Xudong Wu

Prevention of free fatty acid–induced hepatic lipotoxicity by 18β‐glycyrrhetinic acid through lysosomal and mitochondrial pathways

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and affects millions of people worldwide. Despite the increasing prevalence of NAFLD, the exact molecular/cellular mechanisms remain obscure and effective therapeutic strategies are still limited. It is well‐accepted that free fatty acid (FFA)‐induced lipotoxicity plays a pivotal role in the pathogenesis of NAFLD. Inhibition of FFA‐associated hepatic toxicity represents a potential therapeutic strategy. Glycyrrhizin (GL), the major bioactive component of licorice root extract, has a variety of pharmacological properties including anti‐inflammatory, antioxidant, and immune‐modulating activities. GL has been used to treat hepatitis to reduce liver inflammation and hepatic injury; however, the mechanism underlying the antihepatic injury property of GL is still poorly understood. In this report, we provide evidence that 18 β‐glycyrrhetinic acid (GA), the biologically active metabolite of GL, prevented FFA‐induced lipid accumulation and cell apoptosis in in vitro HepG2 (human liver cell line) NAFLD models. GA also prevented high fat diet (HFD)‐induced hepatic lipotoxicity and liver injury in in vivo rat NAFLD models. GA was found to stabilize lysosomal membranes, inhibit cathepsin B expression and enzyme activity, inhibit mitochondrial cytochrome c release, and reduce FFA‐induced oxidative stress. These characteristics may represent major cellular mechanisms, which account for its protective effects on FFA/HFD‐induced hepatic lipotoxicity. Conclusion: GA significantly reduced FFA/HFD‐induced hepatic lipotoxicity by stabilizing the integrity of lysosomes and mitochondria and inhibiting cathepsin B expression and enzyme activity. (HEPATOLOGY 2008.)

Synthesis and anti-inflammatory activities of mono-carbonyl analogues of curcumin

Curcumin has been extensively studied for its anti-inflammatory activities. However, its potential beneficial effects on various disease preventions and treatments are limited by its unstable structure. The beta-diketone moiety renders curcumin to be rapidly metabolized by aldo-keto reductase in liver. In the present study, a series of curcumin analogues with more stable chemical structures were synthesized and several compounds showed an enhanced ability to inhibit lipopolysaccharide (LPS)-induced TNF-alpha and IL-6 synthesis in macrophages.

HIV Protease Inhibitors Induce Endoplasmic Reticulum Stress and Disrupt Barrier Integrity in Intestinal Epithelial Cells

BACKGROUND & AIMS Human immunodeficiency virus (HIV) protease inhibitor (PI)-induced adverse effects have become a serious clinical problem. In addition to their metabolic and cardiovascular complications, these drugs also frequently cause severe gastrointestinal disorders, including mucosal erosions, epithelial barrier dysfunction, and diarrhea. However, the exact mechanisms underlying gastrointestinal adverse effects of HIV PIs remain unknown. This study investigated whether HIV PIs disrupt intestinal epithelial barrier integrity by activating endoplasmic reticulum (ER) stress. METHODS The most commonly used HIV PIs (lopinavir, ritonavir, and amprenavir) were used; their effects on ER stress activation and epithelial paracellular permeability were examined in vitro as well as in vivo using wild-type and CHOP(-)/(-) mice. RESULTS Treatment with lopinavir and ritonavir, but not amprenavir, induced ER stress, as indicated by a decrease in secreted alkaline phosphatase activities and an increase in the unfolded protein response. This activated ER stress partially impaired the epithelial barrier integrity by promoting intestinal epithelial cell apoptosis. CHOP silencing by specific small hairpin RNA prevented lopinavir- and ritonavir-induced barrier dysfunction in cultured intestinal epithelial cells, whereas CHOP(-)/(-) mice exhibited decreased mucosal injury after exposure to lopinavir and ritonavir. CONCLUSIONS HIV PIs induce ER stress and activate the unfolded protein response in intestinal epithelial cells, thus resulting in disruption of the epithelial barrier integrity.

The role of CCAAT enhancer-binding protein homologous protein in human immunodeficiency virus protease-inhibitor-induced hepatic lipotoxicity in mice.

Human immunodeficiency virus (HIV) protease inhibitors (HIV PIs) are the core components of highly active antiretroviral therapy, which has been successfully used in the treatment of HIV‐1 infection in the past two decades. However, benefits of HIV PIs are compromised by clinically important adverse effects, such as dyslipidemia, insulin resistance, and cardiovascular complications. We have previously shown that activation of endoplasmic reticulum (ER) stress plays a critical role in HIV PI–induced dys‐regulation of hepatic lipid metabolism. HIV PI–induced hepatic lipotoxicity is closely linked to the up‐regulation of CCAAT enhancer binding protein homologous protein (CHOP) in hepatocytes. To further investigate whether CHOP is responsible for HIV PI–induced hepatic lipotoxicity, C57BL/6J wild‐type (WT) or CHOP knockout (CHOP−/−) mice or the corresponding primary mouse hepatocytes were used in this study. Both in vitro and in vivo studies indicated that HIV PIs (ritonavir and lopinavir) significantly increased hepatic lipid accumulation in WT mice. In contrast, CHOP−/− mice showed a significant reduction in hepatic triglyceride accumulation and liver injury, as evidenced by hematoxylin and eosin and Oil Red O staining. Real‐time reverse‐transcriptase polymerase chain reaction and immunoblotting data showed that in the absence of CHOP, HIV PI–induced expression of stress‐related proteins and lipogenic genes were dramatically reduced. Furthermore, tumor necrosis factor alpha and interleukin‐6 levels in serum and liver were significantly lower in HIV PI–treated CHOP−/− mice, compared to HIV PI–treated WT mice. Conclusion: Taken together, these data suggest that CHOP is an important molecular link of ER stress, inflammation, and hepatic lipotoxicity, and that increased expression of CHOP represents a critical factor underlying events leading to hepatic injury. (HEPATOLOGY 2013)

S1589 Alcohol Promotes HIV Protease Inhibitor-Induced Hepatoxicity By Activating the ER Stress Response in Hepatocytes

Aim: Cytochrome P450 3A is the most important drug metabolizing enzyme in humans and nearly 40% of its activity is located in the small bowel. We have previously shown that intestinal CYP3A activity is markedly diminished in cirrhotics with transjugular intrahepatic portasystemic shunts (TIPS) and this leads to markedly increased bioavailability of orally administered CYP3A substrates and may lead to adverse effects. We conducted a study to test the hypothesis that oral CYP3A substrates with QT prolonging effect cause abnormal prolongation of QT interval in cirrhotics with TIPS. Methods: 23 subjects (9 controls, 8 cirrhotics with TIPS and 6 cirrhotics without TIPS) participated in this study. Subjects with cirrhosis and TIPSS were matched for age, gender, race and BMI. The mean age ± SD was 52 ± 9 years, 9 females and 6 African American. Oral erythromycin (ERY), a CYP3A substrate known to prolong QT interval in humans, was administered to test the relationship between diminished small bowel CYP3A activity and QT prolongation by oral medications. It was dosed at 500 mg PO BID for 7 days. EKGs (n=32) were obtained from each subject at baseline and after administration of ERY on day 1 and day 7 at scheduled intervals (over 24 hours) on each study day. QT intervals were measured in 3 consecutive beats in two leads and corrected QT interval (QTc) was obtained by fredericia correction and simple regression analysis. Primary outcome measure is maximal QTc change (QTc Max δ) after ERY administration on day 1 and day 7 in comparison to pre-drug administration baseline. The changes in QTc are compared among the three patient groups using ANOVA according to day 1 and day 7. Results: There was no statistically significant difference among the three groups in QTc Max δ following the first dose of ERY (p=0.8) on day 1. However, cirrhotics with TIPS had significantly greater QTc Max δ than other two groups following 7-days of oral ERY administration (p= 0.0271), irrespective of correction formula. Summary: Cirrhotic Patients with TIPS had significantly greater prolongation in QTc following multiple doses of erythromycin than cirrhotics without TIPS. Conclusion: Cirrhotics with TIPS are more prone for adverse events such QT prolongation by oral medications that are metabolized by small bowel CYP3A. The maximal QTc change (mean± SE) (QTc Max δ) milliseconds in each group on day 1 and on day 7 of oral erythromycin 500 mg twice daily.

W1269 HIV Protease Inhibitors Induce ER Stress and Disrupt Barrier Function in Intestinal Epithelial Cells

HIV protease inhibitor (PI)-induced diarrhea is one of the most frequent adverse side effects of these drugs and occurs in 16 to 62% of patients. Recently it has been reported that HIV PIs induce apoptosis and decrease barrier function in intestinal epithelial cells. However, the underlying cellular/molecular mechanisms remain unclear.We have previously demonstrated that HIV PIs induce endoplasmic reticulum (ER) stress, activate the unfolded protein response (UPR), and promote apoptosis in macrophages and hepatocytes. In the present study, we examined whether HIV PI-induced ER stress also plays a role in HIV PI-induced apoptosis and disruption of barrier function in intestinal epithelial cells. Methods: In Vitro studies were conducted in IEC6 cells (normal rat intestinal epithelial cells), differentiated IEC6 (stable Cdx2-transfected, IEC6-Cdx2L1), and stable SEAP-transfected IEC6 cells. HIV PIinduced ER stress was measured by decrease of SEAP activity. The mRNA and protein expression levels of ER stress-related genes were determined by real-time RT-PCR and western blot analysis, respectively. The paracellular permeability was measured by paracellular tracer flux assay and membrane-impermeable molecule, [14C]-mannitol, served as tracer. Lentiviral CHOP siRNA was used to knock down CHOP expression in IEC6 cells. Both C57/B6 wild type and CHOP knock out mice were used in In Vivo studies. Mice were gavaged with individual HIV PI (50 mg/kg) for three weeks. The pathological changes in intestine and colon were determined by histological evaluation. Results: Both ritonavir and lopinavir significantly induced ER stress, increased the expression of GRP78, CHOP, XBP-1 and ATF4, and promoted apoptosis in IEC6 and IEC6-Cdx2L1 cells. Paracellular permeability was increased by ~ 2.5 to 5 fold after cells were treated with 15 μM of ritonavir or lopinavir for 24 h. In contrast, amprenavir neither induced ER stress, nor increased paracellular permeability. Down-regulation of CHOP expression in IEC6 cells reduced HIV PI-induced apoptosis and disruption of barrier function. In Vivo studies demonstrated that ritonavir and lopinavir also significantly induced CHOP, XBP-1 and ATF4 expression in the intestine and colon of wild type mice. Furthermore, histological assessment indicated that ritonavir and lopinavir significantly induced intestinal epithelial damage in wild type mice, but had less effect in CHOP knock out mice. Conclusions: These findings indicate that HIV PI-induced ER stress and subsequent activation of the UPR represents an important cellular/molecular mechanism underlying HIV PI-induced disruption of intestinal barrier function.