Molecular Mechanism Of GPR35 Deletion Inhibiting Aortic Dissection

Objective:To investigate the function of G-protein-coupled receptor 35(GPR35)in aortic dissection(AD)and clarify whether inhibition of inflammatory and aortic smooth muscle apoptosis via restraining the Nuclear factor kappa B(NF-?B)are intrinsic mechanisms which mediate the protective effect of GPR35 deletion on AD.Method:AD model was induced by pumping angiotensin(Ang)-?(4.5mg/kg/d)which continued 14 days.Subcutaneous micropumps were surgically implanted at the scapular region under sterile environment.Detection of GPR35 mRNA and protein expression in aortic tissues through q-PCR and Western-blot.Evaluation of aortic wall and elastic fiber damage by HE staining and VVG staining.Assessment of the inflammatory factors contain of mice via ELISA.Further monocyte macrophage(CD68 positive cells)infiltration was measured through immuno-histological(IHC).meanwhile the mRNA expression of inflammatory factors,Tumor necrosis factor(TNF)-? and Interleukin(IL)-6,were determined by the means of q-PCR.The aortic smooth muscle apoptosis was evaluated through TUNEL staining and active Caspase-3.Finally,to determine the expression of NF-?B P65 protein and phosphorylated NF-? B P65 protein by means of Western-blot.Results:The gross anatomy and HE staining revealed that the formation of classical AD was induced by Ang-II.The expression of GPR35 in AD was significantly increased(P<0.05).The AD was investigated after intervention via Ang-?,and the incidence rate was 53.3%,while the incidence of AD decreased significantly after GPR35 knockout(13.3%),as well as the maximum aortic diameter suppressed significantly(P<0.05).The pathological results indicated the aorta was obviously destructed with the media layer and adventitia thickening,and the elastic fibers were arranged disorderly and even fragmented,meanwhile the formation of false lumens in some aorta.However,the aortic structure was improved notably after GPR35 deficiency.The ELISA results displayed the TNF-? and IL-6 contain in serum were remarkably increased in AD,however,the levels of TNF-? and IL-6 were significantly lessened,and the mononuclear macrophages infiltration decreased,furthermore the IL-6 and TNF-? mRNA expression in the aorta were dramatically repressed after GPR35 deficiency(P<0.05).TUNEL staining results showed that the number of apoptotic cells in AD was notably raised,which reduced significantly after GPR35 inhibition,meanwhile the expression of active Caspase-3 was clearly depressed(P<0.05).The expression of NF-? B P65 protein and phosphorylated NF-? B P65 was upregulated in AD,while after GPR35 deletion,NF-? B P65 and phosphorylated NF-? B P65 protein was remarkably inhibited(P<0.05).Conclusions:GPR35 was involved in AD,and GPR35 deletion restrained inflammatory and smooth muscle cell apoptosis by inhibiting the NF-?B signaling pathway to exert the protective effect of AD.