Chemo-proteomics reveals dihydrocaffeic acid exhibits anti-inflammation effects via Transaldolase 1 mediated PERK-NF-κB pathway
Acute pneumonia is a common inflammatory pathological process . The global epidemic of Corona Virus Disease 2019 manifests itself as acute respiratory disease and pneumonia, in which the inflammatory response plays a key role . Inflammation has become known as a leading etiologic agent of various of disorders and its mediated tissue damage is seen in many organ systems . Overall, the inflammatory response is a critical element in pathological progression of organ illnesses, and clarifying the specific pathogenesis of acute pneumonia will help us to find potential therapeutic targets and new preventive approaches to the disease. Dihydrocaffeic acid (DA) is a kind of metabolite of Chlorogenic acid (CGA), which is one of the major component in coffee . The biological activities of DA include antioxidant activity , anti-inflammatory activity , enzyme inhibitory activity , cytoprotective activity , anticancer activity , antibacterial activity  as well as antiviral activity . It is reported that DA shows anti-inflammatory properties in a variety of diseases and the molecular mechanisms involved are very complex . A recent report has shown that DA improved IL-1β- triggered inflammation through supressing NF-κB pathways . However, the mechanisms of action of DA for treating acute pneumonia -associated inflammation are still not well known. Activity-based chemical proteomic (ABPP) is a widely used method to characterize the interactions between protein targets and small bioactive molecules for consequent characterization of targets . It has become one of the commonly used strategies for analyzing cellular targets of various natural species in recent years. Using ABPP and liquid chromatography liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, we have already succeeded in characterizing the direct targets and specific mechanisms of multiple natural compounds in different disease models . Here, we aimed to investigate the effect of DA in acute pneumonia and uncover the potential mechanisms via ABPP. In this study, we assessed that DA effectively ameliorates lipopolysaccharides (LPS)-activated acute pneumonia, and identified the underlying cellular and molecular mechanisms by using a chemical probe of DA (DA-P) and ABPP technologies. We aimed to provide a novel insight into the molecular mechanisms of the effect of DA on acute pneumonia.   Animal experiments In this study, all animal experiments were approved by the Center of Shenzhen People’s Hospital for the care and use of laboratory animals. Balb/C male mice (25 ± 3 g) were obtained from Vital River Laboratory Animal Technology, and maintained under standard environmental conditions. A total of 40 mice were randomized into five groups: control group, model group (LPS, Lipopolysaccharides, MedChemExpress, Cat. No. HY-D1056, MCE, 10 mg/kg), positive drug group (Dexamethasone, DEX, MedChemExpress, CAS: 50-02-2, Cat. No. HY-14648, 99.86%, MCE, 10 mg/kg), DA-Low dose group (Dihydrocaffeic acid, MedChemExpress, CAS: 1078-61-1, Cat. No. HY-N2406, 99.38%, 10 mg/kg) and DA-High dose group (20 mg/kg). After 14 days of pre-administration of DMSO, DA or DEX, LPS was administered intranasally into mice. Finally, blood and lung tissue samples were collected after euthanasia. Hematoxylin–eosin (H&E) staining Lung tissues from mice were fixed by adding 4% paraformaldehyde (PFA), and embedded in paraffin, sliced and stained with hematoxylin–eosin (H&E). This staining procedure was employed to explore the differences in histological morphology as previous reported . Cell culture RAW 264.7 cells were purchased from Cell Resource Center, Institute of Basic Medicine, China Academy of Medical Sciences, and the resource number is 1101MOU-PUMC000146. Cells were cultured in DMEM containing 10% FBS, and maintained in a 37 °C incubator with 5% (v/v) CO2. Cells grown to 70–90% were collected for the following experiment. Cell viability Cell viability was measured using the CCK-8 assay (C0038, Beyotime) based on manufacturer’s instructions. RAW 264.7 cells were seeded into 96-well plates for overnight and then added with various concentrations of DA or DA-P. Measurement of NO, inflammatory cytokines and Transaldolase 1 RAW 264.7 cells were cultured with LPS (1 µg/ml) for 24 h to develop a model of inflammation, then treated with various concentrations of DA or DA-P. The cell culture supernatant was then collected for further research. Based on the manufacturer’s manuals, Nitric Oxide (NO) Assay Kit and Elisa kits were used to measure the concentrations of NO (Biodee, DEM106), TNF-α (Biorigin, BN50578), IL-1β (Biorigin, BN50543) and IL-6 (Biorigin, BN50553) in culture supernatant, lung tissues and serum. Besides, Elisa kits were used to measure the content (Mlbio, YJ9114701) and enzymatic activity (Mlbio, YJ9114702) of TALDO1 in culture supernatant of cells. Cellular imaging The fluorescence imaging experiments were performed as described previously . RAW 264.7 cells were incubated in glass-bottomed dishes and treated with DMEM containing DA-P and DA. After that, cells were washed twice and immobilized with 4% PFA at RT for 15 min, then transudated with PBS containing 0.1% Triton X-100. Samples were treated with clicked reaction solution (1 mmol/L NaVc, 1 mmol/L CuSO4, 100 mmol/L THPTA and 50 mmol/L TAMRA-N3) with gentle shaking for 1 h at RT. Samples were blocked with 5% BSA and incubated with primary TALDO1 antibodies (Abcam, ab315023, 1:100) at 4℃ overnight. Samples were then incubated with fluorescent secondary antibody (Abcam, Alexa Fluor 488, ab150077) (1:400) for 1 h and Hoechst (1:400) for 10 min. Protein labeling RAW 264.7 cells were treated with LPS overnight, and then treated with or without DA-P for 1.5 h, then all cellular proteins were collected by RIPA buffer (Solarbio, R0020). Proteins were added with the click chemistry reaction cocktail and incubated for 1 h at RT. Labeled protein was precipitated by prechilled acetone (-20 °C) and then visualized by Azure Sapphire. For competitive protein labeling, cells were firstly incubated with or without DA for 3 h, and then treated with DA-P for 1.5 h. For labeling of recombinant TALDO1 protein, proteins were treated with DA for 3 h and then DA-P for 1.5 h. LC-MS/MS based targets identification and pull down experiment As previous reported , RAW 264.7 cells were divided into three groups: control group (DMSO treatment), probe group (DA-P treatment for 1.5 h), and compete group (DA treatment for 3 h and DA-P treatment for 1.5 h). After that, cells were obtained using PBS containing 0.1% triton. The click chemistry (1 mmol/L NaVc, 100 mmol/L THPTA, 1 mmol/L CuSO4 and 50 mmol/L TAMRA-azide) was performed to conjugate biotin tag to proteins. All of the samples were precipitated with pre-cooled acetone at -20 ℃ and dissolved in 0.1% SDS, then incubated with 50 µl neutrAvidin beads for 4 h. The beads were then rinsed three times with 1% SDS, 6 mol/L urea and 1×PBS, respectively. For digestion, beads enriched with proteins were incubated with dithiothreitol (DTT, MedChemExpress, CAS: 3483-12-3, Cat. No. D0632, 99%), and alkylated with iodoacetaamide (IAA, MedChemExpress, CAS: 144-48-9, Cat. No. I6125, 99%), and then digested with trypsin at 37℃ for overnight. The samples were collected and desalted with commercial C18 column, then labeled with dimethylation regents (light: control group; medium: probe group; heavy: conmpete group). Finally, samples were analyzed by LC-MS/MS (Thermo). For pull down assay, after washing beads with PBS, 1x loading buffer (40 µL) were added into beads, then denatured for 5 min at 95 ℃. The next procedures were the same as western blot. GO and KEGG enrichment analysis The abundance changes in the control group (DMSO treatment), probe group (DA-P treatment) and compete groups (DA + DA-P treatment) were used to identify differential proteins based on absolute fold change (probe / compete) > 2 and p-value (FDR) < 0.05, and absolute fold change (probe / control) > 5 and p-value (FDR) < 0.05 . Then screened proteins were analyzed by the volcano plots analysis through the bioladder website ( https://www.bioladder.cn ). Moreover, the selected proteins were analyzed by Veen and GO enrichment using DAVID + version . SiRNA transfection In this topic, complementary oligonucleotides sequences of SiRNA against TALDO1 (GGCAAACACCGACAAGAAATT) were designed and characterized by Ribobio. RAW 264.7 cells were transfected with TALDO1 SiRNA along with a negative SiRNA control according to previous report . Transfected cells were incubated for 48 h and then lysed for further assay. Western blot The detailed experimental procedure is similar as previous report . Proteins were collected with RIPA buffer and then denatured by heating with 1x loading buffer and separated by SDS-PAGE, and electrotransferred to PVDF membranes. The samples were then mixed with the appropriate primary antibodies: rabbit anti–β-actin (20536-1-AP, 1:5000), anti-TNF-α (17590-1-AP, 1:2000), anti-IL-1β (26048-1-AP, 1:2000), anti-p-PERK (29546-1-AP, 1:2000), anti-p65 NF-κB (10745-1-AP, 1:2000), anti-IκBα (10268-1-AP, 1:2000) and anti-TALDO1 (12376-1-AP, 1:2000). Protein contents were visualized by Image J software and standardized according to the corresponding control. Cellular thermal shift assay (CETSA) CETSA experiment was carried out as reported previously . Briefly, RAW 264.7 cell lysis were incubated with or without DA for 3 h. Cell suspensions were then divided into eight PCR tubes and heated at the temperatures from 37 °C to 72 °C for 5 min by PCR analyzer. The lysates were then analyzed via western blot analysis. Plasmids and protein purification The TALDO1 gene was cloned into pet28a vector. The E. coli BL21 was transfected with pet28a-TALDO1, and incubated in LB culture medium containing kanamycin for 4 h at 37 °C. Isopropyl-D-1-thiogalactopyranoside (IPTG, MedChemExpress, CAS: 367-93-1, Cat. No. 5800-OP, 99%) was then incorporated into the sample, and TALDO1 expression was carried out for 17 h at 17 °C. Cell pellets were collected at 4 °C, 12,000 rpm, for 30 min, lysed with lysis buffer. The collected supernatant incorporated recombinant TALDO1 was centrifuged at 4 °C, 12,000 rpm, for 30 min. Protein was amplified through His-tagged Ni-beads columns, and Ni-beads columns containing eluted TALDO1 were purified with elution buffer (200 mmol/L NaCl, 20 mmol/L Tris-HCl and 150 mmol/L imidazole). Imidazole in samples was replaced with PBS and samples were enriched, which could be detected by CBB staining. Preparation of binding site sample of TALDO1 and DA 100 µg of purified TALDO1 recombinant protein was mixed with 10 mM DA and incubated overnight at 4 °C. The protein was then precipitated by adding methanol: chloroform: water solution (4:3:1) and washed once with methanol. Reconstitution was performed in EPPS solution, and then DTT (5 mM) and IAA (20 mM) were mixed sequentially for 30 min. Finally, purified TALDO1 was digested into peptides using 2 µg trypsin, then desalted on commercial C18 columns and examined by mass spectrometry. Molecular docking model The crystal structure of TALDO1 was obtained from the Protein Data Bank. proteins and chemical structures were hydrogenated and dehydrated as well as molecularly docked using Autodock software . Subsequently, Pymol software was utilized to map protein-molecular alignment. Statistical analysis Data were presented as the mean ± SEM for at least three independent assays, and analyzed by one-way ANOVA and two-way ANOVA. Raw data were analyzed by Graph Pad Prism 9.0. The density of WB bands were quantified through Image J software.   In this study, all animal experiments were approved by the Center of Shenzhen People’s Hospital for the care and use of laboratory animals. Balb/C male mice (25 ± 3 g) were obtained from Vital River Laboratory Animal Technology, and maintained under standard environmental conditions. A total of 40 mice were randomized into five groups: control group, model group (LPS, Lipopolysaccharides, MedChemExpress, Cat. No. HY-D1056, MCE, 10 mg/kg), positive drug group (Dexamethasone, DEX, MedChemExpress, CAS: 50-02-2, Cat. No. HY-14648, 99.86%, MCE, 10 mg/kg), DA-Low dose group (Dihydrocaffeic acid, MedChemExpress, CAS: 1078-61-1, Cat. No. HY-N2406, 99.38%, 10 mg/kg) and DA-High dose group (20 mg/kg). After 14 days of pre-administration of DMSO, DA or DEX, LPS was administered intranasally into mice. Finally, blood and lung tissue samples were collected after euthanasia.   Lung tissues from mice were fixed by adding 4% paraformaldehyde (PFA), and embedded in paraffin, sliced and stained with hematoxylin–eosin (H&E). This staining procedure was employed to explore the differences in histological morphology as previous reported .   RAW 264.7 cells were purchased from Cell Resource Center, Institute of Basic Medicine, China Academy of Medical Sciences, and the resource number is 1101MOU-PUMC000146. Cells were cultured in DMEM containing 10% FBS, and maintained in a 37 °C incubator with 5% (v/v) CO2. Cells grown to 70–90% were collected for the following experiment.   Cell viability was measured using the CCK-8 assay (C0038, Beyotime) based on manufacturer’s instructions. RAW 264.7 cells were seeded into 96-well plates for overnight and then added with various concentrations of DA or DA-P.   RAW 264.7 cells were cultured with LPS (1 µg/ml) for 24 h to develop a model of inflammation, then treated with various concentrations of DA or DA-P. The cell culture supernatant was then collected for further research. Based on the manufacturer’s manuals, Nitric Oxide (NO) Assay Kit and Elisa kits were used to measure the concentrations of NO (Biodee, DEM106), TNF-α (Biorigin, BN50578), IL-1β (Biorigin, BN50543) and IL-6 (Biorigin, BN50553) in culture supernatant, lung tissues and serum. Besides, Elisa kits were used to measure the content (Mlbio, YJ9114701) and enzymatic activity (Mlbio, YJ9114702) of TALDO1 in culture supernatant of cells.   The fluorescence imaging experiments were performed as described previously . RAW 264.7 cells were incubated in glass-bottomed dishes and treated with DMEM containing DA-P and DA. After that, cells were washed twice and immobilized with 4% PFA at RT for 15 min, then transudated with PBS containing 0.1% Triton X-100. Samples were treated with clicked reaction solution (1 mmol/L NaVc, 1 mmol/L CuSO4, 100 mmol/L THPTA and 50 mmol/L TAMRA-N3) with gentle shaking for 1 h at RT. Samples were blocked with 5% BSA and incubated with primary TALDO1 antibodies (Abcam, ab315023, 1:100) at 4℃ overnight. Samples were then incubated with fluorescent secondary antibody (Abcam, Alexa Fluor 488, ab150077) (1:400) for 1 h and Hoechst (1:400) for 10 min.   RAW 264.7 cells were treated with LPS overnight, and then treated with or without DA-P for 1.5 h, then all cellular proteins were collected by RIPA buffer (Solarbio, R0020). Proteins were added with the click chemistry reaction cocktail and incubated for 1 h at RT. Labeled protein was precipitated by prechilled acetone (-20 °C) and then visualized by Azure Sapphire. For competitive protein labeling, cells were firstly incubated with or without DA for 3 h, and then treated with DA-P for 1.5 h. For labeling of recombinant TALDO1 protein, proteins were treated with DA for 3 h and then DA-P for 1.5 h.   As previous reported , RAW 264.7 cells were divided into three groups: control group (DMSO treatment), probe group (DA-P treatment for 1.5 h), and compete group (DA treatment for 3 h and DA-P treatment for 1.5 h). After that, cells were obtained using PBS containing 0.1% triton. The click chemistry (1 mmol/L NaVc, 100 mmol/L THPTA, 1 mmol/L CuSO4 and 50 mmol/L TAMRA-azide) was performed to conjugate biotin tag to proteins. All of the samples were precipitated with pre-cooled acetone at -20 ℃ and dissolved in 0.1% SDS, then incubated with 50 µl neutrAvidin beads for 4 h. The beads were then rinsed three times with 1% SDS, 6 mol/L urea and 1×PBS, respectively. For digestion, beads enriched with proteins were incubated with dithiothreitol (DTT, MedChemExpress, CAS: 3483-12-3, Cat. No. D0632, 99%), and alkylated with iodoacetaamide (IAA, MedChemExpress, CAS: 144-48-9, Cat. No. I6125, 99%), and then digested with trypsin at 37℃ for overnight. The samples were collected and desalted with commercial C18 column, then labeled with dimethylation regents (light: control group; medium: probe group; heavy: conmpete group). Finally, samples were analyzed by LC-MS/MS (Thermo). For pull down assay, after washing beads with PBS, 1x loading buffer (40 µL) were added into beads, then denatured for 5 min at 95 ℃. The next procedures were the same as western blot.   The abundance changes in the control group (DMSO treatment), probe group (DA-P treatment) and compete groups (DA + DA-P treatment) were used to identify differential proteins based on absolute fold change (probe / compete) > 2 and p-value (FDR) < 0.05, and absolute fold change (probe / control) > 5 and p-value (FDR) < 0.05 . Then screened proteins were analyzed by the volcano plots analysis through the bioladder website ( https://www.bioladder.cn ). Moreover, the selected proteins were analyzed by Veen and GO enrichment using DAVID + version .   In this topic, complementary oligonucleotides sequences of SiRNA against TALDO1 (GGCAAACACCGACAAGAAATT) were designed and characterized by Ribobio. RAW 264.7 cells were transfected with TALDO1 SiRNA along with a negative SiRNA control according to previous report . Transfected cells were incubated for 48 h and then lysed for further assay.   The detailed experimental procedure is similar as previous report . Proteins were collected with RIPA buffer and then denatured by heating with 1x loading buffer and separated by SDS-PAGE, and electrotransferred to PVDF membranes. The samples were then mixed with the appropriate primary antibodies: rabbit anti–β-actin (20536-1-AP, 1:5000), anti-TNF-α (17590-1-AP, 1:2000), anti-IL-1β (26048-1-AP, 1:2000), anti-p-PERK (29546-1-AP, 1:2000), anti-p65 NF-κB (10745-1-AP, 1:2000), anti-IκBα (10268-1-AP, 1:2000) and anti-TALDO1 (12376-1-AP, 1:2000). Protein contents were visualized by Image J software and standardized according to the corresponding control.   CETSA experiment was carried out as reported previously . Briefly, RAW 264.7 cell lysis were incubated with or without DA for 3 h. Cell suspensions were then divided into eight PCR tubes and heated at the temperatures from 37 °C to 72 °C for 5 min by PCR analyzer. The lysates were then analyzed via western blot analysis.   The TALDO1 gene was cloned into pet28a vector. The E. coli BL21 was transfected with pet28a-TALDO1, and incubated in LB culture medium containing kanamycin for 4 h at 37 °C. Isopropyl-D-1-thiogalactopyranoside (IPTG, MedChemExpress, CAS: 367-93-1, Cat. No. 5800-OP, 99%) was then incorporated into the sample, and TALDO1 expression was carried out for 17 h at 17 °C. Cell pellets were collected at 4 °C, 12,000 rpm, for 30 min, lysed with lysis buffer. The collected supernatant incorporated recombinant TALDO1 was centrifuged at 4 °C, 12,000 rpm, for 30 min. Protein was amplified through His-tagged Ni-beads columns, and Ni-beads columns containing eluted TALDO1 were purified with elution buffer (200 mmol/L NaCl, 20 mmol/L Tris-HCl and 150 mmol/L imidazole). Imidazole in samples was replaced with PBS and samples were enriched, which could be detected by CBB staining.   100 µg of purified TALDO1 recombinant protein was mixed with 10 mM DA and incubated overnight at 4 °C. The protein was then precipitated by adding methanol: chloroform: water solution (4:3:1) and washed once with methanol. Reconstitution was performed in EPPS solution, and then DTT (5 mM) and IAA (20 mM) were mixed sequentially for 30 min. Finally, purified TALDO1 was digested into peptides using 2 µg trypsin, then desalted on commercial C18 columns and examined by mass spectrometry.   The crystal structure of TALDO1 was obtained from the Protein Data Bank. proteins and chemical structures were hydrogenated and dehydrated as well as molecularly docked using Autodock software . Subsequently, Pymol software was utilized to map protein-molecular alignment.   Data were presented as the mean ± SEM for at least three independent assays, and analyzed by one-way ANOVA and two-way ANOVA. Raw data were analyzed by Graph Pad Prism 9.0. The density of WB bands were quantified through Image J software.   DA alleviated LPS-induced acute pneumonia in mice First of all, we examined whether DA could protect mice from acute pneumonia. Lipopolysaccharide (LPS), the major constituent of outer membrane of gram-negative bacteria, could cause lung injury and inflammatory reaction . Here we selected LPS to construct an acute pneumonia model , and we used dexamethasone (DEX), a widely-used nonsteroidal anti-inflammatory drug (NSAID), as a positive drug for the therapy of pneumonia , the detailed procedures were shown in Fig. . Histological examination demonstrated that LPS model group significantly aggravated lung failure, showing obvious lung tissue damage and inflammatory infiltrations. Besides, DA (low dose: 10 mg/kg; high dosen: 20 mg/kg) attenuated these injuries, and its protective effect was similar to that of the positive drug DEX (Fig. B). Moreover, the detection of TNF—α, IL-1β as well as IL-6 levels showed that LPS enhanced the levels of inflammatory factors in lung tissues obviously, which could be significantly alleviated by DA and DEX (Fig. C). Similar conclusions were obtained in serum (Fig. D). Collectively, our data suggest that DA exhibited anti-inflammatory effects in vivo. DA inhibits inflammatory cytokines in RAW 264.7 cells RAW264.7 cells are monocyte/macrophage-like cell line, which is regarded as one of the optimal models for macrophages due to the its cytotoxicity and phagocytosis, and is widely used in inflammation, immunity, apoptosis, and tumor research . LPS has become the gold standard reagent for modeling acute inflammation due to the benefits of easy control, good model reproducibility, and effects that are easy to identify and measure . Considering these, RAW 264.7 cells were induced by LPS to establish an inflammatory model in vitro. The anti-inflammatory effect of DA in RAW 264.7 cells were measured by NO release levels, and the results indicated that DA reduced the release of NO concentration-dependently (Fig. A). Collectively, our data suggest that DA exhibited anti-inflammatory effects in vitro. Synthesis and bioactivity of the DA-Probe Due to the significant anti-inflammation effect of DA, we are interested to explore the protein targets of DA. Thus, we next synthesized a chemical activity-based probe of DA (DA-P) (Fig. B). Subsequently, we first evaluated the cell viability of RAW264.7 cells with CCK-8 kits. The results indicated the influence of DA-P and DA on cell viability was consistent (Fig. C). Next, to verify whether DA-P exhibited similar anti-inflammatory bioactivity as DA, NO assay as well as inflammatory cytokines were examined. The levels of NO indicated that there are no remarkable differences between DA and DA-P treatments (Fig. D). Moreover, the levels of inflammatory factors in RAW 264.7 cells showed that DA-P possesses similar biological activity as DA (Fig. E, Figure ). Besides, WB results also indicated that both DA and DA-P significantly decreased the protein expression levels of TNF-α and IL-6 (Fig. F). Thus, we concluded that there are no significant differences between DA and DA-P in anti-inflammation effects. ABPP-based chemical proteomics to characterize DA targets Next, we applied a chemical proteomics approach to recognize potential targets of DA, detailed procedure is shown in Fig. A. Firstly, we labeled LPS-induced RAW 264.7 cells with various concentrations of DA-P followed by conjugated with a fluorescent dye through click chemistry, numerous capture proteins binding to DA-P in situ was separated by SDS-PAGE electrophoresis and visualized by fluorescence. Besides, the control group is also conducted to ensure the specificity of the visualized bands. The labeling profiles of DA-P with concentrations from 10 to 100 µM was displayed, with fluorescence strength rising with higher concentrations. Besides, incorporation with 50 µM DA-P also produced appropriate levels of labeling (Fig. B), which was selected for further experiments. Moreover, we also observed the competition effect of DA to DA-P in situ in RAW 264.7 cells. After treatment of DA, the labeling profiles of DA-P became much weaker in a concentration-dependent manner, and the profiles even disappeared partly with treatment of 400 µM DA (Fig. C). This data suggests that the DA-P was able to efficiently capture DA targets in cells. Subsequently, we used a chemical proteomics approach to determine DA targets. Cell proteins were added with biotin-alkyne, and protein targets bound to DA were purified by affinity with streptavidin beads. According to previous reported protocols , we performed tandem mass tag quantitation and mass spectrometry (MS) to identify all of the peptide fragments digested from the samples (Table ). We analyzed the protein information labeled by DA-P and these identified protein hits were systematically analyzed and displayed by corresponding scatter plots and volcano plots (Fig. D, Figure ). The ratios of proteins captured by DA-P in the probe/competition group (DA-P/DA- P + 8x DA) and the probe/control group (DA-P/DMSO) were calculated to screen the hits out. After a cut-off of 2 (DA-P/DA- P + 8x DA) and 5 (DA-P/DMSO) in the ratios was applied, the venn results showed there were 81 and 275 highly reliable targets. Among these proteins, 78 proteins were identified in both groups (Fig. E). Gene ontology (GO) analysis suggested these proteins were mostly abundant in biological processes, including the signaling pathways of the metabolic and protein processing in endoplasmic reticulum (Fig. F). Among these potent potential proteins, a protein called transaldolase 1 (TALDO1) was highlighted because of its top positions in these criteria (Top1 in fold change of both DA-P/Compete and DA-P/Control). TALDO1 is a rate-limiting enzyme in the glucose phosphate pathway and is one of the critical players in cellular metabolism as well as proliferation processes . It is reported that lacking of TALDO1 is related to various of clinical symptoms, including coagulopathy, liver dysfunction and tumor , and high levels of TALDO1 have been found in various tumors . However, no prior study has confirmed the relationship between TALDO1 and inflammation. Due to this, we are interested to explore that whether TALDO1 is one of the key target proteins of DA for the anti-inflammatory activity. DA directly targets TALDO1 in LPS-induced RAW264.7 cells Next, a pull-down-immunoblotting assay was conducted to verify the selected DA target TALDO1. The results indicated that DA effectively pulled down TALDO1, and this binding was totally competed by excess DA. In addition, we carried out similar competition experiments on recombinant TALDO1. TALDO1 could be obviously labeled with DA-P and competed by DA in a concentration-dependent manner, further clarifying that DA directly targeted with TALDO1 (Fig. A). Moreover, immunofluorescence staining with antibodies against TALDO1 and DA-P clicked with a Rho tag showed that DA-P co-localized with TALDO1 in LPS-treated RAW 264.7 cells and DA effectively competed the fluorescent signal mediated by DA-P (Fig. B). All these result are considered as a strong evidence of LC-MS/MS. In addition, a cell lysate cellular thermal shift assay (CETSA) was performed to show the direct binding of TALDO1 and DA. Results showed that the thermal stabilization of TALDO1 was increased obviously in DA treatment group (Fig. C). DA covalently binds to Cys250 of TALDO1 and inhibits its enzymatic activity Considering that TALDO1 is a kind of enzyme, we measured the content and enzymatic activity of TALDO1. Results demonstrated that DA suppressed the enzymatic activity of TALDO1 concentration-dependent manner effectively (Fig. A), but the content of TALDO1 didn’t show any difference (Fig. B). The unsaturated groups of DA was reported as a Michael addition acceptor and formed a covalent bond to the protein cysteine residues . It is demonstrated that cysteine residues have a remarkable role in bio-conformation of proteins . Consistent with this, excess iodoacetamide (IAA, a reactive cysteine alkylation reagent) almost totally competed the binding of DA-P to TALDO1 (Fig. C). Besides, DA obviously competed away the binding of IAA-yne to TALDO1 in a concentration-dependent manner (Fig. D), which also indicated that DA could occupy cysteine of TALDO1. To further certify that DA could covalently bind to TALDO1 by the special residue sites, we conducted molecular docking analysis, and the results predicted that CA might interact with different residue sites of ACOD1, including Cys250 (Fig. E). Furthermore, we incubated recombinant TALDO1 protein with DA followed by LC-MS/MS. The MS analysis revaled that the Cys250-containing peptide C 250 ALAGCDFLTISPK had a mass shift (Fig. F). These results suggested that the Cys250 of TALDO1 was covalent modified by DA. DA exhibited anti-inflammation via TALDO1 mediated PERK-NF-ΚB pathway Subsequently, we continued to explore how DA inhibits the enzymatic activity of TALDO1 to produce the anti-inflammatory effect. The activation of nuclear translocation of nuclear factor-κB (NF-κB) is significant for regulating immune and inflammatory responses . Among the proteins in NF-κB pathways, Protein kinase RNA-like ER kinase (PERK) is located on the endoplasmic reticulum (ER) membrane, and it is a membrane receptor protein related to ER stress. PERK is activated in a phosphorylated state (p-PERK) when ER is in a stress condition, which could then inhibit the expression of inflammation inhibitory κB (IκB). It is reported that inflammatory stimuli could accelerate the degradation of IκB proteins and cause nuclear transposition of NF-κB, which induced the transcription of its target genes . Besides, among these IκB proteins, IκBα is a well-known negative feedback regulator limiting the activity of NF-κB . Considering above results in this study, we hypothesized that anti-inflammatory effect of DA is tightly correlated with the PERK-IκBα-NF-κB pathway in LPS-triggered macrophages. Western blot results showed that the levels of TNF-α, p-PERK as well as NF-κB p65 were elevated and the levels of Iκbα was decreased in model group, and DA therapy reversed such influences significantly (Fig. A). Subsequently, we transfected small interfering RNA (SiRNA) against TALDO1 into LPS-induced RAW 264.7 cells to further the significance of TALDO1. Elisa data revealed that silencing of TALDO1 obviously impaired the capacity of DA to reduce the levels of NO and inflammatory cytokines (Fig. B-C, Figure ). In addition, SiRNA against TALDO1 markedly abolished the ability of DA to decrease the enzymatic activity of TALDO1 (Fig. D). Furthermore, western blotting results also revealed that remarkable changes in DA-mediated expressions of TNF-α, p-PERK, IκBα, and NF-κB p65 were significantly suppressed after the addition of TALDO1 SiRNA (Fig. E). In summary, our results verified that DA exhibited anti-inflammation via TALDO1 mediated PERK-IκBα-NF-ΚB pathway.   First of all, we examined whether DA could protect mice from acute pneumonia. Lipopolysaccharide (LPS), the major constituent of outer membrane of gram-negative bacteria, could cause lung injury and inflammatory reaction . Here we selected LPS to construct an acute pneumonia model , and we used dexamethasone (DEX), a widely-used nonsteroidal anti-inflammatory drug (NSAID), as a positive drug for the therapy of pneumonia , the detailed procedures were shown in Fig. . Histological examination demonstrated that LPS model group significantly aggravated lung failure, showing obvious lung tissue damage and inflammatory infiltrations. Besides, DA (low dose: 10 mg/kg; high dosen: 20 mg/kg) attenuated these injuries, and its protective effect was similar to that of the positive drug DEX (Fig. B). Moreover, the detection of TNF—α, IL-1β as well as IL-6 levels showed that LPS enhanced the levels of inflammatory factors in lung tissues obviously, which could be significantly alleviated by DA and DEX (Fig. C). Similar conclusions were obtained in serum (Fig. D). Collectively, our data suggest that DA exhibited anti-inflammatory effects in vivo.   RAW264.7 cells are monocyte/macrophage-like cell line, which is regarded as one of the optimal models for macrophages due to the its cytotoxicity and phagocytosis, and is widely used in inflammation, immunity, apoptosis, and tumor research . LPS has become the gold standard reagent for modeling acute inflammation due to the benefits of easy control, good model reproducibility, and effects that are easy to identify and measure . Considering these, RAW 264.7 cells were induced by LPS to establish an inflammatory model in vitro. The anti-inflammatory effect of DA in RAW 264.7 cells were measured by NO release levels, and the results indicated that DA reduced the release of NO concentration-dependently (Fig. A). Collectively, our data suggest that DA exhibited anti-inflammatory effects in vitro.   Due to the significant anti-inflammation effect of DA, we are interested to explore the protein targets of DA. Thus, we next synthesized a chemical activity-based probe of DA (DA-P) (Fig. B). Subsequently, we first evaluated the cell viability of RAW264.7 cells with CCK-8 kits. The results indicated the influence of DA-P and DA on cell viability was consistent (Fig. C). Next, to verify whether DA-P exhibited similar anti-inflammatory bioactivity as DA, NO assay as well as inflammatory cytokines were examined. The levels of NO indicated that there are no remarkable differences between DA and DA-P treatments (Fig. D). Moreover, the levels of inflammatory factors in RAW 264.7 cells showed that DA-P possesses similar biological activity as DA (Fig. E, Figure ). Besides, WB results also indicated that both DA and DA-P significantly decreased the protein expression levels of TNF-α and IL-6 (Fig. F). Thus, we concluded that there are no significant differences between DA and DA-P in anti-inflammation effects.   Next, we applied a chemical proteomics approach to recognize potential targets of DA, detailed procedure is shown in Fig. A. Firstly, we labeled LPS-induced RAW 264.7 cells with various concentrations of DA-P followed by conjugated with a fluorescent dye through click chemistry, numerous capture proteins binding to DA-P in situ was separated by SDS-PAGE electrophoresis and visualized by fluorescence. Besides, the control group is also conducted to ensure the specificity of the visualized bands. The labeling profiles of DA-P with concentrations from 10 to 100 µM was displayed, with fluorescence strength rising with higher concentrations. Besides, incorporation with 50 µM DA-P also produced appropriate levels of labeling (Fig. B), which was selected for further experiments. Moreover, we also observed the competition effect of DA to DA-P in situ in RAW 264.7 cells. After treatment of DA, the labeling profiles of DA-P became much weaker in a concentration-dependent manner, and the profiles even disappeared partly with treatment of 400 µM DA (Fig. C). This data suggests that the DA-P was able to efficiently capture DA targets in cells. Subsequently, we used a chemical proteomics approach to determine DA targets. Cell proteins were added with biotin-alkyne, and protein targets bound to DA were purified by affinity with streptavidin beads. According to previous reported protocols , we performed tandem mass tag quantitation and mass spectrometry (MS) to identify all of the peptide fragments digested from the samples (Table ). We analyzed the protein information labeled by DA-P and these identified protein hits were systematically analyzed and displayed by corresponding scatter plots and volcano plots (Fig. D, Figure ). The ratios of proteins captured by DA-P in the probe/competition group (DA-P/DA- P + 8x DA) and the probe/control group (DA-P/DMSO) were calculated to screen the hits out. After a cut-off of 2 (DA-P/DA- P + 8x DA) and 5 (DA-P/DMSO) in the ratios was applied, the venn results showed there were 81 and 275 highly reliable targets. Among these proteins, 78 proteins were identified in both groups (Fig. E). Gene ontology (GO) analysis suggested these proteins were mostly abundant in biological processes, including the signaling pathways of the metabolic and protein processing in endoplasmic reticulum (Fig. F). Among these potent potential proteins, a protein called transaldolase 1 (TALDO1) was highlighted because of its top positions in these criteria (Top1 in fold change of both DA-P/Compete and DA-P/Control). TALDO1 is a rate-limiting enzyme in the glucose phosphate pathway and is one of the critical players in cellular metabolism as well as proliferation processes . It is reported that lacking of TALDO1 is related to various of clinical symptoms, including coagulopathy, liver dysfunction and tumor , and high levels of TALDO1 have been found in various tumors . However, no prior study has confirmed the relationship between TALDO1 and inflammation. Due to this, we are interested to explore that whether TALDO1 is one of the key target proteins of DA for the anti-inflammatory activity.   Next, a pull-down-immunoblotting assay was conducted to verify the selected DA target TALDO1. The results indicated that DA effectively pulled down TALDO1, and this binding was totally competed by excess DA. In addition, we carried out similar competition experiments on recombinant TALDO1. TALDO1 could be obviously labeled with DA-P and competed by DA in a concentration-dependent manner, further clarifying that DA directly targeted with TALDO1 (Fig. A). Moreover, immunofluorescence staining with antibodies against TALDO1 and DA-P clicked with a Rho tag showed that DA-P co-localized with TALDO1 in LPS-treated RAW 264.7 cells and DA effectively competed the fluorescent signal mediated by DA-P (Fig. B). All these result are considered as a strong evidence of LC-MS/MS. In addition, a cell lysate cellular thermal shift assay (CETSA) was performed to show the direct binding of TALDO1 and DA. Results showed that the thermal stabilization of TALDO1 was increased obviously in DA treatment group (Fig. C).   Considering that TALDO1 is a kind of enzyme, we measured the content and enzymatic activity of TALDO1. Results demonstrated that DA suppressed the enzymatic activity of TALDO1 concentration-dependent manner effectively (Fig. A), but the content of TALDO1 didn’t show any difference (Fig. B). The unsaturated groups of DA was reported as a Michael addition acceptor and formed a covalent bond to the protein cysteine residues . It is demonstrated that cysteine residues have a remarkable role in bio-conformation of proteins . Consistent with this, excess iodoacetamide (IAA, a reactive cysteine alkylation reagent) almost totally competed the binding of DA-P to TALDO1 (Fig. C). Besides, DA obviously competed away the binding of IAA-yne to TALDO1 in a concentration-dependent manner (Fig. D), which also indicated that DA could occupy cysteine of TALDO1. To further certify that DA could covalently bind to TALDO1 by the special residue sites, we conducted molecular docking analysis, and the results predicted that CA might interact with different residue sites of ACOD1, including Cys250 (Fig. E). Furthermore, we incubated recombinant TALDO1 protein with DA followed by LC-MS/MS. The MS analysis revaled that the Cys250-containing peptide C 250 ALAGCDFLTISPK had a mass shift (Fig. F). These results suggested that the Cys250 of TALDO1 was covalent modified by DA.   Subsequently, we continued to explore how DA inhibits the enzymatic activity of TALDO1 to produce the anti-inflammatory effect. The activation of nuclear translocation of nuclear factor-κB (NF-κB) is significant for regulating immune and inflammatory responses . Among the proteins in NF-κB pathways, Protein kinase RNA-like ER kinase (PERK) is located on the endoplasmic reticulum (ER) membrane, and it is a membrane receptor protein related to ER stress. PERK is activated in a phosphorylated state (p-PERK) when ER is in a stress condition, which could then inhibit the expression of inflammation inhibitory κB (IκB). It is reported that inflammatory stimuli could accelerate the degradation of IκB proteins and cause nuclear transposition of NF-κB, which induced the transcription of its target genes . Besides, among these IκB proteins, IκBα is a well-known negative feedback regulator limiting the activity of NF-κB . Considering above results in this study, we hypothesized that anti-inflammatory effect of DA is tightly correlated with the PERK-IκBα-NF-κB pathway in LPS-triggered macrophages. Western blot results showed that the levels of TNF-α, p-PERK as well as NF-κB p65 were elevated and the levels of Iκbα was decreased in model group, and DA therapy reversed such influences significantly (Fig. A). Subsequently, we transfected small interfering RNA (SiRNA) against TALDO1 into LPS-induced RAW 264.7 cells to further the significance of TALDO1. Elisa data revealed that silencing of TALDO1 obviously impaired the capacity of DA to reduce the levels of NO and inflammatory cytokines (Fig. B-C, Figure ). In addition, SiRNA against TALDO1 markedly abolished the ability of DA to decrease the enzymatic activity of TALDO1 (Fig. D). Furthermore, western blotting results also revealed that remarkable changes in DA-mediated expressions of TNF-α, p-PERK, IκBα, and NF-κB p65 were significantly suppressed after the addition of TALDO1 SiRNA (Fig. E). In summary, our results verified that DA exhibited anti-inflammation via TALDO1 mediated PERK-IκBα-NF-ΚB pathway.   DA has been reported to be one of the major metabolites of chlorogenic acid . To the best of our information, the present study is a systematic exploration of the effects and potential specific molecular mechanisms of DA on actue pneumonia in mice (Fig. ). Pneumonia is a widespread acute infections affecting the alveolar and distant airways . Inflammation is a host protection reaction to infections and tissue injuries, with a range of symptoms including recruitments of immunocytes to the areas of infections or tissue injuries . Our results showed that a dosage of DA obviously reduced inflammatory cytokines in a concentration-dependent manner. However, whether higher concentrations of DA show biotoxicity requires further study. Moreover, it should be noted that we used acute lung injury stimulated by LPS, which is a common model of acute pneumonia. There are other kinds of pneumonia and other methods to build pneumonia model mice need to be researched  which may have a limitation to expand our findings. The clinically used anti-inflammatory agents have the disadvantages of high risk of side effects as well as expensive treatment . The fact that natural products can be used as alternatives to anti-inflammatory drugs offers great promise for identifying biologically active pioneer components and developing them into medicines for the treatment of inflammatory diseases. DA is a valuable chemical compound that is found in fermented foods and drinks, and is one of the major metabolites of chlorogenic acid and caffeic acid-containing foods . In this study, we have revealed that DA shows an anti-inflammatory effect significantly, which is in agreement with prior researches. Our results revealed the anti-inflammation effect of DA in acute pneumonia, which is essential for the drug’s clinical application, while the effec of DA in other inflammation-related diseases needs further study. TALDO1 has been regarded as the speed-limiting enzyme of the pentose phosphate pathway (PPP) and abnormal TALDO1 activity is related to numerous diseases . However, the relationship between TALDO1 and inflammation is still unknown. We demonstrated TALDO1 as a new potent target of DA in anti-inflammation effect through ABPP in this topic. However, it is worth noting that the mechanism of DA anti-inflammation deserves further investigation because it may be multi-targeted. Besides, we found that DA showed a significant effect of anti-inflammation through TALDO1-PERK-NF-κB pathway by silencing the target TALDO1. More relevant studies are still needed to explain whether this pathway play the only role in DA indicated anti-inflammation effect in acute pneumonia.   In conclusion, our findings suggest that DA alleviated acute pneumonia in mice and reduced the generation of inflammatory factors in vivo and in vitro. Besides, we also revealed that DA directly targeted on Cys250 of TALDO1. Moreover, our results indicated that DA significantly suppressed inflammatory response via TALDO1 mediated PERK-IκBα-NF-κB pathway. Thus, our study demonstrated that DA may serve as a new type of drug for the therapy of acute pneumonia and inflammation related diseases, and TALDO1, as a covalent binding protein of DA, may be a novel anti-inflammation target of DA.   Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 2 Supplementary Material 3   