姜黄素对内毒素所致肾脏炎症的抑制作用

目的：肾脏的急慢性炎症与肾脏疾病的进展密切相关,本研究拟探讨姜黄素对内毒素（LPS）所致肾脏炎症的抑制作用。方法：SPF级昆明种小鼠40只,鼠龄6～8周,体重20～25g。小鼠分为3组：（1）正常对照组：腹腔注射生理盐水;（2）模型组（LPS组）：腹腔注射LPS（1mg/kg和5mg/kg）;（3）治疗组（LPS＋姜黄素）：动物先腹腔注射姜黄素（1mg/kg或5mg/kg）3d,然后腹腔注射LPS1mg/kg或5mg/kg。于处理后6h取材,切取部分肾组织用10%甲醛固定,HE染色,进行病理学观察。部分肾组织用于MCP-1mRNA检测。将肾小管上皮细胞（HK-2细胞）培养于KSF培养液中,设正常对照组给予正常培养液,LPS刺激组（1ng/ml,100ng/ml和10μg/ml）,LPS刺激＋姜黄素（5μmol/L和50μmol/L）处理组,分别培养4h和24h后收集细胞,应用Real-timePCR检测各组细胞MCP-1的表达。ELISA检测细胞培养上清液中MCP-1的表达。EMSA检测肾小管上皮细胞NF-κB的活性。结果：腹腔注射LPS（1mg/kg和5mg/kg）虽未引起光镜下肾脏组织的病理改变,但可显著增加小鼠肾脏MCP-1 mRNA的表达,分别增加20倍和26倍,而姜黄素处理可降低LPS所诱导的肾脏MCP-1 mRNA的表达,尤以LPS（1mg/kg）＋姜黄素（1mg/kg）为明显（下降至基础值的2.5倍）。应用不同浓度的LPS刺激HK-2细胞4h,HK-2细胞MCP-1 mRNA表达量显著增加,且呈剂量依赖的效应（分别增加1.60、2.15和14.7倍）。而以100ng/ml的LPS刺激HK-2细胞4h,观察姜黄素的干预作用,可见不同浓度的姜黄素（5μmol/L和50μmol/L）可显著抑制LPS所诱导的HK-2细胞MCP-1 mRNA的表达,且以50μmol/L姜黄素干预组为明显。同时ELSA检测细胞上清液中MCP-1的表达结果相似。EMSA结果显示NF-κB的DNA结合活性随LPS的浓度增加而增加,而应用姜黄素预处理后,由LPS所诱导的增高的NF-κB的DNA结合活性随之下降。结论：早期给予姜黄素治疗,能明显改善LPS诱导的小鼠肾脏趋化因子MCP-1的表达,从而发挥其肾脏损伤的保护功能;而体外研究表明姜黄素可抑制LPS所诱导的肾小管上皮细胞MCP-1表达,其作用可能与抑制细胞NF-κB的活性有关。

Curcumin prevents lipopolysaccharide-induced renal inflammation

Objective：To investigate the effect of curcumin on inflammation in the kidney or renal tubular epithelial cells induced by lipopolysaccharide（LPS）.Methods：Forty male KM mice （weight 20～25 g） were randomly divided into three groups.The mice in group1 were treated by intraperitoneal （i.p.） injection of LPS （1 mg/kg or 5 mg/kg）.And the mice in group 2 were preconditioned by intraperitoneal （i.p.） injection of curcumin （1 mg/kg or 5 mg/kg） for three days and after the third injection of curcumin,one injection （i.p.） of LPS （1 mg/kg or 5 mg/kg）.In group 3,the mice were injected equal volume of vehicle （PBS） as control.At the indicated times,blood and kidney specimen were collected.Kidney tissues were detected by light microscopy and the expression of MCP-1 mRNA were measured by Real-time PCR.In vitro,the renal tubular epithelial cells （HK-2 cells） were cultured and pre-treated by LPS at different concentrations （1 ng/ml,100 ng/ml or 10 μg/ml） with or without curcumin（5 μmol/L or 50 μmol/L）.The expression of MCP-1 mRNA was measured by realtime PCR.The MCP-1 in the supernatant was measured by ELISA.The activity of NF-κB was detected by EMSA.Results：LPS of 1 mg/kg and 5 mg/kg could increase the level of MCP-1 mRNA in renal tissues by 20 times and 26 times,respectively.And the up-regulated MCP-1 expression could be partially inhibited by curcumin.In vitro,the expression of MCP-1 mRNA significantly increased by 1.60,2.15 and 14.7 times after stimulating by LPS of 1 ng/ml,100 ng/ml or 10 μg/ml,respectively.The up-regulated MCP-1 mRNA expression induced by LPS （100 ng/ml） could be significantly inhibited in the pretreated group with curcumin （5 μmol/L and 50 μmol/L）.The upregulated MCP-1 in the supernatant of HK-2 stimulated by LPS could be inhibited by different concentration of curcumin.The activity of NF-κB was up-regulated after LPS stimulation and the effect could be partially inhibited by EMSA method.Conclusion：Intraperitoneal injection of LPS could induce the inflammation and increase the expression of MCP-1 in mice kidney.Curcumin could partially inhibit the effects and it may be used in treatment of kidney inflammation.