Introduction

Atherosclerosis is an important pathological procedure in cardiovascular diseases and underlies the leading cause of death all over the world. The evolving inflammatory reactions induced by oxidized low-density lipoprotein (oxLDL) are essential to the initiation of atherosclerotic plaques and their destabilization [1, 2]. Within the atherosclerotic lesions, macrophages are the key cellular protagonists of the ongoing inflammatory response [3, 4]. Modified lipids engulfed by macrophages lead to the secretion of pro-inflammatory cytokines and further macrophages recruitment, which increases the size and complexity of the atherosclerotic plaques [5]. Focusing on the regulatory mechanisms of macrophage inflammation is important to the prevention and treatment of atherosclerosis.

Adenosine monophosphate-activated protein kinase (AMPK) is an evolutionary conserved energy sensor that regulates energy metabolic homeostasis [6]. Emerging evidences have shown a promising role of AMPK in protecting against atherosclerosis [7]. The activation of AMPK has many underlying anti-atherosclerotic properties, including reducing endothelium apoptosis, monocyte adherence, lipid accumulation, and proliferation of inflammatory cells. Moreover, AMPK can increase antioxidant defenses and nitric oxide generation [8]. There are numerous studies supporting the idea that AMPK can inhibit inflammatory responses through suppressing the activation of nuclear factor-κB (NF-κB) [9, 10].

C1q-TNF-related protein-9 (CTRP9), a member of C1q/TNF-related protein families (CTRPs), circulates in plasma in its globular domain isoform (gCTRP9) [11]. As the closest adiponectin paralog, CTRP9 plays important regulatory roles in many cardiovascular diseases [12–14]. It is generally accepted that AMPK is the most important intracellular signaling molecule mediating adiponectin biological functions [15]. Similar to adiponectin, CTRP9 exerts beneficial effects on endothelium relaxation, monocyte adhesion, cardiovascular inflammation, and myocardial apoptosis through AMPK [12, 13, 16, 17]. A very recent study showed that CTRP9 was a protective factor of atherosclerosis independent of adiponectin in human [18]. Moreover, we recently demonstrated that CTRP9 might contribute to stabilization of atherosclerotic plaques [19]. 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.

Materials and methods

Reagents and antibodies

Recombinant mouse gCTRP9 protein was purchased from Aviscera Bioscience (Santa Clara, CA). OxLDL was ordered from Yiyuan biotechnology (Guangzhou, China). Antibodies for p-AMPK α (Th172), AMPK α, p-NF-κB p65 (Ser 536), NF-κB p65, and Histone H3 were all obtained from Cell Signaling Technology (Danvers, MA). Antibodies for TNF-α and AdipoR1 were acquired from Abcam (Cambridge, UK) and MCP-1 antibody was obtained from Novus (Littleton, CO). Antibody for GAPDH was bought from Bioworld Technology (Minneapolis, MN). Compound C was obtained from Sigma-Aldrich (St Louis, MO). Cell culture medium was purchased from HyClone Laboratories (Logan, UT).

Cell culture

RAW 264.7 macrophages were obtained from the American Type Culture Collection (ATCC), which is cultured in DMEM medium with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin at 37 °C and 5 % CO2 in a humidified incubator. Macrophages were pretreated with gCTRP9 for 2 h, then incubated with oxLDL for the indicated times.

Real-time PCR

Total RNA was extracted using the TRIzol reagent (Invitrogen), and then reverse transcribed into cDNA by the PrimeScriptTM RT Reagent Kit (TaKaRa). All gene transcripts were measured by quantitative PCR with the SYRB Premix Ex TaqTM kit (TaKaRa). Results were calculated by 2−ΔΔCt general method, and β-actin was used as an internal control. The sequences of the primers used were as follows: 5′-ACCCTCACACTCAGATCATCTTC-3′ (forward) and 5′-TGGTGGTTTGCTACGACGT-3′ (reverse) for TNF-α; 5′-CACAACCACCTCAAGCACT-3′ (forward) and 5′-AGGCATCACAGTCCGAGTCA-3′ (reverse) for MCP-1; 5′-GGCTGTATTCCCCTCCATCG-3′ (forward) and 5′-CCAGTTGGTAACAATGCCATGT-3′ (reverse) for β-actin.

Immunofluorescent staining

Cells were washed three times with PBS and fixed in 4 % paraformaldehyde for 10 min, washed again, then treated with 0.1 % Triton X-100 for 20 min to increase antigen accessibility. After blocking with 1 % BSA for 30 min, and incubating with the indicated antibodies overnight at 4 °C, we then stained with the appropriate secondary antibodies for 1 h at 37 °C in the dark. Finally, DAPI was used to stain the cell nuclear. Coverslips were mounted on slides and immunofluorescent staining was evaluated under a confocal microscope.

Protein extraction

Cells were washed with PBS and lysed with lysis buffer according to the manufacturer’s instructions. After centrifugation at 12,000×g for 15 min at 4 °C, the supernatants were collected as whole cell lysates. Nuclear and cytoplasmic proteins were extracted using the nuclear and cytoplasmic protein extraction kit (Beyotime). Briefly, cells were dissolved with cytoplasmic protein extraction agent A and incubated on the ice after vortexing at maximum speed. Next, cytoplasmic protein extraction agent B was added, and the cells were incubated on ice after vortexing at maximum speed. Then the samples were centrifuged at 12,000×g for 5 min at 4 °C and the supernatants including the cytoplasmic extracts were collected. Nuclear protein extraction agent was added into the pellet and then shaken violently for 15–20 times for 30 min during incubation on ice. After centrifugation at 12,000×g for 5 min at 4 °C, the supernatants containing nuclear extracts were obtained. Protein concentrations were quantified and normalized by the bicinchoninic acid (BCA) protein assay.

Western blotting

Protein samples were separated by 10 % SDS-PAGE and transferred onto a PVDF membrane (Millipore). After blocking with 5 % non-fat milk in TBST for 1 h, the blocked membranes were separately incubated with the specific antibodies overnight at 4 °C, including TNF-α (diluted 1:1000), MCP-1 (diluted 1:1000), p-NF-κB p65 (diluted 1:1000), NF-κB p65 (diluted 1:1000), p-AMPK (diluted 1:1000), AMPK (diluted 1:1000), AdipoR1 (diluted 1:1000), Histone H3 (diluted 1:1000), GAPDH (diluted 1:1000). Membranes were washed three times with TBS buffer containing 0.1 % Tween (TBST), 10 min each. The secondary antibody conjugated to horseradish peroxidase (1:5000 dilutions with 1 % non-fat milk in TBST) against the primary antibody was added, incubated, and washed as described in the steps above at room temperature with shaking. An ECL Western blotting detection kit (Millipore) was used for detection. Relative protein levels were quantified by using the Image J.

RNA interference

Cells cultured with antibiotic-free medium were transfected with AdipoR1 siRNA or negative control siRNA (GenePharma) along with Lipofectamine 2000 according to the manufacturer’s instructions. After transfection for 48 h, cells were collected and the efficiency of gene silencing was determined by Western blot.

Statistical analysis

SPSS software 13.0 was used for statistical analysis. Data were presented as mean ± SD of at least 3 independent experiments. The differences were determined by one-way ANOVA with LSD post hoc test. P < 0.05 was considered to be statistically significant.

Results

GCTRP9 suppressed oxLDL-stimulated inflammation in macrophages

To explore the effects of gCTRP9 on the expression of TNF-α and MCP-1 in oxLDL-induced RAW 264.7 macrophages, cells were pretreated with different concentrations (0.1, 0.3, and 1 μg/ml) of gCTRP9 for 2 h, and then stimulated with oxLDL (100 μg/ml) for 24 h. The results demonstrated that 100 μg/ml oxLDL markedly increased the expression of TNF-α and MCP-1, and gCTRP9 could reduce TNF-α and MCP-1 protein levels in a dose-dependent manner (Fig. 1a). Based on this result, we used 1 μg/ml gCTRP9 for the subsequent experiments. Confocal images and real-time PCR showed that pretreatment with gCTRP9 significantly suppressed TNF-α and MCP-1 protein and mRNA expression in response to oxLDL (Fig. 1b, c). Furthermore, we are intended to explore the underlying mechanism of gCTRP9 in macrophages.

Fig. 1
figure 1

GCTRP9 suppressed inflammatory response to oxLDL in a dose-dependent manner in RAW 264.7 macrophages. a Western blot was used to detect the expression of TNF-α and MCP-1 in cell lysates pretreated with different concentrations (0.1, 0.3, and 1 μg/ml) of gCTRP9 for 2 h and then 100 μg/ml oxLDL for 24 h. b Confocal pictures of TNF-α and MCP-1 protein (green) in macrophages stimulated with 100 μg/ml oxLDL for 24 h in the presence or absence of 1 μg/ml gCTRP9 pretreatment for 2 h. DAPI (blue) was used to stain nuclei. Scale bar = 20 μm. c Real-time PCR was used to detect the mRNA expression level of TNF-α and MCP-1 in macrophages. *p < 0.05 versus control group, # p < 0.05 versus oxLDL group. (Color figure online)

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GCTRP9 inhibited NF-κB p65 activation in oxLDL-induced macrophage inflammation

NF-κB is a ubiquitous transcription factor triggered by oxLDL and other inflammatory stimulus [20, 21]. Firstly, the phosphorylation of p65 on serine 536 (S536) was analyzed by Western blotting as in Fig. 2a and we can see p65 was activated by oxLDL in 8 h; however, gCTRP9 markedly diminished its activation compared with the oxLDL group. Then, we extracted nuclear protein and tested the NF-κB p65 nuclear translocation. Figure 2b displayed that the p65 nuclear translocation was induced by oxLDL in 24 h, and gCTRP9 significantly inhibited its nuclear translocation. Next, we verified the nuclear localization by immunostaining, indicating that p65 was mainly localized in the cytoplasm in untreated cells. Under the oxLDL stimulus for 24 h, there was a remarkable accumulation of p65 in the nucleus, and gCTRP9 exerted the ability to suppress the nuclear translocation induced by oxLDL (Fig. 2c). Above all, these data clearly showed that gCTRP9 deactivated NF-κB p65 under the stimulation of oxLDL in macrophages.

Fig. 2
figure 2

Inhibition of gCTRP9 on oxLDL-induced NF-κB p65 phosphorylation and nucleus translocation. a Western blot was used to detect the NF-κB p65 phosphorylation in macrophages pretreated with gCTRP9 for 2 h before stimulation with oxLDL for 8 h. b Western blot for NF-κB p65 in the nuclear fraction of the cells treated with gCTRP9 for 2 h in presence or absence of oxLDL for 24 h. c Confocal analysis for NF-κB p65 (green) translocated to the nucleus in macrophages. The positions of the nuclei were determined by DAPI staining (blue). Scale bar = 20 μm. *p < 0.05 versus control group, # p < 0.05 versus oxLDL group. (Color figure online)

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Activation of AMPK by gCTRP9 contributed to its anti-inflammatory effects

AMPK pathway was correlated with the chronic inflammatory process in atherosclerosis [7]. However, it remains unclear whether gCTRP9 is also capable to activate AMPK in macrophages. To elucidate the effect of gCTRP9 on AMPK activation, we treated macrophages with gCTRP9 in the circumstance of oxLDL. As shown in Fig. 3a, compared with control group or oxLDL group, gCTRP9 significantly activated Thr172 phosphorylation of AMPK in macrophages. Moreover, Compound C (Com C) effectively restrained the Thr172 phosphorylation after gCTRP9 treatment (Fig. 3a).

Fig. 3
figure 3

AMPK contributed to the anti-inflammatory reactions of gCTRP9 in RAW 264.7 macrophages. a Western blot for AMPK phosphorylation level in presence or absence of 10 uM Com C for 1 h, then pretreated with gCTRP9 for 2 h and oxLDL for another 8 h. b Western blot for NF-κB p65 from nuclear extracts in cells in presence or absence of Com C for 1 h, then pretreated with gCTRP9 for 2 h and oxLDL for another 24 h. c Western blot for TNF-α and MCP-1 protein expression in oxLDL-stimulated cells treated with gCTRP9 in the presence or absence of Com C. *p < 0.05 versus control group, # p < 0.05 versus oxLDL group, & p < 0.05 versus gCTRP9 + oxLDL group

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We further investigated the role of AMPK in the anti-inflammatory effects. The NF-κB p65 nuclear translocation was restored when AMPK was inhibited by Com C (Fig. 3b). Also, Com C dramatically reversed the suppressive effect of gCTRP9 on inflammatory responses (Fig. 3c). These results indicated that the anti-inflammatory effects of gCTRP9 were dependent on the activation of AMPK in macrophages.

AdipoR1 mediated the anti-inflammatory effects of gCTRP9

Adiponectin receptor 1 (AdipoR1) may play a role in mediating the effects of CTRP9 on vascular endothelial cells [12] and cardiomyocytes [13, 17]. Moreover, the anti-inflammatory effects of adiponectin is largely dependent on AdipoR1 [22, 23], and AdipoR1 is linked to the activation of AMPK [24]. Thus, we explored the role of AdipoR1 in the anti-inflammatory reactions of gCTRP9. We examined the expression of AdipoR1 under the stimulation of gCTRP9 and oxLDL by Western blot (Fig. 4a) and immunostaining (Fig. 4b). The results demonstrated that gCTRP9 could obviously increase the expression of AdipoR1 on the plasma membrane of macrophages. However, the expression of AdipoR1 was not altered by oxLDL alone.

Fig. 4
figure 4

AdipoR1 is involved in gCTRP9-mediated anti-inflammatory effects in RAW 264.7 macrophages. a Western blot analysis for AdipoR1 in cells. b Confocal analysis for AdipoR1 protein (green). Nuclei were stained by DAPI (blue). Scale bar = 20 μm. c Screening of AdipoR1 siRNA sequences. Cells were transfected by AdipoR1 siRNA of different sequences. AdipoR1 expression was determined by Western blot in each group. d Western blot analysis for total and phosphorylated AMPK in cells transfected with negative control siRNA or AdipoR1 siRNA, and treated with gCTRP9 for 2 h and oxLDL for another 8 h. e Western blot was used to detect TNF-α and MCP-1 expression in macrophages transfected with NC or AdipoR1 siRNA, and treated with gCTRP9 for 2 h and oxLDL for another 24 h. *p < 0.05 versus control group, # p < 0.05 versus oxLDL group, & p < 0.05 versus gCTRP9 + oxLDL in NC group. (Color figure online)

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Then, we detected the participation of AdipoR1 on the anti-inflammatory effects of gCTRP9. With three AdipoR1 siRNA transfected in cells, Western blot demonstrated that the AdipoR1-homo-879 showed the highest inhibition efficiency (Fig. 4c) and was chosen for the subsequent experiments. Macrophages pretreated with negative control siRNA or AdipoR1 siRNA were exposed to oxLDL in the presence or absence of gCTRP9 for the indicated time. As shown in Fig. 4d, the activation of AMPK by gCTRP9 was abolished in AdipoR1 knockdown macrophages. Moreover, knockdown of AdipoR1 reversed the inhibitory effects of gCTRP9 on the expression of TNF-α and MCP-1 induced by oxLDL (Fig. 4e). It is demonstrated that gCTRP9 activated AMPK and its downstream effects through AdipoR1 in macrophages.

Discussion

The present study firstly provided evidence that the anti-inflammatory effects of gCTRP9 were dependent on activating of AMPK and increasing the expression of AdipoR1 in macrophages. GCTRP9-treated macrophages suppressed the expression of TNF-α and MCP-1 by inhibiting the activation of NF-κB in response to oxLDL. Blockade of AMPK or ablation of AdipoR1 could reverse the anti-inflammatory effects of gCTRP9 (Fig. 5).

Fig. 5
figure 5

The anti-inflammatory mechanism of gCTRP9 in macrophages. In resisting the inflammatory response induced by oxLDL in macrophages, gCTRP9 increased the expression of AdipoR1 on the plasma membrane of macrophages, which further raised the AMPK activation of gCTRP9. Activated AMPK blocked the nuclear translocation of NF-κB p65, thus suppressing the transcription of inflammatory factors

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In the development of atherosclerosis, oxLDL is a main pro-inflammatory factor that induces the macrophage inflammation [25]. Numerous experimental and clinical studies suggested that NF-κB signaling was involved in the inflammatory reactions in the whole process of atherosclerosis [26]. Stimulation with oxLDL results in uncontrolled NF-κB activation and the release of inflammatory factors. Among the inflammatory mediators, TNF-α plays an important role in inflammatory cascades, which can not only regulate the activation and maturation of macrophages, but also control the release of pro-inflammatory cytokines and chemokines. Moreover, the production of reactive oxygen and nitrogen intermediates is under the regulation of TNF-α [27]. In addition, TNF-α produced by inflammatory macrophages could induce smooth muscle cell apoptosis, thereby accelerating plaque rupture [28]. Also, as a key chemokine, MCP-1 induces the recruitment of monocytes and accelerates the development of atherosclerotic lesions [29]. Thus, anti-inflammatory agents and inhibitors of NF-κB are effective in suppressing the initiation and development of atherosclerosis.

A very recent study showed that CTRP9 underwent proteolytic cleavage to generate gCTRP9 in cardiac tissues during and/or after its secretion [11]. Different from adiponectin, which circulates as full-length multimers in the plasma, the dominant circulatory isoform of CTRP9 is the biologically active globular domain. Although the plasma CTRP9 level is lower than adiponectin, the potential function of CTRP9 might be more effective. In our previous study, we have demonstrated that overexpression of CTRP9 markedly decreased the expression of TNF-α and MCP-1 in vivo and vitro [19]. In current study, we further revealed that gCTRP9 suppressed NF-κB activation by inhibiting S536 phosphorylation of p65 and p65 nuclear translocation. S536 is a dominant regulatory site of p65 that has been considered to promote its nuclear localization, strengthen its association with co-activators, decrease its association with co-repressors, and thus enhance transcriptional potential [26]. Taken together, gCTRP9 may have a key role in suppressing NF-κB p65 activation, thereby inhibiting the inflammatory response in macrophages.

AMPK is a multi-subunit protein constitutive of three subunits (α, β, γ), and the α subunit determines the activity of the protein. The primary activation site of α subunit is Thr172 [30]. Emerging evidences indicated that AMPK signaling molecule can inhibit the inflammatory responses induced by NF-κB. AMPK inhibits the activation of NF-κB through its signaling networks including direct phosphorylation targets such as SIRT1, FoxOs, p53, and PGC-1α. AMPK is also capable of suppressing endoplasmic reticulum and oxidative stresses which can trigger NF-κB signaling [31]. It has been confirmed that CTRP9 activates AMPK signaling pathway in myotubes, endothelial cells, and cardiomyocytes [12, 13, 32]. In our study, we found gCTRP9 had the ability to activate AMPK at the site of Thr172 in macrophages. What is more, inhibition of AMPK by Com C reversed the suppressive effects of gCTRP9 on inflammation and NF-κB nuclear translocation upon oxLDL in macrophages. The anti-inflammatory effects of CTRP9 in endothelial cells and cardiomyocytes were consistent with our results, which indicated that the anti-inflammatory effects of CTRP9 are partially dependent on AMPK.

It has been proposed that increasing adiponectin receptors may be a useful strategy to enhance adiponectin sensitivity and improve cardiovascular function [33, 34]. AdipoR1 is one of the adiponectin receptors, which protects against atherosclerosis largely through the activation of AMPK signaling pathway [24, 35]. Recent studies revealed that AdipoR1 might be a binding protein which mediated the effects of CTRP9 on vascular endothelial cells [12] and cardiomyocytes [13, 17]. In current study, we found that gCTRP9 increased the expression of AdipoR1 on the plasma membrane of macrophages. Based on this discovery, we speculated that gCTRP9 might promote adiponectin sensitivity. Furthermore, we elucidated that AdipoR1 was partly participated in the anti-inflammatory process of gCTRP9 in macrophages through its intracellular signal pathways including AMPK. Further investigation is needed to clarify whether CTRP9 could interact with adipoR1 on the plasma membrane and how CTRP9 increased the expression of AdipoR1.

In summary, this study uncovers that gCTRP9 potently inhibits the inflammatory response and NF-κB activation by activating AMPK in an AdipoR1-dependent manner in macrophages. Furthermore, gCTRP9 owns the potential ability to induce AdipoR1 expression on the plasma membrane of macrophages. Together with the fact that CTRP9 exhibits advantageous effects on cardiovascular diseases, our findings further suggest that supplement with CTRP9 may be a therapeutic strategy for atherosclerosis.