Original Article Effects of Methylene Blue on the Nitric Oxide-Soluble Guanylate Cyclase-Cyclic Guanylyl Monophosphate Pathway and Cytokine Levels in Rats with Sepsis

Int J Clin Exp Med 2019;12(10):12203-12211 www.ijcem.com /ISSN:1940-5901/IJCEM0093681

Original Article Effects of methylene blue on the nitric oxide-soluble guanylate cyclase-cyclic guanylyl monophosphate pathway and cytokine levels in rats with sepsis

Pengfei Pan*, Yi Wang*, Xiaopeng Li, Chunbo Yang, Hua Zhong, Xinxin Du, Xiaoli Hua, Xiangyou Yu

Department of Critical Care Medicine, The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang, China. *Equal contributors. Received March 11, 2019; Accepted August 9, 2019; Epub October 15, 2019; Published October 30, 2019

Abstract: Background: Methylene blue (MB) inhibits the production of nitric oxide (NO) and cyclic guanylyl monophosphate (cGMP) and thus reverses septic hypotension, but the specific effects of MB on the NO-soluble guanylate cyclase (sGC)-cGMP pathway in different organs of septic rats are unclear. The present study aimed to elucidate the effects of MB on the NO-sGC-cGMP pathway and inflammatory reactions in rats with sepsis. Methods: A Wistar rat model of sepsis was established by cecal ligation and puncture (CLP). The rats were given an injection of 15 mg/ kg of MB via the caudal vein at 6, 12, or 18 h after CLP. The rats were sacrificed at 6 h after the injection. Then, mRNA and protein expressions of sGC alpha 1 (sGCα1) in lung, liver, and kidney tissues were measured by real-time polymerase chain reaction and western blotting, respectively. Levels of cGMP in the lung, liver, and kidney tissues were assessed by enzyme-linked immunosorbent assays (ELISAs). Levels of serum lactic acid, procalcitonin (PCT), interleukin-6 (IL-6), IL-10, and tumor necrosis factor alpha (TNF-α) were also detected by ELISAs. A NO reduction method was used to detect the level of serum NO. Results: The levels of sGCα1 and cGMP in lung, liver, and kidney tissues were upregulated in the septic rats and were downregulated after MB administration (P < 0.01 or P < 0.05). Additionally, the levels of serum NO were increased in septic rats and were decreased after MB administration (P < 0.01 or P < 0.05). In a separate set of experiments, the levels of PCT, IL-6, IL-10, and TNF-α were increased (P < 0.01) in septic rats but were unchanged after MB administration. Conclusion: The NO-sGC-cGMP pathway is activated in different organs of septic rats. Moreover, MB inhibits the NO-sGC-cGMP pathway, but does not affect systemic inflammation.

Keywords: Sepsis, nitric oxide, soluble guanylate cyclase alpha 1, cyclic guanylyl monophosphate, methylene blue, cytokine

Introduction 2) and heme-containing β (1 and 2) subunits [3]. Excessive NO leads to increased expres- Sepsis, a life-threatening organ dysfunction, sion of sGC in vascular smooth muscle cells, results from a dysregulated host response to and aggregation of intracellular cGMP, which invading pathogens [1]. As a current major results in decreased vascular tension, decrea- cause of death, sepsis represents a substantial sed sensitivity to catecholamines, decreased health care burden [2]. Previous research has blood pressure, and even recalcitrant hypoten- shown that the nitric oxide (NO)-soluble guanyl- sion. Thus, the inhibition of the NO-sGC-cGMP ate cyclase (sGC)-cyclic guanylyl monophos- pathway may constitute a way to control the phate (cGMP) pathway plays significant roles in occurrence and development of sepsis and the process of refractory hypotension and septic shock. organ failure in sepsis. Lipopolysaccharides (LPSs) induced by pathogenic microorganisms Methylene blue (MB), which has antioxidant, prompt the host to produce a large number of anti-inflammatory, neuroprotective, and mito- inducible NO synthases (iNOSs), which results chondrial-protective effects, has been widely in the mass production of NO. The major target used as a dye and medication [4]. MB inhibits of NO is sGC, which is a heterodimer of α (1 and both iNOS and sGC. By competitively blocking MB on NO-sGC-cGMP pathway and cytokine levels the target enzyme of NO, MB inhibits the group was further subdivided into 6, 12, and responsiveness of vessels to cGMP-dependent 18-h subgroups according to the time after vasodilators and restores vascular tone [5]. In operation, with six rats included in each sub- 1992, Schneider [6] first applied MB in the group. Rats in the CLP+MB group were given a treatment of septic patients and found that, in 15 mg/kg injection of MB (Jumpcan, Inc., septic shock patients, an intravenous injection Taizhou, Zhejiang, China) via the caudal vein at of MB (2 mg/kg) could improve low vascular the aforementioned time points after CLP, while resistance and increase mean arterial pres- the remaining rats were given the same dose of sure (MAP). A study by Kirov et al. [7] also normal saline at the corresponding time points. showed that, in septic patients, MB could sig- nificantly increase MAP, maintain systemic Preparation of the sepsis model hemodynamic stability, improve tissue perfu- sion, increase cardiovascular responsiveness The CLP approach, as modified by Rittirsch et to vasopressin, and reduce the use of va- al. [9], was used to establish the sepsis model sopressin. used in the present study. In the sham group, only laparotomy and isolation of the caecum MB has been shown to increases MAP and mesentery were performed without ligation or maintain hemodynamic stability in animal perforation. All of the rats were injected subcu- experiments and clinical studies. However, the taneously with normal saline (30 mL/kg) after specific effects of MB on the NO-sGC-cGMP the operation. The rats were sacrificed at 6 h pathway in multiple organs of septic rats remain after the intravenous injection of MB or normal unclear. In addition, a previous study has sug- saline. The blood, lung, liver, and kidney tissues gested that MB may not reduce inflammatory were collected, immediately placed in liquid responses in septic patients [8]. Thus, in order nitrogen, and were then stored at -80°C until to explore the value of MB in the treatment of future use. sepsis, we observed the effects of MB on the RNA isolation and real-time polymerase chain NO-sGC-cGMP pathway in different organs and reaction (RT-PCR) on indices of systemic inflammatory responses in septic rats. Total RNA was extracted from lung, liver, and Materials and methods kidney tissues with TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), and the Animals total RNA level was measured by a nucleic-acid quantometer. RNA was used to synthesize com- Adult female Wistar rats (180-220 g) were used plementary DNA with a reverse transcription kit for all experiments in the present study. The (Thermo Fisher Scientific, Waltham, MA, USA). animals were housed in individual cages under Real-time polymerase chain reaction (RT-PCR) controlled temperature (23°C ± 2°C) and a was performed using an Applied Biosystems 12-h light-dark cycle, with free access to food 7500 Fast Real-Time PCR system with a and distilled water. All of the experimental pro- QuantiNova SYBR Green PCR Kit (KIAQEN, cedures were approved by the ethics commit- Hamburg, Germany). The conditions of RT-PCR tee of the First Affiliated Hospital of Xinjiang were as follows: 95°C for 10 min, followed by Medical University. This study was conducted in 40 cycles of denaturation at 95°C for 10 s, and accordance with the guidelines for the care and annealing and extension at 60°C for 30 s. The use of experimental animals, and the proce- primers for sGCα1 were designed and synthe- dure or animal disposal was completed in sized by Sango Biotech (Shanghai, China). The accordance with the relevant animal ethical sequences were as follows: forward 5’-CGC- requirements. TTTGACCAACAGTGTGGA-3’ and reverse 5’-GG- GCCATCAGTGCTATCTGGA-3’, and the expected Experimental design size of the PCR amplification product was 127 bp. Then, 3-phosphate glyceraldehyde dehydro- A total of 136 rats were randomly separated genase (GAPDH) was employed as an internal into a sham group, a sepsis (cecal ligation and control, and the primers of GAPDH were pro- puncture, CLP) group, and a CLP+MB group, duced by AST (Holly, MI, USA). The sequen- according to a random-number table. Each ces were as follows: accession number NM_

12204 Int J Clin Exp Med 2019;12(10):12203-12211 MB on NO-sGC-cGMP pathway and cytokine levels

017008.4, forward 5’-GACATGGCCGCCTGGA- Armonk, NY, USA) was used for data analysis. GAAAC-3’ and reverse 5’-AGCCCAGGATGCCC- The Kolmogorov-Smirnov method was used to TTTAGT-3’, and the expected size of the PCR test the normality of each sample distribution. amplification product was 92 bp. The expres- Normally distribution data are expressed as the sion level of sGCα1 was calculated by the ΔΔCT mean ± standard deviation (SD), and one-way calculation. analysis of variance was used for comparisons of multiple groups. The least significant differ- Western blotting ence test was used to evaluate pairwise comparisons if the variance was homogeneous, After being fully ground in liquid nitrogen, sam- while the TamhaneT2 test was adopted when ple tissues (i.e., lung, liver, and kidney) were the variance was not homogeneous. Non- fully lysed via a protein lysate. The extracted normal distributed data are expressed as the proteins were separated by 10% sodium dodec- median (interquartile range), with the lower yl sulfate-polyacrylamide gel electrophoresis, quartile (QL) and upper quartile (QU) being transferred to polyvinylidene fluoride mem- reported as follows: [M (QL, QU)]. The Kruskal- branes, and were blocked with 5% skimmed Wallis H test was used for comparisons of mul- milk. Subsequently, the membranes were incu- tiple groups of non-normally distributed data, bated overnight at 4°C with primary antibodies whereas the Mann-Whitney U test was used for against sGCα1 (1:1000; Abcam, Cambridge, such comparisons between only two groups. P UK) and GAPDH (1:1000; CST, MA, USA). The < 0.05 was considered to be statistically membranes were incubated with a horseradish significant. peroxidase (HRP)-conjugated secondary anti- Results body for 1 h at 37°C. The protein bands were visualized, and the gray values were scanned. Effects of MB on sGCα1 mRNA levels and pro- The ratio of gray values of sGCα1 to GAPDH tein levels in lung, liver, and kidney tissues in was used to indicate the relative expression septic rats level of sGCα1 protein in each tissue sample. The expression of sGCα1 mRNA in lung tissues Enzyme-linked immunosorbent assay (ELISA) was upregulated for all of the subgroups in the CLP group compared to that in the sham group Levels of cGMP in the lung, liver, and kidney tis- and for all of the subgroups in the CLP+MB sues were assessed using the Cyclic GMP EIA group as compared with that of the CLP group Kit (581021, Cayman Chemical Company, Ann (Figure 1A). In a separate set of experiments, Arbor, MI, USA) according to the manufacturer’s the expression of sGCα1 in lung tissues was instructions. Levels of serum lactic acid, procal- downregulated for all subgroups in the CLP citonin (PCT), interleukin-6 (IL-6), IL-10, and group versus that in the sham group and was tumor necrosis factor alpha (TNF-α) were de- downregulated for all subgroups in the CLP+MB tected using commercially available ELISA kits group in comparison with that in the CLP group (Elabscience, Wuhan, Hubei, China) in accor- (Figure 1B and 1C). dance with the manufacturer’s instructions. Additionally, the expression of sGCα1 mRNA in Serum nitric-oxide detection liver tissues was upregulated for subgroups (except for the 12-h group) in the CLP group as A NO reduction method was used to detect the compared with that in the sham group and for - level of serum NO. NO3 is specifically reduced all subgroups in the CLP+MB group versus that - to NO2 by nitrate reductase, which can be used in the CLP group (Figure 2A). The expression of to measure NO levels by determining the chro- sGCα1 in liver tissues was downregulated for mogenic depth. NO assay kits (A012-1; Nanjing all subgroups (except for the 12-h group) in the Jiangcheng Bioengineering Institute, Nanjing, CLP group in comparison with that in the sham Jiangsu, China) were used for the detection. group and was downregulated for all of the subgroups in the CLP+MB group as compared with Statistical analysis that in the CLP group (Figure 2B and 2C).

The statistical package for the social sciences The expression of sGCα1 mRNA in kidney tis- version 17.0 software program (IBM Corp., sues was upregulated for all of the subgroups

12205 Int J Clin Exp Med 2019;12(10):12203-12211 MB on NO-sGC-cGMP pathway and cytokine levels

Figure 1. sGCα1 expressions and cGMP concentrations in the lung tissues of septic rats. A. The expressions of sGCα1 mRNA; sGCα1 mRNA in the lung was measured via RT-PCR. B and C. Protein expression levels of sGCα1; protein levels of sGCα1 in the lung were measured via western blotting. D. The cGMP concentration; cGMP in the lung was measured using ELISAs. Data are expressed as the mean ± SD; n = 6. *P < 0.05 and **P < 0.01 vs. Sham group; #P < 0.05 and ##P < 0.01 vs. CLP group.

Figure 2. sGCα1 expressions and cGMP concentrations in the liver tissues of septic rats. A. The expressions of sGCα1 mRNA; sGCα1 mRNA in the lung was measured using RT-PCR. B and C. Protein expression levels of sGCα1; protein levels of sGCα1 in the lung were measured via western blotting. D. The cGMP concentration; cGMP in the lung was measured using ELISAs. Data are expressed as the mean ± SD; n = 6. *P < 0.05 and **P < 0.01 vs. Sham group; #P < 0.05 and ##P < 0.01 vs. CLP group.

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Figure 3. sGCα1 expressions and cGMP concentrations in the kidney tissues of septic rats. A. The expressions of sGCα1 mRNA; sGCα1 mRNA in the lung was measured using RT-PCR. B and C. Protein expression levels of sGCα1; protein levels of sGCα1 in the lung were measured via western blotting. D. The cGMP concentration; cGMP in the lung was measured using ELISAs. Data are expressed as the mean ± SD; n = 6. *P < 0.05 and **P < 0.01 vs. Sham group; #P < 0.05 and ##P < 0.01 vs. CLP group. in the CLP group in comparison with that in the subgroups in the CLP+MB group versus those sham group and was similarly upregulated for in the CLP group (Figure 2D). all of the subgroups in the CLP+MB group versus that in the CLP group (Figure 3A). The The cGMP concentrations in kidney tissues expression of sGCα1 in kidney tissues was were upregulated for all of the subgroups in the downregulated for all of the subgroups in the CLP group as compared with those in the sham CLP group as compared with that in the sham group and were upregulated for all of the sub- group and was downregulated for all of the subgroups in the CLP+MB group in comparison groups in the CLP+MB group as compared with with those in the CLP group (Figure 3D). that in the CLP group (Figure 3B and 3C). Effects of MB on serum NO levels in sepsis Effects of MB on cGMP concentrations in lung, rats liver, and kidney tissues in septic rats The levels of serum NO were increased for all of The cGMP concentrations in lung tissues were the subgroups in the CLP group as compared upregulated for all subgroups (except for the with those in the sham group and were de- 6-h group) in the CLP group as compared with creased for all of the subgroups in the CLP+MB those in the sham group, and were upregulated group as compared with those in the CLP group for all subgroups (except for the 6-h group) in (Figure 4A). the CLP+MB group as compared with those in the CLP group (Figure 1D). Effects of MB on serum lactic acid, PCT, IL-6, IL-10 and TNF-α levels in septic rats Furthermore, the cGMP concentrations in liver tissues were upregulated for all of the sub- The levels of serum lactic acid were increased groups in the CLP group versus those in the for all of the subgroups in the CLP group versus sham group and were upregulated for all of the those in the sham group and were decreased

12207 Int J Clin Exp Med 2019;12(10):12203-12211 MB on NO-sGC-cGMP pathway and cytokine levels

Figure 4. Serum NO levels and blood lactic acid levels in septic rats. A. Serum NO levels; the NO level in serum was measured by the NO reduction method. B. Blood lactic acid level; the lactic acid level in the blood was measured via ELISAs. Data are expressed as the mean ± SD; n = 6. *P < 0.05 and **P < 0.01 vs. Sham group; #P < 0.05 and ##P < 0.01 vs. CLP group.

Table 1. Comparison of PCT in serum [M (QL, QU)] for all of the subgroups in the PCT (pg/mL) CLP+MB group versus those in Group the CLP group (Figure 4B). Sham (n = 6) CLP (n = 6) CLP+MB (n = 6) 6-h (n = 6) 39.25 (24.07) 166.13 (87.07) 137.98 (92.64) Additionally, the levels of serum 12-h (n = 6) 21.15 (17.71)** 201.02 (133.91)** 228.96 (53.21)** PCT, IL-6, IL-10, and TNF-α were 18-h (n = 6) 11.49 (8.70) 152.57 (141.57) 208.83 (132.17) increased for all of the subgroups **P < 0.01 vs. Sham group. in the CLP group as compared with those in the sham group, but remained unchanged for all of Table 2. Comparison of IL-6 in serum [M (QL, QU)] the subgroups in the CLP+MB IL-6 (pg/mL) group versus those in the CLP Group Sham (n = 6) CLP (n = 6) CLP+MB(n = 6) group (Tables 1-4). 6-h (n = 6) 13.55 (9.28) 80.94 (70.78) 96.66 (57.05) Discussion 12-h (n = 6) 22.56 (21.06)** 39.70 (16.90)** 58.35 (17.31)** 18-h (n = 6) 12.92 (9.38) 61.40 (51.00) 63.77 (52.75) Systemically, large amounts of **P < 0.01 vs. Sham group. NO lead to hypotension, microcir- culatory dysfunction, and refrac- toriness to vasopressor catechol- Table 3. Comparison of IL-10 in serum [M (QL, QU)] amines [10]. Additionally, exces- IL-10 (pg/mL) sive NO levels and activation of Group Sham (n = 6) CLP (n = 6) CLP+MB (n = 6) the sGC-cGMP pathway are im- 6-hour (n = 6) 13.42 (14.93) 386.79 (273.08) 302.32 (78.44) portant in sepsis-induced organ 12-hour (n = 6) 16.37 (11.28)** 209.31 (64.89)** 272.3 (161.80)** damage. One study of a rat CLP model of sepsis showed that sGC 18-hour (n = 6) 10.57 (6.35) 192.53 (78.68) 183.19 (79.42) α1 and β1 subunit mRNA and **P < 0.01 vs. Sham group. protein levels were increased in the lungs and that sodium nitro- Table 4. Comparison of TNF-α in serum [M (QL, QU)] prusside-stimulated cGMP accumulation was higher in the lungs TNF-α (pg/mL) Group at 48 h after CLP [11]. Gill et al. Sham (n = 6) CLP (n = 6) CLP+MB (n = 6) [12] found that septic apoptosis 6-h (n = 6) 9.93 (5.65) 70.14 (25.77) 71.10 (16.32) of pulmonary microvascular en- 12-h (n = 6) 11.97 (8.92)** 60.81 (25.37)** 67.45 (9.76)** dothelial cells is a result of leuko- 18-h (n = 6) 10.70 (7.38) 58.06 (27.71) 54.72 (19.39) cyte activation and iNOS-depen- **P < 0.01 vs. Sham group. dent signaling andwhich, in turn,

12208 Int J Clin Exp Med 2019;12(10):12203-12211 MB on NO-sGC-cGMP pathway and cytokine levels may contribute to pulmonary microvascular clinical study of sepsis showed that MB barrier dysfunction as well as albumin hyper- increased MAP but had no effect on oxygen permeability in sepsis-induced lung injury. It delivery [7]. Another clinical study of sepsis was also revealed that iNOS upregulation con- suggests that a short-term infusion of MB tributes to liver microvascular dysfunction and increases MAP, decreases NO production, and liver damage induced by LPSs in a murine attenuates the urinary excretion of renal tubu- model of endotoxemia [13]. LPSs impair NO- lar-injury markers [24]. However, the effects of dependent modulation of intrahepatic resis- MB on different organs in sepsis have remained tance, which increases vascular inflammation unclear. and hepatic oxidative stress [14]. A study of endotoxemia in mice showed that serum NO in- In the present study of a rat model after CLP, an creased in a time-dependent manner, reaching intravenous injection of MB (15 mg/kg) inhibit- the highest levels at 24 h, and that sGC and ed the expression of sGCα1 mRNA and protein cGMP increased in renal cortical slices [15]. In in lung, liver, and kidney tissues and also down- a study of human endotoxemia and sepsis, the regulated the expression of cGMP protein in upregulation of renal iNOS and subsequent these same tissues. The inhibition of the sGC- higher urinary excretion of NO metabolites were cGMP pathway may partially account for the associated with renal proximal tubule damage MB effects on various tissues and organs. Our [16]. A study by Oliveira-Pelegrin et al. [17] sug- present study also found that the administra- gested a role of the NO-cGMP pathway in hor- tion of MB induced an increase in serum NO monal synthesis in the supraoptic and paraven- levels but failed to reduce pro/anti-cytokine lev- tricular nuclei during polymicrobial sepsis. els. Endotoxin and pro-inflammatory cytokines increase iNOS expression and NO production Targeting the NO-sGC-cGMP pathway is accept- [25]. In turn, NO stimulates the synthesis of ed as a way to treat sepsis. MB, a United States pro-inflammatory cytokines via the activation of Food and Drug Administration approved phar- the nuclear factor kappa B pathway. Therefore, maceutical drug, has been safely used to treat MB may affect the release of cytokines. Curr- methemoglobinemia and cyanide poisoning ently, there are only two known randomized [18]. Unlike L-arginine analogs that non-selec- controlled trials that have evaluated the use of tively inhibit NOS, MB is a selective iNOS inhibi- MB in patients with sepsis [7, 26]. Neither of tor. MB attenuates transcription-factor binding these two studies showed a difference in cyto- to iNOS promoters amid iNOS mRNA transcrip- kine levels between the MB group and the con- tion [19]. MB also directly inhibits the activity of trol group. Although MB could restore vascular iNOS and reduces the synthesis of NO [20, 21]. tone, it did not change serum cytokine levels, As for sGC, MB binds to its iron heme moiety of which may account for why MB does not have sGC and prevents the accumulation of cGMP an effect on mortality. In a consecutive study of [5]. Some evidence has suggested that MB 20 patients with refractory septic shock, Park exerts its acute vasopressor effect mainly via et al. [8] reported that MB elevated the MAP sGC inhibition over NOS inhibition [8]. without altering the productions of NO, IL-1, IL-10, or TNF-α. In our present study, the By inhibiting the excessive production of iNOS decrease of NO levels was not accompanied by and cGC, MB blocks the acute hemodynamic a decrease of serum cytokine levels. It is likely effect and other effects of NO, which reverses that a relatively large dose of MB was adminis- sepsis-associated vasoplegia and possibly alle- tered in the present study. Moreover, the pres- viates organ injury. The administration of MB ent study still did not identify a difference in clearly increased MAP and systemic vascular PCT level with MB administration. A limitation resistance in patients with septic shock, al- of the present study is that we only explored the though the effects of MB on mortality are still NO level and the expression of sGCα1/cGMP in unknown [22]. MB can also increase myocardi- three organs. In addition, we did not monitor al contractility depressed by cGMP. Evgenov et any hemodynamic parameters. al. [23] found that MB decreased the enhanced lung fluid filtration by reducing pulmonary capil- In conclusion, we found that the NO-sGC-cGMP lary pressure and permeability during the early pathway was activated in a rat CLP model of phase of endotoxemia in sheep. A separate sepsis and that MB decreased serum NO lev-

12209 Int J Clin Exp Med 2019;12(10):12203-12211 MB on NO-sGC-cGMP pathway and cytokine levels els, inhibited the sGC-cGMP pathway in differ- methylene blue in human septic shock: a pilot, ent organs, and alleviated lactic acidosis, but randomized, controlled study. Crit Care Med did not lower serum levels of PCT or pro-inflam- 2001; 29: 1860-1867. matory cytokines. Future investigations are [8] Park BK, Shim TS, Lim CM, Lee SD, Kim WS, required to further elucidate the mechanisms Kim DS, Kim WD and Koh Y. The effects of methylene blue on hemodynamic parameters and clinical application of MB in the treatment and cytokine levels in refractory septic shock. of sepsis. Korean J Intern Med 2005; 20: 123-128. [9] Rittirsch D, Huber-Lang MS, Flierl MA and Ward Acknowledgements PA. Immunodesign of experimental sepsis by cecal ligation and puncture. Nat Protoc 2009; This study was supported by grants from the 4: 31-36. National Natural Science Foundation of China [10] Schweighöfer H, Rummel C, Mayer K and (No. 81560310). We thank LetPub (www.let- Rosengarten B. Brain function in iNOS knock pub.com) for its linguistic assistance during the out or iNOS inhibited (l-NIL) mice under endo- preparation of this manuscript. toxic shock. Intensive Care Med Exp 2014; 2: 24. Disclosure of conflict of interest [11] Fernandes D, Sordi R, Pacheco LK, Nardi GM, Heckert BT, Villela CG, Lobo AR, Barja-Fidalgo None. C and Assreuy J. Late, but not early, inhibition of soluble guanylate cyclase decreases mortal- Address correspondence to: Dr. Xiangyou Yu, De- ity in a rat sepsis model. J Pharmacol Exp Ther partment of Critical Care Medicine, The First Affi- 2009; 328: 991-999. liated Hospital of Xinjiang Medical University, 138 [12] Gill SE, Taneja R, Rohan M, Wang L and Mehta Liyushan South Road, Urumqi 830054, Xinjiang, S. Pulmonary microvascular albumin leak is China. Tel: +86-0991-4366083; E-mail: yu2796@ associated with endothelial cell death in mu- 163.com rine sepsis-induced lung injury in vivo. PLoS One 2014; 9: e88501. References [13] La Mura V, Pasarín M, Rodriguez-Vilarrupla A, Garcia-Pagán JC, Bosch J and Abraldes JG. [1] Heming N, Sivanandamoorthy S, Meng P, Bou- Liver sinusoidal endothelial dysfunction after nab R and Annane D. Immune effects of corti- LPS administration: a role for inducible-nitric costeroids in sepsis. Front Immunol 2018; 9: oxide synthase. J Hepatol 2014; 61: 1321- 1736. 1327. [2] Kim HI and Park S. Sepsis: early recognition [14] La Mura V, Pasarín M, Meireles CZ, Miquel R, and optimized treatment. Tuberc Respir Dis Rodríguez-Vilarrupla A, Hide D, Gracia-Sancho (Seoul) 2019; 82: 6-14. J, García-Pagán JC, Bosch J and Abraldes JG. [3] Chawla LS, Davison DL, Brasha-Mitchell E, Effects of simvastatin administration on ro- Koyner JL, Arthur JM, Shaw AD, Tumlin JA, Tre- dents with lipopolysaccharide-induced liver vino SA, Kimmel PL and Seneff MG. Develop- microvascular dysfunction. Hepatology 2013; ment and standardization of a furosemide 57: 1172-1181. stress test to predict the severity of acute kid- [15] Knotek M, Esson M, Gengaro P, Edelstein CL ney injury. Crit Care 2013; 17: R207. and Schrier RW. Desensitization of soluble [4] Ahn H, Kang SG, Yoon SI, Ko HJ, Kim PH, Hong guanylate cyclase in renal cortex during endo- EJ, An BS, Lee E and Lee GS. Methylene blue toxemia in mice. J Am Soc Nephrol 2000; 11: inhibits NLRP3, NLRC4, AIM2, and non-canon- 2133-2137. ical inflammasome activation. Sci Rep 2017; 7: [16] Heemskerk S, Pickkers P, Bouw MP, Draisma 12409. A, van der Hoeven JG, Peters WH, Smits P, Rus- [5] Hosseinian L, Weiner M, Levin MA and Fischer sel FG and Masereeuw R. Upregulation of re- GW. Methylene blue: magic bullet for vasople- nal inducible nitric oxide synthase during hu- gia? Anesth Analg 2016; 122: 194-201. man endotoxemia and sepsis is associated [6] Schneider F, Lutun P, Hasselmann M, Stoclet with proximal tubule injury. Clin J Am Soc JC and Tempé JD. Methylene blue increases Nephrol 2006; 1: 853-862. systemic vascular resistance in human septic [17] Oliveira-Pelegrin GR, Aguila FA, Basso PJ and shock. Intensive Care Med 1992; 18: 309- Rocha MJ. Role of central NO-cGMP pathway 311. in vasopressin and oxytocin gene expression [7] Kirov MY, Evgenov OV, Evgenov NV, Egorina during sepsis. Peptides 2010; 31: 1847-1852. EM, Sovershaev MA, Sveinbjørnsson B, Ne- [18] Rodriguez P, Zhao J, Milman B, Tiwari YV and dashkovsky EV and Bjertnaes LJ. Infusion of Duong TQ. Methylene blue and normobaric hy-

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