Protective effect of luteolin against oxidative stress­mediated cell injury via enhancing antioxidant systems.

Physiological stress such as excessive reactive oxygen species (ROS) production may contribute normal fibroblasts activation into cancer­associated fibroblasts, which serve a crucial role in certain types of cancer such as pancreatic, breast, liver and lung cancer. The present study aimed to examine the cytoprotective effects of luteolin (3',4',5,7­tetrahydroxyflavone) against hydrogen peroxide (H2O2)­generated oxidative stress in lung fibroblasts. To examine the effects of luteolin against H2O2­induced damages, cell viability, sub­G1 cell population, nuclear staining with Hoechst 33342, lipid peroxidation and comet assays were performed. To evaluate the effects of luteolin on the protein expression level of apoptosis, western blot assay was performed. To assess the antioxidant effects of luteolin, detection of ROS using H2DCFDA staining, O2­ and ·OH using electron spin resonance spectrometer and antioxidant enzyme activity was performed. In a cell­free chemical system, luteolin scavenges superoxide anion and hydroxyl radical generated by xanthine/xanthine oxidase and the Fenton reaction (FeSO4/H2O2). Furthermore, Chinese hamster lung fibroblasts (V79­4) treated with H2O2 showed a significant increase in cellular ROS. Intracellular ROS levels and damage to cellular components such as lipids and DNA in H2O2­treated cells were significantly decreased by luteolin pretreatment. Luteolin increased cell viability, which was impaired following H2O2 treatment and prevented H2O2­mediated apoptosis. Luteolin suppressed active caspase­9 and caspase­3 levels while increasing Bcl­2 expression and decreasing Bax protein levels. Additionally, luteolin restored levels of glutathione that was reduced in response to H2O2. Moreover, luteolin enhanced the activity and protein expressions of superoxide dismutase, catalase, glutathione peroxidase, and heme oxygenase­1. Overall, these results indicated that luteolin inhibits H2O2­mediated cellular damage by upregulating antioxidant enzymes.

Preventive effect of 7,8-dihydroxyflavone against oxidative stress induced genotoxicity.

We elucidated the protective effect of 7,8-dihydroxyflavone against hydrogen peroxide (H(2)O(2))-induced DNA damage. We found that 7,8-dihydroxyflavone scavenges 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and intracellular reactive oxygen species (ROS). 7,8-Dihydroxyflavone with antioxidant effect prevented the H(2)O(2)-induced cellular DNA damage, as evidenced by comet tail, 8-hydroxy-2'-deoxyguanosine (8-OHdG) content, and phospho-histone H2A.X protein expression. Hence, 7,8-dihydroxyflavone was shown to protect cell via the inhibition of apoptosis induced by H(2)O(2). This was substantiated by decreased apoptotic nuclear fragmentation, decreased sub-G(1) cell population, and decreased DNA fragmentation. Furthermore, 7,8-dihydroxyflavone activated the protein kinase B (PKB, Akt) signal pathway, which is a major survival signal pathway. In addition, LY294002, which is phosphatidylinositol 3 kinase (PI3K, upstream of Akt) inhibitor, attenuated the protective effect of 7,8-dihydroxyflavone against H(2)O(2)-induced cell damage. In conclusion, 7,8-dihydroxyflavone was shown to possess cytoprotective properties against oxidative stress by scavenging intracellular ROS and enhancing Akt activity.

Effect of KIOM-79 against mitochondrial damage induced by streptozotocin in pancreatic beta-cells.

The present study examined the effects of KIOM-79 on streptozotocin (STZ)-induced mitochondrial oxidative stress in rat pancreatic beta-cells (RINm5F). KIOM-79 is a mixture of plant extracts from parched Puerariae radix, gingered Magnoliae cortex, Glycyrrhizae radix, and Euphorbiae radix. A marked increase in mitochondrial reactive oxygen species (ROS) was observed in STZ induced diabetic cells, which returned to control conditions after KIOM-79 treatment. Mitochondrial manganese superoxide dismutase (Mn SOD) activity and its protein expression were downregulated by STZ treatment but upregulated by KIOM-79 treatment. In addition, KIOM-79 treatment restored the loss of the mitochondrial membrane potential (Deltapsi) produced by STZ treatment. KIOM-79 induced an increase in Bcl-2 and a decrease in phospho Bcl-2 and Bax, which are related to permeability of the mitochondrial membrane. Further, KIOM-79 inhibited the translocation of cytochrome c from the mitochondria to the cytosol and elevated the ATP level, which was reduced by STZ treatment. These results suggest that KIOM-79 exhibits a protective effect through activation of antioxidant defense mechanisms and by attenuation of mitochondrial dysfunction in STZ-induced diabetic cells.

Protective effect of esculetin against oxidative stress-induced cell damage via scavenging reactive oxygen species.

AIM: To investigate the anti-oxidant properties of esculetin (6,7-dihydroxycoumarin) against H2O2-induced Chinese hamster lung fibroblast (V79-4) damage. METHODS: The radical scavenging activity was assessed by 1,1-diphenyl- 2-picrylhydrazyl (DPPH) radical, hydroxyl radical, and intracellular reactive oxygen species (ROS). In addition, lipid peroxidation was assayed by the measure of related substances which react with thiobarbituric acid. The amount of carbonyl formation in protein was determined using a protein carbonyl ELISA kit. As well, cellular DNA damage was detected by Western blot and immunofluorescence image. Cell viability was assessed by 3-(4,5-dimethylthiazole-2- yl)-2,5-diphenyltetrazolium bromide assay. RESULTS: Esculetin exhibited DPPH radical scavenging, hydroxyl radical scavenging, and intracellular ROS scavenging activities. The radical scavenging activity of esculetin resulted in the protection of cells from lipid peroxidation, protein carbonyl, and DNA damage induced by H2O2. Therefore, esculetin recovered cell viability exposed to H2O2. CONCLUSION: Esculetin efficiently attenuated the oxidative stress induced cell damage via its anti-oxidant properties. As a result, esculetin may be useful in the development of functional food and raw materials of medicine.

Hyperoside prevents oxidative damage induced by hydrogen peroxide in lung fibroblast cells via an antioxidant effect.

We elucidated the cytoprotective effects of hyperoside (quercetin-3-O-galactoside) against hydrogen peroxide (H2O2)-induced cell damage. We found that hyperoside scavenged the intracellular reactive oxygen species (ROS) detected by fluorescence spectrometry, flow cytometry, and confocal microscopy. In addition, we found that hyperoside scavenged the hydroxyl radicals generated by the Fenton reaction (FeSO4)+H2O2) in a cell-free system, which was detected by electron spin resonance (ESR) spectrometry. Hyperoside was found to inhibit H2O2-induced apoptosis in Chinese hamster lung fibroblast (V79-4) cells, as shown by decreased apoptotic nuclear fragmentation, decreased sub-G(1) cell population, and decreased DNA fragmentation. In addition, hyperoside pretreatment inhibited the H2O2-induced activation of caspase-3 measured in terms of levels of cleaved caspase-3. Hyperoside prevented H2O2-induced lipid peroxidation as well as protein carbonyl. In addition, hyperoside prevented the H2O2-induced cellular DNA damage, which was established by comet tail, and phospho histone H2A.X expression. Furthermore, hyperoside increased the catalase and glutathione peroxidase activities. Conversely, the catalase inhibitor abolished the cytoprotective effect of hyperoside from H2O2-induced cell damage. In conclusion, hyperoside was shown to possess cytoprotective properties against oxidative stress by scavenging intracellular ROS and enhancing antioxidant enzyme activity.

Induction of heme oxygenase-1 by plant extract KIOM-79 via Akt pathway and NF-E2 related factor 2 in pancreatic beta-cells.

The objective of the present study was to determine the mechanism by which KIOM-79 induced heme oxygenase-1 (HO-1) in rat pancreatic beta-cells (RINm5F). A mixture of plant extracts (KIOM-79) was obtained from Magnolia officinalis, Pueraria lobata, Glycyrrhiza uralensis, and Euphorbia pekinensis. HO-1, an antioxidant phase 2 enzyme, was previously reported to possess cytoprotective properties in pancreatic beta-cells. KIOM-79 induced heme oxygenase-1 (HO-1) expression at the mRNA and protein levels, leading to increased HO-1 activity. The transcription factor, NF-E2 related factor 2 (Nrf2), regulates the antioxidant response element (ARE) of the phase 2 detoxifying and antioxidant enzymes, resulting in modulation of HO-1 expression. KIOM-79 increased nuclear translocation, ARE binding, and transcriptional activity of Nrf2. Furthermore, KIOM-79 also elicited activation of Akt (protein kinase B) and LY294004 (inhibitor of Akt)-suppressed KIOM-79-induced activation of Nrf2, which subsequently decreased HO-1 protein levels. Taken together, these data suggest that KIOM-79 augments the cellular antioxidant defense capacity through induction of HO-1 via the Akt-Nrf2-ARE signaling pathway, thereby protecting cells from streptozotocin-induced oxidative stress.

Protective effect of butin against hydrogen peroxide-induced apoptosis by scavenging reactive oxygen species and activating antioxidant enzymes.

The antioxidant property of butin was investigated for cytoprotective effect against H(2)O(2)-induced cell damage. This compound showed intracellular reactive oxygen species (ROS) scavenging, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, inhibition of lipid peroxidation, and DNA damage. This radical scavenging activity of butin protected cell damage exposed to H(2)O(2). Also, butin reduced the apoptotic cells induced by H(2)O(2), as demonstrated by the decreased DNA fragmentation, apoptotic body formation, and caspase 3 activity. In addition, butin restored the activity and protein expression of cellular antioxidant enzymes, superoxide dismutase (SOD), and catalase (CAT) in H(2)O(2)-treated cells. Taken together, these findings suggest that butin protected cells against H(2)O(2)-induced cell damage via antioxidant property.

Eckol protects V79-4 lung fibroblast cells against gamma-ray radiation-induced apoptosis via the scavenging of reactive oxygen species and inhibiting of the c-Jun NH(2)-terminal kinase pathway.

The radioprotective effect of eckol against gamma-ray radiation-induced oxidative stress and its possible protective mechanisms were investigated. Eckol was found to reduce the intracellular reactive oxygen species generated by gamma-ray radiation. Moreover, eckol also protected against radiation-induced cellular DNA damage and membrane lipid peroxidation, which are the main targets of radiation-induced damage. In addition, eckol recovered the cell viability damaged by radiation via the inhibition of apoptosis. Irradiated cells with eckol treatment reduced the expression of bax, the activation of caspase 9 and caspase 3, which were induced by radiation. However, irradiated cells with eckol recovered the expression of bcl-2 and mitochondrial cytochrome c which were decreased by radiation. The anti-apoptotic effect of eckol exerted via the inhibition of mitogen-activated protein kinase kinase-4 (MKK4/SEK1)-c-Jun NH(2)-terminal kinase (JNK)-activator protein 1 (AP-1) cascades induced by radiation. In summary, the results suggest that eckol protects cells against the oxidative stress induced by radiation via the reduction of reactive oxygen species and the attenuation of activation in SEK1-JNK-AP-1 pathway.

Cytoprotective activity of annphenone against oxidative stress-induced apoptosis in V79-4 lung fibroblast cells.

We have elucidated the cytoprotective effect of annphenone (2,4-dihyroxy-6-methoxy-acetophenone 4-O-beta-D-glucopyranoside) against oxidative stress-induced apoptosis. Annphenone scavenged intracellular reactive oxygen species (ROS) and increased antioxidant enzyme activities. It thereby prevented lipid peroxidation and DNA damage, which was demonstrated by the inhibition of the formation of thiobarbituric acid reactive substance (TBARS), inhibition of the comet tail and decreased phospho-H2A.X expression. Annphenone protected Chinese hamster lung fibroblast (V79-4) cells from cell death via the inhibition of apoptosis induced by hydrogen peroxide (H2O2), as shown by decreased apoptotic nuclear fragmentation, decreased sub-G1 cell population and inhibited mitochondrial membrane potential (Deltapsi) loss. Taken together, these findings suggest that annphenone exhibits antioxidant properties by inhibiting ROS generation and thus protecting cells from H2O2-induced cell damage.

Inhibitory effects of triphlorethol-A on MMP-1 induced by oxidative stress in human keratinocytes via ERK and AP-1 inhibition.

Oxidative stress is known to generate reactive oxygen species (ROS) in cells, which subsequently induce the synthesis of matrix metalloproteinases (MMP) and an aging phenomenon. The protective effects of triphlorethol-A, derived from Ecklonia cava, were investigated against hydrogen peroxide (H(2)O(2))-induced damage using human skin keratinocytes. Data showed that triphlorethol-A inhibited ROS formation, induced catalase expression, inhibited DNA damage, and increased cell viability in keratinocytes. Triphlorethol-A treatment significantly reduced MMP-1 expression and production, compared to H(2)O(2)-treated cells. In addition, triphlorethol-A abrogated the activation of extracellular signal regulated protein kinase (ERK), which originates upstream of MMP-1 expression, and was induced by H(2)O(2) treatment. Moreover, triphlorethol-A inhibited DNA binding activity of activator protein-1 (AP-1), a downstream transcription factor of ERK. Data indicate that the antioxidative properties of triphlorethol-A involve the inhibition of MMP-1 via ERK and AP-1 inhibition.

Protective effect of irisolidone, a metabolite of kakkalide, against hydrogen peroxide induced cell damage via antioxidant effect.

The protective properties of irisolidone (a metabolite of kakkalide by intestinal bacteria) against hydrogen peroxide (H(2)O(2)) induced cell damage were investigated. Irisolidone was found to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and the intracellular reactive oxygen species (ROS), thereby preventing lipid peroxidation and DNA damage. Irisolidone inhibited apoptosis in Chinese hamster lung fibroblast (V79-4) cells induced by H(2)O(2) via radical scavenging activity. This was achieved by the activation of the extracellular signal regulated kinase (ERK) and DNA binding activity of activator protein-1 (AP-1) (a downstream transcription factor of ERK) by irisolidone.