Effect of glycogen synthase kinase 3beta-mediated autophagy in acute liver failure

doi:10.3877/cma.j.issn.1674-6880.2017.06.001

Abstract

Abstract:

Objective

To study the effect of the intracellular signaling molecule glycogen synthase kinase 3beta (GSK3β)-mediated autophagy in the pathogenesis of acute liver failure (ALF).

Methods

The primary hepatocytes from C57BL/6 mice were transfected with green fluorescent protein (GFP)-LC3 plasmid following 12 h starvation treatment by amino acid deficiency medium. The control group was treated with 0.2% dimethyl sulfoxide (DMSO); the experiment groups were treated with the different concentrations of SB216763 to inhibit GSK3β activity, following 12 h starvation treatment. The proportion of GFP-LC3 positive cell was counted. Moreover, the autophagy related protein (LC3 Ⅱ, p62, Atg5, Atg7 and Beclin 1) expression levels of mouse hepatocytes were detected at different periods of starvation treatment after given 10 μM SB216763. The mouse ALF model was induced by D-galactosamine (D-GalN) plus lipopolysaccharide (LPS), the autophagy-related mRNA and protein expression levels in mouse liver tissue were detected after given 25 mg/kg SB216763 to inhibit GSK3β activity.

Results

Compared with the DMSO + starvation group, the proportions of GFP-LC3 positive cell increased significantly in the two intervention groups treated with SB216763 (5 μM, 10 μM) [(11.6 ± 2.9) %, (44.0 ± 9.8) %, (59.2 ± 13.7)%; all P < 0.05]. Western-blotting results showed that compared with the DMSO + starvation groups of corresponding periods of starvation treatment (3 h, 6 h, 12 h), the LC3Ⅱ, Atg5, Atg7 and Becilin-1 protein expressions significantly increased, and p62 protein expressions significantly decreased in mouse hepatocytes in SB216763 + starvation groups (all P < 0.05). In the D-GalN/LPS induced mouse ALF model, the expressions of autophagy-related genes Atg5 [(0.626 ± 0.024) vs. (0.243 ± 0.029)], Atg7 [(1.70 ± 0.10) vs. (0.33 ± 0.08)], Beclin-1 [(1.24 ± 0.16) vs. (0.49 ± 0.04)], and proteins LC3Ⅱ[(0.173 ± 0.031) vs. (0.093 ± 0.009)], Atg5 [(1.53 ± 0.11) vs. (0.32 ± 0.05)], Atg7 [(0.92 ± 0.14) vs. (0.58 ± 0.12)], Becilin-1 [(0.263 ± 0.050) vs. (0.130 ± 0.022)] in liver tissue of the SB216763 + D-GalN/LPS group were all significantly higher than the D-GalN/LPS model group (all P < 0.05).

Conclusion

Inhibition of GSK3β activity may play a protective role in ALF by mediating autophagy.

Key words: Glycogen synthase kinase 3beta, Autophagy, Acute liver failure

Xiangying Zhang, Linlin Wei, Hongbo Shi, Sujun Zheng, Yu Chen, Dexi Chen, Xiuhui Li, Zhongping Duan, Feng Ren. Effect of glycogen synthase kinase 3beta-mediated autophagy in acute liver failure[J]. Chinese Journal of Critical Care Medicine(Electronic Edition), 2017, 10(06): 361-366.

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Figures/Tables 4

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组别	细胞计数（个/视野）	GFP-LC3阳性比例（%）
DMSO组	50	3.3 ± 1.7^a
DMSO +饥饿组	50	11.6 ± 2.9
SB216763（2 μM）+饥饿组	50	15.9 ± 1.7
SB216763（5 μM）+饥饿组	50	44.0 ± 9.8^a
SB216763（10 μM）+饥饿组	50	59.2 ± 13.7^a
F值	?	18.744
P值	?	< 0.001

组别	细胞计数（个/视野）	GFP-LC3阳性比例（%）
DMSO组	50	3.3 ± 1.7^a
DMSO +饥饿组	50	11.6 ± 2.9
SB216763（2 μM）+饥饿组	50	15.9 ± 1.7
SB216763（5 μM）+饥饿组	50	44.0 ± 9.8^a
SB216763（10 μM）+饥饿组	50	59.2 ± 13.7^a
F值	?	18.744
P值	?	< 0.001

组别	细胞数（个/孔）	LC3Ⅱ	p62	Atg5	Atg7	Beclin-1
DMSO组	1.5 × 10⁶	0.700 ± 0.081	0.173 ± 0.033	0.083 ± 0.021	0.007 ± 0.005	0.007 ± 0.005
DMSO +饥饿3 h组	1.5 × 10⁶	0.717 ± 0.057	0.920 ± 0.024	0.440 ± 0.062	0.087 ± 0.017	0.303 ± 0.017
DMSO +饥饿6 h组	1.5 × 10⁶	1.170 ± 0.037	1.017 ± 0.029	0.227 ± 0.033	0.130 ± 0.022	0.280 ± 0.024
DMSO +饥饿12 h组	1.5 × 10⁶	1.760 ± 0.051	0.430 ± 0.037	1.063 ± 0.062	0.313 ± 0.033	2.907 ± 0.039
SB216763（10 μM）组	1.5 × 10⁶	0.890 ± 0.054	0.210 ± 0.037	0.033 ± 0.017	0.005 ± 0.004	0.190 ± 0.016
SB216763（10 μM）+饥饿3 h组	1.5 × 10⁶	1.017 ± 0.029^a	0.267 ± 0.056^a	0.137 ± 0.037^a	2.547 ± 0.057^a	1.200 ± 0.115^a
SB216763（10 μM）+饥饿6 h组	1.5 × 10⁶	1.423 ± 0.069^a	0.220 ± 0.033^a	0.890 ± 0.033^a	2.727 ± 0.316^a	2.133 ± 0.128^a
SB216763（10 μM）+饥饿12 h组	1.5 × 10⁶	2.113 ± 0.102^a	0.187 ± 0.033^a	2.017 ± 0.074^a	2.040 ± 0.115^a	3.160 ± 0.051^a
F值	?	125.608	179.407	429.875	202.213	763.610
P值	?	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

组别	细胞数（个/孔）	LC3Ⅱ	p62	Atg5	Atg7	Beclin-1
DMSO组	1.5 × 10⁶	0.700 ± 0.081	0.173 ± 0.033	0.083 ± 0.021	0.007 ± 0.005	0.007 ± 0.005
DMSO +饥饿3 h组	1.5 × 10⁶	0.717 ± 0.057	0.920 ± 0.024	0.440 ± 0.062	0.087 ± 0.017	0.303 ± 0.017
DMSO +饥饿6 h组	1.5 × 10⁶	1.170 ± 0.037	1.017 ± 0.029	0.227 ± 0.033	0.130 ± 0.022	0.280 ± 0.024
DMSO +饥饿12 h组	1.5 × 10⁶	1.760 ± 0.051	0.430 ± 0.037	1.063 ± 0.062	0.313 ± 0.033	2.907 ± 0.039
SB216763（10 μM）组	1.5 × 10⁶	0.890 ± 0.054	0.210 ± 0.037	0.033 ± 0.017	0.005 ± 0.004	0.190 ± 0.016
SB216763（10 μM）+饥饿3 h组	1.5 × 10⁶	1.017 ± 0.029^a	0.267 ± 0.056^a	0.137 ± 0.037^a	2.547 ± 0.057^a	1.200 ± 0.115^a
SB216763（10 μM）+饥饿6 h组	1.5 × 10⁶	1.423 ± 0.069^a	0.220 ± 0.033^a	0.890 ± 0.033^a	2.727 ± 0.316^a	2.133 ± 0.128^a
SB216763（10 μM）+饥饿12 h组	1.5 × 10⁶	2.113 ± 0.102^a	0.187 ± 0.033^a	2.017 ± 0.074^a	2.040 ± 0.115^a	3.160 ± 0.051^a
F值	?	125.608	179.407	429.875	202.213	763.610
P值	?	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

组别	只数	Atg5	Atg7	Beclin-1
对照组	10	0.400 ± 0.035^a	0.64 ± 0.05^a	0.84 ± 0.04^a
D-GalN/LPS组	10	0.243 ± 0.029	0.33 ± 0.08	0.49 ± 0.04
SB216763 + D-GalN/LPS组	10	0.626 ± 0.024^a	1.70 ± 0.10^a	1.24 ± 0.16^a
F值	?	83.729	162.639	28.944
P值	?	< 0.001	< 0.001	< 0.001

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