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23Basing genome-wide expression profiling technology identifies premature ovarian failure potential molecular biomarkers and pharmacodynamics study of American ginseng treats premature ovarian failure
Pengling Ge a' *, Jiaming Xie a' *, Jing Zhao a, Yuna Kan a, Yongxin Yan a, Dongwei Han a'#
department of Pharmacology, School of Basic Medical Sciences, Heilongjiang University of
Chinese Medicine, Harbin 150040, China
*These two authors contributed equally to this work.
#Corresponding authors: Dongwei Han, Department of Pharmacology, School of Basic Medical Sciences, Heilongjiang University of Chinese Medicine, 24 Heping Road, Harbin 150040, China; E-mail: handongwei516@126.com, Tel.: +86-451-82196314
Abstracts: Premature ovarian failure (POF) is a cryptogenic gynecological affliction defined as deprivation of ovarian physiological function before 40 years old by World Health Organization. Due to POF can accelerate the body aging, leading to infertility and a variety of serious diseases, research on POF is becoming more and more important. In this study, we established premature ovarian failure rat model induced by VCD; screening of differentially expressed gene in the ovarian tissue of POF rat and computational bioinformatics analysis; validated the results of microarray profiling and researched the pharmacodynamics of American ginseng treats premature ovarian failure.
Key words: Premature Ovarian Failure, VCD, Genome-wide Expression Profiling, Biomarker,American ginseng
Background:
Premature ovarian failure (POF), also known as premature ovarian insufficiency, is present known one of the important reasons in women aging, typical symptoms for POF are atrophyofovary, amenorrhea and menostasia, which affect roughly 1% of the population worldwide[1]. There are some direct evidences demonstrate that POF is closely related to the infertility of young women. More and more researches revealed that POF had been found to increase the risks of malignant complications, such as Alzheimer's disease, Cardiovascular disease, Osteoporosis, even cancer of the tissues or organs of reproductive system, and so on[2-5]. But, at present, the diagnosis, treatment and the cause for POF is still not clear, there is no effectively treatment for POF patients in the clinical process. Therefore, there is an urgent need to clarify pathogenesis and mechanism of POF, and to establish a complete set of POF diagnostic criteria and research effective therapeutic medicine of POF.
Part I: The establishment of premature ovarian failure rat model induced by VCD.
Objective: Establish suitable animal model of premature ovarian failure, and validated the consistency of manifestations of animal models and clinical POF women; success to gain dysfunctional ovary, as well as the material preparation for further genome-wide expression profiling analysis.
Methods: Wild-type adult female Sprague Dawley rats (SD, 2 months) were intraperitoneally (i.p.) injected with a dose of 4-vinylcyclohexene diepoxide(VCD) 80 mg/kg continuous 14 days, the equal volumes of sesame oil in vehicle. After the last injection, the estrus and general situation of rats was observed for a period of 5 days regularly. Rats continued to feed for 30 days, and then sacrificed to get the ovarian tissue and serum for detection. HE staining was used to determine the pathology analysis of ovarian tissue, and observed the situation of ovarian follicles at different levels. Estradiol (E2), Follicle Stimulating Hormone (FSH) and Luteinzing Hormone (LH) was determined available ELISA kit.
Results:® After intraperitoneally (i.p.) injection of VCD, POF model group rats shown body hair stiff and lackluster, chase and bite each other behavior increased significantly. ® Observation of oestrous cycle shown that the POF model group rats appeared oestrous cycle disorders within 30 days after the last VCD injection. © Ovarian tissue section showed, in POF model group rats ovaries, the number of primordial follicles and primary follicles were decreased significantly. ® Results of serum hormone factors shown that E2 level declined obviously, whereas FSH and LH levels considerably increased in POF model group rats.
Conclusion: The rat model induced by VCD successfully replicated the typical clinical symptoms of POF women, and conforms to the requirements of the experimental study on the stability and reproducibility of animal model.
Part II: Screening of differentially expressed gene in the ovarian tissue of POF rat and computational bioinformatics analysis
Objective: To detect the changes of gene expression levels, investigate the molecular mechanism and screening potential biomarkers of premature ovarian failure, to offer possible targets for research new therapeutic medicine of POF.
Methods: ©Using genome-wide expression profiling technology identifies preliminary screening differentially expressed gene in ovarian tissue of premature ovarian failure; ® Gene ontology (GO) databases analyses[6] were conducted on the obtained list of significantly different genes, and predict its function or possibility participate in regulation of biological processes.
Results: ® Results of gene expression microarray analysis suggested that 651 differentially expressed genes in the ovarian tissue of POF, of which 617 genes were up-regulated and 34 genes were down-regulated; ® Differentially expressed genes were sorted according to Gene Ontology annotations, up-regulated genes involved in GO biological processes displayed that prostaglandin biosynthetic and metabolic process, unsaturated fatty acid biosynthetic and metabolic process and sertoli cell development had the noteworthy enrichment score, down-regulated genes related to GO biological processes exhibited that developmental process, protein homotrimerization and response to wounding process had the significant enrichment score. © Results of GO analysis indicated that HPGDS etc. 9 differentially expressed genes may be involved in regulation of some important biological processes.
Conclusion: Our experimental results demonstrated that prostaglandin biosynthetic, positive regulation of apoptotic process and response to hormone stimulus and so on were tightly related to POF. As well as HPGDS etc. conspicuously differential expressed genes may be the key regulators which may play an important role in the occurrence and development of POF, may serve as potential biomarkers for POF.
Part III: The pharmacodynamics study of American ginseng treats premature ovarian failure and to validate the results of microarray profiling
Objective: To study whether or not American ginseng to play a role of treatment in POF process, and using qRT-PCR assay to validate the consistency of the selected potential biomarkers and the results of microarray profiling.
Methods: Adult female Sprague Dawley rats (SD, 2 months) were intraperitoneally (i.p.) injected with a dose of VCD (80 mg/kg) continuous 14 days, at the same time, treatment group drenched American ginseng (2.25g/kg), model group drenched equal volumes of normal saline. After the last injection, each group continued to drench for 30 days, in addition, normal control group do not do any intervention. Quantitative real-time PCR assay was used to detect the expression of the genes, which were significantly differential expressed and participated in the important function in POF ovary (potential biomarkers of POF), to validation the data of microarray analysis
Results: ® American ginseng treatment group rats showed body hair smooth, clean and tidy, activity of agile and stable mental state, compared with POF model group rats; ® American
ginseng 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:
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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
