Effect Of Electroacupuncture On Gene Expression Profiles In Heart Of Stress - Induced Hypertension Rats

Stress-induced hypertension refers to high blood pressure that caused by the long-term tension and stress.A moden life style with fast pace can cause psychological and emotional stress, which may develop into a gradual increase in hypertension. Many young adults may gradual develop into hypertension from pre-hypertension.Pre-hypertension is a transition stage during which the blood pressure rises from normal to diagnostic hypertension. In this stage, the systolic blood pressure ranges between 120-139 mm Hg and (or) diastolic blood pressure between 80-89 mm Hg.The study found that incidence of cardiovascular events in people with pre-hypertension in future time was as two times as people with normal blood pressure. It is also proved that stress-induced hypertension is preceded by damage of target organs like blood vessels, heart, brain, and changes of the expression of relative proteins and genes. More and more studies begin to focus on the treatment of pre-hypertension, studies showed that treatment in the pre-hypertension stage can significantly reduce admission rate and mortality of hypertensive patients, and enhance the protection of target organs of the disease.Electro-acupuncture therapy was widely applied in the treatment of various kinds of hypertension-related cardiovascular diseases in clinical practices.Lots of researches confirmed that acupuncture reduces blood pressure by immune system, nervous system, and vascular endothelial cell.Gene chip technology, characterized by high sensitivity and throughput, is a new method to analyze gene expression. It could detect multiple gene expression at the same time which provides a platform for the study of multiple genes influenced hypertension. However, previous researches on treatment of hypertension were more concentrated on anti-hypertensive treatment and protection of target organs, which are applied in the middle and late stages. Fewer researches were focused on gene expression and intervention of pre-hypertension.Objective:Around the theme of electro-acupuncture regulate blood pressure of stress-induced pre-hypertension and protect the heart. This paper in accordance with the principle of "prevention before disease", use the tress-induced pre-hypertension rats as model,treat them with electro-acupuncture, using the gene chip technology, bioinformatics analysis technology and RT-PCR technology to screening of differentially expressed genes, further select the differential expressed genes HSPA1A, HSPB1,OLR1, PLA2G4A, COX-2, FGF16, IGFBP3 closely related to hypertension to study. We will explore the acupuncture’s effect on stress-induced pre-hypertension, and try to systematically reveal its biological mechanisms, providing a new idea for the prevention and treatment of hypertension by acupuncture.Methods:"Experiment 1":The observation of electro-acupuncture’s effects on stress-induced pre-hypertension rats’ heart damage and blood pressure.thirty-three,9-week-old Specified Pathogen Free (SPF)Wistar male rats of 220±30g were randomly divided into 3 groups by random number table method:blank control group, model control group, and model+EA group (n=11/group).The stress-induced hypertensive rat (SWR) model was established by electric foot-shocks combined with generated noise.Molding cycle lasted for 14 days and EA intervene was applied on rats in model+EA group during model preparation. Taichong (LR3) is located in the dorsum of the foot, in the depression anterior to the junction of the first and second metatarsals. Quchi (LI11) is located at the proximal end of radius, in the depression lateral and anterior to the elbow joint. The needles (0.16mm*7mm, purchased from Zhong Yan Tai He Acupuncture Goods Co. Ltd.) were directly inserted into LR3 and LI11 bilaterally for about 1.5-2 mm and 4mm respectively. After the insertion, Hans LH202H was connected with Quchi and Taichong to form a circulation of 1mA with a frequency of 2 Hz. The needles were then withdrawn after a 20 minutes’ retention. The acupuncture intervention took once a day (from 5pm) since the first day of model preparation for 14 days. All the intervention was given by the same person. Blank control group and model control group:rats were immobilized in the same restrainer for 20 min without acupuncture intervention. The morphological changes of the heart and the blood pressure were observed after the application of electro-acupuncture on acupoints. Blood pressure was recorded one day before and at the 3rd,5th,7th,9th,11th,13th and 15th day after modeling."Experiment 2":The observation of electro-acupuncture’s effects on stress-induced pre-hypertension rats’ heart gene expression profile.Microarray analysis of stress-induced pre-hypertension rats’ heart was performed with Gene Chip Rat Gene 2.0 ST Array. The changed genes were selected by the criteria:P< 0.05 and Fold Changes ≥1.5. The DAVID 6.7b of DAVID Bioinformatics Resources (http://david.abcc.ncifcrf.gov/) was used to perform gene ontology (GO) enrichment analysis to find out the differential expression genes related main biological function between the groups. Pathway analysis was used to determine the significant pathway of the differential genes according to Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www. genome.adjp/kegg). To find out the differential expression genes related main signal pathway and biochemical metabolic pathways between the groups."Experiment 3":The observation of electro-acupuncture’s effects on stress-induced pre-hypertension rats’ heart expression of HSPA1A, HSPB1, OLR1, PLA2G4A and COX-2.According differentially expression genes of the two screening, to choose the genes closely related to hypertension:HSPA1A, HSPB1, OLR1, PLA2G4A and COX-2 for RT-PCR verification.Use the RT-PCR technology to confirm the heart expression of HSPA1A, HSPB1, OLR1, PLA2G4A and COX-2.Result:"Experiment 1":Systolic blood pressure of Wistar rats in blank control group stayed normal through the whole process of the experiment. Systolic blood pressure in model control group reached higher than 120 mmHg (P<0.01) at the 3rd day, and kept increasing to a significantly higher level when measured at the 3rd,5th,7th,11th,13th and 15th days during model preparation (p<0.01), compared with that of the blank control group. In the modeling process, model control group were of elevated blood pressure, irritation, squealing, gnawing, hard stool, yellow urination, rough hair and bloodshot eyes. With the value kept in the range between 120-139mmHg, The results of HE staining also showed the myocardial damage of model group, cells arranged in disorder, part of the nuclear changed morphologically, cell nucleus becomed bigger or dissolution, a gap is arranged between the muscle cells. Among the muscle fibers scattered bleeding, bleeding around the infiltration of inflammatory cells. the model was successfully established.Compare with model control group, systolic blood pressure in model+EA group remarkably decreased at the 5th,7th,9th,11th,13th and 15th days during EA intervention(p<0.05), indicating that EA at Taichong (LR3) and Quchi(LI11) can significantly lower the blood pressure of stress-induced pre-hypertension rats with better short-term effect"Experiment 2":Gene microarray analysis showed that there were 390 gene expressions changed in model control group compared to blank control group. Of these,182 were down-regulated and 208 were up-regulated. EA intervention changed 330 gene expressions compared with model control group. Among these genes,169 were down-regulated and 161 were up-regulated. (p<0.05 and fold change> 1.5 were identified). Enriched GO terms that were significantly enriched in our gene set were response to stimulus, biological adhesion, cellular process, immune system process, membrane part, receptor activity and molecular transducer activity and so on.Further functional pathway analysis showed that compared with blank control group, changed genes in model control group were involved in 92 KEGG pathways (p<0.05).Compared with model control group, changed genes in model+EA group were mainly related to 144 KEGG pathways."Experiment 3":The expression of HSPA1A(P<0.01), HSPB1(P<0.01), OLR1(P=0.035), PLA2G4A(P<0.01) and COX-2(P=0.041) was significantly higher in model control group compared to blank control group.HSPA1A(P<0.01), HSPB1(P<0.01), OLR1(P<0.01), PLA2G4A (P<0.01) and COX-2(P=0.015) expression was significantly lower in model+EA group when compared to model control group. To some extent, the identified results confirmed the reliability of the array analysis. It indicated that the mechanism of electroacupuncture for regulating blood pressure of stress-induced pre-hypertension rats may be related to the hypertension related genes:HSPA1A, HSPB1, OLR1,PLA2G4A and COX-2 mRNA expression.Conclusion:1. EA at Taichong(LR3) and Quchi(LI11) can significantly lower the blood pressure of stress-induced pre-hypertension rats.2. EA at Taichong(LR3) and Quchi(LI11) can affect its gene expression profile in heart.3. Genes:HSPA1、HSPB1、OLR1、PLA2G4A、COX-2、FGF16 and their signal transduction pathway that related to the MAPK signal pathway, PPAR signal pathway and VEGF signal pathway may be involved in EA’s anti-hypertensive mechanism.