内毒素对小鼠胆汁酸转运体Ntcp mRNA和Bsep mRNA表达及肝功能的影响

The mRNA expression of Ntcp and Bsep in hepatic injury induced by lipopolysaccharide in mice

目的探讨脂多糖（LPS）建立内毒素血症小鼠模型中，LPS剂量和作用时间对肝脏胆汁酸转运体钠离子／牛磺胆酸共转运蛋白（Ntcp）和胆盐输出泵（Bsep）mRNA表达，以及肝功能的影响。方法128只雄性C57BL／6小鼠，腹腔注射LPS（剂量分别是5、10、20、40mg／kg），制作内毒素血症模型；并设生理盐水（NS）对照组、健康对照组；按给药后取材时间进一步分为24h、48h、72h组，每组8只。留取小鼠血液样本测定血清TBIL、TBA、ALT、AST水平，留取肝脏标本，实时荧光定量聚合酶链反应技术（RT-qPCR）测胆汁酸转运体NtcpmRNA和BsepmRNA表达量，苏木精．伊红（HE）染色观察肝组织病理学变化。各组间比较采用单因素方差分析，组间两两比较采用LSD-t检验进行统计。结果（1）内毒素血症各组小鼠NtcpmRNA、BsepmRNA相对表达量在24—72h内下降，24h表达最低[LPS5、10、20、40ms／kg组NtcpmRNA相对表达量分别为（0．64±0．02）、（O．53±0．14）、（0．25±0．09）、（0．15±0．07），BsepmRNA相对表达量分别为（0．74±0．12）、（0．58±0．11）、（0．41±0．09）、（0．27±0．11）]，并随着LPS剂量增加逐渐降低，24h各组差异均有统计学意义（NtcpmRNA、BsepmRNA对应F=5．29、F=9．31，P=0．04、P=0．02）。（2）内毒素血症小鼠各组总胆红素（TBIL）、总胆汁酸（TBA）、谷丙转氨酶（ALT）、谷草转氨酶（AST）较对照组均升高（P〈0．05），24h达峰值；其中40mg／kgLPS处理后72h较24h均有显著降低，TBIL浓度为（1．29±0．25）μmol／L和（1．71±0．22）μmol／L，TBA浓度为（6．97±0．98）μmol／L和（8．96±1．01）μmol／L，ALT水平为（120．17±21．08）u／L和（179．22±16．57）U／L，AST水平为（360．34±35．31）U／L和（510．97±34．70）U／L，均P〈0．05。（3）5、10ms／kgLPS组小鼠24h见肝组织疏松淡染，间质炎性细胞浸润，48h及72h见肝细胞嗜酸性变、空泡样变，炎性细胞浸润，胆小管增生；20、40mg／kgLPS组小鼠各时间点均见肝组织结构改变，灶性坏死，间质炎症细胞浸润，胆小管增生、扩张。结论内毒素血症肝损伤模型24h时即出现NtcpmRNA和BsepmRNA表达下调，且随LPS剂量增加而加重，可能是内毒素肝损伤的重要机制。

Objective To investigate the effects of lipopolysaccharide （LPS） on the expressions of sodium taurocholate co-transporting polypeptide （Ntcp） and bile salt export pump （Bsep）, as well as the liver function markers in the serum including total bilirubin （TBIL）, total bile acids （TBA）, alanine aminotransferase （ALT）, aspartate aminotransferase （AST） in mice. Methods One hundred and twenty- eight C57BL/6 mice were intra-peritoneally injected with different doses of 5, 10, 20 or 40 mg/kg LPS （n = 24）, respectively. No treatment or treated with 0. 9% NaCI in mice as controls. Serum TBIL, TBA, ALT and AST levels were measured at 24 h, 48 h and 72 h after LPS injection in each group. The mRNA expressions of Ntcp and Bsep were detected by reverse transcription quantitative polymerase chain reaction （RT-qPCR）. The liver histological sections were stained with haematoxylin and eosin （H＆E）. Results The Ntcp and Bsep mRNA expressions in mice liver were significantly lower in livers of LPS-treated mice within 24 -72 h compared with control group, and the lowest level was reached at 24 h in a dose-dependent manner. And the relative expressions of Ntep mRNA and Bsep mRNA were （ 0. 64 ± 0. 02 ）, （ 0. 53 ± 0.14）, （0.25±0.09）, （0.15±0.07） and （0.74±0.12）, （0.58±0.11）, （0.41±0.09）, （0.27 ± 0. 11 ） in livers of mice injected with LPS in the different doses of 5, 10, 20, 40 mg/kg, respectively. In addition, serum levels of TBIL, TBA, ALT, and AST were significantly increased in mice of LPS-treated group compared with control group, particularly within 24 h after LPS treatment. Serum levels of TBIL, TBA, ALT, and AST were significantly decreased in mice of 40 mg/kg LPS-treated 72 h group compared with 24 h group presenting them with （1.29 ±0.25） μ mol/L vs. （1.71 ±0.22） μ mol/L, （6.97 ± 0.98）μmol/Lvs. （8.96±1.01） μ mol/L, （120.17±21.08） U/Lvs. （179.22±16.57） U/L, （360. 34 ±35.31） U/L vs. （510.97 ± 34. 70） U/L, respectively. Furthermore, histological changes in liver depend on dose and the course of LPS treatment. Cytoplasm rarefaction and inflammatory cells infiltration were detected at 24 h after treatment with 5 or I0 mg/kg LPS. Acidophilic and vacuolar degeneration, neutrophils infiltration in the hepatic sinusoid and portal area, the proliferation of bile ductulus were observed at 48 h, 72 h after treatment with 5 or 10 mg/kg LPS. In the 20 or 40 mg/kg LPS treatment groups, focal necrosis, infiltration with inflammatory cells, proliferation of bile ductulus and expansion of duct were observed at 24 h, 48 h and 72 h after LPS treatment. Conclusions LPS decreases the mRNA expressions of Ntcp and Bsep in a dose dependent manner in mice, contributing to mechanism of liver injury induced by endotoxin.