Effects of PPAR-γ Agonist Treatment on LPS-Induced Mastitisin Rats

Ding Mingfeng,1 Ming Xiaodong,1 Liu Yue,2 Piao Taikui,3 Xiao Lei,1 and Liu Ming1,4

Abstract—PPAR-γ, a member of the nuclear receptor superfamily, plays an important role in lipid m-etabolism and inflammation. The aim of this study was to investigate the preventive effects of syntheticPPAR-γ agonist rosiglitazone on lipopolysaccharide (LPS)-inducedmastitis in rats. Themouse model ofmastitis was induced by the injection of LPS through the duct of the mammary gland. Rosiglitazone wasinjected 1 h before the induction of LPS intraperitoneally. The results showed that rosiglitazone atten-uated the infiltration of inflammatory cells, the activity of myeloperoxidase (MPO), and the productionof tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in a dose-depen-dent manner. Additionally, Western blotting showed that rosiglitazone inhibited the phosphorylation ofIκB-α and NF-κB p65. These results indicated that rosiglitazone has a protective effect on mastitis, andthe anti-inflammatory mechanism of rosiglitazone on LPS-induced mastitis in rats may be due to itsability to inhibit NF-κB signaling pathways. PPAR-γ may be a potential therapeutic target againstmastitis.

KEYWORDS: PPAR-γ; mastitis; rats; myeloperoxidase; nuclear factor-kappaB (NF-κB).

INTRODUCTION

Mastitis is one of the most epidemic, frequent, andcostly infectious disease affecting the dairy cattle industryworldwide [1, 2]. Bacterial lipopolysaccharide (LPS), themajor constituent of the outer membrane of Gram-negativebacteria, has been referred to be an important risk factor formastitis [3]. LPS induces the synthesis of pro-inflammato-ry cytokines and mediators such as TNF-α, IL-6, and IL-1β [4]. These inflammatory cytokines lead to inflamma-tion and other various clinical manifestations. Nowadays,treatments of bovine mastitis mainly depend on antibiotic

[5]. However, the residues of antibiotic in the milk andmeat are harmful to the health of humans [6]. Therefore,the development of novel therapies for mastitis is urgentlyneeded.

Peroxisome proliferators-activated receptor gamma(PPAR-γ), which belongs to the nuclear receptor super-family, is a ligand-activated transcription factor that isexpressed in various cell types and tissues [7]. Studiesshowed that both mammary gland tissues and mammaryepithelial cells express PPAR-γ [8]. PPAR-γ has beenshown to be an important regulator of target genes in-volved in glucose and lipid metabolism [9]. Much datahave shown that PPAR-γ plays a crucial role in regulatingmilk fat synthesis [10]. Controlling PPAR-γ activationmay prove useful in regulating milk fat production [10].In addition, PPAR-γ also plays an important role in theregulation of inflammatory and immune responses [11,12]. PPAR-γ agonists could negatively regulate inflamma-tory responses through inhibiting NF-κB signaling path-way [13, 14]. PPAR-γ agonist was found to reduce boneerosion and inflammatory bone loss in adjuvant-inducedarthritis [15]. PPAR-γ agonist pioglitazonewas found to beas effective as dexamethasone in a mouse model of asthma[16]. However, the effect of PPAR-γ agonists on LPS-induced mastitis has been reported. In this study, we sought

Ding Mingfeng and Ming Xiaodong contributed equally to this article.

1 Department of General Surgery, The Fourth Hospital of Harbin MedicalUniversity, Harbin, Heilongjiang Province 150001, People’s Republic ofChina

2 Department of General Surgery, the Fifth Hospital of Harbin MedicalUniversity, Daqing, Heilongjiang Province 163316, People’s Republic ofChina

3 Children’s Hospital of Harbin, Harbin, Heilongjiang Province 150010,People’s Republic of China

4 To whom correspondence should be addressed at Department of GeneralSurgery, The Fourth Hospital of Harbin Medical University, Harbin,Heilongjiang Province 150001, People’s Republic of China. E-mail:liumingyxf@163.com

0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York

Inflammation (# 2014)DOI: 10.1007/s10753-014-9924-z

to assess the anti-inflammatory effects of PPAR-γ agonistson LPS-induced mastitis in rats and elucidate the potentialanti-inflammatory mechanism.

MATERIALS AND METHODS

Material

E. coli Lipopolysaccharide (LPS, Escherichia coli055:B5) was purchased from Sigma (St. Louis, MO,USA). TNF-α, IL-6, and IL-1β ELISA kits were pur-chased from the Institute of Radiation of Science andTechnology Development Center of the General Hospitalof People’s Liberation Army (Beijing, China). Themyeloperoxidase (MPO) determination kits were providedby the Jiancheng Bioengineering Institute of Nanjing(Jiangsu, China). Rabbit monoclonal antibodies includingphosphorylated and non-phosphorylated forms of IκB-αand NF-κB were obtained from Cell Signaling Technolo-gy, Inc. (Beverly, MA, USA).

Animals

Seventy-two pregnant SD rats (weighing 300–350 g)were purchased from the Experimental Animal Center ofthe Harbin Medical University (Harbin, China). They werehoused in individual cages and received food and water adlibitum. All animal experimental protocols followed theguidelines for the Care and Use of Laboratory Animalsestablished by the US National Institutes of Health.

Experimental Design

Seventy-two rats were randomly divided into sixgroups and each group contained twelve rats: blank controlgroup, LPS group, three treatment groups which wereinfused LPS at 1 h after administered rosiglitazone (0.1,0.2, 0.3 mg/kg intravenously), and DEX (5 mg/kg) group,respectively. The treatment groups were respectively ad-ministered 0.1, 0.2, 0.3 mg/kg rosiglitazone based on theirweight at 1 h before LPS induction, and DEX group wasregarded as a positive control. The concentrations of dexa-methasone and rosiglitazone used in this study wererefereed to previous reports [17, 18]. Seventy-two hoursafter parturition, the rats were infused with 10 μg LPS,dissolved in 100 μl sterile pyrogen-free, physiologicalsaline, directly into the inguinal mammary glands (leftand right fourth glands). Intramammary infusion was per-formed according to the method of Zou et al. [19]. Theblank control group and LPS group were supplied with an

equal volume of physiological saline i.p. At 12 h afterinfused LPS, the rats were killed with CO2 inhalation,and then, the fourth pair of mammary tissues were collect-ed and stored at −80 °C.

Histopathologic Evaluation of Mammary Gland Tissue

After the rats were killed, the mammary gland tissueswere excised. They were fixed with 10 % neutral phos-phate-buffered formalin. After imbedded in paraffin,stained with hematoxylin and eosin (H&E), pathologicalchanges in the mammary glands tissues were observedunder a light microscope.

Measurement of MPO

Mammary gland tissues were frozen in lipid nitrogenand then homogenized in PBS. The homogenates of mam-mary tissues were used to determine MPO acitivity usingtest kits purchased fromNanjing Jiancheng BioengineeringInstitute (China) according to the instructions.

Determination of TNF-α, IL-1β and IL-6

Remove and dissolved the mammary gland tissuehomogenate samples at −20 °C refrigerator. Concentrationof the cytokines, including TNF-α, IL-6, and IL-1β in thesupernatant, was measured using sandwich enzyme-linkedimmunosorbent assay (ELISA) kits according to the man-ufacture’s protocol.

Western Blot Analysis

Proteins were extracted from the mammary tissuesusing T-PERTissue Protein Extraction Reagent Kit accord-ing to the manufacturer’s instructions. Protein concentra-tion was determined by BCA protein assay kit. The pro-teins were separated on 12 % SDS polyacrylamide gel andtransferred onto the PVDF membrane. After blocking thenonspecific site with blocking solution (5 % nonfat drymilk), the membrane was incubated overnight with specificprimary antibody at 4 °C. Subsequently, the membranewaswashed with PBS-T followed by incubation with the sec-ondary antibody conjugated with horseradish peroxidase atroom temperature for 1 h. Blots were again washed withPBS-T and then developed with the ECL Plus WesternBlotting Detection System (Amersham Life Science, UK).

Statistical Analysis

Data are presented as mean ± SEM. Comparisonbetween groups was made with ANOVA followed by

Mingfeng, Xiaodong, Yue, Taikui, Lei, and Ming

Dunnett’s test. P values of 0.05 or less were consideredstatistically significant.

RESULTS

Effects of Rosiglitazone on Histopathological Changesin Mammary Gland Tissues

Mammary gland specimens from the LPS group,compared to the control group, displayed evident histo-pathologic abnormalities, including inflammatory cells in-filtration, mammary gland alveolus congestion, andmarked thickening of the alveolus walls (Fig. 1b). Howev-er, LPS-induced pathological changes were significantlyattenuated by rosiglitazone (0.1, 0.2, 0.3 mg/kg) (Fig. 1d–f)and DEX (5 mg/kg) treatment (Fig. 1c).

Effects of Rosiglitazone on MPO Activity

MPO activity reflected neutrophils accumulation inthe mammary gland. As shown in Fig. 2, LPS challengeresulted in a significant increase of MPO activity in mam-mary gland compared with the control group (P<0.01).Meanwhile, the MPO activity induced by LPS in mamma-ry gland tissues was significantly decreased in the grouptreated with rosiglitazone (0.1, 0.2, 0.3 mg/kg) comparedwith the LPS group (P<0.05 or P<0.01). The levels of

MPO activity in the DEX group were marked lower thanthat in the LPS group (**P<0.01).

Effects of Rosiglitazone on TNF-α, IL-1β and IL-6Levels in Mammary Gland

The levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were measured by ELISA. Compared withthe control group, the expression of TNF-α, IL-6, and IL-1β was significantly increased in the LPS group. Thelevels of TNF-α, IL-6, and IL-1βwere efficiently inhibitedby rosiglitazone (0.1, 0.2, 0.3 mg/kg) compared to the LPSgroup (Fig. 3).

Effects of Rosiglitazone on LPS-Induced NF-κBActivation

NF-κB plays an important role in the inflammatoryresponse and regulates inflammatory cytokines gene ex-pression. Western blot analysis showed that NF-kB signal-ing pathways were activated after LPS administration.Rosiglitazone (0.1, 0.2, 0.3 mg/kg) significantly inhibitedthe phosphorylation of IκB and NF-κB P65 (Fig. 4).

DISCUSSION

PPAR-γ, belongs to the nuclear receptor superfamily,has been reported to be an important regulator of target

Fig. 1. Histological assessment of the effect rosiglitazone on LPS-induced mastitis. Twelve hours after LPS instillation, the mammary tissues were inflatedand fixed with 10% buffered formalin; samples were embedded in paraffin and then stained with H&E (×400). aControl group. b LPS group. c LPS + DEXgroup. d LPS + rosiglitazone 0.1 mg/kg. e LPS + rosiglitazone 0 mg/kg. f LPS + rosiglitazone 0.3 mg/kg.

Effects of PPAR-γ Agonist Treatment

genes involved in glucose and lipid metabolism [20, 21].Many studies have demonstrated that PPAR-γ also wasan important modulator of inflammation [22, 23]. How-ever, little is known about whether PPAR-γ agonistsmodulate inflammation in mastitis. Thus, we evaluatedthe anti-inflammatory properties of rosiglitazone in LPS-induced mastitis in rats. Previous reports showed thatdexamethasone was used to treat mastitis, and in thisstudy, dexamethasone was used as a positive control[24]. The results showed that rosiglitazone attenuatedmammary gland tissue damage induced by LPS anddecreased the MPO activity, pro-inflammatory cytokineproduction, and the activation of NF-κB. On the compar-ative potency of dexamethasone and rosiglitazone inhibi-tion on LPS-stimulated cytokine production, we foundthat the anti-inflammatory effect of rosiglitazone on LPS-induced mastitis was a little weaker than dexamethasone.The results suggested that PPAR-γ agonists may be auseful agent for preventing and treating LPS-inducedmastitis.

Mastitis is characterized by the mammary gland ede-ma, disruption of endothelial and epithelial integrity, ex-tensive inflammatory cells infiltration, and release of in-flammatory mediators [25]. In this study, the mammaryhistological examination showed that rosiglitazone re-markably attenuated mammary gland pathological chang-es. MPO activity, a marker of neutrophil influx into tissue,is directly proportional to the number of neutrophils intissue [26]. In this study, we detected the effects ofrosiglitazone onMPO activity induced by LPS. The resultsshowed that rosiglitazone significantly inhibited MPO ac-tivity in the LPS-induced mastitis. These data suggested

that rosiglitazone has a protective effect on LPS-inducedmastitis.

Cytokines, an important group of inflammatory me-diators, play an important role in the process of mastitis[27]. Elevated TNF-α, IL-1β, and IL-6 levels were ob-served in LPS-induced mastitis [19]. These cytokines, aswell as other inflammatory mediators, initiate, amplify, andperpetuate the inflammatory response in mastitis. Thus, wedetected the effects of rosiglitazone on cytokines produc-tion. Our results showed that rosiglitazone significantly

Fig. 2. Effects of rosiglitazone on MPO activity in the mammary gland ofLPS-induced mastitis. Rats were given an intraperitoneal injection of ro-siglitazone (0.1, 0.2, 0.3 mg/kg) 1 h before LPS instillation, respectively.MPO activity was determined at 24 h after LPS administration. The valuespresented are the mean ± SEM. #P<0.01 vs. control group, *P<0.05,**P<0.01 vs. LPS group (n=6–8).

Fig. 3. The levels of TNF-α (a), IL-1β (b), and IL-6 (c) in the homoge-nate of rats mammary tissues including control group, LPS group, and t-reatment groups with DEX and rosiglitazone (0.1, 0.2, 0.3 mg/kg). Datarepresent the contents of 1 g mammary tissue, and are presented as mean ±SEM. #P<0.01 significantly different from control group; *P<0.05, and**P<0.01 significantly different from LPS group (n=6–8).

Mingfeng, Xiaodong, Yue, Taikui, Lei, and Ming

decreased the production of TNF-α, IL-1β, and IL-6 inLPS-induced mastitis.

It has been reported that the expressions of cytokinesare modulated by NF-κB. NF-κB is a ubiquitous heterodi-meric transcription factor that exists in an inactive form inthe cytoplasm bound to the inhibitory proteins referred toas IκB [28]. Once stimulated by LPS, NF-κB is activatedby phosphorylation, enters the nucleus and regulates theexpression of inflammatory cytokines, such as TNF-α, IL-1β, and IL-6 [29]. Studies showed that LPS could induceNF-κB activation in many tissues such as mammary gland,lungs, and livers [30–32]. Previous reports demonstratedthat PPAR-γ agonist rosiglitazone reduced pulmonary in-flammatory response in a rat model of endotoxemia byinhibiting NF-κB phosphorylation [18]. Furthermore, inactivated M1 macrophages, the PPARγ-dependenttransrepression pathway is initiated by the sumoylation ofthe liganded PPARγ ligand-binding domain maintainingthe corepressor complex on NF-κB response elements[33]. In this study, we investigated the effects ofrosiglitazone on LPS-induced NF-κB activation in mam-mary gland. The results of the present study showed thatrosiglitazone markedly suppressed NF-κB phosphoryla-tion and IκB degradation in the LPS-induced mastitis.

In conclusion, the present study demonstrates thatrosiglitazone reduces LPS-induced mammary gland injuryand inflammatory cell infiltration, decreases the MPO ac-tivation in the mammary gland, and downregulates theproduction of TNF-α, IL-6, and IL-1β in a dose-dependentmanner. The mechanisms of this action for rosiglitazonemay associate with the inhibition of NF-κB activation. Itsuggested that PPAR-γ activation can serve as a newtherapeutic strategy for mastitis.

Conflict of Interest. All authors declare that they have noconflict of interest.

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