Effect of Simvastatin on Proinflammatory Cytokines Production

7/28/2019 Effect of Simvastatin on Proinflammatory Cytokines Production

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Efect o simvastatin on proinammatory cytokines productionduring lipopolysaccharide-induced inammation in rats

Lana Nei1, Ranko krbi1, Silva Dobri2, Milo P. Stojiljkovi3, Svjetlana S. atara1,Zoran A. Milovanovi2 and Nataa Stojakovi1

1 Department o Pharmacology, oxicology and Clinical Pharmacology, Medical Faculty, University o Banja Luka, SaveMrkalja 14, 78000 Banja Luka, Republic o Srpska, Bosnia and Herzegovina

2 National Poison Control Centre, Medical Military Academy, Crnotravska 11, 11000 Belgrade, Serbia3 Department o Pharmacology, Medical Faculty, University o East Sarajevo, 51000 Foa, Republic o Srpska, Bosnia and

Herzegovina

Abstract. Te eect o simvastatin applied in a short-term pretreatment on proinammatory cytokinesproduction in acute systemic inammation induced by endotoxin lipopolysaccharide (LPS) in ratswas investigated. Both LPS and simvastatin doses were established in separate experiments in whichincreasing doses o both compounds were given to obtain the LD50 LPS and the maximally protectivedose o simvastatin against LD50 LPS. o determine the anti-inammatory eect, simvastatin wasgiven orally or 5 days, ollowed by a single intraperitoneal non-lethal dose o LPS (0.25 LD50).Plasmaconcentrations o tumor necrosis actor alpha (NF-), interleukin (IL)-1 and IL-6 were measured byenzyme-linked immunosorbent assay. Te acute i.p. LD50 LPS amounted to 22.15 mg/kg. Simvastatin o20 mg/kg p.o. was maximally protective against LD50 LPS, and this dose was used or studying its eectson LPS-induced cytokines production. Cytokines concentrations were signifcantly increased uponchallenge o non-lethal dose o LPS. Te peak levels o NF- and IL-1 were signifcantly suppressedby simvastatin, compared to control rats only treated with dimethylsuloxide beore LPS. In contrast,simvastatin did not aect IL-6 levels at all timepoints. Simvastatin pretreatment givenorally producedacute anti-inammatory eects by inhibiting NF- and IL-1, but no IL-6 production.

Key words: Simvastatin Endotoxin Inammation Cytokines Rat

Correspondence to: Lana Nei, Department o Pharmacol-ogy, oxicology and Clinical Pharmacology, Medical Faculty,Save Mrkalja 14, 78000 Banja Luka, Republic o Srpska, Bosniaand Herzegovina

E-mail: [email protected]

Introduction

Several clinical trials showed that statins reduce the risk ocardiovascular events even in the absence o a signifcant

drop o blood cholesterol levels (Ridker et al. 1998; Davignon2004), suggesting that the benefts o statin therapy may alsobe ascribed to their action on nonlipid actors involved ininammation-fbroprolieration, an important eature oatherosclerosis (Marz and Wieland 2000; Bonetti et al. 2003).Tere is growing evidence that statins have additional anti-in-ammatory properties by reducing inammatory parameters

such as C-reactive protein (Albert et al. 2001; Joukhadar et al.2001), tumor necrosis actor (NF-) and interleukin (IL)-1 in patients with hypercholesterolemia and heart transplantrecipients (Holm et al. 2001; Koh et al. 2004), unrelated to their

lipid-lowering activity. Moreover, recent studies have shownthat prior statins therapy prevented vascular hyporeactivityduring acute systemic inammation in humans (Pleiner etal. 2004) and was associated with a reduction o severe sepsisdevelopment (Almog et al. 2004).

Statins are 3-hydroxy-3-methylglutaryl-coenzyme A(HMG-CoA) reductase inhibitors, which reduce low-densitylipoprotein (LDL) cholesterol levels by blocking the meval-onate pathway and increase LDL cholesterol receptor expres-sion in the liver. By inhibiting HMG-CoA reductase, statinsmight directly inuence the cellular events other than choles-terol synthesis, because mevalonate, the product o HMG-CoA

reductase, is the precursor o not only cholesterol, but also o

Gen. Physiol. Biophys. (2009), Special Issue, 28, 119126


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many non-steroidal isoprenoid compounds. Te isoprenoidsarnesyl pyrophosphate and geranylgeranyl pyrophosphateare known to play an important role in signal transduction

pathways by their attachment to signalling proteins, such asRas and Rho (Kwak et al. 2000; Cordle et al. 2005).

Inhibition o HMG-CoA reductase activity in monocytes(erkeltaub et al. 1994) and rat mesangial cells (Kim et al.1995) treated with lipopolysaccharide (LPS) and granulocyte-macrophage-colony stimulating actor reduced the productiono IL-8, IL-6, and MCP-1, (monocyte chemotactic protein-1)responsible or leukocyte recruitment at the inection site.Statins have been shown as eective endothelium-protectiveagents that reduced leukocyte-endothelial cell interactions andimproved endothelial unction via increasing endothelial nitricoxide synthase (eNOS) (Laus et al. 2002; Prueer et al. 2002).

Recently, using a common model o acute local inammation(carrageenan-induced rat paw oedema), we also showed thatsimvastatin administered orally in the single dose produced sig-nifcant anti-inammatory activity (ootpad swelling inhibitionand reduction o polymorphonuclear leukocytes infltration)comparable to that o indomethacin (Nei et al. 2009). Studyby Wagner et al. (2002) showed that acute pretreatment withatorvastatin inhibits NF- plus intereron- stimulated tran-scription actor activation in native endothelial cells in situ andthe subsequent expression o inducible nitric oxide synthase(iNOS) involved in vascular inammation and atherosclerosis.Also, cerivastatin administered intraperitoneally (i.p.) signif-cantly improved the survival o mice with LPS-induced sepsisand reduced NF-, IL-1 and IL-6 overproduction (Ando etal. 2000; Chaudhry et al. 2008).

In the present study, we investigated i simvastatin, a widelyused statin, when givenorally to imitate the regular route ostatins use in humans, may attenuate systematic inammationby inhibition o proinammatory cytokines production. Tefrst part o this study consisted o an experiment examin-ing both lethal and protective doses o LPS and simvastatin,respectively. Te second part o the study presented in vivoexperiments examining anti-inammatory eects o simvas-tatin administered in short-term pretreatment prior to non-lethal, but inammatory dose o LPS, on the pro-inammatory

cytokines (NF-, IL-1 and IL-6) production.Endotoxin or LPS is a component o the outer cell wall

o Gram-negative bacteria. Systemic injection o LPS toexperimental animals is a widely used in vivo model orthe study o endotoxic shock and acute systemic inamma-tion. LPS activates the immune system leading to releaseo endogenous proinammatory cytokines such as NF-,IL-1 and IL-6 (Ando et al. 2000; Dogan et al. 2002). Teimportant eature o this model is that simvastatin givenin short-term treatment does not aect plasma lipid levels(cholesterol-lowering activities takes at least two weeks otherapy), and thereore the results may be interpretedwithout

this conounding variable (Rosenson 1999).

Materials and Methods

Animals

Adult male Wistar rats were obtained rom Military MedicalAcademy Research Laboratories (Belgrade, Serbia) and kept inthe animal unit 7 days beore the experiment. Animals were giv-en standard laboratory diet and tap water ad libitumand housedin an air-conditioned room with an ambient temperature o2224C and a 12 h light/dark cycle (light on at 07:00 a. m.). Atthe start o the experiment, rats weighing 200220 g were placedin individual cages. All experimental procedures on animalswere conducted in accordance with the Guidelines on humancare o experimental animals adopted by the Ethical Committeeand other corresponding national legal codes.

Experimental design

Endotoxin-induced lethality in rats

Te animals were divided into three groups (n = 6 per group),given saline orally (p.o.) and challenged with one o thethree various doses o LPS (10, 20, 30 mg/kg b.w.) given i.p.Te lethality was then monitored over the next 7 days. Tenumber o dead rats resulting rom LPS was recorded andthe median lethal dose (LD50) o LPS (i.p.) was calculated(Litchfeld and Wilcoxon 1949).

Protective eect o simvastatin on lethality induced byendotoxin

o determine the eect o short-term pretreatment o simv-astatin on the survival o rats injected with LD50 o LPS, theanimals were divided into three groups (n = 6 per group);simvastatin was given to rats p.o. in one o the three vari-ous doses (5, 10, and 20 mg/kg b.w.) per day or 5 days, and1.5 h aer the last dose o simvastatin the single LD50 o LPSwas injected i.p. Te survival o animals was monitored ora period o the next 7 days. Simvastatin alone had no eecton the survival o the animals. Te doses o simvastatin

used were comparable to those that had previously beenused in rat/murine studies in vivo (typically 10100 mg/kg/day) and were higher than those used in humans becauseo a signifcant up-regulation (3- to 8-old) o HMG-CoAreductase induced by statin treatment in rodents (Kita etal. 1980; Sparrow et al. 2001; Leung et al. 2003).

Efect o simvastatin on proinammatory cytokinesproduction

In this experiment, simvastatin was used in a dose o 20 m/kgp.o. (the maximally protective dose o simvastatin against

the single LD50 o LPS in rats). o induce acute systemic


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121Statin eect on inammatory cytokines

inammation and proinammatory cytokines production,the animals were challenged with a non-lethal dose oendotoxin i.p. (0.25 LD50 o LPS).

Te rats were randomized into the control and sim-vastatin-treated experimental groups (n = 8 per group).Simvastatin (20 mg/kg) was given p.o. via oral gavage or5 days, and 1.5 h aer the last dose o simvastatin, LPS(Escherichia coli serotype 0127:B8; Sigma Aldrich, Munich,Germany) at a non-lethal single dose (5.5 mg/kg b.w.) wasi.p. administered. Te control animals received the samevolume o 0.1% dimethylsuloxide (DMSO) or 5 days, asa vehicle, beore LPS injection. Te rats were restrainedusing a restraint tube and blood was collected by tail veinpuncture at various time points (0, 60, 90, 120, 180, 240min) aer LPS injection. Blood samples were collected

into Eppendor tubes containing EDA (ethylenediaminetetraacetic acid) solution and centriuged (1000 g, 20 min,4C). Te plasma was stored at 70C until assay. Plasmalevels o NF-, IL-1 and IL-6 were determined usingrat ELISA (enzyme-linked immunoassay) kits in accord-ance with to the manuacturers recommended protocols(R&D Systems, UK). Sensitivity o detection was 20 pg/mlor cytokines.

Drugs

Simvastatin (donated by Krka, Novo Mesto, Slovenia) wasdissolved in 0.1% DMSO as a 10 mg/ml stock. LPS romE. coli serotype 0127:B8 (Sigma Aldrich) was injected i.p.aer dilution with sterile pyrogen-ree physiologic salinesolution, at an injection volume o 1 ml/kg.

Statistical analysis

Te median lethal dose o endotoxin that was lethal to 50%o the rats (LD50 o LPS) was calculated by the Lichfeld andWilcoxon procedure (Lichfeld and Wilcoxon 1949).

Dierences in plasma NF-, IL-1 and IL-6 level be-tween the simvastatin treated and the control animals weredetermined with nonparametric Mann-Whitney U-test and

Kruscal-Wallis test. Results were expressed as mean SE(standard errors),p < 0.05 was considered signifcant.

Results

Determination o LD50o LPS and protective doseo simvastatin

In able 1, LPS dose-dependently increased lethality orats during 7 days ater injection is shown. he LD50 oLPS administered i.p. was calculated to be 22.15 mg/kg

b.w.

Te ecacy o various doses o simvastatin administeredp.o. in the short-term treatment prior to LD50 o LPS chal-lenge resulted in a dose-dependent increase in survival. As

shown in able 1, complete lethality protection was observedat dose o 20 mg/kg b.w. (p.o.) o simvastatin.

Efect o simvastatin on proinammatory cytokinesproduction

For the purpose o this part o the experiment we useda single dose o LPS (5.5 mg/kg b.w., i.p.) as a non-lethaldose (0.25 LD50 o LPS) to induce acute systemic inam-mation. Te dose o 20 mg/kg b.w. (p.o.) o simvastatin thatprotected the animals rom LPS LD50 induced lethality wasused to determine the eect o the drug on proinammatory

cytokines production induced by LPS.All the animals survived this experiment. In addition,simvastatin itsel neither increased serum levels o cy-tokines nor aected the clinical status and the behavior orats, such as their dietary intake or body weight gain (datanot shown).

TNF- response

Te time course o plasma NF- level

Intraperitoneal injection o LPS induced a marked increasein plasma NF- level. Te plasma level increased at 60 minand peaked at 90 min aer injection (p < 0.002) comparedto baseline, with no signifcant dierence in plasma NF-levels between these timepoints (p = 0.52), and thereaer

Table 1. Te eect o simvastatin pretreatment on LPS-inducedlethality in rats

Simvastatin pretreatment(mg/kg/day p.o)b

LPS(mg/kg i.p)a

No. o rats(dead/total)

none1020

30

0/63/6

4/65

1020

LD50

3/61/60/6

a Adult male Wistar rats were divided into three groups (n = 6 pergroup), challenged with one o the various doses o LPS (10, 20,30 mg/kg b.w.) i.p. Lethality was monitored over the 7 days. Te doseo LPS that was lethal to 50% o the rats, i.e. LD50 o LPSadmin-istered i.p. was calculated to be 22.15 mg/kg b.w. by the Litchfeldand Wilcoxon procedure (Litchfeld and Wilcoxon 1949). b Ratswere divided into three groups (n = 6) and were given simvastatinp.o. in doses o 5, 10, and 20 mg/kg/day or 5 days, aer which thesingle LD50 o LPS was administered i.p. Survival o animals was

monitored or a period o the 7 days.


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gradually decreased to near basal levels by 240 min (Figure 1,panel A).

Te efects o simvastatin on LPS-induced plasma NF-elevation

As shown in Figure 1A, in rats pretreated with simvastatin priorto LPS injection, the plasma NF- levels signifcantly increased

at 60 min and 90 min, post LPS injection (p < 0.002) compared

to baseline, with no signifcant dierence in NF- plasma levelsbetween these timepoints (p > 0.05). Posthocanalysis revealedthat simvastatin signifcantly reduced the plasma NF- levels

compared to the control group, with maximal eects at 60 and90 min (p < 0.05) (Figure 1, panel B).

IL-1 response

Te time course o plasma IL-1level

Plasma IL-1 levels increased signifcantly with peak valueat 120 min aer LPS injection (p < 0.002), remained elevatedat 180 min (p < 0.002) and slightly decreased thereaer, butstill present aer 240 min timepoint (Figure 2, panel A).

Te efects o simvastatin on LPS-induced plasma IL-1elevation

As shown in Figure 2A, in rats pretreated with simvastatinprior to LPS injection, the plasma IL-1 levels signifcantlyincreased at 120 min and 180 min post LPS (p < 0.002),compared to baseline, with no signifcant dierences in IL-1 levels aer 120 min (p > 0.05). Posthoc analysis revealedthat simvastatin signifcantly reduced the plasma IL-1 levelscompared to the control group, with maximal eects at 120min (p < 0.01) and 180 min (p < 0.05) (Figure 2, panel B).

IL-6 response

Te time course o plasma IL-6 level

Plasma IL-6 levels increased signifcantly at 120 min (p

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Effect of Simvastatin on Proinflammatory Cytokines Production