Eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone) has many pharmacological activities including anti-inflammation, anti-oxidant and anti-cancer effects. Autophagy is the basic cellular machinery involving the digestion of damaged cellular components. In the present study, we investigated the protection effects of eupatilin against arachidonic acid (AA) and iron-induced oxidative stress in HepG2 cells and tried to elucidate the molecular mechanisms responsible. Eupatilin increased cell viability against AA + iron in a concentration-dependent manner and prevented mitochondrial dysfunction and reactive oxygen species (ROS) production. In addition, AA + iron increased the levels of pro-apoptotic proteins and these changes were prevented by eupatilin. Eupatilin also induced autophagy, as evidenced by the accumulation of microtubule-associated protein 1 light chain3-II and the detection of autophagic vacuoles. Furthermore, the protective effects of eupatilin on mitochondrial dysfunction and ROS production were significantly abolished by autophagy inhibitors. Eupatilin also increased the mRNA level of sestrin-2 and its promoter-driven reporter gene activity, which resulted in the up-regulation of sestrin-2 protein. Finally, gene silencing using sestrin-2 siRNA and the ectopic expression of recombinant adenoviral sestrin-2 indicated that sestrin-2 induction by eupatilin was required for autophagy-mediated cytoprotection against AA + iron. Our results suggest that eupatilin activates sestrin-2-dependent autophagy, thereby preventing oxidative stress induced by AA + iron.
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.
Artemisia apiacea Hance is one of the most widely used herbs for the treatment of malaria, jaundice, and dyspeptic complaint in oriental medicine. This study investigated the effects of methanol extracts of A. apiacea Hance (MEAH) on the induction of inducible nitric oxide synthase (iNOS) and proinflammatory mediators by lipopolysaccharide (LPS) in Raw264.7 macrophage cells and also evaluated the in vivo effect of MEAH on carrageenan-induced paw edema in rats. MEAH treatment in Raw264.7 cells significantly decreased LPS-inducible nitric oxide production and the expression of iNOS in a concentration-dependent manner, while MEAH (up to 100 μg/mL) had no cytotoxic activity. Results from immunoblot analyses and ELISA revealed that MEAH significantly inhibited the expression of cyclooxygenase-2, tumor necrosis factor-α, interleukin-1β, and interleukin-6 in LPS-activated cells. As a plausible molecular mechanism, increased degradation and phosphorylation of inhibitory-κBα and nuclear factor-κB accumulation in the nucleus by LPS were partly blocked by MEAH treatment. Finally, MEAH treatment decreased the carrageenan-induced formation of paw edema and infiltration of inflammatory cells in rats. These results demonstrate that MEAH has an anti-inflammatory therapeutic potential that may result from the inhibition of nuclear factor-κB activation, subsequently decreasing the expression of proinflammatory mediators.
Lipopolysaccharides (LPS) strongly stimulate immune cells, and unabated activation of immune system by LPS may lead to an exacerbation of sickness and depression. In this study, stigmasta-3,5-dien-7-one (ST) was isolated from Phragmitis rhizoma as a negative regulator of LPS-induced inflammation in macrophages. ST effectively reduced nitric oxide (NO), prostaglandin E2, and pro-inflammatory cytokine levels, which were markedly raised by LPS treatment. In addition, ST blocked the nuclear factor-kappa B (NF-κB) signaling pathway via down-regulation of phospho-p38 mitogen-activated protein kinase and phosphorylation and degradation of the inhibitor of NF-κB α. To our knowledge, this is the first study showing anti-inflammatory activities of ST isolated from Phragmitis rhizoma.
Pyungwi-san (PWS) is a traditional basic herbal formula. We investigated the effects of PWS on induction of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), pro-inflammatory cytokines (interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)) and nuclear factor-kappa B (NF-κB) as well as mitogen-activated protein kinases (MAPKs) in lipopolysaccharide-(LPS-) induced Raw 264.7 cells and on paw edema in rats. Treatment with PWS (0.5, 0.75, and 1 mg/mL) resulted in inhibited levels of expression of LPS-induced COX-2, iNOS, NF-κB, and MAPKs as well as production of prostaglandin E2 (PGE2), nitric oxide (NO), IL-6, and TNF-α induced by LPS. Our results demonstrate that PWS possesses anti-inflammatory activities via decreasing production of pro-inflammatory mediators through suppression of the signaling pathways of NF-κB and MAPKs in LPS-induced macrophage cells. More importantly, results of the carrageenan-(CA-) induced paw edema demonstrate an anti-edema effect of PWS. In addition, it is considered that PWS also inhibits the acute edematous inflammations through suppression of mast cell degranulations and inflammatory mediators, including COX-2, iNOS and TNF-α. Thus, our findings may provide scientific evidence to explain the anti-inflammatory properties of PWS in vitro and in vivo.
Licorice, Glycyrrhizae radix, is one of the herbal medicines in East Asia that has been commonly used for treating various diseases, including stomach disorders. This study investigated the effect of licorice on arsenite (As)-induced cytotoxicity in H4IIE cells, a rat hepatocyte-derived cell line. Cell viability was significantly diminished in As-treated H4IIE cells in a time and concentration-dependent manner. Furthermore, results from flow cytometric assay and DNA laddering in H4IIE cells showed that As treatment induced apoptotic cell death by activating caspase-3. Licorice (0.1 and 1.0 mg ml−1) treatment significantly inhibited cell death and the activity of caspase-3 in response to As exposure. These results demonstrate that licorice induced a cytoprotective effect against As-induced cell death by inhibition of caspase-3.
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