Figure 6.

Hepatocyte HSPA12A modulated HMGB1 secretion to paracrinally regulate macrophage chemotaxis and activation. A. HMGB1 knockdown decreased HMGB1 exosomal secretion of hepatocytes. HMGB1 was knockdown in primary hepatocytes (Si-Hmgb1) and scramble RNA treatment served control. After exposed to H/R, culture medium was collected, exosome isolated, and immunoblotted for HMGB1. Blotting for HSP70 and Calnexin served as positive and negative markers, respectively. n = 3/group. B. Hepatocyte HMGB1 knockdown inhibited macrophage chemotaxis. After exposed to H/R, primary hepatocytes were cocultured with primary macrophages in Transwell plate. After coculture for 24 h, macrophage chemotaxis towards hepatocytes was stained with crystal violet. Scale bar = 50 μm. n = 4/group. C. Hepatocyte HMGB1 knockdown inhibited macrophage activation. After exposed to H/R, culture medium of primary hepatocytes was collected as hepatocyte conditioned medium (CM). The CM was then added to macrophages. After treated with hepatocyte CM for 24 h, macrophages were collected to examine the indicated gene expression using immunoblotting. n = 4/group. D. Hepatocyte HMGB1 knockdown decreased ALT and AST leakage when coculture with macrophages. Primary hepatocytes were exposed to H/R, followed by coculture with primary macrophages in Transwell plate. After coculture for 24 h, medium was collected for ALT and AST activity examination. n = 5/group. Data are mean ± SD, * P < 0.05 and ** P < 0.01 by two-way ANOVA followed by Tukey's test. ns, no significance.