Gandouling (肝豆灵) alleviates Wilson disease-associated hepatic fibrosis through inhibiting DNA methyltransferase 3 alpha/dynamin-related protein 1 mediated mitochondrial fission

doi:10.19852/j.cnki.jtcm.2026.05.008

Abstract

Abstract:

OBJECTIVE: To systematically investigate the molecular mechanism by which Gandouling (肝豆灵, GDL) ameliorates Wilson disease (WD)-associated hepatic fibrosis by modulating the dynamin-related protein 1 (Drp1)-mediated mitochondrial fission pathway, using network pharmacology, molecular docking, and experimental validation.

METHODS: A WD rat model was established using copper-overload induction. The experimental groups included a control group, model group, GDL intervention groups (low-, medium-, and high-dose), and a penicillamine (PCA) group. Masson's trichrome staining was performed to assess collagen deposition and hepatic fibrosis severity. Transmission electron microscopy (TEM) was used to evaluate pathological alterations in hepatocyte mitochondria. Enzyme-linked immunosorbent assay was conducted to measure liver function markers [aspartate aminotransferase (AST), alanine amino-transferase (ALT)] and fibrosis biomarkers [hyaluronic acid (HA), laminin (LN), procollagen type Ⅲ (PC-Ⅲ), collagen Ⅳ (C-Ⅳ)]. Western blot analysis was used to detect activation markers of hepatic stellate cells (HSCs) [α-smooth muscle actin (α-SMA), type Ⅰ collagen (collagen Ⅰ)] and to quantify DNA methyltransferase 3 alpha (DNMT3A), phosphorylated DNMT3A (p-DNMT3A), and Drp1 protein levels. Reverse transcription-quantitative polymerase chain reaction was performed to analyze the mRNA expression of DNMT3A and Drp1. Network pharmacology was applied to screen the active components of GDL and identify its core targets and pathways involved in regulating mitochondrial fission in WD-associated hepatic fibrosis. Molecular docking was subsequently conducted to validate the binding affinity between active components and core targets.

RESULTS: Compared with the control group, the model group exhibited significant collagen fiber deposition in liver tissue and mitochondrial structural damage (including cristae rupture, swelling, and reduced matrix density). Compared with the model group, all GDL-treated groups demonstrated significant reductions in serum levels of AST, ALT, HA, LN, PC-Ⅲ, and C-Ⅳ (P < 0.05), except ALT in the GDL-L group. Protein expression levels of α-SMA and collagen Ⅰ were significantly decreased (except in the GDL-L group) (P <0.05). mRNA expression of DNMT3A and Drp1 was significantly downregulated (P <0.05), accompanied by reduced protein levels of DNMT3A, p-DNMT3A, and Drp1 (except in the GDL-L group) (P < 0.05). Network pharmacology identified 103 major active components in GDL (e.g., sennoside D, luteolin, hederagenin), with potential mechanisms involving the advanced glycation end product -receptor for advanced glycation end products, mitogen-activated protein kinase, and phosphate-dylinositol-3-kinase-protein kinase B signaling pathways, targeting core nodes such as AKT serine-threonine kinase 1, albumin, and caspase 3. Molecular docking demonstrated strong binding affinity between active components and core targets.

CONCLUSION: GDL alleviates WD-associated hepatic fibrosis by modulating the DNMT3A/Drp1 signaling axis, thereby suppressing mitochondrial hyperfission and inhibiting hepatic stellate cell activation.

Key words: hepatolenticular degeneration, fibrosis, mitochondrial dynamics, DNA methyltransferase 3 alpha, dynamin I, Gandouling

XUAN Qiaoyu, HUA Daiping, SUN Lanting, GE Shufan, YANG Wenming, WANG Han. Gandouling (肝豆灵) alleviates Wilson disease-associated hepatic fibrosis through inhibiting DNA methyltransferase 3 alpha/dynamin-related protein 1 mediated mitochondrial fission[J]. Journal of Traditional Chinese Medicine, 2026, 46(5): 1139-1148.

Figures/Tables 9

Table 1 Top 5 active component chemical compositions of GDL

MOL ID	Degree	Molecule name	Molecular formula	CAS	Source
MOL002293	71	Sennoside D	C₄₂H₄₀O₁₉	37271-17-3	DH
MOL000006	66	Luteolin	C₁₅H₁₀O₆	491-70-3	DS, JXT
MOL000296	62	Hederagenin	C₃₀H₄₈O₄	465-99-6	EZ, JXT
MOL013352	56	Obacunone	C₂₆H₃₀O₇	751-03-1	HL
MOL000493	54	campesterol	C₂₈H₄₈O	474-62-4	JH, JXT

Figure 1 Masson staining for detection of fibrosis in liver tissue A: Control; B: Model; C: GDL-L; D: GDL-M; E: GDL-H; F: PCA (magnification × 200, scale bar = 50 μm). Control: equal-volume distilled water. Model: modeling plus intragastric administration of distilled water. GDL-L/M/H: 0.24, 0.48, 0.96 g/kg GDL, respectively. PCA: 90 mg/kg PCA. GDL: Gandouling; GDL-L: low-dose GDL group; GDL-M: medium-dose GDL group; GDL-H: high-dose GDL group; PCA: penicillamine.

Figure 2 GDL-associated effects on mitochondrial changes in WD model rats A: Control; B: Model; C: GDL-L; D: GDL-M; E: GDL-H; F: PCA (mitochondria are indicated by red arrows. × 25 000 magnification, scale bar: 500 nm). Control: equal-volume distilled water; Model: modeling plus intragastric administration of distilled water; GDL-L/M/H: 0.24, 0.48, 0.96 g/kg GDL, respectively; PCA: 90 mg/kg PCA. GDL: Gandouling; GDL-L: low-dose GDL group; GDL-M: medium-dose GDL group; GDL-H: high-dose GDL group; PCA: penicillamine.

Table 2 Comparisons of serum AST, ALT, HA, LN, PC-III, and C-IV in rats among different groups ($\stackrel{-}{\mathit{x}}\mathrm{ }$± s)

Groups	n	AST	ALT	HA	LN	PC-III	C-IV
Control	6	31.74±9.20	29.44±3.66	15.78±2.60	201.53±14.97	3129.93±155.76	5.57±0.33
Model	6	148.04±16.23^a	101.80±10.29^a	67.71±3.27^a	637.18±11.08^a	7336.30±193.59^a	19.38±0.51^a
GDL-L	6	115.36±10.07^b	95.07±3.34	60.47±0.58^b	559.79±2.96^b	6672.49±93.04^b	17.62±0.55^b
GDL-M	6	100.04±8.99^b	89.00±7.74^b	53.50±2.11^b	480.62±15.72^b	5297.46±179.90^b	14.02±0.63^b
GDL-H	6	59.20±4.02^b	59.35±3.21^b	31.88±2.00^b	413.65±24.58^b	4219.81±164.06^b	10.45±0.25^b
PCA	6	62.46±6.82^b	62.12±4.82^b	29.94±1.71^b	424.93±5.13^b	4352.91±132.90^b	11.28±0.76^b
F value		110.88	121.37	508.84	652.01	620.59	536.49
P value		< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

Figure 3 Western blotting analysis of α-SMA and collagen I protein expression 1: Control; 2: Model; 3: GDL-L; 4: GDL-M; 5: GDL-H; 6: PCA. Control: equal-volume distilled water; Model: modeling plus intragastric administration of distilled water; GDL-L/M/H: 0.24, 0.48, 0.96 g/kg GDL, respectively; PCA: 90 mg/kg PCA. GDL: Gandouling; GDL-L: low-dose GDL group; GDL-M: medium-dose GDL group; GDL-H: high-dose GDL group; PCA: penicillamine; α-SMA: α-smooth muscle actin; collagen Ⅰ: type Ⅰ collagen; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Table 3 Relative protein expression levels of α-SMA and collagen I in different groups ($\stackrel{-}{\mathit{x}}\mathrm{ }$± s)

Groups	n	α-SMA	collagen I
Control	3	0.140±0.022	0.212±0.049
Model	3	1.102±0.028^a	0.763±0.042^a
GDL-L	3	0.990±0.049	0.654±0.054
GDL-M	3	0.736±0.075^b	0.549±0.045^b
GDL-H	3	0.541±0.050^b	0.356±0.072^b
PCA	3	0.503±0.043^b	0.322±0.088^b
F value		162.401	36.928
P value		< 0.001	< 0.001

Table 4 Relative mRNA expression levels of Drp1 and DNMT3A in different groups ($\stackrel{-}{\mathit{x}}\mathrm{ }$± s)

Groups	n	Drp1	DNMT3A
Control	6	1.00±0.07	1.00±0.04
Model	6	2.88±0.14^a	1.62±0.04^a
GDL-L	6	1.87±0.04^b	1.43±0.03^b
GDL-M	6	1.50±0.03^b	1.34±0.04^b
GDL-H	6	1.33±0.08^b	1.16±0.05^b
PCA	6	1.30±0.07^b	1.16±0.05^b
F value		405.93	175.26
P value		< 0.001	< 0.001

Figure 4 Western blotting analysis of Drp1, DNMT3A and p-DNMT3A protein expression 1: Control; 2: Model; 3: GDL-L; 4: GDL-M; 5: GDL-H; 6: PCA. Control: equal-volume distilled water; Model: modeling plus intragastric administration of distilled water; GDL-L/M/H: 0.24, 0.48, 0.96 g/kg GDL, respectively; PCA: 90 mg/kg PCA. GDL: Gandouling; GDL-L: low-dose GDL group; GDL-M: medium-dose GDL group; GDL-H: high-dose GDL group; PCA: penicillamine; Drp1: dynamin-related protein 1; DNMT3A: DNA methyltransferase 3 alpha; p-DNMT3A: phosphorylated DNMT3A; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Table 5 Relative protein expression levels of Drp1, DNMT3A and p-DNMT3A in different groups ($\stackrel{-}{\mathit{x}}\mathrm{ }$± s)

Group	n	Drp1	DNMT3A	p-DNMT3A
Control	3	0.214±0.014	0.205±0.073	0.078±0.024
Model	3	0.810±0.012^a	0.850±0.064^a	0.823±0.025^a
GDL-L	3	0.699±0.042	0.745±0.051	0.723±0.050
GDL-M	3	0.581±0.059^b	0.613±0.081^b	0.631±0.037^b
GDL-H	3	0.476±0.070^b	0.470±0.092^b	0.416±0.107^b
PCA	3	0.411±0.027^b	0.463±0.079^b	0.459±0.058^b
F value		72.501	28.686	64.508
P value		< 0.001	< 0.001	< 0.001

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