| Backgrounds Atherosclerosis is a chronic disease involving complex immunological and inflammatory reactions. MicroRNA-146(miR-146), including two subsets such as miR-146a and miR-146b, is the first microRNA (miRNA) found to play a regulatory role in immune system. Recently, some studies revealed that there was an overexpression of miR-146a in atherosclerotic plaque, suggesting miR-146a might be associated with immunological reactions of atherosclerosis. TOLL-like receptor-4(TLR4) is a transmembrane signal transduction receptor mediating natural immune reaction through activating nuclear factor-KB (NF-κB) and initiating inflammatory reactions subsequently. By this action, it actively involves into the formation, progression and plaque rupture of atherosclerosis. Both probucol and rosuvastatin are anti-atherosclerotic medications. Previous studies showed that both of them have pleiotropic function besides of their lipid lowering effects. However, the role of miR-146a in the sign path of TLR4/NF-κB in atherosclerosis, and the effects of probucol/rosuvastatin on miR-146a still remains unclear.Objectives The present study was aimed to investigate whether miR-146a regulate the signal path of TLR4/NF-κB in atherosclerosis through its target gene TNF receptor related factor-6(TRAF6), and whether probucol/rosuvastatin have anti-atherosclerotic effects by mediating miR-146a.Methods1. Inflammatory injury cell model was constructed by administrating lipopolysaccharide (LPS)(1μg/ml) to in vitro cultured human THP-1mononuclear cell. Then, the cells were divided into6groups as follows:(1) control group:did not add any intervention;(2) model group:LPS added;(3) miR-146a mimic group: transfection of miR-146a mimic+LPS;(4) miR-146a inhibitor group:transfection of miR-146a inhibitor+LPS;(5) miR-146a mimic negative control group:transfection of miR-146a mimic negative control+LPS;(6) miR-146a inhibitor negative control group:transfection of miR-146a inhibitor negative control+LPS. The mRNA and protein levels of miR-146a, TLR4, myeloid differentiation factor88(MyD88), TRAF6, NF-κB were determined by using RT-PCR and Western-blotting methods. The concentration of interleukin-6(IL-6) and tumor necrosis factor-α (TNF-α) of cell cultural supernatants were determined with ELISA method.2. In vitro cultured human THP-1mononuclear cell was administrated with LPS (1μg/ml) and medications. Then the cells were divided into5groups as follows:(1) control group:absolute ethyl alcohol+dimethyl sulfoxide (DMSO);(2) model group: LPS+absolute ethyl alcohol+DMSO;(3) probucol group:LPS+absolute ethyl alcohol+DMSO+probucol;(4) rosuvastatin group:LPS+absolute ethyl alcohol+DMSO+rosuvastatin;(5) combination group:LPS+absolute ethyl alcohol+DMSO+probucol+rosuvastatin. Similarly, the mRNA and protein levels of miR-146a, TLR4, MyD88, TRAF6, NF-κB were determined by using RT-PCR and Western-blotting methods. And, the concentration of IL-6and TNF-a of cell cultural supernatants were determined with ELISA method.3. Atherosclerotic model was constructed by feeding high-fat diet to aplipoprotein E gene knockout mouse (ApoE-/-). The mouse models were divided into5groups as follows:(1) control group:wild type C57BL/6J mouse was fed with normal diet;(2) model group:ApoE-/-mouse was fed with high-fat diet;(3) probucol group:probucol was given in atherosclerotic models;(4) rosuvastatin group:rosuvastatin was given in atherosclerotic models;(5) combination group:probucol and rosuvastatin were given in atherosclerotic models. After feeding for12weeks, lipid levels were determined, and aorta was collected by execute the mouse. The mRNA expression levels of miR-146a, TLR4and TRAF6of atherosclerosis plaque were determined with RT-PCR method. And the protein levels of TRAF6and NF-κB of atherosclerosis plaque were determined with western-blotting method.Results1. After stimulating human THP-1mononuclear cell with LPS, as compared with control group, the model group had a significantly higher level of miR-146a, higher mRNA expression of TLR4mRNA, and higher protein expression of TRAF6and NF-κB (P<0.05). mRNA expression levels of MyD88, TRAF6and NF-κB were reach to be higher than control group, although without statistical significance. The concentration of IL-6and TNF-a in cell cultural supernatants were significantly higher as compared with control group (P<0.05). miR-146a mimic group had a significantly higher expression of miR-146a (P<0.01) with lower TRAF6mRNA and protein expression (P<0.05), meanwhile, the mRNA expression of TLR4, MyD88, NF-κB and the protein expression of NF-κB were significantly higher compared to model group (P<0.01). miR-146a inhibitor group showed a lower expression level of miR-146a (P<0.01) with higher TRAF6mRNA and protein expression (P<0.01), meanwhile, the mRNA expression of TLR4and the protein expression of NF-κB was significantly decreased (P<0.05). mRNA expression levels of NF-κB and MyD88were reach to be lower than model group, although without statistical signicance. The level of miR-146a in miR-146a mimic group increased about1,145times to that in miR-146a mimic negative control group. The level of miR-146a in miR-146a inhibitor group decreased about5times to that in miR-146a inhibitor negative inhibitor group.2. After stimulating human THP-1mononuclear cell with LPS, as compared with control group, model group had significanlty higher levels of miR-146a, and mRNAs of TLR4, MyD88, TRAF6, NF-κB and protein of TRAF6and NF-κB (P<0.01). The concentration of IL-6and TNF-a in cell cultural supernatants of model group were significantly higher as compared with control group (P<0.01). Compared with model group, miR-146a level and NF-κB mRNA, TRAF6protein were significantly lower in the probucol group than that in the model group (P<0.05). Meanwhile, mRNA expression levels of TLR4, MyD88and TRAF6were reach to be lower than model group, although without statistical signicance. The concentration of IL-6was significantly lower in the probucol group as compared with the model group (P<0.05). Compared with the model group, the rosuvastatin group showed lower levels of miR-146a, TLR4mRNA, TRAF6mRNA and TRAF6protein, NF-κB mRNA and NF-κB protein(P<0.05). The concentrations of IL-6and TNF-a were also significantly lower in the rosuvastatin group as compared with the model group (P<0.05). In addition, as compared with the model group, the combination group showed lower levels of miR-146a, and mRNAs of TLR4, MyD88, TRAF6, NF-κB, and protein of TRAF6and NF-κB (P<0.05). The concentrations of IL-6, TNF-α were also significantly lower in the combination group as compared with the model group (P<0.01). 3. Atherosclerotic models were successfully constructed after feeding high-fat diet to8-week ApoE-/-mice for12weeks. Probucol, rosuvastatin, or both could effectively modulate the lipid levels of ApoE-/-mice and decrease the plaque area of aorta. Compared with the control group, model group showed higher levels of miR-146a, mRNAs of TLR4, TRAF6, NF-κB and protein of TRAF6and NF-κB (P<0.01). The levels of miR-146a, mRNA of TLR4, TRAF6, NF-κB and protein of NF-κB were significantly lower in the probucol group than in the model group (P<0.05). Compared with the model group, the rosuvastatin group had lower levels of miR-146a, TLR4and NF-κB mRNA, and TRAF6protein and NF-κB (P<0.05). As compared with the model group, the combination group showed significantly lower levels of miR-146a, mRNAs of TLR4, TRAF6, NF-κB and protein of TRAF6and NF-κB (P<0.05).Conclusions:1. Stimulating in vitro cultured human THP-1mononuclear cell with LPS (1μg/ml) for24hours could successfully construct the immune inflammatory cell model. Stimulating in vitro cultured human mononuclear cell with LPS could result in the increased expression of miR-146a and enhanced function of TLR4/NF-κB signal path.2. miR-146a could inhibit its target gene TRAF6. Beside this, miR-146a may have another unknown target gene, which plays an important inhibitory role in the signal path of TLR4/NF-κB. miR-146a may regulating the signal path of TLR4/NF-κB through its target genes such as TRAF6and another unknown gene.3. The combination use of probucol and rosuvastatin could effectively inhibit the expression of miR-146a and the signal transduction of TLR4/NF-κB in LPS induced human THP-1mononuclear cell, which subsequently inhibits the progression of atherosclerosis by reducing the release of inflammatory factors such as IL-6and TNF-a.4. Feeding high-fat diet to8-week ApoE-/-mice for12weeks could successfully construct the atherosclerotic model. The expression level of miR-146a was increased in the plaques of ApoE-/-mice.5. After12-week use of probucol and rosuvastatin in ApoE-/-mice, the levels of TC, LDL-c were significantly decreased. This combination therapy might have anti-atherosclerotic effect through lowering the expression of miR-146a and blocking the signal path of TLR4/NF-kB. |
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