1)9 Treatment of primary cultures of rat Kupffer

1).9 Treatment of primary cultures of rat Kupffer PLX3397 concentration cells with gAcrp for

18 hours suppressed LPS-stimulated responses in Kupffer cells isolated from both pair-fed and ethanol-fed rats (Fig. 1).9 In other cellular model systems, adiponectin exerts its anti-inflammatory actions through induction of IL-10.11 Therefore, we tested whether knockdown of IL-10 expression with siRNA ameliorated the ability of gAcrp to suppress LPS-stimulated TNF-α expression in Kupffer cells. Transfection of Kupffer cells with siRNA against IL-10 effectively suppressed IL-10 mRNA accumulation (Supporting Fig. 1A) and prevented the suppression of LPS-stimulated TNF-α mRNA accumulation by gAcrp (Fig. 1). Scrambled siRNA had no effect on IL-10 mRNA (Supporting Fig. 1A) or the response to gAcrp (Fig. 1). Primary cultures of Kupffer cells from ethanol-fed rats are more sensitive than cells from pair-fed rats to the anti-inflammatory actions of both gAcrp and full-length adiponectin to suppress LPS-dependent responses.9 Because IL-10 is required for gAcrp to suppress LPS-stimulated TNF-α mRNA accumulation in Kupffer cells, the more potent effects of adiponectin after ethanol feeding may be attributable to increased gAcrp-stimulated expression of IL-10 or increased sensitivity of the Kupffer cells to stimulation by IL-10. To test these hypotheses, isolated Kupffer cells were treated with increasing concentrations of gAcrp for 18 hours, and IL-10

protein secreted in the media was measured by enzyme-linked immunosorbent assay. Accumulation of IL-10 protein was higher check details in Kupffer cells from ethanol-fed rats compared with cells from pair-fed controls (Fig. 2A). Similarly, IL-10 mRNA expression was also higher in Kupffer cells from ethanol-fed rats compared with cells from pair-fed rats when 上海皓元 incubated with gAcrp (Fig. 2B) or full-length adiponectin (Fig. 2C). These data suggested that increased gAcrp-stimulated IL-10 expression

may contribute, at least in part, to the higher sensitivity of Kupffer cells from ethanol-fed rats to gAcrp. Small interfering RNA knockdown of adiponectin receptors (AdipoR) indicated that the effects of gAcrp on IL-10 mRNA were dependent on the expression of AdipoR1, but not AdipoR2 (Fig. 2D). IL-10 mediates its anti-inflammatory effects through interactions with IL-10 receptors and activation of specific signaling pathways; STAT3 activation is required for IL-10–mediated signaling.2 Surface expression of the IL-10 receptor subunit A, the ligand binding subunit of the IL-10 receptor, on Kupffer cells was not affected by either ethanol feeding or gAcrp treatment (Fig. 3). Stimulation with IL-10 increased the phosphorylation of JAK1 within 30 minutes in Kupffer cells from ethanol-fed, but not pair-fed, rats (Fig. 4). Phosphorylation of STAT3 in response to IL-10 was both more rapid (within 10 minutes) and more robust in Kupffer cells from ethanol-fed rats compared with pair-fed rats (Fig. 4).

This entry was posted in Antibody. Bookmark the permalink.

Leave a Reply

Your email address will not be published. Required fields are marked *

*

You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>