Publications by authors named "Xunlong Shi"

28 Publications

  • Page 1 of 1

Baicalin Inhibits Influenza A Virus Infection Promotion of M1 Macrophage Polarization.

Front Pharmacol 2020 6;11:01298. Epub 2020 Oct 6.

Department of Biological Medicines & Shanghai Engineering Research Center of Immunotherapeutics, Fudan University School of Pharmacy, Shanghai, China.

Background And Aims: The natural compound baicalin (BA) possesses potent antiviral properties against the influenza virus. However, the underlying molecular mechanisms of this antiviral activity and whether macrophages are involved remain unclear. In this study, we, therefore, investigated the effect of BA on macrophages.

Methods: We studied macrophage recruitment, functional phenotypes (M1/M2), and the cellular metabolism flow cytometry, qRT-PCR, immunofluorescence, a cell culture transwell system, and GC-MS-based metabolomics both in H1N1 A virus-infected mice and .

Results: BA treatment drastically reduced macrophage recruitment (CD11b, F4/80) by approximately 90% while maintaining the proportion of M1-polarized macrophages in the bronchoalveolar lavage fluid of infected mice. This BA-stimulated macrophage M1 phenotype shift was further verified in ANA-1 and primary peritoneal macrophages by measuring macrophage M1 polarization signals (CD86, iNOS, TNF-α, ratio, and IL-1β cleavage). Meanwhile, we observed an activation of the IFN pathway (upregulation of and ), an inhibition of influenza virus replication (as measured by the gene), and distinct cellular metabolic responses in BA-treated cells.

Conclusion: BA triggered macrophage M1 polarization, IFN activation, and other cellular reactions, which are beneficial for inhibition of H1N1 A virus infection.
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http://dx.doi.org/10.3389/fphar.2020.01298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574031PMC
October 2020

Baicalin down-regulating hepatitis B virus transcription depends on the liver-specific HNF4α-HNF1α axis.

Toxicol Appl Pharmacol 2020 09 17;403:115131. Epub 2020 Jul 17.

Department of Biological Medicines & Shanghai Engineering Research Center of Immunotherapeutics, School of Pharmacy, Fudan University, Shanghai 201203, PR China. Electronic address:

Baicalin (BA) inhibits hepatitis B virus (HBV) RNAs production and reduces levels of the related hepatocyte nuclear factors (HNFs), although the underlying mechanism is unclear. In this study, we investigated the specific pathway by which BA regulates HBV transcription through the HBV-related HNFs. Following transfection of HepG2 cells with pHBV1.2, we observed that BA inhibited the production of HBV RNAs and viral proteins in a time- and dose-dependent manner. These effects were consistent with the downregulation of HNF1α, which was abolished by HNF1α-shRNA. The shRNA of HNF4α, the upstream gene of HNF1α, also remarkedly reduced HNF1α expression and impaired the anti-HBV efficacy of BA, indicating that this function of BA depended on HNF4α/HNF1α axis. Furthermore, chromatin immunoprecipitation assay showed that BA significantly reduced HNF4α-HNF1α transactivation activity. The similar effects of BA were observed in entecavir (ETV)-resistant HBV transfected HepG2 cells. Thus, we proposed a mechanism for the anti-HBV activity of BA in an HNF4α-HNF1α-dependent manner, which impaired HNF4α and HNF1α transactivation, and effectively inhibited HBV transcription and viral replication.
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http://dx.doi.org/10.1016/j.taap.2020.115131DOI Listing
September 2020

Chlorogenic acid protects PC12 cells against corticosterone-induced neurotoxicity related to inhibition of autophagy and apoptosis.

BMC Pharmacol Toxicol 2019 09 9;20(1):56. Epub 2019 Sep 9.

Department of Microbiological and Biochemical Pharmacy, School of Pharmacy, Fudan University, Shanghai, 201203, China.

Background: There are evidences that chlorogenic acid (CGA) has antidepressant effects, however the underlying molecular mechanism has not been well understood. The aim of the study was to explore the neuroprotective effect of CGA on corticosterone (CORT)-induced PC 12 cells and its mechanism, especially the autophagy pathway.

Methods: PC12 cells were incubated with CORT (0, 100, 200, 400 or 800 μM) for 24 h, cell viability was measured by MTT assay. PC12 cells were cultured with 400 μM of CORT in the absence or presence of CGA (25 μg/ml) for 24 h, morphologies and specific marker of autophagosome were observed by transmission electron microscope (TEM) and confocal immunofluorescence microscopy, respectively. In addition, PC12 cells were treated with different doses of CGA (0, 6.25, 12.5, 25 or 50 μg/ml) with or without CORT (400 μM) for 24 h, cell viability and changes in the morphology were observed, and further analysis of apoptotic and autophagic proteins, and expression of AKT/mTOR signaling pathway were carried out by Western blot. Specific inhibitors of autophagy 3-Methyladenine (3-MA) and chloroquine (CQ) were added to the PC12 cells cultures to explore the potential role of autophagy in CORT-induced neuronal cell apoptosis.

Results: Besides decreasing PC12 cell activity, CORT could also induce autophagy and apoptosis of PC12 cells, while CGA could reverse these effects. In addition, CGA treatment regulated AKT/mTOR signaling pathway in PC12 cells. CGA, similar to 3-MA and QC, significantly inhibited CORT-induced apoptosis in PC12 cells.

Conclusions: Our results provide a new molecular mechanism for the treatment of CORT-induced neurotoxicity by CGA, and suggest CGA may be a potential substance which is can alleviate depression.
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http://dx.doi.org/10.1186/s40360-019-0336-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6734305PMC
September 2019

New metabolites from the biotransformation of ginsenoside Rb1 by sp.229 and activities in inducing osteogenic differentiation by Wnt/β-catenin signaling activation.

J Ginseng Res 2018 Apr 22;42(2):199-207. Epub 2017 Mar 22.

School of Pharmacy, Fudan University, Shanghai, China.

Background: Ginseng is a well-known traditional Chinese medicine that has been widely used in a range of therapeutic and healthcare applications in East Asian countries. Microbial transformation is regarded as an effective and useful technology in modification of nature products for finding new chemical derivatives with potent bioactivities. In this study, three minor derivatives of ginsenoside compound K were isolated and the inducing effects in the Wingless-type MMTV integration site (Wnt) signaling pathway were also investigated.

Methods: New compounds were purified from scale-up fermentation of ginsenoside Rb1 by sp. 229 through repeated silica gel column chromatography and high pressure liquid chromatography. Their structures were determined based on spectral data and X-ray diffraction. The inductive activities of these compounds on the Wnt signaling pathway were conducted on MC3T3-E1 cells by quantitative real-time polymerase chain reaction analysis.

Results: The structures of a known 3-keto derivative and two new dehydrogenated metabolites were elucidated. The crystal structure of the 3-keto derivative was reported for the first time and its conformation was compared with that of ginsenoside compound K. The inductive effects of these compounds on osteogenic differentiation by activating the Wnt/β-catenin signaling pathway were explained for the first time.

Conclusion: This study may provide a new insight into the metabolic pathway of ginsenoside by microbial transformation. In addition, the results might provide a reasonable explanation for the activity of ginseng in treating osteoporosis and supply good monomer ginsenoside resources for nutraceutical or pharmaceutical development.
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http://dx.doi.org/10.1016/j.jgr.2017.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5925613PMC
April 2018

Houttuynia cordata polysaccharides ameliorate pneumonia severity and intestinal injury in mice with influenza virus infection.

J Ethnopharmacol 2018 May 19;218:90-99. Epub 2018 Feb 19.

Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai, China. Electronic address:

Ethnopharmacological Relevance: Hottuynia cordata is an important traditional Chinese medicine for the treatment of respiratory diseases including bacterial and viral infections. Polysaccharides isolated from Houttuynia cordata (HCP), as its main ingredients, have been demonstrated to ameliorate the LPS-induced acute lung injury in mice. The study aimed to determine the protective effects of HCP on multiple organ injury in influenza A virus (IAV) H1N1 infected mice and its primary mechanisms in anti-inflammation and immune regulation.

Materials And Methods: Mice were inoculated with IAV H1N1 and then treated with 20 or 40 mg/kg/d of HCP for survival test and acute lung-gut injury test.

Results: The treatment with HCP resulted in an increase in the survival rate of H1N1 infected mice and the protection from lung and intestine injury, accompanied with the reduced virus replication. HCP markedly decreased the concentration of pulmonary proinflammatory cytokines/chemokines and the number of intestinal goblet cells, and strengthened the intestinal physical and immune barrier, according to the increase of sIgA and tight junction protein (ZO-1) in intestine. At the same time, the inhibition of inflammation in lung and gut was related to the suppressing of the expression of TLR4 and p-NFκB p65 in lung.

Conclusions: These results indicated that HCP ameliorated lung and intestine injury induced by IAV attack. The mechanisms were associated with inhibition of inflammation, protection of intestinal barrier and regulation of mucosal immunity, which may be related to the regulation of gut-lung axis. As an alternative medicine, HCP may have clinical potential to treat IAV infection in human beings.
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http://dx.doi.org/10.1016/j.jep.2018.02.016DOI Listing
May 2018

Material basis studies of anti-Influenza A active ingredients in Tanreqing Injection.

Biomed Chromatogr 2018 Feb 10;32(2). Epub 2017 Oct 10.

Shanghai Institutes of Materia Medica, Chinese Academy of Sciences, Shanghai, China.

Tanreqing Injection (TRQ) has been used primarily in treating infections of the upper respiratory tract and serious influenza in China, as a classical compound herbal recipe. TRQ had been demonstrated to have effects of clearing heat, eliminating phlegm, detoxification, reducing inflammation and alleviating cough. The survival rate, histopathology of lungs and viral titers in mice were evaluated in this study to verify the curative effect of TRQ. However, there is not enough information about the components. In the present study, a high-performance and practical LC/QTOF/MS method was developed for characterization and identification of the natural ingredients in TRQ. A total of 60 compounds, including 10 amino acids, 10 iridoid glucosides, 14 flavonoids, 13 other phenolic compounds, 10 steroid acids and three other compounds, were characterized and identified. We also confirmed the material basis of anti-Influenza A active ingredients in TRQ. Therefore, we have developed an accurate analytical method. LC/QTOF/MS could be applied for identification the complex components in traditional Chinese medicine.
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http://dx.doi.org/10.1002/bmc.4097DOI Listing
February 2018

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

Toxicol Appl Pharmacol 2017 05 18;323:36-43. Epub 2017 Mar 18.

Department of Microbiology and Biopharmacy, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China. Electronic address:

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 HBV transfected hepG2 cells. Our study further revealed that BA mainly inhibited the production of HBV RNAs (3.5, 2.4, 2.1kb), 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 (HNF1α and HNF4α). Thus, BA may benefit the anti-HBV therapy via inhibiting HBV viral RNAs.
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http://dx.doi.org/10.1016/j.taap.2017.03.016DOI Listing
May 2017

The Modified JiuWei QiangHuo Decoction Alleviated Severe Lung Injury Induced by H1N1 Influenza Virus by Regulating the NF- κ B Pathway in Mice.

Evid Based Complement Alternat Med 2015 18;2015:790739. Epub 2015 May 18.

Department of Biosynthesis, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China.

A new approach to treat infections of highly pathogenic influenza virus is to inhibit excessive innate immune response. JiuWei QiangHuo decoction has been used for centuries for the treatment of pulmonary disorders in China. In this study, we evaluated the anti-inflammatory activities of the modified JiuWei QiangHuo (MJWQH) decoction in the treatment of influenza A (H1N1) virus-induced severe pneumonia in mice. The results showed that MJWQH significantly increased the survival rate of H1N1-infected mice and suppressed the production of TNF-α, IL-1, IL-6, MCP-1, RANTES, and IFN-α on day 4 after infection. Moreover, oral administration of MJWQH efficiently inhibited virus replication and alleviated the severity of lung injuries. The results also showed that MJWQH may have potential therapeutic effect on severe lung injury induced by H1N1 virus by regulating the NF-κB pathway. Our study suggested that MJWQH might be an alternative therapy for the treatment of viral pneumonia.
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http://dx.doi.org/10.1155/2015/790739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4451524PMC
June 2015

Combination of apolipoprotein A1-modi liposome-doxorubicin with autophagy inhibitors overcame drug resistance in vitro.

J Pharm Sci 2014 Dec 29;103(12):3994-4004. Epub 2014 Oct 29.

Department of Biosynthesis, School of Pharmacy, Fudan University, Shanghai 201203, People's Republic of China. Electronic address:

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.
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http://dx.doi.org/10.1002/jps.24216DOI Listing
December 2014

Effect analysis of mineral salt concentrations on nosiheptide production by Streptomyces actuosus Z-10 using response surface methodology.

Molecules 2014 Sep 26;19(10):15507-20. Epub 2014 Sep 26.

School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China.

The objective of this study was to develop an optimal combination of mineral salts in the fermentation medium for nosiheptide (Nsh) production using statistical methodologies. A Plackett-Burman design (PBD) was used to evaluate the impacts of eight mineral salts on Nsh production. The results showed that among the no-significant factors, CaCO3, and K2HPO4·3H2O had positive effects, whereas FeSO4·7H2O, CuSO4·5H2O, and ZnSO4·7H2O had negative effects on Nsh production. The other three significant factors (Na2SO4, MnSO4·H2O, and MgSO4·7H2O) were further optimized by using a five-level three-factor central composite design (CCD). Experimental data were fitted to a quadratic polynomial model, which provided an effective way to determine the interactive effect of metal salts on Nsh production. The optimal values were determined to be 2.63, 0.21, and 3.37 g/L, respectively. The model also ensured a good fitting of scale-up Nsh batch fermentation with a maximum production of 1501 mg/L, representing a 1.56-fold increase compared to the original standard condition. All these results revealed that statistical optimization methodology had the potential to achieve comprehensive optimization in Nsh fermentation behaviors, which indicates a possibility to establish economical large-scale production of Nsh.
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http://dx.doi.org/10.3390/molecules191015507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6270855PMC
September 2014

Anti-inflammatory effect of thalidomide on H1N1 influenza virus-induced pulmonary injury in mice.

Inflammation 2014 Dec;37(6):2091-8

Department of Biosynthesis, School of Pharmacy, Fudan University, 826 Zhangheng Road, 201203, Shanghai, China.

The purpose of this study is to investigate the anti-inflammatory effect of thalidomide (Thd) on H1N1-induced acute lung injury in mice. BALB/C mice were infected intranasally with influenza A virus (H1N1) and then treated with Thd at a dose of 100 or 200 mg/kg/day for 7 days. Weight loss and survival of mice were monitored for 14 days after virus challenge, and the serum and lung tissues were collected at 4 days for histological and biochemical analysis. The results showed that Thd significantly improved the survival rate, reduced the infiltration of inflammatory cells and cytokine (e.g., IL-6, TNF-α) and chemokine (e.g., RANTES, IP-10) levels, and inhibited activated p-NFκB p65 in infected mice. These findings suggested that Thd may attenuate H1N1-induced pulmonary injury and thus may find use in the treatment of viral diseases.
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http://dx.doi.org/10.1007/s10753-014-9943-9DOI Listing
December 2014

Ability of recombinant human catalase to suppress inflammation of the murine lung induced by influenza A.

Inflammation 2014 Jun;37(3):809-17

Department of Drug Biosynthesis, School of Pharmacy, Fudan University, 826 Zhang Heng Road, Shanghai, 201203, China,

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.
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http://dx.doi.org/10.1007/s10753-013-9800-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7087580PMC
June 2014

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

Crit Care 2013 Dec 27;17(6):R301. Epub 2013 Dec 27.

Introduction: Factors 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.

Methods: In 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.

Results: The 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.

Conclusions: These 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.
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http://dx.doi.org/10.1186/cc13171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4057515PMC
December 2013

Tumor necrosis therapy antibody interleukin-2 fusion protein elicits prolonged and targeted antitumor effects in vivo.

Appl Microbiol Biotechnol 2014 May 26;98(9):4053-61. Epub 2013 Nov 26.

Department of Biosynthesis & Key Lab of Smart Drug Delivery MOE, School of Pharmacy, Fudan University, 826 Zhang Heng Road, Shanghai, 201203, China.

Interleukin-2 (IL-2) is one of the most successful cytokines applied in tumor immunotherapy because of its ability to stimulate potent cellular immune response. The life-threatening toxicity of vascular leak syndrome (VLS) associated with the high-dose IL-2 treatment regimen has limited its use in tumor immunotherapy. To reverse this situation, a tumor-targeted fusion protein, recombinant human TNT-IL2 (rhTNT-IL2), was generated with both the cytokine activity of IL-2 and the tumor-targeting ability of TNT antibody. TNT is a human tumor necrosis therapy monoclonal antibody capable of binding intracellular antigens which are accessible and abundant in necrotic regions of tumors. The immunotherapeutic potential of this fusion protein was tested in murine melanoma and lung cancer models, and tumor-bearing mice showed satisfied tumor regressions after rhTNT-IL2 immunotherapy. Immunohistochemical study showed a distinct penetration of IL-2 in tumors in mice treated with rhTNT-IL2, indicating its evident tumor-targeting activity. Moreover, the rhTNT-IL2 was well tolerated in cynomolgus monkeys in a 12-week long-term repeated toxicity study. These studies indicate that the targeting of IL-2 to necrotic areas of tumors might be a new approach for the immunotherapy of solid tumors.
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http://dx.doi.org/10.1007/s00253-013-5349-0DOI Listing
May 2014

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

Biomed Chromatogr 2014 Apr 17;28(4):468-74. Epub 2013 Oct 17.

Department of Chinese Medicine, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai, 200092, People's Republic of China.

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.
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http://dx.doi.org/10.1002/bmc.3055DOI Listing
April 2014

Recombinant lipoproteins reinforce cytotoxicity of doxorubicin to hepatocellular carcinoma.

J Drug Target 2014 Jan 4;22(1):76-85. Epub 2013 Oct 4.

School of Pharmacy, Fudan University , Shanghai , China.

Cancer nanotherapeutics are changing the landscape of tumor treatment and used to circumvent limitations of conventional chemotherapy, such as non-specificity and low bioavailability. Reconstituted high density lipoproteins (rHDL) system is one of the most promising targeting delivery systems of chemotherapeutic drugs toward tumors. Here, we developed recombined high-density lipoprotein which can be functionalized to deliver doxorubicin intracellular with a higher efficiency. The cellular viability assay showed that the rHDL/Dox nanovectors had an enhanced efficiency in inhibiting the cell viability of hepatocellular carcinoma cell lines HepG2 and SMMC-7721. FACS and confocal microscopy was used to observe the doxorubicin delivery into cancer cells. Intracellular drug accumulation analysis confirmed that treatment of rHDL/Dox nanovectors resulted in higher intracellular doxorubicin concentration to the levels exceeding that of free drug. On the premise of efficient drug delivery, rHDL/Dox nanovectors have been preliminarily demonstrated effective inducing of cytotoxic effect and cell apoptosis to both of the cell lines in vitro. Tissue distribution experiment showed that rHDL/Dox nanovectors could also deliver doxorubicin to liver effectively. So, we proposed that this lipoprotein-based strategy holds promise for a safer and more efficient delivery of chemotherapeutic agents in the treatment of hepatocellular carcinoma.
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http://dx.doi.org/10.3109/1061186X.2013.839687DOI Listing
January 2014

Pharmacokinetics and pharmacodynamics of recombinant human EPO-Fc fusion protein in vivo.

PLoS One 2013 19;8(8):e72673. Epub 2013 Aug 19.

Department of Biosynthesis, School of Pharmacy, Fudan University, Shanghai, China.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0072673PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3747110PMC
January 2015

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

Appl Microbiol Biotechnol 2014 Mar 6;98(6):2485-94. Epub 2013 Aug 6.

Department of Biosynthesis, School of Pharmacy, Fudan University, Shanghai, China.

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.
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http://dx.doi.org/10.1007/s00253-013-5118-0DOI Listing
March 2014

PEGylated human catalase elicits potent therapeutic effects on H1N1 influenza-induced pneumonia in mice.

Appl Microbiol Biotechnol 2013 Dec 26;97(23):10025-33. Epub 2013 Mar 26.

Department of Drug Biosynthesis, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai, 201203, China.

Therapeutic recombinant human catalase (rhCAT) can quench infection-induced reactive oxygen species (ROS), thereby alleviating the associated tissue damage. Although the intranasal route is efficient to deliver native rhCAT to the lung, the therapeutic effect is limited by rapid elimination from the blood. In this study, we modified rhCAT with the active polymer, polyethylene glycol monomethyl ether (PEG)-5000, and analyzed the pharmacokinetics of PEGylated rhCAT in mice. The high tetra-PEGylation ratio was about 60%, and PEGylation prolonged the half-life of rhCAT in serum (75 vs. 13.5 min for native rhCAT). The protective effects of PEG-rhCAT were investigated in a mouse model of influenza virus A (H1N1)-associated pneumonia. PEG-rhCAT was more effectively delivered than native rhCAT and was associated with higher survival ratio, less extensive lung injuries, reduced ROS levels, and lower viral replication. Collectively, these findings indicate that PEGylation can enhance the therapeutic efficacy of native rhCAT and suggest that PEGylated rhCAT may represent a novel complement therapy for H1N1 influenza-induced pneumonia.
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http://dx.doi.org/10.1007/s00253-013-4775-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079947PMC
December 2013

Inhibitory effect of apolipoprotein A-I on matrix metalloproteinase-2 expression in vivo and in vitro.

Acta Biochim Biophys Sin (Shanghai) 2013 Mar 7;45(3):194-202. Epub 2013 Jan 7.

Department of Pharmacology, Fudan University, Shanghai 201203, China.

In the present study, we investigated the effects of apolipoprotein A-I (apoA-I) on matrix metalloproteinase-2 (MMP-2) expression in vivo and in vitro. First, we detected the effects of apoA-I on aorta MMP-2, peroxisome proliferator-activated receptor α/γ (PPAR α/γ), cyclooxygenase-2 (COX-2), and nuclear factor kappa B (NF-κB) expressions in atherosclerotic rabbit models using immunohistochemical methods. The results showed that the expressions of MMP-2, COX-2, and NF-κB were decreased in aortas of atherosclerotic rabbits treated with apoA-I, while PPAR α/γ expression was increased. Then, we chose the important inflammation cells, macrophages to testify those effects in vitro. Macrophages were divided into six groups and treated with different concentrations of apoA-I, the mRNA expressions of MMP-2, PPAR α/γ, and COX-2 were then determined using reverse-transcription polymerase chain reaction, and protein expression of PPAR γ, NF-κB were detected by western blot analysis. The levels of MMP-2 and PPAR α in cultured supernatants were determined using enzyme-linked immunosorbent assays. Interestingly, the in vitro results were similar to the results of the in vivo study. After incubation with apoA-I for 24 h, the expressions of MMP-2, COX-2, and NF-κB were decreased, while PPAR α/γ expression was increased. In consideration of their particular roles in the process of making plaque stable in vivo and in vitro, we speculate that the inhibitory effect of apoA-I on MMP-2 expression may have a close relationship with the effects of apoA-I on PPAR α/γ, COX-2, and NF-κB expressions. Although further research is needed to clarify the underlying mechanisms of these effects, our findings provide a novel insight into the anti-atherosclerotic plaque rupture effects of apoA-I.
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http://dx.doi.org/10.1093/abbs/gms121DOI Listing
March 2013

Effects of external calcium on the biotransformation of ginsenoside Rb1 to ginsenoside Rd by Paecilomyces bainier 229-7.

World J Microbiol Biotechnol 2012 Mar 16;28(3):857-63. Epub 2011 Sep 16.

School of Pharmacy, Fudan University, 826 Zhangheng Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China.

Calcium is a known signalling molecule in eukaryotic cells and plays a central role in the regulation of many cellular processes. In the following study, we report on the effect of external calcium treatments on the biotransformation of ginsenoside Rb1 to ginsenoside Rd by Paecilomyces bainier 229-7. We observed that the intracellular calcium content of P. bainier 229-7 mycelia was increased in response to exposure to high external Ca(2+) concentrations. Both ginsenoside Rd biotransformation and β-glucosidase activity were both found to be dependent on the external calcium concentration. At an optimal Ca(2+) concentration of 45 mM, maximal ginsenoside Rd bioconversion rate of 92.44% was observed and maximal β-glucosidase activity of 0.1778 U was reached in a 72-h biotransformation. The Ca(2+) channel blocker Verapamil blocked the trans-membrane influx of calcium and decreased ginsenoside Rd biotransformatiom. In addition, β-glucosidase activity and ginsenoside Rd content decreased by 36.0 and 29.2% respectively after a 72-h incubation in the presence of 0.05 mM Calmodulin (CaM) antagonist Perphenazine. These results suggest that both Ca(2+) channels and CaM are involved in ginsenoside Rd biotransformation via regulation of β-glucosidase activity. This is the first report regarding the effects of calcium signal transduction on biotransformation and enzyme activity in fungi.
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http://dx.doi.org/10.1007/s11274-011-0882-4DOI Listing
March 2012

Efficient transposition of IS204-derived plasmids in Streptomyces coelicolor.

J Microbiol Methods 2012 Jan 3;88(1):67-72. Epub 2011 Nov 3.

School of Pharmacy, Fudan University, Yi Xue Yuan Road 138, Shanghai 200032, China.

In order to study functional gene expression in Streptomyces coelicolor, a mini-transposon encoding the apramycin resistance gene aac(3)IV within its inverted repeat (IR) boundaries was constructed based on IS204, which was previously identified in the genome of Nocardia asteroides YP21. The mini-transposon and IS204 transposase gene were then put on a kanamycin-resistant conjugative plasmid pDZY101 that can only replicate in Escherichia coli. After mating with S. coelicolor A3(2) M145, resistant colonies arose efficiently on both apramycin and kanamycin plates. Plasmid rescue indicated that entire plasmids were inserted into the M145 genome with cleavage at an inverted repeat junction formed by the right inverted repeat (IRR) and the last 18bp of the transposase gene, while the left inverted repeat (IRL) was untouched. Southern blot analysis of the mutants using an aac(3)IV gene probe showed that transposition of plasmid pDZY101 was genetically stable, with a single-copy insertion within the S. coelicolor M145 genome. Several mutagenesis libraries of S. coelicolor M145 were constructed using plasmid pDZY101 derivatives and the transposon insertion site was determined. The correlation between novel mutant phenotypes and previously uncharacterized genes was established and these transposon locations were widely scattered around the genome.
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http://dx.doi.org/10.1016/j.mimet.2011.10.018DOI Listing
January 2012

Recombinant high-density lipoprotein complex as a targeting system of nosiheptide to liver cells.

J Drug Target 2008 Jul;16(6):502-8

School of Pharmacy, Fu Dan University, Shanghai, P.R. China.

Nosiheptide is a lipophilic peptide of significant anti-hepatitis B virus (anti-HBV) activity in cell culture, but has poor distribution to liver in vivo. In this study, recombinant high-density lipoprotein (rHDL) complexes of nosiheptide were constructed to target this anti-HBV agent to hepatocytes. The optimized rHDL-nosiheptide complex had a high drug-loading efficiency (>80%) and a diameter smaller than 30 nm. The concentration of nosiheptide in an optimized rHDL-nosiheptide complex to achieve 50% virus inhibition (IC(50)) in HepG(2) 2.2.15 cells was 0.63 microg/ml, which was 40 times lower than the IC(50) of nosiheptide in control liposome (2.5 microg/ml) and 200 times lower than the IC(50) of the free nosiheptide (12.5 microg/ml). The complex targeted most of the administered nosiheptide to the liver within 30 min after i.v. injection to male Wistar rats. Together, this report provides early evidence that it is feasible to develop efficient, HDL-based drug delivery systems against HBV, utilizing apolipoprotein A-I as the targeting moiety.
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http://dx.doi.org/10.1080/10611860802200938 DOI Listing
July 2008

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

Antiviral Res 2008 Aug 7;79(2):114-20. Epub 2008 Apr 7.

Department of Biosynthetic Medicinal Chemistry, School of Pharmaceutical Sciences, Fudan University, Shanghai 200032, PR China.

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.
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http://dx.doi.org/10.1016/j.antiviral.2008.03.001DOI Listing
August 2008

Overexpression of yeast S-adenosylmethionine synthetase metK in Streptomyces actuosus leads to increased production of nosiheptide.

Appl Microbiol Biotechnol 2008 Apr 11;78(6):991-5. Epub 2008 Mar 11.

School of Pharmacy, Fudan University, Shanghai, China.

S-Adenosylmethionine (SAM) is synthesized via the metabolic reaction involving adenosine triphosphate and L-methionine that is catalyzed by the enzyme S-adenosyl-L-methionine synthetase (SAM-s) and encoded by the gene metK. In the present study, metK with the absence of introns from Saccharomyces cerevisiae was introduced into Streptomyces actuosus, a nosiheptide (Nsh) producer. Intracellular SAM levels were determined by high-pressure liquid chromatography. Through optimizing the nutrient content of the medium, it was shown that increased SAM production induced by the overexpression of SAM-s leads to an increase in the intracellular cysteine pool and overproduction of Nsh in S. actuosus. This investigation shows that increased SAM promotes the elevated production of the non-ribosomal thiopeptide Nsh in Streptomyces sp.
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http://dx.doi.org/10.1007/s00253-008-1394-5DOI Listing
April 2008

Overexpression, purification and characterization of a recombinant secretary catalase from Bacillus subtilis.

Biotechnol Lett 2008 Jan 18;30(1):181-6. Epub 2007 Sep 18.

Department of Drug Biosynthesis, School of Pharmacy, Fudan University, Shanghai 200032, PR China.

A recombinant Bacillus subtilis strain (KN25) was generated for the large-scale preparation of catalase. The B. subtilis katA gene encoding for catalase was cloned into the shuttle vector PRB374, downstream of the constitutively active vegII promoter, followed by transformation of the B. subtilis strain WB600 with the plasmid. The transformant strain, KN25 secretes high levels (3,500 U/ml) of catalase, which facilitates its purification. Three simple purification steps yielded nearly homogeneous catalase, with approximately 70% recovery. The purified recombinant catalase has a specific activity of 34,600 U/mg under optimal conditions, and is more resistant to acidic conditions than bovine liver catalase.
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http://dx.doi.org/10.1007/s10529-007-9510-7DOI Listing
January 2008

A novel class of potent influenza virus inhibitors: polysubstituted acylthiourea and its fused heterocycle derivatives.

Bioorg Med Chem Lett 2006 Jan 10;16(1):162-6. Epub 2005 Oct 10.

Bio-X Life Science Research Center, Department of Life Science and Technology, Shanghai Jiaotong University, Shanghai 200030, China.

A series of polysubstituted and fused heterocycle derivatives of acylthiourea was prepared and the biological activity against influenza virus was evaluated. Of the analogues that demonstrated IC(50)s<0.1 microM, acylthiourea derivatives 16 and 50 were further investigated as candidates with the most potential for future development. The SAR of these compounds are discussed and they represent a novel class of highly potent and selective inhibitors of influenza virus.
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http://dx.doi.org/10.1016/j.bmcl.2005.09.033DOI Listing
January 2006

Expression, purification, and characterization of His-tagged penicillin G acylase from Kluyvera citrophila in Escherichia coli.

Protein Expr Purif 2004 Nov;38(1):24-8

Department of Drug Biosynthesis, School of Pharmacy, Fudan University, Shanghai 200032, China.

The DNA fragment encoding Kluyvera citrophila penicillin G acylase (KcPGA) was amplified and cloned into the vector pET28b to obtain a C-terminus His-tagged fusion expression plasmid. The fusion protein KcPGA was successfully overexpressed in Escherichia coli BL21(DE3). The optimal induction concentration of isopropylthio-beta-D-galactoside (IPTG) was found to be 5 microM. The fusion protein was purified in a single step by Ni-IDA affinity chromatograph to a specific activity of 35.3U/mg protein with a final yield of 89% representing a 23-fold purification. The data presented here suggest that the purified fusion protein is stable with respect to pH and temperature. The optimal pH and temperature of recombinant KcPGA are 8.5 and 55 degrees C, respectively. The Km and Vmax are 17.6 microM and 23.8 U/mg, respectively. Therefore, the high yield and high specific activity of recombinant KcPGA produced in E. coli, together with other kinetic parameters, represent an excellent basis for further development of recombinant KcPGA as an immobilized biocatalyst for industrial applications.
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http://dx.doi.org/10.1016/j.pep.2004.05.015DOI Listing
November 2004