Luyong Zhang

Naturally Occurring Pentacyclic Triterpenes as Inhibitors of Glycogen Phosphorylase : Synthesis, Structure-Activity Relationships, and X-ray Crystallographic Studies

Twenty-five naturally occurring pentacyclic triterpenes, 15 of which were synthesized in this study, were biologically evaluated as inhibitors of rabbit muscle glycogen phosphorylase a (GPa). From SAR studies, the presence of a sugar moiety in triterpene saponins resulted in a markedly decreased activity ( 7, 18- 20) or no activity ( 21, 22). These saponins, however, might find their value as potential natural prodrugs which are much more water-soluble than their corresponding aglycones. To elucidate the mechanism of GP inhibition, we have determined the crystal structures of the GPb-asiatic acid and GPb-maslinic acid complexes. The X-ray analysis indicates that the inhibitors bind at the allosteric activator site, where the physiological activator AMP binds. Pentacyclic triterpenes represent a promising class of multiple-target antidiabetic agents that exert hypoglycemic effects, at least in part, through GP inhibition.

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.)

Triptolide: Progress on research in pharmacodynamics and toxicology

ETHNOPHARMACOLOGICAL RELEVANCE Tripterygium wilfordii Hook. f. (Tripterygium wilfordii), also known as Huangteng and gelsemium elegan, is a traditional Chinese medicine that has been marketed in China as Tripterygium wilfordii glycoside tablets. Triptolide (TP), an active component in Tripterygium wilfordii extracts, has been used to treat various diseases, including lupus, cancer, rheumatoid arthritis and nephritic syndrome. This review summarizes recent developments in the research on the pharmacodynamics, pharmacokinetics, pharmacy and toxicology of TP, with a focus on its novel mechanism of reducing toxicity. This review provides insight for future studies on traditional Chinese medicine, a field that is both historically and currently important. MATERIALS AND METHODS We included studies published primarily within the last five years that were available in online academic databases (e.g., PubMed, Google Scholar, CNKI, SciFinder and Web of Science). RESULTS TP has a long history of use in China because it displays multiple pharmacological activities, including anti-rheumatism, anti-inflammatory, anti-tumor and neuroprotective properties. It has been widely used for the treatment of various diseases, such as rheumatoid arthritis, nephritic syndrome, lupus, Behcet׳s disease and central nervous system diseases. Recently, numerous breakthroughs have been made in our understanding of the pharmacological efficacy of TP. Although TP has been marketed as a traditional Chinese medicine, its multi-organ toxicity prevents it from being widely used in clinical practice. CONCLUSIONS Triptolide, a biologically active natural product extracted from the root of Tripterygium wilfordii, has shown promising pharmacological effects, particularly as an anti-tumor agent. Currently, in anti-cancer research, more effort should be devoted to investigating effective anti-tumor targets and confirming the anti-tumor spectrum and clinical indications of novel anti-tumor pro-drugs. To apply TP appropriately, with high efficacy and low toxicity, the safety and non-toxic dose range for specific target organs and diseases should be determined, the altered pathways and mechanisms of exposure need to be clarified, and an early warning system for toxicity needs to be established. With further in-depth study of the efficacy and toxicity of TP, we believe that TP will become a promising multi-use drug with improved clinical efficacy and safety in the future.

Conjugated bile acids activate the sphingosine-1-phosphate receptor 2 in primary rodent hepatocytes.

Bile acids have been shown to be important regulatory molecules for cells in the liver and gastrointestinal tract. They can activate various cell signaling pathways including extracellular regulated kinase (ERK)1/2 and protein kinase B (AKT) as well as the G‐protein–coupled receptor (GPCR) membrane‐type bile acid receptor (TGR5/M‐BAR). Activation of the ERK1/2 and AKT signaling pathways by conjugated bile acids has been reported to be sensitive to pertussis toxin (PTX) and dominant‐negative Gαi in primary rodent hepatocytes. However, the GPCRs responsible for activation of these pathways have not been identified. Screening GPCRs in the lipid‐activated phylogenetic family (expressed in HEK293 cells) identified sphingosine‐1‐phosphate receptor 2 (S1P2) as being activated by taurocholate (TCA). TCA, taurodeoxycholic acid (TDCA), tauroursodeoxycholic acid (TUDCA), glycocholic acid (GCA), glycodeoxycholic acid (GDCA), and S1P‐induced activation of ERK1/2 and AKT were significantly inhibited by JTE‐013, a S1P2 antagonist, in primary rat hepatocytes. JTE‐013 significantly inhibited hepatic ERK1/2 and AKT activation as well as short heterodimeric partner (SHP) mRNA induction by TCA in the chronic bile fistula rat. Knockdown of the expression of S1P2 by a recombinant lentivirus encoding S1P2 shRNA markedly inhibited the activation of ERK1/2 and AKT by TCA and S1P in rat primary hepatocytes. Primary hepatocytes prepared from S1P2 knock out (S1P2−/−) mice were significantly blunted in the activation of the ERK1/2 and AKT pathways by TCA. Structural modeling of the S1P receptors indicated that only S1P2 can accommodate TCA binding. In summary, all these data support the hypothesis that conjugated bile acids activate the ERK1/2 and AKT signaling pathways primarily through S1P2 in primary rodent hepatocytes. (HEPATOLOGY 2012)

A novel genotype H9N2 influenza virus possessing human H5N1 internal genomes has been circulating in poultry in eastern China since 1998.

ABSTRACT Many novel reassortant influenza viruses of the H9N2 genotype have emerged in aquatic birds in southern China since their initial isolation in this region in 1994. However, the genesis and evolution of H9N2 viruses in poultry in eastern China have not been investigated systematically. In the current study, H9N2 influenza viruses isolated from poultry in eastern China during the past 10 years were characterized genetically and antigenically. Phylogenetic analysis revealed that these H9N2 viruses have undergone extensive reassortment to generate multiple novel genotypes, including four genotypes (J, F, K, and L) that have never been recognized before. The major H9N2 influenza viruses represented by A/Chicken/Beijing/1/1994 (Ck/BJ/1/94)-like viruses circulating in poultry in eastern China before 1998 have been gradually replaced by A/Chicken/Shanghai/F/1998 (Ck/SH/F/98)-like viruses, which have a genotype different from that of viruses isolated in southern China. The similarity of the internal genes of these H9N2 viruses to those of the H5N1 influenza viruses isolated from 2001 onwards suggests that the Ck/SH/F/98-like virus may have been the donor of internal genes of human and poultry H5N1 influenza viruses circulating in Eurasia. Experimental studies showed that some of these H9N2 viruses could be efficiently transmitted by the respiratory tract in chicken flocks. Our study provides new insight into the genesis and evolution of H9N2 influenza viruses and supports the notion that some of these viruses may have been the donors of internal genes found in H5N1 viruses.

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.

A Panel of Serum MicroRNAs as Specific Biomarkers for Diagnosis of Compound- and Herb-Induced Liver Injury in Rats

Background Drug-induced liver injury (DILI) has been a public, economic and pharmaceutical issue for many years. Enormous effort has been made for discovering and developing novel biomarkers for diagnosing and monitoring both clinical and preclinical DILI at an early stage, though progress has been relatively slow. Additionally, herb-induced liver injury is an emerging cause of liver disease because herbal medicines are increasingly being used worldwide. Recently, circulating microRNAs (miRNAs) have shown potential to serve as novel, minimally invasive biomarkers to diagnose and monitor human cancers and other diseases at early stages. Methodology/Principal Findings In order to identify candidate miRNAs as diagnostic biomarkers for DILI, miRNA expression profiles of serum and liver tissue from two parallel liver injury Sprague-Dawley rat models induced by a compound (acetaminophen, APAP) or an herb (Dioscorea bulbifera, DB) were screened in this study. The initial screens were performed on serum using a MicroRNA TaqMan low-density qPCR array and on liver tissue using a miRCURY LNA hybridization array and were followed by a TaqMan probe-based quantitative reverse transcription-PCR (qRT-PCR) assay to validate comparison with serum biochemical parameters and histopathological examination. Two sets of dysregulated miRNA candidates in serum and liver tissue were selected in the screening phase. After qRT-PCR validation, a panel of compound- and herb- related serum miRNAs was identified. Conclusions/Significance We have demonstrated that this panel of serum miRNAs provides potential biomarkers for diagnosis of DILI with high sensitivity and specificity.

Sex differences in subacute toxicity and hepatic microsomal metabolism of triptolide in rats

Triptolide, a major active component of Tripterygium wilfordii Hook F (TWHF), has multiple pharmacological activities. However, its clinical use is often limited by its severe toxicity. In the present study, we evaluated the oral toxicity of triptolide in Sprague-Dawley rats for 28 days at the dosages of 0, 200 and 400microg/kg/day, respectively. Significant difference in the toxicity of triptolide at 400microg/kg was found between different sexes. The triptolide-treated female rats showed many abnormalities, including anorexia, diarrhea, leanness, suppression of weight gain and food intake, fatty liver, splenomegaly and atrophy of ovaries. In contrast, no such abnormalities were observed in male rats except for the significant reproductive toxicity. Furthermore, the metabolism of triptolide in liver microsomes from both sexes was investigated by HPLC. A greater rate of triptolide metabolism was observed in male rat hepatic microsomes, suggesting that one of the cytochrome P450s (CYPs) responsible for triptolide metabolism is male-specific or predominant at least. The inhibition experiments with CYP inhibitors showed that CYP3A and CYP2B were mainly involved in the metabolism of triptolide. In addition, since CYP3A2 is a male-predominant form in rats, significant sex difference in the metabolism of triptolide disappeared in vitro after anti-rat CYP3A2 antibody pretreatment. Results suggested that CYP3A2 made an important contribution to the sex-related metabolism of triptolide, which may result in the sex differences in triptolide toxicity.

Global patterns of genetic diversity and signals of natural selection for human ADME genes

Genetic polymorphisms in many genes related to drug absorption, distribution, metabolism and excretion (ADME genes) contribute to the high heterogeneity of drug responses in humans. However, the extent to which genetic variation in ADME genes may contribute to differences among human populations in drug responses has not been studied. In this work, we investigate the global distribution of genetic diversity for 31 core and 252 extended ADME genes. We find that many important ADME genes are highly differentiated across continental regions. Additionally, we analyze the genetic differentiation associated with clinically relevant, functional polymorphism alleles, which is important for evaluating potential among-population heterogeneity in drug treatment effects. We find that ADME genes show significantly greater variation in levels of population differentiation, and we find numerous signals of recent positive selection on ADME genes. These results suggest that genetic differentiation at ADME genes could contribute to population heterogeneity in drug responses.

Nogo-66 inhibits adhesion and migration of microglia via GTPase Rho pathway in vitro.

J. Neurochem. (2012) 120, 721–731.