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1.  Nafamostat mesilate attenuates neuronal damage in a rat model of transient focal cerebral ischemia through thrombin inhibition 
Scientific Reports  2014;4:5531.
Evidence suggests that thrombin, a blood coagulation serine protease, mediates neuronal injury in experimental cerebral ischemia. Here, we test the hypothesis that nafamostat mesilate, a serine protease inhibitor, may ameliorate ischemia-induced neuronal damage through thrombin inhibition after ischemic stroke. Focal ischemia was induced in adult Sprague-Dawley rats by occlusion of the middle cerebral artery for 2 hours followed by 22 hours of reperfusion. The administration of nafamostat mesilate during ischemia and reperfusion reduced the brain infarct volume, edema volume and neurological deficit. Thrombin expression and activity in the ipsilateral striatum were increased after ischemia, whereas the administration of nafamostat mesilate significantly inhibited thrombin expression and activity. Immunostaining showed that the majority of thrombin was expressed in neurons. TUNEL staining showed that nafamostat mesilate reduced the number of dying cells during ischemia. A rat behavioral test showed that nafamostat mesilate treatment significantly improved the learning ability of ischemic rats. These results suggest that nafamostat mesilate may have a potential therapeutic role for neuroprotection against focal cerebral ischemia through thrombin inhibition.
doi:10.1038/srep05531
PMCID: PMC4078306  PMID: 24985053
2.  Reduction of the HIV Protease Inhibitor-Induced ER Stress and Inflammatory Response by Raltegravir in Macrophages 
PLoS ONE  2014;9(3):e90856.
Background
HIV protease inhibitor (PI), the core component of highly active antiretroviral treatment (HAART) for HIV infection, has been implicated in HAART-associated cardiovascular complications. Our previous studies have demonstrated that activation of endoplasmic reticulum (ER) stress is linked to HIV PI-induced inflammation and foam cell formation in macrophages. Raltegravir is a first-in-its-class HIV integrase inhibitor, the newest class of anti-HIV agents. We have recently reported that raltegravir has less hepatic toxicity and could prevent HIV PI-induced dysregulation of hepatic lipid metabolism by inhibiting ER stress. However, little information is available as to whether raltegravir would also prevent HIV PI-induced inflammatory response and foam cell formation in macrophages.
Methodology and Principal Findings
In this study, we examined the effect of raltegravir on ER stress activation and lipid accumulation in cultured mouse macrophages (J774A.1), primary mouse macrophages, and human THP-1-derived macrophages, and further determined whether the combination of raltegravir with existing HIV PIs would potentially exacerbate or prevent the previously observed activation of inflammatory response and foam cell formation. The results indicated that raltegravir did not induce ER stress and inflammatory response in macrophages. Even more interestingly, HIV PI-induced ER stress, oxidative stress, inflammatory response and foam cell formation were significantly reduced by raltegravir. High performance liquid chromatography (HPLC) analysis further demonstrated that raltegravir did not affect the uptake of HIV PIs in macrophages.
Conclusion and Significance
Raltegravir could prevent HIV PI-induced inflammatory response and foam cell formation by inhibiting ER stress. These results suggest that incorporation of this HIV integrase inhibitor may reduce the cardiovascular complications associated with current HAART.
doi:10.1371/journal.pone.0090856
PMCID: PMC3953206  PMID: 24625618
3.  The role of C/EBP homologous protein in HIV protease inhibitor-induced hepatic lipotoxicity 
Hepatology (Baltimore, Md.)  2013;57(3):1005-1016.
HIV protease inhibitors (HIV PIs) are the core components of highly active antiretroviral therapies (HAART), which has been successfully used in 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 dysregulation of hepatic lipid metabolism. HIV PI-induced hepatic lipotoxicity is closely linked to the upregulation of C/EBP 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 H&E staining and Oil Red O staining. Real-time RT-PCR and immunoblot data showed that in the absence of CHOP, HIV PI-induced expression of stress-related proteins and lipogenic genes was dramatically reduced. Furthermore, TNF-α and IL-6 levels in serum and livers 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 increased expression of CHOP represents a critical factor underlying events leading to hepatic injury.
doi:10.1002/hep.26107
PMCID: PMC3566321  PMID: 23080229
HAART; ER Stress; Lipid metabolism; CHOP; inflammation
4.  Conjugated Bile Acids Activate the Sphingosine-1-Phosphate Receptor 2 in Primary Rodent Hepatocytes 
Hepatology (Baltimore, Md.)  2011;55(1):267-276.
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 the extracellular regulated kinase (ERK)1/2 and AKT as well as the G-protein coupled receptor (GPCR), TGR5/M-BAR. Activation of the ERK1/2 and AKT signaling pathways by conjugated bile acids has been reported to be pertussis toxin (PTX) and dominant negative Gαi sensitive 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. Knock down 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 via S1P2 in primary rodent hepatocytes.
doi:10.1002/hep.24681
PMCID: PMC3245352  PMID: 21932398
G protein coupled receptor; homology modeling; S1P receptor 2 knockout mice; cell signaling
5.  Tolerance of neurite outgrowth to Rho kinase inhibitors decreased by cyclooxygenase-2 inhibitor☆ 
Neural Regeneration Research  2012;7(34):2705-2712.
In this study, PC12 Adh cells and Neuro-2a cells were treated with Rho-associated kinase inhibitors (Y27632 and Fasudil), a cyclooxygenase-1 selective inhibitor (SC560), and a cyclooxygenase-2 inhibitor (NS398). We found that these cells became tolerant to Rho-associated kinase inhibitors, as neurite outgrowth induced by these inhibitors diminished following more than 3 days of exposure in either cell line. The proteins cyclooxygenase-2 and cytosolic prostaglandin E synthetase were upregulated at day 3. NS398 decreased the tolerance to neurite outgrowth induction in both cell lines, whereas SC560 had almost no effect. These findings indicate that cells become tolerant to neurite outgrowth induced by Rho-associated kinase inhibitors, this is at least partly associated with upregulation of proteins involved in the cyclooxygenase-2 pathway, and cyclooxygenases-2 inhibition prevents this tolerance.
doi:10.3969/j.issn.1673-5374.2012.34.008
PMCID: PMC4200739  PMID: 25337117
Rho-associated kinase inhibitors; Y27632; fasudil; neurite; cyclooxygenase 2 inhibitors; drug tolerance
6.  BlyS is up-regulated by hypoxia and promotes migration of human breast cancer cells 
Background
The role of B Lymphocyte Stimulator (BLyS) in the survival of malignant B cells and the maintenance of normal B cell development and homeostasis has been intensively studied in the literature. However, the influence of BLyS on breast cancer progression remains unclear. The study aimed to investigate the effect of hypoxia on BLyS regulation, cell migratory response to BLyS and the possible molecular mechanisms.
Methods
In this study, we examined the role of BLyS in the migration of human breast cancer cells by transwell assay. We also explored whether BLyS and its receptors expressed in human breast cancer cell lines by immunofluorescence and Western Blotting. Then we detected the expression level of BLyS in both normoxic and hypoxic conditions by real time-PCR and Western Blotting. Pathways involved were confirmed by Western Blotting, immunofluorescence, transwell assay and luciferase assay.
Results
According to our study, the expression level of BlyS was increased in human breast cancer cell lines in hypoxic conditions. Up-regulation of this protein led to activation and nuclear translocation of NF-kappa B p65. We also found that the number of migrated cells was increased in the presence of BLyS and inhibition of phosphorylation of Akt attenuated the enhanced migratory response.
Conclusions
It suggested that better understanding of BLyS, an immunopotentiator, may offer a potential therapeutic target for the treatment of human breast cancers. In addition, BLyS promoted breast cancer cells migration, underscoring the necessity of appropriate applications of immunopotentiators to cancer treatment.
doi:10.1186/1756-9966-31-31
PMCID: PMC3349571  PMID: 22463935
Hypoxia; BLyS; Cell migration
7.  Efficient synthesis and biological evaluation of epiceanothic acid and related compounds 
Epiceanothic acid (1) is a naturally occurring, but very rare pentacyclic triterpene with a unique pentacyclic triterpene (PT) structure. An efficient synthesis of 1 starting from betulin (3) has been accomplished in 12 steps with a total yield of 10% in our study. Compound 1 and selected synthetic intermediates were further evaluated as anti-HIV-1 agents, inhibitors of glycogen phosphorylase (GP), and cytotoxic agents. Compound 1 exhibited moderate HIV-1 inhibition. Most importantly, compound 5, with an opened A-ring, showed significant GP inhibitory activity with an IC50 of 0.21 μM, suggesting a potential for development as an anti-diabetic agent. On the other hand, compound 12, with a closed A-ring, showed potent cytotoxicity against A549 and MCF-7 human tumor cell lines, with IC50 values of 0.89 and 0.33 μM, respectively. These results suggest that the A-ring of PTs is an important pharmacophore that could be modified to involve different biological activities.
doi:10.1016/j.bmcl.2010.11.004
PMCID: PMC3010304  PMID: 21123066
epiceanothic acid; pentacyclic triterpene; anti-HIV agents; glycogen phosphorylase inhibitors; cytotoxic agents
8.  Evaluation of Mitochondrial Toxicity in Marmota himalayana Treated with Metacavir, a Novel 2′,3′-Dideoxyguanosine Prodrug for Treatment of Hepatitis B Virus▿ 
Metacavir (PNA) is a novel synthetic nucleoside analogue for the treatment of hepatitis B virus (HBV). Our recent studies showed that PNA, a prodrug of 2′,3′-dideoxyguanosine (ddG), exhibited lower mitochondrial toxicity in long-term cultures of HepG2 cells. In the current study, we examined the long-term effects of PNA on mitochondrial toxicity in Marmota himalayana (Himalayan marmot). Himalayan marmots were treated daily with oral PNA (50 or 100 mg/kg), ziduvidine (AZT) (100 mg/kg), or water (control) for 90 days. PNA treatment did not alter the body weight or plasma lactate acid level. In livers from the animals treated with PNA at 100 mg/kg/day, histopathology showed mild steatosis or small focal liver cell necrosis. Electron microscopy also showed minor proliferation and partial mitochondrial swelling with crista reduction. Measurement of respiratory chain complex enzyme activity and mitochondrial DNA (mtDNA) content revealed no significant differences in skeletal muscle, liver, and kidney tissues between animals treated with PNA and controls. In contrast, in Himalayan marmots treated with AZT we observed delayed toxicity, including lactic acidosis, severe hepatic steatosis, obvious mitochondrial damage, and significant decreases in respiratory chain complex enzyme activity and mtDNA content. This is similar to the delayed toxicity syndrome observed previously in animals and humans. In summary, PNA treatment did not alter mitochondrial enzyme activity or mtDNA content. This suggests that PNA could pose a very low risk for adverse mitochondrion-related effects. However, long-term hepatotoxic effects of PNA were observed, and this indicates a need for continued monitoring of PNA-associated hepatotoxicity in clinical trials.
doi:10.1128/AAC.01520-10
PMCID: PMC3088222  PMID: 21282436
9.  Development of a novel self micro-emulsifying drug delivery system (SMEDDS) for reducing HIV protease inhibitor-induced intestinal epithelial barrier dysfunction 
Molecular pharmaceutics  2010;7(3):844-853.
The development of HIV protease inhibitors (PIs) has been one of the most significant advances of the past decade in controlling HIV infection. Unfortunately, the benefits of HIV PIs are compromised by serious side effects. One of the most frequent and deleterious side effects of HIV PIs is severe gastrointestinal (GI) disorders including mucosal erosions, epithelial barrier dysfunction, and leak-flux diarrhea, which occurs in 16–62% of patients on HIV PIs. Although the underlying mechanisms behind HIV PI-associated serious adverse side effects remain to be identified, our recent studies have shown that activation of endoplasmic reticulum (ER) stress response plays a critical role in HIV PI-induced GI complications. The objective of this study was to develop a novel self micro-emulsifying drug delivery system (SMEDDS) using various antioxidants as surfactants and co-surfactants to reduce the GI side effects of the most commonly used HIV PI, ritonavir. The biological activities of this SMSDDS of ritonavir were compared with that of Norvir®, which is currently used in the clinic. Rat normal intestinal epithelial cells (IEC-6) and mouse Raw 264.7 macrophages were used to examine the effect of new SMEDDS of ritonavir on activation of ER stress and oxidative stress. The Sprague-Dawley rats and C57/BL6 mice were used for pharmacokinetic studies and in vivo studies. The intracellular and plasma drug concentrations were determined by HPLC analysis. Activation of ER stress was detected by western blot analysis and secreted alkaline phosphatase (SEAP) reporter assay. Reactive oxygen species (ROS) was measured using dichlorodihydrofluorescein diacetate as a probe. Cell viability was determined by Roche’s cell proliferation reagent WST-1. Protein levels of inflammatory cytokines (TNF-α and IL-6) were determined by Enzyme-linked immunosorbent assays (ELISA). The intestinal permeability was assessed by luminal enteral administration of fluorescein isothiocyanate conjugated dextran (FITC-dextran, 4 kDa). The pathologic changes in intestine were determined by histological examination. The results indicated that incorporation of antioxidants in this new SMEDDS not only significantly reduced ritonavir-induced ER stress activation, ROS production and apoptosis in intestinal epithelial cells and macrophages, but also improved the solubility, stability and bioavailability of ritonavir, and significantly reduced ritonavir-induced disruption of intestinal barrier function in vivo. In conclusion, this new SMEDDS of ritonavir has better bioavailability and less GI side effects as compared to Norvir®. This new SMEDDS can be used for other HIV PIs and any insoluble anti-viral drug with serious GI side effects.
doi:10.1021/mp100003r
PMCID: PMC2882528  PMID: 20349948
Ritonavir; antioxidants; SMEDDS; ER stress; inflammation
10.  In Vitro Mitochondrial Toxicity of Metacavir, a New Nucleoside Reverse Transcriptase Inhibitor for Treatment of Hepatitis B Virus ▿  
Antimicrobial Agents and Chemotherapy  2010;54(11):4887-4892.
Therapy with nucleoside reverse transcriptase inhibitors (NRTIs) can be associated with mitochondrial toxicity. In vitro studies have been used to predict the predisposition for and characterize the mechanisms causing mitochondrial toxicity. Metacavir (PNA) is a novel synthetic nucleoside analog for oral administration with potent and specific antiviral activity against hepatitis B virus (HBV). We assessed the potential for mitochondrial toxicity of PNA in long-term cultures of HepG2 hepatoma cells by measuring mitochondrial function (through lactate secretion), levels of mitochondrial DNA (mtDNA), and the activities of respiratory-chain complexes I to IV. Exposure of HepG2 cells to PNA at concentrations up to 50 μM for 15 days resulted in no deleterious effect on cell proliferation, levels of lactate or mtDNA, or enzyme activities of respiratory-chain complexes I to IV. In contrast, dideoxycytosine at 10 μM and zidovudine at 50 μM have significant effects on cell proliferation, levels of lactate and mtDNA, and enzyme activities of respiratory-chain complexes I to IV. However, PNA at a supratherapeutic concentration of 250 μM could result in significant alterations in the levels of mtDNA and the activities of respiratory-chain complex enzymes, revealing evidence of the potential mitochondrial toxicity of PNA. In summary, these in vitro results indicate that the potential for PNA to interfere with mitochondrial functions is low.
doi:10.1128/AAC.00794-10
PMCID: PMC2976109  PMID: 20805401
11.  HIV Protease Inhibitor Lopinavir-induced TNF-a and IL-6 Expression is Coupled to the Unfolded Protein Response and ERK Signaling Pathways in Macrophages 
Biochemical pharmacology  2009;78(1):70-77.
HIV Protease inhibitor (PI)-associated cardiovascular risk, especially atherosclerosis, has become a major concern in the clinic. Macrophages are key players in the inflammatory response and atherosclerosis formation. We have previously shown that HIV PIs induce endoplasmic reticulum (ER) stress, activate the unfolded protein response (UPR), and increase the synthesis of the inflammatory cytokines, TNF-α and IL-6, by regulating the intracellular translocation of RNA binding protein HuR in macrophages. However, the underlying signaling mechanisms remain unclear. We show here that the HIV PI lopinavir significantly activated the extracellular-signal regulated protein kinase (ERK), but not c-Jun N-terminal kinase (JNK) and p38 MAPK. Lopinavir-induced cytosolic translocation of HuR and TNF-α and IL-6 synthesis were attenuated by specific chemical inhibitor of MEK (PD98058) or over-expression of dominant negative mutant of MEK1. In addition, we demonstrated that lopinavir-induced ERK activation and TNF-a and IL-6 expression were completely inhibited in macrophages from CHOP null mice. Taken together, these results indicate activation of the UPR plays an essential role in HIV PI-induced inflammatory cytokine synthesis and release by activating ERK, which increases the cytosolic translocation of HuR and subsequent binding to the 3′UTR of TNF-α and IL-6 mRNAs in macrophages.
doi:10.1016/j.bcp.2009.03.022
PMCID: PMC2704357  PMID: 19447225
HIV protease inhibitor; inflammatory cytokines; ERK; ER stress; the UPR; RNA binding protein
12.  A Novel Genotype H9N2 Influenza Virus Possessing Human H5N1 Internal Genomes Has Been Circulating in Poultry in Eastern China since 1998 ▿ †  
Journal of Virology  2009;83(17):8428-8438.
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.
doi:10.1128/JVI.00659-09
PMCID: PMC2738149  PMID: 19553328
13.  Conjugated bile acids promote cholangiocarcinoma cell invasive growth through activation of sphingosine 1-phosphate receptor 2 
Hepatology (Baltimore, Md.)  2014;60(3):908-918.
Cholangiocarcinoma (CCA) is an often fatal primary malignancy of the intra- and extrahepatic biliary tract that is commonly associated with chronic cholestasis and significantly elevated levels of primary and conjugated bile acids (CBAs), which are correlated with bile duct obstruction (BDO). BDO has also recently been shown to promote CCA progression. However, whereas there is increasing evidence linking chronic cholestasis and abnormal bile acid profiles to CCA development and progression, the specific mechanisms by which bile acids may be acting to promote cholangiocarcinogenesis and invasive biliary tumor growth have not been fully established. Recent studies have shown that CBAs, but not free bile acids, stimulate CCA cell growth, and that an imbalance in the ratio of free to CBAs may play an important role in the tumorigenesis of CCA. Also, CBAs are able to activate extracellular signal-regulated kinase (ERK)1/2- and phosphatidylinositol-3-kinase/protein kinase B (AKT)-signaling pathways through sphingosine 1-phosphate receptor 2 (S1PR2) in rodent hepatocytes. In the current study, we demonstrate S1PR2 to be highly expressed in rat and human CCA cells, as well as in human CCA tissues. We further show that CBAs activate the ERK1/2- and AKT-signaling pathways and significantly stimulate CCA cell growth and invasion in vitro. Taurocholate (TCA)-mediated CCA cell proliferation, migration, and invasion were significantly inhibited by JTE-013, a chemical antagonist of S1PR2, or by lentiviral short hairpin RNA silencing of S1PR2. In a novel organotypic rat CCA coculture model, TCA was further found to significantly increase the growth of CCA cell spheroidal/“duct-like” structures, which was blocked by treatment with JTE-013. Conclusion: Our collective data support the hypothesis that CBAs promote CCA cell-invasive growth through S1PR2.
doi:10.1002/hep.27085
PMCID: PMC4141906  PMID: 24700501

Results 1-13 (13)