Lipidomic analysis to reveals therapeutic effects of herbal cake-separated moxibustion on high-fat diet-induced hyperlipidemia rabbits

Lipidomic analysis to reveals therapeutic effects of herbal cake-separated moxibustion on high-fat diet-induced hyperlipidemia rabbits | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Lipidomic analysis to reveals therapeutic effects of herbal cake-separated moxibustion on high-fat diet-induced hyperlipidemia rabbits Yuan Fang, Xinyu Chen, Huijuan Liu, Honghua Liu, Lizhi Ouyang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4740592/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Herbal cake-separated moxibustion (HM) is one of the characteristic therapies for the prevention and treatment of hyperlipidemia (HLP). However, the effect of HM on plasma lipid metabolism in HLP rabbits is not clear. Methods New Zealand rabbits were fed with high-fat diet for 8 weeks to induce HLP model, and then HM was intervened for 8 weeks. The level of blood lipid in serum of rabbits was detected by full biochemical analyzer, and the pathological changes of liver tissue were observed by oil red O staining. Then we used ultra-high performance liquid chromatography / quadrupole time-of-flight mass spectrometry combined with multivariate statistical analysis for non-targeted lipidomic analysis. Results HM ameliorated hyperlipidemia induced the abnormal blood lipid level and improved liver lipid deposition induced by high cholesterol diet. Non-targeted lipidomic analysis showed that HM changed the lipid metabolism profile of HLP rabbits. herbal cake-separated moxibustion hyperlipidemia lipidomic blood lipid glycerophospholipid Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Hyperlipidemia (HLP) is a disease characterized by abnormal levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol in the blood caused by lipid metabolic disorders[ 1 , 2 ]. Lipid metabolism disorder refers to a state in which one or more blood lipids are beyond the normal range caused by abnormal heredity, metabolism, or transport. HLP is one of the important risk factors for atherosclerotic cardiovascular disease[ 3 – 5 ]. According to statistics, about 17 million people in the world suffer from cardiovascular diseases related to HLP[ 6 ].Therefore, the effective prevention treatment of the disease is an urgent problem to be solved. At present, conventional drugs for HLP have a rapid effect, but long-term use may cause side effects (such as muscle pain, abnormal liver function, etc.), which limits its clinical application[ 7 – 9 ]. Acupuncture and moxibustion therapy has attracted the attention of researchers because of its wide adaptation, stable curative effect, two-way regulation, psychosomatic co-treatment, less side effects and so on. As a characteristic therapy, acupuncture and moxibustion has unique advantages in the prevention and treatment of HLP [ 10 – 12 ], especially moxibustion with herbal–cake-separated moxibustion (HM), which combines traditional moxibustion therapy with acupoints and traditional Chinese medicine (TCM), plays the role of multi-target and multi-channel system and regulatory effects, and has precise curative effect, simple operation and low price in treating hyperlipidemia [ 13 ]. Previous studies have shown that HM could alleviate HLP by affecting IGF-1/Sp1 signal pathway and regulating cholesterol reverse transport, LDL-C uptake and transport. And it is possible to regulate the levels of homocysteine and insulin-like growth factor-1, regulate hemorheology and repair endothelial function to improve hyperlipidemia. And it may be able to prevent and cure HLP by regulating the structure of microflora and reducing inflammation[ 14 – 16 ]. At present, the lipid-regulating effect of HM has not been reported by liposome. The study of lipid metabolism is of great significance to clarify the mechanism of HM in regulating blood lipids. Lipoomics is a subject that systematically analyzes various lipids and their interactions in cells and tissues, and elucidates their functions and changes under different physiological conditions[ 17 , 18 ]. It has been widely used to study metabolic diseases, such as HLP, diabetes and hyperuricemia[ 18 – 20 ]. Liposome is consistent with the characteristics of multi-target and multi-pathway action of herbal cake-separated moxibustion, which is a powerful tool to study the effect of HM on regulating blood lipid. Ultra-high performance liquid chromatography-high resolution mass spectrometry provides the possibility for high-throughput analysis of lipids and promotes the process of lipid research[ 18 ]. In the study, we mainly studied the effect of HM on diet-induced hyperlipidemia. HM ameliorates abnormal lipid metabolism by altering the pathway of glycerophospholipids metabolism, and improves high-fat diet-related dyslipidemia and liver injury. HM is expected to become a promising lipid-lowering therapy, which opens up a new way for the study of its treatment of metabolic diseases. Materials and methods Animals and ethical statement A total of 36 healthy male New Zealand rabbits, weighing 2.0 ~ 2.5kg, were purchased from Hunan Taiping Biological Co., Ltd. [ordinary class, License No: SCXK (Hunan) 2020-0005]. The animals were raised in a single cage in the Animal Center Laboratory of Hunan University of Chinese Medicine, where they were fed rationally and drank freely. It is raised in an environment with a relative humidity of 50%-70%, a temperature of 20°C-25°C and a day-night cycle of 12 hours. The whole experiment scheme has been approved by Hunan University of Chinese Medicine (License No.: LLBH-202005220001) and implemented in accordance with Animal Research: in vivo experiment report (REACH) Guide[ 21 ]. Experimental protocol After one week of adaptive feeding, all rabbits were randomly divided into three groups(n = 12): control group (NC group), HLP model group (HLP group) and herbal cake separated moxibustion group (HM group). The control group was fed with normal diet, and the rest of the rabbits were fed with high-fat diet to establish HLP model. High-fat diet contains 1% cholesterol, 10% egg yolk powder, 5% lard, 84% basic feed, propylthiouracil (10mg/ kg d). The total daily food intake of each rabbit was about 120g, drinking water was free, and it was fed continuously for 8 weeks. The rabbits in the HM group received the intervention of moxibustion separated by medicinal cake, while the rabbits in the control group and HLP group did not do any intervention except for binding and fixation. Model evaluation: the normal serum TC content of New Zealand rabbits was 1 ~ 2mmol/L. When the serum TC value of each model group was more than 3 times higher than this value (or TC value of the control group), and the serum TG and LDL-C contents of the model group were higher than those of the control group, the HLP model was judged to be valid. Intervention methods Based on the previous research basis[ 13 ], acupoints were selected and divided into two groups and moxibustion was applied alternately. Group Ⅰ: bilateral Tianshu (ST25), Fenglong (ST40), Juque (RN14), 5 acupoints, group Ⅱ: bilateral Xinshu (BL15), Ganshu (BL18), Pishu (BL20), 6 acupoints. The location of acupoints is formulated with reference to Experimental Acupuncture and moxibustion (2nd edition, 2016) edited by Yu Shuguang and Xu Bin and anthropomorphic comparison. The same amount of Hawthorn, salvia miltiorrhiza, rhubarb, turmeric and alisma were broken into foam, mixed with vinegar, and then pressed into a medicinal cake with a diameter of about 1cm and a thickness of about 0.3cm. Fix the rabbit, shave the acupoint area, apply appropriate amount of Vaseline on the acupoint, place the medicine cake, moxa, and light it. Burn out and change moxa sticks, 4 sticks per point, once a day, two groups of acupuncture points alternate moxibustion, 1 day a week off, consecutive 8 weeks. Sample collection After the intervention, the rabbits were anesthetized with 2% pentobarbital sodium solution, laparotomy was performed, and 4ml of abdominal aorta blood was taken from ordinary tubes and EDTA tubes respectively, and the supernatant was obtained by centrifugation at 3000rpm for 10min, and stored in a refrigerator at -80℃ for detection. At the same time, about 500mg of fresh liver group was taken into the frozen storage tube and put into the refrigerator at -80℃ for detection. Biochemical analysis The Olympus AU2700 automatic biochemical analyzer was used to detect the contents of TC, TG and LDL-C in serum according to the instructions of the kit. Oil red O staining Frozen slices were dried for 30 min, fixed with 4% neutral formaldehyde at 4 ℃ for 10 min, fully washed with distilled water and washed with 60% isopropanol. Oil red O dye solution shielded from light for 10 min, 60% isopropanol differentiated into interstitial cleaning; distilled water washing; hematoxylin staining for 3 min, distilled water washing for 3 min, glycerol gelatin sealing tablets, microscopic observation and photography. Plasma lipidomics detection Plasma sample preparation Thaw the sample at 4 ℃, vortex for 30 s100µL of blank rabbit plasma was taken, and chloroform: methanol (2:1) was added, followed by 10µL of the internal standard solution 1 to 10 Lipid Mix, 5uL1mg/mL C17:0, vortex for 1min. and 10µL extraction at -20 ℃.Then, samples were centrifuged at 4 ℃/13000 rpm for 5 min, 300µL of the lower solution was taken, and dried with nitrogen. The mixture was resolubilized with 20µL chloroform: methanol (1:1), then diluted three times with isopropanol: acetonitrile: water (2:1:1) (60µL; vortexed for 1 min). Finally, samples were centrifuged at 4 ℃/13000 rpm for 5 min, and all supernatants were taken and detected. For quality control (QC), the same volume from the prepared samples was mixed into large samples and evenly divided into QC samples to monitor the precision and stability of the instrument. The first three QC samples were used to monitor the precision, and one QC sample was collected at intervals of six samples to monitor the stability. Lipid profiling Liquid chromatography conditions Chromatographic separation was accomplished in an Ultimate 3000 system with an ACQUITY UPLC® BEH C18 (150 × 2.1 mm, 1.8 µm; Waters). The flow rate was 0.3 mL/min, and the column was maintained at 35 ℃. The gradient elution of analytes was carried out with acetonitrile: water (3:2) (0.1% formic acid + 10 mM ammonium formate) (A) and isopropanol: acetonitrile (9:1) (0.1% formic acid + 10 mM ammonium formate) (B). Two µL of each sample was injected after equilibration. The gradient elution procedure was as follows: 0–2 min, 60 − 57% A; 2-2.1 min, 57 − 50% A; 2.1–12 min, 50 − 40% A; 12-12.1 min, 40 − 25% A; 12.1–18 min, 25 − 1% A, 19–20 min, 1–60% A; 20–25 min, 60% A[ 22 ]. Mass spectrum conditions High-resolution MS QE Xactive (Thermo Fisher Scientific, USA) collected positive and negative ion modes. In the positive ion mode, the electrospray voltage was 3.3 kV; in the negative ion mode, the electrospray voltage was 2.8 kV. The capillary temperature was 320°C, the sheath temperature was 40 Arb, and the atomizer temperature was 350°C. A full scan was performed with a mass resolution of 35 000 and a scanning m/z range of 150–2000. HCD is used for two-stage cracking, and the collision voltage is 30 eV. At the same time, dynamic elimination is used to remove unnecessary MS/MS information[ 23 ]. The mass spectrometry data were denoised and normalized, and then imported into SIMCA-P software for multivariate statistical analysis. According to principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) models, the potential biomarkers with variable projection importance (VIP) > 1.0 and t test P < 0.01were screened. Then, KEGG pathway enrichment analysis was performed on the list of different substances by MetaboAnalyst [ 24 ]. Statistical analysis The experimental data were statistically analyzed by SPSS25.0, all the data were expressed by 𝑥̅±s, and plotted and analyzed by Graphad Prisim5. One-way ANOVA method was used to compare among groups, LSD was used to analyze the variance when the variance was uniform, and Dunnet T3 was used to analyze the difference. Results General situation of experimental rabbits In the normal group, the rabbit had a moist coat, good spirit, and rapid response to stimulation. The rabbits in the model group showed varying state changes, mainly characterized by low spirits, slow response, and rapid body weight growth. The condition of the rabbits in the herbal cake-separated moxibustion group was improved and the body weight was reduced. HM reduces the content of lipid in serum of HLP rabbits After eight weeks of intervention, serum TC, LDL-C, and TG contents were significantly higher in the HLP group than in the NC group ( P < 0.001), suggesting the rabbit model was successfully established. Serum TC, TG, and LDL-C contents decreased to different degrees in the HM group compared to the HLP group ( P < 0.001, Fig. 1). These results showed that the herbal cake-separated moxibustion intervention could improve the dyslipidemia of hyperlipidemic rabbits. HM reduces liver Lipid deposition in HLP Rabbits Hematoxylin dyes the nucleus blue and lipid droplets orange. No histological abnormality was found in the liver tissue of the NC group, while the accumulation of lipid droplets was obvious in the HLP group, and the lipid deposition increased significantly. After the HM intervention, lipid droplets were significantly reduced and effectively improved the pathological state of the liver (Fig. 2). Screening and identification of differential lipid metabolites The total ion flow maps of all plasma QC samples are superimposed, and the spectra overlap well, and the retention time and peak signal intensity fluctuate little, indicating that the instrument is in good condition in the whole sample analysis process, which is suitable for the detection and analysis of this project (Fig. 3a and b). The metabolites of NC group, HLP group and HM group were significantly separated by principal component analysis and partial least square-discriminant analysis, indicating that there were differences in plasma lipid metabolism profile among each group, and the metabolic profile of HM group was closer to that of NC group (Fig. 4a and 4b). The cross-validation result of the PLS-DA model is shown in Fig. 4c. The Abscissa represents the number of components selected by the model, and the ordinate is the value of R 2 , Q 2 and Accuracy. R 2 = 0.928, Q 2 = 0.881, R 2 is the accuracy of model establishment, Q 2 is the accuracy of model prediction, R 2 and Q 2 are all greater than 0.5, indicating that the established model has a good explanation and prediction for the distinction among NC group, HLP group and HM group. Figure 4d is the VIP (Variable Importance in Projection) diagram of PLS-DA, which shows the importance of variables and their contribution to sample differentiation. The ordinate represents the first 15 differential metabolites (Table 1 ), the Abscissa is the VIP value, and the right color coding reflects the concentration of the metabolite in different groups. Heat plots can further visualize the differential lipid metabolites. Figure 4e shows the changes of differential lipid metabolites (Table 1 ). The concentration of differential lipid metabolites in NC group is the lowest, followed by HM group, and the highest in HLP group, which is consistent with the results of VIP map. It is suggested that HM can reverse the disorder of lipid metabolism in plasma of HLP rabbits. Table 1 List of differential lipid metabolites Metabolite m/z(Da) t R /min VIP P Chemical formula PC 28:0 678.503 7.654 1.914 5.37×10 − 9 C 36 H 72 NO 8 P SM 52:13;2O 931.682 7.593 1.872 2.44×10 − 12 C 57 H 91 N 2 O 6 P LPC 25:0/0:0 662.479 7.656 1.868 7.52×10 − 11 C 33 H 68 NO 7 P PC 36:6 778.532 7.169 1.808 1.86×10 − 10 C 44 H 76 NO 8 P SM 56:13;2O 1003.706 2.591 1.802 9.73×10 − 11 C 61 H 99 N 2 O 7 P SM 32:0;2O 693.550 7.529 1.773 7.08×10 − 9 C 37 H 77 N 2 O 7 P PC 37:6 792.548 7.870 1.759 1.36×10 − 12 C 45 H 78 NO 8 P LPC O-24:0 594.481 7.861 1.758 6.27×10 − 9 C 32 H 68 NO 6 P SM 36:3;2O 727.569 7.871 1.686 1.65×10 − 11 C 41 H 79 N 2 O 6 P PC 42:4 866.658 14.772 1.676 1.57×10 − 9 C 50 H 92 NO 8 P LPC O-24:1 592.469 7.137 1.676 3.75×10 − 7 C 32 H 66 NO 6 P SM 33:2;2OSM 1712O 687.540 7.522 1.673 1.69×10 − 7 C 38 H 75 N 2 O 6 P PC 32:3 728.519 7.205 1.667 2.14×10 − 7 C 40 H 74 NO 8 P PC 42:10 854.566 7.146 1.657 1.81×10 − 13 C 50 H 80 NO 8 P PC 34:4 754.532 7.657 1.626 5.85×10 − 11 C 42 H 76 NO 8 P The VIP value was obtained from PLS-DA with a threshold of 1.0. The p-values were calculated from the ANOVA In order to further study the potential biomarkers of HM anti-HLP, ROC curve analysis based on PLS-DA was carried out. As shown in the figure, the AUC values of 12 differential lipid metabolites were all greater than 0.85. among them, SM(52: 13;2O)、LPC(25༚0/0༚0)、PC(36༚6)、PC(37༚6)、LPC(O-24༚0)、SM(36༚3༛2O)、PC(42༚4)、LPC(O-24༚1) and PC(42༚10) had high specificity (> 85%) and sensitivity (> 85%) (Fig. 5 ). The results suggest that nine lipid components such as SM(52༚13༛2O) play a key role in improving the disorder of plasma lipid metabolism in HM rabbits. These differential lipid metabolites include 4 phosphatidylcholine (PC), 2 sphingomyelin (SM) and 3 lysophosphatidylcholine (LPCs), suggesting that PC, SM and LPC are potential biomarkers of the hypolipidemic effect of HM. Metabolic pathway analysis In order to further study the effect of lipid metabolites on HLP, the pathway analysis of differential lipid metabolites in rabbit plasma was carried out. The results showed that nine pathways were involved (Fig. 6 ), including glycerophospholipid metabolism (Fig. 7 ), linoleic acid metabolism, alpha-linolenic acid metabolism, arachidonic acid metabolism, GPI-anchor biosynthesis, glycerolipid metabolism, fatty acid biosynthesis, fatty acid prolongation, fatty acid degradation (Fig. 6 , Table 2 ). According to its impact, p -value, and the value of hits (≥ 3), it is determined that glycerophospholipid metabolism ( P = 3.10×10 9 ) plays a key role in the occurrence and development of HLP. Table 2 Effect of HM on Plasma Lipid Metabolism Pathway in HLP Rabbits Pathway Name Total/ Hits Raw p log( p ) Holm p FDR Impact Glycerophospholipid metabolism 36/3 3.10E-09 19.593 2.79E-08 1.97E-08 0.264 Linoleic acid metabolism 5/2 5.89E-09 18.951 4.71E-08 1.97E-08 0.968 alpha-Linolenic acid metabolism 13/1 6.56E-09 18.842 4.71E-08 1.97E-08 0 Arachidonic acid metabolism 36/2 3.39E-08 17.2 2.03E-07 7.62E-08 0.281 GPI-anchor biosynthesis 15/1 6.34E-08 16.573 3.17E-07 1.14E-07 0 Glycerolipid metabolism 16/1 6.42E-03 5.049 2.57E-02 9.62E-03 0.026 Fatty acid biosynthesis 46/1 5.31E-01 0.633 1 5.31E-01 0 Fatty acid elongation 38/1 5.31E-01 0.633 1 5.31E-01 0 Fatty acid degradation 39/1 5.31E-01 0.633 1 5.31E-01 0 “Total”: the number of all metabolites in the metabolic pathway. “Hits”: the number of differentiated metabolites selected in the metabolic pathway. “Raw p ”: the original p -value of the enrichment analysis. “Holm p ” adjusted p- value by Holm–Bonferroni method. FDR: the FDR value in multiplex checking. Impact: the influence value calculated by path topology analysis. Discussion 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. Conclusions In summary, serum TC, TG, and LCL-C levels were significantly increased in hyperlipidemic rabbits induced by a high-fat diet, and the lipid deposition in liver tissue was significantly increased after oil red O staining related to the significant changes in plasma concentrations of PC, SM and LPC. These changes are mainly reflected by disorders in glycerophospholipid metabolism, linoleic acid metabolism, α-linolenic acid metabolism, arachidonic acid metabolism, GPI anchor biosynthesis, and glycerol ester metabolism in diet-induced hyperlipidemia. HM can significantly improve the level of blood lipids and related abnormal metabolic pathways in HLP, so as to exert the effect of anti-HLP. Further research will explore the signal pathways and therapeutic targets associated with these lipid biomarkers. Abbreviations HM Herbal cake-separated moxibustion HLP Hyperlipidemia TC Total cholesterol TG Triglyceride LDL-C Low density lipoprotein cholesterol PC Phosphatidylcholine SM Sphingomyelin LPC Lysophosphatidylcholine Declarations Acknowledgments The authors would like to thank all participants for their participation in the study and their contributions to the study and the writing of the article. Author contribution Mailan Liu designed the study, Yuan Fang, Huijuan Liu, Lizhi Ouyang and Xinyu Chen completed the animal experiment. Yuan Fang and Xinyu Chen analyzed the data and wrote articles, Honghua Liu and Lizhi Ouyang made a statistical analysis of the data. Mailan Liu revised the first draft. All the authors participated in the revision of the paper and determined the final version of the manuscript. Funding The study was supported by grants from the National Natural Science Foundation of China (No.82074559)，Natural Science Foundation of Hunan Province Nos.2018JJ3097, 2020JJ5414, 2021JJ40401), Excellent Youth Fund Project of Hunan Provincial Education Department (Nos. 19B435, 19B428), Training Program for Excellent Young Innovators of Changsha (No. kq1905036) and the science and technology innovation Program of Hunan Province. Data availability The data used to support the results of this study can be obtained from the corresponding authors according to the requirements. Ethics statement The animal experiment was approved by the Hunan University of Traditional Chinese Medicine (LLBH-202005220001), and the disposal of animals was in line with the guidance on being kind to Experimental Animals issued by the Ministry of Science and Technology in 2006. Consent for publication Not applicable. Competing Interests The authors declare that they have no conflict of interest. Author details 1 College of Acupuncture &Tuina and Rehabilitation, Hunan University of Chinese Medicine, Changsha 410006, China. 2 College of Chinese Medicine and College of Life Sciences, Jiangxi University of Chinese Medicine, Nanchang 330103, China. 3 College of Nursing, Hunan University of traditional Chinese Medicine, Changsha 410006, China. References Jia X, Xu W, Zhang L, Li X, Wang R, Wu S. Impact of Gut Microbiota and Microbiota-Related Metabolites on Hyperlipidemia. Front Cell Infect Microbiol 2021 11634780. https://doi.org/10.3389/fcimb.2021.634780 . Al-Tamimi H, Al-Dawood A, Awaishesh S, Abdalla T. Resveratrol mitigates hypercholesterolemia exacerbated hyperthermia in chronically heat-stressed rats. Vet World. 2019;12(2):337–44. https://doi.org/10.14202/vetworld.2019.337-344 . 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4740592","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":330732038,"identity":"6100a3f1-974a-44fa-944b-729247883671","order_by":0,"name":"Yuan Fang","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Fang","suffix":""},{"id":330732039,"identity":"381251b5-5f6c-4db9-8e6d-18805d7239b2","order_by":1,"name":"Xinyu Chen","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xinyu","middleName":"","lastName":"Chen","suffix":""},{"id":330732041,"identity":"fb441834-842e-41aa-aa6a-654f342adb67","order_by":2,"name":"Huijuan Liu","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Huijuan","middleName":"","lastName":"Liu","suffix":""},{"id":330732045,"identity":"47599b8d-0091-4ac9-b643-cfeac07a312f","order_by":3,"name":"Honghua Liu","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Honghua","middleName":"","lastName":"Liu","suffix":""},{"id":330732047,"identity":"3df04db8-80d2-4a36-8738-a18fa8dfdb6c","order_by":4,"name":"Lizhi Ouyang","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Lizhi","middleName":"","lastName":"Ouyang","suffix":""},{"id":330732048,"identity":"bd7d54bb-983e-47e6-a33a-b19d360749d0","order_by":5,"name":"Mailan Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYBACNmaGhMN//9gkQPnMhLXwszc8PMDbkEaCFsmeg4+BWg6ToMXgRnLCAckd5/PMJZKfbmCosE5sYD97gICWtIQDhmduF1vOSDO7wXAmPbGBJy+BgJachAMJbLcTN9zIYbvB2HY4sUGCx4CAlvwPBw6wnYNq+UeEFsmeAwkHG9sOQLU0EKEFGMgJhxnOJCduOPPM7EbCsXTjNp4c/FqAUZn8maHCLnHD8eRnNz7UWMv2s5/BrwUVJIAMIUH9KBgFo2AUjAIcAACzE1H43bSIFgAAAABJRU5ErkJggg==","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Mailan","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-07-15 03:56:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4740592/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4740592/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62022415,"identity":"c56c9082-f146-4d7f-9f2f-12bb5c6c3f08","added_by":"auto","created_at":"2024-08-08 10:01:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":162433,"visible":true,"origin":"","legend":"\u003cp\u003eLevels of TC、TG and LDL-C in rabbit serum (x̅ ±s, n=10)\u003c/p\u003e\n\u003cp\u003eNote: compared to the normal group,\u003csup\u003e **\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; compared to the model group, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4740592/v1/c8555954fbfb375d03c5b68e.png"},{"id":62022421,"identity":"b42b82c2-dd81-45bb-9e9f-bbe936da19ba","added_by":"auto","created_at":"2024-08-08 10:01:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":984243,"visible":true,"origin":"","legend":"\u003cp\u003ePathomorphological changes of liver tissue.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4740592/v1/9dc7ef2d0ed0a2de9c061cb8.png"},{"id":62022899,"identity":"5d09af67-1c82-4dfb-a728-1751a8875338","added_by":"auto","created_at":"2024-08-08 10:09:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":159939,"visible":true,"origin":"","legend":"\u003cp\u003eTotal ion flow diagram of plasma lipids in positive and negative ion modes (a, b).\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4740592/v1/8adbdf77a56ad54e0d91bc0f.png"},{"id":62022902,"identity":"a8f7ccd8-826f-488c-b6db-63455825f382","added_by":"auto","created_at":"2024-08-08 10:09:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":217539,"visible":true,"origin":"","legend":"\u003cp\u003ePCA score plots (a) and PLS-DA score plots (b) of plasma samples. A.NC group, B.HLP group and C.HM group. PLS-DA model cross-validation plots (c), VIP plots (d) and differential metabolite heat plots (e).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4740592/v1/d9bbba97dd5ede5b0b1e3b1d.png"},{"id":62023966,"identity":"604b484a-4d7c-4547-8e71-09c592b6cbfd","added_by":"auto","created_at":"2024-08-08 10:25:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":262947,"visible":true,"origin":"","legend":"\u003cp\u003eROC curves of 15 differential lipid metabolites based on PLS-DA were used to select the biomarkers of the anti-hypolipidemic effect of HM. The related AUC, specificities, and sensitivities are pointed out.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4740592/v1/111fd88d85185776af6b875a.png"},{"id":62022418,"identity":"1b38e74d-6b7a-4d2f-b47f-bda80d644bfc","added_by":"auto","created_at":"2024-08-08 10:01:37","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":46798,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of identified lipid metabolic pathways\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4740592/v1/7ea1282ea7cc9b4b9c3cfff2.jpg"},{"id":62023347,"identity":"648e6fe0-d5d6-4b2b-a204-124dd4d4e697","added_by":"auto","created_at":"2024-08-08 10:17:37","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":21523,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of glycerophospholipid metabolism\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4740592/v1/fcf7aff927454144255d0a1b.jpg"},{"id":62949397,"identity":"346e11d7-6112-4afb-827c-b5f96a0c77ed","added_by":"auto","created_at":"2024-08-21 10:58:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2811766,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4740592/v1/1e852524-e2ff-4491-b04d-509ba3ba091f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lipidomic analysis to reveals therapeutic effects of herbal cake-separated moxibustion on high-fat diet-induced hyperlipidemia rabbits","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHyperlipidemia (HLP) is a disease characterized by abnormal levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol in the blood caused by lipid metabolic disorders[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Lipid metabolism disorder refers to a state in which one or more blood lipids are beyond the normal range caused by abnormal heredity, metabolism, or transport. HLP is one of the important risk factors for atherosclerotic cardiovascular disease[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. According to statistics, about 17\u0026nbsp;million people in the world suffer from cardiovascular diseases related to HLP[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].Therefore, the effective prevention treatment of the disease is an urgent problem to be solved. At present, conventional drugs for HLP have a rapid effect, but long-term use may cause side effects (such as muscle pain, abnormal liver function, etc.), which limits its clinical application[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Acupuncture and moxibustion therapy has attracted the attention of researchers because of its wide adaptation, stable curative effect, two-way regulation, psychosomatic co-treatment, less side effects and so on.\u003c/p\u003e \u003cp\u003eAs a characteristic therapy, acupuncture and moxibustion has unique advantages in the prevention and treatment of HLP [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], especially moxibustion with herbal\u0026ndash;cake-separated moxibustion (HM), which combines traditional moxibustion therapy with acupoints and traditional Chinese medicine (TCM), plays the role of multi-target and multi-channel system and regulatory effects, and has precise curative effect, simple operation and low price in treating hyperlipidemia [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Previous studies have shown that HM could alleviate HLP by affecting IGF-1/Sp1 signal pathway and regulating cholesterol reverse transport, LDL-C uptake and transport. And it is possible to regulate the levels of homocysteine and insulin-like growth factor-1, regulate hemorheology and repair endothelial function to improve hyperlipidemia. And it may be able to prevent and cure HLP by regulating the structure of microflora and reducing inflammation[\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. At present, the lipid-regulating effect of HM has not been reported by liposome.\u003c/p\u003e \u003cp\u003eThe study of lipid metabolism is of great significance to clarify the mechanism of HM in regulating blood lipids. Lipoomics is a subject that systematically analyzes various lipids and their interactions in cells and tissues, and elucidates their functions and changes under different physiological conditions[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. It has been widely used to study metabolic diseases, such as HLP, diabetes and hyperuricemia[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Liposome is consistent with the characteristics of multi-target and multi-pathway action of herbal cake-separated moxibustion, which is a powerful tool to study the effect of HM on regulating blood lipid. Ultra-high performance liquid chromatography-high resolution mass spectrometry provides the possibility for high-throughput analysis of lipids and promotes the process of lipid research[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the study, we mainly studied the effect of HM on diet-induced hyperlipidemia. HM ameliorates abnormal lipid metabolism by altering the pathway of glycerophospholipids metabolism, and improves high-fat diet-related dyslipidemia and liver injury. HM is expected to become a promising lipid-lowering therapy, which opens up a new way for the study of its treatment of metabolic diseases.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and ethical statement\u003c/h2\u003e \u003cp\u003eA total of 36 healthy male New Zealand rabbits, weighing 2.0\u0026thinsp;~\u0026thinsp;2.5kg, were purchased from Hunan Taiping Biological Co., Ltd. [ordinary class, License No: SCXK (Hunan) 2020-0005]. The animals were raised in a single cage in the Animal Center Laboratory of Hunan University of Chinese Medicine, where they were fed rationally and drank freely. It is raised in an environment with a relative humidity of 50%-70%, a temperature of 20\u0026deg;C-25\u0026deg;C and a day-night cycle of 12 hours. The whole experiment scheme has been approved by Hunan University of Chinese Medicine (License No.: LLBH-202005220001) and implemented in accordance with Animal Research: in vivo experiment report (REACH) Guide[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eExperimental protocol\u003c/h2\u003e \u003cp\u003eAfter one week of adaptive feeding, all rabbits were randomly divided into three groups(n\u0026thinsp;=\u0026thinsp;12): control group (NC group), HLP model group (HLP group) and herbal cake separated moxibustion group (HM group). The control group was fed with normal diet, and the rest of the rabbits were fed with high-fat diet to establish HLP model. High-fat diet contains 1% cholesterol, 10% egg yolk powder, 5% lard, 84% basic feed, propylthiouracil (10mg/ kg d). The total daily food intake of each rabbit was about 120g, drinking water was free, and it was fed continuously for 8 weeks. The rabbits in the HM group received the intervention of moxibustion separated by medicinal cake, while the rabbits in the control group and HLP group did not do any intervention except for binding and fixation.\u003c/p\u003e \u003cp\u003eModel evaluation: the normal serum TC content of New Zealand rabbits was 1\u0026thinsp;~\u0026thinsp;2mmol/L. When the serum TC value of each model group was more than 3 times higher than this value (or TC value of the control group), and the serum TG and LDL-C contents of the model group were higher than those of the control group, the HLP model was judged to be valid.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eIntervention methods\u003c/h2\u003e \u003cp\u003eBased on the previous research basis[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], acupoints were selected and divided into two groups and moxibustion was applied alternately. Group Ⅰ: bilateral Tianshu (ST25), Fenglong (ST40), Juque (RN14), 5 acupoints, group Ⅱ: bilateral Xinshu (BL15), Ganshu (BL18), Pishu (BL20), 6 acupoints. The location of acupoints is formulated with reference to \u003cem\u003eExperimental Acupuncture and moxibustion\u003c/em\u003e (2nd edition, 2016) edited by Yu Shuguang and Xu Bin and anthropomorphic comparison.\u003c/p\u003e \u003cp\u003eThe same amount of Hawthorn, salvia miltiorrhiza, rhubarb, turmeric and alisma were broken into foam, mixed with vinegar, and then pressed into a medicinal cake with a diameter of about 1cm and a thickness of about 0.3cm. Fix the rabbit, shave the acupoint area, apply appropriate amount of Vaseline on the acupoint, place the medicine cake, moxa, and light it. Burn out and change moxa sticks, 4 sticks per point, once a day, two groups of acupuncture points alternate moxibustion, 1 day a week off, consecutive 8 weeks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003eAfter the intervention, the rabbits were anesthetized with 2% pentobarbital sodium solution, laparotomy was performed, and 4ml of abdominal aorta blood was taken from ordinary tubes and EDTA tubes respectively, and the supernatant was obtained by centrifugation at 3000rpm for 10min, and stored in a refrigerator at -80℃ for detection. At the same time, about 500mg of fresh liver group was taken into the frozen storage tube and put into the refrigerator at -80℃ for detection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical analysis\u003c/h2\u003e \u003cp\u003eThe Olympus AU2700 automatic biochemical analyzer was used to detect the contents of TC, TG and LDL-C in serum according to the instructions of the kit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eOil red O staining\u003c/h2\u003e \u003cp\u003eFrozen slices were dried for 30 min, fixed with 4% neutral formaldehyde at 4 ℃ for 10 min, fully washed with distilled water and washed with 60% isopropanol. Oil red O dye solution shielded from light for 10 min, 60% isopropanol differentiated into interstitial cleaning; distilled water washing; hematoxylin staining for 3 min, distilled water washing for 3 min, glycerol gelatin sealing tablets, microscopic observation and photography.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePlasma lipidomics detection\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003ePlasma sample preparation\u003c/h2\u003e \u003cp\u003eThaw the sample at 4 ℃, vortex for 30 s100\u0026micro;L of blank rabbit plasma was taken, and chloroform: methanol (2:1) was added, followed by 10\u0026micro;L of the internal standard solution 1 to 10 Lipid Mix, 5uL1mg/mL C17:0, vortex for 1min. and 10\u0026micro;L extraction at -20 ℃.Then, samples were centrifuged at 4 ℃/13000 rpm for 5 min, 300\u0026micro;L of the lower solution was taken, and dried with nitrogen. The mixture was resolubilized with 20\u0026micro;L chloroform: methanol (1:1), then diluted three times with isopropanol: acetonitrile: water (2:1:1) (60\u0026micro;L; vortexed for 1 min). Finally, samples were centrifuged at 4 ℃/13000 rpm for 5 min, and all supernatants were taken and detected.\u003c/p\u003e \u003cp\u003eFor quality control (QC), the same volume from the prepared samples was mixed into large samples and evenly divided into QC samples to monitor the precision and stability of the instrument. The first three QC samples were used to monitor the precision, and one QC sample was collected at intervals of six samples to monitor the stability.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLipid profiling\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eLiquid chromatography conditions\u003c/h2\u003e \u003cp\u003eChromatographic separation was accomplished in an Ultimate 3000 system with an ACQUITY UPLC\u0026reg; BEH C18 (150 \u0026times; 2.1 mm, 1.8 \u0026micro;m; Waters). The flow rate was 0.3 mL/min, and the column was maintained at 35 ℃. The gradient elution of analytes was carried out with acetonitrile: water (3:2) (0.1% formic acid\u0026thinsp;+\u0026thinsp;10 mM ammonium formate) (A) and isopropanol: acetonitrile (9:1) (0.1% formic acid\u0026thinsp;+\u0026thinsp;10 mM ammonium formate) (B). Two \u0026micro;L of each sample was injected after equilibration. The gradient elution procedure was as follows: 0\u0026ndash;2 min, 60\u0026thinsp;\u0026minus;\u0026thinsp;57% A; 2-2.1 min, 57\u0026thinsp;\u0026minus;\u0026thinsp;50% A; 2.1\u0026ndash;12 min, 50\u0026thinsp;\u0026minus;\u0026thinsp;40% A; 12-12.1 min, 40\u0026thinsp;\u0026minus;\u0026thinsp;25% A; 12.1\u0026ndash;18 min, 25\u0026thinsp;\u0026minus;\u0026thinsp;1% A, 19\u0026ndash;20 min, 1\u0026ndash;60% A; 20\u0026ndash;25 min, 60% A[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMass spectrum conditions\u003c/h2\u003e \u003cp\u003eHigh-resolution MS QE Xactive (Thermo Fisher Scientific, USA) collected positive and negative ion modes. In the positive ion mode, the electrospray voltage was 3.3 kV; in the negative ion mode, the electrospray voltage was 2.8 kV. The capillary temperature was 320\u0026deg;C, the sheath temperature was 40 Arb, and the atomizer temperature was 350\u0026deg;C. A full scan was performed with a mass resolution of 35 000 and a scanning m/z range of 150\u0026ndash;2000. HCD is used for two-stage cracking, and the collision voltage is 30 eV. At the same time, dynamic elimination is used to remove unnecessary MS/MS information[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe mass spectrometry data were denoised and normalized, and then imported into SIMCA-P software for multivariate statistical analysis. According to principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) models, the potential biomarkers with variable projection importance (VIP)\u0026thinsp;\u0026gt;\u0026thinsp;1.0 and t test \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01were screened. Then, KEGG pathway enrichment analysis was performed on the list of different substances by MetaboAnalyst [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe experimental data were statistically analyzed by SPSS25.0, all the data were expressed by \u0026#119909;̅\u0026plusmn;s, and plotted and analyzed by Graphad Prisim5. One-way ANOVA method was used to compare among groups, LSD was used to analyze the variance when the variance was uniform, and Dunnet T3 was used to analyze the difference.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003eGeneral situation of experimental rabbits\u003c/h2\u003e\n\u003cp\u003eIn the normal group, the rabbit had a moist coat, good spirit, and rapid response to stimulation. The rabbits in the model group showed varying state changes, mainly characterized by low spirits, slow response, and rapid body weight growth. The condition of the rabbits in the herbal cake-separated moxibustion group was improved and the body weight was reduced.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003eHM reduces the content of lipid in serum of HLP rabbits\u003c/h2\u003e\n\u003cp\u003eAfter eight weeks of intervention, serum TC, LDL-C, and TG contents were significantly higher in the HLP group than in the NC group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), suggesting the rabbit model was successfully established. Serum TC, TG, and LDL-C contents decreased to different degrees in the HM group compared to the HLP group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;1). These results showed that the herbal cake-separated moxibustion intervention could improve the dyslipidemia of hyperlipidemic rabbits.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003eHM reduces liver Lipid deposition in HLP Rabbits\u003c/h2\u003e\n\u003cp\u003eHematoxylin dyes the nucleus blue and lipid droplets orange. No histological abnormality was found in the liver tissue of the NC group, while the accumulation of lipid droplets was obvious in the HLP group, and the lipid deposition increased significantly. After the HM intervention, lipid droplets were significantly reduced and effectively improved the pathological state of the liver (Fig.\u0026nbsp;2).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003eScreening and identification of differential lipid metabolites\u003c/h2\u003e\n\u003cp\u003eThe total ion flow maps of all plasma QC samples are superimposed, and the spectra overlap well, and the retention time and peak signal intensity fluctuate little, indicating that the instrument is in good condition in the whole sample analysis process, which is suitable for the detection and analysis of this project (Fig.\u0026nbsp;3a and b).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cp\u003eThe metabolites of NC group, HLP group and HM group were significantly separated by principal component analysis and partial least square-discriminant analysis, indicating that there were differences in plasma lipid metabolism profile among each group, and the metabolic profile of HM group was closer to that of NC group (Fig.\u0026nbsp;4a and 4b). The cross-validation result of the PLS-DA model is shown in Fig.\u0026nbsp;4c. The Abscissa represents the number of components selected by the model, and the ordinate is the value of R\u003csup\u003e2\u003c/sup\u003e, Q\u003csup\u003e2\u003c/sup\u003e and Accuracy. R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.928, Q\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.881, R\u003csup\u003e2\u003c/sup\u003e is the accuracy of model establishment, Q\u003csup\u003e2\u003c/sup\u003e is the accuracy of model prediction, R\u003csup\u003e2\u003c/sup\u003e and Q\u003csup\u003e2\u003c/sup\u003e are all greater than 0.5, indicating that the established model has a good explanation and prediction for the distinction among NC group, HLP group and HM group.\u003c/p\u003e\n\u003cp\u003eFigure 4d is the VIP (Variable Importance in Projection) diagram of PLS-DA, which shows the importance of variables and their contribution to sample differentiation. The ordinate represents the first 15 differential metabolites (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), the Abscissa is the VIP value, and the right color coding reflects the concentration of the metabolite in different groups. Heat plots can further visualize the differential lipid metabolites. Figure\u0026nbsp;4e shows the changes of differential lipid metabolites (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The concentration of differential lipid metabolites in NC group is the lowest, followed by HM group, and the highest in HLP group, which is consistent with the results of VIP map. It is suggested that HM can reverse the disorder of lipid metabolism in plasma of HLP rabbits.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eList of differential lipid metabolites\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMetabolite\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003em/z(Da)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003et\u003csub\u003eR\u003c/sub\u003e/min\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eVIP\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eChemical\u003c/p\u003e\n\u003cp\u003eformula\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePC 28:0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e678.503\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.654\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.914\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.37\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eC\u003csub\u003e36\u003c/sub\u003eH\u003csub\u003e72\u003c/sub\u003eNO\u003csub\u003e8\u003c/sub\u003eP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSM 52:13;2O\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e931.682\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.593\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.872\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.44\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;12\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eC\u003csub\u003e57\u003c/sub\u003eH\u003csub\u003e91\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003eP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLPC 25:0/0:0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e662.479\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.656\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.868\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.52\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;11\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eC\u003csub\u003e33\u003c/sub\u003eH\u003csub\u003e68\u003c/sub\u003eNO\u003csub\u003e7\u003c/sub\u003eP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePC 36:6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e778.532\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.169\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.808\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.86\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eC\u003csub\u003e44\u003c/sub\u003eH\u003csub\u003e76\u003c/sub\u003eNO\u003csub\u003e8\u003c/sub\u003eP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSM 56:13;2O\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1003.706\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.591\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.802\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.73\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;11\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eC\u003csub\u003e61\u003c/sub\u003eH\u003csub\u003e99\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003eP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSM 32:0;2O\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e693.550\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.529\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.773\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.08\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eC\u003csub\u003e37\u003c/sub\u003eH\u003csub\u003e77\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003eP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePC 37:6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e792.548\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.870\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.759\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.36\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;12\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eC\u003csub\u003e45\u003c/sub\u003eH\u003csub\u003e78\u003c/sub\u003eNO\u003csub\u003e8\u003c/sub\u003eP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLPC O-24:0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e594.481\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.861\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.758\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.27\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eC\u003csub\u003e32\u003c/sub\u003eH\u003csub\u003e68\u003c/sub\u003eNO\u003csub\u003e6\u003c/sub\u003eP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSM 36:3;2O\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e727.569\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.871\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.686\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.65\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;11\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eC\u003csub\u003e41\u003c/sub\u003eH\u003csub\u003e79\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003eP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePC 42:4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e866.658\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14.772\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.676\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.57\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eC\u003csub\u003e50\u003c/sub\u003eH\u003csub\u003e92\u003c/sub\u003eNO\u003csub\u003e8\u003c/sub\u003eP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLPC O-24:1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e592.469\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.137\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.676\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.75\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eC\u003csub\u003e32\u003c/sub\u003eH\u003csub\u003e66\u003c/sub\u003eNO\u003csub\u003e6\u003c/sub\u003eP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSM 33:2;2OSM 1712O\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e687.540\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.522\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.673\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.69\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eC\u003csub\u003e38\u003c/sub\u003eH\u003csub\u003e75\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003eP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePC 32:3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e728.519\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.205\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.667\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.14\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eC\u003csub\u003e40\u003c/sub\u003eH\u003csub\u003e74\u003c/sub\u003eNO\u003csub\u003e8\u003c/sub\u003eP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePC 42:10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e854.566\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.146\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.657\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.81\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;13\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eC\u003csub\u003e50\u003c/sub\u003eH\u003csub\u003e80\u003c/sub\u003eNO\u003csub\u003e8\u003c/sub\u003eP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePC 34:4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e754.532\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.657\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.626\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.85\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;11\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eC\u003csub\u003e42\u003c/sub\u003eH\u003csub\u003e76\u003c/sub\u003eNO\u003csub\u003e8\u003c/sub\u003eP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe VIP value was obtained from PLS-DA with a threshold of 1.0.\u003c/p\u003e\n\u003cp\u003eThe p-values were calculated from the ANOVA\u003c/p\u003e\n\u003cp\u003eIn order to further study the potential biomarkers of HM anti-HLP, ROC curve analysis based on PLS-DA was carried out. As shown in the figure, the AUC values of 12 differential lipid metabolites were all greater than 0.85. among them, SM(52: 13;2O)、LPC(25༚0/0༚0)、PC(36༚6)、PC(37༚6)、LPC(O-24༚0)、SM(36༚3༛2O)、PC(42༚4)、LPC(O-24༚1) and PC(42༚10) had high specificity (\u0026gt;\u0026thinsp;85%) and sensitivity (\u0026gt;\u0026thinsp;85%) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). The results suggest that nine lipid components such as SM(52༚13༛2O) play a key role in improving the disorder of plasma lipid metabolism in HM rabbits. These differential lipid metabolites include 4 phosphatidylcholine (PC), 2 sphingomyelin (SM) and 3 lysophosphatidylcholine (LPCs), suggesting that PC, SM and LPC are potential biomarkers of the hypolipidemic effect of HM.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003eMetabolic pathway analysis\u003c/h2\u003e\n\u003cp\u003eIn order to further study the effect of lipid metabolites on HLP, the pathway analysis of differential lipid metabolites in rabbit plasma was carried out. The results showed that nine pathways were involved (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e), including glycerophospholipid metabolism (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e), linoleic acid metabolism, alpha-linolenic acid metabolism, arachidonic acid metabolism, GPI-anchor biosynthesis, glycerolipid metabolism, fatty acid biosynthesis, fatty acid prolongation, fatty acid degradation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). According to its impact, \u003cem\u003ep\u003c/em\u003e-value, and the value of hits (\u0026ge;\u0026thinsp;3), it is determined that glycerophospholipid metabolism (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.10\u0026times;10\u003csup\u003e9\u003c/sup\u003e) plays a key role in the occurrence and development of HLP.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eEffect of HM on Plasma Lipid Metabolism Pathway in HLP Rabbits\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePathway Name\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTotal/ Hits\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRaw \u003cem\u003ep\u003c/em\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003elog(\u003cem\u003ep\u003c/em\u003e)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eHolm \u003cem\u003ep\u003c/em\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFDR\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eImpact\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGlycerophospholipid metabolism\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e36/3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.10E-09\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e19.593\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.79E-08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.97E-08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.264\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLinoleic acid metabolism\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5/2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.89E-09\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e18.951\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.71E-08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.97E-08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.968\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ealpha-Linolenic acid metabolism\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13/1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.56E-09\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e18.842\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.71E-08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.97E-08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eArachidonic acid metabolism\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e36/2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.39E-08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e17.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.03E-07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.62E-08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.281\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGPI-anchor biosynthesis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15/1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.34E-08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e16.573\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.17E-07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.14E-07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGlycerolipid metabolism\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e16/1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.42E-03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.049\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.57E-02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.62E-03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.026\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFatty acid biosynthesis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e46/1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.31E-01\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.633\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.31E-01\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFatty acid elongation\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e38/1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.31E-01\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.633\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.31E-01\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFatty acid degradation\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e39/1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.31E-01\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.633\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.31E-01\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026ldquo;Total\u0026rdquo;: the number of all metabolites in the metabolic pathway. \u0026ldquo;Hits\u0026rdquo;: the number of differentiated metabolites selected in the metabolic pathway. \u0026ldquo;Raw \u003cem\u003ep\u003c/em\u003e\u0026rdquo;: the original \u003cem\u003ep\u003c/em\u003e-value of the enrichment analysis. \u0026ldquo;Holm \u003cem\u003ep\u003c/em\u003e\u0026rdquo; adjusted \u003cem\u003ep-\u003c/em\u003evalue by Holm\u0026ndash;Bonferroni method. FDR: the FDR value in multiplex checking. Impact: the influence value calculated by path topology analysis.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn 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[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. 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[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eThe 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[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eGlycerophospholipid 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[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], 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[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], 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[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003ePC is closely related to the occurrence of HLP [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. 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[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. 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[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. It was found that the level of PC in plasma of obese mice with dyslipidemia induced by high-fat diet was significantly increased[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and the lipid level returned to / or close to normal after intervention. This is consistent with the results of this study.\u003c/p\u003e \u003cp\u003eLPC 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[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. 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[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. 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[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSM 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[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. These specific SM have been found to be positively correlated with insulin resistance, LDL-C, total cholesterol and TG[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. 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[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. One study described that inhibition of SM can reduce plasma levels of TC and TG in mice, thereby preventing atherosclerosis[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. 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.\u003c/p\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eLimitations\u003c/h2\u003e \u003cp\u003eWe 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.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, serum TC, TG, and LCL-C levels were significantly increased in hyperlipidemic rabbits induced by a high-fat diet, and the lipid deposition in liver tissue was significantly increased after oil red O staining related to the significant changes in plasma concentrations of PC, SM and LPC. These changes are mainly reflected by disorders in glycerophospholipid metabolism, linoleic acid metabolism, α-linolenic acid metabolism, arachidonic acid metabolism, GPI anchor biosynthesis, and glycerol ester metabolism in diet-induced hyperlipidemia. HM can significantly improve the level of blood lipids and related abnormal metabolic pathways in HLP, so as to exert the effect of anti-HLP. Further research will explore the signal pathways and therapeutic targets associated with these lipid biomarkers.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eHM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Herbal cake-separated moxibustion\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHLP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Hyperlipidemia\u003c/p\u003e\n\u003cp\u003eTC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Total cholesterol\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTG \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Triglyceride\u003c/p\u003e\n\u003cp\u003eLDL-C \u0026nbsp; \u0026nbsp; Low density lipoprotein cholesterol\u003c/p\u003e\n\u003cp\u003ePC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Phosphatidylcholine\u003c/p\u003e\n\u003cp\u003eSM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Sphingomyelin\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLPC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Lysophosphatidylcholine\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank all participants for their participation in the study and their contributions to the study and the writing of the article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMailan Liu\u0026nbsp;designed the study,\u0026nbsp;Yuan Fang, Huijuan Liu,\u0026nbsp;Lizhi Ouyang\u0026nbsp;and\u0026nbsp;Xinyu Chen\u0026nbsp;completed the animal experiment.\u0026nbsp;Yuan Fang\u0026nbsp;and\u0026nbsp;Xinyu Chen\u0026nbsp;analyzed the data and wrote articles,\u0026nbsp;Honghua Liu and Lizhi Ouyang\u0026nbsp;made a statistical analysis of the data. Mailan Liu\u0026nbsp;revised the first draft.\u0026nbsp;All the authors participated in the revision of the paper and determined the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was supported by grants from the National Natural Science Foundation of China (No.82074559)，Natural Science Foundation of Hunan Province Nos.2018JJ3097, 2020JJ5414, 2021JJ40401), Excellent Youth Fund Project of Hunan Provincial Education Department (Nos. 19B435, 19B428), Training Program for Excellent Young Innovators of Changsha (No. kq1905036) and the science and technology innovation Program of Hunan Province.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used to support the results of this study can be obtained from the corresponding authors according to the requirements.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experiment was approved by the Hunan University of Traditional Chinese Medicine (LLBH-202005220001), and the disposal of animals was in line with the guidance on being kind to Experimental Animals issued by the Ministry of Science and Technology in 2006.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eCollege of Acupuncture \u0026amp;Tuina and Rehabilitation, Hunan University of Chinese Medicine, Changsha 410006, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eCollege of Chinese Medicine and College of Life Sciences, Jiangxi University of Chinese Medicine, Nanchang 330103, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003eCollege of Nursing, Hunan University of traditional Chinese Medicine, Changsha 410006, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJia X, Xu W, Zhang L, Li X, Wang R, Wu S. 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However, the effect of HM on plasma lipid metabolism in HLP rabbits is not clear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eNew Zealand rabbits were fed with high-fat diet for 8 weeks to induce HLP model, and then HM was intervened for 8 weeks. The level of blood lipid in serum of rabbits was detected by full biochemical analyzer, and the pathological changes of liver tissue were observed by oil red O staining. Then we used ultra-high performance liquid chromatography / quadrupole time-of-flight mass spectrometry combined with multivariate statistical analysis for non-targeted lipidomic analysis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHM ameliorated hyperlipidemia induced the abnormal blood lipid level and improved liver lipid deposition induced by high cholesterol diet. Non-targeted lipidomic analysis showed that HM changed the lipid metabolism profile of HLP rabbits.\u003c/p\u003e","manuscriptTitle":"Lipidomic analysis to reveals therapeutic effects of herbal cake-separated moxibustion on high-fat diet-induced hyperlipidemia rabbits","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-08 10:01:31","doi":"10.21203/rs.3.rs-4740592/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b721e387-47e9-4e7b-ad5c-a0a925de682c","owner":[],"postedDate":"August 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-21T10:49:50+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-08 10:01:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4740592","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4740592","identity":"rs-4740592","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}