Xiaofeng Tian

Dietary flavonoid genistein induces Nrf2 and phase II detoxification gene expression via ERKs and PKC pathways and protects against oxidative stress in Caco-2 cells

SCOPE Flavonoids have well-known antioxidant, anti-inflammatory, and anti-cancer activities. Isoflavone genistein is considered a potent antioxidant agent against oxidative stress. Although several mechanisms have been proposed, a clear antioxidant mechanism of genistein is still remained to be answered. METHODS AND RESULTS In this study, we focused on the concerted effects on expression of Nrf2 and phase II enzyme pathway components. Transient transfection assays, RT-PCR and immunoblot analysis were performed to study its molecular mechanisms of action. In Caco-2 cells, treatment with genistein markedly attenuated H(2)O(2) -induced peroxide formation; this amelioration was reversed by buthionine sulfoximine(GCLC inhibitor) and zinc protoporphyrin(HO-1 inhibitor). Genistein increased HO-1 and GCLC mRNA and protein expression. Genistein treatment activated the ERK1/2 and PKC signaling pathway; therefore increased Nrf2 mRNA and protein expression. The roles of the ERK1/2 and PKC signaling pathway were determined using PD98059 (ERK1/2 inhibitor) and GF109203X (PKC inhibitor) and RNA interference directed against Nrf2. Both inhibitors and siNrf2 abolished genistein-induced HO-1 and GCLC protein expression. These results suggest the involvement of ERK1/2, PKC, and Nrf2 in inducing HO-1 and GCLC by genistein. CONCLUSION Our studies show that genistein up-regulated HO-1 and GCLC expression through the EKR1/2 and PKC /Nrf2 pathways during oxidative stress.

Novel role of resveratrol: suppression of high-mobility group protein box 1 nucleocytoplasmic translocation by the upregulation of sirtuin 1 in sepsis-induced liver injury.

ABSTRACT Background: High-mobility group protein box 1 (HMGB1) is essential in the response to injury during sepsis. We hypothesized that resveratrol (RESV) administration would inhibit nuclear-cytoplasmic HMGB1 translocation in hepatocytes, which is associated with sirtuin 1 (SIRT1) upregulation. We investigated the regulatory role of SIRT1 in HMGB1 nucleocytoplasmic translocation and its effect on sepsis-induced liver injury. Methods: Rats were randomly assigned to pretreatment with RESV (60 mg/kg per day), nicotinamide (60 mg/kg per day), or vehicle (olive oil), which was administered by gavage for 3 days directly before cecal ligation and puncture was performed to induce sepsis. Parallel control groups were established. Rats were killed 24 h after surgery, and cytokine production, histology, apoptosis, SIRT1, serum HMGB1, nuclear and cytoplasmic HMGB1/ac-HMGB1, and the interaction between SIRT1 and HMGB1 were evaluated. In vitro evaluations were performed in human liver L02 cells subjected to lipopolysaccharide-induced injury, and siRNA-mediated SIRT1 knockdown experiments were performed. Results: Sepsis-induced serum aminotransferase activities and proinflammatory chemokine levels were reduced by RESV pretreatment, which also improved liver histological parameters in association with SIRT1 upregulation. Resveratrol inhibited HMGB1 cytoplasmic translocation. Nicotinamide, an SIRT1 inhibitor, reduced the SIRT1-mediated suppression of HMGB1 translocation and aggravated cecal ligation and puncture–induced liver damage. Sirtuin 1 knockdown in vitro confirmed that RESV increased the SIRT1-mediated repression of HMGB1 translocation. In vivo, SIRT1 and HMGB1 physically interacted in the nucleus, and SIRT1 regulated HMGB1 acetylation in response to septic liver injury. Conclusions: Resveratrol protects against sepsis-induced liver injury through the SIRT1-mediated HMGB1 nucleocytoplasmic translocation pathway, a new potential therapeutic target in sepsis-induced liver injury.

Salvianolic acid B protects against acute ethanol-induced liver injury through SIRT1-mediated deacetylation of p53 in rats.

Salvianolic acid B (SalB) is isolated from the traditional Chinese medical herb salvia miltiorrhiza. It has many biological and pharmaceutical activities. This study aimed to investigate the effect of SalB on acute ethanol-induced hepatic injury in rats and to explore the role of SIRT1 in this process. The results showed that pretreatment with SalB significantly reduced ethanol-induced elevation in aminotransferase activities, decreased hepatotoxic cytokine levels such as Interleukin-6 (IL-6), and increased the antioxidant enzyme activity. Moreover, SalB pretreatment reversed the increase in NF-κB, cleaved caspase-3 and decrease in B-cell lymphoma-extra large (Bcl-xL) caused by ethanol exposure. Importantly, SalB pretreatment significantly increased the expression of SIRT1, a NAD(+)-dependent deacetylase, whereas the increase in SIRT1 was accompanied by decreased acetyl-p53 expression. In HepG2 cells, SalB pretreatment increased SIRT1 expression in a time and dose-dependent manner and such an increase was abrogated by siRNA knockdown of SIRT1. Additionally, inhibition of SIRT1 significantly increased the acetylation of p53, and blocked SalB-induced acetylation of p53 down-regulation. Collectively, this study indicated that SalB can alleviate acute ethanol-induced hepatocyte apoptosis through SIRT1-mediated deacetylation of p53 pathway.

Inhibition of HMGB1 release via salvianolic acid B-mediated SIRT1 up-regulation protects rats against non-alcoholic fatty liver disease.

The inflammatory mediator high-mobility group box 1 (HMGB1) plays a critical role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). However, the regulation of HMGB1 in NAFLD, particularly through sirtuin 1 (SIRT1), remains unclear. In this study, we investigated the role of SIRT1-mediated inhibition of HMGB1 release in NAFLD and the effect of salvianolic acid B (SalB), which is a water-soluble phenolic acid extracted from Radix Salvia miltiorrhiza, on NAFLD through SIRT1/HMGB1 signaling. In vivo, SalB treatment significantly attenuated high-fat diet (HFD)-induced liver damage, hepatic steatosis, and inflammation. Importantly, SalB significantly inhibited HMGB1 nuclear translocation and release, accompanied by SIRT1 elevation. In HepG2 cells, palmitic acid (PA)-induced pro-inflammatory cytokines release were blocked by HMGB1 small interfering RNA (siRNA) transfection. Moreover, pharmacological SIRT1 inhibition by Ex527 induced HMGB1 translocation and release, whereas SIRT1 activation by resveratrol or SalB reversed this trend. SIRT1 siRNA abrogated the SalB-mediated inhibition of HMGB1 acetylation and release, suggesting that SalB-mediated protection occurs by SIRT1 targeting HMGB1 for deacetylation. We are the first to demonstrate that the SIRT1/HMGB1 pathway is a key therapeutic target for controlling NAFLD inflammation and that SalB confers protection against HFD- and PA-induced hepatic steatosis and inflammation through SIRT1-mediated HMGB1 deacetylation.

Salvianolic acid B protects against acetaminophen hepatotoxicity by inducing Nrf2 and phase II detoxification gene expression via activation of the PI3K and PKC signaling pathways

Acetaminophen (APAP) is used drugs worldwide for treating pain and fever. However, APAP overdose is the principal cause of acute liver failure in Western countries. Salvianolic acid B (SalB), a major water-soluble compound extracted from Radix Salvia miltiorrhiza, has well-known antioxidant and anti-inflammatory actions. We aimed to evaluate the ability of SalB to protect against APAP-induced acute hepatotoxicity by inducing nuclear factor-erythroid-2-related factor 2 (Nrf2) expression. SalB pretreatment ameliorated acute liver injury caused by APAP, as indicated by blood aspartate transaminase levels and histological findings. Moreover, SalB pretreatment increased the expression of Nrf2, Heme oxygenase-1 (HO-1) and glutamate-l-cysteine ligase catalytic subunit (GCLC). Furthermore, the HO-1 inhibitor zinc protoporphyrin and the GCLC inhibitor buthionine sulfoximine reversed the protective effect of SalB. Additionally, siRNA-mediated depletion of Nrf2 reduced the induction of HO-1 and GCLC by SalB, and SalB pretreatment activated the phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC) signaling pathways. Both inhibitors (PI3K and PKC) blocked the protective effect of SalB against APAP-induced cell death, abolishing the SalB-induced Nrf2 activation and decreasing HO-1 and GCLC expression. These results indicated that SalB induces Nrf2, HO-1 and GCLC expression via activation of the PI3K and PKC pathways, thereby protecting against APAP-induced liver injury.

Activation of the SIRT1/p66shc antiapoptosis pathway via carnosic acid-induced inhibition of miR-34a protects rats against nonalcoholic fatty liver disease

Recent studies have demonstrated that miR-34a expression is significantly upregulated and associated with apoptosis in nonalcoholic fatty liver disease (NAFLD). Carnosic acid (CA) is a novel antioxidant and a potential inhibitor of apoptosis in organ injury, including liver injury. This study aimed to investigate the signaling mechanisms underlying miR-34a expression and the antiapoptotic effect of CA in NAFLD. CA treatment significantly reduced the high-fat diet (HFD)-induced elevations in aminotransferase activity as well as in serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and malondialdehyde (MDA) levels but increased serum high-density lipoprotein cholesterol (HDL-C) and hepatic superoxide dismutase (SOD) levels. Moreover, CA treatment ameliorated the increase in cleaved caspase-3 caused by HFD exposure and completely reversed the HFD-induced decreases in manganese superoxide dismutase (MnSOD) and B-cell lymphoma-extra large expression. CA also counteracted the HFD- or palmitic acid (PA)-induced increases in caspase-3 and caspase-9 activity. Mechanistically, CA reversed the HFD- or PA-induced upregulation of miR-34a, which is the best-characterized regulator of SIRT1. Importantly, the decrease in miR-34a expression was closely associated with the activation of the SIRT1/p66shc pathway, which attenuates hepatocyte apoptosis in liver ischemia/reperfusion injury. A dual luciferase assay in L02 cells validated the modulation of SIRT1 by CA, which occurs at least partly via miR-34a. In addition, miR-34a overexpression was significantly counteracted by CA, which prevented the miR-34a-dependent repression of the SIRT1/p66shc pathway and apoptosis. Collectively, our results support a link between liver cell apoptosis and the miR-34a/SIRT1/p66shc pathway, which can be modulated by CA in NAFLD.

miR-34a-5p Inhibition Alleviates Intestinal Ischemia/Reperfusion-Induced Reactive Oxygen Species Accumulation and Apoptosis via Activation of SIRT1 Signaling.

AIMS Reactive oxygen species (ROS) generation and massive epithelial apoptosis are critical in the pathogenesis of intestinal ischemia/reperfusion (I/R) injury. We previously found that the Sirtuin 1 (SIRT1)-mediated antioxidant pathway was impaired in the intestine after I/R. Here, we investigate the potential role of SIRT1-targeting microRNAs (miRNAs) in regulating ROS accumulation and apoptosis in intestinal I/R, and the important role SIRT1 involved in. RESULTS C57BL/6 mice were subjected to intestinal I/R induced by occlusion of the superior mesenteric artery followed by reperfusion. Caco-2 cells were incubated under hypoxia/reoxygenation condition to mimic I/R in vivo. We find that SIRT1 is gradually repressed during the early reperfusion, and that this repression results in intestinal ROS accumulation and apoptosis. Using bioinformatics analysis and real-time PCR, we demonstrate that miR-34a-5p and miR-495-3p are significantly increased among the 41 putative miRNAs that can target SIRT1. Inhibition of miR-34a-5p, but not miR-495-3p, attenuates intestinal I/R injury, as demonstrated by repressing p66shc upregulation, manganese superoxide dismutase repression, and the caspase-3 activation in vitro and in vivo; it further alleviates systemic injury, as demonstrated by reducing inflammatory cytokine release, attenuating lung and liver lesions, and improving survival. Interestingly, SIRT1 plays an indispensable role in the protection afforded by miR-34a-5p inhibition. INNOVATION This study provides the first evidence of miRNAs in regulating oxidative stress and apoptosis in intestinal I/R. CONCLUSION miR-34a-5p knockdown attenuates intestinal I/R injury through promoting SIRT1-mediated suppression of epithelial ROS accumulation and apoptosis. This may represent a novel prophylactic approach to intestinal I/R injury. Antioxid. Redox Signal. 24, 961-973.

Salvianolic acid A preconditioning confers protection against concanavalin A-induced liver injury through SIRT1-mediated repression of p66shc in mice

Salvianolic acid A (SalA) is a phenolic carboxylic acid derivative extracted from Salvia miltiorrhiza. It has many biological and pharmaceutical activities. The purpose of this study was to investigate the effect of SalA on concanavalin A (ConA)-induced acute hepatic injury in Kunming mice and to explore the role of SIRT1 in such an effect. The results showed that in vivo pretreatment with SalA significantly reduced ConA-induced elevation in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and decreased levels of the hepatotoxic cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Moreover, the SalA pretreatment ameliorated the increases in NF-κB and in cleaved caspase-3 caused by ConA exposure. Whereas, the pretreatment completely reversed expression of the B-cell lymphoma-extra large (Bcl-xL). More importantly, the SalA pretreatment significantly increased the expression of SIRT1, a NAD(+)-dependent deacetylase, which was known to attenuate acute hypoxia damage and metabolic liver diseases. In our study, the increase in SIRT1 was closely associated with down-regulation of the p66 isoform (p66shc) of growth factor adapter Shc at both protein and mRNA levels. In HepG2 cell culture, SalA pretreatment increased SIRT1 expression in a time and dose-dependent manner and such an increase was abrogated by siRNA knockdown of SIRT1. Additionally, inhibition of SIRT1 significantly reversed the decreased expression of p66shc, and attenuated SalA-induced p66shc down-regulation. Collectively, the present study indicated that SalA may be a potent activator of SIRT and that SalA can alleviate ConA-induced hepatitis through SIRT1-mediated repression of the p66shc pathway.

PROTEASOME INHIBITOR LACTACYSTIN ABLATES LIVER INJURY INDUCED BY INTESTINAL ISCHAEMIA–REPERFUSION

1 The aim of the present study was to investigate the role of proteasome in the pathogenesis of liver injury induced by intestinal ischaemia–reperfusion (I/R) and the effect of the proteasome inhibitor lactacystin on neutrophil infiltration, intracellular adhesion molecule (ICAM)‐1 and nuclear factor (NF)‐κB expression in the liver tissues of rats. 2 Thirty‐two Wistar rats were randomly divided into four groups (n = 8 in each group) as follows: (i) a control, sham‐operated group; (ii) an I/R group subjected to 1 h intestinal ischaemia and 4 h reperfusion; (iii) a group pretreated with 0.2 mg/kg lactacystin 1 h before intestinal I/R; and (iv) a group pretreated with 0.6 mg/kg lactacystin 1 h before intestinal I/R. Liver and intestine histology were observed. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH), as well as 20S proteasome activity in circulating white blood cells, were measured. Myeloperoxidase (MPO) activity in liver tissues and the immunohistochemical expression of liver NF‐κB and ICAM‐1 were assayed. In addition, a western blot of liver NF‐κB was performed. 3 Compared with the sham‐operated control group, liver and intestine injury was induced by intestinal I/R, characterized as histological damage including oedema, haemorrhage and infiltration by inflammatory cells, as well as a significant increase in serum AST (365 ± 121 vs 546 ± 297 IU/L, respectively; P < 0.05), ALT (65 ± 23 vs 175 ± 54 IU/L, respectively; P < 0.01) and LDH levels (733 ± 383 vs 1434 ± 890 IU/L, respectively; P < 0.05). Compared with the control group, MPO activity in the liver tissues increased significantly in the I/R group (2.05 ± 0.69 vs 3.42 ± 1.11 U/g, respectively; P < 0.05). Strong positive expression of liver ICAM‐1 and NF‐κB p65 was observed. 4 Compared with the intestinal I/R group, administration of 0.6 mg/kg lactacystin markedly reduced 20S proteasome activity in circulating white blood cells (15.47 ± 4.00 vs 2.07 ± 2.00 pmol 7‐amino‐4‐methylcoumarin (AMC)/s per mg, respectively; P < 0.01) and ameliorated liver injury, which was demonstrated by decreased levels of serum AST (546 ± 297 vs 367 ± 86 IU/L, respectively; P < 0.05), ALT (175 ± 54 vs 135 ± 26 IU/L, respectively; P < 0.05) and LDH (1434 ± 890 vs 742 ± 218 IU/L, respectively; P < 0.05) and a reduced liver pathological score (2.13 ± 0.64 vs 1.25 ± 0.46, respectively; P < 0.01). Compared with the intestinal I/R group, MPO activity in liver tissues decreased significantly following lactacystin pretreatment (3.42 ± 1.11 vs 2.58 ± 0.61 U/g, respectively; P < 0.05) and the expression of liver NF‐κB and ICAM‐1 was markedly ameliorated. 5 The present study reveals that the proteasome inhibitor lactacystin ablates liver injury induced by intestinal I/R. One possible mechanism responsible for this effect is the inhibition of enhanced ICAM‐1 and neutrophil infiltration by inhibition of NF‐κB activity. The results suggest the feasibility of using proteasome inhibitor clinically in the treatment of intestinal I/R.

The protective effects of curcumin on experimental acute liver lesion induced by intestinal ischemia-reperfusion through inhibiting the pathway of NF-κB in a rat model.

Objective. In this study, we investigated the protective effect and mechanism of curcumin on a rat model of intestinal ischemia/reperfusion (I/R), which induces an acute liver lesion. Methods. Curcumin was injected into rats in the curcumin groups through left femoral vein. The same volume of vehicle (0.9% normal saline) was injected into sham and I/R groups. Blood and liver tissue were gathered for serological and histopathological determination. Results. Intestinal I/R led to severe liver injury manifested as a significant increase in serum AST and ALT levels; all of those were reduced by treatment with curcumin. Simultaneously, the activity of SOD in liver decreased after intestinal I/R, which was increased by curcumin treatment. On the other hand, curcumin reduced MPO activity of liver tissue, as well as serum IL-6 and TNF-α levels observably. This is in parallel with the decreased level of liver intercellular cell adhesion molecule-1 (ICAM-1) and nuclear factor-κB (NF-κB) expression. Conclusion. Our findings suggest that curcumin treatment attenuates liver lesion induced by intestinal I/R, attributable to the antioxidative and anti-inflammatory effect via inhibition of the NF-κB pathway.