Tanshinone ΙΙ A-Incubated Mesenchymal Stem Cells Inhibit Lipopolysaccharide-Induced Inflammation of N9 Cells through TREM2 Signaling Pathway

Abstract

Our previous study found that incubating mesenchymal stem cells (MSC) with tanshinone IIA (TIIA) before transplantation could significantly increase the inhibitory effect of MSC on neuroinflammation. Here, we investigated the possible mechanism of this effect. N9 cells and MSC were inoculated at a ratio of 1 : 1 into a Transwell coculture system. MSC were inoculated into the upper chamber, and N9 cells were inoculated into the lower chamber. In this experiment, N9 cells were treated with 1 μg/mL lipopolysaccharide (LPS) for 24 hours to induce inflammation, MSC were treated with 10 μM TIIA for 48 hours to prepare TIIA-incubated MSC (TIIA-MSC), and TREM2 siRNA was used to silence the TREM2 gene in MSC. The changes in IL-1β, IL-6, and TNF-α were evaluated by Western blotting. We found that LPS significantly increased the levels of IL-1β, IL-6, and TNF-α. While both MSC and TIIA-MSC downregulated the levels of (P = 0.092, P = 0.002), IL-6 (P = 0.014, P < 0.001), and TNF-α (P = 0.044, P = 0.003), TIIA-MSC downregulated IL-6 more significantly (P = 0.026). In addition, silencing TREM2 reduced the ability of TIIA-MSC to attenuate IL-6 (P = 0.005) and TNF-α (P = 0.033). These data suggest that the enhanced anti-inflammatory effect of TIIA-MSC on LPS-induced N9 cells may be mediated through the TREM2 signaling pathway.

Figure 1

The identification of MSC by flow cytometry and the schematic representation of the study design. (a, b) Flow cytometry was used to detect the surface antigens of MSC: CD29 (97.78%), CD90 (98.01%), and CD45 (0.54%). (c) Brief descriptions of the experimental procedures and experimental conditions. (d) Transwell system used in the present study.

Figure 2

RT-qPCR and Western blotting analyses of TREM2 siRNA transfection efficiency. (a) Relative expression levels of TREM2 mRNA. (b) Expression levels of TREM2 protein. ^aP < 0.05 vs. control, ^bP < 0.05 vs. siRNA-NC, mean ± SEM, n = 3.

Figure 3

Inflammatory factor (IL-1β, IL-6, and TNF-α) protein expression. (a) Representative Western blotting results for IL-1β, IL-6, TNF-α, and GAPDH. (b) Relative protein expression of IL-6. (c) Relative protein expression of IL-1β. (d) Relative protein expression of TNF-α. ^aP < 0.05 vs. control, ^bP < 0.05 vs. LPS, ^cP < 0.05 vs. LPS + MSC, ^dP < 0.05 vs. LPS + TIIA − MSC, mean ± SEM, n = 3.