Discovery of caffeoylisocitric acid as a Keap1-dependent Nrf2 activator and its effects in mesangial cells under high glucose.

Diabetic nephropathy (DN) is one of the severe microvascular complications of diabetes mellitus. Oxidative stress resulting from aberrant metabolism of glucose mediates renal inflammation and fibrosis in the progression of DN. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor regulating the expression of antioxidant enzymes. Activating Nrf2 will give a promising therapy for DN. To discover novel Nrf2 activators, we have investigated caffeoylisocitric acid using mesangial cells under high glucose. The results showed at 10 µM, caffeoylisocitric acid significantly inhibited the self-limited proliferation of mesangial cells induced by high glucose. Further assessments have disclosed caffeoylisocitric acid mitigated oxidative stress, inflammation and accumulation of extracellular matrix resulting from high glucose via inactivating MAPK signalling. Meanwhile activation of Nrf2 was observed and involved in these effects through the interaction between Keap1 and caffeoylisocitric acid to disrupt Keap1-Nrf2 complex. Therefore, caffeoylisocitric acid is a promising Nrf2 activator targeting DN.

Discovery of a coumarin derivative as Nrf2 activator mitigating oxidative stress and fibrosis in mesangial cells under high glucose.

Diabetic nephropathy (DN) is a severe microvascular complication of diabetes mellitus. Oxidative stress and fibrosis largely contribute to the progression of DN. Recently, Nrf2 was found to be a potential target preventing DN. In the discovery of novel Nrf2 activators for the treatment of DN, we have evaluated coumarin derivatives from Wikstroemi indiaca. Molecular docking results have shown compound 4 could bind to Keap1 and activate Nrf2 significantly. Cell-based assays have revealed compound 4 activated Nrf2 and attenuated oxidative stress and fibrosis induced by high glucose in mesangial cells. Meanwhile, it was validated that disruption of the interaction between Keap1 and Nrf2 was involved in the activation of Nrf2 by compound 4 in mesangial cells under high glucose.

Discovery of polypodiside as a Keap1-dependent Nrf2 activator attenuating oxidative stress and accumulation of extracellular matrix in glomerular mesangial cells under high glucose.

Diabetic nephropathy (DN) is a severe microvascular complication of diabetes mellitus. High glucose has resulted in oxidative stress and following renal fibrosis as the crucial nodes of this disease. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor regulating transcription of many antioxidant genes and suppressing synthesis of extracellular matrix. To discover Nrf2 activators targeting DN, we have evaluated polypodiside using cell-based assays. The results showed polypodiside inhibited the high glucose-induced self-limited proliferation of glomerular meangial cells. Activation of Nrf2 and enhanced transcription to antioxidant response elements were observed in the presence of polypodiside. Oxidative stress and accumulation of extracellular matrix induced by high glucose in glomerular meangial cells have been ameliorated by polypodiside. Further investigations revealed the effects of polypodiside on glomerular meangial cells were associated with activation of Nrf2. Co-immunoprecipitation of Nrf2 disclosed polypodiside disrupted the Kelch-like ECH-associated protein-1 (Keap1)-Nrf2 interaction. Molecular docking elucidated polypodiside could enter the Nrf2 binding cavity of Keap1 via interacting with the residues encompassing that cavity. These findings indicate polypodiside is a Keap1-dependent Nrf2 activator affording the catabatic effects against oxidative stress and accumulation of extracellular matrix in glomerular meangial cells under high glucose.

Correction to: Mammalian Target of Rapamycin 2 (MTOR2) and C-MYC Modulate Glucosamine-6-Phosphate Synthesis in Glioblastoma (GBM) Cells Through Glutamine: Fructose-6-Phosphate Aminotransferase 1 (GFAT1).

The original version of this article unfortunately contained an error in author group. The authors Yi-Xiang See, Xin Chen, Zi-Kai Chen and Ze-Bin Huang were inadvertently included in the article.

Mammalian Target of Rapamycin 2 (MTOR2) and C-MYC Modulate Glucosamine-6-Phosphate Synthesis in Glioblastoma (GBM) Cells Through Glutamine: Fructose-6-Phosphate Aminotransferase 1 (GFAT1).

Glucose and glutamine are two essential ingredients for cell growth. Glycolysis and glutaminolysis can be linked by glutamine: fructose-6-phosphate aminotransferase (GFAT, composed of GFAT1 and GFAT2) that catalyzes the synthesis of glucosamine-6-phosphate and glutamate by using fructose-6-phosphate and glutamine as substrates. The role of mammalian target of rapamycin (MTOR, composed of MTOR1 and MTOR2) in regulating glycolysis has been explored in human cancer cells. However, whether MTOR can interact with GFAT to regulate glucosamine-6-phosphate is poorly understood. In this study, we report that GFAT1 is essential to maintain the malignant features of GBM cells. And MTOR2 rather than MTOR1 plays a robust role in promoting GFAT1 protein activity, and accelerating the progression of glucosamine-6-phosphate synthesis, which is not controlled by the PI3K/AKT signaling. Intriguingly, high level of glucose or glutamine supply promotes MTOR2 protein activity. In turn, up-regulating glycolytic and glutaminolytic metabolisms block MTOR dimerization, enhancing the release of MTOR2 from the MTOR complex. As a transcriptional factor, C-MYC, directly targeted by MTOR2, promotes the relative mRNA expression level of GFAT1. Notably, our data reveal that GFAT1 immunoreactivity is positively correlated with the malignant grades of glioma patients. Kaplan-Meier assay reveals the correlations between patients' 5-year survival and high GFAT1 protein expression. Taken together, we propose that the MTOR2/C-MYC/GFAT1 axis is responsible for the modulation on the crosstalk between glycolysis and glutaminolysis in GBM cells. Under the condition of accelerated glycolytic and/or glutaminolytic metabolisms, the MTOR2/C-MYC/GFAT1 axis will be up-regulated in GBM cells.

Phytochemicals from fern species: potential for medicine applications.

Ferns are an important phytogenetic bridge between lower and higher plants. Historically they have been used in many ways by humans, including as ornamental plants, domestic utensils, foods, and in handicrafts. In addition, they have found uses as medicinal herbs. Ferns produce a wide array of secondary metabolites endowed with different bioactivities that could potentially be useful in the treatment of many diseases. However, there is currently relatively little information in the literature on the phytochemicals present in ferns and their pharmacological applications, and the most recent review of the literature on the occurrence, chemotaxonomy and physiological activity of fern secondary metabolites was published over 20 years ago, by Soeder (Bot Rev 51:442-536, 1985). Here, we provide an updated review of this field, covering recent findings concerning the bioactive phytochemicals and pharmacology of fern species.

[Analysis of Volatile Components of Polypodium hastatum by GC-MS].

OBJECTIVE: To further reveal the chemical constituents of Polypodium hastatum, volatile components from this plant were investigated. METHODS: The volatile components were extracted under reflux from the whole plant of Polypodium hastatum, and then analyzed qualitatively and quantitatively by GC-MS. RESULTS: 60 volatile components were detected and of all components detected, the structures and relative contents of 34 volatile compounds were elucidated. CONCLUSION: In the volatile components identified, most are fatty acid esters, especially methyl and ethyl esters, which compose the major volatile chemical constituents of Polypodium hastatum.

Study the interactions between human serum albumin and two antifungal drugs: fluconazole and its analogue DTP.

Binding affinities of fluconazole and its analogue 2-(2,4-dichlorophenyl)-1,3-di(1H-1,2,4-triazol-yl)-2-propanol (DTP) to human serum albumin (HSA) were investigated under approximately human physiological conditions. The obtained result indicated that HSA could generate fluorescent quenching by fluconazole and DTP because of the formation of non-fluorescent ground-state complexes. Binding parameters calculated from the Stern-Volmer and the Scatchard equations showed that fluconazole and DTP bind to HSA with binding affinities of the order 10(4)L/mol. The thermodynamic parameters revealed that the binding was characterized by negative enthalpy and positive entropy changes, suggesting that the binding reaction was exothermic. Hydrogen bonds and hydrophobic interaction were found to be the predominant intermolecular forces stabilizing the drug-protein. The effect of metal ions on the binding constants of fluconazole-HSA complex suggested that the presence of Mg(2+) and Zn(2+) ions could decrease the free drug level and extend the half-life in the systematic circulation. Docking experiments revealed that fluconazole and DTP binds in HSA mainly by hydrophobic interaction with the possibility of hydrogen bonds formation between the drugs and the residues Arg 222, Lys 199 and Lys 195 in HSA.

Natural products targeting human lactate dehydrogenases for cancer therapy: A mini review.

Reprogramming cancer metabolism has become the hallmark of cancer progression. As the key enzyme catalyzing the conversion of pyruvate to lactate in aerobic glycolysis of cancer cells, human lactate dehydrogenase (LDH) has been a promising target in the discovery of anticancer agents. Natural products are important sources of new drugs. Up to now, some natural compounds have been reported with the activity to target LDH. To give more information on the development of LDH inhibitors and application of natural products, herein, we reviewed the natural compounds with inhibition of LDH from diverse structures and discussed the future direction of the discovery of natural LDH inhibitors for cancer therapy.

Swinhoeic acid from Potentilla fragarioides ameliorates high glucose-induced oxidative stress and accumulation of ECM in mesangial cells via Keap1-dependent activation of Nrf2.

OBJECTIVES: Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes mellitus. Oxidative stress resulting from high glucose promotes accumulation of ECM and development of DN. Activation of Nrf2 could attenuate oxidative stress and following accumulation of ECM. To find novel therapy for DN, we explored the effects of swinhoeic acid from Potentilla fragarioides on mesangial cells under high glucose and underlying mechanisms. METHODS: CCK-8 and BrdU incorporation assays for survival of mesangial cells gave the concentration of swinhoeic acid in following investigations. ROS, MDA, SOD and CAT were determined. And ECM proteins and their upstream regulators TGF-ß1 and CTGF were detected using ELISA assays. Activation of Nrf2 was explored by immunofluorescence staining together with luciferase reporter assay. To demonstrate the role of Nrf2 activation, siRNA interference was performed. And co-immunoprecipitation assay was used to elucidate swinhoeic acid affects the interaction between Keap1 and Nrf2. RESULTS: Swinhoeic acid at 10 and 20 µM attenuated oxidative stress and accumulation of ECM in mesangial cells under high glucose. Itactivated Nrf2 in a Keap1-dependent manner, which was involved in its effects. CONCLUSION: Swinhoeic acid ameliorates oxidative stress and accumulation of ECM resulting from high glucose in mesangial cells via activating Nrf2 in Keap1-dependent manner.

[Studies on the chemical constituents from the roots of Kalopanax septemlobus].

OBJECTIVE: To investigate the chemical constituents of Kalopanax septemlobus. METHODS: Chromatographic techniques including silica gel, gel, semi-preparative HPLC and PTLC as well as recrystallization were employed in the isolation and purification, and the structures were elucidated by spectral analysis and physical and chemical properties. RESULTS: 6 compounds were identified as liriodendrin (1), (-) -syringarenol (2), trans-coniferyl aldehyde (3), trans-caffeic acid (4), beta-daucosterol (5), beta-sitosterol (6). CONCLUSION: Compounds 2 -5 are obtained from this genus for the first time.

Phelligridin D from Inonotus obliquus attenuates oxidative stress and accumulation of ECM in mesangial cells under high glucose via activating Nrf2.

Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes mellitus and becomes the financial burden and health problem. Pathogenesis of DN has revealed that high glucose has resulted in the oxidative stress and accumulation of extracellular matrix (ECM). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor regulating the expression of anti-oxidant enzymes. Therefore, activating Nrf2 gives a promising approach for the treatment of DN. In the discovery of bioactive phytochemicals targeting DN, we have identified phelligridin D from Inonotus obliquus and explored its protective effects against oxidative stress and accumulation of ECM using mesangial cells under high glucose and potential mechanisms. In addition to inhibiting the self-limited proliferation of mesangial cells cultured in high glucose, phelligridin D can attenuate oxidative stress through reducing reactive oxygen species (ROS) and malondialdehyde (MDA) as well as elevating the activity of superoxide dismutase (SOD) and catalase (CAT). Meanwhile, the major components of ECM including collagen IV, fibronectin and laminin were decreased by phelligridin D via inhibiting the secretion of transforming growth factor-ß1 (TGF-ß1) and downstream connective tissue growth factor (CTGF). Further investigations have revealed phelligridin D activated Nrf2 in mesangial cells under high glucose, which was involved in its protective effects. These findings can provide evidences for the discovery of novel therapy targeting DN and application of I. obliquus in practice.

Wikstromol from Wikstroemia indica induces apoptosis and suppresses migration of MDA-MB-231 cells via inhibiting PI3K/Akt pathway.

Triple negative breast cancer (TNBC) is the most severe type of breast cancer due to the lack of specific targets and rapid metastasis, which result in the poor prognosis. Recently, phosphatidylinositol 3-kinase (PI3K)/Akt pathway has emerged as a potential target for the treatment of TNBC. In our research interest to discover phytochemicals targeting TNBC, we have investigated wikstromol from Wikstroemia indica using the human TNBC MDA-MB-231 cells. The results showed wikstromol at 10 µM inhibited cell growth of MDA-MB-231 cells which was confirmed by MTT assay. Further DAPI staining has revealed wikstromol at 10 µM induced apoptosis of cancer cells, which was associated with the activation of caspase-3 following down-regulation of Bcl-2 as well as up-regulation of Bax, cleaved PARP and phosphorylated p53. Meanwhile, it was observed at 0.1 µM wikstromol suppressed the migration of the cancer cells via decreasing transcription of NF-κB and reducing activity and secretion of downstream MMP-9. In addition, p-PI3K and p-Akt were down-regulated in MDA-MB-231 cells in the presence of wikstromol at 0.1 µM, which indicated inactivation of PI3K/Akt pathway was involved in these inhibitory effects.

[Studies on the chemical constituents of Wikstroemia indica].

OBJECTIVE: To study the chemical constituents of Wikstroemia indica. METHODS: The constituents were isolated and purified by silica gel chromatography repeatedly, and the structures were determined by spectral data and chemical envidance. RESULTS: Six compounds were isolated from its petroleum ether, dichloromethane, acetone and methanol extracts and identified as: daphnoretin-7-O-beta-D-glucoside (1), aloe-emodin (2), kaempferol (3), 29-nonacosanolide (4), 1-octadecanol (5), beta-sitosterol (6). CONCLUSION: Compounds 2, 4, 5 are isolated from this plant for the first time.

Theaflavic acid from black tea protects PC12 cells against ROS-mediated mitochondrial apoptosis induced by OGD/R via activating Nrf2/ARE signaling pathway.

Cerebral ischemic stroke is a severe disease afflicting people worldwide. Phytochemicals play a pivotal role in the discovery of novel therapeutic approaches for the prevention of ischemic stroke. In our continual search for bioactive natural products for the treatment of ischemic stroke, we have evaluated the protective effects of theaflavic acid (TFA) from black tea using PC12 cells injured by oxygen and glucose deprivation/restoration (OGD/R), and investigated the possible mechanisms. The results showed that TFA can protect PC12 cells against OGD/R through increasing cell viability and decreasing intracellular lactate dehydrogenase (LDH) release. Further investigations found that TFA could inhibit the overproduction of intracellular reactive oxygen species (ROS), reduce malondialdehyde content, and elevate superoxide dismutase activity, which implied that TFA suppresses oxidative stress in PC12 cells induced by OGD/R. In addition, overload of intracellular calcium and collapse of the mitochondrial membrane potential were improved in the presence of TFA, and the activity of caspase-3 was significantly reduced by TFA. Western blot analysis showed that the expression of Bcl-2 was up-regulated while Bax was down-regulated. Therefore, it can be concluded that TFA can inhibit mitochondria-dependent apoptosis of PC12 cells induced by OGD/R. In addition, activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response elements (ARE) signaling pathway was explored to elucidate the mechanism by which TFA inhibits ROS-mediated apoptosis in PC12 cells. The results revealed that TFA promoted the translocation of Nrf2 into nuclei, enhanced the transcriptional activity of ARE, and up-regulated expression of downstream HO-1, which indicates that the Nrf2/ARE signaling pathway is involved in the protection by TFA of PC12 cells injured by OGD/R.

Kalopanaxsaponin A induces reactive oxygen species mediated mitochondrial dysfunction and cell membrane destruction in Candida albicans.

Candidiasis causes high morbidity and mortality among immunocompromised patients. Antifungal drug resistance and cytotoxicity highlight the need of effective antifungal therapeutics. In this study, we found that kalopanaxsaponin A (KPA), a triterpenoid saponin natural product, could inhibit the proliferation of various Candida species, and exerted a fungicidal effect against C. albicans. To further explore its antifungal action mode, spectrofluorophotometer, fluorescence microscopy and transmission electron microscopy were performed, showing that KPA treatment induced the accumulation of intracellular reactive oxygen species (ROS), resulting in mitochondrial dysfunction. Meanwhile, KPA treatment also broke down the membrane barrier of C. albicans causing the leakage of intracellular trehalose, the entrance of extracellular impermeable substance and the decrease of ergosterol content. Both ROS accumulation and membrane destruction contributed to the death of C. albicans cells. Our work preliminarily elucidated the potential mechanisms of KPA against C. albicans on a cellular level, and might provide a potential option for the treatment of clinical candidiasis.

Anticandidal Activity of Kalopanaxsaponin A: Effect on Proliferation, Cell Morphology, and Key Virulence Attributes of Candida albicans.

BACKGROUND: The pathogenicity of Candida albicans is attributed to various virulence factors including adhesion to the surface of epithelial cells or mucosa, germ tube formation, hyphal morphogenesis, development of drug resistant biofilms, and so on. The objective of this study was to investigate the effects of Kalopanaxsaponin A (KPA) on the virulence of C. albicans. METHODS: The effect of KPA on the virulence of C. albicans was characterized by an XTT reduction assay and fluorescent microscopic observation. The action mechanism was further explored using GC/MS system and BioTek Synergy2 spectrofluorophotometry. The cytotoxicity and therapeutic effect of KPA were evaluated by the Caenorhabditis elegans-C. albicans infection model in vivo. RESULTS: The minimum inhibitory concentration (MIC) of KPA was 8∼16 µg/mL for various genotypes of C. albicans. The compound was identified as having remarkable effect on the adhesion, morphological transition and biofilm formation of C. albicans. The results of fluorescent microscopy and GC/MS system suggested that KPA could promote the secretion of farnesol by regulating the expression of Dpp3 and decrease the intracellular cAMP level, which together inhibited morphological transition and biofilm formation. Notably, KPA showed low toxicity in vivo and a low possibility of developing resistance. CONCLUSION: Our results demonstrated that KPA had remarkable efficacy against C. albicans pathogenicity, suggesting that it could be a potential option for the clinical treatment of candidiasis.

Sikokianin A from Wikstroemia indica protects PC12 cells against OGD/R-induced injury via inhibiting oxidative stress and activating Nrf2.

Ischemic cerebral stroke is a severe cause of human death and disability. Natural products play an important role in the discovery of novel therapy for cerebral ischemia. Herein, we investigate the neuroprotective effects of sikokianin A identifiedfrom Wikstroemia indica using PC12 cell exposed to OGD/R. The results revealed sikokianin A can improve the poor viability and release of intracellular LDH in PC12 cells induced by OGD/R. Further studies have demonstrated the increased ROS and MDA together with reduced SOD activity were attenuated by sikokianin A. Meanwhile, decreased mitochondrial membrane potential, activated Caspase-3, down-regulated Bcl-2 and up-regulated Bax were reversed. These results indicate the protective effects of sikokianin A are associated with inhibiting oxidative stress and apoptosis resulting from OGD/R. Additionally, sikokianin A can activate Nrf2 and downstream HO-1 in PC12 cells treated by OGD/R, which implied Nrf2/HO-1 signaling pathway was involved in the protective effects of sikokianin A.

Probing the binding interaction of AKR with human serum albumin by multiple fluorescence spectroscopy and molecular modeling.

Human serum albumin (HSA) is the major transport protein affording endogenous and exogenous substances in plasma. It can affect the behavior and efficacy of chemicals in vivo through the binding interaction. AKR (3-O-α-l-arabinofuranosyl-kaempferol-7-O-α-l-rhamnopyranoside) is a flavonoid diglycoside with modulation of estrogen receptors (ERs). Herein, we investigated the binding interaction between AKR and HSA by multiple fluorescence spectroscopy and molecular modeling. As a result, AKR specifically binds in site I of HSA through hydrogen bonds, van der Waals force, and electrostatic interaction. The formation of AKR-HSA complex in binding process is spontaneously exothermic and leads to the static fluorescence quenching through affecting the microenvironment around the fluorophores. The complex also affects the backbone of HSA and makes AKR access to fluorophores. Molecular modeling gives the visualization of the interaction between AKR and HSA as well as ERs. The affinity of AKR with HSA is higher than the competitive site marker Warfarin. In addition, docking studies reveal the binding interaction of AKR with ERs through hydrogen bonds, van der Waals force, hydrophobic, and electrostatic interactions. And AKR is more favorable to ERß. These results unravel the binding interaction of AKR with HSA and mechanism as an ERs modulator.

Insight into the binding interaction of kaempferol-7-O-α-L-rhamnopyranoside with human serum albumin by multiple fluorescence spectroscopy and molecular modeling.

Human serum albumin (HSA) is a transporting protein that has multiple functions. The binding interaction between HSA and small molecules affects its function and efficacy of small molecules. The present study reports that kaempferol-7-O-α-L-rhamnopyranoside (KR) interacts with HSA as indicated by multiple fluorescence spectroscopy and molecular modeling. KR can quench the intrinsic fluorescence of HSA through the formation of a KR-HSA complex in a static manner. In addition, the binding site is located in subdomain IIA as confirmed by competitive experiments using site-specific warfarin and ibuprofen, and the driving forces include hydrogen bonds, van der Waals forces and electrostatic interaction derived from a thermodynamic analysis. The formation of KR-HSA is exothermic and spontaneous. Although there is no hydrophobic interaction around Tyr and Trp residues, the secondary structure of HSA changes through the formation of the KR-HSA complex. In addition, docking results visualized and further supported these results. Finally, these results can provide more information to further investigate the use of KR on the prevention of diabetic complications.