Haining Tan

Hematopoietic effects and mechanisms of Fufang e׳jiao jiang on radiotherapy and chemotherapy-induced myelosuppressed mice.

ETHNOPHARMACOLOGICAL RELEVANCE Fufang e׳jiao jiang (FEJ), which has been widely used in clinic to replenish qi (vital energy) and nourish blood, is a famous traditional Chinese medicine formula made up of Colla corii asini (donkey-hide gelatin prepared by stewing and concentrating from the hide of Equus asinus Linnaeus.), Radix codonopsis pilosulae (the root of Codonopsis pilosula (Franch.) Nannf.), Radix ginseng rubra (the steamed and dried root of Panax ginseng C.A. Mey.), Fructus crataegi (the fruit of Crataegus pinnatifida Bunge) and Radix rehmanniae preparata (the steamed and sun dried tuber of Rehmannia glutinosa (Gaertn.) Libosch. ex Fisch. & C.A. Mey.). The present study aimed to investigate the hematopoietic effects of FEJ on myelosuppressed mice induced by radiotherapy and chemotherapy systematically and to explore the underlying hematopoietic regulation mechanisms. METHODS The myelosuppressed mouse model was induced by (60)Co radiation, cyclophosphamide and chloramphenicol. FEJ was then administered by i.g. at the dosages of 5, 10, or 20 mL/kg·d for 10d. The numbers of blood cells from peripheral blood and bone marrow nucleated cells (BMNC) were counted. Body weight and the thymus and spleen indices were also measured. The numbers of hemopoietic progenitor cells and colony-forming unit-fibroblast (CFU-F) were measured in vitro. The ratio of hematopoietic stem cells (HSC) in BMNC, cell cycle and apoptosis of BMNC were determined by flow cytometry. The histology of femoral bone was examined by H&E staining. The levels of transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), erythropoietin (EPO), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-6 (IL-6) in serum were measured by ELISA. IL-1β, IL-3, IL-6 mRNA levels in spleen were detected by real-time quantitative PCR (RT-qPCR). In addition, bone marrow stromal cells (BMSC) were cultured in vitro followed by treatment with different doses of FEJ (2.5, 5, 10 μL/mL) for 48 h. Then the levels of cytokines (IL-6, SCF, GM-CSF) in the conditioned media and their mRNA levels in BMSC were determined by ELISA and RT-qPCR, respectively. RESULTS FEJ could significantly increase the numbers of peripheral blood cells and BMNC, and reverse the loss of body weight and the atrophy of thymus and spleen in a dose-dependent manner. The quantities of hemopoietic progenitor cells and CFU-F in bone marrow were also significantly increased in a dose-dependent manner after FEJ administration. A high-dose FEJ of 20 mL/kg·d could significantly increase the ratio of HSC in BMNC, promote bone marrow cells entering the proliferative cycle phase (S+G2/M) and prevent cells from proceeding to the apoptotic phase. FEJ could also improve the femoral bone marrow morphology. Furthermore, FEJ could increase the levels of GM-CSF and IL-3 and reduce the level of TGF-β in serum, and enhance the expressions of IL-1β and IL-3 mRNA in spleen. Lastly, the levels of cytokines (IL-6, SCF, GM-CSF) in the conditioned media and their mRNA levels in BMSC were elevated after treatment with FEJ. CONCLUSIONS FEJ was clearly confirmed to promote the recovery of bone marrow hemopoietic function in a myelosuppressed mouse model, which may be attributed to (i) improving bone marrow hematopoietic microenvironment; (ii) facilitating the cell proliferation and preventing BMNC from apoptosis; (iii) stimulating the expressions of IL-1β, IL-3, IL-6, SCF and GM-CSF and inhibiting the expression of TGF-β.

Research advances of endostatin and its short internal fragments.

Endostatin, the C-terminal fragment of collagen XVIII, is a potent angiogenesis inhibitor. At present, there are a large number of research papers on endostatin. However, the action mechanism of endostatin is still a matter of ongoing discussion. The objective of this review is to elucidate its origin and elementary structure, and to discuss its structure basis of activity and action mechanisms based on the latest research. Furthermore, some published studies reporting the antiangiogenic effects of endostatin-derived peptides were also reviewed. It is proposed that the amino acid sequence of endostatin contains both angiosuppressive and angiostimulatory domains. Short endostatin fragments may be exploited as a new angiogenesis inhibitor for therapeutic applications, in substitution of the full length endostatin. These studies on endostatin fragments also shed light on our understanding of the molecular action mechanisms of endostatin.

Tat PTD-endostatin: A novel anti-angiogenesis protein with ocular barrier permeability via eye-drops.

BACKGROUND Endostatin, a specific inhibitor of endothelial cell proliferation and angiogenesis, has been proved to have effects on ocular neovascular diseases by intraocular injection. In order to increase its permeability to ocular barriers and make it effective on fundus oculi angiogenesis diseases via non-invasive administration (eye drops), endostatin was fused to Tat PTD via a genetic engineering method. METHODS Most of the Tat PTD- endostatin was expressed as inclusion bodies in Escherichia coli, so pure and active Tat PTD-endostatin was prepared by a series of operations, including inclusion body denaturation, refolding and chromatography. The anti-angiogenesis activity of Tat PTD-endostatin was investigated by cell proliferation experiments and chick embryo chorioallantoic membrane assay. In addition, its translocating ability and concrete entry mechanism into cells were also investigated by fluorescence microscope and flow cytometry. The penetrating ability to ocular barriers was also studied by immunohistochemistry. A mouse choroidal neovascularization model was established to investigate the pharmacodynamics of Tat PTD-endostatin. RESULTS The obtained Tat PTD-endostatin had excellent anti-angiogenesis activity and was superior to Es in cellular translocating. Macropinocytosis may be the dominant route of entry of Tat PTD-endostatin into cells. Tat PTD-endostatin could cross ocular barriers and arrive at the retina after eye-drop administration. In addition, it displayed inhibitory effects on choroidal neovascularization via eye drops. CONCLUSIONS Tat PTD-endostatin possessed excellent ocular penetrating ability and anti-angiogenesis effects. GENERAL SIGNIFICANCE Tat PTD is a promising ocular delivery tool, and Tat PTD-endostatin is a potential drug for curing fundus oculi angiogenesis diseases.

Effect and mechanisms of curdlan sulfate on inhibiting HBV infection and acting as an HB vaccine adjuvant

In this study, the effect and mechanisms of curdlan sulfate (CS3) on hepatitis B virus (HBV) infection and promoting immune response of the mice immunized with recombinant hepatitis B surface protein (HBsAg) were investigated. The results showed that CS3 could inhibit HBV infection of HepG2 and HepaRG cells, especially the process of HBV particle binding to the cell surfaces. The surface plasmon response (SPR) technology indicated that CS3 could bind with recombinant HBsAg and the binding ability depended on the content of sulfate groups on the polysaccharide chains. Co-administration of CS3 to BALB/c mice immunized with HBsAg significantly enhanced the influx of macrophages and dendritic cells in spleen, increased antigen-specific CD4+ and CD8+ cell numbers, and promoted splenocyte proliferation. The titer of HBsAg-specific antibodies was also augmented by use of CS3 as a vaccine adjuvant. The higher expression of interferon (IFN)-γ, lower expression of interleukin (IL)-4, and higher IgG2a/IgG1 ratio within the anti-HBsAg antibodies in mice immunized with HBsAg plus CS3 than those in mice receiving HBsAg alone indicated that CS3 induced a shift toward a Th1-biased immune response. These results presented that CS3 could be developed as an immunotherapy agent or vaccine adjuvant for HBV infection treatment or prevention.

Characterization and Secondary Structure Analysis of Endostatin Covalently Modified by Polyethylene Glycol and Low Molecular Weight Heparin

Endostatin (ES), as an angiogenesis inhibitor, has been approved by the State Food and Drug Administration (SFDA) in China for the treatment of patients with non-small-cell lung cancer. However, as a protein drug, there are a lot of obstacles on its clinical application, such as need of high dose to maintain its efficacy, expensive and poor stability, etc and limits its clinical use. In order to overcome these shortcomings, we chemically modified ES by polyethylene glycol and low molecular weight heparin (LMWH), respectively. The changes of the secondary structure of the modified products were studied by Fourier transform infrared spectroscopy and Circular dichroism spectra to obtain better ES derivatives. Our study demonstrated that the modified products have a better heat tolerance than ES towards. The result of secondary structure analysis suggests the percentage of beta-turn in whole protein is an important factor on the activity and heat stability and ES modified by LMWH can maintain higher activity and its secondary structure.

Pharmacokinetic analysis of in vivo disposition of heparin–superoxide dismutase

To improve the half-life and tissue targeting of SOD to suppress reactive oxygen species (ROS)-mediated injury, chemically modified derivative of superoxide dismutase (SOD) with heparin, anionized SOD (Hep-SOD), was designed. In this study, the pharmacokinetics of Hep-SOD had been studied. This study aimed to investigate the pharmacokinetics, tissue distribution and cell targeting. ¹²⁵I-radiolabeled Hep-SOD conjugate was administered to healthy mice by intravenous (i.v.) bolus injection. Compared with native SOD, the half-life of Hep-SOD conjugate, including t(₁/₂α) and of t(₁/₂β), was lengthen and area under the plasma concentration versus time curve (AUC) of Hep-SOD was increased. The study showed that both native SOD and Hep-SOD was rapidly and widely distributed in the livers, kidneys, spleens, hearts and lungs. Furthermore, compared with Hep-SOD, radioactivity of native SOD decreased more sharply over time in most tissues. Compared with native SOD, higher amount of Hep-SOD radioactivity was found in the livers. Since livers are not the known target of ¹²⁵I, the most possible reason is that Hep-SOD binds to its specific targets in the livers.

Synthesis of unsymmetrical 3,6-branched Man5 oligosaccharide: a comparison between one-pot sequential glycosylation and stepwise synthesis.

An expeditious three-component, one-pot sequential glycosylation protocol has been developed for the preparation of 3,6-branched unsymmetrical mannopentaose (Man5), employing a mannose trisaccharide donor, a mannose monosaccharide donor and a mannose monosaccharide acceptor. The high efficiency of this one-pot procedure was demonstrated by comparison study with a stepwise synthesis using the same three building blocks.

Study on glyco-modification of endostatin-derived synthetic peptide endostatin2 (ES2) by soluble chitooligosaccharide

Soluble O-(2-hydroxyl)propyl-3-trimethyl ammonium chitooligosaccharide chloride (HTCOSC) was covalently conjugated to the 11-amino-acid peptide derived from amino terminus of endostatin (endostatin2, ES2, IVRRADRAAVP) to overcome its poor stability, low cell affinity and instable activity. Nuclear magnetic resonance spectroscopy and mass spectrometry was used to study the structure and molecular weight information. The anti-angiogenic activities were evaluated using cell counting Kit-8 assay, flow cytometry assay, wounding migration assay, transwell migration assay, chicken chorioallantoic membrane (CAM) assay and zebra fish angiogenesis assay. In contrast with ES2, the novel carbohydrate-polymer HTCOSC-ES2 displayed improved heat stability, higher cell affinity, better inhibition on endothelial cell proliferation, tube formation, 2-dimensional and 3-dimensional migration in vitro. According to the evaluation in CAM and zebra fish, HTCOSC-ES2 also displayed better anti-angiogenic activity than ES2 in vivo. These results indicate that HTCOSC has good properties as potential candidate for protein/peptide modifier and HTCOSC-ES2 has good potential in angiogenesis related diseases treatment.

Hyaluronic acid-endostatin2-alft1 (HA-ES2-AF) nanoparticle-like conjugate for the target treatment of diseases

ABSTRACT Anti‐flt1 peptide (GNQWFI, AF) specifically binds to Vascular Endothelial Growth Factor Receptor 1 (VEGFR1), thereby inhibiting the interaction of VEGFR1 with a series of ligands. ES2 (IVRRADRAAVP) can effectively inhibit the proliferation and invasion of endothelial cells and play a key role in anti‐angiogenesis. AF and ES2 peptides differ in their activity. To better exploit the advantages of both, we designed a new peptide called ES2‐AF (IVRRADRAAVPGGGGGGNQWFI). Hyaluronic acid (HA) is widely used in the pharmaceutical industry because of its biodegradable and high load performance. The HA‐specific cell surface receptor CD44 was highly expressed in the tumour site during the anti‐tumour study. Therefore, we used HA as a modifier to chemically modify ES2‐AF; it was expected that the modified compound would have preferable solubility, stronger targeting, longer half‐life, and better anti‐angiogenesis effects in vivo. In this study, the anti‐proliferative, anti‐migration and targeting activities of HA‐ES2‐AF in vitro were studied by MTT, ELISA, transwell and SPR assays. Meanwhile, the anti‐neovascularization activity of HA‐ES2‐AF in vivo was studied by CAM assay, and the targeting of HA‐ES2‐AF to tumour tissue was studied by bioimaging techniques. Finally, we also studied the half‐life of HA‐ES2‐AF in vivo. In short, the bioactivity of the new peptide ES2‐AF was enhanced to a certain extent, and ES2‐AF modified by HA had higher anti‐neovascularization activity in vitro and in vivo, had stronger targeting to tumour tissue, and had a significantly prolonged half‐life in vivo. These results laid the foundation for its further development into targeting anti‐tumour drugs. Graphical abstract Figure. No Caption available.