General Information. Reaction mixtures at temperatures higher than ambient temperature were heated in an oil bath. All general chemicals were used directly without further purification. Petroleum ether (PE) refers to the fraction boiling in the 60°C–90°C range. Melting points were detected with an RY-I apparatus and uncorrected. NMR spectra of new compounds were recorded on a Bruker AV 400 spectrometer at 400 MHz (1H NMR), and 101 MHz (13C NMR). Chemical shifts were reported in ppm relative to internal TMS for 1H NMR data, and deuterated solvent for 13C NMR data, respectively. Data are recorded in the following format: chemical shift, multiplicity, coupling constant in hertz (Hz), and signal area integration in natural numbers. High-resolution mass spectra (HRMS) were obtained on Bruker Maxis impact instrument with ESI ion source and TOF mass analyzer.

To the solution of myricitrin (0.8 g, 1.72 mmol) in MeCN (40 mL), was added K₂CO₃ (1.8 g, 12.9 mmol) and dimethyl sulfate (1.2 mL, 12.9 mmol). The reaction mixture was refluxed for 24 h, then filtrated and concentrated in a vacuum. The obtained residue was dissolved in EtOH (40 mL), then was added conc. HCl (1.4 mL). The solution was stirred at 70°C for 12 h, then concentrated in a vacuum to remove EtOH. The residue was dissolved in DCM (40 mL) and washed with a saturated NaCl solution. The organic phase was dried over Na₂SO₄ and then concentrated in vacuo to yield crude product 4.

Compound 5 was synthesized according to the reported procedure (lit.). To the solution of compound 4 (620 mg, 1.6 mmol) and 5 (767 mg, 1.68 mmol) in DCM, was added EDCI (460 mg, 2.4 mmol) and DMAP (20 mg, 0.16 mmol). The solution was stirred at 25°C for 12 h. The contents were adsorbed on silica gel. The product was purified by column chromatography with EA to give the product CMyrH (595 mg, 45% yield) as a light yellow solid. MP 182°C–184°C. ¹H NMR (400 MHz, CDCl₃) δ 7.53–7.43 (m, 3H), 7.41 (d, J = 11.1 Hz, 1H), 7.05 (s, 2H), 6.63 (d, J = 11.3 Hz, 1H), 6.55 (d, J = 2.2 Hz, 1H), 6.51 (s, 1H), 6.40 (d, J = 2.2 Hz, 1H), 4.71–4.64 (m, 1H), 3.95 (s, 3H), 3.94 (s, 3H), 3.92 (s, 3H), 3.91 (s, 3H), 3.89 (s, 3H), 3.88 (s, 6H), 3.62 (s, 3H), 3.08 (t, J = 7.4 Hz, 2H), 2.46 (dd, J = 13.3, 6.5 Hz, 1H), 2.36 (td, J = 12.9, 6.7 Hz, 1H), 2.28–2.18 (m, 1H), 1.98 (s, 3H), 1.94–1.80 (m, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 175.47, 170.48, 170.07, 168.84, 164.74, 161.30, 159.34, 153.84, 153.40, 153.04, 151.45, 151.42, 151.24, 141.66, 140.70, 139.11, 134.68, 134.08, 130.99, 126.95, 124.81, 123.68, 108.75, 108.33, 107.32, 105.77, 104.98, 96.41, 92.88, 61.56, 61.45, 61.15, 60.56, 56.55, 56.48, 56.26, 56.04, 52.68, 38.51, 37.25, 33.31, 30.21, 23.01, 14.34. HRMS (m/z): calculated for C₄₄H₄₆N₂NaO₁₄ [M + Na]⁺) 849.2841, found 849.2859.

Colchicine (Q107209-15g) was purchased from Chemxyz Bio-technology Co., Ltd. (Shanghai, China), and myricetin (MED80097-25g) was from MedBio Pharmaceutical Technology Inc., (Shanghai, China). Bleomycin (B5507-15un) was from Sigma (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and other cell culture supplements were from Gibco (Grand Island, NY, USA).

Human bronchial epithelial cells (BEAS-2B cells, purchased from Procell, Wuhan, China) were incubated in DMEM with 10% FBS under the conditions of 5% CO2 at 37°C. BEAS-2B cells were passaged (dilution, 1:3) when attaining 90% confluency. For the establishment of models, BEAS-2B cells were disposed of diverse concentrations (0.01, 0.1, 0.3, 1, 3, 10, and 30 μg/mL) of BLM for 24 h. For the evaluation of protection of CmyrH, myricetin and colchicine, cells were pre-treated with CMyrH (0.01 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM), myricetin (0.01 μM, 0.1 μM, 1 μM, 3 μM, 10 μM, 30 μM, 100 μM) or colchicine (0.01 nM, 0.1 nM, 1 nM, 0.01 μM, 0.1 μM, 0.3 μM) for 2 h, and then exposed to BLM for 24 h.

CCK-8 assay was applied to evaluate the cell viability of BEAS-2B cells. Briefly, BEAS-2B cells (2500 cells/well) were seeded into 96-well plates. The next day, cells were pre-treated with CMyrH, myricetin or colchicine for 2 h, and therewith undergo BLM for 24 h. At the end of treatment, CCK-8 solution (Fluorescence, Beijing, China) was directly pipetted into the culture medium (100 μL per well) and cells were further incubated at 37°C for 1 h. The absorbance was measured at 562 nm using a microplate reader.

Adult male-specific pathogen-free Sprague-Dawley rats (200 ± 20 g, 6–8 weeks) were purchased from the Guangdong VRLAT Co., Ltd. (Certificate: SYXK (Guangdong) 2022–0063). All rats were housed in standard cages in a climate-controlled room (24°C) under 12/12 h of light/dark cycles for 5 days. The experimental protocol was approved by the Animal Experimentation Ethics Committee of the Shenzhen Second People’s Hospital, protocol number 202300110. All the experimental procedures were carried out in accordance with the international guidelines for the care and use of laboratory animals.

Acute lung injury was induced by airway delivery of bleomycin (BLM, 5 mg/kg) in rats, applying a previously described method (Albert et al., 2020; Zhou et al., 2020). Briefly, the trachea was exposed by making a small midline incision on the neck following anesthesia with isoflurane, and inoculated with 5 mg/kg bleomycin solution in 0.1 mL of PBS using a sterile syringe with a 28-gauge needle. Survival of rats were then checked three times daily for a period of 3 days.

Rats were weighed and randomly divided into six groups (9 rats per group). Rats from Col-treated groups were treated with different doses of colchicine (1.0 and 1.5 mg/kg, i.p.) and the CMyrH-treated groups with CMyrH (1.0 and 3.0 mg/kg, i.p.). Control and ALI-model rats were treated with saline only. Rats were euthanized with an overdose of 5% isofluorane inhalation 3 days after treatment, followed by cervical dislocation after lung tissues and blood samples harvesting.

At the endpoint, rats were sacrificed, the right upper lungs were removed, weighed (wet weight), and dried at 55°C for 72 h. The resulting dry lungs were weighed (dry weight), and the ratio between the two values was determined, without correction for blood content.

The extracted lungs were histopathologically examined and assessed for inflammatory changes. For each group of rats, the left lobe of the lung of 4/8 rats was fixed in 4% of paraformaldehyde, and fixed sections of paraffin-embedded lungs were stained with hematoxylin and eosin (H&E). Lung lesions were scored at four levels (from 0 (normal), 1 (mild), 2 (moderate) to 3 (severe)) by the % of the affected tissue, as previously published (Glineur et al., 2014; Abdel-Daim et al., 2018; Qin et al., 2020).

Immunohistochemical staining was performed on 4 mum paraffin sections prepared from the abovementioned resected specimens. Sections were incubated at 4°C overnight with a rabbit anti-caspase-1 polyclonal antibody (1:200, 22915-1-AP; Proteintech, Chicago, USA), a rabbit anti-ASC polyclonal antibody (1:100, GTX55818; Genetex, USA), a rabbit anti-NLRP-3 polyclonal antibody (1:200, bs10021R; Biosis, Beijing, China), a rabbit anti-GSDMD polyclonal antibody (1:200, 20770-1-AP; Proteintech, Chicago, USA) and a rabbit anti-NE polyclonal antibody (1:100, A13015; Abclonal, Wuhan, China) according to the manufacturer’s protocol. The IHC images were analyzed using a TEKsqray Digital Slide Scanner (SQS-40P, Shenzhen, China) and ImageJ software.

Lung tissue lysates were prepared by homogenizing tissue in ice-cold lysis buffer containing 1 mM phenylmethane sulfonyl fluoride (PMSF), protease and phosphatase inhibitor cocktails (Servicebio, Wuhan, China), using a high-speed low-temperature tissue grinding machine (Servicebio, Wuhan, China), and then centrifuged at 13,000 rpm for 15 min at 4°C. The supernatant was collected and used to measure total protein concentration with the BCA assay (Solarbio, Beijing, China). The levels of inflammatory cytokines IL-1β, IL-6, IL-18 and TNF-α were measured by ELISA, using a Quantikine ELISA Kit (Spbio, Wuhan, China) and a microplate reader (Molecular devic, Guangzhou, China) at 450 nm, according to the manufacturer’s protocol, specific for each investigated cytokine.

The crystal structure of neutrophil elastase (PDB ID: 6SMA) was obtained from the RSCB PDB database (http://www.rcsb.org/) and saved as a PDB format file. The structures of the proposed ligands were drawn through ChemDraw 15.0 and then converted to a 3D structure following energy minimization by Chem3D 16.0. The molecular docking was performed by Autodock 4.2, and binding energy was predicted to evaluate the affinity of the target proteins and active components. The best binding conformations were selected from 50 lowest energy docked structures based on a criterion combining the lowest binding energy and root-mean-square deviation values (RMSD <1) (Bahuguna et al., 2022), and were displayed by PyMOL. Here, if the binding energy of the ligand <0, the receptors bind spontaneously, and if the binding energy is ≤ −5.0 kcal/mol, they form a stable docking (Guo et al., 2022).

Statistical analyses were performed using GraphPad Prism Software (version 7.0 and 8.0). One-way ANOVA followed by posthoc tests, incorporating a Bonferroni correction for multiple comparisons, was employed, where p < 0.05 was considered significant. The cell viability and inflammatory cytokines were presented as mean ± SEM, and the lung W/D ratio was expressed as means ± SD. Cumulative survival rates were compared using the log-rank statistical analysis. Histological scores were expressed as median (interquartile range) and analyzed by the Kruskal–Wallis test followed by the Mann-Whitney U test.

The synthetic method was shown in Figure 2. The myricitrin 3) was firstly methylated with excess dimethyl sulfate and Potassium carbonate. After being refluxed in MeCN for 24 h, the crude product was directly treated with HCl to afford the intermediate 4. Then compound 5 was synthesized from (−)-colchicine 1) in one step according to our reported procedure (Pu et al., 2022). Compounds 4 and 5 underwent an esterification reaction with EDCI and DMAP to provide the new hybrid CMyrH in moderate yield.

Since BLM at the dose of 10 μM resulted in approximately 50% cell death (Figure 3A), then 10 μM inducer was chosen for the subsequent experiments. To evaluate the protective effects of CMyrH, colchicine or myricetin against BLM-induced ALI, BEAS-2B cells were pretreated for 2 h with different concentrations of these compounds, then exposed to 10 μM BLM. It was found that CMyrH at 3 μM, colchicine at 0.01 nM, and myricetin at 30–100 μM protected BEAS-2B cells from BLM-induced injury (Figures 3C–E).

After an intratracheal BLM challenge, the survival rates of the model and CMyrH (1.0 mg/kg and 3.0 mg/kg, i.p.) groups remained at 100% within 72 h. However, we found that 6 out of 9 rats at colchicine doses of 1.5 mg/kg (vs. CMyrH 3.0 mg/kg i.p. group, log-rank test, p = 0.004) and 2 out of 9 rats at colchicine doses of 1.0 mg/kg (vs. CMyrH 1.0 mg/kg i.p. group, log-rank test, p = 0.145) were dead during 3 days of drug treatment (Figure 4A).

The lung wet/dry weight ratio (W/D) was elucidated to estimate pulmonary edema. Lung edema formation was significantly enhanced in the model group, as shown by an increased lung W/D (p < 0.001). All CMyrH and colchicine treatments led to a reduced lung W/D as compared to the model (p < 0.001), but no differences in the lung W/D were detected between COL- (1.0 mg/kg) and CMyrH- (3.0 mg/kg) treatment groups (Figure 4B).

Histologically, the lungs of BLM-induced rats showed moderate-to-severe pneumonia. CMyrH- (1.0 mg/kg) treated rats showed less severe (moderate) histopathological changes in the lungs. CMyrH- (3.0 mg/kg) and COL- (1.0 mg/kg) showed multifocal bronchiolitis and bronchitis to mild to moderate bronchointerstitial pneumonia (Figure 5). As shown in Figure 4C, median scoring of the severity of lung tissue damage at the end of the study demonstrated substantially reduced pathological scores in rats treated with 3.0 mg/kg CMyrH (p < 0.01) and 1.0 mg/kg colchicine (p < 0.05). The CMyrH- (1.0 mg/kg) treated rats also had reduced pathological median scores in lung tissues compared to the ALI-model rats, but this difference was not statistically significant (p < 0.05).

As shown in Figures 4D–G, the lung cytokine productions of IL-1β, IL-6, IL-18 and TNF–α in ALI-model rats were significantly increased postoperative administration of BLM. However, Treatments with both drugs (CMyrH and colchicine) mitigated the cytokine overproductions of IL-1β, IL-6, IL-18 and TNF-α, as compared to the ALI-model rats. The higher dose of CMyrH more effectively alleviated the inflammatory cytokines levels than the low dose.

According to our research, the ALI-model group, when compared with the control group, showed elevated expression of NLRP3, ASC, caspase-1 and GSDMD, with an increase in the levels of IL-1β and IL-18. However, the groups which received CMyrH (1.0 and 3.0 mg/kg) exhibited a weak expression of caspase-1, ASC, GSDMD and NLRP3 accompanied by a reduction in IL-1β and IL-18 secretion in a dose-dependent manner than ALI-model rats. The present IHC analysis results suggested the ability of CMyrH to suppress pyroptosis caused by BLM in the rats’ lung tissues.

The docking results provided the first evaluation as to which CMyrH exhibited a stronger binding affinity (higher docking score value) to NE, namely, CMyrH-b, which exhibited a higher affinity to NE than CMyrH-a, -c, and -d. Furthermore, silico studies showed that CMyrH inhibited NE protein by forming both a covalent bond between α-/β-unsaturated carbonyl groups in the colchicine fragment of CMyrH-b and Ser195, a hydrogen bond between the amino groups outside the 7-membered ring in colchicine fragment of CMyrH-b and Ser214, and a hydrogen bond between the ketone carbonyl groups in myricetin fragment of CMyrH-b and Gly216. Results showed that CMyrH has the highest binding energy values, −13.54 kcal/mol for NE protein (Ki value was 118.49 pM), followed by colchicine and myricetin with docking scores of −5.86 kcal/mol and −1.89 kcal/mol (Ki values were 51.03 μM and 40.93 mM), respectively.

As for some adverse reactions, we altered the chemical structure of Colchicine to enhance its clinical application. Colchicine-CD44-Targeted Peptide conjugate decreased the cytotoxic effect of colchicine while maintaining anti-inflammatory against gout (Zoghebi et al., 2019). However, there were few studies about the hybridization of Colchicine with bioactive natural molecules to exert its efficient biological effects with low toxicity in ALI. In this current study, we designed a new kind of C-10-modified Colchicinoid which was a heterozygous molecule of Colchicine and Myricetin with a simple amino acid as the linker (Figure 2).

Subsequently, we used human lung epithelial BEAS-2B cells to estimate the effects of CMyrH and its parent molecules on BLM-induced cell injury. As shown in Figure 3, CMyrH antagonized significantly BLM-induced BEAS-2B cells injury, in a dose-dependent manner, especially in the 3 μM group; myricetin revised BLM-induced injury in increased dose-manner at 30 μM; colchicine reversed BLM-induced injury at 0.01 nM, but the safety range between the upper and lower limits was extremely narrow and even overlap, which creates difficulties when setting therapeutic margins. Thus, these results suggested that a novel hybrid CMyrH exhibited a better and safer anti-inflammatory effect on BLM-induced BEAS-2B cell injury compared with colchicine.

In the end, the CMyrH’s ability to improve ALI was further verified by using a rat BLM-induced acute lung injury model. As shown in Figures 4B–C and Figures 5H, E staining of lung tissue sections revealed less lung injury in rats administered CMyrH (at 1.0 mg/kg and 3.0 mg/kg) for 3 days post-challenge, with significantly lower lung injury score and W/D, indicating mild lung inflammation and pulmonary edema. CMyrH 1.0 mg/kg i.p. was relatively safe in rats up to 72 h, in addition, an acute toxicity study of CMyrH showed no signs of toxicity or cause mortality in rats even at doses = 3.0 mg/kg (Figure 4A). For reference, colchicine at 1.0 mg/kg reduced histopathological end scores and W/D in their lung tissues compared to the model rats (Figures 4B,C), however, colchicine administration for 72 h showed severe toxic effects with a 22.2%, 66.7% mortality rate at the doses of 1.0 mg/kg and 1.5 mg/kg i.p. (Figure 4A). Furthermore, multiple cytokines in lung tissue dramatically declined after treatment with the diverse concentration of CMyrH (1.0 mg/kg and 3.0 mg/kg), importantly, a high dose of CMyrH remarkably reversed proinflammatory cytokines including IL-1β, IL-6, IL-18 and TNF-α more than that of a low dose (Figures 4D–G). These findings indicated that threefold doses of CMyrH had already achieved anti-inflammation efficacy comparable or even superior to colchicine, without severe toxicity or treatment-related deaths.

Pyroptosis is a form of cell death triggered by the innate immune system that has been implicated in the pathogenesis of acute lung injury (Hsu et al., 2022; Liang et al., 2022). Activated NLRP3 recruits ASC and caspase-1 to form the NLRP3 inflammasome, resulting in the cleavage of gasdermin D (GSDMD) and membrane pores formation (pyroptosis) accompanied by the proinflammatory cytokines IL-1β and IL-18 release (Faria et al., 2021; Tummers and Green, 2022). In the current study, the immunohistochemical expression of caspase-1, ASC, GSDMD and NLRP3 was investigated in lung sections to reflect on the anti-pyroptosis potential of CMyrH. Our findings showed a decrease in caspase-1, ASC, GSDMD and NLRP3 immunoreactivity in lung sections accompanied by a reduction in cytokine production of IL-1β and IL-18 affiliated to both CMyrH-treated (1.0 mg/kg and 3.0 mg/kg) groups. These evidences suggested that CMyrH might protect rats against ALI caused by BLM by alleviating lung inflammation through the inhibition of pyroptosis.

Human neutrophil elastase (HNE) is a potent serine protease secreted by activated polymorphonuclear neutrophils (PMNs), whose uncontrolled production can result in inflammatory-derived disease conditions. (González-Fernández et al., 2018.). Besides its crucial role in powerful host defense, HNE is well known as one of the most destructive enzymes in the human body (Thierry, 2020). It is reported that excessive HNE activity is involved in the pathogenesis of ALI, thereby administering anti-NE might prevent lung tissue damage caused by inflammation (Conrad and Eltzschig, 2020). Analyzing the resultant in silico interactions of the CMyrH-b molecule with the residues of host HNE protein showed that CMyrH-b molecule significantly hit most of the active site residues of the HNE protein with strong interactions, such as covalent bond and hydrogen bonding. The docking score with NE indicated that CMyrH was the highest affinity binding with the lowest energy value −13.54 kcal/mol (Ki value was 118.49 pM) as compared to other docking complexes. Immunohistochemical studies confirmed this result, with lung tissues from CMyrH-treated rats analyzed showing a decrease in immunoreactivity for antibodies specific to HNE (Figure 6 Figure 7). It is suggested that CMyrH-b had a strong inhibitory effect on neutrophil elastase, which might also be an important mechanism for CMyrH-b to antagonize ALI.