The protective role of HMW HA in inflammatory

The protective role of HMW-HA in inflammatory diseases and pulmonary damage led us to investigate whether PM2.5-induced ALI could be attenuated by HMW-HA and the underlying mechanisms. In this study, we used a rat model to evaluate the effect of HMW-HA on pulmonary histology, lung inflammation, oxidative stress, lung edema and epithelial apoptosis after PM2.5 exposure, and an in vitro model to study whether HMW-HA would repress PM2.5-induced apoptosis through its anti-oxidative property. The study demonstrated that PM2.5 stimulated reactive oxygen species (ROS) production and subsequent activation of ASK1 and p38/JNK MAPK pathway, resulting in airway epithelial cell apoptosis and consequent ALI, and HMW-HA could act as an antioxidant to mitigate PM2.5-associated airway epithelial apoptosis, lung inflammation and ALI.
Materials and methods
Results
Discussion PM2.5 has been documented as a pollution criterion in China since 2012, and many compounds such as selenium, vitamin E, omega-3 fatty acids and essential oils have been tested whether they are potential interventions to PM2.5-related health issue (He et al., 2017; Liu et al., 2017; Du et al., 2017; Wang et al., 2017). The present study explored the function of HMW-HA against PM2.5 toxicity in the lungs of the rats, and disclosed that HMW-HA could alleviate PM2.5-induced neutrophil infiltration, pro-inflammatory cytokine production, oxidative damage, lung edema, histological injury and alveolar epithelial apoptosis. To further investigate the protective mechanism of HMW-HA, a cell model was applied. We observed that HMW-HA mitigated PM2.5-increased oxidative stress, ASK1 activation, p38 and JNK phosphorylation and subsequent apoptosis in BEAS-2B abk (Fig. 7). Epithelial injury during ALI is associated with apoptotic death of airway epithelial cells, and bronchial epithelium is a primary target of inhaled PM2.5. In this study, we reported that ROS, ASK1 and downstream p38/JNK MAPK pathways were involved in PM2.5-induced BEAS-2B apoptosis, and PM2.5 activated ASK1 through ROS-mediated phosphorylation at Thr845. Several in vitro studies have documented the importance of ROS in PM2.5-associated cellular changes as well. For example, Soberanes et al. (2009) illustrated that ROS generated by PM2.5 from mitochondrial complex III activated intrinsic apoptotic pathway via ASK1-JNK-p53, and Longhin et al. (2013) clarified that augmented ROS formation by PM2.5 led to DNA damage and G2/M cell cycle arrest. ASK1, a member of MAPK kinase kinase (MAP3K) family, plays a pivotal role in apoptotic signaling, and phosphorylation is a main mechanism to regulate ASK1 activity. Phosphorylation of ASK1 at Thr845 is necessary for ASK1 activity as dephosphorylation at this site results in inactivation of ASK1, and on the contrary, phosphorylation at Ser83 and Ser967 impairs the activity of ASK1 (Saito et al., 2007; Goldman et al., 2004). Similar to our finding, ASK1 phosphorylation at Thr845 ha s been observed after H2O2 exposure and nickel poisoning, triggering cell apoptosis (Saito et al., 2007; Pan et al., 2010). JNK and p38 are classical targets of ASK1 that have been identified mostly as pro-apoptotic proteins. Besides apoptosis, ASK1-p38/JNK signaling pathway also regulates inflammation and neovascularization (Cai et al., 2014). In our preliminary study, we also found that ERK MAPK pathway was activated by PM2.5, and PM2.5-upregulated ERK phosphorylation could be restrained by HMW-HA (data not shown). The regulation of ERK after PM2.5 insult and HMW-HA treatment should be further examined in the future. The administration of PM2.5 in our in vivo study was 8 mg/kg b.w. for consecutive three days, and was sufficient to induce lung injury and inflammation in SD rats. Li et al., (2015a,b) showed that intratracheal instillation of 1.5 mg/kg b.w. PM2.5 five times every two days could cause mitochondrial damage in lung tissue of SD rats, and a higher concentration of PM2.5 was asscoiated with a more serious damage. As for in vitro study, the dose 100 μg/ml was chosen based on the dose-response experiments of PM2.5 on BEAS-2B cell viability, and 100 μg/ml is a common concentration used in the studies investigating the toxicological mechanisms of PM2.5 in epithelial cells (Jin et al., 2017).