| Objective: In recent years,cerebrovascular diseases have become one of the major diseases threatening human health.Among all cerebrovascular diseases,intracranial aneurysms are of great concern due to their high prevalence and lethality.In a study that included 21 countries,the prevalence of unruptured aneurysms was shown to be approximately 3.2%.The annual rupture rate of unruptured aneurysms was 0.95%.In Asian populations,the morbidity and mortality rate of patients with ruptured aneurysms is approximately 26.7-35.8%.Even among patients who can achieve self-care after surgical treatment,40-70% still have cognitive impairment.Factors that promote the creation,growth,and rupture of cerebral aneurysms may involve genetics,injury,hemodynamics,environmental factors,and inflammation.Among these,macrophage-induced inflammatory responses are thought to play a central role in the overall disease history of cerebral aneurysms.Toll-like receptors are type I transmembrane proteins that play a key role in innate immunity and various inflammatory responses.In humans and mice,Toll-like Receptor 4(TLR4)can be widely expressed in a variety of cells,mainly in monocytes-macrophages,where it is involved in inflammatory responses through myeloid differentiation primary response gene 88 signaling pathways that activate nuclear factor κB,leading to tissue damage.Tumor necrosis factor-stimulated Gene 6 Protein(TSG-6)is an endogenously secreted anti-inflammatory protein that can act on macrophages through an autocrine mode and plays an important role in regulating the inflammatory response.This study focused on the role of tumor necrosis factor α-stimulated gene-6 protein in regulating intracranial aneurysm disease and clarified its possible mechanism of action,namely,TSG-6 negatively regulates TLR4-NF-κB signaling pathway to inhibit macrophage inflammation and smooth muscle phenotype modulation.Methods: 1.Tissue samples of Ruptured Intracranial Aneurysm(RIA)and Unruptured Intracranial Aneurysm(UIA)walls removed during microsurgical craniotomy and Superficial Temporal Artery(STA)vessels were collected as controls,along with Clinical information and cerebral angiographic images were collected from the above patients.2.The tissue specimens were stained with HE and MASSON to compare the main pathological changes of the aneurysm tissue.The distribution and expression of Macrophage marker CD68,vascular smooth muscle cell contraction phenotype markerα-SMA and synthetic phenotype vascular smooth muscle cell marker MYH were detected using Western Blot(WB)and immunohistochemical staining.The distribution and expression of TSG-6 in tissue specimens were detected using WB and immunofluorescence staining.3.Computational Fluid Dynamics(CFD)models were constructed,and the cerebral angiography data were imported to obtain Wall Shear Stress(WSS)measurements.Shear Stress(WSS)blood flow parameters were characterized and the relationship between wall shear and aneurysm wall inflammation was analyzed.4.To investigate the effect of TSG-6 on cerebral aneurysm disease,a mouse cerebral aneurysm model was constructed and divided into sham-operated group(Sham),aneurysm model group(IA),AAV-TSG-6 overexpression group(OE),AAV-TSG-6 overexpression control group(OE-GFP),AAV-TSG-6 knockdown group(KD),AAV-TSG-6 knockdown control group(KD-GFP),and the distribution of aneurysm formation,survival curve and blood pressure monitoring curve of mice were plotted.5.Mice Cranial base vessels were stained with HE and Masson;the expression of TSG-6,TLR4,MCP-1,i NOS,CD206,α-SMA and MYH in mice Cranial base vessels were detected by immunohistochemistry,immunofluorescence and WB.6.To investigate the role of TLR4 in cerebral aneurysm in cerebral aneurysm,a mouse cerebral aneurysm model was constructed and divided into sham-operated group(Sham),aneurysm model group(IA),AAV-TLR4 knockout group(KD),and AAV-TLR4 knockout control group(Control),and the distribution of aneurysm formation,survival curve and blood pressure monitoring curve of mice were plotted.7.Immunohistochemistry detected the expression of TLR4 in the Cranial base vessels of mice;WB detected the expression of TLR4,My D88,P-NF-κB p65,NF-κB p65 in mouse cranial base vessels;immunohistochemistry,immunofluorescence and WB detected the expression of MCP-1,i NOS,CD206,α-SMA,MYH in mouse cranial base vessels.Results: 1.A total of 48 specimens were included.Among them,24 cases were in the superficial temporal artery group(STA group),12 cases were in the unruptured Intracranial aneurysm group(UIA group),and 12 cases were in the ruptured Intracranial aneurysm group(RIA group).2.HE and Masson staining showed that the vascular tissue in the STA group had clearly arranged vascular structures in all layers,with intact elastic membranes;muscle fibers were closely arranged in bands;in the UIA group,endothelial cell structures were broken,elastic membranes disappeared,and smooth muscle cells were abnormally proliferated and arranged.The endothelial cell structure was broken,the elastic membrane disappeared,and smooth muscle cells proliferated abnormally and were disorganized in the RIA group,the wall structure was further damaged,the extracellular matrix was decomposed and vitreous degeneration was observed,and disordered collagen fibers were visible,while smooth muscle cells were almost invisible and only appeared as loose and diffuse speckled staining areas.The vascular smooth muscle content was significantly higher in the UIA group(p <0.05)and lower in the RIA group(p <0.05).CD68 expression levels were significantly higher in the UIA and RIA groups(p <0.05);TLR4 expression levels were significantly higher in the UIA group(p<0.05)and lower in the RIA group(p <0.05).In contrast to STA,in UIA tissues,vascular smooth muscle gradually lost its original contractile phenotype and shifted to a synthetic phenotype;in RIA tissues,both phenotypes were significantly reduced(p <0.05).TSG-6could be co-localized with MYH in immunofluorescence staining and was significantly expressed in the UIA group(p <0.05).3.Parent artery and aneurysm necks have high WSS values,which contribute to vasodilation.In patients with ruptured aneurysms,WSS values were not uniformly distributed within the aneurysm walls,with lower WSS at the location of rupture;in patients with unruptured aneurysms,WSS values within the aneurysm walls were uniformly and consistently lower;comparing the mean values of WSS within the UIA and RIA aneurysms during the cardiac cycle,the RIA group had lower WSS within the aneurysms(p <0.05);the mean optical density values of CD68 immunohistochemical staining correlation with the mean value of WSS during the cardiac cycle suggested that WSS was negatively correlated with macrophage infiltration(r=-0.8489,p<0.05).4.The TSG-6 intervention model was divided into six groups:sham-operated group(Sham),aneurysm model group(IA),AAV-TSG-6 overexpression group(OE),AAV-TSG-6 overexpression control group(OE-GFP),AAV-TSG-6 knockdown group(KD),and AAV-TSG-6 knockdown control group(KD-GFP).the difference between IA group and Sham group,OE and KD groups were statistically different in tumorigenic distribution(p <0.05).Compared with the IA group,the OE group could significantly delay the time to aneurysm rupture although it did not significantly improve the survival rate(p>0.05);the KD group could reduce the survival rate,but it was not statistically significant(p>0.05).blood pressure in the Sham group was significantly lower than the rest of the groups(p<0.05).5.WB and immunohistochemistry detected significantly higher expression of TSG-6,TLR4,MCP-1,i NOS,CD206,MYH in IA group(p<0.05)and significantly lower expression of α-SMA(p<0.05).no significant difference in protein expression between IA group and OE-GFP and KD-GFP groups(p>0.05).expression of TSG-6,CD206,α-SMA in OE group was significantly higher than that in IA group(p<0.05);expression of TLR4,MCP-1,i NOS,MYH was significantly lower than that in IA group(p<0.05).CD206 and α-SMA in the OE group were significantly higher than those in the IA group(p<0.05);the expression of TLR4,MCP-1,i NOS,and MYH were significantly lower than those in the IA group(p<0.05).the expression of TLR4,MCP-1,i NOS,and MYH in the KD group were significantly higher than those in the IA group(p<0.05),and the expression of TSG-6,CD206,and α-SMA were significantly lower than those in the IA group(p <0.05).6,The TLR4 intervention model was divided into 4 groups: sham-operated group(Sham),aneurysm model group(IA),AAV-TLR4 knockout group(KD),and AAV-TLR4 knockout control group(Control).there was a statistical difference in the distribution of tumor formation between IA group and Sham group and KD group(p<0.05).Compared with the IA group,the KD group significantly improved survival(p <0.05).blood pressure in the Sham group was significantly lower than that in the remaining groups(p<0.05).7.WB and immunohistochemistry detected the expression of TLR4,My D88,P-NF-κB p65,MCP-1,i NOS,CD206,MYH in IA group was significantly higher than that in Sham group;the expression of NF-κB p65 was not significantly different(p>0.05)and the expression of α-SMA was significantly lower than that in Sham group(p<0.05).the expression of TLR4,My D88,P-NF-κB p65,MCP-1,i NOS,CD206,MYH in IA group was significantly higher than that in Sham group(p<0.05).The expression of CD206 and α-SMA in the KD group was significantly higher than that in the IA group(p<0.05);the expression of TLR4,My D88 and P-NF-κB p65 was significantly lower than that in the IA group(p <0.05),and the expression of NF-κB p65 was not significantly different(p > 0.05).Conclusions: 1.With the induction of hemodynamics,the vascular structure is significantly damaged with the infiltration of macrophages.During the process of aneurysm formation,it relies on smooth muscle proliferation and phenotypic transformation to complete vascular remodeling.As the inflammatory infiltration of macrophages gradually increases,the synthetic and contractile phenotypes of smooth muscle are further lost,and the smooth muscle content in the ruptured aneurysm wall decreases significantly,making it difficult to maintain the original morphology of the aneurysm wall and leading to rupture.2.TSG-6,an endogenously secreted anti-inflammatory protein,can be expressed on vascular smooth muscle in response to macrophage inflammation,TSG-6 regulates atherogenesis and progression through negative regulation of TLR4 receptors,inhibition of macrophage chemotaxis,regulation of macrophage polarization to M2-like cells,and inhibition of VSMC proliferation and phenotype modulation.3.Inhibition of TLR4 receptor expression resulted in a significant decrease in the probability of aneurysm formation and rupture.Negatively regulating the activation of TLR4-NFκB pathway could reduce MCP-1 secretion and attenuate the infiltration of macrophages into the vessel wall;induce and regulate the polarization of macrophages to M2-like cells and attenuate the inflammation of the vessel wall;and also inhibit the conversion of VSMC from contractile to synthetic phenotype,delaying the occurrence,development and rupture of aneurysms. |
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