br Methods br Results br Discussion

Methods
Results
Discussion It is almost certain that PHMG-p caused interstitial fibrosis in humans due to inhalation exposure. Many epidemiological and toxicological studies have demonstrated that PHMG-p causes inflammatory and fibrotic responses in cells, animals, and humans [1,2,4,9,10]. In addition, PHMG-p has been used to establish a mouse model of pulmonary inflammation and fibrosis [11]. In contrast, there has been no study of the relationship between PHMG-p and respiratory diseases other than interstitial fibrosis. MUC5AC is a deleterious and dispensable component of airway mucus. Koeppen et al. [12] revealed that MUC5AC played a detrimental role in acute lung injury. In addition, overexpression of MUC5AC is associated with various respiratory diseases by plugging or tethering the airway. A quantitative study of fatal order GSK-3 Inhibitor IX found that more than 98% of airways were occluded to some extent by mucus [13]. The level of MUC5AC was significantly and consistently increased in asthma [14]. Thus, MUC5AC was suggested as a suitable target to alter this obstruction in asthma [15]. The present results confirm that PHMG-p stimulates the expression and secretion of MUC5AC (Fig. 1, Fig. 2), which suggests a link between PHMG-p and different obstructive respiratory diseases, in addition to interstitial fibrosis. Many studies have already suggested that the EGFR/ERK system is required to induce MUC5AC as the classical pathway [16,17]; 2001; Zhen et al., 2007). Cigarette smoke, which is well known to cause COPD, triggers the overexpression of MUC5AC [18]. In addition, acrolein, which is a component in tobacco smoke, induces MUC5AC hypersecretion and mucus plugging [8,19]. The mechanism of MUC5AC stimulation by cigarette smoke has been explained to involve the EGFR/ERK system. In the present study, PHMG-p activated p38 and JNK, but not ERK (Fig. 3). Pretreatment with p38 or JNK inhibitors attenuated the upregulation of MUC5AC mRNA and protein induced by PHMG-p, which indicates that PHMG-p-induced MUC5AC expression occurs via the p38 and JNK pathways (Fig. 4). The p38 inhibitor, SB203580, attenuated the activation of both p38 and JNK by PHMG-p (Fig. 4A). Clerk et al. [20] reported that SB203580 could inhibit SAPK/JNK isoforms, despite being known as a specific inhibitor of p38. Hence, in the present study, SB203580 reduced the activation of p38 and JNK simultaneously, which resulted in an effective decrease in MUC5AC levels (Fig. 4B and C). Unlike pretreatment with SP600125 (JNK inhibitor) at a low concentration (10 μM), PHMG-p-induced MUC5AC expression was increased at a high concentration (40μ M) (Fig. 4D and E) and the phosphorylation of p38 was also increased. The connection between SP600125 and the activation of p38 is currently unknown. It was already known that major MAPKs, including p38 and JNK, are activated in parallel [21], because they share upstream mechanisms. Bennet et al. [22] reported the partial inhibitory effect of SP600125 on p38, whereas Vaishnav et al. [23] confirmed that this inhibitor increased the activation of p38 slightly. As SP600125 can affect the upstream kinases of JNK and p38, such as MAPK kinase (MKK)3/6 and MKK4/7, respectively [22], a contradictory phenomenon may occur. Ultimately, we assumed in this study that activation of JNK played a role in MUC5AC upregulation by PHMG-p, although MUC5AC expression at high concentrations of SP600125 was higher than that at low concentrations of SP600125 (Fig. 4A and E). Collectively, we suggest that a specific mechanism involving p38 and JNK contributed to the upregulation of MUC5AC by PHMG-p. TLRs link many downstream pathways, including MAPK pathways. When TLRs are activated, MyD88, a TLR adaptor protein, induces the activation of other proteins such as interleukin-1 receptor-associated kinase 1/4 and MAPK kinase kinase, which leads to MAPK phosphorylation. Among TLRs, TLR2 and 4 are widely known to activate MUC5AC [24,25]. In addition, TLR6, which is usually dimerized with TLR2 or TLR4, contributes to the activation of MAPK [[26], [27], [28]]. As TLRs have been shown to contribute to the production of mucin [29] and MAPK activation [30,31] in several studies, we decided to knockdown TLRs to elucidate whether they are associated with MUC5AC upregulation by PHMG-p. MUC5AC production by PHMG-p was reduced in TLR2-, 4-, or 6-silenced cells, and silencing of TLR6 attenuated the expression of MUC5AC to the greatest extent (Fig. 5C and D). The TLR6 of dendritic cells protected airway inflammation and mucus secretion via interleukin (IL)-17A secretion from T cells [32]. Moreover, TLR6 could initiate immune responses against Brucella abortus, whereas TLR2 had no effect on resistance to this bacterium [33]. Therefore, it is assumed that TLR6 has a protective role in the overexpression of MUC5AC by PHMG-p treatment. A widely accepted theory is that each TLR has affinity for a specific pathogen or substance, or is affected by pathogen-associated molecular patterns (PAMPs) and DAMPs. Because TLR6 is a member of the TLR family and forms heterodimers with TLR2 or TLR4 [28,34], it is suggested that both heterodimers contribute to the increase in MUC5AC by PHMG-p as part of their upstream mechanisms. Thus, TLR6 may have recognized PHMG-p or DAMPs induced by PHMG-p, thereby playing a defense system role via the airway secretion of MUC5AC. However, the direct interaction of PHMG-p with TLR6 should be confirmed in future studies.