Salvia-Nelumbinis naturalis improves lipid metabolism of NAFLD by regulating the SIRT1/AMPK signaling pathway

Abstract

Background: Salvia-Nelumbinis naturalis (SNN), the extract of Chinese herbal medicine, has shown effects on NAFLD. This study aims to explore the underlying mechanism of SNN for regulating the lipid metabolism disorder in NAFLD based on the SIRT1/AMPK signaling pathway.

Methods: Male C57BL/6J mice fed with a high-fat diet (HFD) were used to establish the NAFLD model. Dynamic changes of mice including body weight, liver weight, serological biochemical indexes, liver histopathological changes, and protein level of AMPK and SIRT1 were monitored. After18 weeks, SNN treatment was administrated to the NAFLD mice for another 4 weeks. Besides the aforementioned indices, TC and TG of liver tissues were also measured. Western blot and quantitative RT-PCR were used to detect the expression and/or activation of SIRT1 and AMPK, as well as the molecules associated with lipid synthesis and β-oxidation. Furthermore, AML12 cells with lipid accumulation induced by fatty acids were treated with LZG and EX527 (SIRT1 inhibitor) or Compound C (AMPK inhibitor ) to confirm the potential pharmacological mechanism.

Results: Dynamic observation found the mice induced by HFD with gradually increased body and liver weight, elevated serum cholesterol, hepatic lipid accumulation, and liver injury. After 16 weeks, these indicators have shown obvious changes. Additionally, the hepatic level of SIRT1 and AMPK activation was identified gradually decreased with NAFLD progress. The mice with SNN administration had lower body weight, liver weight, and serum level of LDL-c and ALT than those of the NAFLD model. Hepatosteatosis and hepatic TG content in the liver tissues of the SNN group were significantly reduced. When compared with control mice, the NAFLD mice had significantly decreased hepatic expression of SIRT1, p-AMPK, p-ACC, ACOX1, and increased total Acetylated-lysine, SUV39H2, and SREBP-1c. The administration of SNN reversed the expression of these molecules. In vitro experiments showed the effect of SNN in ameliorating hepatosteatosis and regulating the expression of lipid metabolism-related genes in AML12 cells, which were diminished by EX527 or Compound C co-incubation.

Conclusions: Taken together, the SIRT1/AMPK signaling pathway, involved in hepatic lipid synthesis and degradation, plays a pivotal role in the pathogenesis of NAFLD development. The regulation of SIRT1/AMPK signaling greatly contributes to the underlying therapeutic mechanism of SNN for NAFLD.

Keywords: AMPK; Lipid metabolism; Nonalcoholic fatty liver disease; SIRT1; Salvia-Nelumbinis naturalis.

Fig. 1

Dynamic change of liver histopathology and lipid content of mice fed with HFD. A HE staining of liver tissues; CV indicates lobular central vein; yellow arrows indicate inflammatory cells, blue arrows indicate ballooning degeneration; the original magnification is 200×. B Histological assessment of NAFLD activity score; C The dynamic change of liver TC and TG content. n = 3; *P < 0.05, vs. Control

Fig. 2

Dynamic change of SIRT1 expression and AMPK activation in liver tissues of mice. A The hepatic SIRT1 protein and mRNA expression level. B The hepatic expression level of AMPK and P-AMPK. n = 3; *P < 0.05, **P < 0.01 vs. Control

Fig. 3

SNN improved HFD-induced mice NAFLD. A The body weight and liver weight. B The expression of serum ALT (C) serum TG, TC, and LDL-c. D HE staining and (E) Oil Red O staining of liver sections of mice; CV indicates lobular central vein; yellow arrows indicate inflammatory cells, blue arrows indicate ballooning degeneration; the original magnification is 200×. F Histological assessment of NAFLD activity score. G The content of liver TG and TC. n = 5-8; *P < 0.05, **P < 0.01, ***P < 0.001vs. Control; ^#P < 0.05, ^##P < 0.01 vs. Model

Fig. 4

Hepatic expression of SIRT1 and activation of AMPK of mice. A Hepatic level of SIRT1 and SUV39H2 expression. B The level of acetylated-Lysine in liver tissues was evaluated by Western blot analysis. C The expression and activation of AMPK of mouse liver tissues. β-actin was determined as the loading control. n = 3-6; *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control; ^#P < 0.05, ^##P < 0.01, ^###P < 0.01vs. Model

Fig. 5

SNN regulated the hepatic expression of molecules associated with the lipid metabolism in NAFLD mice. A The level of ACC activation in liver tissues. B The hepatic protein expression of SREBP-1c, FASN. C The expression of PPARα and ACOX1 in liver tissues. β-actin was determined as the loading control. n = 3-6; *P < 0.05, **P < 0.01 vs. Control; ^#P < 0.05 vs. Model

Fig. 6

Inhibition of SIRT1 prevented the improvement of SNN on FFA-induced steatosis in AML12 cells. A DAPI and Nile Red double staining showed the level of lipid accumulation in AML12 cells (200×). B The quantification of Nile Red staining. C The mRNA level of molecules associated with lipid metabolism including FASN and ACOX1. n = 3-6; *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control; ^#P < 0.05, ^###P < 0.01vs. Model

Fig. 7

Inhibition of AMPK attenuated the improvement of SNN on FFA-induced steatosis in AML12 cells. A DAPI and Nile Red double staining showed the level of lipid accumulation in AML12 cells (200×). B The quantification of Nile Red staining. C The mRNA level of FASN and ACOX1. n = 3-6; *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control; ^#P < 0.05, ^###P < 0.01vs. Model