Cordycepin Ameliorates Nonalcoholic Steatohepatitis by Activation of the AMP-Activated Protein Kinase Signaling Pathway

Abstract

Background and aims: Nonalcoholic fatty liver disease, especially nonalcoholic steatohepatitis (NASH), has become a major cause of liver transplantation and liver-associated death. NASH is the hepatic manifestation of metabolic syndrome and is characterized by hepatic steatosis, inflammation, hepatocellular injury, and different degrees of fibrosis. However, there is no US Food and Drug Administration-approved medication to treat this devastating disease. Therapeutic activators of the AMP-activated protein kinase (AMPK) have been proposed as a potential treatment for metabolic diseases such as NASH. Cordycepin, a natural product isolated from the traditional Chinese medicine Cordyceps militaris, has recently emerged as a promising drug candidate for metabolic diseases.

Approach and results: We evaluated the effects of cordycepin on lipid storage in hepatocytes, inflammation, and fibrosis development in mice with NASH. Cordycepin attenuated lipid accumulation, inflammation, and lipotoxicity in hepatocytes subjected to metabolic stress. In addition, cordycepin treatment significantly and dose-dependently decreased the elevated levels of serum aminotransferases in mice with diet-induced NASH. Furthermore, cordycepin treatment significantly reduced hepatic triglyceride accumulation, inflammatory cell infiltration, and hepatic fibrosis in mice. In vitro and in vivo mechanistic studies revealed that a key mechanism linking the protective effects of cordycepin were AMPK phosphorylation-dependent, as indicated by the finding that treatment with the AMPK inhibitor Compound C abrogated cordycepin-induced hepatoprotection in hepatocytes and mice with NASH.

Conclusion: Cordycepin exerts significant protective effects against hepatic steatosis, inflammation, liver injury, and fibrosis in mice under metabolic stress through activation of the AMPK signaling pathway. Cordycepin might be an AMPK activator that can be used for the treatment of NASH.

FIG. 1

Effects of cordycepin on lipid accumulation and inflammation in hepatocytes. (A) Oil red O staining showing the degrees of lipid accumulation in L02 cells treated with DMSO, 50 μM or 100 μM cordycepin in response to BSA or PO (0.5 mM PA and 1.0 mM OA) stimulation for 12 hours. (B) TG and TC contents in L02 cells in the indicated groups stimulated with BSA or PO (0.5 mM PA and 1.0 mM OA) for 12 hours. n = 3 per group. The data are presented as the mean ± SD. Significant differences between the DMSO‐BSA group and the DMSO‐PO group: *P < 0.05, **P < 0.01; significant differences between the DMSO‐PO group and the cordycepin‐PO group: ^# P < 0.05, ^## P < 0.01. (C) Relative mRNA levels of the indicated lipid metabolism–related genes (Fasn, Scd1, Accα, Pparγ, and Pparα) and inflammatory factors (Il6, Tnfα, Ccl5, and Cxcl10) and in L02 cells treated with PA (0.5 mM). The relative mRNA expression was normalized to that of the β‐actin‐encoding gene Actb. n = 3 per group. The data are presented as the mean ± SD. Significant differences between the DMSO‐BSA group and the DMSO‐PA group: **P < 0.01; significant differences between the DMSO‐PA group and the cordycepin‐PA group: ^# P < 0.05, ^## P < 0.01. (D) PCA of RNA‐seq data for L02 cells treated with PA and cordycepin. (E,F) GSEA of pathways related to inflammation, lipid metabolism, and apoptosis. The font colors of the pathways related to inflammation and lipid metabolism are blue and yellow, respectively. n = 4 per group. (G) Heatmap of lipid metabolism–related, inflammation‐related, and apoptosis‐related gene expression profiles based on the RNA‐seq data set. Abbreviations: BSA, bovine serum albumin; Cordy, cordycepin; NES, normalized enrichment score; PC, principal component.

FIG. 2

HFD‐induced hepatic steatosis and inflammation are alleviated in mice treated with cordycepin. (A) LWs and (B) LW/BW ratios of the mice. n = 10 per group. (C) Hepatic lipid (TG and TC) levels. n = 10 per group. (D) Hepatic steatosis and lipid accumulation were measured by H&E, oil red O staining, and NAFLD activity score in the indicated groups. n = 6 per group. Scale bar, 100 µm. (E) Quantitative real‐time PCR analysis of the transcript levels of genes related to lipid metabolism (Hmgcr, Fasn, Scd1, Pparγ, Cd36, and Fabp1). Gene expression was normalized to Actb mRNA levels. n = 6 per group. (F) Western blotting of proteins involved in lipid metabolism in the livers of the mice. GAPDH served as a loading control. n = 3 per group. (G) Immunofluorescence staining of Cd11b (red) in the livers of HFD‐fed mice. Nuclei were labeled with DAPI (blue). n = 4 per group. Scale bar, 50 µm. (H) Quantitative real‐time PCR analysis of the transcript levels of genes related to inflammation (Il1b, Cxcl2, Cxcl10, Ccl2, and Ccl5). Gene expression was normalized to Actb mRNA levels. n = 6 per group. (I) Western blotting of proteins associated with inflammation: p‐IKKβ, IKKβ, IKBα, p‐p65, p65, and GAPDH. GAPDH served as a loading control. n = 3 per group. (J) Levels of serum ALT and AST were measured in mice after 24 weeks of normal chow or HFD feeding. n = 10 per group. The data are presented as the mean ± SD. Significant differences between the control‐normal chow group and the control‐HFD group for (A‐C) and (J): *P < 0.05, **P < 0.01; significant differences between the control‐HFD group and the cordycepin‐HFD group: ^# P < 0.05, ^## P < 0.01; significant differences between the control‐HFD group and the cordycepin‐HFD group for (D‐F): *P < 0.05, **P < 0.01. Abbreviations: Cordy, cordycepin; HPF, high‐power field; NC, normal chow.

FIG. 3

Cordycepin attenuated hepatic steatosis and injury in mice fed the HFHC diet. (A) LW and LW/BW ratios of the mice. n = 10 per group. (B) Hepatic lipid (TG and TC) levels. n = 10 per group. (C) Hepatic steatosis and lipid accumulation were measured by H&E, oil red O staining, and NAFLD activity score in the indicated groups. n = 6 per group. Scale bar, 100 µm. (D) Quantitative real‐time PCR analysis of the transcript levels of genes related to lipid metabolism (Hmgcr, Srebp1, Fasn, Scd1, Pparγ, Cd36, and Fabp1). Gene expression was normalized to Actb mRNA levels. n = 6 per group. (E) Western blotting of proteins involved in lipid metabolism (FASN and PPARγ). GAPDH served as a loading control. The data are presented as the mean ± SD. Significant differences between the control group and the cordycepin group: *P < 0.05, **P < 0.01. Abbreviation: Cordy, cordycepin.

FIG. 4

Cordycepin attenuated hepatic inflammation and fibrosis in mice fed the HFHC diet. (A) Relative mRNA levels of inflammatory genes (Tnfα, Il1b, Cxcl10, Ccl2, and Ccl5) in the livers of the indicated mice fed the HFHC diet for 16 weeks. n = 6 mice per group. (B) Immunofluorescence staining of Cd11b (red) in the livers of the indicated mice fed the HFHC diet for 16 weeks. Nuclei were labeled with DAPI (blue). n = 4 mice per group. Scale bar, 50 μm. (C) Immunoblot analyses of IKBα, GAPDH, and total and phosphorylated IKKβ and p65 protein in the liver tissues of vehicle‐treated or cordycepin‐treated mice fed the HFHC diet for 16 weeks (n = 3 mice per group). (D) Relative mRNA levels of profibrotic genes (Cola1, Col3a1, a‐Sma, Ctgf, Tgfb, and Timp1) in the livers of the indicated mice fed the HFHC diet for 16 weeks. The relative mRNA expression was normalized to that of Actb. n = 6 mice per group. (E) Representative images showing PSR staining in the livers of the indicated mice fed the HFHC diet for 16 weeks. n = 6 mice per group. Scale bar, 100 μm. (F) Immunoblot analyses of total and phosphorylated Smad2 and Smad3 proteins in the liver tissues of vehicle‐treated and cordycepin‐treated mice fed the HFHC diet for 16 weeks (n = 3 mice per group). (G) Serum ALT and AST levels of the mice in the indicated groups. n = 10 per group. The data are presented as the means ± SD. Significant differences between the control group and the cordycepin group: *P < 0.05, **P < 0.01. Abbreviations: Cordy, cordycepin; HPF, high‐power field; n.s., no significant difference between the control group and the cordycepin group (P > 0.05).

FIG. 5

RNA‐seq and proteomics analyses revealed key differential targets between the vehicle‐treated and cordycepin‐treated HFHC diet–fed mice. (A) PCA and unsupervised hierarchical clustering analysis of the RNA‐seq data from the mice fed the HFHC diet (n = 5 mice per group) for 16 weeks. (B,C) GSEA pathway enrichment analysis of pathways related to inflammation, lipid metabolism, apoptosis, and fibrosis. (D) Heatmap of lipid metabolism–related, fibrosis‐related, inflammation‐related, and apoptosis‐related gene expression profiles based on the RNA‐seq data set. n = 5 per group. (E) Protein levels in the livers of HFHC diet–fed mice treated with vehicle and cordycepin based on a proteomics assay. The font colors of the protein related to lipid metabolism and inflammation are yellow and blue, respectively. (F,G) Heatmap of representative differentially expressed proteins related to lipid metabolism and inflammation in the livers of HFHC diet–fed mice treated with vehicle and cordycepin based on a proteomics assay. Abbreviations: ECM, extracellular matrix; FA, fatty acid; MAPK, mitogen‐activated protein kinase; NES, normalized enrichment score; PC, principal component; Rig‐I, retinoic acid–inducible gene I.

FIG. 6

Association analysis of the transcriptomes and phosphoproteomics revealed that the AMPK pathway may be the target of cordycepin. (A) GSEA and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of the transcriptomes of cells (in purple) and HFHC diet–fed mouse liver samples (in green). (B) Venn diagram and the corresponding scores of the intersecting pathways based on the transcriptomic data from L02 cell and HFHC diet–fed mouse liver samples. (C) Core kinases in the AMPK pathway based on the available kinase‐substrate databases. (D) Western blotting of proteins involved in the AMPKα‐mediated signaling cascade in cells. (E,F) Western blotting of proteins involved in the AMPKα‐mediated signaling cascade in HFD‐fed and HFFC‐fed mice, respectively. GAPDH served as a loading control. The data are presented as the mean ± SD. Significant differences between the DMSO/control group and the cordycepin group: *P < 0.05, **P < 0.01. Abbreviations: Cordy, cordycepin; ERBB, erythroblastic leukemia viral oncogene homolog; FDR, false discovery rate; FOXO, forkhead box O; HIF‐1, hypoxia‐inducible factor 1; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK, mitogen‐activated protein kinase; MTOR, mammalian target of rapamycin; NES, normalized enrichment score; PI3K, phosphoinositide 3‐kinase.

FIG. 7

AMPK activation is responsible for the protective effects of cordycepin on lipid accumulation and inflammation in hepatocytes under metabolic stress. (A) Oil red O staining showing the degrees of lipid accumulation in DMSO‐treated and cordycepin (50 μM)–treated primary hepatocytes after PO (0.2 mM PA and 0.4 mM OA) stimulation for 12 hours in the presence or absence of CC. (B) TC and TG levels in primary hepatocytes in the indicated groups stimulated with PO (0.5 mM PA and 1.0 mM OA) for 12 hours. n = 3 per group. (C) Relative mRNA levels of the indicated lipid metabolism–related genes (Fasn, Cd36, Scd1, Pparγ, Ppara, and Cpt1a) and inflammatory factors (Il6, Tnfα, Il1b, and Ccl2) in primary hepatocytes treated with PA. The relative mRNA expression was normalized to that of Actb. n = 4 per group. (D) Western blot analysis of the total and phosphorylated protein levels of AMPKα and ACC after treatment with PA for 0 or 12 hours. GAPDH was the loading control for (D). n = 3 per group. The data are presented as the mean ± SD. Significant difference between the control‐DMSO group and the control‐cordycepin group for (B), ^## P < 0.01; n.s., no significant difference between the CC‐DMSO group and the CC‐cordycepin group for (B); significant difference between the control‐DMSO group and the control‐cordycepin group for (C): *P < 0.05, **P < 0.01; n.s., no significant difference between the CC‐DMSO group and the CC‐cordycepin group for (C). Abbreviation: Cordy, cordycepin.

FIG. 8

The AMPK pathway is required for cordycepin to improve HFD‐induced liver inflammation and steatosis in mice. (A) LWs and LW/BW ratios of the indicated groups. n = 10 per group. (B) Hepatic lipid levels in the indicated groups. n = 10 per group. (C) Representative H&E‐stained and oil red O–stained liver sections from the indicated HFD‐fed mice. n = 6 mice per group. Scale bar, 100 μm. (D,E) Quantitative real‐time PCR analysis of the expression of lipid‐related genes (Cd36, Fasn, Scd1, and Pparα) and inflammation‐related factors (Ccl2, Il1b, and Il6) in the indicated groups. The relative mRNA expression was normalized to that of Actb. n = 4 per group. (F) Serum ALT and AST concentrations in the indicated groups. n = 10 per group. (G) Western blot analysis of the total and phosphorylated protein levels of AMPKα and ACC in the indicated groups. n = 3 per group. GAPDH served as a loading control. The data are presented as the mean ± SD. Significant difference between the phosphate‐buffered saline‐control‐HFD group and the phosphate‐buffered saline‐cordycepin‐HFD group: *P < 0.05, **P < 0.01. Abbreviation: Cordy, cordycepin; n.s., no significant difference between the CC‐control‐HFD group and the CC‐cordycepin‐HFD group; PBS, phosphate‐buffered saline.