miR-21 attenuates lipopolysaccharide-induced lipid accumulation and inflammatory response: potential role in cerebrovascular disease

Abstract

Background: Atherosclerosis constitutes the leading contributor to morbidity and mortality in cardiovascular and cerebrovascular diseases. Lipid deposition and inflammatory response are the crucial triggers for the development of atherosclerosis. Recently, microRNAs (miRNAs) have drawn more attention due to their prominent function on inflammatory process and lipid accumulation in cardiovascular and cerebrovascular disease. Here, we investigated the involvement of miR-21 in lipopolysaccharide (LPS)-induced lipid accumulation and inflammatory response in macrophages.

Methods: After stimulation with the indicated times and doses of LPS, miR-21 mRNA levels were analyzed by Quantitative real-time PCR. Following transfection with miR-21 or anti-miR-21 inhibitor, lipid deposition and foam cell formation was detected by high-performance liquid chromatography (HPLC) and Oil-red O staining. Furthermore, the inflammatory cytokines interleukin 6 (IL-6) and interleukin 10 (IL-10) were evaluated by Enzyme-linked immunosorbent assay (ELISA) assay. The underlying molecular mechanism was also investigated.

Results: In this study, LPS induced miR-21 expression in macrophages in a time- and dose-dependent manner. Further analysis confirmed that overexpression of miR-21 by transfection with miR-21 mimics notably attenuated lipid accumulation and lipid-laden foam cell formation in LPS-stimulated macrophages, which was reversely up-regulated when silencing miR-21 expression via anti-miR-21 inhibitor transfection, indicating a reverse regulator of miR-21 in LPS-induced foam cell formation. Further mechanism assays suggested that miR-21 regulated lipid accumulation by Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) pathway as pretreatment with anti-TLR4 antibody or a specific inhibitor of NF-κB (PDTC) strikingly dampened miR-21 silence-induced lipid deposition. Additionally, overexpression of miR-21 significantly abrogated the inflammatory cytokines secretion of IL-6 and increased IL-10 levels, the corresponding changes were also observed when silencing miR-21 expression, which was impeded by preconditioning with TLR4 antibody or PDTC.

Conclusions: Taken together, these results corroborated that miR-21 could negatively regulate LPS-induced lipid accumulation and inflammatory responses in macrophages by the TLR4-NF-κB pathway. Accordingly, our research will provide a prominent insight into how miR-21 reversely abrogates bacterial infection-induced pathological processes of atherosclerosis, indicating a promising therapeutic prospect for the prevention and treatment of atherosclerosis by miR-21 overexpression.

Figure 1

LPS-induced expression of miR-21 in macrophages. After stimulation with the indicated times (A) and doses (B) of LPS, the mRNA levels of miR-21 in macrophages were detected by quantitative RT-PCR. *P < 0.05 versus control group. **P < 0.01.

Figure 2

Effect of miR-21 on lipid-laden foam cell formation in LPS-stimulated macrophages. To investigate the function of miR-21 on LPS-induced lipid accumulation, cells were respectively transfected with miR-21 mimics, scrambled control microRNA, anti-miR-210 inhibitor and anti-microRNA control inhibitor for 6 h. (A) Overexpression of miR-21 in miR-21 transfected cells. *P < 0.05 versus control microRNA group. (B) Down-regulation of miR-21 in macrophages. *P < 0.05 versus anti-microRNA control inhibitor group. (C-D) After exposure to ox-LDL (μg/ml) for 24 h, the ratio of CE/TC and foam cell formation was assessed by HPLC assay (C) and Oil red O staining (D). *P < 0.05.

Figure 3

miR-21 inhibited lipid accumulation by TLR4-NF-κB pathway. (A) After overexpressed or silenced the expression levels of miR-21, the activation of TLR4 and intra-nuclear NF-κB p65 was demonstrated by western blotting analysis. (B-C) Before stimulation with 100 ng/ml LPS for 24 h, cells were stimulated with anti-TLR4 antibody (10 μg/ml), or 30 μM NF-κB inhibitor PDTC for 4 h. The silencing effect of TLR4 (B) and intra-nuclear NF-κB p65 (C) was evaluated. (D) The association between miR-21 and the TLR4-NF-κB pathway was analyzed by western blotting. *P < 0.05.

Figure 4

miR-21 regulated the secretion of inflammatory cytokines in LPS-induced macrophages. (A) Following transfection with miR-21, levels of IL-6 and IL-10 was detected by ELISA assay. *P < 0.05 versus LPS-untreated group. ^#P < 0.05 versus LPS plus miR-con group. (B-C) The effect of miR-21 silencing on IL-6 and IL-10 levels in macrophages exposed to 100 ng/ml LPS. (D) Cells were pretreatment with anti-TLR4 antibody (10 μg/ml) or 30 μM NF-κB inhibitor PDTC for 4 h, and then LPS-induced IL-6 and IL-10 levels were detected in anti-miR-21 inhibitor-transfected cells. *P < 0.05.