2A). Then we investigated whether STAT3 phosphorylation could be inhibited by sgp130Fc treatment using immunofluorescence staining. While the expression Alvespimycin of STAT3PY705 was highly up-regulated in the colon tissues of the 3% DSS-treated group, it was notably repressed in the group of mice treated with 3% DSS and a subsequent injection of sgp130Fc (Fig. 2B). Remarkably, a significant reduction in the expression of S100A9 mRNA (Fig. 2C) and protein (Fig. 2D) was observed in CECs from the 3% DSS + sgp130Fc group compared to mice receiving DSS alone, and was associated with STAT3PY705 expression (Fig. 2B). Figure 2 Effect of soluble gp130-Fc (sgp130Fc) injection on the expression of S100A9 in colonic epithelial cells (CECs) from mice with dextran sulfate sodium (DSS)-induced colitis.
Next, to provide direct evidence that the increased S100A9 expression by IL-6 was mediated through STAT3 activation, we introduced a delivery system of si-STAT3 using CH-NPs [44]. si-negative/CH-NPs or si-STAT3/CH-NPs were injected intravenously twice on days 2 and 3 into the 3% DSS-treated group. Then we also investigated whether STAT3 knockdown could affect disease development using si-STAT injection. Disease activities were significantly decreased on day 6 of DSS exposure in the STAT3 suppression group compared to the negative si-RNA-treated mice (Fig. 3A). The expression levels of S100A9 mRNA (Fig. 3B) were notably suppressed in the group of mice treated with si-STAT3/CH-NPs, resulting that STAT3 regulated the expression level of S100A9 in the CECs in DSS-induced colitis (Fig. 3C).
Figure 3 Effect of small interfering STAT3 chitosan nanoparticle (si-STAT3/CH-NP) injection on the expression of S100A9 in colonic epithelial cells (CECs). IL-6/STAT3 Signaling Cascades Induce S100A9 Expression in Caco-2 Cells In Vitro To further confirm whether IL-6 directly triggers S100A9 expression through STAT3 activation, in vitro experiments were performed using a human IEC line (Caco-2). Sustained STAT3 activation was observed until 60 min (Fig. 4A) and S100A9 expression was significantly increased at mRNA levels after IL-6 stimulation for 3 or 6 h (Fig. 4B). S100A9 was released in the cell culture supernatant up to 24 h after stimulation of these cells with IL-6 (Fig. 4B). Thus, IL-6 can be postulated to mediate phosphorylated STAT3 and up-regulate S100A9 expression in Caco-2 cells.
Figure 4 The role of the interleukin-6 (IL-6)/STAT3 axis in the expression of S100A9 in the Caco-2 cell line. IL-6-mediated S100A9 expression was further investigated to determine whether it is dependent on STAT3 activation using various agents that inhibit STAT3 signaling cascades. S3I and the STAT3 inhibitor, which inhibit phosphorylation by targeting a tyrosine residue or by dimerizing STAT3, respectively were incubated for 3 Entinostat h prior to IL-6 stimulation. After 6 h, the expression of S100A9 at both mRNA and protein levels was measured.
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