a similar genetic organization and high nucleotide sequence identity to the ifnq gene cluster from Streptomyces sp. RI-77, a producer of the naphthoquinones JBIR-76 and JBIR-77. In this study, the overexpression of the Streptomyces antibiotic regulatory protein (SARP) in Nocardia sp. CS682DR (nargenicin gene-deleted mutant) explicitly produced new fnqs, namely, NOC-IBR1 and NOC-IBR2. Subsequently, the role of the SARP regulator was confirmed by gene inactivation using CRISPR-Cas9 and complementation studies. Furthermore, antioxidant, antimicrobial, and cytotoxicity assays were performed for the isolated compounds, and it was found that NOC-IBR2 exhibited superior activities to NOC-IBR1. In addition, a flexible methyltransferase substrate, ThnM3, was found to be involved in terminal methylation of NOC-IBR1, which was confirmed by in vitro enzyme assays. Thus, this study supports the importance of genome mining and genome editing approaches for exploring new specialized metabolites in a rare actinomycete called Nocardia.

Bioactive Naphthoquinone and Phenazine Analogs from the Endophytic Streptomyces sp. PH9030 as α-Glucosidase Inhibitors.

A talented endophytic Streptomyces sp. PH9030 is derived from the medicinal plant Kadsura coccinea (Lem.) A.C. Smith. The undescribed naphthoquinone naphthgeranine G (5) and seven previously identified compounds, 6-12, were obtained from Streptomyces sp. PH9030. The structure of 5 was identified by comprehensive examination of its HRESIMS, 1D NMR, 2D NMR and ECD data. The inhibitory activities of all the compounds toward α-glucosidase and their antibacterial properties were investigated. The α-glucosidase inhibitory activities of 5, 6, 7 and 9 were reported for the first time, with IC50 values ranging from 66.4 ± 6.7 to 185.9 ± 0.2 µM, as compared with acarbose (IC50 = 671.5 ± 0.2 µM). The molecular docking and molecular dynamics analysis of 5 with α-glucosidase further indicated that it may have a good binding ability with α-glucosidase. Both 9 and 12 exhibited moderate antibacterial activity against methicillin-resistant Staphylococcus aureus, with minimum inhibitory concentration (MIC) values of 16 µg/mL. These results indicate that 5, together with the naphthoquinone scaffold, has the potential to be further developed as a possible inhibitor of α-glucosidase.

Transient efficacy of buparvaquone against Theileria haneyi in chronically infected horses.

BACKGROUND: Theileria haneyi is one of the three known causative agents of equine piroplasmosis. While imidocarb is generally effective in the clearance of the highly pathogenic Theileria equi, it is ineffective in the treatment of T. haneyi. Moreover, co-infection with T. haneyi has been shown to impede the successful treatment of T. equi. Furthermore, tulathromycin and diclazuril have demonstrated inefficacy in eradicating T. haneyi. The absence of an effective therapeutic agent against this parasite represents a significant obstacle in managing equine piroplasmosis. METHODS: To address this issue, we evaluated the efficacy of buparvaquone in the treatment of T. haneyi in chronically infected horses. RESULTS: Our findings showed that treatment of horses with the recommended dose of 2.5 mg/kg of buparvaquone led to a rapid abatement of T. haneyi levels, to a level where the parasites were not detectable by nested PCR. Following treatment, the horses remained PCR negative for a minimum of seven weeks until recrudescence occurred. Subsequent re-administration of buparvaquone at an increased dosage of 6 mg/kg upon recrudescence failed to exert a theilericidal effect on T. haneyi. Throughout the treatment regimen, the hematological parameters of the horses and most components of the chemistry panel remained within the normal range, except for blood urea nitrogen levels, which fell below the normal range in certain instances. CONCLUSIONS: BPQ at 2.5 mg/kg and 6 mg/kg had a robust theilericidal effect but was ineffective in the clearance of the T. haneyi infection in persistently infected animals.

Tetramerization of PKM2 Alleviates Traumatic Brain Injury by Ameliorating Mitochondrial Damage in Microglia.

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Microglial activation and neuroinflammation are key cellular events that determine the outcome of TBI, especially neuronal and cognitive function. Studies have suggested that the metabolic characteristics of microglia dictate their inflammatory response. The pyruvate kinase isoform M2 (PKM2), a key glycolytic enzyme, is involved in the regulation of various cellular metabolic processes, including mitochondrial metabolism. This suggests that PKM2 may also participate in the regulation of microglial activation during TBI. Therefore, the present study aimed to evaluate the role of PKM2 in regulating microglial activation and neuroinflammation and its effects on cognitive function following TBI. A controlled cortical impact (CCI) mouse model and inflammation-induced primary mouse microglial cells in vitro were used to investigate the potential effects of PKM2 inhibition and regulation. PKM2 was significantly increased during the acute and subacute phases of TBI and was predominantly detected in microglia rather than in neurons. Our results demonstrate that shikonin and TEPP-46 can inhibit microglial inflammation, improving mitochondria, improving mouse behavior, reducing brain defect volume, and alleviating pathological changes after TBI. There is a difference in the intervention of shikonin and TEPP-46 on PKM2. Shikonin directly inhibits General PKM2; TEPP-46 can promote the expression of PKM2 tetramer. In vitro experiments, TEPP-46 can promote the expression of PKM2 tetramer, enhance the interaction between PKM2 and MFN2, improve mitochondria, alleviate neuroinflammation. General inhibition and tetramerization activation of PKM2 attenuated cognitive function caused by TBI, whereas PKM2 tetramerization exhibited a better treatment effect. Our experiments demonstrated the non-metabolic role of PKM2 in the regulation of microglial activation following TBI. Both shikonin and TEPP-46 can inhibit pro-inflammatory factors, but only TEPP-46 can promote PKM2 tetramerization and upregulate the release of anti-inflammatory factors from microglia.

Survivin inhibition attenuates EGF-induced epithelial mesenchymal transformation of human RPE cells via the EGFR/MAPK pathway.

PURPOSE: The abnormal growth factors-induced epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells was known as a vital pathogenesis of proliferative vitreoretinopathy (PVR). This study aims to explore how survivin inhibition affects EMT induced by epidermal growth factor (EGF) in RPE cells. METHODS: Human primary RPE cells were identified in vitro. EMT in RPE cells was induced by EGF. Inhibition of survivin in RPE cells was accomplished through the use of a survivin inhibitor (YM155) and survivin siRNA. The viability, proliferation and migration of RPE cells was detected by methylthiazol tetrazolium assay, bromodeoxyuridine labeling assay, and wound healing assay, respectively. The EGF receptor /mitogen-activated protein kinase (EGFR/MAPK) proteins and EMT-related proteins were measured by western blot and immunofluorescence assay. RESULTS: EGF induced significant EMT in RPE cells, activated the phosphorylation of EGFR/MAPK signaling proteins, and caused changes to EMT-related proteins. YM155 suppressed RPE cells' viability, proliferation, and migration; induced the phosphorylation of EGFR, JNK, and P38MAPK; and down regulated EGFR and phosphorylated ERK. YM155 also increased expression of E-cadherin and ZO-1 proteins and reduced expression of N-cadherin, Vimentin, and α-SMA proteins. The EGF-induced increase of RPE cell proliferation and migration was constrained by survivin inhibition. Moreover, survivin inhibition in RPE cells suppressed the EGF-caused phosphorylation of EGFR/MAPK proteins and attenuated the EGF-induced reduction of E-cadherin and ZO-1 proteins and increase of N-cadherin, Vimentin, and α-SMA proteins. CONCLUSIONS: Survivin inhibition attenuates EGF-induced EMT of RPE cells by affecting the EGFR/MAPK signaling pathway. Survivin might be a promising target for preventing PVR.

SIRT1 improves lactate homeostasis in the brain to alleviate parkinsonism via deacetylation and inhibition of PKM2.

Sirtuin 1 (SIRT1) is a histone deacetylase and plays diverse functions in various physiological events, from development to lifespan regulation. Here, in Parkinson's disease (PD) model mice, we demonstrated that SIRT1 ameliorates parkinsonism, while SIRT1 knockdown further aggravates PD phenotypes. Mechanistically, SIRT1 interacts with and deacetylates pyruvate kinase M2 (PKM2) at K135 and K206, thus leading to reduced PKM2 enzyme activity and lactate production, which eventually results in decreased glial activation in the brain. Administration of lactate in the brain recapitulates PD-like phenotypes. Furthermore, increased expression of PKM2 worsens PD symptoms, and, on the contrary, inhibition of PKM2 by shikonin or PKM2-IN-1 alleviates parkinsonism in mice. Collectively, our data indicate that excessive lactate in the brain might be involved in the progression of PD. By improving lactate homeostasis, SIRT1, together with PKM2, are likely drug targets for developing agents for the treatment of neurodegeneration in PD.

Shikonin from lithospermum erythrorhizon induces pyroptosis in trophoblast cells by activating the CTSB-NLRP3 inflammasome.

BACKGROUND: With the decline of global fertility, drug therapeutic of ectopic pregnancy is of great significance. Lithospermum erythrorhizon is using for embryo killing as herbal medicine. Shikonin is the critical nucleus of Lithospermum erythrorhizon; however, the mechanism is still unclear. The study aimed to explore the mechanism of shikonin against ectopic pregnancy. MATERIAL AND METHODS: In this study, we examined the viability and LDH release of HTR-8/SVneo cells by assays, observed pore formation in cell membranes by microscopy imaging and PI staining, and IL-1ß release by WB and ELISA assay kit. Then, we used network pharmacology to analyse the potential interaction between shikonin, ectopic pregnancy and pyroptosis and used molecular docking techniques to verify interactions between shikonin and core common targets. Finally, western blotting and immunofluorescence assay were used to explore the mechanism of shikonin-inducing pyroptosis of HTR-8/SVneo cells. RESULTS: Shikonin could cause a significant inhibition of HTR-8/SVneo cell viability in a concentration- and time-dependent manner. In HTR-8/SVneo cells, shikonin-induced cell swelling, bubble formation, an increase in the release of lactate dehydrogenase (LDH) and up-regulation of several pyroptosis-associated factors. And network pharmacology showed that The main targets of shikonin-ectopic pregnancy-pyroptosis were IL-1ß and caspase-1, and molecular docking results showed that shikonin can closely bind to IL-1ß, caspase-1 and GSDMD. Additionally, the necroptosis inhibitor GSK'872 could not suppress the expression of mature-IL-1ß and prevent the pyroptosis phenotype from developing. However, the nucleotide oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inhibitor MCC-950 could downregulate the expression of pyroptosis-associated factors and prevent the pyroptosis phenotype from developing. Shikonin led to an elevation in the expression of cathepsin B (CTSB), and the CTSB inhibitor CA-074 abolished pyroptosis induced by shikonin; however, the NLRP3 inhibitor MCC-950 could not inhibit the expression of CTSB. CONCLUSIONS: Our results suggest that shikonin activates CTSB to induce NLRP3-dependent pyroptosis in HTR-8/SVneo cells. This study has important clinical implications for the treatment of ectopic pregnancy.

Brain-targeting redox-sensitive micelles for codelivery of TMZ and ß-lapachone for glioblastoma therapy.

Glioblastoma (GBM) is a central nervous system cancer with high incidence and poor survival rates. Enhancing drug penetration of the blood-brain barrier (BBB) and targeting efficacy is crucial for improving treatment outcomes. In this study, we developed a redox-sensitive targeted nano-delivery system (HCA-A2) for temozolomide (TMZ) and ß-lapachone (ß-Lapa). This system used hyaluronic acid (HA) as the hydrophilic group, arachidonic acid (CA) as the hydrophobic group, and angiopep-2 (A2) as the targeting group. Control systems included non-redox sensitive (HDA-A2) and non-targeting (HCA) versions. In vitro, HCA-TMZ-Lapa micelles released 100 % of their payload in a simulated tumor microenvironment within 24 h, compared to 43.97 % under normal conditions. HCA-A2 micelles, internalized via clathrin-mediated endocytosis, showed stronger cytotoxicity and better BBB penetration and cellular uptake than controls. In vivo studies demonstrated superior tumor growth inhibition with HCA-A2 micelles, indicating their potential for GBM treatment.