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19ginseng treatment group rats serum hormone levels closer to normal control group than POF model group; © Results of qRT-PCR indicated that the expression levels of HPGDS etc. 3 genes were correlated positively with ovarian aging; the expression levels of PAPPA etc. 6 genes were negative correlated with ovarian failure. These results were consistent with our microarray results.
Conclusion: American ginseng can alleviate ovarian failure, reduce VCD on ovarian damage. HPGDS etc. selected genes may serve as potential biomarkers applied to clinical diagnosis and prognostic evaluation of POF. References:
[1] S. Chatterjee, D. Modi, A. Maitra, S. Kadam, Z. Patel, J. Gokrall, and P. Meherji, Screening for FOXL2 gene mutations in women with premature ovarian failure: an Indian experience. Reprod Biomed Online 15 (2007) 554-560.
[2] Matthews, K.A., et al., Are changes in cardiovascular disease risk factors in midlife women due to chronological aging or to the menopausal transition? [J]. J Am Coll Cardiol, 2009. 54(25): p. 2366-73.
[3] Rogers, N.H., et al., Loss of ovarian function in mice results in abrogated skeletal muscle PPARdelta and FoxOl-mediated gene expression [J]. Biochem Biophys Res Commun, 2010. 392(1): p. 1-3.
[4] Janssen, I., et al., Menopause and the metabolic syndrome: the Study of Women's Health Across the Nation [J]. Arch Intern Med, 2008. 168(14): p. 1568-75.
[5] Pietschmann, P., et al., Osteoporosis: an age-related and gender-specific disease--a minireview [J]. Gerontology, 2009. 55(1): p. 3-12.
[6] M. Ashburner, C.A. Ball, J.A. Blake, D. Botstein, H. Butler, J.M. Cherry, A.P. Davis, K. Dolinski, S.S. Dwight, J.T. Eppig, M.A. Harris, D.P. Hill, L. Issel-Tarver, A. Kasarskis, S. Lewis, J.C. Matese, J.E. Richardson, M. Ringwald, G.M. Rubin, and G. Sherlock, Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 25 (2000) 25-29.
EffectofTangXinKang on Diabetic Cardiomyogathy Rats with Myocardial
Tissues Gene—annexinA4 Expression
Wang Bing Jiang De-youA Liu Chun-hong Liu Cheng-gang Guo Jia-li Chen Qiang Jiang Zheng-long Zhang Hai-li
Heilongjiang University of Chinese medicine,Neijing Department ,Harbin,China
Abstract:Objective:To study the difference ofmyocardial tissues gene-expression between diabetic rats and rats treated by herb prescription. Tomeasure annexin A4 mRNA-expression ofmyocardial tissueswith eve green real time RT-PCR.Methods:(1) Choice the different expression gene; (2) Measure the productionwith Eve green real timeRT-PCR.Results:Getdifferent expressionGene-annexinA4. The expression ofdiabetic cardiomyopathy diabetesmellitusgene-annexinA4 is decreased, while the expression of ratsmyocardialgene- annexinA4 mRNA treated byTangxinkang is in-creased.Conclusion:The study reported one gene-annexinA4mRNA of ratmyocardialgene expression profiles. Myo-cardial tissues gene- annexinA4 mRNA is also expressed when suffering from diabetic and the number of expression is lessthan normal. While TangxinKang canmake the numberof this gene expression increase. This is one ofprotectionmechanisms of diabeticmyocardial tissues damage therapied by herb prescription. Key words: Tangxinkang Diabetitesmellitus Myocardial damange AnnexinA4
Myocardial damage diabetes diabetes (DM) is one of the most common chronic complications, the disease may be primary, but also secondary to coronary blood disorder, and
myocardial lesions appeared earlier, mainly for cardiac diastolic and systolic dysfunction, prone to congestive heart failure. The exact pathological mechanisms are not yet completely clarified.There is no clinical effects of drugs, and the prevention and treatment of the disease in terms of Chinese medicine is promising.Because of its complex pathological mechanisms, the state description and herbal compound multi-component, multi-link, multi-target mechanism of action.In addition the traditional single-factor theory and research methods are difficult to solve, this means of molecular biology research methods, using gene chip technology, the use of internationally recognized academic currently produced by Affymetrix GeneChip Rat 2302.0 find diabetic cardiomyopathy disease-related genes to explore the molecular mechanisms of diabetic cardiomyopathy, candy hearts interpret traditional Chinese medicine treatment of diabetic cardiomyopathy Kang mechanism of action, right in the heart of STZ-induced DM rats were studied gene expression profiles, and screened differentially expressed genes Live with Eve green fluorescence was detected by RT-PCR in rat cardiac tissue content and expression level, this paper reports only sugar Xin on diabetic cardiomyopathy myocardial tissue gene expression of annexin A4mRNA.
1 Materials and methods
1.1 Reagents and drugs
Streptozotocin (STZ, CALBIOCHEM, USA); Tang-Xinkang Heilongjiang University of Chinese Medicine, First Affiliated Hospital of preparation room production; Rat 2302.0 gene chip (AFFYMETRIX, USA).
1.2 Animal model and grouping
Male Wistar rats,weighing 200 ~ 230 g, were given 25 mg • kg-1 STZ left intraperitoneally injected once every morning, continuous 5d except the blank group. 12h fasting before modeling can not help but water; modeling 1h, the rats free access to water, food; modeling 1W measured after the blood sugar level in 16.7mmol / L or more, are considered successful modeling. Then randomly divided into model group, treatment group, both groups were fed a high-calorie diet (high-calorie diet from 1% cholesterol, 10% lard, 10% egg yolk powder, 1% sucrose, 78% of the basal diet made) 13W, Total feeding 26W.
1.3 Method of administration
Treatment group intervented by Tang-Xinkang,while the blank group and model group with saline.
1.4 Specimen Collection
Quick with 180 °C, 12 h and treated surgical scissors removal of the heart, immediately rinse with cold saline, rinsed in ice operations, cut along the long axis of ventricular myocardium, frozen in liquid nitrogen to prepare extracts using RNA.
2 Myocardial Screening of differentially expressed genes
2.1 Extraction of total RNA
the heart of rats was harvested, then using QIAGEN's RNA easy Total RNA Isolation Kit extraction of total RNA, total RNA synthesis by the purified double-stranded cDNA, by PLG extraction, ethanol precipitation, the double-stranded cDNA was purified with a BioArray High Yield RNA Transcript Labeling Kit methods biotinylated cRNA synthesis, QIAGEN Rneasy Columns transcribed in vitro by purified biotinylated cRNA, RNA concentration by spectrophotometric analysis of a quality control, gel electrophoresis IVT product, fragmented cRNA, synthetic cRNA probes.
2.2 Microarray hybridization, elution, staining and detection
The synthesized cRNA probes used by fragmentation after treatment with gene microarray. Affymetrix Rat2302.0 gene chip microarray chip hybridization, elution, staining and detection using Affymetrix company produces special equipment "Microarray Workstation" (work station) and the operating process according to the conditions recommended by proceed.
2.3 Microarray data processing
Microarray data applications "Affymetrix Microarray Suite software 5.0" for data processing.
2.4 Screening criteria Note: differential gene screening criteria for the change as I or MI, ratio> or = 1, the experimental group Detection of P for up-regulated genes, change to D or MD, ratio <or = -1,
Detection of P in the control group is down-regulated genes.
Scatter X-axis signal value for the control group, Y axis signal values for the experimental group, the figure of the red dot experimental group and control group are the gene Detection P, blue has a value other than the two groups P, and yellow dots to both groups a (Fig. 1).
Figure 1 a. Model group / control group grayscale scans; b. Model group / control group scatterplot;c. treatment group / model group grayscale scans; d. treatment group / model group scatterplot.
In accordance with the more stringent conditions for screening differentially expressed genes differentially expressed gene lists more than 2 times, including gene name, GenBank number, Uni number and chromosomal localization. Which, BM385237: annexin A4(anxa4), in diabetic rat heart downregulated, candy hearts Kang upregulated treatment groups.
3 Results
3.1 Fasting glucose, glycated hemoglobin levels (see Table 1) Experimental results show that, fasting blood glucose (FBG) and glycosylated hemoglobin (HbA1C) levels were significantly elevated compared with the normal group.There is significant differencebetweentreatment group and model group (P <0.01), but is still high compared with the normal group. Description candy hearts Kangyou Liang good hypoglycemic, reducing non-enzymatic protein glycosylation role. Table 1The level of the fasting FBG and HbA1c (x ± s)
Group n FBG(mmol/L) HbA1c(%)
Normal group 10 5.19±0.49 3.75±0.51
Model group 10 20.53±3.55* 8.62±1.77*
Treatment group 10 10.74±2.54A 6.86±1.10A
Note: Compared with normal group * P <0.01 compared with model group AP <0.01
3.2Fluorescence quantitative RT-PCR for the expression of annexin A4mRNA myocardial tissue detection
Using Eve green method, sense primer: 5 \ CAAACACACGTGGTCTGCCTA-3 \, length: 21 bp, Tm value: 58 °C; anti-sense primer: 5 \ TTGTACCCTGCCACCATTTTC-3 \, length: 21 bp, Tm value: 58 ° ; product length: 101 bp, product Tm value: 78 ° . 3.2.1 RNA samples reverse transcription
Use TAKARA's RNA PCR kit reverse transcription kit, according to the reaction system provided with the kit for reverse transcription reaction. Reverse Transcription System 10 ^L, 30 °C reaction
10 min, 42 °C reaction 30 ~ 50min, 95 °C heat 5 min, 4 °C reaction 5 min aborted. 3.2.2 Myocardial tissue expression levels of annexin A4mRNA test results (Table 2) In real-time quantitative PCR technique, in order to quantify and compare the convenience, the introduction of fluorescent threshold (threshold) and CT (threshold value) value concept. Fluorescence threshold is set artificially fluorescence amplification curve of a value that can be set in the exponential amplification phase of the fluorescence signal anywhere, CT value of the relationship with the starting template CT value of each template with the template starting copy number of the linear relationship between the number, starting from the more copies, CT value is smaller. Model rats myocardial tissue annexin A4mRNACT value increases, indicating that annexin A4mRNA reduction in cardiac tissue expression, with a significant difference between the control group P <0.05, and after treatment by Tangxinkang .
Myocardial tissue annexin A4RNA CT value downward, indicating sugar can increase heart health annexin A4mRNA expression in cardiac tissue, compared with the model group were significantly different P <0.05.
Table 2 Expression levels of annexin A4mRNA groups Comparison
Group n CT Mean
Normal group 10 21.7292
Model group 10 28.9007^
Treatment group 10 23.7969A
Note: the treatment group compared with model group AP <0.05; model group and blank group ^ P <0.05.
4 Discussion
In this study, the "Golden" Treatment of Diabetes Pathology Act and related expositions, and clinical experience, according to Qi and blood stasis disease intricate network-based nature of the lesions, should advocate with the syndromes associated with multiple methods, thus identified as a nourishing blood disease main treatment method, developed traditional Chinese medicine Tangxinkang by heterophylla, Astragalus, Radix, Polygonatum, wolfberry, salvia, Eupolyphaga other drugs. Prescription to participate Astragalus Prince compatibility, enhanced Qi effect, together wolfberry, Radix, Polygonatum total revenue gas Yin of power. Salvia Ng woodlouse, enhance blood circulation of power. Consistency of various drugs, complement rather than stagnation greasy, pass without injury, AIDS and not help the wet, warm but not Shangyin, played a total of nourishing, meridians circulation effect.
BM385237: annexin A4, annexin A4 (anxa4). Annexin family (annexins) are a class of Ca2 + may be further combined with a calcium-dependent phospholipid binding membrane phospholipid binding proteins, to date, from fungi, protists, to plants, higher vertebrates other than 65 different species ranges , which has been found in more than 160 kinds of annexins member. In mammals, there are 12 species, named ANXA 1-A 11, A 13, which is widely present in the cell membrane, the reservoir near a Ca2 + cells and in the nucleus or in the extracellular matrix. From the immunological cross-reaction and protein sequence analysis and cDNA that are similar in structure, function also has many of the same points. Annexins family are highly conserved C-terminal domain and a unique center of the N-terminus. Center is a homologous repeats 4-8 form a slight bend, disk structure having uneven sides, each repeat sequence is about 70 amino acids in five a-helix domain closely connected. C-terminal proteolytic sites, phosphorylation sites and the binding sites of other proteins, the annexins regulatory regions. Annexins N-terminal sequence of the members of different lengths, only the protein kinase phosphorylation sites similar to the sequence. Experiments prove that the N-terminal discoid slight change does not affect the overall structure, but the functional impact of convex Ca2 +-dependent phospholipid binding function, thereby changing its molecular characteristics [1]. The biological function for annexins currently just some preliminary studies, they play in the cell such as the inhibition of phospholipase A2 activity, participation in cytoskeleton activities involved in phospholipid membrane receptor function and
regulation, anticoagulation, conduction mitosis information, and promote cell secretion and other important biological functions. Early studies focused on annexins involved in the inflammatory mechanism in its response, in recent years, more and more studies have found that in the process of tumor development and may play an important role, such as mediated apoptosis and differentiation. And with annexin A1, annexin A2, annexin A5 related reports more, while traditional Chinese medicine for diabetes state regulation of annexin A4 no special report. Annexin A4 (anxa4), belonging to annexin family, whose gene is located on 2p13, the protein exists in various tissues and organs of animals and plants, accounting for the total amount of intracellular protein 2%. Which is a Ca2 +-dependent phospholipid binding protein, a phospholipase inhibitor, combined with calcium ions, and calcium-dependent phospholipid binding activity can promote membrane fusion, in cyst transport, a calcium ion channel-forming, cytoskeletal activity, phospholipid and regulation and function of membrane receptor endocytosis, exocytosis and secretion of other important biological functions [2]. Recently reported in the literature annexins A4 may regulate cell growth and differentiation, regulation of apoptosis and other biological functions [3], and these biological behavior disorders may be associated with diabetes incidence of myocardial injury has a strong correlation. Studies have shown that annexins A4 can inhibit the intracellular Ca2 + levels, the maintenance of intracellular Ca2 + levels of stability [4], thus avoiding myocardial contractile dysfunction, but also may increase the resistance of cells to apoptosis.
The experimental results show that annexins A4 gene chip in the diabetic state downregulated myocardial tissue, suggesting that diabetic cardiomyopathy may occur with annexins A4 gene regulation related to its mechanism may be that: (1) regulation of calcium imbalance caused by myocardial contractile dysfunction, cause or aggravate diabetic cardiomyopathy myocardial injury; (2) is not conducive to the formation of the skeleton structure of myocardial cells; (3) myocardial cell growth, differentiation or apoptosis causes abnormal myocardial injury. 5.Conclusions
Our results showed that diabetic cardiomyopathy annexins A4mRNA expression was low.However ,its expression increased after treatment indicating candy hearts. Concord diabetic rat heart by upregulating the expression of annexins A4mRNA protect cardiac tissue , delaying further development of the disease.
References
[1] Gerke V, Moss SE.Annexins :From Structure to Function [J]. Physiol Rev.2002,82(2):331.
[2] Mattin I,Dominguez F,Avila S,etal. Human endometrial receptivity,gene regulation[J]. Reprod Immunol.2002,55:131-139.
[3]Piston P, Ferrari D, Rapizzi E,etal The Ca2+ concentration of the endoplasmic reticulum is a key determinant of ceramide-induced apoptosis:significance for the molecular mechanism of Bcl-2 action. [J].Embo .2001,20(11):2690-2701.
[4]OkuseK,Malik-HallM,Baker MD,etal.Annexin II light chainregulates sensory neuron-specific sodium channel expression. [J] Nature.2002,417(6889):653-656.
The Animal Model of Insulin Resistance Induced by High-fat Diet
SONG Ying-xing
(Heilongjiang University of Chinese Medicine,Harbin,150040,China)
Abstract: Insulin resistance (IR) is type 2 diabetes mellitus (T2DM), impaired glucose tolerance(IGT), hyperlipidemia, hyperuricemia, cardiovascular disease and metabolic syndrome common disease foundation. In lab studies, the establishment of animal model of insulin resistance is the primary condition, The resistance way about it can be divided into four kinds: genotype, drug injection type, diet induced type, various ways mixed. This article mainly discusses on the type high
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