Phospho-ROCK2 (Ser1366) Antibody - #AF7143

FIGURE 5 9-MF exerts anti-neuroinflammatory and anti-neurotoxic effects through the inhibition of ROCK2 and GSK3β, respectively. (a) The expression of p-ROCK2 and p-GSK3β in the hippocampal region of APP/PS1 transgenic mice was evaluated by Western blotting analysis. The quantification of (b) p-ROCK2 and (c) p-GSK3β in (a) was shown. The design of experiments in (d) Aβ-treated BV2 cells and (e) GA-treated SH-SY5Y cells was demonstrated, respectively. BV2 cells were treated with fasudil or 9-MF at various concentrations for 1 h. Aβ at 3 μM was further added. After 24 h, the secretion of pro-inflammatory cytokines was evaluated by ELISA. 9-MF significantly prevented Aβ-induced secretion of (f) IL-1β and (g) TNF-α in BV2 cells. ROCK2 was knocked down by siRNA in BV2 cells. (h) The expression of ROCK2 was evaluated by Western blotting analysis. (i) The quantification of ROCK2 expression in (h) was shown. ROCK2-knockdown BV2 cells were treated with fasudil or 9-MF for 2 h. Aβ at 3 μM was further added. After 24 h, the secretion of pro-inflammatory cytokines was evaluated by ELISA. The knockdown of ROCK2 abolished the reduction of (j) IL-1β and (k) TNF-α by 9-MF in Aβ-treated BV2 cells. SH-SY5Y cells were treated with IO or 9-MF at various concentrations for 2 h. GA at 0.7 mM was further added. After 24 h, FDA/PI double staining and MTT assay were performed. (s) Representative FDA/PI double staining of SH-SY5Y cells was shown. (n) FDA-positive living cells in (s) were analyzed. (o) Cell viability was analyzed by MTT assay. 9-MF significantly prevented GA-induced neurotoxicity in SH-SY5Y cells. (l) The expression of p-GSK3β was evaluated by Western blotting analysis. (m) The quantification of p-GSK3β expression in (l) was shown. 9-MF significantly prevented GA-induced decrease of p-GSK3β expression in SH-SY5Y cells. GSK3β was knocked down by siRNA in SH-SY5Y cells. (p) The expression of GSK3β was evaluated by Western blotting analysis. (q) The quantification of GSK3β expression in (p) was shown. GSK3β-knockdown SH-SY5Y cells were treated with IO or 9-MF for 2 h. GA at 0.7 mM was further added. After 24 h, FDA/PI double staining was performed. (t) Representative FDA/PI double staining of SH-SY5Y cells was shown. (r) FDA-positive living cells in (t) were analyzed. The knockdown of GSK3β abolished the neuroprotective effects of 9-MF against GA in SH-SY5Y cells. GA: Glyceraldehyde. The data were expressed as mean ± SD. n = 3 in (b, c, f–m, p, q), and 6 in (n, o, r). Statistical analysis was performed using One-way ANOVA and Tukey's test.

Fig. 2. Mechanical tension promoted the osteogenic differentiation of pre-osteoblasts by activated RhoA/ROCK.