Xunlong Shi

Biotransformation of ginsenoside Rb1 to ginsenoside Rd by highly substrate-tolerant Paecilomyces bainier 229-7

Paecilomyces bainier 229-7 was obtained after UV irradiation for 8 min in the presence of 0.4% LiCl and selection on potato dextrose media containing 30 mg/mL saponin from Panax notoginseng leaves (SPNL). The mutant produces ginsenoside Rd from ginsenoside Rb1 with a bioconversion rate as high as 94.9% under optimized culture conditions in shake flasks when supplied with 20 mg/mL of SPNL. Scale-up in 10-L fermenter resulted in an 89% bioconversion rate. Ginsenoside Rd was purified from the culture medium by a macroporous resin with a chromatographic purity of 92.6%. These results suggest that P. bainier 229-7 could be useful for the preparation of ginsenoside Rd in the pharmaceutical industry.

In vitro and in vivo anti-hepatitis B virus activities of a plant extract from Geranium carolinianum L.

Natural products provide a large reservoir of potentially active agents with anti-hepatitis B virus (HBV) activity. We examined the effect of the polyphenolic extract from Geranium carolinianum L. (PPGC) on HBV replication both in vitro and in vivo. In the human HBV-transfected liver cell line HepG(2) 2.2.15, PPGC effectively suppressed the secretion of the HBV antigens in a dose-dependent manner with IC(50) values of 46.85 microg/ml for HBsAg and 65.60 microg/ml for HBeAg at day 9. Consistent with the HBV antigen reduction, PPGC (100 microg/ml) also reduced HBV DNA level by 35.9%. In the duck hepatitis B virus (DHBV) infected ducks, after PPGC was dosed intragastricly (i.g.) once a day for 10 days, the plasma DHBV DNA level was reduced, with an ED(50) value of 47.54 mg/kg. In addition, Southern blot analysis confirmed the in vivo anti-HBV effect of PPGC in ducks and PPGC also reduced the plasma and the liver DHBV DNA level in a dose-dependent manner. Furthermore, significant improvement of the liver was observed after PPGC treatment, as evaluated by the histopathological analysis.

Inhibition of the inflammatory cytokine tumor necrosis factor-alpha with etanercept provides protection against lethal H1N1 influenza infection in mice

IntroductionFactors implicated in influenza-mediated morbidity and mortality include robust cytokine production (cytokine storm), excessive inflammatory infiltrates, and virus-induced tissue destruction. Tumor necrosis factor-alpha (TNF-α) is an important pro-inflammatory cytokine present during influenza infection, but it is unclear whether direct inhibition of TNF-α can elicit protection against influenza infection.MethodsIn this study, the commercially available TNF-α inhibitor etanercept was used to inhibit TNF-α induced by lethal A/FM/1/47 (H1N1) influenza virus infection of mice. The effects of TNF-α inhibition on mouse survival, pathologic changes, immune cell infiltration, inflammatory cytokine secretion, Toll-like receptor expression, and activation of the NF-κB (nuclear factor kappa B) signaling pathway were evaluated.ResultsThe intranasal delivery of etanercept provided significant protection against mortality (30% of mice survived up to 14 days after infection) in mice treated with etanercept. In contrast, no survivors were found beyond 6 days in mice treated with saline after lethal challenge with H1N1 influenza virus. It was observed that etanercept significantly reduced inflammatory cell infiltration (for example, macrophages and neutrophils), inflammatory cytokine secretion (for example, interleukin-6, TNF-α, and interferon gamma), and expression of Toll-like receptors (TLR-3, TLR-4, and TLR-7). Etanercept also downregulated and inhibited the cascade proteins of the NF-κB signaling pathway (for example, MyD88, TRIF, NF-κB, and p65), as well as enhanced host control of virus replication.ConclusionsThese findings indicate that etanercept, by blocking TNF-α, can significantly downregulate excessive inflammatory immune responses and provide protection against lethal influenza infection, making its use a novel strategy for controlling severe influenza-induced viral pneumonia.

Modified Jiu Wei Qiang Huo decoction improves dysfunctional metabolomics in influenza A pneumonia-infected mice

In order to study the effective mechanism of a traditional Chinese medicine (TCM), modified Jiu Wei Qiang Huo decoction (MJWQH), against H1N1-induced pneumonia in mice, we chose a holistic approach. A reverse-phase liquid chromatography with quadruple time-of-flight mass spectrometry (LC-Q-TOF-MS) was developed to determine metabolomic biomarkers in mouse serum for the MJWQH effects. Thirteen biomarkers of H1N1-induced pneumonia in mice serum were identified, which comprised l-valine, lauroylcarnitine, palmitoyl-l-carnitine, l-ornithine, uric acid, taurine, O-succinyl-l-homoserine, l-leucine, l-phenylalanine, PGF2α, 20-ethyl-PGE2, arachidonic acid, and glycerophospho-N-arachidonoyl ethanolamine. Among them, metabolites of amino acids, fatty acids and arachidonic acid had the most relevant changes in mice with H1N1-induced pneumonia. MJWQH effectively improved weight loss, lung index, biomarkers and inflammatory mediators such as prostaglandin E2 and phospholipase A2 in the infected mice. Importantly, MJWQH reversed the elevated biomarkers to the control levels from the infection, which provided a systematic view and a theoretical basis for its prevention or treatment. The results suggest that the protective effect of MJWQH against H1N1-induced pneumonia is possibly through regulation of pathways for amino acid, fatty acid and arachidonic acid metabolism. They also suggest that the LC-MS-based metabolomic strategy is a powerful tool for elucidation of the mechanisms of TCM.

Baicalin inhibits autophagy induced by influenza A virus H3N2.

Baicalin, a natural product isolated from Scutellariaradix, has been reported to have significant in vivo and in vitro anti-influenza virus activity, but the underlying mechanism remains poorly understood. In this study, we found that baicalin inhibited autophagy induced by influenza virus A3/Beijing/30/95 (H3N2) in both A549 and Ana-1 cells. The results showed that H3N2 induced autophagy by suppressing mTOR signaling pathway, which however could be significantly inhibited by baicalin. Baicalin could suppress the expression of Atg5-Atg12 complex and LC3-II, and attenuate autophagy induced by starvation. Thus, the inhibition of autophagy induced by virus may account for the antiviral activities of baicalin against H3N2. Autophagy may be a potential marker in developing novel anti-influenza drugs.

Involvement of autophagy in recombinant human arginase-induced cell apoptosis and growth inhibition of malignant melanoma cells

Recombinant human arginase (rhArg) has been developed for arginine derivation therapy of cancer and is currently in clinical trials for a variety of malignant solid tumors. In this study, we reported for the first time that rhArg could induce obvious autophagy in human melanoma cells; inhibition of autophagy by chloroquine (CQ) significantly increased rhArg-induced cell apoptosis and growth inhibition of A375 cells. A significant increase in mitochondrial membrane potential loss and elevated intracellular reactive oxygen species (ROS) levels were detected in A375 cells after rhArg treatment when compared with control. Membrane transition inhibitor cyclosporine A blocked autophagy and accelerated cell death induced by rhArg, indicating that rhArg induced autophagy via mitochondria pathway. Furthermore, antioxidant N-acetyl-l-cysteine suppressed rhArg-induced autophagy and rescued cells from cell growth inhibition, suggesting that ROS played an important role in rhArg-induced A375 cell growth inhibition and autophagy. Akt/mTOR signaling pathway was involved in autophagy induced by rhArg in a time-dependent manner. Moreover, rhArg could induce ERK1/2 activation in a dose- and time-dependent manner and rhArg-induced autophagy was attenuated when p-ERK1/2 was inhibited by MEK 1/2 inhibitor, U0126. Taken together, this study provides new insight into the molecular mechanism of autophagy involved in rhArg-induced cell apoptosis and growth inhibition, which facilitates the development of rhArg in combination with CQ as a potential therapy for malignant melanoma.

Pharmacokinetics and Pharmacodynamics of Recombinant Human EPO-Fc Fusion Protein In Vivo

In this study, the in vivo pharmacokinetics and pharmacodynamics of a novel recombinant human erythropoietin (rhEPO) Fc fusion protein, rhEPO-Fc, were studied in both rodents and rhesus monkeys. Animal models of anemia induced by irradiation, cyclophosphamide and partial renal ablation were used to evaluate therapeutic effects of rhEPO-Fc. We have demonstrated that serum half-life of rhEPO-Fc was 29.5 to 38.9 h at doses of 8, 25, 80 µg/kg in rhesus monkeys and 35.5 to 43.5 h at doses of 16, 50, 160 µg/kg in rats. In anemia animal models, rhEPO-Fc dose-dependently (7.5–30.0 µg/kg in mice, 5.4–21.4 µg/kg in rats and 5.0–10.0 µg/kg in rhesus monkeys) increased reticulocyte level, followed by an increase of RBC count, hemoglobin and hematocrit levels. At reduced intervention frequency of weekly treatments, rhEPO-Fc showed similar hematopoietic effects as compared with rhEPO given three times a week. These results indicated that rhEPO-Fc could potentially be used in treatment of anemia and warrants future clinical trials.

Combination of Apolipoprotein A1‐Modi Liposome–Doxorubicin with Autophagy Inhibitors Overcame Drug Resistance In Vitro

Multidrug resistance (MDR) represents the major drawback in chemotherapy. Liposome-based approaches could reverse MDR to some extent through circumventing the active efflux effect of P-glycoprotein. However, the reverse power of liposome is very limited because the nontargeting liposome is inefficient to deliver drugs to tumor actively. Besides, autophagy could reinforce the resistance of tumor cells to the cytotoxicity of intracellular drugs. Here, liposomal doxorubicin (Lipodox) that was conjugated with apolipoprotein A1-apo-Lipodox, was employed in tumor targeting delivery of doxorubicin. In the experiments, apo-Lipodox could enter cells effectively and reverse MDR more efficiently than their nontargeting counterpart. Autophagy occurred in this process and contributed to the survival of tumor cells. Combination use of autophagy inhibitors could enhance the cytotoxicity of apo-Lipodox and reverse drug resistance to a higher degree. We propose that this strategy holds promise to overcome MDR in human cancer.

Ability of Recombinant Human Catalase to Suppress Inflammation of the Murine Lung Induced by Influenza A

Influenza A virus pandemics and emerging antiviral resistance highlight the urgent need for novel generic pharmacological strategies that reduce both viral replication and inflammation of the lung. We have previously investigated the therapeutic efficacy of recombinant human catalase (rhCAT) against viral pneumonia in mice, but the protection mechanisms involved were not explored. In the present study, we have performed a more in-depth analysis covering survival, lung inflammation, immune cell responses, production of cytokines, and inflammation signaling pathways in mice. Male imprinting control region mice were infected intranasally with high pathogenicity (H1N1) influenza A virus followed by treatment with recombinant human catalase. The administration of rhCAT resulted in a significant reduction in inflammatory cell infiltration (e.g., macrophages and neutrophils), inflammatory cytokine levels (e.g., IL-2, IL-6, TNF-α, IFN-γ), the level of the intercellular adhesion molecule 1 chemokine and the mRNA levels of toll-like receptors TLR-4, TLR-7, and NF-κB, as well as partially maintaining the activity of the antioxidant enzymes system. These findings indicated that rhCAT might play a key protective role in viral pneumonia of mice via suppression of inflammatory immune responses.

Baicalin benefits the anti-HBV therapy via inhibiting HBV viral RNAs

Background: Although current antiviral treatments (nucleoside analogs, NAs) for chronic hepatitis B virus (HBV) infection are effective in suppressing HBV‐DNA replication, their clinical outcomes can be compromised by the increasing drug resistance and the inefficiency in promoting HBsAg/HBeAg seroconversion. Objectives: In this study, we will explore possible effects and mechanism of a natural product baicalin (BA) with the anti‐HBV efficacy of entecavir (ETV), a first‐line anti‐HBV drug, in HBV‐DNA, HBsAg/HBeAg seroconversion and drug‐resistance. Methods: The co‐effects of BA and ETV were conducted in wild‐type/NA‐resistance mutant HBV cell lines and DHBV‐infected duckling models. HBV‐DNA/RNAs, HBsAg/HBeAg, host factors (hepatocyte nuclear factors) were explored for possible anti‐HBV mechanism. Results and discussion: BA could significantly enhance and reduced HBsAg and HBeAg in hepG2.2.15, a wild‐type HBV cell line. Co‐treatment of BA and ETV had a more dramatic effect in NA‐resistant HBVrtM204V/rtLl80M transfected hepG2 cells. Our study further revealed that BA mainly inhibited the production of HBV RNAs (3.5, 2.4, 2.1 kb), the templates for viral proteins and HBV‐DNA synthesis. BA blocked HBV RNAs transcription possibly by down‐regulating transcription and expression of HBV replication dependent hepatocyte nuclear factors (HNF1&agr; and HNF4&agr;). Thus, BA may benefit the anti‐HBV therapy via inhibiting HBV viral RNAs. HIGHLIGHTSBaicalin benefits the anti‐HBV therapy.Baicalin enhances ETV antiviral efficacy and overcomes NA‐resistant HBV mutation.The anti‐HBV effect of baicalin is achieved by inhibiting HBV RNAs.Baicalin down‐regulates HBV replication‐dependent host factors HNF 1&agr; and HNF 4&agr;.