Adra1d Mediates The Contraction And Relaxation Responses Of Rat VSMCs Regulated By Farrerol

Objective:1.In order to determine the effect of farrerol on the relaxation of vascular smooth muscle cells?VSMCs?which were contracted by Angiotensin II?AngII?.2.Elucidate the molecular mechanism of Adra1d mediates the relaxation responses of rat VSMCs regulated by farrerol.Methods:1.Primary rat thoracic aorta VSMCs were cultured by tissue block adhered method.VSMCs morphology was observed under microscope.VSMCs were identified by immunohistochemical staining with anti-?-actin antibody.2.Collagen contraction experiment:A three-dimensional collagen containing no cells was prepared,and the degree of VSMCs contraction was measured by macroscopically measuring the size of the collagen area.3.Cell surface dynamic observation experiments:For the same field of vision,take photos at different times,measuring the size of the same cell surface.The degree of VSMCs contraction was measured by t the size of cell.4.Cytoskeletal staining experiments:the cytoskeleton was stained with Rhodamine-Phalloidin,photographs of different experimental groups were taken,and the size of different cell surface areas was measured to represent the degree of cell contraction.5.Cell length measurement experiment:the cytoplasm was stained with eosin,and the axial length of cells was measured by microscopy to represent the degree of cell contraction.6.Calcium imaging experiments:the concentration of intracellular calcium in each group to represent the degree of cell contraction 7.Western blot:the protein expression levels of MLCK and SM22?and the phosphorylation levels of MLCP and MLC in different groups of VSMCs were detected.8.Real-time PCR:the mRNA expression of MLCK and SM22?in different groups of VSMCs was detected.9.Lentiviral transfection experiment:A lentivirus containing shRNA of Adra1d was produced and transfected into VSMCs to inhibit the expression of Adra1d gene.Results:1.The primary rat VSMCs were cultured successfully.The single cells were fusiform and the cells showed“peak-like”growth.Immunohistochemistry results of anti-?-actin antibody were positive and the purity of VSMCs was above 95%.2.The results of morphological experiments?collagen contraction experiment,cell surface dynamic observation experiment,cytoskeletal staining experimen,and cell length determination experimen?showed:compared with the control group,VSMCs after treated with 10?mol/L Ang II for 1 h?contractile group?were significantly contracted;compared with the contractile group,VSMCs after treated with 60?mol/L farrerol for 4 h?relaxed group?were significantly relaxed.3.The results of calcium imaging experiments showed:compared with the control group,the intracellular Ca²⁺in the contractile group increased significantly;compared with the contractile group,the intracellular Ca²⁺in the relaxed group were significantly reduced.4.There were many contractile proteins changes occurred:compared with the control group,the expression of MLCK,SM22?and the phosphorylation of MLC,MLCP in the contractile group were significantly increased;compared with the contractile group,the expression of MLCK,SM22?and the phosphorylation of MLC,MLCP in the relaxed group were significantly decreased.5.The lentivirus containing Adra1d shRNA were successfully constructed.The expression of Adra1d mRNA and protein was significantly reduced in VSMCs after transfected.The results of morphological experiment,intracellular Ca²⁺concentration,and contractile protein of VSMCs treated with farrerol after Adr-shRNA interference showed that:since the reduced of expression of Adra1d,the ability of farrerol to relax VSMCs was significantly reduced.Conclusion:Ang II can induce the contraction of VSMCs,while farrerol can relax those contracted VSMCs.Farrerol may exert its relaxing effect by upregulating the expression of Adra1d.