The Role And Mechanism Of Purinergic Receptor P2X7 Regulating Macrophage Pyroptosis In The Initiation And Development Of Abdominal Aortic Aneurysm

Background and objective:Abdominal aortic aneurysm（AAA）is a chronic aortic disease characterized by irreversible aneurysmal dilation of the abdominal aorta with an increase of more than 50%in diameter.Its early clinical symptoms are not typical and easy to be misdiagnosed.If not treated timely and effectively,it will rupture and leads to high risk of death.At present,there are no effective drugs to treat and delay the progress of AAA except for open surgery and endovascular aneurysm repair（EVAR）.Therefore,it is urgent to study the molecular mechanism of AAA formation and find new targets for the prevention and treatment of AAA.P2X7 is a member of purinergic receptor family.As a non-specific cation channel,activated P2X7 can induce Ca²⁺influx and K⁺outflow.In recent years,P2X7-mediated inflammatory response has been associated with the development of cardiovascular diseases,such as atherosclerosis,hypertension and myocardial infarction and so on.It is gradually becoming a new research hotspot in the field of cardiovascular inflammation.However,whether P2X7 is involved in the occurrence and development of AAA has not been reported so far.The aim of this study is to explore the mechanism of P2X7-mediated macrophage pyroptosis in AAA according to clinical samples,animal models,cells and molecular levels,so as to provide a theoretical basis for the prevention and treatment of AAA in clinic and find potential intervention targets for drug treatment.Method:（1）The normal abdominal aortic tissue and AAA tissue were collected,the protein of tissue was extracted,and the expression level of P2X7 was detected by Western blot;The tissue samples were sectioned and stained with H&E and EVG to evaluate the histopathological changes and elastic fiber breakage between the two groups.The tissue sections were stained with immunofluorescence and immunohistochemistry to detect the expression levels of P2X7,NLRP3,Caspase-1,GSDMD and the co-localization of P2X7 and macrophages in the two groups;Blood samples from AAA patients and non-AAA patients were collected,peripheral blood mononuclear cells（PBMC）were extracted,and the m RNA expression levels of P2X7,NLRP3,Caspase-1 and GSDMD were detected by real-time quantitative PCR（RT-q PCR）.（2）The AAA model induced by Ca Cl₂in C57BL/6J mice and the AAA model induced by AngiotensinⅡ（AngⅡ）in Apo E^-/-mice were established.The AAA tissue protein was extracted and the expression level of P2X7 was detected by Western blot;Immunofluorescence and immunohistochemical staining were performed in the normal abdominal aorta and AAA tissue of AAA mice induced by Ca Cl₂.The expression levels of P2X7,NLRP3,Caspase-1 and GSDMD and the co-localization of P2X7 and macrophages were detected;P2X7agonist（ATP-γS）and P2X7 antagonist（A438079）were injected intraperitoneally into the mice of the AAA model induced by Ca Cl₂,and the lumen diameter of the abdominal aorta was measured by vernier caliper and color Doppler ultrasound;The AAA tissue sections were stained with H&E and EVG to evaluate the histopathological changes and elastic fiber breakage;MMP activity was detected in the AAA tissues of the two groups;Caspase-1 activity was detected by FLICA staining;The levels of ROS in the two groups were detected by DHE staining;P2X7 knockout(P2X7^-/-)mice were bred.The AAA was induced in P2X7^-/-mice and WT mice by Ca Cl₂.The lumen diameter of the abdominal aorta in the two groups was measured,H&E and EVG staining were performed in the AAA samples of two groups,and MMP activity,Caspase-1 activity and ROS level in the AAA samples of the two groups were detected.（3）The macrophage pyroptosis model was established.Bone marrow-derived macrophages（BMDM）from P2X7^-/-mice and wild type mice were extracted and stimulated with LPS+ATP and LPS+Nigericin respectively.The cell and supernatant proteins were extracted.The expression levels of NLRP3,Caspase-1 and GSDMD were detected by Western blot;PI staining and FLICA staining were used to detect the changes of cell membrane integrity and Caspase-1 activity of BMDM after pyroptosis stimulation.Result:（1）P2X7,NLRP3,Caspase-1 and GSDMD were significantly upregulated in human AAA lesions and P2X7 co-located with macrophages.（2）The expression levels of P2X7,NLRP3,Caspase-1 and GSDMD in PBMC of AAA patients were significantly higher than those in control group.（3）P2X7,NLRP3,Caspase-1 and GSDMD were significantly upregulated in experimental murine AAA lesions induced by Ca Cl₂,and P2X7 co-located with macrophages;The expression of P2X7was also significantly increased in experimental murine AAA lesions induced by AngⅡ.（4）P2X7 agonist（ATP-γS）can significantly increase the AAA diameter,the elastic fiber breakage index,Caspase-1 activity,MMP activity and ROS level in AAA tissue induced by Ca Cl₂,and promote the progression of AAA in mice,while P2X7 antagonist A438079 can significantly decreased AAA diameter,the elastic fiber breakage index,Caspase-1 activity,MMP activity and ROS level in abdominal aorta tissue,and inhibit the AAA formation.（5）Compared with wild type mice,the AAA diameter and elastic fiber breakage index of P2X7^-/-mice induced by Ca Cl₂ decreased significantly,and the Caspase-1 activity,MMP activity and ROS level in AAA lesions of P2X7^-/-mice decreased significantly.（6）The knockout of P2X7 significantly inhibited the activation of Caspase-1and GSDMD in BMDM stimulated by LPS+ATP.（7）The knockout of P2X7 significantly inhibited the destruction of cell membrane integrity of BMDM stimulated by LPS+ATP.Conclusion:Our study demonstrated that the purinergic receptor P2X7 activated by ATP may mediate the macrophage pyroptosis by regulating NLRP3-Caspase1-GSDMD pathway,so as to promote the up-regulation of ROS level and the increase of MMP activity,and finally lead to the formation or development of AAA,which may serve as a potential intervention target for the prevention and treatment of AAA.