Inflamm. Res. (2013) 62:9–15 DOI 10.1007/s00011-012-0545-4
Inflammation Research
ORIGINAL RESEARCH PAPER
Salidroside attenuates inflammatory responses by suppressing nuclear factor-jB and mitogen activated protein kinases activation in lipopolysaccharide-induced mastitis in mice Depeng Li • Yunhe Fu • Wen Zhang • Gaoli Su • Bo Liu • Mengyao Guo • Fengyang Li • Dejie Liang • Zhicheng Liu • Xichen Zhang • Yongguo Cao • Naisheng Zhang • Zhengtao Yang
Received: 2 April 2012 / Revised: 3 July 2012 / Accepted: 6 August 2012 / Published online: 23 August 2012 Ó Springer Basel AG 2012
Abstract Background and objective Mastitis is defined as inflammation of the mammary gland in domestic dairy animals and humans. Salidroside, a major component isolated from Rhodiola rosea L., has potent anti-inflammatory properties, but whether it can be used in mastitis treatment has not yet been investigated. The aim of this study was to assess the protective effects of salidroside against lipopolysaccharide (LPS)-induced mastitis in mice and the mechanism of action. Methods and results We used a mouse mastitis model in which mammary gland inflammation was induced by LPS challenge. Salidroside administered 1 h before LPS infusion significantly attenuated inflammatory cell infiltration, reduced the activity of myeloperoxidase in mammary tissue, and decreased the concentration of tumor necrosis factor-a, interleukin (IL)-1b, and IL-6 in a dose-dependent manner. Further studies revealed that salidroside downregulated phosphorylation of LPS-induced nuclear transcription factor-kappaB (NF-jB) p65 and inhibitor of NF-jB a (IjBa) in the NF-jB signal pathway, and suppressed phosphorylation of p38, extracellular signalregulated kinase (ERK) and c-jun NH2-terminal kinase (JNK) in MAPKs signal pathways.
Responsible Editor: Graham Wallace. D. Li and Y. Fu contributed equally to this work. D. Li Y. Fu W. Zhang G. Su B. Liu M. Guo F. Li D. Liang Z. Liu X. Zhang Y. Cao N. Zhang Z. Yang (&) Department of Clinical Veterinary Medicine, College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, Jilin, People’s Republic of China e-mail:
[email protected]
Conclusions This study demonstrates that salidroside is an effective suppressor of inflammation and may be a candidate for the prophylaxis of mastitis. Keywords Salidroside Lipopolysaccharide (LPS) Mastitis Cytokine Nuclear factor-kappaB (NF-jB) Mitogen activated protein kinases (MAPKs)
Introduction Bovine mastitis, a major disease affecting dairy cattle worldwide, results from the inflammation of the mammary gland, and is a costly disease for dairy producers [1]. During the course of the pathology, the quality and production of milk are reduced and the somatic cell count is increased in the milk. At first sight, the worldwide implementation of mastitis control programs has led to a significant decrease in the prevalence of the disease [2]. But in recent years, conventional methods have frequently been unable to prevent intramammary infection; antibiotics are not very effective in the treatment of mastitis, and have a negative impact on human health. Therefore, alternative drugs are needed to replace antibiotics. Mastitis caused by Escherichia coli is relatively common in high-milk-yield cows, particularly around parturition or during early lactation [3]. Lipopolysaccharide (LPS), a virulence factor of coliform bacteria, can elicit transient mastitis characterized by recruitment of blood neutrophils into the alveolar and duct space [4]. Inoculation of the mammary glands with bacterial LPS is sufficient to induce an inflammatory response [5]. LPSinduced models of mastitis are recognized as valuable tools for studying the effects of mastitis because they mimic the responses observed during natural mastitis without the risks associated with a bacterial infection [5, 6].
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Fig. 1 Chemical structure of salidroside
Rhodiola rosea L. has been widely used for a long time in Traditional Chinese Medicine. Salidroside (p-hydroxyphenethyl-b-D-glucoside, C14H20O7, molecular weight 300.30), shown in Fig. 1, has been identified as the most potent ingredient in this medicinal plant [7]. Salidroside has been reported to have many pharmacological properties, including anti-inflammatory effects [8]. However, there is little information available on whether salidroside could exert protective effects against LPS-induced mastitis. The present study was designed to investigate the antiinflammatory effects and mechanism of action of salidroside in LPS-induced mastitis in mice.
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antibodies IjBa, p65, p38, ERK, and JNK, and mouse monoclonal antibodies p-IjBa, p-p65, p-p38, p-ERK, and p-JNK were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). HRP-conjugated goat anti-rabbit and goat–mouse antibodies were provided by GE Healthcare (Buckinghamshire, UK). T-PER tissue protein extraction reagent (78510) was purchased from Thermo. All other chemicals were of reagent grade. Mastitis mouse model and treatment regimen
BALB/c mice, 6–8 weeks old (36 female and 18 male), were purchased from the Center of Experimental Animals of Baiqiuen Medical College of Jilin University (Jilin, China). Mice were housed for 2–3 days to adapt them to the environment, and then two female and one male mice were housed in each cage with water and food supplied ad libitum. All mice were maintained under pathogen-free conditions, with a 12-h light/dark cycle. The experiments followed the guidelines for the care and use of laboratory animals published by the US National Institutes of Health.
The lactating mice, 5–7 days after birth of the offspring, were randomly divided into six groups: blank control group, LPS group, salidroside (10, 20, and 40 mg/kg) ? LPS groups, and dexamethasone (DEX) ? LPS group. Each group contained six mice. The pups were removed 1 h before inducing inflammation of the mammary gland, then salidroside (10, 20, and 40 mg/kg) was given by intraperitoneal (i.p.) injection, and DEX (0.5 mg/kg) was used as a positive control. The protocol of salidroside administration was carried out in accordance with many previous studies [9–11]. Blank control and LPS group mice were given an equal volume of distilled water i.p. The lactating mice were anesthetized by urethane (15 g urethane dissolved in 150 ml physiological saline, 1.5 g/kg i.p.). A 100-ll syringe with a 30-gauge blunt needle was used to inoculate both L4 (on the left) and R4 (on the right) abdominal mammary glands. The anesthetized mice were placed on their backs under a binocular microscope. The teats and the surrounding area were disinfected with 70 % ethanol. Each udder canal was exposed by a small cut at the near end of the teat and then challenged via teat canal catheterization with 10 lg of LPS dissolved in 50 ll nonpyrogenic phosphate-buffered saline (PBS) [12]. At 12 h after LPS infusion, the mice were killed using CO2 inhalation and mammary tissues were collected and stored at -80 °C until analysis.
Reagents
Histopathological examination
LPS (Escherichia coli 055:B5) was purchased from Sigma Chemical Co. (L-2880, St. Louis, MO, USA). Salidroside (purity 99.8 %) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (110818 Beijing, China). Dexamethasone (DEX) sodium phosphate injection (no. H41020055) was purchased from Changle Pharmaceutical Co. (Xinxiang, Henan, China). Mouse tumor necrosis factor (TNF)-a, interleukin (IL)-6, and IL-1b enzyme-linked immunosorbent assay (ELISA) kits were purchased from Biolegend (USA). The myeloperoxidase (MPO) determination kit was purchased from the Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu Province, China). Rabbit monoclonal
Mammary glands for histopathological examination were fixed in 10 % formalin, dehydrated with graded alcohol and embedded in paraffin, and then stained with hematoxylin and eosin (H&E).
Materials and methods Animals
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Preparation of tissue homogenates The whole right mammary gland tissues were weighed and homogenized with PBS (1:9, w/v) on ice and then centrifuged at 2,000g for 40 min at 4 °C. Lipid was removed and the supernatant was collected. The supernatant was centrifuged again at 2,000g for 20 min at 4 °C to remove any remaining lipid [13].
Salidroside attenuates inflammation in mastitis
Myeloperoxidase assay The activity of MPO reflects the parenchymal infiltration of neutrophils and macrophages. MPO activity in homogenates of mammary tissue was evaluated following the manufacturer’s protocols. Cytokine assays The homogenate of mammary gland tissue samples was centrifuged. The supernatant obtained was assayed for TNF-a, IL-1b, and IL-6 levels using ELISA kits in accordance with the manufacturer’s instructions. Western blot analysis Total protein was extracted from mammary tissue, and its concentration was determined by BCA protein assay kit. For Western blot analyses, equal amounts of protein were loaded in each well and fractionated on a 10 % sodium dodecyl sulphate–polyacrylamide gel. Subsequently, proteins were transferred onto polyvinylidene difluoride membranes. The membranes were then washed in Tris–Tween buffered saline [TTBS, 20 mM Tris-HCl buffer, pH 7.6, containing 137 mM NaCl and 0.05 % (v/v) Tween 20], and blocked with 5 % skim milk in TTBS for 2 h at room temperature with gentle agitation. Incubation with primary antibodies in diluent buffer [5 % (w/v) BSA and 0.1 % Tween 20 in TBS] was performed overnight at 4 °C (1:1,000 dilution) with gentle agitation. The membranes were washed in TTBS three times for 5 min each time with gentle agitation and then incubated with a 1:7,000 dilution (v/v) of secondary antibody for 1 h at room temperature with gentle agitation. Membranes were washed in TTBS three times for 5 min each time with gentle agitation, and processed with SuperSignal West Pico Chemiluminescent Substrate. b-Actin was used as a control for equal protein loading. Statistical analysis All values are expressed as mean ± SEM. Differences between the mean values of normally distributed data were assessed by one-way ANOVA (Dunnett’s t test) and the two-tailed Student’s t test. Statistical significance was set at P \ 0.05.
Results Effect of salidroside on histopathological changes Mammary gland tissues were harvested at 12 h after LPS challenge. To evaluate the histopathological changes after
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salidroside treatment in LPS-treated mice, mammary gland sections were subjected to H&E staining. There was no pathological change observed in the mammary gland controls (Fig. 2a). In the LPS group (Fig. 2b), the mice exhibited marked increases in inflammatory cell infiltration, mostly including neutrophils, macrophages, etc. In addition, mammary gland tissues from the experimental group administered LPS alone were significantly damaged, with interstitial edema and thickening of the alveolus wall. However, these histopathological changes were ameliorated in the DEX group (Fig. 2c) and salidroside treatment groups with the doses of 10 mg/kg (Fig. 2d), 20 mg/kg (Fig. 2e), and 40 mg/kg (Fig. 2f). Effect of salidroside on MPO activity MPO activity is another measure of mammary gland parenchymal phagocyte infiltration. LPS challenge resulted in significant increases of MPO activity in mammary glands compared with the control group (P \ 0.01). Intraperitoneal injection of salidroside at a dose of 10 mg/ kg did not noticeably reduce the MPO activity, while the doses of 20 and 40 mg/kg caused the MPO activity to decline significantly compared with the LPS group (P \ 0.05 and P \ 0.01, respectively). Meanwhile, the MPO activity induced by LPS in mammary gland tissue was significantly decreased in the group treated with DEX compared with the LPS group (P \ 0.01) (Fig. 3). Effect of salidroside on cytokine production To evaluate the levels of cytokines in the tissue homogenate of LPS-induced mammary gland, the mammary gland was collected 12 h after LPS challenge. The expression levels of TNF-a, IL-1b, and IL-6 were measured by ELISA. TNF-a, IL-1b, and IL-6 levels of LPS-stimulated mammary glands were significantly increased compared with those in control group. Salidroside inhibited the production of TNF-a, IL-6, and IL-1b in LPS-induced mouse mastitis in a dose-dependent manner, and DEX significantly reduced TNF-a, IL-1b, and IL-6 production compared to that in the LPS group (Fig. 4). Effect of salidroside on LPS-induced NF-jB and MAPKs activation As shown in Figs. 5 and 6, the levels of p65 and IjBa phosphorylation in the NF-jB signal pathway and the levels of p38, ERK, and JNK phosphorylation in the MAPKs signal pathway were significantly increased in the LPS group, but decreased in those groups pretreated with salidroside.
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Fig. 2 Histopathology of mammary tissue after infusion with LPS (9100). Mammary tissue of control group (a), LPS group (b) and treatment groups administered DEX 5 mg/kg (c), salidroside 10 mg/kg (d), 20 mg/kg (e), and 40 mg/kg (f)
Fig. 3 Myeloperoxidase (MPO) activity in mammary tissue from control group, LPS group, and treatment groups administered 10, 20, and 40 mg/kg salidroside and 5 mg/kg DEX. Data are presented as mean ± SEM (n = 6). #P \ 0.01 significantly different from control group; *P \ 0.05 and **P \ 0.01 significantly different from LPS group
Discussion Mastitis research in dairy animals has been hampered by high costs, a long gestation period, an uncertain health status, and lack of uniform genetic backgrounds [14]. The mouse mastitis model offers an adequate alternative due to the similarities between the mammary glands of these species [12, 15]. For these reasons, the mouse model of
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induced mastitis is commonly used as a practical approach to the study of bovine intramammary infections [12, 15]. Salidroside, which is regarded as one of the most important bioactive components, has been widely used in Traditional Chinese Medicine [7]. The acute and sub-acute toxicity of salidroside extracted from Rhodiola imbricata root were studied in rats, and these results showed that the oral LD50 of the extract was [10 g/kg, indicating an adequate margin of safety in the treated animals [16]. MPO activity, a marker of neutrophil influx into tissue, is directly proportional to the number of neutrophils in the tissue [17]. MPO plays an important role in neutrophil antimicrobial responses [18]. MPO activity, an important index of tissue damage, was measured to evaluate the protective effects of salidroside. As shown in Fig. 3, we confirmed that pretreatment with salidroside significantly improved LPS-induced mastitis in mice and was associated with a reduction in MPO activity of mammary gland and decreased mammary neutrophilia. Histopathological observation indicated that no pathological changes were present in mice mammary tissues without infusion of LPS (Fig. 2a). However, in the LPS group (Fig. 2b), mice exhibited marked increases in inflammatory cell infiltration, mostly including neutrophils, macrophages, etc. Salidroside markedly inhibited the infiltration of inflammatory cells and decreased mammary damage. These results may be due to LPS infusion
Salidroside attenuates inflammation in mastitis
Fig. 4 a TNF-a levels in mammary tissue from control group, LPS group and treatment groups administered salidroside 10, 20, and 40 mg/kg salidroside and 5 mg/kg DEX. Data represent the contents of 1 ml supernatant of mammary homogenate, and are presented as mean ± SEM (n = 6). #P \ 0.01 significantly different from control group; *P \ 0.05 and **P \ 0.01 significantly different from LPS group. b IL-1b levels in mammary tissue from control group, LPS group and treatment groups administered salidroside 10, 20, and 40 mg/kg salidroside and 5 mg/kg DEX. Data represent the contents
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of 1 ml supernatant of mammary homogenate, and are presented as mean ± SEM (n = 6). #P \ 0.01 significantly different from control group; **P \ 0.01 significantly different from LPS group. c IL-6 levels in mammary tissue from control group, LPS group and treatment groups administered salidroside 10, 20, and 40 mg/kg salidroside and 5 mg/kg DEX. Data represent the contents of 1 ml supernatant of mammary homogenate, and are presented as mean ± SEM (n = 6). #P \ 0.01 significantly different from control group; *P \ 0.05 and **P \ 0.01 significantly different from LPS group
Fig. 5 Western blot analysis in mammary tissues derived from mice infused with LPS (10 lg/ gland) indicated that salidroside could inhibited phosphorylation of NF-jB p65 and IjBa. Lane 1 mammary tissues from control group; lane 2 from LPS group; lanes 3, 4, 5 from treatment groups with 10, 20, and 40 mg/kg salidroside, respectively. b-Actin was used as a control. Values are presented as mean ± SEM (n = 6). #P \ 0.01 significantly different from control group; *P \ 0.05 and **P \ 0.01 significantly different from LPS group
triggering the release of cytokines in the mammary gland. Some cytokines such as TNF-a and IL-1b can stimulate vascular endothelial cells to induce vascular endothelial intercellular adhesion molecule expression and up-regulation, which results in endothelial cell and leukocyte adhesion, leukocyte migration, granulocyte degranulation, capillary permeability increase, and migration to inflammatory positions, leading to mammary damage [19]. This is consistent with a previous report that macrophages encountering LPS in tissues are triggered to release cytokines that increase the permeability of blood vessels, allowing polymorphonuclear neutrophils to pass into the tissues [20]. Accordingly, it is proposed that salidroside could reduce the inflammatory cell infiltration and decrease mammary damage through inhibiting the production of pro-inflammatory cytokines.
Cytokines are chemicals released by cells that allow them to communicate with each other [21]. The pivotal role of cytokines in host defense and pathophysiological development during infectious and inflammatory diseases has been well established [22]. LPS is a strong innate immune cell activator and induces the secretion of proinflammatory cytokines [23, 24]. Among these cytokines, TNF-a, IL-1b, and IL-6 are known to be important inflammatory mediators. TNF-a is the earliest and primary endogenous mediator of the process of an inflammatory reaction [10]. Significant expression of TNF-a has been shown in many different types of inflammatory processes, including mastitis [25]. Many of the actions of IL-1 are pro-inflammatory and include stimulation of the acute phase response [26]. IL-1 has also been demonstrated to stimulate production of several secondary cytokines that
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Fig. 6 Western blot analysis in mammary tissues derived from mice infused with LPS (10 lg/ gland) indicated that salidroside could inhibited phosphorylation of p-p38, p-ERK, and p-JNK. Lane 1 mammary tissue from control group; lane 2 from LPS group; lanes 3, 4, 5 from treatment groups with 10, 20, and 40 mg/kg salidroside, respectively. b-Actin was used as a control. Values are presented as mean ± SEM (n = 6). #P \ 0.01 significantly different from control group; *P \ 0.05 and **P \ 0.01 significantly different from LPS group
amplify inflammation, such as IL-6 [27]. IL-6 is an important cytokine mediator released in many aspects of the immunological and inflammatory responses [28]. Among the stimuli which trigger the release of IL-6 are other cytokines, particularly TNF and IL-1 [29, 30]. In this study, TNF-a, IL-1b, and IL-6 were studied at the local level in the LPS-induced mouse mastitis model. The concentrations of TNF-a, IL-1b, and IL-6 were markedly increased in the mammary gland tissue 12 h after LPS induction, and salidroside could inhibit production of TNF-a, IL-6, and IL-1b in LPS-induced mouse mastitis in a dose-dependent manner. A previous study had demonstrated that the concentrations of TNF-a, IL-1, and IL-6 in serum were unchanged at 3, 6, 9, 12, and 24 h after LPS induction [31]. These results show that there is a local inflammation 12 h after LPS induction, rather than development into systemic inflammation. To further characterize the nature of the inhibitory effect of salidroside on cytokine production, we examined the effects of salidroside on the activation of the transcription factor NF-jB, which regulates the expression of many immune and inflammatory genes and the production of
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cytokines. NF-jB is essential for host defense and inflammatory responses to microbial and viral infections [32], and it is an important transcription factor required for the expression of a number of pro-inflammatory cytokines [33]. In the majority of cells, NF-jB exists in an inactive form in the cytoplasm, bound to the inhibitory IjB proteins. Treatment of cells with various inducers results in the degradation of IjB proteins via their phosphorylation and subsequent translocation of NF-jB p65 into the nucleus, where NF-jB activates appropriate target genes. We also examined the effects of salidroside on the phosphorylation of p38, ERK, and JNK in the MAPKs signal pathway, which is well recognized to control the synthesis and release of pro-inflammatory mediators by activated macrophages during the inflammatory response. In the present study, we demonstrated that NF-jB and MAPKs were activated in LPS-induced mouse mastitis, and, more importantly, that pretreatment with salidroside significantly suppressed the phosphorylation of NF-jB p65, IjB a, p38, ERK, and JNK. In the previous study, it had been proved that salidroside could inhibit LPS-induced production of pro-inflammatory cytokines TNF-a, IL-6, and IL-1b both in RAW 264.7
Salidroside attenuates inflammation in mastitis
macrophages and in a murine model of endotoxemia, and that the anti-inflammatory effects were exerted by preventing the activation of the NF-jB and ERK/MAPKs signal pathways [11]. Our results are consistent with this report, and we demonstrated the precise protective effects of salidroside on LPS-induced mastitis in mice from three aspects, histopathological change, MPO activity, and cytokines, and we not only explained the mechanism of salidroside in the NF-jB signal pathway, but also illustrated it in the MAPKs signal pathway. In summary, pretreatment with salidroside could significantly reduce inflammatory cell infiltration into mammary tissue, down-regulate the concentration of TNF-a, IL-1b, and IL-6 in homogenate of mammary gland, suppress phosphorylation of NF-jB p65, IjBa, p38, ERK, and JNK, and block the up-regulation of pro-inflammatory cytokines. Accordingly, our research shows that salidroside has protective effects against LPS-induced mastitis in mice, and can be a potential agent for prophylaxis of mastitis. Further and more comprehensive studies are required before the full clinical application of this drug. Acknowledgments 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).
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