CTS (purity ≥ 98%) were obtained from the Laboratory of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University (Guangzhou, China). LPS was purchased from Sigma-Aldrich (St. Louis, USA). Compound C were purchased from Selleck (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco (NY, USA). Fetal bovine serum (FBS) was abtained from HyClone (Logan, USA). Myeloperoxidase (MPO) and lactic acid assay kits were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The antibodies against CD86 (A1199) and Arg-1 (A4923) were obtained from ABclonal (Wuhan, China). The antibodies against CD206 (ab64693) were purchased from Abcam (Cambridge, MA). The antibody against iNOS (AF0199) was from Affinity Biosciences (OH, United States). The antibodies against β-actin (6600-1-Ig), GLUT1 66290-1-Ig) and PFKFB3 (3763-1-AP) were purchased from Proteintech (Chicago, USA). The antibodies against PKM2 (D78A4), HIF-1α (D1S7W), p-AMPK (40H9) and AMPK (2532) were obtained from Cell Signaling Technology (Danvers, USA). The antibodies for flow cytometry, including phycoerythrin (PE) anti- CD86 (105007) and allophycocyanin (APC) anti-CD206 (141708) were obtained from BioLegend (San Diego, USA).

The animal procedures were approved by the Research Ethics Committee of Sun Yat-sen University and conducted following the Guide for the Care and Use of Laboratory (NIH Publication No. 85-23, revised 1996). Sprague-Dawley rats (SD rats, male, SPF grade, 6-8 weeks, weighing 200-230 g) were supplied by the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China) and the certification No. 44008500024762. LPS (5 mg/kg) was dissolved in normal saline (NS) and administrated by intratracheal instillation to SD and the acute lung injury model was established by LPS administration for 24 h. SD rats were randomly divided into several groups (10 rats in each group): the control group, the LPS-induced acute lung injury model group and the CTS treatment groups at three different concentrations. CTS was dissolved in sodium carboxymethylcellulose (CMC-Na, 5%, W/V) at different concentrations (15, 30 and 60 mg/kg/day). Before LPS treatment, the CTS treatment group was pre-administered intragastrically for 5 days, while the control and model groups were given the same volume of solvent solution. The rats in each drug administration group were given drug intervention once at 6 hours, 12 hours and 18 hours after modeling.

Pulmonary function was measured by using a whole-body plethysmograph (Emka Technologies, Paris, France) for rats. The parameters of pulmonary function included enhanced pause (Penh), relaxation time (RT), end inspiratory pause (EIP), end expiratory pause (EEP) and minute ventilation volume (MV). Rats were placed in a plethysmograph chamber and 10 min was used for acclimation before 5 min of assessing respiratory parameters.

At the end of the in vivo experiment, all rats were anesthetized and sacrificed. The whole lung of the rat was quickly removed and weighed. The lung weight to body weight ratios were calculated according to the following formula: Lung to Body weight ratio = (Lung weight (g))/(Body weight (g)) × 100%. Subsequently, left lung tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectionalized, and stained with hematoxylin and eosin (HE). HE scores were calculated by light microscopic analysis of four parameters including alveolar septal thickness, interstitial edema, infiltration of inflammatory cells, and alveolar congestion/collapse. Each parameter was categorized into four grades: 0 = normal; 1 ≤ 25%; 2 = 25–50%; 3 = 50–75%; and 4 ≥ 75%, and the mean score of the four parameters was used to represent the overall lung injury (25). Histopathological images were captured and analysis by using light microscope at 400x magnification (EVOS FL Auto Cell Imaging System, USA). The right lung was excised and weighed to assay wet weight, followed by drying at 80 ? for 48 h to obtain dry weight. The lung wet/dry (W/D) weight ratio W/D weight ratio was calculated to indicate pulmonary edema formation.

MPO activity of lung tissue was measured by a commercial kit according to the manufacturer’s instructions (A044, Nanjing Jiancheng Bioengineering Institute, China).

By intratracheal injection of 5 mL sterile saline and then slowly withdrawn, repeated irrigation three times. The collected bronchoalveolar lavage fluid was centrifuged at 300 g for 10 min at 4°C, and the supernatant was extracted for subsequent cytokine and total protein inspection. Cytokine levels in the supernatants of BALF were determined using commercially available TNF-α, IL-1β, IL-6, IL-10 ELISA kits (Wuhan Huamei Biotech Co., Ltd., Wuhan, China) according to the manufacturer’s instructions. Total protein concentration in the supernatant BALF was determined using the BCA protein quantification kit (Thermo Fisher Scientific, Waltham, USA).

RAW264.7 cell line was obtained from ProCell (Wuhan, China) and maintained in 37°C incubators with 5% CO₂. The cultured media was DMEM media with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin. RAW264.7 were pretreated with the indicated concentrations of CTS (2.5, 5, 10 μM) for 2 h before being stimulated with LPS (1 μg/mL) for another 24 h.

Glucose uptake of RAW264.7 cells were assayed by using the Glucose Uptake-Glo Assay kit from Promega (Wisconsin, USA). The level of lacticte acid was detected by using a commercial kit (Nanjing Jiancheng Bioengineering Institute, China).

Frozen lung tissue sections were fixed in acetone for 20 min, then permeabilized by 0.3% Triton X-100 (Sigma, St. Louis, USA) for 15 min and blocked by 10% goat serum for 30 min. Subsequently, sections were incubated with anti-CD68 antibody (BIO-RAD, MCA341GA 1:100 dilution) overnight at 4°C, anti-CD86 antibody (ABclonal, A11991, 100 dilution), and anti-CD206 antibody respectively (Abcam, ab64693, 1:100 dilution). Sections were washed with PBS followed by incubation with fluorescent secondary antibodies (Abcam, ab150116, ab150077) in dark for 1 h at room temperature. Finally, the sections were re-stained with 4’,6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature. Fluorescent images were captured under a fluorescent microscope (EVOS FL Auto Cell Imaging System, USA).

For immunohistochemistry (IHC) analysis, paraffin-embedded lung tissues were deparaffinized, rehydrated through an alcohol series followed by antigen retrieval with sodium citrate buffer. Tumor sections were blocked with 5% normal goat serum with 0.3% Triton X-100 and 3% H₂O₂ in PBS for 60 min at room temperature and then incubated with anti-iNOS antibody (Affinity Biosciences, AF0199, 1:100 dilution) or anti-Arg-1 antibody (ABclonal, A4923, 1:100 dilution) at 4°C overnight. Then, HRP-conjugated goat anti-rabbit IgG polyclonal antibody (Abcam, ab6721, 1:1000) were used. Alternatively, sections were stained with DAB and restained with hematoxylin, and then photographed using a microscope (EVOS FL Auto Cell Imaging System, USA). The area of the positive area was calculated using Image-Pro Plus 6.0 software (Media Cyber??netics, Silver Spring, USA).

RAW264.7 cells were grown in 96-well plates at a density of 10000 cells per well and cultured overnight in the incubator. Different concentrations of CTS (2.5, 5, 10 μM) were administrated to cells for 24 h with or without LPS (1 μg/mL) stimulation. Subsequently, 10 μL of CCK-8 solution was added into each well and incubated for another 4 h. The absorbance values of each well were measured at 450 nm using a microplate reader (Bio-Tek, Winooski, USA).

RAW264.7 cell suspension was collected and incubated with anti-CD16/32 (BioLegend, San Diego, USA) at 4 ? for 20 min to block Fc receptor. And then, the cells were washed twice in staining buffer (BioLegend, San Diego, USA) and stained with anti-CD86-PE antibody (BioLegend, San Diego, USA)) or anti-CD206-APC antibody (BioLegend, San Diego, USA) for 30 min. Followed by washing twice with staining buffer (BioLegend, San Diego, USA), resuspended the RAW264.7 cells in 300 μL staining buffer. Flow cytometry data were obtained using a CytoFLEX S flow cytometer (Beckman Coutler, Brea, USA) and analyzed using FlowJo software (Ashland, USA).

OCR and ECAR were measured by using XF-96 Extracellular flux analyzer (Seahorse Bioscience, North Billerica, MA, USA) to assess mitochondrial oxidative phosphorylation and glycolysis capacity respectively. CTS at different concentrations (2.5, 5, 10 μM) were pre-incubated with RAW264.7 cells for 2 h with co-stimulation of LPS (1 μg/mL) for 24 h. For the ECAR assay, the medium was replaced with XF solution containing 2 mmol/L L-glutamine prior to analysis. Glucose (10 mM), oligomycin (1 μM) and 2-DG (50 mM) were used to determine the glycolysis rate, glycolysis capacity and glycolysis reserve capacity of cells. For the OCR assay, the medium was replaced with XF solution containing glucose (2.5 M), pyruvate (1 mM) and glutamine (1 mM) prior to analysis. Oligomycin (1 μM), FCCP (0.75 μM), rotenone (0.5 μM) and antimycin A (0.5 μM) were used to determine basal respiration, mitochondrial ATP production and maximum respiration.

Total protein was extracted from lung tissues or RAW264.7 cells using RIPA lysis buffers containing protease inhibitors and phosphatase inhibitors. The concentrations of proteins were measured using the BCA protein quantification kit (Thermo Fisher Scientific, Waltham, USA). Equal amount of protein samples was boiled and loaded in 10% SDS-PAGE for separation and transferred to PVDF membrane (Meck Millipore, Burlington, USA). The PVDF membranes were blocked with 5% skim milk at room temperature for 1 hour and then incubated with different primary antibodies at 4°C overnight. The membranes were incubated with the corresponding secondary antibodies for 1 h at room temperature. The proteins were visualized by chemiluminescence using an ECL system (GE Healthcare, Pittsburgh, USA) and the images were captured using an imaging system (Tanon, Shanghai, China).

Total RNA was extracted from RAW264.7 cells using Trizol reagent (Invitrogen, Carlsbad, USA) and cDNA was synthesized using the QuantiTect reverse transcription kit (QIAGEN, Valencia, USA) according to the manufacturer’s protocol. The relative mRNA expression level was determined using the 2-delta delta Ct analysis method, where GAPDH was used as a home keeper gene. The primer sequences used in this experiment were listed in Table 1.

The results were expressed as mean ± standard error of mean (SEM) from at least three independent experiments and analyzed by using GraphPad Prism 8.0 software (San Diego, CA, USA). Student’s t-test was used to compare differences between two groups. Differences between groups were compared using one-way analysis of variance (ANOVA) followed by Post hoc Bonferroni’s test. P < 0.05 was considered statistically significant.

To determine the effects of CTS on pulmonary function of rats, non-invasive lung function tests were performed in rats. Penh is an indicator of airway resistance to positively reflect the constriction degree of internal bronchi. Compared with control group, a single dose of LPS (5 mg/kg) administration through intratracheal instillation significantly increased the value of enhanced pause (Penh) (Figure 1A), shortened the time period of end-inspiratory pause time (EIP) and relaxation time (RT) (Figures 1B, C), prolonged end-expiratory pause time (EEP) (Figure 1D), and finally decreased minute ventilation volume (MV) (Figure 1E). These results indicated that LPS administration successfully induced acute lung injury of rats. In contrast, CTS treatment at 15, 30 and 60 mg/kg effectively improved pulmonary function of rat in a dose-dependent manner (Figures 1A–E).

Additionally, pathological changes of lung tissue were analyzed by HE staining. As Figures 1F, G shown, the pulmonary structure was destroyed following LPS-stimulation as evidenced by disorganized pulmonary alveoli structure, obvious perivascular edema, widened space and significantly thickened alveolar walls accompanied by a large number of inflammatory cell infiltration, all of which were effectively relieved by CTS. Furthermore, LPS-induced increase in lung coefficient and right lung wet to dry weight ratio (Lung W/D ratio) were also relieved by CTS treatment in a dose dependent manner (Figures 1H, I). Total protein concentration in BALF and neutrophil infiltration was used to assess permeability of lung and the severity of pulmonary edema. Compared with the control group, LPS significantly induced the total protein concentration of BALF (Figure 1J). while CTS decreased the level of BALF in dose-dependent manner. In addition, LPS induced the neutrophil infiltration as indicated by the increased activity of MPO in lung tissue (Figure 1K). Moreover, CTS treatment relieved the protein concentration of BALF and neutrophil infiltration. All these results revealed that CTS alleviated lung pathology and inflammatory cell infiltration in a dose-dependent manner.

Macrophages are sentinel cells of the lung innate immune system and can be differentiated into the proinflammatory (M1) phenotype or anti-inflammatory (M2) phenotype macrophages according to different stimulations (26, 27). Excessive activation of M1 phenotype macrophages or deficiency of M2 phenotype macrophages is the key factors causing uncontrolled lung inflammation in acute lung injury (28). To explore the in vivo effects of CTS on inflammatory response and macrophage polarization, we measured the levels of different cytokines in alveolar lavage fluid and the changes of macrophage polarization subtypes in lung tissue. Our ELISA results showed that LPS significantly induced the secretion of pro-inflammatory cytokines (such as IL-1β, IL-6 and TNF-α) and inhibited the secretion of anti-inflammatory factors (IL-10) in alveolar lavage fluid (Figure 2A). However, CTS significantly inhibited the secretion of IL-1β, IL-6 and TNF-α, and increased the IL-10 levels in alveolar lavage fluid (Figure 2A). Cluster of differentiation 68 (CD68) was used as a pan-macrophage marker, cluster of differentiation 86 (CD86) was used as a specific marker for M1 macrophages, and cluster of differentiation 206 (CD206) was used as a specific marker for M2 macrophages (29). Our immunofluorescence results showed that the number of CD68⁺CD86⁺ macrophages significantly increased during LPS-induced acute lung injury rats (Figures 2B, C). Western blotting and immunohistochemical also showed that the expression of iNOS was also increased following LPS-stimulation (Figures 2D, E). All these results indicated that LPS promoted macrophage differentiated into the proinflammatory type. However, we found that CTS could significantly promote the trans-differentiation of M1 macrophages into M2 macrophages, evidence by decreased CD68⁺CD86⁺ and iNOS level and increased the protein levels of CD68⁺CD206⁺ and Arg1 level.

Our in vivo results suggested that CTS inhibited LPS-induced inflammatory response of lung tissues by regulating macrophage polarization. Therefore, we further validated the effects of CTS on macrophage polarization by using RAW264.7 cell line. Firstly, CCK-8 results showed that CTS treatment with or without LPS did not alter the cell viability of RAW264.7 cell line (Figure 3A). According to our previous studies (30), CTS was used at different concentrations (2.5, 5, 10 μM) to inhibit inflammation. CTS could dose-dependently relieved LPS-induced expression of iNOS and CD86 (Figure 3B), and increased expression of Arg-1 and CD206 (Figure 3C). Our flow cytometry results furthermore showed that CTS dose-dependently decreased the proportion of CD86⁺ M1 macrophages and increased the proportion of CD206⁺ M2 macrophages (Figures 3D, E). Collectively, both in vitro and in vivo results consistently showed that CTS inhibited the polarization of macrophage and relieved inflammation in vitro.

Phenotypic transformation of macrophages are closely related to the metabolism pattern (31). Based on metabolic characteristics of different macrophages phenotypes, M1 phenotype macrophages mainly rely on glycolysis, while M2 phenotype macrophages rely on fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS) (32). Therefore, we further detected the effects of CTS on macrophage metabolism. Our results showed that CTS abrogated LPS-induced glucose uptake and lactic acid production in macrophage in a dose-dependent manner (Figures 4A, B). Extracellular acidification rate (ECAR) is a key indicator for measuring glycolysis flux and mitochondrial oxygen consumption rate (OCR) is the gold standard for detecting oxidative phosphorylation. Subsequently, we detected ECAR and OCR respectively in macrophage by using XF-96 extracellular flux analyzer. As shown in Figures 4C, D, LPS significantly increased glycolysis rate, glycolytic capacity and higher glycolysis reserve capacity, whereas basal respiration, mitochondrial related ATP production and maximum respiration rate were significantly inhibited in macrophage following LPS stimulation (Figures 4E, F). Conversely, CTS effectively relieved glycolysis and improved mitochondrial oxidative phosphorylation of macrophage (Figures 4C–F). Moreover, we detected the expression of proteins closely related to glycolysis such as pyruvate kinase M2 (PKM2), and 6-phosphofructo-2kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and glucose transporter 1 (GLUT1). As shown in Figure 4G, the expression of PKM2, PFKFB3 and GLUT1 were significantly increased by LPS, whereas CTS treatment effectively inhibited the expression of these proteins. These results indicated that CTS might block LPS-induced metabolic dysfunction in macrophage.

As a sensor of intracellular energy metabolism, AMP-activated protein kinase (AMPK) plays an important role in oxidative phosphorylation, cell growth and regulation of immune responses (33). AMPK has been shown to be a metabolic regulator of macrophage polarization (34), which was inhibited in LPS-induced M1 type macrophage and meant increased glycolysis as major metabolism pathway (35). However, activation of AMPK could switch the metabolism pattern from glycolysis to aerobic oxidation and promote the transformation of macrophages from M1 to M2 phenotype (36, 37). According to previous studies (38), CTS is an activator of AMPK pathway. Whether it could relieve LPS induced inflammation and metabolism dysfunction by activating AMPK remains unknown. Therefore, we detected the phosphorylation at ser172 and total protein level of AMPK in macrophage with CTS and LPS co-treatment. As shown in Figures 5A, B, both the in vivo and in vitro results showed that the phosphorylation of AMPK was decreased following LPS stimulation, whereas CTS treatment significantly augmented AMPK ser127 phosphorylation (Figures 5A, B). These results indicated that CTS activated AMPK during ALI.

Compound C, a specific pharmacological inhibitor for AMPK, was used to further verify the involvement of AMPK on the protective of CTS. And then the type of macrophage polarization was measured. As Figure 5C shown, compound C (5 μM) treatment effectively blocked the phosphorylation of AMPK. Moreover, the expression of iNOS and CD86 were inhibited and the expression of Arg-1 and CD206 were augmented by CTS, which were deprived following compound C stimulation (Figure 5D). Flow cytometry results also revealed that compound C inhibited CTS induced M2 type macrophage and promoted polarization of macrophages to M1 type (Figure 5E). These results suggest that AMPK was closely involved in the regulation of CTS on macrophage polarization.

Since changes of macrophage polarization phenotype are closely related to cell metabolism, we further explored whether AMPK is involved in the regulation on metabolism of macrophage. Our results showed that LPS sharply increased rate of glycolysis and deterioration of mitochondrial oxidative phosphorylation of macrophage, while CTS antagonized these results (Figures 6A–D). However, inhibition of AMPK partially blocked the effects of CTS on macrophage metabolism dysfunction. There is ample evidence that AMPK acts as a negative regulator of the “Warburg” effect, by inhibiting HIF-1α-mediated glycolysis (39–41). The LPS-induced metabolic switch from OXPHOS to aerobic glycolysis in macrophages is associated with HIF1α activation (42). This metabolic pattern switch depends on the stabilization of HIF-1α and resulting in in the expression of key glycolytic proteins, such as GLUT1, PKM2, and PFKFB3 (43). Here, our results showed that LPS significantly promoted the expression of HIF-1α, which was ameliorated by CTS treatment (Figure 6E). However, inhibition of AMPK by compound C blocked the effect of CTS on HIF-1α and the mRNA expression of GLUT1 and PFKFB3 (Figures 6E, F). FAO, an important provider of acetyl CoA that fuels the TCA cycle and OXPHOS, is significantly increased in M2 macrophages (44). AMPK is a key regulator of FAO, which promotes the increase of intracellular FAO by increasing the expression of fatty acid metabolism enzymes, such as carnitine palmitoyltransferase 1A (CPT1A), carnitine palmitoyltransferase 2 (CPT2) and medium-chain acyl-coA dehydrogenase (MCAD) (45, 46). Therefore, we speculated that CTS enhanced mitochondrial oxidative phosphorylation through activation of AMPK-mediated increases in FAO. The effect of CTS on the expression of fatty acid oxidation genes were determined. Our results showed that the expressions of CPT1A, CPT2 and MCAD were significantly inhibited by LPS while promoted by CTS (Figure 6G). However, Compound C blocked the mRNA expression of CPT1A, CPT-2 and MCAD. These results suggested that AMPK was involved in role of CTS on regulation of macrophage polarization and metabolism reprogramming.