ABSTRACT
Type V collagen is an essential component of the extracellular matrix (ECM), and its remodeling releases specific protein fragments that can specifically inhibit endothelial cell responses such as proliferation, migration, and invasion. In this study, we have successfully constructed two engineered strains of Pichia pastoris capable of producing recombinant collagen through a new genetic engineering approach. Through high-density fermentation, the expression of 1605 protein and 1610 protein could reach 2.72 g/L and 4.36 g/L. With the increase of repetition times, the yield also increased. Bioactivity analysis showed that recombinant collagen could block the angiogenic effect of FGF-2 on endothelial cells by eliminating FGF-2-induced endothelial cell migration and invasion. Collectively, the recombinant proteins we successfully expressed have a wide range of potential for inhibiting angiogenesis in the biomaterials and biomedical fields.
Subject(s)
Recombinant Proteins , Recombinant Proteins/pharmacology , Recombinant Proteins/genetics , Humans , Collagen/chemistry , Collagen/pharmacology , Cell Movement/drug effects , Repetitive Sequences, Amino Acid , Amino Acid Sequence , Human Umbilical Vein Endothelial Cells/drug effects , Angiogenesis Inhibitors/pharmacology , Angiogenesis Inhibitors/chemistry , Fibroblast Growth Factor 2/genetics , Fibroblast Growth Factor 2/pharmacology , Fibroblast Growth Factor 2/metabolism , Fibroblast Growth Factor 2/chemistry , Gene Expression , Fermentation , Saccharomycetales/genetics , Saccharomycetales/metabolismABSTRACT
BACKGROUND: The anti-oxidative and anti-inflammatory activities of electro-acupuncture (EA), a traditional clinical method, are widely accepted, but its mechanisms are not yet well defined. In this study, we investigated the role of extracellular signal-regulated kinases1/2 (ERK1/2) pathways on electro-acupuncture - mediated up-regulation of heme oxygenase-1 (HO-1) in rabbit lungs injured by LPS-induced endotoxic shock. MATERIAL/METHODS: Seventy rabbits were randomly divided into 7 groups: group C, group M, group D, group SEAM, group EAM, group EAMPD, and group PD98059. Male New England white rabbits were given EA treatment on both sides once a day on days 1-5, and then received LPS to replicate the experimental model of injured lung induced by endotoxic shock. Then, they were killed by exsanguination at 6 h after LPS administration. The blood samples were collected for serum examination, and the lungs were removed for pathology examination, determination of wet-to-dry weight ratio, MDA content, SOD activity, serum tumor necrosis factor-α, determination of HO-1 protein and mRNA expression, and determination of ERK1/2 protein. RESULTS: The results revealed that after EA treatment, expression of HO-1and ERK1/2 was slightly increased compared to those in other groups, accompanied with less severe lung injury as indicated by lower index of lung injury score, lower wet-to-dry weight ratio, MDA content, and serum tumor necrosis factor-α levels, and greater SOD activity (p<0.05 for all). After pretreatment with ERK1/2 inhibitor PD98059, the effect of EA treatment and expression of HO-1 were suppressed (p<0.05 for all). CONCLUSIONS: After electro-acupuncture stimulation at ST36 and BL13, severe lung injury during endotoxic shock was attenuated. The mechanism may be through up-regulation of HO-1, mediated by the signal transductions of ERK1/2 pathways. Thus, the regulation of ERK1/2 pathways via electro-acupuncture may be a therapeutic strategy for endotoxic shock.
Subject(s)
Electroacupuncture/methods , Gene Expression Regulation, Enzymologic/physiology , Heme Oxygenase-1/metabolism , Lipopolysaccharides/adverse effects , Lung/enzymology , MAP Kinase Signaling System/physiology , Shock, Septic/chemically induced , Analysis of Variance , Animals , Blotting, Western , DNA Primers , Histological Techniques , Lung/pathology , Male , Polymerase Chain Reaction , Rabbits , Shock, Septic/therapy , Superoxide Dismutase/blood , Tumor Necrosis Factor-alpha/bloodABSTRACT
OBJECTIVE: To develop a method of quality control for Hugan qingzhi tablets. METHODS: Fructus Crataegi, Rhizoma Alismatis and Radix Notoginseng were identified by TLC. HPLC was used for the determination of ursolic acid in Hugan qingzhi tablets. RESULTS: The chromatographic spots were identified without the interference of negative control. Ursolic acid had a good linearity over the concentration range of 40-200 microg/mL (r = 1.000). The average recoveries was 99.05% with relatively standard deviations of 1.3%. CONCLUSION: This method is reliable, accurate and specific and can be used for the quality control of Hugan qingzhi tablets.
Subject(s)
Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/standards , Hypolipidemic Agents/chemistry , Hypolipidemic Agents/standards , Plants, Medicinal , Triterpenes/analysis , Alisma/chemistry , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Crataegus/chemistry , Drug Combinations , Drugs, Chinese Herbal/administration & dosage , Hypolipidemic Agents/administration & dosage , Panax notoginseng/chemistry , Plants, Medicinal/chemistry , Quality Control , Reproducibility of Results , Rhizome/chemistry , Tablets , Ursolic AcidABSTRACT
OBJECTIVE: To study the purification and isolation of polysaccharides from Salvia miltiorrhiza. METHODS: The root was extracted by water purified preliminarily by alcohol precipitation, and then four different types macroporous adsorption resin and one ion-exchange resin were comparatively investigated in the purification and isolation of Salvia polysaccharides. RESULTS: The total polysaccharides of crude extracts was 40.35%, and protein content amounted to 8.96%. Compared with the traditional methods, AB-8, DB-301 type of resin used in purification of polysaccharides could simplify the working process and obtain better effect. Then the obtained crude polysaccharides through AB-8 resin were purified by ion-exchange resin DEAE-52. Three portions of powder were obtained through lyophilization and named as SMP1, SMP2, SMP3. CONCLUSION: Purification of Salvia polysaccharides can be conducted by adoping AB-8, DB-301 type of resin and DEAE-52 ion-exchange resin.
