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. 2020 Dec 14:2020:3183714.
doi: 10.1155/2020/3183714. eCollection 2020.

Gandouling Tablets Inhibit Excessive Mitophagy in Toxic Milk (TX) Model Mouse of Wilson Disease via Pink1/Parkin Pathway

Jing Zhang  1   2 , Lu-Lu Tang  2 , Liang-Yong Li  2 , Shen-Wei Cui  2 , Shan Jin  2 , Huai-Zhen Chen  2 , Wen-Ming Yang  2 , Dao-Jun Xie  2 , Gu-Ran Yu  1
Affiliations

Gandouling Tablets Inhibit Excessive Mitophagy in Toxic Milk (TX) Model Mouse of Wilson Disease via Pink1/Parkin Pathway

Jing Zhang et al. Evid Based Complement Alternat Med. .

Abstract

Objective: Gandouling (GDL) tablet is a Chinese patent medicine approved by the National Medical Product Administration, which is used to treat Wilson disease (WD) in China. In this study, we aimed to investigate the effects of GDL on mitophagy in the hippocampus in the toxic milk (TX) mouse model of WD.

Methods: Mice were randomly divided into the following four groups: control, Wilson (model group), D-penicillamine (DPA), and GDL groups. The animal behaviors were evaluated by the water maze experiment, traction test, and pole test. Transmission electron microscopy was used for the detection of mitochondrion structure. An enzyme-linked immunosorbent assay (ELISA) was performed for the analysis of the changes in liver function. Colocalization of mitophagy-related proteins was detected by fluorescence microscopy. Western blotting (WB) and reverse transcription-polymerase chain reaction (RT-PCR) were conducted for the detection of protein expression and mRNA levels, respectively.

Results: Significant reduction in neurological impairments was observed in the WD model group. All of these results were significantly reversed by GDL intervention. Compared with the levels in the Wilson group, the levels of alanine aminotransferase (ALT), aspartate transaminase (AST), total bilirubin (TBIL), and albumin (ALB) changed obviously. Colocalization between mitophagy-related proteins pink1, parkin, and mitochondria was changed significantly. The mitophagy-related mRNA (pink1, parkin, and LC3II) and protein expression levels (pink1, parkin, and the rate of LC3II/LC3I) were decreased significantly, while p62 was remarkably increased after GDL intervention.

Conclusion: Our findings indicated that the neuroprotective mechanism of GDL may occur via the inhibition of excessive mitophagy through the regulation of the pink1/parkin pathway in the TX mouse brain of WD.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
The effects of GDL on behavior in TX mouse model of Wilson disease. (a) The times of escape latencies in all mice. (b) The frequency of crossing the platform in all mice. (c) The scores of the traction test in all mice. (d) The times of pole test in all mice. Data are expressed as the mean ± SD (n = 12 per group) and were analyzed by one-way analysis of variance followed by the least significant difference test (Figure 1(a), 1(b), and 1(d)) and Tamhane's T2 test (Figure 1(c)). Control: control group; Wilson: Wilson group; DPA: D-penicillamine group; GDL: Gandouling group; ∗p < 0.01 versus the control group; △p < 0.01 versus the Wilson group.
Figure 2
Figure 2
Mitochondrial ultrastructural changes in the hippocampal tissue of mouse model of Wilson disease (2 μm, 500 nm). (a) The control group with normal mitochondrial ultrastructures. (e) Enlargement of the boxed area in (a) and abundant normal mitochondrial ultrastructure were observed. (b) Abundant mitochondria appeared to be dissolved, swollen, show vacuolization, and obvious mitochondrial autophagosomes were observed in the Wilson group. (f) Enlargement of the boxed area in (b). (c) Few mitochondrial vacuolizations were observed in the DPA group. (g) Enlargement of the boxed area in (c). (d) Lysosomes were observed in neurons and no obvious abnormalities were found in the GDL group, and few mitochondria appeared to have vacuolization. (h) Enlargement of the boxed area in (d) and many lysosomes were observed obviously.
Figure 3
Figure 3
The effects of GDL on the indicators, including ALT, AST, TBIL, and ALB, in TX mouse model of Wilson disease. Data are expressed as the mean ± SD (n = 12 per group) and were analyzed by one-way analysis of variance followed by the least significant difference test. (a) The indicators ALT and AST in all mice. (b) The indicators TBIL and ALB in all mice. Control: control group; Wilson: Wilson group; DPA: D-penicillamine group; GDL : Gandouling group; ∗p < 0.01 versus the control group; △p < 0.05, △△p < 0.01 versus the Wilson group.
Figure 4
Figure 4
The effects of GDL on parkin and p62 mitophagy-related immunofluorescence expression levels in TX mouse model of Wilson disease (500×, 20 μm). Data are expressed as the mean ± SD (n = 3 per group) and were analyzed by one-way analysis of variance followed by the least significant difference test. The blue color is DAPI staining, and the red is neurons. (a) Parkin-positive neurons. (b) p62-positive neurons. (c) Mean fluorescence intensity for parkin and p62. Control: control group; Wilson: Wilson group; DPA: D-penicillamine group; GDL: Gandouling group; DAPI: 4′,6-diamidino-2-phenylindole. ∗p < 0.01 versus the control group; △p < 0.05 versus the Wilson group.
Figure 5
Figure 5
The effects of GDL on pink1, parkin, p62, and LC3II mitophagy-related gene expression levels in TX mouse model of Wilson disease. Data are expressed as the mean ± SD (n = 3 per group) and were analyzed by one-way analysis of variance followed by the least significant difference test. Control: control group; Wilson: Wilson group; DPA: D-penicillamine group; GDL: Gandouling group; ∗p < 0.01 versus the control group, △p < 0.05, △△p < 0.01 versus the Wilson group.
Figure 6
Figure 6
The effects of GDL on pink1, parkin, p62, and LC3 mitophagy-related protein expression levels in TX mouse model of Wilson disease. (a) Western blot assay for pink1, parkin, p62, and LC3. (b) pink1, parkin, p62, and LC3 mitophagy-related protein expression levels with mitochondria. Data are expressed as the mean ± SD (n = 3 per group) and were analyzed by one-way analysis of variance followed by the least significant difference test. Control: control group; Wilson: Wilson group; DPA: D-penicillamine group; GDL : Gandouling group; ∗p < 0.05, ∗∗p < 0.01 versus the control group; △p < 0.05, △△p < 0.01 versus the Wilson group.
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