The Role of Marein During Endothelial-Mesenchymal Transition in Diabetic Kidney Disease / Rol de la Mareína Durante la Transición Endotelial-Mesenquimatosa en la Enfermedad Renal Diabética

SUMMARY: Marein is a flavonoid compound that reduces blood glucose and lipids and has a protective effect in diabetes. However, the effect and mechanism(s) of marein on renal endothelial-mesenchymal transition in diabetic kidney disease (DKD) have not been elucidated. In this study, single-cell sequencing data on DKD were analyzed using a bioinformation method, and the data underwent reduced dimension clustering. It was found that endothelial cells could be divided into five subclusters. The developmental sequence of the subclusters was 0, 1, 4, 2, and 3, of which subcluster 3 had the most interstitial phenotype.The expression of mesenchymal marker protein:Vimentin(VIM), Fibronectin(FN1), and fibroblast growth factor receptor 1 (FGFR1) increased with the conversion of subclusters. In db/db mice aged 13-14 weeks, which develop DKD complications after 8-12 weeks of age, marein reduced blood levels of glucose, creatinine, and urea nitrogen, improved structural damage in kidney tissue, and reduced collagen deposition and the expression of FN1 and VIM. Marein also up-regulated autophagy marker:Light chain 3II/I(LC3II/I) and decreased FGFR1 expression in renal tissue. In an endothelial-mesenchymal transition model, a high glucose level induced a phenotypic change in human umbilical vein endothelial cells. Marein decreased endothelial cell migration, improved endothelial cell morphology, and decreased the expression of VIM and FN1. The use of the FGFR1 inhibitor, AZD4547, and autophagy inhibitor, 3-Methyladenine(3-MA), further demonstrated the inhibitory effect of marein on high glucose-induced endothelial-mesenchymal transition by reducing FGFR1 expression and up-regulating the autophagy marker protein, LC3II/I. In conclusion, this study suggests that marein has a protective effect on renal endothelial- mesenchymal transition in DKD, which may be mediated by inducing autophagy and down-regulating FGFR1 expression.

La mareína es un compuesto flavonoide que reduce la glucosa y los lípidos en sangre y tiene un efecto protector en la diabetes. Sin embargo, no se han dilucidado el efecto y los mecanismos de la mareína sobre la transición endotelial- mesenquimatosa renal en la enfermedad renal diabética (ERD). En este estudio, los datos de secuenciación unicelular sobre DKD se analizaron utilizando un método de bioinformación y los datos se sometieron a una agrupación de dimensiones reducidas. Se descubrió que las células endoteliales podían dividirse en cinco subgrupos. La secuencia de desarrollo de los subgrupos fue 0, 1, 4, 2 y 3, de los cuales el subgrupo 3 tenía el fenotipo más intersticial. La expresión de la proteína marcadora mesenquimatosa: vimentina (VIM), fibronectina (FN1) y receptor del factor de crecimiento de fibroblastos. 1 (FGFR1) aumentó con la conversión de subgrupos. En ratones db/db de 13 a 14 semanas de edad, que desarrollan complicaciones de DKD después de las 8 a 12 semanas de edad, la mareína redujo los niveles sanguíneos de glucosa, creatinina y nitrógeno ureico, mejoró el daño estructural en el tejido renal y redujo la deposición y expresión de colágeno de FN1 y VIM. Marein también aumentó el marcador de autofagia: Cadena ligera 3II/I (LC3II/I) y disminuyó la expresión de FGFR1 en el tejido renal. En un modelo de transición endotelial-mesenquimal, un nivel alto de glucosa indujo un cambio fenotípico en las células endoteliales de la vena umbilical humana. Marein disminuyó la migración de células endoteliales, mejoró la morfología de las células endoteliales y disminuyó la expresión de VIM y FN1. El uso del inhibidor de FGFR1, AZD4547, y del inhibidor de la autofagia, 3-metiladenina (3-MA), demostró aún más el efecto inhibidor de la mareína en la transición endotelial-mesenquimal inducida por niveles altos de glucosa al reducir la expresión de FGFR1 y regular positivamente la proteína marcadora de autofagia. , LC3II/I. En conclusión, este estudio sugiere que la mareína tiene un efecto protector sobre la transición endotelial-mesenquimatosa renal en la ERC, que puede estar mediada por la inducción de autofagia y la regulación negativa de la expresión de FGFR1.

Marein Alleviates Doxorubicin-Induced Cardiotoxicity through FAK/AKT Pathway Modulation while Potentiating its Anticancer Activity.

Doxorubicin (DOX) is an effective anticancer agent, yet its clinical utility is hampered by dose-dependent cardiotoxicity. This study explores the cardioprotective potential of Marein (Mar) against DOX-induced cardiac injury and elucidates underlying molecular mechanisms. Neonatal rat cardiomyocytes (NRCMs) and murine models were employed to assess the impact of Mar on DOX-induced cardiotoxicity (DIC). In vitro, cell viability, oxidative stress were evaluated. In vivo, a chronic injection method was employed to induce a DIC mouse model, followed by eight weeks of Mar treatment. Cardiac function, histopathology, and markers of cardiotoxicity were assessed. In vitro, Mar treatment demonstrated significant cardioprotective effects in vivo, as evidenced by improved cardiac function and reduced indicators of cardiac damage. Mechanistically, Mar reduced inflammation, oxidative stress, and apoptosis in cardiomyocytes, potentially via activation of the Focal Adhesion Kinase (FAK)/AKT pathway. Mar also exhibited an anti-ferroptosis effect. Interestingly, Mar did not compromise DOX's efficacy in cancer cells, suggesting a dual benefit in onco-cardiology. Molecular docking studies suggested a potential interaction between Mar and FAK. This study demonstrates Mar's potential as a mitigator of DOX-induced cardiotoxicity, offering a translational perspective on its clinical application. By activating the FAK/AKT pathway, Mar exerts protective effects against DOX-induced cardiomyocyte damage, highlighting its promise in onco-cardiology. Further research is warranted to validate these findings and advance Mar as a potential adjunctive therapy in cancer treatment.

Marein, a novel natural product for restoring chemo-sensitivity to cancer cells through competitive inhibition of ABCG2 function.

The pivotal roles of ATP-binding cassette (ABC) transporters in drug resistance have been widely appreciated. Here we report that marein, a natural product from Coreopsis tinctoria Nutt, is a potent chemo-sensitizer in drug resistant cancer cells overexpressing ABCG2 transporter. We demonstrate that marein can competitively inhibit efflux activity of ABCG2 protein and increase the intracellular accumulation of the chemotherapeutic drugs that belong to substrate of this transporter. We further show that marein can bind to the conserved amino acid residue F439 of ABCG2, a critical site for drug-substrate interaction. Moreover, marein can significantly sensitize the ABCG2-expressing tumor cells to chemotherapeutic drugs such as topotecan, mitoxantrone, and olaparib. This study reveals a novel role and mechanism of marein in modulating drug resistance, and may have important implications in treatment of cancers that are resistant to chemotherapeutic drugs that belong to the substrates of ABCG2 transporters.

UPLC-quadrupole time-of-flight-tandem mass spectrometry combined with chemometrics and network pharmacology to differentiate Coreopsis tinctoria Nutt.

Coreopsis tinctoria Nutt. (C. tinctoria) is a traditional medicinal plant, primarily found in plateau areas with altitudes exceeding 3000 m. The efficacy of C. tinctoria appears to be intricately tied to its quality. However, there is a scarcity of studies focused on evaluating the quality of C. tinctoria from diverse geographical locations. In this study, we used ultra-performance liquid chromatography-quadrupole time-of-flight-tandem mass spectrometry to analyze and identify the prevalent chemical components in 12 batches of C. tinctoria sourced from Xinjiang, Qinghai, Tibet, and Yunnan provinces in China. By using cluster analysis and discriminant analysis of partial least squares, we assessed the similarity and identified varying components in the 12 batches of C. tinctoria. Subsequently, their quality was further evaluated. Utilizing network pharmacology, we identified potential active components for the treatment of diabetes mellitus. The findings revealed the presence of 16 flavonoids, 3 phenylpropanes, 2 sugars, 2 amino acids, and 7 hydrocarbons in the analyzed samples. Through variable importance screening, 17 constituents were identified as quality difference markers. Marein and flavanomarein emerged as pivotal markers, crucial for distinguishing variations in C. tinctoria. In addition, network pharmacology predicted 187 targets for 9 common active components, including marein and flavanomarein. Simultaneously, 1747 targets related to diabetes mellitus were identified. The drug-component-disease target network comprised 91 nodes and 179 edges, encompassing 1 drug node, 9 component nodes, and 81 target nodes. In summary, marein and flavanomarein stand out as key biomarkers for assessing the quality of C. tinctoria, offering a scientific foundation for the quality evaluation of C. tinctoria Nutt.

Protective effect of marein against alcoholic fatty liver and its mechanism / 中国药房

OBJECTIVE To explore the protective effect of marein against alcoholic fatty liver （AFL） and its potential mechanisms. METHODS AFL mice model was established with strong wine by gavage. The mice were randomly divided into normal control group （n＝9， 0.5% sodium carboxymethyl cellulose solution）， model group （n＝10， 0.5% sodium carboxymethyl cellulose solution） and marein 75 and 150 mg/kg groups （n＝9）. Mice were given relevant medicine intragastrically， once a day， for consecutive 30 days. After the last medication， the levels of triglyceride （TG）， malondialdehyde （MDA）， and superoxide dismutase （SOD） in liver tissue were determined， and hepatic histopathological changes of liver tissue were observed； the protein expression levels of peroxisome proliferator-activated receptor α （PPARα）， carnitine palmitoyltransferase-1 （CPT-1）， and diacylglycerol acyltransferase （DGAT） were determined in liver tissue. BRL hepatocytes injury model was induced by ethanol combined with ferrous sulfate and oleic acid； after treatment with 3， 6 and 12 μmol/L of marein for 24 h， the distribution of lipid droplets， the levels of TG， MDA and SOD and protein expressions of PPARα， CPT-1 and DGAT in hepatocytes were examined. After pretreatment with MK886 （PPARα inhibitor， 10 μmol/L），modeled hepatocytes were treated with 12 μmol/L of marein for 24 h， and the protein expressions of PPARα， CPT-1 and DGAT were determined. RESULTS As the results showed in vivo， compared with the model group， after treatment with 75 and 150 mg/kg of marein， the degree of steatosis was significantly reduced， and the levels of TG and MDA and protein expression of DGAT were significantly decreased（P＜0.05 or P＜0.01）； the levels of SOD， protein expressions of PPARα and CPT-1 were significantly increased（P＜0.05 or P＜0.01）. As the results showed in vitro， after treatment with 3， 6 and 12 μmol/L of marein， the lipid accumulation of hepatocytes was significantly inhibited， and the levels of TG and MDA， protein expression of DGAT were significantly decreased（P＜0.05 or P＜0.01）， while the levels of SOD， protein expressions of PPARα and CPT-1 were significantly increased（P＜0.05 or P＜0.01）. After MK886 pretreatment， the effects of marein on the above protein expressions were abolished. CONCLUSIONS Marein might exert a protective effect against AFL. The mechanisms might be related to inhibiting oxidative stress-mediated injury and improving PPARα-mediated lipid metabolism signaling pathway.

Marein alleviates myocardial fibrosis in diabetic mice / 基础医学与临床

Objective To study the effect of marein on myocardial fibrosis in diabetic mice.Methods Ten lep-tin receptor gene defective heterozygous(db/m)mice aged 5-6 weeks were selected as the control group and 30 diabetic mice with leptin receptor gene defective db/db were divided into:db/db group(db/db,n=10),metformin(Met)positive group(280 mg/kg daily,n=10)and marein drug intervention group(50 mg/kg,n=10).After continuous administration for 8 weeks,the cardiac morphological changes were observed by HE staining and Masson staining.The distribution and expression of vimentin were detected by immunohistochemis-try method.The expression of fibronectin,vimentin,and transforming growth factor-β1(TGF-β1)protein in cardiac tissue was detected by Western blot.Results Myocardial fiber hypertrophy was observed in db/db group,and myocardial structural damage was improved in metformin group and marein group.Compared with db/m group,the expression of myocardial collagen fiber in db/db group increased(P＜0.01),while the expression of myo-cardial collagen fiber in metformin group and marein group decreased(P＜0.01).Compared with the control group,the expression of vimentin in myocardial tissue of db/db group was significantly increased(P＜0.01),while the expression of vimentin in metformin group and marein group was significantly decreased(P＜0.01).The expression of fibronectin,vimentin and TGF-β1 in db/db group was significantly increased as compared with those in db/m group(P＜0.01),while the expression of fibronectin,vimentin and TGF-β1 in metformin group and marein group were significantly decreased(P＜0.01).Conclusions Marein improves myocardial fibrosis in diabetic db/db mice.

Marein Ameliorates Myocardial Fibrosis by Inhibiting HIF-1α and TGF-ß1/Smad2/3 Signaling Pathway in Isoproterenol-stimulated Mice and TGF-ß1-stimulated Cardiac Fibroblasts.

BACKGROUND: Myocardial fibrosis significantly contributes to the pathogenesis and progression of heart failure. OBJECTIVE: We probe into the impact of marein, a key bioactive compound in functional food Coreopsis tinctoria, on isoproterenol-stimulated myocardial fibrotic mice and transforming growth factor ß1 (TGF-ß1)-stimulated cardiac fibroblasts (CFs). METHODS: Isoproterenol was administered to the experimental mice via subcutaneous injection, and simultaneous administration of marein (25-100 mg/kg) was performed via oral gavage. CFs were stimulated with TGF- ß1 to trigger differentiation and collagen synthesis, followed by treatment with marein at concentrations of 5-20 µM. RESULTS: Treatment with marein in mice and CFs resulted in a significant reduction in the protein expression levels of α-smooth muscle actin, collagen type I, and collagen type III. Additionally, marein treatment decreased the protein expression levels of TGF-ß1, hypoxia-inducible factor-1α (HIF-1α), p-Smad2/3, and Smad2/3. Notably, molecular docking analysis revealed that marein directly targets HIF-1α. CONCLUSION: Marein might exert a protective function in isoproterenol-stimulated myocardial fibrotic mice and TGF-ß1-stimulated CFs, which might result from the reduction of TGF-ß1 induced HIF-1α expression, then inhibiting p-Smad2/3 and Smad2/3 expressions.

Marein Alleviates High Glucose-Induced Human Retinal Microvascular Endothelial Cell Injury by Up-Regulating SNHG7 Expression.

BACKGROUND: Marein has been shown to possess therapeutic effects against diabetic retinopathy, but whether it can protect against high glucose (HG)-induced human retinal microvascular endothelial cell (HRMEC) injury remains unclear. Our study aimed to explore the effect of marein on HG-induced HRMEC injury and the mechanism underlying this purported therapeutic effect. METHODS: HRMEC was divided into normal glucose group, high glucose (HG) group, HG+marein low, medium and high (L, M, H) concentrations group, HG+pcDNA group, HG+pcDNA-small nucleolar RNA host gene 7 (SNHG7) group, HG+marein+si-negative control (NC) group, and HG+marein+si-SNHG7 group. Flow cytometry and Western blotting were performed to determine apoptosis rate and apoptosis-related protein levels. Superoxide dismutase (SOD) activity, lactate dehydrogenase (LDH) level and malondialdehyde (MDA) content were detected to assess cellular oxidative stress. SNHG7 expression was examined using real-time quantitative PCR. RESULTS: After treatment with low, medium and high concentrations of marein, apoptosis rate, Bax level, LDH level and MDA content were decreased, while B-cell lymphoma-2 (Bcl-2) level, SOD activity, and SNHG7 expression were promoted in HG-induced HRMEC injury in a concentration-dependent manner (p < 0.05). After overexpression of SNHG7, apoptosis rate, Bax level, LDH level and MDA content were decreased, while Bcl-2 level and SOD activity were enhanced in HG-induced HRMEC injury (p < 0.05). In contrast, SNHG7 knockdown reversed the effect of marein on HG-induced HRMEC injury. CONCLUSIONS: Marein could alleviate HG-induced HRMEC injury by up-regulating SNHG7 expression.

Marein Prevented LPS-Induced Osteoclastogenesis by Regulating the NF-κB Pathway In Vitro.

Many bone diseases such as osteolysis, osteomyelitis, and septic arthritis are caused by gram-negative bacterial infection, and lipopolysaccharide (LPS), a bacterial product, plays an essential role in this process. Drugs that inhibit LPS-induced osteoclastogenesis are urgently needed to prevent bone destruction in infective bone diseases. Marein, a major bioactive compound of Coreopsis tinctoria, possesses anti-oxidative, anti-inflammatory, anti-hypertensive, anti-hyperlipidemic, and anti-diabetic effects. In this study, we measured the effect of marein on RAW264.7 cells by CCK-8 assay and used TRAP staining to determine osteoclastogenesis. The levels of osteoclast-related genes and NF-κB-related proteins were then analyzed by western blot, and the levels of pro-inflammatory cytokines were quantified by ELISA. Our results showed that marein inhibited LPS-induced osteoclast formation by osteoclast precursor RAW264.7 cells. The effect of marein was related to its inhibitory function on expressions of pro-inflammatory cytokines and osteoclast-related genes containing RANK, TRAF6, MMP-9, CK, and CAII. Additionally, marein leads to markedly inhibited NF-κB signaling pathway activation in LPS-induced RAW264.7 cells. Concurrently, when the NF-κB signaling pathway was inhibited, osteoclast formation and pro-inflammatory cytokine expression were decreased. Collectively, marein could inhibit LPS-induced osteoclast formation in RAW264.7 cells via regulating the NF-κB signaling pathway. Our data demonstrate that marein might be a potential drug for bacteria-induced bone destruction disease. Our findings provide new insights into LPS-induced bone disease.

Mechanisms of Coreopsis tinctoria Nutt in the treatment of diabetic nephropathy based on network pharmacyology analysis of its active ingredients / Mecanismos de Coreopsis tinctoria Nutt. En el tratamiento de la nefropatía diabética basado en el análisis farmacológico de red de sus principios activos

SUMMARY: Coreopsis tinctoria Nutt. (C. tinctoria Nutt.) can protect diabetic kidneys, but the mechanisms are unclear. This work is to investigate the potential mechanisms of C. tinctoria Nutt. in the treatment of diabetic nephropathy based on network pharmacology analysis of its active ingredients. Twelve small molecular compounds of C. tinctoria Nutt. and targets related to diabetic nephropathy were docked by Discovery Studio 3.0. DAVID database was used for GO enrichment and KEGG pathway analysis. Cytoscape 3.6.1 was used to construct active ingredient-target network. Cell viability was detected with MTT. Glucose consumption was analyzed with glucose oxidase method. Protein expression was measured with Western blot and immunofluorescence. Electron microscopy observed autophagosomes. The core active ingredients of C. tinctoria Nutt. included heriguard, flavanomarein, maritimein, and marein. Twenty-one core targets of the 43 potential targets were PYGM, TLR2, RAF1, PRKAA2, GPR119, INS, CSF2, TNF, IAPP, AKR1B1, GSK3B, SYK, NFKB2, ESR2, CDK2, FGFR1, HTRA1, AMY2A, CAMK4, GCK, and ABL2. These 21 core targets were significantly enriched in 50 signaling pathways. Thirty- four signaling pathways were closely related to diabetic nephropathy, of which the top pathways were PI3K/AKT, insulin, and mTOR, and insulin resistance. The enriched GO terms included biological processes of protein phosphorylation, and the positive regulation of PI3K signaling and cytokine secretion; cellular components of cytosol, extracellular region, and extracellular space; and molecular function of protein kinase activity, ATP binding, and non-membrane spanning protein tyrosine kinase activity. In vitro experiments found that marein increased the expression of phosphorylated AKT/AKT in human renal glomerular endothelial cells of an insulin resistance model induced by high glucose, as well as increased and decreased, respectively, the levels of the microtubule-associated proteins, LC3 and P62. C. tinctoria Nutt. has many active ingredients, with main ingredients of heriguard, flavanomarein, maritimein, and marein, and may exert anti-diabetic nephropathy effect through various signaling pathways and targets.

RESUMEN: Coreopsis tinctoria Nutt. (C. tinctoria Nutt.) puede proteger riñones diabéticos, sin embargo los mecanismos son desconocidos. Este trabajo se realizó para investigar los potenciales mecanismos de C. tinctoria Nutt. en el tratamiento de la nefropatía diabética basado en el análisis de farmacología en red de sus principios activos. Doce compuestos moleculares pequeños de C. tinctoria Nutt. y los objetivos relacionados con la nefropatía diabética fueron acoplados por Discovery Studio 3.0. La base de datos DAVID se utilizó para el enriquecimiento GO y el análisis de la vía KEGG. Se usó Cytoscape 3.6.1 para construir una red de ingrediente-objetivo activa. La viabili- dad celular se detectó mediante MTT. El consumo de glucosa se analizó con el método de glucosa oxidasa. La expresión proteica fue determinada mediante Western blot e inmunofluorescencia. En la microscopía electrónica se observó autofagosomas. Los principales ingredientes activos de C. tinctoria Nutt. incluyeron heriguard, flavanomarein, maritimin y marein. Veintiún de los 43 objetivos potenciales fueron PYGM, TLR2, RAF1, PRKAA2, GPR119, INS, CSF2, TNF, IAPP, AKR1B1, GSK3B, SYK, NFKB2, ESR2, CDK2, FGFR1, HTRA1, AMY2A, CAMK4, GCK y ABL2. Estos 21 objetivos principales se enriquecieron significativamente en 50 vías de señalización. Treinta y cuatro vías de señalización estuvieron estrechamente relacionadas con la nefropatía diabética, de las cuales las principales vías fueron PI3K/ AKT, insulina y mTOR, y resistencia a la insulina. Los términos GO enriquecidos incluyeron procesos biológicos de fosforilación proteica, la regulación positiva de la señalización de PI3K y la secreción de citoquinas; componentes celulares del citosol, región extracelular y espacio extracelular; y la función molecular de la actividad de la proteína quinasa, la unión de ATP y la actividad de la proteína tirosina quinasa que no se extiende por la membrana. Los experimentos in vitro encontraron que la mareína aumentaba la expresión de AKT/AKT fosforilada en células endoteliales glomerulares renales humanas en un modelo de resistencia a la insulina inducida por niveles elevados de glucosa, así como aumentaron y disminuyeron respectivamente, los niveles de las proteínas asociadas a los microtúbulos, LC3 y P62. C. tinctoria Nutt. tiene muchos principios activos, con ingredientes principales de heriguard, flavanomarein, maritimain y marein, y puede ejercer un efecto de nefropatía antidiabética a través de distintass vías de señalización y objetivos.

Marein regulates insulin resistance in diabetic nephropathy mice by inducing autophagy / Marein regula la resistencia a la insulina en ratones con nefropatía diabética induciendo la autofagia

SUMMARY: Marein is the main active substance of Coreopsis tinctoria nutt. It not only has anti-oxidation and anti-tumor effects, but also can lower blood lipid, prevent high blood glucose, improve insulin resistance, inhibit gluconeogenesis and promote glycogen synthesis. However, the exact mechanism of its action is still unclear. Here, we explored the effect and mechanism of Marein on insulin resistance. The mice were divided into db/m, db/db, metformin+db/db, and marein+db/db groups. The body weight and kidney weight were recorded. Serum biochemical and renal function tests were measured after 8 weeks of continuous administration. Kidney tissues were subjected to HE staining, PAS staining, and Masson staining. The effect of marein on PI3K/Akt signal and autophagy pathway was detected by Western blot. After 8 weeks of Marein intervention, the body weight and kidney weight of mice did not change significantly, but the fasting blood glucose and blood lipid levels were significantly reduced than db/db group. Marein significantly improved the insulin resistance index, increased serum adiponectin and improved glucose and lipid metabolism disorders of db/db mice. Moreover, marein improved the basement membrane thickness of glomeruli and tubules, improved glomerular sclerosis and tubular fibrosis, as well as renal insufficiency, thereby protecting kidney function and delaying the pathological damage. Furthermore, marein increased the expression of PI3K and the phosphorylation of Akt/Akt (Ser473), and promoted the expression of LC3II/I, Beclin1 and ATG5. Additionally, it promoted the expression of FGFR1 in the kidney of db/db mice, and promoted the increase of serum FGF21 and FGF23. Marein has a protective effect on the kidneys of diabetic mice. It protects diabetic nephropathy by regulating the IRS1/PI3K/Akt signaling pathway to improve insulin resistance. Therefore, marein may be an insulin sensitizer.

RESUMEN: Marein es la principal sustancia activa de Coreopsis tinctoria nutt. No solo tiene efectos antioxidantes y antitumorales, sino que también puede reducir los lípidos en sangre, prevenir la glucemia alta, mejorar la resistencia a la insulina, inhibir la gluconeogénesis y promover la síntesis de glucógeno. Sin embargo, el mecanismo exacto de su acción aún no está claro. Se analizó el efecto y el mecanismo de Marein sobre la resistencia a la insulina. Los ratones se dividieron en grupos db / m, db / db, metformina + db / db y mareína + db / db. Se registró el peso corporal y el peso de los riñones. Se midieron las pruebas de función renal y bioquímica sérica después de 8 semanas de administración continua. Los tejidos renales se sometieron a tinción HE, tinción PAS y tinción Masson. El efecto de la mareína sobre la señal de PI3K / Akt y la vía de autofagia se detectó mediante Western blot. Al término de 8 semanas de tratamiento con mareína, el peso corporal y el peso de los riñones de los ratones no cambiaron significativamente, pero los niveles de glucosa en sangre y lípidos en sangre en ayunas se redujeron significativamente en relación a los del grupo db / db. Marein mejoró significativamente el índice de resistencia a la insulina, aumentó la adiponectina sérica y mejoró los trastornos del metabolismo de la glucosa y los lípidos de los ratones db / db. Además, la mareína mejoró el grosor de la membrana basal de los glomérulos y túbulos, mejoró la esclerosis glomerular y la fibrosis tubular, así como la insuficiencia renal, protegiendo la función renal y retrasando el daño patológico. Además, la mareína aumentó la expresión de PI3K y la fosforilación de Akt / Akt (Ser473), y promovió la expresión de LC3II / I, Beclin1 y ATG5. Además, promovió la expresión de FGFR1 en el riñón de ratones db / db y el aumento de FGF21 y FGF23 en suero. Marein tiene un efecto protector sobre los riñones de ratones diabéticos. Protege la nefropatía diabética regulando la vía de señalización IRS1 / PI3K / Akt para mejorar la resistencia a la insulina. Por tanto, la mareína puede ser un sensibilizador a la insulina.

Marein ameliorates Ang II/hypoxia-induced abnormal glucolipid metabolism by modulating the HIF-1α/PPARα/γ pathway in H9c2 cells.

The objectives of this study were to investigate the effects of marein, a major bioactive compound in functional food Coreopsis tinctoria, in hypertrophic H9c2 cells. Treating angiotensin II/hypoxia-stimulated H9c2 cells with marein led to decreasing cell surface area, intracellular total protein, atrial natriuretic peptide, and free fatty acids levels, but increasing glucose level. Marein treatment decreased hypoxia inducible factor-1α (HIF-1α), peroxisome proliferator activated receptor γ (PPARγ), medium chain acyl-coenzyme A dehydrogenase, glucose transporter-4, and glycerol-3-phosphate acyltransferase protein expressions, and increased PPARα, fatty acid transport protein-1, carnitine palmitoyltransferase-1, and pyruvate dehydrogenase kinase-4 protein expressions. Similar results were observed in HIF-1α-overexpressing H9c2 cells, whereas these effects were abolished in siRNA-HIF-1α-transfected H9c2 cells. It was concluded that marein could ameliorate abnormal glucolipid metabolism in hypertrophic H9c2 cells, and the effects could be attributable to reduction of HIF-1α expression and subsequent regulation PPARα/γ-mediated lipogenic gene expressions.

Marein ameliorates diabetic nephropathy by inhibiting renal sodium glucose transporter 2 and activating the AMPK signaling pathway in db/db mice and high glucose-treated HK-2 cells.

Marein, an active component of the Coreopsis tinctoria Nutt. plant, is known to improve diabetic nephropathy (DN). However, its anti-diabetic functions in DN and potential mechanisms remain unclear. The aim of this study was to elucidate the effects and mechanisms of Marein in diabetic db/db mice with DN, and in high glucose-treated HK-2 cells. In vivo, treating diabetic db/db mice with Marein for 12 consecutive weeks restored diabetes-induced hyperglycemia and dyslipidemia, and ameliorated renal function deterioration, glomerulosclerosis, and renal ectopic lipid deposition. Marein exerted renoprotective effects by directly inhibiting renal tubule sodium glucose transporter 2 (SGLT2) expression, and then activating the AMP-activated protein kinase (AMPK)/acetyl CoA carboxylase (ACC)/peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) pathway in db/db mice. Meanwhile, Marein ameliorated fibrosis and inflammation by suppressing the pro-inflammatory factors interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1), and expression of the extracellular matrix proteins, fibronectin (FN) and collagen 1 (COL1) in diabetic mice. In vitro, MDCK monolayer cells were established to explore the characteristics of Marein transmembrane transport. Marein was found to be absorbed across the membrane at a medium level that involved active transport and this was mediated by SGLTs. In HK-2 cells, Marein decreased uptake of the fluorescent glucose analog, 2-NBDG, by 22 % by inhibiting SGLT2 expression. In high glucose-treated HK-2 cells, Marein decreased SGLT2 expression and increased phosphorylated (p)-AMPK/p-ACC to improve high glucose-induced cellular dysfunction. Furthermore, Marein treatment decreased SGLT2 expression in SGLT2-overexpressing HK-2 cells. In addition, molecular docking and dynamics analysis revealed that SGLT2 was a direct target of Marein. Collectively, our results demonstrated that Marein ameliorates DN by inhibiting renal SGLT2 and activating p-AMPK, suggesting Marein can potentially prevent DN by suppressing renal SGLT2 expression directly.

Marein protects human nucleus pulposus cells against high glucose-induced injury and extracellular matrix degradation at least partly by inhibition of ROS/NF-κB pathway.

Intervertebral disc degeneration (IDD), a major cause of discogenic low back pain, is a musculoskeletal disorder involving the apoptosis of nucleus pulposus cells (NPCs) and extracellular matrix (ECM) degradation. Marein is a major active flavonoid ingredient extracted from the hypoglycemic plant Coreopsis tinctoria with several beneficial biological activities including anti-diabetic effects. Nevertheless, there are no reports concerning the effects of marein on IDD. Our study aimed to evaluate the effects of marein on high glucose (HG)-induced injury and ECM degradation in human NPCs (HNPCs). CCK-8 assay was applied to evaluate cell viability. Flow cytometry analysis, a cell death detection ELISA, and caspase-3 activity assay were used to assess apoptosis. The mRNA expression of ECM-related proteins matrix metalloproteinase (MMP)-3, MMP-13, Collagen II, and aggrecan were determined by qRT-PCR. The changes of the nuclear factor-kappa B (NF-κB) pathway were examined by western blot. Stimulation with HG significantly reduced cell viability and induced apoptosis in HNPCs. Moreover, HG exposure increased MMP-3 and MMP-13 expression and decreased Collagen II and aggrecan expression in HNPCs. Notably, marein effectively alleviated HG-induced viability reduction, apoptosis and ECM degradation in HNPCs. We also found that marein inhibited HG-induced ROS generation and NF-κB activation in HNPCs. Inhibition of NF-κB pathway reinforced HG-induced injury and ECM degradation in HNPCs. In summary, marein protected HNPCs against HG-induced injury and ECM degradation at least partly by inhibiting the ROS/NF-κB pathway.

Marein inhibited oxidative stress and inflammation of high glucose induced HBZY-1 cells via AMPK signaling pathway / 中国药理学通报

Aim To study the role of marein mediated AMPK signaling pathway in delaying oxidative stress, inflammation and fibrotic protein expression in diabetic nephropathy ( DN ). Methods In vitro diabetic nephropathy model was established by HG + PA induced rat mesangial cells ( HBZY-1 ) , and the cultured HBZY-1 cells were divided into normal control group (NG), HG + PA( GlucoselOO mmol • L'1 + Palmitic acid 250 [imo\ • L'1, HG +PA) model group, HG + PA + marein with different doses of 25 p,mol • L"1, 50 p,mol • L"1, 100 jimol • L"1, and 200 junol • L"1 groups. MTS was used to detect the effect of marein on HBZY-1 cell proliferation, and the optimal concentration was selected. Western blot was used to test the protein expression of NOX4, TGF-fU, MCP-1, a-SMA, FN, Collagen VI. Adenosine monophosphate activated( AMPK) protein kinase family of AMPK7I, p-AMPK a expression were measured. Results Marein inhibited high glucose palmitate-induced proliferation of HBZY-1 cells, down-regulated NOX4, TGF-(31, MCP-1, cx-SMA, FN and Collagen VI expression in model cells. Meanwhile, marein up-regulated both AMPK 7I and p-AMPKa expression. Conclusions Marein may inhibit the HBZY-1 cell proliferation, oxidative stress, inflammation and fibrosis factors expression in HG + PA induced HBZY-1 cell by activating of both AMPK 7I and AMPK signaling pathway, thus delaying renal injury in diabetic nephropathy.

Protective effects of flavonoids from Coreopsis tinctoria Nutt. on experimental acute pancreatitis via Nrf-2/ARE-mediated antioxidant pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Oxidative stress is a prominent feature of clinical acute pancreatitis (AP). Coreopsis tinctoria has been used traditionally to treat pancreas disorders like diabetes mellitus in China and Portugal and its flavonoid-rich fraction contain the main phytochemicals that have antioxidant and anti-inflammatory activities. AIM OF THE STUDY: To investigate the effects of flavonoids isolated from C. tinctoria on experimental AP and explore the potential mechanism. MATERIALS AND METHODS: LC-MS based online technique was used to analyse and isolate targeted flavonoids from C. tinctoria. Freshly isolated mouse pancreatic acinar cells were treated with taurocholic acid sodium salt hydrate (NaT, 5 mM) with or without flavonoids. Fluorescence microscopy and a plate reader were used to determine necrotic cell death pathway activation (propidium iodide), reactive oxygen species (ROS) production (H2-DCFDA) and ATP depletion (luminescence) where appropriate. AP was induced by 7 repeated intraperitoneal caerulein injections (50 µg/kg) at hourly interval in mice or retrograde infusion of taurolithocholic acid 3-sulfate disodium salt (TLCS; 5 mM, 50 µL) into the pancreatic duct in mice or infusion of NaT (3.5%, 1 mL/kg) in rats. A flavonoid was intraperitoneally administered at 0, 4, and 8 h after the first caerulein injection or post-operation. Disease severity, oxidative stress and antioxidant markers were determined. RESULTS: Total flavonoids extract and flavonoids 1-6 (C1-C6) exhibited different capacities in reducing necrotic cell death pathway activation with 0.5 mM C1, (2 R,3 R)-taxifolin 7-O-ß-D-glucopyranoside, having the best effect. C1 also significantly reduced NaT-induced ROS production and ATP depletion. C1 at 12.5 mg/kg and 8.7 mg/kg (equivalent to 12.5 mg/kg for mice) significantly reduced histopathological, biochemical and immunological parameters in the caerulein-, TLCS- and NaT-induced AP models, respectively. C1 administration increased pancreatic nuclear factor erythroid 2-related factor 2 (Nrf2) and Nrf2-medicated haeme oxygenase-1 expression and elevated pancreatic antioxidant enzymes superoxide dismutase and glutathione peroxidase levels. CONCLUSIONS: Flavonoid C1 from C. tinctoria was protective in experimental AP and this effect may at least in part be attributed to its antioxidant effects by activation of Nrf2-mediated pathways. These results suggest the potential utilisation of C. tinctoria to treat AP.

Inhibition of metabolic disorders in vivo and in vitro by main constituent of Coreopsis tinctoria / 中草药·英文版

Objective: To investigate the effects of the ethyl acetate extract of Coreopsis tinctoria (EAEC) on insulin resistance (IR) in rats fed a high-fat diet. Methods: Male Sprague-Dawley (SD) rats were fed a HFD (60% fat) supplemented with EAEC for 8 weeks. The administration of EAEC to the rats with HFD-induced insulin resistance reduced hyperglycemia, plasma levels of insulin, and steatosis in the liver. Metabolomic study was used to analyze the metabolic levels of the high glucose-treated cells, control cells and marein-treated cells. Results: High glucose and high fat conditions caused a significant increase in blood glucose, insulin, serum TC, TG and LDL-C levels, leading to abnormal IR in rats. However, treatment with EAEC protects against HFD-induced IR by improving the fasting serum glucose homeostasis and lipid homeostasis. The high glucose conditions significantly decreased glycogen synthesis and increased PEPCK, G6Pase and Krebs cycle-related enzyme protein levels, leading to an abnormal metabolic state in HepG2 cells. However, treatment with marein improved IR by increasing glucose uptake and glycogen synthesis and by downregulating PEPCK and G6Pase protein levels. The statistical analysis of the HPLC/MS data demonstrated that marein could restore the normal metabolic state. Conclusion: The results revealed that EAEC ameliorates IR in rats, and marein has the potential to improve IR by ameliorating glucose metabolism disorders.

Marein protects against methylglyoxal-induced apoptosis by activating the AMPK pathway in PC12 cells.

Diabetic encephalopathy, which is characterized by cognitive decline and dementia, commonly occurs in patients with long-standing diabetes. Previous studies have suggested that methylglyoxal (MG), an endogenous toxic compound, plays an important role in diabetic complications such as cognitive impairment. MG induces neuronal apoptosis. To clarify whether marein, a major compound from the hypoglycemic plant Coreopsis tinctoria, prevents PC12 cell damage induced by MG, we cultured PC12 cells in the presence of MG and marein. Marein attenuated MG-induced changes in the mitochondrial membrane potential (ΔΨm), mitochondrial permeability transition pores (mPTPs), intracellular Ca2+ levels, the production of reactive oxygen species (ROS), glutathione (GSH)/glutathione disulfide (GSSG) and adenosine triphosphate (ATP), and the increase in the percentage of apoptotic cells. Marein also increased glyoxalase I (Glo1) activity, phospho-AMPKα (Thr172) and Bcl-2 expression and diminished the activation of Bax, caspase-3 and inhibitor of caspase-activated deoxyribonuclease (ICAD). Importantly, pretreatment of cells with marein diminished the compound C-induced inactivation of p-AMPK. Molecular docking simulation showed that marein interacted with the γ subunit of AMPK. In conclusion, we found for the first time that the neuroprotective effect of marein is due to a reduction of damage to mitochondria function and activation of the AMPK signal pathway. These results indicate that marein may be a potent compound for preventing/counteracting diabetic encephalopathy.

Protective effects of marein on high glucose-induced glucose metabolic disorder in HepG2 cells.

BACKGROUND: Our previous study has shown that Coreopsis tinctoria increases insulin sensitivity and regulates hepatic metabolism in high-fat diet (HFD)-induced insulin resistance rats. However, it is unclear whether or not marein, a major compound of C. tinctoria, could improve insulin resistance. Here we investigate the effect and mechanism of action of marein on improving insulin resistance in HepG2 cells. METHODS: We investigated the protective effects of marein in high glucose-induced human liver carcinoma cell HepG2. In kinase inhibitor studies, genistein, LY294002, STO-609 and compound C were added to HepG2 cells 1h before the addition of marein. Transfection with siRNA was used to knock down LKB1, and 2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG), an effective tracer, was used to detect glucose uptake. RESULTS: The results showed for the first time that marein significantly stimulates the phosphorylation of AMP-activated protein kinase (AMPK) and the Akt substrate of 160kDa (AS160) and enhanced the translocation of glucose transporter 1 (GLUT1) to the plasma membrane. Further study indicated that genistein (an insulin receptor tyrosine kinase inhibitor) altered the effect of marein on glucose uptake, and both LY294002 (a phosphatidylinositol 3-kinase inhibitor) and compound C (an AMP-activated protein kinase inhibitor) significantly decreased marein-stimulated 2-NBDG uptake. Additionally, marein-stimulated glucose uptake was blocked in the presence of STO-609, a CaMKK inhibitor; however, marein-stimulated AMPK phosphorylation was not blocked by LKB1 siRNA in HepG2 cells. Marein also inhibited the phosphorylation of insulin receptor substrate (IRS-1) at Ser 612, but inhibited GSK-3ß phosphorylation and increased glycogen synthesis. Moreover, marein significantly decreased the expression levels of FoxO1, G6Pase and PEPCK. CONCLUSIONS: Consequently, marein improved insulin resistance induced by high glucose in HepG2 cells through CaMKK/AMPK/GLUT1 to promote glucose uptake, through IRS/Akt/GSK-3ß to increase glycogen synthesis, and through Akt/FoxO1 to decrease gluconeogenesis. Marein could be a promising leading compound for the development of hypoglycemic agent or developed as an adjuvant drug for diabetes mellitus.