Formononetin attenuates psoriasiform inflammation by regulating interferon signaling pathway.

BACKGROUND: Psoriasis is a long-lasting, inflammatory, continuous illness caused through T cells and characterized mainly by abnormal growth and division of keratinocytes. Currently, corticosteroids are the preferred option. However, prolonged use of traditional topical medication can lead to adverse reactions and relapse, presenting a significant therapeutic obstacle. Improved alternative treatment options are urgently required. Formononetin (FMN) is a representative component of isoflavones in Huangqi (HQ) [Astragalus membranaceus (Fisch.) Bge.]. It possesses properties that reduce inflammation, combat oxidation, inhibit tumor growth, and mimic estrogen. Although FMN has been shown to ameliorate skin barrier devastation via regulating keratinocyte apoptosis and proliferation, there are no reports of its effectiveness in treating psoriasis. OBJECTIVE: Through transcriptomics clues and experimental investigation, we aimed to elucidate the fundamental mechanisms underlying FMN's action on psoriasis. MATERIALS AND METHODS: Cell viability was examined using CCK8 assay in this study. The results of analysis of differentially expressed genes (DEGs) between FMN-treated HaCaT cells and normal HaCaT cells using RNA-sequencing (RNA-seq) were presented on volcano plots and heatmap. Enrichment analysis was conducted on DEGs using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO), and results were validated through RT-qPCR verification. After 12 days of FMN treatment in psoriasis mouse model, we gauged the PASI score and epidermis thickness. A variety of techniques were used to assess FMN's effectiveness on inhibiting inflammation and proliferation related to psoriasis, including RT-qPCR, HE staining, western blot, and immunohistochemistry (IHC). RESULTS: The findings indicated that FMN could suppress the growth of HaCaT cells using CCK8 assay (with IC50 = 40.64 uM) and 20 uM FMN could reduce the level of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) to the greatest extent. FMN-treated HaCaT cells exhibited 985 up-regulated and 855 down-regulated DEGs compared to normal HaCaT cells. GO analysis revealed that DEGs were linked to interferon (IFN) signaling pathway. Furthermore, FMN improved pathological features, which encompassed decreased erythema, scale, and thickness scores of skin lesions in psoriasis mouse model. In vivo experiments confirmed that FMN down-regulated expression of IFN-α, IFN-ß, IFN-γ, decreased secretion of TNF-α and IL-17 inflammatory factors, inhibited expression of IFN-related chemokines included Cxcl9, Cxcl10, Cxcl11 and Cxcr3 and reduced expression of transcription factors p-STAT1, p-STAT3 and IFN regulatory factor 1 (IRF1) in the imiquimod (IMQ) group. CONCLUSIONS: In summary, these results suggested that FMN played an anti-inflammatory and anti-proliferative role in alleviating psoriasis by inhibiting IFN signaling pathway, and FMN could be used as a potential therapeutic agent.

Unraveling the anti-breast cancer activity of Cimicifugae rhizoma using biological network pathways and molecular dynamics simulation.

Cimicifugae is a commonly used treatment for breast cancer, but the specific molecular mechanisms underlying its effectiveness remain unclear. In this research, we employ a combination of network pharmacology, molecular docking, and molecular dynamics simulations to uncover the most potent phytochemical within Cimicifugae rhizoma in order to delve into its interaction with the target protein in breast cancer treatment. We identified 18 active compounds and 89 associated targets, primarily associated to various biological processes such as lipid metabolism, the signaling pathway in diabetes, viral infections, and cancer-related pathways. Molecular docking analysis revealed that the two most active compounds, Formononetin and Cimigenol, exhibit strong binding to the target protein AKT1. Through molecular dynamics simulations, we found that the Cimigenol-AKT1 complex exhibits greater structural stability and lower interaction energy compared to the stigmasterol-AKT1 complex. Our study demonstrates that Cimicifugae rhizoma exerts its effects in breast cancer treatment through a multi-component, multi-target synergistic approach. Furthermore, we propose that Cimigenol, targeting AKT-1, represents the most effective compound, offering valuable insights into the molecular mechanisms underpinning its role in breast cancer therapy.

Formononetin protects against Aspergillus fumigatus Keratitis: Targeting inflammation and fungal load.

PURPOSE: To investigate the potential treatment of formononetin (FMN) on Aspergillus fumigatus (A. fumigatus) keratitis with anti-inflammatory and antifungal activity. METHODS: The effects of FMN on mice with A. fumigatus keratitis were evaluated through keratitis clinical scores, hematoxylin-eosin (HE) staining, and plate counts. The expression of pro-inflammatory factors was measured using RT-PCR, ELISA, or Western blot. The distribution of macrophages and neutrophils was explored by immunofluorescence staining. The antifungal properties of FMN were assessed through minimum inhibitory concentration (MIC), propidium iodide (PI) staining, fungal spore adhesion, and biofilm formation assay. RESULTS: In A. fumigatus keratitis mice, FMN decreased the keratitis clinical scores, macrophages and neutrophils migration, and the expression of TNF-α, IL-6, and IL-1ß. In A. fumigatus-stimulated human corneal epithelial cells (HCECs), FMN reduced the expression of IL-6, TNF-α, IL-1ß, and NLRP3. FMN also decreased the expression of thymic stromal lymphopoietin (TSLP) and thymic stromal lymphopoietin receptor (TSLPR). Moreover, FMN reduced the levels of reactive oxygen species (ROS) induced by A. fumigatus in HCECs. Furthermore, FMN inhibited A. fumigatus growth, prevented spore adhesion and disrupted fungal biofilm formation in vitro. In vivo, FMN treatment reduced the fungal load in mice cornea at 3 days post infection (p.i.). CONCLUSION: FMN demonstrated anti-inflammatory and antifungal properties, and exhibited a protective effect on mouse A. fumigatus keratitis.

Formononetin and Dihydroartemisinin Act Synergistically to Induce Apoptosis in Human Acute Myeloid Leukemia Cell Lines.

OBJECTIVE: Enhanced cell survival and drug resistance in tumor cells have been linked to the overexpression of antiapoptotic members of the Bcl-2 family proteins, including Bcl-2 and Mcl-1. The aim of this study was to explore the impact of formononetin and dihydroartemisinin combination on the growth and apoptosis of acute myeloid leukemia (AML) cells. MATERIALS AND METHODS: In this experimental study, the cell survival and cell proliferation were tested by MTT assay and trypan blue staining. The evaluation of cell apoptosis was conducted using Hoechst 33342 staining and a colorimetric assay to measure caspase-3 activity. To determine the mRNA levels of Mcl-1, Bcl-2, Bax, and Cyclin D1, a quantitative real-time polymerase chain reaction (qRT-PCR) was performed. RESULTS: We showed that treatment with either formononetin or dihydroartemisinin alone, led to significant decrease in the cell survival and growth, and triggered apoptosis in U937 and KG-1 AML cell lines. Moreover, treatment with each of the compounds alone significantly decreased the mRNA levels of Mcl-1, Bcl-2 and Cyclin D1 mRNA, while, the expression level of Bax mRNA was enhanced. Combination of two compounds showed a synergistic anti-cancer effect. CONCLUSION: The anti-leukemic potential of formononetin and dihydroartemisinin is exerted through the effect on cell cycle progression and intrinsic pathway of apoptosis. Therefore, they can be considered as a potential anti-leukemic agent alone or along with existing chemotherapeutic drugs.

Formononetin Restrains Tumorigenesis of Breast Tumor by Restraining STING-NF-κB and Interfering with the Activation of PD-L1.

BACKGROUND: Breast cancer (BC), a common tumor in women, has high morbidity and mortality. Formononetin, an active ingredient in red clover and Astragalus membranaceus, has a wide range of pharmacological applications, including as an anticancer agent. Since immunotherapy is a hot topic in the treatment strategy of BC, it was dedicated to appraising the specific mechanism of formononetin in BC immunotherapy in this research. METHODS: Different formononetin concentrations (0, 20, 40, 60, 80, 100 µM) were used to treat BC cells transfected with pcDNA3.1-Programmed death ligand 1 (PD-L1) or Short-hairpin RNA (sh)-PD-L1. Cells were separated into four subgroups: CTRL, pcDNA3.1-PD-L1, sh-CTRL, and sh-PD-L1. Cell viability and cell cycle were assessed through Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and flow cytometry. Programmed death ligand 1 (PD-L1) mRNA concentration was validated via quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Cell metastasis was evaluated via cloning assay and transwell assay. The p-STING/stimulator of interferon genes (STING), p-p65/p65, and PD-L1 concentrations were determined by western blot. RESULTS: Formononetin restrained the proliferation of MCF-7 and MDA-MB-468 cells, and reduced PD-L1 mRNA, p-STING/STING, and p-p65/p65 protein concentrations. Whereas PD-L1 inhibition restrained the viability of BC cells, pcDNA3.1-PD-L1 intervention had the opposite result. STING pathway inhibitor C-176 combined with formononetin treatment further restrained cell proliferation, colony formation, and cell invasion, in contrast to cells treated with formononetin alone. CONCLUSION: Formononetin can restrain the proliferation of BC cells, which may be mediated through the interference of PD-L1 and suppression of the activation of the STING-NF-κB pathway.

Formononetin alleviates cerebral ischemia-reperfusion injury in rats by targeting the PARP-1/PARG/Iduna signaling pathway.

Formononetin has been demonstrated to protect against cerebral ischemia-reperfusion injury, however its mechanism has to be further researched. This study examined the effect of formononetin on cerebral ischemia-reperfusion injury in rats using the PARP-1/PARG/Iduna signaling pathway. In male SD rats, a model of cerebral ischemia-reperfusion injury was developed. Animals were randomly assigned to one of eight groups: Sham operation, Sham operation + formononetin, MCAO, MCAO + formononetin, PARP inhibitor (PJ34) + MCAO, formononetin + PJ34 + MCAO, PARG inhibitor (Ethacridine lactate) + MCAO, and ethacridine lactate + formononetin. The neurological deficit test, TTC staining, HE staining, Nissl staining, TUNEL staining, and western blotting were utilized to assess formononetin's protective effects in MCAO rats. The data show that formononetin can effectively alleviate neurological dysfunction and pathological changes in brain tissue in rats with cerebral ischemia-reperfusion injury, reduce the area of cerebral infarction and neuronal apoptosis, decrease the protein levels of PARP-1, PARG, Caspase-3, P53, and AIF in brain tissue, and increase the protein levels of Iduna and p-AKT. As a result, we concluded that formononetin improves brain ischemia-reperfusion injury in rats by modulating the PARP-1/PARG/Iduna signaling pathway.

Formononetin reverses Treg/Th17 imbalance in immune-mediated bone marrow failure mice by regulating the PI3K/Akt signaling pathway.

BACKGROUND: Severe aplastic anemia (SAA) is a syndrome of bone marrow failure which is life-threatening. Recent studies have demonstrated that CD4 + T cell subsets, including T regulatory (Treg) and T helper 17 (Th17) cells, play a pivotal role in the pathogenesis of SAA. Formononetin (FMN) is a natural compound extracted from the traditional Chinese medicine Huangqi, which has the ability to regulate the imbalance of Treg/Th17 cells in some inflammatory diseases. Nevertheless, the therapeutic effect of FMN in SAA has yet to be definitively established. Therefore, the objective of this research was to investigate the effect of FMN on SAA and elucidate its underlying mechanism. METHODS: In vivo experiments, the mice were divided into the following five groups: control, model, low-dose FMN, high-dose FMN, and positive control cyclosporine A group. The immune-mediated bone marrow failure (BMF) mouse model was established by the total body X-ray radiation and lymphocyte infusion. After 10 days of continuous administration of FMN, the numbers of Treg/Th17 cells in the bone marrow and spleen were assessed by flow cytometry. The protein expressions of PI3K/Akt pathway in the bone marrow and spleen was assessed by immunohistochemistry and western blotting. In vitro, the impact of FMN on the differentiation of naive CD4 + T cells into Treg cells was investigated by flow cytometry and ELISA. RESULTS: In comparison with the control group, the model group showed a reduction in bone marrow nucleated cells, a significant decrease in peripheral blood cells, and an altered CD8 + /CD4 + T cell ratio. These findings indicate the successful establishment of a mouse model of immune-mediated BMF. After FMN treatment, there were the increased levels of red blood cells and hemoglobin. In addition, FMN mitigated the bone marrow destruction and restored the CD8 + /CD4 + T cell ratio. Furthermore, in comparison with the control group, the model group showed the decreased levels of Treg cells and the increased levels of Th17 cells. After FMN treatment, there was a significantly increased number of Treg cells and a decreased number of Th17 cells. Additionally, FMN remarkably down-regulated the expression levels of PI3K and Akt proteins in immune-mediated BMF mice. CONCLUSIONS: FMN alleviates immune-mediated BMF by modulating the balance of Treg/Th17 cells through the PI3K/Akt signaling pathway.

Identification of Dalbergiae Odoriferae Lignum active ingredients and potential mechanisms in the treatment of adriamycin-induced cardiotoxicity based on network pharmacology and experimental verification.

The purpose of this study is to investigate the ingredients and mechanisms through which Dalbergiae Odoriferae Lignum (DOL) reduces adriamycin-induced cardiotoxicity. DOL's ingredients and drug targets were acquired from Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP), and adriamycin-induced cardiotoxicity disease targets were gathered from GeneCards and National Center for Biotechnology Information (NCBI). The therapeutic targets of DOL against adriamycin-induced cardiotoxicity were identified by intersecting drug and disease targets. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) were conducted using R. Subsequently, core targets were determined and used for molecular docking with DOL ingredients. In vitro and in vivo experiments validated DOL's primary ingredients against adriamycin-induced cardiotoxicity efficacy. Western blot and immunohistochemistry verified its impact on target protein. After intersecting 530 drug targets and 51 disease targets, 19 therapeutic targets for DOL alleviated adriamycin-induced cardiotoxicity were received. Molecular docking demonstrated that DOL primary ingredient formononetin had a robust binding affinity for nitric oxide synthase 3 (NOS3). Experimental results showed that formononetin effectively mitigated adriamycin-induced cardiotoxicity. Additionally, western blot and immunohistochemistry showed that formononetin improved NOS3 expression. The network pharmacology and experimentation suggest that the primary ingredient of DOL, formononetin, may target NOS3 to act as a therapeutic agent for adriamycin-induced cardiotoxicity.

Elucidating the mechanisms of formononetin in modulating atherosclerotic plaque formation in ApoE-/- mice.

BACKGROUND: Atherosclerosis(AS) poses a pressing challenge in contemporary medicine. Formononetin (FMN) plays a crucial role in its prevention and treatment. However, the detailed impact of FMN on the stability of atherosclerotic plaques and its underlying mechanisms remain to be elucidated. METHODS: An intervention consisting of FMN was given along with a high-fat food regimen in the ApoE-/- mouse model. The investigation included the evaluation of the degree of atherosclerotic lesion, the main components of the plaque, lipid profiles, particular markers indicating M1/M2 macrophage phenotypes, the quantities of factors related to inflammation, the infiltration of macrophages, and the identification of markers linked to the α7nAChR/JAK2/STAT3 axis effect molecules. RESULTS: The evaluation of aortic morphology in ApoE-/-mice revealed that FMN significantly improved the plaque area, fibrous cap protrusion, lipid deposition, and structural alterations on the aortic surface, among other markers of atherosclerosis,and there is concentration dependence. Furthermore, the lipid content of mouse serum was assessed, and the results showed that the low-, medium-, and high-dosage FMN groups had significantly lower levels of LDL-C, ox-LDL, TC, and TG. The results of immunohistochemical staining indicated that the low-, medium-, and high-dose FMN therapy groups had enhanced CD206 expression and decreased expression of CD68 and iNOS. According to RT-qPCR data, FMN intervention has the potential to suppress the expression of iNOS, COX-2, miR-155-5p, IL-6, and IL-1ß mRNA, while promoting the expression of IL-10, SHIP1, and Arg-1 mRNA levels. However, the degree of inhibition varied among dosage groups. Western blot investigation of JAK/STAT signaling pathway proteins and cholinergic α7nAChR protein showed that p-JAK2 and p-STAT3 protein expression was suppressed at all dosages, whereas α7nAChR protein expression was enhanced. CONCLUSIONS: According to the aforementioned findings, FMN can reduce inflammation and atherosclerosis by influencing macrophage polarization, blocking the JAK/STAT signaling pathway, and increasing α7nAChR expression.

Combination of Formononetin and Sulforaphane Natural Drug Repress the Proliferation of Cervical Cancer Cells via Impeding PI3K/AKT/mTOR Pathway.

Natural substances have been demonstrated to be an unrivalled source of anticancer drugs in the present era of pharmacological development. Plant-based substances, together with their derivatives through analogues, play a significant character in the treatment of cancer by altering the tumor microenvironment and several signaling pathways. In this study, it was investigated whether the natural drugs, formononetin (FN) and sulforaphane (SFN), when combined, assess the efficacy of inhibiting cervical cancer cell proliferation by impeding the PI3K/Akt/mTOR signaling pathway in HeLa cells. The cells were treated with the combination of FN and SFN (FN + SFN) in various concentrations (0-50 µM) for 24 h and then analyzed for various experiments. The combination of FN + SFN-mediated cytotoxicity was analyzed by MTT assay. DCFH-DA staining was used to assess the ROS measurement, and apoptotic changes were studied by dual (AO/EtBr) staining assays. Protein expressions of cell survival, cell cycle, proliferation, and apoptosis protein were evaluated by flow cytometry and western blotting. Results showed that the cytotoxicity of FN and SFN was determined to be around 23.7 µM and 26.92 µM, respectively. Combining FN and SFN causes considerable cytotoxicity in HeLa cells, with an IC50 of 21.6 µM after 24-h incubation. Additionally, HeLa cells treated with FN and SFN together showed increased apoptotic signals and considerable ROS generation. Consequently, by preventing the production of PI3K, AKT, and mToR-mediated regulation of proliferation and cell cycle-regulating proteins, the combined use of FN + SFN has been regarded as a chemotherapeutic medication. Further research will need to be done shortly to determine how effectively the co-treatment promotes apoptosis to employ them economically.

Formononetin attenuates cigarette smoke-induced COPD in mice by suppressing inflammation, endoplasmic reticulum stress, and apoptosis in bronchial epithelial cells via AhR/CYP1A1 and AKT/mTOR signaling pathways.

Chronic obstructive pulmonary disease (COPD) is a chronic, progressive, and lethal lung disease with few treatments. Formononetin (FMN) is a clinical preparation extract with extensive pharmacological actions. However, its effect on COPD remains unknown. This study aimed to explore the effect and underlying mechanisms of FMN on COPD. A mouse model of COPD was established by exposure to cigarette smoke (CS) for 24 weeks. In addition, bronchial epithelial BEAS-2B cells were treated with CS extract (CSE) for 24 h to explore the in vitro effect of FMN. FMN significantly improved lung function and attenuated pathological lung damage. FMN treatment reduced inflammatory cell infiltration and pro-inflammatory cytokines secretion. FMN also suppressed apoptosis by regulating apoptosis-associated proteins. Moreover, FMN relieved CS-induced endoplasmic reticulum (ER) stress in the mouse lungs. In BEAS-2B cells, FMN treatment reduced CSE-induced inflammation, ER stress, and apoptosis. Mechanistically, FMN downregulated the CS-activated AhR/CYP1A1 and AKT/mTOR signaling pathways in vivo and in vitro. FMN can attenuate CS-induced COPD in mice by suppressing inflammation, ER stress, and apoptosis in bronchial epithelial cells via the inhibition of AhR/CYP1A1 and AKT/mTOR signaling pathways, suggesting a new therapeutic potential for COPD treatment.

Mechanisms analysis for Formononetin counteracted-Osimertinib resistance in non-small cell lung cancer cells: From the insight into the gene transcriptional level.

Formononetin is one of the main isoflavone components, which has strong anti-cancer effects in non-small cell lung cancer (NSCLC). However, the potentials and the mechanisms of Formononetin to counteract the Osimertinib resistance in NSCLC are unclear. In this study, Formononetin-induced cell apoptosis, cell proliferation, and clonal formation were detected in Osimertinib-resistant NSCLC cells (H1975_OR). RNA sequencing analysis was conducted to study the gene expression profiles of Formononetin-induced H1975_OR cells. The results indicated that Formononetin could significantly induce cell apoptosis, whereas dramatically inhibited cell proliferation and clonal formation on H1975_OR cells. Furthermore, a total of 4309 differentially expressed genes (DEGs) between Formononetin-treated and nontreated H1975_OR cells were had been detected. Gene Ontology (GO) annotation enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and the Gene Set Enrichment Analysis (GSEA) showed that Formononetin affected the expression of genes involving in anatomical structure morphogenesis, anatomical structure development, and multicellular organism development via regulating inflammation- and metabolism-related signaling pathways. Taken together, our study preliminarily revealed the mechanisms of Formononetin to counteract the Osimertinib resistance in NSCLC cells from the transcriptional level and provided a potential treatment method for Osimertinib-resistant NSCLC patients.

Formononetin ameliorates cisplatin-induced hair cell death via activation of the PI3K/AKT-Nrf2 signaling pathway.

Cisplatin (CDDP) stands as a highly effective chemotherapeutic agent; however, its ototoxicity remains a perplexing challenge in the field. Formononetin (FMNT), a potent flavonoid isolated from Astragalus membranaceus, displays a diverse range of promising pharmacological activities, encompassing antioxidant, anti-apoptotic, and anti-inflammatory effects. Nonetheless, the advantageous effects of FMNT on cisplatin-induced cochlear hair cell injury demand further investigation. This study aimed to assess the protective properties of FMNT against cisplatin-induced hair cell damage by conducting in vitro assays on explant-cultured cochlear hair cells. The findings revealed that FMNT exhibited a notable reduction in cisplatin-induced hair cell apoptosis. Also, FMNT effectively mitigated the accumulation of reactive oxygen species and mitochondrial damage in cochlear explants exposed to cisplatin, while also restoring the turnover of the reduced glutathione (GSH)/glutathione disulfide (GSSG) ratio. Furthermore, our study demonstrated that FMNT protects hair cells against CDDP injury through the activation of the PI3K/AKT-Nrf2 signaling pathway. Consequently, formononetin emerges as a potential therapeutic agent for the treatment of cisplatin-induced ototoxicity.

Formononetin promotes fatty acid ß-oxidation to treat non-alcoholic steatohepatitis through SIRT1/PGC-1α/PPARα pathway.

BACKGROUND: Non-alcoholic steatohepatitis (NASH), the progressive form of non-alcoholic fatty liver disease (NAFLD), carries a high risk of cirrhosis and hepatocellular carcinoma. With the increasing incidence of NASH, the accompanying medical burden is also increasing rapidly, so the development of safe and reliable drugs is urgent. Formononetin (FMNT) has a variety of pharmacological effects such as antioxidant and anti-inflammation, and plays a major role in regulating lipid metabolism, reducing hepatic steatosis and so on, but the mechanism for alleviating NASH is unclear. MATERIALS AND METHODS: We firstly established a mouse model on NASH through methionine-choline deficient (MCD) diet to investigate the improvement of FMNT as well as the effects of fatty acid ß oxidation and SIRT1/PGC-1α/PPARα pathway. Then, we explored the mechanisms of FMNT regulation in SIRT1/PGC-1α/PPARα pathway and fatty acid ß oxidation based on genes silencing of SIRT1 and PGC1A. In addition, SIRT1 agonist (SRT1720) and inhibitor (EX527) were used to verify the mechanism of FMNT on improvement of NASH. RESULTS: Our study found that after FMNT intervention, activities of ALT and AST and TG level were improved, and liver function and hepatocellular steatosis on NASH mice were significantly improved. The detection of ß oxidation related indicators showed that FMNT intervention up-regulated FAO capacity, level of carnitine, and the levels of ACADM and CPT1A. The detection of factors related to the SIRT1/PGC-1α/PPARα pathway showed that FMNT activated and promoted the expression of SIRT1/PGC-1α/PPARα pathway, including up-regulating the expression level of SIRT1, improving the activity of SIRT1, promoting the deacetylation of PGC-1α, and promoting the transcriptional activity of PPARα. Furthermore, after genes silencing of SIRT1 and PGC1A, we found that FMNT intervention could not alleviate NASH, including improvement of hepatocellular steatosis, enhancement of ß oxidation, and regulation of SIRT1/PGC-1α/PPARα pathway. Afterwards, we used SRT1720 as a positive control, and the results indicated that FMNT and SRT1720 intervention had no significant difference on improving hepatocellular steatosis and promoting fatty acid ß oxidation. Besides, we found that when EX527 intervention inhibited expression of SIRT1, the improvement of FMNT on NASH was weakened or even disappeared. CONCLUSION: In summary, our results demonstrated that FMNT intervention activated SIRT1/PGC-1α/PPARα pathway to promote fatty acid ß oxidation and regulate lipid metabolism in liver, ultimately improved hepatocellular steatosis on NASH mice.

The effect of different LED wavelengths on the components and biosynthesis of isoflavonoid in sprout Astragalus membranaceus.

An artificial light source is the optimal element for studying the usability of the medicinal plant Astragalus membranaceus as a sprout vegetable. Based on artificial light source conditions, formononetin (FO) level was the highest (2.6 mg/L) in A. membranaceus exposed to white light emitting diode (LED) light, and calycosin (CA) level was the highest (3.09 mg/L) in the plant exposed to red LED light. According to the publicly available transcriptome data of LED-exposed sprout A. membranaceus LED, reference genes related to the content enhancement of FO, an isoflavone compound, and those related to the content enhancement of CA were selected. The expression patterns of these genes were assayed using qPCR. Among the genes related to FO enhancement, Gene-225190T showed the highest mRNA levels in cells of LED-white light-exposed sprout A. membranaceus; among the genes related to CA enhancement, Gene_042770T showed the highest expression under red LED light. Most genes related to the overall biosynthesis regulation of flavonoids of the upper concept of isoflavone were highly expressed in response to red LED light, and the transcriptional level of 4CL in response to red LED light was the highest. Based on these results, the artificial light sources that regulated the FO and CA contents in sprouts A. membranaceus were white and red LED lights, and the selected reference genes were capable of regulating isoflavone biosynthesis.

Formononetin ameliorates isoproterenol induced cardiac fibrosis through improving mitochondrial dysfunction.

Formononetin, an isoflavone compound, has been extensively researched due to its various biological activities, including a potent protective effect on the cardiovascular system. However, the impact of formononetin on cardiac fibrosis has not been investigated. In this study, C57BL/6 mice were used to establish cardiac fibrosis animal models by subcutaneous injecting of isoproterenol (ISO) and formononetin was orally administrated. The results showed that formononetin reversed ISO-induced heart stiffness revealed by early-to-atrial wave ratio (E/A ratio). Masson staining, western blot, immunohistochemistry and real-time PCR exhibited that the cardiac fibrosis and fibrosis-related proteins (collage III, fibronectin, TGF-ß1, α-SMA, and vimentin) and genes (Col1a1, Col3a1, Acta2 and Tgfb1) induced by ISO were significantly suppressed by formononetin. Furthermore, by combining metabolomics and network pharmacology, we found three important targets (ALDH2, HADH, and MAOB), which are associated with mitochondrial function, were involved in the beneficial effect of formononetin. Further validation revealed that these three genes were more abundance in cardiomyocyte than in cardiac fibroblast. The mRNA expression of ALDH2 and HADH were decreased, while MOAB was increased in cardiomyocyte upon ISO treatment and these phenomena were reversed by formononetin. In addition, we investigated mitochondrial membrane potential and ROS production in cardiomyocytes, the results showed that formononetin effectively improved mitochondrial dysfunction induced by ISO. In summary, we demonstrated that formononetin via regulating the expressions of ALDH2, HADH, and MAOB in cardiomyocyte to improve mitochondrial dysfunction and alleviate ß-adrenergic activation cardiac fibrosis.

Neuroprotective potential of formononetin, a naturally occurring isoflavone phytoestrogen.

The increased prevalence of neurological illnesses is a burgeoning challenge to the public healthcare system and presents greater financial pressure. Formononetin, an O-methylated isoflavone, has gained a lot of attention due to its neuroprotective potential explored in several investigations. Formononetin is widely found in legumes and several types of clovers including Trifolium pratense L., Astragalus membranaceus, Sophora tomentosa, etc. Formononetin modulates various endogenous mediators to confer neuroprotection. It prevents RAGE activation that results in the inhibition of neuronal damage via downregulating the level of ROS and proinflammatory cytokines. Furthermore, formononetin also increases the expression of ADAM-10, which affects the pathology of neurodegenerative disease by lowering tau phosphorylation, maintaining synaptic plasticity, and boosting hippocampus neurogenesis. Besides these, formononetin also increases the expression of antioxidants, Nrf-2, PI3K, ApoJ, and LRP1. Whereas, reduces the expression of p65-NF-κB and proinflammatory cytokines. It also inhibits the deposition of Aß and MAO-B activity. An inhibition of Aß/RAGE-induced activation of MAPK and NOX governs the protection elicited by formononetin against inflammatory and oxidative stress-induced neuronal damage. Besides this, PI3K/Akt and ER-α-mediated activation of ADAM10, ApoJ/LRP1-mediated clearance of Aß, and MAO-B inhibition-mediated preservation of dopaminergic neurons integrity are the major modulations produced by formononetin. This review covers the biosynthesis of formononetin and key molecular pathways modulated by formononetin to confer neuroprotection.

Formononetin, a bioactive isoflavonoid constituent from Astragalus membranaceus (Fisch.) Bunge, ameliorates type 1 diabetes mellitus via activation of Keap1/Nrf2 signaling pathway: An integrated study supported by network pharmacology and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Type 1 diabetes mellitus (T1DM) results from insulin deficiency due to the destruction of pancreatic ß-cells. Previously, our studies showed that inhibition of Keap1/Nrf2 signaling pathway promoted the onset of T1DM, which suggests that finding drugs that can activate the Keap1/Nrf2 signaling may be a promising therapeutic strategy for the T1DM treatment. Astragalus membranaceus (Fisch.) Bunge is a common traditional Chinese medicine that has been frequently applied in Chinese clinics for the treatment of diabetes and other diseases. Formononetin (FMNT), one of the major isoflavonoid constituents isolated from this herbal medicine, possesses diverse pharmacological benefits and T1DM therapeutic potential. However, the exact molecular mechanisms underlying the action of FMNT in ameliorating T1DM have yet to be fully elucidated. AIMS OF THE STUDY: This study is to investigate the regulation of FMNT on the Keap1/Nrf2 signaling pathway to ameliorate T1DM based on network pharmacology approach combined with experimental validation. MATERIALS AND METHODS: A mouse-derived pancreatic islet ß-cell line (MIN6) was used for the in vitro studies. An alloxan (ALX)-induced T1DM model in wild-type and Nrf2 knockout (Nrf2-/-) C57BL/6J mice were established for the in vivo experiments. The protective effects of FMNT against ALX-stimulated MIN6 cell injury were evaluated using MTT, EdU, apoptosis and comet assays. The levels of blood glucose in mice were measured by using a blood monitor and test strips. The protein expression was detected by Western blot analysis. Furthermore, the binding affinity of FMNT to Keap1 was evaluated using cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) assay, and solvent-induced protein precipitation (SIP) assay. The interaction pattern between FMNT and Keap1 was assessed by molecular docking and molecular dynamics simulation techniques. RESULTS: Network pharmacology analysis revealed that FMNT exerted its therapeutic effect against T1DM by mainly regulating oxidative stress response-associated signaling molecules and pathways, such as Nrf2 regulating anti-oxidant/detoxification enzymes and Keap1-Nrf2 signaling pathway. The in vivo results showed that FMNT significantly deceased the ALX-induced high blood glucose levels and conversely increased the ALX-induced low insulin contents. In vitro, FMNT markedly protected MIN6 cells from ALX-induced cytotoxicity, proliferation inhibition and DNA damage and reduced the ALX-stimulated cell apoptosis. FMNT also inhibited ALX-induced overproduction of intracellular ROS to alleviate oxidative stress. In addition, FMNT could bind to Keap1 to notably activate the Keap1/Nrf2 signaling to upregulate Nrf2 expression and promote the Nrf2 translocation from the cytoplasm to the nucleus, resulting in enhancing the expression of antioxidant proteins HO-1 and NQO1. Inhibition of Keap1/Nrf2 signaling by ALX was also markedly abolished in the cells and mice exposed to FMNT. Moreover, these effects of FMNT in ameliorating T1DM were not observed in Nrf2-/- mice. CONCLUSIONS: This study demonstrates that FMNT could bind to Keap1 to activate the Keap1/Nrf2 signaling to prevent intracellular ROS overproduction, thereby attenuating ALX-induced MIN6 cell injury and ameliorating ALX-stimulated T1DM. Results from this study might provide evidence and new insight into the therapeutic effect of FMNT and indicate that FMNT is a promising candidate agent for the treatment of T1DM in clinics.

Formononetin attenuates high glucose-induced neurotoxicity by negatively regulating oxidative stress and mitochondrial dysfunction in Schwann cells via activation of SIRT3.

High glucose induces Schwann cells death and neurotoxicity. Formononetin was originally found in Astragalus membranaceus and showed anti-tumor and anti-neuroinflammation properties. The aim of this study is to explore the molecular mechanism underlying the neuroprotective effects of formononetin and identify its direct protein target. The effects of formononetin on oxidative stress and mitochondrial dysfunction in Schwann cells induced by high glucose were investigated. High glucose treatment significantly induced oxidative stress, mitochondrial dysfunction and apoptosis in Schwann cells, while these effects were partially or completely prevented by co-treatment with formononetin. Mechanistically, we found that SIRT3/PGC-1α/SOD2 pathway was activated by formononetin under high glucose conditions as evidenced by western blotting. Knockdown of SIRT3 by siRNA delivery reversed the protective effects of formononetin on high glucose-induced Schwann cells injury and changes in expression profile of SIRT3 downstream target genes. Molecular docking, thermal shift assay and surface plasmon resonance assay revealed a direct binding between formononetin and SIRT3. Taken together, we identified a novel SIRT3 activator formononetin and revealed its beneficial effects on high glucose-induced neurotoxicity, suggesting that targeting SIRT3 in Schwann cells may be a new approach for treatment of peripheral nerve regeneration related diseases such as diabetic peripheral neuropathy.

Synthesis, molecular docking studies of formononetin derivatives as potent Bax agonists for anticancer activity.

Formononetin as a Bax agonist exhibits anticancer effects. To identify novel Bax agonist, 18 new structurally modified formononetin derivatives were synthesised and their anticancer activities were evaluated in the A549 and Beas-2b cell lines. The results indicated that 7a elicited the most potent inhibitory effect against the A549 cell line, with an IC50 value of 0.87 µM, and no obvious toxicity to Beas-2b cells. These results indicated that 7a was 40-fold and 6.94-fold more efficacious than Formononetin and Doxorubicin, respectively. Additionally, western blot and immunofluorescence assays demonstrated that 7a downregulated the protein expression of Bcl-2 and upregulated the expressions of Bax to promote A549 apoptosis, the obtained results also suggested that 7a had the potential to be developed into a lead compound that can be applied in the prevention and treatment of lung cancer.