Separation and physicochemical properties of polysaccharides from Morinda officinalis and their effects on human liver cells / 国际药学研究杂志

Objective: To separate the fractions and components of crud polysaccharides MOP-60 and MOP-60-100 from Morinda officinalis, characterize their physicochemical properties and effects on the LO2 cell proliferation as well as on the ConA- and H2O2-induced LO2 cell death. Methods: The five fractions(MOP-60-A, MOP-60-B, MOP-60-100-A, MOP-60-100-B and MOP-60- 100-C)were obtained by column chromatographic separation of MOP-60 or MOP-60-100 on a DEAE-DEAE-cellulose column. A component MOP-60-Ⅰ was obtained by dialysis of MOP-60-A. The further separation and purification of MOP-60-100-B by the Sephadex G-100 column chromatography afforded the other two components MOP-60-100-Ⅰ and MOP-60-100-Ⅱ. The molecular distribution was determined with gel filtration chromatography. The monosaccharide compositions were analyzed with capillary electrophoresis after acid hydrolysis and PMP derivation. The effects of the fractions and components of polysaccharides on the human LO2 liver cell proliferation and on the ConA- and H2O2-induced LO2 cell damage were evaluated by the MTT method. Results: MOP-60-A, MOP-60-100-A and MOP-60-Ⅰ were composed of fructose(fructosan), and the peak relative molecular mass of MOP-60-Ⅰ was 2339. MOP-60-B, MOP-60-100-B, MOP-60-100-C, MOP-60-100-Ⅰ and MOP-60-100-Ⅱ were composed of multiple monosaccharides and heteroglycans. The peak relative molecular mass of MOP-60-100-Ⅰ and MOP-60-100-Ⅱ were 62 828 and 7783, respectively. MOP-60-B, MOP-60-100-B and MOP-60-100-C increased human LO2 hepatocyte proliferation and reduced the ConA-induced LO2 cell death at 250 mg/L(P<0.01, compared to solvent or ConA alone group). MOP-60-100-C also reduced the H2O2-induced LO2 cell death at 100 and 250 mg/L(P<0.05 and P<0.01, compared to H2O2 alone group). Conclusion: The acidic fractions MOP-60-100-B and MOP- 60-100-C from M. officinalis significantly promoted LO2 cell proliferation and inhibited the ConA- and H2O2-induced cell damage in human liver LO2 cells.

Protective effect of raspberry extract on ConA-induced acute liver injuryin mice / 中国中药杂志

In this experiment,the antioxidant capacity of raspberry extract and the protective effect on liver injury induced by ConA in mice were investigated. Balb/C male mice were randomly divided into six groups: normal group,model group,bicyclol control group( 200 mg·kg~(-1)),low-dose raspberry extract group( 200 mg·kg~(-1)),middle-dose raspberry extract group( 400 mg·kg~(-1)),and highdose raspberry extract group( 800 mg·kg~(-1)). Each group was intragastrically administered with drugs according to the body weight once a day. Seven days later,all of the groups except for the normal group were treated with ConA( 20 mg·kg~(-1)) through tail vein injection to establish the acute liver injury model. The mice were put to death 8 hours later. The organ indexes were calculated. These rum levels of ALT,AST and LDH and the activities of SOD,CAT,GSH and MDA in liver tissue were detected. HE staining was used to observe the pathological changes of liver tissue in mice. Western blot was used to detect the expressions of Bax,Bcl-2,Nrf2 and Keap-1. The antioxidant capacity of raspberry extract was measured by CAA assay. The results showed that,raspberry extract had a strong antioxidant capacity. Simultaneously,compared with the model group,raspberry extract can significantly improve the pathological conditions of liver,and significantly reduce ALT,AST and LDH activities in serum of liver injury mice( P<0. 01). The activities of SOD,CAT in liver homogenate supernatant were significantly increased in the high-dose group,the content of GSH increased,while the content of MDA was sharply declined in the high-dose group( P<0. 01). Meanwhile,raspberry extract down-regulated the expressions of Bax and Keap-1 and up-regulated the expressions of Bcl-2 and Nrf2. CAA showed that the compound raspberry extract had a strong antioxidant capacity. Therefore,raspberry extract has an obvious protective effect on acute liver injury induced by ConA in mice.

Mechanism of Fuzheng Quxie Prescription Against Liver Injury of ConA Model Mice / 中国实验方剂学杂志

Objective:To study the protective effect of Fuzheng Quxie prescription on liver injury by influencing the expressions of tumor necrosis factor (TNF)-α, apoptosis factor (Fas), apoptosis factor ligand (FasL), and macrophages (CD68) in the liver tissues of concanavalin A(ConA) model mice. Method:The sixty mice were randomly divided into normal group, model group,bicyclol group (62.5 mg·kg-1), and low, medium and high-dose Fuzheng Quxie prescription groups(50.3,67.0,83.8 g·kg-1). The normal group and the model group were given the equal volume of pure water for 7 d. They were fasted for 12 hours before the last administration. At 2nd hour after the last administration, phosphate buffer(PBS) was injected into the caudal vein of the normal group, and ConA (20 mg·kg-1) was injected into the caudal vein of the other groups for modeling. The animals were put to death six hours later after the injection, and the expressions of TNF-α, Fas, FasL and CD68 in liver tissues were observed by immunohistochemistry. Result:Compared with normal group, the expressions of TNF-α, Fas, FasL and CD68 in the liver tissues of the model group were significantly increased(Pα, FasL and CD68 in liver tissues of the bicyclol group were significantly decreased compared with the model group(Pα, FasL and CD68 in liver tissues were significantly decreased in medium and high-dose Fuzheng Quxie prescription groups (PPConclusion:Fuzheng Quxie prescription can effectively reduce the apoptosis of liver cells in ConA model mice by inhibiting Kupffer cells and Fas/FasL system activation.

Determination and Quality Evaluation of Glycoprotein in Recombinant Human Albumin from Different Expression Systems / 中国药学杂志

OBJECTIVE: To determine glycoprotein content in recombinant human albumin from different expression systems with different methods. METHODS: Recombinant human albumin samples from Saccharomyces cerevisiae expression system, Pichia pastoris expression system, Oryza sativa expression system as well as plasma derived human albumin were investigated by phenol sulfuric acid method, HPLC peak area method and ConA combining elution HPLC method. RESULTS: For 10 batches of samples expressed in pichia pastoris expression system, the total contents of mannose were 2.7 mg•g(Pro)-1 (A manufacturer, n = 4) and 1.7 mg•g (Pro)-1 (B manufacturer, n = 6), respectively. The HPLC peak area percentages of ConA binding protein in recombinant human albumin from Pichia pastoris expression system were the highest, which showed 2.65% (A manufacturer) and 0.71% (B manufacturer) respectively, the peak area percentage of ConA binding protein in Oryza sativa expression system was 0.05% (E manufacturer, n = 3), and the ConA binding protein peak area of plasma derived human albumin was 0.01% (W manufacturer). The results of ConA binding and elution analysis with HPLC method for Quantitative determination of ConA binding protein showed that the ConA binding protein contents in the samples from pichia pastoris expression system were much higher: 27.58 mg•g(Pro)-1 (A manufacturer), 21.48 mg•g(Pro)-1 (B manufacturer), 32.02 mg•g(Pro)-1 (C manufacturer); the ConA binding protein content in the sample from Saccharomyces cerevisiae expression system was lower, 2.29 mg•g(Pro)-1 (D manufacturer); the ConA binding protein content in the samples from oryza sativa expression system was the lowest, 1.27 mg•g(Pro)-1 (E manufacturer); the plasma derived human albumin ConA binding protein content was 31.16 mg•g(Pro)-1 (S manufacturer). CONCLUSION: In terms of the results of the samples and methods involved in this study, there were glycosylated or glycol forms of protein in all recombinant human albumin samples from different expression systems; the glycosylated protein content in samples of Pichia pastoris expression system is higher than Saccharomyces cerevisiae expression system, while the glycoformed protein in samples of Oryza sativa expression system is the lowest. Plasma derived human albumin also contains glycoprotein or glycosylated protein.

The effection of heat stress on cell immunology in rats / 重庆医学

Objective To investigate the effect of heat stress on the expression of Toll-like receptor 4 (TLR4) and peripheral blood T regulatory cells (Treg) in splenic cells of rats.Methods Senventy-two SD rats were divided into 20 ℃ control group and 37 ℃ group.Each group was divided into non stimulation,bacterial lipopolysaccharide (LPS) stimulation and concanavin A (Con-A) stimulation subgroup.Each subgroup had 1,12,48 h and 168 h observation points,and flow cytometry was used to determine the level of TLR4 and Treg.Results The TLR4+ immunocompetent cells in the spleen was decreased from 1 h to 168 h in every subgroup of the 37 ℃ group compared to the 20 ℃ control group (P＜0.05).The level of CD4+CD25+ Treg in the peripheral blood in rats from 1 to 48 h was significantly decreased (P＜0.05) and slightly increased at 168 h in LPS stimulation subgroup in the 37 ℃ group compared to the 20 ℃ control group.The level of CD4+ CD25+ Foxp3+ Treg in the peripheral blood in rats at 12 h were significantly increased in non-stimulation and concanavalin A (Con-A) stimulation subgroups in the 37 ℃group,and were significantly decreased at 168 h in every subgroup of the 37 ℃ group compared with 20 ℃ control group (P＜0.05).The level of CD8+CD25+ Treg in the peripheral blood in rats was significantly increased at 1 h and 168 h in the 37 ℃ hot and humid group in the every subgroup whencompared with the 20 ℃ control group (P＜0.05).The level of CD8+ CD25+Foxp3+ Treg in the peripheral blood in rats was significantly decreased at 1 h and 48 h in the every subgroup,and was significantly increased at 12 h and 168 h in the non-stimulation and LPS stimulation subgroups in the 37 ℃ group compared to the 20 ℃ control group (P＜0.05).Conclusion High temperature and damp heat can destroy the innate immunity and alter the functional state of adaptive immunity in rats.

Glycoproteomics analysis of plasma proteins associated with Opisthorchis viverrini infection-induced cholangiocarcinoma in hamster model

Objective To apply lectin affinity chromatography and glycoproteomics-based LC-MS/MS to preliminarily investigate the possible potential plasma biomarkers of Opisthorchis viverrini (OV)-associated CCA in OV/dimethylnitrosamine (DMN)-induced CCA hamster model. Methods Nine Syrian hamsters were divided into 3 groups as follows (n = 3 each): normal (healthy control group); OV group; and OV/DMN group (CCA group). Pooled plasma samples collected from animals in each group at the 6th month post-infection with OV metacercarae were subjected to glycoproteomics analysis. Glycoproteins in the pooled sample from each group were initially isolated by concanavalin A (ConA)-based affinity chromatography. The expression of glycoproteins isolated by both enrichment methods were determined using LC-MS/MS. Results Among the 24 ConA-binding glycoproteins isolated, two proteins, N-myc downstream regulated gene 1 (NDRG1) and fetuin-B (FETUB) were found up-regulated only in the samples from the OV and control groups, but not in the OV/DMN (CCA) groups. On the other hand, one protein, i.e., NSFL1 cofactor p47 isoform ×3 (NSFL1C) was found only in the samples from OV/DMN (CCA) and control groups, but not in the OV group. The remaining 21 proteins were upregulated in the samples from all groups. Conclusions NDRG1, FETUB and NSFL1C glycoproteins isolated by ConA-based affinity chromatography could be potential biomarkers for CCA. Plasma samples with negative for NDRG1 and FETUB proteins but positive for NSFL1C are likely to be OV-associated CCA. Nevertheless, this conclusion remains to be confirmed whether this battery test can discriminate OV-associated CCA from other risk factors.

Glycoproteomics analysis of plasma proteins associated with Opisthorchis viverrini infection-induced cholangiocarcinoma in hamster model

OBJECTIVE@#To apply lectin affinity chromatography and glycoproteomics-based LC-MS/MS to preliminarily investigate the possible potential plasma biomarkers of Opisthorchis viverrini (OV)-associated CCA in OV/dimethylnitrosamine (DMN)-induced CCA hamster model.@*METHODS@#Nine Syrian hamsters were divided into 3 groups as follows (n = 3 each): normal (healthy control group); OV group; and OV/DMN group (CCA group). Pooled plasma samples collected from animals in each group at the 6th month post-infection with OV metacercarae were subjected to glycoproteomics analysis. Glycoproteins in the pooled sample from each group were initially isolated by concanavalin A (ConA)-based affinity chromatography. The expression of glycoproteins isolated by both enrichment methods were determined using LC-MS/MS.@*RESULTS@#Among the 24 ConA-binding glycoproteins isolated, two proteins, N-myc downstream regulated gene 1 (NDRG1) and fetuin-B (FETUB) were found up-regulated only in the samples from the OV and control groups, but not in the OV/DMN (CCA) groups. On the other hand, one protein, i.e., NSFL1 cofactor p47 isoform ×3 (NSFL1C) was found only in the samples from OV/DMN (CCA) and control groups, but not in the OV group. The remaining 21 proteins were upregulated in the samples from all groups.@*CONCLUSIONS@#NDRG1, FETUB and NSFL1C glycoproteins isolated by ConA-based affinity chromatography could be potential biomarkers for CCA. Plasma samples with negative for NDRG1 and FETUB proteins but positive for NSFL1C are likely to be OV-associated CCA. Nevertheless, this conclusion remains to be confirmed whether this battery test can discriminate OV-associated CCA from other risk factors.

Study on the protective effect in immunological liver injury in mice of Zhizi Baipi Soup and its disassembled prescription / 中国药理学通报

Aim To investigate the effect of Zhizi Baipi soup and its disassembled prescription containing Zhizi on protecting concanavalin (ConA)-induced immuno-logical liver injury in mice and explore the possible protective mechanism.Methods The model of mouse immunological liver injury induced by (ConA,20 mg ·kg -1 )was used to observe the effects of Zhizi Baipi soup and its disassembled prescription containing Zhizi by gavage administration.Results Zhizi Baipi Soup and its disassembled prescription containing Zhizi were able to reduce the level of serum ALT,AST and MDA content,improve liver tissue SOD activity and reduce the expression of NF-κB-p65 and NF-κB-p-p65,in which all parties of Zhizi Baipi Soup Group had best effect on immunological liver injury in mice.Conclu-sion Zhizi Baipi Soup and its disassembled prescrip-tion containing Zhizi have remarkably protective effect on ConA-induced immunological liver injury in mice, and the potential mechanism may be related to the reg-ulation of the immune response pathway.

Effect of Lectin on Fine Structure and Extracellular Matrix Protein of Cancer Cell Lines / 대한해부학회지

Lectins are glycoproteins that bind specifically to carbohydrates. Considerable interests in the lectins were encouraged by several reports that certain members of the family bind to the extracellular matrix proteins (ECM), such as fibronectin and laminin. However, the relations between lectin and ECM protein remain unclear. To elucidate the relations of lectin-matrix-cell, we treated three cancer cell lines, HeLa, L929, and EATC with ConA and PHA-P at low dose (4 microgram/ml) and high dose (20 microgram/ml) for 1, 3, 5 days. 1. Whether or not lectins significantly regulate the cell proliferation was evaluated by MTT assays. 2. Whether the amount of fibronectin and laminin which of cancer cells can be influenced by lectins was confirmed by immunocytochemical staining. 3. Whether, in turn, the lectins which can change the morphology were observed under inverted and electron microscopes. ConA and PHA-P inhibited cell proliferation rate of all cell lines in a dose- and time- dependent manner. The amount of fibronectin and laminin considerably reduced in the three cell lines after the lectins treatment in a dose- and time-dependent manner. The cancer cell lines showed various morphological changes such as cell aggregation, irregular-shaped cellular processes, rounded cells, cytoplasmic vacuolation, swollen RERs, dilation of mitochondria, margination of chromatin and cell death. In conclusion, our results showed ConA and PHA-P caused damages of the three cancer cell lines, but the effect of PHA-P was much stronger than ConA. Taken together, the present data strongly indicate that ConA and PHA-P influence the cell proliferation rate, reduce the amount of fibronectin and laminin and induce cell injuries of HeLa, L929, and EATC cell lines. Our results also suggest that the cancer cell proliferation and the morphological changes might be modulated by the specific interaction between lectins and ECM proteins associated with the cell surface.

Preparation of Concanavalin A-ß-galactosidase conjugate and its application in lactose hydrolysis.

A Concanavalin A-β-galactosidase conjugate was prepared using glutaraldehyde as the crosslmking reagent. The conjugate bound to Sephadex G-50 beads was more thermostable and hydrolyzed lactose faster than the free enzyme. The immobilized enzyme may prove useful in the preparation of low lactose milk which is required by persons suffering from lactose intolerance.

Effect of conditioned medium with concanavalin A-activated spleen cells supernatant on cultured rabbit corneal endothelial cells / 西安交通大学学报(医学版)

Objective To evaluate the effect of conditioned medium made up of ConA-activated spleen cells supernatant on cultured rabbit corneal endothelial cells (RCECs).Methods RCECs were primarily cultured in vitro by revealing descemet membrane and endothelium combining tissues masses.Culture medium was RPMI 1640 with 10% FBS and varied concentrations of conA-activated spleen cells supernatant.Experimental groups were divided by different concentrations of ConA-conditioned supernatant:5%,10%,15%,and 20%.In the control group only RPMI1640 and 10% FBS were used.Methyl thiazolyl tetrazolium colorimetry analysis was used to evaluate the proliferation of RCECs cellular population.The cultured cells were identified by immunohistochemical staining for neuronal specific enolase (NSE).Results The RCECs were cultured successfully.RCECs presented positive expression in cytoplasm with NSE immunohistochemical assay.There were significant differences in the experimental groups compared with the control group (P

THE STUDY OF T LYMPHOCYTE SUBSETS AND SUPPRESSOR CELL ACTIVITY IN CHILDREN WITH PRIMARY NEPHRITIC SYNDROME / 西安交通大学学报(医学版)

The peripheral blood T lymphocyte subsets and ConA-induced suppressor cell activity (ConA-SCA) were determined in 37 patients with primary nephritic syndrome(PNS). The results showed that the patients in initial onset had the abnormal number and function of T lymphocytes, which returned to a normal level with treatment with steroid and improvement of patients'condition. The indings suggest that the immunoregulatory imbalance of this disease may be a primary change and play an important role in pathogenesis of PNS.