| Objective: The main pathological basis of antiphospholipid syndrome(APS)is hypercoagulable state and the presence of a large number of antiphospholipid antibodies(aPL).β2 glycoprotein I(β2GPI),a key target antigen of aPL,can bind to the oxidized lipoprotein specifically,and the anti-β2 glycoprotein I antibody(β2GPI-Ab)binds to the β2GPI-oxidized lipoprotein complex could interfere the lipid metabolism.Atherosclerosis(As)mainly affects the large and middle arteries in the body.There are complex pathological changes in atherosclerotic plaques,and vascular smooth muscle cells(VSMC)play an important role in this process.aPL promotes inflammation and procoagulant effects on endothelial cells,leading to endothelial cell dysfunction and inflammatory response,suggesting a correlation between APS and As.In recent years,studies have found the phenotypes of VSMC in atherosclerotic plaques undergo “macrophage-like” change,and can phagocytose large amounts of lipids to form “foam cells”,then reducing the stability of plaques.Previous studies of our research team found that Toll-like receptor 4(TLR4)plays an important role in the accumulation of lipids in rat thoracic aorta smooth muscle cell line(A7r5)with oxLDL/β2GPI/β2GPI-Ab complex stimulation.But the specific mechanism is not yet clarified.This article explores the role of TLR4 in the oxLDL/β2GPI/β2GPI-Ab complex induced phenotypic transformation of A7r5 to “macrophage-like” cells at first.Then we further explore TLR4-c-Jun N-terminal kinase 1/2(JNK1/2)signaling pathway in the expression of lipid transport-associated molecules induced by oxLDL/β2GPI/β2GPI-Ab complex.Methods:(1)Real-time quantitative PCR(RT-q PCR)and immunofluorescence(IF)were used to detect oxLDL,oxLDL/β2GPⅠ complex,oxLDL/β2GPⅠ-Ab complex,β2GPⅠ/β2GPⅠ-Ab complex and oxLDL/β2GPⅠ/β2GPⅠ-Ab complex respectively inducing the m RNA and protein expression of smooth muscle cytoskeletal protein α-smooth muscle actin(α-SMA),macrophage surface marker CD68 and galectin-3(LGALS3)in A7r5 cells.The cells were pretreated with the TLR4-specific inhibitor TAK-242 to confirm the involvement of TLR4 in the process.(2)Western blot was used to analyze oxLDL,oxLDL/β2GPⅠ complex,oxLDL/β2GPⅠ-Ab complex,β2GPⅠ/β2GPⅠ-Ab complex and oxLDL/β2GPⅠ/β2GPⅠ-Ab complex induce A7r5 cells t-JNK1/2 and p-JNK1/2(Thr183/Tyr185)protein expression.(3)oxLDL,oxLDL/β2GPⅠ complex,oxLDL/β2GPⅠ-Ab complex,β2GPⅠ/β2GPⅠ-Ab complex and oxLDL/β2GPⅠ/β2GPⅠ-Ab complex stimulate A7r5 cells to observe the m RNA and protein expression of CD36,ABCA1 and ABCG1.The cells were pretreated with the TLR4-specific inhibitor TAK-242 or the JNK1/2 inhibitor SP600125 to investigate whether TLR4 or JNK1/2 were involved in the process.Results:(1)oxLDL(30 μg/m L)/ β2GPⅠ(100 μg/m L)/ β2GPⅠ-Ab(100 μg/m L)complex can significantly promote the expression of macrophage molecular markers CD68 and LGALS3 in vascular smooth muscle cells,and decrease the expression of smooth muscle cytoskeletal protein α-SMA.TAK-242(5 μM)significantly inhibited the phenotype change induced by oxLDL/β2GPⅠ/β2GPⅠ-Ab complex.(2)The oxLDL/β2GPI/β2GPI-Ab complex can increase the phosphorylation level of JNK1/2 in A7r5 cells.At the same time,it can significantly increase the m RNA and protein expression of CD36,and inhibit the expression of ABCA1 and ABCG1 in A7r5 cells.(3)After pretreatment with TAK-242(5 μM)or SP600125(90 n M),the addition of CD36 and decrease of ABCA1 and ABCG1 induced by oxLDL/β2GPI/β2GPI-Ab complex were reversed in A7r5 cells.Conclusions:(1)The oxLDL/β2GPI/β2GPI-Ab complex induced the transformation of A7r5 cells from contractile phenotype to “macrophage-like” cells,which can be reversed by using TLR4 inhibitor TAK-242.(2)The oxLDL/β2GPI/β2GPI-Ab complex induced the increase in the phosphorylation level of JNK1/2 in A7r5 cells,and promoted the expression of CD36 while inhibiting the expression of ABCA1 and ABCG1.(3)After pretreatment with TAK-242 or SP600125,the increase of CD36 and inhibition of ABCA1 and ABCG1 expression were reversed which induced by oxLDL/β2GPI/β2GPI-Ab complex in A7r5 cells. |
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