| BACKGROUNDAtherosclerosis, a chronic systematic inflammatory disease, represents the leading cause of morbidity and mortality throughout industrialized societies. Actually, inflammation has become recognised as a hallmark throughout all stages of atherogenesis, from endothelial dysfunction to onset of atherosclerotic plaque rupture. Furthermore, it is also well accepted that atherosclerosis is a lipid disorder disease, characterized by accumulation of oxidized low-density lipoprotein (ox-LDL) and macrophages in the arterial wall. During the formation of atherosclerotic plaque, circulating monocytes infiltrate into the subendothelial space, where they differentiate into macrophages and subsequently internalize modified lipoproteins and further transform into foam cells. Macrophage inflammation results in enhanced oxidative stress and cytokine/chemokine secretion, causing more ox-LDL formation, endothelial cell activation, monocyte recruitment, and foam cell formation. Thus, deeper understanding of the interaction between ox-LDL and inflammatory mediators involved in foam cells formation is a vital step to effectively prevent plaque rupture and subsequently clinical life-threatening complications of atherosclerosis.The insulin-like growth factor (IGF), an autocrine/paracrine growth factor, is pivotal to numerous functions such as cellular growth, proliferation, metabolism, glucose homeostasisis. The IGF system is comprised of the IGF ligands (IGF1 and IGF2), cell surface receptors that mediate the biological effects of the IGFs, including the IGF1 receptor (IGF-1R), the IGF2 receptor (IGF-2R), and the insulin receptor (IR), as well as a family of IGF-binding proteins (IGFBPs). To date, a series of clinical and experimental studies have highlighted that IGF, particularly IGF1, might exert anti-inflammatory and anti-oxidant effects. However, IGF2 has received little attention in the study of atherosclerosis. In addition, our group have demonstrated that IGF2 expression could be up-regulated by ox-LDL in THP-1 macrophages. Remarkably, little is known about the direct downstream target nuclear transcription factor and pro-inflammation cytokine of IGF2 on atherosclerosis. Taken together, these unsolved issues suggest that more in-depth researches were needed to clarify the detailed mechanism and effect of ox-LDL and IGF2 on atherosclerosis initiation and progression.Interleukin6 (IL-6), a pro-inflammatory cytokine, can be activated in monocytes, or macrophages, hepatocytes and endothelial cells via the membrane-bound receptor, resulting in classical signalling and downstream initiation of pro-inflammatory cascades that increase hepatic production of CRP, fibrinogen and other acute-phase reactants. To our knowledge, numerous studies provide solid evidence for a prominent role of the pro-inflammatory IL-6 pathway in determining risk of coronary heart disease. Additionally, increasing studies have showed that ox-LDL could markedly stimulated IL-6 expression in macrophages. However, the detailed mechanism and effect of how ox-LDL contribute to IL-6 up-regulation remains ambiguous.Nuclear factor kappa B (NF-κB), the first reported inflammatory transcription factor, is thought to be involved in multiple steps in the initiation and progression of atherosclerosis, including initiation of monocyte adhesion, foam cell formation, and inflammation. Once activated, the NF-κB dimers translocate to the nucleus, bind specific DNA sequences and promote the transcription of IL-6 and other pro-inflammatory genes. Many studies have demonstrated that ox-LDL can activate or inactivate NF-κB and pro-inflammatory signalling in vivo and in vitro. Furthermore, a finding showed that many biological effects of ox-LDL are mediated through signaling pathways, especially via the activation of transcription factors, which in turn will stimulate the expression of genes involved in the inflammation. But even so, the detailed relation between NF-κB and ox-LDL in THP-1 macrophages remains poorly defined. Moreover, how NF-κB is regulated by upstream signalling, especially in macrophages, is not clear.In the present study, we found that ox-LDL up-regulated IGF2, NF-κB and IL-6 expressions. Over-expression of IGF2 significantly up-regulated NF-κB and IL-6 expressions. Additionally, the up-regulation of NF-κB and IL-6 expressions were significantly abolished by IGF2 siRNA in THP-1 macrophages. Furthermore, NF-κB siRNA could abolished the up-regulation of IL-6 induced by ox-LDL in THP-1 macrophages.MATERIALS AND METHODSCell cultureHuman monocytic THP-1 cells was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The THP-1 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal calf serum (FCS) and 1%penicillin/streptomycin. Cells were incubated at 37℃ in an atmosphere of 5% CO2. Cells were seeded in 6-or 12-well plates or 60-mm dishes.Preparation of ox-LDLNative LDL was purchased from Sigma-Aldrich, St. Louis, MO, USA. Native LDL (200mg protein/ml) was oxidized by exposure to CuSO4 (5mmol/1 free Cu2+) in PBS (phosphate-buffered saline) at 37℃ for 24 h. Control incubations were treated with 200mmol/L EDTA. The freshly prepared ox-LDL was dialyzed at 37℃ for 48h against 500 volumes of PBS to remove Cu2+and was sterilized by passage through a 0.45mm filter. Oxidation of LDL was confirmed by the measurement of TBARS (thiobarbituric acid-reactive substances) with MDA [malonaldehyde bis (dimethyl acetal)] as the standard. The TBARS content of ox-LDL was 6.03 ±0.15 versus 0.31 ±0.12 nmol/100mg protein in the native LDL preparation (P＜0.01). Protein content was determined by BCA protein assay kit (KeyGen Biotechnologies,Nanjing, China) with the use of BSA (bovine serum albumin) as the standard. The ox-LDL was kept in 50 mmol/L Tris-HCl,0.15 mol/L NaCl and 2 mmol/L EDTA at pH 7.4 and was used within ten days of preparation.RNA Isolation and Real-Time Quantitative PCR AnalysisTotal RNA from THP-1 macrophages was extracted using TRIzol reagent purchased from Invitrogen Corporation, according to the manufacturer’s instructions. Real-time quantitative PCR (qPCR) with SYBR Green detection chemistry was performed on an ABI 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) with the following primers:IGF2 forward,51-GGAACCCACATTGGCCTGA-3’;IGF2reverse,5’-CCGGCGAGGCAGAATATAACAC-3’; NF-κB forward,5’-GCCTCCACAAGGCAGC AAATA-3’ NF-κB reverse,5’-CACCACTGGTCAGAGACTCGGTAA-3’IL-6 forward,5’-AAGCCAGAGCTGTGCAGATGAGTA-3’;IL-6reverse,5’-TGTCCTGCAGC CACTGGTTC-3’; Melt curve analyses of all real-time quantitative PCR products were performed and shown to produce a single DNA duplex. All samples were measured in triplicate and the mean value was considered for comparative analysis. Quantitative measurements were determined using the △△Ct method and GAPDH expression was used as the internal control.Western Blot AnalysisProteins were extracted from THP-1 macrophages using protein extraction kit (KeyGen Biotechnologies, Nanjing, China) and were quantified using the BCA protein assay kit (KeyGen Biotechnologies, Nanjing, China). Western blot analysis were performed (12%-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 30μg protein per lane) in order to obtain the interest proteins. Rabbit polyclonal anti-IL-6 antibodies were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; Rabbit polyclonal anti-IGF2, anti-NF-κB and β-actin-specific antibodies were purchased from Abcam, Cambridge, MA, USA; The proteins were visualized using a chemiluminescence method (ECL Plus Western Blot Detection System; Amerisham Biosciences, Foster City, CA, USA).Transfection with small interfering RNA (siRNA)The siRNAs against IGF2 (IGF2-siRNA), NF-κB (NF-κB-siRNA) and an irrelevant 21-nucleotide control siRNA (Negative Control) were purchased from Ribo Biotechnology. THP-1 cells (2×106 cells/well) were transfected using Lipofectamine 2000 (Invitrogen).48h after transfection, real-time quantitative PCR and western blot analysis were performed.Construction of recombinant plasmidsThe PIRES2-EGFP and PCR-XL-TOPO vectors were purchased from Invitrogen Corporation, which contains IGF2 and were assembled by the chemically synthesized oligos through PCR). The fragment of EcoRI-IGF2-IRES-EGFP-XhoI was amplified using the template of the PCR-XL-TOPO and PIRES2-EGFP vectors, respectively. By using overlap PCR, EcoRI-IGF2-IRES-EGFP-XhoI was joined by the two above-mentioned segments. Gel electrophoresis was performed and the relevant band was excised from the gel, double enzyme-digested by EcoRI/XhoI, incorporated into the pcDNA3.1 (+) vector, and then transformed into competent E. coli DH5a cells for further amplification and use. The recombinant plasmids were verified by sequencing and named pcDNA3.1-IGF2. The plasmid transfection process was performed using Lipofectamine 2000 transfection reagent according to the manufacturer’s instructions.ELISA assays of cytokines concentration in the supernatantCommercially available specific human IL-6 ELISA Kits were purchased from YanJi Biotechnologies, Shanghai, China. The concentration of IL-6 of cells stimulated with ox-LDL in cultured supernatant was measured following the manufacturer instructions. The optical density (OD) value was detected using ELISA plate reader using 450 nm wavelengths. The concentration of IL-6 in the samples is then determined by comparing the O.D. of the samples to the standard curve. Standard and sample dilutions were added in duplicate wells for each time point to each ELISA plate and the average concentration was used as protein level (pg/mL) in cell culture supernatant.Statistical analysesData are expressed as means ± standard deviations (SDs). The date were compared by one-way analysis of variance and the Student’s t-test, using the Statistical Package for the Social Sciences (version13.0) software (SPSS, Inc., Chicago, IL, USA). A two-tailed probability (P) value <0.05 was considered statistically significant.RESULTS1. Ox-LDL Up-regulated IGF2, NF-κB and IL-6 Expressions inTHP-1MacrophagesIt is well-accepted that the expressions of trascription factor NF-κB and IL-6 could be remarkably increased by ox-LDL. Moreover, our group have demonstrated IGF2 expression was up-regulated when treated with ox-LDL in THP-1 macrophages. In this part, we observed the effect of ox-LDL on IGF2, NF-κB and IL-6 expressions in THP-1 macrophages by qPCR and Western blot analyses.Ox-LDL obviously increased IGF2, NF-κB and IL-6 messenger RNA (mRNA) and protein expressions in THP-1 macrophages in a dose-dependent manner.Concentration level of protein IL6 in the supernatant of the ox-LDL-stimulated THP-1 is also increased significantly, when treated with 50,100 μg/mL ox-LDL for 48h.2 s The Role of IGF2 in the Up-regulation of NF-κB and IL-6 in Ox-LDL-stimulated THP-1 MacrophagesTreatment with recombinant plasmids overexpressing IGF2 (pcDNA3.1-IGF2) increased IGF2 protein expression by 887% in THP-1 macrophages. Furthermore, pcDNA3.1-IGF2 markedly enhanced NF-κB and IL-6 protein expressions in ox-LDL-stimulated THP-1 macrophages. Subsequently, we examined the effect of IGF2 siRNA on the up-regulation of NF-κB and IL-6 expressions. On the other hand, IGF2 protein expression decreased by 88% when treated with IGF2 siRNA in THP-1 macrophages, in comparison to the control siRNA. Treatment with IGF2 siRNA completely abolished the up-reguation of NF-κB and IL-6 protein expressions, which were induced by ox-LDL in THP-1 macrophages.3n The Role of NF-κB in the Up-regulation of IL-6 in Ox-LDL-stimulated THP-1 MacrophagesTo confirm the role of NF-kB in ox-LDL-stimulated IL-6 up-regulation, we investigated the effect of NF-κB siRNA in THP-1 Macrophages. NF-κB protein expression decreased by 89% when treated with siRNA targeting NF-κB in THP-1 macrophages, in comparison to the control siRNA. Suppression of NF-κB expression by siRNA markedly abolished ox-LDL-stimulated up-regulation of IL-6 protein expression, while ox-LDL-induced up-regulation of IGF2 protein expression was independent of siRNA NF-κB in THP1 macrophages.Conclusions1. Ox-LDL obviously increased IGF2, NF-κB and IL-6 expression at both transcriptional levels and translational levels in a dose-dependent manner in THP-1 Macrophages.2. Over-expression of IGF2 markedly enhanced NF-κB and IL-6 protein expressions in ox-LDL-stimulated THP-1 macrophages. On the other hand, Suppression of IGF2 expression by siRNA completely abolished the up-reguation of NF-κB and IL-6 protein expressions, which were stimulated by ox-LDL in THP-1 macrophages.3. Treatment with NF-kB siRNA markedly abolished ox-LDL-stimulated up-regulation of IL-6 protein expression, while IGF2 protein expression was independent of siRNA NF-κB in ox-LDL-stimulated THP1 macrophages. |
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