Endotoxin exacerbates liver injury by down-regulating HNF4α expression

doi:10.11958/20212081

Abstract

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

CLC Number:

R575.3

Figures/Tables 7

References 21

[1]	SOLE C, SOLA E. Update on acute-on-chronic liver failure[J]. Gastroenterol Hepatol, 2018, 41(1):43-53. doi: 10.1016/j.gastrohep.2017.05.012.
[2]	TANG Y J, CHEN H, YI Y, et al. Inhibition of eIF2α dephosphorylation protects hepatocytes from apoptosis by alleviating ER stress in acute liver injury[J]. Biomed Res Int, 2020, 2020:2626090. doi: 10.1155/2020/2626090.
[3]	PEREGO J, BOURBON C, CHASSON L, et al. Guanabenz prevents d-Galactosamine/Lipopolysaccharide-induced liver damage and mortality[J]. Front Immunol, 2017, 8: 679. doi: 10.3389/fimmu.2017.00679.
[4]	PEREA L, COLL M, SANJURJO L, et al. Pentraxin-3 modulates lipopolysaccharide-induced inflammatory response and attenuates liver injury[J]. Hepatology, 2017, 66(3):953-968. doi: 10.1002/hep.29215.
[5]	LIU H, ZHANG W, DONG S, et al. Protective effects of sea buckthorn polysaccharide extracts against LPS/d-GalN-induced acute liver failure in mice via suppressing TLR4-NF-κB signaling[J]. J Ethnopharmacol, 2015, 176:69-78. doi: 10.1016/j.jep.2015.10.029.
[6]	GABALLAH H H, EL-HORANY H E, HELAL D S. Mitigative effects of the bioactive flavonol fisetin on high-fat/high-sucrose induced nonalcoholic fatty liver disease in rats[J]. J Cell Biochem, 2019, 120(8):12762-12774. doi: 10.1002/jcb.28544.
	WANG B, CAI S R, GAO C, et al. Lipopolysaccharide results in a marked decrease in hepatocyte nuclear factor 4 alpha in rat liver[J]. Hepatology, 2001, 34(5):979-989. doi: 10.1242/jcs.114.6.1205.
[8]	BULLA G A, GIVENS E, BROWN S, et al. A common regulatory locus affects both HNF4/HNF1alpha pathway activation and sensitivity to LPS-mediated apoptosis in rat hepatoma cells[J]. J Cell Sci, 2001, 114(Pt 6):1205-1212. doi: 10.1053/jhep.2001.28885.
[9]	SIMMONDS R C. Bioethics and animal use in programs of research,teaching,and testing[M]. In: WEICHBROD R H, THOMPSONG A, NORTONJ N. Management of animal care and use programs in research,education,and testing. 2nd ed. Boca Raton(FL): CRC Press/Taylor & Francis; 2018:35-62. doi: 10.1201/9781315152189-4.
[10]	BRAHMANIA M, LIU S, WAHED A S. Alcohol,tobacco and coffee consumption and liver disease severity among individuals with chronic hepatitis B infection in North America[J]. Ann Hepatol, 2020, 19(4):437-445. doi: 10.1016/j.aohep.2020.01.005.
[11]	FERNANDEZ J, ACEVEDO J, WIEST R, et al. Bacterial and fungal infections in acute-on-chronic liver failure:prevalence,characteristics and impact on prognosis[J]. Gut, 2018, 67(10):1870-1880. doi: 10.1136/gutjnl-2017-314240.
[12]	SCHWABE R F, LUEDDE T. Apoptosis and necroptosis in the liver:a matter of life and death[J]. Nat Rev Gastroenterol Hepatol, 2018, 15(12):738-752. doi: 10.1038/s41575-018-0065-y.
[13]	WANG P P, HUANG X, YANG M W, et al. Effects of non-drug treatment on liver cells apoptosis during hepatic ischemia-reperfusion injury[J]. Life Sci, 2021, 275:119321. doi: 10.1016/j.lfs.2021.119321.
[14]	CHOI Y M, LEE S Y, KIM B J. Naturally occurring hepatitis B virus mutations leading to endoplasmic reticulum stress and their contribution to the progression of hepatocellular carcinoma[J]. Int J Mol Sci, 2019, 20(3):597. doi: 10.3390/ijms20030597.
[15]	ZHAO X C, LIVINGSTON M J, LIANG X L, et al. Cell apoptosis and autophagy in renal fibrosis[J]. Adv Exp Med Biol, 2019, 1165:557-584. doi: 10.1007/978-981-13-8871-2_28.
[16]	DUBOIS V, STAELS B, LEFEBVRE P, et al. Control of cell identity by the nuclear receptor HNF4 in organ pathophysiology[J]. Cells, 2020, 9(10):2185. doi: 10.3390/cells9102185.
[17]	LIU W T, JING Y Y, GAO L, et al. Lipopolysaccharide induces the differentiation of hepatic progenitor cells into myofibroblasts constitutes the hepatocarcinogenesis-associated microenvironment[J]. Cell Death Differ, 2020, 27(1):85-101. doi: 10.1038/s41418-019-0340-7.
[18]	CHEN S N, TAN Y, XIAO X C, et al. Deletion of TLR4 attenuates lipopolysaccharide-induced acute liver injury by inhibiting inflammation and apoptosis[J]. Acta Pharmacol Sin, 2021, 42(10):1610-1619. doi: 10.1038/s41401-020-00597-x.
[19]	SUN J, ZHANG J, WANG X, et al. Gut-liver crosstalk in sepsis-induced liver injury[J]. Crit Care, 2020, 24(1):614. doi: 10.1186/s13054-020-03327-1.
[20]	KUDRU C U, ESHWARA V K, NAGIRI S K, et al. Spectrum of bacterial infections and predictors of mortality in adult cirrhotic patients[J]. Med Pharm Rep, 2019, 92(4):356-361. doi: 10.15386/mpr-1387.
[21]	CHENG Y J, YANG B C, LIU M Y. Lead increases lipopolysaccharide-induced liver-injury through tumor necrosis factor-alpha overexpression by monocytes/macrophages:role of protein kinase C and P42/44 mitogen-activated protein kinase[J]. Environ Health Perspect, 2006, 114(4):507-513. doi: 10.1289/ehp.8550.

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^＊＊

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^＊＊

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^＊＊

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^＊＊

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^＊＊