Ganggang Shi

A poly(L-lysine)-based hydrophilic star block co-polymer as a protein nanocarrier with facile encapsulation and pH-responsive release.

A hydrophilic star block co-polymer was synthesized, characterized, and evaluated as a protein nanocarrier. The star block co-polymer was composed of a hyperbranched polyethylenimine (PEI) core, a poly(L-lysine) (PLL) inner shell, and a poly(ethylene glycol) (PEG) outer shell. The model protein insulin can be rapidly and efficiently encapsulated by the synthesized polymer in aqueous phosphate buffer at physiological pH. Complexation between PEI-PLL-b-PEG and insulin was investigated using native polyacrylamide gel electrophoresis. The uptake of enhanced green fluorescent protein into Ad293 cells mediated by PEI-PLL-b-PEG was also investigated. The encapsulated insulin demonstrated sustained release at physiological pH and showed accelerated release when the pH was decreased. The insulin released from the star block co-polymer retained its chemical integrity and immunogenicity.

Self-assembled nanoparticles of glycyrrhetic acid-modified pullulan as a novel carrier of curcumin.

Glycyrrhetic acid (GA)-modified pullulan nanoparticles (GAP NPs) were synthesized as a novel carrier of curcumin (CUR) with a degree of substitution (DS) of GA moieties within the range of 1.2–6.2 groups per hundred glucose units. In the present study, we investigated the physicochemical characteristics, release behavior, in vitro cytotoxicity and cellular uptake of the particles. Self-assembled GAP NPs with spherical shapes could readily improve the water solubility and stability of CUR. The CUR release was sustained and pH-dependent. The cellular uptake of CUR-GAP NPs was confirmed by green fluorescence in the cells. An MTT study showed CUR-GAP NPs with higher cytotoxicity in HepG2 cells than free CUR, but GAP NPs had no significant cytotoxicity. GAP is thus an excellent carrier for the solubilization, stabilization, and controlled delivery of CUR.

The protective effects of N-n-butyl haloperidol iodide on myocardial ischemia-reperfusion injury in rats by inhibiting Egr-1 overexpression.

Aims: Our previous studies have shown that N-n-butyl haloperidol iodide (F2) can antagonize myocardial ischemia/reperfusion (I/R) injury by blocking intracellular Ca2+ overload. The present study is to test the hypothesis that the protective effects of F2 on myocardial I/R injury is mediated by downregulating Egr-1 expression. Methods: The Sprague–Dawley rat myocardial I/R model and cardiomyocyte hypoxia/reoxygenation (H/R) model were established. With antisense Egr-1 oligodeoxyribonucleotide (ODN), the relationship between Egr-1 expression and myocardial I/R injury was investigated. Hemodynamic parameters, myeloperoxidase (MPO), cardiac troponin I (cTnI) and tumor necrosis factor-α (TNF-α) were measured to assess the degree of injury and inflammation of myocardial tissues and cells. Egr-1 mRNA and protein expressions were examined by Northern-blot and Western-blot analyses. Results: Treatment with antisense Egr-1 ODN significantly reduced Egr-1 protein expression and attenuated injury of myocardial tissues and cells. Meanwhile, treatment with F2 significantly inhibited the overexpression of Egr-1 mRNA and protein in myocardial tissues and cells. Consistent with downregulation of Egr-1 expression by F2, inflammation and other damages were significantly relieved evidenced by the amelioration of hemodynamics, the reduction in myocardial MPO activity as well as the decrease in leakage of cTnI and release of TNF-α from cardiomyocyte. Conclusions: These results suggested that the overexpression of Egr-1 was causative in myocardial I/R or H/R injury, and F2 could protect myocardial tissues and cells from I/R or H/R injury, which was largely due to the inhibition of Egr-1 overexpresssion.

Anti-Inflammatory Activity of N-Butanol Extract from Ipomoea stolonifera In Vivo and In Vitro

Ipomoea stolonifera (I. stolonifera) has been used for the treatment of inflammatory diseases including rheumatism and rheumatoid arthritis in Chinese traditional medicine. However, the anti-inflammatory activity of I. stolonifera has not been elucidated. For this reason, the anti-inflammatory activity of n-butanol extract of I. stolonifera (BE-IS) was evaluated in vivo by using acute models (croton oil-induced mouse ear edema, carrageenan-induced rat paw edema, and carrageenan-induced rat pleurisy) and chronic models (cotton pellet-induced rat granuloma, and complete Freund’s adjuvant (CFA)-induced rat arthritis). Results indicated that oral administration of BE-IS significantly attenuated croton oil-induced ear edema, decreased carrageenan-induced paw edema, reduced carrageenan-induced exudates and cellular migration, inhibited cotton pellet-induced granuloma formation and improved CFA-induced arthritis. Preliminary mechanism studies demonstrated that BE-IS decreased the levels of myeloperoxidase (MPO) and malondialdehyde (MDA), increased the activity of anti-oxidant enzyme superoxide dismutase (SOD) in vivo, and reduced the production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 in lipopolysaccharide-activated RAW264.7 macrophages in vitro. Results obtained in vivo and in vitro demonstrate that BE-IS has considerable anti-inflammatory potential, which provided experimental evidences for the traditional application of Ipomoea stolonifera in inflammatory diseases.

The Protective Effect of Egr-1 Antisense Oligodeoxyribonucleotide on Myocardial Injury Induced by Ischemia-reperfusion and Hypoxia- reoxygenation

Aims: Our previous studies have shown that myocardial ischemia-reperfusion (I/R) injury is related closely with early growth response (Egr)-1 overexpression. The present study is to confirm thoroughly the effects of Egr-1 on the occurrance and development of myocardial I/R injury. Methods: The Sprague-Dawley rat myocardial I/R model and cultured cardiomyocyte hypoxia-reoxygenation (H/R) model were established. The synthesized Egr-1 antisense oligodeoxyribonucleotide (AS-ODN) was transfected into myocardial tissues and cells. Hemodynamic parameters, myeloperoxidase (MPO), cardiac troponin I (cTnI), tumor necrosis factor-α (TNF-α), morphology, spontaneous beat and cell viability were measured to assess the degree of injury and inflammation of myocardial tissues and cells. Results: In vivo, Egr-1 AS-ODN significantly attenuated injury and inflammation of myocardial tissues caused by I/R evidenced by the amelioration of hemodynamics and the reduction in MPO activity. In vitro, Egr-1 AS-ODN significantly relieved injury and inflammation of cultured cardiomyocyte caused by H/R evidenced by the improvement in morphology, structure and beat as well as the decrease in leakage of cTnI and release of TNF-α from cultured cardiomyocyte. Conclusions: These data suggest that Egr-1 plays a vital role in the pathogenesis of myocardial I/R injury and Egr-1 AS-ODN could protect the myocardium from I/R injury.

Effect of N -n-butyl haloperidol iodide on ROS/JNK/Egr-1 signaling in H9c2 cells after hypoxia/reoxygenation

Reactive oxygen species (ROS)-induced oxidative stress in cells is an important pathophysiological process during myocardial ischemia/reperfusion (I/R) injury, and the transcription factor Egr-1 is a master switch for various damage pathways during reperfusion injury. An in vitro model of myocardial I/R injury and H9c2 cardiomyoblast cells hypoxia/reoxygenation (H/R) was used to assess whether there is abnormal intracellular ROS/JNK/Egr-1 signaling. We also assessed whether N-n-butyl haloperidol (F2), which exerts protective effects during myocardial I/R injury, can modulate this pathway. H/R induced ROS generation, JNK activation, and increased the expression of Egr-1 protein in H9c2 cells. The ROS scavengers edaravone (EDA) and N-acetyl-L-cysteine (NAC) reduced ROS level, downregulated JNK activation, and Egr-1 expression in H9c2 cells after H/R. The JNK inhibitor SP600125 inhibited Egr-1 overexpression in H9c2 cells caused by H/R. F2 could downregulate H/R-induced ROS level, JNK activation, and Egr-1 expression in H9c2 cells in a dose-dependent manner. The ROS donor hypoxanthine-xanthine oxidase (XO/HX) and the JNK activator ANISO antagonized the effects of F2. Therefore, H/R activates ROS/Egr-1 signaling pathway in H9c2 cells, and JNK activation plays an important role in this pathway. F2 regulates H/R-induced ROS/JNK/Egr-1 signaling, which might be an important mechanism by which it antagonizes myocardial I/R injury.

Purification, partial characterization and bioactivity of sulfated polysaccharides from Grateloupia livida

Purification, preliminary characterization and bioactivity of polysaccharides from Grateloupia livida (GL) were investigated. Three water-soluble sulfated polysaccharide fractions (GLP-1, GLP-2 and GLP-3) were isolated and purified from the edible and medicinal red seaweed, Grateloupia livida (Harv.) Yamada by DEAE Sepharose CL-6B and Sephadex G-100 column chromatography, and chemical characterization was performed by HPGPC, GC-MS, FT-IR and SEM. In addition, anticoagulant activities were determined by activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) using normal human plasma in vitro. The antioxidant activities against DPPH and ABTS+ radicals were evaluated and compared. The molecular weights of GLP-1, GLP-2 and GLP-3 were 39.5, 60.4 and 3.36kDa, respectively. Monosaccharide analysis revealed that three polysaccharide fractions were homopolysaccharides and comprised of galactose only. Anticoagulant assays indicated that crude GLP, and purified GLP-1 and GLP-2 effectively prolonged APTT and TT, but not PT. All polysaccharide fractions exhibited significant in vitro antioxidant activities in a dose-dependent manner. GLP-2 showed consistently better anticoagulant and antioxidant activities compared with GLP, GLP-1 and GLP-3. These results demonstrate that sulfated polysaccharides isolated from Grateloupia livida can serve as readily available alternative natural sources of anticoagulant and antioxidant agents.

Effect of Pullulan Nanoparticle Surface Charges on HSA Complexation and Drug Release Behavior of HSA-Bound Nanoparticles

Nanoparticle (NP) compositions such as hydrophobicity and surface charge are vital to determine the presence and amount of human serum albumin (HSA) binding. The HSA binding influences drug release, biocompatibility, biodistribution, and intercellular trafficking of nanoparticles (NPs). Here, we prepared 2 kinds of nanomaterials to investigate HSA binding and evaluated drug release of HSA-bound NPs. Polysaccharides (pullulan) carboxyethylated to provide ionic derivatives were then conjugated to cholesterol groups to obtain cholesterol-modified carboxyethyl pullulan (CHCP). Cholesterol-modified pullulan (CHP) conjugate was synthesized with a similar degree of substitution of cholesterol moiety to CHCP. CHCP formed self-aggregated NPs in aqueous solution with a spherical structure and zeta potential of −19.9±0.23 mV, in contrast to −1.21±0.12 mV of CHP NPs. NPs could quench albumin fluorescence intensity with maximum emission intensity gradually decreasing up to a plateau at 9 to 12 h. Binding constants were 1.12×105 M−1 and 0.70×105 M−1 to CHP and CHCP, respectively, as determined by Stern-Volmer analysis. The complexation between HSA and NPs was a gradual process driven by hydrophobic force and inhibited by NP surface charge and shell-core structure. HSA conformation was altered by NPs with reduction of α-helical content, depending on interaction time and particle surface charges. These NPs could represent a sustained release carrier for mitoxantrone in vitro, and the bound HSA assisted in enhancing sustained drug release.

Egr-1, the Potential Target of Calcium Channel Blockers in Cardioprotection with Ischemia/Reperfusion Injury in Rats

Aims: In this study, we tested whether Egr-1 is a potential target of calcium channel blockers in cardioprotection with I/R injury. Methods: We treated rats in vivo I/R and rat cultured cardiomyocytes in vitro hypoxia/reoxygenation (H/R) models with three types of classical calcium channel blockers (verapamil, diltiazem and nifedipine). Activity of creatine kinase (CK), lactate dehydrogenase (LDH), myeloperoxidase (MPO) superoxide dismutase (SOD) and level of malondialdehyde (MDA) in plasma and culture medium were measured to assess the degree of injury and inflammation of myocardial tissues and cells. Egr-1 mRNA and protein expressions were examined by RT-PCR and Western-blot analyses. Results: Calcium channel blockers (verapamil, diltiazem and nifedipine) significantly attenuated myocardial injury, as shown by reduced release of CK and LDH, preserved SOD activity and decreased MDA production and MPO activity. Concomitant with cardioprotection by calcium channel blockers, the mRNA and protein expression of Egr-1 increased with I/R and H/R injury was significantly reduced in myocardial tissue and cultured cardiomyocytes. Conclusions: These results suggested that the cardioprotective effects of calcium channel blockers with I/R or H/R injury might be mediated by downregulating Egr-1 expression. Egr-1 might be the potential target of calcium channel blockers in cardioprotection with ischemia/reperfusion injury.

N -n-butyl haloperidol iodide protects cardiomyocytes against hypoxia/reoxygenation injury by inhibiting autophagy

N-n-butyl haloperidol iodide (F2), a novel compound derived from haloperidol, protects against the damaging effects of ischemia/reperfusion (I/R) injury in vitro and in vivo. In this study, we hypothesized the myocardial protection of F2 on cardiomyocyte hypoxia/reoxygenation (H/R) injury is mediated by inhibiting autophagy in H9c2 cells. The degree of autophagy by treatment with F2 exposed to H/R in H9c2 cell was characterized by monodansylcadaverine, transmission electron microscopy, and expression of autophagy marker protein LC3. Our results indicated that treatment with F2 inhibited autophagy in H9c2 cells exposed to H/R. 3-methyladenine, an inhibitor of autophagy, suppressed H/R-induced autophagy, and decreased apoptosis, whereas rapamycin, a classical autophagy sensitizer, increased autophagy and apoptosis. Mechanistically, macrophage migration inhibitory factor (MIF) was inhibited by F2 treatment after H/R. Accordingly, small interfering RNA (siRNA)-mediated MIF knockdown decreased H/R-induced autophagy. In summary, F2 protects cardiomyocytes during H/R injury through suppressing autophagy activation. Our results provide a new mechanistic insight into a functional role of F2 against H/R-induced cardiomyocyte injury and death.