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. 2023 Jan-Dec:37:3946320231154995.
doi: 10.1177/03946320231154995.

Resveratrol suppresses lipopolysaccharide-mediated activation of osteoclast precursor RAW 264.7 cells by increasing miR-181a-5p expression

Hai-Yan Xue  1 , Ming-Wei Liu  2 , Guang Yang  1
Affiliations

Resveratrol suppresses lipopolysaccharide-mediated activation of osteoclast precursor RAW 264.7 cells by increasing miR-181a-5p expression

Hai-Yan Xue et al. Int J Immunopathol Pharmacol. 2023 Jan-Dec.

Abstract

Resveratrol (Res) has anti-inflammation and antiosteoporosis functions. We evaluated the effect of Res on osteoclast differentiation by releasing inflammatory cytokines from osteoclast precursor RAW 264.7 cells stimulated by lipopolysaccharide (LPS). In the study, LPS (1 ng/L) was used to induce the Raw 264.7 inflammatory injury model in vitro. A total of 25 ng/mL M-CSF + 30 ng/mL RANKL or plus 1 μg/L LPS was used to induce osteoclastogenesis in the experiments. We utilized the Cell Counting Kit-8 assay to measure the relative cell survival of RAW 264.7 cells. Then, enzyme-linked immunosorbent assays were utilized to measure the abundance of inflammatory markers, such as interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and IL-6. Subsequently, Western blot analysis was applied to assess the abundance of phosphorylated transforming growth factor beta-activated kinase 1 (P-TAK1) protein, TNF receptor-associated factor 6 (TRAF6), nuclear factor-κB inhibitor protein (IκB), phosphorylated IκB-α (P-IκB-α), and nuclear factor κB65 (NF-κB65). mRNA expression levels of miR-181a-5p, TRAF6, specific gene calcitonin receptor (CTR), activated T nuclear factor 1 (NFATC1), cathepsin K (CTSK), and matrix metalloproteinase (MMP)-9 were determined via a real-time polymerase chain reaction. Osteoclast bone resorption function was determined. Finally, tartrate-resistant acid phosphatase (TRAP) staining was performed.The results found that Compared with the model group, the degrees of expressions of supernatant inflammatory factors TNF-α, IL-1β, and IL-6 were substantially attenuated in the Res treatment group (p < 0.05). Furthermore, the extent of miR-181a-5p expression in the RAW 264.7 cells significantly increased, whereas P-IκB-α, P-TAK1, NF-κB65, and TRAF6 expressions significantly decreased in the Res treatment group as opposed to the model group (p < 0.05). The CTR, NFATC1, MMP-9, CTSK, and TRAP mRNA expression levels were substantially reduced during osteoclast differentiation and bone resorption in the Res treatment group.The results suggest that Res can reduce the RAW 264.7 cell differentiation into osteoclasts and relieve LPS-stimulated osteoporosis, and the underlying mechanism may be associated with the Res-inhibited activity of the TRAF6/TAK1 pathway through the increased miR-181a-5p expression.

Keywords: lipopolysaccharide; miR-181a-5p; osteoclast; raw 264.7 cells; resveratrol.

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Conflict of interest statement

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Effects of miR-181a-5p mimic on LPS-elicited RAW 264.7 cell inflammation. Transfection of RAW 264.7 cells with either an miR-181a-5p mimic or incubation with an miR-181a-5p inhibitor for 48 h when exposed to l ng/mL LPS. ELISA was utilized to measure the abundance of IL-6, IL-1β, and TNF-α in the supernatant. Results from two separate experimental tests are shown as mean ± SD. *p < 0.05 versus control subgroup. #p < 0.05 vs the model subgroup.
Figure 2.
Figure 2.
Effects of miR-181a-5p mimic on the TRAF6/NF-kB/IkB and RANKL/RANK signaling pathway activities of LPS-stimulated RAW 264.7 cells. Transfection of RAW 264.7 cells with either an miR-181a-5p mimic or their incubation with an miR-181a-5p inhibitor for 48 h when exposed to l ng/mL LPS or plus 25 ng/mL M-CSF + 30 ng/mL RANKL or 100 nmol/L 1,25(OH)2D3. The extent of p-IkB-α, RANKL, RANK, NF-kB65, and TRAF6 protein expressions in the RAW 264.7 cells was assayed via Western blotting. Results from two separate experimental tests are shown as mean ± SD. **p < 0.01 versus the control subgroup. #p < 0.05 versus the model subgroup.
Figure 3.
Figure 3.
Influence of miR-181a-5p mimic on the LPS-mediated capacity of RAW 264.7 cells to differentiate into osteoclasts and the levels of MMP-9, CTSK, and NFATC1 protein expressions. Transfection of RAW 264.7 cells with either an miR-181a-5p mimic or their incubation with an miR-181a-5p inhibitor for 48 h when exposed to l ng/mL LPS+25 ng/mL M-CSF + 30 ng/mL RANKL. The levels of NFATC1, MMP-9, and CTSK expressions were assayed via Western blot. After TRAP staining, the number of osteoclasts was determined. Results from two separate experimental tests are shown as mean ± SD. *p < 0.05 versus the control group. #p < 0.05 versus the model subgroup.
Figure 4.
Figure 4.
Effects of miR-181a-5p inhibitor and TRAF6 inhibitor (C25-140) on NFATC1, CTSK, and MMP-9 expression levels in LPS-stimulated RAW 264.7 cells. Transfection of RAW 264.7 cells was performed with an miR-181a-5p inhibitor for 48 h with 25 ng/mL M-CSF + 30 ng/mL RANKL or plus 1 μg/L LPS stimulation. The degrees of NFATC1, CTSK, TAK1, NF-kB, and MMP-9 mRNA expressions were assayed via RT-PCR. Results from two separate experimental tests are shown as mean ± SD. **p < 0.01, *p < 0.05 versus the control subgroup. #p < 0.05 versus the model subgroup. △p < 0.05 versus the miR-181a-5p inhibitor and TRAF6 inhibitor (C25-140) subgroup.
Figure 5.
Figure 5.
Effect of miR-181a-5p inhibitor and TRAF6 inhibitor (C25-140) on LPS-mediated RAW 264.7 cell inflammation and differentiation into osteoclasts. Transfection of RAW 264.7 cells was performed with an miR-181a-5p inhibitor for 48 h with 25 ng/mL M-CSF + 30 ng/mL RANKL or plus 1 μg/L LPS stimulation. ELISA was utilized to quantify the TNF-α, IL-6, and IL-1β levels in the supernatant. The number of osteoclasts was determined after TRAP staining. Results from two separate experimental tests are shown as mean ± SD. **p < 0.01, *p < 0.05 versus the control subgroup. #p < 0.05 versus the model subgroup. △p < 0.05 versus the miR-181a-5p inhibitor and TRAF6 inhibitor (C25-140) subgroup.
Figure 6.
Figure 6.
Effects of Res on viability, inflammation, and miR-181a-5p expression in RAW 264.7 cells or LPS-induced RAW 264.7 cell inflammation. Incubation of RAW 264.7 cells with or without 1 mg/L LPS and varying doses of Res for 24 h. CCK-8 assay was utilized to determine the viability of RAW 264.7 cells. ELISA was utilized to determine the levels of IL-6, IL-1β, and TNF-α in the supernatant in LPS-induced RAW 264.7 cells. RT-PCR was utilized to determine the miR-181a-5p expression in LPS-induced RAW 264.7 cells. Results from two separate experimental tests are expressed as mean ± SD. **p < 0.01,*p < 0.05 versus the control group. ##p < 0.01, #p < 0.05 versus the model group.
Figure 7.
Figure 7.
Effects of Res on the activities of TRAF6/TAK1 and RANKL/RANK signaling pathway in LPS-stimulated RAW 264.7 cells. Incubation of RAW 264.7 cells with 1 mg/L LPS or plus 25 ng/mL M-CSF + 30 ng/mL RANKL and different dosages of Res for 24 h. Levels of protein expressions of RANKL, RANK, p-IkB-α, p-TAK1, NF-kB65, and TRAF6 in the RAW 264.7 cells were assayed via Western blotting. Results from two separate experimental tests are expressed as mean ± SD. **p < 0.01 versus the control subgroup. ##p < 0.01, #p < 0.05 versus the model subgroup.
Figure 8.
Figure 8.
Effects of Res on the expression of related osteoclast genes in LPS-stimulated RAW 264.7 cells. Incubation of RAW 264.7 cells was performed with 1 mg/L LPS plus 25 ng/mL M-CSF + 30 ng/mL RANKL and distinct dosages of Res for 96 h. Levels of mRNA expressions of RANKL, RANK, TRAF6, MMP-9, CTSK, and NFATC1 in RAW 264.7 cells were assayed via RT-PCR. Results from two separate experimental tests are shown as mean ± SD. **p < 0.01 versus the control subgroup. ##p < 0.01, #p < 0.05 versus the model subgroup.
Figure 9.
Figure 9.
Effects of Res on the LPS-mediated ability of RAW 264.7 cells to differentiate into osteoblasts and their osteoclast bone resorption function. Incubation of RAW 264.7 cells was performed with 1 mg/L LPS plus 25 ng/mL M-CSF + 30 ng/mL RANKL and different doses of Res for 96 h. After TRAP staining, the proportion of osteoclasts and their capacity for bone resorption were determined. The bone resorption area and the number of bone lacunae were measured. Results from two separate experimental tests are shown as mean ± SD. *p < 0.01 versus the control subgroup. ##p < 0.01, #p < 0.05 versus the model subgroup.
Figure 10.
Figure 10.
TRAF6 is an miR-181a-5p downstream target. A) Overexpression of miR-181a-5p considerably decreased the TRAF6 mRNA levels in the RAW 264.7 cells stimulated by LPS (p < 0.05). miR-181a-5p downregulation substantially elevated the levels of TRAF6 (p < 0.05). B and C) Overexpressed miR-181a-5p substantially reduced the abundance of TRAF6 protein in the RAW 264.7 cells stimulated by LPS relative to that of the empty vector control (p < 0.05). miR-181a-5p downregulation considerably elevated the levels of TRAF6 protein in RAW 264.7 cells. E) miR-181a-5p bound to the 3′-UTR domain of TRAF6. This bond was disrupted in mutant TRAF6. F) Dual-luciferase reporter test revealed the binding of the miR-181a-5p mimic to the 3′-UTR domain of WT TRAF6 instead of the MUT TRAF6 (p < 0.05).
Figure 11.
Figure 11.
Mechanisms of the effects of Res on LPS-mediated activation of osteoclast precursor RAW 264.7 cells.
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