2.1 Animal grouping and treatment

A total of 20 male wild-type C57BL/6 mice (age, 8–10 weeks) were obtained from the Experimental Center of China Second Military Medical University and randomly allocated into one of two groups (n = 10): control group and LPS-induced ALI model group.

Murine model of ALI was established by intratracheal instillation of LPS, as previously described.^{10, 11} Briefly, the mice were weighted and anesthetized with 5% chloral hydrate (intraperitoneally, 100 mg/kg). The cervical region was shaved and disinfected with 75% alcohol. After careful dissection, the trachea was exposed. LPS (from Escherichia coli 055:B5; L2880; Sigma-Aldrich; 15 mg/kg) was subsequently slowly injected into trachea of the mice. Following LPS administration for 12 h, mice were euthanized through cervical dislocation, and Lung tissue samples were then harvested for conventional hematoxylin and eosin (H&E) and immunohistochemical staining. Bronchoalveolar lavage fluid (BALF) were collected and centrifuged at 4°C, and the supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA).

2.2 Immunohistochemistry (IHC)

The IHC staining was performed on formalin-fixed, paraffin-embedded sections. Slides (5 μm) were treated with 0.3% hydrogen peroxide for 30 min, followed by incubation overnight with anti-ROR2 (Abcam) using a 1:100 working dilution. The sections were visualized using diaminobenzidine for 10 min, counterstained with hematoxylin, and examined by microscopy.

2.3 Quantitative polymerase chain reaction (qPCR) analysis

Total RNA of lung tissues was extracted by Trizol (Invitrogen), and cDNA was synthesized using First Strand cDNA Synthesis Kit (Thermo Scientific). The qPCR was performed using a 40-cycle two-step PCR with sequence-specific primer pairs on the ABI 7900 fast real-time detection system (Invitrogen). Primers were designed using Primer 5.0 software and the sequences of ROR2 were as follows: forward, 5′-GTCCAACGCACAGCCCAAATC-3′ and reverse, 5′-CCGGTTGCCAATGAAGCGTG-3′. The primer sequences of glyceraldehyde phosphate dehydrogenase (GAPDH) as the housekeeping control was forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′.

2.4 Cell culture and transfection

Human alveolar epithelial A549 cells (ATCC® Number: CRM-CCL-185™) were purchased from Institute of Shanghai Biochemistry and Cell Biology, Chinese Academy of Sciences. A549 cells were cultured in Roswell Park Memorial Institute 1640 medium supplemented with 5% fetal bovine serum (FBS; Sijiqing Biological Engineering Material Co., Ltd.) according to the instructions of American Type Culture Collection (ATCC). A549 cells were grown in six-well culture plates and allowed to adhere for 12 h at 37°C in a humidified atmosphere with 5% CO₂. After serum starvation for 18 h, cells were transfected with ROR2-targeting siRNA (2.9 × 10⁸ plague forming unit/mL) or negative control siRNA. Transfection mixtures were premixed in 250 μL serum-free medium before addition to the cells. After 48 h of transfection, the cells were then treated with 10 μg/mL LPS for 24 h.

2.5 Cell proliferation assay

Cell proliferation assay was performed using cell counting kit-8 (CCK-8; Beyotime Biotechnology). In brief, 100 μL cell suspension was prepared in a 96-well plate at 1 × 10⁴ cells/well, and incubated for 24, 48, or 72 h. Then, 10 μL CCK-8 solution was added to each well and incubated for 1 h. The absorbance at 450 nm was measured with a microplate analyzer.

2.6 Cell cycle and apoptosis analysis

Cell cycle was examined with the Cell Cycle and Apoptosis Analysis Kit (Beyotime Biotechnology), while cell apoptosis was examined with the Annexin V-FITC Apoptosis Detection Kit (Invitrogen) according to the instructions. After staining, samples were analyzed using flow cytometer (Beckman Coulter). Hoechst 33342 staining was also used to evaluate cell apoptosis as previous described.¹⁴

2.7 ELISA

Culture supernatants were collected and centrifuged at 3000g for 15 min. The supernatants were used for ELISA analysis. Measurement of inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-8 in culture supernatants and BALF were performed using commercially available ELISA kits.

2.8 Western blot analysis

After exacting from cultured cells using radio immuno precipitation assay (Thermo Scientific), 30 μg protein were subjected to electrophoresis. The protein was transferred to polyvinylidene fluoride (PVDF) membranes at 250 mA at 4°C for 2 h. After incubation with following indicated antibodies overnight at 4°C: ROR2, GAPDH, cell division cycle protein 2 (CDC2), cyclin B1 (CCNB1), caspase-3, caspase-9, JNK, p-JNK, ERK1/2 and p-ERK1/2 (1:1000 dilution; Cell Signaling Technology), the membranes were washed with tris buffered saline tween (TBST) and stained with secondary antibody (1:5000 dilution) at room temperature for 2 h. After adding the Chemiluminescent Substrates (Invitrogen), the bands were scanned.

2.9 Statistical analysis

Values were expressed by the mean ± standard deviation (SD). SPSS 18.0 software (SPSS, Inc.) was used for data analysis using Student's t test or analysis of variance with the Dunnett's multiple comparison test. p < .05 was regarded as statistically significance.

3.1 ROR2 mRNA and protein are highly expressed in murine models of LPS-induced ALI

To determine whether ROR2 was involved in the pathogenesis and development of ALI, murine models of ALI was constructed by intratracheal administration of LPS; the results of histopathological examination indicate that murine models of LPS-induced ALI are constructed successfully (Figure 1A). Furthermore, ROR2 mRNA and protein expression in the lung tissues were examined by qPCR with sequence-specific primers and IHC, respectively. As illustrated in Figure 1B, ROR2 mRNA expression in lung tissues of LPS-challenged mice was significantly higher than that of the control group. Immunohistochemical staining of ROR2 protein was scarcely detected in the lung tissues of control group, whereas, widespread pale brown staining was observed in the lung tissues of LPS-treated mice (Figure 1C). Colorimetric sandwich ELISA kit detection of TNF-α, IL-1β, and IL-8 in BALF in LPS-stimulated mice showed that these inflammatory cytokines were significantly upregulated in BALF (Figure 1D).

3.2 Downregulation of ROR2 enhances proliferation, promotes cell cycle process, and inhibits apoptosis of LPS-treated A549 cells in vitro

To determine the involvement of ROR2 in LPS-induced A549 cell injury, we first transfected ROR2 siRNA into A549 cells, which was further induced by LPS for 24 h. Western blot analysis revealed that the expression level of ROR2 was dramatically decreased by ROR2 siRNA (Figure 2A). We found that cell proliferation was markedly increased in A549 cells with ROR2 knockdown (Figure 2B). In addition, ROR2 inhibition effectively inhibited LPS-induced the increased expression of ROR2 (Figure 2C). The effects of ROR2 downregulation on proliferation, cell cycle, and apoptosis of LPS-treated A549 cells in vitro were further investigated. Compared with control group, LPS administration notably decreased the cell viability; nevertheless, downregulation of ROR2 markedly attenuated the decrease in proliferation of LPS-challenged A549 cells; there was no significant difference in cell proliferation between LPS treatment and negative control siRNA treatment (Figure 3). LPS treatment notably blocked the entry of A549 cells from G1 phage into S phage of cell cycle; on the contrary, ROR2 downregulation markedly attenuated LPS-treated A549 cell arrest at G1 phage (Figure 4A). As illustrated in Figure 4B, expression of cell cycle-related proteins, CDC2 and CCNB1, was significantly decreased by LPS; no statistically significant difference was found between LPS treatment and negative control siRNA treatment; however, ROR2-targeting siRNA treatment markedly upregulated the expression of CDC2 and CCNB1. Following LPS administration, higher rate of apoptosis was observed in A549 cells, while the increase in the apoptosis rate of LPS-challenged A549 cells was remarkably attenuated by downregulating ROR2 (Figure 5A,B). In addition, LPS administration significantly increased the expression of caspase-3 and caspase-9, and the increase in the expression of caspase-3 and caspase-9 was significantly attenuated by downregulation of ROR2 (Figure 5C).

3.3 Downregulation of ROR2 inhibits pro-inflammatory cytokine release from LPS-treated A549 cells in vitro

It is well known that pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-8, are key mediators in LPS-induced inflammatory responses. Effect of ROR2 downregulation on pro-inflammatory cytokine release from LPS-treated A549 cells in vitro is illustrated in Figure 6. In comparison with control group, LPS-challenged A549 cells with or without negative control siRNA treatment released higher levels of TNF-α, IL-1β, and IL-8. In contrast, the levels of TNF-α, IL-1β, and IL-8 were significantly decreased by downregulation of ROR2 when compared with LPS treatment.

3.4 Downregulation of ROR2 inhibits JNK and ERK signaling pathway in LPS-treated A549 cells in vitro

As shown in Figure 7, the phosphorylation of JNK, ERK1/2, and p38 was markedly increased after LPS administration in A549 cells when compared with control group. The phosphorylation levels of JNK and ERK1/2 were significantly decreased by ROR2 downregulation in comparison with LPS treatment. The findings suggest that downregulation of ROR2 inhibits JNK and ERK signaling pathways in A549 cells treated with LPS.