Study On The Immune Response And The Intervention Effects Mediated By TLR4Pathway In Atherosclerotic Mouse Models Complicated By Lupus

BackgroundAtherosclerosis (AS) is a chronic inflammatory disease charactered withautoimmune inflammation. Systemic lupus erythematosus (SLE) is a typicalautoimmune disease. The morbidity and mortality rates of cardiovascular andcerebrovascular diseases are dramatically increased in SLE. Recently, it is reportedhigh mobility group protein (HMGB1) and toll like receptor (TLRs) are the keymolecules to exacerbate AS and SLE. But the roles of HMGBI-TLR4axis in the micemodels of SLE complicated with AS, still remain unclear.ObjectiveThe ApoE-/-mice were intraperitoneally administered pristane to establish themice model of SLE complicated with AS. HMGB1, TLR4and the downstream keysignal molecules expression in aortic plaque were determined. TLR4-RNAinterference (RNAi) was performed to further validate the role of TLR4signal in thismodel. Our study was aimed to provide a theoretical basis for AS relatedautoimmune inflammatory mechanism and for its potential therapy method.Methods1. Selection of effective siRNA vector for TLR4: The efficiency of lentivirusestransfecting mouse smooth muscle cells was determined by the expression intensity ofGFP fluorescence. Real time PCR was performed to select effective lentivirusestargeting mouse TLR4small interfering RNA (shRNA).2. Mice were randomized into four groups：ApoE-/-pristane (AS)，ApoE-/-control(A), B6pristane (S) and B6control group (C),30in each group. The pristanemice received a single intraperitoneal (i.p) injection of0.5mL of pristane(Sigma-Aldrich), while control mice isovolumetric saline. Mice were treated withhigh-fat diet for12weeks. Then they were further divided into the carotid arterycatheter subgroup, TLR4i subgroup and non interference subgroup. Anther12-weekhigh fat diet was administered. 3. Histological and morphological analysisThe specimens of aortic tree, brachiocephalic artery plaques, left renal artery, HE,oil red were collected. H.E, oil red O staining, immunohistochemistry andimmunofluorescence staining were performed to investigate the following indexes:（1）The efficiency of lentiviral transfection: the expression of GFP in plaquesand renal tissue were detected with fluorescent microscope, The expression of TLR4mRNA was assayed by quantitative real-time PCR analysis;（2）Neointima formation: The thickness of neointimal formation was measuredin all the mice following collar placement;（3）The distribution and expression of BAFF in abdominal aortic adventitia wasevaluated by immunohistochemical analysis. And the macrophage phagocytic lipidfunction was assessed through peritoneal macrophages primary culture and oil redstaining;（4）The size, number, the percentage of plaque area in aortic tree weredetermined with En face quantification of atherosclerotic lesions after oil-red Ostaining.（5）For plaque vulnerability analysis, immunohistochemistry was performed todetect macrophage marker F4/80and smooth muscle α-actin. The content of type Icollagen was assessed by Picro-sirius Red staining, and deposition of lipids wasdetermined with Oil Red O staining. The plaque vulnerability index was calculated aspositive staining area of (macrophages+lipid) divided by positive staining area of(smooth muscle α-actin+collagen).（6）Prussian blue staining was performed to detect the occurrence ofhemorrhage in plaque. Apoptosis was analyzed with terminal deoxynucleotidyltransferase end-labeling (TUNEL) kit.（7）The glomerular injury, the glomerular deposition of IgG and C3wereassayed by HE staining and immunofluorescence respectively.4. Molecular biological analysis（1）The proteins expressions of HMGB1, TLR4，IRF3， MyD88，TRIF， NF- κB in aorta were determined with western blotting.（2）The mRNA levels of TLR4, IL-4, IL-12, IFN-β,MCP-1and TNF-αweredetected with real-time PCR.5. Lipid profile and auto-antibodies in serum.The levels of anti-double-stranded deoxyribonucleic acid (anti-dsDNA) antibodyand anti-Sm were measured by ELISA. Serum total cholesterol and triglyceridesconcentrations were measured by enzymatic assay.Results1. The silencing rate of TLR4gene expression was89.5%.2. Pristane promoted the formation of neointima after following collar placemen.The intima thickness in AS group was1.25-fold higher than A group, and S group was1.55-fold higher than C-p group.3. Pristane induced high expression of vascular adventitial BAFF, and abdominalmacrophage phagocytosis was enhanced.4. The percentage of plaque area was1.83times higher in AS mice than that in Agroup. And it was reduced by26.5%in AST group while by29.6%in AT as comparedwith their corresponding controls.5. The vulnerability index in AS, A, AST and AT groups were3.41±0.44,2.71±0.38,2.13±0.25,1.38±0.19respectively, the difference showed statisticalsignificance.6. Pristane accelerated intraplaque: the incidence of hemorrhage was40.5%inAS empty vector group, significantly higher than that in A group (2%). Pristanepromotes plaque apoptosis: the percentage of TUNEL positive cells in AS group wassignificantly higher than the A group (41.8±3.9%vs24.9±3.1%). The percentage ofTUNEL positive cells in group AST and group AT plaques were decreased by22.2%,29.3%respectively following RNAi treatment.7. Pristane and apoE gene knockout accelerated renal tissue damage and IgGand C3deposition. The antibody level of anti ds-DNA antibody, anti Sm in serum inAS group were significantly higher than that of C, A and S group. The titers ofds-DNA autoantibody in A group, and anti-Sm in S group were significantly higher as compared with C group (P<0.05). And pristane had no significant effect on the levelsof serum lipid.8. Western blot analysis showed pistane caused varying degrees of upregulationMGB1-TLR4, MyD88and TRIF, NF-κ B and IRF3protein expressions.9.The levels of TLR-4, TNF-α, IFN-β, MCP-1, IL-4, IL-12mRNA wereincreased significantly in AS group, which were attenuated by RNA interference.Conclusions1. Pristane accelerates AS, as evidenced by thickened neointima, elevated lesionsburden, increased plaque vulnerability, increased adventitial BAFF content andapoptosis.2. ApoE-/-increases the production of anti ds-DNA antibodies and anti-Smantibodies levels; And it elevates glomerular IgG deposition and C3depositions.3. Aortic HMGBI-TLR4-MyD88-NF-κ B pathway activation maybe theunderlying pathogenesis for the SLE complicated with AS. Our results provide a newtheoretical basis for in-depth study of the pathogenesis of AS and potential therapeuticapproaches.