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Oxymatrine attenuates lipopolysaccharide-induced acute lung injury by activating the epithelial sodium channel and suppressing the JNK signaling pathway

The epithelial sodium channel (ENaC) and mitogen-activated protein kinase (MAPK) pathway have been reported to be associated with the progression of acute lung injury (ALI). Oxymatrine (OMT) alone or combined with other drugs can ameliorate paraquat- or oleic acid-induced lung injury. However, the effect of OMT on lipopolysaccharide (LPS)-induced ALI remains unknown. The aim of the present study was to evaluate whether OMT can attenuate LPS-induced ALI through regulation of the ENaC and MAPK pathway using an ALI mouse model. Histological assessment of the lung and inflammatory cell counts in bronchoalveolar lavage fluid (BALF) were performed by H&E and Wright-Giemsa staining. The lung wet/dry (W/D) weight ratio and the levels of tumor necrosis factor-α (TNF-α), C-reactive protein (CRP), ENaC subunits, and the MAPK pathway members were determined. Isolated type II rat alveolar epithelial cells were incubated with OMT 30 min before LPS stimulation to investigate the activation of ENaC and the MAPK pathway. The results showed that OMT remarkably alleviated histopathologic changes in lung and pulmonary edema, reduced inflammatory cell counts in BALF, and decreased TNF-α and CRP levels in a dose-dependent manner. OMT significantly increased the three subunits of ENaC proteins in vivo and in vitro, while it decreased p-ERK/ERK, p-p38/p38, and p-JNK/JNK ratios in vivo. However, only the JNK pathway was markedly inhibited in vitro following pretreatment with OMT. Collectively, the results suggested that OMT might alleviate LPS-induced ALI by elevating ENaC proteins and inhibiting the JNK signaling pathway.


Introduction
Acute lung injury (ALi) and its most severe form, acute respiratory distress syndrome (ARDS), are clinically severe respiratory disorders characterized by gas exchange impairment, hemorrhage, and pulmonary edema [38]. LPS is a major component of the outer membrane of Gram-negative bacteria, including Pseudomo-nas aeruginosa (P. aeruginosa). LPS stimulation can activate a variety of immune cells and induce the release of pro-inflammatory and immunoregulatory cytokines from the host, thereby affecting the immune function of the body [44]. When delivered into animals and humans, LPS can cause typical symptoms of lung infection, including pulmonary leukocyte accumulation, edema, severe inflammation, and mortality [29].
Oxymatrine (OMT), a quinolizidine alkaloid, is a bioactive ingredient of the traditional Chinese herb Sophorae flavescentis radix (the dried roots of Sophora flavescens Ait) [27]. OMT has been demonstrated to possess antiviral, anticancer, anti-inflammatory, and anti-fibrotic activities [11]. Studies revealed that OMT can alleviate LPS-induced mastitis and acute intestinal inflammation through regulation of the nuclear factor-κB (NF-κB) signaling pathway [13,35]. A recent study demonstrated that the combination of OMT and sodium ferulate protects mice against ALi. However, the possible mechanism of OMT in LPS-induced ALi remains unclear.
The mitogen-activated protein kinase (MAPK) pathway participates in the release of inflammatory cytokines/mediators in the pathogenesis of inflammatory diseases, including ALi [3,5]. A recent study reported that OMT can suppress inflammation responses in LPSactivated microglia through the MAPK pathway [9]. The MAPK pathway is also inactivated in the protection of OMT against sepsis-induced myocardial injury and intracerebral hemorrhage (iCH) [18,37]. Xu et al. demonstrated that OMT can attenuate oleic acid-induced ALi by inhibiting the p38 MAPK pathway [34]. The above evidence suggests that the MAPK pathway may be involved in protection of OMT against LPS-induced ALi.
Pulmonary edema is a primary pathological feature of ALI/ARDS, and the epithelial sodium channel (ENaC) plays an important role in relieving pulmonary edema in ALI/ARDS [8,14]. In lung tissue, ENaC could transport excess edematous fluid out of the alveolar cavity through the channel transport mechanism [10,43], whereas abnormally low expression of ENaC leads to impaired fluid clearance [30]. Dagenais and colleagues demonstrated that P. aeruginosa and its secreted LPS reduce the expression and activity of ENaC in alveolar epithelial cells in vivo and in vitro [1,6,7,28]. Moreover, inhibition of the MAPK pathway abrogated LPS-induced ENaC downregulation [28]. Thus, we speculate that OMT may prevent the downregulation of ENaC and protect against LPS-induced ALi through regulation of the MAPK pathway.
in the present study, an animal model of ALi was generated in C57BL/6 mice by intratracheal injection of LPS from P. aeruginosa. We evaluated the effect of OMT on pathological changes, inflammation in BALF and lung tissues, and pulmonary edema in vivo and clarified the possible mechanisms both in vivo and in vitro.

Ethics statement
Experimental procedures were approved by the Animal Care and Use Committee of China Medical University and performed in accordance with relevant guidelines (approval number: 2015PS361K).

Animals and groups
Male C57BL/6 mice (age, 8-10 weeks; weight, 18-22 g) were purchased from Charles River (Beijing, China) and randomly divided into 5 groups (the control group, LPS group, LPS + 12.5 mg/kg OMT group, LPS + 25 mg/kg OMT group, and LPS + 50 mg/kg OMT group) (18 mice/group). After anesthetization, the mice in the LPS group were intratracheally administered 50 µg LPS from P. aeruginosa serotype 10 (product no.: L9143) (Sigma-Aldrich Corp., St. Louis, MO, USA) in 70 µl PBS to establish the ALi model. The mice in the control group received the same volume of PBS in the same way as described above. The mice in the LPS + OMT groups were intraperitoneally injected with 12.5, 25, or 50 mg/ kg OMT (Meilunbio, Dalian, China) 30 min before LPS administration. Meanwhile, the mice in the control group and the LPS group were pretreated with an equal volume of sterile saline.

Bronchoalveolar lavage fluid (BALF) cell count
Six hours after LPS administration, the mice were anesthetized. The trachea was cannulated and lavaged three times with 1.5 ml normal saline (0.5 ml normal saline each time). BALF was centrifuged at 2,000 rpm for 10 min, and the pellet was resuspended in 0.5 ml PBS. Inflammatory cells (white blood cells, macrophages, neutrophils, and lymphocytes) in 10 µl cell suspensions were fixed in methanol and then stained with Wright-Giemsa stain (nanjing Jiancheng Bioengineering Institute, Nanjing, China). The inflammatory cells were counted under a microscope (Olympus, Tokyo, Japan).

H&E staining
At 6 h post-LPS exposure, the lung tissues were excised, fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin. The tissues were cut into 5 µm thick sections. After deparaffinization and rehydration, the sections were stained with hematoxylin and eosin. The pathological changes were photographed under a microscope (Olympus).

ELISA
The levels of C-reactive protein (CRP) in lung tissues were determined by ELISA kits purchased from USCN Business Co., Ltd. (Wuhan, China), according to the manufacturer's instructions. The tumor necrosis factor (TNF)-α level in the supernatant of tissue homogenate was measured with a Mouse TNF-α ELISA Kit (Boster, Wuhan, China). The optical density at 450 nm was read on a BioTek microplate reader (BioTek, Winooski, VT, USA).

Lung wet/dry (W/D) weight ratio
Pulmonary edema was evaluated by measuring the lung wet/dry (W/D) weight ratio. The lung tissues were removed and weighed. Subsequently, they were placed in an incubator for 72 h to obtain their dry weight. The lung W/D weight ratio was calculated.

Isolation and identification of type II rat alveolar epithelial cells
Lungs from male Sprague-Dawley rats were washed twice with PBS and were filled with elastase (4.2 U/ml) via the trachea. After digestion at 37°C for 15 min, lung tissues were minced, and elastase was neutralized with 0.25% DNase I and FBS in a shaker for 10 min. The minced tissues were filtered through filters, and the resultant cell suspension was centrifuged at 1,000 rpm for 10 min at 4°C. After discarding the supernatant, the pellet was resuspended in DMEM and purified by the differential adhesion method to remove fibroblasts. Immunofluorescence was performed to identify type II alveolar epithelial cells by using an antibody against surfactant protein-C (SP-C). The cells were mounted on slides, fixed in 4% paraformaldehyde for 15 min, immersed in 0.1% Triton X-100 for 30 min, and treated with goat serum for 15 min at room temperature. Subsequently, the slides were incubated with anti-SP-C antibody (Santa Cruz Biotechnology, Dallas, Texas, USA ; 1:50 dilution) at 4°C overnight followed by 60 min of incubation with Cy3-conjugated goat anti-rabbit antibody (Beyotime Institute of Biotechnology, Haimen, China; 1:400 dilution) at room temperature. The nuclei were counterstained with DAPi, and the slides were photographed under a fluorescence microscope (Olympus).

Cell culture and treatment
Type ii rat alveolar epithelial cells were cultured in DMEM containing 10% FBS in a 5% CO 2 incubator.
The cells were divided into 8 groups (the control group, 250 µg/ml OMT group, 500 µg/ml OMT group, 1,000 µg/ml OMT group, LPS group, LPS + 250 µg/ml OMT group, LPS + 500 µg/ml OMT group, and LPS + 1,000 µg/ml OMT group). The cells in the corresponding groups were pretreated with 250, 500, and 1,000 µg/ml OMT (Meilunbio), respectively. The cells cultured with vehicle were used as the controls. After pretreatment for 30 min, the cells in the corresponding groups were incubated with 15 µg/ml LPS (Sigma-Aldrich Corp.) at 37°C until they were subjected to analyses.

MTT assay
Cell viability was measured by MTT assay at 6, 12, and 24 h. After treatment with OMT and/or LPS, the cells were treated with 5 mg/ml MTT (Sigma-Aldrich Corp.) at 37°C for 4 h. The supernatant was discarded. The optical density was determined at 490 nm after dissolving the crystal violet with 150 µl DMSO (Sigma-Aldrich Corp.).

Statistical analysis
Data are expressed as the mean ± SD. Results were analyzed by one-way ANOVA followed by Bonferroni post hoc test. A P value<0.05 was considered statistically significant.

OMT attenuates histopathological changes in lung tissues
We evaluated histopathological changes in mice by H&E staining and assessed lung injury with a 9-score system. As shown in Fig. 1, the mice in the control group showed a normal structure of lung tissues and no histo- pathological changes. There were obvious histopathological changes in the lung tissues of the mice in the LPS group, including obvious inflammatory cell infiltration, thickened alveolar walls, severe hemorrhage in the alveolus, and alveolus collapse. After treatment with dif-ferent doses of OMT, the histopathological changes of the lung were significantly ameliorated in a dose-dependent manner when compared with the LPS group.

OMT attenuates inflammation in ALI induced by LPS in mice
The severity of inflammation was measured by inflammatory cell (white blood cells, macrophages, neutrophils, and lymphocytes) counts in BALF and TNF-α and CRP levels in lung tissues. LPS stimulation significantly increased inflammatory cell counts in BALF ( Fig. 2A) and the levels of TNF-α and CRP (Fig. 2B) compared with the control group. Compared with the LPS group, OMT markedly reduced inflammatory cell counts and lung TNF-α and CRP levels in a dose-dependent manner.

OMT attenuates pulmonary edema in mice
Pulmonary edema can be assessed by lung W/D weight ratio. The results showed that the lung W/D weight ratio was dramatically increased in the LPS group compared with the control group (Fig. 3). OMT lowered the lung W/D weight ratio in a dose-dependent manner compared with the LPS group.

OMT activates the epithelial Na + channel (ENaC) in mice
Real-time PCR and Western blotting results showed that the levels of expression of α-ENaC, β-ENaC, and  γ-ENaC at mRNA (Fig. 4A) and protein (Fig. 4B) levels were significantly lower in the LPS group than those in the control group. However, OMT administration obviously upregulated the reduced levels of α-ENaC, β-ENaC, and γ-ENaC, indicating the activation of ENaC.

OMT inhibits the activation of the mitogen-activated protein kinase (MAPK) pathway in mice
Extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 are members of the MAPK pathway. To examine the involvement of the MAPK pathway, we measured the levels of MAPK pathway members. We found that the expression levels of phosphorylated forms of ERK, p38, and JNK were notably increased in the LPS group compared with the control group (Fig. 5). However, OMT treatment resulted in remarkable decreases in p-ERK, p-p38, and p-JNK expression compared with the LPS group.

Identification of type II alveolar epithelial cells and cytotoxicity of OMT
in our in vitro study, we firstly identified the isolated cells using immunofluorescence staining for SP-C. As shown in Fig. 6A, the results showed that type ii rat alveolar epithelial cells were successfully isolated. Then, we examined the cytotoxicity of OMT in type ii rat alveolar epithelial cells by MTT assay. The results showed that OMT (250, 500, and 1,000 µg/ml) had no cytotoxicity in type II rat alveolar epithelial cells (Fig. 6B). The addition of LPS significantly reduced the cell viability of type ii alveolar epithelial cells. OMT preconditioning, especially at the dose of 500 µg/ml, slightly abolished the effect of LPS at 24 h.

OMT activates ENaC in alveolar epithelial cells
We further confirmed whether ENaC was activated in type ii alveolar epithelial cells using real-time PCR and Western blotting. In the LPS group, the mRNA (Fig. 7A) and protein (Fig. 7B) levels of α-ENaC, β-ENaC, and γ-ENaC were decreased compared with the control group. However, OMT injection restored the levels of α-ENaC, β-ENaC, and γ-ENaC.

OMT inhibits the activation of the JNK pathway in type II alveolar epithelial cells
The expression levels of MAPK pathway members and their phosphorylated forms in type ii alveolar epi-  thelial cells were examined by Western blotting. As shown in Fig. 8, the protein levels of p-ERK, p-p38, and p-JNK were shown to be elevated by LPS exposure. OMT preconditioning significantly reduced the ratios of p-JNK/JNK in LPS-stimulated cells. The differences were not statistically significant, although p-ERK/ERK and p-p38/p38 ratios were decreased by OMT preconditioning.

Discussion
LPS is the pathogenic constituent of P. aeruginosa and can induce strong immune responses in bodies [2]. LPS has been widely used to establish the experimental model of ALi [32,42]. Our present study investigated the protective effect of oxymatrine (OMT) preconditioning on acute lung injury (ALi) and further elucidated the possible mechanisms.
Xu et al. demonstrated that OMT attenuates oleic acid-induced ALi by inhibiting pulmonary edema and inflammatory cell infiltration in alveolar spaces [34]. Yuan et al. found that sodium ferulate combined with OMT decreases inflammatory cells in BALF and alleviates pulmonary edema in a LPS-induced ALi mouse model [36]. Additionally, OMT has been applied in the treatment of acute pancreatitis and acute intestinal inflammation due to its anti-inflammatory effect [13,41]. in our study, Wright-Giemsa staining results consistently showed that OMT dose-dependently decreased LPS-induced increases of inflammatory cell (white blood cells, macrophages, neutrophils, and lymphocyte) numbers in BALF. C-reactive protein (CRP) is an important indicator of inflammation and correlates with the severity of diseases, which is markedly increased after bacterial infection [21]. TNF-α is a vital pro-inflammatory cytokine that can activate other cytokines and chemokines to aggravate lung tissue injury. Zhang et al. found that OMT pretreatment inhibits the expression of TNF-α and IL-1β in LPS-stimulated macrophages [40]. Moreover, we found that OMT significantly reduced the up- regulated levels of CRP and TNF-α in lung tissues induced by LPS exposure. The data suggest that OMT administration may protect against LPS-induced ALi via its anti-inflammatory effect. ALI is characterized by inflammation, pulmonary edema, and alveolar-capillary barrier damage. The lung W/D weight ratio is an index of pulmonary edema [39]. In our study, we found that OMT significantly alleviated histopathological changes and inhibited pulmonary edema induced by LPS.
The epithelial sodium channel (ENaC) consists of three subunits (α, β, and γ) [26]. Alpha-ENaC forms the channel pore, while β-ENaC and γ-ENaC are associated with the channel activity [24,26]. ENaC plays a major role in sodium and water homeostasis [4]. Knockdown of α-ENaC by siRNA transfection inhibits lung fluid absorption in SD rats [23]. Hummler et al. found that α-ENaC-deficient neonatal mice fail to clear lung fluid, develop respiratory distress, and die within 40 h of birth [19]. Roux et al. reported that IL-1β reduces α-ENaC expression and activity, inhibits lung epithelial sodium absorption, and ultimately contributes to alveolar edema via the p38 MAPK signaling pathway [31]. We found that LPS administration decreased the expression levels of α-ENaC, β-ENaC, and γ-ENaC in lung tissues and alveolar epithelial cells, whereas OMT treatment increased α-ENaC, β-ENaC, and γ-ENaC levels. The data suggest that OMT administration may alleviate pulmo-nary edema via ENaC.
MAPKs, including extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase (JNK), are members of the serine/threonine kinase family, which participate in signal transduction in eukaryotic organisms [22]. MAPKs play important roles in inflammation, oncogenesis, cell proliferation, apoptosis, differentiation, and stress responses [17,20]. The MAPK signaling pathway is inhibited by OMT administration in LPS-induced mastitis in mice [35]. Moreover, OMT protects rats against myocardial injury in a rat model of sepsis via suppression of the p38 MAPK signaling pathway [37]. We found that OMT administration significantly inhibited LPS-induced activation of the MAPK signaling pathway in a mouse model of ALi. As expected, we consistently observed suppression of the JNK signaling pathway in OMT-treated alveolar epithelial cells in vitro. LPS-induced activation of the ERK and p38 pathways was slightly inhibited by OMT; however, the difference was not statistically significant. The data suggest that OMT administration may protect against LPS-induced ALi in vivo and in vitro by inhibiting the JNK signaling pathway. Toll-like receptor 4 (TLR4) can recognize and interact with LPS and subsequently activate the downstream MAPK pathways, eventually leading to an inflammatory response [12,15]. Previous studies reported that LPS-induced ALi can be attenuated by other drugs through the TLR4-mediated MAPK pathway lowing OMT (500 µg/ml) treatment, the cells were exposed to LPS (15 µg/ml) for 30 min. The expression levels of p-ERK, ERK, p-p38, p38, p-JNK, and JNK were quantified by Western blotting analysis. The ratios of p-ERK/ERK, p-p38/p38, and p-JNK/JNK were calculated. Data are expressed as the mean ± SD. Results were analyzed by one-way ANOVA followed by Bonferroni post hoc test. *P<0.05 compared with the control group. **P<0.01 compared with the control group. # P<0.05 compared with the LPS group. [16,33]. OMT may also ameliorate ALi induced by LPS by inhibiting the TLR4-mediated signaling pathways; however, the precise mechanism requires further investigation and verification.
in summary, the results demonstrated that pretreatment with OMT attenuated LPS-induced ALi in both C57BL/6 mice and rat alveolar epithelial cells. The inhibitory effects of OMT on ALI were associated with the ENaC and JNK signaling pathway.