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Lidocaine attenuates lipopolysaccharide-induced inflammatory responses and protects against endotoxemia in mice by suppressing HIF1α-induced glycolysis
International Immunopharmacology 80 (2020) 106150
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Lidocaine attenuates lipopolysaccharide-induced inflammatory responses and protects against endotoxemia in mice by suppressing HIF1α-induced glycolysis
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Shengwei Lina, Peipei Jina, Chao Shaob, Wenbin Lua, Qian Xianga, Zhengyu Jianga, Yan Zhanga, ⁎ Jinjun Biana, a b
Faculty of Anesthesiology, Changhai Hospital, Second Military Medical University/Naval Medical University, Shanghai 200433, China Department of Anesthesiology, Urumqi General Hospital of Lanzhou Military Command, Urumqi 830000, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Lidocaine Cytokine Glycolysis HIF1α Endotoxemia
Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infections. Previous studies have indicated that lidocaine, an amide local anesthetic, has anti-inflammatory properties; however, the underlying mechanism remains unclear. In this study, we have shown that lidocaine dose-dependently inhibits lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in macrophages and that lidocaine protects mice from LPS-induced inflammation. Moreover, we have demonstrated that lidocaine reduces the release of TNF-α and IL-6 through the reduction of the expression of GLUT1 and HK2 to further suppress HIF1α-induced aggravation of inflammatory cascades. Lidocaine can inhibit the enhanced glycolysis and glycolytic capacity induced by LPS in the macrophages. As an inhibitor of PHDs (prolyl hydroxylases), Dimethyloxalylglycine (DMOG) can reduce the anti-inflammatory effects of lidocaine. In conclusion, the present study indicates that lidocaine can be used as a potential therapeutic agent for sepsis.
1. Introduction Sepsis is defined as a life-threatening organ dysfunction caused by dysregulated host response to infections and is the leading cause of death in critically ill patients [1]. The in-hospital mortality caused by sepsis still remains high with a percentage probability of 25–30%; this remains a huge problem, globally [2]. One of the most notable features of sepsis is that immune cells are activated to secrete large amounts of inflammatory factors. As the main source of inflammatory factors, the metabolism in macrophages is closely related to its function [3]. Previous investigations have shown that LPS can activate macrophages resulting in their altered metabolism [4]. Recent studies indicate that there might be a potential link between macrophage glycolysis and inflammation [5]. The metabolism in macrophages has been recognized as a critical mechanism for regulating inflammatory responses [6]. Glucose, which is metabolized through anaerobic glycolysis and aerobic oxidative phosphorylation, has been reported to play a crucial role in regulating immune responses in macrophages. LPS-induced glucose transport in macrophages is mainly
mediated by glucose transporter 1 (GLUT1), which controls glucose uptake [7]. In addition, Hexokinase (HK) is a key enzyme for glycolysis as it controls the initial rate-limiting step of this process. LPS strongly enhanced the expression of HK2 [8]. Perturbing glucose uptake and its subsequent metabolic events can alter, both, glucose and lactate homeostasis. As a product of glycolysis, lactate leads to the acidification of the extracellular matrix. The alterations in extracellular matrix can be used as indirect readouts of glucose metabolism. Acidification of the extracellular matrix can also be measured as the extracellular acidification rate (ECAR) [9]. The transcriptional factor hypoxia-inducible factor-1 (HIF-1) is a key player in hypoxic or inflammatory conditions. It consists of an oxygen-sensitive α subunit and a constitutive β subunit [10]. Under the hypoxic or inflammatory environment, the α subunit escapes prolyl hydroxylation and binds to the β subunit. The heterodimer then translocates to the nucleus and activates the transcription of genes involved in cell metabolism, cell growth, and apoptosis. A previous study showed that HIF1α is one of the key molecules upstream of glycolysis; HIF1α could regulate the activity of several glycolytic proteins
⁎ Corresponding author at: Faculty of Anesthesiology, Changhai Hospital, Second Military Medical University/Naval Medical University, 168 Changhai Road, Shanghai 200433, China. E-mail address: [email protected] (J. Bian).
https://doi.org/10.1016/j.intimp.2019.106150 Received 24 August 2019; Received in revised form 20 December 2019; Accepted 21 December 2019 1567-5769/ © 2020 Elsevier B.V. All rights reserved.
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2.3. Cell culture
including GLUT1 and HK2 [11,12]. Over-expression of HIF1α enhances glycolysis by regulating the expression of the glycolytic gene like GLUT1 in the LPS-stimulated macrophages [11]. In pulmonary cells, HK2 expression is regulated by HIF1α -dependent mechanism [12]. HIF1α is hydroxylated at conserved proline residues by PHDs, thereby marking it for rapid proteasomal degradation. Dimethyloxalylglycine (DMOG), a cell penetrant analog, inhibits PHDs and stabilizes HIF1α in cultured cells, thereby influencing the subsequent processes [13]. It has been evidenced in previous studies that DMOG can be safely administered to the experimental animals [14]. Lidocaine, a common local anesthetic drug, has been reported to have anti-inflammatory effects. Intravenous lidocaine can reduce inflammation and relief the pain after bimaxillary surgery [15]. Previous studies have suggested that lidocaine exerts its anti-inflammatory effects by inhibiting the NF- κ B signaling, which modulates the release of the inflammatory factors [16]. Studies have shown that one of the upstream events of HIF-1α signaling is the NF-κ B pathway; it serves as a metabolic switch in macrophages [17]. However, whether lidocaine can exert anti-inflammatory effects by inhibiting HIF1α remains unknown. In the current study, we have investigated the effects of lidocaine on macrophages under inflammation in endotoxemia mice as well as the related mechanism. Our data suggests that lidocaine can inhibit glycolysis in macrophages of the endotoxemia mice, thus, reducing the inflammatory response.
Thioglycollate (TG)-elicited peritoneal macrophages were isolated from C57BL/6 mice as previously described [20]. The peritoneal macrophages (PMs) were incubated with different concentrations of lidocaine (1 to 10 μM) at 37 °C for 6 h. The cell viability was measured by the cell counting kit-8 assay (DOJNDO, SHANGHAI, CHINA). Bone marrow-derived cells from C57BL/6 mice were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin; they were differentiated into the macrophages (bone marrow-derived macrophages [BMDMs]) by applying recombinant murine GM-CSF (25 ng/ml; Miltenyi Biotech) for 7 days. 2.4. ELISA assay PMs were seeded in 24-well plates at 2–5 × 105 cells/well a day before the experiment. Thirty minutes after lidocaine treatment the cells were exposed to 100 ng/ml LPS. The supernatant was collected 6 h after LPS stimulation. The media were collected and centrifuged at 10,000 rpm for 5 min. The TNF-α and IL-6 contents were determined by a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) using the mouse ELISA kits for TNF-α (Roche) and IL-6 (Roche) according to the manufacturer's instructions. 2.5. Real-Time polymerase chain reaction analysis
2. Materials and methods
Total RNA was isolated using TRIZOL reagent and chloroform extraction according to the manufacturer's protocol (Sangon, China). Reverse transcription reactions were performed using the PrimeScript RT Reagent Kit (Takara, Japan). The parameters used for PCR reaction are as follows: 94 °C for 3 min, denaturation (94 °C for 30 s), annealing (60 °C for 45 s), and extension (72 °C for 30 s) for 30 cycles, then a final extension at 72 °C for 10 min. PCR products were analyzed using 1.5% agarose gel electrophoresis. The optical intensity of gel bands was estimated using ImageJ software (NIH, USA). All experiments were performed in triplicates. The sequences of the specific primers used are as follows: HIF-1α, 5′-GAAACGACCACTGCTAAGGCA-3′(forward) and 5′-GGCAGACAGCTTAAGGCTCCT-3′ (reverse)
2.1. Animal experiments C57BL/6 mice (7 weeks old) were obtained from the Animals Experimentation Center of Naval Medical University (Shanghai, China) and were maintained in micro isolator cages; the mice received food and water ad libitum. All procedures were performed in accordance with the requirements of Provisions and General Recommendation of Chinese Experimental Animals Administration Legislation and were approved by the Animal Care and Use Committee of Changhai Hospital. The C57BL/6 mice were divided into three groups (n = 6): control (saline, 200 ul), LPS (8 mg/kg, 200 ul), and LPS with lidocaine (6 mg/ kg, 200 ul). Endotoxemia was induced by injecting LPS intraperitoneally (i.p.). Subsequently, the lidocaine(sigma-Aldrich) or saline were given i.p. 30 min prior to the LPS challenge. After 8 h, the serum was separated for analysis from heart blood by centrifugation. The livers and lungs were harvested and fixed in 10% formalin for hematoxylin and eosin(H&E) staining.
2.6. Western blot analysis Western blot analysis was performed as previously described [21]. Briefly, post treatment PMs were harvested and lysed, and the cleared lysate was separated by SDS-PAGE. After electrophoresis, proteins were transferred to the PVDF membranes. The membranes were first hybridized with primary antibodies and then were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit IgG secondary Ab (Santa Cruz). The human HIF1α monoclonal antibody was from Millipore (USA) and b-actin antibody was from EnoGene (China). The immune blots were visualized by the BioshineChemiQ 4800 mini Imaging System.
2.2. Histopathological test The lungs and livers were harvested 8 h after LPS challenge in each group. After fixation in 10% formaldehyde for > 8 h, the organs were prepared for slicing and staining with hematoxylin and eosin. The sections were examined and scored for using the light microscope by two independent pathologists. The scoring system has been described in previous studies [18,19]. Generally, for lung inflammation: grade 0, normal; grade 1, minimal inflammatory changes; grade 2, mild to moderate inflammatory changes (no obvious damage to the lung architecture); grade 3, moderate inflammatory injury (thickening of the alveolar septa); grade 4, moderate to severe inflammatory injury (formation of nodules or areas of pneumonitis that distorted the normal architecture); 5, severe inflammatory injury with total obliteration of the field. For liver necrosis: 0, normal; 1, individual cell necrosis; 2, up to 30% lobular necrosis; 3, up to 60% lobular necrosis; 4, > 60% lobular necrosis.
2.7. XF bioenergetic analysis Metabolic analyses were performed using Seahorse XFe96 Analyzers (Agilent Technologies, USA), which enable the real-time measurements of extracellular acidification rate (ECAR) by creating a transient microchamber within each well of the specialized microplate (Agilent Technologies). BMDMs were seeded at a density of 3 × 106/well onto the 96-well microplate for 2 h prior to the assay. Glycolysis is the ECAR after the addition of glucose. Glycolytic capacity is the increase in ECAR after the injection of oligomycin following glucose. Data were normalized with the cell number and expressed as mpH/min/103 cells (ECAR). 2
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Fig. 1. The effect of lidocaine on the production of pro-inflammatory cytokines in macrophages. (A) For cytotoxicity assessment of lidocaine, macrophages were treated with different concentrations of lidocaine for 6 h and then the cytotoxicity was measured by CCK-8 assay. (B) ELISA experiments to illustrate the effect of lidocaine on the production of pro-inflammatory cytokines in macrophage. (C) The effect of lidocaine on mRNA expression of pro-inflammatory cytokines was measured by qRT-PCR. Results are expressed as means ± SEM from n = 4 independent experiments. *** P < 0.001, **P < 0.01, * P < 0.05.
treatment. The release of TNF-α and IL-6 was lower in the lidocaine (5 μmol/L) treatment group as compared with lidocaine (1 μmol/L) treatment group (p < 0.05, Fig. 1B). Furthermore, qPCR analysis showed that LPS upregulated TNF-α and IL-6 expression at mRNA level after 1 h treatment. Treatment with lidocaine inhibited LPS-induced TNF-α and IL-6 mRNA expression in a dose-dependent manner. The mRNA expression of TNF-α and IL-6 was lower in the lidocaine (5 μmol/L) treatment group than that in the lidocaine (1 μmol/L) treatment group (p < 0.05, Fig. 1C).
2.8. Statistical analysis All data are expressed as mean ± SEM and were analyzed using Prism 5.0 statistical program (GraphPad Software). Comparisons among three or more experimental groups were performed using ANOVA with Tukey’s post hoc test to determine the significance. A value of p < 0.05 was considered as significant. 3. Results 3.1. Lidocaine inhibits LPS-induced inflammation in macrophages.
3.2. Lidocaine alleviates LPS-induced inflammation in mice.
To investigate the influence of lidocaine in vitro, we treated macrophages with different concentrations of lidocaine. The results suggest that at concentrations of 1–5 μmol/L, lidocaine does not influence the viability of cells significantly suggesting the low cytotoxicity of lidocaine (p > 0.05, Fig. 1A). LPS induced a marked release of TNF-α and IL-6, which was significantly attenuated by lidocaine (5 μmol/L)
We then investigated the anti-inflammatory activity of lidocaine in an animal model. We treated the mice with LPS to induce endotoxemia; this treatment induces an increased secretion of cytokines (Fig. 2A). Administration of lidocaine (6 mg/kg, i.p.) before the LPS treatment reduced the secretion of cytokines (Fig. 2B). The pathological observations demonstrated that lidocaine apparently reduced LPS-induced 3
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Fig. 2. The effect of lidocaine in LPS-induced endotoxemia and H&E staining of liver and lung tissue from endotoxemia mice. Each group of mice was pretreated with 6 mg/kg of lidocaine (i.p.) or saline. Thirty minutes after lidocaine treatment, animals were administered 8 mg/kg of LPS (i.p.). (A) Schematic timeline of in vivo experiment. (B) The results indicate the level of pro-inflammatory cytokines in the serum of mice. (C) the H&E staining of mice liver and lung tissue sections. (D) quantification of the tissue damage. Results are expressed as means ± SEM from n = 6 independent experiments. *** P < 0.001, ** P < 0.01, * P < 0.05, # P < 0.05(LPS compared with LPS + LIDO).
3.3. Lidocaine reduces glycolysis in LPS-treated macrophages.
lung injury in mice, which can be tracked by measuring alveolar wall thickness, edema, bleeding, immune cell infiltration, the damage of blood vessels, and alveolar structure; the necrosis area in the mice liver was also reduced by lidocaine treatment (Fig. 2C). The semiquantitative scoring of the pathological changes also showed that lidocaine reduced the lung injury when compared with control mice (Fig. 2D, p < 0.05). These data suggest that lidocaine is effective in preventing LPS induced injury in LPS-induced endotoxemia models.
An inflammatory response is usually accompanied with modulation of metabolic changes [6]. To investigate the role of lidocaine on metabolism in macrophages, we examined the mRNA expression of glycolytic enzymes. Our data showed that LPS treatment increased the expression of GLUT1 and HK2 in macrophages at the transcriptional level. Furthermore, treatment with lidocaine significantly reduced the mRNA expression level of GLUT1 and HK2 in macrophages (p < 0.05, Fig. 3A). We used GM-CSF (100 ng/ml) to enhance the glycolysis to further delineate the mechanism by which lidocaine inhibits the
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Fig. 3. The effect of lidocaine on glycolysis in macrophages. Peritoneal macrophages (PMs) from WT mice were stimulated with LPS (100 ng/mL) for 1–3 h and the indicated mRNA expression was determined with qPCR. (A) GLUT1 and HK2 mRNA expressions were determined by qPCR. (B) Macrophages were pretreated with GM-CSF for 3 h and subsequently GLUT1 and HK2 mRNA expressions were determined by qPCR. (C) Macrophages were pretreated with GM-CSF for 3 h and production of TNF-α and IL-6 was determined by ELISA. (D) A representative graph output from XFe96 showing ECAR response to glucose (10 mM), oligomycin (10 mM), and 2-DG (100 mM). BMDMs were primed by 25 ng/ml GM-CSF for 24 h. BMDMs were seeded in 96-well plate for 2 h and (E) glycolytic activities (glycolysis and glycolytic capacity) were examined using XF Analyzer. Results are expressed as means ± SEM from n = 4 independent experiments. *** P < 0.001, ** P < 0.01, * P < 0.05.
We further examined the glycolysis and glycolytic capacity in macrophages by using the XFe96 analyzer. After adding glucose, ECAR was significantly increased in the LPS-stimulated macrophages. When oligomycin was added to inhibit oxidative phosphorylation in the cells,
inflammatory responses in LPS-treated macrophages. After pretreatment with GM-CSF, the concentrations of TNF-α and IL-6 as well as the mRNA expression of GLUT1 and HK2 showed no difference between lidocaine treatment group and the LPS group (p < 0.05, Fig. 3B,3C).
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Fig. 4. HIF1α is required for regulating the anti-inflammatory effect of lidocaine. (A) HIF-1α mRNA level in PMs was examined by qRT-PCR (left) and HIF-1α protein level in PMs was examined by Western blot (right). (B) PMs were stimulated with LPS (100 ng/mL) for 6 h followed by DMOG treatment for 3 h. Supernatants were collected and assayed for TNF-α and IL-6 production by ELISA. (C) PMs were stimulated with LPS (100 ng/mL) for 1–3 h followed by DMOG treatment for 3 h. GLUT1 and HK2 mRNA expression levels were determined by qPCR. Results are expressed as means ± SEM from n = 4 independent experiments. ** P < 0.01,* P < 0.05.
lidocaine significantly attenuated the upregulation of HIF1α induced by LPS (p < 0.05, Fig. 4A). However, after pretreatment with DMOG (0.5 mmol/L), the concentrations of TNF-α and IL-6 were found to be similar between the lidocaine and LPS groups (p＞0.05, Fig. 4B). Since the expression level of GLUT1 and HK2 positively correlates with HIF1α expression level in macrophages, we subsequently examined the effect of DMOG on the expression of GLUT1 and HK2 at mRNA level using qRT-PCR. DMOG dramatically inhibited the influence of lidocaine. Meanwhile, the expression of GLUT1 and HK2 showed no difference at the mRNA level between the two groups (Fig. 4C). These results indicate that lidocaine can inhibit the expression of HIF1α and glycolytic pathway in LPS-treated macrophages, thus, resulting in anti-inflammatory effects.
the ECAR level was further increased in LPS-stimulated macrophages suggesting a higher level of glycolytic capacity (Fig. 3D). Lidocaine pretreated macrophages responded less to the stimulation as compared with the response of LPS-stimulated macrophages (Fig. 3D). Glycolytic capacity and glycolysis rate were significantly higher in LPS-stimulated macrophages as compared to that in lidocaine pre-treated macrophages (Fig. 3E). These results suggest that lidocaine pretreatment could inhibit the enhanced glycolysis induced by LPS in macrophages. 3.4. Lidocaine inhibits the expression of HIF-1α in LPS-treated macrophages. A previous study has shown that HIF1α is one of the key molecules upstream of glycolysis [22]. To investigate whether lidocaine inhibits glycolysis by down-regulation of HIF1α, we performed a series of experiments to examine the effect of lidocaine in the LPS-treated macrophages. Our data demonstrated that the LPS treatment increased the expression of HIF1α in macrophages. Furthermore, the treatment of
4. Discussion This study demonstrates beneficial inhibitory effects of lidocaine on the expression and secretion of HIF1α and HIF1α-enhanced production 6
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inflammatory cytokine production from coronary artery disease monocytes and macrophages [32]. The use of drugs that are specifically activated in macrophages could be considered for future therapies [33]. Further work will be needed to explore the utility of targeting specific metabolic events in macrophages for therapeutic gain.
of pro-inflammatory factors such as TNF-α and IL-6. In vivo investigation indicated that lidocaine protects mice from LPS-induced inflammation. The present data provide evidence that lidocaine exerts anti-inflammatory effects to inhibit the expression of GLUT1 and HK2 through suppressing HIF1α activity. Previous studies have indicated that lidocaine can reduce mortality and protect against renal and hepatic dysfunction in murine septic peritonitis [23]. However, more importantly, a double-blinded, prospective, randomized clinical trial showed that lidocaine reduces neutrophil recruitment by abolishing chemokine-induced arrest and transendothelial migration in septic patients. This indicates that lidocaine might play a possible therapeutic role in decreasing the inappropriate activation, positioning, and recruitment of leukocytes during sepsis [24]. The above-mentioned studies provide strong evidence for lidocaine’s ability to inhibit the inflammation caused by sepsis. Therefore, not surprisingly, our data demonstrate that lidocaine is protective against inflammation as well as the inflammatory infiltration in the lung and liver. It was shown that lidocaine protects septic rats via inhibition of HMGB1 expression and activation of NF- κ B [25]. Lidocaine was demonstrated to inhibit the release of HMGB1 in LPS-stimulated macrophages [26]. Lidocaine was also reported to attenuate LPS-induced inflammatory responses in microglia, which may be mediated by blockade of p38 MAPK and NF- κ B signaling pathways [27]. Ahmed Tawakol et al. showed that macrophage glycolysis and proinflammatory activation mainly depend on HIF due to their crucial role in glucose metabolism [28]. As a result, hypoxia potentiates inflammation and glycolysis mainly via these pathways. Therefore, we investigated whether lidocaine affects the inflammation through the abovementioned pathways. We found that lidocaine can significantly inhibit the mRNA expression of GLUT1 and HK2 in LPS-stimulated macrophages. Granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances anaerobic glycolysis while exerting a mild pro-inflammatory effect [29]. After pretreatment with GM-CSF, lidocaine partially reduced the release of TNF-α and IL-6 caused by LPS treatment. The upregulation effect of LPS on GLUT1 and HK2 mRNA could also be reversed by lidocaine in macrophages. Based on the previous studies, ECAR is an important and irreplaceable indicator for assessing the level of glycolysis. We found that ECAR level was further increased in LPSstimulated macrophages suggesting a higher level of glycolysis. Glycolytic capacity and glycolysis rate were significantly higher in LPSstimulated macrophages as compared to those in lidocaine pre-treated macrophages. In general, HIF1 is considered to promote and enhance inflammatory responses to maintain the host defense not only under hypoxia but also in normoxia. Previous studies have indicated that HIF1α expression in macrophages affects their intrinsic inflammatory profile and promotes the development of atherosclerosis [30]. Wang et al. showed that HIF1α promotes peritoneal macrophage glycolysis metabolism with high expression of some glycolytic genes including GLUT1 and PKM2 [31]. Our data shows that lidocaine suppresses HIF1α expression and inflammation in macrophages, while the antiinflammatory effect could be reversed by DMOG. Our study has several drawbacks. There is a lack of relevant experiments using gene knockout mice and clinical relevance of the LPS model of sepsis. Taken together, our results show that lidocaine can significantly inhibit the secretion of LPS-induced inflammatory cytokines in macrophages by inhibiting HIF1α-mediated glycolytic pathway, as well as, exert anti-inflammatory effects in endotoxemia mice model. It may provide a new method for treating inflammation and sepsis. Since a very significant metabolic reprogramming occurs in macrophages under sepsis, ultimately it might be possible to target metabolic changes in macrophages therapeutically. Approaches might include inhibition of PKM2, which would prevent inflammatory macrophage activation while supporting anti-inflammatory pathways [5]. Interfering PKM2 may be particularly interesting, since it has been shown to block
5. Authors’ contributions Shengwei Lin and Peipei Jin contributed equally to the work. Jinjun Bian designed the experiments and provided financial support. Shengwei Lin, Chao Shao, and Wenbin Lu performed Seahorse XF Glycolysis Stress Test. Yan Zhang, Qian Xiang, and Peipei Jin contributed to the statistical analysis. Shengwei Lin and Zhengyu Jiang wrote the paper. CRediT authorship contribution statement Shengwei Lin: Resources, Data curation, Methodology, Writing original draft, Visualization. Peipei Jin: Formal analysis, Methodology, Software. Chao Shao: Methodology, Software. Wenbin Lu: Methodology, Software. Qian Xiang: Methodology, Software. Zhengyu Jiang: Writing - review & editing. Yan Zhang: Supervision, Formal analysis, Validation. Jinjun Bian: Conceptualization, Funding acquisition, Supervision. Acknowledgements This work was supported by the National Natural Science Foundation of China (grant numbers 81671939 and 81871579). References [1] M. Singer, et al., The third international consensus definitions for sepsis and septic shock, (Sepsis-3) 315 (8) (2016) 801–810. [2] Jonathan C., et al. Sepsis: a roadmap for future research. 2015. 15(5): p. 581-614. [3] B. Kelly, L.A. O'neill, Metabolic reprogramming in macrophages and dendritic cells in innate immunity, Cell Res. 25 (7) (2015) 771. [4] J.-C. Rodríguez-Prados, et al., Substrate fate in activated macrophages: a comparison between innate, classic, and alternative activation, J. Immunol. 185 (1) (2010) 605–614. [5] E.M. Palsson-McDermott, et al., Pyruvate kinase M2 regulates Hif-1α activity and IL-1β induction and is a critical determinant of the warburg effect in LPS-activated macrophages, Cell Metabol. 21 (1) (2015) 65–80. [6] L.A. O'Neill, R.J. Kishton, J. Rathmell, A guide to immunometabolism for immunologists, Nature Rev. 16 (9) (2016) 553. [7] A.J. Freemerman, et al., Metabolic reprogramming of macrophages glucose transporter 1 (GLUT1)-mediated glucose metabolism drives a proinflammatory phenotype, J. Biolog. Chem. 289 (11) (2014) 7884–7896. [8] L. Perrin-Cocon, et al., Toll-like receptor 4-induced glycolytic burst in human monocyte-derived dendritic cells results from p38-dependent stabilization of HIF1α and increased hexokinase II expression, J. Immunol. 201 (5) (2018) ji1701522. [9] C. Wei, et al., Evaluating the efficacy of Glut inhibitors using a seahorse extracellular flux analyzer, Glucose Transport, Springer, 2018, pp. 69–75. [10] G.L. Wang, B.H. Jiang, E.A. Rue, G.L. Semenza, Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension, Proceedings of the National Academy of Sciences, 1995, pp. 5510–5514, , https:// doi.org/10.1073/pnas.92.12.5510. [11] Ting Wang, Huiying Liu, Guan Lian, Song-Yang Zhang, Xian Wang, Changtao Jiang, HIF1α-induced glycolysis metabolism is essential to the activation of inflammatory macrophages, Mediat. Inflammat. 2017 (2017) 1–10, https://doi.org/10.1155/ 2017/9029327. [12] Suzette R. Riddle, Aftab Ahmad, Shama Ahmad, Samir S. Deeb, Mari Malkki, B. Kelly Schneider, Corrie B. Allen, Carl W. White, Hypoxia induces hexokinase II gene expression in human lung cell line A549, American J. Physiol.-Lung Cell. Mol. Physiol. 278 (2) (2000) L407–L416, https://doi.org/10.1152/ajplung.2000.278.2. L407. [13] P. Jaakkola, et al., Targeting of HIF-α to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation, Science 292 (5516) (2001) 468–472. [14] J. Peng, et al., Dimethyloxalylglycine prevents bone loss in ovariectomized C57BL/ 6J mice through enhanced angiogenesis and osteogenesis, PLoS One 9 (11) (2014) e112744. [15] U. Lee, et al., Intravenous lidocaine for effective pain relief after bimaxillary surgery, Clin. Oral Invest. 21 (9) (2017) 2645–2652. [16] F. Guang, et al., Lidocaine attenuates lipopolysaccharide-induced acute lung injury through inhibiting NF-kappaB activation, Pharmacology 81 (1) (2007) 32–40.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Lidocaine attenuates lipopolysaccharide-induced inflammatory responses and protects against endotoxemia in mice by suppressing HIF1α-induced glycolysis
T
Shengwei Lina, Peipei Jina, Chao Shaob, Wenbin Lua, Qian Xianga, Zhengyu Jianga, Yan Zhanga, ⁎ Jinjun Biana, a b
Faculty of Anesthesiology, Changhai Hospital, Second Military Medical University/Naval Medical University, Shanghai 200433, China Department of Anesthesiology, Urumqi General Hospital of Lanzhou Military Command, Urumqi 830000, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Lidocaine Cytokine Glycolysis HIF1α Endotoxemia
Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infections. Previous studies have indicated that lidocaine, an amide local anesthetic, has anti-inflammatory properties; however, the underlying mechanism remains unclear. In this study, we have shown that lidocaine dose-dependently inhibits lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in macrophages and that lidocaine protects mice from LPS-induced inflammation. Moreover, we have demonstrated that lidocaine reduces the release of TNF-α and IL-6 through the reduction of the expression of GLUT1 and HK2 to further suppress HIF1α-induced aggravation of inflammatory cascades. Lidocaine can inhibit the enhanced glycolysis and glycolytic capacity induced by LPS in the macrophages. As an inhibitor of PHDs (prolyl hydroxylases), Dimethyloxalylglycine (DMOG) can reduce the anti-inflammatory effects of lidocaine. In conclusion, the present study indicates that lidocaine can be used as a potential therapeutic agent for sepsis.
1. Introduction Sepsis is defined as a life-threatening organ dysfunction caused by dysregulated host response to infections and is the leading cause of death in critically ill patients [1]. The in-hospital mortality caused by sepsis still remains high with a percentage probability of 25–30%; this remains a huge problem, globally [2]. One of the most notable features of sepsis is that immune cells are activated to secrete large amounts of inflammatory factors. As the main source of inflammatory factors, the metabolism in macrophages is closely related to its function [3]. Previous investigations have shown that LPS can activate macrophages resulting in their altered metabolism [4]. Recent studies indicate that there might be a potential link between macrophage glycolysis and inflammation [5]. The metabolism in macrophages has been recognized as a critical mechanism for regulating inflammatory responses [6]. Glucose, which is metabolized through anaerobic glycolysis and aerobic oxidative phosphorylation, has been reported to play a crucial role in regulating immune responses in macrophages. LPS-induced glucose transport in macrophages is mainly
mediated by glucose transporter 1 (GLUT1), which controls glucose uptake [7]. In addition, Hexokinase (HK) is a key enzyme for glycolysis as it controls the initial rate-limiting step of this process. LPS strongly enhanced the expression of HK2 [8]. Perturbing glucose uptake and its subsequent metabolic events can alter, both, glucose and lactate homeostasis. As a product of glycolysis, lactate leads to the acidification of the extracellular matrix. The alterations in extracellular matrix can be used as indirect readouts of glucose metabolism. Acidification of the extracellular matrix can also be measured as the extracellular acidification rate (ECAR) [9]. The transcriptional factor hypoxia-inducible factor-1 (HIF-1) is a key player in hypoxic or inflammatory conditions. It consists of an oxygen-sensitive α subunit and a constitutive β subunit [10]. Under the hypoxic or inflammatory environment, the α subunit escapes prolyl hydroxylation and binds to the β subunit. The heterodimer then translocates to the nucleus and activates the transcription of genes involved in cell metabolism, cell growth, and apoptosis. A previous study showed that HIF1α is one of the key molecules upstream of glycolysis; HIF1α could regulate the activity of several glycolytic proteins
⁎ Corresponding author at: Faculty of Anesthesiology, Changhai Hospital, Second Military Medical University/Naval Medical University, 168 Changhai Road, Shanghai 200433, China. E-mail address: [email protected] (J. Bian).
https://doi.org/10.1016/j.intimp.2019.106150 Received 24 August 2019; Received in revised form 20 December 2019; Accepted 21 December 2019 1567-5769/ © 2020 Elsevier B.V. All rights reserved.
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2.3. Cell culture
including GLUT1 and HK2 [11,12]. Over-expression of HIF1α enhances glycolysis by regulating the expression of the glycolytic gene like GLUT1 in the LPS-stimulated macrophages [11]. In pulmonary cells, HK2 expression is regulated by HIF1α -dependent mechanism [12]. HIF1α is hydroxylated at conserved proline residues by PHDs, thereby marking it for rapid proteasomal degradation. Dimethyloxalylglycine (DMOG), a cell penetrant analog, inhibits PHDs and stabilizes HIF1α in cultured cells, thereby influencing the subsequent processes [13]. It has been evidenced in previous studies that DMOG can be safely administered to the experimental animals [14]. Lidocaine, a common local anesthetic drug, has been reported to have anti-inflammatory effects. Intravenous lidocaine can reduce inflammation and relief the pain after bimaxillary surgery [15]. Previous studies have suggested that lidocaine exerts its anti-inflammatory effects by inhibiting the NF- κ B signaling, which modulates the release of the inflammatory factors [16]. Studies have shown that one of the upstream events of HIF-1α signaling is the NF-κ B pathway; it serves as a metabolic switch in macrophages [17]. However, whether lidocaine can exert anti-inflammatory effects by inhibiting HIF1α remains unknown. In the current study, we have investigated the effects of lidocaine on macrophages under inflammation in endotoxemia mice as well as the related mechanism. Our data suggests that lidocaine can inhibit glycolysis in macrophages of the endotoxemia mice, thus, reducing the inflammatory response.
Thioglycollate (TG)-elicited peritoneal macrophages were isolated from C57BL/6 mice as previously described [20]. The peritoneal macrophages (PMs) were incubated with different concentrations of lidocaine (1 to 10 μM) at 37 °C for 6 h. The cell viability was measured by the cell counting kit-8 assay (DOJNDO, SHANGHAI, CHINA). Bone marrow-derived cells from C57BL/6 mice were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin; they were differentiated into the macrophages (bone marrow-derived macrophages [BMDMs]) by applying recombinant murine GM-CSF (25 ng/ml; Miltenyi Biotech) for 7 days. 2.4. ELISA assay PMs were seeded in 24-well plates at 2–5 × 105 cells/well a day before the experiment. Thirty minutes after lidocaine treatment the cells were exposed to 100 ng/ml LPS. The supernatant was collected 6 h after LPS stimulation. The media were collected and centrifuged at 10,000 rpm for 5 min. The TNF-α and IL-6 contents were determined by a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) using the mouse ELISA kits for TNF-α (Roche) and IL-6 (Roche) according to the manufacturer's instructions. 2.5. Real-Time polymerase chain reaction analysis
2. Materials and methods
Total RNA was isolated using TRIZOL reagent and chloroform extraction according to the manufacturer's protocol (Sangon, China). Reverse transcription reactions were performed using the PrimeScript RT Reagent Kit (Takara, Japan). The parameters used for PCR reaction are as follows: 94 °C for 3 min, denaturation (94 °C for 30 s), annealing (60 °C for 45 s), and extension (72 °C for 30 s) for 30 cycles, then a final extension at 72 °C for 10 min. PCR products were analyzed using 1.5% agarose gel electrophoresis. The optical intensity of gel bands was estimated using ImageJ software (NIH, USA). All experiments were performed in triplicates. The sequences of the specific primers used are as follows: HIF-1α, 5′-GAAACGACCACTGCTAAGGCA-3′(forward) and 5′-GGCAGACAGCTTAAGGCTCCT-3′ (reverse)
2.1. Animal experiments C57BL/6 mice (7 weeks old) were obtained from the Animals Experimentation Center of Naval Medical University (Shanghai, China) and were maintained in micro isolator cages; the mice received food and water ad libitum. All procedures were performed in accordance with the requirements of Provisions and General Recommendation of Chinese Experimental Animals Administration Legislation and were approved by the Animal Care and Use Committee of Changhai Hospital. The C57BL/6 mice were divided into three groups (n = 6): control (saline, 200 ul), LPS (8 mg/kg, 200 ul), and LPS with lidocaine (6 mg/ kg, 200 ul). Endotoxemia was induced by injecting LPS intraperitoneally (i.p.). Subsequently, the lidocaine(sigma-Aldrich) or saline were given i.p. 30 min prior to the LPS challenge. After 8 h, the serum was separated for analysis from heart blood by centrifugation. The livers and lungs were harvested and fixed in 10% formalin for hematoxylin and eosin(H&E) staining.
2.6. Western blot analysis Western blot analysis was performed as previously described [21]. Briefly, post treatment PMs were harvested and lysed, and the cleared lysate was separated by SDS-PAGE. After electrophoresis, proteins were transferred to the PVDF membranes. The membranes were first hybridized with primary antibodies and then were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit IgG secondary Ab (Santa Cruz). The human HIF1α monoclonal antibody was from Millipore (USA) and b-actin antibody was from EnoGene (China). The immune blots were visualized by the BioshineChemiQ 4800 mini Imaging System.
2.2. Histopathological test The lungs and livers were harvested 8 h after LPS challenge in each group. After fixation in 10% formaldehyde for > 8 h, the organs were prepared for slicing and staining with hematoxylin and eosin. The sections were examined and scored for using the light microscope by two independent pathologists. The scoring system has been described in previous studies [18,19]. Generally, for lung inflammation: grade 0, normal; grade 1, minimal inflammatory changes; grade 2, mild to moderate inflammatory changes (no obvious damage to the lung architecture); grade 3, moderate inflammatory injury (thickening of the alveolar septa); grade 4, moderate to severe inflammatory injury (formation of nodules or areas of pneumonitis that distorted the normal architecture); 5, severe inflammatory injury with total obliteration of the field. For liver necrosis: 0, normal; 1, individual cell necrosis; 2, up to 30% lobular necrosis; 3, up to 60% lobular necrosis; 4, > 60% lobular necrosis.
2.7. XF bioenergetic analysis Metabolic analyses were performed using Seahorse XFe96 Analyzers (Agilent Technologies, USA), which enable the real-time measurements of extracellular acidification rate (ECAR) by creating a transient microchamber within each well of the specialized microplate (Agilent Technologies). BMDMs were seeded at a density of 3 × 106/well onto the 96-well microplate for 2 h prior to the assay. Glycolysis is the ECAR after the addition of glucose. Glycolytic capacity is the increase in ECAR after the injection of oligomycin following glucose. Data were normalized with the cell number and expressed as mpH/min/103 cells (ECAR). 2
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Fig. 1. The effect of lidocaine on the production of pro-inflammatory cytokines in macrophages. (A) For cytotoxicity assessment of lidocaine, macrophages were treated with different concentrations of lidocaine for 6 h and then the cytotoxicity was measured by CCK-8 assay. (B) ELISA experiments to illustrate the effect of lidocaine on the production of pro-inflammatory cytokines in macrophage. (C) The effect of lidocaine on mRNA expression of pro-inflammatory cytokines was measured by qRT-PCR. Results are expressed as means ± SEM from n = 4 independent experiments. *** P < 0.001, **P < 0.01, * P < 0.05.
treatment. The release of TNF-α and IL-6 was lower in the lidocaine (5 μmol/L) treatment group as compared with lidocaine (1 μmol/L) treatment group (p < 0.05, Fig. 1B). Furthermore, qPCR analysis showed that LPS upregulated TNF-α and IL-6 expression at mRNA level after 1 h treatment. Treatment with lidocaine inhibited LPS-induced TNF-α and IL-6 mRNA expression in a dose-dependent manner. The mRNA expression of TNF-α and IL-6 was lower in the lidocaine (5 μmol/L) treatment group than that in the lidocaine (1 μmol/L) treatment group (p < 0.05, Fig. 1C).
2.8. Statistical analysis All data are expressed as mean ± SEM and were analyzed using Prism 5.0 statistical program (GraphPad Software). Comparisons among three or more experimental groups were performed using ANOVA with Tukey’s post hoc test to determine the significance. A value of p < 0.05 was considered as significant. 3. Results 3.1. Lidocaine inhibits LPS-induced inflammation in macrophages.
3.2. Lidocaine alleviates LPS-induced inflammation in mice.
To investigate the influence of lidocaine in vitro, we treated macrophages with different concentrations of lidocaine. The results suggest that at concentrations of 1–5 μmol/L, lidocaine does not influence the viability of cells significantly suggesting the low cytotoxicity of lidocaine (p > 0.05, Fig. 1A). LPS induced a marked release of TNF-α and IL-6, which was significantly attenuated by lidocaine (5 μmol/L)
We then investigated the anti-inflammatory activity of lidocaine in an animal model. We treated the mice with LPS to induce endotoxemia; this treatment induces an increased secretion of cytokines (Fig. 2A). Administration of lidocaine (6 mg/kg, i.p.) before the LPS treatment reduced the secretion of cytokines (Fig. 2B). The pathological observations demonstrated that lidocaine apparently reduced LPS-induced 3
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Fig. 2. The effect of lidocaine in LPS-induced endotoxemia and H&E staining of liver and lung tissue from endotoxemia mice. Each group of mice was pretreated with 6 mg/kg of lidocaine (i.p.) or saline. Thirty minutes after lidocaine treatment, animals were administered 8 mg/kg of LPS (i.p.). (A) Schematic timeline of in vivo experiment. (B) The results indicate the level of pro-inflammatory cytokines in the serum of mice. (C) the H&E staining of mice liver and lung tissue sections. (D) quantification of the tissue damage. Results are expressed as means ± SEM from n = 6 independent experiments. *** P < 0.001, ** P < 0.01, * P < 0.05, # P < 0.05(LPS compared with LPS + LIDO).
3.3. Lidocaine reduces glycolysis in LPS-treated macrophages.
lung injury in mice, which can be tracked by measuring alveolar wall thickness, edema, bleeding, immune cell infiltration, the damage of blood vessels, and alveolar structure; the necrosis area in the mice liver was also reduced by lidocaine treatment (Fig. 2C). The semiquantitative scoring of the pathological changes also showed that lidocaine reduced the lung injury when compared with control mice (Fig. 2D, p < 0.05). These data suggest that lidocaine is effective in preventing LPS induced injury in LPS-induced endotoxemia models.
An inflammatory response is usually accompanied with modulation of metabolic changes [6]. To investigate the role of lidocaine on metabolism in macrophages, we examined the mRNA expression of glycolytic enzymes. Our data showed that LPS treatment increased the expression of GLUT1 and HK2 in macrophages at the transcriptional level. Furthermore, treatment with lidocaine significantly reduced the mRNA expression level of GLUT1 and HK2 in macrophages (p < 0.05, Fig. 3A). We used GM-CSF (100 ng/ml) to enhance the glycolysis to further delineate the mechanism by which lidocaine inhibits the
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Fig. 3. The effect of lidocaine on glycolysis in macrophages. Peritoneal macrophages (PMs) from WT mice were stimulated with LPS (100 ng/mL) for 1–3 h and the indicated mRNA expression was determined with qPCR. (A) GLUT1 and HK2 mRNA expressions were determined by qPCR. (B) Macrophages were pretreated with GM-CSF for 3 h and subsequently GLUT1 and HK2 mRNA expressions were determined by qPCR. (C) Macrophages were pretreated with GM-CSF for 3 h and production of TNF-α and IL-6 was determined by ELISA. (D) A representative graph output from XFe96 showing ECAR response to glucose (10 mM), oligomycin (10 mM), and 2-DG (100 mM). BMDMs were primed by 25 ng/ml GM-CSF for 24 h. BMDMs were seeded in 96-well plate for 2 h and (E) glycolytic activities (glycolysis and glycolytic capacity) were examined using XF Analyzer. Results are expressed as means ± SEM from n = 4 independent experiments. *** P < 0.001, ** P < 0.01, * P < 0.05.
We further examined the glycolysis and glycolytic capacity in macrophages by using the XFe96 analyzer. After adding glucose, ECAR was significantly increased in the LPS-stimulated macrophages. When oligomycin was added to inhibit oxidative phosphorylation in the cells,
inflammatory responses in LPS-treated macrophages. After pretreatment with GM-CSF, the concentrations of TNF-α and IL-6 as well as the mRNA expression of GLUT1 and HK2 showed no difference between lidocaine treatment group and the LPS group (p < 0.05, Fig. 3B,3C).
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Fig. 4. HIF1α is required for regulating the anti-inflammatory effect of lidocaine. (A) HIF-1α mRNA level in PMs was examined by qRT-PCR (left) and HIF-1α protein level in PMs was examined by Western blot (right). (B) PMs were stimulated with LPS (100 ng/mL) for 6 h followed by DMOG treatment for 3 h. Supernatants were collected and assayed for TNF-α and IL-6 production by ELISA. (C) PMs were stimulated with LPS (100 ng/mL) for 1–3 h followed by DMOG treatment for 3 h. GLUT1 and HK2 mRNA expression levels were determined by qPCR. Results are expressed as means ± SEM from n = 4 independent experiments. ** P < 0.01,* P < 0.05.
lidocaine significantly attenuated the upregulation of HIF1α induced by LPS (p < 0.05, Fig. 4A). However, after pretreatment with DMOG (0.5 mmol/L), the concentrations of TNF-α and IL-6 were found to be similar between the lidocaine and LPS groups (p＞0.05, Fig. 4B). Since the expression level of GLUT1 and HK2 positively correlates with HIF1α expression level in macrophages, we subsequently examined the effect of DMOG on the expression of GLUT1 and HK2 at mRNA level using qRT-PCR. DMOG dramatically inhibited the influence of lidocaine. Meanwhile, the expression of GLUT1 and HK2 showed no difference at the mRNA level between the two groups (Fig. 4C). These results indicate that lidocaine can inhibit the expression of HIF1α and glycolytic pathway in LPS-treated macrophages, thus, resulting in anti-inflammatory effects.
the ECAR level was further increased in LPS-stimulated macrophages suggesting a higher level of glycolytic capacity (Fig. 3D). Lidocaine pretreated macrophages responded less to the stimulation as compared with the response of LPS-stimulated macrophages (Fig. 3D). Glycolytic capacity and glycolysis rate were significantly higher in LPS-stimulated macrophages as compared to that in lidocaine pre-treated macrophages (Fig. 3E). These results suggest that lidocaine pretreatment could inhibit the enhanced glycolysis induced by LPS in macrophages. 3.4. Lidocaine inhibits the expression of HIF-1α in LPS-treated macrophages. A previous study has shown that HIF1α is one of the key molecules upstream of glycolysis [22]. To investigate whether lidocaine inhibits glycolysis by down-regulation of HIF1α, we performed a series of experiments to examine the effect of lidocaine in the LPS-treated macrophages. Our data demonstrated that the LPS treatment increased the expression of HIF1α in macrophages. Furthermore, the treatment of
4. Discussion This study demonstrates beneficial inhibitory effects of lidocaine on the expression and secretion of HIF1α and HIF1α-enhanced production 6
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inflammatory cytokine production from coronary artery disease monocytes and macrophages [32]. The use of drugs that are specifically activated in macrophages could be considered for future therapies [33]. Further work will be needed to explore the utility of targeting specific metabolic events in macrophages for therapeutic gain.
of pro-inflammatory factors such as TNF-α and IL-6. In vivo investigation indicated that lidocaine protects mice from LPS-induced inflammation. The present data provide evidence that lidocaine exerts anti-inflammatory effects to inhibit the expression of GLUT1 and HK2 through suppressing HIF1α activity. Previous studies have indicated that lidocaine can reduce mortality and protect against renal and hepatic dysfunction in murine septic peritonitis [23]. However, more importantly, a double-blinded, prospective, randomized clinical trial showed that lidocaine reduces neutrophil recruitment by abolishing chemokine-induced arrest and transendothelial migration in septic patients. This indicates that lidocaine might play a possible therapeutic role in decreasing the inappropriate activation, positioning, and recruitment of leukocytes during sepsis [24]. The above-mentioned studies provide strong evidence for lidocaine’s ability to inhibit the inflammation caused by sepsis. Therefore, not surprisingly, our data demonstrate that lidocaine is protective against inflammation as well as the inflammatory infiltration in the lung and liver. It was shown that lidocaine protects septic rats via inhibition of HMGB1 expression and activation of NF- κ B [25]. Lidocaine was demonstrated to inhibit the release of HMGB1 in LPS-stimulated macrophages [26]. Lidocaine was also reported to attenuate LPS-induced inflammatory responses in microglia, which may be mediated by blockade of p38 MAPK and NF- κ B signaling pathways [27]. Ahmed Tawakol et al. showed that macrophage glycolysis and proinflammatory activation mainly depend on HIF due to their crucial role in glucose metabolism [28]. As a result, hypoxia potentiates inflammation and glycolysis mainly via these pathways. Therefore, we investigated whether lidocaine affects the inflammation through the abovementioned pathways. We found that lidocaine can significantly inhibit the mRNA expression of GLUT1 and HK2 in LPS-stimulated macrophages. Granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances anaerobic glycolysis while exerting a mild pro-inflammatory effect [29]. After pretreatment with GM-CSF, lidocaine partially reduced the release of TNF-α and IL-6 caused by LPS treatment. The upregulation effect of LPS on GLUT1 and HK2 mRNA could also be reversed by lidocaine in macrophages. Based on the previous studies, ECAR is an important and irreplaceable indicator for assessing the level of glycolysis. We found that ECAR level was further increased in LPSstimulated macrophages suggesting a higher level of glycolysis. Glycolytic capacity and glycolysis rate were significantly higher in LPSstimulated macrophages as compared to those in lidocaine pre-treated macrophages. In general, HIF1 is considered to promote and enhance inflammatory responses to maintain the host defense not only under hypoxia but also in normoxia. Previous studies have indicated that HIF1α expression in macrophages affects their intrinsic inflammatory profile and promotes the development of atherosclerosis [30]. Wang et al. showed that HIF1α promotes peritoneal macrophage glycolysis metabolism with high expression of some glycolytic genes including GLUT1 and PKM2 [31]. Our data shows that lidocaine suppresses HIF1α expression and inflammation in macrophages, while the antiinflammatory effect could be reversed by DMOG. Our study has several drawbacks. There is a lack of relevant experiments using gene knockout mice and clinical relevance of the LPS model of sepsis. Taken together, our results show that lidocaine can significantly inhibit the secretion of LPS-induced inflammatory cytokines in macrophages by inhibiting HIF1α-mediated glycolytic pathway, as well as, exert anti-inflammatory effects in endotoxemia mice model. It may provide a new method for treating inflammation and sepsis. Since a very significant metabolic reprogramming occurs in macrophages under sepsis, ultimately it might be possible to target metabolic changes in macrophages therapeutically. Approaches might include inhibition of PKM2, which would prevent inflammatory macrophage activation while supporting anti-inflammatory pathways [5]. Interfering PKM2 may be particularly interesting, since it has been shown to block
5. Authors’ contributions Shengwei Lin and Peipei Jin contributed equally to the work. Jinjun Bian designed the experiments and provided financial support. Shengwei Lin, Chao Shao, and Wenbin Lu performed Seahorse XF Glycolysis Stress Test. Yan Zhang, Qian Xiang, and Peipei Jin contributed to the statistical analysis. Shengwei Lin and Zhengyu Jiang wrote the paper. CRediT authorship contribution statement Shengwei Lin: Resources, Data curation, Methodology, Writing original draft, Visualization. Peipei Jin: Formal analysis, Methodology, Software. Chao Shao: Methodology, Software. Wenbin Lu: Methodology, Software. Qian Xiang: Methodology, Software. Zhengyu Jiang: Writing - review & editing. Yan Zhang: Supervision, Formal analysis, Validation. Jinjun Bian: Conceptualization, Funding acquisition, Supervision. Acknowledgements This work was supported by the National Natural Science Foundation of China (grant numbers 81671939 and 81871579). References [1] M. Singer, et al., The third international consensus definitions for sepsis and septic shock, (Sepsis-3) 315 (8) (2016) 801–810. [2] Jonathan C., et al. Sepsis: a roadmap for future research. 2015. 15(5): p. 581-614. [3] B. Kelly, L.A. O'neill, Metabolic reprogramming in macrophages and dendritic cells in innate immunity, Cell Res. 25 (7) (2015) 771. [4] J.-C. Rodríguez-Prados, et al., Substrate fate in activated macrophages: a comparison between innate, classic, and alternative activation, J. Immunol. 185 (1) (2010) 605–614. [5] E.M. Palsson-McDermott, et al., Pyruvate kinase M2 regulates Hif-1α activity and IL-1β induction and is a critical determinant of the warburg effect in LPS-activated macrophages, Cell Metabol. 21 (1) (2015) 65–80. [6] L.A. O'Neill, R.J. Kishton, J. Rathmell, A guide to immunometabolism for immunologists, Nature Rev. 16 (9) (2016) 553. [7] A.J. Freemerman, et al., Metabolic reprogramming of macrophages glucose transporter 1 (GLUT1)-mediated glucose metabolism drives a proinflammatory phenotype, J. Biolog. Chem. 289 (11) (2014) 7884–7896. [8] L. Perrin-Cocon, et al., Toll-like receptor 4-induced glycolytic burst in human monocyte-derived dendritic cells results from p38-dependent stabilization of HIF1α and increased hexokinase II expression, J. Immunol. 201 (5) (2018) ji1701522. [9] C. Wei, et al., Evaluating the efficacy of Glut inhibitors using a seahorse extracellular flux analyzer, Glucose Transport, Springer, 2018, pp. 69–75. [10] G.L. Wang, B.H. Jiang, E.A. Rue, G.L. Semenza, Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension, Proceedings of the National Academy of Sciences, 1995, pp. 5510–5514, , https:// doi.org/10.1073/pnas.92.12.5510. [11] Ting Wang, Huiying Liu, Guan Lian, Song-Yang Zhang, Xian Wang, Changtao Jiang, HIF1α-induced glycolysis metabolism is essential to the activation of inflammatory macrophages, Mediat. Inflammat. 2017 (2017) 1–10, https://doi.org/10.1155/ 2017/9029327. [12] Suzette R. Riddle, Aftab Ahmad, Shama Ahmad, Samir S. Deeb, Mari Malkki, B. Kelly Schneider, Corrie B. Allen, Carl W. White, Hypoxia induces hexokinase II gene expression in human lung cell line A549, American J. Physiol.-Lung Cell. Mol. Physiol. 278 (2) (2000) L407–L416, https://doi.org/10.1152/ajplung.2000.278.2. L407. [13] P. Jaakkola, et al., Targeting of HIF-α to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation, Science 292 (5516) (2001) 468–472. [14] J. Peng, et al., Dimethyloxalylglycine prevents bone loss in ovariectomized C57BL/ 6J mice through enhanced angiogenesis and osteogenesis, PLoS One 9 (11) (2014) e112744. [15] U. Lee, et al., Intravenous lidocaine for effective pain relief after bimaxillary surgery, Clin. Oral Invest. 21 (9) (2017) 2645–2652. [16] F. Guang, et al., Lidocaine attenuates lipopolysaccharide-induced acute lung injury through inhibiting NF-kappaB activation, Pharmacology 81 (1) (2007) 32–40.
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