环境内分泌干扰物双酚A对非酒精性脂肪性肝病大鼠模型肠道黏膜屏障的影响

摘要

目的 研究环境内分泌干扰物双酚A(BPA)对非酒精性脂肪性肝病(NAFLD)大鼠炎症反应的影响,以及对肠道黏膜屏障的损害作用.方法 将18只成熟雄性SD大鼠随机分为3组:正常组、NAFLD组和NAFLD+BPA组,每组6只.光镜下观察肝脏病理改变,ELISA检测血清TNFα、IL-1β、IL-6和IL-8等炎症因子,鲎试剂终点比色法检测内毒素水平,免疫荧光法观察肠道黏膜Occludin蛋白的表达情况,及实时聚合酶链式反应测定肠道黏膜外周蛋白ZO-1 mRNA的改变.计量资料多组间比较采用单因素方差分析,进一步两两比较用SNK-q检验.结果 肝脏病理学证实NAFLD模型建模成功.100 nmol/L BPA摄入8周后,TNFα、IL-6、IL-8炎症因子表达上调[分别为(127.65±22.40)pg/ml、(199.34±17.46)pg/ml和(258.79±12.82)pg/ml]伴内毒素水平增高[(0.88±0.26)EU/ml],与正常组[分别为(64.87±10.83)pg/ml、(91.27±9.82)pg/ml、(123.76±19.68)pg/ml和(0.27±0.09)EU/ml]及NAFLD组[分别为(92.34±10.68)pg/ml、(181.93±20.11)pg/ml、(201.64±22.34)pg/ml和(0.63±0.15)EU/ml]相比,差异均有统计学意义(P值均<0.05).NAFLD组和NAFLD+BPA组中的IL-1β水平明显高于正常组[(186.31±20.06)pg/ml、(208.78±13.77)pg/ml)vs(112.84±23.12)pg/ml,P值均<0.05],但NAFLD组和NAFLD+BPA组间差异无统计学意义.此外,与正常组比较,NAFLD组和NAFLD+BPA组中肠道紧密连接蛋白Occludin水平下降,肠道黏膜外周蛋白ZO-1 mRNA表达水平呈显著降低,BPA干预促进ZO-1 mRNA进一步下调(68.03±11.73、45.24±6.98、33.25±11.04,两两组间比较,P值均<0.05).结论 在NAFLD背景下,长期低剂量BPA的暴露能加重炎症因子释放,促进内毒素血症,进而损伤肠道黏膜屏障.%Objective To investigate the influence of bisphenol A ( BPA) , an environmental endocrine disruptor , on inflammatory response in rats with nonalcoholic fatty liver disease ( NAFLD) , as well as its adverse effect on intestinal mucosal barrier .Methods A total of 18 mature male rats were randomly divided into normal group , NAFLD group, and NAFLD+BPA group, with 6 rats in each group.liver patho-logical changes were observed under a light microscope;ELISA was used to measure the serum levels of tumor necrosis factor -α( TNF-α), interleukin-1β(IL-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8);the terminal colorimetric analysis with Limulus a-mebocyte lysate was used to measure the level of endotoxin;an immunofluorescence assay was used to measure the expression of occluding protein in the intestinal mucosa;real-time PCR was used to measure the mRNA expression of ZO -1 in the intestinal mucosa .A one-way analysis of variance was used for comparison of continuous data between multiple groups , and the SNK-q test was used for further compari-son between two groups .Results Liver pathological examination showed that the rat model of NAFLD was established successfully .After the 8-week treatment with 100 nmol/L BPA, compared with the normal group and the NAFLD group , the NAFLD+BPA group had signifi-cant increases in the expression of TNF -α(127.65 ±22.4 pg/ml vs 64.87 ±10.83 pg/ml and 92.34 ±10.68 pg/ml, P<0.05), IL-6 (199.34 ±17.46 pg/ml vs 91.27 ±9.82 pg/ml and 181.93 ±20.11 pg/ml, P<0.05), and IL-8 (258.79 ±12.82 pg/ml vs 123.76 ± 19.68 pg/ml and 201.64 ±22.34 pg/ml, P<0.05) and the level of endotoxin (0.88 ±0.26 EU/ml vs 0.27 ±0.09 EU/ml and 0.63 ± 0.15 EU/ml, P<0.05).The NAFLD group and the NAFLD+BPA group had a significantly higher level of IL -1βthan the normal group (186.31 ±20.06 pg/ml and 208.78 ±13.77 pg/ml vs 112.84 ±23.12 pg/ml, both P<0.05), but there was no significant difference be-tween these two groups .Compared with the normal group , the NAFLD group and the NAFLD+BPA group had significant reductions in the expression of occluding protein and the mRNA expression of ZO -1 in the intestinal mucosa , and the NAFLD+BPA group had a significantly greater reduction in the mRNA expression of ZO -1 than the control group and the NAFLD group (33.25 ±11.04 vs 68.03 ±11.73/45.24 ± 6.98, P<0.05).Conclusion In the background of NAFLD, long-time exposure to low-dose BPA can increase the release of some in-flammatory factors, promote endotoxemia , and thus damage intestinal mucosal barrier .