Research on the impact of in vivo liver transfection of carboxylesterase 1f gene on acute liver failure

doi:10.3969/j.issn.1674-8115.2025.11.007

Abstract

Abstract:

Objective ·To explore the effects of in vivo gene transfection of Carboxylesterase 1f (Ces1f) on mice with acute liver failure (ALF) induced by lipopolysaccharide (LPS) in combination with D - galactosamine (D - GalN), as well as its impact on the expression of hepatic endocannabinoid (EC) and their specific receptors, cannabinoid receptor 1 (Cb1r) and cannabinoid receptor 2 (Cb2r). Methods ·Twenty male C57BL/6J mice were randomly divided into four groups (n=5 per group): the blank control group, the Ces1f over expression group, the ALF model group (LPS/D-GalN), and the Ces1f over expression ALF model group. Recombinant adeno-associated virus 8 (rAAV8) containing the Ces1f sequence was injected via the tail vein, while the control group received an injection of the empty viral vector. Three weeks after virus injection, an ALF animal model was established by intraperitoneal injection of LPS+D-GalN. Immunofluorescence was used to observe the transfection efficiency of Ces1f in the mouse liver. Quantitative real-time polymerase chain reaction (qPCR) and Western blotting were employed to determine the expression levels of CES1F, CB1R, and CB2R in mouse liver tissue. Hematoxylin-eosin (H-E) staining was used to observe the pathological changes in the mouse liver tissue. The enzyme-linked immunosorbent assay (ELISA) was utilized to detect the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), arachidonoylethanolamide (AEA), and 2-arachidonoylglycerol (2-AG). Results ·The AAV8 virus had high transfection efficiency and significant hepatotropism in the mouse liver. Compared with the control group, the ALF model group showed decreased mRNA (P<0.001) and protein expression levels of Ces1f and Cb2r (P=0.039, P=0.012). The pathological injury score of liver tissue, the levels of serum ALT, AST, IL-1β, and TNF-α, as well as the levels of Il-1β mRNA, Tnf-α mRNA, AEA, 2-AG, Cb1r mRNA, and CB1R protein in liver tissue were significantly increased (P<0.001). Compared with the ALF model group, the Ces1f over expression ALF model group had significantly increased Ces1f mRNA (P<0.001) and CES1F protein (P=0.002) expression levels, decreased serum AST level (P=0.011), significantly decreased pathological injury scores of liver tissue (P=0.001), decreased levels of serum ALT, IL-1β, and TNF-α, and decreased levels of Il-1β mRNA, Tnf-α mRNA, AEA, and 2-AG in liver tissue (P<0.001). The levels of Cb1r mRNA (P=0.024) and CB1R protein (P=0.027) were also decreased. Conclusion ·Liver-targeted Ces1f gene transfection exerts a protective effect in the ALF mouse model induced by LPS/D-GalN. This protective effect may be achieved by downregulating the levels of hepatic endocannabinoids AEA and 2 - AG and inhibiting the expression of CB1R.

Key words: acute liver failure, carboxylesterase 1f, in vivo liver transfection, endocannabinoid system, mice

CLC Number:

R575.3

BIAN Shu, YU Qian, ZHANG Yuting, LI Dingjia, ZHANG Qiaoting, LIU Liangming. Research on the impact of in vivo liver transfection of carboxylesterase 1f gene on acute liver failure[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(11): 1480-1489.

Figures/Tables 8

Fig 1 Schematic diagram of the vector structure

Tab 1 Primer sequences for qPCR

Gene	Primer	Sequence (5′→3′)	Product/bp
Ces1f	F	TGGAGAGTCAGCAGGAGGTTACAG	290
	R	AGCACACTTGTCACGAACTGGTC	290
Cb1r	F	GCCTTGCAGATACCACCTT	103
	R	TTGAGCCCACGTAGAGGA	103
Cb2r	F	GAAAGCCCTCGTACCTGTT	121
	R	GGTCACACTGCCGATCTT	121
Il-1β	F	AGTTGACGGACCCCAAA	104
	R	TCTTGTTGATGTGCTGCTG	104
Tnf-a	F	CGCTGAGGTCAATCTGC	110
	R	GGCTGGGTAGAGAATGGA	110
Gapdh	F	GACATGCCGCCTGGAGAAAC	244
	R	GTCCACCACCCTGTTGCTGTAG	244

Fig 2 AAV8 transfection in mouse liver demonstrates liver tropism

Fig 3 Expression of Ces1f gene and protein in liver tissues of mice from each group

Fig 4 Serum transaminase levels and liver pathology with scoring in mice from each group

Fig 5 Changes in the levels of inflammatory factors TNF-α and IL-1β in each group of mice

Fig 6 Changes in the levels of AEA and 2-AG in the liver tissues of each group of mice

Fig 7 Expression of Cb1r and Cb2r genes and proteins in the liver tissues of each group of mice

TrendMD

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