Jichao Cao

Anti-metastatic and anti-angiogenic activities of sulfated polysaccharide of Sepiella maindroni ink.

A previous study demonstrated that SIP-SII, a sulfated Sepiella maindroni ink polysaccharide, suppressed the invasion and migration of cancer cells via the inhibition of the proteolytic activity of matrix metalloproteinase-2 (MMP-2). Therefore, this study investigated the anti-metastatic effect of SIP-SII in vivo. SIP-SII (15 and 30 mg/kg d) markedly decreased B16F10 pulmonary metastasis in mice models by 85.9% and 88.0%, respectively. Immunohistochemistry showed that SIP-SII decreased the expression of the intercellular adhesion molecule 1 (ICAM-1) and basic fibroblast growth factor (bFGF) in lung metastasis nodules. In addition, SIP-SII inhibited neovascularization in chick chorioallantoic membrane assay at 0.08-2 mg/mL. In the in vitro experiments, SIP-SII (0.8-500 μg/mL) significantly decreased the protein and mRNA expression of ICAM-1 and bFGF in SKOV3 and EA.hy926 cells, respectively. These results suggested that SIP-SII might suppress melanoma metastasis via the inhibition of the tumor adhesion mediated by ICAM-1 and the angiogenesis mediated by bFGF, as well as resulting in depression of the invasion and migration of carcinoma cells.

Characterization and stability investigation of Cu,Zn-superoxide dismutase covalently modified by low molecular weight heparin

Cu,Zn-superoxide dismutase (SOD) was chemically modified with low molecular weight heparin (LMWH). To characterize the conjugate, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (native PAGE) with protein staining and polysaccharide staining were employed. The stabilities of the modified enzyme to heat, acid, alkali, and trypsin treatment were also investigated. SDS-PAGE of the conjugate presented two major bands, and native PAGE of the conjugate showed similar banding position with protein staining and polysaccharide staining, which was different from that of the unmodified SOD and LMWH/SOD mixture. Moreover, the conjugate migrated faster with increasing extent of the modification. Enhanced heat stability, acid resistance, alkali resistance, and anti-trypsin stability of the modified enzyme were observed compared with those of the unmodified enzyme. Results of the study suggest that covalent linkage in LMWH-SOD can be effectively characterized by electrophoretic techniques and the chemical modification of SOD with LMWH can enhance the stabilities of the enzyme. In addition, native PAGE with protein staining can be used to evaluate the extent of the modification.

Efficacy of oral colon-specific delivery capsule of low-molecular-weight heparin on ulcerative colitis

Low-molecular-weight heparin has the potential for the treatment of ulcerative colitis, and targeted drug delivery to the colon is important for topical treatment of this disease, so low-molecular-weight heparin oral colon-specific delivery capsule was prepared, and the in vitro and in vivo drug release behavior was investigated. The macroscopical and histological scoring systems, wet colon mass index and myeloperoxidase activity were assessed to evaluate the efficacy of the capsule after administered orally to experimental colitis mice. Serum levels, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and a link factor of blood coagulation and inflammation factor Xa (FXa) were assayed by enzyme-linked immunosorbent assay. The expression of Musashi-1 (as an intestinal stem cell marker) in the colons was assessed by immunohistochemical analysis. The in vitro and in vivo drug release studies clearly indicated that the specific coated capsules were capable of protecting low-molecular-weight heparin from releasing in stomach and small intestine, while specifically delivering at colon. The oral colon-specific delivery capsule of low-molecular-weight heparin could attenuate macroscopic and histological features of colitis. The results showed that low-molecular-weight heparin oral colon-specific delivery capsule significantly decreased the serum levels of TNF-α, IL-6 as well as FXa, while increased the expression of Musashi-1 in colon compared with acetic acid-induced ulcerative colitis model group. The results showed that low-molecular-weight heparin oral colon-specific delivery capsule had the potential for treatment of inflammatory bowel disease.

Attenuation effects of heparin-superoxide dismutase conjugate on bleomycin-induced lung fibrosis in vivo and radiation-induced inflammatory cytokine expression in vitro.

In this study, the effects of heparin-superoxide dismutase conjugate (heparin-SOD) on bleomycin-induced pulmonary fibrosis in vivo and on inflammatory cytokine expression in vitro were evaluated. To investigate the effects of heparin-SOD on pulmonary fibrosis, heparin-SOD was administered to bleomycin (BLM)-treated mice by intraperitoneal injection once a day and the hydroxyproline content in lung was determined per 7days. The degree of fibrosis was assessed quantitatively using histopathologic features. The results showed that heparin-SOD inhibited BLM-induced lung fibrotic lesions as reflected by the decrease of lung hydroxyproline content and lung fibrosis grade 28days after BLM instillation. The in vitro effects on the cytokine level expressed by irradiated 3T3 fibroblasts showed that heparin-SOD significantly lowered the levels of transforming growth factor-beta1 and interleukin-1beta. These results strongly demonstrated that heparin-SOD might be useful in the prevention and treatment of pulmonary fibrosis.

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.

Expression of thymosin α1-thymopentin fusion peptide in Pichia pastoris and its characterization

Thymopentin plays an important role in improving imbalanced immune systems of patients, however, it has a limited half-life in plasma. To get more stable and active thymopentin analogs, a fusion thymosin α1-thymopentin (Tα1-TP5) gene was synthesized and cloned into vector pGAPZαA. Tα1-TP5 fusion peptide was expressed in pichia pastoris and purified by metal chelating chromatography and gel filtration chromatography. The circular dichroism spectra (CD) indicated that the secondary structure of Tα1-TP5 fusion peptide is dominated by a-helix and random coil. In vitro analysis showed that the plasma half-life of Tα1-TP5 fusion peptide is 140 ± 14 min, which is longer than that of TP5 (5.6±0.7 min) and Tα1 (127±11 min). The in vitro activity assay presented that Tα1-TP5 fusion peptide has greater activity in promoting proliferation of Kunming mouse splenocytes, and in vivo experiment it showed better activity in promoting the phagocytosis of macrophages and secretion of IL-2 than both Tα1 and TP5. Our findings suggest that Tα1-TP5 fusion peptide might be a potential therapeutic agent.

A novel conjugate of low-molecular-weight heparin and Cu,Zn-superoxide dismutase: study on its mechanism in preventing brain reperfusion injury after ischemia in gerbils.

Low-molecular-weight heparin (LMWH) and Cu,Zn-superoxide dismutase (SOD) were extensively investigated on preventing brain reperfusion injury after ischemia (BRII) in the past few years and both exhibited some advantages as well as limits in practice. To explore whether chemical modification for LMWH and SOD can lead to improved bioactivity,in our present study, we examined the efficacy of LMWH conjugated SOD (LMWH−SOD) in the model of BRII gerbils. Ischemia/reperfusion was performed for 5 min by clamping the bilateral common carotid arteries of gerbils. LMWH−SOD, SOD and the mixture of LMWH and SOD (LMWH+SOD) were administered intravenously to corresponding animals just before ischemia. 24 h after reperfusion, serum malondialdehyde (MDA) content and SOD activity were measured, the expression of intercellular adhesion molecule-1 (ICAM-1) was examined by immunohistochemistry, and the brain sections were processed for Nissl staining and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling.The results showed that LMWH−SOD significantly lowered MDA content (P<0.001, versus SOD and LMWH+SOD) and elevated SOD activity (P<0.05, versus SOD and LMWH+SOD) in the serum of BRII gerbils. Immunohistochemical results indicated ICAM-1 positive staining was lighter, pyramidal cells of hippocampal CA1 region were more regular and the changes in cell edema were minor, and apoptosis of hippocampal cells was milder in LMWH−SOD treated animals than in SOD or LMWH+SOD treated animals, untreated BRII animals and sham-operated animals. The results suggest that the novel LMWH−SOD conjugate can inhibit upregulation of ICAM-1 and prevent neuronal cell apoptosis in BRII gerbils, and the LMWH−SOD conjugate has better anti-inflammatory and neuroprotective effects in BRII than native SOD and the mixture of LMWH and SOD.

Stability Profiles and Therapeutic Effect of Cu/Zn Superoxide Dismutase Chemically Coupled to O-Quaternary Chitosan Derivatives against Dextran Sodium Sulfate-Induced Colitis

Superoxide dismutase (SOD) has attracted considerable attention on treatment of reactive oxygen species (ROS)-related disorders. We previously conjugated Cu/Zn SOD to O-quaternary chitosan derivatives (O-HTCC) to yield a polymer–enzyme conjugate O-HTCC-SOD that demonstrated superior therapeutic effect to native SOD. The present study demonstrated that O-HTCC-SOD had wider pH activity range, better thermal stability, excellent long-term stability for storage, as well as unique reinstatement of activity exposure to proteolytic degradation that was helpful for longer half-life in vivo. O-HTCC-SOD exerted significant anti-inflammatory effects on lipopolysaccharides (LPS)-stimulated mouse peritoneal macrophages by down-regulating production of pro-inflammatory cytokines and intracellular ROS. O-HTCC-SOD significantly attenuated dextran sodium (DSS)-induced colitis in mice as observed by the colitis severity, neutrophil infiltration and histopathological damage, whereas native SOD failed to do so. In conclusion, conjugation of O-HTCC conferred SOD with better stability and enhanced therapeutic potential, offering a promising option in treatment of inflammatory bowel disease.