Silencing of Peroxiredoxin-4 in Anticancer Activity of Gamma-Tocotrienol

Peroxiredoxin-4 (PRDX4) is known to have a role in protecting cells from oxidative stress. It has been previously reported to increase in HepG2 liver cancer cells treated with gamma-tocotrienol (GTT). As GTT treatment potentially kills the cancer cell by regulating multiple signaling pathways, this study aims to determine the involvement of PRDX4 in GTT anticancer activity by silencing the PRDX4 gene. The efficiency of PRDX4 silencing is achieved by optimizing HepG2 cell density, effect of serum presence in transduction media, incubation time of the cells with lentivirus, polybrene concentration, puromycin dose, functional titer, and multiplicity of infection (MOI) of the lentivirus. Silenced HepG2-PRDX4 cells (HepG2-shRNA-PRDX4) were treated with 70 μM of GTT for 48 h. GTT treatment significantly decreased the HepG2-shRNA-PRDX4 cell viability, increased apoptosis rate, and reduced free radical production compared to untreated HepG2-shRNA-PRDX4 cells. These findings are further supported by proteomic analysis, which showed that pro-apoptotic and DNA damage proteins were upregulated, and proteins involved in cell cycle arrest, carcinogenesis, and anti-apoptotic signaling pathways were downregulated in HepG2-shRNA-PRDX4 cells treated with GTT compared to control. In conclusion, PRDX4 plays a role in GTT anticancer activity by increasing free radical production and oxidative damage to induce apoptosis in HepG2 cell

Manuka honey protects middle-aged rats from oxidative damage

OBJECTIVE: This study aimed to determine the effect of manuka honey on the oxidative status of middle-aged rats. METHOD: Twenty-four male Sprague-Dawley rats were divided into young (2 months) and middle-aged (9 months) groups. They were further divided into two groups each, which were either fed with plain water (control) or supplemented with 2.5 g/kg body weight of manuka honey for 30 days. The DNA damage level was determined via the comet assay, the plasma malondialdehyde level was determined using high performance liquid chromatography, and the antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase, glutathione peroxidase and catalase) were determined spectrophotometrically in the erythrocytes and liver. The antioxidant activities were measured using 1,1-diphenyl-2-picrylhydrazyl and ferric reducing/antioxidant power assays, and the total phenolic content of the manuka was analyzed using UV spectrophotometry and the Folin-Ciocalteu method, respectively. RESULTS: Supplementation with manuka honey reduced the level of DNA damage, the malondialdehyde level and the glutathione peroxidase activity in the liver of both the young and middle-aged groups. However, the glutathione peroxidase activity was increased in the erythrocytes of middle-aged rats given manuka honey supplementation. The catalase activity was reduced in the liver and erythrocytes of both young and middle-aged rats given supplementation. Manuka honey was found to have antioxidant activity and to have a high total phenolic content. These findings showed a strong correlation between the total phenolic content and antioxidant activity. CONCLUSIONS: Manuka honey reduces oxidative damage in young and middle-aged rats; this effect could be mediated through the modulation of its antioxidant enzyme activities and its high total phenolic content. Manuka honey can be used as an alternative supplement at an early age to improve the oxidative status

Protective Effects of Gelam Honey against Oxidative Damage in Young and Aged Rats

Aging is characterized by progressive decline in physiological and body function due to increase in oxidative damage. Gelam honey has been accounted to have high phenolic and nonphenolic content to attenuate oxidative damage. This study was to determine the effect of local gelam honey on oxidative damage of aged rats. Twenty-four male Spraque-Dawley rats were divided into young (2 months) and aged (19 months) groups. Each group was further divided into control (fed with plain water) and supplemented with 2.5 mg/kg body weight of gelam honey for 8 months. DNA damage level was determined by comet assay and plasma malondialdehyde (MDA) by high performance liquid chromatography (HPLC). The activity of blood and cardiac antioxidant enzymes was determined by spectrophotometer. The DNA damage and MDA level were reduced in both gelam honey supplemented groups. Gelam honey increases erythrocytes CAT and cardiac SOD activities in young and cardiac CAT activity in young and aged groups. The DNA damage was increased in the aged group compared to young group, but reduced at the end of the study. The decline of oxidative damage in rats supplemented with gelam honey might be through the modulation of antioxidant enzyme activities

Tocotrienol-rich Fraction Modulates Cardiac Metabolic Profile changes in Isoprenaline-Induced Myocardial Infarction rats

In myocardial infarction (MI), the occurrence of energy depletion, oxidative stress, and decreased amino acids metabolism alter tissue metabolites. Evidence has shown that tocotrienol-rich fraction (TRF) prevents myocardial injury in MI. However, the protective mechanism at the metabolite level is unknown. Male Sprague-Dawley rats were grouped into control, isoprenaline (ISO)-induced MI (MI), healthy rats receiving 200 mg/kg TRF (200TRF), and MI rats receiving 200 mg/kg TRF (200TRF+MI) groups. TRF was administered via oral gavage daily for 12 weeks followed by intraperitoneal ISO injection (85 mg/kg) for two consecutive days at a 24-hour interval to induce MI. High-performance liquid chromatography was performed to analyze serum α-tocopherol and tocotrienol concentration whereas ultrahigh-performance liquid chromatography-mass spectrometry was used for the untargeted metabolomic study. Serum α-tocopherol but not tocotrienol was increased in the 200TRF (p=0.121) and 200TRF+MI (p<0.05) following TRF supplementation. Multivariate analysis by Orthogonal Projections to Latent Structures Discriminant Analysis showed high predictability of the group comparison models for MI vs control and 200TRF+MI vs MI (cross-validation: Q2 >0.7, R2 Y>0.8, p<0.05). A total of 84 and 37 metabolites [when covariance of p≥|0.05| (magnitude) and p(corr)≥|0.5| (reliability)] were significantly different in the myocardial homogenates of MI vs control and 200TRF+MI vs MI, respectively. MI rats had reduced S-adenosylmethionine and L-cystathionine that might worsen MI by disturbing glutathione metabolism; decreased phosphoribosyl-pyrophosphate and purine salvage process that might impair DNA synthesis, and elevated glucose-6-phosphate suggesting enhanced anaerobic glycolysis possibly for rapid production of energy. Conversely, TRF supplementation reversed the impaired metabolic pathways caused by MI

Tocotrienol-rich fraction modulates cardiac metabolic profile changes in isoprenaline-induced myocardial infarction rats

In myocardial infarction (MI), the occurrence of energy depletion, oxidative stress, and decreased amino acids metabolism alter tissue metabolites. Evidence has shown that tocotrienol-rich fraction (TRF) prevents myocardial injury in MI. However, the protective mechanism at the metabolite level is unknown. Male Sprague-Dawley rats were grouped into control, isoprenaline (ISO)-induced MI (MI), healthy rats receiving 200 mg/kg TRF (200TRF), and MI rats receiving 200 mg/kg TRF (200TRF+MI) groups. TRF was administered via oral gavage daily for 12 weeks followed by intraperitoneal ISO injection (85 mg/kg) for two consecutive days at a 24-hour interval to induce MI. High-performance liquid chromatography was performed to analyze serum α-tocopherol and tocotrienol concentration whereas ultra-high-performance liquid chromatography-mass spectrometry was used for the untargeted metabolomic study. Serum α-tocopherol but not tocotrienol was increased in the 200TRF (p=0.121) and 200TRF+MI (p0.7, R2Y>0.8, p<0.05). A total of 84 and 37 metabolites [when covariance of p≥|0.05| (magnitude) and p(corr)≥|0.5| (reliability)] were significantly different in the myocardial homogenates of MI vs control and 200TRF+MI vs MI, respectively. MI rats had reduced S-adenosylmethionine and L-cystathionine that might worsen MI by disturbing glutathione metabolism; decreased phosphoribosyl-pyrophosphate and purine salvage process that might impair DNA synthesis, and elevated glucose-6-phosphate suggesting enhanced anaerobic glycolysis possibly for rapid production of energy. Conversely, TRF supplementation reversed the impaired metabolic pathways caused by MI

Inhibitory effects of Gynura procumbens ethanolic extract on nitric oxide production and inducible nitric oxide synthase (iNOS) protein expression in macrophages

Nitric oxide (NO) overproduction by inducible nitric oxide synthase (iNOS) may be associated with acute and chronic inflammations. Macrophages as important cells in the innate immune system are able to be stimulated and can lead to iNOS activation and excessive NO production. Gynura procumbens is a medicinal plant traditionally used in treating various ailments including inflammation but the mechanism of anti-inflammatory activity of this plant is still elusive. This study was carried out to investigate the anti-inflammatory therapeutic effects of Gynura procumbens ethanolic extract on NO production and iNOS protein expression in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). Cell viability of RAW 264.7 macrophages treated with Gynura procumbens ethanolic extract was determined by MTT assay. NO production was determined by Griess assay following Gynura procumbens ethanolic extract treatment alone or in combination with LPS stimulation. Protein expression of iNOS was determined by western blot. RAW 264.7 macrophages viability of more than 90% was observed after 24 h treatment with Gynura procumbens ethanolic extract concentration range of 3.9 μg/mL to 500 μg/mL. Significant inhibition of NO production level has been identified in LPS-stimulated RAW 264.7 cells pre-treated with 250 μg/mL Gynura procumbens ethanolic extract (p<0.05) while all selected concentrations of Gynura procumbens ethanolic extract showed no significant alteration of NO production in the absence of LPS stimulation. Pre-treatment of 250 μg/mL Gynura procumbens ethanolic extract also demonstrated significant suppression of iNOS protein expression in LPS-stimulated RAW 264.7 cells (p<0.05). In conclusion, this study demonstrates that Gynura procumbens ethanolic extract exhibits anti-inflammatory potential through inhibition of NO production and iNOS protein expression in LPS-stimulated macrophages, suggesting that this plant could be further researched for its beneficial use in inflammatory disorders

Palm tocotrienol-rich fraction protects neonatal rat cardiomyocytes from H2 O2 - induced oxidative damage

Oxidative stress plays an important role in the pathogenesis of heart disease. Tocotrienol-rich fraction (TRF) is an antioxidant and that has the potential to reduce the risk of heart disease. This study is to determine the protective effects of palm TRF against H2 O2 -induced oxidative damage in neonatal rat cardiomyocytes (NRCM). The NRCM were divided into control, treated with TRF (10 µg/mL), H2 O2 (0.5 mM) and treated with TRF prior to H2 O2 induction (pre-treatment). Cell viability was determined by the MTS assay,while the presence of reactive oxygen species (ROS) was determined using fluorescent dihydroethidium (DHE) and 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2 DCFDA) dye. Mitochondrial integrity and cell death were determined using JC-1 and Annexin V-FITC staining, respectively. Lactate dehydrogenase (LDH) and superoxide dismutase (SOD) activity were determined by colorimetric assay kit. The concentration of H2 O2 from 0.5 to 5 mM reduced the cell viability and the H2 O2 IC50 value of 0.5 mM was used in the experiment. H2 O2 induction increased the intensity of carboxy-H2 DCFDA and DHE-stains; and also the intensity of green fluorescence of J-monomers in JC-1 staining compared to the control group. The activity of LDH increased while the activity of SOD decreased in the H2 O2 group. Pre-treatment with TRF reduced the intensities of carboxy-H2 DCFDA and DHE-stains, as well as the green fluorescence of J-monomers in JC-1. Meanwhile, the LDH activity was reduced in the pre-treatment group but no changes were recorded in SOD activity compared to the H2 O2 group. Palm TRF protects cardiomyocytes from oxidative damage by reducing ROS production and maintaining the mitochondrial membrane integrity thus reducing cell death

Gelam Honey Protects against Gamma-Irradiation Damage to Antioxidant Enzymes in Human Diploid Fibroblasts

The present study was designed to determine the radioprotective effects of Malaysian Gelam honey on gene expression and enzyme activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) of human diploid fibroblasts (HDFs) subjected to gamma-irradiation. Six groups of HDFs were studied: untreated control, irradiated HDFs, Gelam honey-treated HDFs and HDF treated with Gelam honey pre-, during- and post-irradiation. HDFs were treated with 6 mg/mL of sterilized Gelam honey (w/v) for 24 h and exposed to 1 Gray (Gy) of gamma rays at the dose rate of 0.25 Gy/min. Gamma-irradiation was shown to down-regulate SOD1, SOD2, CAT and GPx1 gene expressions (p &lt; 0.05). Conversely, HDFs treated with Gelam honey alone showed up-regulation of all genes studied. Similarly, SOD, CAT and GPx enzyme activities in HDFs decreased with gamma-irradiation and increased when cells were treated with Gelam honey (p &lt; 0.05). Furthermore, of the three different stages of study treatment, pre-treatment with Gelam honey caused up-regulation of SOD1, SOD2 and CAT genes expression and increased the activity of SOD and CAT. As a conclusion, Gelam honey modulates the expression of antioxidant enzymes at gene and protein levels in irradiated HDFs indicating its potential as a radioprotectant agent