Paeonia japonica root extract protects hepatocytes against oxidative stress through inhibition of AMPK-mediated GSK3β

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

Cho

et al. 2017

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Endoplasmic reticulum (ER) stress is characterized by an accumulation of misfolded proteins, and ER stress reduction is essential for maintaining tissue homeostasis. However, the molecular mechanisms that protect cells from ER stress are not completely understood. The present study investigated the role of sestrin 2 (SESN2) on ER stress and sought to elucidate the mechanism responsible for the hepatoprotective effect of SESN2 in vitro and in vivo. Treatment with tunicamycin (Tm) increased SESN2 protein and mRNA levels and reporter gene activity. Activating transcription factor 6 (ATF6) bound to unfolded protein response elements of SESN2 promoter, transactivated SESN2, and increased SESN2 protein expression. In addition, dominant negative mutant of ATF6α and siRNA against ATF6α blocked the ER stress-mediated SESN2 induction, whereas chemical inhibition of PERK or IRE1 did not affect SESN2 induction by Tm. Ectopic expression of SESN2 in HepG2 cells inhibited CHOP and GRP78 expressions by Tm. Moreover, SESN2 decreased the phosphorylations of JNK and p38 and PARP cleavage, and blocked the cytotoxic effect of excessive ER stress. In a Tm-induced liver injury model, adenoviral delivery of SESN2 in mice decreased serum ALT, AST and LDH activities and the mRNA levels of CHOP and GRP78 in hepatic tissues. Moreover, SESN2 reduced numbers of degenerating hepatocytes, and inhibited caspase 3 and PARP cleavages. These results suggest ATF6 is essential for ER stress-mediated SESN2 induction, and that SESN2 acts as a feedback regulator to protect liver from excess ER stress.

Section: Histopathology and Immunohistochemistrymentioning

confidence: 99%

Activating transcription factor 6-dependent sestrin 2 induction ameliorates ER stress-mediated liver injury

Jegal

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

Cho

et al. 2017

Self Cite

“…HepG2 cells (a human hepatocyte-derived cell line) and HeLa cells (a human cervical cancer cell line) were purchased from American Type Culture Collection (Rockville, MD, USA) and cultured in Dulbecco's modified Eagle's medium containing 100 U/mL of penicillin, 100 μ g/mL of streptomycin, and 10% fetal bovine serum at 37°C with 5% CO 2 . For all experiments, the cells were grown to 80-90% confluency and starved serum for 12 h. HepG2 cells were treated with AA and iron, as described previously [ 9 ]. Briefly, cells were incubated with 10 μ M of AA for 12 h and then subsequently exposed to 5 μ M of iron for 1 h. 30-1000 μ g/mL of SDT was pretreated 1 h before AA treatment.…”

Section: Methodsmentioning

confidence: 99%

“…The level of reduced GSH in cell homogenates was determined by using a GSH BIOXYTECH GSH-400 kit (Oxis International Inc., Portland, OR, USA), as previously described [ 9 ]. GSH content was measured at 405 nm using a microplate reader (Infinite 200 PRO, Tecan, Männedorf, Switzerland) and was normalized by cell number.…”

Section: Methodsmentioning

confidence: 99%

Sipjeondaebo-tang Alleviates Oxidative Stress‐Mediated Liver Injury through Activation of the CaMKK2‐AMPK Signaling Pathway

Evidence-Based Complementary and Alternative Medicine

Kim

Jung

et al. 2018

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Sipjeondaebo-tang (SDT) is used frequently as a herbal prescription to treat deficiency syndromes in traditional Korean medicine. We investigated the hepatoprotective effects of SDT against oxidative stress and attempted to clarify the underlying molecular mechanisms. SDT pretreatment reduced arachidonic acid (AA) plus iron-mediated cytotoxicity in a concentration-dependent manner and prevented changes in apoptosis-related protein expression. In addition, SDT pretreatment significantly reduced glutathione depletion, hydrogen peroxide production, and mitochondrial dysfunction via treatment with AA plus iron. SDT increased the phosphorylation of AMP-activated protein kinase (AMPK) in accordance with the phosphorylation of Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2). Experiments using an AMPK chemical inhibitor (Compound C) or CaMKK2 chemical inhibitor (STO-609) suggested that the CaMKK2-AMPK signaling pathway contributes to SDT-mediated protection of mitochondria and cells. Moreover, administration of SDT for 4 consecutive days to mice significantly reduced the alanine aminotransferase and aspartate aminotransferase activities induced by carbon tetrachloride, and the numbers of degenerated hepatocytes, infiltrated inflammatory cells, nitrotyrosine-positive cells, and 4-hydroxynonenal-positive cells in liver tissue. Therefore, SDT protects hepatocytes from oxidative stress via CaMKK2-dependent AMPK activation and has the therapeutic potential to prevent or treat oxidative stress-related liver injury.

“…After treatments, viable cells were stained with MTT (0.5 mg/mL, 4 h), and viabilities were assessed as previously described [8].…”

Section: Methodsmentioning

confidence: 99%

“…AMPK activation in liver reduces lipid synthesis by inducing the phosphorylation of acetyl-CoA carboxylase (ACC), decreases protein metabolism by inhibiting mammalian target of rapamycin complex 1 (mTORC1), induces autophagy by activating unc-51-like protein kinase 1, and protects cells and mitochondria from oxidative stress [7], [8], [9]. Thus, AMPK activation in liver relieves metabolic imbalances caused by alcohol intake or high calorie diet and protects tissues from toxic stimuli.…”

Section: Introductionmentioning

confidence: 99%

20 S -Protopanaxadiol, an aglycosylated ginsenoside metabolite, induces hepatic stellate cell apoptosis through liver kinase B1–AMP-activated protein kinase activation

Journal of Ginseng Research

Jung

Kim

et al. 2017

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BackgroundPreviously, we reported that Korean Red Ginseng inhibited liver fibrosis in mice and reduced the expressions of fibrogenic genes in hepatic stellate cells (HSCs). The present study was undertaken to identify the major ginsenoside responsible for reducing the numbers of HSCs and the underlying mechanism involved.MethodsUsing LX-2 cells (a human immortalized HSC line) and primary activated HSCs, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assays were conducted to examine the cytotoxic effects of ginsenosides. H2O2 productions, glutathione contents, lactate dehydrogenase activities, mitochondrial membrane permeabilities, apoptotic cell subpopulations, caspase-3/-7 activities, transferase dUTP nick end labeling (TUNEL) staining, and immunoblot analysis were performed to elucidate the molecular mechanism responsible for ginsenoside-mediated cytotoxicity. Involvement of the AMP-activated protein kinase (AMPK)-related signaling pathway was examined using a chemical inhibitor and small interfering RNA (siRNA) transfection.Results and conclusionOf the 11 ginsenosides tested, 20S-protopanaxadiol (PPD) showed the most potent cytotoxic activity in both LX-2 cells and primary activated HSCs. Oxidative stress-mediated apoptosis induced by 20S-PPD was blocked by N-acetyl-l-cysteine pretreatment. In addition, 20S-PPD concentration-dependently increased the phosphorylation of AMPK, and compound C prevented 20S-PPD-induced cytotoxicity and mitochondrial dysfunction. Moreover, 20S-PPD increased the phosphorylation of liver kinase B1 (LKB1), an upstream kinase of AMPK. Likewise, transfection of LX-2 cells with LKB1 siRNA reduced the cytotoxic effect of 20S-PPD. Thus, 20S-PPD appears to induce HSC apoptosis by activating LKB1–AMPK and to be a therapeutic candidate for the prevention or treatment of liver fibrosis.