| Objective:Use gene chip technology to screen the related target gene of steroid hormone metabolism in rats,which preconditioned by acupuncture at Jiaji points in myocardial ischemia reperfusion injury(MIRI).And use Real Time-PCR method to validate the reliability of the related target gene,to provid Genetics evidence for the protection of acupuncture preconditioning at Jiaji points in MIRI in Rats.Methods:30 rats were divided into 3 groups:blank group,model group,Jiaji group.Ten rats for each group.Jiaji group were pretreated for 7 days.Then used the model group and Jiaji group to copy MIRI model with the method of ligating the anterior descending coronary artery.Removed the cardiac muscle tissue of ischemia reperfusion injury,and the total RNA was isolated from the tissue by Trizol,and kept at low temperature.Fluorescent probe labeled each group,and hybrided with Agilent rat 4x44k gene expression profile chip.Scanned the image,and analysed the date.Then compared blank group with model group,and model group with Jiaji group.Screened the related target gene of steroid hormone metabolism in rats,which preconditioned by acupuncture at Jiaji points in MIRI.Designed primers of the related target gene of steroid hormone metabolism in rats,which preconditioned by acupuncture at Jiaji points in MIRI.Each of these three group’s total RNA of cardiac muscle tissue of ischemia reperfusion injury was reverse-transcripted into cDNA.Configured the Real Time-PCR system and hatched,detected the mRNA expression,and validate the reliability of the related target gene.Results:Electrophoresis run in denatured agarose gel,and detection with nucleic acid protein analyzer showed that the quality of the total RNA isolated from these rats’ cardiac muscle tissue by Trizol is good for further use.The signal intensity of gene chip scan image is high and correspond to the standards of data acquisition.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)were respectively used to find out the 4 differential expression genes related to steroid hormone metabolism.They were CYP11B2、CYP1B1、HSD3B1、UGT1A9.Compare to blank group,these 4 genes were up-regulated in model group.Numerical values are as follow(model group/blank group):CYP11B2 is 1.73,CYP1B1 is 2.33,HSD3B1 is 3.66,UGT1A9 is 3.45.After preconditioned by acupuncture at Jiaji points,compared to the model group,these 4 genes were down-regulated in Jiaji group.Numerical values are as follow(Jiaji group/model group):CYP11B2 is 1.36,CYP1B1 is 1.43,HSD3B1 is 3.27,UGT1A9 is 2.22.KEGG signaling pathway analysis revealed that the protein coded by CYP11B2 is aldosterone synthase which is key enzyme of aldosterone synthesis,the protein coded by HSD3B1 is 3-Beta-Hydroxy-Delta(5)-Steroid Dehydrogenase which is related to the myocardial local aldosterone synthesis,the enzyme protein encoded by CYP1B1 is an important member of the cytochrome P450 superfamily which can oxidize 17beta-estradiol and synthesize 6-beta hydroxy testosterone,and UGT-9 encoded by UGT1A9 plays a part in inactivate and control the estrogenic activity.Real Time-PCR have produced results essentially in agreement with the results of gene expression chip.The mRNA expression of CYP11B2,CYP1B1,HSD3B1,and UGT1A9 regularly increases in sequence of blank group,Jiaji group,and model group.Compare to blank group,in model group the mRNA expression of CYP11B2,CYP1B1,HSD3B1,and UGT1A9 of cardiac muscle tissue of ischemia reperfusion injury significant increased(P<0.01).Compare to model group,in Jiaji group the mRNA expression of CYP1132,CYP1B1,HSD3B1,and UGT1A9 of cardiac muscle tissue of ischemia reperfusion injury significant decreased(P<0.01).Conclusion:During ischemia reperfusion,the up-regulated genes,CYP11B2,CYP1B1,HSD3B1 and UGT1A9,are related to myocardial regional Steroid Hormone Metabolism.After acupuncture preconditioning at Jiaji points,those 4 genes significantly down-regulated.It shows that the possible protection mechanism of the acupuncture preconditioning at Jiaji points in myocardial ischemia reperfusion injury are myocardial regional aldosterone synthesis,regulate the activity of estrogen hand androgen. |
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