Curcumin prevents high glucose damage in retinal pigment epithelial cells through ERK1/2-mediated activation of the Nrf2/HO-1 pathway

To study the effects of curcumin on human retinal pigment epithelial (RPE) cells exposed to high glucose (HG) insult, we performed in vitro studies on RPE cells cultured both in normal and HG conditions to assess the effects of curcumin on the cell viability, nuclear factor erythroid 2-related factor 2 (Nrf2) expression, HO-1 activity, and ERK1/2 expression. RPE cells exposed to HG insult were treated with curcumin. The cell viability, apoptosis, HO-1 activity, ERK, and Nrf2 expression were evaluated. The data indicated that treatment with curcumin caused a significant decrease in terms of apoptosis. Further, curcumin was able to induce HO-1 expression via Nrf2 activation and counteracts the damage elicited by HG. The present study demonstrated that curcumin provides protection against HG-induced damage in RPE cells through the activation of Nrf2/HO-1 signaling that involves the ERK pathway, suggesting that curcumin may have therapeutic value in the treatment of diabetic retinopathy.     

Tagitinin C induces ferroptosis through PERK-Nrf2-HO-1 signaling pathway in colorectal cancer cells

Rationale: Colorectal cancer (CRC) is a common malignant tumor of the digestive system. However, the efficacy of surgery and chemotherapy is limited. Ferroptosis is an iron- and reactive oxygen species (ROS)-dependent form of regulated cell death (RCD) and plays a vital role in tumor suppression. Ferroptosis inducing agents have been studied extensively as a novel promising way to fight against therapy resistant cancers. The aim of this study is to investigate the mechanism of action of tagitinin C (TC), a natural product, as a novel ferroptosis inducer in tumor suppression.Methods: The response of CRC cells to tagitinin C was assessed by cell viability assay, clonogenic assay, transwell migration assay, cell cycle assay and apoptosis assay. Molecular approaches including Western blot, RNA sequencing, quantitative real-time PCR and immunofluorescence were employed as well.Results: Tagitinin C, a sesquiterpene lactone isolated from Tithonia diversifolia, inhibits the growth of colorectal cancer cells including HCT116 cells, and induced an oxidative cellular microenvironment resulting in ferroptosis of HCT116 cells. Tagitinin C-induced ferroptosis was accompanied with the attenuation of glutathione (GSH) levels and increased in lipid peroxidation. Mechanistically, tagitinin C induced endoplasmic reticulum (ER) stress and oxidative stress, thus activating nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). As a downstream gene (effector) of Nrf2, heme oxygenase-1 (HO-1) expression increased significantly with the treatment of tagitinin C. Upregulated HO-1 led to the increase in the labile iron pool, which promoted lipid peroxidation, meanwhile tagitinin C showed synergistic anti-tumor effect together with erastin.Conclusion: In summary, we provided the evidence that tagitinin C induces ferroptosis in colorectal cancer cells and has synergistic effect together with erastin. Mechanistically, tagitinin C induces ferroptosis through ER stress-mediated activation of PERK-Nrf2-HO-1 signaling pathway. Tagitinin C, identified as a novel ferroptosis inducer, may be effective chemosensitizer that can expand the efficacy and range of chemotherapeutic agents.     

Trigonelline induces autophagy to protect mesangial cells in response to high glucose via activating the miR-5189-5p-AMPK pathway

BackgroundDiabetic nephropathy (DN) is a primary cause of end‐stage renal disease. Increasing evidence indicates that microRNAs (miRNAs) are involved in DN pathogenesis. Trigonelline (TRL) has been shown to lower blood sugar and cholesterol levels, promote nerve regeneration, and exert anti-cancer and sedative properties.MethodThe effect of TRL on human mesangial cell (HMC) growth was assessed using the MTT assay. Differentially expressed miRNAs were validated using real-time quantitative polymerase chain reaction (real-time PCR). Bioinformatics, cell transfection, and Western blot analyses were utilized to confirm the binding of miR-5189-5p to HIF1AN. The effects of miR-5189-5 expression on cell proliferation were also assessed. Western blot analysis was used to determine the activation of multiple signaling molecules including phosphorylated-(p)-AMPK, SIRT1, LC3B, p62, and Beclin-1 in the autophagy pathway.ResultsTRL improved proliferation, increased the expression of miR-5189-5p, reduced HIF1AN, and restored the inhibition of autophagy in HMCs induced by high glucose. MiR-5189-5p mimics inhibited HIF1AN expression, and the miR-5189-5p inhibitor increased HIF1AN expression. MiR-5189-5p mimics significantly improved the proliferation of HMCs induced by high glucose, reduced the relative protein expression of p-AMPK, SIRT1, LC3B, and Beclin-1, and significantly increased the relative protein expression of p62.ConclusionWe showed that TRL up-regulated miR-5189-5p expression, activated the AMPK pathway, and activated autophagy in HMCs. Our study demonstrates that TRL could be a new treatment strategy to protect mesangial cells in response to high glucose.     

S1P2 receptor mediates sphingosine-1-phosphate-induced fibronectin expression via MAPK signaling pathway in mesangial cells under high glucose condition

Accumulation of extracellular matrix including fibronectin in mesangium is one of the major pathologic characteristics in diabetic nephropathy. In the current study, we explored role of sphingosine-1-phosphate (S1P) receptor in fibronectin expression and underlying molecular mechanism. Among five S1P receptors the mRNA level of S1P2 receptor was the most abundant in kidney of diabetic rats and mesangial cells under high glucose condition. S1P augmentation of fibronectin was significantly inhibited by S1P2 receptor antagonist JTE-013 and S1P2-siRNA. S1P-stimulated fibronectin expression was remarkably blocked by ERK1/2 inhibitor PD98059 and p38MAPK inhibitor SB203580. Phospho-ERK1/2 and phospho-p38MAPK level induced by S1P were markedly abrogated by JTE-013 and S1P2-siRNA. In conclusion, S1P2 receptor was significantly up-regulated under diabetic condition. S1P2 receptor mediated fibronectin expression through the activation of S1P-S1P2-MAPK (ERK1/2 and p38MAPK) axis in mesangial cells under high glucose condition, suggesting that it might be a potential therapeutic target for diabetic nephropathy treatment.     

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.     

Aldosterone promotes fibronectin production through a Smad2-dependent TGF-beta1 pathway in mesangial cells

Accumulating evidence demonstrates that aldosterone can cause extra-cellular matrix (ECM) accumulation, in addition to regulating sodium and potassium homeostasis. Increased extra-cellular matrix production by renal glomerular mesangial cells has been suggested to be involved in pathogenesis of glomerular sclerosis. The present studies examine whether aldosterone is also produced in renal mesangial cells, and the effect of aldosterone on ECM accumulation in these cells. In cultured renal mesangial cells, aldosterone synthase (CYP11B2), mineralocorticoid receptor (MR), and 11beta-HSD2 mRNA expressions were detected by RT-PCR. The ability of renal mesangial cells to produce aldosterone was confirmed by directly detecting aldosterone in culture medium via radioimmunoassay. Real-time RT-PCR showed that the expression of CYP11B2 mRNA in mesangial cells was significantly enhanced by AngII (P<0.001) and by potassium (P<0.05). Exposure of the cultured mesangial cells to aldosterone significantly increased fibronectin production from 12.4+/-1.9 to 74.6+/-16.8ng/ml (P<0.05). The aldosterone induced fibronectin production was abolished by aldosterone receptor antagonist spironolactone. Aldosterone also increased the TGF-beta1 reporter luciferase activity from 0.8+/-0.1 to 1.7+/-0.1 (P<0.05). Immunoblot showed TGF-beta1 protein expression was increased following aldosterone treatment. Blocking TGF-beta1 signaling pathway by knocking down Smad2 significantly blunted the aldosterone induced fibronectin production. The present studies indicate that renal mesangial cell is a target of local aldosterone action, which promotes ECM protein fibronectin production via TGF-beta1/Smad2 signaling pathway.     

Shizukaol A exerts anti-inflammatory effect by regulating HMGB1/Nrf2/HO-1 pathway

BackgroundSarcandra glabra (Thunb.) Makino (Chloranthaceae) has a long history of being used in Traditional Chinese medicines (TCMs) to treat painful joints, fractures, arthritis, and other diseases caused by inflammation. It has been reported that lindenane-type sesquiterpenoid dimers are main anti-inflammatory ingredient of S. glabra. Meanwhile, shizukaol A, the precursor of these sesquiterpene dimers, possesses a good inhibitory effect on nitric oxide (NO) in our previous study. But its anti-inflammatory mechanism is still unclear.PurposeThis study aimed to explore the possible anti-inflammatory mechanism and potential targets of shizukaol A in lipopolysaccharide (LPS)-induced RAW 264.7 cells.MethodsThe release of NO and inflammatory cytokines in LPS-stimulated RAW 264.7 cells were measured by Griess reagent and ELISA, respectively. The relevant proteins including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor kappa B (NF-κB) p65, High mobility group box 1 (HMGB1) were detected by western blot. Nuclear translocation of p65, HMGB1 and nuclear factor E2-related factor 2 (Nrf2) were examined by immunofluorescence. The level of reactive oxygen species (ROS) was tested by flow cytometry. The target of shizukaol A was investigated by molecular docking and Drug Affinity Responsive Target Stability (DARTS).ResultsShizukaol A had a good inhibitory effect on NO with half maximal inhibitory concentration (IC₅₀) of 13.79 ± 1.11 μM. Shizukaol A could down-regulate the expression of iNOS and COX-2. Further studies demonstrated that shizukaol A can significantly inhibit phosphorylation and nuclear translocation of NF-κB. Meanwhile, shizukaol A decreased the level of ROS and enhanced the expression of heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1). Furthermore, shizukaol A up-regulated the expression of Nrf2 and its nuclear translocation. More importantly, shizukaol A could inhibit activation of HMGB1 by targeting HMGB1.ConclusionShizukaol A inhibited inflammation by targeting HMGB1 to regulate the Nrf2/HO-1 signaling pathway. Thus, shizukaol A may be an attractive therapeutic candidate for inflammatory diseases.     

Effect of autophagy on ferroptosis in foam cells via Nrf2

Molecular and Cellular Biochemistry - The progression of atherosclerotic plaque is accelerated by death of foam cells during the development of the plaque. There are several forms of foam cell...     

Saikosaponin A alleviates Staphylococcus aureus-induced mastitis in mice by inhibiting ferroptosis via SIRT1/Nrf2 pathway

Mastitis is a common and serious bacterial infection of the mammary gland. Saikosaponin A (SSA) is a triterpenoid saponin isolated from Bupleurum falcatum that has the ability to treat various diseases. However, little is known about the role of SSA in achieving mastitis remission. Here, we found that SSA alleviated Staphylococcus aureus (S. aureus)-induced mastitis by attenuating inflammation and maintaining blood-milk barrier integrity. Furthermore, S. aureus activated nuclear factor kappa B (NF-κB) pathway by upregulated p-p65 and p-IκB. S. aureus also induced ferroptosis in mammary gland in mice, mainly characterized by excessive iron accumulation, mitochondrial morphological changes and impaired antioxidant production. However, S. aureus-induced NF-κB activation and ferroptosis were prevented by SSA. Moreover, SAA could upregulate the expression of SIRT1, Nrf2, HO-1 and GPX4. And the inhibitory effects of SAA on inflammation and ferroptosis were reversed by SIRT1 inhibitor EX-527. In conclusion, SAA protected S. aureus-induced mastitis through suppressing inflammation and ferroptosis by activating SIRT1/Nrf2 pathway.     

Sphingosine kinase-1 pathway mediates high glucose-induced fibronectin expression in glomerular mesangial cells

Diabetic nephropathy is characterized by accumulation of glomerular extracellular matrix proteins, such as fibronectin (FN). Here, we investigated whether sphingosine kinase (SphK)1 pathway is responsible for the elevated FN expression in diabetic nephropathy. The SphK1 pathway and FN expression were examined in streptozotocin-induced diabetic rat kidney and glomerular mesangial cells (GMC) exposed to high glucose (HG). FN up-regulation was concomitant with activation of the SphK1 pathway as reflected in an increase in the expression and activity of SphK1 and sphingosine 1-phosphate (S1P) production in both diabetic kidney and HG-treated GMC. Overexpression of wild-type SphK1 (SphK(WT)) significantly induced FN expression, whereas treatment with a SphK inhibitor, N,N-dimethylsphingosine, or transfection of SphK1 small interference RNA or dominant-negative SphK1 (SphK(G82D)) abolished HG-induced FN expression. Furthermore, addition of exogenous S1P significantly induced FN expression in GMC with an induction of activator protein 1 (AP-1) activity. Inhibition of AP-1 activity by curcumin attenuated the S1P-induced FN expression. Finally, by inhibiting SphK1 activity, both N,N-dimethylsphingosine and SphK(G82D) markedly attenuated the HG-induced AP-1 activity. Taken together, these results demonstrated that the SphK1 pathway plays a critical role in matrix accumulation in GMC under diabetic condition, suggesting that the SphK1 pathway could be a potential therapeutic target for diabetic nephropathy.     

Eriodictyol regulated ferroptosis,mitochondrial dysfunction,and cell viability via Nrf2/HO-1/NQO1 signaling pathway in ovarian cancer cells

This study aimed to investigate the antitumor effect and the underlying molecular mechanism of eriodictyol on ovarian cancer cells. CaoV3 and A2780 were exposed to eriodictyol at different concentrations of 0−800 μM. Cell apoptosis and viability were determined by TdT-mediated dUTP Nick-End Labeling (TUNEL) assay and Cell Counting Kit-8 (CCK-8) assay, respectively. Mitochondrial membrane potential was evaluated by flow cytometers with a JC-1 detection kit. Fe²⁺ content was evaluated using an iron assay kit. The section of tumor tissues was observed using hematoxylin-eosin (H&E) staining and nuclear factor erythroid 2-related factor 2 (Nrf2) expression was detected by immunohistochemistry (IHC) staining. Eriodictyol suppressed cell viability and induced cell apoptosis of CaoV3 and A2780 cells. Half maximal inhibitory concentration (IC₅₀) value of CaoV3 at 24 and 48 h was (229.74 ± 5.13) μM and (38.44 ± 4.68) μM, and IC₅₀ value of A2780 at 24 and 48 h was (248.32 ± 2.54) μM and (64.28 ± 3.19) μM. Fe²⁺ content and reactive oxygen species production were increased and protein levels of SLC7A11 and GPX4 were decreased by eriodictyol. Besides, eriodictyol reduced the ratio of JC-1 fluorescence ratio, glutathione and malondialdehyde contents but elevated Cytochrome C level. Nrf2 phosphorylation were obviously downregulated by eriodictyol. Finally, eriodictyol suppressed tumor growth, aggravated mitochondrial dysfunction and downregulated Nrf2 expression in tumor tissue in mice. Eriodictyol regulated ferroptosis, mitochondrial dysfunction and cell viability via Nrf2/HO-1/NQO1 signaling pathway in ovarian cancer.     

Acetaminophen sensitizing erastin-induced ferroptosis via modulation of Nrf2/heme oxygenase-1 signaling pathway in non-small-cell lung cancer

Growing evidence confirms that ferroptosis plays an important role in tumor growth inhibition. However, some non-small-cell lung cancer (NSCLC) cell lines are less sensitive to erastin-induced ferroptotic cell death. Elucidating the mechanism of resistance of cancer cells to erastin-induced ferroptosis and increasing the sensitivity of cancer cells to erastin need to be addressed. In our experiment, erastin and acetaminophen (APAP) cotreatment inhibited NSCLC cell viability and promoted ferroptosis and apoptosis, accompanied with attenuation of glutathione and ectopic increases in lipid peroxides. Erastin and APAP promoted NSCLC cell death by regulating nucleus translocation of nuclear factor erythroid 2-related factor 2 (Nrf2); and the ferroptosis induced by erastin and APAP was abrogated by bardoxolone methyl (BM) with less generation of reactive oxygen species and malondialdehyde. As a downstream gene of Nrf2, heme oxygenase-1 expression decreased significantly with the cotreatment of erastin and APAP, which could be rescued by BM. In vivo experiment showed that the combination of erastin and APAP had a synergic therapeutic effect on xenograft of lung cancer. In short, the present study develops a new effective treatment for NSCLC by synergizing erastin and APAP to induce ferroptosis.