key: cord-1001693-rb5z1ha6 authors: An, Lulu; Zhao, Jianxiong; Sun, Xiao; Zhou, Yingying; Zhao, Zhongxi title: S-allylmercaptocysteine inhibits mucin overexpression and inflammation via MAPKs and PI3K-Akt signaling pathways in acute respiratory distress syndrome date: 2020-06-20 journal: Pharmacol Res DOI: 10.1016/j.phrs.2020.105032 sha: 9f400f59178e6d53437baf25107e4481c68bdaf8 doc_id: 1001693 cord_uid: rb5z1ha6 Cytokine storm is an important cause of acute respiratory distress syndrome and multiple organ failure. Excessive secretion and accumulation of mucins on the surface of airway cause airway obstruction and exacerbate lung infections. MUC5AC and MUC5B are the main secreted mucins and overexpressed in various inflammatory responses. S-allylmercaptocysteine, a water-soluble organic sulfur compound extracted from garlic, has anti-inflammatory and anti-oxidative effects for various pulmonary diseases. The aim of this work was to investigate the therapeutic effects of SAMC on mucin overproduction and inflammation in 16HBE cells and LPS-induced ARDS mice. Results show that SAMC treatment ameliorated inflammatory cell infiltration and lung histopathological changes in the LPS-induced ARDS mice. SAMC also inhibited the expressions of MUC5AC and MUC5B, decreased the production of pro-inflammatory markers (IL-6, TNF-α, CD86 and IL-12) and increased the production of anti-inflammatory markers (IL-10, CD206 and TGF-β). These results confirm that SAMC had potential beneficial effects on suppressed hyperinflammation and mucin overexpression. Furthermore, SAMC exerted the therapeutic effects through the inhibition of phosphorylation of MAPKs and PI3K-Akt signaling pathways in the 16HBE cells and mice. Overall, our results demonstrate the effects of SAMC on the LPS-induced mucin overproduction and inflammation both in the 16HBE cells and mice. Acute respiratory distress syndrome (ARDS) is a clinical manifestation of the lung injury caused by infectious, non-infectious, and other damaging events, which is characterized by hypoxic respiratory failure and often accompanied by severe pulmonary inflammation [1, 2] . Lung inflammation imbalance can induce cytokine storm, leading to worsening of ARDS. When severe acute respiratory syndrome (SARS) caused by coronavirus (SARS-CoV) swept China in 2003, the cytokine storm was the direct cause of deaths for many patients [3] . In 2009, hyperinflammation was observed in patients with ARDS caused by H1N1 virus infection [4] . In coronavirus disease 2019 , the cytokine storm syndrome was also an important cause of respiratory failure in many patients, especially young patients [5, 6] . There are currently no effective drugs to cure the cytokine storm in patients with ARDS. Therefore, there is an urgent need to explore alternative and complementary medicines. In normal physiology, mucins play a critical role in defending the respiratory tract against pathogens and toxins [7] . However, the excessive production of bronchial J o u r n a l P r e -p r o o f mucins blocks the airway, restricts airflow, and accelerates the decline of lung functions [8] . In addition, it also endangers mucociliary functions and decreases mucus clearance, leading to aggravated lung infections [9] . MUC5AC and MUC5B, two major secreted mucins, are secreted by goblet/mucous cells including many specialized epithelial cells, which are closely related to the viscoelasticity of sputum [10, 11] . Mei-Juan Liu et al. demonstrate that lipopolysaccharides (LPS) could induce the high expression of MUC5AC and epidermal growth factor receptor (EGFR) in 16HBE cells [12] . After the intratracheal administration, LPS activates the EGF receptor and subsequently triggers downstream signaling cascades such as mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase-Akt (PI3K-Akt) signaling pathways [13] . MAPK members are the critical signal transduction molecules that involve in inflammatory responses and regulate the synthesis of MUC5AC, including p38 MAP kinases, c-jun N-terminal kinases (JNK), extracellular signal-related kinases 1/2 (ERK1/2) [14] . Besides, Akt-mTOR-STAT3, as a downstream of PI3K, also regulates inflammation and the MUC5AC expression [15] . Therefore, drugs regulating MAPKs and PI3K-Akt signaling pathways may have the therapeutic potential for mucus hypersecretion and pulmonary cytokine storm. Aged garlic extract (AGE) is an odorless product created through prolonged extraction of fresh garlic, which has a strong anti-oxidative, free radical scavenging, anti-inflammatory and other activities [16] [17] [18] . S-allylmercaptocysteine (SAMC), as the major water-soluble garlic component of AGE, has shown the prospect in the treatment of mucus hypersecretion and anti-inflammation in an in-vitro COPD model [19] . However, the therapeutic effects of SAMC on LPS-induced mucin overexpression and cytokine storm in the ARDS model have not yet evaluated. In this study, we evaluated the effect of SAMC on mucin overproduction and J o u r n a l P r e -p r o o f inflammatory responses in 16HBE cells and mice. LPS was used to induce MUC5AC overexpression and inflammation. MAPKs and PI3K-Akt signaling pathways involved in the protective effect of SAMC were also examined both in vitro and in vivo. Control mice received the same volume of saline. All mice were sacrificed at 24 hours. The lung tissues were fixed in 4% paraformaldehyde for 24 h and then embedded in paraffin wax and sectioned (at 4-μm thickness). The tissue sections were stained with hematoxylin and eosin (HE) following the standard protocols. The slides were then examined with a morphometric microscope (Olympus Corporation, Tokyo, Japan). Lung injury scoring criteria of the ARDS mouse model were as following: alveolar and interstitial hemorrhage, pulmonary edema, alveolar or interstitial inflammatory cells infiltration or aggregation, and thickness of alveolar wall/hyaline membrane formation. Lung injury was scored according to the severity of the damage range of 0 to 4 with 0 for no damage and 4 for the maximum damage. The lung tissues were fixed in 4% paraformaldehyde for 24 h and then embedded in paraffin wax and sectioned (at 4-μm thickness). The tissue sections were stained with Alcian blue-periodic acid Schiff (AB-PAS) following the standard protocols. The slides were observed under a morphometric microscope at 400× to reflect the localization of Table 1 . Relative expressions of the different genes were determined with the 2 −ΔΔCT method using GAPDH as the endogenous control. All experimental data are presented as mean ± SD of at least three experiments. The one-way analysis of variance (ANOVA) was used to determine whether there were any statistically significant differences between the means of independent groups with a post hoc test for between-group comparisons using GraphPad Prism 5.0. P < 0.05 was considered to be statistically significant. Cell cytotoxicity was detected by the MTT assay. 16HBE cells were treated with various doses of SAMC or LPS for 12 h. As shown in Fig. 1A and B, no cell cytotoxicity was found under the tested concentrations of SAMC and LPS on 16HBE cells. To examine the airway morphologic changes, we performed the AB-PAS staining. As seen in Fig. 3 , the airway surface mucin secretion was increased, small airways were thickened and alveolar space was collapsed after exposure to LPS. SAMC administration notably alleviated these histopathological changes. Immunohistochemical experiments showed that the MUC5AC positive staining in the airway epithelium was increased in the lung tissues of the LPS-induced mice, and this J o u r n a l P r e -p r o o f augmentation was decreased in mice treated with SAMC (Fig. 3) . represented as means ± SD. To determine the anti-inflammatory activity of SAMC, we detected typical inflammatory cytokines in the bronchoalveolar lavage fluid (BALF) by ELISA kits. Results show that SAMC treatment suppressed the expressions of pro-inflammatory cytokines IL-6 ( Fig. 5A ) and IL-12 (Fig. 5B) , and elevated expressions of antiinflammatory cytokines IL-10 ( Fig. 5C ) and TGF-β (Fig. 5D) . We also evaluated mRNA expression changes in the pro-inflammatory markers (IL-6, TNF-α, CD86 and IL-12) and anti-inflammatory markers (IL-10, CD206 and TGF-β) in 16HBE cells and lung tissues by the RT-PCR analysis ( Fig. 5E and 5F ). These results consistently suggest that SAMC inhibited the cytokines, and helped to reconstitute inflammatory Previous studies reveal that the EGFR activation plays a key role in mediating mucin synthesis and anti-inflammation. In this study, the RT-PCR analysis was used to detect the gene expression of EGFR in 16HBE cells and mice after the treatment with or without SAMC. As presented in Fig. 6A , the treatment with SAMC reduced EGFR levels compared to the LPS stimulated group both in 16HBE cells and in mice. In order to examine MAPKs and Akt-mTOR-STAT3 pathways in the downstream targeting of the EGFR signaling, the phosphorylation status of p38, JNK, Akt, mTOR and STAT3 were determined both in 16HBE cells and mice after the treatment with SAMC and LPS by the western blot analysis. Our results clearly show that the SMAC treatment reduced the phosphorylation of these proteins compared with the LPS groups in vitro ( Fig. 6B and 6C ) and in vivo ( Fig. 6D and 6E ). ARDS is characterized by severe pulmonary gas exchange and pulmonary dynamic injury [1] . The most common causes are local or systemic inflammation, including pneumonia and sepsis [20] . Cytokine storm that rapidly produces a variety of cytokines such as IL-6, IL-12, TNF-α, IFN-γ, MCP-1 and IL-8 results in cell death, disruption of the epithelial and endothelial barriers, and edema [21] which is an important cause of acute respiratory distress syndrome and multiple organ failure [22] . Pseudomonas aeruginosa LPS is an immunocompromised opportunistic pathogen, which generally causes infectious diseases of the lung such as ARDS, pneumonia and sepsis. Yanagihara et al. report that a single instillation of LPS (P. aeruginosa lipopolysaccharide) into the lungs of mice induces hyperinflammation and leads to the hyperplasia and metaplasia of secretory cells [23] . Therefore, the LPS-induced 16HBE cell and ARDS mouse models were used in the present study. It has reported that aged garlic extract (AGE) has an immunoregulatory ability, which can improve the inflammation-related processes [24, 25] . In this study, we demonstrate that SAMC In recent years, the issue of mucin hypersecretion increasingly attracts people's attention. The airway mucus layer is composed of water, ions, mucins, proteins, and lipids, and its major function is to capture foreign allergens, pathogens and pollutant, and to clear these stimulus from the lung by ciliary transport or coughing [26] . Mucins are the major components of the mucus layer and characterized by the presence of a large number of O-glycans and an extensive number of tandem repeats rich in serine and threonine residues [27] . In general, the presence of mucin production can protect the airway surface, however, in the case of sustained overproduction of mucins caused by secretory cell hyperplasia, it will block the airway, reduce lung function, and even threaten life [28] . Several reports have shown that SAMC, a water-soluble organic sulfur compound, has beneficial effects in mucin overproduction and inflammatory disorders [29] . In the present study, we confirmed that the SMAC treatment ameliorated the bronchial mucus hypersecretion as indicated by the AB-PAS and IHC staining, and decreased the expressions of MUC5AC and MUC5B both in vitro and in vivo. EGFR is a member of the ErbB family of receptor tyrosine kinases, and whose activation can cause the activation of multiple downstream signaling pathways [30] . In summary, our study suggested that SAMC ameliorated the inflammation and mucus hypersecretion via MAPKs and PI3K-Akt signaling pathways in the LPSinduced 16HBE cells and mice. These data show that SAMC could be considered as a potential therapeutic candidate for mucin overproduction and various inflammatory diseases. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial supports for this work that could have influenced its outcome. Acute lung injury and the acute respiratory distress syndrome: pathophysiology and treatment The acute respiratory distress syndrome: from mechanism to translation SARS-CoV virushost interactions and comparative etiologies of acute respiratory distress syndrome as determined by transcriptional and cytokine profiling of formalin-fixed paraffinembedded tissues Three fatal cases of pandemic 2009 influenza A virus infection in Shenzhen are associated with cytokine storm COVID-19: consider cytokine storm syndromes and immunosuppression Detectable serum SARS-CoV-2 viral load (RNAaemia) is closely correlated with drastically elevated interleukin 6 (IL-6) level in critically ill COVID-19 patients Mucins, Mucus, and Goblet Cells Treatment of airway mucus hypersecretion Role of cilia, mucus, and airway surface liquid in mucociliary dysfunction: lessons from mouse models The biology of mucus: Composition, synthesis and organization Roles of mucus adhesion and cohesion in cough clearance EGFR plays an essential role in lipopolysaccharide (LPS)-induced MUC5AC hypersecretion in human bronchial epithelial cells Naringenin attenuates mucous hypersecretion by modulating reactive oxygen species production and inhibiting NF-κB activity via EGFR-PI3K-Akt/ERK MAPKinase signaling in human airway epithelial cells Organization and regulation of mitogen-activated protein kinase signaling pathways Pathway as a Downstream Target of EGFR Signaling to Regulate PD-L1 Expression on NSCLC cells Antioxidant and radical scavenging effects of aged garlic extract and its constituents Comparison of Anti-Oxidant and Anti-Inflammatory Effects between Fresh and Aged Black Garlic Extracts Anti-inflammatory activity of sulfur-containing compounds from garlic S-allylmercapto-l-cysteine modulates MUC5AC and AQP5 secretions in a COPD model via NF-кB signaling pathway Cytokine-mediated inflammation in acute lung injury Agents to reduce cytokine storm The cytokine storm and factors determining the sequence and severity of organ dysfunction in multiple organ dysfunction syndrome Lipopolysaccharide Induces Mucus Cell Metaplasia in Mouse Lung Immunomodulatory Effects of Glutathione, Garlic Derivatives, and Hydrogen Sulfide Mucus clearance as a primary innate defense for mammalian airways Structure and function of the polymeric mucins in airways mucus Association of chronic mucus hypersecretion with FEV1 decline and chronic obstructive pulmonary disease morbidity. Copenhagen City Heart Study Group The immunomodulation and anti-inflammatory effects of garlic organosulfur compounds in cancer chemoprevention Peroxiredoxin 6 suppresses Muc5ac overproduction in LPS-induced airway inflammation through H(2)O(2)-EGFR-MAPK signaling pathway Metalloproteinases mediate mucin 5AC expression by epidermal growth factor receptor activation The authors declare that there are no competing interests associated with the manuscript.