Weaning stress decrease growth performance and damage the immune function of piglets [1, 2, 17]. Supplementation of plant extracts improve growth performance, alleviate oxidant stress and improve immune function, thereby reduce the negative effects of weaning stress on piglets[18, 19]. LE exhibits antioxidant function [12–14] and immunomodulatory function on RAW264.7 cells and mice [11, 20–22], thus is a potential potential alternatives protection against early weaned stress in piglets.
Serum biochemical indicators reflects the metabolism and deposition of substances in the animal body. We investigated dietary LE supplementation on serum biochemical indicators of piglets. LE supplement reduced AST level in the serum of piglets (Table 2). Transaminase plays an important role in the process of amino acid metabolism, and AST acts as a indicator intracellular enzyme relative to liver function and be used as the index of liver damage [23]. Reduced serum AST level indicated improvement of the liver function of weaned piglets. Studies have revealed that licorice extract or its active ingredient glycyrrhizic acid have hepatoprotective function, and licorice extract inhibited the increase of serum AST and ALT in a liver injury rat model induced by carbon tetrachloride [24]. Glycyrrhetinic acid, a decomposition product of glycyrrhizic acid, improves the liver index of piglets and promoted liver development [25],and prevents the toxic accumulation of bile acids, and protect the liver from cholic acid [24], protects liver cells from oxidant damage induced by tert-butyl hydroperoxide and prevents cell death by inhibiting the reduction of intracellular glutathione and reactive oxygen species and depolarizing mitochondrial membrane [26]. Therefore, it not strange that LE increased the liver index of weaned piglets (Table 1), indicating a protective effect of LE on the liver function [27]. The activity of alkaline phosphatase (AKP) in serum reflects the metabolic efficiency of lipids. AKP is an isozyme with genetic markers. Animal performance are affected by AKP activity and the daily body weight gain is positively correlated with AKP activity [28]. In present study LE increased the serum AKP activity (Table 2), which is consistent with improvement of the growth performance of weaned piglets. Serum cholesterol reflects the absorption and metabolism of lipids by the animal body. 50 mg/kg LE reduced TC and HDL-C in serum (Table 2), similar results are found that supplementation of LE through drinking water reduces the serum glucose, low density lipoprotein cholesterol (L-DLC) and total cholesterol in broilers [29]. This decreases of serum TC and HDL-C may be related to the inhibition of lipid peroxidation by licorice, the active ingredient of licorice inhibits the formation of lipid peroxides, and in the subsequent liver clearance process increases the rate of conversion of cholesterol into bile acids, thereby reducing cholesterol and increased liver bile acid content [30]. LE had no effect on the spleen index and kidney index of weaned piglets (Table 1). Studies shown that licorice extract mainly affects liver and intestine of weaned piglets [20, 31].
Weaning stress results in oxidant stress, which induces a variety of diseases. We explored the effect of dietary LE on the antioxidant capacities in serum, liver and spleen. Dietary LE supplementation moderately increased activity of GSH-Px, SOD and T-AOC, and decreased MDA level in those tissues (Table 3). The antioxidant activity of LE may be attributed to its chemical components, including glycyrrhizin flavonoids, glycyrrhizin and polysaccharides [32, 33], which have multiple phenolic hydroxyl groups and benzene ring structures. Chalcone C attenuates the inflammation induced by lipopolysaccharide and interferon by reducing the expression of inducible NO and regulating the activity of antioxidant enzymes such as glutathione peroxidase [34] Glycyrrhizin inhibit mitochondrial lipid peroxidation and scavenges free radicals [12], Chalcone has a strong inhibitory effect on the production of superoxide anions, and also has a strong scavenging activity on free radicals[14]. The mechanism of antioxidant capacity of flavonoids compounds include inhibiting the generation of ROS by affect their conformation and inhibiting enzyme activity involved in their production, scavenging of ROS, or up-regulation or protection of antioxidant defense [35, 36]. Furthermore, flavonoids obviously inhibit the formation of malondialdehyde (MDA) and has a strong scavenging effect on hydroxyl radical and superoxide anion radical [33]. The LE used in present study contains 60% flavonoids, therefore supplementation of dietary LE enhanced the antioxidant capacity of weaned piglets.
To investigate whether LE exhibits immune-regulatory and anti-inflammatory properties in weaned piglets, we measured expression of 9 inflammation-related genes (ICAM-1, IL-1β, IL-2, IL-6, IL-8, IL-10, MCP-1, TNF-α and INOS) in liver, spleen and thymus. LE down-regulated the mRNA levels of 3 inflammatory genes (IL-6, TNF-α, ICAM-1) in liver(Fig. 1), TNF-α in spleen (Fig. 2) and 7 genes (IL-6, IL-8, IL-10, IL-1β, TNF-α, MCP-1 and ICAM-1) in thymus (Fig. 3). IL-6 and TNF-α were important mediator of inflammatory response, which were commonly used as markers for systemic pro-inflammatory cytokine activation [37], and TNF-a, IL-1β and IL-6 are also important inducers of acute phase proteins [38]. IL-2 is known to be associated with cellular immunity and play important roles in animal immune function [39]. IL-8 plays a key role in the recruitment and activation of neutrophils during inflammation [40]. The immune response can be regulated by the expression of INOS, ICAM-1 and MCP-1[41, 42], and ICAM-1 is an adhesion molecule that promotes the firm adhesion of leukocytes to endothelial cells [43], ICAM-1 and MCP-1 have a low basal expression in epithelial cells but is up-regulated in response to a variety of inflammatory mediators [44]. The down-regulated of those inflammation-related genes in liver,spleen and thymus indicated enhancement of immunity function of the weaned piglets by LE. Similar results are found that licorice extract reduces the secretion and mRNA levels of TNF-α, IL-6 and IL-1β in macrophages, and inhibits skin swelling and the expression of INOS and COX-2 in a mouse inflammation model [45]. Ural Glycyrrhiza reduces the release of NO and PGE2 in LPS-treated macrophages, and down-regulates mRNA levels of IL-6, TNF-α, IL-1β and COX-2 [11]. Chalcone A (LCA), the active ingredients of flavonoids in licorice extract, inhibits the release of cytokines in mouse [46]. IL-10 is an anti-inflammatory cytokine associated with humoral immunity [47]. The down-regulation on expression of IL-10 in thymus by LE (Fig. 3), may indicated that piglets were in a health condition and not need to expression more IL-10 to maintain the immune homeostasis (Fig. 3).
We further investigated the serum immunoglobulin levels of piglets. LE increased the serum IgG and had a tendency to increase the serum IgM (Table 4). IgG is accounting for about 80% of the total serum immunoglobulin, and play roles in resisting the invasion of viral infections [48]. The increase of serum IgG indicated enhancement of the serum immunity of weaned piglets by dietary LE. The active ingredients in licorice extract, such as glycyrrhizin flavonoids and glycyrrhizic acid, increase the phagocytic function of phagocytic cells and regulate the number and function of lymphocytes [49], thus prevent some inflammatory mediators by regulating immune-related signaling pathways or enzyme activities produced to activate macrophages, indirectly inhibit platelet aggregation [30], inflammatory cytokine secretion [30, 50] and neutrophil adhesion [43, 51], thus regulate the inflammatory response and improve immune function of piglets.