Resolvin D1 activates the sphingosine-1-phosphate signaling pathway in murine livers with ischemia/reperfusion injury.

Resolvins (Rvs) are endogenous lipid mediators that promote resolution of inflammation and return to homeostasis. We previously reported that RvD1 both facilitates M2 macrophage polarization of Kupffer cells (KCs) and efferocytosis and modulates thioredoxin 2-mediated mitochondrial quality control in liver ischemia/reperfusion (IR) injury. However, the specific cellular or molecular targets of RvD1 remain poorly understood. Sphingosine-1-phosphate (S1P), the natural sphingolipid ligand for a family of G protein-coupled receptors (S1P1-S1P5), regulates lymphocyte circulation and various immune responses. Here we investigated the role of RvD1 in IR-induced hepatocellular damage with a focus on S1P signaling. Male C57BL/6 mice were subjected to partial hepatic ischemia for 60 min, followed by reperfusion. Mice were pretreated with RvD1 (15 µg/kg, i.p.) 1 h prior to ischemia and immediately before reperfusion. To deplete KCs, liposome clodronate was administered (100 µL/mice, i.v.) 24 h prior to ischemia. Mice were pretreated with VPC23019 (100 µg/kg, i.p.), an antagonist for S1P1/S1P3 10 min prior to initial RvD1 treatment. Exogenous RvD1 attenuated IR-induced hepatocellular damage as evidenced by serum HMGB1 release. RvD1 attenuated the decrease in hepatic S1P concentration induced by IR. KC depletion by liposome clodronate did not alter the effect of RvD1 on sphingosine kinases (SKs) and S1P receptors, suggesting independency of KCs. Moreover, in purified hepatocytes of mice exposed to IR, mRNA expression of SK1, SK2, S1P1, and S1P3 decreased significantly, and this was attenuated by RvD1. Finally, VPC23019 pretreatment abolished the hepatoprotective effects of RvD1 in serum HMGB1 release. Our findings suggest that RvD1 protects the liver against IR injury by activating S1P signaling.

BST106 Protects against Cartilage Damage by Inhibition of Apoptosis and Enhancement of Autophagy in Osteoarthritic Rats.

Chrysanthemum zawadskii var. latilobum (CZ) has been used as a traditional medicine in Asian countries for the treatment of inflammatory diseases. Recently, CZ extract was shown to inhibit differentiation of osteoclasts and provide protection against rheumatoid arthritis. The aim of this study was to investigate the molecular mechanisms of BST106, the ethanol extract of CZ, for cartilage protection in monosodium iodoacetate (MIA)-induced osteoarthritis (OA), particularly focusing on apoptosis and autophagy. BST106 (50, 100, and 200 mg/kg) was orally administered once daily to MIA-induced OA rats. Swelling, limping, roentgenography, and histomorphological changes were assessed 28 d after MIA injection. Biochemical parameters for matrix metalloproteinase (MMP), apoptosis, and autophagy were also assessed. BST106 ameliorated the severity of swelling and limping after MIA injection. Roentgenographic and histomorphological examinations revealed that BST106 reduced MIA-induced cartilage damage. BST106 decreased MIA-induced increases in MMP-2 and MMP-13 mRNA levels. Increased levels of serum cartilage oligomeric matrix protein and glycosaminoglycan release were attenuated by BST106. Furthermore, BST106 suppressed the protein expression of proapoptotic molecules and increased the protein expression of autophagosome- and autolysosome-related molecules. These findings indicate that BST106 protects against OA-induced cartilage damage by inhibition of the apoptotic pathway and restoration of impaired autophagic flux.

Overexpression of HO-1 Contributes to Sepsis-Induced Immunosuppression by Modulating the Th1/Th2 Balance and Regulatory T-Cell Function.

Background: Countervailing anti-inflammatory response and immunosuppression can cause death in late sepsis. Depletion and dysfunction of T cells are critical for developing sepsis-induced immunosuppression. Heme oxygenase-1 (HO-1) has a regulatory effect on differentiation and function of T cells and anti-inflammatory properties. We therefore investigated the immunosuppressive role of HO-1 in sepsis with a focus on its effects on helper T-cell (Th) differentiation and regulatory T cells (Treg). Methods: Sepsis was induced by cecal ligation and puncture (CLP). Mice were intraperitoneally injected with zinc protoporphyrin (ZnPP; 25 mg/kg), an HO-1 inhibitor, or hemin (20 mg/kg), an HO-1 inducer, at 24 and 36 hours post-CLP. Splenocytes were isolated 48 hours post-CLP. Mice were intranasally infected with Pseudomonas aeruginosa 4 days post-CLP as a secondary pneumonia infection model. Results: ZnPP improved survival and bacterial clearance, whereas hemin had the opposite effect in septic mice. CLP induced lymphocyte apoptosis and a proinflammatory Th1 to anti-inflammatory Th2 shift, which was attenuated by ZnPP. ZnPP attenuated the CLP-induced Treg population and protein expression of inhibitory costimulatory molecules. Furthermore, ZnPP improved survival in the secondary pneumonia infection model. Conclusions: Our findings suggest that HO-1 overexpression contributes to sepsis-induced immunosuppression during late phase sepsis by promoting Th2 polarization and Treg function.

Resolvin D1 protects the liver from ischemia/reperfusion injury by enhancing M2 macrophage polarization and efferocytosis.

Resolution of inflammation is an active process involving a novel category of lipid factors known as specialized pro-resolving lipid mediators, which includes Resolvin D1 (RvD1). While accumulating evidence suggests that RvD1 counteracts proinflammatory signaling and promotes resolution, the specific cellular targets and mechanisms of action of RvD1 remain largely unknown. In the present study, we investigated the role and molecular mechanisms of RvD1 in ischemia/reperfusion (IR)-induced sterile liver inflammation. Male C57BL/6 mice underwent 70% hepatic ischemia for 60min, followed by reperfusion. RvD1 (5, 10, and 15µg/kg, i.p.) was administered to the mice 1h before ischemia and then immediately prior to reperfusion. RvD1 attenuated IR-induced hepatocellular damage and the proinflammatory response. In purified Kupffer cells (KCs) from mice exposed to IR, the levels of M1 marker genes (Nos2a and Cd40) increased, while those of M2 marker genes (Arg1, Cd206, and Mst1r) decreased, demonstrating a proinflammatory shift. RvD1 markedly attenuated these changes. Depletion of KCs by liposome clodronate abrogated the effects of RvD1 on proinflammatory mediators and macrophage polarization. In addition, RvD1 attenuated increases in myeloperoxidase activity and Cxcl1 and Cxcl2 mRNA expression. RvD1 markedly augmented the efferocytic activity of KCs, as indicated by increases in F4/80(+)Gr-1(+) cells in the liver. However, antagonist pretreatment or gene silencing of the RvD1 receptor, ALX/FPR2, abrogated the anti-inflammatory and pro-resolving actions of RvD1. These data indicate that RvD1 ameliorates IR-induced liver injury, and this protection is associated with enhancement of M2 polarization and efferocytosis via ALX/FPR2 activation.

Genipin protects the liver from ischemia/reperfusion injury by modulating mitochondrial quality control.

Hepatic ischemia and reperfusion (IR) injury is closely linked to oxidative mitochondrial damage. Since mitochondrial quality control (QC) plays a pivotal role in the recovery of impaired mitochondrial function, mitochondrial QC has emerged as a potential therapeutic target. Genipin, an iridoid compound from Gardenia jasminoides, has been showed antioxidant and anti-inflammatory properties. In this study, we investigated the hepatoprotective mechanism of genipin against IR-induced hepatic injury, particularly focusing on mitochondrial QC. Male C57BL/6 mice underwent liver ischemia for 60min, followed by reperfusion for 6h. Genipin (100mg/kg, i.p.) or vehicle (10% Tween 80 in saline) was administrated to mice 1h before ischemia. Liver and blood samples were collected 6h after reperfusion. Hepatic IR increased hepatocellular oxidative damage and induced mitochondrial dysfunction. These phenomena were ameliorated by genipin. Hepatic IR also increased the level of mitochondrial fission, such as dynamin-related protein 1 and the level of PINK1 protein expression. In contrast, hepatic IR decreased the levels of mitochondrial biogenesis related proteins (e.g., peroxisome proliferator-activated receptor gamma coactivator 1α, nuclear respiratory factor 1, and mitochondrial transcription factor A), mitophagy related proteins (e.g., Parkin), and fusion related protein (e.g., mitofusin 2). Furthermore, hepatic IR decreased the levels of sirtuin1 protein and phosphorylation of AMP-activated protein kinase. Genipin alleviated these IR-induced changes. These data indicate that genipin protects against IR-induced hepatic injury via regulating mitochondrial QC. (225/250).

Role of necroptosis in autophagy signaling during hepatic ischemia and reperfusion.

Ischemia and reperfusion (I/R) is a complex phenomenon involving massive inflammation and cell death. Necroptosis refers to a newly described cell death as "programmed necrosis" that is controlled by receptor-interacting protein kinase (RIP) 1 and RIP3, which is involved in the pathogenesis of several inflammatory diseases. Autophagy is an essential cytoprotective system that is rapidly activated in response to various stimuli and involves crosstalk between different modes of cell death and inflammation. In this study, we investigated pattern changes in necroptosis and its role in autophagy signaling during hepatic I/R. Male C57BL/6 mice were subjected to 60min of ischemia followed by 3h reperfusion. Necrostatin-1 (Nec-1, a necroptosis inhibitor; 1.65mg/kg) was administered intraperitoneally 5min before reperfusion. Hepatic I/R significantly increased the level of RIP3, phosphorylated RIP1 and RIP3 protein expression, and RIP1/RIP3 necrosome formation, which were attenuated by Nec-1. I/R also significantly increased serum levels of alanine aminotransferase, tumor necrosis factor-α, and interleukin-6, which were attenuated by Nec-1. Meanwhile, hepatic I/R activated autophagy and mitophagy, as evidenced by increased LC3-II, PINK1, and Parkin, and decreased sequestosome 1/p62 protein expression. Nec-1 attenuated these changes and attenuated the increased levels of autophagy-related protein (ATG) 3, ATG7, Rab7, and cathepsin B protein expression during hepatic I/R. Moreover, hepatic I/R activated the extracellular signal-regulated kinase (ERK) pathway, and Nec-1 attenuated this increase. Taken together, our findings suggest that necroptosis contributes to hepatic damage during I/R, which induces autophagy via ERK activation.

The role of heme oxygenase-1 in drug metabolizing dysfunction in the alcoholic fatty liver exposed to ischemic injury.

This study was designed to investigate the role of heme oxygenase-1 (HO-1) in hepatic drug metabolizing dysfunction after ischemia/reperfusion (IR) in alcoholic fatty liver (AFL). Rats were fed a Lieber-DeCarli diet for five weeks to allow for development of AFL and were then subjected to 90min of hepatic ischemia and 5h of reperfusion. Rats were pretreated with hemin (HO-1 inducer) or ZnPP (HO-1 inhibitor) for 16h and 3h before hepatic ischemia. After hepatic IR, ethanol diet (ED)-fed rats had higher serum aminotransferase activities and more severe hepatic necrosis compared to the control diet (CD)-fed rats. These changes were attenuated by hemin and exacerbated by ZnPP. The activity and gene expression of HO-1 and its transcription factor (Nrf2) level increased significantly after 5h of reperfusion in CD-fed rats but not in ED-fed rats. After reperfusion, cytochrome P450 (CYP) 1A1, 1A2, and 2B1 activities were reduced to levels lower than those observed in sham group, whereas CYP2E1 activity increased. The decrease in CYP2B1 activity and the increase in CYP2E1 activity were augmented after hepatic IR in ED-fed animals. These changes were significantly attenuated by hemin but aggravated by ZnPP. Finally, CHOP expression and PERK phosphorylation, microsomal lipid peroxidation, and levels of proinflammatory mediators increased in ED-fed rats compared to CD-fed rats after reperfusion. These increases were attenuated by hemin. Our results suggest that AFL exacerbates hepatic drug metabolizing dysfunction during hepatic IR via endoplasmic reticulum stress and lipid peroxidation and this is associated with impaired HO-1 induction.

Enhanced nitric oxide-mediated autophagy contributes to the hepatoprotective effects of ischemic preconditioning during ischemia and reperfusion.

Ischemic preconditioning (IPC) protects against liver ischemia/reperfusion (I/R) injury. Autophagy is an essential cytoprotective system that is rapidly activated by multiple stressors. Nitric oxide (NO) acts as an inducer of IPC. We examined the impact of autophagy in liver IPC and its regulation by NO. Male C57BL/6 mice were subjected to 60 min of hepatic ischemia followed by 6 h of reperfusion. IPC was achieved for 10 min of ischemia followed by 10 min of reperfusion prior to sustained ischemia. N(ω)-Nitro-l-arginine methyl ester (L-NAME, 15 mg/kg, i.v., all NOS inhibitor) and aminoguanidine (AG, 10 mg/kg, i.v., iNOS inhibitor) were injected 10 min before IPC. SB203580 (10 mg/kg, i.p., p38 inhibitor) was injected 30 min before IPC. I/R increased serum alanine aminotransferase activity. IPC attenuated this increase, which was abolished by L-NAME, but not AG. Microtubule-associated protein-1 light chain 3-II levels increased and p62 protein levels decreased after I/R; these changes were augmented by IPC and abolished by L-NAME. I/R increased liver protein expression of autophagy-related protein (Atg)12-Atg5 complex and lysosome-associated membrane protein-2. IPC augmented the expression of these proteins, which were abolished by L-NAME, but not AG. IPC also augmented the level of phosphorylated p38 MAPK induced by I/R and this phosphorylation was abolished by L-NAME. Our findings suggest that IPC-mediated NO protects against I/R-induced liver injury by enhancing autophagic flux.

Melatonin enhances mitophagy and mitochondrial biogenesis in rats with carbon tetrachloride-induced liver fibrosis.

Liver fibrosis leads to liver cirrhosis and failure, and no effective treatment is currently available. Growing evidence supports a link between mitochondrial dysfunction and liver fibrogenesis and mitochondrial quality control-based therapy has emerged as a new therapeutic target. We investigated the protective mechanisms of melatonin against mitochondrial dysfunction-involved liver fibrosis, focusing on mitophagy and mitochondrial biogenesis. Rats were treated with carbon tetrachloride (CCl4) dissolved in olive oil (0.5 mL/kg, twice a week, i.p.) for 8 wk. Melatonin was administered orally at 2.5, 5, and 10 mg/kg once a day. Chronic CCl4 exposure induced collagen deposition, hepatocellular damage, and oxidative stress, and melatonin attenuated these increases. Increases in mRNA and protein expression levels of transforming growth factor ß1 and α-smooth muscle actin in response to CCl4 were attenuated by melatonin. Melatonin attenuated hallmarks of mitochondrial dysfunction, such as mitochondrial swelling and glutamate dehydrogenase release. Chronic CCl4 exposure impaired mitophagy and mitochondrial biogenesis, and melatonin attenuated this impairment, as indicated by increases in mitochondrial DNA and in protein levels of PTEN-induced putative kinase 1 (PINK1); Parkin; peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α); nuclear respiratory factor 1 (NRF1); and transcription factor A, mitochondrial (TFAM). CCl4-mediated decreases in mitochondrial fission- and fusion-related proteins, such as dynamin-related protein 1 (DRP1) and mitofusin 2, were also attenuated by melatonin. Moreover, melatonin induced AMP-activated protein kinase (AMPK) phosphorylation. These results suggest that melatonin protects against liver fibrosis via upregulation of mitophagy and mitochondrial biogenesis, and may be useful as an anti-fibrotic treatment.

Expression levels of chaperones influence biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and Pseudomonas putida Baeyer-Villiger monooxygenase.

We demonstrated for the first time that the archaeal chaperones (i.e., γ-prefoldin and thermosome) can stabilize enzyme activity in vivo. Ricinoleic acid biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and the Pseudomonas putida KT2440 Baeyer-Villiger monooxygenase improved significantly with co-expression of γ-prefoldin or recombinant themosome originating from the deep-sea hyperthermophile archaea Methanocaldococcus jannaschii. Furthermore, the degree of enhanced activity was dependent on the expression levels of the chaperones. For example, whole-cell biotransformation activity was highest at 12 µmol/g dry cells/min when γ-prefoldin expression level was approximately 46% of the theoretical maximum. This value was approximately two-fold greater than that in E. coli, where the γ-prefoldin expression level was zero or set to the theoretical maximum. Therefore, it was assumed that the expression levels of chaperones must be optimized to achieve maximum biotransformation activity in whole-cell biocatalysts.

Role of Kupffer cells in ischemic injury in alcoholic fatty liver.

BACKGROUND: This study was designed to evaluate the role of Kupffer cells (KCs) in hepatic drug metabolizing dysfunction after hepatic ischemia-reperfusion (IR) in alcoholic fatty liver. MATERIALS AND METHODS: Rats were fed the Lieber-DeCarli diet for 5 wk to develop alcoholic fatty liver, then were subjected to 90 min of hepatic ischemia and 5 h of reperfusion. For ablation of KCs, rats were pretreated with gadolinium chloride (GdCl3) 48 and 24 h before the IR procedure. RESULTS: After the IR procedure, ethanol diet (ED)-fed rats had higher serum aminotransferase activity compared with the control diet-fed rats. These changes were attenuated by GdCl3. The ED-fed rats exhibited increased hepatic microsomal total cytochrome P450 (CYP) content and nicotinamide adenine dinucleotide phosphate-CYP reductase and CYP1A1, 1A2, 2B1, and 2E1 isozyme activity. After hepatic IR, these increases were reduced to lower levels than observed in the sham group, except CYP2E1 activity. Increases in CYP2E1 activity and its expression were augmented after hepatic IR in ED-fed animals, but were attenuated by GdCl3. Finally, toll-like receptor 4 and myeloid differentiation primary response gene 88 protein expression, nuclear translocation of nuclear factor-κB and activator protein 1, and levels of proinflammatory mediators were further increased in ED-fed animals compared with control diet-fed animals after IR. These increases were attenuated by GdCl3. CONCLUSIONS: We suggest that KCs contribute to hepatic drug metabolizing dysfunction during hepatic IR in alcoholic fatty liver via the toll-like receptors 4-mediated inflammatory response.

Stimulation of the α7 nicotinic acetylcholine receptor protects against sepsis by inhibiting Toll-like receptor via phosphoinositide 3-kinase activation.

BACKGROUND: The Toll-like receptor (TLR) plays an important role in the induction of the hyperinflammatory response and tissue injury in sepsis. The cholinergic antiinflammatory pathway serves as a link between the parasympathetic and innate immune systems. We examined the antiinflammatory effect of nicotine, a potent α7 nicotinic acetylcholine receptor (α7nAChR) agonist, with regard to TLR expression and signaling during sepsis. METHODS: Polymicrobial sepsis was induced in mice by cecal ligation and puncture (CLP). The subjects received intraperitoneal nicotine (400 µg/kg) immediately after CLP for the biochemical study and 0, 24, 48, and 72 hours after CLP for the survival test. Intraperitoneal methyllycaconitine (MLA; 5 mg/kg), an α7nAChR antagonist, was administered 5 minutes prior to nicotine treatment. We evaluated the effects of nicotine using α7nAChR and phosphoinositide 3-kinase (PI3K) inhibitors in lipopolysaccharide-stimulated RAW264.7 cells. RESULTS: Nicotine improved sepsis-induced mortality, attenuated organ failure, and suppressed inflammatory cytokines, which were abolished by MLA. Nicotine enhanced PI3K/Akt activation and reduced PU.1 activity and TLR4 expression. MLA and PI3K inhibitors blocked this effect of nicotine. CONCLUSIONS: Our findings suggest that stimulation of the cholinergic antiinflammatory pathway by nicotine protects against septic injury and that this may be associated with inhibition of TLR4 expression via α7nAChR/PI3K signaling.

Melatonin inhibits mTOR-dependent autophagy during liver ischemia/reperfusion.

BACKGROUND: Autophagy is a self-digestion system responsible for maintaining cellular homeostasis and interacts with reactive oxygen species produced during ischemia/reperfusion (I/R). Melatonin (MLT) is a potent and endogenous anti-oxidant that has beneficial effects in liver I/R injury. In this study, we examined the cytoprotective mechanisms of MLT in liver I/R, focusing on autophagic flux and associated signaling pathways. METHODS: Male C57BL/6 mice were subjected to 70% liver ischemia for 60 min followed by reperfusion. MLT (10 mg/kg, i.p.) was injected 15 min prior to ischemia and again immediately before reperfusion. Rapamycin (Rapa, 1 mg/kg, i.p.), which induces autophagy, was injected 1.5 h before ischemia. RESULTS: Liver I/R increased autophagic flux as indicated by the accumulation of LC3-II and degradation of sequestosome1/p62. This increase was attenuated by MLT. Likewise, electron microscopic analysis showed that autophagic vacuoles were increased in livers of mice exposed to I/R, which was attenuated by MLT. I/R decreased phosphorylation of mammalian target of rapamycin (mTOR) and 4E-BP1 and 70S6K, downstream molecules of the mTOR pathway, but increased expression of calpain 1 and calpain 2. MLT attenuated the decrease in mTOR, 4E-BP1 and 70S6K phosphorylation. Pretreatment of Rapa reversed the effect of MLT on autophagic flux as well as mTOR pathway. CONCLUSION: Our findings suggest that MLT downregulates autophagy via activation of mTOR signaling, which may in turn contribute to its protective effects in liver I/R injury.

Impaired expression of caveolin-1 contributes to hepatic ischemia and reperfusion injury.

Caveolae are membrane structures enriched in glycosphingolipids and cholesterol, and caveolin-1 (Cav-1) has been recognized to be pivotal in ischemic tolerance. Sphingosine-1-phosphate (S1P), one of the sphingolipid metabolites, is well known for its anti-apoptotic properties, counteracting ischemia and reperfusion (IR) injury. Here, we investigated the cytoprotective mechanism of Cav-1 against IR injury. Male C57BL/6 mice underwent 70% hepatic ischemia for 60 min, followed by reperfusion. Mice were pretreated with methyl-beta-cyclodextrin (MßCD, 10, 25 and 50mg/kg, i.p.), a caveolae disruptor, or saline 48 and 24h before ischemia. Serum and liver tissues were collected at the end of ischemia, at 0, 1, 4 and 24h of reperfusion. Decreases in the expression of Cav-1 protein and in the number of caveolae of the liver ultrastructure were observed during IR, which were augmented by pretreatment with MßCD. MßCD also augmented the IR-induced increases in serum alanine aminotransferase and tumor necrosis factor-α levels. IR decreased the levels of sphingosine kinase 2 (SK2) and S1P receptor 2 (S1P2) mRNA expressions, while MßCD also augmented these decreases. Moreover, IR resulted in increases of mitochondrial cytochrome c release, caspase 3, 8 activities and Bax/Bcl-xL ratio, and MßCD augmented all of these apoptotic parameters. MßCD also increased p38 MAPK and JNK phosphorylation, but did not affect ERK and PI3K/Akt. Our findings demonstrate that downregulation of Cav-1 mediates IR-induced liver damage by inhibiting SK2/S1P2 signaling and enhancing the apoptotic pathway.

Protective effects of lupeol against D-galactosamine and lipopolysaccharide-induced fulminant hepatic failure in mice.

This study examined the hepatoprotective effects of lupeol (1, a major active triterpenoid isolated from Adenophora triphylla var. japonica) against d-galactosamine (GalN) and lipopolysaccharide (LPS)-induced fulminant hepatic failure. Mice were orally administered 1 (25, 50, and 100 mg/kg; dissolved in olive oil) 1 h before GalN (800 mg/kg)/LPS (40 µg/kg) treatment. Treatment with GalN/LPS resulted in increased levels of serum alanine aminotransferase, tumor necrosis factor (TNF)-α, and interleukin (IL)-6, as well as increased mortality, all of which were attenuated by treatment with 1. In addition, levels of toll-like receptor (TLR)4, myeloid differentiation primary response gene 88, TIR-domain-containing adapter-inducing interferon-ß (TRIF), IL-1 receptor-associated kinase (IRAK)-1, and TNF receptor associated factor 6 protein expression were increased by GalN/LPS. These increases, except TRIF, were attenuated by 1. Interestingly, 1 augmented GalN/LPS-mediated increases in the protein expression of IRAK-M, a negative regulator of TLR signaling. Following GalN/LPS treatment, nuclear translocation of nuclear factor-κB and the levels of TNF-α and IL-6 mRNA expression increased, which were attenuated by 1. Together, the present findings suggest that lupeol (1) ameliorates GalN/LPS-induced liver injury, which may be due to inhibition of IRAK-mediated TLR inflammatory signaling.

Protective mechanisms of acacetin against D-galactosamine and lipopolysaccharide-induced fulminant hepatic failure in mice.

This study examined the hepatoprotective effects of acacetin (1), a flavonoid isolated from Agastache rugosa, against d-galactosamine (GalN) and lipopolysaccharide (LPS)-induced fulminant hepatic failure. Mice were given an intraperitoneal injection of 1 (25, 50, and 100 mg/kg), or the vehicle alone (5% dimethyl sulfoxide-saline), 1 h before GalN (800 mg/kg)/LPS (40 µg/kg) treatment and sacrificed at 6 h after GalN/LPS injection. GalN/LPS markedly increased mortality and serum aminotransferase activity, and these increases were attenuated by 1. GalN/LPS increased serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels, while 1 attenuated TNF-α levels and further increased IL-6 levels. GalN/LPS increased protein expression of toll-like receptor 4, phosphorylation of extracellular signal-related kinase, and p38 and c-Jun N-terminal kinase and increased nuclear protein expression of nuclear factor κB; these increases were attenuated by 1. GalN/LPS increased Atg5 and Atg7 protein expressions, and these increases were augmented by 1. GalN/LPS activated autophagic flux as indicated by decreased microtubule-associated protein 1 light chain 3-II and sequestosome1/p62 protein expression. This activation was enhanced by 1. These findings suggest that 1 protects against GalN/LPS-induced liver injury by suppressing TLR4 signaling and enhancing autophagic flux.

Chlorogenic acid attenuates high mobility group box 1 (HMGB1) and enhances host defense mechanisms in murine sepsis.

Sepsis is a complex, multifactorial, rapidly progressive disease characterized by an overwhelming activation of the immune system and the countervailing antiinflammatory response. In the current study in murine peritoneal macrophages, chlorogenic acid suppressed endotoxin-induced high mobility group box 1 (HMGB1) release in a concentration-dependent manner. Administration of chlorogenic acid also attenuated systemic HMGB1 accumulation in vivo and prevented mortality induced by endotoxemia and polymicrobial sepsis. The mechanisms of action of chlorogenic acid included attenuation of the increase in toll-like receptor (TLR)-4 expression and suppression of sepsis-induced signaling pathways, such as c-Jun NH2-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB, which are critical for cytokine release. The protection conferred by chlorogenic acid was achieved through modulation of cytokine and chemokine release, suppression of immune cell apoptosis and augmentation of bacterial elimination. Chlorogenic acid warrants further evaluation as a potential therapeutic agent for the treatment of sepsis and other potentially fatal systemic inflammatory disorders.

Activation of protein kinase C delta reduces hepatocellular damage in ischemic preconditioned rat liver.

BACKGROUND: Liver ischemic preconditioning (IPC), pre-exposure of the liver to transient ischemia, has been applied as a useful surgical method to prevent liver ischemia and reperfusion (I/R) injury. Although activation of protein kinase C (PKC), especially novel PKCs, has been known as central signaling responsible for the liver protection of IPC, determination of the involved isozyme in strong protection afforded by IPC has not been elucidated. MATERIALS AND METHODS: Rats were subjected to 90 min of partial liver ischemia followed by 3, 6, and 24 h of reperfusion. IPC was induced by 10 min of ischemia after 10 min of reperfusion before sustained ischemia. Rottlerin, a PKC-δ selective inhibitor; PKC-εV1-2 peptide, a selective PKC-ε inhibitor; and 3,7-dimethyl-1-[2-propargyl] xanthine, an adenosine A2 receptor antagonist, were intravenously injected before IPC. N-acetyl-L-cysteine, a strong antioxidant, and Nω-nitro-L-arginine methyl ester, a nonselective nitric oxide synthase inhibitor, were injected intraperitoneally before IPC. RESULTS: IPC resulted in strong protection against liver I/R injury as evidenced by biochemical and histologic analyses. Inhibition of PKC-δ strongly attenuated the IPC-induced liver protection, whereas PKC-ε inhibition did not exert any effect on IPC-induced protection. Although inhibition of reactive oxygen species, adenosine, and nitric oxide attenuated the beneficial effects of IPC, inhibition of adenosine only attenuated PKC-δ and -ε translocation. CONCLUSIONS: Our findings suggest that IPC protects against I/R-induced hepatic injury through activation of PKC-δ.

Protective mechanism of anethole on hepatic ischemia/reperfusion injury in mice.

The aim of this study was to investigate the hepatoprotective effect of anethole (trans-anethole, 1), a major component of Foeniculum vulgare, and its molecular mechanism during ischemia/reperfusion (I/R). Mice were subjected to 60 min of partial hepatic ischemia followed by 1 and 6 h of reperfusion. Compound 1 (50, 100, and 200 mg/kg) or the vehicle alone (10% Tween 80-saline) was orally administered 1 h prior to ischemia. After 1 and 6 h of reperfusion, serum alanine aminotransferase, tumor necrosis factor-α, and interleukin 6 levels significantly increased, but these increases were attenuated by 1. Nuclear translocation of interferon regulatory factor (IRF)-1, release of high mobility group box (HMGB) 1 into the extracellular milieu, and the interactions between IRF-1 and histone acetyltransferase p300 increased after I/R. These increases were attenuated by 1. Compound 1 suppressed increases in toll-like receptor (TLR) 4, myeloid differentiation primary response gene 88 protein expression, phosphorylation of p38, extracellular signal-regulated kinase, c-Jun N-terminal kinase, nuclear translocation of nuclear factor kappa B, and phosphorylated c-Jun. The present findings suggest that 1 protects against hepatic I/R injury by suppression of IRF-1-mediated HMGB1 release and subsequent TLR activation.

Genipin attenuates sepsis by inhibiting Toll-like receptor signaling.

The pathogenesis of sepsis is characterized by overwhelming inflammatory responses that lead to tissue damage and organ failure. Toll-like receptor (TLR) signaling is crucial for induction of hyperinflammatory responses and tissue injury during sepsis. Genipin, an aglycon of geniposide, has antiinflammatory and antimicrobial activities. The purpose of this study was to test the hypothesis that genipin reduces multiple organ dysfunction and mortality during sepsis through inhibition of TLR signaling. Male ICR were subjected to sepsis by cecal ligation and puncture (CLP) or endotoxemia by lipopolysaccharide (LPS). Various doses of genipin (1, 2.5 and 5 mg/kg) or a vehicle were administered intravenously immediately after CLP or intraperitoneally after LPS treatment. In another set of survival tests, mice were treated with 2.5 mg/kg of genipin 0 and 24 h after CLP. Genipin was found to improve survival and to attenuate multiple organ dysfunction. Genipin attenuated production of proinflammatory cytokines and release of high-mobility group box 1 (HMGB1). Genipin prevented TLR2 and TLR4, myeloid differentiation factor 88 and the Toll/interleukin-1 receptor domain-containing adaptor protein, inducing interferon-ß overexpression. Phosphorylation of mitogen-activated protein kinases and interferon regulatory factor 3 and translocation of nuclear factor (NF)-κB were prevented by genipin. Moreover, genipin attenuated increases in serum tumor necrosis factor-α and HMGB1 in LPS-induced endotoxemia. Pam3CSK4- and LPS-mediated production of nitrites and proinflammatory cytokines was suppressed by genipin in RAW264.7 cells. Genipin attenuated mortality and organ injuries during sepsis through interference with TLR signaling. Therefore, genipin might be useful as a potential therapeutic agent for treatment of sepsis.