Regulation Of NOD/RIP2 Signal Pathway In Macrophage Inflammatory Activation

Aim To explore the role of NOD1/RIP2 signal pathway in the initiation and progression of atherosclerosis,the effects of NOD1/RIP2 on macrophage inflammatory activation and phenotypic variation were observed by the human monocytic cell line THP-1.Methods Macrophages were incubated with different concentrations of ox-LDL(10,25,50 mg/L)for 24 h and were incubated with 50 mg/L for different time(8,16,24 h).The expression of NOD1 and RIP2 was detected by real-time PCR and Western blot.ELISA was used to detected the Tumor Necrosis Factor-α(TNF-α)and monocyte chemotactic protein 1(MCP-1).The RIP2 si RNA was designed and transfected into macrophages by hiperfict transfection reagent.The most efficient si RNA was selected by real-time PCR and Western blot.After transfection,cells was treated with 50 mg/L ox-LDL.TNF-α and MCP-1 was detected by ELISA.FACS was used to detect membrane molecule CD86.The expression of IL-10/IL-12 was detected by real-time PCR.Results ox-LDL could up-regulate the expression of NOD1/RIP2 signal pathway as a dose-dependent manner and time-dependent manner in THP-1 derived macrophages.The expression of NOD1 m RNA was up-regulated by the increasing concentrations of ox-LDL(control:1 ± 0;10 mg/L:2.32 ± 0.7;25 mg/L:4.37 ± 1.36;50 mg/L:15.6 ± 1.95,P<0.01).RIP2 m RNA also up-regulated(control:1±0;10 mg/L:3.45±0.98;25 mg/L:7.62±0.92;50mg/L:10.21 ± 1.13,P<0.01).ox-LDL could also up-regulate the expression of NOD1protein(control:0.14 ± 0.01;10 mg/L:0.30 ± 0.03;25 mg/L:0.93 ± 0.03;50 mg/L;10.21 ±1.13,P<0.01)and RIP2 protein(control:0.2±0.02;10 mg/L:0.21±0.01;25 mg/L:0.85±0.07;50 mg/L:1.52±0.09,P<0.01).The expression of NOD1 m RNA was up-regulated by the increasing time of ox-LDL(control:1±0;8 h:7.39±1.59;16 h:11.2±2.51;24 h:16.1±3.59,P<0.01),and RIP2 m RNA also up-regulated(control:1±0;8 h:1.46±0.29;16 h:2.54±0.37;24 h:4.27±0.69,P<0.01).ox-LDL could also up-regulate the expression of NOD1protein(control:0.41±0.03;8 h:1.63±0.12;16 h:2.27±0.11;24 h:2.86±0.15,P<0.01)and RIP2 protein(control:0.39 ± 0.03;8 h:1.23 ± 0.14;16 h:4.42 ± 0.28;24 h:5.93 ± 0.64,P<0.01).The expression of TNF-α(control:18.48 ± 0.76 pg/m L;10mg/L:24.6 ± 2.78pg/m L;25 mg/L group:30.56 ± 3.58 pg/m L;50mg/L:61.52 ± 3.91 pg/m L,P<0.01)and MCP-1(control:49.10±4.03 pg/m L;10mg/L:108.54±4.96 pg/m L;25 mg/L:127.91±4.12pg/m L;50mg/L:163.4±7.22 pg/m L,P<0.01)increased with the increasing concentrations of ox-LDL.The expression of TNF-α(control:18.51 ± 0.77 pg/m L;8 h:27.01 ± 0.95pg/m L;16 h:44.71 ± 2.05 pg/m L;24 h:71.53 ± 2.35 pg/m L,P<0.01)and MCP-1(control:49.10 ± 4.03 pg/m L;8 h:74.51± 3.93 pg/m;16 h:77.76 ± 7.24 pg/m L;24h:163.4 ± 7.22 pg/m L,P<0.01)increased with the increasing time of ox-LDL.After transfection,real-time PCR showed that 50 nmol/L was the most efficient concentration(0.22±0.08 vs.1±0,P<0.01).The most efficient si RNA was selected by Western blot(0.38 ± 0.05 vs.2.22 ± 0.08,P<0.01).The most efficient si RNA was transfected into macrophages.After transfection,cells was treated with 50 mg/L ox-LDL.Compared with control,the group incubate with ox-LDL increased the expression of TNF-α(30.68 ± 1.31 pg/m L vs.12.77 ± 0.68 pg/m L,P<0.01)and MCP-1(53.42 ± 2.99pg/m L vs.18.06 ± 2.11 pg/m L,P<0.01).Compared with the the group incubate with ox-LDL,the group si RNA+ ox-LDL decreased the expression of TNF-α(30.68 ± 1.31pg/m L vs.12.77±0.68 pg/m L,P<0.01)and MCP-1(28.62±0.88 pg/m L vs.53.42±2.99pg/m L,P<0.01).Real-time PCR and FACS was used to detect IL-10/IL-12/membrane molecule CD86.Compared with control,the group incubate with ox-LDL decreased the expression of CD86(15821±525.8 vs.20415.9±389.3,P<0.01)/ IL-12(0.3±0.07 vs.1±0,P<0.01),and increased the expression of IL-10(4.26±0.22 vs.1±0,P<0.01).Compared with the the group incubate with ox-LDL,the group si RNA + ox-LDL did not change the expression of CD86(16064.2±631.8 vs.15821±525.8,P>0.05)/IL-12(0.3±0.07 vs.0.3 1±0.08,P>0.05)/IL-10(4.31±0.28 vs.4.26±0.22,P>0.05).Conclusion ox-LDL can up-regulate the expression of NOD1/RIP2 signal pathway as a dose-dependent manner and time-independent in macrophages.NOD1/RIP2 signaling pathway can regulate the inflammatory activation of macrophages induced by ox-LDL,but it has no significant effect on macrophage phenotypic variation induced by ox-LDL.