CTRP9 Induces Macrophage Derived Foam Cells Necrotosis Through AMPK Pathway

Objective:1.Effects of CTRP9 on the necroptosis of macrophage derived foam cells.2.Whether the necroptosis of foam cells prevented by CTRP9 is related to the AMPK pathway.Methods:1.Cell culture and foam cell model establishment(1)RAW264.7 cells of mice were cultured in 10% complete medium and cultured in 37 C and 5% carbon dioxide incubator.(2)The final concentration of 50μg/ml oxidized low density lipoprotein(ox-LDL) was used to establish foam cell model for 24 h.(3)To induce the foam cells necroptosis,the cells cultured in serum-free medium and ZDEVD and ZIETD(ZDEVD,20μM,ZIETD,20μM)at suitable concentration for 24 h.2.The experimental group:(1)Control group.(2)CTRP9 protein(0,0.1,0.3,1 μg/ml)intervention group(24 hours).(3)AMPK/NF-kappa B pathway inhibitor Compound C(10mM)intervention group.(4)AMPK/NF-kappa B pathway inhibitor Compound C(10mM)+CTRP9(1μg/ml)intervention group.3.MTT assay was used to detect cell viability.RT-PCR was used to detect the expression of RIP1 and RIP3.Western blot was used to detect the expression of RIP1 and RIP3.Results:1.CTRP9 can significantly improve the survival rate of foam cells: after adding different concentrations of CTRP9(0,0.1,0.3,1 μg/ml),MTT method was used to detect the survival rate of RAW264.7 derived foam cells(P < 0.05).2.CTRP9 inhibited the necroptosis of foam cells in a concentration dependent manner.Western Blot and qRT-PCR detected that compared with the control group,the relative expression levels of RIP1,RIP3 mRNA and its protein decreased after adding CTRP9 group(P < 0.05).With the increase of CTRP9 concentration(0,0.1,0.3,1μg/ml),the relative expression levels of RIP1,RIP3 and its protein decreased gradually.3.CTRP9 inhibits the expression of RIP1 and RIP3 via AMPK signaling pathway:Western Blot and qRT-PCR detection showed that,compared with CTRP9 group,the Compound C(AMPK inhibitor)inhibited the relative expression of RIP1,RIP3 and their proteins(P < 0.05).Conclusing:1.CTRP9 increased the expression of RIP1 and RIP3 in RAW264.7 derived foam cells in a concentration dependent manner,and inhibited necroptosis.2.Compound C can prevent CTRP9 from decreasing the expression of RIP1 and RIP3 in RAW264.7 derived foam cells,that is,CTRP9 inhibits necroptosis of RAW264.7derived foam cells and is related to AMPK pathway.