Role Of β₃ Adrenoceptor In Rat Thoracic Aorta Tone And MicroRNAs Expression

Objective: 1. Exploring the function and the possible signaling pathway of β₃ adrenergic receptor（β₃-AR） in rat thoracic aorta of Sham rats and chronic heart failure（CHF） rats using vascular ring perfusion system. 2. Exploring the effect of β₃-AR inhibition on vascular endothelial function, vascular contraction response, micro RNAs（mi RN As） expression and NF-κB expression in Sham and CHF rat thoracic aorta after 5 weeks intraperitoneal injection of SR.Methods: 1. 200 Sprague Dawley（SD） rats weighting 180-220 g were randomly divided into Sham（70 rats） and CHF（130 rats） groups. Left ventricle anterior descending coronary arterywas ligated to establish C HF model, normal EC G tracings were recorded before surgery, and continuous electrocardiograph was recorded during the surgery. Penicillin of 200 thousands units was injected intraperitoneally for three consecutive days to prevent infection. 2. In vitro experiment was carried out after 4 weeks of surgery. Ecocardiograph was used to determine the success of CHF. Rats in Sham group and CHF group were used to perform aorta ring perfusion to observe the relaxant effect of BRL 37344（BRL, the specific β₃-AR agonist） on rat thoracic aorta. The mechanism of the relaxant effect of β₃-AR were detected by pre-treatment of L-NAME（the NOS antagonist） and H-89（the PKA antagonist）. Immunohistochemistry staining was used to locating and confirme the expression of β₃-AR in rat thoracic aorta.3. To clarify the role of β₃-AR on vascular function in vivo, a β₃-AR specific blocker named SR 59230A（SR） was injected intraperitoneally twice a day at a dose of 85 nmol in 1 m L saline. One week after the liga tion surgery, rats for in vivo experiment were divided into 4 groups. The Sham rats were further divided into Sham control（10 rats） and Sham+SR（10 rats） groups, and CHF rats were divided into CHF control（10 rats） and CHF+SR（10 rats） groups. The control groups were given 0.9% of saline; SR groups were injected with SR for 5 weeks. Vascular endothelial function and vascular contraction response were measured by vascular relaxing response to acetylcholine（Ach） and contracting response to Noradrenaline bitartrate（NA）. H&E staining was used to observe the morphological structure of rat thoracic arota in four groups. The effect of SR in vivo administration on the expression of mi RN As in rat thoracic arota was also determined by RT-PCR. The expression of NF-κB was detected by immunohistochemistry and Western blot.Results: 1. In aorta rings pre-constricted with 30 mmol/L KC l, BRL produced relaxant effect in Sham rats, and the effect was stronger in endothelium- intact vascular than in endothelium- free vascular（P<0.05）. The relaxant effect was antagonized by SR（P<0.01）, and was also partially attenuated by L-NAME（P<0.01）, a nitric oxide（NO） synthase inhibitor, but not changed by Propranolol（Pra）, the β1, β2 adrenoceptors inhibitor. The relaxant effect caused by BRL on thoracic aorta smooth muscle of chronic cardiac failure rats was bigger than the Sham rats（P<0.01）. Immunohistochemistry staining showed that β₃-AR located both in the endothelial layer and the vascular smooth muscle layer, and the expression o f β₃-AR was increased in CHF rat thoracic arota smooth muscle. 2. After 5 weeks injection of SR, the Sham+SR group showed a reduced endothelium-dependent relaxation response to Ach compared with those observed in Sham control group, while the CHF control group showed a reduced endothelium-dependent relaxation response to Ach（had statistical significance）. TheCHF+SR group showed an increased Ach endothelium-dependent relaxation compared to the C HF control group, but had no statistical significance. The haematoxylin and eosin staining support this result by showing the changes of vessel structure. Besides, the SR groups showed an increased contraction response to NA（10-8-10-5 mol/L） compared to the control groups. 3. The mi Script mi RN A PC R Arrays indicated that the Sham+SR group has 48 differentially expressed mi RN As compared to the Sham control group, and the CHF+SR group has 42 differentially expressed mi RN As compared to the C HF control group. In Sham+SR and CHF+SR groups, 9 mi RN As were up-regulated, they were rno- mi R-142-3p, rno- mi R-144-3p, rno- mi R-146a-5p, rno- mi R-223-3p, rno- mi R-224-5p, rno-mi R-342-3p, rno- mi R-451-5p, rno- mi R-497-5p, rno- mi R-92a-3p, 16 mi RN As were down-regulated, they were rno-let-7b-e-5p, rno- mi R-103/107-3p, rno-mi R-130a-3p, rno-mi R-181b-5p, rno- mi R-185-5p, rno- mi R-183-5p, rno- mi R-214-3p, rno-mi R-320-3p, rno- mi R-352, rno- mi R-494-3p, rno- mi R-93-5p, rno- mi R-99a-5p. After literature comparison, it appeared that 19 of them were related to NF-κB pathway, in which rno- let-7c-5p and rno- mi R-451-5p had statistical significance. 4. The immunohistochemistry results indicated that the expression of NF-κB in Sham+SR group was a little higher than in Sham control group, it was increased obviously in CHF control group. After in vivo SR application, NF-κB expression decreased, the Western blot results showed similar result.Conclusion: 1. β₃-AR located both on endothelial and smooth muscle layers. Stimulation of β₃-AR relaxed rat thoracic aorta and the relaxant effect was related to the activation of NO synthase pathway. The expression of β₃-AR was up-regulated in CHF rat thoracic aortic smooth muscle, and the relaxant effect of β₃-AR activation on thoracic aorta smooth muscle was increased. After in vivo inhibition of β₃-AR, the contraction response of rat thoracic arota increased both in Sham and CHF group.2. Vascular endothelium function was injuryed in Sham+SR group. The structure and function of thoracic arota endothelium in CHF rat was injuried, but SR injection postponed the damage of vascular. 3. SR application altered expression of mi RN As in Sham and CHF groups, in which rno- let-7c-5p and rno- mi R-451-5p are related to NF-κB pathway. 4. The expression of NF-κB in CHF rat choracic aorta was increased, the role of β₃-AR on vascular function was related to NF-κB pathway.