肿瘤坏死因子-α对急性肝功能衰竭大鼠肠黏膜Claudin-1、Zonula Occludens-1和肌球蛋白轻链激酶表达的影响及肿瘤坏死因子α拮抗剂的干预效果

Impact of tumor necrosis factor-α and its antagonist on intestinal mucosa claudin-1, Zonula Occludens-1 and myosin light chain kinase in rat models of acute liver failure

目的研究TNF-α对急性肝功能衰竭（ALF）大鼠肠黏膜Claudin-1、Zonula Occludens（ZO）-1蛋白和肌球蛋白轻链激酶（MLCK）表达的影响及TNF-α拮抗剂干预效果。方法健康雄性SD大鼠270只，重复造模5次，每次54只，按照随机数字表法将54只大鼠分为正常对照组、模型组和干预组。正常对照组6只仅腹腔注射0．9％氯化钠溶液12mL／kg；模型组和干预组各24只，均腹腔注射口GalN 1200mg／kg，建立ALF模型；干预组在给予D～GalN 24h前腹腔注射TNF-α拮抗剂（rhTNFR：Fc）12．5mg／kg。模型组与干预组于造模后8、24、48、72h各时间点处死6只大鼠，正常对照组于72h同时处死动物。应用生物化学方法检测肝功能；ELISA法检测血清TNF-α水平；肝组织和末段回肠组织经HE染色检测；免疫组织化学和Western印迹法检测各组末段回肠Claudin-1、ZO-1蛋白和MLCK的表达。组内两两比较采用t检验。结果用D-GalN成功诱导ALF模型。模型组72h转氨酶开始下降时，TBil继续上升，出现胆酶分离现象，而干预组未出现该现象。模型组72h末段回肠可见绒毛倒伏，部分绒毛尖端脱落，黏膜层可见大量中性粒细胞浸润；干预组末段回肠结构完整，黏膜层可见少量中性粒细胞浸润。模型组、干预组血清TNF-α水平均在24h升至高峰，分别为（239．83±15．81）、（182．71±17．08）ng／L；均较正常对照组[（24．19±3．57）ng／L]显著升高，差异均有统计学意义（t值分别为22．68、15．73，均P〈0．01）；而干预组显著低于模型组，差异有统计学意义（t=4．58，P〈0．01）。模型组大鼠回肠黏膜Claudin-1、ZO-1相对表达量在早期均逐渐降低，24h下降至最低，分别为0．355±0．068、0．387±0．091，均显著低于正常对照组（1．640±0．188、1．015±0．150），差异有统计学意义（t值分别为12．87、7．14，均P〈0．01）；干预组大鼠回肠黏膜Claudin-1、ZO-1蛋白相对表达量亦于24h下降至最低，分别为1．051±0．370、0．642±0．082，明显低于正常对照组（t值分别为2．84、4．36，均P〈0．05），但均高于模型组，差异有统计学意义（t值分别为3．70、4．15，均P〈0．01）。模型组、干预组大鼠回肠MLCK相对表达量在早期均逐渐升高，24h均上升至高峰，分别为1．298±0．194、1．033±0．073，明显高于对照组的0．460±0．069，差异有统计学意义（t值分别为8．16、11．44，均P〈0．01），但干预组低于模型组，两者差异有统计学意义（t=2．56，P〈0．05）。结论ALF大鼠模型的血清TNF-α水平升高，Claudin-1和ZO-1蛋白表达下降、MLCK含量增加，使得细胞收缩加剧及细胞间隙增大。而TNF—α拮抗剂通过抑制MLCK含量的增加、阻止Claudin-1和ZO-1蛋白表达的下降，使肝功能明显改善、降低细胞炎性反应和肝细胞凋亡。

Objective To study the impact of tumor necrosis factor-α （TNF-α） and its antagonist on the expressions of intestinal mucosa claudin-1, Zonula Occludens-1 （ZO-1） and myosin light chain kinase （MLCK） in rat models of acute liver failure. Methods Fifty four healthy male SpragueDawley （SD） rats were randomly divided into normal control group, model group and intervention group according to a random number table. Rats in normal control （n= 6） group were intraperitoneally injected with 0. 9% saline （12 mL/kg）. Rats in model group （n=24） and intervention group （n= 24） were intraperitoneally injected with a full dose of D-galactosamine （D-GaIN） at a dose of 1 200 mg/kg to establish model of acute liver failure, while rats in intervention group were intraperitoneally injected with TNF-α antagonists （rhTNFR, Fc） at a dose of 12.5 mg/kg before 24 hours given D-GaIN. At each time point of hour 8, 24, 48 and 72, six rats in both model group and the intervention group were sacrificed, respectively, while the normal control group were all anesthetized and sacrificed at 72 h. Models were repeated five times. Serum liver function was detected by biochemical method, and serum TNF-α level was detected by enzyme-linked immunosorbent assay （ELISA）. Hematoxylin-eosin （HE） stained sections of liver and terminal ileum were examined under an optical microscope for pathological changes and protein expression of the terminal ileum Claudin-1, ZO-1 protein and MLCK were determined by immunohistochemistry and Western blot. Means among groups were compared with t test. Results Acute liver failure was successfully induced in the D-GalN injected rats. In the model group, alanine aminotransferase （ALT） began to decline, total bilirubin continued to rise, and enzyme-jaundice separation developed at hour 72. But total bilirubin in intervention group at hour 72 was decreased. Light microscope showed that at hour 72, villus lodged at terminal ileum in the model group with part of villus tip failing off in the model group. Villus mucosa and submucosa interstitial were edema and infiltrated with numerous neutrophils. The terminal ileum kept integrate in the intervention group, and villus mucosa and submucosa were mild edema and only infiltrated with a small amount of neutrophil. Expressions of tumor necrosis necrosis factor （TNF）-α in rats of model group and intervention group were gradually increased and peaked at hour 24 （[239. 83 ± 15. 81] and [182.71±17.08] ng/L, respectively）, which were significantly higher than that of the control group （[24. 19±3.57] ng/L, t=22.68 and 15.73, respectivelyboth P〈0.01）. Expression of serum TNF-α in the intervention group was significantly lower than that of model group （t= 4. 58, P〈0. 01）. Expressions of Claudin-1 and ZO-1 in model group decreased gradually at an early stage and reached the lowest level at hour 24 （0. 355 ± 068 and 0. 387 ±0. 091, respectively）, which were both significantly lower than that of control group （1. 640±0. 188 and 1. 015±0. 150, respectively; t= 12.87 and 7. 14, respectively both P〈0.01）. In the intervention group, expressions of Claudin-1 and ZO-1 also decreased to the lowest level at hour 24 （1. 051±0. 370 and 0. 642 ±0. 082, respectivley）, which were both significantly lower than that of control group （t= 2.84 and 4. 36, respectively both P〈0.05）, but significantly higher than model group with stastically difference （t= 3.70 and 4.15, respectively; both P〈0.01）. MLCK protein levels in the model and intervention group were gradually increased, which peaked at hour 24 （1. 298±0. 194 and 1. 033 ±0. 073, respectively）, significantly higher than the control group （0. 460±0. 069,t=8.16 and 11.44, both P〈0.01）; and MLCK in the intervention group was lower than that in the model group with statistically difference （t=2.56, P〈0.05）. Conclusions Expression of serum TNF-α in rat model of acute liver failure increases, which leads to decreased expression of Claudin-1 and ZO-1, and increased expression of MLCK, makes cell shrunk and cell gap increased. TNF-α antagonist could significantly reduce the inflammation and liver cell apoptosis, improve liver function by inhibiting MLCK expression and preventing decrease of Claudin-1 and ZO-1 proteins.