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Cyclocarya paliurus (Batal.) leaves, which contain a range of bioactive compounds, have been used as a traditional Chinese medicine homologous food since ancient times. However, there is a paucity of literature on comprehensive studies of alkaloids in the leaves of Cyclocarya paliurus (Batal.). For the first time, this study aimed to discover and identify alkaloids extracted from Cyclocarya paliurus (Batal.) leaves by ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UPLC-QTOF-MS). A total of ten alkaloids have been identified from Cyclocarya paliurus (Batal.) leaves based on accurate mass spectra (mass accuracy, isotopic spacing and distribution) and comparison to fragmentation spectra reported in the literature. In vitro, alkaloids alleviated insulin resistance by increasing glucose consumption and glycogen content in insulin resistance HepG2 cells. The RNA-seq and western blotting results showed that alkaloids could upregulate the expression of phosphatidylinositol 3-kinase (PI3K), and increase the phosphorylation of insulin receptor protein kinase B (AKT). This study not only clarified the chemical constituents and revealed that diverse alkaloids also presented from Cyclocarya paliurus (Batal.) leaves, also, it will provide chemical information on potential compounds for developing new drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Advanced glycation end products (AGEs) are characterized diabetic metabolites inducing macrophage M1 polarization which is crucial in diabetes-exacerbated atherosclerosis. Matrine was proved anti-atherosclerotic. The current study was aimed to investigate the inhibitory effects of matrine on AGEs- induced macrophage M1 polarization and underlying molecular mechanisms. Primary mouse macrophages were exposed to AGEs. Receptor for AGEs (RAGE) and toll-like receptor 4 (TLR4) were over-expressed by vectors. Matrine was used to treat these cells. Inducible nitric oxide synthase (iNOS) expression and pro-inflammatory cytokine production were used to evaluate macrophage M1 polarization. Oxidative stress was assessed by intracellular reactive oxygen species (ROS) generation, total antioxidant capacity (TAC) and malondialdehyde (MDA) contents. Relative mRNA expression level was determined by real-time PCR. Western blotting was used to evaluate protein and protein phosphorylation levels. Bisulfite sequencing PCR (BSP) was used to evaluate DNA methylation. Matrine reduced AGEs exposure-elevated expressions of DNA methyltransferase (DNA MTase, DNMT)3a and DNMT3b in macrophages which were not affected by RAGE or TLR4 over expressions. DNA methylation rate of GPX1 promoter was reduced from 97.22% to 66.67% in AGEs- exposed macrophages treated by matrine. GPX1 expression was up-regulated by matrine, which further suppressed AGEs/RAGE-mediated oxidative stress. Thus, the activation of down-stream TLR4/STAT1 signaling pathway was inhibited by matrine treatment which eventually suppressed AGEs- induced macrophage M1 polarization. However, these effects of matrine were impaired by RAGE and TLR4 overexpression. Results from this study suggested that matrine inhibited AGEs- induced macrophage M1 polarization by suppressing RAGE-induced oxidative stress-mediated TLR4/STAT1 signaling pathway. Matrine exerted anti-oxidant effects via increasing GPX1 expression by inhibiting DNMT3a/b-induced GPX1 promoter DNA methylation., (Copyright © 2022. Published by Elsevier B.V.)

Advanced glycation end products (AGEs) induce vascular smooth muscle cells (VSMCs) contractile-synthetic phenotypic conversion which plays roles in aggravated atherosclerosis in diabetes. Matrine has been proved to suppress AGEs-induced phenotypic conversion which is governed by Notch pathway. Endoplasmic reticulum stress was associated with Notch pathway. Cultured human coronary smooth muscle cells (HCSMCs) were incubated with AGE-BSA at 0, 5 and 10 μmol/l. Specific siRNA was used to silence Protein kinase RNA-like ER kinase (PERK). Matrine at 0, 0.5 and 1.0 mmol/l were used to pre-treat the cells. Immunofluorescent staining of Smooth muscle myosin heavy chain 11 (MYH11) and smooth muscle α-actin 2 (ACTA2) were used to identify the contractile phenotype of HCSMCs. Protein phosphorylation and expression levels were evaluated by Western Blotting. AGE-BSA exposure facilitated the contractile-synthetic phenotypic conversion of HCSMCs in a concentration-dependent manner. AGE-BSA exposure increased expression levels of glucose-regulated protein 78 (GRP78), Delta-like 4 (Dll4), Notch intracellular domain (NICD1), Hes family basic helix-loop-helix (bHLH) transcriptional factor 1 (HES1), as well as the phosphorylation level of PERK. Specific perk-siRNA transfection dramatically lowered PERK phosphorylation and resulted in down-regulation of Dll4, NICD1 and HES1 in HCSMCs exposed to AGE-BSA. Pre-treatment of matrine suppressed AGE-BSA-induced phenotypic conversion of HCSMCs in a concentration-dependent manner. Moreover, matrine pre-treatment reduced expression level of GRP78, NICD1, HES1 and the phosphrylation level of PERK in AGE-BSA-exposed HCSMCs in a concentration-dependent manner. These results suggested that matrine suppressed AGE-BSA-induced HCSMCs phenotypic conversion via attenuating ER stress PERK signaling-dependent Dll4- Notch pathway activation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The fibrotic response of vascular smooth muscle cells (VSMCs) takes responsibilities in atherosclerosis. Advanced glycation end products (AGEs) induce and promote the fibrotic responses of VSMCs. Matrine shows potent anti-fibrotic and cardio-protective effects. This study was aimed to investigate the underlying mechanisms of matrine's inhibitory effects on AGEs-induced VSMCs fibrotic responses. Cultured human coronary smooth muscle cells (HCSMCs) were pre-treated with matrine and exposed to AGEs. Specific siRNA was used to silence polymerase delta interacting protein 2 (Poldip2) expression. Sircol collagen assay was used to assess collagen content. Protein expression and phosphorylation levels were determined by Western blotting. Matrine pre-treatment significantly reduced collagen content, increased smooth muscle myosin heavy chain 11 (MYH11) and Poldip2 expression, decreased expressions of collagen I, β1-integrin, phsphoinositide-3-kinase (PI3K) and nuclear phosphorylated p70S6k, and reduced phosphorylation levels of protein kinase B (Akt) and mechanistic target of rapamycin (mTOR) in HCSMCs exposed to AGEs in a concentration dependent manner. Specific siRNA effectively silenced Poldip2 expression and impaired matrine's effect on collagen content, expressions of MYH11, collagen I, β1-integrin, PI3K, nuclear p-p70S6k and phosphorylation levels of Akt and mTOR in HCSMCs exposed to AGEs. Matrine suppresses AGEs- induced fibrotic responses in HCSMCs via regulating Poldip2/mTOR signaling pathway., (Copyright © 2019 Elsevier B.V. All rights reserved.)

BACKGROUND The objective of this study was to study the anti-inflammatory effect and possibly involved molecular mechanisms of matrine on oxidized low-density lipoprotein (ox-LDL)-exposed macrophages. MATERIAL AND METHODS Cultured human macrophages (THP-1 cell line) were exposed to ox-LDL at final concentrations of 0, 25, 50, and 100 μg/mL. Several cells were then treated with matrine at serial diluted concentrations. 2,7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA) staining was used to evaluate reactive oxygen species (ROS) production; a colorimetric method was used to determine the cellular antioxidant capacity; production of pro-inflammatory cytokines interleukin (IL)18 and tumor necrosis factor (TNF)alpha were determined by enzyme-linked immunosorbent assay (ELISA); and immunoblot assay was used to assess the relative protein phosphorylation and expression. RESULTS ox-LDL exposure significantly elevated intracellular ROS level and supernatant IL18 and TNFalpha concentrations, but impaired total antioxidant capacity (TAC) of macrophages. The relative phosphorylations of MAPK kinase kinases (MKK)6, MKK3, and p38 mitogen-activated protein kinases (MAPK) were increased by ox-LDL exposure. The expression levels of IL18 and TNFalpha were also increased in ox-LDL-treated macrophages. The matrine treatment reduced intracellular ROS level and supernatant IL18 and TNFalpha concentrations and increased TAC in a concentration- dependent manner. The relative phosphorylations of MKK6, MKK3, and p38 MAPK were reduced after matrine administration. Moreover, the expression levels of IL18 and TNFalpha were also decreased by matrine treatment, in a concentration-dependent manner. CONCLUSIONS ox-LDL increases inflammatory response in macrophages by activating the ROS-mediated MKKs/p38 MAPK-induced inflammatory signaling pathway. Matrine suppresses ox-LDL-induced inflammatory by inhibiting the MKKs/p38 MAPK signaling pathway.

Featured by heart dysfunction, the diabetic cardiomyopathy is causing mortality and morbidity in type 2 diabetes mellitus patients. Matrine was considered as a cardioprotective agent. This study was aimed to investigate the therapeutic effects and molecular mechanisms of matrine on advanced glycation end products (AGEs)- induced cardiac dysfunctions. Rats and isolated primary myocytes were exposed to AGEs. Left ventricular hemodynamic parameters were used to assess the cardiac function. Cell apoptosis was detected with TUNEL assay. Calcium indicator was used to determine the intracellular calcium concentration ([Ca 2+ ] i ). The molecular coupling between FK506-binding protein 12.6 (FKBP12.6) and ryanodine receptor 2 (RyR2) was evaluated by immunoprecipitation. Apoptotic protein expressions were measured by western blotting. The activity of RyR2 was measured by [ 3 H]-ryanodine binding assay. AGEs exposure impaired systolic and diastolic functions and induced apoptosis in myocardium. AGEs exposure also elevated [Ca 2+ ] i , decreased mitochondrial membrane potential (MMP) and induced cell apoptosis in myocardium and cultured myocytes. AGEs impaired association between FKBP12.6 and RyR2 and further increased RyR2 activity in vivo and in vitro. The expression levels of cytochrome c and active caspase3 were elevated by AGEs exposure. Matrine treatment improved cardiac function in AGEs- exposed hearts. Matrine inhibited the disassociation of FKBP12.6 and RyR2, decreased the activity of RyR2, [Ca 2+ ] i, apoptosis, expression levels of cytochrome c and active caspase3 in vivo and in vitro. Matrine attenuated AGEs- induced cardiac dysfunctions by regulating RyR2- mediated calcium overload- triggered myocardial apoptosis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Vascular smooth muscle cells (VSMCs) contractile- synthetic phenotypic conversion takes responsibility in the atherosclerotic plaque formation by abnormal synthesis, secretion and deposition of extracellular matrix (ECM). Matrine exerts therapeutic effects on both cardiovascular diseases and organ fibrosis. In this study, we investigated matrine's inhibitory effect and mechanisms on AGEs- induced VSMC contractile- synthetic phenotypic conversion. Cultured human coronary smooth muscle cells (HCSMCs) were exposed to AGEs. Matrine at serially diluted concentrations were used to treat the cells. HCSMCs phenotype was identified by immunofluorescent staining of contractile phenotypic markers including mooth muscle myosin heavy chain (MYH11) and smooth muscle α-actin (ACTA2). Sircol collagen assay was used to assess the collagen secretion level. Notch signaling activation was determined by luciferase assay. Western blotting was used to evaluate expression levels of collagen I, collagen VIII, Delta-like (Dll)1, Dll3, Dll4, Jagged1, Jagged2, Notch intracellular domain (NICD)1 and Hes family basic helix-loop-helix (bHLH) transcription factor1 (HES1). Matrine pre-treatment recovered the AGEs- induced contractile- synthetic phenotypic conversion by increasing MYH11 and ACTA2 in HCSMCs. Matrine reduced AGEs- mediated activation of Notch signaling, down-regulated expression levels of NICD1, HES1, collagen I and collagen VIII and collagen secretion contents in HCSMCs. Matrine inhibited expression level of Dll4 without affecting other Notch ligands including Dll1, Dll3, Jagged1 and Jagged2 in HCSMCs exposed to AGEs. These results suggested that AGEs exposure facilitated the contractile- synthetic phenotypic conversion of HCSMCs. Matrine blocked this phenotypic conversion by suppressing Dll4- Notch signaling pathway activation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Diabetes is characterized by a high level of advanced glycation end products (AGEs), which induce damage in the arterial endothelium. Matrine has been shown to have cardioprotective effects. This study's aim was to investigate the protective effects and possible molecular mechanisms of matrine in AGE-induced human aortic endothelial cell (HAEC) injury. Cultured HAECs were treated with AGEs and/or serially diluted matrine. Cell viability was evaluated by MTT assay. Intracellular reactive oxygen species production was determined by flow cytometry. The expression levels of nucleotide-binding, leucine-rich repeat, and pyrin-domain-containing 3 (NLRP3), adaptor molecule apoptosis-associated speck-like protein, cleaved caspase-1 and interleukin-1β (IL-1β) were assessed by Western blotting; the concentration of IL-1β in culture supernatants was determined by enzyme-linked immunosorbent assay (ELISA). In a concentration-dependent manner, matrine co-treatment with AGEs substantially inhibited the reduction in cell viability and the increase in intracellular reactive oxygen species induced by AGEs. Co-treatment with matrine significantly inhibited the AGE-induced increase in NLRP3, ASC, caspase-1, p20 and IL-1β expression in HAECs in a concentration-dependent manner. Moreover, the AGE-mediated increase in IL-1β expression in cell culture supernatants was also reduced by co-treatment with matrine in a concentration-dependent manner. AGEs induced HAEC injury by inducing reactive oxygen species -mediated NLRP3 inflammasome activation. Matrine recovered HAEC viability by inhibiting reactive oxygen species -mediated NLRP3 inflammasome activation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cardiac fibrosis is one of the pathological characteristics of diabetic cardiomyopathy (DbCM). Matrine treatment has proven to be effective in cases of organ fibrosis and cardiovascular diseases. In the present study, the anti-fibrosis-associated cardioprotective effects of matrine on DbCM were investigated. Rats with experimental DbCM were administered matrine orally. Cardiac functions were evaluated using invasive hemodynamic examinations. Cardiac compliance was assessed in isolated hearts. Using Sirius Red and fluorescence staining, the collagen in diabetic hearts was visualized. MTT assay was used to select non‑cytotoxic concentrations of matrine, which were subsequently used to treat isolated cardiac fibroblasts incubated under various conditions. Western blotting was performed to assess activation of the transforming growth factor‑β1 (TGF‑β1)/Smad signaling pathway. Rats with DbCM exhibited impaired heart compliance and left ventricular (LV) functions. Excessive collagen deposition in cardiac tissue was also observed. Furthermore, TGF‑β1/R‑Smad (Smad2/3) signaling was revealed to be markedly activated; however, the expression of inhibitory Smad (I‑Smad, also termed Smad7) was reduced in DbCM. Matrine administration led to a marked recovery in LV function and heart compliance by exerting inhibitory effects on TGF‑β1/R‑Smad signaling pathway‑induced fibrosis without affecting I‑Smad. Incubation with a high concentration of glucose triggered the TGF‑β1/R‑Smad (Smad2/3) signaling pathway and suppressed I‑Smad signaling transduction in cultured cardiac fibroblasts, which led to an increase in the synthesis of collagen. After cardiac fibroblasts had been treated with matrine at non‑cytotoxic concentrations without affecting I‑Smad, matrine blocked TGF‑β1/R‑Smad signaling transduction to repress collagen production and deposition. In conclusion, the results of the present study demonstrated that TGF‑β1/Smad signaling‑associated cardiac fibrosis is involved in the impairment of heart compliance and LV dysfunction in DbCM. By exerting therapeutic effects against cardiac fibrosis via its influence on TGF‑β1/Smad signaling, matrine exhibited cardioprotective effects in DbCM.

Background: The matrine-type alkaloids are bioactive components extracted from Sophora flavescens , which is used in treatment of diabetes mellitus in traditional Chinese medicine. Advanced glycation end products mediate diabetic vascular complications. This study was aimed to investigate the protective effects and molecular mechanisms of matrine-type alkaloids on advanced glycation end products-induced reactive oxygen species-mediated endothelial apoptosis., Methods and Results: Rats aorta and cultured rat aortic endothelial cells were exposed to advanced glycation end products. Matrine-type alkaloids, p38 mitogen-activated protein kinase (MAPK) inhibitor, and small interference RNAs against p38 MAPK kinases MAPK kinase kinase (MKK)3 and MKK6 were administrated. Intracellular reactive oxygen species production, cell apoptosis, phosphorylation of MKKs/p38 MAPK, and expression levels of heme oxygenase/NADPH quinone oxidoreductase were assessed. The nuclear factor erythroid 2-related factor 2 nuclear translocation and the binding activity of nuclear factor erythroid 2-related factor 2 with antioxidant response element were also evaluated. Matrine-type alkaloids suppressed intracellular reactive oxygen species production and inhibited endothelial cell apoptosis in vivo and in vitro by recovering phosphorylation of MKK3/6 and p38 MAPK, nuclear factor erythroid 2-related factor 2 nuclear translocation, and antioxidant response element binding activity, as well as the expression levels of heme oxygenase/NADPH quinone oxidoreductase. p38 MAPK inhibitor treatment impaired the effects of matrine-type alkaloids in vivo and in vitro. MKK3/6 silencing impaired the effects of matrine-type alkaloids in vitro., Conclusions: Matrine-type alkaloids exert endothelial protective effects against advanced glycation end products induced reactive oxygen species-mediated apoptosis by targeting MKK3/6 and enhancing their phosphorylation., (© 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.)

Cardiac function and compliance impairments are the features of cardiac fibrosis. Matrine shows therapeutic effects on cardiovascular diseases and organ fibrosis. In this study, we examined the therapeutic effects and mechanisms of matrine on cardiac fibrosis of DbCM. Matrine was administrated orally to rats with DbCM. Cardiac functions and compliance were evaluated. The collagen deposition was visualized by sirius red staining. Real-time PCR was used to determine the expression level of miRNA. Western blotting was performed to assess the protein expression. NFAT nuclear translocation was evaluated by fluorescent immunochemistry staining and Western blotting. Intracellular calcium level was assessed by fura-2/AM staining. A colorimetric method was used to determine calcineurin enzymatic activity. Impaired cardiac function and compliance were observed in rats with DbCM. Increased collagen deposition in cardiac tissue was found. Furthermore, ATF6 signaling was activated, leading to intracellular calcium accumulation and NFAT activation which further initiated ECM gene expressions. Matrine administration recovered cardiac function and improved compliance by exerting inhibitory effects against ATF6 signaling- induced fibrosis. The high- glucose incubation induced ATF6 signaling activation in cultured CFs to increase the synthesis of ECM. Matrine blocked the ATF6 signaling in CFs to inhibit ECM synthesis within non- cytotoxic concentrations. ATF6 signaling induced cardiac fibrosis was one of the mechanisms involved in DbCM, which was characterized by loss of cardiac compliance and functions. Matrine attenuated cardiac compliance and improved left ventricular functions by exerting therapeutic effects against cardiac fibrosis via affecting ATF6 signaling pathway., (Copyright © 2017 Elsevier B.V. All rights reserved.)