Ox-LDL/β2GPI/ANTI-β2GPI Complex Promotes Apoptosis And Inflammatory Factors Expression In RAW264.7 Cells

Objective:Patients with antiphospholipid syndrome(APS)are prone to have complications of atherosclerosis(AS),which progresses faster and more severely than primary AS.The previous results of our research group show that ox-LDL/β2GPI/anti-β2GPI complex can promote macrophages foaming through the TLR4 pathway,but its effects on the apoptosis of macrophages have not been explored.Therefore,the purpose of this study is to explore the effects of ox-LDL/β2GPI/anti-β2GPI complex on the apoptosis of mouse mononuclear macrophage RAW264.7 cells,as well as the roles of inflammatory factors,TLR4 pathway and m TOR pathway during the process.Methods:(1)RAW264.7 were treated with ox-LDL/β2GPI/anti-β2GPI complex for different durations.The apoptosis level of RAW264.7 was detected by Annexin V-FITC/PI apoptosis kit,and the m RNA expression levels of inflammatory factors IL-1β and TNF-α were detected by RT-q PCR.(2)RAW264.7 were treated with medium,ox-LDL,β2GPI/anti-β2GPI complex,ox-LDL/β2GPI complex,ox-LDL/anti-β2GPI complex and ox-LDL/β2GPI/anti-β2GPI complex respectively.The apoptosis levels of RAW264.7 were detected by Annexin V-FITC/PI apoptosis kit.The protein expression levels of Cleaved-Caspase-3,Bcl-2,IL-β,TNF-α,p/T-m TOR,TLR4 and p/T-NF-κB were detected by Western Blot.The m RNA expression levels of inflammatory factors were detected by RTq PCR.(3)RAW264.7 were treated with medium,ox-LDL/β2GPI/anti-β2GPI complex,TLR4 inhibitor(TAK-242,1 μg/m L)combined with oxLDL/β2GPI/anti-β2GPI complex and m TOR inhibitor(rapamycin,50 n M)combined with ox-LDL/β2GPI/anti-β2GPI complex,the m RNA expression levels of IL-β and TNF-α were detected by RT-q PCR.The protein expression levels of Cleaved-Caspase-3,Bcl-2,IL-β and TNF-α were observed by Western Blot.Annexin V-FITC/PI apoptosis kit was used to analyse the apoptosis levels of cells.(4)TNF-α at different concentrations(0,50,100 and 200 ng/m L)were used to stimulate RAW264.7 for 36 h,and the apoptosis levels of cells were detected by Annexin V-FITC/PI apoptosis kit.The protein expression levels of Cleaved-Caspase-3 and Bcl-2 were detected by Western Blot.Results(1)After treatment with ox-LDL/β2GPI/anti-β2GPI complex,the apoptosis rates of RAW264.7 increased with time prolongation,and there were significant differences at 36 h and 48 h compared with 0 h(P<0.05).Among the six groups,the increasement of apoptotic rate and Cleaved-Caspase-3 and the decreasement of Bcl-2 were significantly in ox-LDL/β2GPI/anti-β2GPI complex group(P<0.05).(2)After treatment with ox-LDL/β2GPI/anti-β2GPI complex,the m RNA expression of IL-1β and TNF-α reached the peak at 12 h.The m RNA and protein expression levels of two inflammatory factors in oxLDL/β2GPI/anti-β2GPI complex group were higher than that of other groups(P<0.05).(3)After treated with ox-LDL/β2GPI/anti-β2GPI complex,TLR4 and m TOR signaling pathway molecules were activated,and the protein expression of those molecules was significantly increased(P<0.05).(4)After pre-treatment with TLR4 inhibitor or m TOR inhibitor,the m RNA and protein levels of IL-1β and TNF-α were partially decreased.At the same time,the increased apoptotic rates of cells were decreased,and the induced apoptotic protein expression was also reversed to a certain extent(P<0.05).(5)When the TNF-α concentration reached 100 ng/m L,the apoptosis rate was significantly increased(P<0.05).Conclusions:(1)The ox-LDL/β2GPI/anti-β2GPI complex can induce the apoptosis of RAW264.7 cells and promote the expression of inflammatory factors.TLR4 inhibitor or m TOR inhibitor can partially alleviate these changes,suggesting that TLR4/NF-κB pathway and m TOR pathway play a great role during the process.(2)A certain concentration of TNF-α can induce apoptosis of macrophages,suggesting that the complex promotes the level of apoptosis by promoting the expression of inflammatory factors.