Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, United States.
World J Gastroenterol. 2011 Nov 21;17(43):4772-8. doi: 10.3748/wjg.v17.i43.4772.
To examine the activation of the Nalp3 inflammasome and its downstream targets following lipopolysaccharide (LPS)-induced stimulation in the liver.
Six-to-eight-week-old C57BL/6 chow fed mice were injected intraperitoneally with 0.5 μg/g bodyweight LPS and sacrificed 2, 4, 6, 18 or 24 h later. LPS-induced liver damage was confirmed by a biochemical assay to detect alanine aminotransferase (ALT) levels. To determine if LPS stimulation in the liver led to activation of the inflammasome, real-time quantitative polymerase chain reaction was used to evaluate the mRNA expression of components of the Nalp3 inflammasome. Enzyme-linked immunosorbent assays were used to determine the protein expression levels of several downstream targets of the Nalp3 inflammasome, including caspase-1 and two cytokine targets of caspase-1, interleukin (IL)-1β and IL-18.
We found that LPS injection resulted in liver damage as indicated by elevated ALT levels. This was associated with a significant increase in both mRNA and protein levels of the proinflammatory cytokine tumor necrosis factor (TNF)-α in the liver, as well as increased levels of TNFs in serum. We showed that LPS stimulation led to upregulation of mRNA levels in the liver for all the receptor components of the inflammasome, including Nalp3, Nalp1, pannexin-1 and the adaptor molecule apoptosis-associated speck-like, caspase recruitment domain-domain containing protein. We also found increased levels of mRNA and protein for caspase-1, a downstream target of the inflammasome. In addition, LPS challenge led to increased levels of both mRNA and protein in the liver for two cytokine targets of caspase-1, IL-1β and IL-18. Interestingly, substantial baseline expression of pre-IL-1β and pre-IL-18 was found in the liver. Inflammasome and caspase-1 activation was indicated by the significant increase in the active forms of IL-1β and IL-18 after LPS stimulation.
Our results show that the Nalp3 inflammasome is upregulated and activated in the liver in response to LPS stimulation.
研究脂多糖（LPS）诱导刺激后肝脏中 Nalp3 炎性小体及其下游靶标的激活情况。
将 6-8 周龄的 C57BL/6 常规饮食喂养的小鼠经腹腔注射 0.5μg/g 体重 LPS，2、4、6、18 或 24 小时后处死。通过检测丙氨酸转氨酶（ALT）水平的生化检测来确认 LPS 诱导的肝损伤。为了确定 LPS 刺激肝脏是否导致炎性小体激活，采用实时定量聚合酶链反应评估 Nalp3 炎性小体成分的 mRNA 表达。酶联免疫吸附试验测定 Nalp3 炎性小体的几个下游靶标（包括 caspase-1 和 caspase-1 的两种细胞因子靶标白细胞介素（IL）-1β和 IL-18）的蛋白表达水平。
我们发现 LPS 注射导致肝损伤，表现为 ALT 水平升高。这与肝脏中促炎细胞因子肿瘤坏死因子（TNF）-α的 mRNA 和蛋白水平显著增加以及血清中 TNFs 水平升高相关。我们表明，LPS 刺激导致肝脏中炎性小体的所有受体成分（包括 Nalp3、Nalp1、pannexin-1 和衔接子分子凋亡相关斑点样、含 caspase 募集域域的蛋白）的 mRNA 水平上调。我们还发现炎性小体的下游靶标 caspase-1 的 mRNA 和蛋白水平升高。此外，LPS 刺激导致肝脏中 caspase-1 的两个细胞因子靶标 IL-1β和 IL-18 的 mRNA 和蛋白水平升高。有趣的是，在肝脏中发现了大量的前体 IL-1β和前体 IL-18 的基础表达。LPS 刺激后 IL-1β和 IL-18 的活性形式显著增加，表明炎性小体和 caspase-1 激活。
我们的结果表明，Nalp3 炎性小体在 LPS 刺激后在肝脏中上调和激活。
