H2O2-induced Cytotoxicity Research Articles

Design, synthesis and biological evaluation of dual-function inhibitors targeting NMDAR and HDAC for Alzheimer’s disease

Histone deacetylases (HDACs) have been indicated important roles in neurodegenerative disorders including Alzheimer’s disease (AD). Herein, a series of novel compounds that contain a memantine moiety were designed to target HDACs and N-methyl-d-aspartate receptor (NMDAR) which are related to the treatment of AD. Biological characterization established that compound 9d exhibited a balanced inhibitory activity on NMDAR and HDACs. This compound is relatively selective to HDAC6 with IC50 of 0.18 μM and also maintains comparable activity on NMDAR (Ki = 0.59 μM) as memantine. Functionally, treatment with 9d increased the level of AcTubulin in MV4-11 cells and rescued PC-12 cells from H2O2-induced cytotoxicity with EC50 of 0.94 μM. Studies in mice also demonstrated that compound 9d efficiently penetrates the blood brain barrier to reach the brain tissue. Collectively, the results strongly encourage further development of 9d as a potential therapeutic agent for AD.

A Novel Compound YS-5-23 Exhibits Neuroprotective Effect by Reducing β-Site Amyloid Precursor Protein Cleaving Enzyme 1's Expression and H2O2-Induced Cytotoxicity in SH-SY5Y Cells.

The abnormally accumulated amyloid-β (Aβ) and oxidative stress contribute to the initiation and progression of Alzheimer's disease (AD). β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the rate-limiting enzyme for the production of Aβ. Furthermore, Aβ was reported to increase oxidative stress; then the overproduced oxidative stress continues to increase the expression and activity of BACE1. Consequently, inhibition of both BACE1 and oxidative stress is a better strategy for AD therapy compared with those one-target treatment methods. In the present study, our novel small molecule YS-5-23 was proved to possess both of the activities. Specifically, we found that YS-5-23 reduces BACE1's expression in both SH-SY5Y and Swedish mutated amyloid precursor protein (APP) overexpressed HEK293 cells, and it can also suppress BACE1's expression induced by H2O2. Moreover, YS-5-23 decreases H2O2-induced cytotoxicity including alleviating H2O2-induced apoptosis and loss of mitochondria membrane potential (MMP) because it attenuates the reactive oxygen species (ROS) level elevated by H2O2. Meanwhile, PI3K/Akt signaling pathway is involved in the anti-H2O2 and BACE1 inhibition effect of YS-5-23. Our findings indicate that YS-5-23 may develop as a drug candidate in the prevention and treatment of AD.

Purification and characterization of antioxidant peptides from enzymatic hydrolysate of mungbean protein.

In this study, the antioxidant activity of mungbean protein hydrolysate (MPH) was systematically investigated. MPH was fractionated by ultrafiltration into two major fractions (MPH-1 <3kDa, MPH-2 >3kDa). Fraction MPH-1, which exhibited the highest antioxidant activity, was further fractionated by gel column into three fractions (MPH-1A, MPH-1B, and MPH-1C). The antioxidant activity of the MPH-1B fraction was stronger than that of the other fractions. Eight mungbean peptides (P1-P8) were identified in fraction MPH-1B by UPLC-Q-TOF-MS. Among them, peptides Trp-Gly-Asn (WGN, P2), Ala-Trp (AW, P4), Arg-Gly-Trp-Tyr-Glu (RGWYE, P5), and Gly-Val-Pro-Phe-Trp (GVPFW, P7) had high antioxidant activity. Moreover, these four peptides exerted protective effects against H2 O2 -induced cytotoxicity and regulated the MDA content, CAT activity, and total GSH content in HepG2 cells with specific observation. This study demonstrated the potential of MPH as a source of antioxidant peptides. This provides a scientific basis for the preparation of antioxidant peptides from mungbean protein. PRACTICAL APPLICATION: This study demonstrated the potential of the hydrolysate of mungbean protein as a source of antioxidant peptides and provided a scientific basis for the preparation of antioxidant peptides from mungbean protein.

Inhibition of bee pollen on acetylcholine esterases and cytotoxicity of human neuroblastoma, cerebral glioma, and macrophages

Inhibition of Actinidia argute, Quercus dentate, the mixed plant‐bee pollen extracts on acetylcholineesterase (AChE) and butyrylcholineesterase (BChE), which are indicators of anti‐Alzheimer’s activity, were investigated. Actinidia argute, Quercus dentate, the mixed plant‐bee pollen extracts (0–3mg/ml) did not show inhibitory activity of AChE and BChE. Additionally, the inhibitory activities of cathechin, rutin, hesperidin, and quercetin, which are flavonoids in bee pollen, on AChE and BChE were examined. Catehchin and rutin inhibited about 54 and 86% of AChE activity at a concentration of 1 mg/ml, respectively. Also, cathechin and rutin(1 mg/ml) inhibited about 42 and 86% of BChE, respectively. Quercetin was effective in inhibiting AChE and BChE, but the inhibitory pattern was not observed in proportion to the treatment concentration. However, hesperidin did not show a decrease for AChE and BChE activity. In addition, we investigated the inhibitory activity of Actinidia argute, Quercus dentate, the mixed plant‐bee pollen extracts against H2O2‐induced cytotoxicity for human neuroblastoma(SK‐N‐SH), cerebral glioma(C6), and macrophages(RAW 264.7). Cytotoxicity of SK‐N‐SH, C6, and RAW 264.7 by Actinidia argute, Quercus dentate, the mixed plant‐bee pollen extracts were examined by MTT in the range of 0–3 mg/ml. For SK‐N‐SH, C6, and RAW 264.7, there was a difference in the toxic concentrations by Actinidia argute, Quercus dentate, the mixed plant‐bee pollen extracts. Cytotoxicity by H2O2(0–10 mM) was partially improved by Actinidia argute, Quercus dentate, the mixed plant‐bee pollen extracts.

Novel hepatoprotective peptides derived from protein hydrolysates of mealworm (Tenebrio molitor)

In the present study, we hypothesized that protein hydrolysates of mealworm (Tenebrio molitor) which is known to exert significant scavenging activity toward reactive oxygen species (ROS) might protect liver cells against ROS-induced cytotoxicity. Therefore, hepatoprotective effects of protein hydrolysates of mealworm and their underlying mechanisms were investigated in AML12 mouse liver cells and the responsible peptides were further identified. Pretreatment with the mealworm alcalase hydrolysate (MAH; <1 kDa) showed the highest protective effect against H2O2-induced cytotoxicity in AML12 cells among three mealworm hydrolysates produced by different proteases (alcalase, flavourzyme, and neutrase). Further mechanistic studies demonstrated that MAH reduces ROS levels through increasing NF-E2-related factor 2-mediated expression of catalase, heme oxygenase-1, and genes involved in glutathione synthesis. Moreover, two novel hepatoprotective peptides, Ala-Lys-Lys-His-Lys-Glu and Leu-Glu, which shared similar mechanisms of action with MAH were identified. These results suggest that MAH and the two peptides represent potential sources of natural hepatoprotective agents.

Anti-inflammatory and Anti-oxidative Effects of Phytohustil® and Root Extract of Althaea officinalis L. on Macrophages in vitro.

IntroductionThe medicinal plant marshmallow Althaea officinalis L. (A. officinalis), is used for the treatment of cough since centuries. Application of medicinal extracts of marshmallow roots shows immediate effects like a protective film on the inflamed mucosa. Because the soothing layer reduce irritation of the mucous system, a faster regeneration is supported by defense mechanisms required to protect the respiratory tract from environmental injury. Macrophages (MΦ), which belong to a group of multipurpose defensive cells, provide the first line of defense against mucosal invasive pathogens. The present study was performed to investigate, whether the herbal medicinal product has anti-inflammatory or anti-oxidative effects on pro-inflammatorily activated MΦ or after oxidative stress induction. Special attention should be payed to elucidate the effects of A. officinalis on the mechanism of intracellular defense as well as on migratory capacity of the MΦ.ResultsTreatment of PMA-differentiated human THP-1 MΦ with Phytohustil® increased their viability without affecting the cell number. Phytohustil® or root extracts of A. officinalis (REAo) – an active component of Phytohustil® – were able to protect human MΦ against H2O2-induced cytotoxicity and H2O2-induced ROS production. Phytohustil®, REAo or diclofenac used as anti-inflammatory reference substance, inhibited the LPS-induced release of tumor necrosis factor-alpha (TNF-α) as well as of IL6 in MΦ. Treatment with Phytohustil®, its excipients or REAo did not impair the mitochondrial membrane potential (MMP). Finally, Phytohustil® and REAo activated the migratory capacity of MΦ.ConclusionThe present in vitro investigations indicate protective, i.e., anti-oxidative and anti-inflammatory effects of REAo and Phytohustil®, additionally improving the migratory capacity of MΦ. These antiinflammatory effects were similar or even better than diclofenac. Thus, our data support and may explain the positive effect of Phytohustil® observed in patients during the therapy of inflamed buccal mucosal membranes or treatment of cough.

Gastrodin Inhibits H2O2-Induced Ferroptosis through Its Antioxidative Effect in Rat Glioma Cell Line C6.

Ferroptosis is a form of necrosis caused by iron-induced accumulation of lipid hydroperoxide, involving several molecular events, and has been implicated in Parkinson's disease. Gastrodin is a component of Gastrodia elata Blume with strong antioxidant activity. We examined whether gastrodin can prevent H2O2-induced cytotoxicity in rat glioma cell line C6. For this purpose, C6 cells were pretreated with gastrodin (1, 5, 25 µM) and then exposed to 100 µM H2O2. Results showed that pretreatment of C6 cells with gastrodin decreased H2O2-induced lactate dehydrogenase (LDH) release and cell death. Moreover, gastrodin decreased intracellular malondialdehyde (MDA) level, whereas increased glutathione peroxidase (GPX) activity and glutathione (GSH) level after H2O2 treatment. In addition, treatment of deferoxamine (DFO), ferrostatin-1, and liproxstatin-1 abolished ferroptosis induced by H2O2 or erastin pretreatment. Treatment with gastrodin attenuated H2O2-induced ferroptosis and decreased lipid reactive oxygen species (ROS) (C11-BODIPY) production in C6 cells. Moreover, gastrodin increased the protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2), GPX4, ferroportin-1 (FPN1), and heme oxygenase-1 (HO-1) in C6 cells treated with H2O2. RSL3, a GPX4 inhibitor, inhibited GPX4 protein level in cells co-treated with gastrodin and 100 µM H2O2. These findings indicate that gastrodin can inhibit H2O2-induced ferroptosis through its antioxidative effect in C6 cells.

Cistanche deserticola polysaccharide induces melanogenesis in melanocytes and reduces oxidative stress via activating NRF2/HO-1 pathway.

As a main part of pigmentation disorders, skin depigmentation diseases such as vitiligo and achromic naevus are very common and get more attention now. The pathogenesis of depigmentation includes melanocyte dysfunction and loss, which are possibly caused by heredity, autoimmunity and oxidative stress. Among them, oxidative stress plays a key role; however, few clinical treatments can deal with oxidative stress. As reported, Cistanche deserticola polysaccharide (CDP) is an effective antioxidant; based on that, we evaluated its role in melanocyte and further revealed the mechanisms. In this study, we found that CDP could promote melanogenesis in human epidermal melanocytes (HEMs) and mouse melanoma B16F10 cells, it also induced pigmentation in zebrafish. Furthermore, CDP could activate mitogen‐activated protein kinase (MAPK) signal pathway, then up‐regulated the expression of microphthalmia‐associated transcription factor (MITF) and downstream genes TYR, TRP1, TRP2 and RAB27A. Otherwise, we found that CDP could attenuate H2O2‐induced cytotoxicity and apoptosis in melanocytes. Further evidence revealed that CDP could enhance NRF2/HO‐1 antioxidant pathway and scavenge intracellular ROS. In summary, CDP can promote melanogenesis and prevent melanocytes from oxidative stress injury, suggesting that CDP helps maintain the normal status of melanocytes. Thus, CDP may be a novel drug for the treatment of depigmentation diseases.

Circular RNA HIPK3 downregulation mediates hydrogen peroxide-induced cytotoxicity in human osteoblasts.

Hydrogen peroxide (H2O2) induces oxidative injury to human osteoblasts. The expression and potential function of circular RNA HIPK3 (circHIPK3) in H2O2-treated human osteoblasts were tested. We show that H2O2 significantly downregulated circHIPK3 in OB-6 cells and primary human osteoblasts. Furthermore, circHIPK3 levels were decreased in the necrotic femoral head tissues of dexamethasone-treated patients. In OB-6 osteoblastic cells and primary human osteoblasts, forced overexpression of circHIPK3 by a lentiviral construct alleviated H2O2-induced viability reduction, cell death and apoptosis. Contrarily, circHIPK3 silencing by targeted shRNA potentiated H2O2-induced cytotoxicity in OB-6 cells and primary human osteoblasts. Moreover, circHIPK3 downregulation by H2O2 induced miR-124 accumulation in OB-6 cells and primary human osteoblasts. On the contrary, miR-124 inhibition by transfection of the miR-124 inhibitor protected human osteoblasts from H2O2. Importantly, forced overexpression of miR-124 by transfection of the miR-124 mimic induced significant cytotoxicity in OB-6 cells and primary human osteoblasts. H2O2 downregulated miR-124’s targets, cyclin dependent kinase 6 and Rho-Associated Protein Kinase 1, in human osteoblasts. In conclusion circHIPK3 downregulation mediates H2O2-induced cytotoxicity in human osteoblasts.

Cytoprotective effects of berry anthocyanins against induced oxidative stress and inflammation in primary human diabetic aortic endothelial cells

Type 2 diabetes is associated with oxidative stress and low-grade inflammation resulting in endothelial dysfunction (ED). This study determined to explore the protective effects of berry-derived anthocyanins (AC) with potent antioxidant and anti-inflammatory activities in human diabetic endothelial cells upon oxidative and inflammatory stressors. Cultured healthy human aortic endothelial cells (HAEC) and diabetic human aortic endothelial cells (D-HAEC) exposed to oxidative stress by hydrogen peroxide (H2O2, 75 μM) and lipopolysaccharide (LPS, 1 μg/mL) as an inflammatory inducer before treatment with AC (50 μl/ml). The results from cytotoxicity assays showed that AC had no significant effects in cell viability (P-value < 0.0001), and exposure to H2O2 75 μM had a less toxic effect (P-value < 0.05). Although, AC significantly decreased H2O2-induced cytotoxicity and oxidative stress in both HAEC and D-HAEC cell lines (P-value < 0.0001), no positive impact of AC was found on the GSSG/GSH ratios (P-value < 0.05). Exposure to the LPS increased the production of IL-6 in both HAEC and D-HAEC cell lines (P-value < 0.0001), whereas AC treatment reduced LPS‐induced IL‐6 production in both cell lines with a more robust impact on D-HAEC (P-value < 0.0001). While LPS increased inflammasome assembling and caspase-1 activation, AC treatment inhibited caspase-1 activation in D-HAEC (P ≤ 0.05). This study indicated that berry anthocyanins reduced oxidative stress and inflammation via the inhibition of the NF-ƙB signaling pathway, which contributes to mitigating the diabetes-induced up-regulation of NF-ƙB.

Resveratrol induces SIRT1-Dependent autophagy to prevent H2O2-Induced oxidative stress and apoptosis in HTR8/SVneo cells

IntroductionPre-eclampsia (PE) is a serious complication of pregnancy, and the likely pathogenic basis of early onset PE are placental dysfunction and increased oxidative stress. Resveratrol (RES) is a potent antioxidant which has shown beneficial effects in many diseases. The aim of this study was to investigate the protective effects of RES against oxidative stress-induced damage in trophoblasts, and elucidate the potential mechanisms. MethodsWe established an in vitro model of oxidative stress by exposing the human first-trimester extravillous trophoblast cell line HTR8/SVneo to H2O2. The level of oxidative stress was reflected by ROS, MDA and SOD. The viability of cells was determined by the MTS assay. Apoptosis was detected using Annexin V-FITC staining and flow cytometry. Levels of SIRT1(sirtuin 1) and autophagy-related proteins (LC3, Beclin-1, p62) were detected by western blot. Autophagosomes were observed by transmission electron microscopy (TEM). ResultsPre-treatment with RES significantly ameliorated H2O2-induced cytotoxicity, morphological damage, oxidative stress and apoptosis. Mechanistically, RES restored the levels of SIRT1 and autophagy-related proteins including LC3-II, Beclin-1 and p62 that were dysregulated by H2O2. Blocking autophagy by 3-methyladenine (3-MA) completely abolished the protective effects of RES, as did knocking down SIRT1. ConclusionRES may protect human trophoblasts against H2O2-induced oxidative stress by activating SIRT1-dependent autophagy, and therefore has therapeutic potential in PE.

Scopoletin: Antiamyloidogenic, Anticholinesterase, and Neuroprotective Potential of a Natural Compound Present in Argyreia speciosa Roots by In Vitro and In Silico Study.

Alzheimer’s disease (AD) is characterized by depositions of amyloid β (Aβ) peptides aggregates resulting in plaques formation in the central nervous system (CNS). This study evaluates the disease-modifying potential of scopoletin against multiple factors associated with AD such as cholinesterase enzymes, Aβ peptides, and neuroprotective properties against Aβ- and H2O2-induced cytotoxicity under in vitro conditions. Scopoletin was identified and quantified using UPLC-QTOF (ultra-high performance liquid chromatography-quadrupole time-of-flight) and high-performance liquid chromatography (HPLC), respectively. The antiamyloidogenic potential was evaluated by thioflavin T and congo red binding assay. Inhibition of key enzymes, that is, acetylcholinesterase and butyrylcholinesterase, was investigated by Ellman’s assay. UPLC-QTOF analysis showed that most abundant phytoconstituent present in Argyreia speciosa hydroalcoholic root extract was scopoletin followed by festuclavine and ergometrine. Scopoletin was further quantified using novel reverse phase (RP)-HPLC method developed in this study. The neuroprotective potential of scopoletin was found to be 69% against Aβ42-induced neurotoxicity and 73% against H2O2-induced cytotoxicity in PC12 cell culture at 40 μM final concentration. At the same concentration, scopoletin inhibited Aβ42 fibril formation up to 57%. The IC50 concentration for AChE and BuChE enzyme inhibition by scopoletin was 5.34 and 9.11 μM, respectively. The antiaggregation and enzyme inhibition results were complemented with strong molecular interactions of scopoletin with target proteins validated by in silico molecular docking analysis. Based on this study, it can be concluded that scopoletin can be used as a lead for amelioration of symptoms and disease-modifying effects in AD.

Synthesized 2-Trifluoromethylquinazolines and Quinazolinones Protect BV2 and N2a Cells against LPS- and H2O2-induced Cytotoxicity.

Microglia are associated with neuroinflammation, which play a key role in the pathogenesis of neurodegenerative diseases. It has been reported that some quinazolines and quinazolinones possess anti-inflammatory properties. However, the pharmacological properties of certain quinazoline derivatives are still unknown. The antioxidant, cytotoxic, and protective effects of a series of synthesized 2- trifluoromethylquinazolines (2, 4, and 5) and quinazolinones (6-8) in lipopolysaccharide (LPS)- murine microglia (BV2) and hydrogen peroxide (H2O2)-mouse neuroblastoma-2a (N2a) cells were investigated. The antioxidant activity of synthesized compounds was evaluated with ABTS and DPPH assays. The cytotoxic activities were determined by MTS assay in BV2 and N2a cells. The production of nitric oxide (NO) in LPS-induced BV2 microglia cells was quantified. The highest ABTS and DPPH scavenging activities were observed for compound 8 with 87.7% of ABTS scavenge percentage and 54.2% DPPH inhibition. All compounds were noncytotoxic in BV2 and N2a cells at 5 and 50 μg/mL. The compounds which showed the highest protective effects in LPS-induced BV2 and H2O2-induced N2a cells were 5 and 7. All tested compounds, except 4, also reduced NO production at concentrations of 50 μg/mL. The quinazolinone series 6-8 exhibited the highest percentage of NO reduction, ranging from 38 to 60%. Compounds 5 and 8 possess balanced antioxidant and protective properties against LPS- and H2O2-induced cell death, thus showing great potential to be developed into anti-inflammatory and neuroprotective agents. Compounds 5 and 7 were able to protect the BV2 and N2a cells against LPS and H2O2 toxicity, respectively, at a low concentration (5 μg/mL). Compounds 6-8 showed potent reduction of NO production in BV2 cells.

Selenium nanoparticles alleviate hyperlipidemia and vascular injury in ApoE-deficient mice by regulating cholesterol metabolism and reducing oxidative stress.

Atherosclerosis and related cardiovascular diseases represent the greatest threats to human health worldwide. This study was designed to investigate the anti-atherosclerotic activity of selenium nanoparticles (SeNPs) in apolipoprotein E deficient (ApoE-/-) mice fed a high-cholesterol, high-fat diet. The results demonstrated that animals either treated with SeNPs (50 μg Se per kg per day) or with atorvastatin (10 mg per kg per day) alone showed significant relief of vascular injury after 8 weeks of treatment. SeNPs could obviously decrease the level of serum total cholesterol, triglyceride and low-density lipoprotein cholesterol, whereas increase serum high-density lipoprotein cholesterol. At the same time, SeNPs regulated the expression levels of key genes associated with cholesterol metabolism in the liver. Furthermore, SeNPs significantly reduced the lipid peroxidation level, but increased the NO level and the activities of glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase in the serum and liver. SeNPs also increased the expression levels of antioxidant selenoenzymes or selenoproteins in the liver. In addition, SeNPs could alleviate H2O2-induced cytotoxicity and oxidative stress by upregulating the activities of SOD and GPx in endothelial cells cultured in vitro. These results suggested that SeNPs could significantly alleviate hyperlipidemia and vascular injury in ApoE-/- mice, possibly by regulating cholesterol metabolism and reducing oxidative stress through antioxidant selenoenzymes/selenoproteins. SeNPs might be a potential candidate for the prevention of atherosclerosis.

Characterization and antioxidant activities of intracellular polysaccharides from Agaricus bitorquis (QuéL.) Sacc. Chaidam ZJU-CDMA-12

The antioxidant activities of polysaccharides from the fruiting body (PFB), extracellular polysaccharides (EPS) and intracellular polysaccharides (IPS) from Agaricus bitorquis (QuéL.) Sacc. Chaidam ZJU-CDMA-12 in vitro were compared. IPS showed stronger antioxidant activities than PFB and EPS in vitro. Further purification and structure analyses indicated that IPS mainly consisted of three fractions (IPS-I, IPS-II and IPS-III). FT-IR and NMR data indicated that IPS was mainly composed of (1 → 6)-linked α-d-glucose. There are significant differences of antioxidant activities among IPS-I, IPS-II and IPS-III fractions in vitro, and IPS-III showed stronger antioxidant activity than IPS-I and IPS-II. IPS-III also possesses a potent antioxidant ability inside HepG2 cells, and it could protect HepG2 cells from H2O2-induced cytotoxicity by scavenging overproduced cellular ROS and inhibiting SOD, CAT and GSH depletion to weaken lipid peroxidation. These findings suggested that IPS-III could be a novel antioxidant and that it could afford protection against H2O2-induced cytotoxicity and oxidative stress.

Protective Effects of Aqueous Extract of Mentha suaveolens against Oxidative Stress-Induced Damages in Human Keratinocyte HaCaT Cells.

Mentha suaveolens is an aromatic herb that has a wide range of biological activities, including antimicrobial, antifungal, anti-inflammatory, and hepatoprotective properties. Although there are a few reports on the antioxidant property of M. suaveolens, its cytoprotective activity against oxidative stress has not been reported yet. The objective of this study was to determine the protective activity of M. suaveolens aqueous extract (MSAE) against hydrogen peroxide- (H2O2-) induced oxidative stress and apoptosis in human keratinocyte HaCaT cells. MSAE pretreatment decreased H2O2-induced cytotoxicity and suppressed H2O2-induced intracellular ROS generation. Furthermore, MSAE suppressed expression levels of H2O2-induced apoptotic genes such as cleaved caspase-3, caspase-9, and cleaved poly (ADP-ribose) polymerase (PARP). Pretreatment with MSAE induced expression of phase II enzyme such as HO-1 through translocation of NF-E2-related factor (Nrf2) upon H2O2 exposure. These results revealed that the cytoprotective effect of MSAE against oxidative stress-induced cell death was associated with activation of Nrf2-mediated phase II enzyme expression.

Functional Rescue of Cataract-Causing αA-G98R-Crystallin by Targeted Compensatory Suppressor Mutations in Human αA-Crystallin.

The G98R mutation in αA-crystallin is associated with the onset of presenile cataract and is characterized biochemically by an increased oligomeric mass, altered chaperone function, and loss of structural stability over time. Thus, far, it is not known whether the inherent instability caused by gain-of-charge mutation could be rescued by a compensatory loss of charge mutation elsewhere on the protein. To answer this question, we investigated whether αA-G98R-mediated instability could be rescued through suppressor mutations by introducing site-specific "compensatory" mutations in αA-G98R-crystallin, αA-R21Q/G98R, αA-G98R/R116C, and αA-R157Q/G98R. The recombinant proteins were expressed, purified, characterized, and evaluated by circular dichroism (CD), intrinsic fluorescence, and bis-ANS-binding studies. Chaperone-like activities of recombinant proteins were assessed using alcohol dehydrogenase (ADH) and insulin as unfolding substrates. Far-UV CD studies revealed an increased α-helical content in αA-G98R in comparison to αA-WT, αA-R21Q, R157Q, and the double mutants, αA-R21Q/G98R, and αA-R157Q/G98R. Compared to αA-WT, αA-R21Q, and αA-G98R, the double mutants showed an increased intrinsic tryptophan fluorescence, whereas the highest hydrophobicity (bis-ANS-binding) was shown by αA-G98R. Introduction of a second mutation in αA-G98R reduced its bis-ANS-binding activity. Both αA-R21Q/G98R and αA-R157Q/G98R showed greater chaperone-like activity against ADH aggregation than αA-G98R. However, among the three G98R mutants, only αA-R21Q/G98R protected ARPE-19 cells from H2O2-induced cytotoxicity. These results suggest that the lost chaperone-like activity of αA-G98R-crystallin can be rescued by another targeted mutation and that substitution of αA-R21Q-crystallin at the N-terminal region can rescue a deleterious mutation in the conserved α-crystallin domain of the protein.

Neuroprotective effects of glucomoringin-isothiocyanate against H2O2-Induced cytotoxicity in neuroblastoma (SH-SY5Y) cells

Neurodegenerative diseases (NDDs) are pathological conditions characterised by progressive damage of neuronal cells leading to eventual loss of structure and function of the cells. Due to implication of multi-systemic complexities of signalling pathways in NDDs, the causes and preventive mechanisms are not clearly delineated. The study was designed to investigate the potential signalling pathways involved in neuroprotective activities of purely isolated glucomoringin isothiocyanate (GMG-ITC) against H2O2-induced cytotoxicity in neuroblastoma (SH-SY5Y) cells. GMG-ITC was isolated from Moringa oleifera seeds, and confirmed with NMR and LC-MS based methods. Gene expression analysis of phase II detoxifying markers revealed significant increase in the expression of all the genes involved, due to GMG-ITC pre-treatment. GMG-ITC also caused significant decreased in the expression of NF-kB, BACE1, APP and increased the expressions of IkB and MAPT tau genes in the differentiated cells as confirmed by multiplex genetic system analysis. The effect was reflected on the expressed proteins in the differentiated cells, where GMG-ITC caused increased in expression level of Nrf2, SOD-1, NQO1, p52 and c-Rel of nuclear factor erythroid factor 2 (Nrf2) and nuclear factor kappa-B (NF-kB) pathways respectively. The findings revealed the potential of GMG-ITC to abrogate oxidative stress-induced neurodegeneration through Nrf2 and NF-kB signalling pathways.

The antitoxic effects of quercetin and quercetin-conjugated iron oxide nanoparticles (QNPs) against H2O2-induced toxicity in PC12 cells.

BackgroundWe recently showed that quercetin-conjugated iron oxide nanoparticles (QNPs) promoted the bioavailability of quercetin (Qu) in the brain of rats and improved the learning and memory of diabetic rats. In this study, we characterized the modifications in the antitoxic effects of Qu after conjugation.Materials and methodsWe conjugated Qu to dextran-coated iron oxide nanoparticles (DNPs) and characterized DNPs and QNPs using FTIR, XRD, DLS, Fe-SEM, and EDX analyzes. The antiradical properties of Qu, DNPs, and QNPs were compared by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity assay. Catalase-like activities of DNPs and QNPs were estimated using catalase activity assay kit, and the antitoxic effects of Qu and QNPs were evaluated with spectrophotometry, MTT assay, flow cytometry, and real-time q-PCR.ResultsQu had a stronger anti-radical activity than DNPs and its activity decreased after being conjugated to DNPs. The catalase-like activity of DNPs remained intact after conjugation. DNPs had less toxicity on PC12 cells viabilities as compared to free Qu, and the conjugation of Qu with DNPs attenuated its cytotoxicity. Furthermore, MTT assay results indicated 24 h pretreatment with Qu had more protective effects than QNPs against H2O2-induced cytotoxicity, while Qu and QNPs had the same effects for 48 and 72 h incubation. Although the total antioxidant capacity of Qu was attenuated after conjugation, the results of flow cytometry and real-time q-PCR confirmed that 24 h pretreatment with the low concentrations of Qu and QNPs had the similar antioxidant, anti-inflammatory, and anti-apoptotic effects against the cytotoxicity of H2O2.ConclusionQu and QNPs showed the similar protective activities against H2O2-induced toxicity in PC12 cells. Given the fact that QNPs have magnetic properties, they may serve as suitable carriers to be used in neural research and treatment.

Effects of hepatocyte growth factor gene-transfected mesenchymal stem cells on dimethylnitrosamine-induced liver fibrosis in rats

Nowadays, transplantation of human mesenchymal stem cells (MSCs) has emerged as a potential cellular therapy for liver cirrhosis. Hepatocyte growth factor (HGF) plays an important role in the regeneration of the liver. The objective of the study was to investigate the therapeutic effect of HGF-transfected human umbilical cord blood-derived MSCs on dimethylnitrosamine (DMN)-induced liver fibrosis in rats. HGF-transfected MSCs were transplanted into rats with DMN-induced liver fibrosis. H2O2-induced cytotoxicity, apoptosis and intracellular reactive oxygen species were reduced in HGF-transfected MSCs in HGF-transfected MSCs. Pro-apoptotic proteins, such as cleaved poly (ADP-ribose) polymerase and cleaved caspase-3, were decreased in HGF-transfected MSCs. Biochemical analysis showed that the levels of aspartate aminotransferase and alanine aminotransferase were decreased after transplantation of HGF-transfected MSCs in rat fibrosis. Trichrome staining showed that HGF-transfected MSCs reduced liver damage. Taken together, our study indicated that HGF-transfected MSCs have therapeutic effects on DMN-induced liver fibrosis in rats.