ROS Generation Levels Research Articles

Cycloastragenol exerts protective effects against UVB irradiation in human dermal fibroblasts and HaCaT keratinocytes

BackgroundCycloastragenol (CAG) is a triterpene aglycone of astragaloside IV that possesses various pharmacological actions including improving telomerase activity, inhibiting inflammation and cell proliferation, inducing apoptosis. ObjectiveCAG has also shown effect to significantly improve the appearance of aging skin but, its molecular mechanism of protective effect against UVB induced-damage have not been elucidated. We investigated the potential effect of CAG on UVB wrinkle promoting activities and skin-moisturizing effects in human dermal fibroblasts (HDF) and HaCaT keratinocytes. MethodsAfter UVB irradiation or H2O2 treatment, the levels of matrix metalloproteinases (MMPs) and ROS generation were measured in CAG-treated HDF cells. In addition, after UVB irradiation, hyaluronic acid and skin hydration factors (filaggrin and SPT) were also analyzed in CAG (0–0.5–1–2 µM)-treated HDF and HaCaT cells. ResultsWe found that CAG caused a significant decrease in the levels of UVB-induced MMP-1, MMP-9, MMP-13 and ROS generation, also increased UVB-damaged Collagen Ⅰ. We also noted that CAG increased cell viability and can regulate MMP-1, MMP-9, MMP-13and Collagen Ⅰ in H2O2-damaged HDF cells. Moreover, we noticed that CAG effectively enhanced levels of hyaluronic acid and expression of skin hydration factors (filaggrin and serine palmitoyltransferase (SPT)) in UVB-damaged HDF and HaCaT cells. ConclusionThis is first report indicating that CAG can exhibit protective effect against UVB and H2O2-induced damages and can contribute in maintenance of healthy skin.

Azadiradione (AZD) neem biomass derived limonoid: extraction, characterization, and potential biological activities with special reference to anti-microbial and anti-cancer activities

The resistance of conventional drugs to bacterial infections and cancer poses a significant threat to the healthcare system causing increased mortality. Metabolites derived from plant biomass mainly medicinal plants exhibit a wide spectrum of biological activities with high efficacy and biocompatibility. Given the rising importance of using natural compounds as an antimicrobial and anticancerous agent, the current study aimed to determine the antibacterial and anticancerous properties of Azadiradione (AZD), neem-derived limonoid. The antibacterial property of AZD was determined against two important nosocomial pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa and the anticancer activity of AZD was analysed through evaluation of cytotoxicity in human colon cancer cell line (HCT-116). The Minimum inhibitory concentration (MIC) of AZD was found to be ≥6.25 µM and ≥25 µM for S. aureus and P. aeruginosa, respectively. The fluorescent assisted cell sorter (FACS) and scanning electron microscope (SEM) analysis have also revealed that AZD (100 µM) exhibits effective antibacterial activity. The AZD has also exhibited cytotoxicity, inhibited cell migration, increased ROS generation and superoxide levels, reduces mitochondrial membrane potential (MMP), and induces apoptosis in a concentration-dependent manner (25 µM - 100 µM) in human colon cancer cell lines. The highest concentration (100 µM) of AZD was found to arrest the Sub G1 and G0/G1 phase of the cell cycle, upregulates mRNA expression of pro-apoptotic proteins such as Bax, caspase-3, caspase-9 and downregulate anti-apoptotic protein- Bcl-2. Thus, AZD could be regarded as an effective anticancer antibiotic in future therapeutics.

The Relationship between Lifespan of Marine Bivalves and Their Fatty Acids of Mitochondria Lipids.

Marine bivalves belonging to the Mytilidae and Pectinidae Families were used in this research. The specific objectives of this study were: to determine the Fatty Acids (FAs) of mitochondrial gill membranes in bivalves with different lifespans, belonging to the same family, and to calculate their peroxidation index; to compare the levels of ROS generation, malondialdehyde (MDA), and protein carbonyls in the mitochondria of gills, in vitro, during the initiation of free-radical oxation; to investigate whether the FAs of mitochondria gill membranes affect the degree of their oxidative damage and the maximum lifespan of species (MLS). The qualitative membrane lipid composition was uniform in the studied marine bivalves, regardless of their MLS. In terms of the quantitative content of individual FAs, the mitochondrial lipids differed significantly. It is shown that lipid matrix membranes of the mitochondria of long-lived species are less sensitive to in vitro-initiated peroxidation compared with the medium and short-lived species. The differences in MLS are related to the peculiarities of FAs of mitochondrial membrane lipids.

Oxidative stress-mediated mitochondrial apoptosis induced by the acaricide, fenpyroximate, on cultured human colon cancer HCT 116 cells

Fenpyroximate (FEN) is an acaricide that inhibits mitochondrial electron transport at the NADH-coenzyme Q oxidoreductase (complex I). The present study was designed to investigate the molecular mechanisms underling FEN toxicity on cultured human colon carcinoma cells (HCT116). Our data showed that FEN induced HCT116 cell mortality in a concentration dependent manner. FEN arrested cell cycle in G0/G1 phase and increased DNA damage as assessed by comet assay. Induction of apoptosis was confirmed in HCT116 cells exposed to FEN by AO-EB staining and Annexin V-FITC/PI double staining assay. Moreover, FEN induced a loss in mitochondrial membrane potential (MMP), increased p53 and Bax mRNA expression and decreased bcl2 mRNA level. An increase in caspase 9 and caspase 3 activities was also detected. All toghether, these data suggest that FEN induce apoptosis in HCT116 cells via mitochondrial pathway. To check the implication of oxidative stress in FEN-induced cell toxicity, we examined the oxidative stress statue in HCT116 cells exposed to FEN and we tested the effect of a powerful antioxidant, N-acetylcystein (NAC), on FEN-caused toxicity. It was observed that FEN enhanced ROS generation and MDA levels and disturbed SOD and CAT activities. Besides, cell treatment with NAC significantly protected cells from mortality, DNA damage, loss of MMP, and caspase 3 activity induced by FEN. To the best of our knowledge, this is the first study showing that FEN induced mitochondrial apoptosis via ROS generation and oxidative stress.

An Magnetic-Targeting Nano-Diagnosis and Treatment Platform for TNBC.

In this experiment, we constructed a magnetic targeting nano-diagnosis and treatment platform of doxorubicin (DOX) combined with iron nanoparticles, and explored their application value and mechanism in the treatment of Triple Negative Breast Cancer (TNBC), as well as its new diagnosis and treatment mode in Magnetic Resonance Imaging (MRI). Hollow mesoporous nanoparticles (HFON) were synthesized by solvothermal method, and loaded the drug DOX (DOX@HFON) to treat TNBC. The experiments in vivo and in vitro were carried out according to the characteristics of the materials. In vitro experiments, the killing effect of the drug on cells was verified by cell viability CCK8, ROS generation level, LPO evaluation and flow cytometry; the MRI effect and targeted anti-tumor therapy effect were studied by in vivo experiments; then the tumor tissue sections were detected by Ki-67, CD31, ROS, LPO and TUNEL immunofluorescence detection; H&E staining and blood biochemical tests were used to evaluate the biosafety of the materials. Through a series of characterization tests, it is confirmed that the nano-materials prepared in this experiment have positive drug loading properties. MDA-MB-231 cells had great phagocytic ability to DOX@HFON under Confocal Laser Scanning Microscope (CLSM). Experiments in vitro confirmed that DOX and Fe were released and concentrated in cells, and a large number of ROS production and induction of LPO were detected by DCFH-DA and C11-BODIPY probes in cells. Apoptosis experiments further confirmed that DOX@HFON induced apoptosis, autophagy and ferroptosis. In the vivo experiment, the anti-tumor therapy effect of MAGNET@DOX@HFON group was the most significant, and in MRI also proved that the drug had great tendency and imaging ability in tumor tissue. The new magnetic targeting nano-diagnosis and treatment platform prepared in this experiment is expected to become a new treatment model for TNBC.

Celastrol promotes apoptotic cell death in children neuroblastoma cells through caspases dependent pathway.

To investigate the in-depth pharma-cological mechanisms of celastrol in children neuro-blastoma treatment. In the current study, we examined the effects of celastrol on children neuroblastoma cells viability and proliferation by cell counting kit-8 assay and colony formation assay. Annexin V-FTIC and PI staining were applied to determine cell apoptosis after celastrol treatment. ROS generation levels were examined by 2', 7'-dichloroflfluorescin diacetate. We found that celastrol could suppress the proliferation of children neuroblastoma cells with few effects on normal cell lines . Further mechanisms studies have shown that celastrol inhibited cell cycle progression and induced cell apoptosis in QDDQ-NM and SH-SY5Y cells. In addition, ROS production might involve in celastrol-mediated apoptotic cell death in children neuroblastoma cells by activating caspase death pathway. Our findings demonstrated that celastrol could promote ROS generation-induced apoptosis in neuroblastoma cell by activating caspase death pathway. These findings suggested that celastrol might be a potential novel anti-neuroblastoma agent with minor cytotoxicity.

Screening and differential oxidative stress responses of hot pepper (Capsicum annuum L.) genotypes under cold stress

Cold stress (CS) anomalies have recently increased, major abiotic threat that delimit chilli production and yield. Cold tolerance is differing between genotypes, especially in commercial crops like hot pepper (chilli). specific investigations are required to understand biochemical and physiological changes in response to Cold stress (CS). Crop selection for increased tolerance needs traits with high sensitivity, fast and cost effective. This study aimed to select chilli genotypes which revealing differential tolerance to CS at seedling stage. Accordingly, 12 chilli genotypes were examined under two cold regimes including control (35/25 °C – day/night) and CS (10/8 °C – day/night). Multivariate cluster analysis of 14 traits grouped the genotypes into three clusters under stress condition, indicating that CS influencing physiology and oxidative potential of chilli seedlings. Chlorophyll, Membrane stability, Hydrogen peroxide content, lipid peroxidation and proline, phenolic contents are the influential traits for evaluating the impact of cold stress. ROS and ROS mediated traits showed a differential performance among the genotypes tested. Enzymatic antioxidants were significantly increased across all genotypes. Decrements of pungency levels in chilli fruits were significant only in CA-173 and F-02 genotypes. Altogether, the antioxidant profile has explored there is an association between capsaicinoid levels and antioxidant activities, antioxidants may involve preserving capsaicinoid metabolism to maintain the quality of chilli fruits during CS. Grouping of genotypes by cluster analysis is correlated with cold injury index and chlorophyll stability index under CS conditions. These indices can be employed as proper screening indices for cold tolerance in chilli genotypes. . • Simulated short-term cold stress has differential negative impacts among Chilli genotypes. • Differential levels of ROS generation and antioxidant potential among the genotypes tested. • ROS-induced oxidation of capsaicinoids was countered by antioxidant enzymes and chilli fruit quality was maintained under short-term cold stress. • 12 genotypes were grouped into 3 clusters: 1 – Sensitive; 2 – Moderately tolerant; 3 – Tolerant. • Cold injury index and Chlorophyll stability index can be used as cost-effective screening indices for selection of cold-tolerant chilli genotypes.

Overcoming Hypoxia‐Induced Ferroptosis Resistance via a19F/1H‐MRI Traceable Core‐Shell Nanostructure

AbstractLipid peroxides accumulation induced ferroptosis is an effective cell death pathway for cancer therapy. However, the hypoxic condition of tumor microenvironment significantly suppresses the efficacy of ferroptosis. Here, we design a novel nanoplatform to overcome hypoxia‐induced ferroptosis resistance. Specifically, we synthesize a novel kind of perfluorocarbon (PFOB)@manganese oxide (MnOx) core‐shell nanoparticles (PM‐CS NPs). Owing to the good carrier of O2as fuel, PM‐CS NPs can induce higher level of ROS generation, lipid peroxidation and GSH depletion, as well as lower activity of GPX4, compared with MnOx NPs alone. Moreover, the supplement of O2can relieve tumor hypoxia to break down the storage of intracellular lipid droplets and increase expression of ACSL4 (a symbol for ferroptosis sensitivity). Furthermore, upon stimulus of GSH or acidity, PM‐CS NPs exhibit the “turn on”19F‐MRI signal and activatable T1/T2‐MRI contrast for correlating with the release of Mn. Finally, PM‐CS NPs exert high cancer inhibition rate for ferroptosis based therapy via synergetic combination of O2‐mediated enhancement of key pathways of ferroptosis.

Overcoming Hypoxia-Induced Ferroptosis Resistance via a 19 F/1 H-MRI Traceable Core-Shell Nanostructure.

Lipid peroxides accumulation induced ferroptosis is an effective cell death pathway for cancer therapy. However, the hypoxic condition of tumor microenvironment significantly suppresses the efficacy of ferroptosis. Here, we design a novel nanoplatform to overcome hypoxia-induced ferroptosis resistance. Specifically, we synthesize a novel kind of perfluorocarbon (PFOB)@manganese oxide (MnOx) core-shell nanoparticles (PM-CS NPs). Owing to the good carrier of O2 as fuel, PM-CS NPs can induce higher level of ROS generation, lipid peroxidation and GSH depletion, as well as lower activity of GPX4, compared with MnOx NPs alone. Moreover, the supplement of O2 can relieve tumor hypoxia to break down the storage of intracellular lipid droplets and increase expression of ACSL4 (a symbol for ferroptosis sensitivity). Furthermore, upon stimulus of GSH or acidity, PM-CS NPs exhibit the "turn on" 19 F-MRI signal and activatable T1 /T2 -MRI contrast for correlating with the release of Mn. Finally, PM-CS NPs exert high cancer inhibition rate for ferroptosis based therapy via synergetic combination of O2 -mediated enhancement of key pathways of ferroptosis.

The Effect of Blocking Neurokinin-1 Receptor by Aprepitant on the Inflammatory and Apoptosis Pathways in Human Ovarian Cancer Cells.

Ovarian cancer is the seventh most common cancer globally, and the second most common cancer among women with significant mortality. Toward this end, it is shown that substance P (SP) is involved in tumor initiation and progression through the neurokinin-1 receptor (NK1R). However, the exact molecular mechanism of the SP/NK1R system in ovarian cancer is not yet fully clarified. In this in vitro study, we decided to investigate the effect of the SP/NK1R system and blockage of NK1R by its specific antagonist (Aprepitant) on the proliferation of ovarian cancer cells as well as the alteration of inflammatory pathways. Our results revealed that Aprepitant stimulated apoptotic cell death and attenuated inflammation of ovarian cancer cells through the NF-kB and P53 signaling pathways. After treatment with Aprepitant, the expression of downstream anti-apoptotic genes related to the NF-kB pathway (survivine and bcl2) was decreased. However, we indicated the positive effect of SP on the proliferation of ovarian cancer cells by inducing the expression of NF-kB protein and NF-kB anti-apoptotic target genes. Moreover, Pro-apoptotic p53 target genes (P21 and Bax) were increased through aprepitant treatment, while SP attenuated these genes' expression. Besides, ROS generation in ovarian cancer cells after treatment with SP induced, while blocking of NK1R with Aprepitant reduced the level of ROS generation. Given this, our data suggest that this NK1R might be used as an important therapeutic target in ovarian cancer and Aprepitant could be considered a new drug in ovarian cancer therapy.

Brassinin inhibits proliferation and induces cell cycle arrest and apoptosis in nasopharyngeal cancer C666-1 cells

Nasopharyngeal cancer is a tumor that occurs in the mucous epithelium of the nasopharynx. Due to its rapid growth and early metastatic nature, the successful treatment of nasopharyngeal cancer is highly challenging. Here, we intended to assess the in vitro anticancer property of brassinin against the nasopharyngeal cancer C666-1 cells. The in vitro free radical scavenging property of the brassinin was assessed by various free radical scavenging activities such as FRAP, DPPH, chemiluminescence (CL), and ORAC assays. The cytotoxic level of the brassinin (1–50 µM) against the nasopharyngeal cancer C666-1 cells and normal Vero cells were assessed by the MTT cytotoxicity assay. The levels of TBARS, GSH, and the SOD activity was assessed using kits. The level of ROS generation, MMP, and apoptosis were investigated by the respective fluorescent staining techniques. The flow cytometry analysis was done to scrutinize the cell cycle arrest. The Bax/Bcl-2 level and caspase activities were examined using respective kits. The brassinin treatment effectively scavenged the free radicals, which are assessed by the FRAP, DPPH, chemiluminescence (CL), and ORAC assays. The proliferation of brassinin treated C666-1 cells were decreased remarkably, while the same concentration of brassinin did not disturbed the Vero cell viability. The 30 µM of brassinin effectively increased the ROS production, depleted the MMP, and stimulated the apoptosis in the C666-1 cells. The brassinin increased the TBARS and depleted the GSH and SOD in the C666-1 cells. The flow cytometry analysis revealed that the brassinin administration improved the G0/G1 ratio and decreased the proportion of cells with ‘S’ and ‘G2/M’ phase. The Bax, caspase-3 and −9 were elevated and Bcl-2 level was decreased in the brassinin administered C666-1 cells. Our findings discovered that the brassinin has the capacity to prevent the proliferation and stimulate the apoptotic cell death C666‐1 cells via blocking cell cycle and increasing oxidative stress and apoptotic markers. Hence, it can be a talented therapeutic agent to treat the nasopharyngeal cancer in the future.

Anticancer property and normal cell toxicity profile of pyrrolidine‐based platinum (II) complexes: Their DNA, BSA interaction, and molecular docking

The bioactivity and cytotoxic property of cis‐[Pt (AEP)Cl2] (C‐1); [Pt (AEP)(OH2)2](NO3)2 (C‐2); [Pt (AEP)(GSH) (C‐3)]; and [Pt (AEP)(DL‐meth)]2+(C‐4) (where AEP = 1‐[2‐Aminoethyl] pyrrolidine; GSH = Glutathione; DL‐meth = DL‐methionine) were extensively investigated to find out the more active and less toxic anticancer lead molecules. Spectroscopically characterized Pt (II) complexes (C‐1 to C‐4) were under investigation for their anticancer activity and bio‐molecular interaction with DNA and BSA. The binding property of the complexes toward DNA and BSA were evaluated by UV–Vis, fluorescence spectroscopy, gel electrophoresis, cyclic voltammetry (CV), circular dichroism (CD), and viscometric methods for their pharmacokinetic and pharmacodynamics importance. The BSA binding activity as well as their “Drug reservoir” property of Pt (II) complexes were highlighted for their biological significance. Furthermore, to assess the manner of interaction and also binding strength of the complexes with DNA and BSA molecule, a theoretical approach by molecular docking was performed, utilizing the optimized structure of the complexes. Structural optimization has been performed with density functional theory (DFT) correlation functional (B3LYP) at 6‐31G + (d, p) and LANL2DZ basis set was used for C, N, H, O, S, and metal, respectively. In vitro anticancer activity and level of ROS generation in presence of each complex were explored on A549 cancer cell as well as normal human cell lines HEK293, and their potential cytotoxic property was observed as compared to well‐known recognized anticancer cisplatin.

Investigation of the optimal culture time for warmed bovine ovarian tissues before transplantation†.

Ovarian tissue cryopreservation by vitrification is an effective technique, but there are still many unresolved issues related to the procedure. The aim of this study was to investigate the optimal culture time of postwarmed ovarian tissues and their viability before ovarian tissue transplantation. The bovine ovarian tissues were used to evaluate the effect of postwarming culture periods (0, 0.25, 0.5, 1, 2, 5, and 24h) in the levels of residual cryoprotectant, LDH release, ROS generation, gene and protein abundance, and follicle viability and its mitochondrial membrane potential. Residual cryoprotectant concentration decreased significantly after 1h of culture. The warmed ovarian tissues that underwent between 0 and 2h of culture time showed similar LDH and ROS levels compared with fresh nonfrozen tissues. The anti-Mullerian hormone transcript abundance did not differ in any of the groups. No increase in the relative transcript abundance and protein level of Caspase 3 and Cleaved-Caspase 3, respectively, in the first 2h of culture after warming. On the other hand, an increased protein level of double stranded DNA breaks (gamma-H2AX) was observed in postwarmed tissues disregarding the length of culture time, and a temporary reduction in pan-AKT was detected in postwarming tissues between 0 and 0.25h of culture time. Prolonged culture time lowered the percentage of viable follicles in warmed tissues, but it did not seem to affect the follicular mitochondrial membrane potential. In conclusion, 1-2h of culture time would be optimal for vitrified-warmed tissues before transplantation.

Melatonin in cryopreservation media improves transplantation efficiency of frozen–thawed spermatogonial stem cells into testes of azoospermic mice

BackgroundCryostorage of spermatogonial stem cells (SSCs) is an appropriate procedure for long-term storage of SSCs for fertility preservation. However, it causes damage to cellular structures through overproduction of ROS and oxidative stress. In this study, we examined the protective effect of melatonin as a potent antioxidant in the basic freezing medium to establish an optimal cryopreservation method for SSCs.MethodsSSCs were obtained from the testes of neonatal male mice aged 3–6 days. Then, 100 μM melatonin was added to the basic freezing medium containing DMSO for cryopreservation of SSCs. Viability, apoptosis-related markers (BAX and BCL2), and intracellular ROS generation level were measured in frozen–thawed SSCs before transplantation using the MTT assay, immunocytochemistry, and flow cytometry, respectively. In addition, Western blotting and immunofluorescence were used to evaluate the expression of proliferation (PLZF and GFRα1) and differentiation (Stra8 and SCP3) proteins in frozen–thawed SSCs after transplantation into recipient testes.ResultsThe data showed that adding melatonin to the cryopreservation medium markedly increased the viability and reduced intracellular ROS generation and apoptosis (by decreasing BAX and increasing BCL2) in the frozen–thawed SSCs (p < 0.05). The expression levels of proliferation (PLZF and GFRα1) and differentiation (Stra8 and SCP3) proteins and resumption of spermatogenesis from frozen–thawed SSCs followed the same pattern after transplantation.ConclusionsThe results of this study revealed that adding melatonin as an antioxidant to the cryopreservation medium containing DMSO could be a promising strategy for cryopreservation of SSCs to maintain fertility in prepubertal male children who suffer from cancer.

Combination of nicotinamide mononucleotide and troxerutin induces full protection against doxorubicin-induced cardiotoxicity by modulating mitochondrial biogenesis and inflammatory response.

Clinical application of doxorubicin (DOX) is restricted due to its cardiotoxicity, reinforcing the significance of exploring new strategies to counteract DOX-induced cardiotoxicity. The present work aimed to investigate the ameliorative impact of combination therapy with nicotinamide mononucleotide (NMN) and troxerutin (TXR) on DOX-induced cardiotoxicity, with mitochondrial function/biogenesis and inflammatory response approach. Male Wistar rats (n = 30, 250-300g) were divided into groups with/without DOX and/or NMN and TXR, alone or in combination. Rats underwent 6 consecutive intraperitoneal injections of DOX (cumulative dose: 12mg/kg). NMN (100mg/kg/day; intraperitoneally) and/or TXR (150mg/kg/day; orally) were administered for 28 days before DOX challenge. Seven days following the last injection of DOX, evaluation of cardiac histopathological changes, BNP and LDH levels, mitochondrial function (membrane potential, ROS generation, and ATP levels), expression of proteins involved in mitochondrial biogenesis (PGC-1α, NRF1, and TFAM), and inflammatory cytokines (TNF-α, IL-1β, and IL-6) was performed. Combination of NMN and TXR significantly decreased the severity of histopathological damages, and BNP and LDH levels (P < 0.01 to P < 0.001). It also restored mitochondrial functional endpoints, and expression of proteins involved in mitochondrial biogenesis (P < 0.05 to P < 0.001), and decreased inflammatory cytokines (P < 0.01 to P < 0.001). The positive impacts of this combination therapy were more potent as compared to monotherapies. These findings shed new light on the understanding of additive properties of NMN/TXR combination therapy in protecting against DOX-induced cardiotoxicity. The cardioprotective effect of this combination therapy may be mediated in part through the restoration of mitochondrial function/biogenesis associated with the PGC-1α/NRF1/TFAM pathway, and suppression of inflammatory response.

Pulegone ameliorates inflammation and oxidative stress in L-arginine-induced acute pancreatitis in mice by regulating the activation of p38 MAPK pathway

Purpose: To evaluate the effects of pulegone (PLG) on inflammation and oxidative stress in L-arginineinduced acute pancreatitis (AP), and determine its molecular mechanism.Methods: Hematoxylin-Eosin (H &amp; E) staining assay were performed, and histopathological score, blood glucose concentration, and serum amylase level were evaluated in order to assess the effects of PLG on pancreatic injury in L-arginine-induced AP mice. Enzyme linked immunosorbent assay (ELISA) was conducted to assess the levels of myeloperoxidase (MPO), malonaldehyde (MDA) and inflammatory factors (IL-6, TNF-α and IL-1β) in L-arginine-induced AP mice. Serum lactate dehydrogenase (LDH) and relative levels of ROS generation in L-arginine-induced AP mice were determined using an LDH kit and immunofluorescence assay, respectively. The effect of pulegone (PLG) on the activation of p38 MAPK pathway in L-arginine-induced AP mice was evaluated by Western blot.Results: Significant pancreatic tissue injury occurred in L-arginine-induced AP mice was revealed. PLG alleviated the pathological injury of pancreatitis, and decreased the blood glucose concentration and serum amylase level in L-arginine-induced AP mice. In addition, PLG inhibited oxidative stress and inflammatory responses, and was enabled to inhibit the activation of p38 MAPK pathway in L-arginineinduced AP mice. Furthermore, PLG exhibited protective effect against the development of pancreatic injury in L-arginine-induced AP mice.Conclusion: PLG ameliorates L-arginine-induced inflammation and oxidative stress in AP mice in vivo by inhibiting p38 MAPK pathway, and therefore, is a potential therapeutic agent for the management of acute pancreatitis.

Reduction of phosphorylated α-synuclein through downregulation of casein kinase 2α alleviates dopaminergic-neuronal function

Among the post-translational modifications of α-synuclein, phosphorylation has been reported to modulate the protein's nuclear localization, gene-expression and cytotoxicity. However, its effect on the functional performance of dopaminergic-neurons is not known. We aimed to evaluate the effect of siRNA-silencing of casein kinase (CK)2α in SH-SY5Y-cells overexpressing A53T α-synuclein, in alleviating phosphorylated α-synuclein serine129 (pSyn-129)-induced changes in intracellular Ca2+ ([Ca2+]i) response to physiological stimuli and vesicular-dopamine release. A53T transfection showed distinct increase in basal pSyn-129 expression with simultaneous nuclear localization, and CK2α siRNA decreased ROS-generation and pSyn-129 levels. A significant reduction was observed in KCl-induced ([Ca2+]i) response and vesicular-dopamine release in the A53T-transfected cells with a corresponding decrease in immunopositive-population of resting-vesicles (VMAT2). CK2α siRNA treatment showed recovery in [Ca2+]i rise with a corresponding upregulation of expression of voltage-gated Ca2+-channels (VGCC) CaV1.3 and CaV2.2 and RyR1 responsible for Ca2+ induced Ca2+ release from ER, VMAT2 expression and vesicular-dopamine release. Thus, using CK2α siRNA to reduce phosphorylation improved cellular-pathology in terms of ROS generation and pSyn-129 levels, as well as functional performance of DA-neuronal cells.

Development of 5-hydroxyl-1-azabenzanthrone derivatives as dual binding site and selective acetylcholinesterase inhibitors

A series of novel 5-hydroxyl-1-azabenzanthrone derivatives were designed, synthesized and evaluated as dual binding site acetylcholinesterase inhibitors for the treatment of Alzheimer's disease (AD). The most effective Compound 16 showed selective inhibition of acetylcholinesterase (eeAChE IC50 = 0.045 μM; eeBuChE IC50 = 19.68 μM; SI = 437.33). Most of the compounds showed cytoprotective effects on PC12 cells damaged by hydrogen peroxide, which might be related to their antioxidant activity. Further experiments confirmed that 16 exhibited anti-apoptotic effects at low concentrations and reduced the relative level of ROS generation in PC12 cells. The expression level of proteins related to antioxidant stress pathway in PC12 cells was relatively increased after administrated with 16, which may be beneficial to delay the progression of the disease. Moreover, 16 was evaluated to be safe in vivo and in vitro, and showed good overall pharmacokinetic performance and high bioavailability (Foral = 55.5%). Besides, 16 showed comparable performance in ameliorating the scopolamine-induced cognition impairment to donepezil. In addition, in vitro BBB permeability experiments confirmed that 16 had high BBB permeability.

Skin damage induced by zinc oxide nanoparticles combined with UVB is mediated by activating cell pyroptosis via the NLRP3 inflammasome\u2013autophagy\u2013exosomal pathway

BackgroundZinc oxide nanoparticles (ZnONPs) are widely used nanomaterial in personal cosmetics, such as skin creams and sunscreens, due to their whitening properties and strong UV light absorption. However, the safety issues and the hazards of ZnONPs, which can be taken up by the skin and cause skin toxicity, are still unclear. From a chemoprevention point of view, pterostilbene (PT) has been reported to prevent skin damage effectively by its anti-inflammatory and autophagy inducer effect. This study aims to determine the skin toxicity and the potential mechanisms of UVB and ZnONPs exposure and the preventive effect of PT.ResultsThe co-exposure of UVB and ZnONPs elicit NLRP3 inflammasome activation and pyroptosis in keratinocytes. Furthermore, exposure to both UVB and ZnONPs also disrupts cellular autophagy, which increases cell exosome release. In vivo UVB and ZnONPs exposure triggers skin toxicity, as indicated by increased histological injury, skin thickness and transepidermal water loss. Notably, the NLRP3 inflammasome-mediated pyroptosis are also activated during exposure. Topical application of pterostilbene attenuates NLRP3 inflammasome activation and pyroptosis by decreasing ROS generation and mitochondrial ROS (mtROS) levels. In addition to its antioxidant effect, PT also reversed autophagy abnormalities by restoring normal autophagic flux and decreasing NLRP3 inflammasome-loaded exosome release.ConclusionsOur findings reveal that ZnONPs induce skin damage in conjunction with UVB exposure. This process involves an interplay of inflammasomes, pyroptosis, autophagy dysfunction, and exosomes in skin toxicity. PT alleviates skin inflammation by regulating the inflammasome–autophagy–exosome pathway, a finding which could prove valuable when further evaluating ZnONPs effects for cosmetic applications.

Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation.

Background & AimsNADPH oxidase 1 (NOX1) has emerged as a prime regulator of intestinal mucosa immunity and homeostasis. Dysregulation of NOX1 may cause inflammatory bowel disease (IBD). It is not clear how NOX1 is regulated in vivo under inflammatory conditions. We studied the role of CK2 in this process.MethodsThe NOX1 organizer subunit, NADPH oxidase organizer 1 (NOXO1), was immunoprecipitated from cytokine-treated colon epithelial cells, and bound proteins were identified by mass spectrometry analysis. Sites on NOXO1 phosphorylated by CK2 were identified by nanoscale liquid chromatography coupled to tandem mass spectrometry. NOX1 activity was determined in colon epithelial cells and colonoids in the presence or absence of CX-4945, a CK2 specific inhibitor. Acute colitis was induced by administration of trinitrobenzenesulfonic acid in mice treated or not with CX-4945. Colon tissues were analyzed by histologic examination, quantitative polymerase chain reaction, and Western blots. CK2 activity, markers of inflammation, and oxidative stress were assessed.ResultsWe identified CK2 as a major partner of NOXO1 in colon epithelial cells under inflammatory conditions. CK2 directly binds NOXO1 at the C-terminus containing the Phox homology domain and phosphorylates NOXO1 on several sites. CX-4945 increased ROS generation by NOX1 in human colon epithelial cells and organoids. Strikingly, CK2 activity was reduced in trinitrobenzenesulfonic acid–induced acute colitis, and CX-4945 exacerbated colitis inflammation as shown by increased levels of CXCL1, ROS generation, lipid peroxidation, and colon damage.ConclusionsThe ubiquitous protein kinase CK2 limits NOX1 activity via NOXO1 binding and phosphorylation in colonic epithelial cells and lessens experimental colitis. Loss of CK2 activity during acute colitis results in excessive ROS production, contributing to the pathogenesis. Strategies to activate CK2 could be an effective novel therapeutic approach in IBD.