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1.
China Journal of Chinese Materia Medica ; (24): 1054-1065, 2023.
Article in Chinese | WPRIM | ID: wpr-970577

ABSTRACT

This study aims to examine the effect of superfine powder and aqueous extract of Polygonati Rhizomaon on natural perimenopausal syndrome in rats and explore the underlying mechanism. To be specific, a total of 60 female SD rats(14-15 months old) with estrous cycle disorder were screened by the vaginal smear and randomized into model control group, β-estradiol 3-benzoate group(0.1 mg·kg~(-1)), superfine powder of Polygonati Rhizoma group(0.25, 0.5 g·kg~(-1)) and aqueous extract of Polygonati Rhizoma group(0.25, 0.5 g·kg~(-1)), and another 10 female SD rats(14-15 months old) were selected as the youth control group. The administration lasted 6 weeks. Then the perimenopausal syndrome-related indexes such as body temperature, microcirculatory blood flow of face and ear, vertigo period, salivary secretion, grip force, and bone strength were determined and open field test was conducted. The immune system-related indexes such as the wet weight and index of thymus and spleen, percentage of T lymphocytes and subgroups in peripheral blood, and hematological indexes were measured. In addition, the ovary-related indexes such as estrous cycle, the wet weight and index of uterus and ovary, ovarian tissue morphology, and cell apoptosis were determined. Moreover, hypothalamus-pituitary-ovary axis(HPO)-related indexes such as serum sex hormone levels, cytochrome P450 family 11 subfamily A member 1(CYP11A1), cytochrome P450 family 19 subfamily A member 1(CYP19A1), and cytochrome P450 family 17 subfamily A member 1(P450 17A1) in ovarian tissue were measured. The results showed that the superfine powder and aqueous extract of Polygonati Rhizoma significantly decreased body temperature(anal, facial and dorsal temperature), microcirculatory blood flow in the ear, and vertigo period, increased salivary secretion, grip force, bone strength, total distance and total speed in the open field test, wet weight and index of thymus and spleen, lymphocyte ratio, CD3~+ level, and CD4~+/CD8~+ ratio, reduced neutrophil number and ratio, estrous cycle disorder ratio, and number of ovarian apoptotic cells, raised wet weight and index of uterus, wet weight of ovary, levels of inhibin B(INHB), estradiol(E_2), anti-müllerian hormone(AMH), and ovarian CYP11A1 and CYP19A1, decreased follicle-stimulating hormone(FSH) and luteinizing hormone(LH) content, and improved ovarian tissue morphology. It is suggested that the superfine powder and aqueous extract of Polygonati Rhizoma can improve the symptoms associated with natural perimenopausal syndrome in rats and enhance ovarian function and immune function. The mechanism is that they regulate HPO axis function by increasing estrogen synthesis.


Subject(s)
Female , Animals , Rats , Rats, Sprague-Dawley , Microcirculation , Cholesterol Side-Chain Cleavage Enzyme , Perimenopause , Powders , Cytochrome P-450 CYP1A1
2.
China Journal of Chinese Materia Medica ; (24): 1214-1219, 2014.
Article in Chinese | WPRIM | ID: wpr-321336

ABSTRACT

WRKY transcription factor is one of the Zinc finger proteins which contains a highly conserved WRKY domain and is a family of the plant-specific transcription factor. The plasmid pET28a-SmWRKY1 harboring Salvia miltiorrhiza WRKY1 (SmWRKY1) gene was successfully transformed and expressed in Escherichia coli BL21 (DE3). The conditions on protein expression of SmWRKY1 in E. coli, including induction duration, temperature, IPTG concentration and the E. coli concentration were optimized. The results showed that the highest protein expression of SmWRKY1 was obtained at 24 hours after the E. coli was cultured in the presence of 0.2 mol x L(-1) IPTG at 20 degrees C with A600 values of 1.0-1.5. This recombinant histidine-tagged protein was expressed at 2.454 g x L(-1) as inclusion body, which was first extracted using urea, and then purified by Ni2+ affinity chromatography and identified by SDS-PAGE. The expression of SmWRKY1 in E. coli was further confirmed by western blotting analysis.


Subject(s)
Blotting, Western , Cloning, Molecular , DNA-Binding Proteins , Chemistry , Genetics , Metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Chemistry , Genetics , Metabolism , Molecular Weight , Plant Proteins , Chemistry , Genetics , Metabolism , Recombinant Proteins , Chemistry , Genetics , Metabolism , Salvia miltiorrhiza , Genetics
3.
Acta Pharmaceutica Sinica ; (12): 1618-1623, 2013.
Article in Chinese | WPRIM | ID: wpr-298035

ABSTRACT

Tanshinones are the bioactive components of the Chinese medicinal herb Salvia miltiorrhiza, while its biosynthetic pathway remains to be characterized. Rapid identification and characterization of the genes correlated to tanshinones biosynthesis is very important. As one of the intermediates of tanshinones biosynthesis, the ferruginol content is relative low in both root and engineered bacteria. It is urgent to construct an efficient system for conversion of miltiradiene to ferruginol to obtain large amount of ferruginol as the substrates for further identifying other downstream genes involved in tanshinones biosynthesis. In this study, we constructed the whole-cell yeast biocatalysts co-expressing miltiradiene oxidase CYP76AH1 and cytochrome P450 reductases (SmCPR1) from Salvia miltiorrhiza, and then characterized it with RT-PCR. After permeabilization, the yeast whole-cell could catalyze turnover of miltiradiene to ferruginol efficiently through single-step biotransformation with a conversion efficiency up to 69.9%. The yeast whole-cell biocatalyst described here not only provide an efficient platform for producing ferruginol in recombinant yeast but also an alternative strategy for identifying other CYP genes involved in tanshinones biosynthesis.


Subject(s)
Biosynthetic Pathways , Biotransformation , Cytochrome P-450 Enzyme System , Genetics , Metabolism , Diterpenes , Metabolism , Abietanes , Chemistry , Electrophoresis, Agar Gel , Gene Amplification , NADPH-Ferrihemoprotein Reductase , Genetics , Metabolism , Open Reading Frames , Plasmids , Saccharomyces cerevisiae , Genetics , Metabolism , Salvia miltiorrhiza , Chemistry
4.
China Journal of Chinese Materia Medica ; (24): 957-961, 2013.
Article in Chinese | WPRIM | ID: wpr-350682

ABSTRACT

<p><b>OBJECTIVE</b>A SmERF1 gene was isolated from Salvia miltiorrhiza, and expression patterns to different stress condition were analysed in the root tissues of S. miltiorrhiza.</p><p><b>METHOD</b>The cDNA of SmERF1 gene from S. miltiorrhiza was isolated by RTPCR, and the phylogenetic tree using the neighbour-joining tree method in Mega 5 was obtained. To confirm the protein is likely to localize in the nucleus, the SmERF1 coding region was fused to the N-terminus of the GFP gene under the control of the CaMV 35S promoter and transferred into onion epidermal cells using the particle bombardment method. Semi-quantitative RT-PCR analysis revealed different expression pattern of SmERF1 gene in response to exogenous ABA, MeJA and SA.</p><p><b>RESULT</b>The phylogenetic tree analysis revealed that SmERF1 is most similar to AP2/ERF VII subgroup members. The transient expression of the SmERF1::GFP fusion protein indicated that the SmERF1 was exclusively localized to the nucleus. The transcript of SmERF1 highly accumulated when the plants were treated with MeJA, while accumulated slightly in response to exogenous ABA, salicylic acid.</p><p><b>CONCLUSION</b>These results suggest hormone such as ABA, MeJA and SA signaling pathways can be involved in the activation and inhibition of the SmERF1.</p>


Subject(s)
Cloning, Molecular , DNA-Binding Proteins , Genetics , Metabolism , Gene Expression Regulation, Plant , Molecular Sequence Data , Phylogeny , Plant Proteins , Genetics , Metabolism , Protein Transport , Salvia miltiorrhiza , Classification , Genetics , Metabolism
5.
China Journal of Chinese Materia Medica ; (24): 3712-3716, 2012.
Article in Chinese | WPRIM | ID: wpr-346852

ABSTRACT

<p><b>OBJECTIVE</b>Accumulation of tanshinton in Salvia miltiorrhiza are enhanced by exogenous application of jasmonates. The core JA signaling module COI1/JAZ/MYC2 play a central role on control of downstream gene expression in the JA pathway. To obtained the antibody of SmJAZ, SmJAZ recombinant protein was expressed in Escherichia coli and optimal expression was performed.</p><p><b>METHOD</b>The full-length SmJAZ1 ORF was sub-cloned in a prokaryotic expression vector pET32a. The recombinant fusion protein had high expression level in BL21 (DE3) strain of E. coli, and SDS-PAGE analysis showed its molecular weight was about 24 kDa.</p><p><b>RESULT</b>The induction of E. coli [pET32-JAZ1] in different temperature, induction time, IPTG concentrations and IPTG adding time of E. coli were performed. The induction time and the induction temperature are positively related trends with SmJAZ1 protein expression, and IPTG concentration had no significant impact in protein expression, whereas IPTG adding time had significant impact on protein expression.</p><p><b>CONCLUSION</b>Shaking the culture at 30 degrees C until the A600 is approximately 0.9 (2 h in LB), and add IPTG to a final concentration of 0.1 mmol x L(-1), and then the optimal expression of SmJAZ1 recombinant protein were accumulated after the induction time of 20 h.</p>


Subject(s)
Electrophoresis, Polyacrylamide Gel , Escherichia coli , Genetics , Gene Expression , Plant Proteins , Genetics , Metabolism , Plasmids , Genetics , Metabolism , Recombinant Proteins , Metabolism , Repressor Proteins , Genetics , Metabolism , Salvia miltiorrhiza , Genetics , Metabolism , Time Factors
6.
Chinese Journal of Contemporary Pediatrics ; (12): 459-461, 2010.
Article in Chinese | WPRIM | ID: wpr-347571

ABSTRACT

<p><b>OBJECTIVE</b>To study serum insulin-like growth factor 1 (IGF-1) levels and their association with growth and development in infants aged 1-24 mouths.</p><p><b>METHODS</b>A total of 525 healthy infants (125 preterm, 400 term) were enrolled. Serum IGF-1 levels were measured using ELISA 1.5, 4, 6, 8, 12, 18 and 24 months after birth. The body weight and body length were simultaneously measured.</p><p><b>RESULTS</b>Serum IGF-1 levels were the lowest in preterm infants 1.5 months after birth (86+/-60 ng/mL). Thereafter, serum IGF-1 levels increased, and were significantly higher than those in term infants between 4 and 12 months after birth. Serum IGF-1 levels in term infants were the highest (116+/-52 ng/mL) 1.5 months after birth during their life of 12 months old. Thereafter, serum IGF-1 levels decreased and reached to a nadir (69+/-58 ng/mL) 8 months after birth. IGF-I levels were positively correlated with the weight and the height (SDS) in both preterm and term infants.</p><p><b>CONCLUSIONS</b>Serum IGF-1 levels are closely associated with growth and development in infants.</p>


Subject(s)
Female , Humans , Infant , Infant, Newborn , Male , Body Height , Body Weight , Child Development , Infant, Premature , Insulin-Like Growth Factor I
7.
Journal of Southern Medical University ; (12): 1837-1839, 2009.
Article in Chinese | WPRIM | ID: wpr-336072

ABSTRACT

<p><b>OBJECTIVE</b>To identify and characterize the polypeptides specifically binding to human B type natriuretic peptide (BNP) screened from 12TM phage display peptide library.</p><p><b>METHODS</b>The BNP-binding peptides were screened from 12TM phage display peptide library and identified by ELISA.</p><p><b>RESULTS</b>After 4 rounds of screening, 10 of the 16 phage clones were identified as the positive clones which could bind to BNP. Five amino acid sequences were obtained in the 10 positive clones. Dose-dependent ELISA results demonstrated that the screened polypeptides could specifically bind to BNP.</p><p><b>CONCLUSION</b>These screened polypeptides can bind specifically to BNP, which provides a basis for further research on expression and purification of anti-BNP polypeptides and the development of the detection kit of BNP.</p>


Subject(s)
Humans , Natriuretic Peptide, Brain , Metabolism , Peptide Library , Peptides , Metabolism , Protein Binding
8.
China Biotechnology ; (12)2006.
Article in Chinese | WPRIM | ID: wpr-686095

ABSTRACT

Chemokine receptor 5 (CCR5 ),as a member of the G protein-coupled receptor(GPCR)superfamily,is a membrane protein on cell surface and one of the major of coreceptors for HIV-1infection.CCR5 has became a molecular target for the novel drugs against HIV-1,and antagonists for CCR5 could be grouped as following, chemokine derivatives,small molecule non-peptide compounds,molecular antibodies and peptides. These compounds with high anti-viral activity and affinity are at different stages, and some have been under clinical studies. Therefore, the development research in the different kind of CCR5 antagonists is reviewed.

9.
China Biotechnology ; (12)2006.
Article in Chinese | WPRIM | ID: wpr-685597

ABSTRACT

Survivin is a protein that inhibits apoptosis and regulates cell division.The cDNA sequence of survivin was amplified by RT-PCR and sub-cloned into the prokaryotic expression vector pET-21b(+),followed by transformation into E.coli strain BL21(DE3) and induction with IPTG.The recombinant survivin protein fusing with 6?His tag was expressed in E.coli in the form of inclusion body at the expression level over 60% of the total cell protein.Results of Western blotting showed that recombinant survivin reacted specifically with anti-human survivin antibody.After gel filtration,the recombinant protein reached the purity over 95%,which facilitate the study of diagnosing and inhibitor agents targeting survivin.

10.
Acta Physiologica Sinica ; (6): 128-134, 2003.
Article in English | WPRIM | ID: wpr-318929

ABSTRACT

The purposes of this study was to determine the effects of recombinant human interleukin-10 (rhIL-10) on proliferation of vascular smooth muscle cells (VSMCs) stimulated by advanced glycation end products (AGE) and neointima hyperplasia after rat carotid arterial injury. Rat aortic VSMCs were cultured and treated with rhIL-10 or AGE respectively, and then co-treated with rhIL-10 and AGE. Proliferation of VSMCs was quantified by colormetric assay. Cell cycle analysis was performed by flow cytomertry. Sprague-Dawley rats were treated with recombinant human IL-10 (rhIL-10) for 3 d after carotid arteries injury. The ratio of neointima to media area at the site of arterial injury was measured 28 d after balloon injury. The p44/42 MAPK activity was evaluated by the immunoblotting technique using anti-p44/42 phospho-MAPK antibody. Compared to control, AGE stimulated VSMCs proliferation. rhIL-10 alone had no effect on VSMCs growth. With AGE stimulation, rhIL-10, at dose as low as 10 ng/ml, inhibited VSMCs growth (P<0.05). The cell number in G(0)/G(1) phase of AGE and rhIL-10 co-treatment group was higher than that of AGE treatment alone (P<0.01) by flow cytometry analysis. Compared with the control group of neointima hyperplasia in rats, the ratio of neointima to media area of recombinant human IL-10 group was reduced by 45% (P<0.01). The p44/42 MAPK activity was significantly enhanced by AGE. The AGE effects were opposed by rhIL-10. The anti-inflammatory cytokine rhIL-10 inhibits AGE-induced VSMCs proliferation. Recombinant human IL-10 also inhibited neointima hyperplasia after carotid artery injury in rats. The results suggest the possibility that recombinant human IL-10, as a potential therapeutic approach, prevents neointimal hyperplasia.


Subject(s)
Animals , Male , Rats , Aorta, Thoracic , Cell Biology , Atherosclerosis , Carotid Artery Injuries , Pathology , Carotid Intima-Media Thickness , Cell Proliferation , Cells, Cultured , Glycation End Products, Advanced , Pharmacology , Hyperplasia , Interleukin-10 , Pharmacology , Muscle, Smooth, Vascular , Cell Biology , Myocytes, Smooth Muscle , Neointima , Drug Therapy , Rats, Sprague-Dawley , Recombinant Proteins , Pharmacology , Tunica Intima , Pathology
11.
Acta Physiologica Sinica ; (6): 79-82, 2002.
Article in Chinese | WPRIM | ID: wpr-272979

ABSTRACT

Vessel injury provokes a release in proinflammatory cytokines that influence vascular smooth muscle cell (VSMC) proliferation. The purposes of this study was to determine the effects of recombinant human interleukin-10 (rhIL-10) on rat vascular smooth muscle cell proliferation and the activity of p44/p42 mitogen-activated protein kinase (MAPK) promoted by tumor necrosis factor-alpha (TNF-alpha). Rat aortic VSMCs were cultured and treated with rhIL-10 or TNF-alpha respectively, and then cotreated with rhIL-10 and TNF-alpha. The proliferation of VSMCs was quantified by colormetric assay. Cell cycle analysis was performed by flow cytometry. The p44/42 MAPK activity was evaluated by the immunoblotting technique using anti-p44/42 phospho-MAPK antibody. Compared to control group, TNF-alpha stimulated significantly VSMC proliferation in TNF-alpha group. rhIL-10 alone had no effect on VSMC growth, but significantly inhibited VSMC proliferation induced by TNF-alpha at a dose of 10 ng/ml. The cell number in G(0)/G(1) phase of TNF-alpha and rhIL-10 co-treatment group was higher than that of TNF- alpha group (P<0.01) by flow cytometry analysis. The p44/42 MAPK activity was significantly enhanced by TNF-alpha and the TNF-alpha effect was opposed by rhIL-10. It is suggested that rhIL-10 can inhibit TNF-alpha induced VSMC proliferation and phosphorylation of p44/42 MAPK.


Subject(s)
Animals , Male , Rats , Cell Cycle , Cell Division , Cells, Cultured , Interleukin-10 , Pharmacology , Mitogen-Activated Protein Kinases , Muscle, Smooth, Vascular , Cell Biology , Rats, Sprague-Dawley , Recombinant Proteins , Pharmacology , Tumor Necrosis Factor-alpha , Pharmacology
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