Chlorogenic acid reduces body weight gain and affects TG, TC, LDL, ALT, and AST in the serum
Regular monitoring of the rat weights over the experimental period revealed the difference between the final and initial weights, employed as the metric of weight gain to assess the obesity levels in the rats (Figure 1A). Chlorogenic acid was found to mitigate weight gain in NASH-afflicted rats. Serum levels of TG, TC, LDL, ALT, and AST in each group are demonstrated in Figure 1B-F. HFD rats exhibited heightened serum TG and TC levels compared to the Nd group. However, the chlorogenic acid treatment reduced serum TG and TC concentrations in the Cha group compared to the HFD group. The chlorogenic acid intervention also resulted in lower serum LDL concentrations in rats than in the HFD group. The changes in the ALT and AST levels were congruent with the alterations observed in the LDL levels.
Chlorogenic acid improves liver histopathology and reduces lipid accumulation
Hepatic steatosis is one of the main features of NASH. The liver surface in Nd rats was consistently dense and exhibited a deep red hue. In contrast, the livers of HFD rats demonstrated a higher pore count, increased hypertrophy, and yellowish coloration. However, upon chlorogenic acid treatment, both the Cha and Piog groups exhibited a decrease in surface pores, and their livers appeared denser and redder (Figure 2A). Histological examination of H&E stained samples revealed that the hepatocytes of Nd rats were orderly, the hepatic lobules structure was lucid, and the cells presented an intact structure with the nucleus centralized and a homogeneous cytoplasm, devoid of steatosis, ballooning degeneration, or infiltration of inflammatory cells. In contrast, HFD hepatocytes exhibited severe steatosis and swelling, with an array of lipid vesicle sizes and nuclei displaced to one cell side. However, the Cha group exhibited a reduction in hepatocyte steatosis and ballooning degeneration compared to the HFD group. Oil red O staining showed abundant lipid droplets in the HFD liver cells, pointing towards an aberrant lipid metabolism induced by HFD in rat liver. Notably, chlorogenic acid intervention significantly reduced lipid accumulation in rat liver tissue (Figure 2B).
Chlorogenic acid improves liver lipid metabolism
Lipidomic Profiling Reveals Significant Alterations and the Efficacy of chlorogenic Acid Lipidomic analysis was performed on liver tissues from HFD, Nd, Cha, and Piog groups, and a total of 756 lipid metabolites were identified. These included 322 triacylglycerols (TAG), 164 Phosphatidylcholines (PC), 43 Phosphatidylethanolamines (PE), 36 Sphingomyelins (SM), 28 Diacylglycerols (DAG), 28 Ceramides (Cer/NS), 22 Diacylglyceryl trimethylhomoserines (DGTS), 19 Cholesteryl esters (CE), 16 Lysophosphatidylcholines (LPC), 4 Lysophosphatidylethanolamines (LPE), 14 Hexosylceramides (HexCer/NS), 10 Acylcarnitines (Acar), and others.
Notably, the lipid metabolism disruption in NASH rats was ameliorated by chlorogenic acid. A principal component analysis was performed to explore and elucidate group differences. As illustrated in Figure 3A-B, marked distinctions were detected between HFD, Nd, and Cha groups, suggesting that chlorogenic acid intake ameliorated lipid metabolism disruption, reverting it closer to a normal state. Furthermore, the Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) score plots exhibited clear separation between Nd and HFD groups as well as between HFD and Cha groups, implying that chlorogenic acid was effective in mitigating lipid metabolism disruption (Figure 3C-D).
The thermogram further demonstrated that the lipid metabolite patterns in the HFD group varied considerably from those in the Nd group. However, these patterns were partly restored upon chlorogenic acid supplementation. Volcano plot analysis was employed to screen potential lipid biomarkers explaining these differences. Significant disparities in lipid species were identified between Nd and HFD as well as between Cha and HFD using criteria of Fold Change (FC) ≥ 2 or ≤ 0.5 and Variable Importance in Projection (VIP) ≥ 1322 (Figure 4A-B).
In the volcano plots, each point signifies a metabolite, encompassing all metabolites measured in the experiment. The x-axis denotes the fold changes of each metabolite (log2 transformed), while the y-axis represents the P-value (log10 transformed) of the Student's t-test. The scatter size reflects the VIP value from the OPLS-DA model, with a larger scatter signifying higher VIP values. The scatter color reflects the final screening result, with red indicating significantly up-regulated lipids, blue indicating significantly downregulated lipids, and gray indicating lipids without significant differences.
Lipid histograms (Figure 4C-D) and lipid bubble diagrams (Figure 4E-F) display lipid variations. CE, Cer-NS, LPC, LPE, PC, PE, and SM were significantly downregulated, and DAG, DGTS, and TAG were markedly up-regulated. Compared to HFD, 11 lipids in the Cha group demonstrated significant alterations, where Acar, CE, Cer-NS, HexCer-NS, PC, PG, and SM were markedly up-regulated, and DAG, DGTS, and TAG were significantly downregulated. These findings suggest that chlorogenic acid can reverse the liver lipid metabolism disorder in NASH rats.
Each point represents a lipid type in the lipid group bubble diagram (Figure 4E-F). The size of the point represents the P-value of the Student's t-test (negative log10 transformed), with larger points indicating smaller P-values. Colored points represent lipids with significant differences (P < 0.05, marked with different colors based on lipid classification), while gray points represent lipids without significant differences (P ≥ 0.05). The x-axis of the lipid group bubble chart denotes the relative percentage change in each lipid's content, and the black line at the bottom denotes the distribution density of metabolites. The denser the black lines, the higher the number of lipids.
Potential molecular markers of chlorogenic acid improve lipid metabolism
In addition, lipid molecule expression analysis showed differences in potential lipid biomarker levels among the three groups. There were 7 kinds of TAG: TAG(18:2/20:5/22:6),TAG(18:0/19:0/20:6),TAG(18:2/18:3/22:6),TAG(17:2/18:2/18:2),TAG(15:0/17:2/19:0),TAG(14:0/18:2/20:5),TAG(18:2/18:2/20:4) There were 3 kinds of DGTS: DGTS(25:0/22:1),DGTS(19:1/26:2),DGTS(25:0/22:2), 4 kinds of PC PC(16:0/22:3),PC(16:1/26:4),PC(14:0e/22:4),PC(14:0e/22:3). These results suggest that the above 14 lipids may be potential biomarkers of chlorogenic acid in improving NASH rats.
Pathway Analysis
Pathway enrichment analysis was conducted to understand the functional roles of the different lipid metabolites. Figure 6 shows that compared with the Nd group, significant impairments were identified in regulating several metabolic pathways in the HFD rats. These included pathways of glycerolipid metabolism, cholesterol metabolism, lipid digestion and absorption, thermogenesis, and lipolysis.
The effect of chlorogenic acid on liver lipid metabolism-related genes
To further discern potential mechanisms through which chlorogenic acid might ameliorate abnormal lipid metabolism, we evaluated the transcription levels of genes implicated in lipid metabolism using qRT-PCR (Figure 7). Sterol regulatory element-binding protein 1c (SREBP-1c) is a key transcription factor involved in adipogenesis, and its activity can promote the accumulation of triglycerides in the liver. Stearoyl-CoA desaturase-1 (SCD1) is a crucial lipogenic enzyme that facilitates fat production within the liver. Compared to the Nd group, the expression levels of the SREBP-1c and SCD1 genes in the HFD group were significantly elevated (p<0.01). Following intervention with chlorogenic acid, the transcription levels of the SREBP-1c and SCD1 genes in the Cha group were significantly reduced compared to those in the HFD group (p<0.01).