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. 2019 Jan 7;19(1):8.
doi: 10.1186/s12906-018-2424-1.

The traditional Chinese formulae Ling-gui-zhu-gan decoction alleviated non-alcoholic fatty liver disease via inhibiting PPP1R3C mediated molecules

Yanqi Dang  1 , Shijun Hao  2 , Wenjun Zhou  1 , Li Zhang  1 , Guang Ji  3
Affiliations

The traditional Chinese formulae Ling-gui-zhu-gan decoction alleviated non-alcoholic fatty liver disease via inhibiting PPP1R3C mediated molecules

Yanqi Dang et al. BMC Complement Altern Med. .

Abstract

Background: Ling-gui-zhu-gan decoction (LGZG), a classic traditional Chinese medicine formula, has been confirmed to be effective in improving steatosis in non-alcoholic fatty liver disease (NAFLD). However, the mechanism under the efficacy remains unclear. Hence, this study was designed to investigate the mechanisms of LGZG on alleviating steatosis.

Methods: Twenty four rats were randomly divided into three groups: normal group, NAFLD group, fed with high fat diet (HFD) and LGZG group (fed with HFD and supplemented with LGZG). After 4 weeks intervention, blood and liver were collected. Liver steatosis was detected by Oil Red O staining, and blood lipids were biochemically determined. Whole genome genes were detected by RNA-Seq and the significant different genes were verified by RT-qPCR. The protein expression of Protein phosphatase 1 regulatory subunit 3C (PPP1R3C) and key molecules of glycogen and lipid metabolism were measured by western blot. Chromophore substrate methods measured glycogen phosphorylase (GPa) activity and glycogen content.

Results: HFD can markedly induce hepatic steatosis and promote liver triglyceride (TG) and serum cholesterol (CHOL) contents, while liver TG and serum CHOL were both markedly decreased by LGZG treatment for 4 weeks. By RNA sequencing, we found that NAFLD rats showed significantly increase of PPP1R3C expression and LGZG reduced its expression. RT-qPCR and Western blot both verified the alteration of PPP1R3C upon LGZG intervention. LGZG also promoted the activity of glycogen phosphorylase liver type (PYGL) and inhibited the activity of glycogen synthase (GS) in NAFLD rats, resulting in glycogenolysis increase and glycogen synthesis decrease in the liver. By detecting glycogen content, we also found that LGZG reduced hepatic glycogen in NAFLD rats. In addition, we analyzed the key molecules in hepatic de novo lipogenesis and cholesterol synthesis, and indicated that LGZG markedly inhibited the activity of acetyl-CoA carboxylase (ACC), sterol receptor element-binding protein-1c (SREBP-1c) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), resulting in lipid synthesis decrease in the liver.

Conclusion: Our data highlighted the role of PPP1R3C targeting pathways, and found that hepatic glycogen metabolism might be the potential target of LGZG in preventing NAFLD.

Keywords: Glycogen metabolism; Ling-gui-zhu-Gan decoction; Lipogenesis; Non-alcoholic fatty liver disease; PPP1R3C.

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Conflict of interest statement

Ethics approval and consent to participate

The study protocol (PZSHUTCM18101801) was approved by the Institutional Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine and in accordance with the principles outlined in the NIH Guide for the Care and Use of Laboratory Animals.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
LGZG decoction alleviated hepatic steatosis and dyslipidemia in NAFLD rats. Liver sections were stained with oil Red O (a) (image magnification × 200), (b) liver TG content was detected (n = 4 per group), (c) Serum CHOL, (d) TG, (e) NEFA were analyzed (n = 8 per group). Data were presented as means ± SEM. *P < 0.05, ***P < 0.001
Fig. 2
Fig. 2
PPP1R3C was the target of LGZG decoction in NAFLD rats. RNA-Sequence identified (a) significantly different genes between NAFLD vs Normal group, (b) significantly different genes between LGZG vs NAFLD group. RT-qPCR further verified, (c) SLC38A2 mRNA expression, (d) ZFP189 mRNA expression, (e) OSGIN1 mRNA expression, and (f) PPP1R3C mRNA expression. Western blot detected (g) PTG protein expression. Data were presented as means ± SEM (n = 8 per group). *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 3
Fig. 3
LGZG regulated key molecules of Glycogen metabolism in NAFLD rats. Western blot detected (a) p-GSK3β and GSK3βprotein expression, (b) p-GS and GS protein expression, (c) PYGL protein expression, (d) GPa activity was analyzed by ELISA. Data were presented as means ± SEM (n = 8 per group). *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 4
Fig. 4
LGZG reduced hepatic glycogen in NAFLD rats. a The level of hepatic glycogen, (b) The level of muscle glycogen. Data were presented as means ± SEM (n = 8 per group). **P < 0.01, ***P < 0.001
Fig. 5
Fig. 5
LGZG inhibited hepatic de novo lipogenesis and cholesterol synthesis. a SREBP-1c protein expression in nucleus. b p-ACC and ACC protein expression. c HMGCR protein expression. Data were presented as means ± SEM (n = 8 per group). *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 6
Fig. 6
Summary of the study. Traditional Chinese formulae LGZG inhibited PPP1R3C expression. Low PPP1R3C expression could reduce GS activity, promote PYGL expression, and reduce glycogen storage via inhibiting glycogenesis and accelerating glycogenolysis. In addition, low PPP1R3C expression decreased the nucleic SREBP-1c and the ACC activity, which might induce suppressed lipogenesis in the liver. Thus by inhibiting PPP1R3C mediated molecules, LGZG alleviated NAFLD and related complications
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