The Role Of CTRP9 In Inflammatory Cytokines Expression And Its Molecular Mechanisms In Macrophages

1 BackgroundAtherosclerotic cardiovascular disease (ASCVD) are the leading cause of the mortality and disability in our country and around the world. The globular burden of ASCVD will increase at a higher rate in future years. Atherosclerosis (AS) is the main pathologic basis of ASCVD. However, the pathophysiological mechanism of AS has not been completely elucidated so far. Numerous evidences show that atherosclerosis is an inflammatory disease of the arteries associated with lipid and other metabolic alterations. The pathologic factors of lipid metabolism disorder and inflammation interact with each other in the pathologic process of AS. Within the atherosclerotic lesions, macrophages are the key cellular protagonists in the whole process of AS. Modified lipids engulfed by monocyte-derived macrophages lead to the secretion of pro-inflammatory cytokines. Focusing on the regulatory mechanisms of macrophage inflammation is important to the prevention and treatment of atherosclerosis.C1q-TNF-related protein-9 (CTRP9) is a novel adipokine which belongs to the protein family of C1q/TNF-related proteins. CTRP9 plays important regulatory roles in metabolism, myocardial protection, vasodilation and diabetic cardiomyopathy. In previous study, we demonstrated that CTRP9 was able to protect against atherosclerotic vulnerable plaques by reducing pro-inflammatory cytokines in macrophages. However, the underlying mechanism by which CTRP9 may exert its anti-inflammatory effects in macrophages has not been elucidated. In the present study, we aim to clarify the possible mechanism of gCTRP9 on anti-inflammatory response induced by oxLDL in RAW 264.7 macrophages.2 Objective(1) To investigate the effects of CTRP9 on the expression of inflammatory factors in oxLDL induced inflammation in macrophages;(2) To elucidate the underlying molecular mechanisms of CTRP9 mediated anti-inflammatory effects in macrophages.3 Methods3.1 Cell culture and treatment(1) RAW 264.7 macrophages was cultured in DMEM medium with 10%fetal bovine serum and 1% penicillin/streptomycin at 37℃ and 5% CO2 in a humidified incubator.(2) OxLDL (100 ug/ml) was used to stimulate and induce inflammatory responses in RAW264.7 macrophages. Macrophages were pretreated with gCTRP9 for 2 h, then incubated with oxLDL for the indicated times. And then cells were harvested for measurement.3.2 Quantitative real-time PCRTotal RNA was extracted from RAW264.7 macrophages to detect the expression level of TNF-a and MCP-1 mRNA.3.3 Western blotting analysisTotal proteins were extracted from macrophages. The protein expression of TNF-a, MCP-1, NF-κB, AMPK and AdipoRl were analyzed by western blot.3.4 Immunofluorescent stainingCoverslips were mounted on slides and immunofluorescence were performed in cells. The expression of TNF-a, MCP-1, AdipoRl and the nuclear translocation of NF-κB was evaluated under a confocal microscope.3.5 AdipoRl siRNA transfectionThe highest efficiency sequence was screened by western blot. After transfection with Lipofectamine 2000 according to the manufacturer’s instructions, cells were used in the following experiment.3.6 Statistical AnalysisSPSS software 13.0 was used for statistical analysis. Data were presented as mean±SD. The differences were determined by one-way ANOVA. P< 0.05 was considered to be statistically significant.4 Results4.1 CTRP9 suppressed oxLDL-stimulated inflammation in macrophagesThe protein expression of TNF-a and MCP-1 was increased under the treatment of oxLDL (p< 0.05). Compared with the oxLDL group, gCTRP9 could reduce TNF-a and MCP-1 protein levels in a dose-dependent manner (p< 0.05). Also, real-time PCR and confocal images showed that pretreatment with gCTRP9 significantly suppressed TNF-a and MCP-1 protein and mRNA expression in response to oxLDL (p< 0.05).4.2 CTRP9 inhibited NF-κB p65 activation in oxLDL-induced macrophage inflammationThe phosphorylation level of NF-κB p65 was increased by oxLDL (p< 0.05). However, gCTRP9 markedly diminished its activation compared with the oxLDL group (p< 0.05). Western blot and confocal images showed that oxLDL increased the nuclear localization of NF-κB p65 (p< 0.05), and gCTRP9 exerted the ability to suppress the nuclear translocation induced by oxLDL (p< 0.05).4.3 Activation of AMPK by CTRP9 contributed to its anti-inflammatory effectsCompared with control group or oxLDL group, gCTRP9 significantly activated the phosphorylation of AMPK in macrophages (p< 0.05). Moreover, Compound C (Com C) effectively restrained the phosphorylation after gCTRP9 treatment (p< 0.05). Com C dramatically reversed the suppressive effect of gCTRP9 on the expression of TNF-a and MCP-1 (p< 0.05). Also, the NF-κB nuclear translocation was restored when AMPK was inhibited by Com C (p< 0.05).4.4 AdipoRl mediated the anti-inflammatory effects of CTRP9The expression of AdipoRl was not altered by oxLDL. Compared with the control group or oxLDL group, the expression of AdipoRl was increased under the treatment of gCTRP9 (p< 0.05). Confocal images demonstrated that gCTRP9 could obviously increase the expression of AdipoRl on the plasma membrane of macrophages compared with the two groups (p< 0.05). The activation of AMPK by gCTRP9 was abolished in AdipoRl knockdown macrophages compared with the negative control (p<0.05). Moreover, knockdown of AdipoRl reversed the inhibitory effects of gCTRP9 on the expression of TNF-a and MCP-1 induced by oxLDL compared with the negative control (p<0.05).5 Conclusions(1) CTRP9 increases the expression of AdipoRl in macrophages;(2)CTRP9 suppresses the inflammatory responses in macrophages through AdipoRl/AMPK/NF-κB pathway.