Combination of baicalein and miR-106a-5p mimics significantly alleviates IL-1β-induced inflammatory injury in CHON-001 cells

Abstract

Osteoarthritis (OA) induces inflammation and degeneration of all joint components, and as such, is a considerable source of disability, pain and socioeconomic burden worldwide. Baicalein (BAI) and microRNA (miR)-106a-5p suppress the progression of OA; however, the effects of BAI and miR-106a-5p for the combined treatment of OA are not completely understood. An in vitro OA model was established by treating CHON-001 cells with 20 ng/ml interleukin (IL)-1β. Cell Counting Kit-8 and flow cytometry assays were conducted to evaluate cell viability and apoptosis, respectively. Western blotting was performed to determine the expression levels of Bax, active caspase-3, Bcl-2, collagen I, collagen III, aggrecan, matrix metallopeptidase (MMP)-13, MMP-9, active Notch1 and transcription factor hes family bHLH transcription factor 1 (Hes1). The levels of IL-6 and tumor necrosis factor-α in the cell culture medium were quantified via ELISA. The present study revealed that treatment with BAI or miR-106a-5p mimic alleviated IL-1β-induced apoptosis, and BAI + miR-106a-5p combination treatment exerted enhanced anti-inflammatory effects compared with monotherapy. Furthermore, IL-1β-induced accumulation of collagen, collagen III, MMP-13 and MMP-9 in CHON-001 cells was reversed to a greater degree following combination treatment compared with monotherapy. Likewise, IL-1β-induced aggrecan degradation was markedly reversed by combination treatment. IL-1β-induced upregulation of active Notch1 and Hes1 in CHON-001 cells was also significantly attenuated by combined BAI + miR-106a-5p treatment. In conclusion, the results of the present study revealed that the combination of BAI and miR-106a-5p mimic significantly decreased IL-1β-induced inflammatory injury in CHON-001 cells, which may serve as a novel therapeutic strategy for OA.

Keywords: CHON-001 cells; baicalein; interleukin-1β; microRNA-106a-5p; osteoarthritis.

Figure 1

Establishment of an in vitro osteoarthritis model. (A) Cell viability was assessed by conducting the Cell Counting Kit-8 assay. CHON-001 cell apoptosis was (B) determined by flow cytometry and (C) quantified. Levels of (D) TNF-α and (E) IL-6 were evaluated by performing ELISAs. ^**P<0.01 vs. control (0 ng/ml IL-1β). TNF-α, tumor necrosis factor-α; IL, interleukin; PI, propidium iodide.

Figure 2

Combination of BAI and miR-106a-5p mimics significantly alleviated IL-1β-induced reductions in cell viability. The effect of (A) BAI and (B) BAI + IL-1β on cell viability. ^**P<0.01 vs. control; ^##P<0.01 vs. IL-1β. (C) Transfection efficiency of miR-106a-5p mimics. ^**P<0.01 vs. NC (D) The effect of IL-1β and miR-106a-5p mimics on cell viability. ^**P<0.01 vs. control; ^#P<0.05 and ^##P<0.01 vs. IL-1β. The effect of different concentrations of (E) BAI and (F) miR-106a-3p mimics on cell viability in IL-1β-treated CHON-001 cells. ^**P<0.01 BAI, baicalein; miR, microRNA; IL, interleukin; NC, negative control.

Figure 3

Combination of BAI and miR-106a-5p mimics significantly alleviated IL-1β induced cell apoptosis. Cell apoptosis was (A) determined by flow cytometry and (B) quantified. Protein expression levels were (C) determined by western blotting and semi-quantified for (D) Bax, (E) Bcl-2 and (F) active caspase-3. ^**P<0.01 vs. control; ^##P<0.01 vs. IL-1β_20 ng/ml; ^{^^}P<0.01 vs. IL-1β + miR-106a-5p; ^&&P<0.01 vs. IL-1β + BAI. BAI, baicalein; miR, microRNA; IL, interleukin; PI, propidium iodide.

Figure 4

Combination of BAI and miR-106a-5p mimics significantly alleviated the osteoarthritis phenotype and reversed the inflammatory response. Protein expression levels were (A) determined by western blotting and semi-quantified for (B) collagen I, (C) collagen III, (D) aggrecan, (E) MMP-13 and (F) MMP-9. (G) IL-6 and (H) TNF-α levels were evaluated using ELISAs. ^**P<0.01 vs. control; ^##P<0.01 vs. IL-1β_20 ng/ml; ^{^^}P<0.01 vs. IL-1β + miR-106a-5p; ^&&P<0.01 vs. IL-1β + BAI. BAI, baicalein; miR, microRNA; MMP, matrix metallopeptidase; IL, interleukin; TNF-α, tumor necrosis factor-α.

Figure 5

Combination of BAI and miR-106a-5p mimics reversed IL-1β-mediated upregulation of active Notch1 and Hes1. Protein expression levels were (A) determined by western blotting and semi-quantified for (B) active Notch1 and (C) Hes1. ^**P<0.01 vs. control; ^##P<0.01 vs. IL-1β_20 ng/ml; ^{^^}P<0.01 vs. IL-1β + miR-106a-5p; ^&&P<0.01 vs. IL-1β + BAI. BAI, baicalein; miR, microRNA; IL, interleukin; Hes1, hes family bHLH transcription factor 1.