Cyanidin-3-O-β-glucoside inhibits lipopolysaccharide-induced inflammatory response in mouse mastitis model.

Cyanidin-3-O-β-glucoside (C3G) (CAS number 7084-24-4), a typical anthocyanin pigment that exists in the human diet, has been reported to have anti-inflammatory properties. However, the effect of C3G on lipopolysaccharide (LPS)-induced mastitis and the molecular mechanisms have not been investigated. In this study, we detected the protective effects of C3G on a LPS-induced mouse mastitis model and investigated the molecular mechanisms in LPS-stimulated mouse mammary epithelial cells (MMECs). Our results showed that C3G could attenuate mammary histopathologic changes and myeloperoxidase activity, and inhibit TNF-α, interleukin (IL)-1β, and IL-6 production caused by LPS. Meanwhile, C3G dose-dependently inhibited TNF-α and IL-6 in LPS-stimulated MMECs. C3G suppressed LPS-induced nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3) activation. Furthermore, C3G disrupted the formation of lipid rafts by depleting cholesterol. Moreover, C3G activated liver X receptor (LXR)-ABCG1-dependent cholesterol efflux. Knockdown of LXRα abrogated the anti-inflammatory effects of C3G. In conclusion, C3G has a protective effect on LPS-induced mastitis. The promising anti-inflammatory mechanisms of C3G are associated with upregulation of the LXRα-ABCG1 pathway which result in disrupting lipid rafts by depleting cholesterol, thereby suppressing toll-like receptor 4-mediated NF-κB and IRF3 signaling pathways induced by LPS.

surrounding area were disinfected with 70% ethanol. LPS (10 g dissolved in 50 l sterile PBS) was infused into the mammary gland through the duct of the mammary gland ( 18 ).
Seventy-two female mice were randomly divided into six groups: the blank control group, the LPS group, the LPS + C3G groups (10, 20, or 40 mg/kg), and the LPS + DEX (5 mg/kg) group. The treatment groups were respectively administered 10, 20, and 40 mg/kg C3G ip at 1 h before and 12 h after LPS infusion based on our preliminary experiment. The DEX group was administered 5 mg/kg DEX ip at 1 h before and 12 h after LPS infusion. The blank control group and LPS group were supplied with an equal volume of distilled water ip. At 24 h after LPS inoculation, the mice were euthanized with CO 2 inhalation and then the four pairs of mammary glands were collected and stored at Ϫ 80°C until analysis.

Histopathologic evaluation of the mammary tissue
The mammary tissues were collected and fi xed with 10% buffered formalin, imbedded in paraffi n, and sliced. After hematoxylin and eosin staining, pathological changes of the mammary tissues were observed under a light microscope.

Myeloperoxidase activity analysis
Myeloperoxidase (MPO) activity represents the parenchymal infi ltration of neutrophils and macrophages. MPO activity in homogenates of mammary tissue was determined using test kits purchased from Nanjing Jiancheng Bioengineering Institute (China) according to the instructions.

Cell culture and treatment
Mouse mammary epithelial cells (MMECs) were prepared as previously described. Briefl y, the mammary glands from pregnant female mice were minced and digested at 37°C with a collagenase I/II/trypsin mixture (Invitrogen, Carlsbad, CA). Undissociated tissues and debris were removed after fi ltration. Then the cells were collected by centrifugation at 250 g for 5 min three times . Cell pellets were resuspended in DMEM/F12 containing 10% FCS and incubated for 1 h at 37°C, and then the supernatant was collected. This step was repeated three times to clear away fibroblasts. After the last incubation, cells were resuspended in DMEM/F12 containing 10% FCS, 0.5% transferrin, 0.1% T3, and 0.5% EGF and cultured at 37°C with 5% CO 2 . Medium was changed once every 48 h. In all experiments, MMECs were incubated in the presence or absence of various concentrations of C3G that was always added 12 h prior to LPS (0.1 g/ml) treatment.

MTT assay for cell viability
Cell viability was determined by MTT assay. In brief, 1 × 10 4 MMECs were isolated and seeded in 96-well plates, then the cells were treated with 50 l of C3G at different concentrations (0-100 M) for 1 h, followed by stimulation with 50 l LPS. After 18 h of LPS stimulation, 20 l MTT (5 mg/ml) was added to each well, and the cells were further incubated for an additional 4 h. The supernatant was removed and the formation of formazan was resolved with 150 l/well of DMSO. The optical density (OD) was measured at 570 nm on a microplate reader (TECAN, Austria).

ELISA assay
The levels of infl ammatory cytokines, TNF-␣ , IL-1 ␤ , IL-6, and RANTES, were measured by ELISA kits according to the manufacturer's instructions (BioLegend).The OD of the microplate was read at 450 nm.

Western blot analysis
Total proteins from cells were extracted by mammalian protein extraction reagent (M-PER, Thermo). Briefl y, culture medium Anthocyanins are a large group of phytochemicals occurring in a wide range of fruits and vegetables. Cyanidin-3-O-␤ -glucoside (C3G) ( Fig. 1 ), one of the most investigated anthocyanins, has been reported to have anti-infl ammatory effects. C3G was found to inhibit infl ammatory mediators, including iNOS and COX-2 expression in LPS-stimulated THP-1 cells ( 14 ). It has been reported that C3G has specifi c inhibitory effects on NF-B activation ( 15 ). However, the molecular mechanism of the anti-infl ammatory actions of C3G in LPS-induced infl ammatory response remains unclear. Although a number of studies have addressed the therapeutic potential of C3G ( 16,17 ), its ability to protect against LPS-induced mastitis remains poorly understood. Herein, we report the preventive effects of C3G on LPSinduced mastitis in mice and elucidate the potential antiinfl ammatory mechanism.

Mouse model of LPS-induced mastitis
Seventy-two female mice were purchased from the Center of Experimental Animals of Baiqiuen Medical College of Jilin University (Jilin, China) 5-7 days after parturition. All animals were housed in microisolator cages and fed with standard laboratory chow and water ad libitum; the mice were kept at a temperature of 24 ± 1°C and 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. Both L4 (on the left) and R4 (on the right) abdominal mammary glands were infused with LPS using a 100 l syringe with a 30-gauge blunt needle. Lactating mice were anesthetized by ethyl ether and put on their back under a binocular. The teats and the cholesterol (84 g/ml) for 30 min ( 21 ). The cells were exposed to LPS. The translocation of TLR4 to lipid rafts was analyzed as mentioned above.

LXR gene assay
For LXR activation studies, 0.75 g of LXRE-driven luciferase reporter vector (LXRE-tk-Luc) and 0.75 g of ␤ -galactosidase control vector (Promega) were used. The MMECs were transfected with vectors using FuGENE HP transfection reagent (Roche Applied Science, Indianapolis, IN) according to the manufacturer's instructions. Six hours after transfection, cells were treated with C3G for 12 h. The ␤ -galactosidase enzyme activity was determined using the ␤ -galactosidase enzyme system (Promega) according to the manufacturer's instructions. Luciferase activity was normalized by ␤ -galactosidase activity.

Transient transfection of siRNA against LXR ␣
The plasmid containing siRNA against LXR ␣ (si-LXR ␣ ; ON-TARGETplus SMART pool), nontargeting siRNA (si-control), and the DharmaFECT transfection reagent were purchased from Thermo Scientifi c Dharmacon (USA). si-LXR ␣ and si-control stock solutions (20 M) were diluted with diethyl pyrocarbonate water to form 5 M solutions. The DharmaFECT transfection reagent was mixed with 5 M si-LXR ␣ or si-control, incubated for 20 min, and then added to the culture medium at a fi nal concentration of 25 M. The MMECs were incubated with si-LXR ␣ and si-control for 48 h.

Statistical analysis
All data are expressed as mean ± SEM. The differences among the various experimental groups were analyzed by one-way ANOVA. P < 0.05 was considered to be statistically signifi cant.

C3G improved the LPS-induced histopathologic changes
Histopathologic changes of mammary gland tissues from each experimental group were examined after hematoxylin and eosin staining. There was no infl ammatory reaction in the control group ( Fig. 2A ). Compared with the control group, apparent histopathologic changes could be seen in was carefully removed from cells. Then, 200 l M-PER reagent was added to the 6-well plate and shaken gently for 5 min. The lysate was collected and transferred to a microcentrifuge tube and centrifuged at 14,000 g for 7 min to pellet the cell debris. Then the supernatant was transferred for analysis .
Protein concentration was determined through the BCA method. Equal amounts of protein were loaded in each well and separated by 10% SDS-PAGE, which subsequently was transferred onto a polyvinylidene difl uoride membrane. The membrane was blocked for 2 h with 5% skim milk in TBST on the shaker at room temperature and then washed three times (10 min each) with Tri-Tween buffered saline [20 mM Tris-HCl buffer (pH 7.6), 137 mM NaCl, and 0.05% Tween 20]. The membrane was placed on primary antibody diluted at a 1:1,000 proportion in diluent buffer [5% (w/v) BSA and 0.1% Tween 20 in TBS] and incubated overnight at 4°C on the shaker. Subsequently, the membrane was washed with PBS-T followed by incubation with the secondary antibody conjugated with HRP at room temperature for 1 h . The membrane was again washed three times (10 min each), as above, and fi nally the results were generated by using an ECL Western blotting kit.

Isolation of lipid rafts
Lipid rafts were isolated as described previously ( 19 ). Briefl y, MMECs were lysed in ice-cold MBS buffer [25 mM MES (pH 6.5), 150 mM NaCl, 1 mM Na 3 VO 4 , 1% Triton X-100, and protease inhibitors]. Lysates were mixed with 4 ml of 40% sucrose by mixing with 2 ml of 80% sucrose and overlaid with 4 ml of 35% sucrose and 4 ml of 5% sucrose in MBS buffer. Samples were ultracentrifuged at 39,000 rpm for 18 h and fractionated into 12 subfractions. The translocation of TLR4 to lipid rafts was measured by Western blot analysis.

Quantifi cation of cholesterol levels in lipid rafts of MMECs
Lipid rafts were isolated as described above. The cholesterol level of lipid rafts was assayed by gas-liquid chromatography as previously described ( 20 ).

Cholesterol replenishment experiment
MMECs were treated with culture medium alone or medium containing C3G (10, 20, or 40 M), or M ␤ CD (10 mM) at 37°C for 60 min. Subsequently, the cells were washed with PBS and incubated with medium alone or medium containing water-soluble ( 5 ). In this study, we detect whether the inhibition of infl ammatory response by C3G is mediated through the NF-B and IRF3 pathways. NF-B and IRF3 protein were determined by Western blotting . The results showed that C3G signifi cantly inhibits the phosphorylation of NF-B and IRF3 ( Fig. 7 ).

C3G inhibits translocation of TLR4 to lipid rafts
Lipid rafts play an important role in the TLR4 signaling pathway. LPS induced TLR4 to recruit into lipid the LPS group, represented by thickening of the alveolar wall, interstitial patchy hemorrhage, hyperemia, edema, and the extensive existence of infl ammatory cells in alveolar spaces ( Fig. 2B ). In the LPS + C3G groups with the dose of 10 ( Fig. 2D ), 20 ( Fig. 2E ), and 40 mg/kg ( Fig. 2F ), LPSinduced histopathologic changes were markedly attenuated in a dose-dependent manner. Treatment with DEX (5 mg/kg, Fig. 2C ) also signifi cantly reduced the injury of mammary gland tissues compared with the LPS group.

Effects of C3G on the MPO activity
As shown in Fig. 3 , LPS signifi cantly increased the MPO activity in mammary tissue ( P < 0.01) compared with the control group. MPO activity was reduced in the treatment groups, especially the treatment groups of 20 mg/kg and 40 mg/kg which had markedly decreased MPO activity in comparison with the LPS group ( P < 0.05 and P < 0.01).

C3G decreased the levels of pro-infl ammatory cytokines
The levels of pro-infl ammatory cytokines, TNF-␣ , IL-1 ␤ and IL-6, were measured by ELISA. Compared with the control group, LPS signifi cantly increased the levels of TNF-␣ , IL-1 ␤ , and IL-6. In contrast, these increases induced by LPS were signifi cantly decreased by C3G ( Fig. 4 ).

Effects of C3G on cell viability
The potential cytotoxicity of C3G was evaluated by the MTT assay after incubating cells for 18 h in the absence or presence of LPS; the result showed that cell viabilities were not affected by the C3G at the concentrations used (10, 20, or 40 M) ( Fig. 5 ). Thus, the effects of C3G on MMECs were not attributable to cytotoxic effects.

Effects of C3G on cytokine production in LPS-stimulated MMECs
The expression of TNF-␣ , IL-6, and RANTES was detected by ELISA. The results showed that C3G suppressed TNF-␣ , IL-6, and RANTES production in LPS-stimulated MMECs in a dose-dependent manner ( Fig. 6 ).

C3G suppresses LPS-induced NF-B and IRF3 activation
NF-B and IRF3 play a critical role in regulating of infl ammatory cytokine expression. Once stimulated by LPS, NF-B and IRF3 phosphorylation activates these proteins    4. The levels of TNF-␣ , IL-1 ␤ , and IL-6 in the homogenate of mouse mammary tissues including control group, LPS group, and treatment groups with C3G (10, 20, or 40 mg/kg). Data represent the contents of 1 g mammary tissue, and are presented as mean ± SEM (n = 6). # P < 0.01 signifi cantly different from control group; * P < 0.05 and ** P < 0.01 signifi cantly different from LPS group. by Western blotting. As shown in Fig. 8A , GM1 was lost from the rafts in C3G-treated cells, suggesting that C3G could destroy lipid rafts. The results also showed that LPS stimulation induced localization of TLR4 to raft fractions. This effect was prevented by pretreatment with C3G or M ␤ CD ( Fig. 8A ). The results ( Fig. 8B ) showed that the cholesterol amounts in rafts were decreased by C3G or M ␤ CD. Meanwhile, the cholesterol amounts in total fractions were also decreased by C3G or M ␤ CD.

C3G decreases lipid raft cholesterol levels in MMECs
Lipid rafts are plasma membrane microdomains that contain high concentrations of cholesterol and glycosphingolipids. Studies have shown that treatment with raftdisrupting drugs can inhibit TLR4 translocation into lipid rafts and LPS-induced NF-B activation and TNF-␣ production in macrophages ( 23 ). Thus, we determined whether C3G decreased cholesterol levels of lipid rafts in MMECs . As shown in Fig. 9 , the cholesterol levels of lipid rafts were decreased by C3G in a dose-dependent manner .

Cholesterol replenishment prevents the anti-infl ammatory effect of C3G
Cholesterol replenishment experiments were carried out to detect the anti-infl ammatory mechanism of C3G. As shown in Fig. 10 , the inhibition effect of C3G on LPSinduced cytokines was abolished.

C3G upregulates the expression of LXR ␣ and ABCG1 in MMECs
LXR ␣ plays an important role in cholesterol homeostasis by regulating the sensors of cholesterol levels in cells . Activation of LXR ␣ induces expression of genes involved in cholesterol effl ux, such as ABCG1. In this study, we performed a luciferase reporter gene assay to test whether C3G could enhance transcriptional activity of LXR ␣ . As rafts ( 22 ). To further address the potential anti-infl ammatory effects of C3G, we determined the effects of C3G on the translocation of TLR4 to lipid rafts. We isolated raft fractions and examined the translocation of TLR4

Knockdown of LXR ␣ abrogated the effects of C3G on lipid raft cholesterol levels, and LPS induced infl ammatory response in MMECs
To detect whether the anti-infl ammatory effects of C3G is LXR ␣ dependent, LXR ␣ was silenced in MMECs by its specifi c siRNA . When LXR ␣ was silenced, the effects of C3G on cholesterol levels and cytokines expression induced by LPS were reversed ( Fig. 12 ) ( Fig. 13 ).

DISCUSSION
Bovine mastitis is a highly prevalent and important infectious disease of dairy cattle. It is characterized by mammary gland edema, mammary alveolar damage, and infl ammatory cell infi ltration. LPS, a main component of the outer membrane of gram-negative bacteria, has been identifi ed as an important risk factor for mastitis ( 24 ). C3G, a typical anthocyanin pigment that exists in the human diet, has been reported to have anti-infl ammatory properties. In the present study, we detected the effects of C3G on LPSinduced mastitis. The results showed that C3G obviously decreased mammary gland injury and MPO activity. These results indicated that C3G had a protective effect of LPSinduced mastitis.
shown in Fig. 11A , C3G dose-dependently increased expression of the LXR luciferase reporter gene. Meanwhile, the expression of LXR ␣ and ABCG1 were detected by Western blotting. As shown in Fig. 11B , C3G upregulated the expression of LXR ␣ and ABCG1 in a dose-dependent manner . Fig. 8. A: The recruitment of TLR4 to lipid rafts was inhibited by C3G. MMECs were pretreated with C3G or M ␤ CD, followed by treatment with 0.1 g/ml LPS. The cells were lysed and subjected to discontinuous sucrose density gradient centrifugation as described in the Experimental Procedures. The fractions were analyzed by Western blotting using CTxB conjugated to HRP (GM1) or anti-TLR4 primary antibody . Fractions 3 and 4 correspond to lipid rafts. Representative blots of three separate experiments are shown. GM-1 and TLR4 content of macrophage lipid rafts was calculated as the amount of GM1 and the amount of TLR4 per total fractions. B: Cholesterol levels in each fraction were measured. The values presented are the mean ± SEM of three independent experiments and differences between mean values were assessed by Students's t -test. # P < 0.05 versus control group; ** P < 0.01 versus LPS group . activation. The results showed that C3G inhibited LPSinduced cytokine production by preventing NF-B and IRF3 phosphorylation. However, the molecular targets of C3G have not been identifi ed.
TLR4 is the major receptor for LPS. LPS signaling is initiated by binding to TLR4, leading to activation of downstream targets. Recently, reports have shown that lipid rafts play an important role in the TLR4 signaling pathway ( 12 ). Lipid rafts provide platforms for the formation of receptor complexes and play fundamental roles in signaling transduction . Upon stimulation by LPS, TLR4 is recruited to lipid rafts and induces downstream signaling activation ( 28 ). To further clarify the mechanism of C3G on LPSinduced infl ammatory response, we investigated the effects of C3G on cholesterol levels in lipid rafts. The results showed that C3G could decrease the levels of cholesterol in lipid rafts. Meanwhile, our cholesterol replenishment Mammary epithelial cells directly contact with the invading pathogens and play an important role in upper immunity ( 25 ). They recognize the pathogen-associated molecular patterns of invading pathogens via pattern recognition receptors such as TLRs, and induce the production of cytokines. Thus, we used the mammary epithelial cells to investigate the mechanism of C3G on LPS-induced mastitis in vitro. Cytokines, an important group of infl ammatory mediators, play an important role in the process of mastitis ( 26 ). Elevated TNF-␣ and IL-6 were observed in LPS-induced mastitis ( 3,27 ). These cytokines, as well as other infl ammatory mediators, initiate, amplify, and perpetuate the infl ammatory response in mastitis. In this study, we found that C3G inhibited the production of TNF-␣ and IL-6 in a dose-dependent manner. It has been reported that the expression of cytokines is modulated by NF-B and IRF3. In this study, we investigated the effects of C3G on LPS-induced NF-B and IRF3 Fig. 10. Cholesterol (Cho) replenishment prevents the antiinfl ammatory effect of C3G. MMECs were treated with culture medium alone or medium containing C3G (10, 20, or 40 M) or M ␤ CD (10 mM) at 37°C for 60 min. Subsequently the cells were washed with PBS and incubated with medium alone or medium containing water-soluble cholesterol (84 g/ml) for 30 min. Cells were treated with 0.1 g/ml LPS for 24 h. Levels of TNF-␣ , RANTES, and IL-6 in culture supernatants were measured by ELISA. The data presented are the mean ± SEM of three independent experiments and differences between mean values were assessed by ANOVA. # P < 0.05 versus control group; * P < 0.05 and ** P < 0.01 versus LPS group. Fig. 11. Effects of C3G on LXR transcriptional activity and LXR ␣ and ABCG1 expression. A: Cells were transfected with LXRE-driven luciferase reporter vector (LXRE-tk-Luc) and ␤galactosidase control vector (Promega). Six hours later, cells were treated with C3G for 12 h. Relative luciferase activity was determined by normalization with ␤ -galactosidase activity (* P < 0.05, ** P < 0.01). B: Effects of C3G on LXR ␣ and ABCG1 expression. Cells were treated with C3G (10, 20, or 40 M) for 12 h. Protein samples were analyzed by Western blot with specifi c antibodies. ␤ -Actin was used as a control. The values presented are the mean ± SEM of three independent experiments and differences between mean values were assessed by Students's t -test (* P < 0.05, ** P < 0.01). results confi rmed that after cholesterol replenishment, the inhibition effect of C3G on LPS-induced cytokines was abolished. Overall, our results demonstrated that C3G disrupted lipid rafts by depleting cholesterol and inhibiting LPS-induced infl ammatory responses.
LXR ␣ and LXR ␤ are members of the nuclear receptor superfamily that regulates cholesterol homeostasis ( 29 ). LXRs regulate intracellular cholesterol levels through mediating the expression of ABCG1 ( 30 ). ABCG1 is known to play an important role in cholesterol and oxysterol effl ux ( 31,32 ). To investigate the reason that C3G decreased the cholesterol levels in lipid rafts, we detected the effects of C3G on LXR ␣ and ABCG1 activation. Our results showed that C3G increased LXR ␣ and ABCG1 expression. These results suggest that C3G activates the LXR ␣ -ABCG1 pathway by mediating cholesterol effl ux to reduce lipid raft cholesterol content. To further confi rm the involvement of the LXR ␣ -ABCG1 pathway in the antiinfl ammatory effect of C3G on mammary epithelial cells, LXR ␣ was silenced by siRNA. We showed that when LXR ␣ was silenced, the effects of C3G on ABCG1 expression, membrane cholesterol levels, and expression of cytokines induced by LPS were reversed. The present results obtained from LXR ␣ knockdown support the critical role of the LXR ␣ -ABCG1 pathway in the anti-infl ammatory effects of C3G.
In conclusion, the studies demonstrate that C3G inhibits LPS-induced infl ammatory cytokine production. The promising anti-infl ammatory effect of C3G on LPS-stimulated primary MMECs is associated with upregulation of the LXR ␣ -ABCG1 pathway, thereby suppressing TLR4-mediated NF-B and IRF3 signaling pathways induced by LPS. Fig. 12. Knockdown of LXR ␣ abrogates the effects of C3G on lipid raft cholesterol levels, and LPS induces infl ammatory response in MMECs. Cells were transfected with a siRNA specifi c for LXR ␣ , or a scrambled siRNA (negative control) as indicated. Then the cells were treated with C3G (40 M) for 12 h. Lipid raft cholesterol levels were detected. Meanwhile, the cells were treated with C3G (40 M) for 12 h and stimulated by 0.1 g/ml LPS for 24 h. Levels of TNF-␣ , RANTES, and IL-6 in culture supernatants were measured by ELISA. The data presented are the mean ± SEM of three independent experiments and differences between mean values were assessed by Students's t -test. # P < 0.05 versus control group; ** P < 0.01 versus LPS group. Fig. 13. Anti-infl ammatory mechanism of C3G is associated with upregulation of the LXR ␣ -ABCG1 pathway which results in disrupting lipid rafts by depleting cholesterol, thereby suppressing the TLR4mediated NF-B and IRF3 signaling pathways induced by LPS .