Glycine Attenuates Lipopolysaccharide-Induced Acute Lung Injury by Regulating NLRP3 Inflammasome and NRF2 Signaling

Glycine supplementation has been reported to alleviate lipopolysaccharide (LPS)-induced lung injury in mice. However, the underlying mechanisms responsible for this beneficial effect remain unknown. In the present study, male C57BL/6 mice were treated with aerosolized glycine (1000 mg in 5 mL of 0.9% saline) or vehicle (0.9% saline) once daily for 7 continuous days, and then were exposed to aerosolized LPS (5 mg in 5 mL of 0.9% saline) for 30 min to induce lung injury. Sera and lung tissues were collected 24 h post LPS challenge. Results showed that glycine pretreatment attenuated LPS-induced decreases of mucin at both protein and mRNA levels, reduced LPS-triggered upregulation of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interferons, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukins. Further study showed that glycine-reduced LPS challenge resulted in the upregulation of nuclear factor κB (NF-κB), nucleotide binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome. In addition, LPS exposure led to the downregulation of NRF2 and downstream targets, which were significantly improved by glycine administration in the lung tissues. Our findings indicated that glycine pretreatment prevented LPS-induced lung injury by regulating both NLRP3 inflammasome and NRF2 signaling.


Introduction
Acute lung injury is a common and severe pulmonary complication of critical illness caused by multiple factors, such as pneumonia, sepsis, shock, and respiratory bacteria or viruses infection [1]. Under physiological conditions, an intracellular homeostasis and a normal function of the lungs are maintained by the host defense systems, including mucus layer and immune response. Mucus layer, which is mainly consisted of secreted mucins, including MUC5AC and MUC5B, is the first defense line that prevents the contact of host cells with lumen contents, such as chemicals, toxins, and pathogens [2]. The dysfunction of mucus is associated with bacteria invasion, which in turn activates immune response, infiltration of macrophage, and the secretion of pro-inflammatory cytokines, such as

Alcian Blue Staining
Lung tissues fixed in 4% formaldehyde were dehydrated, embedded in paraffin, sectioned, and stained with alcian blue solution (blue) and nuclear fast red solution (red) according to manufacturer's instructions (Burlingame, USA). Sections from each mouse were visualized by a blinded observer and pictured using a light microscope equipped with a computer-assisted morphometric system.

Serum Inflammatory Cytokine Analysis
Serum inflammatory cytokines were detected using a commercial mouse inflammation panel kit (BioLegend, San Diego, USA) and the CytExpert Flow Cytometer (Beckman, USA) according to the instructions of manufacturer. Data were analyzed by the LEGENDplex™ software (BioLegend, San Diego, USA).

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from the lung tissues using a TRIzol kit according to the instructions. The cDNA was obtained by reverse transcription of total RNA, which was carried out by a FastQuant RT Kit (with gDNase). RNA concentration and value of OD260/280 were measured using the Nanodrop P330 (Implen, Germany). The integrity of total RNA with an OD260/280 value of 1.8-2.0 was assessed by 1% agarose gel electrophoresis before performing qPCR experiments. qRT-PCR was performed by the SYBR green mix and specific primers for target genes with the ABI-Prism 7500 Sequence Detection System (Applied Biosystems) according to the instructions of manufacturer. The primer sequences used in the present study are listed in Table 1. Gapdh was used as an internal control. The 2 −∆∆CT method was used to determine the fold changes of mRNA levels with the Microsoft Excel software.

Western Blotting
Lung tissues were homogenized in liquid nitrogen, dissolved and vortexed in cold radio-immunoprecipitation assay (RIPA) buffer (10 mm Tris-HCl, pH 7.4; 150 mm NaCl; 10 mm EDTA; 1% NP-40; 0.1% SDS) for protein extraction. Protein abundance was determined by using the Western blotting technique, as described previously [25]. The protein bands were developed by a chemiluminescence kit (Amersham Biosciences) using the Image Quant LAS 4000 mini system (GE Healthcare Bio-sciences). Protein band density was quantified by the ImageJ software (GE Healthcare Life Sciences).

Statistical Analysis
All data are presented as means ± SEM. Results were analyzed by 1-way ANOVA, using the SAS software, version 9.1 (SAS Institute Inc., Cary, North Carolina, USA.). Differences between means were determined by using the Student-Newman-Keuls multiple-comparison test. p < 0.05 was taken to indicate statistical significance.

Glycine Supplementation Restored Mucin Layer in LPS-Treated Mice
Compared with the controls, LPS exposure led to reduced airway mucin at protein level as shown by alcian blue staining, which was remarkably restored by glycine ( Figure 1A). qRT-PCR analysis showed that mice in the LPS treatment group had significant downregulation of Muc5ac and Muc5b at the mRNA level in the lung tissues, as compared with the controls (Figure 1B,C). However, glycine supplementation significantly increased (p < 0.05) the mRNA levels of Muc5ac and Muc5b, as compared with those of mice in the LPS treatment group ( Figure 1B,C).

Western Blotting
Lung tissues were homogenized in liquid nitrogen, dissolved and vortexed in cold radioimmunoprecipitation assay (RIPA) buffer (10 mm Tris-HCl, pH 7.4; 150 mm NaCl; 10 mm EDTA; 1% NP-40; 0.1% SDS) for protein extraction. Protein abundance was determined by using the Western blotting technique, as described previously [25]. The protein bands were developed by a chemiluminescence kit (Amersham Biosciences) using the Image Quant LAS 4000 mini system (GE Healthcare Bio-sciences). Protein band density was quantified by the ImageJ software (GE Healthcare Life Sciences).

Statistical Analysis
All data are presented as means ± SEM. Results were analyzed by 1-way ANOVA, using the SAS software, version 9.1 (SAS Institute Inc., Cary, North Carolina, USA.). Differences between means were determined by using the Student-Newman-Keuls multiple-comparison test. p < 0.05 was taken to indicate statistical significance.

Glycine Supplementation Restored Mucin Layer in LPS-Treated Mice
Compared with the controls, LPS exposure led to reduced airway mucin at protein level as shown by alcian blue staining, which was remarkably restored by glycine ( Figure 1A). qRT-PCR analysis showed that mice in the LPS treatment group had significant downregulation of Muc5ac and Muc5b at the mRNA level in the lung tissues, as compared with the controls (Figure 1B and 1C). However, glycine supplementation significantly increased (p < 0.05) the mRNA levels of Muc5ac and Muc5b, as compared with those of mice in the LPS treatment group ( Figure 1B and 1C).  Red arrows indicate goblet cells. White and black scale bars mean 50 and 20 µm respectively. The mRNA level in the CON group was set as 1.00 to calibrate the relative levels. Gapdh was used as a reference gene. Values are means ± SEM, n = 7. Means for an indicated parameter without a common letter differ, p < 0.05.

Glycine Inhibited Activation of NF-κB in LPS-Stimulated Lung
Western blotting results showed that LPS treatment led to increased (p < 0.05) phosphorylation of P65, without affecting that of TLR4 and MYD88 at protein level ( Figure 3). LPS-induced activation of P65 was significantly suppressed by glycine pretreatment. Of note, protein levels of TLR4 and MYD88 in the lung tissues of mice treated with aerosolized LPS plus glycine were lower (p < 0.05) than that of mice in the control or LPS treatment group ( Figure 3A, 3B, and 3C).

Glycine Inhibited Activation of NF-κB in LPS-Stimulated Lung
Western blotting results showed that LPS treatment led to increased (p < 0.05) phosphorylation of P65, without affecting that of TLR4 and MYD88 at protein level ( Figure 3). LPS-induced activation of P65 was significantly suppressed by glycine pretreatment. Of note, protein levels of TLR4 and MYD88 in the lung tissues of mice treated with aerosolized LPS plus glycine were lower (p < 0.05) than that of mice in the control or LPS treatment group ( Figure 3A-C).

Glycine Inhibited Activation of NF-κB in LPS-Stimulated Lung
Western blotting results showed that LPS treatment led to increased (p < 0.05) phosphorylation of P65, without affecting that of TLR4 and MYD88 at protein level ( Figure 3). LPS-induced activation of P65 was significantly suppressed by glycine pretreatment. Of note, protein levels of TLR4 and MYD88 in the lung tissues of mice treated with aerosolized LPS plus glycine were lower (p < 0.05) than that of mice in the control or LPS treatment group ( Figure 3A, 3B, and 3C).  The quantification of band in the CON group was set as 1.00 to calibrate the relative levels. Actin was used as a loading control. Values are means ± SEM; n = 7. Means without a common letter differ, p < 0.05.

Glycine Pretreatment Blocked Activation of NLRP3 Inflammasome in Lung Tissues of LPS-Challenged Mice
Western blotting results showed that LPS treatment increased (p < 0.05) the protein levels of NLRP3, ASC, and cleaved caspase1-a downstream target of NLRP3-and decreased the protein level of procaspase1 in comparison to the controls, indicating the activation of the NLRP3 inflammasome ( Figure 4). This effect of LPS was remarkably prevented (p < 0.05) by glycine pretreatment.

Glycine Pretreatment Blocked Activation of NLRP3 Inflammasome in Lung Tissues of LPS-Challenged
Mice.
Western blotting results showed that LPS treatment increased (p < 0.05) the protein levels of NLRP3, ASC, and cleaved caspase1-a downstream target of NLRP3-and decreased the protein level of procaspase1 in comparison to the controls, indicating the activation of the NLRP3 inflammasome ( Figure 4). This effect of LPS was remarkably prevented (p < 0.05) by glycine pretreatment.

Glycine Administration Enhanced NRF2 Signaling in Lung Tissues of LPS-Challenged Mice.
Compared with the controls, LPS treatment resulted in decreased (p < 0.05) protein abundance of NRF2, and down-stream targets, such as HO1, NQO1, and GSTA4, which were abolished by glycine ( Figure 5A-E). qRT-PCR results showed that Nrf2 gene expression was down-regulated in response to LPS exposure, along with downstream targets, such as Ho1, Nqo1, and Gsta4. However, these effects of LPS were attenuated (p < 0.05) by glycine supplementation ( Figure 5F-I).

Glycine Administration Enhanced NRF2 Signaling in Lung Tissues of LPS-Challenged Mice
Compared with the controls, LPS treatment resulted in decreased (p < 0.05) protein abundance of NRF2, and down-stream targets, such as HO1, NQO1, and GSTA4, which were abolished by glycine ( Figure 5A-E). qRT-PCR results showed that Nrf2 gene expression was down-regulated in response to LPS exposure, along with downstream targets, such as Ho1, Nqo1, and Gsta4. However, these effects of LPS were attenuated (p < 0.05) by glycine supplementation (Figure 5F-I).

Glycine Increased Protein Abundance of HSP70 and HSP40 in Lung Tissues of LPS-Challenged Mice
Western blotting results indicated that LPS exposure led to the decreased protein abundance of HSP40 and HSP70, which were reversed (p < 0.05) by glycine administration ( Figure 6A, 6B, and 6C). qRT-PCR results showed that mRNA levels of Hsp40 and Hsp70 were reduced by LPS treatment, as compared with that of controls, which were significantly prevented by glycine supplementation (p < 0.05) ( Figure 6D and 6E). , and Gsta4. The protein abundance or mRNA level in the CON group was set as 1.00 to calibrate the relative levels. GAPDH was used as an internal control. Values are means ± SEM; n = 7. Means without a common letter differ, p < 0.05.

Glycine Increased Protein Abundance of HSP70 and HSP40 in Lung Tissues of LPS-Challenged Mice
Western blotting results indicated that LPS exposure led to the decreased protein abundance of HSP40 and HSP70, which were reversed (p < 0.05) by glycine administration (Figure 6A-C). qRT-PCR results showed that mRNA levels of Hsp40 and Hsp70 were reduced by LPS treatment, as compared with that of controls, which were significantly prevented by glycine supplementation (p < 0.05) ( Figure 6D,E).

Glycine Increased Protein Abundance of HSP70 and HSP40 in Lung Tissues of LPS-Challenged Mice
Western blotting results indicated that LPS exposure led to the decreased protein abundance of HSP40 and HSP70, which were reversed (p < 0.05) by glycine administration ( Figure 6A, 6B, and 6C). qRT-PCR results showed that mRNA levels of Hsp40 and Hsp70 were reduced by LPS treatment, as compared with that of controls, which were significantly prevented by glycine supplementation (p < 0.05) ( Figure 6D and 6E).

Autophagy was not Involved in the Beneficial Effect of Glycine on LPS-Induced Acute Lung Injury
As shown by the Western blotting results, the inhalation of aerosolized LPS had no effect on abundance of protein involved in autophagy, such as P62, ATG5, LC3, and Beclin1 (Figure 7). Furthermore, pre-treatment with glycine did not affect autophagic proteins expression. Figure 6. Glycine attenuated LPS-induced downregulation of HSP70 and HSP40 in lung tissues of LPS-challenged mice. (A) Representative protein bands of HSP40, HSP70, and GAPDH; (B, C) statistical analysis of protein abundance; (D, E) relative mRNA levels of Hsp40 and Hsp70. The protein abundance or mRNA level in the CON group was set as 1.00 to calibrate the relative levels. GAPDH was used as an internal control. Values are means ± SEM; n = 7. Means for an indicated parameter without a common letter differ significantly, p < 0.05.

Autophagy was not Involved in the Beneficial Effect of Glycine on LPS-Induced Acute Lung Injury.
As shown by the Western blotting results, the inhalation of aerosolized LPS had no effect on abundance of protein involved in autophagy, such as P62, ATG5, LC3, and Beclin1 (Figure 7). Furthermore, pre-treatment with glycine did not affect autophagic proteins expression.

Discussion
In the present study, we found that glycine prevented aerosolized LPS exposure-induced reduction of mucin and upregulation of pro-inflammatory cytokines. This beneficial effect is associated with the inhibition of the NF-κB and NLRP3 inflammasome signaling pathway, as well as the restoration of NRF2 signaling.
The exposure of airway epithelium to respiratory pathogens, allergens, and toxins is associated with epithelial dysfunction and the development of lung diseases. Mucin produced by goblet cells, airway epithelial cells, and mucous cells in submucosal glands is one of the main components of a defense line that covers the air way cells, therefore preventing the contact of lumen contents with epitheliums [26]. The deregulation of the mucus barrier has been reported to be associated with lung injury and the pathogenesis of multiple lung diseases [27]. In the present study, mice pretreated with glycine were subjected to aerosolized LPS, which has been previously reported to induce lung injury [23]. We found that glycine administration prevented the LPS-induced downregulation of mucin at both protein and mRNA levels, which was consistent with previous studies [28,29]. This reduction of mucin following LPS treatment facilitate contact of risk factors with epithelial cells, thus contributing to the dysfunction of the respiratory barrier and inflammatory responses [30][31][32]. As expected, mice

Discussion
In the present study, we found that glycine prevented aerosolized LPS exposure-induced reduction of mucin and upregulation of pro-inflammatory cytokines. This beneficial effect is associated with the inhibition of the NF-κB and NLRP3 inflammasome signaling pathway, as well as the restoration of NRF2 signaling.
The exposure of airway epithelium to respiratory pathogens, allergens, and toxins is associated with epithelial dysfunction and the development of lung diseases. Mucin produced by goblet cells, airway epithelial cells, and mucous cells in submucosal glands is one of the main components of a defense line that covers the air way cells, therefore preventing the contact of lumen contents with epitheliums [26]. The deregulation of the mucus barrier has been reported to be associated with lung injury and the pathogenesis of multiple lung diseases [27]. In the present study, mice pretreated with glycine were subjected to aerosolized LPS, which has been previously reported to induce lung injury [23]. We found that glycine administration prevented the LPS-induced downregulation of mucin at both protein and mRNA levels, which was consistent with previous studies [28,29]. This reduction of mucin following LPS treatment facilitate contact of risk factors with epithelial cells, thus contributing to the dysfunction of the respiratory barrier and inflammatory responses [30][31][32]. As expected, mice treated with LPS had elevated protein levels of proinflammatory cytokines such as TNF-α, IFN-β, IFN-γ, GM-CSF, and interleukins, such as IL-1β, IL-17A, IL-23, IL-27, IL-12p70, and IL-6 in serum, indicating the occurrence of lung injury in response to aerosolized LPS challenge. Intriguingly, we found these alterations following LPS challenge were remarkably attenuated by glycine administration, indicating the protective effect of glycine on lung injury. NF-κB is a crucial transcriptional factor whose function is associated with the biosynthesis of pro-inflammatory cytokine [33]. To investigate the potential involvement of NF-κB signaling in the beneficial effects as observed, Western blotting was conducted and the result showed that glycine pre-treatment abrogated LPS-induced activation of NF-κB, indicating a regulatory effect of glycine. It is well-known that TLR4/MYD88 is responsible for the upregulation of NF-κB signaling in response to bacterial infection in various conditions [33]. However, this was not the case in our study, because the protein abundance of TLR4 and MYD88 was not affected by LPS. In vivo and in vitro studies have shown that the phosphorylation of p38MAPK is another protein responsible for NF-κB-dependent inflammatory responses and cytokines secretion [34][35][36]. Further study is needed to answer whether p38MAPK is implicated in and contributes to this protective effect.
Recent studies have shown that the activation of the NLRP3 inflammasome is a critical mediator responsible for the maturation of IL-1β for the development of acute lung injury [8,9]. This effect was validated in our study as shown by the enhanced protein levels of NLRP3, ASC, cleaved caspase1, and increased IL-1β. Importantly, these effects were attenuated by glycine administration, indicating a regulatory effect of glycine in LPS-challenged mice. The accumulation of ROS has been reported to activate the NLRP3 inflammasome and downstream targets, including caspase1 and IL-1β, one of the main cytokines associated with lung injury in both clinical patients and experimental animals [12]. Glycine has been reported to alleviate ROS-induced cellular damage by promoting the synthesis of glutathione (GSH), an endogenous antioxidant, in intestinal porcine epithelial cells and other tissues [21,37]. Glycine supplementation might reduce the ROS level due to increased GSH product, therefore contributing to reduced NLRP3 inflammasome and decreases the protein level of IL-1β in the lung tissues.
NRF2 is a transcriptional factor associated with survival by upregulating downstream targets, such as HO1, NQO1, and GSTA4 in response to various stresses [13]. Deficiency of NRF2 signaling leads to severe lung injury of mice in response to LPS exposure [38]. In contrast, enhanced protein levels of NRF2 have been reported to alleviate LPS-induced lung injury in mice [39,40]. In consistent with previous report [41], we found that glycine pre-administration reversed LPS-induced depletion of NRF2 and led to the increased protein level of genes implicated in cellular survival [42]. In a recent study, the authors showed that NRF2 is a repressor of NF-κB [43]. Our results indicated that the glycine-induced downregulation of NF-κB was mediated, at least partially, via NRF2 signaling. Heat shock proteins, including HSP40 and HSP70, are negative regulators of ROS generation [44]. HSP70 knockout mice displayed the excessive activation of NF-κB and increased inflammatory cytokine in the lung tissues [45]. In our study, glycine administration prevented the LPS-induced downregulation of HSP70 and HSP40 to both protein and mRNA level, which might contribute to reduced lung injury through repressing inflammatory responses and inhibiting apoptosis of epithelial cells in the lung tissues, as previously reported [46].
Glycine is traditionally regarded as a nutritionally non-essential amino acid due to its de novo synthesis from serine, choline, threonine, and Glyoxylate [47,48]. Critical roles of glycine on tissue repair, metabolic regulation, and anti-oxidative capacity are recognized in recent years [23,48]. In the present study, we observed a protective effect of glycine on acute lung injury in mice by regulating NLRP3 inflammasome signaling pathway, and survival proteins, including NRF2, HSP40, and HSP70. This is the first study that linked glycine and NLRP3 in lung disorder. Glycine receptor (GlyR), a glycine-gated chloride channel, has been identified in postsynaptic membranes, hepatic and alveolar macrophages, neutrophils, and lymphocytes [49]. Importantly, glycine prevents LPS-induced endotoxemia by activating the GlyR in Kupffer cells [49] or hepatic parenchymal cells [50]. However, both LPS and glycine had no effect on the protein abundance of GlyR, thus excluding an involvement of GlyR and its contribution to the anti-inflammatory effect as observed in the present study.
The dysfunction of endoplasmic reticulum and activation of autophagy have been reported to implicate in lung injury in both humans and animals [51]. However, we found that LPS treatment did not affect the protein levels of autophagy markers, including P62, ATG5, LC3, and Beclin1, as well as protein levels of activating transcription factor 6 (ATF6), inositol-requiring enzyme 1α (IRE1α), and PKR-like ER kinase (PERK)-well-known sensors for endoplasmic reticulum stress signaling. Therefore, both ER stress and autophagy are not related to the protective effect of glycine. More studies are required to uncover the underlying mechanisms in the future, which might advance our understanding of the benefits of glycine in the context of lung injury.

Conclusions
In conclusion, we found that glycine administration ameliorated aerosolized LPS-induced acute lung injury, as shown by increased mucin, decreased protein level of pro-inflammatory cytokines. This beneficial effect of glycine was associated with the modulation of the NLRP3 inflammasome and NRF2 signaling. Our findings provide a novel nutritional strategy for acute lung injury by providing glycine to animals. It should be noted that well-defined studies on efficacy and safety are required prior to its application in humans.

Conflicts of Interest:
The authors declare no conflict of interest.