Acute lung injury (ALI), is a serious inflammatory disease [1–3], and LPS is a key mediator of organ dysfunction and death associated with severe gram-negative infections , which is the most important pathogen causing ALI to occur [25, 26]. Intraperitoneal injection is a widespread method to rodents for administering drugs , and we established the ALI model caused by LPS (i.p., 10 mg/kg).
Recently, basic and clinical researches have shown that Hydrogen gas (H2) is an important physiological regulatory factor with antioxidant, anti-inflammatory and anti-apoptotic properties . In the present study, we investigated the protective effects of H2 treatment in LPS-induced ALI mice and the protective effect might be related to its ability of ameliorating the extent of oxidative stress and preventing the release of pro-inflammatory molecules [15–18]. H2 inhalation can attenuate many kinds of lung injuries caused by ventilator, transplantation, hyperoxia, irradiation, and sepsis [29–32]. The histopathologic evaluation shows that, lung in the LPS group showed a thickened alveolar wall, edema and hemorrhage, less alveolar space and inflammatory cell infiltration after LPS stimulation. H2 inhalation significantly prevented these histopathological changes caused by LPS. The H2 treatment ameliorates LPS-induced lung neutrophil infiltration and inflammation.
To verify the success of the ALI model, we observed the pathological and inflammatory changes of different time points of mice after LPS injection intraperitoneally. The critical feature of ALI is the lung parenchyma injury and acute inflammatory process, including the release of inflammatory mediators such as TNF-α, IL-1β and IL-6 . Proinflammatory cytokines appear in the early stages of inflammation, indicating the severity of ALI in a certain sense . In this study, the inflammatory factors gradually increased at 6 h after LPS administration, which was significantly higher than that in H2 groups at 24 h. These results suggest that the ALI model was successfully established after intraperitoneal injections of LPS, with the lung injury being obvious at 24h point and the H2 markedly reduced the inflammation of ALI mice.
In the vivo study, we found that (1) hydrogen gas inhalation protected mice against ALI lethality (Fig. 1); (2) H2 markedly reduced the inflammation and oxidative damages in ALI mice (Figs. 2 and 3); (3) H2 improved the lung injury caused by LPS stimulation significantly (Fig. 4); and (4) H2 inhibited the TLR4 expression in lung tissues (Fig. 5). In conclusion, these results shows that molecular hydrogen alleviates LPS-induced ALI by reducing lung inflammation and oxidative damages, it may be associated with decreased NF-κB activity.
As well known that LPS causes the decrements of neutrophilic inflammation and pulmonary function, which act via Toll-like receptor 4 to induce the expression of inflammatory cytokines and chemokines [35, 36]. The mechanism of ALI injury may be due to bacterial endotoxin (LPS) activation, promoting the interaction of TLR4 and resulting in activation of NF-κB and release of TNF-α and IL-6 , all of these factors act a pivotal part in lung inflammatory damages. A lot of experiments have proved that inhibiting the activity of NF-κB in different models can alleviated the tissue damage and down-regulated release of cytokines [38, 39]. Therefore, inhibition of NF-κB activation will be an effective choice for protecting ALI.
To explore the beneficial effect of H2 inhalation on LPS-induced ALI by inhibiting NF-κB activation, we raise the question: how can H2 inhibit NF-κB activation? First, it is reported that H2 can directly activate the NF-κB signaling through inhibiting the phosphorylation of IκB-α . Second, research shows that H2 can inhibit the activation of the NF-κB signaling pathway by scavenging the oxygen radicals . Hydrogen gas, a potential antioxidant with rapid gaseous diffusion, is very effective in reducing cytotoxic free radical, such as reactive oxygen species(ROS) and mild enough to prevent interference with metabolic redox reactions or disrupt cell signaling. In our vitro experimental, (1) the elevations of TNF-α, IL-6 and IL-1β mRNA levels were suppressed by H2 (Fig. 6); (2) H2 alleviates the cell damages induced by LPS and reduces the ROS, NO level of cell (Fig. 7); (3) the intracellular ROS levels were significantly reduced in LPS + 60%H2 group (Fig. 8); (4) H2 inhibits TLR4 expression and NF-κB activation in macrophages caused by LPS(Fig. 9).
In this study, LPS administration induced a massive inflammatory cells and the release of cytokines in the serum, which were attenuated by H2 inhalation treatment. Moreover, we assessed the lung oxidative damages by measuring the level of MDA and NO, the result shows that hydrogen gas decreased the lung oxidative damages caused by LPS. On the other hand, we found that H2 dramatically inhibited the release of pro-inflammatory cytokine (TNF-α and IL-1β) of LPS-challenged mice and significantly up-regulated anti-inflammatory cytokines (IL-10). Previous studies reported that H2 inhalation could inhibit the release of pro-inflammatory cytokines , however, there are no studies about its effects on anti-inflammatory cytokines.