Se Jin Hwang

Efficacy of sauchinone as a novel AMPK-activating lignan for preventing iron-induced oxidative stress and liver injury

Iron-overload disorders cause hepatocyte injury and inflammation by oxidative stress, possibly leading to liver fibrosis and hepatocellular carcinoma. This study investigated the efficacy of sauchinone, a bioactive lignan, in preventing iron-induced liver injury and explored the mechanism of sauchinones activity. To create iron overload, mice were injected with phenylhydrazine, and the effects on hepatic iron and histopathology were assessed. Phenylhydrazine treatment promoted liver iron accumulation and ferritin expression, causing hepatocyte death and increased plasma arachidonic acid (AA). Sauchinone attenuated liver injury (EC(50)=10 mg/kg) and activated AMPK in mice. Treatment of hepatocytes with iron and AA simulated iron overload conditions: iron + AA synergistically amplified cytotoxicity, increasing H(2)O(2) and the mitochondrial permeability transition. Sauchinone protected hepatocytes from iron + AA-induced cytotoxicity, preventing the induction of mitochondrial dysfunction and apoptosis (EC(50)=1 microM), similar to the result using metformin. Sauchinone treatment activated LKB1, which led to AMPK activation: these events contributed to cell survival. Evidence of cytoprotection by LKB1 and AMPK activation was revealed in the reversal of sauchinones restoration of the mitochondrial membrane potential by either dominant negative mutant AMPKalpha or chemical inhibitor. In conclusion, sauchinone protects the liver from toxicity induced by iron accumulation, and sauchinones effects may be mediated by LKB1-dependent AMPK activation.

Inhibition of SREBP-1c-mediated hepatic steatosis and oxidative stress by sauchinone, an AMPK-activating lignan in Saururus chinensis

Sauchinone, as an AMP-activated kinase (AMPK)-activating lignan in Saururus chinensis, has been shown to prevent iron-induced oxidative stress and liver injury. Sterol regulatory element binding protein-1c (SREBP-1c) plays a key role in hepatic steatosis, which promotes oxidative stress in obese subjects. Previously, we identified the role of AMPK in liver X receptor-alpha (LXRalpha)-mediated SREBP-1c-dependent lipogenesis. Because sauchinone as an antioxidant has the ability to activate AMPK, this study investigated its effects on SREBP-1c-dependent lipogenesis in hepatocytes and in high-fat diet (HFD)-induced hepatic steatosis and oxidative injury. Sauchinone prevented the ability of an LXRalpha agonist (T0901317) to activate SREBP-1c, repressing transcription of the fatty acid synthase, acetyl-CoA carboxylase, stearoyl-CoA desaturase-1, ATP-binding cassette transporter A1, and LXRalpha genes. Consistent with this, an HFD in mice caused fat accumulation in the liver with SREBP-1c induction, which was attenuated by sauchinone treatment. Also, sauchinone had the ability to inhibit oxidative stress as shown by decreases in thiobarbituric acid-reactive substance formation, nitrotyrosinylation, and 4-hydroxynonenal production. Moreover, it prevented not only the liver injury, but also the AMPK inhibition elicited by HFD feeding. These results demonstrate that sauchinone has the capability to inhibit LXRalpha-mediated SREBP-1c induction and SREBP-1c-dependent hepatic steatosis, thereby protecting hepatocytes from oxidative stress induced by fat accumulation.

Liquiritigenin, a flavonoid aglycone from licorice, has a choleretic effect and the ability to induce hepatic transporters and phase-II enzymes.

Liquiritigenin (LQ), an active component of licorice, has an inhibitory effect on LPS-induced inhibitory nitric oxide synthase expression. This study investigated the effects of LQ on choleresis, the expression of hepatic transporters and phase-II enzymes, and fulminant hepatitis. The choleretic effect and the pharmacokinetics of LQ and its glucuronides were monitored in rats. After intravenous administration of LQ, the total area under the plasma concentration-time curve of glucuronyl metabolites was greater than that of LQ in plasma, which accompanied elevations in bile flow rate and biliary excretion of bile acid, glutathione, and bilirubin. The expressions of hepatocellular transporters and phase-II enzymes were assessed by immunoblots, real-time PCR, and immunohistochemistry. In the livers of rats treated with LQ, the protein and mRNA levels of multidrug resistance protein 2 and bile salt export pump were increased in the liver, which was verified by their increased localizations in canalicular membrane. In addition, LQ treatment enhanced the expression levels of major hepatic phase-II enzymes. Consistent with these results, LQ treatments attenuated galactosamine/LPS-induced hepatitis in rats, as supported by decreases in the plasma alanine aminotransferase, liver necrosis, and plasma TNF-alpha. These results demonstrate that LQ has a choleretic effect and the ability to induce transporters and phase-II enzymes in the liver, which may be associated with a hepatoprotective effect against galactosamine/LPS. Our findings may provide insight into understanding the action of LQ and its therapeutic use for liver disease.

E‐cadherin antagonizes transforming growth factor β1 gene induction in hepatic stellate cells by inhibiting RhoA–dependent Smad3 phosphorylation

Cadherins mediate cell‐cell adhesion and catenin (ctn)‐related signaling pathways. Liver fibrosis is accompanied by the loss of E‐cadherin (ECAD), which promotes the process of epithelial‐mesenchymal transition. Currently, no information is available about the inhibitory role of ECAD in hepatic stellate cell activation. Because of ECADs potential for inhibiting the induction of transforming growth factor β1 (TGFβ1), we investigated whether ECAD overexpression prevents TGFβ1 gene induction; we also examined what the molecular basis could be. Forced expression of ECAD decreased α‐smooth muscle actin and vimentin levels and caused decreases in the constitutive and inducible expression of the TGFβ1 gene and its downstream genes. ECAD overexpression decreased Smad3 phosphorylation, weakly decreased Smad2 phosphorylation, and thus inhibited Smad reporter activity induced by either treatment with TGFβ1 or Smad3 overexpression. Overexpression of a dominant negative mutant of ras homolog gene family A (RhoA) diminished the ability of TGFβ1 to elicit its own gene induction. Consistently, transfection with a constitutively active mutant of RhoA reversed the inhibition of TGFβ1‐inducible or Smad3‐inducible reporter activity by ECAD. Studies using the mutant constructs of ECAD revealed that the p120‐ctn binding domain of ECAD was responsible for TGFβ1 repression. Consistently, ECAD was capable of binding p120‐ctn, which recruited RhoA; this prevented TGFβ1 from increasing RhoA‐mediated Smad3 phosphorylation. In the liver samples of patients with mild or severe fibrosis, ECAD expression reciprocally correlated with the severity of fibrosis. Conclusion: Our results demonstrate that ECAD inhibits Smad3/2 phosphorylation by recruiting RhoA to p120‐ctn at the p120‐ctn binding domain, whereas the loss of ECAD due to cadherin switching promotes the up‐regulation of TGFβ1 and its target genes, and facilitates liver fibrosis. (HEPATOLOGY 2010.)

Transplantation of Human Adipose-Derived Stem Cells in a Rabbit Model of Traumatic Degeneration of Lumbar Discs

OBJECTIVE The purpose of the present study is to assess the possibility of disc regeneration by treatment with adipose-derived stem cells (ADSCs) in a rabbit model of degenerative disc disease, and to evaluate the efficacy of a percutaneous technique for constructing a model of degenerative disc disease in rabbits. METHODS The study sample consisted of 20 mature male New Zealand white rabbits. Intervertebral discs were injured in each rabbit by a percutaneous technique at L2-3, L3-4, and L4-5 under C-arm guidance with a 19-gauge spinal needle. Magnetic resonance images (MRI) were checked at 6, 9, 12, and 15 weeks after injury to evaluate disc degeneration. Nineteen weeks after injury, ADSCs were injected into the L4-5 disc space, with saline injected into the L3-4 disc as a control, using a 21-gauge spinal needle. Histologic confirmations of degenerated discs were performed at 10 and 18 weeks after injury with safranin O and trichrome stains. RESULTS MRI revealed intervertebral disc degeneration from 9 weeks after injury, and full degeneration at 15 weeks after injury, when compared with uninjured control discs. We confirmed the proliferation of ADSCs at the L4-5 level in 10-week rabbits after cell injection. Histologically, the ADSC-injected discs exhibited elevated extracellular matrix secretion and little ossification of damaged cartilage in the nucleus pulposus compared with degenerative control discs. CONCLUSIONS These results suggest that the injection of ADSCs into injured lumbar discs could be an effective treatment for degenerative disc disease by promoting the cartilage regeneration.

Nrf2-mediated liver protection by sauchinone, an antioxidant lignan, from acetaminophen toxicity through the PKCδ-GSK3β pathway

BACKGROUND AND PURPOSE Sauchinone, an antioxidant lignan, protects hepatocytes from iron‐induced toxicity. This study investigated the protective effects of sauchinone against acetaminophen (APAP)‐induced toxicity in the liver and the role of nuclear factor erythroid‐2‐related factor‐2 (Nrf2) in this effect.

Ajoene, A Stable Garlic By-Product, Inhibits High Fat Diet-Induced Hepatic Steatosis and Oxidative Injury Through LKB1-Dependent AMPK Activation

Hepatic steatosis, a hepatic component of metabolic syndrome, is common and may progress to steatohepatitis and cirrhosis. The liver X receptor-α (LXRα)-sterol regulatory element binding protein-1c (SREBP-1c) pathway plays a key role in hepatic steatosis. This study investigated the potential of ajoene, a stable garlic by-product, to inhibit high fat diet (HFD)-induced hepatic steatosis and the underlying mechanism. Ajoene treatment attenuated fat accumulation and induction of lipogenic genes in the liver of HFD-fed mice. Blood biochemical analyses and histopathologic examinations showed that ajoene prevented liver injury with the inhibition of oxidative stress, as evidenced by thiobarbituric acid reactive substances formation and nitrotyrosinylation. Moreover, ajoene treatment inhibited LXRα agonist (T0901317)-mediated SREBP-1c activation, and transactivation of the lipogenic target genes in hepatocytes. Ajoene was found to activate AMP-activated protein kinase (AMPK) via LKB1, responsible for the inhibition of p70 ribosomal S6 kinase-1 (S6K1). The ability of ajoene to repress T0901317-induced SREBP-1c expression was antagonized by inhibition of AMPK or activation of S6K1, supporting the role of these kinases in the antisteatotic effect. Our results demonstrate that ajoene has an effect of activating AMPK through LKB1 and inhibit S6K1 activity, contributing to the prevention of SREBP-1c-mediated hepatic lipogenesis via the inhibition of LXRα activity.

Inhibition of liver X receptor-α-dependent hepatic steatosis by isoliquiritigenin, a licorice antioxidant flavonoid, as mediated by JNK1 inhibition.

Isoliquiritigenin (ILQ), a flavonoid obtained from Glycyrrhizae species, has an antioxidant effect. This study investigated the potential of ILQ for inhibiting liver X receptor-α (LXRα)-mediated lipogenesis and steatosis in hepatocytes and its underlying molecular basis. Treatment with ILQ antagonized the ability of an LXRα agonist (T0901317) to activate sterol regulatory element binding protein-1c (SREBP-1c), thereby repressing transcription of fatty acid synthase, acetyl-CoA carboxylase, ATP-binding cassette transporter-A1, and stearoyl-CoA desaturase-1. ILQ treatment inhibited activating phosphorylation of JNK1 elicited by palmitate or TNFα. JNK1, but not JNK2, increased LXRα phosphorylation at serine residues, promoting LXRα activation. The ability of ILQ to inhibit JNK1 downstream of ASK1-MKK7 led to the repression of T0901317-inducible LXRα and SREBP-1c activation. In mice fed a high-fat diet, ILQ treatment inhibited hepatic steatosis, as shown by a decrease in fat accumulation and repression of lipogenic genes. The results of blood biochemistry and histopathology confirmed attenuation of high-fat diet-induced liver injury by ILQ. Moreover, ILQ inhibited oxidative stress, as indicated by decreases in thiobarbituric acid-reactive substance formation, iNOS and COX2 induction, and nitrotyrosinylation. Our results demonstrate that ILQ has the ability to repress LXRα-dependent hepatic steatosis through JNK1 inhibition and protect hepatocytes from oxidative injury inflicted by fat accumulation.

Inhibition of LXRα-dependent steatosis and oxidative injury by liquiritigenin, a licorice flavonoid, as mediated with Nrf2 activation.

Liver X receptor-α (LXRα) functions as a major regulator of lipid homeostasis through activation of sterol regulatory element binding protein-1c (SREBP-1c), which promotes hepatic steatosis and steatohepatitis. NF-E2-related factor 2 (Nrf2) is the crucial transcription factor that is necessary for the induction of antioxidant enzymes. This study investigated the potential of liquiritigenin (LQ), a hepatoprotective flavonoid in licorice, to inhibit LXRα-induced hepatic steatosis, and the underlying mechanism of the action. LQ treatment attenuated fat accumulation and lipogenic gene induction in the liver of mice fed a high fat diet. Also, LQ had the ability to inhibit oxidative liver injury, as shown by decreases in thiobarbituric acid reactive substances formation and nitrotyrosinylation. Moreover, LQ treatment antagonized LXRα agonist (T0901317)-mediated SREBP-1c activation, and transactivation of the lipogenic target genes. LQ was found to activate Nrf2, and the ability of LQ to inhibit LXRα-mediated SREBP-1c activation was reversed by Nrf2 deficiency, which supports the inhibitory role of Nrf2 in LXRα-dependent lipogenesis. Consistently, treatment with other Nrf2 activators or forced expression of Nrf2 also inhibited LXRα-mediated SREBP-1c activation. Our results demonstrate that LQ has an efficacy to activate Nrf2, which contributes to inhibiting the activity of LXRα that leads to SREBP-1c induction and hepatic steatosis.

Hepcidin inhibits Smad3 phosphorylation in hepatic stellate cells by impeding ferroportin-mediated regulation of Akt

Hepatic stellate cell (HSC) activation on liver injury facilitates fibrosis. Hepatokines affecting HSCs are largely unknown. Here we show that hepcidin inhibits HSC activation and ameliorates liver fibrosis. We observe that hepcidin levels are inversely correlated with exacerbation of fibrosis in patients, and also confirm the relationship in animal models. Adenoviral delivery of hepcidin to mice attenuates liver fibrosis induced by CCl4 treatment or bile duct ligation. In cell-based assays, either hepcidin from hepatocytes or exogenous hepcidin suppresses HSC activation by inhibiting TGFβ1-mediated Smad3 phosphorylation via Akt. In activated HSCs, ferroportin is upregulated, which can be prevented by hepcidin treatment. Similarly, ferroportin knockdown in HSCs prohibits TGFβ1-inducible Smad3 phosphorylation and increases Akt phosphorylation, whereas ferroportin over-expression has the opposite effect. HSC-specific ferroportin deletion also ameliorates liver fibrosis. In summary, hepcidin suppresses liver fibrosis by impeding TGFβ1-induced Smad3 phosphorylation in HSCs, which depends on Akt activated by a deficiency of ferroportin.