| Objective :To clarify the expression of Axl in clinical specimens,animal,and cell models,and to study the effect and mechanism of exogenous Axl on IA rupture,so as to provide a new intervention target for preventing the rupture of intracranial aneurysms.Methods :Part Ⅰ:(1)Healthy control(Healthy),Unruptured intracranial aneurysm(UIA),Ruptured intracranial aneurysm(RIA)serum and tissue samples were collected,and the superficial temporal artery(STA)was used as the control group in healthy control patients.Soluble Axl(sol Axl)expression in serum of patients with Healthy,UIA and RIA was detected by enzyme-linked immunosorbent assay(Elisa).Immunohistochemical staining was performed to detect Axl expression,M1 macrophage and M2 macrophage infiltration in STA,UIA,RIA patients.(2)Systemic hypertension was induced by ligating bilateral posterior renal artery branches +angiotensin II(Ang-II)+8% Na Cl diet,and elastase was injected into right basal pool to weaken the vascular structure,followed by diet containing 0.12% BAPN to induce mouse IA.7.0 MRI was used to observe IA formation.On the 21 st postoperative day,HE and Masson staining were performed to confirm that the IA model was successfully constructed.Immunofluorescence staining was then used to detect normal blood vessels,Axl expression in UIA,RIA and M1 macrophage and M2 macrophage infiltration.(3)IA in rat was induced by ligating left common carotid artery,right external carotid artery,occipital artery,and pterygopalatine artery to increase flow in circle of Willis,followed by diet containing 8% Na Cl+0.12% BAPN diet.Western blot was performed to confirm the Axl expression in normal arteries,UIA,and RIA.Part Ⅱ:(1)After extracting bone marrow-derived macrophages(BMDM)from mice,they were induced to M1 and M2 macrophages by LPS/IFN-γ and IL-4,respectively,to detect the transcription and translation levels of Axl and elucidate the factors affecting Axl expression.(2)Axl-specific agonist rm Gas6(recombined mouse growth arrest-specific protein 6,rm Gas6)and inhibitor R428 were used to promote or inhibit Axl,respectively and detect the changes of inflammatory factors of macrophages in different phenotypes.(3)After transfection with si-STAT1,the role and mechanism of Axl in regulating macrophage phenotypic transformation were clarified.Part Ⅲ: After constructing IA model in mice and rat,0.2ml of PBS,4μg/kg of rm Gas6,75mg/kg of R428 were administered daily through intraperitoneal injection to agitate or inhibit Axl.After 21 days,IA in mice were harvested for immunofluorescence analysis to assess p STAT1 expression and infiltration of M1 and M2 macrophages in the three groups.After 90 days,IA in rats were collected and the expression of p STAT1,M1,and M2 macrophage-like inflammatory factors in the aneurysm wall was quantitatively analyzed by WB.The effect of Axl on the infiltration of M1 and M2 macrophages in IA wall was determined by flow cytometry.Results :Part Ⅰ: 1.In the serum results of patients,sol Axl was increased in UIA,and further increased in RIA compared with UIA.2.In aneurysm wall specimens,Axl expression was increased in the wall of unruptured aneurysm,and further increased in the wall of ruptured aneurysm.Both M1 and M2 were significantly increased in the wall of unruptured aneurysm,and M1 infiltration increased in the wall of ruptured aneurysm,but M2 did not increase.Part Ⅱ: 1.The expression of Axl increased in M1 and M2.The activation of Axl by rm Gas6 promoted the phosphorylation of STAT1 and the expression of HIF-1α,and promoted the secretion of M1 inflammatory mediators Nos2,Il-1β and Mmp9,but inhibited the secretion of M2 inflammatory mediators Arg-1.3.R428 inhibits Axl phosphorylation and the secretion of inflammatory mediators Nos2,Il-1β and Mmp9.4.After STAT1 knocks down,rm Gas6 promotes M1 and the effect of inhibiting M2 is abolished.Part Ⅲ: 1,R428 inhibits phosphorylation of p STAT1 in UIA,whereas rm Gas6 promotes p STAT1.2.rm Gas6 promoted M1(CD86 labeled)macrophage invasion in tumor wall,while R428 inhibited Axl and decreased M1 macrophage invasion.3.rm Gas6 inhibited M2(CD206 labeled)macrophage infiltration in tumor wall,while R428 inhibited Axl and promoted M2 macrophage infiltration.4.rm Gas6 promoted IA rupture in mice,while R428 reduced IA rupture rate.Compared to the Vehicle group,rm Gas6 promotes the progression of IA and increases Grade3,and R428 treatment not only inhibits the IA rupture rate,but also stabilizes IA in Grade2 and Grade1.5.Flow cytometry analysis showed that compared with the control group,M1 cell infiltration increased in UIA tube wall in rm Gas6 treatment group,and R428 inhibited M1 cell infiltration in UIA.Conclusions :1.Patient IA serum results showed that serum sol Axl was significantly elevated in ruptured aneurysm.2.The results of patient and animal IA tissues confirmed that compared with the unruptured aneurysm wall,Axl was significantly increased in ruptured aneurysm,M1 macrophage infiltration was significantly increased,and M2 macrophage infiltration was not significantly different.3.In vitro studies have shown that Axl promotes the transformation of macrophages to M1 and inhibits M2 macrophages through the STAT1/HIF-1α pathway.4.Axl inhibitor R428 can reduce the rate of intracranial aneurysm rupture. |
