Ganoderic acid A from Ganoderma lucidum protects against alcoholic liver injury through ameliorating the lipid metabolism and modulating the intestinal microbial composition.

Alcoholic liver injury is mainly caused by long-term excessive alcohol consumption and has become a global public threat to human health. It is well known that Ganoderma lucidum has excellent beneficial effects on liver function and lipid metabolism. The object of this study was to investigate the hepatoprotective effects of ganoderic acid A (GAA, one of the main triterpenoids in G. lucidum) against alcohol-induced liver injury and reveal the underlying mechanisms of its protective effects. The results showed that oral administration of GAA significantly inhibited the abnormal elevation of the liver index, serum total triglyceride (TG), cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in mice exposed to alcohol intake, and also significantly protected the liver against alcohol-induced excessive lipid accumulation and pathological changes. Besides, alcohol-induced oxidative stress in the liver was significantly ameliorated by the dietary intervention of GAA through decreasing the hepatic levels of lactate dehydrogenase (LDH) and malondialdehyde (MDA), and increasing hepatic activities of catalase (CAT), superoxide dismutase (SOD), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and hepatic levels of glutathione (GSH). In addition, GAA intervention evidently ameliorated intestinal microbial disorder by markedly increasing the abundance of Muribaculaceae, Prevotellaceae, Jeotgalicoccus, Bilophila, Family_XIII_UCG_001, Aerococcus, Ruminococcaceae_UCG_005, Harryflintia, Christensenellaceae, Rumonpcpccaceae, Prevotelaceae_UCG_001, Clostridiales_vadinBB60_group, Parasutterella and Bifidobacterium, but decreasing the proportion of Lactobacillus, Burkholderia_Caballeroria_Paraburkholderia, Escherichia_Shigella and Erysipelatoclostridium. Furthermore, liver metabolomics based on UPLC-QTOF/MS demonstrated that oral administration of GAA had a significant regulatory effect on the composition of liver metabolites in mice exposed to alcohol intake, especially the levels of the biomarkers involved in the metabolic pathways of riboflavin metabolism, glycine, serine and threonine metabolism, pyruvate metabolism, glycolysis/gluconeogenesis, biosynthesis of unsaturated fatty acids, synthesis and degradation of ketone bodies, fructose and mannose metabolism. Moreover, dietary supplementation of GAA significantly regulated the hepatic mRNA levels of lipid metabolism and inflammatory response related genes. Conclusively, these findings demonstrate that GAA has beneficial effects on alleviating alcohol-induced liver injury and is expected to become a new functional food ingredient for the prevention of alcoholic liver injury.

Vigna radiata (L.) R. Wilczek Extract Inhibits Influenza A Virus by Targeting Viral Attachment, Penetration, Assembly, and Release.

Vigna radiata (L.) R. Wilczek (mung bean) is a Chinese functional food with antioxidant, antimicrobial and anti-inflammatory activities. However, little is known about its antiviral activity. We aimed to investigate the antiviral activity and mechanisms of action of Vigna radiata extract (VRE) against influenza virus. HPLC was conducted to analyze the components of the VRE. The anti-influenza viral activity of VRE in Mardin-Darby canine kidney (MDCK) cells was evaluated by virus titration assays, hemagglutination assays, quantitative RT-PCR assays, cellular α-glucosidase activity assays and neuraminidase activity assays. Chromatographic profiling analysis identified two major flavonoids, vitexin and isovitexin, in the ethanol extract of Vigna radiata. Through in vitro studies, we showed that VRE, at concentrations up to 2,000 µg/ml, exhibited no cytotoxicity in MDCK cells. VRE protected cells from influenza virus-induced cytopathic effects and significantly inhibited viral replication in a concentration-dependent manner. A detailed time-of-addition assay revealed that VRE may act on both the early and late stages of the viral life cycle. We demonstrated that 1) VRE inhibits virus entry by directly blocking the HA protein of influenza virus; 2) VRE inhibits virus entry by directly binding to cellular receptors; 3) VRE inhibits virus penetration; 4) VRE inhibits virus assembly by blocking cellular α-glucosidase activity, thus reducing HA protein trafficking to the cell surface; and 5) VRE inhibits virus release by inhibiting viral neuraminidase activity. In summary, Vigna radiata extract potently interferes with two different subtypes of influenza viruses at multiple steps during the infectious cycle, demonstrating its broad-spectrum potential as an anti-influenza preventive and therapeutic agent. Continued development of Vigna radiata-derived products into antiviral therapeutics is warranted.

Antiviral efficacy of nanoparticulate vacuolar ATPase inhibitors against influenza virus infection.

BACKGROUND: Influenza virus infections are a major public health concern worldwide. Conventional treatments against the disease are designed to target viral proteins. However, the emergence of viral variants carrying drug-resistant mutations can outpace the development of pathogen-targeting antivirals. Diphyllin and bafilomycin are potent vacuolar ATPase (V-ATPase) inhibitors previously shown to have broad-spectrum antiviral activity. However, their poor water solubility and potential off-target effect limit their clinical application. METHODS: In this study, we report that nanoparticle encapsulation of diphyllin and bafilomycin improves the drugs' anti-influenza applicability. RESULTS: Using PEG-PLGA diblock copolymers, sub-200 nm diphyllin and bafilomycin nanoparticles were prepared, with encapsulation efficiency of 42% and 100%, respectively. The drug-loaded nanoparticles have sustained drug release kinetics beyond 72 hours and facilitate intracellular drug delivery to two different influenza virus-permissive cell lines. As compared to free drugs, the nanoparticulate V-ATPase inhibitors exhibited lower cytotoxicity and greater in vitro antiviral activity, improving the therapeutic index of diphyllin and bafilomycin by approximately 3 and 5-fold, respectively. In a mouse model of sublethal influenza challenge, treatment with diphyllin nanoparticles resulted in reduced body weight loss and viral titer in the lungs. In addition, following a lethal influenza viral challenge, diphyllin nanoparticle treatment conferred a survival advantage of 33%. CONCLUSIONS: These results demonstrate the potential of the nanoparticulate V-ATPase inhibitors for host-targeted treatment against influenza.

Targeting and Enrichment of Viral Pathogen by Cell Membrane Cloaked Magnetic Nanoparticles for Enhanced Detection.

Attachment to cellular surfaces is a major attribute among infectious pathogens for initiating disease pathogenesis. In viral infections, viruses exploit receptor-ligand interactions to latch onto cellular exterior prior to subsequent entry and invasion. In light of the selective binding affinity between viral pathogens and cells, nanoparticles cloaked in cellular membranes are herein employed for virus targeting. Using the influenza virus as a model, erythrocyte membrane cloaked nanoparticles are prepared and modified with magnetic functionalities (RBC-mNP) for virus targeting and isolation. To maximize targeting and isolation efficiency, density gradient centrifugation and nanoparticle tracking analysis were applied to minimize the presence of uncoated particles and membrane vesicles. The resulting nanoparticles possess a distinctive membrane corona, a sialylated surface, and form colloidally stable clusters with influenza viruses. Magnetic functionality is bestowed to the nanoparticles through encapsulation of superparamagnetic iron-oxide particles, which enable influenza virus enrichment via magnetic extraction. Viral samples enriched by the RBC-mNPs result in significantly enhanced virus detection by multiple virus quantification methods, including qRT-PCR, immunnochromatographic strip test, and cell-based titering assays. The demonstration of pathogen targeting and isolation by RBC-mNPs highlights a biologically inspired approach toward improved treatment and diagnosis against infectious disease threats. The work also sheds light on the efficient membrane cloaking mechanism that bestows nanoparticles with cell mimicking functionalities.

Nanoparticulate vacuolar ATPase blocker exhibits potent host-targeted antiviral activity against feline coronavirus.

Feline infectious peritonitis (FIP), caused by a mutated feline coronavirus, is one of the most serious and fatal viral diseases in cats. The disease remains incurable, and there is no effective vaccine available. In light of the pathogenic mechanism of feline coronavirus that relies on endosomal acidification for cytoplasmic entry, a novel vacuolar ATPase blocker, diphyllin, and its nanoformulation are herein investigated for their antiviral activity against the type II feline infectious peritonitis virus (FIPV). Experimental results show that diphyllin dose-dependently inhibits endosomal acidification in fcwf-4 cells, alters the cellular susceptibility to FIPV, and inhibits the downstream virus replication. In addition, diphyllin delivered by polymeric nanoparticles consisting of poly(ethylene glycol)-block-poly(lactide-co-glycolide) (PEG-PLGA) further demonstrates an improved safety profile and enhanced inhibitory activity against FIPV. In an in vitro model of antibody-dependent enhancement of FIPV infection, diphyllin nanoparticles showed a prominent antiviral effect against the feline coronavirus. In addition, the diphyllin nanoparticles were well tolerated in mice following high-dose intravenous administration. This study highlights the therapeutic potential of diphyllin and its nanoformulation for the treatment of FIP.

Identification of an infectious bronchitis coronavirus strain exhibiting a classical genotype but altered antigenicity, pathogenicity, and innate immunity profile.

Avian coronavirus infectious bronchitis virus (IBV) poses economic threat to the poultry industry worldwide. Pathogenic IBV 3575/08 was isolated from broilers vaccinated with the attenuated viral vaccine derived from a Taiwan strain 2575/98. In this study, extensive investigations were conducted on the genome sequences, antigenicity, pathogenicity, and host immune responses of several IBV strains in specific-pathogen-free chickens. Sequence analyses revealed that 3575/08 and 2575/98 shared high homology in their structural genes, but not in non-structural accessory proteins such as 3a, 3b and 5b. Despite a high degree of homology in their spike protein genes, cross neutralization test showed low cross protection between 3575/08 and 2575/98, suggesting distinct antigenicity for the two strains. Animal challenge experiments exhibited strong respiratory and renal pathogenicity for 3575/08. In addition, early and prolonged viral shedding and rapid viral dissemination were observed. Immune gene expression profiling by PCR array showed chickens infected with 3575/08 had delayed expression of a subset of early innate immune genes, whereas chickens infected with the wild-type or attenuated-type 2575/08 revealed quick gene induction and efficient virus control. In summary, this study reveals a new IBV strain, which harbors a known local genotype but displays remarkably altered antigenicity, pathogenicity and host defenses.

KMUP-1 Attenuates Endothelin-1-Induced Cardiomyocyte Hypertrophy through Activation of Heme Oxygenase-1 and Suppression of the Akt/GSK-3ß, Calcineurin/NFATc4 and RhoA/ROCK Pathways.

The signaling cascades of the mitogen activated protein kinase (MAPK) family, calcineurin/NFATc4, and PI3K/Akt/GSK3, are believed to participate in endothelin-1 (ET-1)-induced cardiac hypertrophy. The aim of this study was to investigate whether KMUP-1, a synthetic xanthine-based derivative, prevents cardiomyocyte hypertrophy induced by ET-1 and to elucidate the underlying mechanisms. We found that in H9c2 cardiomyocytes, stimulation with ET-1 (100 nM) for 4 days induced cell hypertrophy and enhanced expressions of hypertrophic markers, including atrial natriuretic peptide and brain natriuretic peptide, which were all inhibited by KMUP-1 in a dose-dependent manner. In addition, KMUP-1 prevented ET-1-induced intracellular reactive oxygen species generation determined by the DCFH-DA assay in cardiomyocytes. KMUP-1 also attenuated phosphorylation of ERK1/2 and Akt/GSK-3ß, and activation of calcineurin/NFATc4 and RhoA/ROCK pathways induced by ET-1. Furthermore, we found that the expression of heme oxygenase-1 (HO-1), a stress-response enzyme implicated in cardio-protection, was up-regulated by KMUP-1. Finally, KMUP-1 attenuated ET-1-stimulated activator protein-1 DNA binding activity. In conclusion, KMUP-1 attenuates cardiomyocyte hypertrophy induced by ET-1 through inhibiting ERK1/2, calcineurin/NFATc4 and RhoA/ROCK pathways, with associated cardioprotective effects via HO-1 activation. Therefore, KMUP-1 may have a role in pharmacological therapy of cardiac hypertrophy.

[A prospective randomized controlled trial of Sapylin combined with chemotherapy for advanced colon cancer following radical operation].

OBJECTIVE: To evaluate the effect of Sapylin combined with intraperitoneal and systemic chemotherapy for advanced colon cancer following radical operation on local recurrence, hepatic metastasis, and overall survival rate. METHODS: From Jan. 2004 to Dec. 2005,132 patients with stage II or III colon carcinoma after radical operation were randomly divided into two groups: Sapylin combined with chemotherapy(Sapylin) group and the control group. Toxic reaction, local recurrence, hepatic metastasis, and overall survival rate between two groups were compared. RESULTS: Both groups successfully completed the trial. There was no significant difference in toxic reaction between two groups, the recurrence and hepatic metastasis rate in Sapylin group were lower than those in the control group(9/60, 15.0% vs. 22/72, 30.6%; 11/60,18.3% vs. 22/72, 34.7%, respectively), which were statistically significant (P<0.05 respectively). The 3- year survival rate in Sapylin group was higher than that in control group(73.3% vs. 54.2%), which was statistically significant (P<0.05). CONCLUSION: Sapylin combined with intraperitoneal and systemic chemotherapy can effectively decrease local recurrence and hepatic metastasis,and improve the survival in patients with advanced colon cancer following radical operation.

Addition of hepatectomy decreases liver recurrence and leads to long survival in hilar cholangiocarcinoma.

AIM: To evaluate hepatic recurrence and prognostic factors for survival in patients with surgically resected hilar cholangiocarcinoma in a single institution over the last 13 years. METHODS: From 1994 to 2007, all patients with hilar cholangiocarcinoma referred to a surgical clinic were evaluated. Demographic data, tumor characteristics, and outcome were analyzed retrospectively. Outcome was compared in patients who underwent additional liver resection with resection of the tumor. RESULTS: Of the 69 patients submitted to laparotomy for tumor resection, curative resection (R(0) resection) was performed in 40 patients, and palliative resection in 29. Thirty-one patients had only duct resection, and 38 patients had combined duct resection with liver resection including 34 total or part caudate lobes. Curative rates with the combined hepatectomy were significantly improved compared with those without additional hepatectomy (27/38 vs 13/31; chi2 = 5.94, P < 0.05). Concomitant liver resection was associated with a decreased incidence of initial recurrence in liver one year after surgery (11/38 vs 23/31; chi2 = 13.98, P < 0.01). The 3-year survival rate after R(0) resection was 30.7% and was 10.5% for palliative resection. R(0) resection improved the 3-year survival rate (30.7% vs 10.5%; chi2 = 12.47, P < 0.01). CONCLUSION: Hepatectomy, especially including the caudate lobe combined with bile duct resection should be considered standard treatment to cure hilar cholangiocarcinoma.

Effects of l-stepholidine on forebrain Fos expression: comparison with clozapine and haloperidol.

l-Stepholidine (SPD) is a tetrahydroprotoberberine alkaloid and a mixed dopamine D1 agonist/D2 antagonist. Preliminary clinical trials suggest that SPD improves both positive and negative symptoms of schizophrenia without producing significant extrapyramidal side effects. Here, we report that SPD mimics the effect of the atypical antipsychotic drug clozapine, preferentially increasing Fos expression in corticolimbic areas. Thus, at 10 mg/kg (i.p.), SPD induced Fos expression in the medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and lateral septal nucleus (LSN) without significantly affecting the dorsolateral striatum (DLSt). At higher doses (20-40 mg/kg), SPD also increased Fos expression in the DLSt. The increase, however, was less pronounced than the increase seen in the NAc. Within the NAc, SPD also induced more Fos expression in the shell than in the core. In all subcortical areas examined, the Fos expression induced by SPD was mimicked by the D2 antagonist sulpiride and reversed by the D2 agonist quinpirole, suggesting that the effect is due to blockade of D2-like receptors by SPD. In the mPFC, however, the effect was not mimicked by sulpride or reversed by quinpirole. It was also not mimicked by the D1 agonist SKF38393 or SKF38393 plus sulpride, and not reversed by the D1 antagonist SCH23390. These results suggest that, in the mPFC, SPD may induce Fos expression through a non-DA mechanism. Whether the mechanism involves an interaction of SPD with other neurotransmitters such as 5-HT and norepinephrine remains to be determined.

[C-fos expression within PVN and NTS of the rat induced by gastric ischemia/reperfusion injury].

AIM: To investigate the effect of paraventricular nucleus (PVN) stimulation and the c-fos expression within PVN and nucleus tractus solitarius (NTS) of the rat following gastric ischemia/reperfusion injury (GI/RI). METHODS: The rat celiac artery was clamped for thirty minutes and reperfused for sixty minutes, using Fos immunohistochemical method (ABC method) examined the c-fos expression within PVN and NTS. RESULTS: (1) Both electrical and chemical stimulation of the PVN obviously attenuated the GI/ RI. (2) Bilateral electrolytic lesion of NTS could eliminate the protective effect of electrical stimulation of the PVN. (3) The Fos-like immunoreactive neurons were increased in bilateral PVN and NTS by GI/RI. CONCLUSION: The function of PVN and NTS could be affected by the GI/RI noxious stimulation. PVN, NTS were involved in the regulation of GI/RI.