促炎症因子基因表达与长期水砷暴露小鼠肝损伤的关系

The relationship between the gene expression of inflammatory mediator and liver damage of mice chronically expososed to inorganic arsenate

目的探讨促炎症因子在慢性水砷暴露小鼠肝损伤中的作用。方法60只雄性小鼠按体质量随机分成对照组、亚砷酸钠组（iAs孙组，300mg／L）、砷酸钠组（iAs鼻组，300mg／L）。10个月后处死小鼠，肝功能检查血清丙氨酸转氨酶（ALT）、天门冬氨酸氨基转移酶（AST）、球蛋白（GLB）；部分肝组织做HE染色；Trizol-酚-氯仿一步法提取肝组织总RNA，紫外分光光度法测定总RNA及纯度，实时荧光定量PCR法测定肝组织中肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）、环氧化酶2（COX-2）mRNA的表达，以18S基因作为质控。结果ALT、AST、GLB在对照组[（38．0±5．6）U／L、（118．3±9．1）U／L、（20．9±0．6）g／L]、iAs^3＋组[（61．5±5．5）U／L、（530．9±39．0）U/L、（27．2±4．1）g／L]和iAs^5＋组[（48．3±2．6）U／L、（243．9±13．6）U／L、（23．2±2．1）g／L]组间比较，差异均有统计学意义（F值分别为45．653、164．326、9．639，P〈0．05）；ALT、AST组间两两比较差异均有统计学意义（P〈0．05），GLB仅在iAs如组与对照组比较差异有统计学意义（P〈0．05）。肝组织病理检查示有明显的炎症细胞浸润和肝细胞坏死．TNF-α、IL-6、COX-2mRNA的表达在对照组（11．84±3．21、41．08±22．98、26．60±5．84）、iAs^3＋组（48．59±19．65、218．87±70．05、207．86±155．39）、iAs^5＋组（35．56±10．84、84．15±28．38、18．88±6．34）组间比较，差异均有统计学意义（F值分别为24．317、49．611、8．499，P〈0．05）。结论长期砷暴露可导致肝脏的炎症反应、慢性肝损伤。TNF-α、IL-6、COX-2在iAs卦所导致的肝脏损伤中均起作用，其中TNF-α、IL-6炎症因子基因在iAs孙组肝组织的表达较iAss＋组更明显。COX-2在iAs鼻组所导致的肝脏损伤中可能无明显作用。

Objective To investigate the effect of inflammatory mediator in mice chronically exposed to inorganic arsenate. Methods 60 mice were divided into control group, sodium arsenite group （iAs^3＋ 300 mg/L） and sodium arsenite group（iAs^5＋ 300 mg/L） at random, iAs^3＋group and iAs^5＋group were daily given sodium arsenite and sodium arsenate respectively. The mice were sacrificed after 10 monthes for liver function and pathologic examinations. Total RNA was extracted by the Trizol-Phenol-Chlorofor-method from liver tissue. The total RNA extracted from hepatic tissue was detected for its density and purity, as well as the expression of tumor necrosis factor-alpha （TNF-α） mRNA, Interleukin-6 （IL-6） mRNA, cyclooxygenase 2 （ COX-2 ） mRNA with real time fluorescence quantitative PCR. The results were controlled with 18S. Results Serum alanine aminitransperase （ALT）, aspartate aminotransferase（AST）, globulin（GLB） of arsenic exposed mice[iAs^3＋group （61.5 ± 5.5）U/L, （530.9 ± 39.0）U/L, （27.2 ± 4.1）g/L, iAs^5＋group （48.3 ± 2.6）U/L, （243.9 ± 13.6）U/L, （23.2 ± 2.1）g/L] were different from that of the normal mice [（38.0 ± 5.6）U/L, （118.3 ±9.1）U/L, （20.9 ± 0.6）g/L], the difference had statistical significances（F = 45.653,164.326,9.639, P〈 0.05）. Serum ALT and serum AST had statistical significances among the three group（P 〈 0.05）, serum GLB had statistical significance between iAs^3＋group and the control group （P 〈 0.05）. Pathologic examination identified notable inflammation and liver cell necrosis involved in arsenic groups. Compared with the control group （11.84 ± 3.21,41.08 ± 22.98,26.60 ± 5.84）, the expression of TNF-α mRNA, IL-6 mRNA, COX-2 mRNA had statistical significance between iAs^3＋group （48.59 ± 19.65,218.87 ± 70.05,207.86 ± 155.39） and iAs^5＋group （35.56 ± 10.84,84.15 ± 28.38,18.88 ± 6.34）, the difference had statistical significances（F= 24.317,49.611,8.499, P〈 0.05）. Conclusions long-term exposure of mice to inorganic arsenate could result in inflammation and chronic damage of liver. The inflammation induced by iAs^3＋is more severe than that by iAs^5＋