安徽医科大学学报

目的 探究转录因子EB(TFEB)-自噬途径在利福平所致肝损伤中的作用及其可能机制。方法 将40只6～8周龄的C57/BL6雄性小鼠随机分为五组：对照组、模型组、TFEB低剂量激动剂组、TFEB高剂量激动剂组、自噬激动剂组，每组8只。除对照组外，其余四组每天给予利福平200 mg/(kg·d)灌胃；TFEB激动剂在给予利福平后1 h腹腔注射，低剂量为20 mg/kg,高剂量为50 mg/kg,连续7 d;自噬激动剂在第1天给予利福平前6 h予10 mg/kg灌胃。造模7 d后结束实验。实验通过检测肝功能指标和肝脏病理学变化来评估肝损伤程度，采用Western blot定量分析肝脏细胞核/肝组织中总的TFEB、螯合体1(p62)、微管相关蛋白轻链3(LC3)、苄氯素1(Beclin-1)、钠离子-牛磺胆酸钠共转运多肽(NTCP)、胆盐输出泵(BSEP)的水平。结果 与对照组相比，模型组小鼠血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、总胆红素(TBIL)、直接胆红素(DBIL)以及总胆汁酸(TBA)的水平升高，差异有统计学意义(P<0.05),肝脏出现明显病理变化；与模型组相比，TFEB激动剂高剂量、低剂量和自噬激动剂组小鼠上述指标降低(P<0.05);与TFEB激动剂低剂量组相比，高剂量组小鼠上述指标降低(P<0.05)。对照组TFEB入核比例为(1.0±0.10),模型组降至(0.6±0.05)(P<0.05),TFEB低剂量激动剂组恢复至(0.8±0.08),高剂量激动剂组提高至(0.9±0.07),自噬激动剂组为(0.7±0.06)(P<0.05)。与对照组相比，模型组小鼠肝脏NTCP和BSEP的表达水平降低(P<0.05),在TFEB低剂量和高剂量激动剂组中，NTCP和BSEP表达有所恢复，自噬激动剂组NTCP和BSEP表达也提高(P<0.05)。与对照组相比，模型组小鼠肝脏组织中TFEB、LC3-Ⅱ/LC3-Ⅰ和Beclin-1的蛋白表达水平明显降低(P<0.05),而p62的蛋白表达水平明显升高(P<0.05)。与模型组相比，TFEB激动剂高剂量组、低剂量组和自噬激动剂组小鼠肝脏组织中TFEB、LC3-Ⅱ/LC3-Ⅰ和Beclin-1的蛋白表达水平有所升高(P<0.05),而p62的蛋白表达水平有所降低(P<0.05)。结论 TFEB通过激活自噬途径可改善利福平所致的肝损伤，主要机制可能与上调NTCP和BSEP的表达有关。

Objective To investigate the role of transcription factor EB(TFEB)-autophagy pathway in rifampicin-induced liver injury and its possible mechanism. Methods Forty 6-8-week-old C57/BL6 mice were randomly divided into five groups: control group, model group, TFEB low-dose agonist group, TFEB high-dose agonist group, and autophagy agonist group, with 8 mice in each group. Except for the control group, the other four groups were given rifampicin 200 mg/(kg·d) by gavage daily. TFEB agonist was administered intraperitoneally at a low dose of 20 mg/kg and a high dose of 50 mg/kg for 7 days at 1 h after rifampicin administration. Autophagy agonist was administered by gavage at a dose of 10 mg/kg 6 h before rifampicin administration on day 1. The experiment was completed 7 days after modeling. The degree of liver injury was evaluated by detecting liver function indexes and liver pathological changes. Western blot was used to detect the protein expression total TFEB, chelator 1(p62), microtubule-associated protein light chain 3(LC3), benzyl chloride 1(Beclin-1), sodium taurocholate co-transporting polypeptide(NTCP) and bile salt export pump(BSEP) levels in liver nucleus/liver tissue were quantified. Results Compared with the control group, the serum levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), total bilirubin(TBIL), direct bilirubin(DBIL), and total bile acid(TBA) in the model group increased(P<0.05), and obvious pathological changes were observed in the liver. Compared with the model group, the high dose and low dose of TFEB agonist and autophagy agonist groups had reductions in the above indicators(P<0.05). Compared with the low-dose TFEB agonist group, the high-dose TFEB agonist group had reductions in the above indicators(P<0.05). The proportion of TFEB in the nucleus was(1.0±0.10) in the control group,(0.6±0.05) in the model group,(0.8±0.08) in the low-dose agonist group, and(0.9±0.07) in the high-dose agonist group(P<0.05). Autophagy agonist group(0.7±0.06)(P<0.05). Compared with the control group, the levels of NTCP and BSEP in the liver of the model group decreased(P<0.05), and the expression of NTCP and BSEP in the TFEB low-dose and high-dose agonist groups were restored, and the expression of NTCP and BSEP in the autophagy agonist group also increased(P<0.05). Compared with the control group, the protein expression levels of TFEB, LC3-Ⅱ/LC3-Ⅰand Beclin-1 in the liver tissue of the model group significantly decreased(P<0.05), while the protein expression level of p62 significantly increased(P<0.05). Compared with the model group, the protein expression levels of TFEB, LC3-Ⅱ/LC3-Ⅰand Beclin-1 in the liver tissue of the TFEB agonist high-dose group, low-dose group and autophagy agonist group increased(P<0.05), while the protein expression level of p62 decreased(P<0.05). Conclusion TFEB can improve rifampicin-induced liver injury by activating autophagy pathway, and the main mechanism may be related to the up-regulation of NTCP and BSEP expression.