Novel C3-Methylene-Bridged Indole Derivatives with and without Substituents at N1: The Influence of Substituents on Their Hemolytic, Cytoprotective, and Antimicrobial Activity.

Alkaloids are natural compounds useful as scaffolds for discovering new bioactive molecules. This study utilized alkaloid gramine to synthesize two groups of C3-substituted indole derivatives, which were either functionalized at N1 or not. The compounds were characterized by spectroscopic methods. The protective effects of the new compounds against in vitro oxidative hemolysis induced by standard oxidant 2,2'-azobis(2-amidinopropane dihydro chloride (AAPH) on human erythrocytes as a cell model were investigated. Additionally, the compounds were screened for antimicrobial activity. The results indicated that most of the indole derivatives devoid of the N1 substitution exhibited strong cytoprotective properties. The docking studies supported the affinities of selected indole-based ligands as potential antioxidants. Furthermore, the derivatives obtained exhibited potent fungicidal properties. The structures of the eight derivatives possessing indole moiety bridged to the imidazole-, benzimidazole-, thiazole-, benzothiazole-, and 5-methylbenzothiazoline-2-thiones were determined by X-ray diffraction. The C=S bond lengths in the thioamide fragment pointed to the involvement of zwitterionic structures of varying contribution. The predominance of zwitterionic mesomers may explain the lack of cytoprotective properties, while steric effects, which limit multiple the hydrogen-bond acceptor properties of a thione sulfur, seem to be responsible for the high hemolytic activity.

Comparison of the Antioxidant and Cytoprotective Properties of Extracts from Different Cultivars of Cornus mas L.

Cornus mas L. is a rich source of vitamin C and polyphenols. Due to their health-benefit properties, C. mas L. extracts have been used in, e.g., dermatology and cosmetology, and as a food supplement. Peroxisome proliferator-activated receptor gamma (PPARγ) and its co-activator (PGC-1α) are now suspected to be the main target of active substances from C. mass extracts, especially polyphenols. Moreover, the PPARγ pathway is involved in the development of different diseases, such as type 2 diabetes mellitus (DM2), cancers, skin irritation, and inflammation. Therefore, the aim of the present study was to evaluate the PPARγ pathway activation by the most popular water and ethanol extracts from specific C. mas L. cultivars in an in vitro model of the human normal fibroblast (BJ) cell line. We analyzed the content of biologically active compounds in the extracts using the UPLC-DAD-MS technique and revealed the presence of many polyphenols, including gallic, quinic, protocatechuic, chlorogenic, and ellagic acids as well as iridoids, with loganic acid being the predominant component. In addition, the extracts contained cyanidin 3-O-galactoside, pelargonidin 3-O-glucoside, and quercetin 3-glucuronide. The water-ethanol dark red extract (DRE) showed the strongest antioxidant activity. Cytotoxicity was assessed in a normal skin cell line, and positive effects of all the extracts with concentrations ranging from 10 to 1000 µg/mL on the cells were shown. Our data show that the studied extracts activate the PPARγ/PGC-1α molecular pathway in BJ cells and, through this mechanism, initiate antioxidant response. Moreover, the activation of this molecular pathway may increase insulin sensitivity in DM2 and reduce skin irritation.

Artemisinin Confers Cytoprotection toward Hydrogen Peroxide-Induced Cell Apoptosis in Retinal Pigment Epithelial Cells in Correlation with the Increased Acetylation of Histone H4 at Lysine 8.

Increased oxidative stress is one of the critical pathologies inducing age-related macular degeneration (AMD), characterized by retinal pigment epithelial (RPE) cell damage and death. The unbalanced acetylation and deacetylation of histones have been implicated in AMD pathogenesis or hydrogen peroxide (H2O2)-induced cell damage. Therefore, strategies aimed at controlling the balance between acetylation and deacetylation may effectively protect RPE cells from oxidative damage. Artemisinin is an antimalarial lactone drug derived from Artemisia annua, with antioxidant activity known to modulate histone acetylation in the brain, but its effect on the retina is unknown. In this study, we aimed to investigate whether Artemisinin exerts a cytoprotective effect on oxidative stress-induced apoptosis in RPE cells by regulating histone acetylation. We hypothesized that Artemisinin confers cytoprotection toward H2O2-induced apoptosis in RPE cells through this mechanism. In the present study, we found that Artemisinin at a sub-clinic dosage of 20 µM inhibited the H2O2-induced cell viability decrease and B-cell lymphoma 2 (Bcl-2) protein level decrease and attenuated the H2O2-induced decrease in the histone H4 lysine (Lys) 8 acetylation [Acetyl-H4 (Lys 8)] level in the retinal RPE cell line D407. As expected, histone deacetylase inhibitor Trichostatin A at the concentration of 250 nM increased the Acetyl-H4 (Lys 8) level in D407 cells and attenuated the H2O2-induced cell viability decrease and apoptosis. Similar findings were obtained using adult RPE (ARPE)19 cells, another human RPE cell line, and primary human RPE cell cultures. In conclusion, these results confirmed our hypothesis and indicated that Artemisinin attenuated H2O2-induced apoptosis in apparent correlation with the increase in the Acetyl-H4 (Lys 8) level, which is associated with gene transcription and cell survival. By modulating histone acetylation, Artemisinin may restore the balance between acetylation and deacetylation and enhance the resistance and survival of RPE cells under oxidative stress. Our study provides novel mechanistic insights into the effect of Artemisinin on histone acetylation and apoptosis in RPE cells and supports the potential application of Artemisinin in the prevention and/or treatment of AMD.

Letrozole protects against cadmium-induced inhibition of spermatogenesis via LHCGR and Hsd3b6 to activate testosterone synthesis in mice.

The heavy metal cadmium is proposed to be one of the environmental endocrine disruptors of spermatogenesis. Cadmium-induced inhibition of spermatogenesis is associated with a hormone secretion disorder. Letrozole is an aromatase inhibitor that increases peripheral androgen levels and stimulates spermatogenesis. However, the potential protective effects of letrozole on cadmium-induced reproductive toxicity remain to be elucidated. In this study, male mice were administered CdCl2 (4 mg/kg BW) orally by gavage alone or in combination with letrozole (0.25 mg/kg BW) for 30 days. Cd exposure caused a significant decreases in body weight, sperm count, motility, vitality, and plasma testosterone levels. Histopathological changes revealed extensive vacuolization and decreased spermatozoa in the lumen. However, in the Cd + letrozole group, letrozole treatment compensated for deficits in sperm parameters (count, motility, and vitality) induced by Cd. Letrozole treatment significantly increased serum testosterone levels, which were reduced by Cd. Histopathological studies revealed a systematic array of all germ cells, a preserved basement membrane and relatively less vacuolization. For a mechanistic examination, RNA-seq was used to profile alterations in gene expression in response to letrozole. Compared with that in the Cd-treated group, RNA-Seq analysis showed that 214 genes were differentially expressed in the presence of letrozole. Gene ontology (GO) enrichment analysis and KEGG signaling pathway analysis showed that steroid biosynthetic processes were the processes most affected by letrozole treatment. Furthermore, we found that the expression of the testosterone synthesis-related genes LHCGR (luteinizing hormone/choriogonadotropin receptor) and Hsd3b6 (3 beta- and steroid delta-isomerase 6) was significantly downregulated in Cd-treated testes, but these genes maintained similar expression levels in letrozole-treated testes as those in the control group. However, the transcription levels of inflammatory cytokines, such as IL-1ß and IL-6, and oxidative stress-related genes (Nrf2, Nqo1, and Ho-1) showed no changes. The present study suggests that the potential protective effect of letrozole on Cd-induced reproductive toxicity might be mediated by the upregulation of LHCGR and Hsd3b6, which would beneficially increase testosterone synthesis to achieve optimum protection of sperm quality and spermatogenesis.

Formyl peptide receptor 2 determines sex-specific differences in the progression of nonalcoholic fatty liver disease and steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is an important health concern worldwide and progresses into nonalcoholic steatohepatitis (NASH). Although prevalence and severity of NAFLD/NASH are higher in men than premenopausal women, it remains unclear how sex affects NAFLD/NASH pathophysiology. Formyl peptide receptor 2 (FPR2) modulates inflammatory responses in several organs; however, its role in the liver is unknown. Here we show that FPR2 mediates sex-specific responses to diet-induced NAFLD/NASH. NASH-like liver injury was induced in both sexes during choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) feeding, but compared with females, male mice had more severe hepatic damage. Fpr2 was more highly expressed in hepatocytes and healthy livers from females than males, and FPR2 deletion exacerbated liver damage in CDAHFD-fed female mice. Estradiol induced Fpr2 expression, which protected hepatocytes and the liver from damage. In conclusion, our results demonstrate that FPR2 mediates sex-specific responses to diet-induced NAFLD/NASH, suggesting a novel therapeutic target for NAFLD/NASH.

A Major Intestinal Catabolite of Quercetin Glycosides, 3-Hydroxyphenylacetic Acid, Protects the Hepatocytes from the Acetaldehyde-Induced Cytotoxicity through the Enhancement of the Total Aldehyde Dehydrogenase Activity.

Aldehyde dehydrogenases (ALDHs) are the major enzyme superfamily for the aldehyde metabolism. Since the ALDH polymorphism leads to the accumulation of acetaldehyde, we considered that the enhancement of the liver ALDH activity by certain food ingredients could help prevent alcohol-induced chronic diseases. Here, we evaluated the modulating effects of 3-hydroxyphenylacetic acid (OPAC), the major metabolite of quercetin glycosides, on the ALDH activity and acetaldehyde-induced cytotoxicity in the cultured cell models. OPAC significantly enhanced the total ALDH activity not only in mouse hepatoma Hepa1c1c7 cells, but also in human hepatoma HepG2 cells. OPAC significantly increased not only the nuclear level of aryl hydrocarbon receptor (AhR), but also the AhR-dependent reporter gene expression, though not the nuclear factor erythroid-2-related factor 2 (Nrf2)-dependent one. The pretreatment of OPAC at the concentration required for the ALDH upregulation completely inhibited the acetaldehyde-induced cytotoxicity. Silencing AhR impaired the resistant effect of OPAC against acetaldehyde. These results strongly suggested that OPAC protects the cells from the acetaldehyde-induced cytotoxicity, mainly through the AhR-dependent and Nrf2-independent enhancement of the total ALDH activity. Our findings suggest that OPAC has a protective potential in hepatocyte models and could offer a new preventive possibility of quercetin glycosides for targeting alcohol-induced chronic diseases.

Protective Effect of Quercetin 3-O-Glucuronide against Cisplatin Cytotoxicity in Renal Tubular Cells.

Quercetin, a flavonoid with promising therapeutic potential, has been shown to protect from cisplatin nephrotoxicity in rats following intraperitoneal injection, but its low bioavailability curtails its prospective clinical utility in oral therapy. We recently developed a micellar formulation (P-quercetin) with enhanced solubility and bioavailability, and identical nephroprotective properties. As a first aim, we herein evaluated the oral treatment with P-quercetin in rats, which displayed no nephroprotection. In order to unravel this discrepancy, quercetin and its main metabolites were measured by HPLC in the blood and urine after intraperitoneal and oral administrations. Whilst quercetin was absorbed similarly, the profile of its metabolites was different, which led us to hypothesize that nephroprotection might be exerted in vivo by a metabolic derivate. Consequently, we then aimed to evaluate the cytoprotective capacity of quercetin and its main metabolites (quercetin 3-O-glucoside, rutin, tamarixetin, isorhamnetin and quercetin 3-O-glucuronide) against cisplatin toxicity, in HK-2 and NRK-52E tubular cell lines. Cells were incubated for 6 h with quercetin, its metabolites or vehicle (pretreatment), and subsequently 18 h in cotreatment with 10-300 µM cisplatin. Immediately after treatment, cell cultures were subject to the MTT technique as an index of cytotoxicity and photographed under light microscopy for phenotypic assessment. Quercetin afforded no direct cytoprotection and quercetin-3-O-glucuronide was the only metabolite partially preventing the effect of cisplatin in cultured tubule cells. Our results identify a metabolic derivative of quercetin contributing to its nephroprotection and prompt to further explore exogenous quercetin-3-O-glucuronide in the prophylaxis of tubular nephrotoxicity.

4-Phenylbutyrate protects against rifampin-induced liver injury via regulating MRP2 ubiquitination through inhibiting endoplasmic reticulum stress.

Rifampin (RFP), a first-line anti-tuberculosis drug, often induces cholestatic liver injury and hyperbilirubinemia which limits its clinical use. Multidrug resistance-associated protein 2 (MRP2) localizes to the hepatocyte apical membrane and plays a pivotal role in the biliary excretion of bilirubin glucuronides. RFP is discovered to reduce MRP2 expression in liver cells. 4-Phenylbutyrate (4-PBA), a drug used to treat ornithine transcarbamylase deficiency (DILI), is reported to alleviate RFP-induced liver cell injury. However, the underlying mechanism still remains unclear. In the current study, we discovered that RFP induced HepG2 cell viability reduction, apoptosis and MRP2 ubiquitination degradation. Administration of 4-PBA alleviated the effect of RFP on HepG2 cell viability reduction, apoptosis and MRP2 ubiquitination degradation. In mechanism, 4-PBA suppressed RPF-caused intracellular Ca2+ disorder and endoplasmic reticulum (ER) stress, as well as the increases of Clathrin and adapter protein 2 (AP2). ER stress marker protein C/EBP homologous protein took part in the modulation of AP2 and clathrin. Besides, 4-PBA reduced the serum bilirubin level in RFP-induced cholestasis mouse model, along with raised the MRP2 expression in liver tissues. These findings indicated that 4-PBA could alleviate RFP-induced cholestatic liver injury and thereby decreased serum total bilirubin concentration via inhibiting ER stress and ubiquitination degradation of MRP2, which provides new insights into the mechanism of 4-PBA in the treatment of RFP-induced cholestasis and liver damage.

Novel anti-aging herbal formulation Jing Si displays pleiotropic effects against aging associated disorders.

Common characteristics of aging include reduced somatic stem cell number, susceptibility to cardiac injuries, metabolic imbalances and increased risk for oncogenesis. In this study, Pleiotropic anti-aging effects of a decoction Jing Si herbal drink (JS) containing eight Traditional Chinese Medicine based herbs, with known effects against aging related disorders was evaluated. Adipose derived mesenchymal stem cells (ADMSCs) from 16 week old adult and 24 month old aging WKY rats were evaluated for the age-related changes in stem cell homeostasis. Effects of JS on self-renewal, klotho and Telomerase Reverse Transcriptase expression DNA damage response were determined by immunofluorescence staining. The effects were confirmed in senescence induced human ADMSCs and in addition, the potential of JS to maintain telomere length was evaluated by qPCR analysis in ADMSCs challenged for long term with doxorubicin. Further, the effects of JS on doxorubicin-induced hypertrophic effect and DNA damage in H9c2 cardiac cells; MPP+-induced damages in SH-SY5Y neuron cells were investigated. In addition, effects of JS in maintaining metabolic regulation, in terms of blood glucose regulation in type-II diabetes mice model, and their potential to suppress malignancy in different cancer cells were ascertained. The results show that JS maintains stem cell homeostasis and provides cytoprotection. In addition JS regulates blood glucose metabolism, enhances autophagic clearances in neurons and suppresses cancer growth and migration. The results show that JS acts on multiple targets and provides a cumulative protective effect against various age-associated disorders and therefore it is a candidate pleiotropic agent for healthy aging.

MRSA-induced endothelial permeability and acute lung injury are attenuated by FTY720 S-phosphonate.

Disruption of the lung endothelial barrier is a hallmark of acute respiratory distress syndrome (ARDS), for which no effective pharmacologic treatments exist. Prior work has demonstrated that FTY720 S-phosphonate (Tys), an analog of sphingosine-1-phosphate (S1P) and FTY720, exhibits potent endothelial cell (EC) barrier protective properties. In this study, we investigated the in vitro and in vivo efficacy of Tys against methicillin-resistant Staphylococcus aureus (MRSA), a frequent bacterial cause of ARDS. Tys-protected human lung EC from barrier disruption induced by heat-killed MRSA (HK-MRSA) or staphylococcal α-toxin and attenuated MRSA-induced cytoskeletal changes associated with barrier disruption, including actin stress fiber formation and loss of peripheral VE-cadherin and cortactin. Tys-inhibited Rho and myosin light chain (MLC) activation after MRSA and blocked MRSA-induced NF-κB activation and release of the proinflammatory cytokines, IL-6 and IL-8. In vivo, intratracheal administration of live MRSA in mice caused significant vascular leakage and leukocyte infiltration into the alveolar space. Pre- or posttreatment with Tys attenuated MRSA-induced lung permeability and levels of alveolar neutrophils. Posttreatment with Tys significantly reduced levels of bronchoalveolar lavage (BAL) VCAM-1 and plasma IL-6 and KC induced by MRSA. Dynamic intravital imaging of mouse lungs demonstrated Tys attenuation of HK-MRSA-induced interstitial edema and neutrophil infiltration into lung tissue. Tys did not directly inhibit MRSA growth or viability in vitro. In conclusion, Tys inhibits lung EC barrier disruption and proinflammatory signaling induced by MRSA in vitro and attenuates acute lung injury induced by MRSA in vivo. These results support the potential utility of Tys as a novel ARDS therapeutic strategy.

Protective effect of liquiritin on coronary heart disease through regulating the proliferation of human vascular smooth muscle cells via upregulation of sirtuin1.

This study aimed to explore whether liquiritin affects the development of coronary heart disease by regulating the proliferation and migration of human vascular smooth muscle cells (hVSMCs). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) release detection were performed to measure the toxic effects of liquiritin on hVSMCs. An in vitro atherosclerosis model in hVSMCs was established using oxidized low-density lipoprotein (ox-LDL), and cell proliferation and apoptosis were detected using an MTT assay and flow cytometry analysis. Western blotting and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) were used to detect protein and mRNA expressions, respectively. Caspase3 activity and cell migration were measured using an activity detection kit and Transwell assay, respectively. The results indicated that liquiritin at doses <160 µM had no significant effect on cell viability and LDH release in hVSMCs. Ox-LDL significantly induced cell proliferation and migration, and inhibited hVSMCs apoptosis. Liquiritin significantly inhibited cell proliferation and migration, and enhanced cell apoptosis in ox-LDL induced hVSMCs. Sirtuin1 (SIRT1) was lowly expressed in atherosclerotic plaque tissues in coronary heart disease patients and in ox-LDL-induced hVSMCs. Liquiritin improved SIRT1 expression in ox-LDL-induced hVSMCs, whereas the improvement was inhibited by Selisistat (EX 527, an effective SIRT1 inhibitor) treatment. EX 527 reversed the effects of liquiritin on cell proliferation, migration, and apoptosis in ox-LDL-induced hVSMCs In conclusion, liquiritin plays a protective role in coronary heart disease by regulating the proliferation and migration of hVSMCs by increasing SIRT1 expression.

Metabolically induced intracellular pH changes activate mitophagy, autophagy, and cell protection in familial forms of Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder induced by the loss of dopaminergic neurons in midbrain. The mechanism of neurodegeneration is associated with aggregation of misfolded proteins, oxidative stress, and mitochondrial dysfunction. Considering this, the process of removal of unwanted organelles or proteins by autophagy is vitally important in neurons, and activation of these processes could be protective in PD. Short-time acidification of the cytosol can activate mitophagy and autophagy. Here, we used sodium pyruvate and sodium lactate to induce changes in intracellular pH in human fibroblasts with PD mutations (Pink1, Pink1/Park2, α-synuclein triplication, A53T). We have found that both lactate and pyruvate in millimolar concentrations can induce a short-time acidification of the cytosol in these cells. This induced activation of mitophagy and autophagy in control and PD fibroblasts and protected against cell death. Importantly, application of lactate to acute brain slices of WT and Pink1 KO mice also induced a reduction of pH in neurons and astrocytes that increased the level of mitophagy. Thus, acidification of the cytosol by compounds, which play an important role in cell metabolism, can also activate mitophagy and autophagy and protect cells in the familial form of PD.

Activated Phosphoinositide 3-Kinase/Akt/Mammalian Target of Rapamycin Signal and Suppressed Autophagy Participate in Protection Offered by Licochalcone A Against Amyloid-ß Peptide Fragment 25-35-Induced Injury in SH-SY5Y Cells.

OBJECTIVE: To assess effect of licochalcone A (LicA) on amyloid-ß (Aß) peptide fragment 25-35-induced nerve injury and reveal the potential molecular mechanisms involved. METHODS: Viability of SH-SY5Y cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay after treatment with Aß25-35 and/or LicA, following which apoptosis was detected by flow cytometry and Hoechst staining. Then, reactive oxygen species, glutathione, and superoxide dismutase were measured with flow cytometry and spectrophotometry. The ultrastructure of mitochondria was examined by transmission electron microscopy, and the biomarker proteins of autophagy, apoptosis, and phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway were measured with Western blotting. RESULTS: LicA improved cell viability and decreased lactate dehydrogenase leakage remarkably in Aß25-35-induced injury in SH-SY5Y cells. After treatment with LicA, reactive oxygen species, glutathione, and superoxide dismutase levels in cells all were significantly decreased, which indicated that LicA has an antioxidative effect on Aß25-35-induced oxidative injury. LicA could also significantly reduce Aß25-35-induced autophagy in SH-SY5Y cells. In the cells injured by Aß25-35, LicA prevented the transformation from light chain protein 3-I to light chain protein 3-II and reduced the levels of proteins GRP78, GRP94, CHOP, and Bax, but increased the levels of antiapoptotic protein and phosphorylation of PI3K, Akt, and mTOR. These effects of LicA were restored or suppressed by mTOR inhibitor rapamycin or PI3K inhibitor LY294002. CONCLUSIONS: LicA protects SH-SY5Y cells against Aß25-35-induced injury, wherein suppressed autophagy and activated PI3K/Akt/mTOR signaling pathway are involved, and mTOR-dependent autophagy at least plays some role.

SGLT-2 inhibitors as cardio-renal protective agents.

Despite remarkable advances in diabetes care, patients with type 2 diabetes are still burdened by higher morbidity and mortality than non-diabetic individuals. Atherosclerotic cardiovascular disease, heart failure, and chronic kidney disease represent the most relevant causes of morbidity and mortality and sustain each other in a vicious circle. Cardiovascular diseases are the main cause of death in patients with chronic kidney disease, and, in turn, chronic kidney disease is a significant contributor to the risk of major cardiovascular events and hospitalization for heart failure. Cardiovascular outcome trials with SGLT-2 inhibitors in type 2 diabetes yielded unprecedented results on prevention of worsening heart failure and renal disease progression and mortality, further confirmed by randomized controlled trials in patients with baseline heart failure and chronic kidney disease, with or without diabetes, and observations from the real-world setting. However, the evidence regarding SGLT-2 inhibitors benefit on atherosclerotic cardiovascular events is conflicting. Hence, SGLT-2 inhibitors represent a remarkably valuable weapon in diabetes management, to be used in the context of a multi-targeted treatment strategy to address the many issues of this multifaceted disease.

Herceptin induces ferroptosis and mitochondrial dysfunction in H9c2 cells.

Ferroptosis has been previously implicated in the pathological progression of cardiomyopathy. Herceptin (trastuzumab), which targets HER2, is commonly applied for the treatment of HER2+ breast cancer. However, its clinical use is limited by its cardiotoxicity. Therefore, the present study aimed to investigate if targeting ferroptosis could protect against Herceptin­induced heart failure in an in vitro model of H9c2 cells after treatment of Herceptin, Herceptin + ferroptosis inhibitor ferrostatin­1 (Fer­1) or Herceptin + Deferoxamine. H9c2 cell viability was measured by MTT assay. Reactive oxygen species (ROS) levels were detected by measuring the fluorescence of DCFH­DA­A and MitoSOX™ Red. Glutathione (GSH)/oxidized glutathione (GSSG) ratio was measured using the GSH/GSSG Ratio Detection Assay kit. Mitochondrial membrane potential and ATP content were evaluated by JC­1 staining and bioluminescent assay kits, respectively. Protein expressions of glutathione peroxidase 4, recombinant solute carrier family 7 member 11, mitochondrial optic atrophy1­1/2, mitofusin, Acyl­CoA synthetase long chain family member 4, cytochrome c, voltage­dependent anion­selective channel, dynamin­related protein, mitochondrial fission 1 protein and mitochondrial ferritin were evaluated by western blotting. It was found that Herceptin reduced H9c2 cell viability whilst increasing intracellular and mitochondrial ROS levels in a dose­ and time­dependent manner. Furthermore, Herceptin decreased glutathione peroxidase (GPX) protein expression and the GSH/ GSSG ratio in H9c2 cells in a dose­ and time­dependent manner. The Fer­1 abolished this Herceptin­induced reduction in cell viability, GSH/GSSG ratio, mitochondrial membrane potential and ATP content. Fer­1 also reversed the suppressive effects of Herceptin on the protein expression levels of GPX4, recombinant solute carrier family 7 member 11, mitochondrial optic atrophy1­1/2 and mitofusin in H9c2 cells. Subsequently, Fer­1 was found to reverse the Herceptin­induced increase in mitochondrial ROS and iron levels in H9c2 cells, as well as the increased protein expression levels of Acyl­CoA synthetase long chain family member 4, cytochrome c, voltage­dependent anion­selective channel, dynamin­related protein, mitochondrial fission 1 protein and mitochondrial ferritin in H9c2 cells. However, compared with deferoxamine, an iron chelator, the effects of Fer­1 were less effective. Collectively, these findings provided insights into the pathogenic mechanism that underlie Herceptin­induced cardiomyopathy, which potentially provides a novel therapeutic target for the prevention of cardiotoxicity in HER2+ breast cancer treatment.

Monooxygenase System and NO Metabolism in Liver Microsomes of Rats with Toxic Hepatitis and the Effect of Sesquiterpene Lactones.

We analyzed changes in activities of enzymes of phases I and II of xenobiotic biotransformation and parameters of NO metabolism in liver microsomes of rats with toxic CCl4-induced hepatitis after a 14-day course of sesquiterpene lactones from Artemisia leucodes (10 mg/kg). It was found that toxic hepatitis was associated with significant inhibition of NADPH-cytochrome c-reductase, benzo(a)pyrene hydroxylase, and NADPH-diaphorase, reduced cytochrome P-450 content, and enhanced induction of nitrate/nitrite reductase with accumulation of NO metabolites in the liver. Administration of sesquiterpene lactones stimulated activity of the studied components of the cytochrome P-450 system and promoted recovery of the NOergic system components; the effects were most pronounced in 7 and 14 days after treatment.

Role of Amino Acid Arginine and Nitric Oxide in Mechanisms of Cytoprotective Effect of Non-Opiate Leu-Enkephalin Analogue In Vitro.

Incubation of primary culture of pulmonary fibroblasts with non-opiate analogue of leuenkephalin (NALE; Phe-D-Ala-Gly-Phe-Leu-Arg, 0.1 µM) reduced generation of superoxide anion-radical (by 20.7%) and decreased the number of p53+ cells (by 40.2%) induced by exposure to H2O2 (60 µM). The cytoprotective effect of NALE was potentiated by NO synthase inhibitor L-NAME (1 mM): the number of p53+ cells decreased by 65.3% and morphometric parameters of the cell nuclei and nucleoli were improved. Incubation of pulmonary fibroblasts culture with peptide G (Phe-D-Ala-Gly-Phe-Leu-Gly, 0.1 µM) also significantly reduced the damaging effect of H2O2: the number of p53+ cells decreased by 73.5%, the area of cell nuclei returned to normal, and generation of superoxide anion-radical decreased by 18.4%. These results indicate that C-terminal amino acid Arg and activation of NO synthase are not involved in the direct cytoprotective effect of NALE.

Dimethyl Fumarate Ameliorates Nucleus Pulposus Cell Dysfunction through Activating the Nrf2/HO-1 Pathway in Intervertebral Disc Degeneration.

BACKGROUND: Oxidative stress, inflammation, and nucleus pulposus cells (NPCs) apoptosis are involved in pathogenesis of intervertebral disc (IVD) degeneration (IVDD). Dimethyl fumarate (DMF) has been found to effectively depress oxidative stress and inflammation via the Nrf2 pathway. Hence, this project was designed to explore the underlying mechanisms of how DMF protects NPCs from damage by LPS challenge. METHODS AND RESULTS: CCK8 assay and flow cytometry of apoptosis indicated that DMF treatment attenuated LPS-induced NPC damage. Western blot analysis demonstrated that DMF enhanced the expressions of nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in LPS-challenged NPCs. DMF treatment significantly decreased the accumulation of ROS, downregulated inflammatory cytokines (p-NF-κB, IL-1ß, and TNF-α), and ER stress-associated apoptosis proteins (Bip, calpain-1, caspase-12, caspase-3, and Bax) in LPS-challenged NPCs. The level of antiapoptotic protein Bcl-2 was promoted by DMF treatment in LPS-challenged NPCs. Glutathione (GSH) assay showed that DMF treatment improved reduced to oxidized glutathione ratio in LPS-challenged NPCs. Furthermore, the results of western blot analysis indicated that in LPS-challenged NPCs, DMF treatment ameliorated the elevated levels of matrix degradation enzymes (MMP-13, aggrecanase 1) and type I collagen and the reduced levels of matrix composition (type II collagen and ACAN). However, Nrf2 knockdown abolished these protective effects of DMF. CONCLUSION: Our data suggested that treatment with DMF mitigated LPS-induced oxidative stress, inflammation, and ER stress-associated apoptosis in NPCs via the Nrf2/HO-1 signaling pathway, thus reliving LPS-induced dysfunction of NPCs, which offered a novel potential pharmacological treatment strategy for IVDD.

Carbon Monoxide as a Therapeutic for Airway Diseases: Contrast and Comparison of Various CO Delivery Modalities.

The quest to find novel strategies to tackle respiratory illnesses has led to the exploration of the potential therapeutic effects of carbon monoxide (CO) as an endogenous signaling molecule and a cytoprotective agent. Further, several studies have demonstrated the pharmacological efficacy of CO in animal models of respiratory disorders, such as acute lung injury and pulmonary hypertension. Because of the gaseous nature of CO and its affinity for multiple targets, its controlled delivery has been a challenge. Past studies have employed different delivery modalities, including CO gas, HO-1 inducers, and CO donors, sometimes leading to substantive variations in the resulting pharmacological effects for various reasons. Herein, this review summarizes and analyzes the differences among the profiles of various CO-delivery modalities in terms of their efficacy, dosing regimen, and pharmacokinetics in airways models. We believe that analysis of these issues will help in understanding the fundamental roles of CO in airways, and eventually, contribute to its development as a medicine for respiratory diseases.

Triggers for the Nrf2/ARE Signaling Pathway and Its Nutritional Regulation: Potential Therapeutic Applications of Ulcerative Colitis.

Ulcerative colitis (UC), which affects millions of people worldwide, is characterized by extensive colonic injury involving mucosal and submucosal layers of the colon. Nuclear factor E2-related factor 2 (Nrf2) plays a critical role in cellular protection against oxidant-induced stress. Antioxidant response element (ARE) is the binding site recognized by Nrf2 and leads to the expression of phase II detoxifying enzymes and antioxidant proteins. The Nrf2/ARE system is a key factor for preventing and resolving tissue injury and inflammation in disease conditions such as UC. Researchers have proposed that both Keap1-dependent and Keap1-independent cascades contribute positive effects on activation of the Nrf2/ARE pathway. In this review, we summarize the present knowledge on mechanisms controlling the activation process. We will further review nutritional compounds that can modulate activation of the Nrf2/ARE pathway and may be used as potential therapeutic application of UC. These comprehensive data will help us to better understand the Nrf2/ARE signaling pathway and promote its effective application in response to common diseases induced by oxidative stress and inflammation.