Polyene Phosphatidylcholine Ameliorates High Fat Diet-Induced Non-alcoholic Fatty Liver Disease via Remodeling Metabolism and Inflammation

Abstract

Recent years have witnessed a rise in the morbidity of non-alcoholic fatty liver disease (NAFLD), in line with the global outbreak of obesity. However, effective intervention strategy against NAFLD is still unavailable. The present study sought to investigate the effect and mechanism of polyene phosphatidylcholine (PPC), a classic hepatoprotective drug, on NAFLD induced by high fat diet (HFD). We found that PPC intervention reduced the mass of liver, subcutaneous, epididymal, and brown fats in HFD mice. Furthermore, PPC supplementation significantly mitigated liver steatosis and improved glucose tolerance and insulin sensitivity in HFD mice, which was accompanied by declined levels of hepatic triglyceride, serum triglyceride, low density lipoprotein, aspartate aminotransferase, and alanine aminotransferase. Using transcriptome analysis, there were 1,789 differentially expressed genes (| fold change | ≥ 2, P < 0.05) including 893 upregulated genes and 896 downregulated genes in the HFD group compared to LC group. A total of 1,114 upregulated genes and 1,337 downregulated genes in HFD + PPC group were identified in comparison to HFD group. With the help of Gene Ontology (GO) analysis, these differentially expressed genes between HFD+PPC and HFD group were discovered related to "lipid metabolic process (GO: 0006629)," "lipid modification (GO: 0030258)," and "lipid homeostasis (GO: 0055088)". Though Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, we found pathways associated with hepatic homeostasis of metabolism and inflammation. Notably, the pathway "Non-alcoholic fatty liver disease (mmu04932)" (P-value = 0.00698) was authenticated in the study, which may inspire the potential mechanism of PPC to ameliorate NAFLD. The study also found that lipolysis, fatty acid oxidation, and lipid export associated genes were upregulated, while the genes in uptake of lipids and cholesterol synthesis were downregulated in the liver of HFD mice after PPC supplementation. Interestingly, PPC attenuated the metabolic inflammation via inhibiting pro-inflammatory macrophage in the livers of mice fed by HFD. In summary, this study demonstrates that PPC can ameliorate HFD-induced liver steatosis via reprogramming metabolic and inflammatory processes, which inspire clues for further clarifying the intervention mechanism of PPC against NAFLD.

Keywords: high fat diet; inflammation; liver steatosis; metabolic remodeling; non-alcoholic fatty liver disease; polyene phosphatidylcholine.

FIGURE 1

PPC supplementation improves metabolic parameters in the mice fed by HFD. All the mice were sacrificed at 13th week. (A–C) The mass of subcutaneous fat, epididymal fat, and brown fat. (D) The hepatic TG level. (E–I) The serum levels of TG, LDL, HDL, AST and ALT. (J) The body weight gain. n = 8 mice for each group. The differences were analyzed using one-way or two-way ANOVA. Data represent means with SEM. The pound signs indicate statistically significant differences compared to the LC group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001. Asterisks indicate statistically significant differences compared to the HFD group. *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 2

PPC supplementation improves insulin sensitivity and alleviates liver steatosis in the mice fed by HFD. (A) Glycemia changes in every 30 min. (B) AUC for GTT. (C) Fasting glycemia. (D) Blood insulin. (E) HOMA-IR. (F) The mass of liver. (G) Representative liver images and statistical analysis of H&E staining. (H) Representative liver images and statistical analysis of oil red staining. The scale bar is 50 μm. n = 8 mice for each group. The differences were analyzed using ANOVA. Data represent means with SEM. The pound signs indicate statistically significant differences compared to the LC group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001. Asterisks indicate statistically significant differences compared to the HFD group. *P < 0.05, **P < 0.01, ***P < 0.001. ns, no significance.

FIGURE 3

PPC supplementation remodels the transcriptome profile of liver in the mice fed by HFD. (A) The number of DEGs. (B) The volcano plot shows the distributions of DEGs between HFD + PPC and HFD mice. (C) The volcano plot of DEGs between HFD and LC. The x-axis indicates the fold change. red dots, upregulated; green dots, downregulated. (D) Go annotation of DEGs between HFD + PPC and HFD with terms related to hepatic metabolism and inflammation. n = 3 for each group.

FIGURE 4

PPC supplementation modulates KEGG pathways in HFD mice. The bubble chart shows the dysregulated terms in three KEGG levels between HFD + PPC and HFD mice (P < 0.05). The larger the point, the more genes fall into this pathway and the yellower point means the higher significance of enrichment.

FIGURE 5

PPC improves NAFLD by regulating hepatic metabolism and inflammation in HFD mice. The flow chart shows the Non-alcoholic fatty liver disease pathway (mmu04932) in KEGG. Genes identified as DEGs between HFD and HFD + PPC in our study are marked in red frames (upregulated) and green frames (downregulated), while others are marked in blank frames.

FIGURE 6

PPC supplementation reprograms the glucose and lipid metabolism in the liver of mice fed by HFD. (A) The expression of genes in lipid metabolism. (B) The expression of genes in glucose metabolism. n = 3 for each group. The differences were analyzed using two-tailed Student’s t-test. Data represent means with SEM. Asterisks indicate statistically significant differences compared to the HFD group. *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 7

PPC supplementation attenuated the metabolic inflammation via inhibiting pro-inflammatory macrophage polarization in the liver of mice fed by HFD. (A) The expression of genes related to pro-inflammatory macrophage, anti-inflammatory macrophage, and chemokines. n = 3 for each group. (B) Immunohistochemical analysis of F4/80 positive cells in liver. (C) Immunohistochemical analysis of CD206 positive cells in liver. (D) Immunohistochemical analysis of CD11c positive cells in liver. (E) Counting of positive cells via ImageJ. Statistical significance was determined using the ANOVA followed by the post hoc Tukey test for comparisons. Two-tailed Student’s t-test was used for comparison between two groups. Data represent means with SEM. The pound signs indicate statistically significant differences compared to the LC group. ^##P < 0.01, ^###P < 0.001. Asterisks indicate statistically significant differences compared to the HFD group. *P < 0.05, **P < 0.01, ***P < 0.001. The scaleplate of the representative images is 50 μm.

FIGURE 8

PPC supplementation enhances lipolysis and alleviates inflammation in the subcutaneous adipose tissues of mice fed by HFD. (A) Representative adipose tissue images of H&E staining. (B) The statistical results of subcutaneous adipocyte diameter. (C) The statistical results of subcutaneous adipocyte superficial area. (D) The expression of key enzymes in synthesis of fatty acids. (E) The expression of key enzymes related to fatty acid oxidation. (F) The expression of genes related to inflammation. The scaleplate of the representative images is 50 μm. n = 6 for each group. The differences were analyzed using ANOVA. Data represent means with SEM. The pound signs indicate statistically significant differences compared to the LC group.^#P < 0.05, ^##P < 0.01, ^###P < 0.001. Asterisks indicate statistically significant differences compared to the HFD group. *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 9

The overview of hepatic metabolic and inflammatory reprogramming in HFD mice after PPC intervention. HFD resulted in abnormal lipid accumulation and activated pro-inflammatory macrophage (red arrows), while PPC prevented the pathology of NAFLD via remodeling metabolism and inflammation (green arrows).