内毒素血症通过下调HNF4α表达加剧肝损伤

doi:10.11958/20212081

天津医药 ›› 2022, Vol. 50 ›› Issue (10): 1026-1030.doi: 10.11958/20212081

内毒素血症通过下调HNF4α表达加剧肝损伤

唐永静¹(), 陈云芬¹, 廖月¹, 程其娇¹, 张桂娟¹, 王云², 何毅怀¹^,^△()

1 遵义医科大学附属医院感染科（邮编563000）
2 大方县人民医院消化科

收稿日期:2021-11-21 修回日期:2022-05-24 出版日期:2022-10-15 发布日期:2022-10-20
通讯作者: 何毅怀 E-mail:372828980@qq.com;993565989@qq.com
作者简介:唐永静（1995）,女,硕士在读,主要从事肝衰竭的发病机制方面研究。E-mail： 372828980@qq.com
基金资助:
国家自然科学基金资助项目(81560110);贵州省科技厅科技支撑计划项目(黔科合支撑（2019）2803号);贵州省科学技术基金项目（黔科合平台人才[2017]5733-013(黔科合平台人才[2017]5733-013);贵州省卫生健康委科学技术基金项目(gzwjkj2020-1-041)

Endotoxin exacerbates liver injury by down-regulating HNF4α expression

TANG Yongjing¹(), CHEN Yunfen¹, LIAO Yue¹, CHENG Qijiao¹, ZHANG Guijuan¹, WANG Yun², HE Yihuai¹^,^△()

1 Department of Infectious Diseases, the Affiliated Hospital of Zunyi Medical University, Zunyi 563000, China
2 Department of Gastroenterology, Dafang County People's Hospital

Received:2021-11-21 Revised:2022-05-24 Published:2022-10-15 Online:2022-10-20
Contact: HE Yihuai E-mail:372828980@qq.com;993565989@qq.com

唐永静, 陈云芬, 廖月, 程其娇, 张桂娟, 王云, 何毅怀. 内毒素血症通过下调HNF4α表达加剧肝损伤[J]. 天津医药, 2022, 50(10): 1026-1030.

TANG Yongjing, CHEN Yunfen, LIAO Yue, CHENG Qijiao, ZHANG Guijuan, WANG Yun, HE Yihuai. Endotoxin exacerbates liver injury by down-regulating HNF4α expression[J]. Tianjin Medical Journal, 2022, 50(10): 1026-1030.

摘要/Abstract

摘要：

目的探讨内毒素血症下调肝细胞核因子4α（HNF4α）表达介导肝损伤进展的机制。方法 144只BALB/c小鼠采用随机数字表法分组进行以下实验：（1）四氯化碳（CCl₄）诱导小鼠急性肝损伤。对照组和0.5 mL/kg、1.0 mL/kg、2.0 mL/kg CCl₄组。（2）筛选脂多糖（LPS）干预小鼠的剂量。对照组和0.1 mg/kg、0.5 mg/kg、2.5 mg/kg LPS组。（3）LPS干预CCl₄（1.0 mL/kg）诱导急性肝损伤模型小鼠。对照组、CCl₄组、0.1 mg/kg LPS+CCl₄组、0.5 mg/kg LPS+CCl₄组。每组12只。诱导24 h后处死小鼠,采用酶速率法检测血清丙氨酸转氨酶（ALT）,重氮法检测总胆红素（TBil）水平;Western blot法检测肝组织HNF4α、胱天蛋白酶3剪切体（Cleaved caspase-3）蛋白表达;原位末端标记法检测肝细胞凋亡情况。结果 0.5、1.0、2.0 mL/kg CCl₄组的血清ALT、TBil及肝组织HNF4α、Cleaved caspase-3蛋白表达水平高于对照组,且呈剂量依赖性增高。2.5 mg/kg LPS组血清ALT、TBil及肝组织Cleaved caspase-3蛋白表达水平高于对照组和0.1、0.5 mg/kg LPS组,肝组织HNF4α蛋白表达水平低于对照组和0.1、0.5 mg/kg LPS组（P<0.05）。CCl₄组和0.1、0.5 mg/kg LPS+CCl₄组的血清ALT、TBil,肝组织HNF4α、Cleaved caspase-3蛋白表达水平及肝细胞凋亡指数均高于对照组（P<0.05）;0.1、0.5 mg/kg LPS+CCl₄组的血清ALT、TBil,肝组织Cleaved caspase-3蛋白表达水平及肝细胞凋亡指数高于CCl₄组,HNF4α蛋白表达水平低于CCl₄组（P<0.05）。结论内毒素血症通过下调HNF4α表达增加肝细胞凋亡,可能是其介导肝损伤进展的机制之一。

关键词: 肝细胞核因子4α, 内毒素血症, 肝损伤, 脂多糖, 细胞凋亡

Abstract:

Objective To investigate the mechanism of endotoxemia down-regulating the expression of hepatocyte nuclear factor 4α (HNF4α) and mediating the progression of liver injury. Methods A total of 144 BALB/c mice were divided into the following experiments using the digital table method. (1) carbon tetrachloride (CCl₄) induced acute liver injury in mice: the control group, the 0.5 mL/kg, the 1.0 mL/kg and the 2.0 mL/kg groups; (2) screening doses of lipopolysaccharide (LPS) intervention in mice: the control group, the 0.1 mg/kg, the 0.5 mg/kg and the 2.5 mg/kg LPS groups; (3) LPS intervention in CCl₄ (1.0 mL/kg)-induced acute liver injury model mice: the control group, the CCl₄ group, the 0.1 mg/kg LPS + CCl₄ group and the 0.5 mg/kg LPS + CCl₄ group. There were 12 mice in each dose group. Mice were sacrificed after 24 hours induction, and blood samples of mice were collected to detect serum alanine aminotransferase (ALT) by enzyme rate method, and total bilirubin (TBil) level by diazo method. The protein contents of HNF4α and Cleaved caspase-3 in liver tissue were detected by Western blot assay. TUNEL assay was used to detect the apoptosis of hepatocytes. Results The serum levels of ALT, TBil, and liver tissue HNF4α and Cleaved caspase-3 protein expression levels were higher in the 0.5, 1.0 and 2.0 mL/kg CCl₄ groups than those in the control group in a dose-dependent manner. The serum levels of ALT and TBil, and liver tissue Cleaved caspase-3 protein were higher in the 2.5 mg/kg LPS group than that in the control group and the 0.1 and 0.5 mg/kg LPS groups. The expression level of HNF4α protein in liver tissue was lower in the 2.5 mg/kg LPS group than that in the control group and the 0.1, 0.5 mg/kg LPS group (P<0.05). Serum levels of ALT and TBil, liver tissue HNF4α and Cleaved caspase-3 protein expression levels and hepatocyte apoptosis index were higher in the CCl₄ group and the 0.1 and 0.5 mg/kg LPS + CCl₄ groups than those in the control group (P<0.05). The serum ALT, TBil, liver tissue Cleaved caspase-3 protein expression level and hepatocyte apoptosis index were higher in the 0.1, 0.5 mg/kg LPS+CCl₄ groups than those of CCl₄ group. The protein expression level of HNF4α was lower than that of the CCl₄ group (P<0.05). Conclusion Endotoxemia increases hepatocyte apoptosis by downregulating the expression of HNF4α, which may be one of the machanisms mediating the progression of liver injury.

Key words: hepatocyte nuclear factor 4α, endotoxemia, liver injury, lipopolysaccharide, apoptosis

中图分类号:

R575.3

图/表 7

表1 不同剂量CCl4诱导组血清ALT、TBil水平及肝组织HNF4α、Cleaved caspase-3蛋白表达水平比较 ($\bar{x} \pm s$)

Tab. 1 Comparison of serum ALT and TBil levels, and protein expression levels of HNF4α and Cleaved caspase-3 in liver tissue between the four groups of mice induced by different doses of CCl4

组别	n		ALT（U/L）		TBil（μmol/L）
对照组	12		42.08±5.53		1.28±0.15
0.5 mL/kg CCl₄组	12		2 496.25±202.35^a		1.93±0.17^a
1.0 mL/kg CCl₄组	12		4 458.00±506.94^ab		4.45±0.27^ab
2.0 mL/kg CCl₄组	12		6 502.08±599.06^abc		6.35±0.69^abc
F			493.724^＊＊		404.139^＊＊
组别		HNF4α		Cleaved caspase-3
对照组		1.00±0.00		1.00±0.00
0.5 mL/kg CCl₄组		1.95±0.05^a		1.71±0.11^a
1.0 mL/kg CCl₄组		6.46±0.25^ab		5.55±0.21^ab
2.0 mL/kg CCl₄组		11.48±0.43^abc		8.48±0.16^abc
F		4 020.010^＊＊		6 745.702^＊＊

图1 Western blot检测不同剂量CCl4诱导组肝组织HNF4α、Cleaved caspase-3蛋白表达 A：对照组;B：0.5 mL/kg CCl4组;C：1.0 mL/kg CCl4组;D：2.0 mL/kg CCl4组。

Fig.1 Western blot detection of HNF4α and Cleaved caspase-3 protein expression in liver tissue induced by different doses of CCl4

表2 不同剂量LPS组血清ALT、TBil水平及肝组织HNF4α、Cleaved caspase-3蛋白表达水平比较 ($\bar{x} \pm s$)

Tab. 2 Comparison of serum ALT and TBil levels, and protein expression levels of HNF4α and Cleaved caspase-3 in liver tissue between the four groups of mice induced by different doses of LPS

组别	n		ALT（U/L）		TBil（μmol/L）
对照组	12		32.92±5.89		1.18±0.29
0.1 mg/kg LPS组	12		37.67±9.12		1.48±0.21
0.5 mg/kg LPS组	12		53.33±8.51		1.50±0.21
2.5 mg/kg LPS组	12		5 725.50±109.62^abc		5.44±0.22^abc
F			29 112.963^＊＊		836.544^＊＊
组别		HNF4α		Cleaved caspase-3
对照组		1.00±0.00		1.00±0.00
0.1 mg/kg LPS组		0.94±0.10		1.06±0.10
0.5 mg/kg LPS组		0.86±0.07		2.44±0.14
2.5 mg/kg LPS组		0.47±0.03^abc		7.40±0.16^abc
F		156.332^＊＊		7 022.815^＊＊

图2 Western blot检测不同剂量LPS诱导组肝组织HNF4α、Cleaved caspase-3蛋白表达 A：对照组;B：0.1 mg/kg LPS组;C：0.5 mg/kg LPS组;D：2.5 mg/kg LPS组。

Fig.2 Western blot detection of HNF4α and Cleaved caspase-3 protein expression in liver tissue induced by different doses of LPS

表3 各组血清ALT、TBil水平,肝组织HNF4α、Cleaved caspase-3蛋白表达水平及肝细胞凋亡指数比较 ($\bar{x} \pm s$)

Tab. 3 Comparison of serum ALT and TBil levels, protein expression levels of HNF4α and Cleaved caspase-3 in liver tissue and apoptotic index between the four groups

组别		n	ALT（U/L）		TBil（μmol/L）
对照组		12	34.75±2.24		1.26±0.26
CCl₄组		12	3 412.08±122.68^a		3.38±0.29^a
0.1 mg/kg LPS+CCl₄组		12	5 683.00±177.65^ab		5.40±0.20^ab
0.5 mg/kg LPS+CCl₄组		12	6 269.92±98.71^ab		6.28±0.16^ab
F			6 236.269^＊＊		1 011.176^＊＊
组别	HNF4α			Cleaved caspase-3		凋亡指数
对照组	1.00±0.00			1.00±0.00		0.52±0.11
CCl₄组	3.41±0.26^a			2.29±0.15^a		27.58±0.83^a
0.1 mg/kg LPS+CCl₄组	2.45±0.14^ab			2.78±0.23^ab		31.93±0.73^ab
0.5 mg/kg LPS+CCl₄组	1.55±0.08^ab			3.23±0.31^ab		36.71±0.82^ab
F	513.070^＊＊			238.550^＊＊		6 101.197^＊＊

图3 Western blot检测各组小鼠肝脏HNF4α、Cleaved caspase-3蛋白表达水平 A：对照组;B：CCl4 组;C：0.1 mg/kg LPS+CCl4 组;D：0.5 mg/kg LPS+CCl4 组。

Fig.3 Western blot detection of HNF4α and Cleaved caspase-3 protein levels in liver tissue of mice

图4 原位末端标记染色检测各组小鼠肝细胞凋亡情况（×20）

Fig.4 TUNEL detection of hepatocyte apoptosis of mice in each group (×20)

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内毒素血症通过下调HNF4α表达加剧肝损伤

Endotoxin exacerbates liver injury by down-regulating HNF4α expression

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