Tuihuang Mixture improves α‑naphthylisothiocyanate-induced cholestasis in rats by inhibiting NLRP3 inflammasomes via regulating farnesoid X receptor

doi:10.12122/j.issn.1673-4254.2025.04.06

Abstract

Abstract:

Objective To study the therapeutic mechanism of Tuihuang Mixture against cholestasis. Methods Forty-eight Wistar rats were randomized equally into blank group, model group, ursodeoxycholic acid group and Tuihuang Mixture group. Except for those in the blank group, all the rats were given α‑naphthylisothiocyanate (ANIT) to establish rat models of cholestasis, followed by treatments with indicated drugs or distilled water. Serum levels of ALT, AST, ALP, γ-GT, TBA and TBIL of the rats were determined, and hepatic expressions IL-1β, IL-18, FXR, NLRP3, ASC, Caspase-1 and GSDMD were detected using q-PCR, ELISA or Western blotting. Histopathological changes of the liver tissues were observed using HE staining. Results The rat models of cholestasis had significantly increased serum levels of ALT, AST, ALP, γ-GT, TBA and TBIL with increased mRNA and protein expressions of IL-1β and IL-18, decreased protein and mRNA expressions of FXR, and increased protein expressions of NLRP3 and Caspase-1 and mRNA expressions of NLRP3, ASC, Caspase-1 and GSDMD in the liver tissue, showing also irregular arrangement of liver cells, proliferation of bile duct epithelial cells and inflammatory cells infiltration. Treatment of the rat models with Tuihuang Mixture significantly decreased serum levels of ALT, AST, ALP, γ-GT, TBA and TBIL, lowered IL-1β and IL-18 and increased FXR protein and mRNA expressions, and reduced NLRP3, ASC, Caspase-1 and GSDMD proteins and NLRP3, ASC and Caspase-1 mRNA expressions in the liver tissue. Tuihuang Mixture also significantly alleviated hepatocyte injury, bile duct epithelial cell proliferation and inflammatory cell infiltration in the liver of the rat models. Conclusion Tuihuang Mixture can effectively improve cholestasis in rats possibly by inhibiting NLRP3 inflammatosome-mediated pyroptosis via regulating FXR.

Key words: Tuihuang Mixture, cholestasis, farnesoid X receptor, NLRP3 inflammasomes, cell pyroptosis

Zhengwang ZHU, Linlin WANG, Jinghan ZHAO, Ruixue MA, Yuchun YU, Qingchun CAI, Bing WANG, Pingsheng ZHU, Mingsan MIAO. Tuihuang Mixture improves α‑naphthylisothiocyanate-induced cholestasis in rats by inhibiting NLRP3 inflammasomes via regulating farnesoid X receptor[J]. Journal of Southern Medical University, 2025, 45(4): 718-724.

Figures/Tables 7

References 34

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Gene	Primer sequence (5'-3')	Length (bp)
FXR	Forward: CACTGACACGCCCTTTTTGC	86
FXR	Reverse: TGGAGGATAAAACGAGGCGG	86
NLRP3	Forward: CTCACCTCACACTCCTGCTG	122
NLRP3	Reverse: AGAACCTCACAGAGCGTCAC	122
ASC	Forward: CTGCAGATGGACCCCATAGAC	74
ASC	Reverse: GTGAGCTCCAAGCCATACCC	74
Caspase-1	Forward: GACCGAGTGGTTCCCTCAAG	108
Caspase-1	Reverse: GACGTGTACGAGTGGGTGTT	108
GSDMD	Forward: CAGAACCAGTGTCTGGCAGT	86
GSDMD	Reverse: ACGTTGCATGATCTCCCAGG	86
IL-1β	Forward: AGGCTGACAGACCCCAAAAG	178
IL-1β	Reverse: CTCCACGGGCAAGACATAGG	178
IL-18	Forward: ACCACTTTGGCAGACTTCACT	126
IL-18	Reverse: CGTTGGCTGTTCGGTCGATA	126
β-actin	Forward: ATGGATGACGATATCGCTGC	150
β-actin	Reverse: CTTCTGACCCATACCCACCA	150

Gene	Primer sequence (5'-3')	Length (bp)
FXR	Forward: CACTGACACGCCCTTTTTGC	86
FXR	Reverse: TGGAGGATAAAACGAGGCGG	86
NLRP3	Forward: CTCACCTCACACTCCTGCTG	122
NLRP3	Reverse: AGAACCTCACAGAGCGTCAC	122
ASC	Forward: CTGCAGATGGACCCCATAGAC	74
ASC	Reverse: GTGAGCTCCAAGCCATACCC	74
Caspase-1	Forward: GACCGAGTGGTTCCCTCAAG	108
Caspase-1	Reverse: GACGTGTACGAGTGGGTGTT	108
GSDMD	Forward: CAGAACCAGTGTCTGGCAGT	86
GSDMD	Reverse: ACGTTGCATGATCTCCCAGG	86
IL-1β	Forward: AGGCTGACAGACCCCAAAAG	178
IL-1β	Reverse: CTCCACGGGCAAGACATAGG	178
IL-18	Forward: ACCACTTTGGCAGACTTCACT	126
IL-18	Reverse: CGTTGGCTGTTCGGTCGATA	126
β-actin	Forward: ATGGATGACGATATCGCTGC	150
β-actin	Reverse: CTTCTGACCCATACCCACCA	150

Group	Dose (g/kg)	ALT (U/L)	AST (U/L)	ALP (U/L)	γ-GT (U/L)
Blank	-	17.11±3.72	51.53±7.37	136.92±23.27	0.79±0.19
Model	-	309.03±61.60**	340.19±56.20**	428.38±80.54**	9.52±2.19**
UDCA	0.1	263.38±42.35^#	305.22±51.98	385.27±64.91	4.74±1.09^##
THM	17.25	177.98±31.14^##	219.90±45.40^##	359.38±53.84^#	3.93±1.08^##
P<0.05, *P<0.01 vs Blank group; ^#P<0.05, ^##P<0.01 vs Model group.

Group	Dose (g/kg)	ALT (U/L)	AST (U/L)	ALP (U/L)	γ-GT (U/L)
Blank	-	17.11±3.72	51.53±7.37	136.92±23.27	0.79±0.19
Model	-	309.03±61.60**	340.19±56.20**	428.38±80.54**	9.52±2.19**
UDCA	0.1	263.38±42.35^#	305.22±51.98	385.27±64.91	4.74±1.09^##
THM	17.25	177.98±31.14^##	219.90±45.40^##	359.38±53.84^#	3.93±1.08^##
P<0.05, *P<0.01 vs Blank group; ^#P<0.05, ^##P<0.01 vs Model group.

Group	Dose (g/kg)	TBA (μmol/L)	TBIL (μmol/L)
Blank	-	8.50±3.29	0.89±0.19
Model	-	106.28±29.19**	34.95±9.18**
UDCA	0.1	84.60±19.95^#	21.51±5.19^##
THM	17.25	57.62±14.74^##	17.79±4.39^##
P<0.05, *P<0.01 vs Blank group; ^#P<0.05, ^##P<0.01 vs Model group.