Effect Of Electroacupuncture On Gene Expression Profile Of Hypothalamus In Prehypertensive Rats With Stress

In recent years, the prevalence of Stress-induced hypertension has significantly increased in people who always worked in high-pressure environment for a long time. To advance the front of the prevention and treatment of cardiovascular disease, JNC-7 in USA pointed out that 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 120-139mmHg and (or) diastolic blood pressure between 80-89 mm Hg. The incidence of pre-hypertension,15% to 40% in global, is also about 38.9% to 41.3% in China. Studies have shown that prehypertension is often combined with one or more risk factors, not only was positively correlated with the occurrence of cardiovascular events in the long-term, but also lead to the damages of target organs like blood vessels, heart, brain, and kidneys. Therefore, early stage treatment could effectively reduce the incidence of high blood pressure and protect target organs. However, the current studies are more focused on the treatment of hypertension and target organ protection in the middle and late stages, fewer researches have focused on gene expression and intervention of pre-hypertension. Using gene chip technology to study acupuncture’s intervention effect on pre-hypertension and protective effects on target organs has not been reported.Objective:In accordance with the principle of "preventive treatment of disease" in TCM, we will use chip technology, bioinformatics analysis and RT-PCR method to observe electro-acupuncture (EA)’s intervention effect on pre-hypertension and protective effects on target organs, to systematically reveal its biological mechanisms and to provide new evidence and reliable treatment for the prevention and treatment of hypertension.Methods:"Experiment 1":The Effect of electro-acupuncture on the blood pressure and aortic tissue morphology of rats with Stress-induced Pre-hypertension33 SPF wistar male rats of 220±30g were randomly divided into 3 groups:blank control group, model control group, and model+EA group (n=11/group).The stress-induced pre-hypertension rat (SIPHR) model was established by electric foot-shocks combined with generated noise lasting for 14 days and during this period EA intervene was applied on rats in model+EA group. Blood pressure was recorded one day before and at the 3rd,5th,7th,9th, 11th,13th and 15th day after modeling.Animal drawn on the 15th day after treatment and Using HE to observe the aortic tissue morphology of rats. "Experiment 2":Effect of Electro-acupuncture on Gene Expression in Hypothalamus of Rats with Stress-induced Pre-hypertension and PCR validation of the key genesUsing Gene Chip Rat Gene 2.0 ST technology to observe gene expression in hypothalamus of 3 group rats and screening of differentially expressed genes(P<0.05,Fold Changes≥1.5 or Fold Changes(?)-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. After then, using RT-PCR technology to validate the selected key genes.Results:"Experiment 1":Compared with the blank control group, systolic blood pressure of model control group reached higher 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).With the value kept in the range between 120-139mmHg, the model was successfully established. The model group rats were of eye congestion, irritability and other changes in appearance and behavior in the modeling process. HE staining results also showed that rats in the model group appears aortic endothelial cell loss, smooth muscle hyperplasia and other pathological changes. During the EA treatment on the 5th,7th,9th day, systolic blood pressure in model group and model+EA group were of significant statistical difference (P<0.01)while on the 11th,13th and 15th day, systolic blood pressure in model group and model+EA group were of statistical difference (P<0.05).It was indicated that EA at Taichong (LR3) and Qu chi(LI11) can significantly decrease the blood pressure of SIPHR and the short-term effect was better. In the same time, according to the HE staining images, the aorta tissue damage of EA group was lighter, indicating that EA has protective effects on target organs of SIPHR."Experiment 2":Gene microarray analysis showed that EA could significantly affect the gene expression profile in hypothalamus. Compared with the blank control group, there were 307 gene expressions changed, of which 73 were down-regulated and 234 were up-regulated in model control group. Compared with the model control group, EA intervention changed 383 gene expressions, of which 273 were down-regulated and 110 were up-regulated. GO functional enrichment analysis revealed that these differentially expressed genes were mainly related to response to stimulus、cellular process、immune system process、extracellular matrix、 menbrane part、antioxidant activity、receptor activity、molecular transducer activity and so on. KEGG signaling pathway analysis revealed that 24 pathways which were closely correlated with hypertension were activated. In addition, there were 6 common differentially expressed genes among 3 groups,of which 6 genes were identified:HspB1、vtn、BMP-4、p2rx4、 Ppplr14a and TH. They were all up-regulated in model control group comparing with the blank control group. However, after EA intervention, they were all down-regulated in model+ EA group. We speculated that these six key genes played an important role in the development of stress-induced pre-hypertension and the EA’s anti-hypertensive mechanism may be involved in the regulation of the six key genes function expression and their related signal transduction pathways.Conclusion:1. EA at Tai chong (LR3) and Qu chi(LI11) can significantly decrease the blood pressure of SIPHR and has protective effects on target organs of SIPHR.2 EA at Tai chong(LR3) and Qu chi(LI11) could significantly affect the gene expression profile in hypothalamus.3. The mRNA abnormal expression of HspB1、vtn、BMP-4、p2rx4、Ppplr14a and TH genes plays an important role in the process of molecular biology mechanism of stress-induced pre-hypertension4. The EA’s anti-hypertensive mechanism may be involved in down-regulating HSPB1 to regulate the contraction of vascular smooth muscle, down-regulating P2RX4, PPP1R14A to reduce intracellular Ca2+ concentration and down-regulating TH to reduce the excitability of the sympathetic nervous system...