Sage Journals: Discover world-class research

Abstract

Xanthine oxidase activation occurs in sepsis and results in the generation of uric acid (UrAc) and reactive oxygen species (ROS). We aimed to evaluate the effect of xanthine oxidase inhibitors (XOis) in rats stimulated with lipopolysaccharide (LPS). LPS (10 mg/kg) was administered intraperitoneally (i.p.) immediately after allopurinol (Alo, 2 mg/kg) or febuxostat (Feb, 1 mg/kg) every 24 h for 3 days. To increase UrAc levels, oxonic acid (Oxo) was administered by gavage (750 mg/kg per day) for 5 days. Animals were divided into the following 10 groups (n = 6 each): (1) Control, (2) Alo, (3) Feb, (4) LPS, (5) LPSAlo, (6) LPSFeb, (7) Oxo, (8) OxoLPS, (9) OxoLPSAlo, and (10) OxoLPSFeb. Feb with or without Oxo did not aggravate sepsis. LPS administration (with or without Oxo) significantly decreased the creatinine clearance (ClCr) in LPSAlo (60%, P < 0.01) versus LPS (44%, P < 0.05) and LPSFeb (35%, P < 0.05). Furthermore, a significant increase in mortality was observed with LPSAlo (28/34, 82%) compared to LPS treatment alone (10/16, 63%) and LPSFeb (11/17, 65%, P < 0.05). In addition, increased levels of thiobarbituric acid reactive substances (TBARS), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 were observed at 72 h compared to the groups that received LPS and LPSFeb with or without Oxo. In this study, coadministration of Alo in LPS-induced experimental sepsis aggravated septic shock, leading to mortality, renal function impairment, and high ROS and proinflammatory IL levels. In contrast, administration of Feb did not potentiate sepsis, probably because it did not interfere with other metabolic events.

Keywords

allopurinol febuxostat inflammation interleukin lipid peroxidation LPS reactive oxygen species sepsis septic shock uric acid xanthine oxidase inhibitors

Introduction

According to the Third International Consensus 2016, sepsis should be defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection.¹ In addition, septic shock should be defined as a subset of sepsis associated with a greater risk of mortality than sepsis alone,¹ especially when kidney failure is established by acute renal injury and accompanied by multiple organ failure. The Toll-like receptor 4 (TLR4) stimulation caused by lipopolysaccharide (LPS) or TLR 7 and 8 by (+) ssRNA virus induces xanthine oxidase (XO) activation with monosodium urate crystal (i.e. uric acid (UrAc)) and reactive oxygen species (ROS) formation.² XO is also activated by urate crystals that stimulate TLR-2 and 4 and hypoxemia due to hemodynamic changes in sepsis, which is commonly observed in ischemia/reperfusion (I/R) models.^2,3

Prior administration of allopurinol (Alo) has a well-documented protective effect in the first 24 h in animal models of sepsis,^4–11 renal I/R,³ liver,^12,13 intestinal I/R,¹⁴ tourniquets on rat hind-limbs,¹⁵ brain I/R,¹⁶ cocaine-induced diastolic dysfunction,¹⁷ hyperthermia,¹⁸ fructose-fed rats,^19,20 streptozotocin-induced diabetes,²¹ and Crotalus terrificus snake venom.²² However, Alo did not protect against experimental sepsis,^23–25 renal I/R,²⁶ lung I/R,²⁷ zymosan,²⁸ or cell culture with LPS,²⁹ or in animals with infected burns that showed improvement only on day 1,³⁰ as the protection disappeared subsequently. Alo administration for 14 days in patients treated for burns resulted in lower mortality,³¹ but did not protect perioperatively against I/R injury in humans undergoing aortic aneurysm surgery³² (Supplementary Tables 1 and 2).

Although Alo has anti-inflammatory and protective effects in several experimental models, including sepsis, there are still controversial results, especially after 24 h. Its effect has not yet been entirely elucidated following its administration immediately prior to the induction of sepsis, and few studies have compared the effects of diverse xanthine oxidase inhibitors (XOis). Therefore, we conducted experiments to investigate these effects.

Material and methods

Lipopolysaccharide

Bacterial LPS from Escherichia coli (EC) strain 2630: O111: B₄, was obtained from Sigma-Aldrich (St. Louis, MO, USA) and administered intraperitoneally (i.p.) at 10 mg/kg body weight, diluted in distilled water, to rats as one dose every 24 h for 3 days.

Xanthine oxidase inhibitors

The following XOi were administered by gavage every 24 h for 3 days: Alo (2 mg/kg, GlaxoSmithKline do Brasil, RJ, Brazil) and febuxostat (Feb, 1 mg/kg, Takeda, IL, USA).

Oxonic acid

Oxonic acid (Oxo, Sigma-Aldrich), which was used to increase UrAc levels, was administered by gavage (750 mg/kg·per day), diluted in 0.25% methylcellulose in saline, once daily for 5 days before administration of LPS.

Animal ethics

The experimental protocol was approved by the Ethics Committee of the Universidade Federal de São Paulo (0220/12). Each experimental group was composed of six animals.

Experimental groups

Male Wistar rats weighing 200–250 g were divided into 10 groups: (1) Control (received water by gavage), (2) Alo, (3) Feb, (4) LPS, (5) LPSAlo, (6) LPSFeb, (7) Oxo, (8) OxoLPS, (9) OxoLPSAlo, and (10) OxoLPSFeb. At 0 and 17 h of the experimental period, blood samples were collected from the orbital sinus, and the animals were maintained in individual metabolic cages for 24 h for urine collection. Animals were euthanized 72 h after the experiment commenced under anesthesia (ketamine/xylazine, 10:1, 0.2 mL/100 g/kg, i.p.). Blood was collected, and the kidneys were perfused with saline solution, removed, and subsequently histopathologically analyzed.

Biochemical analysis

The blood creatinine (Cr) and UrAc levels were assayed spectrophotometrically according to standard procedures using commercially available diagnostics kits (Labtest Diagnostica, MG, Brazil).

Lipid peroxidation

Lipid peroxidation was measured using the thiobarbituric acid reactive substances (TBARS) assay. The reactive substances combine with thiobarbituric acid to form a red compound. Malondialdehyde (MDA) was used to construct a standard curve, and the results are expressed as MDA (mM)/mg protein. Urine samples were added to a solution of 0.375% thiobarbituric acid, 15% trichloroacetic acid, and 0.25 N HCl (Sigma-Aldrich). The samples were continually agitated while being heated to 95°C for 20 min, and then they were allowed to cool to 22°C. The absorbance was spectrophotometrically determined at 535 nm. The results were expressed as 10⁻⁷ M/mg creatinine.

Cytokine analysis

Immunoassays, based on Luminex xMAP (multi-analyte profiling) technology, were performed to simultaneously detect and quantify multiple cytokines (interleukin (IL)-1α, IL-4, IL-1β, IL-2, IL-6, IL-10, and tumor necrosis factor (TNF)-α) using the Cat#RECTYMAG65K-08 granulocyte-macrophage colony-stimulating factor (GM-CSF) kit. The assays were performed at the beginning (time 0), and at 17 and 72 h in all groups, but for Oxo-treated animals, the assays were also performed 5 days before.

Histopathology

Hematoxylin and eosin (H&E) staining of the paraffin-embedded kidney tissue slices was performed.

Immunohistochemistry

Paraffin-embedded tissues were cut into 4-μm thick sections using a rotary microtome (Leica Microsystems, Herlev, Denmark). The kidney slices were deparaffinized and rehydrated, and then the sections were boiled in a target retrieval solution (1 mmol/L Tris, pH 9.0, with 0.5 mM ethylene glycol tetraacetic acid (EGTA)) for 10 min for antigen retrieval. Nonspecific binding was prevented by incubating the sections in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA), 0.05% saponin, and 0.2% gelatin. Endogenous peroxidase activity was blocked with 5% hydrogen peroxidase (H₂O₂) in absolute methanol for 10 min at 22°C.

The sections were incubated with primary antibodies against proliferating cell nuclear antigen (PCNA, 1:500) or cleaved caspase-3 (1:100), overnight at 4°C. PCNA is a nuclear protein linked to DNA replication, also known as cyclin, which plays a role in initiating cell proliferation by enhancing the DNA polymerase enzyme. After the sections were washed, they were incubated with horseradish peroxidase–labeled polymer conjugated to a secondary antibody (Dako, Denmark) for 1 h at room temperature. The sites of antibody-antigen reactions were visualized using 0.5% 3,3′-diaminobenzidine tetrachloride (Dako) dissolved in distilled water with 0.1% H₂O₂. A total of 10 sections per animal along the kidney cortex were analyzed, and labeled cells (staining light to dark brown) were counted.

Statistical analysis

The results are expressed as the mean ± standard error (SE). The data were analyzed using survival Kaplan–Meier curves and a one-way analysis of variance (ANOVA) followed by Tukey tests using GraphPad Prism 5. P < 0.05 was considered statistically significant.

Results

Coadministration of Alo but not Feb aggravated septic shock by increasing mortality

LPS administration significantly reduced the mean arterial blood pressure (BP) of rats, measured using a tail-cuff method, in all groups. The LPS group at 72 h showed a mean arterial BP of 95.5 ± 13.2 (P < 0.05) and the LPSAlo group at 24, 48, and 72 h showed values of 94.3 ± 12.9, 97.7 ± 13.6, and 96.2 ± 15.1 mmHg, respectively, compared to the control value of 127 ± 7 mmHg (Figure 1(a)). Alo administration immediately before LPS treatment enhanced the septic shock, which manifested as a significant increase in mortality (P < 0.05) in the LPSAlo group (28/34, 82%) compared to the LPS group (10/16, 63%) and LPSFeb group (11/17, 65%, Figure 1(c)).

Figure 1.

Blood pressure (BP) (mmHg) and survival curves. (a) BP in the LPS, LPSAlo, and LPSFeb groups and (b) BP in the Oxo, OxoLPS, OxoLPSAlo, and OxoLPSFeb groups compared to the control group showed significantly lower values (*P < 0.05 and ***P < 0.001) and significantly higher values (#P < 0.05). (c) Survival curves of groups challenged with LPS, LPSAlo, and LPSFeb for 72 h. The LPSAlo group showed significantly lower survival rates than the LPS and LPSFeb groups;*P < 0.05. (d) Survival curves for all groups challenged with Oxo. No deaths were observed in the OxoLPS and OxoLPSFeb groups, but the OxoLPSAlo group had mortality, #P < 0.01. (e) Comparison of survival curves of groups challenged with Alo. Lower mortality was observed in the OxoLPSAlo group than in the LPSAlo group (P = 0.08). LPS: lipopolysaccharide; Alo: allopurinol; Feb: febuxostat; Oxo: oxonic acid.

Oxo pre-treatment increase BP, blood UrAc levels and decreased mortality

After 5 days from Oxo administration, a significant increase in BP occurred (from 124.7 ± 4.3 to 149 ± 20.9 mmHg, P < 0.05, Figure 1(b)) accompanied by a significant increase in blood UrAc levels (from 1.7 ± 0.2 to 3.0 ± 0.7 mg/dL, P < 0.001, Figure 2(b)). There were no deaths in the OxoLPS or OxoLPSFeb groups compared to the OxoLPSAlo group (12/18, 67%; P < 0.02, Figure 1(d)), and we observed a decrease in the number of deaths in the OxoLPSAlo group compared to the LPSAlo group (P = 0.08, Figure 1(e)). We noted significantly lower mean values of creatinine clearance (ClCr, mL/min) in all LPS-treated groups (44%, P < 0.05), which were the LPSAlo (60%, P < 0.01), LPSFeb (35%, P < 0.05, Figure 2(c)), OxoLPS (0.61 ± 0.14, P < 0.01), OxoLPSFeb (0.64 ± 0.10, P < 0.01), and OxoLPSAlo (0.57 ± 0.24, P < 0.01) groups, compared to the control group (1.25 ± 0.19 mL/min, Figure 2(d)).

Figure 2.

Biochemistry. UrAc: (a) LPS, LPSAlo, and LPSFeb groups and (b) OxoLPS, OxoLPSAlo, and OxoLPSFeb groups. UrAc values of treated groups were significantly higher than those of control groups, *P < 0.05 and ***P < 0.001. Creatinine clearance: (c) LPS, LPSAlo, and LPSFeb groups and (d) Oxo, OxoLPS, OxoLPSAlo, and OxoLPSFeb groups. All LPS-treated groups with or without Alo, Feb, or Oxo showed significantly lower values compared to controls,*P < 0.05; **P < 0.01; and ***P < 0.001. TBARS: (e) LPS, LPSAlo, and LPSFeb groups and (f) Oxo, OxoLPS, OxoLPSAlo, and OxoLPSFeb groups. All LPS-treated groups that received Alo, Feb, and/or Oxo showed significantly higher values than the LPS-alone group, **P < 0.01 and ***P < 0.001. UrAc: uric acid (mg/dL); LPS: lipopolysaccharide; Alo: allopurinol; Feb: febuxostat; Oxo: oxonic acid; creatinine clearance: ClCr (mL/min); TBARS: thiobarbituric acid reactive substances (nMol/mg, urinary Cr).

LPS increases ROS especially with coadministration of Oxo

In addition, we observed high levels of ROS (TBARS: nMol/mg urinary Cr, P < 0.001) in all of the groups compared to the control. Furthermore, the values were higher in the Alo-treated groups (LPSAlo, 293.3 ± 35.8 and OxoLPSAlo, 304.3 ± 60.4) than in the LPS (267.8 ± 44.9), LPSFeb (220.0 ± 39.1), OxoLPS (236.7 ± 33.2), and OxoLPSFeb (211.2 ± 43.6) groups (Figure 2(e) and (f)).

Inflammatory cytokine

In all the groups who received LPS, the levels of TNF-α increased about three times in relation to control groups in 17 h and returned to basal levels in all groups, except in the LPSAlo and OxoLPSAlo groups where this late decline was not observed (Figure 3(a) and (b); Table 1).

Figure 3.

Cytokine analysis (pg/mL): TNF-α: (a) LPS, LPSAlo, and LPSFeb groups and (b) Oxo, OxoLPS, OxoLPSAlo, and OxoLPSFeb groups. All groups treated with LPS alone or with drugs showed significantly higher values than the control group ***P < 0.001. IL-6: (c) LPS, LPSAlo, and LPSFeb groups and (d) Oxo, OxoLPS, OxoLPSAlo, and OxoLPSFeb groups. All groups treated with LPS alone or with drugs showed significantly higher values than the control group, *P < 0.05, **P < 0.01, and ***P < 0.001. IL-1α: (e) LPS, LPSAlo, and LPSFeb groups and (f) Oxo, OxoLPS, OxoLPSAlo, and OxoLPSFeb groups. All groups treated with LPS alone or with drugs showed significantly higher values than the control group, **P < 0.01 and ***P < 0.001. IL-1β: (g) LPS, LPSAlo, and LPSFeb groups and (h) Oxo, OxoLPS, OxoLPSAlo, and OxoLPSFeb groups. All groups treated with LPS alone or with drugs showed significantly higher values than the control group, ***P < 0.001 and LPSAlo 72 h versus LPSFeb 72 h, ** P < 0.01. IL-10: (i) LPS, LPSAlo, and LPSFeb groups and (j) Oxo, OxoLPS, OxoLPSAlo, and OxoLPSFeb groups. All groups treated with LPS with drugs showed significantly higher values than groups treated with LPS alone after 17 h: *P < 0.05, **P < 0.01, and ***P < 0.001. IL-2: (k) LPS, LPSAlo, LPSFeb, Oxo, OxoLPS, OxoLPSAlo, and OxoLPSFeb groups. All groups treated with LPS with other drugs showed significantly higher values than the groups treated with LPS alone after 17 h: ***P < 0.001.GM-CSF: (l) LPS, LPSAlo, and LPSFeb, Oxo, OxoLPS, OxoLPSAlo, and OxoLPSFeb groups. All groups treated with LPS with drugs showed significantly higher values than groups treated with LPS alone after 17 h: *P < 0.05, **P < 0.01, and ***P < 0.001. TNF: tumor necrosis factor; IL: interleukin; LPS: lipopolysaccharide; Alo: allopurinol; Feb: febuxostat; Oxo: oxonic acid; GM-CSF, granulocyte-macrophage colony-stimulating factor.

Table1.

Cytokine analysis.

	control	Alo	Feb	LPS	LPSAlo	LPSFeb	Oxo	OxoLPS	OxoLPSAlo	OxoLPSFeb
TNF-α
–5 days							1.3 ± 0.1	1.3 ± 0.1	1.4 ± 0.1	1.3 ± 0.2
0	1.3 ± 0.1	1.4 ± 0.1	1.4 ± 0.1	1.4 ± 0.2	1.3 ± 0.1	1.4 ± 0.1	1.7 ± 0.1	1.6 ± 0.6	1.6 ± 0.2	1.6 ± 0.2
17 h	1.4 ± 0.1	1.3 ± 0.1	1.3 ± 0.2	4.9 ± 1.0*	4.5 ± 1.0*	4.1 ± 0.6*	1.5 ± 0.1	4.8 ± 1.0*	4.6 ± 0.8*	4.2 ± 0.7*
72 h	1.5 ± 0.2	1.1 ± 0.2	1.2 ± 0.1	1.5 ± 0.3	4.2 ± 1.0*	1.4 ± 0.4	1.5 ± 0.1	1.5 ± 0.3	3.4 ± 0.4*	1.2 ± 0.2
IL-6
–5 days							7.8 ± 0.5	8.5 ± 0.5	7.8 ± 0.6	8.7 ± 1.1
0	8.7 ± 1.6	8.2 ± 1.5	9.1 ± 0.7	8.2 ± 1.0	8.4 ± 1.0	8.3 ± 2.0	10.1 ± 0.3	10.0 ± 1.6	10.0 ± 1.3	8.7 ± 1.2
17 h	7.7 ± 1.5	7.9 ± 1.6	9.3 ± 1.2	175.7 ± 40	238.8 ± 28#	199.5 ± 23	10.1 ± 1.3	231.3 ± 19	275.7 ± 52##	229.2 ± 30
72 h	9.6 ± 1.7	7.6 ± 1.5	7.6 ± 1.5	95.0 ± 29.7**	139.2 ± 40&	90.3 ± 21**	9.8 ± 1.5	98.8 ± 9**	142.5 ± 22.6&	92.2 ± 10**
IL-1α
–5 days							1.8 ± 0.4	1.7 ± 0.4	1.9 ± 0.6	1.8 ± 0.4
0	1.7 ± 0.4	1.8 ± 0.5	1.7 ± 0.4	1.7 ± 0.4	1.8 ± 0.4	1.8 ± 0.6	2.2 ± 0.2	2.2 ± 0.3	2.1 ± 0.4	1.9 ± 0.6
17 h	1.8 ± 0.5	1.7 ± 0.4	1.8 ± 0.5	16.2 ± 4.7•	22.2 ± 3.9•	18.3 ± 5.5•	2.0 ± 0.0	20.7 ± 4.8•	27.7 ± 3.6***	18.7 ± 7.0•
72 h	1.7 ± 0.6	1.5 ± 0.6	1.7 ± 0.3	17.4 ± 4.4•	30.7 ± 5.5***	15.8 ± 1.6	2.0 ± 0.3	18.4 ± 3.6•	35.4 ± 6.2***	15.9 ± 2.7•
IL-1β
–5 days							34 ± 3	34 ± 4	36 ± 4	32 ± 5
0	36 ± 6	31 ± 3	33 ± 5	33 ± 3	32 ± 3	34 ± 3	37 ± 6	38 ± 4	39 ± 6	38 ± 5
17 h	38 ± 2	30 ± 4	30 ± 5	594 ± 64*	655 ± 72	588 ± 66*	38 ± 1	650 ± 86*	625 ± 54*	558 ± 42*
72 h	32 ± 4	29 ± 4	29 ± 4	629 ± 47*	632 ± 95*	490 ± 67▪	39 ± 3	662 ± 54*	638 ± 55*	565 ± 59*
IL-10
–5 days							34 ± 7	33 ± 6	31 ± 2	32 ± 2
0	32 ± 5	34 ± 8	35 ± 14	33 ± 3	32 ± 3	35 ± 3	36 ± 11	35 ± 3	39 ± 2	37 ± 9
17 h	35 ± 3	33 ± 5	33 ± 12	475 ± 95*	777 ± 60*	497 ± 70*	35 ± 6	659 ± 101	803 ± 59*	765 ± 92*
72 h	31 ± 3	34 ± 10	34 ± 10	756 ± 60* •	1102 ± 231* •	704 ± 118* •	36 ± 3	786 ± 111*	1148 ± 153* •	825 ± 114*
IL-2
–5 days							5.7 ± 1.0	7.0 ± 1.7	5.6 ± 1.0	7.0 ± 0.6
0	5.2 ± 1.1	5.5	5.3	6.6 ± 0.3	5.5 ± 0.6	5.7 ± 0.5	5.8 ± 1.5	5.8 ± 1.3	5.7 ± 1.8	6.0 ± 1.2
17 h	5.9 ± 1.8	5.3	6.9	152.3 ± 25.8•	241 ± 18.8***	144.3 ± 24.1•	5.9 ± 1.0	160.3 ± 12.6•	264.8 ± 31.0***	162.5 ± 16.3•
72 h	6.3 ± 2.0	6.5	6.5	139.0 ± 12•	208.7 ± 32.5***	120.5 ± 28.1•	6.1 ± 1.0	147.8 ± 27.6•	250.5 ± 33.8***	156.3 ± 16.9•
GM-CSF
–5 days							1.6 ± 0.0	1.6 ± 0.1	1.6 ± 0.2	1.6 ± 0.2
0	1.6 ± 0.1	1.7 ± 0.5	1.6 ± 0.4	1.6 ± 0.1	1.6 ± 0.3	1.6 ± 0.1	2.0 ± 0.3	2.1 ± 0.2	2.1 ± 0.5	1.9 ± 0.2
17 h	1.6 ± 0.3	1.6 ± 0.4	1.5 ± 0.4	11.3 ± 1.4•	17.8 ± 3.5•	13.4 ± 2.5**	2.0 ± 0.4	14.9 ± 2.4•	21.8 ± 4.7***	17.0 ± 2.5*
72 h	1.6 ± 0.1	1.6 ± 0.3	1.6 ± 0.3	8.8 ± 0.9•	12.3 ± 1.6•	10.3 ± 1.0•	2.1 ± 0.7	11.6 ± 1.4•	18.0 ± 5.0**	12.7 ± 1.0•

GM-CSF: granulocyte-macrophage colony-stimulating factor. TNF-α (pg/mL): in relation to controls: significantly higher values ***P < 0.001. IL-6 (pg/mL): in relation to LPS 17 h lower values **P < 0.01 and higher values #P < 0.05 and ##P < 0.01. Higher IL-6 values compared to LPS 72 h &P < 0.05. IL-1α (pg/mL): in relation to LPS 17 h: significantly higher values *P < 0.01 and ***P < 0.001; •P < 0.001 compared to controls. IL-1β (pg/mL): in relation to control: significantly higher values *P < 0.001. LPSAlo 72 h versus LPSFeb 72 h ▪P < 0.01. IL-10 (pg/mL): in relation to L 17 h: *P < 0.05; **P < 0.01; ***P < 0.001 and •P < 0.001 compared to controls. IL-2 (pg/mL): in relation to LPS 17 h: significantly higher values ***P < 0.001; •P < 0.001 compared to controls. GM-CSF (pg/mL): in relation to LPS 17 h: significantly higher values *P < 0.05; **P < 0.01; ***P < 0.001 and •P < 0.001 compared to controls.

The levels of IL-6 in relation with controls were elevated during 17 h, by 19 times the superior values in the LPS group; by 26 times in the LPSAlo group; by 22 times in the LPSFeb group; by 25 times in the OxoLPS group; by 30 times in the OxoLPSAlo group; and by 25 times in the OxoLPSFeb group. After 72 h, lower IL-6 values were present in about 50% of cases in all groups, except in LPSAlo and OxoLPSAlo groups where these values remained higher, both about 15 times in relation to the control group (Figure 3(c) and (d)).

After 17 and 72 h, all animals treated with LPS alone or with LPS with drugs showed significantly higher IL-1α values than those of the control group, (P < 0.001) and LPSAlo, and the OxoLPSAlo group had even higher values than the LPS, LPSFeb, OxoLPS, and OxoLPSFeb groups (P < 0.001; Figure 3(e) and (f)). After 17 h, all animals treated with LPS alone or with Alo or Feb showed significantly higher IL1-β values than those of the control group. After 72 h, no difference between LPS and LPSAlo was observed, but the LPSFeb group showed lower levels of IL-1β than the LPS and LPSAlo groups (P < 0.05 and P < 0.001, respectively; Figure 3(g) and (h)).

Compared to the control group after 17 h, all groups showed significantly higher IL-10 levels, (P < 0.001) and the IL-10 increase was even higher and more significant after 72 h. After 17 and 72 h, the LPSAlo group showed significantly higher IL-10 levels (P < 0.001) than the LPS and LPSFeb groups. Oxo administration further increased the IL-10 levels in all groups. After 17 h there were no differences among the OxoLPS, OxoLPSAlo, and OxoLPSFeb groups, but after 72 h, the OxoLPSAlo group showed significantly higher IL-10 levels (P < 0.001) than the OxoLPS and OxoLPSFeb groups (Figure 3(i) and (j)).

All groups that received LPS alone or with drugs showed significantly higher IL-2 and GM-CSF levels (P < 0.001; Figure 3(k) and (l)).

Histopathological changes, proliferation (PCNA), and apoptosis rates

Minimal alterations were observed in the histopathological analysis, with rare areas that indicated circulatory disruption where vasodilatation and hemorrhage occurred. However, no significant difference (P = 0.85) was observed between the control and all LPS-treated groups, which is in agreement with previously reported data (Figure 4).³³ After 72 h, all groups challenged with LPS alone or with drugs showed significantly (P < 0.001) lower values for PCNA immunohistochemical staining than the control group (1.087 ± 0.66/µm²), which was consistent with the severity of sepsis³⁴ (Figure 5 and Supplementary Table 3). Tissues sections immunohistochemically stained for cleaved caspase-3 demonstrated a significantly higher apoptosis cell number/field in all groups that received LPS alone or with others drugs compared to the control group (P < 0.001; Figure 6 and Supplementary Table 4).

Figure 4.

Histopathological sections of kidney were obtained at 72 h (n = 3 rats/group). (a) LPS, normal and (b) LPS, normal, 100× scale bar = 50 µm. (c) LPS, sector with vasodilatation and hemorrhaging (arrow) and (d) LPS, vacuolar degeneration of proximal renal tubules (arrow). 400× scale bar = 20 µm.

Figure 5.

Immunohistochemical staining of PCNA (1:500 µm²): (a) Control; (b) LPS; (c) LPSAlo; (d) LPSFeb; (e) OxoLPS; (f) OxoLPS; (g) OxoLPSAlo; and (h) OxoLPSFeb. All groups (i) treated with LPS alone or with drugs demonstrated a significantly lower proliferation rate (***P < 0.001) than the control and Oxo groups. There were no significant differences among the LPS, LPSAlo, LPSFeb, OxoLPS, OxoLPSAlo, and OxoLPSFeb groups. PCNA (arrow), LPS: lipopolysaccharide; Alo: allopurinol; Feb: febuxostat; Oxo: oxonic acid. 100× scale bar = 50 µm.

Figure 6.

Staining for cleaved caspase-3 (1:100): apoptotic cell number/field. (a) Control; (b) LPS; (c) LPSAlo; (d) LPSFeb; (e) Oxo; (f) OxoLPS; (g) OxoLPSAlo; and (h) OxoLPSFeb. All groups (i) the number of apoptotic cells (arrow) per field was significantly higher in groups treated with LPS alone or with drugs compared to the control group (P < 0.001). There were no significant differences among the LPS, LPSAlo, LPSFeb, OxoLPS, OxoLPSAlo, and OxoLPSFeb groups. LPS: lipopolysaccharide; Alo: allopurinol; Feb: febuxostat; Oxo: oxonic acid. 100× scale bar = 50 µm.

Discussion

Sepsis has a high mortality rate, mainly due to septic shock, aggravated by renal insufficiency and multiple organ failure.¹ In this study, we would like to be clear that LPS induces endotoxemia and systemic inflammatory response syndrome (SIRS) but not sepsis as defined in the third consensus definition. LPS-treated groups had lower BP, impaired renal function, and higher ROS levels compared to the control group. Furthermore, immunohistochemistry for PCNA revealed less cell proliferation and that for cleaved caspase-3 indicated considerable apoptosis that accompanied the permanently elevated IL-10 levels related to septic shock.^4,34–36 ROS are involved in the caspase-3 apoptosis cascade³⁷ in renal tubular cells in response to systemic sepsis stress, which is a mechanism of acute kidney injury (AKI),³⁵ where increased expression of renal IL-10 has been observed.³⁷

Alo is readily absorbed from the gastrointestinal tract. In our experiment, LPS administration immediately after daily treatment with 2 mg/kg Alo paradoxically worsened the sepsis compared to Alo pre-treatment at doses between 10 and 100 mg/kg body weight from 4 days to 20 min before LPS treatment.^5,38–40 This contrasts with the effects observed in the first 24 h, when Alo protected against experimental sepsis.^5,38–40 It was previously shown that the protective effects of Alo may disappear after 24 h, with mortality rates reaching 100%.²² Matuschak et al.⁴¹ showed the potent suppressive effect of combined hypoxic stress/reperfusion (H/R) and EC stimulation on the activation and nuclear translocation of nuclear factor (NF)-Kb, in contrast with the robust hypoxic stress-induced transactivation of NF-kB without sepsis. However, administration of the anti-inflammatory compound Alo in H/R + EC increased the NF-kB transactivation, which could,⁴¹ in part, explain the paradoxical effect observed in the LPSAlo and OxoLPSAlo groups.

In cell cultures exposed to LPS, Alo blocked caspase-3 activity and ROS formation in the first 24v h, but this effect disappeared within 48 h, inexplicably.¹⁰ No deaths were observed in the OxoLPS and OxoLPSFeb groups, perhaps because the elevated UrAc level functioned as an antioxidant. UrAc is a potent extracellular antioxidant that evolutionarily confers longevity,⁴² maintains BP under conditions with low amounts of dietary sodium, and has a protective effect in multiple sclerosis⁴³ and severe sepsis.⁴⁴ However, it is also an intracellular pro-oxidant, which perhaps explains the increased rates of lipid peroxidation, the levels of proinflammatory ILs and selectively decreases the human blood monocyte production of the natural IL-1 receptor antagonist (IL-1Ra) and changes the production to highly inflammatory IL-1β.⁴⁵ In human vaccines, aluminum hydroxide adjuvant boosts adaptive immunity by inducing UrAc and activating inflammatory dendritic cells.⁴⁶ Oxo administration for 5 days significantly increased UrAc and BP levels, which was expected. UrAc causes endothelial dysfunction and indirectly decreases levels of nitric oxide, which regulates vascular endothelial cell tone.⁴⁷ Furthermore, it activates the renin-angiotensin system and the proliferation of smooth muscle cells by stimulation of inflammatory responses.⁴⁷ In the kidney, it triggers afferent arteriopathy, induction of the epithelial-mesenchymal transition, and interstitial tubular fibrosis.⁴⁷ Hyperuricemia may be an early marker of sepsis severity.⁴⁷

Alo is the drug of choice for reducing UrAc levels, although it is contraindicated in the initial phase of gout treatment. Recent meta-analyses have shown that continuous use of Alo in patients with hyperuricemia preserves renal function, and the subsequent slower induction of proteinuria protects against arterial hypertension and slows the progression to chronic kidney disease and mortality.⁴⁸ Alo thus has a beneficial effect on ischemia, inflammation, and chronic heart failure.⁴⁸ However, some adverse effects such as hepatitis, nephropathies, and hypersensitivity reactions limit the clinical use of Alo.⁴⁹ Although occurring at a frequency of <0.1% in humans, hypersensitivity to Alo increases the risk of death by 27%,⁴⁹ with the main risk factors as follows: male sex with arterial hypertension and/or renal insufficiency and overdosage,⁴⁹ recent initiation of therapy, use of concomitant diuretics or nonhormonal anti-inflammatory drugs, and the presence of HLA-B*5801.⁴⁹

In the LPSAlo group, the shock was possibly triggered by the hemodynamic changes of sepsis evolving into AKI that was aggravated by Alo or the animals developed interstitial nephritis induced by Alo.⁴⁹ However, no substrate such as eosinophilia was found in histopathology. There may be another unknown mechanism underlying the paradoxical beneficial therapeutic effect of Alo and negative effect of renal function, as the use of Feb did not worsen the sepsis. Feb does not have structural similarity to purines such as Alo, which may interfere with other stages of purine metabolism.⁵⁰

Feb is an XOi with a favorable toxicological profile and high bioavailability, and it reduces UrAc levels in a more sustained and potent manner than Alo with adverse effects in less than 2% of patients; such effects include diarrhea and increased hepatic transaminases, without serious cutaneous reactions.^51,52 Feb is primarily metabolized by hepatic oxidation and glucuronidation and eliminated by both the liver and kidney. In contrast to the renal elimination of the active metabolite of Alo (oxypurinol), renal elimination plays a minor role in the elimination of Feb, facilitating safer prescription including for the treatment of tumor lysis syndrome (TLS). Although TLS is classically treated with Alo, studies on Feb in clinical trials have shown a more promising profile.⁵² In this experiment, baseline TNF-α levels were approximately 1 pg/mL and increased significantly (P < 0.001) by least four-fold in the first 17 h of LPS administration. The values returned to baseline levels at 72 h, which represents a successful inflammatory response according to the literature⁵³ as well as the determined IL-2 levels.⁵³

The LPSAlo and OxoLPSAlo groups still showed significantly increased TNF-α levels after 72 h, which is related to the severity of the condition, shock, and higher death rate.^54,55 Similarly, compared to the control levels at 17 and 72 h, all groups treated with LPS with or without XOi or Oxo showed elevation of IL-6, especially the LPSAlo and OxoLPSAlo groups at 72 h. This is related to greater severity of the condition and a worse prognosis, as IL-6 is considered a marker for the early diagnosis, follow-up, and prognosis of sepsis.⁵⁵

Significantly higher IL-1β values were observed in all animal groups treated with LPS with or without XOi than in the control group; however, after 72 h, the IL-1β levels in the LPSFeb group were significantly lower than those in all other groups.⁵⁶ In this study, IL-10 values gradually increased with significant differences in all groups at 17 and 72 h, which could be interpreted as a good anti-inflammatory response.^53,55 This is because IL-10 is the main cytokine dampening the innate immune response. TNF-α and other cytokines stimulate IL-10 synthesis, which by feedback blocks the synthesis of TNF-α, normalizing serum levels of cytokines.^39,55 The peak of IL-10 levels is related to a higher risk of shock, and when IL-10 levels remain high, as in the groups that received Alo, it is a factor for the development of multiple organ failure,^55,57 septic shock, and death.⁵⁷

Here, administration of Feb immediately prior to LPS, with or without previous administration of Oxo, did not aggravate sepsis, mortality rates, or levels of TBARS, TNF-α, IL-6, IL-1α, IL- 2, IL-10, and GM-CSF, with similar values compared to the group that received LPS alone (with or without prior administration of Oxo).

Administration of LPS plus Alo (with or without Oxo) significantly decreased the ClCr values, with a significant increase in mortality and also increased levels of TBARS, TNF-α, IL-6, IL-1α, IL -1β, IL-2, IL-10, and GM-CSF in the first 17 h compared to the levels in groups treated with LPS and LPS plus Feb with or without Oxo. The TNF-α and IL-6 levels remained significantly higher after 72 h in the animals that received LPS plus Alo (with or without Oxo), which was correlated with a higher risk of death.

In this study, LPS administration immediately after Alo in experimental LPS-induced sepsis aggravated septic shock with increased mortality and impaired renal function, with higher values of ROS and proinflammatory ILs. Clinically, the known risk factors for Alo adverse reactions include recent initiation of therapy and renal failure. In contrast, when we administered Feb, no damage to the sepsis-regulating system was observed, probably because it did not interfere with other metabolic events, which probably occurs with Alo. The prescription of Alo requires physician expertise for use in patients with sepsis who at any time might progress to shock and renal failure. In contrast, Feb, a XOi with hepatic metabolism and renal and hepatic excretion, did not aggravate or improve septic shock with renal failure. Further studies are needed to elucidate the role of XOs in septic shock with renal failure.

Supplemental Material

supplements_tables – Supplemental material for Xanthine oxidase inhibitors and sepsis

Supplemental material, supplements_tables for Xanthine oxidase inhibitors and sepsis by Maria Fátima de Paula Ramos, Alceni do Carmo Morais Monteiro de Barros, Clara Versolato Razvickas, Fernanda T Borges and Nestor Schor in International Journal of Immunopathology and Pharmacology

Footnotes

Acknowledgements

Department of Morphology and Genetics, UNIFESP/EPM, São Paulo, Brazil

Declaration of conflicting interests

We have no direct or indirect commercial financial incentive associated with publishing this article. In addition, the authors have no conflicts of interest, and the source of extra-institutional funding is indicated in the manuscript. All authors participated in the design, interpretation of the study, analysis of the data, and review of the manuscript.

Funding

This work was supported by grants from Conselho Nacional de Desenvolvimento Científico Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP), Fundação Oswaldo Ramos (FOR), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

References

Singer

Deutschman

Seymour

et al . (2016) The third international consensus definitions for sepsis and septic shock (sepsis-3). Journal of the American Medical Association 315: 801–810.

Tschopp

Schroder

(2010) NLRP3 inflammasome activation: The convergence of multiple signalling pathways on ROS production? Nature Reviews Immunology 10: 210–215.

Rhoden

Mauri

Leonardo Petteffi

et al . (1998) Efeitos do alopurinol Sobre a Lipoperoxidação de Membranas Celulares Renais na síndrome da isquemia e reperfusão: Estudo experimental EM ratos. Acta Cirúrgica Brasileira Available at: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0102-86501998000200002 (accessed 8 May 2018).

Ogawa

Morita

Kunimoto

et al . (1982) Changes in hepatic lipoperoxide concentration in endotoxemic rats. Circulatory Shock 9: 369–374.

McKechnie

Furman

Parratt

(1986) Modification by oxygen free radical scavengers of the metabolic and cardiovascular effects of endotoxin infusion in conscious rats. Circulatory Shock 1986; 19: 429–439.

Xiang

Klintman

Thorlacius

(2003) Allopurinol inhibits CXC chemokine expression and leukocyte adhesion in endotoxemic liver injury. Inflammation Research 52: 353–358.

Guillory

et al . (1993) Mechanisms of endotoxin-induced intestinal injury in a hyperdynamic model of sepsis. Journal of Trauma 34: 676–623; discussion 682–673.

Chatterjee

Lardinois

Bonini

et al . (2009) Site-specific carboxypeptidase B1 tyrosine nitration and pathophysiological implications following its physical association with nitric oxide synthase-3 in experimental sepsis. Journal of Immunology 183: 4055–4066.

Chatterjee

Ehrenshaft

Bhattacharjee

et al . (2009) Immuno-spin trapping of a post-translational carboxypeptidase B1 radical formed by a dual role of xanthine oxidase and endothelial nitric oxide synthase in acute septic mice. Free Radical Biology and Medicine 46: 454–461.

10.

Chatterjee

Lardinois

Bhattacharjee

et al . (2011) Oxidative stress induces protein and DNA radical formation in follicular dendritic cells of the germinal center and modulates its cell death patterns in late sepsis. Free Radical Biology and Medicine 50: 988–999.

11.

Hung

(2000) Importance of histamine, glutathione and oxyradicals in modulating gastric haemorrhagic ulcer in septic rats. Clinical and Experimental Pharmacology and Physiology 27: 306–312.

12.

Wibbenmeyer

Lechner

Munoz

et al . (1995) Downregulation of E. Coli-induced TNF-alpha expression in perfused liver by hypoxia-reoxygenation. American Journal of Physiology 268: G311–G319.

13.

Loftis

Johanns

Lechner

et al . (2000) Brief hypoxic stress suppresses postbacteremic NF-kappaB activation and TNF-alpha bioactivity in perfused liver. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology 279: R99–R108.

14.

Shafik

(2013) Febuxostat improves the local and remote organ changes induced by intestinal ischemia/reperfusion in rats. Digestive Diseases and Sciences 58: 650–659.

15.

Ward

Maldonado

Vivaldi

(1992) Oxygen-derived free radicals mediate liver damage in rats subjected to tourniquet shock. Free Radical Research Communications 17: 313–325.

16.

Yamaguchi

Okamoto

Kusano

et al . (2015) The effects of xanthine oxidoreductase inhibitors on oxidative stress markers following global brain ischemia reperfusion injury in C57BL/6 mice. PLoS ONE 10: e0133980.

17.

Vergeade

Mulder

Vendeville

et al . (2012) Xanthine oxidase contributes to mitochondrial ROS generation in an experimental model of cocaine-induced diastolic dysfunction. Journal of Cardiovascular Pharmacology 60: 538–543.

18.

Hall

Buettner

Oberley

et al . (2001) Mechanisms of circulatory and intestinal barrier dysfunction during whole body hyperthermia. American Journal of Physiology: Heart and Circulatory Physiology 280: H509–H521.

19.

Zhang

Pan

et al . (2012) Allopurinol, quercetin and rutin ameliorate renal NLRP3 inflammasome activation and lipid accumulation in fructose-fed rats. Biochemical Pharmacology 84: 113–125.

20.

Wang

Ding

et al . (2015) Pterostilbene and allopurinol reduce fructose-induced podocyte oxidative stress and inflammation via microRNA-377. Free Radical Biology and Medicine 83: 214–226.

21.

Romagnoli

Gomez-Cabrera

Perrelli

et al . (2010) Xanthine oxidase-induced oxidative stress causes activation of NF-kappaB and inflammation in the liver of type I diabetic rats. Free Radical Biology and Medicine 49: 171–177.

22.

Frezzatti

Silveira

(2011) Allopurinol reduces the lethality associated with acute renal failure induced by Crotalus durissus terrificus snake venom: Comparison with probenecid. PLOS Neglected Tropical Diseases 5: e1312.

23.

Castillo

Toledo-Pereyra

Gutierrez

et al . (1991) Peritonitis after cecal perforation. An experimental model to study the therapeutic role of antibiotics associated with allopurinol and catalase. The American Surgeon 57: 313–316.

24.

Villa

Ghezzi

(1995) Effect of N-acetyl-L-cysteine on sepsis in mice. European Journal of Pharmacology 292: 341–344.

25.

di Penta

Moreno

Reix

et al . (2013) Oxidative stress and proinflammatory cytokines contribute to demyelination and axonal damage in a cerebellar culture model of neuroinflammation. PLoS ONE 8: e54722.

26.

Bussmann

Marton Filho

Módolo

et al . (2014) Effect of allopurinol on the kidney function, histology and injury biomarker (NGAL, IL 18) levels in uninephrectomised rats subjected to ischaemia-reperfusion injury. Acta Cirúrgica Brasileira 29: 515–521.

27.

Matuschak

Munoz

Johanns

et al . (1998) Upregulation of postbacteremic TNF-alpha and IL-1alpha gene expression by alveolar hypoxia/reoxygenation in perfused rat lungs. American Journal of Respiratory and Critical Care Medicine 157: 629–637.

28.

Deitch

Kemper

Specian

et al . (1992) A study of the relationship among survival, gut-origin sepsis, and bacterial translocation in a model of systemic inflammation. Journal of Trauma 32: 141–147.

29.

Ndengele

Bellone

Lechner

et al . (2000) Brief hypoxia differentially regulates LPS-induced IL-1beta and TNF-alpha gene transcription in RAW 264.7 cells. American Journal of Physiology: Lung Cellular and Molecular Physiology 278: L1289–L1296.

30.

Jones

Barber

Minei

et al . (1991) Differential pathophysiology of bacterial translocation after thermal injury and sepsis. Annals of Surgery 214: 24–30.

31.

Sahib

Al-Jawad

Alkaisy

(2010) Effect of antioxidants on the incidence of wound infection in burn patients. Annals of Burns and Fire Disasters 23: 199–205.

32.

Wijnen

Vader

Roumen

(2002) Multi-antioxidant supplementation does not prevent an increase in gut permeability after lower torso ischemia and reperfusion in humans. European Surgical Research 34: 300–305.

33.

Langenberg

Bagshaw

May

et al . (2008) The histopathology of septic acute kidney injury: A systematic review. Critical Care 12: R38.

34.

Abcejo

Andrejko

Ochroch

et al . (2011) Impaired hepatocellular regeneration in murine sepsis is dependent on regulatory protein levels. Shock 36: 471–477.

35.

Lee

Choi

et al . (2012) Distinct pathophysiologic mechanisms of septic acute kidney injury: Role of immune suppression and renal tubular cell apoptosis in murine model of septic acute kidney injury. Critical Care Medicine 40: 2997–3006.

36.

Zarbock

Gomez

Kellum

(2014) Sepsis-induced acute kidney injury revisited: Pathophysiology, prevention and future therapies. Current Opinion in Critical Care 20: 588–595.

37.

Tinsley

Grayson

Swanson

et al . (2003) Sepsis induces apoptosis and profound depletion of splenic interdigitating and follicular dendritic cells. Journal of Immunology 171: 909–914.

38.

Kunimoto

Morita

Ogawa

et al . (1987) Inhibition of lipid peroxidation improves survival rate of endotoxemic rats. Circulatory Shock 21: 15–22.

39.

Warner

Hasselgren

Fischer

(1986) Effect of allopurinol and superoxide dismutase on survival rate in rats with sepsis. Current Surgery 43: 292–293.

40.

Netea

Kullberg

Blok

et al . (1997) The role of hyperuricemia in the increased cytokine production after lipopolysaccharide challenge in neutropenic mice. Blood 89: 577–582.

41.

Matuschak

Lechner

Chen

et al . (2004) Hypoxic suppression of E. Coli-induced Nf-kappa B and AP-1 transactivation by oxyradical signaling. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology 287: R437–R445.

42.

Ames

Cathcart

Schwiers

et al . (1981) Uric acid provides an antioxidant defense in humans against oxidant- and radical-caused aging and cancer: A hypothesis. Proceedings of the National Academy of Sciences of the United States of America 78: 6858–6862.

43.

Third

Johnson

Foley

et al . (2004) Altered circadian relationship between serum nitric oxide, carbon dioxide, and uric acid in multiple sclerosis. Chronobiology International 21: 739–758.

44.

Giovannini

Chiarla

Giuliante

et al . (2007) Biochemical and clinical correlates of hypouricemia in surgical and critically ill patients. Clinical Chemistry and Laboratory Medicine 45: 1207–1210. Erratum in Clin Chem Lab Med 45: 1570.

45.

Crişan

Cleophas

MCP

Novakovic

et al . (2017) Uric acid priming in human monocytes is driven by the AKT-PRAS40 autophagy pathway. Proceedings of the National Academy of Sciences of the United States of America 114(21): 5485–5490.

46.

Kool

Soullié

van Nimwegen

et al . (2008) Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells. Journal of Experimental Medicine 205(4): 869–882.

47.

Cristóbal-García

García-Arroyo

Tapia

et al . (2015) Renal oxidative stress induced by long-term hyperuricemia alters mitochondrial function and maintains systemic hypertension. Oxidative Medicine and Cellular Longevity 2015: 535686.

48.

Stamp

Chapman

Palmer

(2016) Alopurinol e função renal: uma atualização. Joint Spine Osso 83: 19–24.

49.

Arellano

Sacristán

(1993) Allopurinol hypersensitivity syndrome: A review. Annals of Pharmacotherapy 27: 337–343.

50.

Curiel

Guzman

(2012) Challenges associated with the management of gouty arthritis in patients with chronic kidney disease: A systematic review. Seminars in Arthritis and Rheumatism 42: 166–178.

51.

Tausche

Christoph

Forkmann

et al . (2014) As compared to allopurinol, urate-lowering therapy with febuxostat has superior effects on oxidative stress and pulse wave velocity in patients with severe chronic tophaceous gout. Rheumatology International 34: 101–109.

52.

Khanna

Fitzgerald

Khanna

et al . (2012) American College of Rheumatology guidelines for management of gout. Part 1: Systematic nonpharmacologic and pharmacologic therapeutic approaches to hyperuricemia. Arthritis Care & Research 64: 1431–1446.

53.

Takai

Yamauchi

Ookura

et al . (2014) Febuxostat for management of tumor lysis syndrome including its effects on levels of purine metabolites in patients with hematological malignancies—A single institution’s, pharmacokinetic and pilot prospective study. Anticancer Research 34: 7287–7296.

54.

Netea

van der Meer

van Deuren

et al . (2003) Proinflammatory cytokines and sepsis syndrome: Not enough, or too much of a good thing? Trends in Immunology 24: 254–258.

55.

Burkovskiy

Sardinha

Zhou

et al . (2013) Cytokine release in sepsis. Advances in Bioscience and Biotechnology 4: 860–865.

56.

Nicholas

Bubnov

Yasinska

et al . (2011) Involvement of xanthine oxidase and hypoxia-induciblefactor1 in Toll-like receptor 7/8-mediated activation of caspase 1 and interleukin-1β. Cellular and Molecular Life Sciences 68: 151–158.

57.

Doughty

Carcillo

Kaplan

et al . (1998) A resposta interleucina-10 da citocina anti-inflamatória compensatória na sepse pediátrica induziu falência múltipla de órgãos. Peito 113: 1625–1631.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.39 MB