Calycosin-7-O-β-D-glucoside attenuates palmitate-induced lipid accumulation in hepatocytes through AMPK activation

https://doi.org/10.1016/j.ejphar.2022.174988Get rights and content

Abstract

Calycosin-7-O-β-D-glucoside (CG) is the major component of Astragali Radix (AR), a traditional Chinese drug. As reported, CG could attenuate cerebral ischemia/reperfusion injury, protect blood-brain barrier integrity, and ameliorate myocardial infarction. To date, whether CG has a protective effect on metabolic diseases remains to be elucidated. In the present study, CG could attenuate palmitate-induced lipid accumulation in hepatocytes in a dose-dependent manner, with down-regulation of lipogenesis related genes expression and up-regulation of lipids β-oxidation related genes expression. CG could decrease the triglyceride (TG) content from 0.30 mmol/g protein to 0.21 mmol/g protein and reduce the total cholesterol (TC) content from 0.39 mmol/g protein to 0.26 mmol/g protein. Moreover, CG stimulated the phosphorylation of AMP-activated protein kinase (AMPK), and the protective effect of CG on hepatocytes was partially reversed both by the inhibitor of AMPK signaling pathway and overexpression of AMPK-DN. Our findings revealed that CG could ameliorate palmitate-induced lipids accumulation in hepatocytes via AMPK activation and it may be a promising therapeutic medicine for hepatic steatosis.

Introduction

Metabolic associated fatty liver disease (MAFLD), characterized by excessive fat accumulation in hepatocytes, has become the most prevalent chronic liver disorder worldwide (Lim et al., 2021; Ye et al., 2020; Younossi, 2019). MAFLD is a progressive cascade of liver disorders, ranging from hepatic steatosis to non-alcoholic steatohepatitis (NASH), liver cirrhosis, and eventually hepatocellular carcinoma (HCC) (Cohen et al., 2011; Younossi et al., 2016). Moreover, MAFLD is closely implicated in the development of obesity, type 2 diabetes, hypertension, and coronary heart disease (Cohen et al., 2011; Kim et al., 2021; Semmler et al., 2021).

Liver is the central organ for energy metabolism, which is involved in a series of lipid metabolic processes including fatty acid uptake, de novo lipogenesis (DNL), and fatty acids redistribution to other tissues. Hepatic steatosis results from an imbalance of hepatic TG homeostasis, due to an increase in de novo lipogenesis (DNL) or reduction of fatty acid oxidation and very low-density lipoprotein (VLDL) secretion (Cai et al., 2019; Lambert et al., 2014). Till now, except diet control and exercise, there are few effective treatments for MAFLD. Therefore, pharmacological therapies for MAFLD are urgently required.

It is well established that AMP-activated protein kinase (AMPK) can act as a key energy sensor and widely participate in cellular energy metabolism in most tissues (Cantó et al., 2009; Hardie et al., 2006; Li et al., 2019a). As a heterotrimeric complex, AMPK can be allosterically activated by the increase of AMP/ATP ratio (Cantó et al., 2009). Once activated, AMPK will switch energy-consuming anabolism including lipogenesis and gluconeogenesis, to catabolic process on ATP production. AMPK activation can reduce the expression of Srebp-1c, the key transcription factor in DNL, and inactivate acetyl-CoA carboxylase (ACC), a rate-limiting enzyme in lipogenesis (Herzig and Shaw, 2018). Importantly, the activation of hepatocellular AMPK by metformin or berberine suppresses lipogenesis and lipid accumulation in hepatocytes, which indicates AMPK being a therapeutic target for MAFLD (Huang et al., 2018; Zhu et al., 2019).

Calycosin-7-O-β-D-glucoside (CG) is the major isoflavonoid extracted from Astragali Radix (AR), the dried root of Astragalus membranaceus. As a traditional Chinese herb Astragali Radix is widely used not only in Chinese tea and cooking (Su et al., 2021), but also in the treatment of bacterial infections, inflammation, diabetes, cardiovascular and cerebral vascular diseases, as well as hepatic diseases (Auyeung et al., 2016; Su et al., 2021; Zou et al., 2021). Previous studies have shown that CG could shrink cerebral infarct size and protect blood-brain barrier integrity in a rat model of cerebral ischemia–reperfusion (I/R) injury (Fu et al., 2014). Additionally, CG was reported to attenuate I/R and heat stoke induced myocardial injury (Liu et al., 2020a; Ren et al., 2016; Tsai et al., 2019). Moreover, CG protects against AGEs-induced endothelial cell apoptosis, and exerts a modulatory effect on gut microbiota (Ruan et al., 2015; Tang et al., 2011; Zhang et al., 2014). However, it remains unknown whether CG could alleviate lipid accumulation in hepatocytes. The present study was designed to explore the effect of CG on palmitate-induced lipid accumulation in hepatocytes and its underlying mechanism.

Section snippets

Materials

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), collagenase type.

IV, and other culture reagents were purchased from Gibco Life Technologies (Grand Island,NY). Bovine serum albumin (BSA), calycosin-7-O-β-D-glucoside (CG), compound C, and AICAR were obtained from Sigma (St Louis, MO, USA). Antibodies against ACC, p-ACC, AMPK, p-AMPK, GAPDH, and anti-rabbit IgG conjugated with horseradish peroxidase were purchased from Cell Signaling Technology (Beverly, MA, USA). Cell

CG ameliorates palmitate-induced lipid accumulation in hepatocytes

To determine the working concentration of CG, HepG2 cells were treated with 0.1, 1, 10, 100, and 1000 μM CG for 24 h and 48 h. At a concentration between 0.1 and 100 μm, CG did not affect HepG2 cells’ viability (Fig. 1B). Hence, hepatocytes were then treated with 10 nM CG for 24 h to detect the CG-mediated effect on palmitate-induced lipid accumulation.

Oil Red O staining was performed to detected hepatocellular lipid deposition. As shown in Fig. 2A, palmitate treatment significantly contributed

Discussion

Calycosin-7-O-β-D-glucoside (CG) is the major component of Astragali Radix (AR), a traditional Chinese drug, and it has many benefits in attenuating cerebral ischemia/reperfusion injury, protecting blood-brain barrier integrity, and ameliorating myocardial infarction (Fu et al., 2014; Liu et al., 2020a; Ren et al., 2016). However, it remains unknown whether CG can play a protective role in metabolic diseases. The present study demonstrated that CG could inhibit palmitate-induced lipid

Conclusions

In conclusion, our results firstly corroborated that CG could attenuate palmitate-induced lipid accumulation in hepatocytes by activating AMPK signaling pathway, which may provide a rationale for CG as a novel treatment option for hepatic steatosis.

CRediT authorship contribution statement

Wan Xu: Conceptualization, Methodology, Software. Feiye Zhou: Data curation, Writing – original draft. Qin Zhu: Visualization, Investigation. Mengyao Bai: Supervision. Ruyuan Deng: Software, Validation, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (grant no. 81800684).

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