Glycyrrhizin inhibits the inflammatory response in mouse mammary epithelial cells and a mouse mastitis model Yunhe Fu, Ershun Zhou, Zhengkai Wei, Dejie Liang, Wei Wang, Tiancheng Wang, Mengyao Guo, Naisheng Zhang and Zhengtao Yang Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Jilin University, Changchun, Jilin Province, PR China

Keywords glycyrrhizin; lipid raft; mastitis; nuclear factor-jB; Toll-like receptor 4 Correspondence Z. Yang, Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Jilin University, Changchun, Jilin Province 130062, PR China Fax: +86 431 87835140 Tel: +86 431 87835140 E-mail: [email protected] (Received 20 October 2013, revised 22 March 2014, accepted 31 March 2014) doi:10.1111/febs.12801

Glycyrrhizin, a triterpene glycoside isolated from licorice root, is known to have anti-inflammatory activities. However, the effect of glycyrrhizin on mastitis has not been reported. The purpose of this study was to investigate the anti-inflammatory effect and mechanism of action of glycyrrhizin on lipopolysaccharide (LPS)-induced mastitis in mouse. An LPS-induced mouse mastitis model was used to confirm the anti-inflammatory activity of glycyrrhizin in vivo. Primary mouse mammary epithelial cells were used to investigate the molecular mechanism and targets of glycyrrhizin. In vivo, glycyrrhizin significantly attenuated the mammary gland histopathological changes, myeloperoxidase activity and infiltration of neutrophilic granulocytes and downregulated the expression of tumor necrosis factor-a, interleukin (IL)-1b and IL-6 caused by LPS. In vitro, glycyrrhizin dose-dependently inhibited the LPS-induced expression of tumor necrosis factor-a, IL-6, and RANTES. Western blot analysis showed that glycyrrhizin suppressed LPS-induced nuclear factor-jB and interferon regulatory factor 3 activation. However, glycyrrhizin did not inhibit nuclear factor-jB and interferon regulatory factor 3 activation induced by MyD88-dependent (MyD88, IKKb) or TRIF-dependent (TRIF, TBK1) downstream signaling components. Moreover, glycyrrhizin did not act though affecting the function of CD14 or expression of Toll-like receptor 4. Finally, we showed that glycyrrhizin decreased the levels of cholesterol of lipid rafts and inhibited the translocation of Toll-like receptor 4 to lipid rafts. Moreover, glycyrrhizin activated ATP-binding cassette transporter A1, which could induce cholesterol efflux from lipid rafts. In conclusion, we find that the antiinflammatory effects of glycyrrhizin may be attributable to its ability to activate ATP-binding cassette transporter A1. Glycyrrhizin might be a useful therapeutic reagent for the treatment of mastitis and other inflammatory diseases.

Introduction Mastitis is an inflammatory reaction of the mammary gland that can be caused by > 150 different types of pathogen. Gram-negative bacteria are the most

frequent causative agents of severe mastitis [1]. Lipopolysaccharide (LPS) has been reported to be an important risk factor for mastitis [2]. Toll-like recep-

Abbreviations ABCA1, ATP-binding cassette transporter A1; DEX, dexamethasone; HE, hematoxylin and eosin; HRP, horseradish peroxidase; IFN, interferon; IKKb, IjB kinase b; IL, interleukin; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; MMEC, mouse mammary epithelial cell; MPO, myeloperoxidase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; MbCD, methyl-b-cyclodextrin; NFjB, nuclear factor-jB; SEM, standard error of the mean; TBK1, TANK-binding kinase 1; TLR, Toll-like receptor; TNF, tumor necrosis factor; TRIF, TIR domain-containing adaptor inducing interferon-b.

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tors (TLRs) constitute a large family of type I transmembrane receptors that play an important role in driving host inflammatory responses and promoting adaptive immunity following infection. TLR4 is one of the best characterized TLRs [3–5]. Upon stimulation by LPS, TLR4 is recruited to lipid rafts and interacts with its adaptor molecules, leading to activation of MyD88-dependent and TIR domain-containing adaptor inducing interferon (IFN)-b (TRIF)-dependent signaling pathways. The MyD88-dependent and TRIFdependent pathways could activate nuclear factor-jB (NF-jB) and interferon regulatory factor 3 (IRF3) to induce the production of inflammatory cytokines [6,7]. The activation of TLR4 by LPS induces NF-jB and IRF3 activation, resulting in the release of cytokines [8]. Lipid rafts are membrane microdomains enriched in cholesterol, sphingolipids and glycosylphosphatidylinositol-linked proteins that provide a platform for the initiation of multiple cellular responses to extracellular stimuli. Lipid rafts are essential for TLR4-mediated signal transduction and target gene expression. Studies have shown that LPS-induced expression of inflammatory cytokines can be significantly inhibited by the lipid raft inhibitor methyl-b-cyclodextrin (MbCD). Glycyrrhizin, a triterpene glycoside isolated from licorice root (Fig. 1), is known to be effective as an antiinflammatory agent. Glycyrrhizin inhibits nitric oxide activity and inflammatory cytokine production in LPSactivated macrophages [9] and LPS-induced mouse liver injury [10]. Moreover, studies have shown that the antiinflammatory action of glycyrrhizin is mediated by

interference with membrane-dependent receptor signaling [11]. However, the molecular mechanism of the antiinflammatory actions of glycyrrhizin in the LPS-induced inflammatory response remains unclear. The effects of glycyrrhizin on LPS-induced mastitis remain poorly understood. Herein, we report the effects of glycyrrhizin on LPS-induced mastitis in mice, and elucidate the potential anti-inflammatory mechanism.

Results Morphology and cytoskeleton 18 expression of cultured primary mouse mammary epithelial cells (MMECs) The morphology and cytoskeleton 18 expression of cultured MMECs are shown in Fig. 1B. Purified epithelial cells were obtained by digestion, and identified by the detection of cytoskeleton 18. The epithelial cell morphology and the positive staining for cytokeratin18 indicated that the cultured cells were MMECs. Effects of glycyrrhizin on mammary histopathological changes Mammary tissues, harvested at 24 h after injection of LPS, were subjected to hematoxylin and eosin (HE) staining. As shown in Fig. 2, mammary tissues from mice in the control group showed a normal structure and no histopathological changes under a light microscope (Fig. 2A). Morphological examination of mammary

A

B

C

Fig. 1. (A) Chemical structure of glycyrrhizin. (B) Morphology of cultured primary MMECs. Purified epithelial cells were obtained by digestion (9 400). (C) Immunocytochemistry of primary MMECs (9 400).

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Fig. 2. Histopathological sections of mammary tissues (HE, 9 100): mammary tissue of the control group (A), the LPS group (B), the LPS + DEX group (C), the LPS + glycyrrhizin 12.5 mg
kg 1 group (D), the LSP + glycyrrhizin 25 mg
kg 1 group (E), and the LPS + glycyrrhizin 50 mg
kg 1 group (F).

gland sections after LPS injection showed inflammatory cell infiltration, mammary gland alveolar congestion, and marked thickening of the alveolar walls (Fig. 2B). However, the LPS-induced pathological changes were significantly reduced by glycyrrhizin (50 mg
kg 1) (Fig. 2F) and dexamethasone (DEX) (5 mg
kg 1) treatment (Fig. 2C). Effects of glycyrrhizin on myeloperoxidase (MPO) activity Activation of MPO, which reflects the level of inflammation and oxidative stress, is a functional biomarker of neutrophils. The results of immunohistochemistry are shown in Fig. 3. LPS challenge resulted in significant increases in the activity and distribution of MPO in the LPS group (Fig. 3B) as compared with the control group (Fig. 3A). Treatment with glycyrrhizin (12.5, 25 and 50 mg
kg 1) (Fig. 2D–F) and DEX (5 mg
kg 1) markedly reduced the LPS-induced increases in MPO activity and distribution in the mammary gland tissues. FEBS Journal 281 (2014) 2543–2557 ª 2014 FEBS

Effects of glycyrrhizin on cytokine production As shown in Fig. 4, LPS significantly increased the expression of tumor necrosis factor (TNF)-a, interleukin (IL-6) and IL-1b in the homogenate of the mammary tissues. Glycyrrhizin (12.5, 25 and 50 mg
kg 1) and DEX inhibited the expression of TNF-a (P < 0.01), IL-6 (P < 0.05 or P < 0.01) and IL-1b (P < 0.01) induced by LPS. Effects of glycyrrhizin on cell viability The potential cytotoxicity of glycyrrhizin was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results showed that cell viability was not affected by the glycyrrhizin (50, 100 and 200 lg
mL 1) treatment (Fig. 5). Effects of glycyrrhizin on cytokine production in LPS-stimulated MMECs The levels of TNF-a, IL-6, RANTES and IL-10 in the culture supernatants were measured with ELISA kits.

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Fig. 3. Representative photographs of mammary tissues: MPO immunohistochemical staining (9 400) in mice with LPS treatment. Almost no MPO immunostaining was observed in the mammary tissue from the control group (A) and the LPS + DEX group (C). The intensity in the LPS group (B) was significantly higher, and there was a gradational decrease in intensity in the LPS + glycyrrhizin 12.5 mg
kg 1 group (D), the LPS + glycyrrhizin 25 mg
kg 1 group (E), and the LPS + glycyrrhizin 50 mg
kg 1 group (F).

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B

C

Fig. 4. The levels of TNF-a (A), IL-1b (B) and IL-6 (C) in the homogenates of mouse mammary tissues, including the control group, the LPS group, and the groups treated with DEX and glycyrrhizin (12.5, 25 and 50 mg
kg 1). Data represent the contents of 1 g of mammary tissue, and are presented as mean  SEM (n = 6). #P < 0.01, significantly different from the control group. *P < 0.05 and **P < 0.01, significantly different from the LPS group.

As shown in Fig. 6, treatment of primary MMECs with LPS resulted in a significant increase in cytokine production as compared with the control group. Glycyrrhizin suppressed TNF-a, IL-6 and RANTES expression in LPS-stimulated MMECs. However, glycyrrhizin upregulated IL-10 expression in LPS-stimulated MMECs. 2546

Glycyrrhizin inhibits TLR4-mediated IL-8 production in mouse TLR4/MD-2-cotransfected HEK293T cells To confirm that glycyrrhizin inhibits the inflammatory response by targeting TLR4 signaling pathways, we determined the effect of glycyrrhizin on the production of IL-8 in LPS-stimulated HEK293T-TLR4/MD-2 FEBS Journal 281 (2014) 2543–2557 ª 2014 FEBS

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MyD88 or IKKb in HEK293T cells (Fig. 9A,B). Moreover, glycyrrhizin did not inhibit IRF3 activation induced by TRIF or TBK1 in HEK293T cells (Fig. 9C,D). These results demonstrate that the glycyrrhizin-mediated inhibition of LPS-activated TLR4 signaling does not occur through MyD88 or TRIF and their downstream signaling components (the intracellular components of the TLR4 signaling pathway), and that glycyrrhizin exerts its anti-inflammatory actions through effects on membrane components, and not through a specific block in the downstream signaling cascades of the TLR4 signaling pathway. Glycyrrhizin does not affect CD14 function or TLR4 expression Fig. 5. Effect of glycyrrhizin on the cell viability of primary MMECs. Cells were cultured with different concentrations of glycyrrhizin (0–200 lg
mL 1) in the absence or presence of 1 lg
mL 1 LPS for 24 h. Cell viability was determined with the MTT assay. The values presented are the means  SEMs of three independent experiments. #P < 0.05 versus the control group.

cells. The cells were treated with glycyrrhizin for 12 h, and then treated with or without 1.0 lg
mL 1 LPS for 18 h. Cell-free supernatants were used for the IL-8 assays. The results showed that glycyrrhizin inhibited IL-8 production in LPS-stimulated HEK293T-TLR4/ MD-2 cells in a dose-dependent manner (Fig. 7). Glycyrrhizin inhibits LPS-induced NF-jB and IRF3 activation in MMECS NF-jB and IRF3 play critical roles in the regulation of the inflammatory response. To determine whether glycyrrhizin mediates the inhibition of the inflammatory response through the NF-jB and IRF3 pathways, NF-jB and IRF3 expression was determined by western blotting. As shown in Fig. 8, LPS stimulation significantly increased the phosphorylation of NF-jB and IRF3. However, glycyrrhizin significantly inhibited the activation of NF-jB and IRF3 induced by LPS. Glycyrrhizin does not suppress the induction of NF-jB and IRF3 activation through downstream signaling components of TLR4 In this study, we investigated the inhibitory effects of glycyrrhizin on NF-jB and IRF3 activation induced by the TLR4 downstream signaling components: MyD88, IjB kinase b (IKKb), TRIF, and TANKbinding kinase 1 (TBK1). The results showed that glycyrrhizin did not inhibit NF-jB activation induced by FEBS Journal 281 (2014) 2543–2557 ª 2014 FEBS

The membrane components of LPS signaling include TLR4, CD14, MD-2, lipid rafts, and other molecules. In this study, we explored the effects of glycyrrhizin on different components of the membrane complex. TLR4 is the major receptor for LPS. To investigate whether glycyrrhizin inhibits the LPS-induced inflammatory response through the suppression of TLR4 expression, we determined TLR4 expression by western blotting. The results showed that glycyrrhizin did not affect the LPS-induced upregulation of TLR4 expression (Fig. 10A). Cell membrane CD14 is a glycophosphatidylinositol-linked protein that is part of the LPS receptor complex. In contrast, soluble CD14 circulates as a soluble plasma protein. Soluble CD14 has a similar function to cell memebrane CD14, which is required for the LPS receptor complex. To investigate whether glycyrrhizin affects CD14 function to exert its antiinflammatory actions, HEK293T-TLR4/MD-2 cells (which do not express CD14) were cultured in serumfree medium supplemented with LPS in the presence or absence of various concentrations of glycyrrhizin. The results showed that glycyrrhizin dose-dependently inhibited IL-8 production in LPS-stimulated HEK293T-TLR4/MD-2 cells (Fig. 10 B), and that IL8 expression was three-fold lower than in the presence of serum/CD14. We also determined the effects of glycyrrhizin on CD14 expression by western blotting. The results showed that glycyrrhizin did not affect the LPSinduced upregulation of CD14 expression (Fig. 10A). These results suggested that the anti-inflammatory actions of glycyrrhizin did not involve CD14. Glycyrrhizin reduces TLR4 migration into lipid rafts Lipid rafts are involved in TLR4 signaling. Stimulation of cells with LPS recruits TLR4 to lipid rafts. To

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Fig. 6. Glycyrrhizin inhibits LPS-induced cytokine production in a dose-dependent manner. Cells were treated with 1 lg
mL 1 LPS in the absence or presence of glycyrrhizin (50, 100 and 200 lg
mL 1) for 24 h. The levels of TNF-a, IL-6, RANTES and IL-10 in culture supernatants were measured with ELISA. The data presented are the means  SEMs of three independent experiments, and differences between mean values were assessed with ANOVA. #P < 0.05 versus the control group; *P < 0.05 and **P < 0.01 versus the LPS group.

further address the potential anti-inflammatory effects of glycyrrhizin, we determined the effects of glycyrrhizin on TLR4 migration into lipid rafts. We isolated raft fractions, and examined the translocation of TLR4 by immunoblotting. The results showed that LPS stimulation induced the localization of TLR4 to raft fractions. This effect was inhibited by pretreatment with glycyrrhizin or MbCD (Fig. 11). Glycyrrhizin decreases membrane cholesterol levels in MMECs Previous studies have shown that raft-disrupting agents (depleting cholesterol) can inhibit TLR4 trans2548

location into lipid rafts [12,13]. Thus, the effects of glycyrrhizin on cholesterol levels of lipid rafts in MMECs were determined. As shown in Fig. 12A, glycyrrhizin decreased the cholesterol levels of lipid rafts in a dose-dependent manner. Glycyrrhizin upregulates the expression of ATPbinding cassette transporter A1 (ABCA1) in MMECs ABCA1 is a lipid pump that causes the efflux of cholesterol and phospholipid out of cells [14,15]. Activation of ABCA1 has been shown to induce cholesterol efflux from lipid rafts [16]. To determine why FEBS Journal 281 (2014) 2543–2557 ª 2014 FEBS

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glycyrrhizin decreased cholesterol levels of lipid rafts, the expression of ABCA1 was investigated by western blotting. As shown in Fig. 12B, glycyrrhizin upregulated the expression of ABCA1 in a dose-dependent manner.

Discussion

Fig. 7. Glycyrrhizin inhibits IL-8 production in LPS-treated HEK293T-TLR4/MD-2 cells in a dose-dependent manner. Cells were treated with 1 lg
mL 1 LPS in the absence or presence of glycyrrhizin (50, 100, 200 lg
mL 1) for 24 h. Levels of IL-8 in culture supernatants were measured with ELISA. The data presented are the means  SEMs of three independent experiments, and differences between mean values were assessed with ANOVA. #P < 0.05 versus the control group; *P < 0.05 and **P < 0.01 versus the LPS group.

Mastitis is the most prevalent and costly production disease in dairy herds. It remains a major challenge to the worldwide dairy industry, despite the widespread implementation of mastitis control measures. Mastitis research is limited by high costs, a long gestation period, and an uncertain health status [17]. Glycyrrhizin, a triterpene glycoside isolated from licorice root, has been reported to have anti-inflammatory activities [18]. In the current study, we investigated the effect of glycyrrhizin on LPS-induced mastitis in mice. In vivo, the results showed that glycyrrhizin had a protective effect on LPS-induced mastitis. In vitro, the results showed that glycyrrhizin suppressed the production of cytokines by reducing TLR4 migration into lipid rafts.

Fig. 8. Glycyrrhizin inhibits LPS-induced NF-jB and IRF3 activation. Primary MMECs were preincubated with glycyrrhizin (50, 100 and 200 lg
mL 1) for 12 h, and then treated with 1 lg
mL 1 LPS for 1 h. Protein samples were analyzed by western blotting with specific antibodies. b-Actin was used as a control. The values presented are the means  SEMs of three independent experiments, and differences between mean values were assessed with ANOVA. #P < 0.05 versus the control group; *P < 0.05 and **P < 0.01 versus the LPS group.

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A

B

C

D

Fig. 9. Glycyrrhizin does not suppress NF-jB activation induced by MyD88 or IKKb in MyD88-dependent signaling pathways. (A, B). HEK293T cells were transfected with NF-jB–luciferase reporter plasmid and the expression plasmid of MyD88 or IKKb. Twenty-four hours later, cells were treated with glycyrrhizin (50, 100 and 200 lg
mL 1) for 6 h. Relative luciferase activity was determined by normalization with b-galactosidase activity. The data presented are the means  SEMs (n = 3). Glycyrrhizin did not suppress IRF3 activation induced by TRIF or TBK1 in MyD88-independent signaling pathways. (C, D). HEK293T cells were transfected with IFN-b PRDIII-I–luciferase plasmid and the expression plasmid of TRIF or TBK1. Twenty four hours later, cells were treated with glycyrrhizin (50, 100 and 200 lg
mL 1) for 6 h. Relative luciferase activity was determined by normalization with b-galactosidase activity. The data presented are the means  SEMs (n = 3). Veh, vehicle.

Studies have shown that oral glycyrrhizin is metabolized in the intestine to glycyrrhetinic acid [19]. It is not yet clear whether glycyrrhizin administered intraperitoneally acts as such or through a hydrolyzed/ metabolized product. However, many studies have investigated the anti-inflammatory effects of glycyrrhizin administered by intraperitoneal injection [10,20]. In this study, we found that glycyrrhizin administered by intraperitoneal injection inhibited LPS-induced mastitis in mice. The percentage of neutrophils in the somatic cell count has been proposed as a mastitis indicator [21]. MPO activity, a marker of neutrophil influx into tissue, is assessed for the quantification of neutrophil accumulation in tissues [22]. In this study, we found that glycyrrhizin significantly inhibited MPO activity induced by LPS. Histological observation demonstrated marked thickening of the alveolar walls and significant infiltration of inflammatory cells in LPSinduced mastitis. Administration of glycyrrhizin substantially attenuated mammary gland pathological changes. Both histological observation and MPO activ2550

ity showed that glycyrrhizin had a protective effect against LPS-induced mastitis. LPS has been reported to be an important risk factor for mastitis [2]. It has been demonstrated that LPS can effectively induce mastitis in mouse and bovine models [23–25]. LPS can induce the production of cytokines, and these cytokines can amplify the inflammatory response in mastitis [26]. It has been suggested that TNF-a, IL-1b, IL-6 and RANTES play an important role in mastitist pathogenesis [27]. Increased levels of TNF-a, IL-1b and IL-6 have been noted in Escherichia coli infection and LPS-infused mammary glands. Our results showed that glycyrrhizin significantly inhibited TNF-a, IL-1b and IL-6 expression, both in vivo and in vitro. Therefore, the results suggest that the effect of glycyrrhizin on LPS-induced mastitis in mouse may be attributable to the inhibition of inflammatory cytokines. TLRs constitute a large class of innate immunity receptors [28,29]. LPS activates the TLR4-mediated signaling pathway, resulting in the activation of NFjB and IRF3 to regulate the production of cytokines FEBS Journal 281 (2014) 2543–2557 ª 2014 FEBS

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A

B

Fig. 10. Glycyrrhizin does not affect TLR4 expression and the function of CD14. (A) Primary MMECs were preincubated with glycyrrhizin (50, 100 and 200 lg
mL 1) for 12 h, and then treated with 1 lg
mL 1 LPS for 6 h. The protein samples were analyzed by western blotting with specific antibodies. b-Actin was used as a control. The data are presented as the means  SEMs of three independent experiments, and the differences between the mean values were assessed with ANOVA. #P < 0.05 versus the control group; *P < 0.05 and **P < 0.01 versus the LPS group. (B) Glycyrrhizin inhibits IL-8 production in LPS-treated HEK293T-TLR4/MD-2 cells cultured in serum-free medium in a dose-dependent manner. Cells that do not express CD14 cultured in serum-free medium were treated with 1 lg
mL 1 LPS in the absence or presence of glycyrrhizin (50, 100 and 200 lg
mL 1) for 24 h. The IL-8 levels in the culture supernatants were measured with ELISA. The data are presented as the means  SEMs of three independent experiments, and the differences between the mean values were assessed with ANOVA. #P < 0.05 versus the control group; *P < 0.05 and **P < 0.01 versus the LPS group.

[8,30]. In this study, we tested whether the anti-inflammatory effect of glycyrrhizin is exerted though the TLR4-mediated signaling pathway. Our results showed FEBS Journal 281 (2014) 2543–2557 ª 2014 FEBS

that glycyrrhizin inhibited LPS-induced IL-8 production in HEK293T-TLR4/MD-2 cells, suggesting that glycyrrhizin exerts its anti-inflammatory action by

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Fig. 11. The recruitment of TLR4 to lipid rafts is inhibited by glycyrrhizin. Primary MMECs were pretreated with glycyrrhizin or MbCD, and then treated with 1 lg
mL 1 LPS. The cells were lysed and subjected to discontinuous sucrose density gradient centrifugation as described in Experimental procedures. The fractions were analyzed by western blotting with antibody against cholera toxin subunit B conjugated to HRP (GM1) or primary antibody against TLR4. Fractions 3 and 4 correspond to lipid rafts. Fractions 9–12 correspond to nonlipid rafts. The TLR4 content of lipid rafts was calculated as a percentage of total membrane TLR4 (lipid rafts + nonrafts). The data are presented as the means  SEMs of three independent experiments, and differences between mean values were assessed with Students’s t-test. #P < 0.05 versus the control group; **P < 0.01 versus the LPS group.

interfering with the TLR4 signaling pathway. Furthermore, we found that LPS-induced NF-jB and IRF3 activation were also inhibited by glycyrrhizin. However, the molecular targets of glycyrrhizin remain unclear. Therefore, we determined the effects of glycyrrhizin on the intracellular signaling pathway of TLR4: from MyD88 to NF-jB activation, and from TRIF to IRF3 activation. HEK293T cells (absence of TLR4 and MD-2) were used to investigate the molecular targets [31,32]. Studies have shown that transfection of a MyD88 plasmid into HEK293T cells induces overexpression of MyD88, and MyD88 overexpression in HEK293T cells induces NF-jB activation [33,34]. Our results showed that glycyrrhizin did not inhibit NF-jB and IRF3 activation induced by MyD88 or TRIF and their downstream signaling components. These results suggested that the molecular target of glycyrrhizin is upstream of these signaling molecules. In other words, glycyrrhizin may act on the plasma membrane components of the LPS signaling pathway. We first determined TLR4 expression by western blotting. The results showed that glycyrrhizin did not affect the expression of TLR4 upregulated by LPS, suggesting that the molecular target of glycyrrhizin was not TLR4. Second, we investigated whether glycyrrhizin exerted its anti-inflammatory actions by affecting the 2552

function of CD14. HEK293T-TLR4/MD-2 cells (which do not express CD14) cultured in medium free of serum were stimulated by LPS in the presence or absence of various concentrations of glycyrrhizin. The results showed that glycyrrhizin dose-dependently inhibited IL8 production in LPS-stimulated HEK293T-TLR4/MD2 cells. This indicated that the anti-inflammatory actions of glycyrrhizin did not involve CD14. Lipid rafts are plasma membrane microdomains that contain high concentrations of cholesterol and glycosphingolipids [35]. It is well known that lipid rafts also take part in the process of signal transduction. Studies have shown that lipid rafts play an important role in LPS-induced signaling in macrophages and epithelial cells [36,37]. TLR4 was recruited to lipid rafts after cells were treated with LPS [38]. Treatment with raft-disrupting drugs inhibited LPS-induced NF-jB activation and TNF-a production [12,13]. Our results demonstrated that glycyrrhizin decreased cholesterol levels in lipid rafts and suppressed the translocation of TLR4 to lipid rafts. Moreover, glycyrrhizin activated ABCA1, which induces cholesterol efflux from lipid rafts. Honda showed that glycyrrhizin attenuated the formation of the LPS–TLR4/MD-2 complexes, resulting in inhibition of homodimerization of TLR4 [39]. Both TLR4 oligomerization and translocation of the receptor to lipid FEBS Journal 281 (2014) 2543–2557 ª 2014 FEBS

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A

In conclusion, the results have demonstrated that the protective effect of glycyrrhizin against LPSinduced mastitis may be attributable to its ability to activate ABCA1. These findings suggest that glycyrrhizin may be an agent for preventing and treating LPSinduced mastitis.

Experimental procedures Materials B

Fig. 12. (A) Effects of glycyrrhizin on membrane lipid raft cholesterol levels. MMECs were treated with glycyrrhizin (50, 100 and 200 lg
mL 1) for 12 h. Lipid raft cholesterol levels were measured with GLC, and the results are plotted as lg cholesterol (mg protein 1). The data are presented as the means  SEMs of three independent experiments, and differences between mean values were assessed with ANOVA. *P < 0.05 and **P < 0.01 versus the control group. (B) Effect of glycyrrhizin on ABCA1 expression. Primary MMECs were treated with glycyrrhizin (50, 100 and 200 lg
mL 1) for 12 h. Protein samples were analyzed by western blotting with specific antibodies. b-Actin was used as a control. The data are presented as the means  SEMs of three independent experiments, and differences between mean values were assessed with ANOVA. *P < 0.05 and **P < 0.01 versus the control group.

rafts are important initial events in transmitting ligand engagement to activation of intracellular signaling pathways [31]. It may be that glycyrrhizin decreased the levels of cholesterol in lipid rafts, which resulted in inhibition of homodimerization of TLR4. ABCA1 is a plasma membrane protein that plays an important role in the movement of cholesterol [40]. Reports have shown that macrophage ABCA1 dampens inflammation by reducing TLR4 trafficking to lipid rafts through reduction of lipid raft cholesterol levels [16]. Also, studies have shown that ABCA1 activates protein kinase A and upregulates the anti-inflammatory cytokine IL-10. Elevated protein kinase A levels transform macrophages to an M2-like phenotype [41]. Thus, glycyrrhizin could upregulate the expression of IL-10. FEBS Journal 281 (2014) 2543–2557 ª 2014 FEBS

Glycyrrhizin was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Dimethylsulfoxide and LPS (E. coli 055: B5), were purchased from Sigma Chemical Co. (St Louis, MO, USA). Mouse TNF-a, IL-6 and IL-1b ELISA kits were purchased from Biolegend (San Diego, CA, USA). Mouse RANTES ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). Mouse mAb against phospho-NF-jB, mouse mAb against NF-jB, mouse mAb against phospho-IRF3 and rabbit mAb against IRF3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Goat anti-rabbit-HRP secondary sera was provided by GE Healthcare (Chalfont St Giles, UK). b-Actin was purchased from Tianjin Sungene Biotech Co. (Tianjin, China). All other chemicals were of reagent grade. DMEM, DMEM/F12/1 : 1 and fetal bovine serum were obtained from Hyclone (Logan, UT, USA). Collagenase I and collagenase II were purchased from Invitrogen (Carlsbad, CA, USA). Epidermal growth factor and transferrin were purchased from PeproTech. All other chemicals were of reagent grade. Recombinant vectors encoding TLR4 (NM_021297), MD-2 (NM_001159711), MyD88 (NM_010851), TRIF (BC094338) and IKKb (AF026524) were generated by the PCR-based amplification of RAW264.7 cDNA, followed by subcloning into the pcDNA3.1 eukaryotic expression vector (Invitrogen), as previously described. The TBK1 and IFN-b PRDIII-I luciferase plasmids were obtained from K. Fitzgerald (University of Massachusetts Medical School) via Addgene. The NF-jB–luciferase reporter plasmid was purchased from Stratagene (La Jolla, CA, USA).

Animals and treatment Seventy-two healthy female and 36 male BALB/c mice (6–8 weeks of age) were purchased from the Center of Experimental Animals of Baiqiuen Medical College of Jilin University (Jilin, China). This study was approved by the Jilin University Animal Care and Use Committee. All animals were fed with standard laboratory chow and water ad libitum, and kept at a temperature of 24  1 °C, with a relative humidity of 40–80%. All experiments followed the guidelines for the care and use of laboratory animals published by the US National Institutes of Health. The mice were placed into groups, with one male and one female as

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a couple group. After a week, the pregnant female mice then were randomly divided into six groups: blank control group, LPS group, LPS + glycyrrhizin groups (12.5, 25 and 50 mg
kg 1), and LPS + DEX (5 mg
kg 1) group. For in vitro experiments, glycyrrhizin (20 lg) was dissolved in 100 lL of dimethylsulfoxide and further diluted in NaCl/Pi to give final concentrations of 12.5, 25 and 50 mg
kg 1. The blank control group and LPS group received equal volumes of intraperitoneal NaCl/Pi at 1 h before and 12 h after LPS infusion, on the basis of our preliminary experiment. The DEX group received 5 mg
kg 1 intraperitoneal DEX at 1 h before and 12 h after LPS infusion. The blank control group and LPS group received equal volumes of intraperitoneal NaCl/Pi. At 24 h after LPS inoculation, the mice were killed with CO2 inhalation, and the fourth pairs of mammary glands were collected and stored at 80 °C until analysis.

MPO immunohistochemistry Five-micrometer-thick paraffin-embedded sections were cut for MPO immunohistochemistry. The sections were treated with 3% H2O2 for 10 min, and then incubated with the rabbit IgG against mouse MPO (1 : 200; Thermo; RB-373A0) at 4 °C overnight after three washes with NaCl/Pi. The sections were washed three times, and incubated with HRP-conjugated second antibody. All the sections were analyzed after diaminobenzidine staining.

Histopathological evaluation of the mammary tissue The mammary tissues from six mice were collected and fixed with 10% buffered formalin. The mammary samples were embedded in paraffin, sliced, and then stained with HE reagent.

Cell culture and treatment Primary MMECs were prepared as previously described [42]. Mammary tissues from six gravid BALB/c mice were minced into pastes. The tissues were then digested with a collagenase I/collagenase II/trypsin mixture at 37 °C. The cells were centrifuged three times at 250 g for 5 min each. Cell pellets were resuspended in DMEM/F12, and incubated for 1 h at 37 °C; the supernatant was then collected. To clear away fibroblasts, this step was performed three times. The cells were then resuspended in DMEM/F12 containing 10% fetal bovine serum, 0.5% transferrin, 0.1% triiodothyronine, and 0.5% epidermal growth factor, and cultured at 37 °C with 5% CO2. HEK293T cells were cultured in DMEM containing 10% fetal bovine serum at 37 °C with 5% CO2. The medium was changed once every 48 h. For in vitro experiments, glycyrrhizin (20 lg) was dissolved in 100 lL of dimethylsulfoxide and further diluted in culture 2554

medium to give a final concentration of 20 lg
mL 1 as the stock solution. The working concentrations in DMEM/F12 were prepared to give final concentrations of 50, 100 and 200 lg
mL 1 glycyrrhizin in the cell culture plates. Glycyrrhizin-untreated cells containing dimethylsulfoxide as the solvent at the highest concentration used in the tests were used as a negative control. In all experiments, cells were pretreated with glycyrrhizin for 12 h and then stimulated with LPS (1 lg
mL 1).

Cytokeratin-18 immunocytochemistry The cultured cells (3 9 104 mL 1) were cultured on glass coverslips for 24 h. Then, the coverslips were washed with NaCl/Pi three times, and fixed with 4% formaldehyde for 25 min. The cells were saturated with 3% Triton X-100 for 10 min, and then treated with 5% BSA for 1 h. Then, the cells were treated with anti-cytokeratin-18 IgG and fluorescein isothiocyanate-conjugated goat anti-(rabbit IgG). The nuclei were stained with Hoechst. Cells were visualized with a laser scanning confocal microscope (Olympus; FV1700).

Cell viability assay Cell viability was determined with the MTT assay. Primary MMECS (4 9 105 cells mL 1) were plated in 96-well plates at 37 °C for 1 h. The cells were subsequently treated with 50 lL of glycyrrhizin (0–200 lg
mL 1) for 12 h, and this was followed by stimulation with 50 lL of LPS for 18 h. Then, 20 lL of MTT (5 mg
mL 1) was added for 4 h. The supernatant was removed, and 150 lL per well of dimethylsulfoxide was added. The attenuance was read at 570 nm with a microplate reader (Tecan, Austria).

Cell transfection and luciferase assay HEK293T cells were cotransfected with a luciferase plasmid and various expression plasmids or the corresponding empty plasmids vectors, by use of the FuGENE HD transfection reagent (Roche), according to the manufacturer’s instructions. The b-galactosidase plasmid was cotransfected as an internal control. Luciferase activity was measured with the Luciferase Reporter-Gene Assay (Promega). b-Galactosidase enzyme activity was determined with the b-galactosidase Enzyme System (Promega), according to the manufacturer’s instructions. The luciferase activity was normalized to the b-galactosidase activity.

Cytokine assay TNF-a, IL-1b and IL-6 in mammary gland homogenates were measured with ELISA kits, according to the manufacturer’s instructions (BioLegend, Camino Santa Fe, CA, USA).The attenuance of the microplate was read at 450 nm.

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Glycyrrhizin inhibits the inflammatory response

Isolation of lipid rafts

Author contributions

Lipid rafts were isolated as previously described [43]. Briefly, primary MMECs were lysed in ice-cold MBS buffer. The lysates were mixed with 4 mL of 40% sucrose, obtained by mixing with 80% sucrose (2 mL), and overlaid with 35% sucrose (4 mL) and 5% sucrose (4 mL) in MBS buffer. The samples were ultracentrifuged at 100 000g for 18 h, and fractionated into 12 subfractions. Equal amounts of proteins for all treatments were used. GM1 is a classic marker for lipid rafts. Thus, the effects of glycyrrhizin on GM1 and TLR4 were detected by western blot analysis.

YF and ZY designed experiments, YF, EZ, ZW, DJ, WW, TW and MG performed experiments, ZY and NZ analyzed the data, YF and ZY wrote the paper.

Quantification of cholesterol levels in lipid rafts of MMECs Lipid rafts were isolated as described above. Cholesterol levels in lipid raft were determined with GLC, as previously described [44].

Western blot analysis Total proteins from MMECs were extracted with the M-PER Mammalian Protein Extraction Reagent, according to the manufacturer’s instructions. The concentrations were determined with the BCA protein assay kit. Then, the proteins were separated by SDS/PAGE and electrophoretically transferred onto a poly(vinylidene difluoride) membrane. The membrane was blocked in NaCl/Tris containing 5% nonfat dry milk for 1 h, and incubated with a specific primary antibody at 4 °C for 12 h. Subsequently, the membrane was incubated with a secondary antibody at 37 °C for 1 h. The blots were developed with the ECL Plus Western Blotting Detection System (GE Healthcare, Chalfont St Giles, UK).

Statistical analysis All statistical procedures, means and standard errors of the mean (SEMs) were computed with SPSS 16.0. Data are expressed as mean  SEM. Comparisons between groups were performed with ANOVA followed by Dunnett’s test. Differences were considered to be significant at P < 0.05.

Acknowledgements This work was supported by a grant from the National Natural Science Foundation of China (No. 30972225, 30771596) and the Research Fund for the Doctoral Program of Higher Education of China (No. 20110061130010).

Conflicts of interest All authors declare that they have no conflict of interest.

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