Topical drug administration to the oral mucosa proves to be a promising treatment alternative for inflammatory diseases. However, disease-related changes in the cell barrier must be considered when developing such delivery systems. This study aimed at investigating the changes in the lining mucosa caused by inflammation and evaluating the consequences on drug delivery systems such as nanostructured lipid carriers (NLC). For this, TR146 cells were treated with inflammatory cytokines and bacterial components. Cell viability and integrity, reactive oxygen species (ROS), and interleukin (IL)-8 release were used as endpoints to assess inflammation. Translocation of phosphatidylserine, cytoskeletal arrangement, opening of desmosomes, and cell proliferation were examined. Transport studies with NLC were performed considering active and passive pathways. The results showed that IL-1ß and tumor necrosis factor α induced inflammation by increasing IL-8 and ROS production (22-fold and 2-fold). Morphologically, loss of cell–cell connections and formation of stress fibers and hyperplasia were observed. The charge of the cell membrane shifted from neutral to negative, which increased the absorption of NLC due to the repulsive interactions between the hydrophobic negative particles and the cell membrane on the one hand, and interactions with lipophilic membrane proteins such as caveolin on the other. Oral health is an essential indicator of general health, well-being, and quality of life. Mechanical stress and chemical toxicants might change the homeostasis in the mouth leading to a number of conditions such as tooth decay, periodontal disease, stomatitis, cancer, oral mucositis, infections, xerostomia, and others. It is estimated that oral diseases affect 3.5 billion people worldwide and are among the fourth most common diseases in Europe, with annual costs of €79 billion ,2.A hallmark of oral diseases is inflammation, which occurs as a physiological reaction of the human body to pathogenic organisms, injuries, antigens, and ionizing radiation . It can 2 (PGE2) [The pro-inflammatory reactions after tissue injury are mainly triggered by IL-1, which is associated with the innate immune system. Additionally, IL-1 is involved in the expression of genes encoding IL-8 ,10. Both2 (PGE2) ,15.C. albicans infections. In the studies, a fivefold increased expression of TNF-α was detected after C. albicans infection [In-vivo studies remain the gold standard to study processes in oral pathogenesis. However, such studies are expensive, ethically questionable, and vary between individuals. Therefore, in-vitro cell systems that mimic in-vivo conditions are becoming increasingly important. Cell lines used to mimic the epithelial lining mucosa include primary cells, immortalized cell lines, and human tumor-derived cell lines such as HO-1-u-1 cells and TR146 cells ,17,18. TAlthough a large number of studies have been performed examining TR146 cells for drug transport, interaction studies, and monitoring the inflammatory potential of drug candidates, there is little information whether and how they change during inflammatory processes ,29. HoweThe aim of this study was to investigate morphological cell alterations induced by pro-inflammatory cytokines and LPS and examine their effects on nanoparticle interactions. The concentrations of cytokines and LPS used were selected based on physiological inflammation data from the literature ,31,32. S2O2), ethylenediaminetetraacetic acid (EDTA), Bovine Serum Albumin (BSA), Dynasore hydrate, Chlorpromazine hydrochloride, Genistein, and 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) were purchased from Sigma-Aldrich . Dihydroethidium (DHE), Alexa Fluor 488 Phalloidin, Hoechst 33342, IL-1ß, human protein, recombinant, Gibco™ and TNF-α Recombinant Human Protein were obtained from Thermo Fisher Scientific . Palmitic acid  and oleic acid  were used for the preparation of NLC. Dulbecco’s Modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin streptomycin (Penstrep), Hank’s Balanced Salt Solution (HBSS), phosphate buffered saline , and 0.25% trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA) from Gibco, Life Technologies Corporation  were used for all cell culture experiments. HyClone (DMEM without phenol red) was obtained from GE Healthcare Life Sciences . Ultra purified water , Millipore S.A.S., Molsheim, France) was used for all experiments.Human buccal TR146 cells were obtained from Imperial Cancer Research Technology . IL-1α human, LPS from Escherichia coli O55:B5, MEM Non-essential Amino Acid Solution , Tween 80, oil red o, hydrogen peroxide  in 96-well plates  and cultured for 24 h. Subsequently, the medium was replaced by IL-1α, IL-1β, LPS, and TNF-α diluted with serum-free DMEM and incubated for 24 h (n = 6). For each stimulant, different concentrations were used: (i) IL-1α (100–800 ng/mL), (ii) IL-1β (100–800 ng/mL), (iii) LPS , and (iv) TNF-α (100–800 ng/mL). To determine the cell viability, a CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay  was used according to the manufacturer’s instructions. After an incubation time of 4 h, the absorbance was measured at 490 nm using a UV-/VIS-plate reader . Untreated, blank-corrected wells represented 100% viability. The membrane integrity of TR146 cells was investigated by determining the lactate dehydrogenase (LDH) release using a CytoTox-ONE Homogeneous Membrane Integrity Assay (Promega) according to the manufacturer’s instructions. The fluorescence signal was measured at an excitation wavelength of 560 nm and an emission wavelength of 590 nm with a UV-VIS-plate reader . Control wells representing 100% LDH release were treated with 2 µL of a lysis solution and all data were blank corrected.TR146 cells were seeded (2 × 104 cells/100 µL and incubated for 24 h. After 24 h the medium was replaced by (i) IL-1α , (ii) IL-1β , (iii) TNF-α , and (iv) LPS  diluted in serum-free DMEM. H2O2 diluted with serum-free DMEM to reach a final concentration of 200, 400, and 800 µM was used as reference for the maximum ROS production. Untreated wells served as control for viable cells. After an incubation time of 24 h, 100 µL of DHE diluted in serum-free DMEM at a concentration of 10 µM was added to each well and incubated for 4 h. Subsequently, the fluorescence was measured at an excitation wavelength of 544 nm and an emission wavelength of 612 nm using a Fluostar OPTIMA (BMG Labtech).To determine the generation of ROS, TR146 cells were seeded in 96-well plates  with a seeding density of 2 × 104 cells/well and cultured for 1 week. Five-hundred microliters of IL-1α , IL-1β , LPS , and TNF-α  diluted in serum-free DMEM were applied and incubated for 24 h. Untreated wells served as control. After 24 h, the supernatants of the cells were collected and stored at −80 °C  until further use. The IL-8 release of the samples was measured using enzyme-linked immune assays (ELISA) (n = 4). Human IL-8 ELISA sets (BD OptEIA™) from BD Biosciences  were used according to the manufacturer’s instruction at different concentrations. The absorbance of the samples was measured at 450 nm using a SPECTRAmax . The IL-8 release was calculated using the standard curve prepared for each ELISA.For the determination of the IL-8 release, TR146 cells were seeded in 24-well plates  with a seeding density of 4 × 103 cells/well and cultured for 24 h. Subsequently, the medium was replaced by IL-1ß  and TNF-α  diluted with DMEM. After an incubation time of 1, 3, and 7 days, the cell proliferation was investigated using a CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega) according to the manufacturer’s instructions. After an incubation time of 4 h, the absorbance was measured at 490 m using a UV-/Vis plate reader . Untreated wells were used as control and all wells were blank corrected.For cell proliferation studies, TR146 cells were seeded in 96-well plates with a seeding density of 5 × 104 cells/300 µL and incubated for 2–3 days. Subsequently, medium was removed, (i) IL-1ß  and (ii) TNF-α  diluted in serum-free DMEM were added and incubated for 24 h. After 24 h, cells were washed twice with 1x Annexin binding buffer (ABB) of Vybrant Apoptosis Assay Kit #2 . Five microliters of Annexin V-Alexa Fluor 488 (staining phosphatidylserine (PS) exposing cells) and 2 µL propidium iodide  in 300 µL ABB were added and incubated at room temperature in the dark for 15 min. To remove unbound Annexin V-Alexa Fluor 488, cells were washed twice with ABB and covered with ABB. For imaging, a Leica DMI6000 B with IMC in connection with a Leica DFC360 FX camera and AF 6000 software was used. In addition to the brightfield transmission, the green fluorescence of Annexin V-Alexa Fluor 488 was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. For the red spectral region (PI), an excitation wavelength of 536 nm and an emission wavelength of 617 nm were used. Exposure time, intensity, and gain were fixed for all measurements.TR146 cells were seeded in 8-well chamber slides  with a seeding density of 2 × 104 cells/well and incubated for 1 week. Subsequently, the medium was replaced by IL-1β  and TNF-α  diluted in serum-free DMEM and incubated for 24 h. Untreated cells served as control and were incubated with serum-free DMEM. After incubation, the dilutions were removed, and cells were washed twice with PBS. The cytoskeleton of the cells was stained with Alexa Fluor 488 Phalloidin and the nuclei were counterstained with Hoechst 33342. Images were acquired with a LSM 510 Meta confocal laser scanning microscope  equipped with ZEN2008 software package. Alexa Fluor 488 Phalloidin dyed cytoskeleton was detected at an excitation wavelength of 488 nm using a BP 505-550 nm bandpass detection for the green channel. Hoechst stained nuclei were visualized at 405 nm excitation wavelength with a BP 420–480 nm bandpass detection for the blue channel. Images of randomly chosen areas of the cell monolayers were captured via cLSM. Z-stacks were acquired, and virtual radial sections were documented.For the visualization of morphological changes, TR146 cells were seeded in 24-well plates  with a seeding density of 4 × 104 cells/well) and incubated for 1 week. The medium was replaced by (i) IL-1ß  and (ii) TNF-α  diluted in serum-free DMEM and incubated for 24 h. Untreated cells served as control and were incubated with serum-free DMEM. After an incubation time of 23 h, EDTA diluted with serum-free medium to reach a final concentration of 0.25 mM was added to the control wells and incubated for 1 h. EDTA was used as control for cell destabilization. After a washing, fixation and blocking procedure according to the manufacturer’s instructions, cells were labelled with the primary antibody  diluted with PBS to a final concentration of 4 µg/mL for 3 h at room temperature. Mouse IgG1 kappa isotype control  was used as negative control and was treated the same as the primary antibody. Moreover, PBS was added to control wells, which were not treated with the primary antibody. After 3 h, cells were washed and incubated with the secondary antibody  for 45 min. Images were acquired with cLSM. Goat anti-mouse IgG1 cross-adsorbed secondary antibody, Alexa Fluor 568 conjugate was detected at 543 nm excitation wavelength using a LP 560 nm long pass detection for the red spectral region.Immunofluorescence analysis of goat anti-mouse immunoglobulin 1 (IgG1) cross-adsorbed secondary antibody, Alexa Fluor 568 conjugate  was performed using TR146 cells, which were seeded in 24-well plates  was mixed with palmitic acid and oleic acid  and heated to 70 °C. Subsequently, an aqueous stabilizer solution  that was heated to the same temperature was added. The mixture was stirred at 8000 rpm for 1 min with an Ultra Turrax . The obtained pre-suspension was transferred into a high-pressure homogenizer , which is equipped with a water jacket to control the temperature. The system temperature was kept constant at 70 °C. The suspensions were homogenized running five cycles at 500 bars. Finally, the suspensions were transferred into glass vials and cooled in an ice-bath for 30 min. Blank NLC were produced without the addition of oil red o. If not otherwise stated, all concentrations were given with regard to the total formulation amount.Oil red o labeled NLC were prepared according to Tetyczka et al. ,34. BrieSize analysis was conducted by Photon Correlation Spectroscopy (PCS) using a Zetasizer Nano ZS , which is equipped with a 532 nm laser. The measurements were performed at a measurement angle of 173° (backscatter) at 25 °C with an equilibration time of 30 s. Prior to each measurement, samples were diluted with MQ-water. The refractive indices (RI) of the particles and MQ-water were set to 1.4381  and 1.3325, respectively. PCS yields the polydispersity index (PdI) as a measure of the width of the particle size distribution and the hydrodynamic diameter (z-average).v/v) sodium chloride solution to a pH of 5.5–6 and a conductivity of 50 µS/cm).The zeta potential was investigated via Laser-Doppler-Micro-Electrophoresis coupled with PCS  using a scattering angle of 173° at 25 °C. The calculation was performed according to the Helmholtz-Smoluchovski equation . Prior tTo determine potential effects of blank NLC on the generation of ROS, experiments were conducted. Briefly, blank NLC in concentrations of 500 and 750 µg/mL were applied on TR146 cells, incubated with 10 µM DHE for 4 h and the fluorescence was measured at an excitation wavelength of 544 nm and an emission wavelength of 612 nm using a Fluostar OPTIMA (BMG Labtech). Subsequently, IL-8 release of TR146 cells treated with blank NLC  for 4 h was investigated using ELISA sets from BD Biosciences . After measuring the absorbance of the samples at 450 nm , the IL-8 release was calculated using the standard curve prepared for each ELISA. Moreover, the impact of blank NLC  on PS translocation, morphological changes, and cell–cell contacts was determined. For PS translocation studies, a Vybrant Apoptosis Assay Kit #2 from Molecular Probes™ was used according to the manufacturer’s instructions. Visualization of the samples was performed as described above . Morphol4 cells/well and incubated for 1 week. Next, cells were incubated with IL-1ß  and TNF-α  diluted with serum-free DMEM for 24 h. After incubation, medium was removed and replaced by oil red o labeled NLC dispersions diluted with serum-free DMEM to reach final concentrations of 500 and 750 µg/mL and incubated for 4 h. Untreated cells were used as control and were incubated with serum-free DMEM or with particle dispersions . After 4 h, the dispersions were removed and the cells were washed with PBS. For qualitative and semi-quantitative uptake studies, the cytoskeleton of TR146 cells was stained with Alexa Fluor 488 Phalloidin and the nuclei were counterstained with Hoechst 33342. Images were acquired with a LSM 510 Meta cLSM equipped with ZEN2008 software package. Particles were detected at 633 nm excitation wavelength using a LP 650 nm long pass detection for the red channel. Images of randomly chosen areas of the cell monolayers were captured via cLSM. Z-stacks were acquired, and virtual radial sections were documented. The semi-quantitative particle uptake was further evaluated using the Nikon Software NIS-Elements . For this, a defined number of cells and their respective surface area was calculated. Further, the area of the particles (µm2) was assessed. Based on this, the percentage of particles taken up by the cells was calculated.TR146 cells were seeded in 24-well plates with a seeding density of 4 × 104 cells/well and incubated for 1 week. Next, cells were incubated with IL-1ß  and TNF-α  diluted with serum-free DMEM for 24 h. After incubation, medium was removed and exchanged with Dynasore , Chlorpromazine , Genistein , and EIPA  with the respective inflammatory cytokines diluted in serum-free DMEM and incubated for 40 min at 37 °C. Subsequently, oil red o labeled NLC dispersions at concentrations of 500 and 750 µg/mL were added and incubated for 4 h. Untreated cells were used as control and were incubated with serum-free DMEM or with inhibitors and particle dispersions  for 4 h at 37 °C. Moreover, cells were incubated with particle dispersions  at 4 °C for 4 h. After 4 h, the dispersions were removed, and the cells were washed with PBS. The cytoskeleton of TR146 cells was stained as described in TR146 cells were seeded in 24-well plates with a seeding density of 4 × 10p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.001 (***).The results were presented as mean values ± standard deviation (SD). Statistical analysis (student’s t-test) was performed and data were compared with the respective control. Differences were considered to be significant at a level of To determine if stimulants  alter TR146 cells in a concentration-dependent manner, the cell viability and the membrane integrity were investigated and compared to a cell control. All results are presented in p ≤ 0.001 ***). However, the measured viability was still above 90%. The membrane integrity was comparable to the cell control with no significant alterations in the LDH release. IL-1α at concentrations from 100 to 400 ng/mL showed good comparability to the cell control. The highest concentration of 800 ng/mL significantly decreased the cell viability to 71.61 ± 7.25% . The LDH release at the lowest concentration was 9.56 ± 1.95% and increased with higher concentration to 41.81 ± 8.75% .For LPS, which was tested at concentrations of 300–70,000 ng/mL, a significant decrease in cell viability was observed starting at a concentration of 2000–70,000 ng/mL . At a concentration of 800 ng/mL, the LDH release increased to 38.64 ± 9.66% (p ≤ 0.001 ***), which correlates with a decreased cell viability.Similar to IL-1α, no cytotoxic effects were found at IL-1ß concentrations between 100 and 300 ng/mL. Starting from a concentration of 400 to 800 ng/mL, a significant decrease in cell viability to 92.80 ± 2.57% and 27.74 ± 3.64%, respectively, was obtained . These concentrations also affected the LDH release .The lowest concentration of TNF-α  neithered affect cell viability nor membrane integrity compared to the cell control. Similar to IL-1ß, a concentration of 400 ng/mL already triggered a significant loss in cell viability to 90.88 *** ± 3.91% followed only by a marginal decrease at a concentration of 800 ng/mL , and highly reactive hydroxyl radicals (HO·) [The potential generation of ROS was assessed by the ROS-driven conversion of the non-fluorescent DHE to the fluorescent ethidium. The production of these oxygen species occurs due to an incomplete reduction of molecular oxygen leading to the formation of superoxide anions (ls (HO·) . The cel2O2 (200 to 800 µM) showed a significant increase in ROS from 1.40 ± 0.06 to 6.04 ± 0.26 (p ≤ 0.001 ***) in a concentration-dependent manner. Similar to the positive control, treatment with IL-1ß at concentrations of 100–400 ng/mL showed a significant increase in ROS generation; the same results were found for TNF-α . Taking into account the standard deviations, no differences were found in ROS production within the tested concentration range.After treatment of TR146 cells with LPS and IL-1α, the production of ROS was comparable with the untreated control . By contIL-8 is one of the most important chemokines in inflammatory processes and is released by different cell types. This release occurs after stimulation of epithelial cells with cell components of microorganisms, such as LPS and endogenous mediators including all cytokines or cellular stress ,13. A 3-p ≤ 0.01 **) regardless of the concentration  (p ≤ 0.001 ***).In contrast, IL-1ß showed a 3-fold increased IL-8 release at a concentration of 400 ng/mL  . An evenDue to the morphological alterations, a change in the proliferation rate of cells treated with inflammatory mediators was expected. The cell proliferation studies showed that TR146 cells treated with both IL-1ß and TNF-α revealed a moderate to significantly reduced growth rate .Inflammatory processes lead to changes in the structure of the cell membrane, more specifically to PS translocation from the inner to the outer leaflet. The results of the negative (untreated) control A showed A concentration of 300 ng/mL of TNF-α also induced stress-related blebs C. FigureVisualization of the cell structure of TR146 cells after incubation with the potential inflammatory triggers IL-1ß and TNF-α, revealed inflammation-induced re-organizations. In general, TR146 are epithelial cells with a characteristic squamous morphology, prominent cell nuclei and closed cell–cell contacts  at confluent conditions. After treating the cells with IL-1ß and TNF-α, a structural re-organization of the cytoskeleton was observed B,C. The The effects of inflammatory processes on cell–cell contacts  were visualized. The cell control  showed np ≤ 0.05 *) and 1.26 ± 0.12 (p ≤ 0.001 ***)). In contrast, no increase in IL-8 release was detected and levels were comparable to the control  and 0.93 ± 0.53 (750 µg/mL)). This correlated with the PS translocation studies, as no differences from the untreated cells were evident. Similarly, no morphological changes were observed and cell–cell contacts remained intact (data not shown). A schematic overview of the results is shown in After particle preparation, blank NLC showed a z-average of 276.32 ± 2.49 nm . Oil red o labeled NLC revealed a z-average of 293.44 ± 19.72 nm with a narrow particle size distribution . The zeta potential of blank NLC and labeled NLC was −37.27 ± 0.82 mV and −39.16 ± 4.09 mV, respectively, indicating good physical stability . Howeverp ≤ 0.001 ***), respectively, which was confirmed by a strong increase in red fluorescence intensity , TR146 cells were incubated with 500 and 750 µg/mL particles. Thereby, the NLC uptake was dependent on the degree of inflammation, the cytokine used, and the particle concentration applied. IL-1ß and TNF-α concentrations of 400 and 300 ng/mL showed the highest IL-8 release and ROS production and induced morphological changes. Consequently, treatment with these cytokine concentrations resulted in the highest particle internalization. ntensity C,F.The uptake mechanisms involved, i.e., active and passive, were investigated using relevant conditions and inhibitors at concentrations that did not affect the cells viability. It was found that the uptake mechanism of NLC was independent of the conditions present. Cells incubated at 4 °C showed no NLC uptake regardless of whether inflammation was induced or not A–C. AfteInflammation of the oral mucosa is dependent on the underlying disease and the respective inflammation cascade. The treatment is carried out either by local therapies such as antibacterial mouthwashes, or systemic therapies using highly potent pharmacological drugs . AdminisIn this study, four inducers of inflammation, namely LPS, IL-1α, IL-1ß, and TNF-α were tested. To assess their potential in TR146 cells, parameters including ROS and IL-8 were used to classify whether or not inflammation was induced. Thereby, ROS are the key signaling molecules to be produced during inflammatory processes. Subsequent DNA damage activates the immune system and the production of cytokines . IL-8 isFor LPS, it was found that the inflammatory potential on TR146 cells was negligible. LPS is known to bind to TLR located on epithelial cells, more specifically to TLR-4, inducing IL-8 release. However, TR146 cells mainly express TLR-1, 3, and 6 and TLR-4 only to a small extent . Hence, Moreover, IL-1ß and TNF-α induced cell morphological changes. The cell membrane consists of different proteins and lipids, particularly phospholipids, which determine the charge of the membrane by their arrangement. Thereby, the neutral phospholipids such as phosphatidylcholine and sphingomyelin are located in the outer leaflet. The inner leaflet comprises higher amounts of negatively charged PS and phosphatidylethanolamine . Due to In the oral mucosa, epithelial cells are connected via desmosomes, which are responsible for the elasticity and mechanical stability of the tissue ,64. ThesIn order to investigate how these changes influence interactions with drug delivery systems, we tested lipid nanocarriers, so-called NLC. Initially, comprehensive studies were performed on untreated TR 146 cells to exclude possible influences of lipid particles on changes in cell morphology, influence on membrane integrity, and apoptosis. Interestingly, the ROS production of untreated cells was increased. According to the literature, it is known that nanoparticles can interact with mitochondria, resulting in mitochondrial damage and consequently ROS production . AlthougIn general, the uptake mechanism of lipid particles depends on the cell type, and the toxicity profile is determined by the lipid composition . AnotherInflammatory diseases of the mouth, such as stomatitis and oral mucositis, are chronic conditions pathologically characterized by inflammation and epithelial lesions. Treatment, particularly with locally applied therapeutics, is currently limited, although side effects can be significantly reduced, and application is facilitated for dry mouth and dysphagia. Using human epithelial TR146 cells, it was found that cytokines, in particular IL-1ß and TNF-α, induced an inflammatory event in TR146 cells. This resulted in cell membrane restructuring, formation of stress fibers, loss of cell–cell connections, change in the membrane charge, and decreased cell proliferation, leading to an altered barrier integrity. The changes did not affect the cellular transport mechanism, i.e., caveolin-mediated endocytosis per se, but increased the uptake capacity of negatively charged NLC due to a decrease in membrane integrity. An optimum particle size of approximately 280 nm further promoted hydrophobic forces, which were more pronounced over shorter distances than the electrostatic repulsive forces between the negatively charged membrane and the negative particles. In addition, the use of cationic lipids could reduce these electrostatic repulsive forces to a minimum and thus further enhance the interaction with the cell membrane. Moreover, overexpression of caveolin-1 contributed to an increased NLC uptake in TR146 cells, which will be further investigated. In addition, preliminary studies excluded that the NLC particles exert negative effects on non-treated cells. It must be noted here that the data are based on studies of a simple cell system. To perform even more complex studies and thus build a deeper understanding, a co-culture model is needed to represent the anatomy and physiology of the oral mucosa in the diseased state in more detail.


O-tetradecanoylphorbol-13-acetate (TPA) and co-treatment with 0.1 mg/mL CNDs. This co-treatment significantly increased the expression of CD 206 and CD 68 (key receptors involved in phagocytosis) and increased the expression of CCL2 (a monocyte chemoattractant and pro-inflammatory cytokine). The phagocytic activity of THP-1 monocyte-derived macrophages co-treated with 0.1 mg/mL CNDs also showed a significant increase. Furthermore, this study also examined potential entrance routes of CNDs into macrophages. We have demonstrated an inhibition in the uptake of CNDs in macrophages treated with nocodazole (microtubule disruptor), N-phenylanthranilic acid (chloride channel blocker), and mercury chloride (aquaporin channel inhibitor). Collectively, this research provides evidence that CNDs cause functional changes in macrophages and indicates a variety of potential entrance routes.Atherosclerosis represents an ever-present global concern, as it is a leading cause of cardiovascular disease and an immense public welfare issue. Macrophages play a key role in the onset of the disease state and are popular targets in vascular research and therapeutic treatment. Carbon nanodots (CNDs) represent a type of carbon-based nanomaterial and have garnered attention in recent years for potential in biomedical applications. This investigation serves as a foremost attempt at characterizing the interplay between macrophages and CNDs. We have employed THP-1 monocyte-derived macrophages as our target cell line representing primary macrophages in the human body. Our results showcase that CNDs are non-toxic at a variety of doses. THP-1 monocytes were differentiated into macrophages by treatment with 12- Cardiovascular disease (CVD) has more clinical implications than any other condition worldwide. Globally, CVD accounts for one-third of all deaths . In the Macrophages play a key role in the onset of the disease state. Free oxygen radicals modify low-densitiy lipoprotieins (LDL) into ox-LDL. Upon injury to the endothelium of blood vessels by ox-LDL, circulating monocytes differentiate into pro-inflammatory (M1) or anti-inflammatory (M2) macrophages. In progressing lesions, M1 macrophages engulf excess ox-LDL. In the process, these macrophages become lipid-laden and lose mobility, finally proceeding to settle en masse on the bed of arteries as plaque. Dysregulated plaque build-up in arteries results in a several fatal long-term health issues. Due to their crucial role as mediators in atherogenesis, as well as their involvement in several aspects of the immune response, macrophages are popular targets in vascular research and therapeutic treatment. In recent years, interest in the development of nanoparticles for biological application has risen. A key area of intrigue revolves around the interaction of nanoparticles with some aspects of the immune response, resulting in their induction or repression . Their uCNDs have been synthesized with scavenging properties and proved themselves capable ex vivo scavengers of free radicals, one of which is 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH) . In thisAtherosclerosis is a long-standing inflammatory disease characterized by the narrowing of arteries due to a build-up of plaque. The overproduction of ROS and its subsequent oxidative stress play a key role in its initiation. Macrophages play an essential role as intermediators of the disease state by differentiating into a pro-inflammatory state, secreting cytokines and eventually becoming foam cells. As the concrete source for plaque build-up, macrophages signify an area of interest. CNDs are a prospective choice for biomedical implementation, having shown usefulness in ROS scavenging, biosensing, and drug delivery. However, currently, no account exists that indicates whether or not CNDs have any ability to affect the M1/M2 polarization of macrophages. In this study, we examined the effects of CNDs on the expression of M1/M2 biomarkers and phagocytic activity of macrophages, as well as potential entrance routes.® TIB-202™, Manassas, VA, USA) cells were cultured in Roswell Park Memorial Institute 1640 Medium (RPMI 1640) fortified with 10% Fetal Bovine Serum (FBS) and 1% Penicillin–Streptomycin. This cell line was obtained from the ATCC  and grown in Cellstar® Filter Cap 75 cm2 cell-culture treated, filter screw cap flasks in humidified incubators programmed to 37 °C and 5% CO2. Corresponding media was renewed every 2 days and cells were split into a new passage upon 85–90% confluence.The THP-1 cell line . Upon dilution to 2 mg/mL in DI-H2O, CNDs were measured for fluorescence in a quartz cuvette to determine excitation and emission wavelengths. The surface chemistry of CNDS was characterized by carbon 1s X-ray photoelectron spectroscopy . The CNDs preparation using citric acid and ethylenediamine (EDA) as precursors was synthesized based on an adaption of a previously published microwave-assisted method . In a 10THP-1 cells were cultured in cell plates containing RPMI 1640 Medium  or Hank’s Balanced Salt Solution (HBSS) with calcium, magnesium, and glucose . Monocyte differentiation into macrophages was induced with 3 ng/µL of 12-O-tetra-decanoylphorbol-13-acetate (TPA) in an incubation period of 72 h period. Cells were lifted by cell scraper and the supernatant placed in 50 mL Falcon tubes. Cell plates were rinsed with 2 mL PBS and also added to the supernatant. Then, cells were pelleted by centrifuge.2 incubators. Surrounding media was decanted and replaced with new media, followed by another incubation period of 72 h, after which plates were rinsed with 2 mL PBS and also added to the supernatant. Then, cells were pelleted by centrifuge.For cell viability measured by trypan blue staining and flow cytometry ViaCount assays, THP-1 cells were treated with 3 ng/mL TPA in the presence or absence of 0.01, 0.1, 0.3, or 0.6 mg/mL CNDs in RPMI media for 72 h. The media was refreshed, and then, the cells were incubated for another 72 h. For other experiments, THP-1 monocytes were cultured in cell plates and co-treated with 0.1 mg/mL CNDs and 3 ng/µL TPA for 72 h in RPMI media. Incubation occurred in 37 °C/5% COg for 5 min at 4 °C and resuspended in either PBS or respective media. After resuspension, cells were counted using a hemocytometer. Trypan blue was used to count viable, unstained cells, and the resulting concentration was also calculated.Before and after differentiation and CNDs treatments, cells were counted. Monocytes and macrophages were centrifuged at 300 ® cell culture treated plates . Upon treatment and incubation, adhered cells were lifted sing a cell scraper. The media was extracted into 50 mL tubes. The cell plates were rinsed twice with 1 × PBS to ensure no treatment media remained and also to extract any remaining cells. Then, cells were then centrifuged at 300 g for 5 min at 4 °C. The supernatant media was decanted, and the resulting cell pellets were treated with 1 mL of ambion TRIzol® . The resulting solution was pipetted into 1 mL Eppendorf tubes. Then, 200 µL of chloroform were added, which was followed by agitation, and then the solution was centrifuged at 12,000 rcf for 15 min. The top aqueous phase was transferred to another set of 1 mL Eppendorf tubes and then combined with 500 µL isopropanol and agitated before centrifuging again at 12,000 rcf for 10 min. The resulting pellet (RNA) was washed with 1 mL 75% ethanol and centrifuged at 7400 rcf for 5 min twice. Then, the pellet was resuspended in 10–15 µL of DEPC H2O.THP-1 cells were cultured in appropriate media in Corning2O, and 0.625 µL of MMLV-Reverse Transcriptase. Using Applied Biosystems™ Veriti™ 96-Well Thermal Cycler , the 25 µL solution was converted to cDNA.After RNA extraction, the resulting RNA was quantified by way of a Thermo Scientific™ Nanodrop 2000 . Then, RNA was diluted to a concentration of 500 ng/µL. Then, 2 µL of diluted RNA were mixed with 5 µL of 5× Buffer, 1.25 µL of ddNTP, 1.25 µL of Random Primer, 14.875 µL of DEPC H® Green PCR Master Mix, 2 µL of 5 µM Forward Primer  was employed and ran for 40 cycles. Each individual cycle constituted a 95 °C phase for 15 s, a 58 °C phase for 60 s, and a 60 °C phase for 15 s. Comparative threshold values were evaluated in order to quantify gene expression.Once cDNA was synthesized using the methods above, the resulting cDNA was probed for a selection of M1/M2 biomarkers as mentioned previously, using H_GAPDH as a housekeeping gene. This was performed by mixing 1 µL of cDNA with 10 µL of Power SYBRd Primer , 2 µL ofd Primer , 2 µL of6 THP-1 cells were grown in cell culture plates in corresponding media. Differentiation into macrophages was induced by administering 3 ng/µL TPA with an incubation period of 72 h (with or without co-treatment with a CNDs concentration of 0.1 mg/mL) at 37 °C, 5% CO2. After harvesting and pelleting cells, concentration was re-suspended in HBSS to 2 × 106 cells/mL. Then, 1 mL of control cells were treated with 1000 ng/mL TPA in order to activate cells to serve as a positive control. Next, 100 µL of cell suspension was added to 5 wells per sample, plus 50 µL of HBSS (negative control wells contained 200 µL of HBSS). Cells were left to incubate for 18 h in 35 °C/5% CO2 incubators. This incubation period allows macrophages to settle. Then, HBSS was removed, and 200 µL of fluorescently labeled E. coli suspension was administered for 2 h. Upon the removal of suspension, cells were treated for 60 s with 100 µL of Trypan Blue suspension. Immediate removal of suspension followed. The phagocytic activities of cells were quantified using a BioTek™ Synergy 2.0 plater reader .First, 4 × 106/mL. Then, 80 µL of cells were treated with 20 µL of ViaCount Reagent for 10 min at room temperature, upon which 500 µL of cold PBS was added. The samples were analyzed for viability using a Guava® easyCyte™ Flow Cytometer .Cells were cultured with the necessary incubation times and treatments. Next, cells were harvested by cell scraping and placed into tubes. The cell concentration was adjusted to 5 × 10THP-1 human monocyte-derived macrophages were grown to 85–90% confluence with corresponding media in clear cell plates and pre-treated with or without the following inhibitors for 30 min: Cytochalasin A or D (5 µg/mL), chlorpromazine (10 µg/mL), genistein (200 µM), nocodazole (20 µM), phenylglyoxal (100 µg/mL), amiloride hydrochloride (50 uM), n-phenylanthranilic acid (0.1mM), niflumic acid (10 mM), ebselen (15 µM), amiodarone hydrochloride (10 µM), chlorpromazine HCl (0.1 mg/mL), mercury chloride (0.075 mM), and copper sulfate (100 µM). Then, cells were treated with a concentration of 0.1 mg/mL CNDs for 24 h. Cells were harvested and resuspended in PBS. Fluorescence was read at 360/460 top 400 nm in a well plate reader (Synergy 2.0).2 hybrid regions. The main peak at 350 nm comes from the n–π∗ transitions of C=O moieties  was observed in these TPA-treated cells.THP-1 monocytes were treated with 0, 1, 3, and 10 ng/mL TPA for 72 h in RPMI media so as to stimulate the cells into differentiation. Then, the media was replaced, and cells were allowed to incubate for an additional 72 h. By way of qrt-PCR, expression of CD 206 (a macrophage differentiation marker) was assessed. As demonstrated by p < 0.05) in cell viability only at a concentration of 0.6 mg/mL (p < 0.05) in cell viability at 0.6 mg/mL CNDs (p < 0.05) between untreated cells and cells treated with the same CNDs concentration  macrophages play a crucial intermediary role in the atherosclerosis disease state. The effect of CNDs on the expression of M1 or M2 biomarkers was analyzed by PCR. THP-1 monocytes were co-treated with 3 ng/mL TPA and 0.1 mg/mL CNDs for 72 h. Then, these cells had their media refreshed, followed by an additional incubation period of 72 h. Afterwards, cells were harvested, RNA isolated, cDNA synthesized, and analyzed for expression of genes by qrt-PCR.p < 0.05) in CD 206, CD 68, and CCL2 expression in cells treated with 0.1 mg/mL CNDs. No significant effect was observed in the expression of TNF-alpha, IL-8, and IL-10  in phagocytic activity  of CNDs uptake with the use of Nocodazole, mercury chloride, and N-phenylanthranilic acid , cells were left to incubate for 24 h. Cell viability was tested using previously mentioned Trypan Blue cell count and ViaCount protocols. Our results indicate no significant decrease in the viability of macrophages at any designated concentration of each inhibitor in both the Trypan Blue a and ViaMacrophages play an important role as mediators of atherosclerosis. For this reason, they are highly sought targets when studying the disease state. CNDs are recently discovered carbon-based nanomaterials reported to have sizes of 10 nm or less, and they also exhibit favorable qualities for use in biomedical application . CollectMacrophages play a crucial intermediary role in the atherosclerosis disease state. The overabundance of settling macrophages and foam cells, due to an excess of lipoprotein ingestion, leads to the emergence of plaque. These macrophages exacerbate the inflammatory microenvironment by secreting pro-inflammatory cytokines to different cell types . A side As a model, THP-1 human monocyte-derived macrophages were utilized. These monocytes exhibit a homogenous genetic background and differentiate into adheren macrophages upon exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA). The cell line is resembling of primary monocytes/macrophages, which made it an ideal model for our purposes . These mPhagocytic activity is among the most important functions of macrophages. As a form of endocytosis, phagocytosis is defined by the use of a cell membrane to engulf extracellular particles, allowing them entrance into the cell’s cytoplasm. As key players of the immune system, macrophages ingest a variety of particles including microbes, modified lipids, and even dead cells entirely . The phaThe expressions of macrophage biomarkers, from treatment with CNDs during the differentiation process of THP-1 monocyte-derived macrophages, were analyzed to further understand if this boost in phagocytic function favors M1 or M2 polarization. Circulating monocytes that are activated through receptor-ligand binding differentiate into M1 (pro-inflammatory) or M2 (anti-inflammatory) macrophages. This is typically dependent on the immunological response in need. M1 macrophages typically eliminate xenobiotics through phagocytosis and promote the local inflammatory environment. In an atherogenic state, M1 macrophages aim to phagocytose modified lipoproteins in an effort to clear cholesterol. They also extend the inflammatory response by secreting several pro-inflammatory cytokines such as TNF-alpha, IL-1, IL-6, and IL-12 . These cCells treated with 0.1 mg/mL CNDs demonstrated a significant increase in CCL-2 and CD 68, which are both considered M1 biomarkers b,f. SeveE. coli [Our results also demonstrated a significant increase in the expression of CD 206, which is a prominent M2 biomarker a. This rE. coli . AdditioE. coli . This reIn recent years, interest in the development of various carbon nanoparticles in the biological application has risen. For example, diamond-like carbon (DLC) nanofilm is a promising material for application in medical implants, with high mechanical and chemical inertness and biocompatibility ,43. Both+/H+ exchange [To further deepen our understanding of the interaction of CNDs and macrophages, the final aims of this study examined potential uptake routes of CNDs into THP-1 monocyte-derived macrophages. Previous studies have denoted the involvement of actin, microtubules, and endocytic pathways in the uptake of nanoparticles: (i) Dos Santos et al. showed that the use of chlorpromazine, genistein, nocodazole, and cytochalasin A inhibited the uptake of carboxylated polystyrene nanoparticles via clathrin-mediated endocytosis in various cell lines ,47. Chloexchange . These s2), which is known to inhibit aquaporin channels [2)  . Changesnels [2) . Treatmenels [2) . This li2 and suggest that CNDs may pass through aquaporins in similar fashion. The observed inhibition of CNDs uptake as a result of treatment with nocodazole suggests that CNDs can gain entrance into cells through endocytic pathways. N-phenylanthranilic acid acts as a chloride channel blocker in cell membranes. The CNDs utilized in this study exhibit negatively charged surface functional groups. Given that chloride is a negatively charged molecule, the passage of CNDs through this channel has merit. This result also gives rise to an interesting notion. Though small even in the nanoparticle scale (≈10 nm), CNDs are still relatively large in comparison to chloride ions (≈0.2 nm). Our results suggest that depending on the surface groups tailored to CNDs, size may not be an issue in gaining entrance into cells through ion channels. Nanoparticles have demonstrated the capability of binding to carrier proteins in order to enter plant cells through aquaporins, ion channels, or endocytosis . These fOur results showed that CNDs are taken up by THP-1 monocyte-derived macrophages. However, in vivo, it remains unclear whether CNDs can enter macrophages and accumulate in the aorta or atherosclerotic plaque. In addition, the efficacy of the interaction of nanoparticles and their target cell is judged not just by their ability to enter a cell but also by the time it takes to be metabolized or released. It is not yet clear whether CNDs are released from macrophages or other cells or tissues and whether this nanoparticle can be metabolized into different molecules, which remains to be further examined in the future.In summary, our results provide novel evidence of the interaction of CNDs and macrophages involved in atherosclerosis. CNDs were confirmed to be non-toxic in concentrations that do not exceed 0.3 mg/mL by performing Trypan Blue cell counts and ViaCount flow cytometry. Our PCR results indicate a significant increase in the expression of at least one M2 biomarker (CD 206) and increases in M1 biomarkers CCL2 and CD 68. Two of these biomarkers are involved in the phagocytic function of macrophages. Although no fixed conclusions can yet be assumed regarding how CNDs affect macrophage polarization, our phagocytosis assay results indicate that CNDs treatment during the differentiation process boosts phagocytic activity, which is possibly due to the scavenging of ROS. Lastly, we also determined potential cellular uptake routes of CNDs. Results showcased inhibitions of CNDs uptake in cells treated with nocodazole, n-phenylanthranilic acid, and mercury chloride, providing evidence for entrance routes in the form of endocytosis, chloride, and water channels. Due to their crucial role as mediators in atherogenesis and their involvement in several aspects of the immune response, macrophages are popular targets in vascular research and therapeutic treatment . Upon in


Compound kushen injection (CKI), a Chinese patent drug, is widely used in the treatment of various cancers, especially neoplasms of the digestive system. However, the underlying mechanism of CKI in pancreatic cancer (PC) treatment has not been totally elucidated.Here, to overcome the limitation of conventional network pharmacology methods with a weak combination with clinical information, this study proposes a network pharmacology approach of integrated bioinformatics that applies a weighted gene co-expression network analysis (WGCNA) to conventional network pharmacology, and then integrates molecular docking technology and biological experiments to verify the results of this network pharmacology analysis.NCAPG, BUB1, CDK1, TPX2, DLGAP5, INAVA, MST1R, TMPRSS4, TMEM92 and SFN) associated with the development of PC, and survival analysis found 5 genes  associated with the prognosis and survival of PC. By integrating these results into the conventional network pharmacology study of CKI treating PC, we found that the mechanism of CKI for PC treatment was related to cell cycle, JAK-STAT, ErbB, PI3K-Akt and mTOR signalling pathways. Finally, we found that CDK1, JAK1, EGFR, MAPK1 and MAPK3 served as core genes regulated by CKI in PC treatment, and were further verified by molecular docking, cell proliferation assay, RT-qPCR and western blot analysis.The WGCNA analysis revealed 2 gene modules closely associated with classification, staging and survival status of PC. Further CytoHubba analysis revealed 10 hub genes (Overall, this study suggests that the optimized network pharmacology approach is suitable to explore the molecular mechanism of CKI in the treatment of PC, which provides a reference for further investigating biomarkers for diagnosis and prognosis of PC and even the clinical rational application of CKI.The online version contains supplementary material available at 10.1186/s13020-021-00534-y. Pancreatic cancer (PC) is a common malignant tumour of the digestive tract characterized by concealed onset, high malignancy, and rapid development, and the majority of PC cases have locally advanced or metastatic at the time of diagnosis –3. AccorCompound kushen injection (CKI), is made of Kushen (Radix Sophorae flavescentis) and Baituling (Rhizoma Smilacisglabrae) by modern technology , has beeNetwork pharmacology has become an effective tool to elucidate the complex overall mechanism of traditional Chinese medicine (TCM) and provides a new perspective for analyzing drug effects . In accoIn the present study, we aim to apply WGCNA to optimize network pharmacology methods. Firstly, WGCNA was used to construct a gene co-expression network, find clusters (modules) of highly related genes, modules and correlate with external sample traits. And then, we screened the relevant modules to obtain biomarkers that were closely related to PC development. Survival analyses were employed to identify hub genes affecting the pathogenesis and prognosis of PC patients. In addition, we constructed a “compound-putative target network”, “CKI-PC The protein–protein interaction (PPI) network”, “drug-compound-PPI target-pathway network” through the network pharmacology method to explore the mechanism of CKI in the treatment of PC. This study aimed to reveal the complex mechanism of multi-component, multi-target, and multi-pathway of CKI in the treatment of PC at a system level, and provide a better basis for the diagnosis, treatment and prognosis of PC. Figure RNA sequencing data of pancreatic adenocarcinoma, containing a total of 182 samples, were obtained from The Cancer Genome Atlas (TCGA) database . After rThe WGCNA package was used to construct the gene co-expression networks. To ensure that the connections between genes match the scale-free network distribution, the WGCNA algorithm selects the results that best fit the scale-free network distribution by choosing the weighting parameters. The soft threshold power β selected by the “pickSoftThreshold” function was used to achieve scale-free topology. The soft threshold power β refers to the correlation coefficient between the logarithmic value of the node with a connection degree k (log(k)) and the logarithmic value of the probability of a node with a connection degree k (log(p(k))).In this study, the topological overlap matrix was reconstructed by computing topological overlap measure (TOM), which is a robust measure of network interconnectedness. According to the dissimilarity matrix, which represents the connection relationship of genes, cluster analysis of genes by dissimilarity was performed to construct a hierarchical cluster tree . The dynCytoHubba , a plug-p-values.Then, we selected the data in the GEO database as the external validation dataset. We searched the GEO database using the term “pancreatic cancer” and the criteria for inclusion of PC chip datasets were to meet the following: ① Patients newly diagnosed with pancreatic cancer and not receiving any treatment; ② Datasets with total samples greater than 50. We performed differential expression analysis after normalizing and homogenizing raw data using the limma package in R, followed by Benjamini & Hochberg  correction of P-value were entered into the multivariate Cox proportional hazards regression analysis. Based on the selected gene expression profile, a survival-related linear risk assessment model was constructed. The formula for calculating the prognostic risk score is:In this study, a univariate Cox proportional hazards regression analysis was performed for the genes in the selected modules using the survival package to identify candidate genes strongly correlated with survival. Subsequently, candidate genes that screened according to Using the median prognostic risk score as a cut-off value, samples were divided into high- and low-risk groups. Furthermore, the correlation between gene expression and survival time was confirmed by calculating the hazard ratio (HR) and 95% confidence interval (CI). The Kaplan–Meier and Log-rank test methods were used to evaluate the differences in overall survival rates between the high- and low-risk groups. The survivalROC package analyzed a time-dependent receiver operating characteristic curve (ROC curve) to evaluate the prediction accuracy of the prognostic signature for time-dependent cancer death. Moreover, the predictive ability of the gene signature for clinical outcomes was measured by calculating the area under the curve (AUC).−1; The mobile phase was consisted of 0.1% ammonia in water (A) and carbinol (B). The eluting program was: 5–20% B for 0–1 min, 20–80% B for 1–30 min, 80–60% B for 30–60 min. The ion source was electrospray ionization (ESI); MS was operated in positive/negative mode; The scan mode was Full scan/ddMS2; The scan range was 100–1200 Da; The capillary temperature was 350 ℃; The spray voltage in negative mode was 3800 V; The spray voltage in positive mode was 3200 V; The sheath gas was 35 arb; the aux gas was 15 arb; Three collision energies of low, medium and high were used for MS2. The positive ion mode was 30 eV, 40 eV, 50 eV, and the negative ion mode was 30 eV, 50 eV, 70 eV. The resolution of the Full scan was 70,000 FWHM, and the resolution of MS2 was 17,500 FWHM. The reference marker compounds present in the sample were identified based on retention time, MS fragmentation and UV spectra.CKI (batch number: 20200329) was supplied by Zhendong Pharmaceutical Co. Ltd . Shimadzu Nexera LC-40-QE-Orbitrap-MS Separation was performed on an analytical column of Hypersil BDS . The oven was set at 25 ℃; The injection volume was 5 μL; The flow rate was set at 0.5 mL minhttps://pubchem.ncbi.nlm.nih.gov/). The simplified molecular input line entry specification (SMILES) of these compounds were imported into four databases, Search Tool for Interactions of Chemicals (STITCH) [Then, the structural information of the CKI compounds was retrieved from the PubChem database  (https:/(STITCH) , SuperPr(STITCH) , SwissTa(STITCH) , and Tra(STITCH) , to collThe human genes associated with PC were obtained from three resources: (1) 10 hub genes related to the occurrence and development of PC and 5 potential biomarkers closely related to prognosis obtained from WGCNA; (2) Therapeutic Target Database (TTD) database . The TTD2FC |> 1 and adjust P < 0.05. The PPI data were extracted from the Search Tool for the Retrieval of Interacting Genes  database [Homo sapiens”, and the confidence score greater than 0.7 (high confidence) were selected for further investigation.Data were normalized and analyzed by the edgeR package . Differedatabase . The PPIIn this study, we constructed three networks: (1) compound-putative target network was built by connecting CKI compounds and their related targets; (2) CKI-PC PPI network was constructed by connecting the intersections of the compound targets and PC-related targets and other human proteins interacting with them; (3) drug-compound-PPI target-pathway network was constructed by connecting CKI, compounds, PPI targets, and related pathways. The above networks were visualized using Cytoscape and the topological features of the interaction networks were evaluated by calculating three indices  using the NetworkAnalyzer plug-in.https://david.ncifcrf.gov/, version 6.8) [P < 0.05 and the false discovery rate (FDR) was set at < 0.05.To identify the core clustering module in the CKI-PC PPI network, the plug-in Molecular Complex Detection (MCODE) in Cytoscape was used for module analysis. DAVID (ion 6.8)  was emplhttps://www.RCSB.org/). Then, the protein structures were processed by AutoDock Tools (ADT) [The three-dimensional crystal structures of the core target were extracted from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Database   , includils (ADT) . Finally−1) was diluted with DMEM .Human PC cell lines Panc-1 was purchased from Procell Life Science &Technology Co., Ltd. , and cultured in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum  and 1% penicillin/streptomycin  in a saturated humidity environment at 37 °C and 5% CO2. For CKI incubation , CKI (to5 cells mL−1) and seeded into 96-well plates (100 μL·well−1), and cultured for 24 h, routinely. The cells were then cultured with a drug-containing medium for 24, 48 and 72 h respectively. After drug treatment, the CCK-8 solution was added into 96-well plates (10 μL·well−1) and incubated for 4 h at 37 °C. Optical density (OD) was detected at 450 nm using a microplate reader . EdU proliferation assay was performed by BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488  and strictly followed the manufacturer's protocol.The proliferation of Panc-1 cells was detected by Cell Counting Kit-8  assay and 5‐ethynyl‐20-deoxyuridine (EdU) proliferation assay. In brief, after routine digestion, cells were blown into a single-cell suspension  was applied for total RNA isolation according to the manufacturer’s instruction. The quality of total RNA was accredited by SpectraMax Quick Drop readers . Of the total RNA, 1 μg was used for cDNA synthesis following the ReverTra Ace qPCR RT Kit  instruction. RT-qPCR was performed to measure the relative expression of mRNA, using SYBR Green Realtime PCR Master Mix . GAPDH was used as a control and the 2Human AKT1 ELISA Kit, Human CDK1 ELISA Kit, Human JAK1 ELISA Kit, Human EGFR ELISA Kit, Human MAPK1 ELISA Kit and Human MAPK3 ELISA Kit  were used to detect the protein expression of 6 core targets following the manufacturer's instructions.Panc-1 cells were collected in RIPA lysis buffer and centrifuged at 13,000 rpm and 4 ℃ for 10 min. The supernatants were preserved and used for western blot assay. Total protein concentration was gauged by BCA Protein Assay Kit . 20 µg of total protein was mixed with 5 × sample buffer, boiled at 99 ℃ for 5 min and loaded onto 10% SDS-PAGE gels. Then the protein bands were transferred onto NC membranes and blocked with 5% non-fat milk or 5% bovine serum albumin (BSA) for 2 h at room temperature. The NC membranes with proteins were incubated with diluted primary antibodies  at 4 ℃ overnight, including anti-AKT1 (1:500), anti-phospho-AKT1 , anti-CDK1 (1:500), anti-phospho-CDK1 , anti-JAK1 (1:500), anti-phospho-JAK1 , anti-EGFR (1:500), anti-phospho-EGFR , anti-MAPK3/MAPK1 (1:500), anti-phospho-MAPK3/MAPK1  and anti-β-Tubulin (1:2000) antibodies. Then, membranes were incubated with relative sources of secondary antibodies (1:5000) at room temperature for 1.5 h. At last, the specific protein bands were recognized with immobilon western chemiluminescent HRP substrate . Image J software was used for image analysis and the signals of specific proteins were normalized to β-Tubulin.P < 0.05), two asterisks (**: P < 0.01).Data were presented as mean ± SD and statistical analysis was performed with the two-tailed unpaired Student’s t-test using GraphPad Prism 9.0 software. In all statistical analyses, statistical significance was indicated by a single asterisk (*: 2 = 0.85) was selected according to the scale-free criterion to construct the gene co-expression network  with HR < 1 was identified as a protective prognostic gene ; four genes  with HR > 1 were identified as risky prognostic genes , suggesting that the expression of these 5 genes can be effectively distinguished the high- and low-risk of these PC patients  were assigned to the high-risk group, while the remaining 89 patients were assigned to the low-risk group Fig. A. The Ka.273 werents Fig. C. FigureIn our study, 10 marker ingredients of CKI were identified by HPLC–MS Fig. ; Table 3After analysis of the RNA sequencing data, 623 DEGs were identified. In addition, 71 PC-related genes were obtained from the TTD database. Furthermore, 10 hub genes and 5 prognosis-related genes were integrated with the above genes. After deleting duplicate genes, a total of 702 genes were obtained  . In ordeIn the current study, the biological processes (BP), molecular functions (MF), cellular components (CC) and signalling pathways involved in the three key modules were further explored through GO enrichment and KEGG pathway enrichment analysis. The GO enrichment analysis showed that module 1 was closely related to the cell cycle and cell proliferation; module 2 was closely related to JAK-STAT, MAPK cascade, phosphatidylinositol-mediated signalling, phosphorylation, and negative regulation of the apoptotic process; and module 3 was closely related to protein tyrosine kinase activity and MAPK cascade Fig. A. The KEN-methylcytisine, lamprolobine, 9α-hydroxymatrine and sophoranol) were downloaded from the PubChem database. The docking operation  values of CKI on panc-1 cells at 24 h was found to be 3.38 ± 1.40 mg mL−1. And after 48 h and 72 h, the IC50 values of CKI were 2.20 ± 0.54 mg mL−1 and 1.84 ± 0.38 mg mL−1 respectively. Based on the IC50 values and cellular state, 48 h incubated time was chosen for the EdU incorporation assay. The EdU incorporation assay showed that  in all CKI groups compared with the control group. It confirmed that CKI could restrain the strong proliferation of PC cells.Panc-1 cells were used to observe the anti-proliferation effect of CKI in PC treatment. Figure hat Fig. B, C, EdUAKT1, CDK1, JAK1, EGFR, MAPK1 and MAPK3) to evaluate the mechanism of CKI in PC treatment. As shown in Fig. CDK1, JAK1, EGFR, MAPK1 and MAPK3 . However, AKT1 results showed no significant difference in all CKI groups compared with the control group (P > 0.05). We followed up with validation experiments on the protein expression levels of the core targets. ELISA results showed that there was no significant difference in total protein content of AKT1, CDK1, JAK1, EGFR, MAPK1 and MAPK3 in the cytoplasm after CKI intervention  deserve further exploration in PC pathogenesis in addition to the 5 survival-dependent genes described.Although network pharmacology features high-throughput, systematic and holistic research, it also has many limitations, such as the lack of clinical information . Here, WPC cells . To extePC cells . Based oed genes . Similarg events . ADGRG6 g events . ADGRG6 g events . Converser cells . On the er cells . MoreovePC cells . MAPK si28 genes . TherefoCDK1, JAK1, EGFR, MAPK1, and MAPK3 targeted by CKI were investigated by WGCNA and network pharmacology analysis and then verified by in vitro experiments. MAPKs, a class of serine/threonine protein kinases that has four different types  in mammals [MAPKs to regulate cell survival, proliferation, differentiation and migration [JAK1 and TGF-β [Interestingly, in this study, the 5 core genes  mammals . MAPK1 i mammals –63. MAPK mammals . In addi mammals . Aberran mammals . Bioinfo mammals . In addi mammals . Other s mammals . Fortuna mammals , 71. EGF mammals . ErbB2, igration . EGFR isigration , 75. It igration , 77. It igration . In addiigration . Not onligration . CDK1 isigration . At the igration . Besidesigration . Matrineigration . Oxymatrigration . Coincidigration . Matrineigration . JAK is igration . JAK/STAigration . JAK1 isigration . Ruxolitigration . It can nd TGF-β . Matrinend TGF-β . Oxymatrnd TGF-β . ConsistTaken together, we can speculate that the molecular mechanisms of CKI in PC treatment are as follows: (1) CKI inhibits proliferation of PC cells via inhibition of the EGFR transactivation, and downregulation of the NCR-mediated MAPK pathway; (2) CKI inhibits expansion of PC stem cells via inactivation of the GPR87/JAK/STAT pathway; (3) CKI arrests cell cycle progression of PC cells via inhibition of the FAM111B/p16/CDK pathway; (4) CKI inhibits migration and invasion of PC cells via downregulation of the NCR-mediated MAPK/MMP pathway; (5) CKI reverses chemoresistance of PC cells via inhibition of the ROS1-ADGRG6/EGFR pathway; (6) CKI inhibits tumour angiogenesis of PC via inhibition of the ADGRG6/GATA2/STAT5/VEGF/MAPK pathway. However, these speculations should be supported by further basic and clinical experimental data.CDK1, JAK1, EGFR, MAPK1 and MAPK3), which were related to important signalling pathways  and survival-correlated genes . These findings may be useful for clinical decision-making and guidance for rational clinical use of CKI in PC treatment.In summary, this study demonstrated a strategy to optimize conventional network pharmacology, and then explained that the molecular mechanism of CKI treating PC was closely associated with 5 core genes (including Additional file 1: Figure S1. The Kaplan–Meier survival curves for 10 hub targets.Additional file 2: Figure S2. The external validation for the results of WGCNA.Additional file 3: Table S1. Information about the 16 compounds of CKI.Additional file 4: Table S2. Genes associated with PC.Additional file 5: Table S3. Information of potential key targets in CKI-PC PPI network.


Dysbiosis of the gut microbiome is involved in the pathogenesis of various diseases, but the contribution of gut microbes to the progression of chronic obstructive pulmonary disease (COPD) is still poorly understood.We carried out 16S rRNA gene sequencing and short-chain fatty acid analyses in stool samples from a cohort of 73 healthy controls, 67 patients with COPD of GOLD stages I and II severity, and 32 patients with COPD of GOLD stages III and IV severity. Fecal microbiota from the three groups were then inoculated into recipient mice for a total of 14 times in 28 days to induce pulmonary changes. Furthermore, fecal microbiota from the three groups were inoculated into mice exposed to smoke from biomass fuel to induce COPD-like changes.Prevotella-dominated gut enterotype and lower levels of short-chain fatty acids. After 28 days of fecal transplantation from COPD patients, recipient mice exhibited elevated lung inflammation. Moreover, when mice were under both fecal transplantation and biomass fuel smoke exposure for a total of 20 weeks, accelerated declines in lung function, severe emphysematous changes, airway remodeling and mucus hypersecretion were observed.We observed that the gut microbiome of COPD patients varied from that of healthy controls and was characterized by a distinct overall microbial diversity and composition, a These data demonstrate that altered gut microbiota in COPD patients is associated with disease progression in mice model.The online version contains supplementary material available at 10.1186/s12931-021-01872-z. Chronic obstructive pulmonary disease (COPD) is a worldwide public health concern and a leading cause of morbidity and mortality , 2. CurrThe role of the microbiome, and the gut microbiome in particular, has drawn considerable attention in human health and disease. Emerging evidence has revealed the role of gut microbiota dysbiosis in various chronic human diseases , 9. MoreIn the present study, we performed 16S ribosomal RNA sequencing of stool samples from COPD patients and healthy controls, described the features of the gut microbiota, and analyzed their SCFA profiles. Next, we performed fecal microbiota transplantation into mice, demonstrating the role of disordered gut microbiome in COPD.To investigate the features of the gut microbiota in COPD patients and the contribution of the gut-lung axis in COPD, we designed a systematic and reproducible workflow Fig. a–c. All 1/FVC ratio ≥ 70% and FEV1 ≥ 80%), 67 patients with COPD GOLD severity stages I and II (FEV1/FVC ratio < 70% and FEV1 50–80%), and 32 patients with COPD severity stages III and IV (FEV1/FVC ratio < 70% and FEV1 < 50%).All participants underwent pulmonary function measurements to diagnose COPD according to GOLD guidelines . SpiromeAll individuals underwent medical tests prior to stool sample collection, including chest X-ray, electrocardiography, abdominal ultrasound, and blood, urine, and stool tests. Subjects were required to meet the following criteria to qualify for the study: (1) Able and willing to provide written informed consent and medical records from the preceding year. (2) Male aged 40–80 years. (3) Diagnosed with COPD more than 1 year prior to the study. Subjects who met any of the following criteria prior to enrollment were excluded from the study: (1) Treatment with systemic  corticosteroids within the preceding 8 weeks. (2) History of cystic fibrosis, asthma, and/or other clinically significant lung disease other than COPD. (3) A diagnosis of cancer, heart failure, hypertension, diabetes, infectious diseases, renal or liver dysfunction, gastrointestinal disease, or treatment with antibiotics (inclusive of macrolide antibiotics) within the preceding 8 weeks. All clinical information was collected according to standard procedures by the State Key Laboratory of Respiratory Disease of Guangzhou Medical University .Fresh stool from donors was collected in the morning. The consistency of each sample was graded according to the Bristol Stool Form Scale, and only sample types 2–5 were included . Stool shttp://ccb.jhu.edu/software/FLASH/) [http://qiime.org/) software pipeline was used to cluster high-quality reads into operational taxonomic units (OTUs) at the 97% identity level. OTU search was performed using the GreenGenes 13.8 database. The α-diversity indices (Chao1 index) was calculated by QIIME. The bar diagram of alpha diversity indices and relative abundance were drawn using GraphPad Prism 8 software .16S rRNA gene sequence analysis, including raw sequence filtering and taxonomic classification, was performed as described previously . Briefly/FLASH/) . Sequenchttp://enterotypes.org) as indicated by Costea et al. [−4 and that appeared in at least 50% of samples in each group were considered in the analysis. Pearson’s Chi-squared test was used for testing enterotypes between clinical groups. Significance of community dissimilarity based on Bray–Curtis dissimilarity matrices was tested using a permutational multivariate analysis of variance (PERMANOVA) function (adonis) within the R package vegan.Enterotyping was run with a reference-based online tool  were measured in the stool samples by high-performance gas chromatography  with an autosampler and a flame ionization detector according to the manufacturer’s guidelines.Specific pathogen-free male C57BL/6 mice were purchased from Guangzhou University of Chinese Medicine . The mice were housed five to a cage. All experiments were conducted with mice with 8–10 weeks of age. Temperature and relative humidity in the animal facility were controlled at 23 ± 2 °C and at 40–70%, respectively. Lighting was artificial with a sequence of 12 h of light (06:00–18:00) and 12 h of darkness. Rodent food pellets and water were sterilized and provided ad libitum. The Animal Medical Center of Guangzhou Medical University reviewed and approved all experiments (identification number: GY2018-084).g for 10 min at 4 °C, the supernatant was collected and stored at − 80 °C for subsequent use. Enumeration of inoculum from healthy group, COPD I-II group and COPD III-IV group in 1 mL of initial suspension were performed according to plate count methods [To deplete the gut microbiota, mice were provided with broad-spectrum antibiotics  in drinking water for 3 weeks as previously described . Treatme methods .A total of 60 mice were randomly divided into four groups of 15: Phosphate-buffered saline (PBS) FMT group, healthy FMT group, COPD I–II patients FMT group, and COPD III–IV patients FMT group. After microbiota depletion, the fecal microbiota transplantation was performed by a single oral administration of 100 μL per mouse every other day, for a total of 14 times in 28 days. The mice received fecal transplants from healthy individuals, COPD I–II subjects, or COPD III–IV subjects, and PBS as control.Similar to the previous, a total of 60 mice were also randomly divided into four groups of 15: PBS FMT group, healthy FMT group, COPD I–II patients FMT group, and COPD III–IV patients FMT group. After microbiota depletion, all mice were exposed to smoke produced by smoldering China fir sawdust (40 g/exposure) for two 3-h periods, 5 days per week, for 20 weeks in an inhalation chamber (model INH-WB_NOE (R/M)_CAP (PM2.5)_CS_SP; TSE Systems, Bad Homburg, Germany) . Particu20), FEV20 (forced expiratory volume in 20 s), FRC , PEF (peak expiratory flow), MMEF (mean mid expiratory flow) and minute ventilation volume were measure within 10 min. At least three acceptable maneuvers for each test of every mice were conducted to obtain a reliable mean spirometry data.Spirometry data were obtained as previously described using a Forced Pulmonary Maneuver System  . Mice we2 and lung tissue and blood samples were collected. Whole lungs were cyclically inflated and deflated with 1 mL of phosphate-buffered saline  three times. Cells were isolated by centrifugation at 300×g for 10 min at 4 °C and stained with Diff-Quik stain . Differential cell counts were assessed from 400 cells counted on each slide. Plasma cytokine levels were assayed using Luminex xMap and a commercially available mouse cytokine 6-plex panel  according to the manufacturer’s guidelines and measured on a Bio-Plex 200 Platform.Mice were sacrificed by COg at room temperature for 20 min. We blocked the Fc receptors by incubating cells first with anti-CD16/32 antibodies for 15 min on ice. Cells were stained with the following antibodies for 30 min at room temperature: BB600-conjugated anti-mouse CD3, APC-conjugated anti-mouse CD19, BV421-conjugated anti-mouse CD8, and FITC-conjugated anti-mouse CD4 . After washing, cells were fixed with fluorescence-activated cell sorting lysing solution (BD Biosciences). Appropriate isotype controls were used to determine the specificity of the staining. Flow cytometry data were acquired using FACSVerse (BD Biosciences) and analyzed with the FlowJo software .Flow cytometry immunophenotyping was performed on T cells and B cells in whole blood. Peripheral blood mononuclear cells were isolated from sodium heparin-treated venous blood samples by Ficoll-Hypaque density gradient and centrifugation at 1000×β-tubulin was used as an internal control to assess equal loading of total protein between wells. The bound antibodies were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). The abundance of target proteins was quantified by enhanced chemiluminescence.Lung tissues were homogenized on ice for analysis of MUC2, MUC5AC, the tight junction protein claudin 1, α smooth-muscle actin (a-SMA), matrix metalloproteinase 2 (MMP-2), and neutrophil elastase levels. Total proteins were extracted from 100 mg of lung tissues from each group, and concentrations were determined by the BCA protein assay. Thirty micrograms of total protein were loaded into each well and fractionated on a 10% SDS polyacrylamide gel. The housekeeping gene Lung tissues were fixed with 4% paraformaldehyde solution and embedded in paraffin using standard methods as described previously . Consecup values were corrected for multiple testing using the Bonferroni method. Analyses were adjusted by age and smoking history. Pearson’s χ2 test was used for testing enterotypes between clinical groups. Significance of community dissimilarity from Bray–Curtis dissimilarity matrices was tested using a permutational multivariate analysis of variance function (adonis) within the R package vegan. For animal experiments, comparisons were performed using ANOVA and p values were also corrected using the Bonferroni method. Statistical analysis was performed in SPSS version 24 , and the corrected p < 0.05 was considered significant.In the human cohort, microbiota data and SCFA levels were tested by ANOVA and the Kruskal–Wallis H test. The A total of 172 Chinese participants were enrolled and divided into healthy controls (n = 73), COPD stage I–II (n = 67), and COPD stage III–IV (n = 32). Table p = 0.049 vs. healthy subjects and p = 0.009 vs. COPD I–II group, Fig. p = 0.040 vs. healthy group; p = 0.007 vs. COPD I–II group) but a higher abundance of Firmicutes . Compared with the healthy controls, COPD III–IV subjects exhibited a lower abundance of Bacteroidaceae (p = 0.034) and Fusobacteriaceae (p = 0.002). There were no significant differences between COPD I–II and COPD III–IV subjects.To assess whether gut microbial changes are associated with COPD severity, we performed 16S rRNA gene sequencing of fecal samples. Comparison between the three groups showed that intra-individual diversity, as measured by the number of operational taxonomic units (OTU), was significantly lower in the COPD III–IV group , corresponding to differing enterotypes but not clinical groups .In ups Fig. a. Most hp = 0.012) and COPD I–II (p = 0.109) subjects  and isovaleric acid  were also significantly lower in the COPD III–IV subjects . Total SCFAs levels were significantly lower in the COPD III–IV group than in healthy  and tumor necrosis factor α (p = 0.029) were significantly higher  and COPD III–IV patients (p < 0.01) than from healthy controls : Bacteroidetes, 36.84 , 32.13 , 33.05 , and 29.77 , respectively; Firmicutes, 40.33 , 49.88 , 53.60 , and 54.26 , respectively; Proteobacteria, 8.51 , 3.30 , 1.04 , and 3.24 , respectively; Actinobacteria, 0.06 , 9.97 , 7.11 , and 4.49 , respectively. Relative abundances of bacterial phyla differed (p < 0.05 or p < 0.01) between the PBS FMT group and fecal transplants group in Actinobacteria, Firmicutes, and Proteobacteria. There was no significant difference between the healthy FMT group, COPD I–II FMT group, and COPD III–IV FMT group. Results are expressed as the mean; n = 10 mice. Significance was determined by ANOVA and by Kruskal-Wallis test, and p values were corrected using the Bonferroni method.Additional file 2: Fig. S2. Microbial abundance was measured in feces from human subjects and compared with microbial abundance in feces from recipient mice. At the phylum level, inoculum from healthy subjects, COPD I–II subjects, and COPD III–IV subjects contained four major bacterial phyla (%): Bacteroidetes, 54.50, 51.48 and 43.43, respectively; Firmicutes, 38.50, 39.04 and 46.96, respectively; Proteobacteria, 3.67, 6.22 and 7.53, respectively; Actinobacteria, 0.69, 1.08 and 0.65, respectively. As shown below, the characteristics of the murine gut microbial community post-fecal transplant were not identical to those profiled originally in the human-derived sample (A–C). Results are expressed as the mean.Additional file 3: Fig. S3. The biomass smoke exposure system and particle size distributions during exposure. (a) All mice were exposed to biomass smoke in inhalation chamber systems. (b) Particulate matter concentrations and particle size distributions during biomass smoke exposure. Boxes and the inside line represent means ± SD for particulate matter with an aerodynamic diameter < 2.5 μm (PM2.5).Additional file 4: Supplementary table 1-4. Supplementary table 1. Relative abundance of most abundant phyla (%) observed in stool samples collected in healthy control group and COPD group. Supplementary table 2. Relative abundance of most abundant family (%) observed in stool samples collected in healthy control group and COPD group. Supplementary table 3. The viability of bacteria in the inoculum in different microbial growth medium (CFU/ml). Supplementary table 4. Concentrations of particulate matter (PM) and gaseous pollutants measured during exposure.Additional file 5: Ethics approval and consent to participate.Additional file 6: Animal ethics approval.


TaNramp1, TaNramp5, TaHMA2, TaHMA3, TaLCT1 and TaIRT2 in roots. Taken together, our results suggested that GSH could alleviate Cd toxicity in wheat by increasing GSH synthesis gene expression or suppressing Cd transporter genes expression, and further affecting Cd uptake and translocation in wheat plants.Cadmium (Cd), a toxic heavy metal, is harmful to plants and human health. Glutathione (GSH) could alleviate Cd toxicity of plant species, whereas its mechanism responsible for wheat remains poorly understood. Here, we found that exogenous GSH application significantly increased the fresh and dry weight, root elongation, chlorophyll contents, while decreased the contents of malondialdehyde (MDA) and GSH, and translocation factor of Cd compared with Cd treatment. Moreover, GSH application significantly increased activities of antioxidant enzymes and expression of related genes, which involved in GSH synthesis, especially in roots. In addition, we found that GSH application suppressed Cd-induced expression of metal transporter genes The online version contains supplementary material available at 10.1007/s00128-021-03283-8. Cadmium (Cd), a toxic heavy metal, is particularly dangerous and widely spread in the soil, and its accumulation would inhibit plant growth and development by disrupting the enzyme activities, hormone balance, protein synthesis, and key metabolic reactions  family, Iron-Regulated Transporter 1 (IRT1) homologs, Low affinity Cation Transporter (LCT), and Heavy Metal ATPase (HMA), which were identified and characterized for the function mediated-Cd uptake or translocation in plants  were surface-sterilized and germinated as previously described in our published literature , which could reduced the competition of ions in Hoagland's solution and provided an acidic environment for increasing the activity of Cd2+ , Cd (0.5 mM CaCl2 + 50 μΜ CdCl2), Cd + GSH (0.5 mM CaCl2 + 50 μΜ CdCl2 + 20 μΜ GSH), and GSH (0.5 mM CaCl2 + 20 μM GSH). And the concentrations of Cd and GSH were performed as previously studies  and chlorophyll contents were calculated according to the method of Zheng et al.  and Li ePlant shoots and roots were dried and digested as previous study  and reduced glutathione (GSH) content assays according to our previous study  gene was used as an endogenous control gene in this study. All used primers were listed in Table S1.Total RNA of wheat shoots and roots were extracted by using TransZol RNA Isolation Reagent  and determined the concentration. Then, 1 μg of total RNA was prepared for reverse transcription by using ReverTra Ace qPCR RT Kit . qPCR was performed using AceQ™ Universal SYBR® qPCR Master Mix  as following reaction conditions: one cycle at 95°C for 10 min, 40 cycles of amplification at 95°C for 10 s, 58°C for 15 s, and finally 72°C for 20 s in a QuantStudio 3  system. The relative expression levels were calculated according to the method of 2p < 0.05. All the data in this study was calculated at least three biological replicates.Statistically analyses were performed by using the SPSS software (version 25.0), and the statistical assays were tested with LSD-test to evaluate significant differences between the treatments using a significance threshold of To determine the effect of GSH on Cd toxicity, wheat seedlings grew in different culture medium for 10 days. Compared with non-treated control (CK), the growth of wheat plants was inhibited, the color of leaves were displayed visible chlorosis, and the length of roots were inhibited in Cd treatment, while exogenous GSH significantly alleviated the above Cd toxicity effect . After application with exogenous GSH, the chlorophyll a and b contents restored to 61.6% and 65.9% of CK, respectively; and it was greater 2.35- and 2.42-folds than only treated with Cd , were determined in wheat plants. The results showed that exogenous GSH supplement significantly decreased 1.85- or 1.65-folds of MDA or reduced GSH contents compared with Cd treatment in roots, while they did not significantly changed in shoots Table . MoreoveGSH, GST, GR and APX were significantly induced 1.04, 2.26, 5.94, 2.48-folds in roots , GST (6.19-fold), GR (2.66-fold) in roots  in shoots , while significantly decreased the expression levels of GSH (46.3%), GST (19.8%), GR (79.9%) in shoots , compared with Cd treatment. This suggested that exogenous GSH could differentially regulated the expression profiles of these genes which might induced the changes of GSH content under Cd stress.Transcription of defense gene expression provided more precise estimation than their enzyme activity in response to abiotic stresses  . It is interestingly that the only supplement of exogenous GSH inhibited (75%-95%) the transcription of these transporter genes in roots Below is the link to the electronic supplementary material.


Rhinovirus (RV) is a primary etiologic agent of common cold that can subsequently acutely exacerbate bronchial asthma or chronic obstructive pulmonary disease. Kakkonto (Ge-gen-tang in Chinese), one of the most frequently prescribed traditional Japanese (Kampo) medicines, is used for treating common cold, shoulder stiffness, or inflammatory diseases of the upper body. Previous experimental studies have indicated that kakkonto exerts antiviral and anti-inflammatory effects on the influenza virus and the human respiratory syncytial virus. However, there is a lack of reports investigating the efficacy of kakkonto in RV infection. Hence, the aim of the current study was to investigate the effects of kakkonto on RV infection of human nasal epithelial (HNE) cells. HNE cells obtained via endoscopic sinus surgery were cultured and infected with RV14, with or without kakkonto treatment. The supernatants from the cells were collected, and the RV14 titer and cytokine levels were assessed. Reverse transcription-polymerase chain reaction was performed to determine the amount of viral RNA, while the level of nuclear factor kappa B (NF-κB) subunits in the nucleus was assessed by enzyme-linked immunosorbent assay. Although kakkonto treatment did not reduce RV14 titer or RNA levels, indicating that it did not inhibit RV14 proliferation, it was found to reduce the production of specific pro-inflammatory cytokines, including interleukin (IL)-8, tumor necrosis factor (TNF)-α, and monocyte chemotactic protein-1 (MCP-1). Unlike that observed with the kakkonto extract, none of the crude drugs contained in kakkonto reduced IL-8 level. Furthermore, though kakkonto treatment significantly reduced p50 levels, it did not impact the p65 subunit of NF-κB. These results indicated that kakkonto can inhibit inflammation caused by RV infection and may exert an immunomodulatory effect on HNE cells. This is the first report to elucidate the effects of kakkonto extract on RV infection in primary cultures of HNE cells, providing evidence that kakkonto may act as an effective therapy for RV infection and subsequent airway inflammation. Rhinoviruses (RVs) were the most common cause of the common cold prior to the pandemic outbreak of the severe acute respiratory syndrome coronavirus 2. Specifically, RVs were reported to account for 30–50% of all respiratory illnesses in 2003 . The comin vitro models have reported the antiviral and anti-inflammatory effects of kakkonto  cells, while also elucidating the underlying mechanism of action.Kakkonto (Ge-gen-tang in Chinese), one of the most frequently prescribed Kampo medicines in Japan, is used for the treatment of the common cold, shoulder stiffness, or inflammatory diseases of the upper body . Previoukakkonto . Howeverkakkonto  or on thkakkonto . In murikakkonto  and lungkakkonto , prolongkakkonto . Kakkontkakkonto . Furtherkakkonto . Cumulatl-carbocisteine; 14 (63.6%) were treated with macrolides (13 patients with clarithromycin and 1 with erythromycin); 9 (40.9%) were treated with Montelukast sodium; and 9 (40.9%) were treated with antihistamine agents  were ex-smokers; 13 had allergic disease ; 10 (45.5%) were prescribed nasal corticosteroids; 10 (45.5%) were treated with e agents . All par5 viable cells/mL in round-bottom plastic tubes  coated with human placental collagen. Cells were cultured in a 1 ml mixture of Dulbecco’s modified Eagle medium: nutrient mixture F-12 (DMEM/F-12) medium  containing 2% Ultroser G (USG) , 105 U/L penicillin, 100 mg/L streptomycin, 5 mg/L amphotericin B, and 100 mg/L gentamicin. The tubes, loosely covered with screw caps, were placed at a 5° angle and cultured at 37°C in a 5% CO2 atmosphere.Isolation and culturing of HNE cells from the nasal polyps was performed as described previously . The HNE5 U/L penicillin and 100 mg/L streptomycin). The cells were then plated in 96-well plates or plastic tubes and cultured.HEF cells  were cultured as previously described  with sli5 tissue culture infective dose (TCID50)/mL for 1 h, rinsed with phosphate buffered saline (PBS), and cultured in plastic tubes in 1 ml MEM supplemented with 2% ultra-low IgG FBS or 10% FBS and antibiotics (105 U/L penicillin and 100 mg/L streptomycin) in a 33°C incubator  with rotation (12 rotations per hour). The cells were incubated for approximately 3 days until cytopathic effects were observed. The virus-containing fluid obtained from these cells was frozen in aliquots at −80°C.A stock of clinically isolated RV14 was prepared by infecting HEF cells as described previously . Brieflyth Edition defines kakkonto as being composed of seven crude drugs: 4 g Puerariae Radix, 3 g Ephedrae Herba, 3 g Zizyphi Fructus, 2 g Cinnamomi Cortex, 2 g Paeoniae Radix, 2 g Glycyrrhizae Radix, and 2 g Zingiberis Rhizoma  registry numbers of components in kakkonto extract, shown in 10H15NO: 165.23), 19–57 mg of glycyrrhizic acid (C42H62O16: 822.93), and 14–56 mg of paeoniflorin (C23H28O11: 480.46) per extract prepared according to the previously reported method (The Japanese Pharmacopeia defines the kakkonto formula as containing the following indexed components: 9–27 mg of ephedrine or pseudoephedrine (Cd method .The kakkonto extracts were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/ml by vortexing for 5 min at room temperature (23°C). The mixture was then dispensed in microcentrifuge tubes and frozen at −80°C until experimentation. Kakkonto stock solution was diluted in culture medium (DMEM/F-12 with 2% USG) at a concentration of 100 μg/ml and the concentrations of ephedrine, pseudoephedrine, and glycyrrhizic acid were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS) performed by Sekisui Chemistry, Tokyo, Japan.5 TCID50/mL, 0.13 TCID50/cell of the multiplicity of infection). After 1 h of incubation, the viral solution was removed, and cells were rinsed with PBS. The cells were then cultured in 1 ml fresh medium (2% USG in DMEM/F12) in the presence, or absence, of kakkonto extracts diluted in culture medium. The kakkonto treatments were initiated 1 h after infection and continued until the end of the experimental period. To investigate the concentration-dependent effects, cells were treated with 10, 20, or 50 μg/ml kakkonto. The supernatants (1 ml) were collected before infection, as well as 1, 3, 5, and 7 days after infection, and rapidly frozen in ethanol at −80°C. The specific kakkonto concentrations used in this study were determined based on the detected concentrations of ephedrine/pseudoephedrine and glycyrrhizic acid in the kakkonto solution. The specific details of these calculations can be found in Concentration of Index Components in Kakkonto Extracts.Infection of HNE cells with RV14 was performed using previously described methods . A stock5 U/L penicillin and 100 mg/L streptomycin), as described previously  with TaqMan gene expression master mix , as described previously . The amoIL-6 and IL-8 level were assessed using an enzyme-linked immunosorbent assay (ELISA) performed by LSI Medience Corporation  with a solid phase chemiluminescent ELISA kit , and an IL-8 EIA kit . The levels of other cytokines were determined by a multiplex suspension array  using a Luminex® assay system  with a magnetic Luminex® assay human premixed multi-analyte kit . The assay was performed according to the manufacturer’s instructions. Briefly, all samples were diluted using Calibrator Diluent RD6-52, and 50 μL of the diluted samples were used for the assay customized to detect and quantify TNF-α, IL-1β, IL-17C, IL-17E/IL25, granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemotactic protein-1 (MCP-1), thymic stromal lymphopoietin (TSLP), thymus and activation-regulated chemokine (TARC), eotaxin, and regulated on activation normal T cell expressed and secreted (RANTES). The fluorescence intensity of each sample was measured using a Luminex® 100/200TM system , and the concentrations of each analyte were calculated using MILLIPLEX® Analyst 5.1 .The distribution and fluorescence intensity of acidic endosomes in the cells were measured with LysoSensor DND-189 dye  using live-cell imaging . The celNuclear proteins were extracted from the nuclear extract kit of the epithelial cells . The presence of translocated p50 and p65 subunits in the nuclear extracts was assessed using a TransAM NFκB family kit , according to the manufacturer’s instructions. To examine the effect of kakkonto on the NF-κB subunits in the HNE cells, the cells were pretreated with kakkonto (20 μg/ml) or medium alone at 33°C for 24 h, after which the nuclear proteins were extracted.t-test or Mann-Whitney U-test was used for comparison between two groups according to the distribution patterns of the evaluated variables. Statistical significance was set at p < 0.05. In all experiments, n refers to the number of donors from whom the epithelial cells were obtained. All analyses were performed using SPSS version 21 .Results were expressed as mean ± standard error (SE). Statistical analysis was performed using two-way repeated-measures analysis of variance (ANOVA). Student’s LC-MS/MS analysis revealed that the concentrations of ephedrine/pseudoephedrine and glycyrrhizic acid in the kakkonto solution (100 μg/ml) were 363.0 ± 13.9 ng/ml and 867.3 ± 22.2 ng/ml, respectively. We then estimated the concentration of ephedrine/pseudoephedrine in human serum after administration of kakkonto to be up to 40–60 ng/ml , and thap = 0.001; p = 0.36). However, the RV titer in the supernatants of the cells treated with kakkonto tended to decrease slightly by 24 h post-infection (h.p.i.). We then measured viral titers in the supernatants of HNE cells treated with 50 μg/ml kakkonto (The RV14 titer increased significantly until 7 days after infection (kakkonto  and founp = 0.69).To investigate the effect of kakkonto on the proliferation of RV14, we determined the level of RV14 RNA in the cells 1 day post-infection d.p.i., . Treatmep = 0.027, The treatment of HNE cells with 20 μg/ml kakkonto for 24 h significantly reduced the number of green-fluorescent acidic endosomes , as wellp = 0.19).The concentration of IL-6 in the supernatants of untreated, infected cells increased 24 h.p.i. ; whereasp = 0.013; p = 0.031 and 0.021, respectively). We also assessed IL-8 concentration 3 d.p.i. and found it to 142,667 ± 1,453, 149,333 ± 7,216, and 122,000 ± 7,638 pg/ml in the supernatants collected from uninfected cells, infected and untreated cells, and infected cells treated with kakkonto (20 μg/ml), respectively. Thus, 20 μg/ml kakkonto treatment tended to reduce IL-8 concentration 3.d.p.i compared to RV14 infection alone, although the difference was not statistically significant (p = 0.059).In untreated, RV-infected cells, we also observed that the level of IL-8 significantly increased 24 h.p.i. compared to uninfected controls . RV infection also increased the levels of IL1β, GM-CSF, TSLP, while kakkonto treatment only slightly reduced these effects, however, the differences were not statistically significant. Also, RV infection tended to increase eotaxin levels, however, kakkonto treatment did not impact these effects. The levels of RANTES were below the level of detection in seven of the nine samples.Furthermore, RV14 infection was found to increase the concentration of TNF-α, IL-1β, GM-CSF, and MCP-1 , while kp < 0.05).To investigate the mechanism underlying kakkonto-induced cytokine production, we quantified the nuclear levels of NF-κB subunits (p65 and p50) in HNE cells. Compared to the untreated control, Kakkonto treatment (20 μg/ml) did not impact the levels of p65  but signIn the present study, we investigated the effect of kakkonto extract on RV infection in primary cultures of HNE cells. Kakkonto treatment did not reduce the RV14 titers or RNA levels, however, it did significantly reduce the number, and fluorescence intensity of, acidic endosomes, through which RV RNA enters the cytoplasm of epithelial cells . These rRV infection induces the production of various cytokines, including IL-1β, IL-6, IL-8, TNF-α, GM-CSF, and MCP-1 . These pIL-8 plays an important role in neutrophil migration to sites of inflammation. In fact, IL-8 concentration in sputum is reportedly related to the severity of COPD and BA, as well as accelerated reduction in forced expiratory volume . Meanwhil-carbocisteine (45.5%), ambroxol hydrochloride (9.1%), long-acting β2 agonists (22.7%), or macrolides (63.6%), the use of these drugs may have influenced the RV titer or cytokine levels, as well as the abundance of NF-κB. Indeed, these drugs were previously reported to exert antiviral and anti-inflammatory effects against RV infection  2, on the cell surface, recognizes viral capsid proteins. After entering endosomes, TLR 3 and TLR 7/8 bind to double-and single-stranded RNA, respectively. Interactions with these TLRs trigger activation of the NF-κB pathway and increase production of interferon (IFN)-β, IFN-γ, IL-6, and IL-8 . Geng etnfection . In partPuerariae Radix, Ephedrae Herba, Zizyphi Fructus, Cinnamomi Cortex, Paeoniae Radix, Glycyrrhizae Radix, and Zingiberis Rhizoma. The antiviral and anti-inflammatory effects of each crude drug have been investigated to some extent. For example, Chang et al. reported that ginger  and Paeonia lactiflora inhibit human respiratory syncytial virus-induced plaque formation in the airway epithelium by blocking viral attachment and internalization . Meanwhile, Wang et al. reported that puerarin inhibits the expression of TNF-α, IL-6, and IL-1β in LPS-induced murine lung tissue and reduces IL-8 production in A549 cells . Glycyrrkakkonto . Hence, kakkonto . Kakkontkakkonto . FurtherThe severity of viral infection reflects the regulation of virus proliferation and the extent of virus-associated inflammation . Until nThe present study revealed that kakkonto may exert an anti-inflammatory effect following RV infection via suppression of pro-inflammatory cytokine production. Specifically, kakkonto may reduce inflammation induced by RV infection, thereby reducing chronic airway inflammation. To our knowledge, this is the first study to elucidate the effects of kakkonto extract on RV infection in primary cultures of HNE cells. However, further investigation is needed to elucidate the detailed mechanism of the anti-inflammatory effects and clinical efficacy of kakkonto in chronic respiratory diseases.