The Effect And Mechanism Of Dopamine D4 Receptor On Abdominal Aortic Aneurysm

Abdominal Aortic Aneurysm(AAA)is a kind of disease with insidious onset,low diagnosis rate,and high fatality rate after rupture.Effective drug and preventive strategy are still lacking.Therefore,searching for effective drug may reduce the risk of rupture and save lives.Vascular smooth muscle cell(VSMC)is the main functional executor and component of the abdominal aorta.The reduction of VSMC is the most important pathological change of AAA,while,oxidative stress can induce VSMC apoptosis,inflammation,and accelerate extracellular matrix degradation,which leading to the development of AAA.Therefore,reducing VSMC oxidative stress may be an effective way for AAA treatment.Dopamine receptor D4(DRD4),a kind of G-protein-coupled receptor,is composed of seven trans-membrane regions.Not only widely exist in the central system,DRD4 also exist in peripheral systems such as cardiovascular and renal systems.Moreover,DRD4 is involved in the oxidative stress regulation.Therefore,we infer that DRD4 may alleviate AAA through its antioxidant effect.(1)We explore the expression of DRD4 in VSMC and AAA.The expression of DRD4 in VSMC is confirmed for the first time and the expression of DRD4 is decreased in AAA patients and mouse AAA model by real time quantitative polymerase chain reaction(RT-q PCR),Western blotting and immunofluorescence chemical staining.These results imply that DRD4 is expressed in VSMC and decreased in AAA.(2)We explore the function of DRD4 in elastase perfusion induced AAA.In vivo,DRD4 deficiency increases the incidence and mortality of AAA,amplifies the diameter of abdominal aorta,accompany with more VSMC apoptosis,inflammation and extracellular matrix degradation in elastase perfusion induced AAA by survival analysis,small animal Doppler ultrasound,Hematoxylin-Eosin(H&E)staining,Elastin Van Gieson staining,immunohistochemical staining,in situ zymography,gelatin gel zymography and TUNEL assay.In vitro,DRD4 deficiency activates metal matrix protease activity,and accelerates VSMC apoptosis by gelatin gel zymography and flow cytometry.These results imply DRD4 deficiency AAA promote the formation of elastase-induced AAA.(3)We explore the function of DRD4 on oxidative stress in primary VSMC.In vivo,DRD4 deficiency increases the reactive oxygen species(ROS)and lipid oxidation in elastase perfusion induced AAA by Dihydroethidium(DHE)staining and malondialdehyde(MDA)detected assay.In vitro,The primary VSMC is extracted,then treated with DRD4 agonist and antagonists.Activated DRD4 decrease the ROS production in VSMC,DRD4 deficiency or inhibited DRD4 accelerates aggravate the oxidative stress in VSMC by DCF-DA staining and MDA detected assay.These results imply DRD4 deficiency AAA promote the ROS production in VSMC.(4)We explore the mechanism of DRD4 in AAA.NADPH oxidase4(NOX4)and phosphorylated P38 MAPK are up-regulated after DRD4 knockout in elastin infusion-induced AAA by Western blotting.The primary VSMC is extracted,then incubate with DRD4 agonist or DRD4 antagonist after Angiotensin II(Ang II)treatment.Activated or inhibited DRD4 significantly reduced or increased Ang II-induced phosphorylated P38 and NOX4 expression in primary VSMC.It is confirmed that DRD4 regulates oxidative stress through NOX4 by Si RNA transfection,DHE staining and MDA detected assay.Moreover,DRD4 regulate Ang II-induced NOX4 expression through the P38 signaling pathway,which regulates ROS production and ultimately alleviates AAA progression by Western blotting.(5)We explore the function of DRD4 in different AAA models.Exogenous administration of DRD4-specific agonist PD168077 could significantly reduce the AAA incidence.So did the oxidative stress levels,extracellular matrix degradation,VSMC apoptosis and inflammation in Ang II induced AAA by Western blotting,H&E staining,Van Gieson staining,DHE staining,immunohistochemical staining,in situ zymography and gelatin gel zymography.Meanwhile,activated DRD4 significantly reduce the phosphorylated P38 and NOX4 expression by Western blotting.This study,for the first time,demonstrates that DRD4 regulate ROS production through P38 MAPK pathway mediated NOX4,ultimately regulate the formation of AAA,which might be a novel sight for prevention and treatment of AAA.