Capsaicin Improves Lipid Metabolism Disorders Caused by Immune Stress in Weaned Piglets Induced by LPS

1. Introduction

In modern intensive pig production, the common stress factors are long-distance transportation, vaccination and disease, temperature change, weaning, pregnancy, delivery and piggery management [3]. Environmental pathogenic factors, including lipopolysaccharide (LPS), viral pathogens (e.g. porcine reproductive and respiratory syndrome virus) and conditional pathogenic bacteria (e.g. Escherichia coli) can activate the host immune system through pattern recognition receptors (e.g. TLR4) and induce the overexpression of pro-inflammatory cytokines (e.g. TNF-α, IL-1β and IL-6) [6]. This persistent low-grade inflammatory state is defined as immune stress, which is characterised by the destruction of intestinal mucosal barrier integrity and liver metabolic dysfunction [7]. Lipid metabolism is crucial in energy homeostasis and growth in animals, particularly during critical developmental stages, such as the weaning period. Body fat is the main source of energy during immediate post-weaning days [8]. A striking change in body composition following weaning is a decrease in body fat content [9] and a decrease in backfat thickness, whereas lipogenesis is marginal [10]. Therefore, a functional and efficient lipid metabolism system is essential for the growth and development of piglets. A large number of studies have shown that such inflammation-mediated tissue damage can significantly reduce nutrient absorption efficiency, resulting in a decrease in average daily gain and deterioration of the feed conversion rate [11]. However, there are few studies on lipid metabolism under immune stress. Therefore, it is of great importance for a method to regulate lipid metabolism in weaned piglets under immune stress.

When piglets are weaned, due to the critical period of the body’s development, their own functions are not yet well integrated with changes in the environment, and pathogenic microorganisms and other factors can cause immune stress in piglets. Stress can lead to dramatic changes in the gastrointestinal tract of piglets, which interferes with the intestinal microbiota, host physiology and mucosal immune function [12]. Subsequently, feed intake decreased, and the occurrence and growth of diarrhoea after weaning slowed down [13]. Although feed additives, such as nucleotides, probiotics, prebiotics and fatty acids (FAs), have shown results in alleviating weaning stress, the potential of plant feed additives, such as capsaicin (CAP), has not been fully utilised [11].

CAP is an alkaloid compound isolated from Capsicum plants [1]. It is the main component that gives chili a spicy taste [14]. CAP is widely used in the food industry, pharmaceutical industry and biological science research. It has many biological functions, including antioxidation, anti-cancer, improving digestion, improving fat metabolism, immune regulation and bacteriostasis [2,14,15,16,17,18,19,20]. CAP can also improve fat metabolism. It may be involved in the regulation of energy metabolism, improve the body’s basal metabolic rate and play a role in weight management and resistance to obesity by activating TRPV1 [2]. Yang et al. found that CAP could improve hyperlipidaemia in guinea pigs fed a high-fat diet [18]. Gong et al. observed that CAP could manage lipid metabolism by regulating bile acid (BA)/intestinal microflora metabolism in high-fat-fed Sprague Dawley rats [17]. Dai et al. revealed that CAP could prevent the development of atherosclerosis induced by a high-fat diet in mice by remodelling gut microbiota and improving blood lipids and inflammation [19]. However, it is not clear whether CA supplementation could improve lipid metabolism in weaned piglets.

Therefore, the aim of this study was to investigate the alleviating effect of CAP on lipid metabolism in weaned piglets under immune stress and to explore its potential mechanism.

2. Materials and Methods

2.1. Moral Statement

All procedures were approved by the Animal Care and Use Committee of Nanjing Agricultural University (GB14925-2010, NJAU-CAST-2011-093).

2.2. Preparation of CAP and LPS

CAP was purchased from Guangzhou Lidar Biotechnology Co. Ltd., with a purity of ≥ 0.5 %. In the feeding, CAP and basic materials were mixed at an effective dose of 4 mg/kg. LPS purchased from Sigma-Aldrich Chemical Co. (O55: B5; # L2880; St. Louis, Missouri) was dissolved in sterile saline at a concentration of 0.86 % (w/v) and prepared into a solution of 1 mg/mL before being used for administration.

2.3. Animal and Experimental Design

All experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of Nanjing Agricultural University. Twenty-four Duroc-Landrace-Yorkshire (DLY) weaned piglets with an initial body weight of 9.00 ± 0.30 kg were randomly divided into three groups, with eight replicates per group and one pig per replicate. The control group (CON) and LPS group received a basal diet, while the LCA group was fed a basal diet containing 800 mg/kg CAP for 35 days [5]. Piglets in the LPS and LCA groups were intraperitoneally injected with LPS at a dose of 100 μg/kg body weight 4 h before sampling [7], while those in the CON group were intraperitoneally injected with the same dose of normal saline. In this experiment, the injection dose of LPS was referred to the method used by Zhu et al. [7], while the supply dose of CAP was referred to Long et al.’s study [5]. Basal diet composition and nutrient levels (NRC 2012) are shown in Table 1.

Note: 1Premix for per kilogram diet included: lysine, 4.6 g; methionine, 1.2 g; threonine, 0.5 g; NaCl, 0.3 g; vitamin A, 12000 IU; vitamin E, 80 IU; vitamin D3, 2500 IU; vitamin K3, 3.0 mg; biotin, 0.2 mg; nicotinic acid, 25 mg; pantothenic acid, 30 mg; riboflavin, 3.6 mg; thiamin, 1.0 mg; pyridoxine, 1.5 mg; folic acid, 2.0 mg; cobalamin, 12 μg; choline chloride, 800 mg; iron, 120 mg; zinc, 100 mg; copper, 8.8 mg; manganese, 80 mg; iodine, 0.3 mg; selenium, 0.25 mg; 2All nutrient levels were analyzed values except for the digestible energy and SID lysine.

2.4. Sample Collection

After 35 days of feeding, all piglets were sacrificed by carotid artery bloodletting. Blood samples were collected from the anterior vena cava of each pig, and the blood samples were centrifuged at 3,500 g for 15 min at 4°C. Subsequently, the collected upper serum was stored in a −80° refrigerator for subsequent physiological and biochemical indicators. Slaughter sampling was performed, and appropriate amounts of liver, jejunum and ileum tissues were placed in a cryopreservation tube and stored in a −80° refrigerator for the subsequent determination of various indicators.

2.5. Biochemical Assay of Serum Samples

Commercial assay kits were used to determine triglycerides (TG; #A110-1-1), non-esterified free fatty acids (NEFA; #A042-1-1), total cholesterol (TC; #A111-1-1), total bile acid (TBA; #E003-2-1), high-density lipoprotein cholesterol (HDL-C; #A112-1-1) and low-density lipoprotein cholesterol (LDL-C; #A113-1-1), according to the manufacturer’s instructions (Nanjing JianCheng Bioengineering Institute, Nanjing, Jiangsu, China).

According to the manufacturer’s instructions, the porcine enzyme-linked immunosorbent assay kit of Jiangsu Enzyme Immuno Industry Co. Ltd. (Quanzhou City, Jiangsu Province, China) was used to detect the fatty acid binding protein 4 (FABP4; # MM-78445O2) level.

2.6. Determination of Liver Metabolite Concentration and Enzyme Activity

According to the manufacturer’s instructions (Nanjing Jiancheng Institute of Bioengineering), commercial detection kits were used to determine TG (#A110-1-1), TC (#A111-1-1), TBA (#E003-2-1), alanine aminotransferase (ALT; #C009-3-2), aspartate aminotransferase (AST; #C010-2-1), HDL-C (#A112-1-1), LDL-C (#A113-1-1), lipoprotein lipase (LPL; #A067-1-2) and hepatic lipase (HL; #A067-1-2). The total esterase (TL) content was the sum of the HL and LPL contents.

2.7. RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction (PCR) Analysis

Total RNA was extracted from the ileum (mucosal) and liver samples using a total RNA extraction reagent (Vazyme Biotechnology, Nanjing, Jiangsu, China) and quantified using an ND-2000 micro spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). After the RNA quality and concentration were determined, 1 μg of total RNA was reverse-transcribed into complementary DNA (cDNA) using the HiScript III RT SuperMix Reagent (Vazyme Biotechnology), following the manufacturer’s instructions. The mRNA expression levels of specific genes were quantified via real-time polymerase chain reaction (PCR) using SYBR qPCR Master Mix (Vazyme Biotechnology) and the QuantStudio 5 Real-Time PCR System (Thermo Scientific, Wilmington, DE, USA). The SYBR Green PCR reaction mixture consisted of 10 µL TB Green Premix Ex Taq, 0.4 µL ROX Reference Dye II, 2 µL cDNA template, 0.4 µL of each primer (total 0.8 µL, 10 µmol/L) and 6.8 µL of double-distilled H₂O. The reaction conditions were as follows: pre-run at 95°C for 30 s, 40 denaturation cycles at 95°C for 10 s and annealing at 60°C for 30 s. Each sample was run in triplicate. The relative mRNA expression levels were analysed using the 2^−ΔΔCt method after normalisation against β-actin. The results showed a similar trend when GAPDH served as the housekeeping gene. The primer sequences are shown in Table 2.

2.8. Protein Extraction and Western Blot Assay

TP was isolated from frozen liver samples using a lysis buffer that contained protease inhibitors (Beyotime Institute of Biotechnology, Nantong, Jiangsu, China). The protein concentration was determined using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology). Equal amounts of TP—specifically, 20 µg—were loaded for electrophoresis on a 4%–20% SDS-PAGE gel. Following electrophoresis, the proteins were transferred onto PVDF membranes that had been activated with methanol. The membranes were subsequently blocked at room temperature for 2 hours in TBST containing 5% fat-free milk powder, along with 0.05% Tween-20, 100 mmol/L Tris-HCl, and 150 mmol/L NaCl (pH 7.5). After blocking, the membranes were incubated overnight at 4°C with primary antibodies against specific target proteins, including PPARα (#66826-1-Ig; Proteintech), PGC1α, CPT1α, LXRα, and FXR. The blots were then washed three times with TBST and incubated at room temperature for 1.5 hours with a secondary antibody—either alkaline phosphatase-conjugated goat anti-rabbit IgG or anti-mouse IgG (#BL023A and #BL021A; Biosharp, Hefei, Anhui, China). Finally, the blots underwent three additional washes with TBST before protein detection was performed using an enhanced chemiluminescence reagent (#BL520A; Biosharp). The signals were visualized using a ChemiDoc™ Imaging System (BIO-RAD, Hercules, CA, USA), and the intensity of the protein bands was quantified with ImageJ 1.42q software (NIH, Bethesda, MD, USA).

2.9. Liver Metabolites Analysis

The non-targeted metabolomics detection of liver tissue in this experiment was completed by Shanghai Pasenuo Biotechnology Co, Ltd. (Shanghai, China). Multivariate statistical analysis of liver samples was performed using Ropls software. PLS-DA (partial least squares-discriminant analysis) was selected to obtain the best analysis model. Differential metabolites were screened according to P value, VIP value and |log2FC| value. ClusterProfiler software was used to perform functional pathway enrichment analysis on the screened differential metabolites.

2.10. Statistical Analysis

All data were analyzed using SPSS software (version 21.0; IBM-SPSS, Inc., Chicago, Illinois) with an independent samples t-test. The results are presented as mean ± SEM. To evaluate significant differences between multiple groups, Tukey’s Honestly Significant Difference (Tukey HSD) test was applied. Statistical significance was defined as P < 0.05, while P < 0.01 was considered highly significant. Liver non-targeted metabolomics data were analyzed by orthogonal partial least squares–discriminant analysis (OPLS-DA).According to the criteria of VIP value > 1 and P value < 0.05, metabolites with significant differences between NC group and LPS group, LPS group and LCA group were screened out. Pathway enrichment analysis was performed using the Parson’s gene cloud platform. Pearson correlation analysis was performed on liver antioxidant enzyme activity and oxidative metabolites and liver differential metabolites using SPSS 26.0 software. P < 0.05 indicated that the two were significantly correlated, and then OriginPro 2025b software was used to make the correlation heat map.

3. Results

3.1. Changes in FA Metabolites and the Corresponding Enzymes in Serum and Liver

As shown in Table 3, the level of FABP4 in the LPS group was significantly lower than that in the CON group (P < 0.05), the TG and NEFA levels were higher (P < 0.05), and the liver TG and NEFA levels were higher (P < 0.05), indicating that LPS induced these conditions. However, compared with the LPS group, the addition of CAP in the LCA group resulted in a significant decrease in serum TG levels (P < 0.05), an increase in FABP4 levels (P < 0.05), a significant decrease in the liver TG and NEFA levels (P < 0.05) and an increase in the LPL, HL and TL levels (P < 0.05).

Note: The results are expressed as mean ± SEM (n = 8). NC, piglets fed a basal diet; LPS, piglets fed a basal diet and intraperitoneally injected with LPS before sampling; lCA, piglets fed CAP and injected intraperitoneally with LPS before sampling. * indicates a significant difference compared with the CON group (P < 0.05), and # indicates a significant difference compared with the LPS group (P < 0.05). TG, triglyceride; NEFA, non-esterified fatty acids; HL, liver lipase; LPL, lipoprotein lipase; TL, total lipase; FABP4, fatty acid binding protein 4.

3.2. Gene Expression Involved in FA Uptake, Synthesis and Oxidation

Regarding fatty acid (FA) uptake, the mRNA expression levels of FABP2, FATP4, and FFAR3 were significantly higher in the LPS group than in the CON group (P < 0.05). Furthermore, when compared with piglets in the LPS group, those in the LCA group exhibited markedly reduced mRNA expression of FABP2, FATP4, and FFAR3 (P < 0.05) (Figure 1A).

Regarding fatty acid (FA) synthesis, the mRNA expression levels of FASN, DGAT1, and LXRα were significantly higher in piglets from the LPS group than in those from the CON group (P < 0.05). In contrast, piglets in the LCA group showed significantly lower mRNA expression of FASN, DGAT1, and LXRα compared to those in the LPS group (P < 0.05) (Figure 1B).

Turning to fatty acid oxidation, both PPARα and ATGL mRNA expression levels were significantly reduced in the LPS group relative to the CON group (P < 0.05). However, in the LCA group, ATGL mRNA expression was markedly elevated compared with that in the LPS group (Figure 1C).

3.3. Protein Expression Related to FA Transport and Metabolism

As shown in Figure 1, the mRNA expression levels of PPARα, PGC1α, and CPT1α were significantly lower in the LPS group than in the CON group (P < 0.05). In contrast, piglets in the LCA group—whose diet was supplemented with CAP—exhibited significantly higher expression of PPARα, PGC1α, and CPT1α compared to those in the LPS group (P < 0.05) (Figure 1D).

3.4. Changes in Cholesterol and BA Metabolites in Serum and Liver

As shown in Table 4, the HDL-C level in the LPS group was significantly lower than that in the CON group (P < 0.05). Additionally, serum total bile acid (TBA) levels were significantly elevated in the LPS group compared to the CON group (P < 0.05). Moreover, both liver total cholesterol (TC) and LDL-C concentrations were also significantly higher in the LPS group (P < 0.05). Together, these findings suggest that LPS exposure induced dyslipidemia and altered bile acid metabolism. However, when CAP was added to the diet of piglets in the LCA group, several beneficial effects were observed. Specifically, serum TC, LDL-C, and TBA levels were all significantly reduced compared to those in the LPS group (P < 0.05). Furthermore, liver LDL-C levels also showed a significant decline relative to the LPS group (P < 0.05).

Note: The results are expressed as the mean ± standard error (n = 8). NC, piglets fed a basal diet; LPS, piglets fed a basal diet and intraperitoneally injected with LPS before sampling; LCA, piglets fed CAP and injected intraperitoneally with LPS before sampling. * indicates a significant difference compared with the CON group (P < 0.05), and # indicates a significant difference compared with the LPS group (P < 0.05). TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TBA, total bile acid.

3.5. Expression of Genes Related to Cholesterol and BA Metabolism

With regard to cholesterol metabolism, LPS-treated piglets exhibited significantly higher mRNA expression of NPC1L and ABCA1 genes associated with cholesterol production and efflux than did the piglets in the CON group (P < 0.05), and the mRNA expression of ABCG5 significantly decreased (P < 0.05). Compared with the piglets in the LPS group, those in the LCA group showed a significantly decreased mRNA expression of HMGCR, NPC1L and ACAT2 (P < 0.05) and a significantly increased mRNA expression of ABCG5 (P < 0.05) (Figure 2A).

Regarding BA metabolism, compared with the CON group, the mRNA expression of the BA synthesis-related gene CPY7A1 and BA excretion-related genes BSEP, NTCP, MRP2 and MRP4 in the LPS group significantly decreased (P < 0.05), while the mRNA expression of CYP7A1 and MRP4 in the LCA group was significantly higher than that in the LPS group (P < 0.05) (Figure 2B,C).

3.6. Protein Expression and Enzyme Activity Involved in Cholesterol Metabolism and BA Synthesis

Compared with the CON group, the LPS group showed lower (P < 0.05) LXRα and CYP27A1 expression. However, supplementation of CAP resulted in increased (P < 0.05) LXRα and CYP27A1 expression in LCA piglets compared with LPS piglets (Figure 2D).

3.7. Effects of Capsaicin on Hepatic Metabolites in Immune Stressed Piglets

The orthogonal partial least squares discriminant analysis of the liver metabolites of the three groups of piglets is shown in Figure 3. The results showed that the liver metabolites of NC and LPS groups, LPS and LCA groups were significantly separated. The results showed that there were significant differences in liver metabolites between NC group and LPS group, as well as LPS group and LCA group (P < 0.05).

There were 789 differential metabolites in the results of liver non-target metabolome analysis. According to the standard screening of P1, | log2FC | > 1, the three groups of piglet liver differential metabolites volcano map is shown in Fig.4. The results showed that, compared with the NC group, the LPS group exhibited 138 significantly down-regulated and 109 significantly up-regulated metabolites in the liver (P < 0.05). In contrast, when the LCA group was compared with the LPS group, 22 metabolites were significantly down-regulated and 12 metabolites were significantly up-regulated in the liver (P < 0.05). Furthermore, the 247 differential metabolites identified between the LPS and NC groups could be broadly classified into nine categories, including benzenoids; organic acids and derivatives; alkaloids and derivatives; lipids and lipid-like molecules; organic oxygen compounds; organoheterocyclic compounds; nucleosides, nucleotides, and analogues; phenylpropanoids and polyketides; and others. Similarly, the 34 differential metabolites found by comparing the LCA group with the LPS group also fell into nine categories: benzenoids; lipids and lipid-like molecules; organoheterocyclic compounds; organic oxygen compounds; phenylpropanoids and polyketides; and other compound classes. The changes of differential metabolites of fatty acyl compounds in liver of LCA group and LPS group were as follows: the content of Prallethrin, Latanoprost, 1-linoleoyl-2-oleoyl-rac-glycerol increased; the content of Eicosapentaenoic acid decreased.

Figure 4. Volcano plot of differential metabolites. Note: A: NC group compared with LPS group; B: LPS group compared with LCA group.

Figure 4. Volcano plot of differential metabolites. Note: A: NC group compared with LPS group; B: LPS group compared with LCA group.

3.8. Correlation Analysis Between Enzyme Activity and Product Level Related to Lipid Metabolism in Liver of Piglets and Differential Metabolites

Pearson correlation analysis was performed on liver lipid metabolism-related enzyme activities and product levels with differential metabolites, and the results are shown in Figure 5. Compared with the NC group, the LPS group was significantly up-regulated in Mevalonic acid 5-pyrophosphate, Citrate, Palmitoyl carnitine. Stearoylcarnitine increased, lysophospholipids (LysoPC) increased, Leukotriene D4 was significantly up-regulated, Arachidonic acid and Prostaglandin F2α were significantly up-regulated, Pyruvate and Acetacetate increased, Adipoyl-carnitine increased (Fig.5A). Compared with LPS group, Leukotriene D4, Prostaglandin F2α and Arachidonic acid in LCA group were significantly decreased, and various glycerophospholipids (such as 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine) tended to be normal, Myristoleic acid. Monounsaturated fatty acids such as palmitoleic acid were more stable, and the proportion of Linoleic acid and Oleic acid was improved (Figure 5B). The results of the heat map of lipid metabolism in LPS and LCA groups showed that TC was positively correlated with Mevalonic acid 5-pyrophosphate, Pg 44: 12, Eicosapentaenoic acid; tG was positively correlated with 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, Pe 40.7. HDL-C was negatively correlated with 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine. LDL-C was positively correlated with 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine. TBA was positively correlated with Pe 38.6 and Myristoleic acid (Fig.5C).

4. Discussion

Studies have shown that obesity is associated with chronic systemic inflammation [21]. Excessive release of these inflammatory factors can damage the body’s tissues, causing damage to the structure and function of the intestine and liver, which affects the growth and health of animals [22]. At the same time, studies have also found that the metabolites of gut microbes, such as LPS, may promote tissue inflammation through the ‘gut–liver axis’ and ‘gut–fat axis’ [23]. In this study, high TG levels and NEFA accumulation were observed in the serum and liver samples of piglets in the LPS group. The increase in TG and NEFA content is one of the characteristics of dyslipidaemia, which is closely related to the occurrence of obesity. The cause of obesity is the formation and accumulation of fat, and the formation of fat is the enzymatic process of esterification of glycerol and NEFA to form TG [24]. Fat accumulation in adipose tissue occurs in the late stage of adipogenesis, which is associated with an increase in the TG concentration [25]. More evidence suggests that the accumulation of NEFAs is closely related to a series of health problems, such as obesity, insulin resistance and vascular diseases [26,27,28]. NEFA can induce insulin resistance to accumulate TG in cells and liver and can also produce low-grade inflammation in skeletal muscle, liver and adipose tissue by activating the NF-κB pathway, releasing pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6 [29]. Therefore, piglets injected with LPS may be in an immune stress state and produce inflammation, which increases the level of NEFA and negatively affects lipid metabolism in piglets.

In addition, the serum HDL-C level of piglets in the LPS group was lower, while the serum TBA content was higher, and the liver TC and LDL-C levels were higher. HDL-C is one of the main carriers of cholesterol in the blood and plays a key role in maintaining cholesterol homeostasis between the blood and peripheral tissues. It can transport excess cholesterol from the periphery to the liver for elimination through the reverse cholesterol transfer pathway [30,31,32]. Liver BA synthesis is the main metabolic pathway of cholesterol catabolism [33]. When hepatobiliary diseases occur, serum BA levels significantly increase, and high concentrations of BA are cytotoxic [34]. Primary BA is synthesised by cholesterol and binds to taurine or glycine in the liver, which is secreted into bile and further metabolised by the gut microbiota into secondary BAs [33]. In addition to its washing effect on intestinal lipid absorption, BA also acts as a ligand for a variety of nuclear receptors (NR), such as farnesoid X receptor (FXR, also known as NR1H4) [35,36,37]. Taken together, these findings suggest that the ability of piglets in the LPS group to remove excess lipids was weakened, which could also increase the risk of lipid metabolism-related diseases in piglets.

Conversely, in the LCA group, the addition of CAP resulted in the alleviation of excessive lipid (TG and NEFA) accumulation in the liver and serum, while the increase in HL activity and LPL activity in the liver led to an increase in lipid efflux, a significant decrease in the content of TC, LDL-C and TBA and a significant decrease in the content of LDL-C in the liver. HL plays a key role in the hydrolysis of TGs and promotes the uptake of HDL-C in the circulating blood [38,39]. An increase in HDL-C content can help prevent cardiovascular diseases [32]. The concentration of LDL-C is regulated by Low-Density Lipoprotein （LDLR） levels, and LDL-C in the blood can be absorbed by LDLR on the basal surface of intestinal cells or hepatocytes. Studies have shown that a decrease in LDL-C content can help inhibit body fat accumulation. For example, bamboo leaf extract can improve cholesterol metabolism, reduce TG and LDL-C content and inhibit abdominal fat deposition in broilers [40,41]. In a high-fat diet mouse model, dietary CAP supplementation significantly reduced TC, LDL-C and TG levels while increasing HDL-C [42]. Therefore, as previously mentioned, the addition of CAP to the diet can effectively alleviate the lipid metabolism disorder in weaned piglets under stress.

To further explore the molecular mechanism of CAP using lipid regulation, we determined the levels of genes and proteins related to FA and cholesterol metabolism. The hepatic uptake of circulating FA is largely dependent on three major FA transporters located in the plasma membrane of hepatocytes: fatty acid transporter (FATP), cluster of differentiation 36 (CD36) and caveolin [43,44]. Liver FA uptake was found to decrease in mice lacking FATP2 or FATP5 in the liver [45,46]. In this study, although there was no significant difference in the expression of CD36 between the CON and LPS groups, the mRNA expression of FABP2, FATP4 and FFAR3 in the LPS group was significantly higher than that in the CON group. However, the addition of CAP to the diet significantly decreased the expression of FABP2, FATP4 and FFAR3 in the LCA group. Therefore, it is speculated that the intake of FAs in piglets abnormally increases under LPS-induced immune stress, but the addition of CAP to the diet can effectively alleviate this problem.

The formation of FAS is mainly controlled by the sterol regulatory element-binding protein 1c and its downstream targets, acetyl-CoA carboxylase (ACC) and FASN. Initially, acetyl-CoA is transformed by ACC into malonyl-CoA, followed by the conversion of malonyl-CoA into palmitate by FASN. The new FAs can then undergo a series of desaturation, elongation and esterification steps, leading to storage in the form of TG or output in the form of VLDL particles [47]. Among these, ACC is the first rate-limiting enzyme that can convert acetyl-CoA to malonyl-CoA, and FASN is the key lipogenic enzyme that catalyses the final step of FA synthesis [48,49]. The newly synthesised FA will be used to synthesise TG, which is catalysed by DGAT1 and DGAT2 in the last step [50]. In addition, the liver X receptor (LXR) is a key regulator of mammalian lipid homeostasis. This transcription factor controls the expression of a series of genes involved in cholesterol uptake, transport, efflux and excretion in a tissue-dependent manner. The activation of LXRα can promote the synthesis of FAs [51,52]. In this study, LPS injection resulted in a higher mRNA expression of FASN, DGAT1 and LXRα in piglets, indicating that LPS-induced immune stress catalysed abnormal FA synthesis in piglets. By contrast, the abnormal expression of FASN, DGAT1 and LXRα mRNA in piglets in the LCA group supplemented with CAP in their diet was alleviated, indicating that piglets under immune stress have abnormal FA uptake and synthesis and that feeding CAP can alleviate this situation.

After uptake, FA is utilised by hepatocytes to produce ATP through β-oxidation. PPARα is a transcription factor involved in FA oxidation [47]. As one of the FA-activated NRs, PPARα can up-regulate the gene transcription of lipid catabolism-related proteins and plays a key role in the transcriptional regulation of peroxisome and mitochondrial FA oxidation-related genes (e.g. ACOX) [53,54,55]. As a key regulator of mitochondrial biosynthesis and energy metabolism, PGC-1α regulates many physiological processes, such as glucose metabolism and lipid metabolism, and targets the expression of PPARα [56]. Mitochondrial FA β-oxidation is the main pathway of FA catabolism and plays a vital role in maintaining systemic energy homeostasis [57]. As the rate-limiting enzyme of FA oxidation, carnitine palmitoyltransferase I (CPT1) catalyses the conversion of acetyl-CoA into acylcarnitine, which then penetrates the membrane into the mitochondria. PGC-1 also acts as a coactivator in the thyroid induction of CPT1A in the liver [58], highlighting the pleiotropic role of PGC-1 in lipid metabolism [59]. Different from the increase in FA uptake, the mRNA expression of PPARα in the LPS group was down-regulated, the protein expression of PPARα decreased, and the expression of PGC1α and CPT1α decreased. Therefore, we can infer that the increase in serum NEFA levels may be caused by the decrease in FA uptake and utilisation in LPS group piglets. However, the addition of CAP to the diet resulted in a significant up-regulation of PPARα, PGC1α and CPT1α protein expression in the LCA group, which effectively alleviated the adverse effects of immune stress on β-oxidation. In addition, ATGL can hydrolyse TAG into DAG and FA [60]. In this study, qPCR detection showed that the mRNA expression of ATGL in the LPS group decreased significantly, while the mRNA expression of ATGL in the LCA group increased significantly, indicating that CAP promoted the hydrolysis of TG to a certain extent. Therefore, we concluded that the addition of CAP to the diet greatly improved the FA metabolism of LPS-induced immune-stressed piglets, which could also be the reason for the decrease in NEFA content in serum compared with the LPS group.

In cholesterol metabolism, LXR works together with the sterol regulatory element binding protein 2 (SREBP2) pathway to maintain cellular and systemic sterol levels [51,61]. LXRs can act on target genes ABCG5 and ABCG8 to stimulate bile cholesterol excretion, thereby promoting the elimination of excess cholesterol [62], while SREBP2 can promote cholesterol biosynthesis by activating gene transcription that encodes the rate-limiting enzyme HMGCR [63]. In this study, although SREBP2 between the two groups did not differ significantly compared with the CON group, the mRNA expression of ABCG5 in the LPS group was down-regulated, and the protein expression of LXRα decreased. This indicates that the cholesterol excretion of piglets under immune stress was blocked, resulting in cholesterol accumulation, which could also be the reason for the increase in TC content in the liver of piglets in the LPS group. The addition of CAP in the diet up-regulated the mRNA expression of ABCG5 and the protein expression of LXRα in the LCA group, indicating that the blocked cholesterol excretion of piglets under immune stress was alleviated. In addition, the mRNA expression of HMGCR in piglets in the LCA group was down-regulated, implying that the addition of CAP to the diet could also inhibit the transcription of the HMGCR gene and reduce the synthesis of cholesterol, which could be the reason for the decrease in TC content in the serum of piglets in the LCA group. In addition to endogenous synthesis, another important source of cholesterol is intestinal absorption of cholesterol from the diet and bile [64]. Cholesterol obtained from the diet is absorbed in the intestinal lumen through small intestinal epithelial cells through NPC1L1, located on the surface of the intestinal lumen, and then this dietary cholesterol is released in the form of chylomicrons, which are absorbed by the liver [51,64,65]. Free cholesterol can be absorbed from the diet through the intestinal epithelial cells of the small intestine and by hepatocytes from the bile of the biliary tract. Excessive cholesterol is transported to the blood through ABCA1 or into the intestinal cavity and bile duct through ABCG5 and ABCG8 [32]. Intracellular cholesterol is esterified by ACAT2, mainly expressed in intestinal epithelial cells and liver cells in the endoplasmic reticulum, stored in lipid droplets or secreted as lipoproteins [32,64]. Compared with the CON group, the mRNA expression of ABCA1 and NPC1L in the LPS group was up-regulated, indicating that piglets in the LPS group could up-regulate NPC1L to absorb more dietary cholesterol through the intestine. Due to the high TC content in the liver of piglets in the LPS group under immune stress, more ABCA1 expression is needed to transport excess TC to the blood. Compared with piglets in the LPS group, those in the LCA group had lower mRNA expression of NPC1L, indicating that the addition of CAP to the diet could alleviate the excessive intake of dietary cholesterol in the intestine of piglets under stress. In addition, the mRNA expression of ACAT2 in the LCA group was down-regulated, indicating that the addition of CAP to the diet could reduce the esterification and storage of intracellular cholesterol.

BA is synthesised by a multi-step reaction catalysed by two different pathways in hepatocytes: the ‘classical’ (neutral) pathway and the ‘alternative’ (acidic) pathway [66]. The classical pathway of BA synthesis begins with the 7α-hydroxylation initiation of cholesterol through the rate-limiting enzyme cytochrome P450 family 7 subfamily A member 1 (CYP7A1) [67,68]. The alternative pathway is initiated by sterol 27-hydroxylase (CYP27A1), which oxidises cholesterol into a series of alcohol, aldehyde and carboxylic acid metabolites. The reaction is followed by oxysterol 7α-hydroxylation, mainly mediated by CYP7B1 [68,69]. Finally, the synthesised BA is secreted into the bile duct lumen by two ABC transporters (BSEP and MRP2) through the bile duct membrane [70]. The mRNA expression of CYP7A1, BSEP, NTCP, MRP2 and MRP4 in the LPS group was significantly lower than that in the CON group, which was consistent with the change in the TBA level in the liver. In the LCA group, the mRNA expression of CYP7A1 and MRP4 also increased. These results suggest that the decrease in BA levels in piglets injected with LPS under stress may be the result of impaired classical pathways and that the addition of CAP to the diet can effectively restore BA levels to normal levels by promoting classical pathways. In addition, previous studies have shown that increased CYP7A1 activity can down-regulate cholesterol synthesis through the SREBP pathway and enhance cholesterol efflux and elimination through LXR [71], which is consistent with the up-regulation of LXRα protein expression in the LCA group in a previous study.

Metabolomics is an important research method for analyzing the relationship between physiological state and metabolites, which can reveal the metabolic regulation mechanism of animal life activities. As the metabolic center in the body, the liver is closely related to the metabolism of carbohydrates, proteins and lipids, involving key life activities such as energy metabolism, detoxification, glucose and lipid metabolism [72]. Dai et al. (2022) found that the metabolites of CAP in the cecum of mice were mainly concentrated in steroids and their derivatives, organic oxygen compounds, fatty acyl, carboxylic acids and their derivatives, which was similar to the changes of liver metabolites after CAP treatment in this experiment [19].Among the differential metabolites between NC group and LPS group, Mevalonic acid 5-pyrophosphate was significantly up-regulated, suggesting the activation of cholesterol synthesis pathway; the increase of LysoPC may be caused by the activation of phospholipase A2 in the inflammatory response. Leukotriene D4, as a potent pro-inflammatory factor, is derived from arachidonic acid (AA) metabolism, and its significant up-regulation leads to an increase in inflammation-related lipids. The up-regulation of arachidonic acid is a marker of the initiation of the inflammatory cascade; the increase of Pyruvate and Acetacetate may indicate an increase in glycolysis or a compensatory increase in ketone body formation; the increase of Adipoyl-carnitine may reflect the abnormal oxidation of medium-chain fatty acids. Therefore, LPS induces immune stress, leading to imbalance of fatty acid synthesis and decomposition, phospholipid membrane damage and inflammatory lipid release, mitochondrial dysfunction, and abnormal energy metabolism. Among the differential metabolites between the LCA group and the LPS group, Leukotriene D4, Prostaglandin F2α, and Arachidonic acid were significantly reduced in the LCA group, indicating that capsaicin inhibited the arachidonic acid metabolic pathway and reduced the inflammatory response. A variety of glycerophospholipids (such as 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine) tended to normal levels in the LCA group, indicating the recovery of cell membrane stability; the improvement of the ratio of Linoleic acid and Oleic acid indicated that the n-6 / n-3 balance was improved and the pro-inflammatory environment was reduced. The changes of Taurocholic acid sulfate and Glycochenodeoxycholic acid may be related to the effect of capsaicin on the hepatoenteric axis, which indirectly regulates lipid metabolism.

In order to further determine the mechanism of CAP on liver lipid metabolism in immune-stressed piglets, the correlation between liver lipid metabolism related indicators and differential metabolites was analyzed. The results showed that Mevalonic acid 5-pyrophosphate was positively correlated with TC, TG and LDL-C.Mevalonic acid 5-pyrophosphate is the core node of cholesterol biosynthesis, and its up-regulation directly drives the increase of blood lipids. 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine is also positively correlated with TG, TC and LDL-C.This is an AA-rich phospholipid that is easily hydrolyzed by PLA2 to produce AA, which further promotes inflammation and forms a ' lipid metabolism-inflammation feedback loop '. Pe 40.7 was positively correlated with TG, TC and TBA, suggesting that it may be a new protective metabolite produced by capsaicin intervention, which has the effect of regulating lipid transport.

5. Conclusions

Based on the results of this study, we concluded that capsaicin CAP supplementation in the diet of LPS-induced immune stress weaned piglets can significantly improve lipid metabolism disorders. Due to the role of CAP, the PPARα-PGC1α-CPT1α pathway was activated, ATGL was up-regulated, FASN, DGAT1 and NPC1L were down-regulated, and FA metabolism was improved. By up-regulating the LXRα-ABCG5 pathway and FXR-dependent CYP7A1-MRP4 pathway, cholesterol efflux and bile acid metabolism homeostasis were significantly restored. Thus, the lipid metabolism homeostasis of weaned piglets under immune stress was remodeled. At the same time, in terms of metabolism, CAP achieves beneficial regulation of blood lipid profile (TC, TG, LDL-C) by down-regulating cholesterol synthesis precursors (such as MVA), reducing pro-inflammatory phospholipids (such as PA-PC), and regulating bile acid metabolism, and breaks the 'lipid metabolism-inflammation' interaction cycle, providing a new nutritional intervention strategy for alleviating immune stress-induced lipid metabolism disorders in piglets.