Lomerizine was purchased from Absin Bioscience Inc. (Shanghai, China). Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, United States). Dexamethasone was purchased from Thermo Fisher Scientific (Waltham, MA, United States). Hematoxylin and eosin (H&E) staining kit was purchased from Abcam (Waltham, Boston, United States). Dulbecco’s Modified Eagle Medium (DMEM) and macrophage colony-stimulating factor (M-CSF) was purchased from Thermo Fisher Scientific (Waltham, MA, United States). Penicillin-streptomycin solution (Pen Strep) was purchased from Yeasen Biotechnology Co., Ltd. (Shanghai, China). Fetal Bovine Serum (FBS) was purchased from Cegrogen Biotech (Wupperweg, Germany).

Primers were synthesized by Hua Gene Biotech Co., Ltd. (Shanghai, China). The primary antibody against IκB-α was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Primary antibodies against iNOS, phospho-NF-κB p65 (Ser536) Rabbit mAb, NF-kappaB p65 Rabbit mAb, phospho-p38 MAPK (Thr180/Tyr182) XP Rabbit mAb, p38 MAPK XP Rabbit mAb, phospho-SAPK/JNK (Thr183/Tyr185) Rabbit mAb, SAPK/JNK Antibody, Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) Antibody, p44/42 MAPK (ERK1/2) Rabbit mAb, and β-Actin Rabbit Antibody were purchased from Cell Signaling Technology (Danvers, MA, United States).

C57BL/6 mice were purchased from JST Laboratory Animal Co., Ltd. (Shanghai, China). The mice were bred under sterile and temperature-controlled conditions for 12 h of light and dark cycle. Mice had free access to water and feed in the experimental period. Mice were housed under specific pathogen-free conditions at the Laboratory Animal Center of Shanghai Jiao Tong University. All procedures involving mice were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University (A2018075).

Thirty male mice aged 8–10 weeks were selected to establish acute lung injury models. The mice were randomly divided into six groups: PBS group, LPS treatment group, dexamethasone (DEX) treatment group, low dose lomerizine (LMZ) group (10 mg/kg), medium dose LMZ group (20 mg/kg), and high dose LMZ group (40 mg/kg). In PBS group, the neck skin of mice was dissected and sutured. The other groups were given 5 mg/kg LPS via intratracheal administration. Moreover, DEX or various doses of LMZ were injected intraperitoneally 1 h after LPS was administered.

After 6 h of LPS stimulation, mice were anesthetized, and the trachea was exposed with scissors, then a small cut was gently made in the trachea and a needle was inserted, followed by the injection of 1 mL of cooled PBS, waited for 10 s and then the bronchoalveolar lavage fluid (BALF) was slowly withdrawn. And collected BALF was put on ice and then centrifuged at 4°C for 500 rcf for 5 min (Wu et al., 2021). The total number of cells was counted with a hemocytometer. And the supernatant was analyzed for total protein using a EpiZyme BCA kit (Shanghai, China).

The suppernatant after centrifugation of BALF was added 300 μL red blood cell lysate (Sangon biotech, China) and mixed well, and rested on ice for 5 min, 3 mL PBS was added, followed by 500 rcf centrifugation at 4°C for 5 min. The supernatant was discarded and 100 μL of PBS containing 2% FBS was added to the supernatant for 30 min. The detectable antibodies were then added and incubated for 30 min. At the end of the incubation, the supernatant was mixed with 1 mL of PBS and centrifuged at 500 rcf for 5 min, then the supernatant was discarded and resuspended in 150 μL of PBS. For neutrophils staining, anti-Ly-6G (clone 1A8, Cat. 551460) was used. For alveolar macrophage staining, PerCP/Cy5.5-F4/80, PE-CD11c, and FITC-MHC class II (I-A/I-E) antibodies were used. All flow cytometric antibodies were obtained from BD Bioscience (San Jose, CA, United States).

Supernatants from BALF centrifugation and cell culture supernatants from BMDMs were collected and subsequently assayed for cytokine expression using ELISA kits (R&D System, Minneapolis, MN, United States) according to the manufacturer’s instructions.

The lung tissue was removed from the mouse and soaked in deionized water containing 4% paraformaldehyde for 24 h. The lung tissue was then wrapped in paraffin and cut into 10 μm slices with a slicer after the paraffin had solidified. The slices were stained with H&E staining kits, and images were pictured with an Olympus microscope (BX53, Tokyo, Japan).

Euthanized mice were immersed in 75% ethanol for 2 min, then the skin was cut along the abdominal cavity to expose the lower limbs, and the femur and tibia were removed and transferred to DMEM containing 1% Pen Strep. The bones were held with forceps and then the ends were cut with scissors, and 3 mL of cold PBS was injected into the bones with a syringe, and the cells were collected below in a 50 mL centrifuge tube. The collected cells were centrifuged at 500 rcf for 5 min, the supernatant was discarded and subsequently resuspended in complete medium. Cells were cultured in DMEM supplemented with 10% Fetal Bovine Serum (FBS), 1% Penicillin-streptomycin solution (Pen Strep), and 10 ng/mL M-CSF. After 5 days of culturing, cells were planted. BMDMs were pre-treated with LMZ for 30 min and then challenged with 100 ng/mL LPS for 30 min.

Trizol purchased from Invitrogen (CA, United States) to extract the total RNA from cell and tissue samples, and the Nano-Drop 2000 micro-spectrophotometer was used for RNA quantification. The cDNA was prepared by the reverse transcription according to the instructions of the Toyobo reverse transcription kit (Toyobo, Osaka, Japan) and amplified by SYBR Green RT-PCR Master Mix kit (Toyobo, Osaka, Japan) on the StepOne Plus system (Thermo Fisher Scientific, Waltham, MA, United States). Glyceraldehyde-3-Phosphate Dehydrogenase (Gapdh) to test the gene expression data and normalize mRNA levels of different inflammatory factors. The primer sequences used in the reaction are listed in Table 1.

Protein with equal concentrations in each group were mixed with 5× protein loading buffer (Sigma-Aldrich, United States) and incubated at 99°C for 10 min. Then running the sample by 15% SDS-PAGE gels before being subsequently transferred to NC membranes (GE, United States). The immnunoblots were incubated with 5% milk at room temperature for 0.5–1 h, followed by an incubation at 4°C with primary antibodies overnight. After rinsing the membranes five times with TNET buffer (5 min of each), incubated with IgG (H + L)-HRP secondary antibodies (Santa Cruz, United States) for 1–2 h. The band densities were detected using an LumiBest ECL solution from Shanghai Sharebio Biotechnology (Shanghai, China) and Bio-Rad Laboratories (BIO-RAD, United States). ImageJ Software was using for analysis of the results.

For the purpose of measuring myeloperoxidase (MPO) activity, the lung tissues of mice were weighted and homogenized. Referring to the Myeloperoxidase (MPO) assay kit from Nanjing Jiancheng (Nanjing, China), the activity of MPO was measured.

BMDMs were plated and pre-treated with LMZ 0, 3, 10 or 30 μM for 30 min and challenged with 100 ng/mL LPS for 24 h to collect the supernatant. Nitric oxide (NO) levels in BMDMs were tested by NO Kit (Beyotime, Shanghai, China).

The pre-cultured BMDMs were washed three times with HBSS. The Fluo-4-AM 4 μM working solution (Shanghai Sharebio Biotechnology, China) was added to the cells and incubated at 37°C for 40 min. Then removed Fluo-4-AM green-fluorescent calcium indicator and rewashed the cells with HBSS. The cells were then incubated with or without LMZ (30 μM) at 37°C for 10 min before exposed to LPS (1 μg/mL) and Ca²⁺ channel agonist Bay K8644 (BK, 10 μM). Fluorescent Ca²⁺ was detected under a laser-scanning confocal fluorescence microscope (Zeiss LSM 900, Germany). Fluo-4-AM was performed for 10 min (6s intervals) via fluorescence excited at 494 nm and collect fluorescence emission at 516 nm for calcium imaging.

Data shown mean ± standard deviation (s.d.) of one representative experiment out of three independent in vivo experiments and shown mean ± standard error of the mean (s.e.m) of three independent in vitro experiments. All Data were analyzed using one-way ANOVA (Tukey’s test) with Prism version 9 (GraphPad Software, San Diego, CA, United States). p-values <0.05 were considered to be statistically significant.

To determine the function of lomerizine (LMZ) on acute lung injury (ALI), We established lipopolysaccharide (LPS)-induced ALI mice and treated them with LMZ and dexamethasone (DEX) (Figure 1B). Hematoxylin and eosin (HE) staining of lung tissue showed alveolar wall thickening, lung tissue destruction and infiltration of inflammation cells deteriorated in the LPS-treated lung tissue (Figure 1C). However, LMZ treatment significantly reduced the infiltration of inflammatory cells and protected alveolar structures in LPS-induced ALI mice. The protein concentration in bronchoalveolar lavage fluid (BALF) of ALI mice was increased significantly compared with that of the PBS group (Figure 1D). Meanwhile, the number of cells in BALF of LPS-induced mice was increased significantly (Figure 1E). Myeloperoxidase (MPO) activity was measured as a marker of neutrophil infiltration. LPS-challenged mice showed a significant increase in MPO activity in lung tissue, while LMZ treatment significantly inhibited MPO activity (Figure 1F). We detected Ly6G-positive number to assess the infiltration of neutrophils in lung tissue (Figure 1G). LMZ treatment significantly decreased Ly6G-positive neutrophils in BALF (Figure 1H). These data indicated that LMZ mitigates LPS-induced ALI.

To further investigate the phenotype of LMZ in alleviating LPS-induced ALI, we measured the expression levels of pro-inflammatory cytokines of LPS-induced mice in BALF by RT-PCR. The mRNA expression levels of TNF-α, IL-1β and IL-6 were significantly upregulated by LPS treatment, while suppressed by LMZ treatment dose-dependently, which was similar with dexamethasone (DEX) (Figures 2A–C). Consistently, the enzyme-linked immunosorbent assay (ELISA) results showed that LMZ dose-dependently reduced the protein levels of TNF-α, IL-1β and IL-6 induced by LPS (Figures 2D–F). Macrophages play a critical role in the pulmonary inflammation and are involved in tissue injury and inflammatory recovery processes (Chen et al., 2001). To determine whether LMZ alleviates macrophage activation, we stained the cells from BALF with PE-CD11c, PerCP-Cy5.5-F4/80 and FITC-MHC class II antibodies. Alveolar macrophages can be considered as CD11c⁺ F4/80⁺ cells. MHC class II reflected activation of macrophage after LPS stimulation. Flow cytometry analysis revealed an increased percentage of CD11c⁺ F4/80⁺ MHC class Ⅱ⁺ cells after LPS stimulation in BALF which was restored after LMZ treatment (Figure 2G). Moreover, compared to PBS group, the percentage of MHC class Ⅱ-positive cells were approximately 9-fold increase in LPS group, but only about 2-fold increase in LPS and LMZ co-administration group (Figure 2H). These results indicated that LMZ inhibits macrophage activation in LPS-induced mice.

Toll-like receptor 4 (TLR4), which is a classical pattern recognition receptor, plays a key role in the activation of innate immunity (Vijay, 2018; Sartorius et al., 2021). Mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) are two crucial downstream signaling pathways, which contribute to TLR4-induced pro-inflammatory cytokine production (Kawasaki and Kawai, 2014; Manik and Singh, 2022). To determine the underlying anti-inflammatory mechanism of LMZ, we measured the expression and phosphorylation levels of p65 in NF-κB signal pathway, ERK1/2, p38 MAPK, and JNK in MAPK signal pathway in lung tissues of LPS-induced mice. The phosphorylation level of p65 in lung tissue was remarkably increased after LPS stimulation and gradually decreased after LMZ treatment (Figures 3A,B). To further confirm the negative regulator of LMZ in NF-κB pathway, we investigated the expression levels of NF-κB specific inhibitor IκBα, a classical inhibitor of NF-κB. As shown in Figure 3C, the expression level of IκBα was decreased after LPS stimulation, while enhanced by co-administration with LMZ dose-dependently. Furthermore, the phosphorylated level of p38 MAPK, ERK1/2, and JNK increased significantly after LMZ treatment dose-dependently (Figures 3D–F). These results suggested that LMZ significantly attenuated the production of inflammatory cytokines through MAPK and NF-κB signal pathways in LPS-induced ALI mice.

To determine the role of inflammatory cytokines induced by LPS in macrophages, we detected the expression of inflammatory cytokines using RT-PCR and ELISA in BMDMs. The results showed that the mRNA levels of pro-inflammatory cytokines were dramatically upregulated in LPS-stimulated BMDMs compared to that of the PBS group, while the expression levels were downregulated with LMZ treatment dose-dependently (Figures 4A–C). Similarly, the protein levels of TNF-α, IL-1β and IL-6 in macrophages increased after LPS challenge (Figures 4D–F). Furthermore, we also measured downstream mediators of inflammatory cytokines, inducible nitric oxide synthase (iNOS) and NO, expression levels in BMDMs. The expression levels of iNOS and the concentration of NO were significantly upregulated in BMDMs after LPS stimulation (Figures 4G–I). Compared with the LPS group, pre-treated with LMZ downregulated the expression of iNOS and NO dose-dependently. Furthermore, we measured the activity of NF-κB and MAPK signal pathways in LPS-induced macrophages pretreated with LMZ. Results showed that LMZ significantly inhibited the phosphorylated level of p65 and increased the protein level of IκBα (Figures 5A–C). Similarly, the phosphorylated level of p38 MAPK, JNK, ERK1/2 in MAPK signal pathway were significantly decreased after LMZ pretreatment in LPS-challenged BMDMs (Figures 5D–F). These results show that LMZ alleviated LPS-induced pro-inflammatory response through MAPK and NF-κB pathways.

LPS has been reported to promote Ca²⁺ influx in macrophages (Chen et al., 2001; Letari et al., 1991; Zhou et al., 2006). To determine the association between Ca²⁺ influx and pro-inflammatory cytokines expression in macrophages, we detected Ca²⁺ influx using a Fluo-4 AM green-fluorescent calcium indicator. Compared with PBS group, fluorescence intensity increased sharply within 2 min after LPS or Ca²⁺ channel agonist Bay K8644 (BK) stimulation, which was decreased after LMZ pretreatment in BMDMs (Figures 6A,B). Interestingly, we co-incubated LMZ with BK in LPS-induced BMDMs, the fluorescence intensity increased within 2 min again. Moreover, the expression of Tnfα, Il1β, and Il6 were successfully reduced by LMZ and reversed by BK treatment in BMDMs (Figures 6C–E). Namely, LMZ attenuated LPS-induced inflammation by inhibiting Ca²⁺ influx in BMDMs (Figure 6F). These data suggested that the anti-inflammatory effect of LMZ rely on Ca²⁺ channels activation.