7-3--dihydroxy-4--methoxyisoflavone and Inflammation

Atherosclerosis is one of the underlying causes of cardiovascular disease. Formation of foam cells and necrotic core in the plaque is a hallmark of atherosclerosis, which results from lipid deposition, apoptosis, and inflammation in macrophages. Macrophage autophagy is a critical anti-atherogenic process and defective autophagy aggravates atherosclerosis by enhancing foam cell formation, apoptosis, and inflammation. Hence, enhancing autophagy can be a strategy for atherosclerosis treatment. Calycosin, a flavonoid from Radix Astragali, displays anti-oxidant and anti-inflammatory activities and therefore is potential to reduce the risk of cardiovascular disease. However, the anti-atherogenic effect of calycosin and the involved mechanism remains unclear. In this study, we assessed the potential benefits of calycosin on autophagy and atherosclerosis, and revealed the underlying mechanism.. Calycosin protected against atherosclerosis and enhanced plaque stability via promoting autophagy. Calycosin inhibited foam cell formation, inflammation, and apoptosis by enhancing autophagy. MLKL was demonstrated as a new autophagy regulator, which can be negatively regulated by KLF2. Mechanistically, inhibitory effects of calycosin on atherogenesis were via improved autophagy through KLF2-MLKL signalling pathway modulation.. This study demonstrated the atheroprotective effect of calycosin was through upregulating KLF2-MLKL-mediated autophagy, which not only proposed novel mechanistic insights into t atherogenesis but also identified calycosin as a potential drug candidate for atherosclerosis treatment.

Topics: Animals; Apolipoproteins E; Atherosclerosis; Autophagy; Cardiovascular Diseases; Foam Cells; Inflammation; Isoflavones; Kruppel-Like Transcription Factors; Mice; Mice, Knockout; Plaque, Atherosclerotic; Protein Kinases

Osteoarthritis (OA) is a joint disorder that is associated with chondrocyte damage under inflammatory environment. Calycosin is an astragalus extract with potential anti-inflammatory and anti-tumor activities. The purpose of this research is to explore the activity and mechanism of calycosin in interleukin-1beta (IL-1β)-induced chondrocyte injury. In the present study, the targets of calycosin and OA were analyzed according to HERB, DisGeNet, String, GO terms, and KEGG pathway enrichment assays. Human primary chondrocytes were treated with calycosin, and stimulated with IL-1β. Cell viability was detected by CCK-8 assay. Cell apoptosis was investigated by flow cytometry, and caspase-3 activity analyses. Inflammation was analyzed according to inflammatory cytokines levels by enzyme-linked immunosorbent assay (ELISA). The proteins associated with extracellular matrix (ECM) degradation and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/forkhead box O1 (FoxO1) signaling pathways were measured using Western blotting. The results showed that total of 25 overlapping targets of calycosin against OA were predicted. These targets might drive the FoxO pathway. Calycosin alone induced little cytotoxicity to chondrocytes, and it alleviated IL-1β-induced viability inhibition, cell apoptosis, inflammatory cytokine secretion, and ECM degradation in chondrocytes. Calycosin repressed IL-1β-induced activation of the PI3K/AKT/FoxO1 signaling. Activation of the PI3K/AKT/FoxO1 signaling mitigated the suppressive effect of calycosin on chondrocyte apoptosis, inflammation, and ECM degradation induced by IL-1β. As a conclusion, calycosin prevents IL-1β-induced chondrocyte apoptosis, inflammation, and ECM degradation through inactivating the PI3K/AKT/FoxO1 pathway.

Topics: Apoptosis; Chondrocytes; Forkhead Box Protein O1; Humans; Inflammation; Interleukin-1beta; Isoflavones; Osteoarthritis; Phosphatidylinositol 3-Kinase; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt

Shaoyao Gancao Decoction (SG-Tang), originated from the Treatise on Febrile Diseases, is often used to treat OA pain symptoms. Whereas its efficacy has been verified by several clinical studies, the underlying mechanism remained unclear. Network pharmacology and UPLC-QTOF-MS analysis found that calycosin could be regarded as the active components of SG-Tang in treating OA. However, the effect of calycosin on cartilage destruction and the pathogenesis of OA are not known. Therefore, we evaluated the benefits of calycosin for OA and revealed the underlying mechanisms.. Using network pharmacology, UPLC-QTOF-MS analysis and experiments, the active components of SG-Tang were analyzed to explore their potential therapeutic mechanism in OA.. The components of SG-Tang were detected by UPLC-QTOF-MS, and the possible active components and mechanism of SG-Tang in the treatment of OA were screened by network pharmacology. The OA mouse model was constructed by DMM. In total, 30 mice were randomly divided into three groups: Sham, DMM, and DMM + Calycosin. H&E, safranin O/fast green staining and the OARSI scores were used to evaluate joint injury in mice. In addition, OA models were established using chondrocytes treated with 10 ng/mL IL-1β. Treatment groups were treated with 100, 200 or 400 μM calycosin. CCK-8 assay was used for assessing the cytotoxic effects of calycosin. TUNEL staining and Western blotting were used to detect chondrocyte apoptosis. In addition, PI3K/Akt and NF-κB signaling pathway-related markers and cartilage matrix-related indicators were also detected.. In vivo studies showed that calycosin inhibited IL-1β-induced IL-6 and TNF-α production, as well as iNOS and COX-2 expression. Meanwhile, calycosin could inhibit IL-1β-induced degradation of cartilage matrix, including downregulation of MMP3, MMP-13, collagen II and aggrecan. NF-κB and PI3K/AKT were also inhibited by calycosin in OA chondrocytes. Furthermore, calycosin inhibited IL-1β-induced apoptosis in mouse chondrocytes. In a mouse model of OA, our results suggest that calycosin has a chondroprotective effect.. According to this study, calycosin may act as a protective agent against OA by inhibiting the PI3K/AKT and NF-κB pathways. Furthermore, this study suggested that calycosin is a potential candidate for the treatment of OA.

Topics: Animals; Apoptosis; Chondrocytes; Drugs, Chinese Herbal; Inflammation; Interleukin-1beta; Isoflavones; Mice; Mice, Inbred C57BL; NF-kappa B; Osteoarthritis; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt

Gouty arthritis is an inflammatory disease induced by monosodium urate (MSU), and is closely related to the activation of inflammasomes. Calycosin plays an anti-inflammatory role in arthritis. This study explored the mechanism of Calycosin in MSU-induced gouty arthritis. MSU-induced gouty arthritis mouse models with or without treatment of Calycosin were established, and physiological and pathological indicators were determined. Similarly, peripheral blood mononuclear cells (PBMCs) and THP-1 macrophages were used in vitro. Lactate dehydrogenase (LDH) was tested. The degree of centrifugal infiltration was detected by immunofluorescence. ELISA and quantitative reverse-transcription polymerase chain reaction were conducted to determine the levels of inflammatory factors. Immunohistochemistry, immunofluorescence, and flow cytometry were utilized to detect the content of caspase-1. Protein expressions of NF-κB-, p62-Keap1 pathway-, and pyroptosis-related factors were examined by western blot. In MSU-induced mouse models, calycosin increased mechanical hyperalgesia but decreased the swelling index of the mouse knee joint in a time-dependent manner. MSU treatment increased inflammatory cells and LysM-eGFP

Topics: Animals; Arthritis, Gouty; Caspase 1; Disease Models, Animal; DNA-Binding Proteins; Inflammasomes; Inflammation; Kelch-Like ECH-Associated Protein 1; Leukocytes, Mononuclear; Mice; NF-E2-Related Factor 2; NF-kappa B; Pyroptosis; Uric Acid

The precise etiology and pathogenesis of idiopathic pulmonary fibrosis are not completely understood, and no satisfactory treatment exists. This work aimed to examine the effects of calycosin (CA, an isoflavone compound) on pulmonary fibrosis (PF) and explore the underlying mechanism. In this study, we established a mice model of PF induced by 5 mg/mL bleomycin (BLM), and mice were orally administrated with 7 mg/kg or 14 mg/kg CA once a day for three weeks. In vitro, after pretreated with 80 μM CA, MLE-12 cells were stimulated with 10 ng/mL transforming growth factor-β1 (TGF-β1) for inducing epithelial-mesenchymal transition (EMT). The results showed that CA treatment ameliorated the severity of fibrosis and the lung tissue damage, as well as suppressed the secretion of inflammation factors in a dose-dependent manner of the PF mice model induced by BLM. Subsequently, CA inhibited the BLM-induced PF progression by repressing EMT, evidenced by the reverse of the downregulation of E-cadherin and the upregulation of vimentin, α-SMA, and fibronectin. Moreover, the elevated phosphorylation of AKT and GSK3β induced by BLM (or TGF-β1) was decreased by CA treatment, leading to the rescue of the high expression of β-catenin. CA prevented the translocation of β-catenin from the cytoplasm to the nucleus. The repressed effects of CA on the TGF-β1-induced EMT and the AKT/GSK3β/β-catenin axis, as well as the translocation of β-catenin were all reversed by a AKT activator SC79. Taken together, CA ameliorated PF by the EMT inhibition upon suppressing the AKT/GSK3β/β-catenin signaling pathway.

Topics: Animals; beta Catenin; Bleomycin; Body Weight; Epithelial-Mesenchymal Transition; Glycogen Synthase Kinase 3 beta; Inflammation; Isoflavones; Male; Mice; Mice, Inbred C57BL; Proto-Oncogene Proteins c-akt; Pulmonary Fibrosis; Signal Transduction

Type 2 diabetes mellitus (T2DM) is a disease with a drastically growing worldwide prevalence. It is usually associated with numerous complications of which; diabetic nephropathy (DN); is a main complication of microvasculature and more seriously, a common cause of end-stage renal disease (ESRD). Unfortunately, both the lack of a definitive remedy alongside the economic and the social burden on DN patients enforces considerable impetus for developing alternative therapies. IL-33 is a newly discovered member of the IL-1 cytokine family. IL33/ST2 signaling plays a crucial role in acute and chronic kidney diseases. Calycosin is an isoflavone with reported IL33 signaling inhibitory activity. The present study aimed to investigate if calycosin possess renal protective effect in high-fat diet/STZ-induced T2DM model and to clarify the potential underlying mechanisms. HFD-STZ control rats showed functional and structural renal damage confirmed by increased serum creatinine, blood urea nitrogen and albuminuria associated with marked renal glomerulosclerosis and interstitial fibrosis. Initiation of inflammation, oxidative stress, and fibrosis was evident as depicted by elevated renal levels of IL33/ST2 mRNA as well as increased renal NF-κBp65, TNF-α, IL-1β, MDA, and TGF-β contents with suppressed Nrf2 and TAC. Calycosin treatment markedly improved the aforementioned makers of renal injury and dysfunction, modulated IL33/ST2 signaling, inflammatory cytokines, oxidative stress and fibrotic processes. This was accompanied by improvement of T2DM-induced renal ultramicroscopic and histopathological alterations.

Topics: Animals; Cytokines; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Diet, High-Fat; Fibrosis; Inflammation; Interleukin-33; Isoflavones; Kidney; Male; Oxidative Stress; Protective Agents; Rats; Rats, Sprague-Dawley; Receptors, Interleukin-1; Signal Transduction; Streptozocin

The limitation of doxorubicin (DOX), which is widely used for the treatment of solid tumors and hematologic malignancies, is a vital problem in clinical application. The most serious of limit factors is cardiotoxicity. Calycosin (CA), an isoflavonoid that is the major active component in Radix astragali, has been reported in many bioactivities including antitumor, anti-inflammatory, and cardioprotection. The aim of the study was to investigate the effects and mechanisms of CA on DOX-induced cardiotoxicity in vitro and in vivo. CA increased H9c2 cell viability and reduced apoptosis induced by DOX via Bcl-2, Bax, and the PI3K-Akt signaling pathway. Moreover, CA prevented DOX-induced oxidative stress in cells by decreasing the generation of reactive oxygen species. Similarly, oxidative stress was inhibited by CA through the increased activities of antioxidant enzymes such as glutathione peroxidase, catalase, and superoxide dismutase and decreased the levels of aspartate aminotransferase, lactate dehydrogenase, and malondialdehyde in vivo. Furthermore, the levels of sirtuin 1 (Sirt1)-NOD-like receptor protein 3 (NLRP3) and related proteins were ameliorated by CA in cells and in mice hearts. When H9c2 cells were treated by Ex527 (Sirt1 inhibitor), the effect of CA on expressions of NLRP3 and thioredoxin-interacting protein was suppressed. In conclusion, the results suggested that CA might be a cotreatment with DOX to ameliorate cardiotoxicity by Sirt1-NLRP3 pathway.

Topics: Animals; Antioxidants; Apoptosis; Cardiotoxicity; Cell Line; Doxorubicin; Inflammation; Isoflavones; Male; Mice; Oxidative Stress; Phosphatidylinositol 3-Kinases; Reactive Oxygen Species; Receptors, Cell Surface; Signal Transduction; Sirtuin 1

Allergic asthma is a common chronic lung inflammatory disease and seriously influences public health. We aim to investigate the effects of formononetin (FMN) and calycosin (CAL), 2 flavonoids in Radix Astragali, on allergic asthma and elucidate possible therapeutic targets. A house dust mite (HDM)-induced allergic asthma mouse model and TNF-α and Poly(I:C) co-stimulated human bronchial epithelial cell line (16HBE) were performed respectively in vivo and in vitro. The role of G protein-coupled estrogen receptor (GPER) was explored by its agonist, antagonist, or GPER small interfering RNA (siGPER). E-cadherin, occludin, and GPER were detected by western blotting, immunohistochemistry, or immunofluorescence. The epithelial barrier integrity was assessed by trans-epithelial electric resistance (TEER). Cytokines were examined by enzyme-linked immunosorbent assay (ELISA). The results showed that flavonoids attenuated pulmonary inflammation and hyperresponsiveness in asthmatic mice. These flavonoids significantly inhibited thymic stromal lymphopoietin (TSLP), increased occludin and restored E-cadherin in vivo and in vitro. The effects of flavonoids on occludin and TSLP were not interfered by ICI182780 (estrogen receptor antagonist), while blocked by G15 (GPER antagonist). Furthermore, compared with PPT (ERα agonist) and DPN (ERβ agonist), G1 (GPER agonist) significantly inhibited TSLP, up-regulated occludin, and restored E-cadherin. siGPER and TEER assays suggested that GPER was pivotal for the flavonoids on the epithelial barrier integrity. Finally, G1 attenuated allergic lung inflammation, which could be abolished by G15. Our data demonstrated that 2 flavonoids in Radix Astragali could alleviate allergic asthma by protecting epithelial integrity via regulating GPER, and activating GPER might be a possible therapeutic strategy against allergic inflammation.

Topics: Adherens Junctions; Animals; Asthma; Astragalus propinquus; Cadherins; Cytokines; Drugs, Chinese Herbal; Epithelial Cells; Humans; Hypersensitivity; Inflammation; Isoflavones; Mice, Inbred BALB C; Models, Biological; Occludin; Pneumonia; Pyroglyphidae; Receptors, Estrogen; Receptors, G-Protein-Coupled; Thymic Stromal Lymphopoietin; Tight Junctions; Up-Regulation

Parkinson is the second common neurodegenerative disease. The characteristics of Parkinson's disease (PD) are the dopamin neurons loss caused by neuroinflammation responses. C alycosin, an isoflavone phytoestrogen isolated from Astragalus membranaceus, has multiple pharmacological activities, such as anti-inflammation, anti-tumor, and neuroprotective effects. However, it is unknown whether calycosin can mitigate PD symptoms. This study aims to explore whether calycosin can alleviate PD symptoms and the underlying mechanisms. PD was induced in mice by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injection, and calycosin was given intracerebroventricularly to these mice. A cell model of nerve inflammation was established by BV2 microglia cells injected with lipopolysaccharide (LPS). The motor states were evaluated by stepping, whisker, and cylinder experiments. The states of dopaminergic neurons and microglia were detected by immunostainning of tyrosine hydroxylase and cluster of differentiation molecule 11b (CD11b). The expression levels of inflammatory factors were detected by qPCR. Toll-like receptor (TLR)/nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways were investigated by western blot. We found that calycosin treatment mitigated the behavioral dysfunctions and inflammatory responses in MPTP-induced PD mice. The TLR/NF-κB and MAPK pathways in MPTP-induced PD mice were inhibited by calycosin treatment, which was coincident with experiments in LPS-induced BV2 cells. Above all, calycosin mitigates PD symptoms through TLR/NF-κB and MAPK pathways in mice and cell lines.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Cells, Cultured; Dopaminergic Neurons; Inflammation; Isoflavones; Lipopolysaccharides; Male; MAP Kinase Signaling System; Mice; Microglia; Mitogen-Activated Protein Kinases; Neuroprotective Agents; NF-kappa B; Parkinson Disease, Secondary; PC12 Cells; Rats; Toll-Like Receptors

BACKGROUND Diabetic nephropathy (DN), which is one of the primary causes of end-stage renal disease (ESRD), is increasingly diagnosed in patients due to the continuous increase in the prevalence of diabetic mellitus (DM). Astragali Radix, a traditional Chinese herb, is widely administrated to ameliorate the symptoms of diabetes and diabetic nephropathy, but its mechanism is still not yet fully defined. Calycosin (C₁₆H₁₂O₅) is the major active component of Astragali Radix. In this study, we analyzed the role of calycosin in diabetic nephropathy and explored its underlying mechanism. MATERIAL AND METHODS Cell activation, inflammatory cytokines expression and secretion, and protein levels were analyzed in cultured mouse tubular epithelial cells (mTEC). db/db mice were intraperitoneally injected with 10 mg/(kg·d) calycosin or control saline for 4 weeks, followed by analysis of structure injury, inflammation, and NF-κB signaling activity. RESULTS Our results indicated that TNF-α and IL-1β were significantly induced by advanced glycation end-products (AGEs), but calycosin remarkably reduced the expression of TNF-α and IL-1β in the cultured mouse tubular epithelial cells (mTEC). Calycosin effectively alleviated kidney injury in diabetic kidneys of db/db mice during the progression of diabetic renal injury, indicated by the reduction of histological injury and immunohistochemical of inflammatory cytokines. Mechanistically, we identified calycosin inhibited diabetes-induced inflammation in kidneys by suppressing the phosphorylation of IKBa and NF-κB p65 in vitro and in vivo. CONCLUSIONS Calycosin significantly ameliorated diabetes-induced renal inflammation in diabetic renal injury by inhibition of the NF-κB-dependent signaling pathway in vivo and in vitro.

Topics: Animals; Astragalus propinquus; Cell Culture Techniques; Cytokines; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Models, Animal; Drugs, Chinese Herbal; Epithelial Cells; Inflammation; Interleukin-1beta; Isoflavones; Kidney; Male; Mice; Nephritis; NF-kappa B; Phosphorylation; Signal Transduction; Transcription Factor RelA; Tumor Necrosis Factor-alpha

Objective to find the Calycosin glycoside (CG) potential anti-inflammatory effect. The anti-inflammatory activity of CG was evaluated by lipopolysaccharide (LPS)-induced RAW 264.7 cells, and the IL-6, IL-1β, TNF-α and PGE2 level were measured by ELISA; NO levels in the culture media were determined using the Griess reaction. The iNOS and COX-2 mRNA expressions were measured by qRT-PCR and the phosphorylation of IκBα, p65, ERK, JNK, and p38 was determined by western blotting. The CG could markedly inhibit the productions of NO and PGE

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Glycosides; Inflammation; Isoflavones; Lipopolysaccharides; Macrophages; Mice; RAW 264.7 Cells; Signal Transduction

Neuroinflammation is a key contributor to neuronal damage in neurodegenerative diseases. In our previous work on natural effective neuroinflammatory inhibitors, Alhagi sparsifolia Shap. (Leguminosae), a folk medicine widely distributed in Xinjiang, attracted our attention because of its significant anti-neuroinflammatory effect. Therefore, further investigation of the bioactive material basis was carried out. As a result, 33 major components were characterized and identified by chromatographic and spectral methods, respectively. Furthermore, the anti-neuroinflammatory effects of the extract and purified constituents were evaluated in LPS-induced N9 cells in vitro. The results displayed that compounds 1, 2, 3, 5, 6, 8, 11, 15, 16, 17, 22, 23, 25, 26, 28, 30, 33 could exhibit significant inhibitory activities without obvious cytotoxicities at their effective concentrations. Especially, isorhamnetin (1) (IC

Topics: Cell Line; Fabaceae; Humans; Inflammation; Lipopolysaccharides; Microglia; Neuroprotective Agents; Plant Extracts

The activation of synovial fibroblasts (SFs) and the subsequent production and expression of pro-inflammatory cytokines play a crucial role in the pathogenesis and progression of rheumatoid arthritis (RA). In the current study, rheumatoid arthritis synovial fibroblasts (RASFs) isolated from the joint of the patients were used to evaluate the suppressive effects of calycosin (CAL), a compound derived from the Chinese medicinal herb Radix Astragali, on the expression of pro-inflammatory cytokines in RASFs. The results demonstrated that increased mRNA expression levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-25 (IL-25), interleukin-33(IL-33) were significantly inhibited by CAL. Furthermore, the compound obviously suppressed IL-6 and IL-33 secretion. The key inflammatory mediator, cyclooxygenase-2 (COX-2) was significantly attenuated by CAL. A mechanistic study showed that the antioxidant enzymes heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase quinone 1(NQO1) and Nrf2 of RASFs were markedly activated by CAL. Furthermore, CAL potentiated the accumulation of sequestosome 1 (SQSTM1, p62) and the degradation of Kelch-like ECH-associated protein 1 (Keap1), thereby inducing Nrf2 translocation from the cytoplasm to the nucleus. Thus, CAL suppresses the expression of pro-inflammatory cytokines via p62/Nrf2-linked HO-1 induction in RASFs, which suggests that the compound should be further investigated as a candidate anti-inflammatory and anti-arthritic agent.

Topics: Anti-Inflammatory Agents; Antioxidants; Arthritis, Rheumatoid; Cell Nucleus; Cells, Cultured; Cytokines; Cytoplasm; Fibroblasts; Gene Expression; Heme Oxygenase-1; Humans; Inflammation; Isoflavones; NF-E2-Related Factor 2; RNA-Binding Proteins; RNA, Messenger

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature