Effect of Cardamine violifolia on muscle protein degradation and anti-oxidative capacity in weaned piglets after Lipopolysaccharide challenge

Abstract

This study aimed to investigate the impact of Cardamine violifolia on muscle protein degradation, the inflammatory response and antioxidant function in weaned piglets following LPS challenge. Twenty-four weaned piglets were used in a 2 × 2 factorial experiment with dietary treatment (sodium selenite or Cardamine violifolia) and LPS challenge. After 28 days of feeding, pigs were injected intraperitoneally with 100 μg/kg LPS or saline. Dietary supplementation with Cardamine violifolia mitigated the reduction in insulin and growth hormone levels induced by LPS. It also curbed the LPS-induced elevation of plasma glucagon, urea nitrogen, and creatinine concentrations. Cardamine violifolia reduced muscle damage caused by LPS, as evidenced by increased protein content and protein/DNA ratio and decreased TNF-α and IL-1β mRNA expression. Furthermore, Cardamine violifolia modulated the expression of FOXO1, FOXO4, and MuRF1 in muscle, indicative of the protective effect against muscle protein degradation. Enhanced muscle antioxidant function was observed in the form of increased T-AOC, reduced MDA concentration, and decreased mRNA expression of GPX3, DIO3, TXNRD1, SELENOS, SELENOI, SELENOO, and SEPHS2 in LPS-treated piglets. The findings suggest that Cardamine violifolia supplementation can effectively alleviate muscle protein degradation induced by LPS and enhance the antioxidant capacity in piglets.

Introduction

Skeletal muscle is essential in pig production as it directly influences meat yield, quality, the well-being of the animal, and overall farm profitability. However, a variety of factors including weaning, pathogen infection and immunological stress in the breeding process contribute to rapid loss of muscle mass and myofibrillar proteins (muscle atrophy/wasting), and finally affect the growth performance of pigs.^1–3 Recent studies have indicated that muscle atrophy is strongly correlated with systemic inflammation. For example, patients with skeletal muscle atrophy-related diseases exhibit an elevated level of circulating endotoxins including C-reactive protein (CRP), TNF-α, and IL-6. ⁴ In addition, our previous studies have shown that intraperitoneal injection of lipopolysaccharide (LPS) into piglets for 4 h can upregulate the expression of muscle atrophy F-box (MAFbx) and muscle ring finger 1 (MuRF1) through increasing the secretion of inflammatory cytokines such as TNF-α, IL-1β and IL-6 in the muscle.^5,6 Therefore, the regulation of inflammation response may provide novel strategies for attenuating muscle atrophy and improving growth performance in pigs.

Selenium (Se), an essential trace nutrient for animals and humans, was first discovered by Swedish chemist Jons Jakob Berzelius in 1817. Se is widely distributed in the blood and various tissues, such as liver, heart, kidneys, and muscles. It exerts wide range of biological functions including antioxidant defense, anti-inflammation, cancer prevention, and immunity in animals through the synthesis of selenoproteins. ⁷ At present, 24–25 selenoprotein genes have been identified in humans, pigs and mice. Se deficiency has been linked to various muscle diseases, including muscular dystrophy and white muscle disease.^8–11 Se plays an important role in pig nutrition via participating in selenoprotein synthesis, which is central for the antioxidant system regulation. Compared to inorganic Se (i.e., sodium selenite), organic selenium (i.e., selenomethionine and Se-enriched yeast) has higher absorption and biological effectiveness. ¹² Dietary supplementation with Se-enriched yeast showed a higher Se deposition in muscles than sodium selenite, suggesting a vital role of organic Se in muscle antioxidant function. ¹³ Zhan et al. ¹⁴ reported that maternal selenomethionine intake of 0.3 mg/kg significantly increased the average weight gain of piglets from birth to weaning compared with sodium selenite. Collectively, organic Se exerts various functions in improving antioxidant capacity, immunity, growth, and meat quality in pigs, outperforming inorganic Se.

Cardamine violifolia, a newly discovered Brassicaceae plant, is known for its ability to hyperaccumulate Se. The Se content in its leaves exceed 1400 mg/kg dry weight, of which about 85% of Se is organic Se, such as MeSeCys and SeCys. This plant is also rich in a variety of nutrients, particularly proteins, vitamin C, and minerals. Recent studies have shown that Cardamine violifolia plays a vital role in modulating antioxidant defense, anti-fatigue, metabolism, and inflammation.^15–17 For example, dietary Cardamine violifolia supplementation increased Se concentration and decreased lipid accumulation in muscle of broilers by regulating triglyceride biosynthesis and utilization-related genes. ¹⁸ A study by Xu et al. ¹⁹ reported that Cardamine violifolia supplementation enhanced redness and decreased cooking loss in thigh muscles of boilers. Our previous study has reported that adding 0.3 mg/kg Se from Cardamine violifolia to the diet can improve growth performance, intestinal function, and antioxidant capacity in piglet. ²⁰ However, little research has been conducted to investigate the effects of Cardamine violifolia on muscle function in piglets.

Accordingly, we hypothesized that dietary Cardamine violifolia supplementation would protect against muscle injury by regulating antioxidant function in piglets. In this study, we used LPS to establish a model of muscle atrophy in piglets. The piglet model is regarded as an effective animal model for researching muscle diseases due to its anatomical and physiological parallels with the human gastrointestinal system. ²¹ Therefore, the objectives of this study were to investigate the effects of Cardamine violifolia on muscle protein degradation, the inflammatory response and antioxidant function in weanling piglets challenged by LPS. The experiment will provide important value for the application of Cardamine violifolia in livestock industry and human muscle-related diseases.

Materials and methods

Animal studies

Twenty four weaned castrated barrows (Duroc × Large White × Landrace, 35 ± 1 d of age, 9.96 ± 1.34 kg) were purchased from Hubei Oden Agriculture and Animal Husbandry Technology Co., Ltd and randomly allocated to four treatments: (1) Control group [piglets were fed 0.3 mg/kg Se from sodium selenite-supplemented diet and saline injection]; (2) SeCv group [piglets were fed 0.3 mg/kg Se from Cardamine violifolia-supplemented diet and saline injection]; (3) LPS group [piglets were fed 0.3 mg/kg Se from sodium selenite-supplemented diet and LPS injection]; (4) LPS + SeCv group [piglets were fed 0.3 mg/kg Se from Cardamine violifolia-supplemented diet and LPS injection]. Each treatment had six replicates, and each replicate had one piglet. Piglets were individually housed in a 22–25°C controlled house with free access to feed and water. A corn-soybean diet was formulated to meet the nutrient requirements of National Research Council (2012), which is shown in Supplementary Table 1. Piglets were fed the experimental diets for 28 days and then intraperitoneally injected with LPS (E. coli serotype 055: B5, Sigma) at 100 μg/kg body weight (BW) or the equivalent amount of sterile saline once. The dosage of LPS and challenge time were used in accordance with our previous studies.^5,22 Dried Cardamine violifolia powder (1430 mg/kg total Se content) and sodium selenite were obtained from Enshi Se-Run Material Engineering Technology Co. (Enshi, China) and mixed with other trace element premixes (ZnSO₄, FeSO₄, MnSO₄ and CuSO₄). Then, mineral premixes mixed with other ingredients such as maize and soybean meal to process into pellet feed.

Sample collection

After 28 days feeding trial, piglets were challenged with LPS or sterile saline for 4 h, and blood samples were then collected in heparinized vacuum tubes and centrifuged to obtain plasma for analysis. Following blood collection, all pigs were sacrificed under anesthesia with an intravenous injection of sodium pentobarbital (80 mg/kg BW) and the muscle samples were collected instantly. The longissimus dorsi muscle was collected from the 10th rib (right side of the carcass). All muscle samples were frozen in liquid nitrogen and stored at −80˚C for subsequent analysis.

Plasma hormone parameter determination

The concentrations of insulin (No. F01PZA), cortisol (No. D10PZA), glucagon (No. F03PZA) and growth hormone (GH, No. B12PZA) in plasma were determined by commercial ¹²⁵I RIA assay kits (Beijing North Institute of Biological Technology). Plasma glucose, urea nitrogen, creatinine (CREA) and total protein (TP) were measured using the commercial kits sourced from Shanghai Kehua Bio-Engineering Co. (Shanghai, China) by a Hitachi 7100 Automatic Biochemical Analyzer (Hitachi, Japan).

Protein, DNA and RNA content determination

Muscle samples were processed using a PT-3100D tissue homogenizer (Kinematica, Malters, Switzerland) in a 1:10 (w/v) ratio with ice-cold PBS. The protein concentration in these homogenates was quantified following a recognized protocol, employing a detergent-compatible protein assay from Bio-Rad Laboratories, with bovine serum albumin as a reference standard. Additionally, DNA and RNA concentrations in muscle were measured by UV spectrophotometry method.

Muscle morphologic analysis

Pig longissimus dorsi muscle were initially fixed in 4% paraformaldehyde at room temperature for 24 h. After dehydration in graded ethanol (70%, 80%, 95% and 100%), diaphanization was performed with xylene, and then, the tissues were embedded in paraffin. Finally, the muscle tissue blocks were longitudinally cut into 5 μm sections along the muscle fibers on a rotary microtome Microm HM340E (Thermo Scientific, Waltham, MA, USA). For the histological study, the sections from 3 piglets were stained by hematoxylin and eosin and examined by a Leica DM3000 microsystems (Leica, Milton Keynes, UK). The morphological characteristics of the longissimus dorsi muscle were observed under 400× magnification of microscope.

Antioxidant parameter determination

Quantitative assessment of the muscle's antioxidative properties, specifically total antioxidant capacity (T-AOC, No. A015-1), superoxide dismutase (SOD, No. A001-1), glutathione peroxidases (GSH-PX, No. A005-1), and malondialdehyde (MDA, No. A003-1) concentrations, was executed utilizing commercial assay kits sourced from Nanjing Jiancheng Bioengineering Institute according to the manufacturer's procedures. Total protein concentration in muscle was determined employing the bicinchoninic acid protein assay kit (Beyotime, Beijing). The enzymatic activities of T-AOC, SOD, and GSH-PX were expressed in units per milligram (U/mg) of total protein, and MDA concentration was expressed as nanomoles per milligram (nmol/mg) of total protein in muscle.

RNA extraction and quantitative RT-PCR

Gene expression quantification was executed as delineated in our prior publication. ²³ Total RNA extraction from muscle samples was accomplished using the Trizol reagent from TaKaRa Biotechnology. The integrity and purity of RNA were assessed through agarose gel electrophoresis and spectrophotometric analysis. Subsequently, reverse transcription of RNA to cDNA was performed using the PrimeScript^® RT reagent kit (No. RR047A, TaKaRa), according to the manufacturer's protocol. Quantitative real-time PCR for target genes was conducted using the ABI 7500 Real-Time PCR System and SYBR^® Premix Ex TaqTM kit (No. RR420A, TaKaRa). The PCR cycling protocol included an initial denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 5 s and annealing/extension at 60°C for 34 s. Primers for target genes were designed using Primer Premier 6.0 and shown in Supplementary Table 2. Relative mRNA expression of each target gene was normalized to the housekeeping gene β-actin using the 2^−△△Ct method. ²⁴

Western blot analysis

Total protein extraction from muscle samples was conducted using the KeyGEN commercial kit, following the manufacturer's protocol. The protein samples underwent electrophoresis on a 10% SDS-PAGE gels, after which they were transferred to PVDF membranes. Then, these membranes were blocked with 5% bovine serum albumin solution in TBS/Tween-20 buffer and incubated sequentially with primary and secondary antibodies. Specific primary polyclonal antibodies included rabbit anti-Akt (No. 9272S, Cell Signaling Technology), rabbit anti-phospho-Akt (No. 9271S, Cell Signaling Technology), rabbit anti-FOXO1 (No. 9454S, Cell Signaling Technology), rabbit anti-phospho- FOXO1 (No. 9464S, Cell Signaling Technology), and mouse anti-β-actin (Sigma-Aldrich). The protein bands were visualized with the Clarity Max Western ECL Substrate (Bio-Rad) and quantified in the Image J software. The abundance of phosphorylated target protein was normalized to total protein content.

Statistical analysis

All results are presented as mean ± SEM and analyzed using GraphPad Prism 8. Initially, data were examined with a 2 × 2 factorial ANOVA employing the general linear model procedures in SPSS version 22.0 (SPSS Inc., Chicago, IL, USA). The statistical model included the main effects of challenge (saline or LPS), diet (sodium selenite or Cardamine violifolia), and their interaction. Subsequently, differences between groups were evaluated using Duncan's multiple comparison test. A p-value of ≤ 0.05 was considered statistically significant, while p-values between 0.05 and 0.10 were regarded as trends.

Results

Effects of Cardamine violifolia on plasma hormone parameters in piglets challenged with LPS

To investigate the impact of Cardamine violifolia on muscle function, we pretreated piglets with 0.3 mg/kg Se from sodium selenite or Cardamine violifolia in the basal diet. Throughout the 28-d feeding trial, we found that supplementation with Cardamine violifolia significantly increased the amount of Se in longissimus dorsi muscle and gastrocnemius muscle (Supplementary Figure 1). Then, we intraperitoneally injected LPS into pigs to establish muscle damage model. LPS-challenged piglets exhibited fever, anorexia, inactivity, shivering, diarrhea, and vomiting (data no shown). We analyzed plasma hormone levels associated with muscle function in piglets following LPS challenge. As depicted in Figure 1, pigs subjected to LPS exhibited elevated levels of cortisol, glucagon, urea nitrogen, and CREA (P < 0.05), alongside reduced insulin, GH, glucose, and TP levels (P < 0.05) in plasma, compared to those treated with saline. Pigs fed with SeCv displayed lower plasma glucagon level (P < 0.05) and a tendency towards increased insulin level (P = 0.061) compared to those on the basal diet. We observed no significant LPS × diet interaction for insulin, cortisol, glucagon, GH, glucose, and CREA levels. However, an LPS × diet interaction was noted for urea nitrogen (P < 0.05), and a trend was observed for TP (P = 0.068) in plasma. Dietary supplementation with SeCv resulted in increased plasma insulin and GH levels, and decreased glucagon, urea nitrogen, and CREA levels in LPS-challenged pigs, whereas no effect was observed in saline-injected pigs.

Figure 1.

Effects of Cardamine violifolia on plasma hormone parameters in piglets challenged with LPS. Blood samples were collected for analysis after 4 h challenge with LPS or sterile saline. (A-H) Plasma insulin, cortisol, glucagon, growth hormone (GH), glucose, urea nitrogen, creatinine (CREA) and total protein (TP) concentrations. Values are means ± SEM, n = 6. All data were analyzed using a 2 × 2 factorial ANOVA with the general linear model procedures. Differences among groups were evaluated using Duncan's multiple comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Effects of Cardamine violifolia on muscle protein, DNA and RNA contents in piglets challenged with LPS

We further assessed muscle histopathological changes in piglets using H&E staining. LPS-challenged pigs displayed muscle-fiber-free nuclei, membrane rupture, and muscle-fiber dissolution (Figure 2A), whereas these pathological features were significantly alleviated in LPS + SeCv group. We then investigated muscle protein degradation, a hallmark of cachexia, by measuring the protein, DNA and RNA concentrations in muscle. As depicted in Figure 2B-2F, LPS-challenged pigs showed a lower DNA content (P < 0.05) and tended to have a higher RNA/DNA ratio (P = 0.085) compared to those treated with saline. Pigs receiving SeCv displayed higher protein content and protein/DNA ratio (P < 0.05), and tended to have elevated RNA content (P = 0.064) and RNA/DNA ratio (P = 0.070) in muscle, in comparison to those on the basal diet. No significant LPS × diet interaction was observed for muscle protein, DNA, and RNA contents. Dietary SeCv supplementation counteracted the LPS-induced decrease in muscle protein content and protein/DNA ratio.

Figure 2.

Effects of Cardamine violifolia on muscle protein, DNA and RNA contents in piglets challenged with LPS. (A) Representative morphological characteristics of H&E staining of longissimus dorsi muscle (magnification: 200×, scale bar: 50 μm); (B-F) The concentrations of protein, DNA and RNA, as well as RNA/DNA ratio and protein/DNA ratio in muscle. Values are means ± SEM, n = 6. All data were analyzed using a 2 × 2 factorial ANOVA with the general linear model procedures. Differences among groups were evaluated using Duncan's multiple comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Effects of Cardamine violifolia on muscle inflammatory response in piglets challenged with LPS

The inflammatory response plays a key role in triggering muscle atrophy in cases of cachexia. ²⁵ To investigate the effect of Cardamine violifolia on the inflammatory response in muscle, we measured the mRNA expression of TNF-α, IL-6, and IL-1β in the longissimus dorsi muscle. As shown in Figure 3, LPS-challenged pigs exhibited higher mRNA expression of TNF-α IL-6, and IL-1β in muscle compared to saline-treated pigs (P < 0.05). Pigs fed with SeCv exhibited lower IL-1β mRNA expression in muscle than those fed with the basal diet (P < 0.05). There was an LPS × diet interaction for IL-1β (P < 0.05), and a trend for LPS × diet interaction was observed for TNF-α in muscle (P = 0.067). Dietary SeCv supplementation decreased TNF-α and IL-1β mRNA expression in LPS-treated pigs, whereas no effect was observed in saline-injected pigs.

Figure 3.

Effects of Cardamine violifolia on muscle IL-6, IL-1β and TNF-α mRNA expression in piglets challenged with LPS. (A-C) Relative mRNA expression of TNF-α, IL-6 and IL-1β in muscle. Values are means ± SEM, n = 6. All data were analyzed using a 2 × 2 factorial ANOVA with the general linear model procedures. Differences among groups were evaluated using Duncan's multiple comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Effects of Cardamine violifolia on muscle Akt/FOXO signaling pathway in piglets challenged with LPS

To further confirm the effect of Cardamine violifolia on protein degradation signaling pathway in muscle, we evaluated the key components of the Akt/FOXO signaling pathway. As shown in Figure 4, compared to pigs treated with saline, pigs treated with LPS exhibited higher mRNA expressions of Foxo1 and MuRF1, as well as higher protein level of p-FOXO1/t-FOXO1. Conversely, there was a lower protein expression of p-Akt/t-Akt in muscle. Pigs fed with SeCv showed lower MAFbx mRNA expression and tended to have lower MuRF1 mRNA expression (P = 0.069) in muscle compared to those fed the basal diet. An LPS × diet interaction was observed for Foxo4 mRNA expression and p-FOXO1/t-FOXO1. However, no LPS × diet interaction was noted for mRNA expressions of Akt, Foxo1, MuRF1, and MAFbx in muscle. Dietary SeCv supplementation reduced Foxo1, Foxo4, and MuRF1 mRNA expressions, as well as p-FOXO1/t-FOXO1 protein expression in LPS-treated pigs, while no effect was evident in saline-injected pigs.

Figure 4.

Effects of Cardamine violifolia on muscle Akt/FOXO signaling pathway in piglets challenged with LPS. (A-E) Relative mRNA expression of Akt, Foxo1, Foxo4, MuRF1 and MAFbx in muscle. (F) Representative protein bands of p-Akt, t-Akt, p-FOXO1 and t-FOXO1. (G-H) Protein expression of p-Akt and p-FOXO in muscle. Values are means ± SEM, n = 6. All data were analyzed using a 2 × 2 factorial ANOVA with the general linear model procedures. Differences among groups were evaluated using Duncan's multiple comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Effects of Cardamine violifolia on muscle antioxidant parameters in piglets challenged with LPS

We subsequently evaluated the effect of Cardamine violifolia on the muscle anti-oxidative capacity in piglets after the LPS challenge. As depicted in Figure 5, LPS-challenged pigs showed higher MDA concentration and lower SOD activity in muscle compared to those treated with saline (P < 0.05). Pigs fed with SeCv exhibited higher T-AOC and lower MDA concentration than those on the basal diet (P < 0.05). There was a significant interaction between LPS and diet for both T-AOC and SOD activities (P < 0.05), and a trend for LPS × diet interaction was observed for MDA concentration (P = 0.077). Dietary supplementation with SeCv increased muscle T-AOC and reduced MDA concentration in LPS-treated pigs, but had no such effect in saline-injected pigs. Neither SeCv nor LPS treatment significantly influenced GSH-PX activity.

Figure 5.

Effects of Cardamine violifolia on muscle antioxidant parameters in piglets challenged with LPS. (A) Malondialdehyde (MDA) concentration; (B) Total antioxidant capacity (T-AOC); (C) superoxide dismutase (SOD) activity; (D) glutathione peroxidase (GSH-Px) activity. Values are means ± SEM, n = 6. All data were analyzed using a 2 × 2 factorial ANOVA with the general linear model procedures. Differences among groups were evaluated using Duncan's multiple comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Effects of Cardamine violifolia on muscle selenoprotein mRNA expression in piglets challenged with LPS

As shown in Figure 6, LPS-challenged pigs exhibited higher mRNA expressions of iodothyronine deiodinase 2 (DIO2), DIO3 and thioredoxin reductase 1 (TXNRD1) (P < 0.05) and lower mRNA expression of selenoprotein P (SELENOP) (P < 0.05) compared to saline-treated pigs. These pigs also tended to have higher mRNA expressions of glutathione peroxidase 2 (GPX2) (P = 0.060) and selenoprotein I (SELENOI) (P = 0.079) in muscle. Pigs fed with SeCv displayed lower mRNA expressions of GPX3, TXNRD1, and selenoprotein O (SELENOO) (P < 0.05), and tended to have lower expression of selenoprotein S (SELENOS) (P = 0.097), SELENOI (P = 0.092), and selenophosphate synthetase 2 (SEPHS2) (P = 0.087) compared to those on the basal diet. An LPS × diet interaction was observed for DIO3 mRNA expression (P < 0.05), and a trend for this interaction was observed for SELENOS, SELENOI, and SELENOO mRNA expression. Dietary SeCv supplementation reduced the mRNA expression of GPX3, DIO3, TXNRD1, SELENOS, SELENOI, SELENOO and SEPHS2 in LPS-treated pigs, with no significant differences in saline-treated pigs. Neither LPS nor diet significantly affected the mRNA expressions of GPX1, DIO1, TXNRD2, selenoprotein X (SELENOX), and selenoprotein N (SELENON) in muscle.

Figure 6.

Effects of Cardamine violifolia on muscle selenoprotein mRNA expression in piglets challenged with LPS. (A-O) Relative mRNA expression of GPX1, GPX2, GPX3, DIO1, DIO2, DIO3 TXNRD1, TXNRD2, SELENOS, SELENOI, SELENOO, SELENOX SELENOP, SELENON and SEPHS2 in muscle. Values are means ± SEM, n = 6. All data were analyzed using a 2 × 2 factorial ANOVA with the general linear model procedures. Differences among groups were evaluated using Duncan's multiple comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Discussions

Here, we used weaned piglets as a model to investigate the effect of Cardamine violifolia on muscle protein degradation after LPS challenge. Previous research has demonstrated that LPS challenge can decrease the diameter of myofibers and myotubes, and increase myosin heavy chain degradation, ultimately leading to muscle atrophy. ²⁶ In our experiment, we initially analyzed plasma biochemical parameters, including insulin, glucagon, glucose, GH, and urea nitrogen, which are key indicators for evaluating muscle function. ² Insulin is responsible for glucose uptake, carbohydrate oxidation, and protein synthesis. Blood glucose levels, which are regulated by the balance between glucagon and cortisol levels, are associated with the composition of muscle fibers.^6,27 Our findings indicated that LPS challenge led to a decrease in insulin, GH, and glucose concentrations, while increasing glucagon and cortisol concentrations in plasma. However, independent of LPS treatment, Cardamine violifolia increased insulin concentration and reduced glucagon concentration in plasma. Plasma urea nitrogen is an indirect indicator of muscle protein degradation and is negatively correlated with muscle growth. ²⁸ Our study revealed that LPS increased the plasma urea nitrogen concentration, while Cardamine violifolia decreased it after LPS challenge. Similarly, Wei et al. ²⁹ reported that supplementation with Cardamine violifolia significantly decreased the content of plasma urea nitrogen in the broilers challenged with LPS. Taken together, these results suggest that Cardamine violifolia may attenuate LPS-induced muscle atrophy by protecting muscle protein from degradation.

The protein content, RNA, and DNA levels are commonly used to evaluate the capacity for protein synthesis, translational efficiency, and muscle mass. ³⁰ The protein: DNA ratio is a sensitive indicator of muscle protein mass. ³¹ The RNA: DNA ratio reflects the cell capacity for protein synthesis. ³⁰ In this study, LPS challenge reduced protein content in the muscle, indicating that LPS could lead to muscle atrophy. Independent of LPS treatment, Cardamine violifolia increased the protein content and protein: DNA ratio in the muscle. These data indicates that Cardamine violifolia increased muscle protein mass both before and after LPS challenge. Moreover, independent of LPS treatment, Cardamine violifolia tended to increase RNA concentration and the RNA: DNA ratio in the muscle, suggesting that Cardamine violifolia stimulated the growth of muscle independent of LPS challenge.

Several evidence have demonstrated a strong correlation between muscle atrophy and inflammatory response. ⁴ Pro-inflammatory cytokines can lead to muscle wasting directly or via alterations of AKT/FOXO/UPP pathway. ³¹ Similar to our previous studies, LPS challenge induced inflammatory cell infiltration and stimulated the secretion of pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β. However, dietary supplementation with Cardamine violifolia decreased the mRNA expression of muscle IL-1β in LPS-challenged piglets. It has been reported that Se-enriched peptides from Cardamine violifolia decreased plasma concentrations of IL-6, IL-1β, and TNF-α and alleviated high-fat diet-induced systematic inflammation in mice. ³² Dietary supplementation with Cardamine violifolia also relieved the increase in TNF-α and IL-6 expression caused by D-galactose in the hippocampus. ¹⁵ In addition, our previous study showed that Cardamine violifolia supplementation decreased plasma IL-6 level in LPS-challenged pigs.^33,34 The mechanism by which Se supply suppresses inflammation involves several interconnected biochemical pathways, largely related to its role as an essential component of antioxidant enzymes and its influence on immune cell function. Se is a critical component of selenoproteins, particularly GPXs, TXNRDs, and SELENOP, which play vital roles in reducing oxidative stress. By enhancing antioxidant defense, Se helps to neutralize reactive oxygen species (ROS), thereby preventing the activation of several inflammatory signaling pathways, including the NF-κB pathway. ³⁵ Additionally, Se influences the immune response by affecting the function of immune cells, including macrophages, T-cells, and neutrophils. These immune cells are central to the inflammatory process. Se has been reported to enhance immune cell function by improving the production of anti-inflammatory cytokines and promoting the resolution of inflammation. ³⁶ These data support the notion that dietary Cardamine violifolia supplementation may suppress inflammation primarily through its role in antioxidant defense, regulation of immune cell function.

AKT is a critical signaling protein that stimulates protein synthesis and leads to skeletal muscle hypertrophy. It inhibits muscle protein degradation by phosphorylating and inactivating FOXO transcription factor family members.^37,38 This phosphorylation and inactivation of FOXO results in transcriptional inhibition of FOXO target genes, including MAFbx and MuRF1, leading to a decrease in protein degradation. Although the administration of LPS is known to affect AKT signaling and induce muscle protein degradation.^1,5,39 Our results showed that LPS-induced muscle atrophy is independent of AKT signaling. Notably, LPS challenge increased the mRNA expression of FOXO1 and MuRF1 in the muscle, whereas supplementation with Cardamine violifolia decreased the mRNA expression of MuRF1 and MAFbx. A previous study reported that increased expression of MAFbx and MuRF1 has been observed in several models of muscle atrophy. ⁴⁰ These data suggest that Cardamine violifolia alleviates muscle protein degradation by affecting the UPP signaling pathway. However, its inclusion could have no effect on the AKT/FOXO pathway, as evidenced by the unchanged mRNA expression of AKT and FOXO1/4.

Oxidative stress is closely associated with inflammation, which is a common cause of muscle atrophy and injury. ⁴¹ MDA is a product of lipid peroxidation and is considered as an important indicator for reflecting oxidative stress. Antioxidant enzymes, including SOD, T-AOC, and GSH-PX, are crucial components in maintaining the redox balance. A previous study showed that LPS induced muscle injury by enhancing oxidative stress. ⁴² Similarly, we found that LPS increased the oxidative stress product MDA and decreased SOD activity in the muscle. However, supplementation with Cardamine violifolia improved muscle T-AOC, and reduced MDA concentration in LPS-challenged piglets. Wei et al. ²⁹ reported that Cardamine violifolia ameliorated LPS-induced oxidative stress in broilers, as indicated by increased T-AOC and GSH-PX activity and decreased MDA concentration in plasma. Meanwhile, comparative studies on chicken breast or thigh muscle tissues in broiler fed diets with inorganic Se (Na₂SeO₃), organic Se (Se-enriched yeast, Se-enriched plants) showed that organic Se more effectively enhanced muscle resistance to oxidative stress, delayed meat oxidation, reduced lipid levels, and maintained meat freshness than inorganic Se. ¹⁸ The antioxidant function of selenium mainly depends on the catalytically active sites within selenoprotein families. A total of 25 selenoproteins are present in piglets. Among selenoproteins, SELENON is highly expressed in skeletal muscle and plays a critical role in muscle biology. ¹¹ SELENON is implicated in muscle injury and muscle diseases, especially in relation to muscle degeneration. Its expression in muscle cells suggests a protective role, particularly in the face of oxidative stress and injury. SELENON helps counteract oxidative stress in muscle tissues, particularly in the context of exercise or other stressors that induce muscle damage. ⁴³ This antioxidant property is crucial in preventing muscle fiber degeneration and ensuring the structural integrity of muscle tissues. However, our study revealed that LPS stimulation does not affect the expression of SELENON in muscle, suggesting that other selenoproteins may be involved in LPS-induced muscle injury. Our results showed that LPS challenge upregulated the mRNA expression of DIO2, DIO3, TXNRD1, SELENOI, and downregulated SELENOP in the muscle. Our previous study also has reported that LPS challenge upregulated mRNA expression of SELENOS, DIO2, GPX3, TXNRD1, TXNRD3, SELENOK, SELENOT, SELENOI and GPX1 in the spleen, thymus, and lymph node. ⁴⁴ The increase in selenoprotein expression following LPS exposure can be explained by the body's response to acute inflammation, where selenoproteins are upregulated as part of a protective mechanism to mitigate oxidative stress and maintain redox balance. This is in contrast to the decreased expression of selenoproteins often observed in chronic inflammatory conditions, where sustained inflammation may lead to selenium depletion or dysregulation of selenoprotein synthesis. ⁴⁵ In addition, the effect of LPS on selenoprotein expression might also vary depending on tissue type. While LPS may suppress selenoprotein expression in certain tissues, such as the liver, ⁴⁵ our findings could reflect a muscle-specific response where selenoproteins play a more direct role in protecting cells from inflammation-induced damage. The regulation of selenoproteins can vary based on local tissue requirements, as different tissues may experience different degrees of oxidative stress and inflammation in response to LPS. In addition, Cardamine violifolia supplementation moderately recovered the mRNA expression of DIO3, SELENOS, and SELENOI in LPS-treated piglets. Although these selenoproteins might be involved in LPS-induced oxidative stress, the exact mechanism of Cardamine violifolia regulation on selenoprotein expression remains to be determined. Recently, several studies have indicated that the expression of key selenoprotein family members can significantly affect various intracellular processes of myogenesis. For instance, SELENOW not only contains binding sites for MyoD, which regulate its activity, but also prevents excessive autophagy, apoptosis, and necrosis of broiler myoblasts. These significant finding provides a theoretical reference for the molecular mechanism of how Cardamine violifolia protect against muscle atrophy via enhancing selenoprotein expression.

In summary, dietary supplementation with Cardamine violifolia demonstrates a capacity to mitigate LPS-induced muscle atrophy and injury. The favorable effects observed in muscle tissue may be attributed to the prevention of alterations in the AKT/FOXO signaling pathway and the enhancement of antioxidative capacity. However, our study still has limitations. One notable limitation is that measurements were taken at 4 h post-LPS challenge. While this approach is commonly employed in in vivo studies to capture the peak inflammatory response, it may not fully represent the beneficial effects of Cardamine violifolia. Additionally, while the molecular mechanisms underlying the observed effects were partially elucidated through the analysis of key genes and pathways, further investigation is needed to fully understand the broader regulatory networks involved. Advanced approaches such as transcriptomic or proteomic analyses could be employed to identify additional molecular targets and pathways influenced by Cardamine violifolia. Despite these limitations, our findings support the potential of Cardamine violifolia supplementation as a nutritional intervention strategy to mitigate LPS-induced muscle atrophy and related disorders.

Supplemental Material

sj-docx-1-ini-10.1177_17534259251322589 - Supplemental material for Effect of Cardamine violifolia on muscle protein degradation and anti-oxidative capacity in weaned piglets after Lipopolysaccharide challenge

Supplemental material, sj-docx-1-ini-10.1177_17534259251322589 for Effect of Cardamine violifolia on muscle protein degradation and anti-oxidative capacity in weaned piglets after Lipopolysaccharide challenge by Nianbang Wu, Shunkang Li, Yanling Kuang, Wensheng He, Huiling Zhu, Qingyu Gao, Liping Liu, Shuiyuan Cheng, Yulan Liu, Xin Cong and Dan Wang in Innate Immunity