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胸腺基质淋巴细胞生成素在对乙酰氨基酚诱导的急性肝损伤小鼠模型中的作用机制
DOI: 10.12449/JCH250117
伦理学声明： 本研究方案于2021年2月1日经由广西医科大学动物实验中心实验动物伦理委员会审批，批号：202005022，符合实验室动物管理与使用准则。
利益冲突声明：本文不存在任何利益冲突。
作者贡献声明：陈文赏负责课题设计，资料分析，撰写论文；尹明景参与收集数据，修改论文；朱继金负责拟定写作思路，指导撰写文章并最后定稿。
Mechanism of action of thymic stromal lymphopoietin in a mouse model of acetaminophen-induced acute liver injury
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摘要:
目的 探讨胸腺基质淋巴细胞生成素（TSLP）在对乙酰氨基酚（APAP）诱导的急性肝损伤小鼠模型中的作用及其机制。 方法 16只野生型（WT）雄性C57BL/6J小鼠被随机分为2组，分别为control组和APAP组，每组8只；APAP组按照400 mg/kg的剂量腹腔注射APAP溶液建模，control组注射等体积生理盐水，6 h后进行取材。全自动化学分析仪检测血清ALT及AST，实时定量PCR方法检测肝组织炎症因子TNF-α和IL-6的mRNA表达，试剂盒检测肝组织匀浆中谷胱甘肽（GSH）含量，实时定量PCR、Western Blot方法检测TSLP的转录和蛋白水平的表达。另取22只WT雄性C57BL/6J小鼠，随机分为3组，分别为control组（n=8）、APAP组（n=8）和APAP+rTSLP组（n=6），APAP+rTSLP组先腹腔注射rTSLP溶液，同时control组、APAP组注射溶剂PBS；30 min后APAP+rTSLP组和APAP组注射APAP溶液，control组注射等体积生理盐水。检测3组小鼠血清ALT及AST；通过HE染色观察小鼠肝脏的病理变化；试剂盒检测肝组织匀浆中氧化应激指标丙二醛（MDA）、超氧化物歧化酶（SOD）水平；Western Blot方法检测自噬相关蛋白LC3Ⅰ/Ⅱ、Beclin1、P62，以及核因子E2相关因子2（Nrf2）、蛋白激酶B（Akt）、磷酸化-Akt（p-Akt）、哺乳动物雷帕霉素靶蛋白（mTOR）、磷酸化-mTOR（p-mTOR）等分子的蛋白表达。此外，取16只WT雄性C57BL/6J小鼠和16只沉默TSLP受体（TSLPR-/-）小鼠，分为WT小鼠control组、WT小鼠APAP组、TSLPR-/-小鼠control组和TSLPR-/-小鼠APAP组，每组8只，WT小鼠APAP组和TSLPR-/-小鼠APAP组按照400 mg/kg的剂量腹腔注射APAP溶液建模，WT小鼠control组和TSLPR-/-小鼠control组注射等体积生理盐水。检测4组小鼠血清ALT、AST以及肝组织的MDA含量；Western Blot方法检测LC3Ⅰ/Ⅱ、Akt、p-Akt的蛋白表达。计量资料两组间比较采用成组t检验；多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。 结果 APAP诱导急性肝损伤小鼠建模成功后，肝脏TSLP的mRNA和蛋白表达水平较control组均升高（P值均<0.01）。在应用rTSLP的研究中，相比于control组，APAP组的ALT、AST明显升高（P值均<0.001），肝组织HE染色呈现沿中央静脉放射状坏死，氧化应激指标SOD、Nrf2蛋白表达下降，MDA水平上升（P值均<0.01）；而APAP+rTSLP组较APAP组，ALT、AST下降，肝组织坏死面积减小，SOD、Nrf2蛋白表达升高，MDA下降（P值均<0.05）；APAP+rTSLP组与control组相比，LC3Ⅰ/Ⅱ、Beclin1、P62、p-Akt、p-mTOR蛋白表达差异均有统计学意义（P值均<0.01）。在应用TSLPR-/-小鼠的研究中，建模后，TSLPR-/-小鼠相较于WT小鼠，ALT、AST、MDA升高，LC3Ⅰ/Ⅱ、p-Akt蛋白表达下降（P值均<0.01）。 结论 TSLP能够增加自噬，降低氧化应激，从而改善过量APAP引起的急性肝损伤，并且其作用机制可能与PI3K/Akt信号通路的激活和mTOR的抑制有关。 -
关键词:
- 胸腺基质淋巴细胞生成素 /
- 醋氨酚 /
- 化学性与药物性肝损伤 /
- 小鼠， 近交C57BL
Abstract:Objective To investigate the role and mechanism of thymic stromal lymphopoietin （TSLP） in a mouse model of acetaminophen （APAP）-induced acute liver injury. Methods A total of 16 wild-type （WT） male C57BL/6J mice were randomly divided into control group and APAP group， with 8 mice in each group， and the mice in the APAP group were given intraperitoneal injection of APAP solution at a dose of 400 mg/kg to establish an animal model， while those in the control group were given injection of an equal volume of normal saline， with samples collected after 6 hours. An automatic chemical analyzer was used to measure the serum levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST）； quantitative real-time PCR was used to measure the mRNA expression levels of the inflammatory factors tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in liver tissue； the kit was used to measure the content of glutathione （GSH） in liver tissue homogenate； quantitative real-time PCR and Western blot were used to measure the transcriptional level and protein expression level of TSLP. Furthermore， 22 WT male C57BL/6J mice were randomly divided into control group with 8 mice， APAP group with 8 mice， and APAP+recombination TSLP （rTSLP） group with 6 mice； the mice in the APAP+rTSLP group were given intraperitoneal injection of rTSLP solution， while those in the control group and the APAP group were given injection of the solvent PBS； after 30 minutes， the mice in the APAP+rTSLP group and the APAP group were given injection of APAP solution， while those in the control group were given injection of an equal volume of normal saline. The serum levels of ALT and AST were measured； HE staining was used to observe the pathological changes of the liver； kits were used to measure the levels of the oxidative stress indices malondialdehyde （MDA） and superoxide dismutase （SOD） in liver tissue homogenate； Western blot was used to measure the expression levels of the autophagy-related proteins LC3Ⅰ/Ⅱ， Beclin1， and P62 and the molecules such as nuclear factor erythroid 2-related factor 2 （Nrf2）， protein kinase B （Akt）， phosphorylated Akt （p-Akt）， mammalian target of rapamycin （mTOR）， and phosphorylated mTOR （p-mTOR）. In addition， 16 WT male C57BL/6J mice and 16 TSLP receptor-silenced （TSLPR-/-） mice were divided into WT mouse control group， WT mouse APAP group， TSLPR-/- mouse control group， and TSLPR-/- mouse APAP group， with 8 mice in each group； the mice in the WT mouse APAP group and the TSLPR-/- mouse APAP group were used for modeling by intraperitoneal injection of APAP solution at a dose of 400 mg/kg， and those in the WT mouse control group and the TSLPR-/- mouse control group were given injection of an equal volume of normal saline. The serum levels of ALT and AST and the content of MDA in liver tissue were measured for these four groups， and Western blot was used to measure the protein expression levels of LC3Ⅰ/Ⅱ， Akt， and p-Akt. The independent-samples t test was used for comparison of continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. Results After the mouse model of APAP-induced acute liver injury was established successfully， there were significant increases in the mRNA and protein expression levels of TSLP compared with the control group （both P<0.01）. In the study of rTSLP， compared with the control group， the APAP group had significant increases in ALT and AST （both P<0.001） and radial necrosis along the central vein observed by HE staining of liver tissue， as well as significant reductions in the protein expression levels of the oxidative stress indices SOD and Nrf2 and a significant increase in the level of MDA （all P<0.01）； compared with the APAP group， the APAP+rTSLP group had significant reductions in ALT and AST， a significant reduction in necrotic area of liver tissue， significant increases in the protein expression levels of SOD and Nrf2， and a significant reduction in MDA （all P<0.05）； there were significant differences in the protein expression levels of LC3Ⅰ/Ⅱ， Beclin1， P62， p-Akt， and p-mTOR between the APAP+rTSLP group and the control group （all P<0.01）. In the study of TSLPR-/- mice， compared with the WT mice after modeling， the TSLPR-/- mice had significant increases in the levels of ALT， AST， and MDA and significant reductions in the expression levels of LC3Ⅰ/Ⅱ and p-Akt （all P<0.05）. Conclusion TSLP can increase autophagy， reduce oxidative stress， and thus improve acute liver injury induced by APAP overdose， possibly by activating the PI3K/Akt signaling pathway and inhibiting mTOR. -
注： a、b分别为两组小鼠血清ALT、AST水平；c、d分别为两组小鼠肝组织炎性因子TNF-α和IL-6的mRNA表达；e为两组小鼠肝组织GSH水平；f、g分别为两组小鼠肝组织TSLP的mRNA和蛋白表达水平。
图 1 血清肝酶、肝组织炎症因子和TSLP表达的比较
Figure 1. The comparison of serum liver enzymes， liver tissue inflammatory factors and TSLP expression of mice in two groups
注： a，3组小鼠血清ALT水平比较；b，3组小鼠血清AST水平比较；c，3组小鼠肝组织的代表性HE染色结果（×200）及APAP组与APAP+rTSLP组小鼠肝组织坏死面积量化比较。
图 2 3组小鼠血清肝酶比较及肝组织学表现
Figure 2. Serum enzymes and hepatic histopathology of mice in the three groups
注： a，3组小鼠肝组织MDA水平比较；b，3组小鼠肝组织SOD水平比较；c，3组小鼠肝组织Nrf2蛋白表达水平及其量化比较。
图 3 3组小鼠肝组织氧化应激水平
Figure 3. Oxidative stress levels in liver tissues of mice in the three groups
及P62的表达
图 4 3组小鼠肝组织自噬相关蛋白LC3 Ⅰ/Ⅱ、Beclin1
Figure 4. The level of autophagy-related proteins LC3 Ⅰ/Ⅱ、Beclin1 and P62 in liver tissues of the three groups
图 5 3组小鼠Akt、p-Akt、mTOR及p-mTOR的蛋白表达
Figure 5. Protein expressions of Akt、p-Akt、mTOR and p-mTOR in mice liver of three groups
注： a，血清ALT水平比较；b，血清AST水平比较；c，肝组织MDA水平比较；d，肝组织自噬相关蛋白LC3 Ⅰ/Ⅱ的表达；e，肝组织Akt和p-Akt蛋白表达。
图 6 TSLPR-/-小鼠与WT小鼠的比较
Figure 6. Comparison of TSLPR-/- mice and WT mice after modeling
表 1 引物序列
Table 1. Primers sequence
引物名称 正向引物（5'-3'） 反向引物（5'-3'） GAPDH GACATGCCGCCTGGAGAAAC AGCCCAGGATGCCCTTTAGT TSLP CTCAATCCTATCCCTGGCT GACTTCTTGTGCCATTTCCT TNF-α ACGGCATGGATCTCAAAGAC AGATAGCAAATCGGCTGAGG IL-6 ACAACCACGGCCTTCCCTA TCCACGATTTCCCAGAGAACA 
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