The Effect Of Acupuncture On Gene Expression Profile Of Rat Pre-stress Hypertensive Rats

Hypertension,the most common chronic disease and the most important independent risk factor for cardio-vascular diseases,exists extensively all over the world and threatens the health of human beings severely.Pathogenesis of hypertension correlates closely to environmental factors.Prolonged stressful stimulus could induce the onset/progression of hypertension.Cardiovascular hyperfunction such as an increase of heart rate and a rise of blood pressure might occur in animals or humans who are exposed to prolonged stress,and it tends to progress gradually into stress-induced hypertension.The pathogenesis of hypertension,clinically speaking,usually goes through a slow process starting from normal blood pressure(120 mmHg/80 mmHg)and developing into phase I hypertension(≥140 mmHg/90 mmHg).The concept of prehypertension was first proposed in the Seventh Report of the Joint National Committee on Prevention,Detection,Evaluation,and Treatment of High Blood Pressure(JNC7)in 2003.Prehypertension refers to a stage where the systolic pressure(SP)ranges from 120 to 139 mmHg and/or the diastolic pressure ranges from 80 to 89 mmHg.A large amount of epidemiological evidence suggested that the prevalence of prehypertension was up to 30-50%in some countries.The pathogenesis of stress-induced hypertension also goes through a prehypertension phase whcih called stress-induced prehypertension(SIPH)in the development from a physiological state to a pathological one.Prehypertension is often complicated with traditional risk factors for cardiovascular diseases,such as dyslipidemia and abnormal glucose metabolism,and correlates closely to morbidity in cardiovascular diseases.In the stage of SIPH,potential subclinical damage of target organs and alterations of the expression of corresponding genes and proteins could occur.Timely intervention against SIPH is expected to effectively suppress the occurrence of hypertension,protect target organs and lower the incidence of cardio-cerebral-vascular diseases.Acupuncture,an alternative to drug,is of prominent advantages of convenience,security and less adverse reactions,and has been used to treat multiple cardiac-vascular diseases including hypertension.Hypertension is a polygenic disease that is regulated by multiple genes.Heart is the target organ most vulnerable to hypertension.Acupuncture could reduce blood pressure and protect the heart through its regulation on specific genes’ expression.It has been found in our pilot studies that the abnormal function of vascular endothelium and myocardial damage exist in SIPH modeling rats,and acupuncture may regulate effectively blood pressure of SIPH rats,influencing vascular endothelial factors such as nitric oxide,acetylcholine and neuropeptide Y,and protecting vascular endothelial functions.In this research,gene chip technology was used to observe the effect of acupuncture on the gene expression profiles of rats’ myocardium of SIPH.Objective:To evaluate the effect of acupuncture on genetic expression in the myocardium of rats of SIPH.Thereby revealing the mechanism of the biological effect of acupuncture regulating SIPH,and providing a new approach for prophylaxis and treatment of SIPH.Methods:"Experiment 1":The Effect of acupuncture on the blood pressure and heart tissue morphology of SIPH rats36 Wistar rats were randomized into three groups:the control group,model group and model + acupuncture(AP)group(n = 12/group).During the 11-day modeling period,the SIPH rats were prepared by plantar electric stimulation in combination with noise exposure,and daily acupuncture intervention was applied simultaneously on the model + AP group.Systolic pressure(SP)was measured on the day before modeling,and and on the 3rd,5th,7th,9th,11th day after modeling.On the 12th day histopathological observation of the left ventricle was made with hematoxylin-eosin(HE)staining"Experiment 2":The effect of acupuncture on the gene expression profiles in the myocardium of SIPH ratsGene Chip Rat Gene 2.0 Sense Target was used to detect the gene expression profiles in myocardium.The two screening criteria of differentially expressed genes(DEGs)were "P<0.05" and " | fold-change |>1.5".Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were respectively used to find out the differential expression genes related main biochemical metabolic and the significant pathways between the 3 groups.As to reveal the mechanism of the biological effect of acupuncture regulating SIPH in the different pathogenetic phases,and providing a new approach for prophylaxis and treatment of hypertension."Experiment 3":Effect of acupuncture on genetic expression of HbAlc,Igfbp-3,Pik3ca and Car4 in the myocardium of SIPH ratsThrough an extensive literature search and NCBI search,HbA1c,Igfbp-3,Pik3ca and Car4 which were correlated closely to the the pathogenesis of SIPH and acupuncture treatment for SIPH,were screened out from the 49 common DEGs and regarded as the key genes.And using real-time PCR technology to validate the selected key genes.Through comparing the changes of genetic expression between the three groups,we could explore the regulation mechanism of acupuncture on SIPH.Results:"Experiment 1":During the whole experiment,the SP kept increasing in the model group on the 3rd,5th,7th,9th,11th day with notably significant difference(all,P<0.01)when compared with the data of the control group,which maintained normal levels.During model establishment,the rats were easily irritable with symptoms/signs of screaming,biting,yellow urine,dry stool,coarse hair and bloodshot eyes.Significant alteration occurred in behaviors and signs of rats in the model group,with series of turbulences and defensive performances.In the model group,disorder arrangement of cardiac cells was observed with morphological changes of some of the nucleus including their enlargement or disolution.There were also infiltration of inflammatory cells and proliferation of collagen fibers.All these suggesting the successful establishment of the model.Compared with the model group,the SP of the model+MA group on the 5th and 7th day decreased by a notably significant difference(all,P<0.01),and on the 9th and 11th day by a significant difference(all,P<0.05).These findings illustrated the curative effect of acupuncture at Taichong(LR3)and Quchi(LI11)in lowering the SP of SIPH subjects.In the model + AP group,most of the myocardial cells were in normal structure,and only part of the cells had morphological changes with enlarged nucleus or disolution.The degree of myocardial damage was lower significantly in the model + AP group than in the model group,suggesting acupuncture might ameliorate the pathologic development of cardiac lesion in SIPH rats."Experiment 2":Microarray analysis screened out 192 DEGs in model group compared with control group,including 126 down-regulated and 66 up-regulated.Compared with model group,169 DEGs in model + MA group,including 37 down-regulated and 132 up-regulated.In total,42 genes that were down-regulated in model group versus(VS)control group,were found to be up-regulated in model + MA group VS model group.In addition,7 up-regulated genes in model group VS control group were down-regulated in model + MA group VS model group.So these 49 genes were common DEGs of the three groups.Through an extensive literature search and NCBI search,HbA lc,Igfbp-3,Pik3ca and Car4 which were correlated closely to the the pathogenesis of SIPH and acupuncture treatment for SIPH,were screened out from the 49 common DEGs and regarded as the key genes.Go functional enrichment analysis suggested that the main biological functions of DEGs in model group VS control group were rhythmic process,circadian rhythm,translational elongation,ribosome,etc.The main biological functions of DEGs in model + MA group VS model group were cytosolic ribosome,ribosomal subunit,translational elongation and cytosolic part,etc.KEGG pathway analysis suggested that 6 KEGG pathways were both activated by the DEGs in model group VS control group and the DEGs in model + MA group VS model group.Through an literature search and GenomeNet,KEGG PATHWAY Database search,we found that rno04115:p53 signaling pathway correlates to the pathogenesis of hypertension and heart diseases.Therefore we speculated that rno04115:p53 signaling pathway maybe the key pathway for the regulation of acupuncture on SIPH."Experiment 3":As compared with the control group,expression of HbAlc and Car4 in model group increased significantly(P<0.01),but the expression of Igfbp-3 and Pik3ca decreased significantly(P<0.01,P<0.05).Compared with the model group,expression of HbA1c and Car4 in model + MA group decreased significantly(respectively P<0.01,P<0.05),while expression of Igfbp-3 and Pik3ca increased significantly(P<0.01,P<0.05).The RT-PCR results of the key genes were in consistent with the microarray data,indicating that microarray data was reliable.These results suggested the mechanism of acupuncture lowering blood pressure maybe relate to its regulation effect on the genetic expression of HbA1c,Igfbp-3,Pik3ca and Car4.Conclusion:1.Acupuncture at Tai chong(LR3)and Qu chi(LI11)can significantly decrease the blood pressure of SIPH rats and has protective effects on target organs of SIPH rats.2.Acupuncture at Tai chong(LR3)and Qu chi(LI11)could significantly affect the gene expression profile in the myocardium of SIPH rats.3.The mRNA abnormal expression of HbA1c,Igfbp-3,Pik3ca and Car4 genes plays an important role in the process of molecular biology mechanism of the early to middle stages of SIPH.The acupuncture’s anti-hypertensive mechanism may be involved in regulating the expression of the key genes and their related signaling transduction pathways.