Acacetin, a Natural Flavone with Potential in Improving Liver Disease Based on Its Anti-Inflammation, Anti-Cancer, Anti-Infection and Other Effects.

Liver disease is a global public problem, and the cost of its therapy is a large financial burden to governments. It is well known that drug therapy plays a critical role in the treatment of liver disease. However, present drugs are far from meeting clinical needs. Lots of efforts have been made to find novel agents to treat liver disease in the past several decades. Acacetin is a dihydroxy and monomethoxy flavone, named 5,7-dihydroxy-4'-methoxyflavone, which can be found in diverse plants. It has been reported that acacetin exhibits multiple pharmacological activities, including anti-cancer, anti-inflammation, anti-virus, anti-obesity, and anti-oxidation. These studies indicate the therapeutic potential of acacetin in liver disease. This review discussed the comprehensive information on the pathogenesis of liver disease (cirrhosis, viral hepatitis, drug-induced liver injury, and hepatocellular carcinoma), then introduced the biological source, structural features, and pharmacological properties of acacetin, and the possible application in preventing liver disease along with the pharmacokinetic and toxicity of acacetin, and future research directions. We systemically summarized the latest research progress on the potential therapeutic effect of acacetin on liver disease and existing problems. Based on the present published information, the natural flavone acacetin is an anticipated candidate agent for the treatment of liver disease.

Acacetin is a Promising Drug Candidate for Cardiovascular Diseases.

Phytochemical flavonoids have been proven to be effective in treating various disorders, including cardiovascular diseases. Acacetin is a natural flavone with diverse pharmacological effects, uniquely including atrial-selective anti-atrial fibrillation (AF) via the inhibition of the atrial specific potassium channel currents [Formula: see text] (ultra-rapidly delayed rectifier potassium current), [Formula: see text] (acetylcholine-activated potassium current), [Formula: see text] (calcium-activated small conductance potassium current), and [Formula: see text] (transient outward potassium current). [Formula: see text] inhibition by acacetin, notably, suppresses experimental J-wave syndromes. In addition, acacetin provides extensive cardiovascular protection against ischemia/reperfusion injury, cardiomyopathies/heart failure, autoimmune myocarditis, pulmonary artery hypertension, vascular remodeling, and atherosclerosis by restoring the downregulated intracellular signaling pathway of Sirt1/AMPK/PGC-1[Formula: see text] followed by increasing Nrf2/HO-1/SOD thereby inhibiting oxidation, inflammation, and apoptosis. This review provides an integrated insight into the capabilities of acacetin as a drug candidate for treating cardiovascular diseases, especially atrial fibrillation and cardiomyopathies/heart failure.

Pegylation enhances the anti-osteoporosis activity of acacetin in both ovariectomized and LPS-stimulated mice.

Osteoporosis is a condition of progressive bone loss attributable to excessive osteoclastic activity. Acacetin is a potential candidate for osteoporosis therapy because it specifically suppressing osteoclastic function. However, the application of acacetin was limited by its poor solubility and bad pharmacokinetic behavior. In current work, we examined whether PEGylation of acacetin enhances its anti-osteoporosis activity in ovariectomy-induced osteoporosis and LPS-induced osteolysis. In the current study, three types of PEGylated acacetin (PEG3-A, PEG4-A, PEG5-A) were tested for their effects on the solubility and anti-inflammatory activity of acacetin in vitro. PEG5-Acacetin was selected for further investigation as it demonstrated the strongest anti-inflammatory activity comparable to that of naked acacetin and other two PEGylated acacetin. PEGylation in PEG5-Acacetin increased maximum plasma concentration of acacetin by 620.77% in mice. Furthermore, PEG5-A showed a higher anti-osteoclastogenic capacity in vitro than that of naked acacetin. It was found that PEG5-A treatment in vivo mitigated lipopolysaccharide (LPS)- and ovariectomy (OVX)-induced bone loss in mice. More importantly, the in vivo efficiency of PEG5-Acacetin was significantly better than that of naked acacetin. In summary, PEGylated acacetin possesses a clean advantage over the naked acacetin and would be a potential candidate for the osteoporosis therapy.

Anti-inflammation Mechanisms of Flavones Are Highly Sensitive to the Position Isomers of Flavonoids: Acacetin vs Biochanin A.

Acacetin (ACA) and biochanin A (BCA) are isomeric monomethoxyflavones with different structural positions of the 4'-methoxy-phenyl group. Both of them are present in many commonly consumed foods, such as citrus fruits and vegetables, and have been discovered with anti-inflammatory activities, but their mechanisms of action are not clearly elucidated at the molecular level. Herein, we reported the structure-activity relationship of ACA and BCA regarding their potency in inhibiting nitric oxide (NO) production, proinflammatory enzyme expression, and mRNA expression of proinflammatory cytokines in the lipopolysaccharide (LPS)-induced RAW 264.7 cells. Furthermore, transcriptome analysis was conducted to map out the overall pathways impacted by these two isomers. Our results showed that ACA possessed higher suppressive activity in nitric oxide (NO) production (IC50 = 23.93 ± 1.74 µM for ACA and IC50 = 71.41 ± 8.07 µM for BCA), inducible nitric oxide synthase (iNOS) expression, and proinflammatory interleukin (IL)-1ß mRNA expression, but lower inhibitory activity in IL-6 mRNA expression as compared with BCA. Although two compounds were revealed to bind to c-Src protein according to drug affinity responsive target stability (DARTS) and western blotting results, molecular docking and molecular dynamics (MD) simulation showed that they had different binding sites, which subsequently resulted in different signaling transduction. ACA primarily exerted anti-inflammatory activity through the mitogen-activated protein kinase (MAPK) signaling pathway, the tumor necrosis factor (TNF) signaling pathway, and the hypoxia-inducible factor (HIF)-1 signaling pathway, while BCA was particularly involved in the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway, the nuclear factor kappa B (NF-κB) signaling pathway, the TNF signaling pathway, and the toll-like receptor (TLR) signaling pathway. Moreover, two isomers remained stable in the cell culture medium and did not encounter the biotransformation process in RAW 264.7 cells. However, BCA exhibited insufficient cellular uptake ability and transport efficiency in macrophages compared to ACA. Taken together, ACA is more promising for controlling inflammation and worthy of further study for human use.

Acacetin protects against acute lung injury by upregulating SIRT1/ NF-κB pathway.

Acacetin is one of the natural flavone components found in many plants and possesses diverse pharmacological activities. The anti-inflammatory properties and definite mechanism of acacetin remains incompletely illuminated. Here, we evaluated the efficacy of acacetin on lipopolysaccharide (LPS)-induced acute lung injury in vivo and TNF-α-stimulated cellular injury in vitro. As indicated by survival experiments, acacetin reduced mortality and improved survival time of LPS-induced acute lung injury in mice. 50 mg/kg of acacetin obtained higher survival (about 60 %), and 20 mg/kg of acacetin was about 46.7 %. In addition, 20 mg/kg of acacetin rescued lung histopathologic damage in LPS treated mice, lowered lung-to-body weight and lung wet-to-dry ratios, suppressed myeloperoxidase activity in lung tissue, the contents of protein, the numbers of total cells and neutrophils in bronchoalveolar lavage fluid (BALF), and the contents of inflammatory cytokines such as TNF-α, IL-6, IL-17 and IL-1ß in BALF. Acacetin also increased the activity and expression of SIRT1, thereby suppressing acetylation-dependent activation NF-κB. Similarly, in vitro, acacetin increased cell viability, reduced levels of TNF-α, IL-6, IL-17, and IL-1ß, increased NAD+ levels as well as NAD/NADH ratio, and then up-regulated the activity and expression of SIRT1, and restrained acetylation-dependent activation NF-κB in TNF-α-stimulated A549 cells, which could be abolished by SIRT1 siRNA. Collectively, the current study showed that acacetin exerts a protective effiect on acute lung injury by improving the activity and expression SIRT1, thereby suppressing the acetylation-dependent activation of NF-κB-p65 and the release of downstream inflammatory cytokines.

Metabolic characterization and transcriptional profiling of polyphenols in Cannabis sativa L. inflorescences with different chemical phenotypes.

MAIN CONCLUSION: After the most comprehensive analysis of the phenolic composition in Cannabis reported to date, a total of 211 compounds were identified, phenolic profiles were able to discriminate cannabis varieties and a complex regulatory network for phenolics accumulation in Cannabis chemovars was highlighted. Female inflorescences of Cannabis sativa L. are plenty of secondary metabolites, of which flavonoids and phenolic acids have been investigated by far less than phytocannabinoids and terpenoids. Understanding the biochemical composition in phenylpropanoids of Cannabis inflorescences, the molecular basis of flavonoid synthesis and how their content can be modulated by specific transcription factors will shed light on the variability of this trait in the germplasm, allowing the identification of biologically active metabolites that can be of interest to diverse industries. In this work, an untargeted metabolomic approach via UHPLC-HRMS was adopted to investigate the composition and variability of phenylpropanoids in thirteen Cannabis genotypes differentiated for their profile in phytocannabinoids, highlighting that phenolic profiles can discriminate varieties, with characteristic, unique genotype-related patterns. Moreover, the transcription profile of candidate phenolics regulatory MYB and bHLH transcription factors, analyzed by RT-qPCR, appeared strongly genotype-related, and specific patterns were found to be correlated between biochemical and transcriptional levels. Results highlight a complex regulatory network for phenolic accumulation in Cannabis chemovars that will need further insights from the functional side.

Targeting colorectal cancer with Herba Patriniae and Coix seed: Network pharmacology, molecular docking, and in vitro validation.

BACKGROUND: Herba Patriniae and Coix seed (HC) constitute a widely utilized drug combination in the clinical management of colorectal cancer (CRC) that is known for its diuretic, anti-inflammatory, and swelling-reducing properties. Although its efficacy has been demonstrated in a clinical setting, the active compounds and their mechanisms of action in CRC treatment remain to be fully elucidated. AIM: To identify the active, CRC-targeting components of HC and to elucidate the mechanisms of action involved. METHODS: Active HC components were identified and screened using databases. Targets for each component were predicted. CRC-related targets were obtained from human gene databases. Interaction targets between HC and CRC were identified. A "drug-ingredient-target" network was created to identify the core components and targets involved. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted to elucidate the key pathways involved. Molecular docking between core targets and key components was executed. In vitro experiments validated core monomers. RESULTS: Nineteen active components of HC were identified, with acacetin as the primary active compound. The predictive analysis identified 454 targets of the active compounds in HC. Intersection mapping with 2685 CRC-related targets yielded 171 intervention targets, including 30 core targets. GO and KEGG analyses indicated that HC may influence the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. Molecular docking showed that acacetin exhibited an optimal interaction with AKT1, identifying PI3K, AKT, and P53 as key genes likely targeted by HC during CRC treatment. Acacetin inhibited HT-29 cell proliferation and migration, as well as promoted apoptosis, in vitro. Western blotting analysis revealed increased p53 and cleaved caspase-3 expression and decreased levels of p-PI3K, p-Akt, and survivin, which likely contributed to CRC apoptosis. CONCLUSION: Acacetin, the principal active compound in the HC pair, inhibited the proliferation and migration of HT-29 cells and promoted apoptosis through the PI3K/Akt/p53 signaling pathway.

Effect of acacetin on inhibition of apoptosis in Helicobacter pylori-infected gastric epithelial cell line.

BACKGROUND: Helicobacter pylori (H. pylori) infection can cause extensive apoptosis of gastric epithelial cells, serving as a critical catalyst in the progression from chronic gastritis, gastrointestinal metaplasia, and atypical gastric hyperplasia to gastric carcinoma. Prompt eradication of H. pylori is paramount for ameliorating the pathophysiological conditions associated with chronic inflammation of the gastric mucosa and the primary prevention of gastric cancer. Acacetin, which has multifaceted pharmacological activities such as anti-cancer, anti-inflammatory, and antioxidative properties, has been extensively investigated across various domains. Nevertheless, the impact and underlying mechanisms of action of acacetin on H. pylori-infected gastric mucosal epithelial cells remain unclear. AIM: To explore the defensive effects of acacetin on apoptosis in H. pylori-infected GES-1 cells and to investigate the underlying mechanisms. METHODS: GES-1 cells were treated with H. pylori and acacetin in vitro. Cell viability was assessed using the CCK-8 assay, cell mortality rate via lactate dehydrogenase assay, alterations in cell migration and healing capacities through the wound healing assay, rates of apoptosis via flow cytometry and TUNEL staining, and expression levels of apoptosis-associated proteins through western blot analysis. RESULTS: H. pylori infection led to decreased GES-1 cell viability, increased cell mortality, suppressed cell migration, increased rate of apoptosis, increased expressions of Bax and cle-caspase3, and decreased Bcl-2 expression. Conversely, acacetin treatment enhanced cell viability, mitigated apoptosis induced by H. pylori infection, and modulated the expression of apoptosis-regulatory proteins by upregulating Bcl-2 and downregulating Bax and cleaved caspase-3. CONCLUSION: Acacetin significantly improved GES-1 cell viability and inhibited apoptosis in H. pylori-infected GES-1 cells, thereby exerting a protective effect on gastric mucosal epithelial cells.

The haplotype-resolved genome of diploid Chrysanthemum indicum unveils new acacetin synthases genes and their evolutionary history.

Acacetin, a flavonoid compound, possesses a wide range of pharmacological effects, including antimicrobial, immune regulation, and anticancer effects. Some key steps in its biosynthetic pathway were largely unknown in flowering plants. Here, we present the first haplotype-resolved genome of Chrysanthemum indicum, whose dried flowers contain abundant flavonoids and have been utilized as traditional Chinese medicine. Various phylogenetic analyses revealed almost equal proportion of three tree topologies among three Chrysanthemum species (C. indicum, C. nankingense, and C. lavandulifolium), indicating that frequent gene flow among Chrysanthemum species or incomplete lineage sorting due to rapid speciation might contribute to conflict topologies. The expanded gene families in C. indicum were associated with oxidative functions. Through comprehensive candidate gene screening, we identified five flavonoid O-methyltransferase (FOMT) candidates, which were highly expressed in flowers and whose expressional levels were significantly correlated with the content of acacetin. Further experiments validated two FOMTs (CI02A009970 and CI03A006662) were capable of catalyzing the conversion of apigenin into acacetin, and these two genes are possibly responsible acacetin accumulation in disc florets and young leaves, respectively. Furthermore, combined analyses of ancestral chromosome reconstruction and phylogenetic trees revealed the distinct evolutionary fates of the two validated FOMT genes. Our study provides new insights into the biosynthetic pathway of flavonoid compounds in the Asteraceae family and offers a model for tracing the origin and evolutionary routes of single genes. These findings will facilitate in vitro biosynthetic production of flavonoid compounds through cellular and metabolic engineering and expedite molecular breeding of C. indicum cultivars.

Acacetin targets STING to alleviate the destabilization of the medial meniscus-induced osteoarthritis in mice.

Osteoarthritis (OA) is a common joint disorder affecting about 7% of the global population, primarily characterized by the gradual loss of articular cartilage. This degeneration results from local inflammation, matrix depletion, and direct cartilage damage. A critical element in this process is the activation of the stimulator of the interferon genes (STING) pathway. Emerging evidence highlights its potential as a therapeutic target, with natural products showing promise as inhibitors. Our study centers on Acacetin, a basic unit of polyketides known for its anti-inflammatory properties. Prior research has highlighted its potential interaction with STING based on the structure. Thus, this study aimed to assess the effectiveness of Acacetin as a STING inhibitor and its protective role against OA. In vitro experiments showed that Acacetin pretreatment not only mitigated interleukin-1ß (IL-1ß)-induced cytotoxicity but also decreased the inflammatory response and degeneration in chondrocytes stimulated IL-1ß. In vivo studies revealed that Acacetin administration significantly reduced articular cartilage destruction, abnormal bone remodeling, and osteophyte formation in a model of OA induced by destabilization of the medial meniscus (DMM). Mechanistically, Acacetin was found to interact directly with STING, and inhibit IL-1ß-induced activation of STING, along with the subsequent phosphorylation of the TBK1/NF-κB pathway in chondrocytes. In conclusion, our findings establish Acacetin as an effective inhibitor of STING that protects chondrocytes from IL-1ß-induced damage and slows the progression of OA in mice.

RNA Sequencing Analyses Reveal the Potential Anti-Inflammatory Mechanisms of Acacetin Against ODG/R Injuries in Microglia.

Background: Acacetin is a natural flavonoid known for its anti-tumor, antioxidant, and anti-inflammatory properties. Our previous studies have shown its protective effects against cerebral ischemia-reperfusion injury (IRI), but the underlying molecular mechanisms remain unclear. Purpose: The study delves into acacetin's mechanism in mitigating cerebral IRI, with a focus on transcriptomic insights. Methods: We established the oxygen-glucose deprivation/re-oxygenation (OGD/R) model in BV2 microglia, treating them with 10µM acacetin. Then we assessed cell proliferation using CCK-8 and measured Lactate Dehydrogenase (LDH) release. High-throughput RNA sequencing (RNA-seq) underpinned the analysis of differentially expressed genes (DEGs) and long non-coding RNAs (lncRNAs), functional enrichment, and alternative splicing events (ASEs), validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Results: OGD/R injury significantly impaired cell proliferation and increased LDH release, effects mitigated by acacetin. RNA-seq identified 2148 upregulated and 2135 downregulated DEGs post-OGD/R. In contrast, the acacetin-treated group showed 248 upregulated and 240 downregulated DEGs compared to the OGD/R group. All DEGs were enriched in both Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Overlapping analysis indicated that acacetin treatment reversed the expression of 203 genes affected by OGD/R, including inflammation-related genes such as Isg15, Fcgr1, Il1b, and Parp12. Moreover, the oxidative stress-related gene, Mt2, was downregulated post-OGD/R but upregulated following acacetin treatment. We further found that OGD/R and acacetin treatment could modulate gene splicing events, impacting cell apoptosis or inflammatory responses, such as the A3SS splicing event in the Trim47 gene. RNA-seq also highlighted differential expression of numerous lncRNAs, particularly the upregulation of lncRNA Rmrp and Terc post-OGD/R and their subsequent downregulation post-acacetin treatment. These lncRNAs might regulate cell proliferation through mediating target gene expressions. RT-qPCR validation confirmed these findings. Conclusion: Significant upregulation of genes and ASEs linked to oxidative stress and inflammatory response is observed in cerebral IRI. Acacetin intervention reverses these effects, highlighting its mechanism in alleviating the injury by modulating gene expression and splicing events.

Acacetin Attenuates Sepsis-induced Acute Lung Injury via NLRC3-NF-κB Pathway.

Acacetin, a flavonoid derived compound has been recognized for its diverse biological activities, such as anti-oxidative and anti-inflammatory effects. Acute lung injury (ALI) is a severe condition characterized by respiratory insufficiency and tissue damage, commonly triggered by pneumonia and severe sepsis. These conditions induce an inflammatory response via Toll-like receptor 4 (TLR4) signaling activation. This study explored acacetin's therapeutic potential against lipopolysaccharide (LPS) induced ALI in mice, focusing on its ability to modulate the NF-κB pathway via regulation of the Nod-like receptor family CARD domain containing 3 (NLRC3), a signal sensor that plays an important role in the regulation of inflammation and the maintenance of homeostasis. Our findings revealed that high-dose acacetin reduced the mortality rate of ALI mice, significantly ameliorated LPS-induced lung pathological changes, reduced lung edema, and decreased the expression of inflammatory mediators in lung tissues. This protective impact of acacetin appears to stem form its capacity to enhance NLRC3 expression, which, intern, can inhibit the activation of NF-κB and subsequently inhibit the production of inflammatory mediators. NLRC3 deficiency inhibits the protective effect of acacetin on ALI mice. Molecular docking also verified that acacetin tightly bound acacetin to NLRC3. Additionally, acacetin was found to influence macrophage recruitment dynamics via NLRC3, inhibiting the overactivation of NLRC3-NF-κB related pathways. Taken together, our results indicate that acacetin inhibited LPS-induced acute lung injury and macrophage overrecruitment to the lungs in mice by upregulating NLRC3.

Acacetin inhibits activation of microglia to improve neuroinflammation after subarachnoid hemorrhage through the PERK signaling pathway mediated autophagy.

PURPOSE: To explore the effect of acacetin on subarachnoid hemorrhage (SAH) and its possible mechanism. METHODS: SAH model of rat was established, and intraperitoneally injected with three doses of acacetin. To verify the role of PERK pathway, we used the CCT020312 (PERK inhibitor) and Tunicamycin (activators of endoplasmic reticulum stress). The SAH score, neurological function score, brain edema content, and Evans blue (EB) exudate were evaluated. Western blot was used to determine the expression of inflammation-associated proteins and PERK pathway. The activation of microglia was also determined through Iba-1 detection. TEM and immunofluorescence staining of LC3B were performed to observe the autophagy degree of SAH rats after acacetin. Tunel/NeuN staining, HE and Nissl' staining were performed for neuronal damage. RESULTS: Acacetin increased the neurological function score, reduce brain water content, Evans blue exudation and SAH scores. The microglia in cerebral cortex were activated after SAH, while acacetin could inhibit its activation, and decreased the expression of TNF-α and IL-6 proteins. The pathological staining showed the severe neuronal damage and increased neuronal apoptosis after SAH, while acacetin could improve these pathological changes. We also visualized the alleviated autophagy after acacetin. The expression of Beclin1 and ATF4 proteins were increased, but acacetin could inhibit them. Acacetin also inactivated PERK pathway, which could improve the neuronal injury and neuroinflammation after SAH, inhibit the microglia activation and the overactivated autophagy through PERK pathway. CONCLUSION: Acacetin may alleviate neuroinflammation and neuronal damage through PERK pathway, thus having the protective effect on EBI after SAH.

Acacetin alleviates autoimmune myocarditis by regulating CD4+ T cell mitochondrial respiration.

BACKGROUND: Myocarditis refers to an autoimmune inflammatory response of the myocardium with characterization of self-reactive CD4+ T cell activation, which lacks effective treatment and has a poor prognosis. Acacetin is a natural flavonoid product that has been reported to have anti-inflammatory effects. However, acacetin has not been investigated in myocarditis. METHODS: Oral acacetin treatment was administered in an experimental autoimmune myocarditis model established with myosin heavy chain-alpha peptide. Echocardiography, pathological staining, and RT-qPCR were used to detect cardiac function, myocardial injury, and inflammation levels. Flow cytometry was utilized to detect the effect of acacetin on CD4+ T cell function. RNA-seq, molecular docking, and microscale thermophoresis (MST) were employed to investigate potential mechanisms. Seahorse analysis, mitoSOX, JC-1, and mitotracker were utilized to detect the effect of acacetin on mitochondrial function. RESULTS: Acacetin attenuated cardiac injury and fibrosis as well as heart dysfunction, and reduced cardiac inflammatory cytokines and ratio of effector CD4+ T and Th17 cells. Acacetin inhibited CD4+ T cell activation, proliferation, and Th17 cell differentiation. Mechanistically, the effects of acacetin were related to reducing mitochondrial complex II activity thereby inhibiting mitochondrial respiration and mitochondrial reactive oxygen species in CD4+ T cells. CONCLUSION: Acacetin may be a valuable therapeutic drug in treating CD4+ T cell-mediated myocarditis.

Mechanism of acacetin regulating hepatic stellate cell apoptosis based on network pharmacology and experimental verification.

Background: Hepatic fibrosis is caused by various liver diseases and eventually develops into liver cancer. There is no specific drug approved for the treatment of hepatic fibrosis in the world. Acacetin (AC), a natural flavonoid, is widely present in nature in various plants, such as black locust, Damiana, Silver birch. It has been reported that acacetin can inhibit the proliferation of cancer cells and induce apoptosis. Purpose: In this study, we investigated the effect of acacetin on hepatic stellate cell apoptosis, thereby improving hepatic fibrosis, and combined experimental validation and molecular docking to reveal the underlying mechanism. Result: First, we discovered that acacetin inhibited hepatic stellate cell proliferation as well as the expression of fibrosis-related proteins α-smooth muscle actin (α-SMA) and collagen type I 1 gene (COL1A1) in LX2 cells. Acacetin was then found to promote apoptosis of hepatic stellate cells through the caspase cascade pathway. Network pharmacology screening showed that TP53, CASP3, CASP8, BCL2, PARP1, and BAX were the most important targets related to apoptosis in the PPI network. GO and KEGG analyses of these six important targets were performed, and the top 10 enriched biological processes and related signaling pathways were revealed. Further network pharmacology analysis proved that apoptosis was involved in the biological process of acacetin's action against hepatic stellate cells. Finally, molecular docking revealed that acacetin binds to the active sites of six apoptotic targets. In vitro experiments further confirmed that acacetin could promote the apoptosis of LX2 cells by inducing the activation of P53, thereby improving hepatic fibrosis. Conclusion: acacetin induces P53 activation and promotes apoptosis of hepatic stellate cells thereby ameliorating hepatic fibrosis.

Acacetin inhibits inflammation by blocking MAPK/NF-κB pathways and NLRP3 inflammasome activation.

Objective: Our preliminary research indicates that acacetin modulates the nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) inflammasome, providing protection against Alzheimer's Disease (AD) and cerebral ischemic reperfusion injury. The mechanisms of acacetin to inhibit the activation of the NLRP3 inflammasome remain fully elucidated. This study aims to investigate the effects and potential mechanisms of acacetin on various agonists induced NLRP3 inflammasome activation. Methods: A model for the NLRP3 inflammasome activation was established in mouse bone marrow-derived macrophages (BMDMs) using Monosodium Urate (MSU), Nigericin, Adenosine Triphosphate (ATP), and Pam3CSK4, separately. Western blot analysis (WB) was employed to detect Pro-caspase-1, Pro-Interleukin-1ß (Pro-IL-1ß) in cell lysates, and caspase-1, IL-1ß in supernatants. Enzyme-Linked Immunosorbent Assay (ELISA) was used to measured the release of IL-1ß, IL-18, and Tumor Necrosis Factor-alpha (TNF-α) in cell supernatants to assess the impact of acacetin on NLRP3 inflammasome activation. The lactate dehydrogenase (LDH) release was also assessed. The Nuclear Factor Kappa B (NF-κB) and Mitogen-Activated Protein Kinase (MAPK) signaling pathways related proteins were evaluated by WB, and NF-κB nuclear translocation was observed via laser scanning confocal microscopy (LSCM). Disuccinimidyl Suberate (DSS) cross-linking was employed to detect oligomerization of Apoptosis-associated Speck-like protein containing a Caspase Recruitment Domain (ASC), and LSCM was also used to observe Reactive Oxygen Species (ROS) production. Inductively Coupled Plasma (ICP) and N-(6-methoxyquinolyl) acetoethyl ester (MQAE) assays were utilized to determined the effects of acacetin on the efflux of potassium (K+) and chloride (Cl-) ions. Results: Acacetin inhibited NLRP3 inflammasome activation induced by various agonists, reducing the release of TNF-α, IL-1ß, IL-18, and LDH. It suppressed the expression of Lipopolysaccharides (LPS)-activated Phosphorylated ERK (p-ERK), p-JNK, and p-p38, inhibited NF-κB p65 phosphorylation and nuclear translocation. Acacetin also reduced ROS production and inhibited ASC aggregation, thus suppressing NLRP3 inflammasome activation. Notably, acacetin did not affect K+ and Cl-ions efflux during the activation process. Conclusion: Acacetin shows inhibitory effects on both the priming and assembly processes of the NLRP3 inflammasome, positioning it as a promising new candidate for the treatment of NLRP3 inflammasome-related diseases.

Development of simultaneous quantitative analytical method for three active components of Korean mint (Agastache rugosa (Fisch. & C.A.Mey.) Kuntze) extract in human plasma using ultra-high-performance liquid chromatography-tandem mass spectrometry.

Agastache rugosa contains phenolic compounds and flavonoids, and has been extensively used as a traditional herbal medicine. The major components in Agastache rugosa extract (ARE) are rosmarinic acid, tilianin, and acacetin, for which several analytical techniques have been reported. However, these substances have yet to be simultaneously quantified in human plasma. In this study, we aimed to simultaneously determine the three active components of ARE in human plasma by developing a reliable quantitative analytical method using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Chromatographic separation of the plasma samples was achieved using an ACQUITY UPLC® BEH C18 column with a gradient mobile phase of water and acetonitrile containing 0.1 % formic acid. Mass spectrometric detection was performed using a triple quadrupole tandem mass spectrometer in negative electrospray ionization (ESI-) and multiple reaction monitoring (MRM) modes. The developed quantitative method was validated for the three active components. All three analytes exhibited a linear response over the ranges of 0.5-50 ng/mL for rosmarinic acid, 0.1-20 ng/mL for acacetin, and 0.5-20 ng/mL for tilianin with a weighting factor of 1/x (where x is the concentration). At three quality control (QC) concentration levels (low, medium, and high), including the lower limit of quantitation (LLOQ), acceptable accuracy (±15 %) was achieved in the intra- and interday validations. The concentration of rosmarinic acid was highest in plasma. Tilianin and acacetin appeared and were eliminated earlier in the plasma than rosmarinic acid. This study provides a successfully validated method that can be used in further clinical applications of Agastache rugosa extracts.

Acacetin protects against sepsis-induced acute lung injury by facilitating M2 macrophage polarization via TRAF6/NF-κB/COX2 axis.

Acute lung injury (ALI) is the leading cause of death in patients with sepsis syndrome and without effective protective or therapeutic treatments. Acacetin, a natural dietary flavonoid, reportedly exerts several biological effects, such as anti-tumor, anti-inflammatory, and anti-oxidative effects. However, acacetin's effect and underlying mechanism on sepsis-induced ALI remain unclear. Here, the mouse model was established to explore the impact of acacetin on sepsis-induced ALI. Acacetin significantly increased ALI murine survival and attenuated lung injury in histological examinations. Additionally, acacetin down-regulated myeloperoxidase activity, protein concentration, and number of neutrophils and macrophages in bronchoalveolar lavage fluid. Subsequently, inflammatory cytokines, including TNF-α, IL-1ß, and IL-6, were examined. Results showed that acacetin dramatically suppressed the production of TNF-α, IL-1ß, and IL-6. These above results indicated that acacetin attenuated sepsis-induced ALI by inhibiting the inflammatory response. Moreover, acacetin inhibited the expression of markers for M1-type (iNOS, CD86) macrophages and promoted the expression of markers for M2-type (CD206, Arg1) macrophages by western blot. In addition, acacetin down-regulated the expression TRAF6, NF-κB, and Cyclooxygenase-2 (COX2) by western blot. The high concentration of acacetin had a better effect than the low concentration. Besides, over-expression of TRAF6 up-regulated the expression of COX2, CD86, and iNOS, and the ratio of p-NF-κB to NF-κB increased the mRNA levels of TNF-α, IL-1ß, and IL-6, down-regulated the expression of CD206 and Arg1. The effects of TRAF6 were the opposite of acacetin. And TRAF6 could offset the impact of acacetin. This study demonstrated that acacetin could prevent sepsis-induced ALI by facilitating M2 macrophage polarization via TRAF6/NF-κB/COX2 axis.

[Acacetin protects rats from cerebral ischemia-reperfusion injury by regulating TLR4/NLRP3 signaling pathway].

This study aims to investigate the mechanism of acacetin in protecting rats from cerebral ischemia-reperfusion injury via the Toll-like receptor 4(TLR4)/NOD-like receptor protein 3(NLRP3) signaling pathway. Wistar rats were randomized into sham, model, low-and high-dose acacetin, and nimodipine groups, with 10 rats in each group. The rat model of middle cerebral artery occlusion(MCAO) was established with the improved suture method in other groups except the sham group. The neurological deficit score and cerebral infarction volume of each group were evaluated 24 h after modeling. Enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of interleukin-1ß(IL-1ß), IL-6, tumor necrosis factor-α(TNF-α), malondialdehyde(MDA), supe-roxide dismutase(SOD), and glutathione(GSH). Western blot was employed to determine the expression levels of B-cell lymphonoma-2(Bcl-2), Bcl-2-associated X protein(Bax), and TLR4/NLRP3 signaling pathway-related proteins(TLR4, p-NF-κB/NF-κB, NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1ß, and cleaved IL-1ß) in the rat brain tissue. Hematoxylin-eosin(HE) staining was employed to reveal the histopathological changes in the ischemic area. Compared with the sham group, the modeling of MCAO increased the neurological deficit score and cerebral infarction volume, elevated the IL-1ß, IL-6, TNF-α, and MDA levels and lowered the SOD and GSH levels in the brain tissue(P<0.05). Compared with the MCAO model group, low-and high-dose acacetin and nimodipine decreased the neurological deficit score and cerebral infarction volume, lowered the IL-1ß, IL-6, TNF-α, and MDA levels and elevated the SOD and GSH levels in the brain tissue(P<0.05). Compared with the sham group, the model group showed up-regulated protein levels of Bax, TLR4, p-NF-κB/NF-κB, NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1ß, and cleaved IL-1ß and down-regulated protein level of Bcl-2 in the brain tissue(P<0.05). Compared with the MCAO model group, the acacetin and nimodipine groups showed down-regulated protein levels of Bax, TLR4, p-NF-κB/NF-κB, NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1ß, and cleaved IL-1ß and up-regulated protein level of Bcl-2 in the brain tissue(P<0.05). In conclusion, acacetin regulates the TLR4/NLRP3 signaling pathway to inhibit neuroinflammatory response and oxidative stress, thus exerting the protective effect on cerebral ischemia-reperfusion injury in rats.

Effectiveness of silver nitrate application on plant growth and bioactive compounds in Agastache rugosa (Fisch. & C.A.Mey.) kuntze.

The objective of this study was to determine the optimal dose of silver nitrate (AgNO3) for plant growth and to increase the main bioactive compounds in A. rugosa cultivated in a hydroponic system. The application of soaked diniconazole (120 µmol mol-1) to all plants at 7 days after transplanting (DAT) for dwarfing plant height, optimizing cultivation space in the plant factory. Subsequently, plants were soaked with 50, 100, 200, and 400 µmol mol-1 AgNO3 for 10 min at 25 DAT and harvested at 39 DAT. The results indicated that 200 and 400 µmol mol-1 treatments tended to severely decrease plant growth parameters compared to treatments with lower concentrations. The net photosynthetic rate was significantly reduced by the 200 and 400 µmol mol-1 treatments compared to treatments with other concentrations. The 400 µmol mol-1 treatment led to the lowest concentrations of chlorophyll a, chlorophyll a/b, total carotenoid, chlorophyll b, and the total chlorophyll. However, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was considerably increased in 50, 100, 200, and 400 µmol mol-1 compared to that of the control plants. A higher rosmarinic acid (RA) concentration in the whole plant was noticed with the 400 µmol mol-1 treatment compared with that of the untreated plants. The 100 µmol mol-1 treatment exhibited the highest concentration and content of tilianin in the whole plant. Concentration of acacetin 1 significantly increased in the whole plant with 100 and 200 µmol mol-1 treatments compared with that of the untreated plants. Concentrations of acacetin 2 and 3 in the whole plant were the highest with 100 and 200 µmol mol-1 treatments, respectively. The results demonstrated that 100 µmol mol-1 treatments can be used to increase bioactive compounds without severely limiting the plant growth and reducing chlorophyll concentrations of A. rugosa. Implementing this optimal dose can enable growers and researchers to cultivate A. rugosa more efficiently, enhancing bioactive compound content and overall plant performance, thus harnessing the potential health benefits of this valuable plant species.