The Role Of Alpha1-AR In Acute Sympathetic Stress Induced Cardiac Inflammation

Objective:When the body is stimulated by intense stress,the sympathetic nerve is over-activated,causing an increase in catecholamines released from the sympathetic nerve endings and adrenal glands.This in turn causes elevated levels of catecholamines in the blood,including epinephrine（E）and norepinephrine（NE）.Our previous study demonstrated that catecholamine activated theβ-adrenergic receptors（β-ARs）in the heart to promote aseptic inflammation,and further to promote cardiac fibrosis and cardiac remodeling.β-AR mediates acute heart-induced inflammation in the NLRP3/Caspase-1/IL-18 pathway.However,the AR subtypes expressed in hearts not only includedβ-ARs but alsoα-ARs.At present,it is still unclear whetherα-AR mediates cardiac inflammation caused by catecholamines.The main study purposes of this subject:（1）To investigate the effect of byα₁-AR over-activation on the activation of the NLRP3/Caspase-1/IL-18 pathway.（2）To investigate whetherα₁-AR over-activation causes cardiac inflammation through the NLRP3/Caspase-1/IL-18 pathway.Methods:1.In vivo,we used different doses of NE to mimic acute sympathetic over-activation and detect the inflammation of the heart caused by NE.After a single subcutaneous injection of NE 5 mg/kg and 10 mg/kg for 24 hrs and 72 hrs,the induced cardiac inflammation was observed.Theα-AR selective antagonist Prazosin（Praz）was used to determine whether antagonizingα-AR would alleviate NE-induced cardiac inflammation.The method of histochemical staining was used to identify the inflammatory cell infiltration in the heart.The expression of inflammatory factors in the heart was detected by ELISA.Echocardiography was used to detect the effects on cardiac function and structure.2.α1-AR specific agonist,Phenylephrine（PE）were used to determine the effect ofα1-AR activation on cardiac inflammation.Mice were treated by 5 mg/kg dosage and 10mg/kg dosage of PE treatment for 24 hrs and 72 hrs.Cardiac inflammatory cell infiltration was identified by histochemical staining.The expression of cardiac inflammatory factors was detected by ELISA.Echocardiography was used to detect the effects on cardiac function and structure.3.In vivo,wild type（WT）mice were treated by PE and/or Praz,and the expression of NLRP3 and the cleavation of Caspase-1 and IL-18 were detected by Western Blot.4.Langendorff-perfused hearts and neonatal mouse cardiomyocytes（NMCM）were treated with PE,and the expression of NLRP3 and the cleavage activation of Caspase-1 and IL-18 were detected by Western Blot.5.NLRP3 knockout mice and IL-18 knockout mice were treated by PE,the infiltration of cardiac inflammatory cells was identified by histochemical staining;the expression of NLRP3 and the cleavage of Caspase-1 and IL-18 were detected by Western Blot.The ELISA method was used to detect the levels of inflammatory factors and IL-18 in the blood.Results:（1）The single subcutaneous injection of NE can cause cardiac macrophage infiltration after 72 hrs,and the expression levels of cardiac inflammatory factors IL-6,TNFα,MCP-1,MCP-5 were significantly increased;Pretreatment with Praz significantly alleviated NE-induced increase in cardiac macrophage infiltration and inflammatory factor expression.These results indicate thatα-AR mediates NE-induced heart inflammation.（2）PE induced significant infiltration of macrophages and increase of inflammatory factors following a single subcutaneous injection of different doses and different time.The effects of PE were inhibited by 5 mg/kg Praz.PE at the dose of 10 mg/kg caused cardiac systolic and diastolic dysfunction,and the increase of left ventricular posterior wall thickness.These results indicate that selective agonismα₁-AR can cause heart inflammation.（3）The NLRP3 expression level and the cleavage of Caspase-1 and IL-18 were significantly induced by PE treatment in Langendorff-perfused hearts and cultured NMCMs.The NLRP3 level and the cleavage of Caspase-1 and IL-18（P<0.05）in cardiac tissue of WT mice were significantly increased by different dosage of single subcutaneous injection PE treatment at 24 hrs.Whereas the results can be reversed by Praz pretreatment.These results indicate that activatingα₁-AR can activate the NLRP3/Caspase-1/IL-18 pathway.（4）Cardiac macrophage infiltration were significantly supressed in NLRP3 KO mice and IL-18 KO mice after PE treatment than wild-type mice.These results indicate that the NLRP3/Caspase-1/IL-18 pathway mediates cardiac inflammation induced byα₁-AR over-activation.Conclusions:1.α1-AR over-activation can cause cardiac inflammation in vivo.2.α₁-AR over-activation causes the activation of the NLRP3/Caspase-1/IL-18 pathway.3.The NLRP3/Caspase-1/IL-18 pathway mediates cardiac inflammation induced byα₁-AR activation.