高盐饮食对小鼠肾髓质环氧化酶2表达的影响及其机制

目的探讨高盐饮食对小鼠肾髓质环氧化酶2(COX2)表达和尿钠排泄的影响及其相关机制。方法雄性C57BL/6j小鼠(n=30)被分为4组:(1)正常盐组(0.4%NaCl,n=5);(2)高盐组(8%NaCl,n=5);(3)硼替佐米+正常盐组(n=10);(4)硼替佐米+高盐组(n=10)。各组予生理盐水或硼替佐米预处理后,分别予正常盐或高盐饮食3d,实验最后1d于代谢笼中留取小鼠24h尿液。麻醉处死小鼠,分别留取肾脏皮髓质,提取蛋白,部分组织固定。采用Western印迹法检测各组小鼠肾髓质COX2蛋白表达;用免疫组化法检测肾脏核转录因子κB(NF-κB)p65亚基及COX2表达;自动生化仪测定24h尿钠排泄量。另将携带NF-κB启动子驱动的luciferase报告基因HLL转基因小鼠(HIVlong-terminal-repeat,LTR)(n=8)分为两组:(1)正常盐组:正常盐(0.4%NaCl)饮食;(2)高盐组:高盐(8.0%NaCl)饮食。3d后麻醉处死动物,留取肾脏组织。采用荧光素酶检测试剂盒测定NF-κB的转录活性并以总蛋白量进行校正。结果(1)Western印迹结果显示,与正常盐组比较,高盐组C57BL/6j小鼠肾脏髓质COX2蛋白表达显著增加(P<0.05)。(2)NF-κB荧光素酶活性测定结果显示,与正常盐组比较,高盐组HLL小鼠肾脏髓质NF-κB活性表达显著增加(P<0.05)。免疫组化结果显示NF-κBp65亚基表达定位于肾髓质间质细胞。(3)与高盐组比较,硼替佐米+高盐组小鼠肾髓质COX2蛋白表达降低(P<0.05)。(4)与高盐组比较,硼替佐米+高盐组小鼠24h尿钠排泄量减少(P<0.05),组间尿量的差异无统计学意义。结论高盐饮食可诱导小鼠肾髓质COX2高表达并激活NF-κB活性,硼替佐米能抑制高盐饮食对肾髓质COX2表达的诱导。NF-κB通路可能参与了高盐饮食调节肾髓质COX表达及尿钠排泄。

Effect of high salt diet on the renal medullary cyclooxygenase 2 expression

Objective To examine the effect and mechanism of high salt diet on the renal medullary cyclooxygenase 2 (COX2) expression and urinary sodium excretion. Methods Thirty male C57BL/6j mice were divided into four groups: (1) normal salt diet group (0.4%NaCl, n=5); (2) high salt diet group (8% NaCl, n=5); (3) Bortezomib+normal salt diet group (n=10); (4) Bortezomib+high salt diet group (n=10). The different groups were pre-treated with saline or bortezomib，followed by normal salt diet or high salt diet for three days. All the mice were maintained on metabolic cage at the last day and allowed free access to water. Twenty-four hours urine was collected. Body weight, urine volumes were documented. At the end of experiments, mice were sacrificed under anesthesia and the kidneys were harvested for Western blotting and immunohistochemistry. The transgenic mice carrying a luciferase reporter driven by an NF-κB response promoter, HIV long terminal repeat (LTR) (HLL mice) were used to explore the effect of high salt diet on renal medullary NF-κB activity. HLL mice were fed with normal salt diet or high salt diet for 3 days, after which renal medullary luciferase activity was determined using a commercial luciferase assay kit. Luciferase activity was quantified with a luminometer and adjusted for the total amount of proteins. The cellular location of NF-κB was examined using immunohistochemistry of NF-κB p65 staining. Results (1) Western blotting results showed high salt diet significantly increased the COX2 expression in the renal medulla of C57BL/6j mice (P﹤0.05). (2) High salt diet significantly increased NF-κB luciferase reporter activity in the HLL mice renal medullary tissues when compared to normal salt diet (P﹤0.05). The immunohistochemistry of NF-κB p65 showed the expression of NF-κB was mainly in the renal interstitial cells. (3) Western blotting results showed bortezomib inhibited the renal medullar COX2 expression induced by high salt diet (P﹤0.05). (4) Bortezomib decreased the urinary sodium excretion of high salt diet mice (P﹤0.05), but had no change on urine volume. Conclusions High salt diet induce renal medullary COX2 over-expression and activate the activation of NF-κB in renal medullary. Bortezmoib can inhibit the renal medullar COX2 expression induced by high salt diet. The NF-κB pathway activation may involve in the regulation of renal medullar COX2 expression by high salt diet.