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Gallic acid induces necroptosis via TNF-α signaling pathway in activated hepatic stellate cells.

Chang YJ, Hsu SL, Liu YT, Lin YH, Lin MH, Huang SJ, Ho JA, Wu LC - PLoS ONE (2015)

Bottom Line: The beneficial effect of GA on the reduction of animal hepatofibrosis has been indicated due to its antioxidative property.The results indicated that GA elicited aHSC programmed cell death through TNF-α-mediated necroptosis.GA induced significant oxidative stress through the suppression of catalase activity and the depletion of glutathione (GSH).

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Science and Technology, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Gallic acid (3, 4, 5-trihydroxybenzoic acid, GA), a natural phenolic acid widely found in gallnuts, tea leaves and various fruits, possesses several bioactivities against inflammation, oxidation, and carcinogenicity. The beneficial effect of GA on the reduction of animal hepatofibrosis has been indicated due to its antioxidative property. However, the cytotoxicity of GA autoxidation causing cell death has also been reported. Herein, we postulated that GA might target activated hepatic stellate cells (aHSCs), the cell type responsible for hepatofibrosis, to mitigate the process of fibrosis. The molecular cytotoxic mechanisms that GA exerted on aHSCs were then analyzed. The results indicated that GA elicited aHSC programmed cell death through TNF-α-mediated necroptosis. GA induced significant oxidative stress through the suppression of catalase activity and the depletion of glutathione (GSH). Elevated oxidative stress triggered the production of TNF-α facilitating the undergoing of necroptosis through the up-regulation of key necroptotic regulatory proteins TRADD and receptor-interacting protein 3 (RIP3), and the inactivation of caspase-8. Calmodulin and calpain-1 activation were engaged, which promoted subsequent lysosomal membrane permeabilization (LMP). The TNF-α antagonist (SPD-304) and the RIP1 inhibitor (necrostatin-1, Nec-1) confirmed GA-induced TNFR1-mediated necroptosis. The inhibition of RIP1 by Nec-1 diverted the cell death from necroptosis to apoptosis, as the activation of caspase 3 and the increase of cytochrome c. Collectively, this is the first report indicating that GA induces TNF signaling-triggered necroptosis in aHSCs, which may offer an alternative strategy for the amelioration of liver fibrosis.

No MeSH data available.


Related in: MedlinePlus

GA down-regulates the activity of catalase in aHSCs.(A) The effect of antioxidants on the aHSC mortality rate. Activated HSCs were pre-treated with 75 μM of GA for 24 hrs, followed by treatements with DFX (100μM), SOD (100 U/mL), and CAT (100 U/mL) at different time intervals (0, 0.5, 1, and 2 hrs) after GA treatment. The cell viability was determined by an MTT assay. (B) The effect of catalase activity on viability in GA treated aHSCs. The aHSCs were transfected with catalase genes and incubated with GA for 24 hrs, followed by measurement of the cell survivability by MTT assay. (C) GA inhibits the activity of catalase in aHSCs but not in hepatocytes. Cells were treated with GA (0, 25, and 50 μM) for 24 hrs before the measurement of catalase activity. * P<0.05, **P<0.01.
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pone.0120713.g003: GA down-regulates the activity of catalase in aHSCs.(A) The effect of antioxidants on the aHSC mortality rate. Activated HSCs were pre-treated with 75 μM of GA for 24 hrs, followed by treatements with DFX (100μM), SOD (100 U/mL), and CAT (100 U/mL) at different time intervals (0, 0.5, 1, and 2 hrs) after GA treatment. The cell viability was determined by an MTT assay. (B) The effect of catalase activity on viability in GA treated aHSCs. The aHSCs were transfected with catalase genes and incubated with GA for 24 hrs, followed by measurement of the cell survivability by MTT assay. (C) GA inhibits the activity of catalase in aHSCs but not in hepatocytes. Cells were treated with GA (0, 25, and 50 μM) for 24 hrs before the measurement of catalase activity. * P<0.05, **P<0.01.

Mentions: The accumulation of GA–induced H2O2 in aHSCs could be resulted from impaired intracellular antioxidant system. To further investigate this assumption, the effect of antioxidant system on cell survivability was then determined (Fig. 3). Reagents such as deferoxamine (DFX) (a ferric iron chelator to limit Fenton−like reaction), superoxide dismutase (SOD), and catalase (CAT) were used to reduce oxidative stress. DFX chelates ferric iron to retard Fenton’s reaction and the subsequent radical generation. SOD catalyzes the dismutation of superoxide to oxygen and hydrogen peroxide. Catalase catalyzes the decomposition of hydrogen peroxide to water and oxygen. Activated HSCs were initially incubated with GA, followed by the addition of antioxidants at different time intervals (0, 0.5, 1, and 2 hrs) after GA treatment. After 24 hrs of incubation, the cell viability was determined. Fig. 3A indicates that group treated with catalase showed the greatest cell survival promoting effect compared to other antioxidants. Group treated with DFX showed reduced cytotoxic effect in the first two time periods (0 and 0.5 hr) presumably due to the suppression of hydroxyl radical production catalyzed by iron. However, at the late time period (1 and 2 hrs), the cytotoxcity of DFX and GA co−treatment group was similar to that of GA alone, suggesting the critical role of H2O2 in cytotoxicity. There were significant cytotoxicity and no rescuing effect observed in the groups treated with SOD probably because of the accumulation of H2O2 resulted by the catalyzation of superoxides. On the other hand, cell survival was significantly promoted in groups treated with catalase, indicating the involvement of H2O2 in cytotoxicity.


Gallic acid induces necroptosis via TNF-α signaling pathway in activated hepatic stellate cells.

Chang YJ, Hsu SL, Liu YT, Lin YH, Lin MH, Huang SJ, Ho JA, Wu LC - PLoS ONE (2015)

GA down-regulates the activity of catalase in aHSCs.(A) The effect of antioxidants on the aHSC mortality rate. Activated HSCs were pre-treated with 75 μM of GA for 24 hrs, followed by treatements with DFX (100μM), SOD (100 U/mL), and CAT (100 U/mL) at different time intervals (0, 0.5, 1, and 2 hrs) after GA treatment. The cell viability was determined by an MTT assay. (B) The effect of catalase activity on viability in GA treated aHSCs. The aHSCs were transfected with catalase genes and incubated with GA for 24 hrs, followed by measurement of the cell survivability by MTT assay. (C) GA inhibits the activity of catalase in aHSCs but not in hepatocytes. Cells were treated with GA (0, 25, and 50 μM) for 24 hrs before the measurement of catalase activity. * P<0.05, **P<0.01.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4376672&req=5

pone.0120713.g003: GA down-regulates the activity of catalase in aHSCs.(A) The effect of antioxidants on the aHSC mortality rate. Activated HSCs were pre-treated with 75 μM of GA for 24 hrs, followed by treatements with DFX (100μM), SOD (100 U/mL), and CAT (100 U/mL) at different time intervals (0, 0.5, 1, and 2 hrs) after GA treatment. The cell viability was determined by an MTT assay. (B) The effect of catalase activity on viability in GA treated aHSCs. The aHSCs were transfected with catalase genes and incubated with GA for 24 hrs, followed by measurement of the cell survivability by MTT assay. (C) GA inhibits the activity of catalase in aHSCs but not in hepatocytes. Cells were treated with GA (0, 25, and 50 μM) for 24 hrs before the measurement of catalase activity. * P<0.05, **P<0.01.
Mentions: The accumulation of GA–induced H2O2 in aHSCs could be resulted from impaired intracellular antioxidant system. To further investigate this assumption, the effect of antioxidant system on cell survivability was then determined (Fig. 3). Reagents such as deferoxamine (DFX) (a ferric iron chelator to limit Fenton−like reaction), superoxide dismutase (SOD), and catalase (CAT) were used to reduce oxidative stress. DFX chelates ferric iron to retard Fenton’s reaction and the subsequent radical generation. SOD catalyzes the dismutation of superoxide to oxygen and hydrogen peroxide. Catalase catalyzes the decomposition of hydrogen peroxide to water and oxygen. Activated HSCs were initially incubated with GA, followed by the addition of antioxidants at different time intervals (0, 0.5, 1, and 2 hrs) after GA treatment. After 24 hrs of incubation, the cell viability was determined. Fig. 3A indicates that group treated with catalase showed the greatest cell survival promoting effect compared to other antioxidants. Group treated with DFX showed reduced cytotoxic effect in the first two time periods (0 and 0.5 hr) presumably due to the suppression of hydroxyl radical production catalyzed by iron. However, at the late time period (1 and 2 hrs), the cytotoxcity of DFX and GA co−treatment group was similar to that of GA alone, suggesting the critical role of H2O2 in cytotoxicity. There were significant cytotoxicity and no rescuing effect observed in the groups treated with SOD probably because of the accumulation of H2O2 resulted by the catalyzation of superoxides. On the other hand, cell survival was significantly promoted in groups treated with catalase, indicating the involvement of H2O2 in cytotoxicity.

Bottom Line: The beneficial effect of GA on the reduction of animal hepatofibrosis has been indicated due to its antioxidative property.The results indicated that GA elicited aHSC programmed cell death through TNF-α-mediated necroptosis.GA induced significant oxidative stress through the suppression of catalase activity and the depletion of glutathione (GSH).

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Science and Technology, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Gallic acid (3, 4, 5-trihydroxybenzoic acid, GA), a natural phenolic acid widely found in gallnuts, tea leaves and various fruits, possesses several bioactivities against inflammation, oxidation, and carcinogenicity. The beneficial effect of GA on the reduction of animal hepatofibrosis has been indicated due to its antioxidative property. However, the cytotoxicity of GA autoxidation causing cell death has also been reported. Herein, we postulated that GA might target activated hepatic stellate cells (aHSCs), the cell type responsible for hepatofibrosis, to mitigate the process of fibrosis. The molecular cytotoxic mechanisms that GA exerted on aHSCs were then analyzed. The results indicated that GA elicited aHSC programmed cell death through TNF-α-mediated necroptosis. GA induced significant oxidative stress through the suppression of catalase activity and the depletion of glutathione (GSH). Elevated oxidative stress triggered the production of TNF-α facilitating the undergoing of necroptosis through the up-regulation of key necroptotic regulatory proteins TRADD and receptor-interacting protein 3 (RIP3), and the inactivation of caspase-8. Calmodulin and calpain-1 activation were engaged, which promoted subsequent lysosomal membrane permeabilization (LMP). The TNF-α antagonist (SPD-304) and the RIP1 inhibitor (necrostatin-1, Nec-1) confirmed GA-induced TNFR1-mediated necroptosis. The inhibition of RIP1 by Nec-1 diverted the cell death from necroptosis to apoptosis, as the activation of caspase 3 and the increase of cytochrome c. Collectively, this is the first report indicating that GA induces TNF signaling-triggered necroptosis in aHSCs, which may offer an alternative strategy for the amelioration of liver fibrosis.

No MeSH data available.


Related in: MedlinePlus