Effects of adiponectin on IL-10 expression in rat primary Kupffer cells
目的本研究观察外源性脂联素对体外培养的大鼠原代Kupffer细胞白细胞介素（IL）-10表达的调节作用,以阐明脂联素的抗炎作用机制。方法通过两步胶原酶灌流、密度梯度离心和2 h选择性贴壁方法分离纯化大鼠肝脏Kupffer细胞。酶联免疫吸附试验方法检测脂联素作用下原代Kupffer细胞上清液中IL-10的表达水平。荧光定量PCR法检测脂联素作用下原代Kupffer细胞IL-10 mRNA表达。结果 0.5、1.0、1.5μg/ml脂联素处理Kupffer细胞4、8、12、16 h,与对照组相比,同一浓度的脂联素随着培养时间的延长,Kupffer细胞IL-10分泌逐渐增加;而同一作用时间下,随着脂联素浓度的增加,Kupffer细胞IL-10分泌量并没有逐渐增加。1.0和1.5μg/ml脂联素作用Kupffer细胞16 h,IL-10基因表达量与对照组相比显著增加,各处理组间无明显差异。结论脂联素可上调大鼠原代Kupffer细胞IL-10的表达,这可能是其抗炎作用机制之一。Abstract:
Objective To investigate whether adiponectin exposure can induce expression of the anti-inflammatory cytokine, interleukin-10 (IL-10) , in Kupffer cells, a major source of inflammatory cytokines in liver.Methods Primary Kupffer cells were isolated from male Wistar rats by two-step collagenase perfusion and Percoll density gradient centrifugation, purified by selective adherence in culture, and confirmed by immunofluorescence detection of ED2 expression.The Kupffer cells were treated with 0.5, 1.0 or 1.5 μg/ml of adiponectin.Untreated cells served as controls.At 4, 8, 12 and 16 h of exposure, IL-10 protein levels in supernatant were detected by enzyme-linked immunosorbent assay, and IL-10 mRNA levels were measured by real-time reverse transcription PCR.Results None of the concentrations of adiponectin stimulated IL-10 secretion to a level significantly higher than levels detected in control supernatants when exposed to the cells for 4, 8 or 12 h.However, after 16 hours of exposure, the adiponectin treatments did stimulate the IL-10 secretion to significantly higher levels than that in the control group (all P<0.05) .In addition, after 16 h of exposure, the 1.0 and 1.5 g/ml treatment groups showed significantly higher IL-10 mRNA levels than the control group (both P<0.05) .However, the levels of mRNA stimulation were not significantly different between the two treatment groups.Conclusion Adiponectin up-regulates IL-10 expression in primary cultured rat Kupffer cells, which may act as an anti-inflammatory mechanism of adiponectin.
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