Objective To investigate effects of oxidized low density lipoprotein/β2 glycoprotein-Ⅰ/anti-β2 glycoprotein-Ⅰ antibody(oxLDL/β2GPⅠ/aβ2GPⅠ)complex on the proliferation,migration and angiogenesis of vascular endothelial cells and its mechanism.Methods Human umbilical vein endothelial cells(HUVEC)were cultured to logarithmic growth phase and grouped into the control group(normal culture),the oxLDL group(50 mg/L oxLDL),the oxLDL/β2GPⅠ/aβ2GPⅠ group(50 mg/L oxLDL/100 mg/L β2GPⅠ/100 mg/L aβ2GPⅠ)and the VEGF group(100 μg/L VEGF).The gene expressions of VEGF,vascular endothelial cadherin(VE-cadherin),matrix metalloproteinase(MMP)-2 and MMP-9 were detected by real-time quantitative fluorescent PCR(qPCR).Cell counting kit-8(CCK-8)method was employed to detect cell proliferation.Cell migration and invasion were determined by scratch healing test and Transwell assay.Matrigel tube formation assay was used to observe the angiogenesis of HUVEC.The relative protein expression of TLR4,MyD88 and NF-κB were examined by Western blot assay.Results Compared with the control group,the proliferation activity of cells at 48 h of treatment was increased in the oxLDL/β2GPⅠ/aβ2GPⅠ group(P＜0.05).Moreover,compared with the control group,cell migration and angiogenesis were increased in the oxLDL/β2GPⅠ/aβ2GPⅠ group,and the mRNA levels of VEGF,VE-cadherin,MMP-2 and MMP-9 were elevated(P＜0.05).Compared with the control group,levels of TLR4 and MyD88 were elevated in the oxLDL/β2GPⅠ/aβ2GPⅠ complex group(P＜0.05),as well as levels of p-NF-κB p65/NF-κB p65(P＜0.05).Conclusion oxLDL/β2GPⅠ/aβ2GPⅠ complex may promote the proliferation,migration and tube formation of vascular endothelial cells by regulating TLR4/MyD88/NF-κB signaling pathway.

Objective: To investigate the effects of oxidized low-density lipoprotein/β2 glycoproteinⅠ/β2 glycoproteinⅠantibody (oxLDL/β2GPⅠ/β2GPⅠ-Ab) complex on the phenotypic transformation and lipid transpoters on the surface of rat thoracic aorta smooth muscle cell line (A7r5), and their correlation with toll-like receptor 4 (TLR4) signaling pathway. Methods: A7r5 cells were stimulated by oxLDL, oxLDL/β2GPⅠ complex, oxLDL/β2GPⅠ-Ab complex, β2GPⅠ/β2GPⅠ-Ab complex and oxLDL/β2GPⅠ/β2GPⅠ-Ab complex respectively, and then total RNA and protein were collected. The expressions of α-smooth muscle actin (α-SMA), macrophage surface marker CD68, galectin-3 (LGALS3), scavenger receptor class B member 3 (CD36) and ATP-binding cassette transporter A1/G1 (ABCA1/ABCG1) were detected by real-time quantitative PCR (RT-qPCR), western blot and immunofluorescence (IF) respectively. The roles of TLR4 and its downstream signaling molecules in the phenotypic transformation and expression changes of lipid transporters of A7r5 cells induced by oxLDL/β2GPⅠ/β2GPⅠ-Ab complex were investigated by the pretreatment of TLR4 blocker TAK-242 (5 μmol/L) or c-Jun N-terminal kinases 1/2 (JNK 1/2) blocker SP600125 (90 nmol/L). Results: The oxLDL/β2GPⅠ/β2GPⅠ-Ab complex significantly increased the levels of CD68 and LGALS3, and decreased the level of α-SMA, while TAK-242 could reverse this phenomenon. The oxLDL/β2GPⅠ/β2GPⅠ-Ab complex could promote the expression of CD36 and inhibit the expression of ABCA1/ABCG1, while TAK-242 and SP600125 could reverse this process. Conclusion The oxLDL/β2GPⅠ/β2GPⅠ-Ab complex promotes the phenotypic transformation of A7r5 cells to macrophage-like cells, regulates the expression of lipid transport-related molecules and enhances the ability of lipids transport into cells. TLR4 and JNK1/2 are closely related to this process.

Objective: To investigate the effects of β2 glycoprotein Ⅰ/anti-β2 glycoprotein Ⅰ complex (β2/aβ2) on oxidized low density lipoprotein (oxLDL)-mediated lipid accumulation and focal adhesion kinase (FAK) activation in THP-1 macrophage, as well as the role of Toll-like receptor 4 (TLR4) during the process. Methods: THP-1 cells were differentiated into THP-1 macrophage by PMA (100 ng/mL). THP-1 macrophages were treated with RPMI 1640 medium, oxLDL, oxLDL+β2/aβ2 or oxLDL+lipopolysaccharide (LPS). The mRNA expressions of lipid transportation molecules, ACAT1, ABCA1 and ABCG1 were detected by RT-qPCR. Intracellular total cholesterol (TC) and free cholesterol (FC) in THP-1 macrophages were evaluated by Trinder assay, then the content and proportion of intracellular cholesteryl ester (CE) were calculated. The expression and phosphorylation of FAK were detected by immune fluorescence, RT-qPCR and western blot. To evaluate the role of TLR4, THP-1 macrophages were pre-treated with or without TLR4 inhibitor TAK-242 (1 μg/mL). Results: β2/aβ2 treatment significantly inhibited oxLDL-mediated lipid accumulation and FAK expression and phosphorylation in THP-1 macrophages, which could be reversed by TLR4 blockage. Conclusion β2/aβ2 inhibits the oxLDL-mediated lipid accumulation and FAK activation of THP-1 macrophage, which is related to the function of TLR4.

Objective:To investigate the antioxidant function of Mycoplasma pneumoniae MPN662 and analyze the key active sites, and to explore the role of MPN662 in the regulation of the production of reactive oxygen species (ROS) and superoxide dismutase (SOD) in THP-1 cells. Methods:pET28a(+ )- mpn662, recombinant mutant plasmids pET28a(+ )- mpn662-Ser 66 (the 66 th Cys was mutated to Ser) and pET28a(+ )- mpn662-Ala 66 (the 66 th Cys was mutated to Ala) were constructed, recombinant proteins rMPN662, rMPN662-Ser 66 and rMPN662-Ala 66 were expressed, identified, and purified. DTNB method was employed to analyze the MetO reduction activity of rMPN662 and recombinant mutant protein. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were applied to examine the transcription level of the mpn662 gene and the expression level of MPN662 protein after Mycoplasma pneumoniae were stimulated with different concentrations of hydrogen peroxide (H 2O 2) or tert-butyl hydroperoxide (t-BHP), respectively. Fluorescent probes (DCFH-DA) and the total SOD activity detection kit were used to test the levels of intracellular ROS and SOD in THP-1 cells, which were pretreated with rMPN662, and then stimulated by Mycoplasma pneumoniae lipid-associated membrane proteins (LAMPs). Results:Mycoplasma pneumoniae rMPN662 could reduce MetO to Met, and the enzyme activities of mutant protein were significantly lower than those of rMPN662 protein. mpn662 gene mRNA transcription level and MPN662 protein expression level were significantly increased in a dose-dependent manner when Mycoplasma pneumoniae was stimulated with H 2O 2 and t-BHP. Treatment with rMPN662 before THP-1 cells were exposed to LAMPs could decrease the level of ROS and increase the production of SOD. Conclusions:Mycoplasma pneumoniae MPN662 can reduce MetO to Met, and Cys66 is the key amino acid for this activity. MPN662 can decrease the release of ROS and increase the production of SOD in Mycoplasma pneumoniae LAMPs stimulated THP-1 cells.