Dietary chitosan modulates the growth performance, body composition and nonspecific immunity of juvenile yellow catfish (Pelteobagrus fulvidraco).

This study investigated the effects of feeding different concentrations of chitosan on the growth performance, body composition and non-specific immune function of juvenile yellow catfish (Pelteobagrus fulvidraco). Four kinds of experimental diets were respectively prepared by adding 0 (control group), 5, 10 and 15 g/kg of chitosan to the basal feed and fed to juvenile yellow catfish for 8 weeks. Results show that the body weight gain rate, specific growth rate, survival rate, body protein content, serum superoxide dismutase activity, catalase activity, glutathione peroxidise activity, lysozyme activity and disease resistance ability against Aeromonas hydrophila of the experimental group with chitosan added to its diet were significantly higher than those of the control group optimally by 36.22 %, 14.37 %, 9.46 %, 8.97 %, 50.89 %, 33.15 %, 21.52 %, 40.80 %, 41.09 %, and 79.71 %, respectively (P < 0.05). No significant differences in feed efficiency among all groups (P > 0.05) were observed. The optimum dose of dietary chitosan required for the maximum growth of juvenile yellow catfish was 8.95 g/kg. Therefore, adding an appropriate amount of chitosan (8.95 g/kg) to the feed of yellow catfish can significantly improve its growth performance, ameliorate body composition and enhance its non-specific immunity.

Selective Photokilling of Colorectal Tumors by Near-Infrared Photoimmunotherapy with a GPA33-Targeted Single-Chain Antibody Variable Fragment Conjugate.

Antibody-based near-infrared photoimmunotherapy (NIR-PIT) is an attractive strategy for cancer treatment. Tumor cells can be selectively and efficiently killed by the targeted delivery of an antibody-photoabsorber complex followed by exposure to NIR light. Glycoprotein A33 antigen (GPA33) is highly expressed in most human colorectal cancers (CRCs) and is an ideal diagnostic and therapeutic target. We previously produced a single-chain fragment of a variable antibody against GPA33 (A33scFv antibody). Here, we investigate the efficacy of NIR-PIT by combining A33scFv with the NIR photoabsorber IR700 (A33scFv-IR700). In vitro, recombinant A33scFv displayed specific binding and delivery of an NIR dye to GPA33-positive tumor cells. Furthermore, A33scFv-IR700-mediated NIR-PIT was successful in rapidly and specifically killing GPA33-positive colorectal tumor cells. NIR-PIT treatment induced the release of lactate dehydrogenase from tumor cells, followed by cell necrosis, rather than apoptosis, through the promotion of reactive oxygen species accumulation in tumor cells. In mice bearing LS174T tumor grafts, A33scFv selectively accumulated in GPA33-positive tumors. Following only a single injection of the conjugate and subsequent illumination, A33scFv-IR700-mediated NIR-PIT induced a significant increase in therapeutic response in LS174T-tumor mice compared with that in the non-NIR-PIT groups (p < 0.001). Because the GPA33 antigen is specifically expressed in CRC tumors, A33scFv-IR700 might be a promising antibody fragment-photoabsorber conjugate for NIR-PIT of CRC.

Copper promotes the migration of bone marrow mesenchymal stem cells via Rnd3-dependent cytoskeleton remodeling.

The motility of mesenchymal stem cells (MSCs) is highly related to their homing in vivo, a critical issue in regenerative medicine. Our previous study indicated copper (Cu) might promote the recruitment of endogenous MSCs in canine esophagus defect model. In this study, we investigated the effect of Cu on the motility of bone marrow mesenchymal stem cells (BMSCs) and the underlying mechanism in vitro. Cu supplementation could enhance the motility of BMSCs, and upregulate the expression of hypoxia-inducible factor 1α (Hif1α) at the protein level, and upregulate the expression of rho family GTPase 3 (Rnd3) at messenger RNA and protein level. When Hif1α was silenced by small interfering RNA (siRNA), Cu-induced Rnd3 upregulation was blocked. When Rnd3 was silenced by siRNA, the motility of BMSCs was decreased with or without Cu supplementation, and Cu-induced cytoskeleton remodeling was neutralized. Furthermore, overexpression of Rnd3 also increased the motility of BMSCs and induced cytoskeleton remodeling. Overall, our results demonstrated that Cu enhanced BMSCs migration through, at least in part, cytoskeleton remodeling via Hif1α-dependent upregulation of Rnd3. This study provided an insight into the mechanism of the effect of Cu on the motility of BMSCs, and a theoretical foundation of applying Cu to improve the recruitment of BMSCs in tissue engineering and cytotherapy.

Celastrol mediates Th17 and Treg cell generation via metabolic signaling.

A T helper 17 (Th17) cell/regulatory T (Treg) cell imbalance is involved in many immune disorders and diseases. Celastrol, a Chinese herbal compound that has anti-inflammatory and immunosuppressive properties, has been indicated to suppress T cell proliferation and Th17 cell induction, while facilitating Forkhead box P3 (Foxp3) expression and Treg cell generation. In this study, we explored the impact and mechanism of celastrol on Th17 cell/induced Treg (iTreg) cell induction. CD4+CD25- T cells were purified, stimulated with anti-CD3 and anti-CD28 antibodies, and polarized in vitro to generate Th17 or iTreg cells in the presence or absence of celastrol. Initially, we determined that Interleukin (IL)-17 expression by celastrol-treated Th17 was significantly decreased compared with untreated cells; however, the frequency of Foxp3+ cells was increased in celastrol-treated cells. We verified that celastrol inhibited phospho-STAT3 expression in cultured Th17 cells and up-regulated phospho-STAT5 expression in iTreg cells. Furthermore, T cells treated with celastrol were more likely to participate in FAO metabolism instead of glycolysis. Celastrol suppressed the expression of glucose transporter, Glut1, and the rate-limiting enzyme, HK2, in addition to mTOR, HIF-1α, c-Myc and Akt expression in Th17 cells. Conversely, celastrol promoted FAO of lipids by up-regulating CPT1A and AMPKα expression in iTreg cells. Our results suggest that celastrol suppresses Th17 cell induction, while promoting the generation of iTreg cells. We found that celastrol inhibits glycolysis in Th17 cells and promotes FAO by iTreg cells, suggesting that celastrol could mediate the metabolism of Th17 and iTreg cells.

One new linear C14 polyacetylene glucoside with antiadipogenic activities on 3T3-L1 cells from the capitula of Coreopsis tinctoria.

Based on characteristic UV spectrum of the ene-diyne chromophore, one new polyacetylene glucoside and three known polyacetylene glucosides have been isolated from the EtOH extract of Coreopsis tinctoria. Their chemical structures were determined by detailed spectroscopic analysis and by comparison with literature data. Compounds 1-2 were tested for their antiadipogenic effects on 3T3-L1 adipocytes, and both of them reduced lipid accumulation dose-dependently in 3T3-L1 adipocytes.

9,10-Dihydrophenanthrene derivatives and one 1,4-anthraquinone firstly isolated from Dioscorea zingiberensis C. H. Wright and their biological activities.

Two new phenanthrene derivatives, 2,5,7-trimethoxy-9,10-dihydrophenanthrene-1,4-dione (1) and 2,5,6-trihydroxy-3,4-dimethoxy-9,10-dihydrophenanthrene (3), one new anthracenedione, 2,5,7-trimethoxyanthracene-1,4-dione (2), together with two known 9,10-dihydrophenanthrenes (4-5) were isolated from the rhizomes of Dioscorea zingiberensis C. H. Wright. The structures of these new compounds were established based on extensive NMR spectroscopy. Several isolated compounds were evaluated for the inhibition against nitric oxide (NO) production in the lipopolysaccharide (LPS)-activated RAW 264.7 macrophage cell line, DPPH radical scavenging, and inhibitory activity on Free Fatty Acids (FFAs) induced triglyceride accumulation in HepG2 cells. Compound 2 exhibited moderate anti-inflammatory activity, compound 3 possessed comparable DPPH radical scavenging activity as Vitamin C, compounds 2 and 4 showed potent inhibitory activities on triglyceride accumulation.

Copper stimulates growth of human umbilical vein endothelial cells in a vascular endothelial growth factor-independent pathway.

Studies in vivo have shown that dietary copper (Cu) supplementation reverses pressure overload-induced cardiac hypertrophy in a mouse model, which is vascular endothelial growth factor (VEGF)-dependent and correlates with enhanced angiogenesis. Because Cu stimulation of endothelial cell growth and differentiation would play a critical role in angiogenesis, the present study was undertaken to examine the effect of Cu on growth of human umbilical vein endothelial cells (HUVECs) in cultures. The HUVECs were treated with CuSO(4) at a final concentration of 5 µmol/L Cu element in cultures or with a Cu chelator, tetraethylenepentamine (TEPA), at a final concentration of 25 µmol/L in cultures. Cell growth and Cu effect on cell cycle were determined. In addition, the effect of Cu on VEGF and endothelial nitric oxide synthase (eNOS) mRNA levels was determined, and anti-VEGF antibody and siRNA targeting eNOS were applied to determine the role of VEGF or eNOS in the Cu effect on cell growth. Cu significantly stimulated and TEPA significantly inhibited cell growth, and the TEPA effect was blocked by excess Cu. Cu increased the number of cells in the S phase and correspondingly decreased the number in the G1 phase. Interestingly, Cu did not increase the level of VEGF mRNA, but significantly increased eNOS mRNA. Furthermore, neutralizing VEGF by anti-VEGF antibody did not suppress Cu stimulation of cell growth. However, siRNA targeting eNOS completely blocked Cu reversal of TEPA inhibition of cell growth. The data demonstrate that Cu stimulation of HUVEC cell growth is VEGF-independent, but eNOS-dependent.

Full-length cDNA cloning and protein three-dimensional structure modeling of factor VII of rhesus monkey, Macaca mulatta.

FVII is a vitamin K dependent serine protease that plays a key role in extrinsic coagulation pathway. In this paper, we report the full-length cDNA sequences of rhesus monkey FVII. The full-length cDNA has 2424 bp, and predicts an open reading frame of 1416 bp corresponding to 472 amino acids. The deduced protein sequence of rhesus monkey FVII indicates the functional domains including signal peptide, Gla domain, two EGF domains, and catalytic domain. Rhesus monkey FVII is highly homologous to human FVII with amino acid identity of 91.0%. Comparison of three-dimensional protein structure shows high conservation between them. The important functional sites such as the N-terminal gamma-carboxyglutamic acids of the Gla domain, the Ca(2+) binding region of the EGF I domain, the TF binding region, the active site binding cleft, and the macromolecular substrate binding exosite of trypsin domain are all well conserved in FVII of rhesus monkey. Prothrombin time test shows rhesus monkey FVII has a similar clotting time with that of human. This study of rhesus monkey FVII might be helpful for understanding the function compatibility of human and rhesus monkey FVII, which is beneficial for the study of xenotransplantation.

Full-length cDNA cloning and protein three-dimensional structure modeling of porcine prothrombin.

Prothrombin is a vitamin K-dependent serine protease and plays pivotal roles in both procoagulant and anticoagulant pathway of hemostasis. In this study, we cloned the full-length cDNA of porcine prothrombin by cDNA library screening and SMART RACE technique. The full-length cDNA is 2027 bp, with a 1869 bp Open Reading Frame (ORF) coding 623 amino acids. The deduced protein of porcine prothrombin contains signal peptide, propeptide, Gla domain, two kringle domains and trypsin domain. Porcine prothrombin shares 86.15% nucleotide similarity and 83% amino acid similarity with human prothrombin. The trypsin domain is highly conserved between the two species with 92.1% amino acid identity. Macromolecular interaction sites comparison between porcine and human prothrombin suggests that the Gla domain in porcine prothrombin contains an additional potential gamma-carboxyglutamic acid site. However, a thrombin cleavage site (Arg284-Thr285) in its light chain is lost. When thrombin heavy chain is concerned, the most important functional sites such as catalytic triad DHS, RGD site, Na+ binding site and anion-binding exosite-I and II are highly conserved. However, great differences have been observed between residues 145 and 158 of heavy chain which is associated with thrombomodulin binding. Two important limited proteolysis sites at Ala150 and Lys154 were lost in porcine sequence, which would affect epsilon-thrombin and gammaT-thrombin generation. Comparison on 3-D protein models demonstrates that these proteins are obviously different in autolysis loop (Lys145 to Gly155). Compared with that of human prothrombin, variation at critical recognition sites would likely alter its binding affinity and reaction velocity, which would contribute to coagulation disorder when porcine liver is transplanted into human body.

[Effects of YM injection on oxidative stress injury in renal warm ischemia/reperfusion].

OBJECTIVE: This study was undertaken to determine whether YM injection would attenuate warm I/R injury in mice. METHODS: Renal warm I/R was induced in C57BL/6 males, and the mice were sacrificed 24 hours post-reperfusion. YM was given intraperitoneally 12 hours and 30 min before I/R. The effects of YM on oxidative stress and proinflammatory mediators caused by renal I/R injury were assayed. RESULTS: Renal I/R caused severe injuries in the kidneys of mice. In the presence of YM at doses of 5, 15 and 25 mg/kg, the kidney malondialdehyde (MDA) content reduced by 16.6%, 25.0% and 35.5% respectively, and the kidney superoxide dismutase (SOD) activities were elevated by about 38.7%, 48.3% and 76.1% respectively. ICAM-1 expression was dramatically suppressed in the presence of YM (25 mg/kg). CONCLUSION: YM attenuates the renal warm I/R injury at least partially via decreasing the neutrophils infiltration and suppressing the over-expression of adhesion molecules.

Growth inhibition and apoptosis induction of tanshinone II-A on human hepatocellular carcinoma cells.

AIM: To evaluate the effects of tanshinone II-A on inducing growth inhibition and apoptosis of human hepatocellular carcinoma (HCC) cells. METHODS: The human hepatocellular carcinoma cell line SMMC-7721 was used for the study. The cells were treated with tanshinone II-A at different doses and different times. Cell growth and proliferation were measured by MTT assay, cell count and colony-forming assay. Apoptosis induction was detected by microscopy, DNA ladder electrophoresis and flow cytometry. RESULTS: In MTT assay, the inhibitory effect became gradually stronger with the passage of time, 24, 48, 72 and 96 h after treatment with tanshinone II-A, and the most significant effect was observed at 72 h. On the other hand, the increase of doses (0.125, 0.25, 0.5, 1.0 mg/L tanshinone II-A) resulted in enhanced inhibitory effect. The growth and proliferation of SMMC-7721 cells were obviously suppressed in a dose- and time-dependent manner. The results of cell count were similar to that of MTT assay. In colony-forming assay, the colony-forming rates were obviously inhibited by tanshinone II-A. In tanshinone II-A group, the morphology of cellular growth inhibition and characteristics of apoptosis such as chromatin condensation, crescent formation, margination and apoptotic body were observed under light and transmission electron microscopes. DNA ladder of cells was presented in electrophoresis. The apoptosis index (AI) was 16.9% (the control group was 4.6%) in flow cytometry. The cells were arrested in G(0)/G(1) phase, and the expressions of apoptosis-related genes bcl-2 and c-myc were down-regulated and fas, bax, p53 up-regulated. CONCLUSION: Tanshinone II-A could inhibit the growth and proliferation of HCC cell effectively in vitro by apoptosis induction, which was associated with up-regulation of fas, p53, bax, expression and down-regulation of bcl-2 and c-myc.

The protective mechanism of Yisheng Injection against hepatic ischemia reperfusion injury in mice.

AIM: Hepatic ischemia/reperfusion injury may cause acute inflammatory, significant organ damage or dysfunction, and remains an important problem for liver transplantation. Our previous in vivo and in vitro studies demonstrated that Yisheng injection (YS), a traditional Chinese medicine, had protective effect on ischemia/reperfusion injury. In this study, we examined whether YS had protective effect for hepatic ischemia/reperfusion injury and explored its protective mechanism. METHODS: Hepatic warm ischemia/reperfusion was induced in mice. YS at different doses (5, 10, 20 mg/kg) was injected intraperitoneally 24 h and 1 h before ischemia and a third dose was injected intravenously just before reperfusion. The hepatocellular injury, oxidative stress, neutrophil recruitment, proinflammatory mediators and adhesion molecules associated with hepatic ischemia/reperfusion injury were assayed by enzyme-linked immunosorbent assay (ELISA), immunohistochemical assay and reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Undergoing 90 min of ischemia and 6 h of reperfusion caused dramatical injuries in mouse livers. Administration of YS at doses of 5, 10 and 20 mg/kg effectively reduced serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH), from 3 670+/-463 U/L, 2 362+/-323 U/L and 12 752+/-1 455 U/L in I/R group to 1 172+/-257 U/L, 845+/-193 U/L and 2 866+/-427 U/L in YS (20 mg/kg) treated group, respectively (P<0.01). The liver myeloperoxidase (MPO) and malondialdehyde (MDA) contents were decreased from 1.1+/-0.2 (U/mg protein) and 9.1+/-0.7 (nmol/mg protein) in I/R group to 0.4+/-0.1 (U/mg protein) and 5.5+/-0.9 (nmol/mg protein) in YS (20 mg/kg) treated group, respectively (P<0.01). Moreover, the serum levels of tumor necrosis factor-alpha (TNF-alpha) were reduced from 55+/-9.9 (pg/mL) in I/R group to 16+/-4.2 (pg/mL) (P<0.01). Furthermore, the over-expressions of TNF-alpha and intercellular adhesion molecule-1 (ICAM-1) were suppressed by YS treatment in a dose-dependent manner. CONCLUSION: YS attenuates hepatic warm ischemia/reperfusion injury by reducing oxidative stress and suppressing the over-expression of proinflammatory mediators and adhesion molecules.

[Influence of anoxia/reoxygenation on immunofunction of endothelial cells and effect of intervention with yisheng injection on it].

OBJECTIVE: To explore the influence of ischemia/reperfusion (anoxia/reoxygenation) on immunofunction of endothelial cells (ECs) and effect of intervention with Yisheng injection (YSI, a pure natural medicine) on it. METHODS: Model of ECs induced by anoxia/reoxygenation was established to mimic ECs ischemia/reperfusion injury in vivo with human umbilical vein endothelial cell line ECV304. Then YSI was used to intervene the anoxia/reoxygenation process. Nuclear transcriptional factor-kappa B (NF-kappa B) was exhibited by fluorescent staining, HLA-ABC, HLA-DR, CD86 and CD54 were detected by flow cytometry. Mixed endothelial cell-lymphocyte reaction (MELR) was conducted to examine the proliferation of lymphocyte, production of IL-2 and percentage of apoptotic lymphocyte. RESULTS: Anoxia/reoxygenation made the ECV304 cell became round, shrunk and abscised, with increased plasma NF-kappa B, and changed from positive cytoplasm to positive nucleus. HLA-ABC, HLA-DR and CD86 on surface of cells increased but CD54 showed unchanged. MELR showed the incorporation of 3H-TdR and production of IL-2 increased significantly and the percentage of apoptotic lymphocyte decreased. After YSI intervention, the ECV304 cell shaped recovered, NF-kappa B expression didn't down-regulated, but the percentage of positive cells decreased, changed to positive dominant. Besides, reversal changes were shown in other parameters. CONCLUSION: Anoxia/reoxygenation influences some important immune related molecules in ECV304 cells, YSI could antagonizing these influences to maintain the immune function of endothelial cells in a relative normal manner.

[A study on the protective mechanism of yisheng injection against the anoxia-reoxygenation injury to endothelial cells].

OBJECTIVE: To explore the mechanism of ischemia reperfusion injury (IRI) and the protective effect of Yisheng injection on endothelial cells (ECs). METHODS: Human umbilical veins endothelial cell line ECV304 was cultured in RPMI medium 1640 without glucose under 100% N2 for one and a half hours, and then in RPMI medium 1640 with glucose under normal conditions for 5 hours to mimic ECs' IRI posttransplantaion. A pure nature medicine, Yisheng injection was added into the medium in experimental group. Intracellular calcium and mitochondrial were shown through flourescent staining. The activities of succinic dehydrogenase (SDH) and lactic dehydrogenase (LDH) were detected by cytochemical staining. The concentrations of superoxide dismutase (SOD), malonaldehyde (MDA) and nitrous oxide (NO) in culture medium were determined. RESULTS: After treatment with anoxia-reoxygenation, the ECs' morphologic changes were observed, and intracellular calcium concentration, LDH activity, MDA concentration became higher. Meanwhile, mitochondrial membrane potential, concentrations of SOD and NO were lower. Yisheng injection made these changes disappear or become less. CONCLUSION: Anoxia-reoxygenation induces lipid peroxidation, calcium superrcharge in ECs, and damage to mitochondrial function. Yisheng injection can reverse these changes, thus protecting the endothelial cells.