In this study, the HLP model was induced by high-fat diet. The results of serum biochemical indexes showed that the serum TC, TG and LDL-C in the HLP group were significantly higher than those in the NC group, indicating the success of this experimental model. After the intervention of HM, it significantly improved the level of disordered serum lipids. Which is consistent with the previous results of a large number of studies on lipid regulation by HM in our team[25, 26]. The liver is the key organ of lipid metabolism. Studies have shown that a long-term high-fat diet leads to lipid deposition in the liver and leads to steatosis[27]. In this study, lipid deposition in liver tissue of HLP group was significantly higher than that of NC group after oil red O staining, and decreased significantly after HM intervention. These findings indicate that HM might alleviate lipid metabolism disorders and liver damage induced by a high-fat diet in rabbits, and has lipid-lowering effects, nevertheless, the underlying molecular mechanisms remain obscure.
The sources of lipids in human plasma encompass exogenous digestion and absorption by the small intestine, as well as endogenous lipid synthesis. Dysfunctions in the fat metabolism and degradation pathway may result in the development of HLP[28]. The purpose of plasma lipidomics research based on LC-MS is to identify potential biomarkers and differential metabolic pathways associated with HLP. This study selected four PCs, two SMs, and three LPCs belonging to glycerophospholipids as potential plasma biomarkers in hyperlipidemic rabbits treated with herbal cake moxibustion. They participated in nine lipid pathways related to glycerophospholipids metabolism, linoleic acid metabolism, alpha-linolenic acid metabolism, arachidonic acid metabolism, GPI-anchor biosynthesis, glycerolipid metabolism, fatty acid biosynthesis, fatty acid elongation, and fatty acid degradation. Glycerophospholipid metabolism was the main metabolic pathway.
Glycerophospholipid metabolism is not only one of the important mechanisms for the occurrence and development of HLP, but also one of the key targeted pathways for the treatment of HLP. Recently, it has been found that metabonomics and lipidomics methods were used to analyze the serum and liver tissue of HLP mice and the plasma of patients with HLP, and the results showed that the metabolic process of glycerophospholipid was related to the disorder of lipid metabolism in HLP[27, 29, 30], which is consistent with the results of our experiment. Glycerophospholipids, the most abundant phospholipids in the human body, not only serve as crucial components of cell membranes[31], actively participating in metabolism and cell signaling, but also function as active components of bile. These components play a significant role in hyperlipidemia by triggering inflammation and immune responses[32, 33]. PC, SM, and LPC are glycerophospholipids. In this study, the first 15 differential metabolites shown in the VIP diagram based on PLS-DA included seven PCs, five SMs, and three LPCs. Their concentrations were the lowest in the normal group, the highest in the model group, and between the two in the herbal cake-separated moxibustion group, consistent with heatmap results and indicating that HM could improve lipid metabolism disorder.
PC is closely related to the occurrence of HLP [34]. It plays an important role in the maintenance of cellular environment and cholesterol metabolism homeostasis, and plays the role of vascular "scavour", which can reduce the deposition of cholesterol and TAG in blood vessels, regulate lipid levels, and improve HLP, thus to reduce the risk of atherosclerosis[35, 36]. PC is one of the important signaling molecules involved in atherosclerosis, which can enhance the accumulation of very low-density lipoproteins in hepatocytes and promote the production of bile acid in the liver, thus promoting lipid output to regulate lipid metabolism[36]. It was found that the level of PC in plasma of obese mice with dyslipidemia induced by high-fat diet was significantly increased[37]. At the same time, the level of PC in the liver of male mice with non-alcoholic fatty liver induced by high-fat diet also changed significantly [38], and the lipid level returned to / or close to normal after intervention. This is consistent with the results of this study.
LPC is produced by the hydrolysis of oxidized phosphatidylcholine in low density lipoprotein by phospholipase A2. It is the main active component of oxidized low density lipoprotein and is related to the occurrence of atherosclerosis[39, 40]. Some LPC can also regulate the proliferation and apoptosis of vascular endothelial cells by activating peroxisome proliferator-activated receptor γ. Excessive LPC may also trigger inflammation and autoimmune response, affecting the development of HLP-related diseases[41]. Consistent with the results of this study, many previous studies have found that LPC levels in serum and plasma of HLP rats and obese mice are significantly increased, and LPC levels are significantly decreased after intervention[35, 37, 42, 43].
SM is an important part of cell membrane and plays an important role in all life processes. Blood levels of SM were found to be significantly elevated in both obese and hyperlipidemia patients, particularly those with saturated acyl chains[44]. These specific SM have been found to be positively correlated with insulin resistance, LDL-C, total cholesterol and TG[31, 45]. Notably, several studies have found significant increases in SM levels in both plasma and liver in animal models constructed from a high-fat diet or leptin deficiency[37, 46]. One study described that inhibition of SM can reduce plasma levels of TC and TG in mice, thereby preventing atherosclerosis[47]. In our study, the levels of TC and TG in serum decreased significantly after treatment with HM. Therefore, the anti-HLP effect of HM may be related to the decrease of SM, TC and TG levels.
Limitations
We found that HM can intervene HLP by improving the level of blood lipids in HLP and related abnormal metabolic pathways. However, our study still has some limitations and has not yet detected the level of lipids and metabolic pathways related to differential metabolites in the liver. In the future work, we will carry out more complete and in-depth research.