The effect of capsaicin on circulating biomarkers, soluble tumor necrosis factor and soluble tumor necrosisfactor-receptor-1 and -2 levels in vivo using lipopolysaccharide-treated mice (©Y.Ijiri)

Abstract
The circulating soluble tumor necrosis factor (sTNF) and sTNF-receptor (R) 1 and -R2have known as septic biomarker. The pungent component of capsicum, capsaicin (Cap),has several associated physiological activities, including anti-oxidant, anti-bacterial andanti-inflammatory effects. The aim of this study was to elucidate the effect of Cap on cir-culating sTNF and sTNF-R1 and -R2 in vivo using lipopolysaccharide (LPS)-treated mice.LPS (20 mg/kg, ip)-treated group was significantly increased circulating sTNF, sTNF-R1, and-R2 and TNF-mRNA expression levels compared to the vehicle group. Treatment with LPS(20 mg/kg, ip) + Cap (4 mg/kg, sc)-treated group was significantly decreased both circulatingsTNF levels (after 1 h only) and TNF-mRNA expression (after 6 h) compared to the LPS-treated group. There is an early increase in circulating sTNF, sTNR-R1, and -R2 observed inthe LPS-treated mice. Since Cap inhibits this initial increase as biomarkers, circulating sTNF,it is considered a potent treatment option for TNF-related diseases, such as septicemia.In conclusion, Cap interferes with TNF-mRNA transcription and exerts an inhibiting effecton TNF-release from macrophages in the early phase after LPS stimulation. Thus, Cap isconsidered a potent agent for the treatment of TNF-related diseases, such as septicemia.© 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article underthe CC BY-NC-ND license

1. Introduction

The pungent component of capsicum, capsaicin (Cap),has several associated physiological activities, including anti-oxidant, anti-bacterial and anti-inflammatory effects[1–3].

LPS is an outer membrane component of Gram-negativebacteria and has been reported to activate NF-B viatoll-like receptor 4 (TLR4), which is present on antigen-presenting cells such as dendritic cells or macrophages [4],releasing pro-inflammatory mediators, including TNF-,interleukins (IL-1, IL-6, IL-10) [5,6], and nitric oxide (NO)[7]. Macrophages can also release TNF-(as soluble TNF[sTNF]) [8], which mediates its biological activities throughbinding to type 1 and 2 TNF receptors (TNF-R1 and -R2) [9,10]. In addition, TNF-R2, the principal mediator of theeffects of TNF-on cellular immunity, may cooperate withTNF-R1 in the killing of nonlymphoid cells [11]. When TNF-R1 and/or -R2 are stimulated by TNF-, the extracellularportions of transmembrane proteins are cleaved, solubleectodomains are released from the cell surface by a shed-dase known as TNF-converting enzyme (TACE) [12], andsTNF is neutralized by the sTNF-Rs [13]. After cell stim-ulation by various stimuli, including TNF itself, thesetwo receptors can be proteolytically cleaved by TACE [14]into two soluble forms, sTNF-R1 and sTNF-R2, which showprolonged elevation in the circulation of patients with var-ious inflammatory diseases such as septicemia, leukemia,hepatitis C virus infection, lupus, rheumatoid arthritis, andcongestive heart failure [15–22]. Furthermore, increasedcirculating levels of sTNF-R1 and -2 have been reported ina rat model of CCL4induced-liver injury [23].

The aim of this study was to investigate the effect ofCap on circulating TNF-(sTNF), sTNF-R1, and -R2 levelsin LPS-treated mice. The expression of TNF-, sTNF-R1 and-R2 proteins and mRNA were also examined in blood atdifferent time points.

2. Materials and methods

2.1. MaterialsL

PS (Escherichia coli, 055:B55, Lot No. 114K4107) waspurchased from Sigma–Aldrich, Co. (MO, USA), and Cap(98% purity) was provided by Maruishi Pharmaceutical Co.,Ltd. (Osaka, Japan). Other reagents used were commerciallyavailable extra-pure grade chemicals.

2.2. Animals

Male BALB/c mice (age, 8–10 weeks; weight, 21–26 g;Japan SLC, Inc., Shizuoka, Japan) were used. They werehoused for at least one week under controlled environmen-tal conditions (temperature, 24 ± 1◦C; humidity, 55 ± 10%;light cycle, 6:00–18:00) with free access to solid food(NMF, Oriental yeast Co., Ltd., Tokyo, Japan) and water. Allexperimental procedures were conducted according to theguidelines for the use of experimental animals and animalfacilities established by Osaka University of PharmaceuticalSciences.

2.3. Treatment of mice

LPS was dissolved in 2 mg/ml of physiological saline(Fuso Pharmaceutical Industries Ltd., Osaka, Japan) anddiluted to 1 mg/ml in physiological saline. Cap was dis-solved in 1% ethanol + 1% Tween 20 in physiological saline.LPS (20 mg/kg) was administered intraperitoneally (ip) and4 mg/kg Cap was administered subcutaneously (sc) to thebacks of the mice 5 min after LPS administration.

2.4. Measurement of circulating sTNF, sTNF-R1, andsTNF-R2 levels

Mice were divided into four groups: vehicle group, LPSgroup, Cap group, and LPS + Cap group. The animals weresacrificed under anesthesia for the following proceduresat 1, 3, 6, 9, and 12 h after LPS administration. Wholeblood was taken from the abdominal aorta of the mice. Thesamples were centrifuged, and the supernatant was mea-sured. Measurements were performed using Quantikine®Immunoassay Mouse TNF-, Quantikine®ImmunoassayMouse sTNFRI, and Quantikine®Immunoassay MousesTNFRII (R&D Systems, Inc., MN, USA). Within 30 min,absorbance was measured at 450 nm and 570 nm using aplate reader (Labsystems Multiscan MS; Dainippon Sumit-omo Pharma Co. Ltd., Osaka, Japan). The measured value ofthe vehicle group was defined as the control value. The lim-its of detection of sTNF, sTNF-R1, and sTNF-R2 levels were5.1, 5.0, and 5.0 pg/ml, respectively.

2.5. Measurement of circulating TNF-, TNF-R1, andTNF-R2 mRNA expression (derived from macrophages)levels in whole blood

Whole blood was taken from the abdominal aorta ofthe mice under anesthesia at 0.5, 1, 3, 6, and 9 h after LPSadministration. Total RNA was extracted from 300  l ofwhole blood using a total RNA extraction kit (PureLinkTMTotal RNA Blood Purification Kit for isolating total RNAfrom whole Blood; Invitrogen Corporation, CA, USA). Syn-thesis of cDNA was performed by reverse transcriptionusing total RNA solution (PrimeScriptTMRT reagent Kit;Takara Bio Inc, Shiga, Japan), and mRNA was measuredusing a thermal cycler (LightCycler®, Roche Diagnostics,Basel, Switzerland). The results were adjusted using glyc-eraldehyde 3-phosphate dehydrogenase (GAPDH) and 18srRNA, a housekeeping gene, as the internal standards.

2.6. Data analysis

Values are shown as mean ± standard deviation (SD).Statistical analysis was performed using Tukey’s test. A sig-nificant difference was determined as P < 0.05.3.

Results

3.1. Circulating sTNF, sTNF-R1, and sTNF-R2 levels

The circulating sTNF level significantly increased in theLPS group 1 h after LPS administration compared to boththe vehicle (P < 0.01, Fig. 1A) and LPS + Cap (P < 0.01, Fig. 1A)groups (n = 3–4). There was no significant difference in thecirculating sTNF levels between the vehicle and LPS + Capgroups (Fig. 1A). From 3 h to 12 h after LPS stimulation, cir-culating sTNF levels in the LPS group significantly increasedcompared to the vehicle group (P < 0.05 or 0.01, Fig. 1A).

Both the circulating sTNF-R1 and -R2 levels in the LPSand LPS + CAP groups significantly increased from 0.5 hto 12 h after LPS administration, compared to the vehiclegroup (P < 0.05 or 0.01, Fig. 1B and C). No such differences incirculating sTNF-R1 levels were observed between the LPSgroup and the LPS + Cap group (Fig. 1B and C). But at 0.5 hafter LPS administration, sTNF-R1 levels in the LPS + Capgroup were significantly decreased, compared to the LPSgroup (P < 0.05, Fig. 1B).

At 9 h and 12 h after LPS administration, sTNF-R2 levelsin the LPS + Cap group were significantly decreased com-pared to the LPS group (P < 0.01, Fig. 1C).

3.2. Expression of circulating TNF-, TNF-R1, and TNF-R2mRNA derived from leucocytes

Compared to the vehicle group, no significant changewas observed in the circulating TNF-, TNF-R1, or TNF-R2 mRNA expression levels in the Cap group (data notshown). The circulating TNF- mRNA expression level inthe LPS group was significantly increased 0.5, 1, 3, 6, and9 h after LPS administration (P < 0.05, Fig. 2A) comparedto the vehicle group. Despite this, the circulating TNF-mRNA expression level in the LPS + Cap group significantlydecreased 0.5, 1, 3, and 9 h after LPS administration com-pared to the vehicle group (P < 0.05, Fig. 2A).

The circulating TNF-R1 mRNA expression level in theLPS group significantly decreased 0.5, 1, and 3 h after LPSadministration compared to the vehicle group (P < 0.05 or0.01, Fig. 2B), even though they were significantly increased6 h and 9 h after LPS administration compared to the vehi-cle group (P < 0.05, Fig. 2B). Furthermore, the circulatingTNF-R1 mRNA expression level in the LPS + Cap group sig-nificantly increased 9 h after LPS administration comparedwith the vehicle group (P < 0.05, Fig. 2B).

The circulating TNF-R2 mRNA expression level in theLPS group significantly decreased 0.5 h after LPS adminis-tration compared to the vehicle group (P < 0.05, Fig. 2C).Despite this, the circulating TNF-R2 mRNA expression levelin the LPS + Cap group significantly increased 6 h after LPSadministration compared to the vehicle group (P < 0.01,Fig. 2C).

4. Discussion

Cap has been previously reported to improve the sur-vival rate of LPS-treated mice [24], although the precisemechanism of the effect of Cap was not explained. The aimof this study was to elucidate the effect of Cap on circulatingbiomarkers, sTNF, sTNF-R1, and -R2 levels in LPS-treatedmice.

Increased circulating sTNF-R1 and -R2 levels have beenreported in patients with hepatitis C virus infection [18],and increased circulating sTNF-R2 levels in patients withcongestive heart failure [17], obesity-impaired glucosetolerance [25], and leukemia [20,22]. In this study, we con-firmed that the circulating sTNF-R2 levels in plasma wereapproximately 10-fold higher than the circulating sTNF-R1 levels at each time point [23]. Since the circulatingsTNF, sTNF-R1, and -R2 levels are the initial signals of animmune response, plasma changes in them could representa biomarker detectable at an earlier stage than C-reactiveproteins, leukocytes, and fever during sepsis or systemicinflammatory response syndrome (SIRS). These values thusare known biomarkers of septic shock [15].

At the time to maximum plasma concentration (Tmax;1 h after LPS administration), circulating sTNF level in LPSgroup was significantly increased compared to these levelsin vehicle or LPS + Cap groups. The level declined from 3 h to12 h, but the level in the LPS group significantly increasedcompared to the vehicle group (Fig. 2A). While the TNF- _mRNA expression level derived from blood (including leu-cocytes) in the LPS group also significantly increased from0.5 h to 9 h compared with the vehicle or LPS + Cap groups(Fig. 2A). This difference may be due to the release of storedmembrane-bound TNF-(mTNF) from macrophages 1 hafter LPS stimulation [26]. Following LPS stimulation (ininflammation), TNF- _ is primarily expressed as a 26 kDatype II transmembrane protein, mTNF and is subsequentlycleaved by the metalloproteinase-disintegrin TNF-con-verting enzyme (TACE, also known as ADAM-17) into thesecreted 17 kDa monopeptide TNF-(sTNF) [12,14,27].Similarly, TACE, a member of the ADAM family of zincmetalloproteinases, modulates the generation of sTNF-R1 and -R2 by proteolytically cleaving the TNF-R1 and-R2 ectodomains, respectively [27]. Following a single LPSstimulation, the circulating sTNF level in the LPS groupsignificantly and continuously increased from 3 h to 12 hcompared to the vehicle group. At 1 h after LPS stimu-lation the circulating sTNF was considered to be derivedfrom mTNF. From 3 h onwards after LPS stimulation, thecirculating sTNF level was considered to be derived fromTNF- mRNA induced by LPS. While both sTNF-R1 and -R2mRNA levels were not differences among vehicle, LPS, andLPS + Cap groups from 0.5 h to 12 h after LPS stimulation.

Furthermore, the circulating sTNF-R2 level was approxi-mately 10-fold that of sTNF-R1 in this study, similar to theselevels of carbon tetrachloride-induced liver injury rats [23].TNF-R1 has been reported to bind to sTNF more frequentlythan TNF-R2 [26]; therefore, we assumed that binding withTNF-after LPS stimulation neutralized TNF-R1, resultingin decreased circulation of both sTNF and sTNF-R1.

Regarding the effects of Cap on sTNF, the sTNF level inthe LPS + Cap group was significantly depressed by Cap 1 hafter LPS stimulation compared to the LPS group (Fig. 1A).Cap, therefore, has the potential to depress the productionof sTNF via membrane stability. Furthermore, Cap signifi-cantly depressed TNF-mRNA from 0.5 h to 9 h (Fig. 2A).Cap was assumed to depress the increase in TNF-mRNAin LPS-treated mice. The above-mentioned results showthat Cap has the potential to suppress TNF-productionfollowing LPS-stimulation [28,29].

Our results assume the following two mechanisms forthe anti-TNF-effect of Cap: firstly, Cap exerts a release-inhibiting effect on circulating sTNF from macrophagesin the early phase of septicemia; secondly, Cap interfereswith TNF-mRNA transcription. Since Cap inhibits the ini-tial increase in circulating sTNF, it is considered a potenttreatment option for TNF-related diseases, such as sep-ticemia.

5. Conclusion

There is an early increase in circulating sTNF, sTNR-R1,and -R2 observed in the LPS-treated mice. Cap interfereswith TNF-mRNA transcription and exerts an inhibitingeffect on TNF-release from macrophages in the earlyphase after LPS stimulation. Thus, Cap is considered apotent agent for the treatment of TNF-related diseases,such as septicemia.