The Suppressive Effects of Lanthanum on the Productionof Inflammatory Mediators in Mice Challenged by LPS

Fei Guo & Xiang Guo & An Xie & Yuan Lei Lou &

Yang Wang

Received: 18 June 2010 /Accepted: 22 July 2010 /Published online: 3 August 2010# Springer Science+Business Media, LLC 2010

Abstract Lanthanide ions have been proven to have various biologic effects. Lanthanumwith extremely active physical and chemical property was evidenced to possess antibacterialand immune adjustment effects. In the present study, the anti-inflammatory effects oflanthanum chloride (LaCl3) on lipopolysaccharide (LPS)-challenged mice were examined invivo and in vitro. The results indicated that LaCl3 can greatly decrease the secretion oftumor necrosis factor alpha (TNF-α) and interleukin (IL)-1β as well as TNF-α mRNAexpression in the mice challenged with LPS. To clarify the mechanism involved, the effectsof LaCl3 on the activation of nuclear factor (NF)-κB were examined both in liver and inperitoneal macrophages. The LPS-induced activation of NF-κB was significantly blockedby LaCl3. These findings demonstrate that the inhibition of the LPS-induced inflammatorymedia, such as TNF-α and IL-1β, by LaCl3, is due to the inhibition of NF-κ B activation.

Keywords Lipopolysaccharide . Lanthanum . Tumornecrosis factor . Nuclear factor-kappaB

Introduction

Lipopolysaccharide (LPS; bacterial endotoxin), a major integral structural component of theouter membrane of Gram-negative bacteria, is recognized as the most potent initiator ofinflammation. Nuclear factor κB (NF-κB) family of transcription factors plays an importantrole in the inducible expression of genes described above. NF-κB is activated in response toLPS and pro-inflammatory cytokines. There is increasing evidence that NF-κB is of greatimportance in the pathobiology of disease states such as systemic inflammatory response

Biol Trace Elem Res (2011) 142:693–703DOI 10.1007/s12011-010-8792-0

F. GuoBurns Institute, The First Affiliated Hospital, Nanchang University, Nanchang, China

X. GuoThe Affiliated Hospital, Jiu Jiang University, Jiu Jiang, China

A. Xie : Y. L. Lou :Y. Wang (*)Institute of Urology, The First Affiliated Hospital, Nanchang University, Nanchang, Chinae-mail: wangy63cn@126.com

syndrome and multiple organ dysfunction syndrome [1]; therefore, therapeutic interventionsaimed at limiting NF-κB activation and down-regulation production of inflammatorymediators could prove to be beneficial in decreasing host-derived tissue injury and organdysfunction.

Lanthanum, a representative of lanthanides with extremely active physical and chemicalproperty, has been evidenced to possess antibacterial effects and regulating cellularimmunity. Lanthanide compounds are employed in the treatment of various diseases. Forexample, lanthanum carbonate is well tolerated and is effective for the long-termmaintenance of serum phosphorus control in patients with end-stage renal disease [2, 3].Studies also show the greater efficacy of sulfadiazine combined with cerium nitrate in thetreatment of burns patients [4, 5]. Administration of gadolinium chloride (GdCl3) to rats invivo decreased the release of nitric oxide (NO) and the expression of inducible nitric oxidesynthase by isolated rat Kupffer cells in response to LPS [6]. GdCl3 pretreatment eliminatedthe rise of iNOS activity, and its protein level in lungs thus significantly attenuated theincrease in pulmonary exhaled NO product [7]. It remains unknown whether lanthanum isable to suppress the production of inflammatory mediators in LPS-challenged mice and themechanism by which lanthanum ion modulates LPS-mediated inflammatory response. Wechoose BALB/c, a laboratory-bred strain of the house mouse for the following research.

In the present study, we explored the changes in the plasma levels of TNF-α and IL-1βas well as key molecular and cellular events leading to TNF-α secretion in the liver of LPS-treated mice pretreated with NS (normal saline) or lanthanum chloride (LaCl3) in vivo. Wealso evaluated the in vitro effects of LaCl3 on LPS-induced NF-κB activation and TNF-αexpression in peritoneal macrophages isolated from mice.

Materials and Methods

Reagents

LPS from Escherichia coli (serotype 055:B5) and lanthanum chloride (LaCl3·7H2O, purity:99.9%) were from Sigma (USA). DMEM-F12 (LPS<0.03U ml−1) in ready-to-use formwere bought from Hyclone, USA. Fetus bovine serum (FBS; LPS<0.03 U ml−1) was fromGibco, USA. Rabbit polyclonal anti-IκBα, mouse monoclonal anti-NF-κB/p65, and β-actinantibody were purchased from Santa Cruz Biotechnology Inc., (Santa Cruz, CA, USA).Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and HRP-conjugated goatanti-mouse IgG were also from Santa Cruz, USA. Enhanced chemical-luminescence kit wasbought from Pierce, USA. Protein assay kit was from Bio-Rad, USA. Nuclear Extract kitand TransAM™ NF-κB/P65 transcription factor assay kit were obtained from Active Motif(USA). Total RNA Isolation System (Promega's RNAgents®) was used to obtain total RNAfrom tissues and cultured cells. The Reverse transcriptase kit was also from Promega(USA). All primers, Taq polymerase and DNA marker were from Takara Biotechnology(Dalian) Co., Ltd. SYBR® GreenER™ qPCR SuperMix for ABI PRISM was fromInvitrogen, USA. Radio Immunoassay kits for TNF-α and IL-1β were from AmershamInternational PLC (Buckinghamshire, England).

Animals and Treatment

Female BALB/c mice at the age of 4 weeks (16–22 g), obtained from the Department ofExperimental Animal Science, Nanchang University, were housed under a constant light

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and dark cycle, and allowed free access to standard food and water. All experiments wereapproved by the animal ethics committees of Nanchang University, China and performedaccording to their guidelines.

One hundred sixty eight BALB/c mice were randomly divided into 14 groups of 12 miceeach. LPS dissolved in sterile saline (17.5 mg/kg, LD-50, determined by the Reed-Munchmethod [8], data not shown) was intraperitoneally injected. LaCl3 dissolved in 200 μLsterile, pyrogen-free saline was injected intraperitoneally 30 min prior to LPS injection at 1,2, 5, 10, 20, 40, and 80 mg/kg/day, respectively. Control mice received the same volume ofsterile saline. Mortality was scored up to 7 days after challenge.

To evidence the protective effects of LaCl3 against LPS challenge in vivo, 60 BALB/c micewere randomly divided into four groups of 15 mice each: in LPS group, the mice were treatedwith an intraperitoneal injection of 12.5 mg/kg LPS (12.5 mg/kg LPS challenge caused toxiceffects but did not lead to the death of the mice when given to control animals in preliminaryexperiments); in lanthanum- treated group, 10 mg/kg LaCl3 was administered intra-peritoneally 30 min before an intraperitoneal injection of 12.5 mg/kg LPS; 10 mg/kglanthanum control group and pyrogen-free saline control group were set simultaneously, ateach time point (2 h, 4 h and 24 h after LPS injection), blood samples were collected fromfossa orbitalis into plastic tubes containing a 1:10 volume of 3.8% sodium citrate. Citratedplasma was collected separately by centrifugation and stored at −80°C until use. Theconcentration of TNF-α and IL-1β in the citrated plasma was quantitated using RadioImmunoassay kits according to the manufacturer’s instruction. Whole liver tissues werecollected and stored at −80°C for western blot and real-time PCR analyses [9].

Cell Culture and Sample Treatment

Peritoneal macrophages were obtained by a modification of the method of Denlinger et al. [10].Briefly, mice were sacrificed by cervical dislocation and the abdominal skin was reflected.Phosphate buffered saline (PBS; PH 7.4) containing 10 U of heparin/ml was injected into theperitoneal cavity (5 ml/mouse), and the mice were shaken for 2 min. Peritoneal cells werecollected in a syringe and washed once in PBS-heparin at 4°C. Two volumes of medium wereadded followed by centrifugation at 200×g for 5 min. The cells were re-suspended, plated, andmaintained in DMEM-F12 supplemented with 10% FBS, 100 U of penicillin/ml and 100 mgof streptomycin/ml in a humidified environment at 37°C with 5%CO2. After 2 h of incubation,the adherent peritoneal cells were washed three times with medium and immediately used forexperiment. To prevent interference from FBS (FBS may bind with LPS and/or LaCl3), cellswere washed three times with PBS before incubation with lanthanum, and fresh DMEM-F12,rather than DMEM-F12 containing 10% FBS, was added to the cells.

To observe the effects of lanthanum on TNF-α production and mRNA level in LPS-inducedperitoneal macrophages, the cells were divided randomly into two groups: in the LPS group thecells in each subgroup were incubated with 0, 10, 100, and 1,000 ng/ml LPS for 2 h, respectively.After pretreatment with 2.5 μMof LaCl3 for 30 min, the cells in each subgroup of LaCl3+LPSgroup were treated, respectively, with the indicated concentrations of LPS for another 2 h.

To explore the effects of LaCl3 on activation of NF-κB in LPS-induced macrophages,the cells were divided at random into four groups: LPS group, LaCl3 group, LaCl3+LPSgroup and control group: the cells from LPS group were incubated with 1,000 ng/ml LPSfor 30 min; In LaCl3 group, cells were incubated with 2.5 μM of LaCl3 for 30 min for NF-κB assay; Post-2-h lanthanum exposure at the same concentration mentioned above, thecells in LaCl3+LPS group were washed triplicate with PBS to remove LaCl3 and treatedwith subsequent LPS (1,000 ng/ml) activation for another 30 min; in the control group, the

The Suppressive Effects of Lanthanum 695

cells were cultured with DMEM-F12 only. Then the expression and activation of NF-κB/p65 in nuclear, IκBα level in cytoplasm were detected.

Radio Immunoassay

The TNF-α and IL-1 β level in the serum of mice was measured by radio immunoassay kitsfor murine TNF-α and IL-1β according to the manufacturer"s instruction.

Preparation of Cytoplasmic and Nuclear Fractions

The cytoplasmic and nuclear extracts of cultured cells or frozen liver sections were preparedaccording to the instructions given in the manual. Briefly, 8.8×106 cells or 200 μlhomogenized tissue samples were washed with ice-cold PBS containing phosphatase-inhibitors, gently re-suspended in 500 μl hypotonic buffer and incubated for 15 min. Next,25-μl detergent was added and the suspension was vortexed for 10 s at highest speed andthen centrifuged for 30 s at 14,000×g in a microcentrifuge pre-cooled at 4°C. The supernatant(cytoplasmic fraction) was transferred into a pre-chilled microcentrifuge tube and stored inaliquots at −80°C until ready to use. Subsequently, the nuclear pellet was re-suspended in50 μl of complete lysis buffer and the suspension was incubated for 30 min on ice on arocking platform set at 150 rpm. After 30 s, the sample was vortexed at the highest speed; thesuspension was centrifuged for 10 min at 14,000×g in a microcentrifuge pre-cooled to 4°C.The supernatant (nuclear fraction) was also transferred into a pre-chilled microcentrifuge tubeand stored in aliquots at −80°C until ready to use. The protein concentration was determinedby the Bio-Rad protein assay reagent according to the manufacturer"s instructions.

Western Blot Analysis

The proteins in the Nuclear fractions or cytoplasmic fractions (30 μg total protein/lane)were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred ontonitrocellulose membranes by using a trans-blot apparatus (TRANS-BLOTRSD, Bio-Rad,USA). The immunoblot was incubated overnight with Tween 20/Tris-buffered saline(TTBS) containing 5% (w/v) nonfat milk at 4°C, followed by incubation for 2 h with a1:1,000 dilution of primary antibody (anti-I kB a or anti-p65). The blots were washed threetimes within TTBS and incubated with a 1:2,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody or peroxidase-conjugated rabbit anti-mouse IgG secondary antibody (Santa Cruz Biotechnology Inc.) for 1 h at roomtemperature, respectively. The blots were again washed three times with TTBS and thenvisualized by enhanced chemiluminescence (Pierce, USA), followed by autoradiography atseveral exposures to ensure appropriate grayscale density.

Nuclei p65 Activity Assay

According to a previous report [11], NF-κB activation in the nuclear extracts can bequantified by a TransAM NF-κB assay kit (Active Motif) based on the principle of ELISA.Specifically, an immobilized oligonucleotide containing the NF-κB consensus site (5′-GGGACTTTCC-3′) was bound to microwell plates. The active form of NF-κB present innuclear extracts specifically binding to this oligonucleotide was detected using a primaryantibody that recognizes an epitope on p65 that is accessible only when NF-κB is activatedand bound to its target DNA. Following the addition of an HRP-conjugated secondary

696 Guo et al.

antibody, the plates were read on an automated plate reader, and the level of nuclear NF-κBp65 was expressed as the absorbance at 450 nm (A450).

RNA Preparation and Polymerase Chain Reaction

Total RNA was extracted from peritoneal macrophages and whole liver tissue usingPromega's RNAgents® Total RNA Isolation System and quantitated spectrophotometrically.The expression of TNF-α mRNA was examined using quantitative real-time reversetranscription polymerase chain reaction (qRT-PCR). Mice were sacrificed following theindicated treatments (n=5 at each time point). Whole liver tissues were taken and frozen at−80°C until use. All reagents used for RT-PCR were of molecular grade. All RNA sampleswere confirmed free of DNA contamination by treated with DNaseI. One microgram oftotal RNAwas reverse transcribed using a first strand cDNA synthesis kit (Promega). qRT-PCR was performed using SYBR GreenER™ qPCR SuperMix for ABI PRISM®instrument (Invitrogen). The amplification reaction was carried out in an ABI GeneAmp5700 instrument (Applied Biosystems). Reaction was performed in a 20 μl reaction volumewhich contained 90 ng of cDNA, 10 μl of SYBR® GreenER™ qPCR SuperMix and200 nM of specific primers. The thermal profile for qPCR was 95°C for 2 min followed by40 cycles of 95°C for 25 s and 55°C for 45 s. Relative quantity values were analyzed usingABI 5700 Sequence Detection System software (Applied Biosystems) according to the ΔCtmethod which reflects the difference in threshold for each target gene relative to that ofβ-actin. For TNF-α, the forward primer was 5′-GGC AGG TCT ACT TTG GAG TCATTG-3′, and reverse primer was 5′-ACA TTC GAG GCT CCA GTG AAT TCG G-3′.For β-actin: sense strand: 5_-TAA AAC GCA GCT CAT AAC AGT CGG-3, antisense:5-TGC AATCCT GTG GCATCC ATG AAA C-3.

Statistical Analysis

Data were expressed as mean and standard deviation of the mean. All experiments wererepeated at least three times. χ2 was applied to compare the difference of mortality betweenNS control group and treated groups. And Student"s t test was used to determine thestatistical differences between various experimental and control groups. P value<0.05 wasconsidered as significant.

Results

Pretreatment of LaCl3 Alleviated the LPS-Induced Mortality

We explored whether lanthanum chloride was capable of protection against the LPSchallenge. BALB/c mice pretreated with the indicated dosages of LaCl3 were exposed toLD-50 of LPS (17.5 mg/kg). The proportions of mice surviving after LD-50 of LPS areshown in Table 1. Pretreatment with 5–10 mg/kg LaCl3 prior to LPS injection elevated thesurvival rate of mice by 100% from 33% in LPS alone when examined at 7 days after LPSinjection. At the 7th day post-LPS injection, survival rate of LaCl3 pretreated mice (5 and10 mg/kg) was 100% while that of untreated mice was 33%. It is interesting to note that theLaCl3-treated mice without LPS injection do not show any signs of toxicity, the survivalrates of mice injected intraperitoneally with 1, 2, 5 10, 20, 40, and 80 mg/kg respectively,were all 100%.

The Suppressive Effects of Lanthanum 697

LaCl3 Down-regulated TNF-α mRNA Expression in Livers of LPS-Challenged Mice

The effects of LaCl3 on the expression of TNF-α mRNA in livers of mice treated with LPSwere determined using qRT-PCR. LPS treatment (12.5 mg/kg, i.p.) increased TNF-αmRNA over the basal level at 4 h. Pretreatment of LaCl3 (10 mg/kg) significantly decreasedthe mRNA level (Fig. 1).

LaCl3 Suppressed LPS-Induced TNF-α and IL-1β Plasma Secretion

Since TNF-α and IL-1β, the principal mediators of the responses to LPS, are involved inthe inflammatory process [12, 13], we assessed the effects of LaCl3 on the mediators in

0

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25

copi

es/ µ

gRN

A(×

106 )

#

LPS (12.5 mg/kg, i.p) --+

LaCl3 (10 mg/kg) +

+

- - +

# P< 0.001 vs. NS control;

Fig. 1 Effects of lanthanumchloride on the expressionof TNF-α mRNA in the liverof mice challenged with LPS(mean ± SD, n=5). LPS treatmentincreased TNF-α mRNA overthe basal level at 4 h. Pretreat-ment of lanthanum chloridesignificantly decreased themRNA level. #P<0.001 vs.NS control

Table 1 Effects of different concentration of lanthanum chloride on the survival rate of LPS-challengedmice

Group Dose(mg/kg) Treated animals Dead animals Survival rate (%) Pa

NS Control 0 12 8 33

Treated 1 1 12 7 42 >0.05

Treated 2 2 12 5 58 >0.05

Treated 3 5 12 0 100 <0.01

Treated 4 10 12 0 100 <0.01

Treated 5 20 12 1 92 <0.01

Treated 6 40 12 5 58 >0.05

Trreated 7 80 12 11 8 >0.05

a Compared with control group, lanthanum chloride (0, 1, 2, 5, 10, 20, 40, and 80 mg/kg) was administeredintraperitoneally for 30 min prior to LPS injection. LPS was intravenously injected at the dose of 17.5 mg/kg.Mortality was scored up to 7 days after the challenge (n=12)

698 Guo et al.

**

#

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#

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l

TNF-αIL-1

LPS (12.5 mg/kg, i.p)

LaCl3 (10 mg/kg)

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+

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+

# P < 0.001 vs. NS control (Lacl3-/LPS-) group; * P < 0.001 vs. LPS (Lacl3-/LPS+) group

Fig. 2 Effects of lanthanum chloride on the levels of TNF-α and IL-1β in plasma of mice challenged withLPS(mean ± SD, n=5). The concentration of TNF-α was detected 4 h after LPS challenge, while IL-1βlevels in the plasma were assessed at 24 h after LPS treatment. The LPS-induced elevation of plasma TNF-αand IL-1β was significantly suppressed by the treatments of lanthanum chloride at 10 mg/kg (both P<0.001).#P<0.001 vs. NS control (Lacl3-/LPS-) group; *P<0.001 vs. LPS (Lacl3−/LPS+) group

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0 10 100 1000

LPS (ng/ml)

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LPS

LPS+LaCl3

TN

F-α

(pg

/ml)

* P < 0.01 vs. LPS group

**

*

* * *

a

b

Fig. 3 Effects of lanthanumchloride (2.5 μM) on productions(a) and mRNA levels (b) ofTNF-α from peritonealmacrophages stimulated byvarious dosages of LPS(mean ± SD, n=3). *P<0.01vs. LPS group

The Suppressive Effects of Lanthanum 699

plasma of mice treated with LPS. The concentration of TNF-α was detected by RadioImmunoassay at 4 h post-LPS challenge. The plasma TNF-α level in the mice of LaCl3+LPSgroup was 440±215 pg/ml, and there is no significance between NS control group (470±191 pg/ml), LaCl3 control group (460±147 pg/ml) and LaCl3+LPS group (both p>0.05).Furthermore, the LPS-induced elevation of plasma TNF-α was up to 990±242 ng/ml, whichwas significantly suppressed by the treatments of LaCl3 at 10 mg/kg (P<0.001).

IL-1β levels in the plasma were assessed at 24 h post-LPS treatment, in LPS group, theIL-1β level was 226±31 pg/ml, which was significantly higher than those of NS controlgroup (94±45 pg/ml), LaCl3 control group(91±16 pg/ml) and LaCl3+LPS group (100±15 pg/ml), all p<0.001. Furthermore, there is no difference between LaCl3+LPS group andthe two control groups (p>0.05). All these indicated that the plasma IL-1β secretionenhanced significantly when the mice were challenged by LPS, and the elevation of plasmaIL-1β was suppressed by LaCl3 treatment Fig. 2.

LaCl3 Decreased LPS-Stimulated TNF-α Secretion and mRNA Expressionin Peritoneal Macrophages

As shown in Fig. 3, LPS-stimulated TNF-α secretion from macrophages dose dependently.Treatment with 2.5 μM LaCl3 led to a significant decrease at TNF-α protein (Fig. 3a) andmRNA levels (Table 2, and Fig. 3b).

LaCl3(10 mg/kg) + + -

LPS (12.5 mg/kg) -

-

+ -+

IκBα

β-actin

β-actin

p65

Fig. 4 Lanthanum chlorideinhibited LPS-mediateddegradation of IκBα and p65translocation in liver tissues

Table 2 Effects of lanthanum chloride on LPS-induced TNFα mRNA expression of murine peritonealmacrophages (copies/μgRNA, mean ± SD, n=3)

Groups Concentration of LPS (ng/ml)

0 10 100 1,000

LPS only 6:5� 0:59ð Þ � 105 5:0� 0:43ð Þ � 106 1:8� 0:49ð Þ � 108 7:4� 1:2ð Þ � 108

LPS+LaCl3 5:2� 0:66ð Þ � 105 5:0� 0:23ð Þ � 105»

8:1� 0:71ð Þ � 105»

2:3� 0:96ð Þ � 107»

The murine peritoneal macrophages were divided randomly into two groups. In LPS group, the cells in eachsubgroup were incubated with 0, 10, 100, and 1,000 ng/ml LPS for 2 h, respectively. In LaCl3+LPS group,after pretreated with 2.5 μM of LaCl3 for 30 min, the cells in each subgroup were treated, respectively, withthe indicated concentrations of LPS for another 2 h

*P<0.01 vs. LPS only group

700 Guo et al.

LaCl3 Inhibits LPS-Mediated Degradation of IκBα and p65 Translocation in PeritonealMacrophages and Liver Tissues of Mice

As shown in Figs. 4 and 5, the IκBα protein levels in both livers of LPS-treated mice andLPS-stimulated macrophages were markedly decreased at 2 h or 30 min post-treatment,respectively. Thus, the LPS-stimulated degradation of IκBα was blocked by lanthanum. Tostudy the effects on NF-κB/p65 subunit expression, nuclear extracts of macrophages andliver tissues were also studied by western blot. LPS induced a marked increase of nuclearp65 expression which was prevented by the administration of LaCl3. The following NF-κB–DNA binding activity assay was conducted to further prove the results of western blotanalysis.

LaCl3 Inhibited LPS-Mediated Activation of NF-κB/p65 in Peritoneal Macrophages

NF-κ B–DNA binding activity was measured by TransAm™ NF-κB/p65 transcriptionfactor assay kit, which containing a 96-well plate on which has been immobilizedoligonucletide containing the NF-κB consensus site (5′-GGG ACT TTC C-3′). LPSstimulation of the macrophages resulted in a remarkable increase in NF-κB/p65 activation.In LaCl3 group and LaCl3+LPS group, the nuclear activation level of NF-κB/p65 wasreduced significantly, compared with LPS group, p<0.01. However, the nuclear activationlevel of NF-κB/p65 in LaCl3+LPS group was higher than that of control group (p<0.05)but significantly lower than that of LPS group (p<0.01, Fig. 6).

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# P < 0.01 vs. control (Lacl3-/LPS-) group; * P < 0.01 vs. LPS (Lacl3-/LPS+) group

Fig. 6 The effects of lanthanum chloride on NF-κB–DNA binding activity (mean ± SD, n=3). #P<0.01 vs.control (Lacl3-/LPS-) group; *P<0.01 vs. LPS (Lacl3-/LPS+) group

LaCl3(2.5 µM)

LPS (1000 ng/ml) --++

-++-

β-actin

β-actin

IκBα

p65

Fig. 5 Lanthanum chlorideinhibited LPS-mediateddegradation of IκBα andp65 translocation in peritonealmacrophages

The Suppressive Effects of Lanthanum 701

Discussion

Despite numerous antibiotics, the fact of high case fatality rate in sepsis patients does notchanged. Although antibiotic is capable of suppressing or killing bacteria, it does notpossess antagonism against LPS, a key factor in eliciting the systemic inflammatoryresponse associated with infection, which is largely released with the disintegration ofbacteria. Thus seeking for LPS antagonist is critical to the treatment of sepsis. In the currentstudy, we explored the effects of LaCl3 on LPS-challenged mice in vivo and in vitro.

The NF-κB family is a key player in controlling both innate and adaptive immunity. NF-κB proteins are present in the cytoplasm in association with inhibitory proteins that areknown as inhibitors of NF-κB (IκBs). After activation by a large number of inducers,including pathogens, stress signals and pro-inflammatory cytokines, such as LPS, the IκBproteins become phosphorylated, ubiquitylated and, subsequently, degraded by theproteasome. The degradation of IκB allows NF-κB proteins to translocate to the nucleusand bind their cognate DNA binding sites to regulate the transcription of a large number ofgenes, including antimicrobial peptides, cytokines, chemokines, stress-response proteins,and anti-apoptotic proteins [14]. Finding novel inhibitor of NF-κB pathways will provide aplatform for developing specific therapeutics for inflammatory diseases.

The results of the current experiments showed that LaCl3 can decrease the secretion ofTNF-α and IL-1β as well as TNF-α mRNA expression greatly in the mice challenged withLPS. LaCl3 also inhibited LPS-mediated IκBα degradation and p65 translocation both inliver tissues and in peritoneal macrophages. The inhibitory effects of lanthanum areassociated with suppression of IκB degradation and NF-κB/p65 activation, LPS-inducedactivation of NF-κB was significantly blocked by LaCl3. These findings demonstrate thatthe inhibition of the LPS-induced inflammatory media, such as TNF-α and IL-1 β, byLaCl3, is due to the inhibition of NF-κB activation.

The results of the present study indicate that LaCl3 is a potent inhibitor of LPS-inducedpro-inflammatory factors and the inhibition is caused by blocking NF-κB activation. Thesefindings suggest that LaCl3 is a potential therapeutic agent for the treatment of variousinflammatory diseases.

Acknowledgement The work was supported by the National Natural Science Foundation of China(30660182, 30960405), Natural Science Foundation of Jiangxi Province (2007GZY1132), Scientific Plan ofJiangxi Provincial Department of Public Health (20071041), and Program for Innovative Research Team ofNanchang University.

References

1. Pinsky MR (2007) Sepsis and multiple organ failure. Contrib Nephrol 156:47–632. Shigematsu T (2008) Lanthanum carbonate effectively controls serum phosphate without affecting serum

calcium levels in patients undergoing hemodialysis. Ther Apher Dial 12:55–613. Hutchison AJ, Barnett ME, Krause R et al (2009) Lanthanum carbonate treatment, for up to 6 years, is

not associated with adverse effects on the liver in patients with chronic kidney disease stage 5 receivinghemodialysis. Clin Nephrol 71:286–295

4. Hadjiiski OG, Lesseva MI (1999) Comparison of four drugs for local treatment of burn wounds. Eur JEmerg Med 6:41–47

5. de Gracia CG (2001) An open study comparing topical silver sulfadiazine and topical silver sulfadiazine-cerium nitrate in the treatment of moderate and severe burns. Burns 27:67–74

6. Roland CR, Naziruddin B, Mohanakumar T et al (1996) Gadolinium chloride inhibits Kupffer cell nitricoxide synthase (iNOS) induction. J Leukoc Biol 60:487–492

702 Guo et al.

7. Fujii Y, Goldberg P, Hussain SN (1998) Contribution of macrophages to pulmonary nitric oxideproduction in septic shock. Am J Respir Crit Care Med 157:1645–1651

8. Reed LJ, Munch H (1938) A simple method of estimating fifty percent endpoints. Am J Hyg 27:493–4979. Yao YM, Xu CL, Yao FH et al (2006) The pattern of nuclear factor-kappaB activation in rats with

endotoxin shock and its role in biopterin-mediated nitric oxide induction. Zhonghua Shao Shang Za Zhi22:405–410

10. Denlinger LC, Garis KA, Sommer JA et al (1998) Nuclear translocation of NF-kappaB inlipopolysaccharide-treated macrophages fails to correspond to endotoxicity: evidence suggesting arequirement for a gamma interferon-like signal. Infect Immun 66:1638–1647

11. Zhang P, Martin M, Michalek S et al (2005) Role of mitogen-activated protein kinases and NF-κB in theregulation of proinflammatory and anti-inflammatory cytokines by porphyromonas gingivalis hemag-glutinin B. Infect Immun 73:3990–3998

12. Cogswell JP, Godlevski MM,Wisely GB et al (1994) NF-kappa B regulates IL-1 beta transcription througha consensus NF-kappa B binding site and a nonconsensus CRE-like site. J Immunol 153:712–723

13. Parmentier M, Hirani N, Rahman I et al (2000) Regulation of lipopolysaccharide-mediated interleukin-1beta release by N-acetylcysteine in THP-1 cells. Eur Respir J 16:933–939

14. Li Q, Verma IM (2002) NF-kappaB regulation in the immune system. Nat Rev Immunol 2:725–734

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