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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 induces TNF−α −mediated necroptosis in aHSCs.(A) GA induced low levels of sub-G1 population in aHSCs as analyzed by flow cytometry. (B) Plasma memebrane integrity of aHSCs after GA treatment at designated concentrations was evaulated by LDH assay. (C) Involvement of TNF−α and RIP1 in GA−induced necroptosis. Increased cell viability of aHSCs was obtained through the co−incubation of GA at various concentrations and SPD304 (2μM) or Nec−1 (2μg/mL). (D) GA elicited substantial production of TNF−α as determined by immunoblotting and RT-PCR analysis. Immunoblotting analysis of necroptosis−related factors at various GA concentrations (0, 25, 50, and 75 μM) with or without (E) SPD−304, (F) BSO and TNF−α, co-incubation for 24hrs. Representative immunoblots showed the levels of TRADD, caspase−8, p-RIP3, and RIP3. β−actin was used as an internal control. * P<0.05, **P<0.01.
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pone.0120713.g004: GA induces TNF−α −mediated necroptosis in aHSCs.(A) GA induced low levels of sub-G1 population in aHSCs as analyzed by flow cytometry. (B) Plasma memebrane integrity of aHSCs after GA treatment at designated concentrations was evaulated by LDH assay. (C) Involvement of TNF−α and RIP1 in GA−induced necroptosis. Increased cell viability of aHSCs was obtained through the co−incubation of GA at various concentrations and SPD304 (2μM) or Nec−1 (2μg/mL). (D) GA elicited substantial production of TNF−α as determined by immunoblotting and RT-PCR analysis. Immunoblotting analysis of necroptosis−related factors at various GA concentrations (0, 25, 50, and 75 μM) with or without (E) SPD−304, (F) BSO and TNF−α, co-incubation for 24hrs. Representative immunoblots showed the levels of TRADD, caspase−8, p-RIP3, and RIP3. β−actin was used as an internal control. * P<0.05, **P<0.01.

Mentions: The GA-induced cytotoxic effect on aHSCs was observed in dose-dependent manners (Fig. 1A). We then attempted to further reveal the molecular mechanisms by which GA mediated the death of aHSCs. Our cell cycle analysis showed that GA did not provoke significant apoptotic effects on aHSCs (Fig. 4A, S3 Fig.). The sub G1 phase showed slight change after GA treatment (25, 50, and 75 μM). However, LDH release (P<0.05) appeared with the increase in GA concentrations (25, 50, and 75 μM) (Fig. 4B). This dose−dependent LDH release implies the disruption of the plasma membrane and subcellular organelles. Thus, GA might likely mediate a programmed necrotic effect, necroptosis, on aHSCs. It is known that TNF−α pathway has been suggested to be associated with necroptosis, and RIP1 is one of key factors of necroptosis. The TNF−α antagonist, SPD-304, and RIP1 inhibitor, Nec-1, were then used to examine GA-induced programmed necrotic cell death. The addition of SPD-304 and Nec-1 significantly rescued the survivability of aHSCs (Fig. 4C), reduced the production of lipid hydroxides (Fig. 2E), and increased intracellular GSH (Fig. 2G), indicating the involvement of necroptosis in GA-induced programmed cell death.


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 induces TNF−α −mediated necroptosis in aHSCs.(A) GA induced low levels of sub-G1 population in aHSCs as analyzed by flow cytometry. (B) Plasma memebrane integrity of aHSCs after GA treatment at designated concentrations was evaulated by LDH assay. (C) Involvement of TNF−α and RIP1 in GA−induced necroptosis. Increased cell viability of aHSCs was obtained through the co−incubation of GA at various concentrations and SPD304 (2μM) or Nec−1 (2μg/mL). (D) GA elicited substantial production of TNF−α as determined by immunoblotting and RT-PCR analysis. Immunoblotting analysis of necroptosis−related factors at various GA concentrations (0, 25, 50, and 75 μM) with or without (E) SPD−304, (F) BSO and TNF−α, co-incubation for 24hrs. Representative immunoblots showed the levels of TRADD, caspase−8, p-RIP3, and RIP3. β−actin was used as an internal control. * P<0.05, **P<0.01.
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Related In: Results  -  Collection

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pone.0120713.g004: GA induces TNF−α −mediated necroptosis in aHSCs.(A) GA induced low levels of sub-G1 population in aHSCs as analyzed by flow cytometry. (B) Plasma memebrane integrity of aHSCs after GA treatment at designated concentrations was evaulated by LDH assay. (C) Involvement of TNF−α and RIP1 in GA−induced necroptosis. Increased cell viability of aHSCs was obtained through the co−incubation of GA at various concentrations and SPD304 (2μM) or Nec−1 (2μg/mL). (D) GA elicited substantial production of TNF−α as determined by immunoblotting and RT-PCR analysis. Immunoblotting analysis of necroptosis−related factors at various GA concentrations (0, 25, 50, and 75 μM) with or without (E) SPD−304, (F) BSO and TNF−α, co-incubation for 24hrs. Representative immunoblots showed the levels of TRADD, caspase−8, p-RIP3, and RIP3. β−actin was used as an internal control. * P<0.05, **P<0.01.
Mentions: The GA-induced cytotoxic effect on aHSCs was observed in dose-dependent manners (Fig. 1A). We then attempted to further reveal the molecular mechanisms by which GA mediated the death of aHSCs. Our cell cycle analysis showed that GA did not provoke significant apoptotic effects on aHSCs (Fig. 4A, S3 Fig.). The sub G1 phase showed slight change after GA treatment (25, 50, and 75 μM). However, LDH release (P<0.05) appeared with the increase in GA concentrations (25, 50, and 75 μM) (Fig. 4B). This dose−dependent LDH release implies the disruption of the plasma membrane and subcellular organelles. Thus, GA might likely mediate a programmed necrotic effect, necroptosis, on aHSCs. It is known that TNF−α pathway has been suggested to be associated with necroptosis, and RIP1 is one of key factors of necroptosis. The TNF−α antagonist, SPD-304, and RIP1 inhibitor, Nec-1, were then used to examine GA-induced programmed necrotic cell death. The addition of SPD-304 and Nec-1 significantly rescued the survivability of aHSCs (Fig. 4C), reduced the production of lipid hydroxides (Fig. 2E), and increased intracellular GSH (Fig. 2G), indicating the involvement of necroptosis in GA-induced programmed cell death.

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