Cutibacterium acnes (previously Propionibacterium acnes), plays an important role in the pathogenesis and progression of the dermatological skin disorder acne vulgaris. The methanolic extract of Helichrysum odoratissimum (L.) Sweet (HO-MeOH) was investigated for its ability to target bacterial growth and pathogenic virulence factors associated with acne progression. The gas chromatography–mass spectrometry (GC-MS) analysis of HO-MeOH identified α-humulene (3.94%), α-curcumene (3.74%), and caryophyllene (8.12%) as major constituents, which correlated with previous reports of other Helichrysum species. The HO-MeOH extract exhibited potent antimicrobial activity against C. acnes (ATCC 6919) with a minimum inhibitory concentration (MIC) of 7.81 µg/ml. It enhanced the antimicrobial activity of benzoyl peroxide (BPO). The extract showed high specificity against C. acnes cell aggregation at sub-inhibitory concentrations, preventing biofilm formation. Mature C. acnes biofilms were disrupted at a sub-inhibitory concentration of 3.91 µg/ml. At 100 µg/ml, HO-MeOH reduced interleukin-1α (IL-1α) cytokine levels in C. acnes-induced human keratinocytes (HaCaT) by 11.08%, highlighting its potential as a comedolytic agent for the treatment of comedonal acne. The extract exhibited a 50% inhibitory concentration (IC50) of 157.50 µg/ml against lipase enzyme activity, an enzyme responsible for sebum degradation, ultimately causing inflammation. The extract’s anti-inflammatory activity was tested against various targets associated with inflammatory activation by the bacterium. The extract inhibited pro-inflammatory cytokine levels of IL-8 by 48.31% when compared to C. acnes-induced HaCaT cells at 7.81 µg/ml. It exhibited cyclooxygenase-II (COX-II) enzyme inhibition with an IC50 of 22.87 µg/ml. Intracellular nitric oxide (NO) was inhibited by 40.39% at 7.81 µg/ml when compared with NO production in lipopolysaccharide (LPS)-induced RAW264.7 cells. The intracellular NO inhibition was potentially due to the 2.14 fold reduction of inducible nitric oxide synthase (iNOS) gene expression. The HO-MeOH extract exhibited an IC50 of 145.45 µg/ml against virulent hyaluronidase enzyme activity, which is responsible for hyaluronan degradation and scar formation. This study provides scientific validation for the traditional use of H. odoratissimum as an ointment for pimples, not only due to its ability to control C. acnes proliferation but also due to its inhibitory activity on various targets associated with bacterial virulence leading to acne progression.The Gram-positive bacterium  Cutibacterium acnes proliferation, and activation of inflammatory cascades. The Gram-positive rod, C. acnes is of particular importance in acne progression. This bacterium contributes to disease progression with its ability to modulate keratinocyte proliferation, secrete virulent enzymes involved in sebum degradation (lipase) and tissue injury  and activating skin innate immunity through the activation of keratinocytes, sebocytes, and peripheral blood mononuclear cells resulting in the production of pro-inflammatory cytokines interleukin-1β (IL-1β), IL-6, IL-8, IL-12, IL-17, TNF-α, and GM-CSF (granulocyte-macrophage colony-stimulating factor)  lipase enzyme involved in the degradation of sebum triacylglycerides resulting in the release of glycerol and free fatty acids. Glycerol is used as a nutrient source for the C. acnes bacterium, and the free fatty acids arrange themselves between keratinocytes, increasing bacterial cell adhesion, and enhancing biofilm formation within the pilosebaceous unit (C. acnes have shown increased cyclooxygenase-II (COX-II) expression  through chemotaxis and activation of neutrophil cells. These increased levels of NO production within the pilosebaceous follicles causes irritation and rupture of the follicular wall, ultimately leading to the formation of inflammatory lesions  are distributed widely among Gram-positive microorganisms including Cutibacterium species. Hyaluronidases act by completely degrading HA into disaccharides or by degradation into a mixture of unsaturated oligosaccharides. These enzymes contribute to bacterial virulence through tissue injury, facilitating bacterial spread to deeper tissues (Acne vulgaris (AV) is a chronic inflammatory skin disorder of the pilosebaceous, localized on the face, chest, and back. This skin condition affects approximately 85% of people but is particularly common among adolescents. The disorder is characterized by the formation of non-inflammatory comedone lesions, more commonly referred to as blackheads or whiteheads. In more severe cases, the formation of inflammatory papules, pustules, nodules, and cysts is common. Acne vulgaris persistence and unresponsiveness to antibiotic treatments can result in the formation of atrophic (sunken) and hypertrophic scars. The formation of lesions and scars on visible areas of the skin not only affect aesthetic beauty but contribute to psychological problems affecting self-confidence and self-esteem. The progression of AV arises from four main events, including abnormal keratinocyte proliferation, increases in sebum production, increased  factor) . The bioevere AV . The gensurfaces . The attorganism . Abnormaomedones . The cytomedones . C. acneous unit . It is, ous unit . Sebocytpression . The proferation . C. acne lesions . Hyaluro tissues . The inhHelichrysum odoratissimum (L.) Sweet is a perennial shrub of the genus Helichrysum, consisting of approximately 500–600 species, of which approximately 244–250 are found in South Africa  Ltd. . PrestoBlue was purchased from Thermo Fisher Scientific . TRIzol and molecular grade water were purchased from Thermo Fisher Scientific . Dexamethasone was purchased from Sigma Chemicals Co. . Ultra pure liquid chromatography water and acetonitrile (Romil-UpS™) were purchased from Microsep, South Africa. Formic acid (99% purity) was purchased from Thermo Scientific, South Africa. Fluka® Analytical Ammonium hydroxide ≥ 25% in H2O, eluent additive for liquid chromatography (LC) was purchased from Sigma-Aldrich, South Africa.Tetracycline, erythromycin, benzoyl peroxide, crystal violet, actinomycin D, dimethyl sulfoxide (DMSO), Tris, KCl, 5,5’-dithio-bis-(2-nitrobenzoic acid) (DTNB), DMPTB, Triton X-100, ibuprofen, porcine hematin, L-epinephrine, formic acid, NaC. acnes strain (ATCC 6919), brain heart infusion (BHI) agar, and BHI broth were purchased from Anatech Instruments (Pty) Ltd . Human keratinocytes (HaCaT) were donated by Dr. Lester Davids . Murine macrophages (RAW264.7), glucose, and lipopolysaccharide (LPS) (Escherichia coli 0127: B8) were purchased from Merck SA (Pty) Ltd. . DMEM, trypsin, PBS, FBS, penicillin-streptomycin, and amphotericin B (Fungizone) were purchased from Thermo Fisher Scientific ZA . TrypLE was purchased from Thermo Fisher Scientific .The Streptococcus pyogenes, and lipase from Candida rugosa were all purchased from Merck SA (Pty) Ltd. . The XTT cell proliferation kit II was purchased from Merck SA (Pty) Ltd. . The IL-1α and PGE2 ELISA kits were purchased from Biocom Africa (Pty) Ltd . The CBA Human Inflammatory Cytokine kit was purchased from BD Biosciences . The Promega Griess Reagent System was purchased from Promega Corporation . The complementary DNA (cDNA) reverse transcriptase kit and the SYBR Green PCR Master Mix kits were purchased from Thermo Fisher Scientific . Primers were purchased from Integrated DNA Technologies .Human recombinant COX-II, hyaluronidase from H. odoratissimum L. Sweet was collected in the December from Durban  global positioning system (GPS) coordinates: grid 2931CC 29°00’S, 31°00’E. Taxonomical species identification was performed by Ms. Magda Nel from the H. G. W. J. Schweickerdt Herbarium at the University of Pretoria, and a voucher specimen number was deposited (PRU 118963). Shade-dried plant material (leaves and stems) was ground to a fine powder. Plant material (100 g) was extracted with methanol (1:5 w/v) for 72 h with constant agitation on a shaker. The maceration was filtered through a Buchner funnel (Whatman 1.0 filter). The filtrate was then concentrated under vacuum using a Rotary evaporator (Buchi-R-200). The crude extract was then stored at 4°C until use.H. odoratissimum (L.) Sweet (HO-MeOH)  was performed using a LECO Pegasus 4D GC-TOFMS (LECO Africa (Pty) Ltd., Kempton Park, South Africa) equipped with a GC Rxi-5SilMS  column . The temperature of the injector and the interface were kept at 250°C. The following temperature program was used for analysis: 40°C (hold for 3 min) at 10°C/min to 300°C (hold for 5 min). Helium was used as a carrier gas at a constant flow rate of 1 ml.min−1. Splitless injection with a 30 s splitless time was used. The first 5 min of the analysis was considered as solvent delay and omitted from the final GC-MS chromatograms. The MS transfer line temperature was set at 280°C. The mass spectrometer was operated in electron impact ionization mode (EI +) at 70 eV. Detector voltage was set to 1,750 V. The scan range was set at 40–550 Da with a data acquisition rate of 10 spectra/s. Peaks were identified by mass matching to the NIST05 database.The gas chromatography–mass spectrometry (GC-MS) analysis of methanolic extract of ® Synapt G2 high definition mass spectrometry (HDMS) system . The system includes a Waters Acquity Ultra Performance Liquid Chromatography (UPLC®) system linked with a quadrupole-time-of-flight (QTOF) instrument. MassLynx™ (version 4.1) software  was used for data acquisition and processing. The internal lock mass control standard was a 2 pg/μl solution of leucine enkephalin (m/z 555.2693). Sodium formate clusters and Intellistart functionality was used to calibrate the instrument (mass range 112.936–1.132.688 Da). Resolution of 20,000 at m/z 200 [full width at half maximum (FWHM)] and mass error within 0.4 mDa were obtained.Compound separation and peak detection of HO-MeOH using LC-MS was performed using the Waters® 1.7 µm EVO C18 100 Å (2.1 mm ID x 100 mm length) column was used for separation. A volume of 5 µl was injected into the system using an autosampler. A reverse phase step gradient elution scheme from 97% H2O (0.1% formic acid) to 100% acetonitrile (0.1% formic acid) was used as the mobile phase. The gradient started with an isocratic flow (hold 0.1 min) followed by a linear increase to 100% ACN; subsequently the column was washed for 1 min followed by conditioning and re-establishing of initial conditions to allow for equilibration before the start of the next run for the complete elution scheme. The column temperature was kept constant at 40°C and the flow rate was set at 0.4 ml/min for the each run, giving a total run time of 20 min. The positive and negative ion mass spectra were collected in separate chromatographic runs (employing the same separation conditions). Mass spectral scans were collected every 0.3 s. The raw data was collected in the form of a continuous profile. Mass to charge ratios (m/z) between 50 and 1,200 Da were recorded. Quantitative data-independent acquisition (DIA) was done using two simultaneous acquisition functions with low and high collision energy (MSE approach) with a QTOF instrument. The fragmentation energy was set at 1 and 4 V (positive mode) and 1.8 and 6 V (negative mode) for the trap and collision energies, respectively. The ramping was set from 3 to 4 V and 20 to 30 V for both modes for the trap and transfer collision energy, respectively. The electrospray ionization (ESI) mode conditions were set as follows; capillary voltage for ESI was 2.6 and 2.0 kV for positive and negative mode ionization, respectively. The source temperature for both modes was set at 120°C. the sampling cone voltage at 25 V for negative mode and 40 V for positive mode. The extraction cone voltage was set at 4.0 V for both modes. Cone gas (nitrogen) flow was set at 10.0 L/Hr for both modes. The desolvation temperature was set at 300°C with a gas (nitrogen) flow of 600.0 L/h.The HO-MeOH extracts was prepared to a 1 mg/ml stock concentration and filtered using a 0.2 µm syringe filter to remove any particulates. A KinetexC. acnes (ATCC 6919) strain was tested using the microdilution broth assay as described by C. acnes grown for 72 h were then inoculated in BHI broth at a concentration of 1.5 × 108 CFU/ml (OD600 = 0.132). To each test well 100 µl of bacterial suspension was added. The 96-well plates were then incubated in an Anaerocult® jar with Anaerocult® A for the generation of CO2. Control plates were included and consisted of C. acnes alone, an extract vehicle DMSO treatment (2.5% v/v), and a growth media control only. After 72 h, 20 µl of PrestoBlue® reagent was added. The MIC (minimum inhibitory concentration) was then determined visually after 1 h of incubation. An MIC of less than 100 µg/ml was considered as potent anti-microbial activity.Susceptibility of the C. acnes grown for 72 h were then inoculated in BHI broth at a concentration of 1.5 × 108 CFU/ml (0.132 at OD600). To each test well, 100 µl of bacterial suspension was added. The 96-well plates were then incubated in an Anaerocult® jar with Anaerocult® A for the generation of CO2. Control plates were included and consisted of C. acnes only (no treatment) and a solvent control (2.5% v/v DMSO). After 72 h, 20 µl of PrestoBlue® reagent was added and following an hour incubation the MIC was determined. The MIC of HO-MeOH (component A) and antibiotic (component B) in combination, were then used to determine the sum of the fractional inhibitory concentrations (∑ FIC) using the following equation:The interaction between the HO-MeOH (component A) extract and commercial agents (component B) were determined using the variable ratio method described by An FIC value ≤ 0.5 was considered a synergistic combination, an FIC of 0.5 ≤ 1 was considered as additive, an FIC of 1 < 4 was considered non-interactive, and finally an FIC > 4 was considered antagonistic.C. acnes (ATCC 6919) was previously performed by C. acnes were inoculated in sterile 96-well plates by adding 100 µl (0.132 at OD600) in BHI to each well. Following bacterial plating, 100 µl of plant extract and tetracycline were added to achieve concentrations of 0.24–31.25 and 0.02–3.13 µg/ml, respectively. Controls including 100% adhesion (untreated C. acnes) and 0% adhesion (BHI media only) were included. Plates were then incubated anaerobically for 72 h. Following incubation, the BHI media was removed, and plates were gently washed with 100 µl phosphate buffered saline (PBS) three times. Adhered cells were then fixed using 100 µl of 99% methanol for 15 min. The MeOH was aspirated, and plates were allowed to air dry for 20 min. Quantification of adhered cells was then performed by the addition of 100 µl of a 0.5% crystal violet for 20 min. The plates were then gently rinsed with distilled water to remove the excess crystal violet and allowed to air dry. The bound crystal violet was then dissolved using 160 µl of a 33% acetic acid solution. The optical density was then measured at 590 nm using a BIO-TEK Power-Wave XS multi-well reader . For the biofilm disruption, C. acnes were plated as described in the adhesion protocol. After the 72 h adhesion, BHI media was removed, and the plates were gently washed three times with PBS to remove planktonic cells. Following the wash step, 200 µl of fresh BHI was added, and plates were then incubated for an additional 72 h to allow for biofilm maturation. After incubation, the media was removed and replaced with 200 µl of the test sample in BHI broth. After 24 h of treatment, the biofilm was quantified using crystal violet.The bacterial adhesion assay for 2. Cells were grown to 80% confluency, in vented cell culture flasks (T-75) before subculture. Adhered cells were sub-cultured by cell detachment using trypsin-ethylenediaminetetraacetic acid (EDTA) followed by the addition of fresh media into new growing flasks. Cytotoxicity was determined using the XTT Cell Proliferation Kit II. In a 96-well plate, 100 µl of (1 × 106 cells/ml) cell suspension was added, and cells were incubated for 24 h. The HO-MeOH extract was added at a concentration ranging from 3.125 to 400 µg/ml. Actinomycin D was used as the toxic inducer and was tested from 0.05 to 3.91 µg/ml. Treated cells were then incubated for 72 h. An untreated cell control and a 2% v/v DMSO vehicle control were also included. Following incubation, 50 µl of activated XTT was added to all the test and control wells and further incubated for 2 h. The optical density was read at 490 nm with a reference wavelength of 690 nm to remove background. Tests were performed in triplicate and the percentage viability was then calculated. The 50% inhibitory concentration (IC50) was then calculated using GraphPad Prism 4.0 software (HaCaT were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 µg/ml penicillin, 100 µg/ml streptomycin, and 250 µg/ml fungizone and incubated at 37°C and 5% COsoftware .2. Cells were grown to 85% confluency in 100 mm cell culture dishes to prevent contact inhibition. Adhered cells were then sub-cultured by cell detachment using TrypLE followed by the addition of fresh media into new 100 mm cell culture dishes. Cytotoxicity was determined using the 3--2,5-diphenyltetrazolium bromide (MTT) reagent. In a 96-well plate, 100 µl of a 1 × 105 cells/ml was added and incubated for 24 h. The HO-MeOH extract was tested at a concentration range from 3.91 to 125 µg/ml. A 5% DMSO (v/v) was added as the toxic inducer. An untreated cell control was also included in the assay. Following incubation, 20 µl of MTT (5 mg/ml in PBS) was added to the test and control wells and incubated for 4 h. This was followed by the addition of 200 µl of acidified isopropanol (0.1% HCl). Plates were covered in foil and placed on a rotating platform for 10 min. The optical density was then read at 560 nm with a reference wavelength of 670 nm to remove background. Tests were performed in triplicate, and the percentage viability was then calculated. The IC50 was then calculated using GraphPad Prism 4.0 software (Murine macrophages (RAW264.7) cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and incubated at 37°C and 5% COsoftware .5 cells per well and incubated for 24 h. The cells were stimulated with 100 µg/ml of heat-killed C. acnes and 100 ng/ml of LPS from E. coli, independently. The HO-MeOH was added together with the stimulating factor to determine inhibitory activity for 24 h. An untreated cell control, LPS only and C. acnes only controls were also included. Plates were incubated for 24 h in a humidified incubator with 5% CO2 and then centrifuged (980 rpm for 5 min) before the supernatant was collected, aliquoted, and stored at −80°C until use. The quantification of IL-1α was performed using an Elabscience ELISA kit  according to the manufacturer protocol. The IL-1α concentration was calculated using a standard curve.The effect of HO-MeOH on secreted IL-1α levels in human keratinocytes was investigated at non-lethal concentrations determined from the cell viability assay (3.125–400 µg/ml). Human keratinocytes were plated in a 24-well plate at a seeding density of 1 × 10C. rugosa . A stock solution of 0.5 M EDTA, 10% v/v Triton X-100, and 1 M Tris-Cl were also prepared and made up the reaction mixture. The reaction mixture consisted of the following volumes, 20 μl of 40 mM DTNB, 2 μl of 0.5 M EDTA, 5 μl of 10% v/v Triton X-100, and 50 μl of 1 M Tris-Cl, pH 7.5. The reaction mixture was added to a 15 ml centrifuge tube, and the volume adjusted to 900 μl with distilled water. For the enzyme inhibition assay, 160 μl of the reaction mixture was added to a 96-well plate with 10 μl of inhibitor (test sample in DMSO) and 20 μl of enzyme solution (10 U/ml). The plates were then incubated at 37°C for 5 min to allow for inhibitor-enzyme interaction. The reaction was initiated by the addition of 10 μl of a 4 mM DMPTB solution. For tests with no inhibitor, 10 μl of lipase buffer was added. The assay controls included enzyme and substrate (no inhibitor), DMSO (5% v/v) only, no DTNB, no DMPTB, and no enzyme (substrate only). The plated were then incubated for 30 min at 37°C in a BIO-TEK Power-Wave XS multi-well reader  while performing a kinetic read at 405 nm. The IC50 was then calculated using GraphPad Prism 4.0 software.The inhibitory effect of HO-MeOH against lipase was determined described by C. acnes was determined from cell culture supernatants using flow cytometry. The BD Cytometric Bead Array (CBA) Human Inflammatory Cytokine kit was used to detect cytokine production according to the manufacturer’s protocol (Cat. No. 551811) . HaCaT were seeded in 24-well plates at a density of 1 × 105 cells/well in DMEM supplemented with 10% FBS, 100 µg/ml penicillin, 100 µg/ml streptomycin, and 250 µg/ml fungizone and incubated at 37°C and 5% CO2 for 24 h. After incubation, plating media was removed and replaced with 1 ml fresh culture media. Test samples were prepared in 1 ml of DMEM to yield final concentrations of 7.81 µg/ml (HO-MeOH), 0.78 µg/ml (tetracycline), C. acnes (100 µg/ml), and LPS (100 ng/ml) in test wells. The HO-MeOH was added at the MIC to C. acnes-stimulated cells to determine inhibition of cytokines produced by C. acnes. LPS-stimulation was used as a control for cytokine induction. Untreated cells were also included as a control. Following 24 h of incubation plates were centrifuges at 980 rpm for 5 min, and cell-free supernatants were collected and stored at −80°C until use. Cytokine analysis was performed using the Accuri C6 cytometer . Inhibition of cytokines was calculated as a percentage (%) reduction from cytokine levels in C. acnes-stimulated HaCaT cells.The levels of pro-inflammatory cytokine  production in HaCaT cells induced with heat-killed 546 was then determined immediately using the BioTek Power Wave XS . The IC50 was then calculated using GraphPad Prism 4.0 software.The NO radical scavenging activity of HO-MeOH was determined using the methods described by Murine Macrophage Cells (RAW264.7)]. Cells were then seeded in a 96-well plate, at a density of (1×104 cells per well) and allowed to attach for 24 h prior to treatment. The HO-MeOH was tested at non-lethal concentrations obtained from the MTT cell viability assay (3.91–31.25 µg/ml). The positive control dexamethasone was tested at 4 µg/ml (10 µM), and the negative control was DMSO (0.1% v/v). The cells were pre-treated with test samples for 2 h before the induction of NO with 1 µg/ml of LPS (E. coli 0127: B8). Plates were then incubated overnight at 37°C in a humidified incubator with 5% CO2. The amount of NO produced in treated cells was detected using 50 µl of cell supernatant using the manufacturer’s protocol described in the Promega Griess Reagent System . The optical density was then read at 530 nm using a BioTek Synergy H1 microplate reader. The IC50 was then calculated using GraphPad Prism 4.0 software.The cellular NO scavenging activity of HO-MeOH using stimulated murine macrophages (RAW264.7) was performed according to the methods described by 2EDTA in a 96-well plate. Stock concentrations of HO-MeOH and Ibuprofen were prepared in DMSO, and 10 µl of inhibitor was added to obtain final concentrations of 2.5–160 µg/ml for HO-MeOH and 0.4–10 µM (0.01–0.15 µg/ml) for ibuprofen, the positive control. After 5 min incubation with the test inhibitors, 5 µl of 10 µM arachidonic acid substrate was added to initiate the reaction. The test plate was incubated at 37°C for 20 min. Following incubation, 10 µl of 10% formic acid was added as a stop solution. The production of PGE2 was quantified using the PGE2 ELISA kit according to the manufacturer’s protocol (Cat. No. ADI-900-001) . The quantification of PGE2 in test inhibitors and controls was determined from the PGE2 standard curve. The IC50 values for test inhibitors were calculated using GraphPad Prism 4.0 software.Inhibition of human recombinant COX-II activity was investigated using the methods described by Murine Macrophage Cells (RAW264.7). Cells were seeded in a 24-well plate at a cell density of 5 × 105 cells/ml and incubated for 24 h in a humidified incubator with 5% CO2. Cells were then treated with non-lethal concentrations of HO-MeOH (3.91–31.25 µg/ml) and dexamethasone was used as the positive control tested at 4 µg/ml (10 µM). Treatments with the extract and positive control were performed for 2 h before the addition of 1 µg/ml of LPS. Controls with untreated cells, 0.1% v/v DMSO and LPS only were also included. Plates were then incubated overnight. The total RNA was then isolated using the TRIzol reagent. Briefly, 330 µl of TRIzol was added to each replicate and combined in a sterile Eppendorf tube. Samples were then frozen at −20°C overnight. Following incubation, samples were thawed before the addition of 200 µl chloroform and vortexing for 30 s. Eppendorf tubes were then centrifuged  for 15 min. Samples were then carefully removed and the clear upper layer (~400 µl) was pipetted and transferred to a clean, sterile Eppendorf tube, without disturbing the pink TRIzol layer. To the supernatant layer, 500 µl of ice-cold isopropanol was added, vortexing briefly and storing on ice for 10 min. Samples were then centrifuged and supernatants discarded. Pellets were washed with 1 ml of 70% ethanol (using absolute ethanol and molecular grade water) followed by brief vortexing and storage on ice for 10 min before centrifuging  for 5 min. The supernatant was decanted without disturbing the pelleted RNA pellet. The Eppendorf tubes were air-dried for 15–20 min to allow residual ethanol to evaporate. Samples were stored in 40 µl of molecular grade water and stored at −80°C until use. RNA was quantified using the BioTek Synergy H1/Take 3 spectrophotometer to determine the OD260/OD280 ratio. The cDNA was synthesized using 2 µg for each test sample and the controls using the cDNA Reverse Transcriptase Kit  on the ABI Applied Biosystems GeneAMP 9700 thermal cycler . Synthesized cDNA was amplified in duplicate using real-time quantitative PCR using SYBR green PCR Master Mix . The effects of genomic DNA contamination were overcome by selective use of intron-overlapping primers selected using Primer Express version 2.0 software . The quantitative PCR amplifications were performed using the ABI Applied Biosystems 7500 Fast Real-Time PCR System  with 1 cycle at 50°C for 2 min and 1 cycle at 95°C for 10 min, subsequently followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Dissociation curves were completed with a single cycle of 1 min at 95°C, 30 s at 55°C, and finally 30 s at 95°C. The mRNA expression was determined using ∆∆CT analysis with data normalized against the β-actin housekeeping gene using the 7500 Fast System SDS software v1.3.0. Inhibition if gene expression was compared with LPS-stimulated gene expression, where values < 1 indicated inhibition of gene expression and values > 1 indicated the overexpression of the gene of interest in excess of that of LPS stimulation. Melting curve profiles for the specific transcripts were also obtained. The forward primers sequences for each gene were as follows: β-actin (5′-AAC CGT GAA AAG ATG ACC CAG AT-3′), COX-II (5′-TGG TGC CTG GTC TGA TGA TG-3′), and inducible nitric oxide synthase (iNOS) (5′-CCC TCC TGA TCT TGT GTT GGA-3′). The reverse primers for each gene were as follows: β-actin (5′-CAC AGC CTG GAT GGC TAC GT-3′), COX-II (5′-GTG GTA ACC GCT CAG GTG TTG-3′), and iNOS (5′-TCA ACC CGA GCT CCT GGA A-3′) .Murine macrophages (RAW264.7) were maintained as in the section 2HPO4 with 0.1 M NaCl and 0.1 M citric acid with 0.1 M NaCl to pH 5.0. The hyaluronic acid substrate stock solution was prepared to 2 mg/ml (in distilled water). A stock solution of 0.2 mg/ml (in distilled water) bovine serum albumin (BSA) was also prepared. The hyaluronidase from Streptococcus pyogenes was optimized to 1 U/ml. In a 96-well plate, 136 μl of citrate-phosphate buffer was added to the first row. To all the subsequent wells 70 μl of buffer was added. The HO-MeOH extract was diluted in DMSO, and 4 μl was added to the first row in triplicate, followed by a two-fold serial dilution to obtain final concentrations ranging from 1.95 to 250 µg/ml. Ten microliters (10 μl) of BSA and 10 μl of hyaluronidase enzyme were then added. The negative control was DMSO without sample. The plates were then incubated for 15 min at 37°C. The reaction was then initiated by the addition of 10 μl of hyaluronic acid and further incubation for 30 min at 37°C. The reaction was then stopped with the addition of 200 μl of 2.5% (w/v) cetyltrimethylammonium bromide (CTAB) in 0.5 M NaOH and incubated at room temperature for 20 min. The turbidity was then measured by obtaining the optical density at 355 nm using the BioTek PowerWave XS . Additional controls included an extract color control, a substrate only control (100% enzyme inhibition), an enzyme and substrate only control (0% inhibition), and a no substrate control. The IC50 was then calculated using GraphPad Prism 4.0 software.The turbidometric assay described by 50.Results for each experiment were determined using data obtained in triplicate with three independent assay repeats. One-way analysis of variance (ANOVA) was used to determine the differences in means and the Dunnett’s multiple comparison post-test was used to determine significant differences between the means of treated and untreated groups as identified in each bioassay. For enzyme assays a four parameter logistic equation with a 95% confidence interval was performed to determine the ICα-sitosterol (4.28%), α-humulene (3.94%), α-curcumene (3.74%), and valencen (3.23%) , linoleic acid (9.16%), palmitic acid (8.52%), caryophyllene (8.13%), oxalic acid allyl nonyl ester (8.13%), 5,5-diethoxy-2-pentanone (4.74%), stigmasterol (4.36%),  (3.23%) Table 1  (3.23%) . This covulgaris .Helichrysum species, including Helichrysum aureonitens (α-humulene and para-cymene). Hydrodistilled Helichrysum leucocephalum and Helichrysum artemisiodes led to the identification of β-caryophyllene and α-humulene as major components of the oils of both species. These species also contained other constituents like hexadecanoic acid and E, E-farnesene present in HO-MeOH  and β-caryophyllene (9.6%) as a major components. Valencene (1.9%), para-cymene (0.3%), and α-cubebene (0.1%) were also present in the oil showing good correlation with the HO-MeOH components. H. odoratissimum L. Sweet species, found that α-humulene (5.6%) and β-caryophyllene (4.7%) were the major sesquiterpene compounds present.The HO-MeOH shared common compounds with other  HO-MeOH . A studyHelichrysum species, including Helichrysum italicum, Helichrysum populifolium, and Helichrysum stoechas  exhibited an MIC of 7.81 µg/ml against C. acnes, while the positive control tetracycline, exhibited an MIC of 0.78 µg/ml. The minimum bactericidal concentration (MBC) activity was also determined with the HO-MeOH exhibiting a bactericidal effect at 250 µg/ml, compared with tetracycline at 1.56 µg/ml. The effect of the combination of HO-MeOH and the macrolide antibiotic, erythromycin, was also antagonistic to a much larger degree. The general trend observed for the combination of HO-MeOH and benzoyl peroxide (BPO) was that lower concentrations of HO-MeOH (sub-MIC) meant that less BPO was needed to produce an antimicrobial effect. The MIC of BPO alone was 31.25 µg/ml, but with the addition of the HO-MeOH extract in three combinations, antimicrobial activity of BPO was observed at concentrations as low as 5.47, 6.25, and 7.03 µg/ml  leaf and stem extract of H. odoratissimum exhibited no activity against S. epidermidis, another microorganism which has been implicated in the pathogenesis of acne with an MIC of 4 mg/ml but was active against S. aureus with an MIC of 20 µg/ml , has therefore been prescribed as a solution for using antibiotic monotherapies or concurrent topical and oral antibiotic therapies in isolation. This compound is often used when long-term antibiotic use is prescribed for the treatment of acne patients. While macrolide antibiotics are the most common topical antibiotics, cyclines are the most common orally prescribed antibiotics. The burden of antibiotic resistance is escalated by the long treatment regimens often lasting between 3 and 6 months , which is particularly more susceptible to antimicrobial compounds. The potent antimicrobial activity of erythromycin can also be used to explain the susceptibility of the tested strain because clinical isolates of C. acnes commonly have higher resistance toward both erythromycin and clindamycin  of Gingko biloba seed nut extract against C. acnes at 256 µg/ml and an MIC of 64 µg/ml.Gram-negative and Gram-positive bacterial populations are known to cooperate, communicate, and grow symbiotically to form complex social communities in order to enhance invasion and persistence through a process known as quorum sensing. The biofilm structure forms an integral part of this process and is, therefore, an essential target to consider when developing acne therapies, considering the ineffectiveness of systematic antibiotics in this regard . There wnal role   completely inhibited cell proliferation (data not shown)   and 170 µM (43.33 µg/ml) for COX-II and COX-I, respectively . Inhibition of NO was compared with LPS-induced RAW264.7 cells. The HO-MeOH extract showed significant inhibition of NO at 7.81, 15.625, and 31.25 µg/ml. The concentration of nitrite was reduced from 19.46 µM in the LPS-stimulated cells to 14.86, 12.31, and 11.60 µM, respectively. The positive control, dexamethasone showed significant inhibition of NO at 4 µg/ml (10 µM). The negative control 0.1% DMSO showed no inhibitory effect on NO production, indicating that the observed inhibition was likely due to pre-incubation with HO-MeOH. The intracellular NO scavenging activity showed a reduction of 40.39 and 36.74% reduction in NO production levels at 31.25 and 15.625 µg/ml, respectively  (H. odoratissimum for anti-inflammatory activity. The NO scavenging activity and intracellular NO inhibitory activity justified further investigation into possible mechanisms of this anti-inflammatory activity.The inhibition of cellular NO was tested using murine macrophages (RAW264.7) where induction of NO was achieved with treatment of the cells with LPS. The induction of NO by LPS is said to occur disorder . The inhisorder 2−. Inhibi13 µg/ml . A studyrysetin) . These c2 (PGE2) and NO are among the most prominent effects of LPS induction (β-actin gene was used as the reference (housekeeping) gene. The gene expression levels of treated cells were normalized relative to the gene expression of β-actin. Cells induced with LPS only were set to 1 as the maximum level of overexpression. Therefore, values <1 indicated inhibition of gene expression and values >1 indicated gene overexpression relative to LPS stimulation. The melting curves and amplification plot indicated that there was no formation of primer-dimers with the PCR reaction. In an attempt to correlate gene expression data to the anti-inflammatory bioassays, only the COX-II and iNOS gene expression were reported. The HO-MeOH exhibited inhibition of COX-II expression. However, there was no significant difference between the COX-II expression levels in the HO-MeOH treatments when compared to those observed in LPS-induced expression. The positive control, dexamethasone exhibited a significant reduction in COX-II expression with a 2.63 reduction in fold change from 1 to 0.38  in the investigation of anti-inflammatory activity of natural compounds is common practice. These cells behave much like human macrophages, with the induction of LPS resulting in the activation of signal pathways and gene expression. The overexpression of both cyclooxygenase-II and inducible iNOS resulting in the formation of prostaglandin-Enduction . The gen to 0.38 Figure 850 of 145.45 ± 6.22 µg/ml  and 0.06 mM (28 µg/ml), respectively. Inhibition was also observed against Streptococcus equisimilis HYAL activity with IC50 of 1.02 mM (839 µg/ml) and 0.26 mM (122 µg/ml). Flavonoids rutin and silybin were also identified as inhibitors of S. agalactiae HYAL activity. The inhibitory activity of HO-MeOH, therefore, compares well with previously published data on bacterial hyaluronidase inhibitors  (Grant ID: 98334 & 105169) for funding the research.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Streptomyces albulus CICC 11022 by using the kasOp∗ promoter and the ribosome binding site from the capsid protein of phage ϕC31, which resulted in a genetically engineered strain Q-PL2. The titers of ε-PL produced by Q-PL2 were 88.2% ± 8.3% higher than that produced by the wild strain in shake flask fermentation. With the synergistic effect of 2 g/L sodium citrate, the titers of ε-PL produced by Q-PL2 were 211.2% ± 17.4% higher than that produced by the wild strain. In fed-batch fermentations, 20.1 ± 1.3 g/L of ε-PL was produced by S. albulus Q-PL2 in 72 h with a productivity of 6.7 ± 0.4 g/L/day, which was 3.2 ± 0.3-fold of that produced by the wild strain. These results indicate that ε-PL synthase is one of the rate-limiting enzymes in ε-PL synthesis pathway and lays a foundation for further improving the ε-PL production ability of S. albulus by metabolic engineering.ε-Poly-L-lysine (ε-PL) is a natural amino acid polymer produced by microbial fermentation. It has been mainly used as a preservative in the food and cosmetics industries, as a drug carrier in medicines, and as a gene carrier in gene therapy. ε-PL synthase is the key enzyme responsible for the polymerization of L-lysine to form ε-PL. In this study, the ε-PL synthase gene was overexpressed in  Streptomyces (ε-Poly-L-lysine (ε-PL) is a kind of polymer composed of 25–35 L-lysine residues connected by α-amino group and ε-carboxyl group to form an amide bond. It was first separated and purified by Japanese scholars Shima and Sakai in the fermentation broth of ptomyces . ε-PL haptomyces . In addiptomyces .Streptomyces albulus NBRC 14147  requires its own promoter, which is likely regulated in secondary metabolism. The pls gene from S. albulus was heterologously expressed in S. lividans, and the recombinant strain was capable of synthesizing ε-PL. The own promoter of pls is still used in their study . Escherichia coli ET12567/pUZ8002  medium, which contained 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L sodium chloride. S. albulus was cultured at 30°C. The medium used to culture the spores of S. albulus was MS solid medium, which contains 20 g/L of mannitol, 20 g/L of soybean powder, and 20 g/L of agar powder. M3G medium was used for the seed culture of S. albulus, which is composed of 50 g/L of glucose, 10 g/L of (NH4)2SO4, 5 g/L of yeast extract, 0.5 g/L of MgSO4⋅7H2O, 0.8 g/L of K2HPO4, 1.36 g/L of KH2PO4, 0.03 g/L of FeSO4⋅7H2O, 0.04 g/L of ZnSO4⋅7H2O and has an initial pH of 6.8. For shake flask fermentations of S. albulus, M3G medium was used as the fermentation medium after appropriate modification according to the experimental requirements. For fed-batch fermentations of S. albulus, 25 g/L glucose and 25 g/L glycerin were used as the mixed carbon source, 5 g/L sodium citrate was added, and the other components were the same as M3G medium without glucose. When required, antibiotics were used at the following concentrations: 50–80 μg/mL apramycin, 25–50 μg/mL chloramphenicol, 40–50 μg/mL kanamycin, and 25 μg/mL nalidixic acid.S. albulus strains were streaked onto MS solid medium, and spores were collected after 5–6 days of cultivation at 30°C. Then, 400-μL spore solution was inoculated into 300-mL conical flask containing 50 mL seed medium, and seed culture was obtained after 48 h of shaking cultivation at 30°C and 220 rpm unless otherwise specified. Then, the seed culture was inoculated into conical flasks containing 50 mL of fermentation medium at 10% (volume ratio). After 72 h of cultivation at 30°C and 220 rpm, the concentration of ε-PL and other parameters in the fermentation broth were determined.In the shake flask fermentations of ε-PL, the S. albulus were conducted in a 2-L bioreactor  containing 1 L of fermentation medium. The preparation of the seed culture was the same as that in shake flask fermentation. The seed culture was inoculated into the fermenter at 10% volume ratio, and the temperature was controlled at 30°C. The aeration was 3 vvm, and the dissolved oxygen level was controlled at approximately 30% by adjusting the stirring speed at 200–800 rpm. According to Fed-batch fermentations of ε-PL by pls gene . Plasmid DNA was isolated using the Easypure® Plasmid Miniprep Kit . The EasyPure® Quick Gel Extraction Kit  was used for DNA purification. Oligonucleotides were prepared by Invitrogen . The Gibson Assembly® Cloning Kit  was used to integrate the vector and the DNA inserts.Genomic DNA was extracted using the TIANamp Bacteria DNA Kit . Phanta Super-Fidelity DNA Polymerase was used to amplify the pls gene overexpression plasmids are shown in pro-rbs2 of the strong promoter kasOp∗ and the ribosome-binding site (RBS2) from the capsid protein of phage ϕC31 were obtained by annealing oligonucleotides pro1, pro2, and pro4. The primers pls-F2 and pls-R were used to amplify the pls gene from the genome of S. albulus CICC 11022. The PCR conditions were as follows: 94°C for 30 s, 55°C for 30 s, and 72°C for 2 min, for 30 repeated cycles each. The DNA fragments of pro-rbs2 and pls gene were mixed as the template to perform overlapping PCR using primers pro1 and pls-R, and a complete expression element, namely, pro-rbs2-pls—containing the strong promoter kasOp∗, RBS2, and pls gene—was finally obtained. The PCR conditions wer as follows: 94°C for 30 s, 55°C for 30 s, and 72°C for 2 min, for 30 repeated cycles each. The expression element pro-rbs2-pls was ligated with XbaI and EcoRI double-digested vector pSET152 by Gibson reaction and transformed into E. coli Top10. The correctly ligated transformants were screened and verified by sequencing. The sequence of pls gene in S. albulus CICC 11022 shows 100% identity to the pls gene in S. albulus PD-1 (accession no. JF427577). The recombinant overexpression plasmid was named pSET152-pro-rbs2-pls . The cDNA was reverse transcribed using EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix , and qRT-PCR was performed using a QuantiNova SYBR Green PCR kit  on a LightCycler 96 instrument . The PCR conditions were as follows: 95°C for 5 s and 60°C for 10 s, for 40 repeated cycles. The RNA polymerase sigma factor (hrdB) was selected as the reference gene , respectively. After 72 h of fermentation, 20.1 ± 1.3 g/L of ε-PL was produced with a productivity of 6.7 ± 0.4 g/L/day by strain Q-PL2  into the chromosome of S. albulus PD-1 to alleviate oxygen limitation during fermentation. Finally, the production of ε-PL was increased from 22.7 to 34.2 g/L with a productivity of 4.9 g/L/day. vgb gene and S-adenosylmethionine synthetase gene (metK) into the chromosome of S. albulus NK660 for expression, and the ε-PL titer increased from 0.6 to 0.76 g/L. Nitrogen source is another key factor restricting the synthesis of ε-PL. Each molecule of lysine contains two nitrogen atoms. Therefore, the synthesis of intracellular lysine requires more nitrogen sources. In order to solve this problem, the ammonium transporter gene (amtB) was overexpressed in S. albulus PD-1, and the production of ε-PL increased from 22.7 to 34.2 g/L with a productivity of 5.1 g/L/day  that was completely unregulated by feedback inhibition and expressed the mutant protein rAsk in S. albulus CR1. As a result, the concentration of ε-PL increased from 12 to 15 g/L with a productivity of 2.1 g/L/day. In our research, the final concentration of ε-PL increased from 6.3 to 20.1 g/L by overexpressing pls gene in S. albulus and the synergistic effect of citrate. The productivity of ε-PL reached 6.7 g/L/day, which was much higher than other genetically engineered strains.Genetic engineering is one of the important methods to increase the fermentation ability of industrial production strains. But, up to now, fewer researches on enhancing ε-PL biosynthesis by genetic manipulation have been reported. Among these reports, most are focused on alleviating the restriction of environmental factors on fermentation, and very few studies have focused on the key enzymes in the ε-PL metabolic pathway. Dissolved oxygen is one of the key factors affecting ε-PL production.  g/L/day . AspartaermE∗ to overexpress Pls have failed (kasOp∗ (pls gene in S. albulus CICC 11022. During fermentation, the expression levels of the pls gene in the genetically engineered strain Q-PL2 were always 5-fold higher than those in the wild strain, but the final titer of ε-PL was only increased by 88.9%. These results indicate that the lack of intracellular L-lysine is likely to be a key factor limiting the production capacity of the pls gene overexpressing strain Q-PL2. We tried to add various metabolic intermediates to the fermentation medium and found that only the addition of citrate significantly increased the production of ε-PL by the pls gene overexpressing strain Q-PL2 (S. albulus PD-1. They deduced that citric acid feeding resulted in metabolic flux redistribution at the node of phosphoenolpyruvate. The metabolic pathway from phosphoenolpyruvate to tricarboxylic acid cycle was weakened and from phosphoenolpyruvate to oxaloacetate and L-aspartate was enhanced. ε-PL production was improved consequently because L-aspartate is the precursor of L-lysine synthesis (pls gene overexpression and citrate on ε-PL production by S. albulus Q-PL2.ε-PL synthase is the last enzyme in the ε-PL synthesis pathway. The function mechanism of this enzyme is well characterized , but whee failed . In this (kasOp∗  was usedin Q-PL2 . Xia et ynthesis . Additioynthesis . Therefopls gene was overexpressed in S. albulus CICC 11022 by using a non-native promoter, and an enhanced ε-PL production was obtained with the synergistic effect of the gene and citrate. The results indicate that Pls is one of the rate-limiting enzymes in ε-PL synthesis pathway and lay a foundation for further improving the ε-PL production ability of S. albulus by metabolic engineering.In summary, The datasets generated for this study are available on request to the corresponding author.BY and JQ designed the research. AW and WT performed the experiments. XW, LC, and YX analyzed the data. XW and JQ wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tissue engineering technology has been applied extensively for clinical research and human amnion mesenchymal stem cells (hAMSCs) could cause mesenchymal stem cells to differentiate into the bone tissue. However, it is necessary to develop and identify the safer appropriate amount of osteogenic inducer. The objective of this study is to investigate the effect of icariin (ICA) on the proliferation and osteogenic differentiation of hAMSCs.The morphology and phenotype of hAMSCs were discovered by flow cytometry and immunocytochemical staining. The osteogenic differentiation of hAMSCs under the influence of different concentrations of ICA were assessed by alkaline phosphatase (ALP) activity substrate assay and alizarin red staining.− 6 mol/L. The combined analysis of ALP activity and ARS staining showed that ICA could significantly promote the osteogenic differentiation of hAMSCs, and the effect was most significant when the concentration of ICA was 1 × 10− 6 mol/L.MTT assay revealed that the hAMSCs pretreated with ICA exhibited increased proliferation when compared with the control group, and the most optimum concentration of ICA was 1 × 10− 6 mol/L.All the above results implied that ICA could significantly increase proliferation and enhance the osteogenic differentiation of hAMSCs, especially when the concentration of ICA was 1 × 10 Bone defects/losses is a serious problem in orthopedics due to low healing rate using traditional treatment and increases patients’ pain and heavily burdens families , 2. OverEpimedium pubescens, was reportedly used to cure bone diseases in ancient China [Previous findings showed that some drugs including statins , isoflavnt China . And it nt China  and repant China . It has nt China  and stimnt China . TherefoHuman amniotic mesenchymal stem cells (hAMSCs), derived from ditched amniotic membrane (AM) , 16, are2 and then separated and repeatedly rinsed using phosphate-buffered saline (PBS) containing 100 μg/mL streptomycin and 100 U/mL penicillin . Amnion fragments were collected in a 50 mL centrifuge tube and digested for 35 min at 37 °C in a double volume of digestion solution I (0.05% trypsin containing 0.02% EDTA-2Na). After centrifugation at 180 rpm for 2 h at 37 °C, the remaining amnion in the centrifuge tube was filtered through a filter screen and digested by a double volume of digestion solution II (0.75 mg/mL collagenase II containing 0.075 mg/mL DNase I) to acquire hAMSCs. The sample was shaken at a speed of 180 r/min and digested for approximately 2 h at 37 °C. After filter and centrifuge, the resultant hAMSCs were transferred to low-glucose (LG)-DMEM complete medium, supplemented with 10% heat-inactivated fetal bovine serum (FBS) to rest the cell suspension in a humid atmosphere of 5% CO2 for 5 min at 37 °C to precipitate hAMSC .The placental amniotic tissue was collected from normal pregnant women undergoing full-term cesarean section after gaining the informed consent of the pregnant women or her relatives. This project was approved by the medical ethics committee of Zunyi Medical University . After removing residual blood under sterile conditions, the amniotic tissue was cut into small pieces of 1–2 cmThe resultant hAMSCs  were collected in a 50-mL centrifuge tube and digested for 3 min at 37 °C in a double volume of digestion solution I (0.05% trypsin containing 0.02% EDTA-2Na). When the cells reached 80% confluence, they were digested and subcultured into flasks. Cells from passage 3 (P3) were used for further analysis in this study. The cell morphology was constantly observed under a light microscope . In addition, these hAMSCs were analyzed using the immunocytochemical staining method according to the protocols as described in a previous study .6 cells/mL, and then incubated with and HLA-DR for 25 min in the dark. After washing again with D-PBS containing 0.1% BSA, the cell suspension was centrifuged at 1000 rpm for 5 min and the supernatant was discarded. Finally, the labeled cells were analyzed using the flow cytometry system  and CellQuest software  after fixation with 1% paraformaldehyde.For the phenotypic characterization of hAMSCs, P3 hAMSCs were trypsinized and subsequently washed with D-PBS containing 0.1% BSA, adjusted to a density of 1.5 × 104 cells/mL in 96-well plates. After incubation for 24 h, the medium was changed to ICA-containing media at a concentration of 0 (blank and control), 1 × 10− 4 (ICA-1), 1 × 10− 5 (ICA-2), 1 × 10− 6 (ICA-3), 1 × 10− 7 (ICA-4), and 1 × 10− 8 (ICA-5) mol/L accordingly. Cells were incubated at 37 °C in a 5% CO2 incubator for 72 h, and 20 μL 5 mg/mL MTT was then added to each well, followed by incubation at 37 °C in a 5% CO2 incubator for 4 h. Later, the medium was discarded, and 150 μL of dimethylsulfoxide (DMSO) was added to each well. After incubation at 37 °C for 15 min, the OD value of each well was determined at a wavelength of 490 nm by a microplate reader .This experiment was divided into three groups: blank group (no cells and no ICA), control group (with cells but no ICA), and ICA group (with cells and ICA). The P3 hAMSCs were seeded at a density of 1 × 105 cells/mL in 96-well plates in LG-DMEM culture medium under sterile conditions. After 24 h of incubation, the medium was replaced with LG-DMEM culture medium with 10% FBS. The experiment was divided into 5 groups according to the different substances added to the medium, namely, the classic group , 50 mg/L vitamin C , and 10 mmol/L β-glycerophosphate ), the blank group (without additives), the ICA-1 group (1 × 10− 4 mol/L ICA), the ICA-2 group (1 × 10− 5 mol/L ICA), and the ICA-3 group (1 × 10− 6 mol/L ICA). Cells were incubated at 37 °C in a 5% CO2 incubator for 72 h, and 20 μL 5 mg/mL MTT was then added to each well, followed by incubation at 37 °C in a 5% CO2 incubator for 4 h. The culture medium was changed every 3 days, and the intervention was continuously induced for 21 days. Meanwhile, during the induction period, the cell morphology of the five periods of 1, 3, 7, 14, and 21 days were photographed for morphological observation, and the samples were preserved. ALP  was extracted and detected with the ALP assay kit  as directed by the manufacturer. The cells on the 21st day were stained with alizarin red staining (ARS), and statistical analysis was performed based on the number of stained calcium nodules.The P3 hAMSCs were seeded at a density of 1 × 10χ2 test. P < 0.05 was considered as statistically significant.Experimental data were expressed as mean ± standard deviation (SD) and analyzed using the SPSS 19.0 statistical software. One-way analysis of variance was used for comparison between groups, and the rate comparison was performed by The microscopic examination of P0 hAMSCs cultured in vitro revealed a typical adherent growth state Fig. a. After Flow cytometry revealed that hAMSCs highly expressed MSC surface markers CD29, CD44, CD73, and CD166 . OD absorbance values of samples cultured for different times are measured to evaluate the proliferation kinetics of osteoblast-like cells. MTT assay revealed that the hAMSCs pretreated with ICA exhibited increased proliferation when compared with the control group. Meanwhile, in the presence of ICA, the proliferation fold of hAMSCs increases first and then decreases with the increase of ICA concentration. For example, ICA increased the proliferation from 0.524 ± 0.033-fold in the control group to 0.728 ± 0.048 (P < 0.05), 0.719 ± 0.044 (P < 0.05), 0.688 ± 0.057 (P < 0.05), and 0.659 ± 0.041-fold (P < 0.05) at 1 × 10− 4, 1 × 10− 5, 1 × 10− 7, and 1 × 10− 8 mol/L, respectively. It is noteworthy that ICA at the optimum concentration of 1 × 10− 6 mol/L could increase the proliferation of hAMSCs from 0.524 ± 0.033-fold to 0.807 ± 0.080-fold (P < 0.05) compared with the control group  and the amount of mineralization was quantified by eluting ARS staining from differentiated osteoblasts. As shown in Fig. P < 0.01), 43.073 ± 0.422 (P < 0.01), 45.534 ± 1.072 (P < 0.0he 5), and 38.598 ± 0.6444 (P < 0.01). ALP activity in the ICA-1, ICA-2, ICA-3 group , and the classic group (63.583 ± 0.615) were significantly higher than that in the blank group (7.457 ± 0.377) on the 14th day. Following a 21-day culture period, the ALP activity of hAMSCs in ICA-1, ICA-2, ICA-3, and the classic group reached 92.819 ± 0.509 (P < 0.01), 139.955 ± 1.428 (P < 0.01), 145.102 ± 2.384 (P < 0.01) and 122.056 ± 1.676 (P < 0.01) compared to the blank group (11.175 ± 0.799)  and could promote bone formation by stimulating osteogenic differentiation of MSC recently [Osteogenic inducers are very important for effective stem cell-based treatment of bone defects/losses. Recently, many researches showed that traditional Chinese medicines could induce directional MSC differentiation into osteoblasts –25. Thess/losses , 26. Icarecently , 27–33. recently , 29. Somrecently –33. Base− 4mol/L, 1 × 10− 5 mol/L, 1 × 10− 6 mol/L, 1 × 10− 7 mol/L, and 1 × 10− 8 mol/L. MTT assay indicated that the hAMSCs pretreated with ICA exhibited increased proliferation compared with the control group, and the proliferation fold of hAMSCs decreases with the increase of ICA concentration  is one of the biochemical markers for osteoblast activity. It is an enzyme in which osteoblasts secrete into the extracellular matrix . ALP can− 6 mol/L, it is most significant to increase proliferation and promote the osteogenic differentiation of hAMSCs. Therefore, an appropriate amount of icariin might be used as a potential candidate compound for stem cell-based therapy of bone disease.Bone defects/losses is a serious problem in orthopedics, and osteogenic inducers play central roles in effective stem cell-based treatment during the processes. In this study, we have demonstrated that icariin is a safe, effective, and novel natural osteogenic inducer for hAMSCs. When the concentration of ICA was 1 × 10Additional file 1: Table S1. Number of calcified nodules on days 21 in each group.


Both NGs were several hundred nanometers in size, had negative zeta potentials, and several % (w/w) drug contents. They released PD gradually at pH 7.4 and 6. They exhibited fairly good retention in the systemic circulation. In the efficacy examination using rats with adjuvant-induced arthritis, both NGs showed the stronger and more prolonged suppression of paw inflammation than PD alone. These suggested that the present NGs should be possibly useful as anti-arthritis targeting therapeutic systems.Although prednisolone (PD) is used as an anti-arthritis drug due to its rapid and strong anti-inflammatory potential, its frequent and large dosing often brings about adverse effects. Therefore, targeting therapy has attracted increasing attention to overcome such adverse effects. In the present study, nanogels (NGs) composed of macromolecule–PD conjugates were developed as a novel targeting delivery system, and their anti-inflammatory potential was examined. Conjugates were prepared by carbodiimide coupling between glycyl-prednisolone (GP) and the natural anionic polysaccharides, alginic acid (AL) and hyaluronic acid (HA). NGs were produced by the evaporation of organic solvent from the conjugate solution. The obtained NGs, named AL-GP-NG and HA-GP-NG, respectively, were examined for particle characteristics,  In general, a NG is a kind of nanoparticle. NGs are three-dimensional nano-sized hydrogels formed by physical or chemical crosslinking of polymers  and hyaluronic acid (HA), which are actively examined in medical fields because of their highly biocompatible properties. AL is used as a biomaterial for wound dressings, etc. , 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (WSC), and sodium alginate ), cat no. 154725, lot no. QR13046) were purchased from FUJIFILM Wako Pure Chemical Corp. . N-Trityl-glycine (Tr-G) was obtained from Sigma-Aldrich Co., LLC. . Hyaluronic acid (viscosity of 1% aqueous solution at 25 °C = 1.2 ± 0.1 mPa·s (n = 3), product code H0595, lot no. 8FUKM-CI) was purchased from Tokyo Chemical Industry Co., Ltd. . Non-viable and desiccated Mycobacterium tuberculosis H37 Ra was obtained from Becton, Dickinson and Company  and used in the preparation of adjuvants. All other chemicals were of reagent grade.Prednisolone, carbonyl-diimidazole (CDI), dimethyl-aminopyridine (DMAP), 2.2.ad libitum. Regarding the conditions of the animal room, temperature was set at 23 ± 1 °C, relative humidity was maintained at 60 ± 1%, and the light-dark cycle was 12 h. The experimental protocol was approved by the Committee on Animal Research of Hoshi University , in which the approval code was 30-076 . Animal experiments were conducted based on the Guiding Principles for the Care and Use of Laboratory Animals of Hoshi University .Female Lewis rats (160–170 g) were purchased from Charles River Laboratories Japan, Inc. . They were bred on the breeding diet MF  with water 2.3.1H NMR) and electron ionization mass spectrometry (EI-MS). 1H NMR (DMSO-d6), δ (ppm): 7.20–7.42 , 6.15–6.18 , 5.92 , 5.02–5.05 , 4.72–4.75 , 4.29 , 2.97–2.99 . EI-MS m/z: 659 (M+).The conjugates of GP with AL and HA, named AL-GP and HA-GP, respectively, were synthesized as shown in 1H NMR and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). 1H NMR (DMSO-d6), δ (ppm): 7.31–7.33 , 6.15–6.17 , 5.92 , 4.97–5.00 , 4.84–4.87 , 4.27–4.38 . MALDI-TOF-MS m/z: 418.54 ([M + H]+).Next, GP was obtained by detritylation of Tr-GP: soon after Tr-GP (300 mg) was dissolved in 75% (v/v) aqueous acetic acid (10 mL) by heating at 80 °C for 10 min, triphenylcarbinol was precipitated. Then, the mixture underwent ice cooling and filtrated. The filtrate was evaporated with a rotary evaporator, and the residue was dissolved in the chloroform/methanol mixture. The resultant solution was chromatographed with a silica gel column using a chloroform/methanol mixture  as the elution solvent. The fractions of the major product (GP) were collected and evaporated to dryness. The structure of GP was confirmed by the measurements of Finally, the conjugates of GP with the polysaccharides were prepared as follows. As to AL-GP, after GP (30 mg) and AL-Na (60 mg) were dissolved in 10 mL of the THF/water  mixture, WSC (500 mg), and NHS (300 mg) were added, and the mixture was stirred at room temperature overnight. The resultant mixture was chromatographed with a Sephadex G50 gel column (2.6 cm (inner diameter)×19 cm (length)) using a mixture of 0.1 M NaCl aqueous solution and methanol  as the elution solvent. After the macromolecular fractions had been gathered, the solution obtained was dialyzed against water at 4 °C. The medium remaining in the dialysis bag was lyophilized to yield AL-GP powder. HA-GP was prepared in the same manner, except for the use of HA (60 mg) instead of AL-Na (60 mg).in vacuo until THF was removed completely from the solvent. The NG was obtained as a white suspension in water.The NGs of AL-GP and HA-GP, named AL-GP-NG and HA-GP-NG, respectively, were obtained by the solvent evaporation technique . After t2.4.The drug contents of AL-GP-NG and HA-GP-NG were examined by the complete liberation of PD from NGs followed by spectrophotometric measurements. The NGs, AL-Na, and HA (3 mg) were placed in 5 mL of 0.1 M NaOH aqueous solution and incubated at 45 °C for 10 min. After the media were centrifuged, supernatants were measured spectrophotometrically at 246 nm. The difference in absorbance at 246 nm between that supernatant and polysaccharide solution was obtained as a net absorbance by the conjugated PD. The amount of the conjugated PD was calculated by comparing the net absorbance (246 nm) with the absorbance of PD itself in 0.1 M NaOH aqueous solution (246 nm).The particle size and zeta potential of each NG were analyzed using the dynamic light scattering apparatus, ELS-Z2 .2.5.AL-GP-NG and HA-GP-NG were suspended in phosphate-buffered saline of pH 7.4 (PBS) and 0.1 M acetate buffer of pH 6.0 at a concentration of 2 mg/mL. The suspension (4 mL) was placed in a cellulose dialysis tube  made by Viskase Companies, Inc. . The tube containing the NG sample was completely immersed in 16 mL of PBS or acetate buffer, and incubated at 37 °C by shaking horizontally at 60 rpm for 48 h. Aliquot samples (50 µL) were taken at appropriate time points. Immediately after sampling, 100 µL of 0.1 M acetate buffer (pH 4) was added to the sample to suppress further release, because the carboxy ester is very stable around pH 4. The resultant mixture was analyzed by HPLC to measure the concentration of released PD.2.6.AL-GP-NG and HA-GP-NG were suspended in saline at 2.5 mg PD eq./mL. The NG suspension was injected intravenously via the jugular vein into normal rats at 2.5 mg PD eq./kg. Blood samples (each 300 µL) were taken immediately before and 0.5, 1, 3, and 7 h after administration. After each sampling, centrifugation was performed at 1500 rpm to obtain plasma. Plasma (50 µL), 20% trichloroacetic acid (10 µL), and methanol (35 µL) were mixed and shaken vigorously for 5 min. After centrifugation of the mixture, the supernatant was analyzed by HPLC to assess the concentration of free PD.The concentration of total (free + conjugated) PD was analyzed as follows: plasma (50 µL) and 0.1 M NaOH aqueous solution (50 µL) were mixed, and the mixture was incubated at 45 °C for 10 min. Then, 0.1 M HCl aqueous solution (50 µL) was added to neutralize the sample pH. Furthermore, 20% trichloroacetic acid (30 µL) and methanol (105 µL) added, and the mixture underwent vigorous shaking for 5 min. After centrifugation of the sample, the supernatant was analyzed by HPLC to determine the concentration of PD.As the control, PD alone, dissolved in 50% (w/v) polyethylene glycol 400 (PEG400) saline solution at 2.5 mg/mL, was administered intravenously to normal rats via the jugular vein at 2.5 mg/kg. Blood sampling and analyses of the plasma concentration of PD were performed as in the case of the free PD in the NG suspension.Pharmacokinetic parameters were calculated using the program MULTI produced by Yamaoka et al. .2.7.In vivo experiments of efficacy and adverse effects were performed using rats with adjuvant-induced arthritis. The in vivo experimental schedules are shown in M. tuberculosis H37 Ra (20 mg) was suspended in 4 mL of liquid paraffin and ground in a mortar. The resultant suspension (100 µL) was injected intracutaneously into the foot pad of the right hind paw as the adjuvant. Body weights and both hind paw volumes were measured every day after the injection of the adjuvant.Drugs  were administered intravenously at 2.5 mg PD eq./kg 15 and 16 d after the adjuvant injection; the drug administration was conducted in the manner of consecutive dosing at 2.5 mg PD eq./kg × 2 d. The control group received no treatment. Body weights and the volumes of both hind paws were monitored every day until 28 d after the adjuvant injection. Changes in body weight were examined as an index of adverse effects. The hind paw volume was investigated as an index of the degree of inflammation.2.8.A HPLC analysis was performed using an apparatus made by Shimadzu Corp. ; A LC-20AD pump, SPD-20A UV/VIS detector, SIL-20AC autosampler, SLC-10A system controller, and LabSolutions system were used at room temperature. An YMC-Pack ODS-AM column (6 mm (inner diameter)×150 mm (length)) was used as an analytical column. The detector was set at 246 nm.in vitro studies, a 30% (v/v) 2-propanol aqueous solution containing trifluoroacetic acid at 0.1% (w/v) was used as the mobile phase, and flowed at 1 mL/min. The sample (20 µL) was injected onto the column.In in vivo experiments, a mixture of acetonitrile and 50 mM sodium citrate–phosphoric acid buffer of pH 4.1  was used as the mobile phase, and its flow rate was set at 1 mL/min. Each sample (20 µL) was injected on the column. The concentration of PD was calculated by the absolute calibration method.As to the analysis of samples in 2.9.p< .05.Statistical analyses were performed with a one-way ANOVA followed by Tukey’s post hoc test, and the significance of differences was set as 3.3.1.Carboxy esterification between Tr-G and PD was conducted as described previously , with CDI in dry THF using DMAP as a catalyst, in which esterification at the position of C21 of PD was confirmed from the chemical shift at the C21 protons:  in PD,  in Tr-GP. Furthermore, the integrated intensities of the protons of Tr-GP showed that the ratio of glycine to PD was 1:1 (mol/mol). These indicated the ester formation at C21 of PD and the composition ratio of PD/Tr-G at 1:1 (mol/mol). GP, obtained by detritylation of Tr-GP, showed similar features for the chemical shift and proton integrated intensities. GP was confirmed to be a glycine monoester of PD, in which the ester was formed at the C21 position.Conjugates of GP with the natural anionic polysaccharides, AL and HA, called AL-GP and HA-GP, respectively, were prepared by carbodiimide coupling . NanogelThe PD content of the NG was determined from the absorbance at 246 nm after the alkaline hydrolysis of the ester bond. Although the treatment of PD in the alkaline solution gave some degradation of PD, the absorbance of the treated sample at 246 nm was hardly changed. Therefore, even if the PD was degraded to a fair extent in the complete liberation of PD from NG at the alkaline conditions, the drug content could be determined from the absorbance at 246 nm. The PD contents, particle sizes, and zeta potentials were obtained as shown in 3.2.The release profiles of PD from both NGs were obtained as shown in 3.3.In order to find out the pharmacokinetic features of AL-GP-NG and HA-GP-NG, they were intravenously injected into normal rats, and the plasma concentration–time profiles of free PD and total (conjugated + free) PD were investigated.Total PD was assessed from PD liberated from NGs by hydrolysis with 0.1 M NaOH. However, liberated PD was not fully recovered by HPLC because PD was decomposed at the strong basic pH. When AL-GP-NG and HA-GP-NG were treated in 0.1 M NaOH aqueous solution at 45 °C for 10 min, the mean recovery ratios of intact PD were 55 and 70%, respectively. The recovery ratios were used as correction coefficients to calculate total PD concentrations.Plasma concentration–time profiles were obtained as shown in Regarding the i.v. injection of AL-GP-NG, the total PD concentration was markedly higher than the free PD concentration. The total PD concentration was higher than 40 µg/mL immediately after its administration, and was still higher than 10 µg/mL at 7 h. The free PD concentration was maximal at 0.5 h, being 1.64 µg/mL, and then the free PD concentration gradually decreased.As to the i.v. injection of HA-GP-NG, total PD concentrations were also higher than free PD concentrations. The total PD level at 0 h was calculated at 24.03 µg/mL. Overall, total PD levels were lower with HA-GP-NG than with AL-GP-NG. At 7 h, the total PD concentration decreased to 0.39 µg/mL. The concentration of free PD was maximal at 0.5 h, being 1.05 µg/mL, and was not detected at 7 h. The levels of free PD were also lower with HA-GP-NG than with AL-GP-NG.Cmax, AUC (0–7 h), MRT (0–7 h), and VRT (0–7 h) than free PD by PD alone and NG. Higher MRT (0–7 h) and VRT (0–7 h) were observed in free PD with AL-GP-NG than in free PD by PD alone. HA-GP-NG also showed similar pharmacokinetic features to those of AL-GP-NG. Since both NGs gave higher total plasma concentrations and more prolonged systemic retention, suggesting that the NGs should work well as a targeting delivery system to the sites of inflammation.The pharmacokinetic parameters of AL-GP-NG and HA-GP-NG are shown in 3.4.Efficacy studies were performed using diseased model rats with adjuvant-induced arthritis. Changes in body weight after i.v. administration were examined as adverse effects. The anti-inflammatory effectiveness of each drug was evaluated from changes in hind paw volumes  drug contents. They gradually released PD at approximately 30-40% per day at pH 7.4 and more slowly at pH 6. Furthermore, both NGs exhibited fairly good retention in the systemic circulation. These


These findings suggest that reversine could be a good candidate as an alternative or supplementary drug for CCA treatment.Reversine is a selective inhibitor of mitotic kinase monopolar spindle 1 (MPS1) and has been reported as an anticancer agent in various cancers. The effects of reversine on bile duct cancer, cholangiocarcinoma (CCA), a lethal cancer in Northeastern Thailand, were investigated. This study reports that reversine inhibited cell proliferation of CCA cell lines in dose- and time-dependent manners but had less inhibitory effect on an immortalized cholangiocyte cell line. Reversine also triggered apoptotic cell death by decreasing anti-apoptotic proteins, Bcl-XL and Mcl-1, increasing Bax pro-apoptotic protein and activating caspase-3 activity. Moreover, reversine induced autophagic cell death by increasing LC3-II and Beclin 1 while decreasing p62. Reversine activated autophagy via the AKT signaling pathway. Additionally, this study demonstrated for the first time that reversine could diminish the expression of Hypoxia-Inducible Factor 1- alpha (HIF-1 Opisthorchis viverrini, is a major contributing cause of Thai CCA patients (Cholangiocarcinoma (CCA) is a cancer of bile duct epithelia. Problems of CCA are a high mortality rate related to late diagnoses and cancer metastasis. The highest incidence of CCA has been reported in Northeastern Thailand . The livpatients . 5-Fluorpatients . Therefoα expressions; modulation of this signaling pathway led to cellular starvation including reduction of glucose uptake and ATP production , a mitotic checkpoint protein . This syoduction .Upregulation of MPS1 was reported in OV-associated CCA tissues , therefoIn the current study, the suppressive effects of reversine on viability of CCA cell lines were determined using the MTT assay, the cell cycle was analyzed by flow cytometry and the related molecular mechanisms of autophagy and apoptosis were investigated using Western blot analysis. The glucose uptake and ATP production were also measured. Results revealed that reversine induced G2/M arrest, apoptosis and autophagy via glucose and ATP deprivation in CCA cell lines.2 atmosphere. At 70–80% confluence, cells were detached from the culture flask using 0.25% w/v trypsin/EDTA and processed according to the particular assay. Reversine was purchased from Cayman Chemical  and prepared as a 100 mM stock in DMSO.CCA cell lines  were obtained from the Japanese Collection of Research Bioresources Cell Bank, Osaka, Japan and established as previously described . An immo50) by the dose–response inhibition mode of the GraphPad Prism® 7.0 software .Cells were seeded in 96 well plates and treated with 0.1% DMSO as a vehicle control and various concentrations of reversine at 0.1, 0.5, 1, 10 and 20 µM, for 24, 48, and 72 h. MTT reagent was added into each well to a final concentration of 0.5 mg/ml and incubated for 4 h. DMSO was added to dissolve the insoluble formazan complex and the absorbance at 540 nm was measured. The data were analyzed at a half maximal inhibitory concentration (IC5 cells) were seeded into a 6-well plate and treated with reversine  for 24 h. Cells were collected, fixed with cold 70% ethanol overnight at 4 °C and treated with 0.1 mg/ml of RNase A at 37 °C for 1 h. Cells were stained with 10 µg/ml propidium iodide (PI) at 4 °C for 30 min in the dark. Cell cycle analysis was determined using a flow cytometer (BD LSR II™). FlowJo™ software was used to generate the acquisition and analysis plots of 20,000 cells.KKU-213A and KKU-213B cell lines , Bcl-XL, and Bax , Beclin 1 and p62 . GAPDH,  was used as an internal control. The immunoreactivity signals were captured and analyzed by ImageQuant™ TL analysis software .CCA cells were lysed in buffer  containing phosphatase and protease inhibitors. Protein lysates were applied on a 12% SDS-PAGE for Western blot analysis. Proteins were detected by antibodies including PI3K, AKT, phospho-AKT, GLUT1, LC3-II, Mcl-1, and cleaved caspase-3 , HIF-15 cells) were seeded onto 6-well plates and treated with a sublethal dose (IC25) at 4 µM, of reversine for 12 and 24 h. Glucose levels in the collected media were determined using a Glucose Colorimetric/Fluorometric Assay kit  following the manufacturer’s protocol. Absorbance was measured at 570 nm and the glucose standard provided with the kit was used to calculate glucose levels. The glucose uptake was calculated by comparing the difference in leftover glucose concentrations between the vehicle and the reversine treatment. The amounts of ATP in cell lysates were measured and calculated from the standard curves using the ATP Colorimetric/Fluorometric Assay Kit according to the manufacturer’s protocol . The glucose uptake and ATP production of vehicle control were set as 100%.KKU-213A and KKU-213B cell lines  and SPSS 17.0 software . A P-value less than 0.05 was considered as statistically significant.Data were presented as mean ± SD. Statistical comparisons between groups were tested using the Student’s P < 0.05) (50) of reversine at different time points for the 3 CCA cell lines were in the 0.62–10 µM range which were significantly lower than those of the MMNK1 cell line (7.05–20 µM range) as shown in 50 of reversine was lower than 5-FU at least 6–12 times  for 24, 48 and 72 h. Cell viability was significantly decreased in dose- and time-dependent manners in all cell lines (*  < 0.05) –1C. The 12 times . This inP < 0.05), whereas Bax pro-apoptotic protein and cleaved caspase-3 were increased noticeably in reversine treatment for 48 and 72 h in both CCA cell lines  (α) and glucose transporter 1 (GLUT1) by Western blot assay were further analyzed. The results showed markedly decreased protein expression of HIF-1α and GLUT1 in reversine treated cells compared with vehicle control  –3D. Thes control –3D. Collα catalytic subunit of PI3K (p110α) and the phosphorylation of serine 473 of AKT (s473) in reversine-treated CCA cell lines.The PI3K/AKT signaling pathway has been reported to regulate the autophagy. Therefore, the activation of PI3K/AKT after reversine treatment was determined by Western blot analysis. α, GLUT1 and p62 expression with the concurrence of decreased Beclin 1 and LC3-II in reversine combined with SC-79 treatment (#P < 0.05).To further confirm that reversine induced autophagy via the AKT signaling pathway, SC-79, an AKT activator was used to activate the phosphorylation of AKT (s473). A combination of reversine and SC-79 recovered the cells from reversine-mediated autophagy –4B. Westreatment . Among t50 of reversine at 72 h in 50 ∼ 5 µM) (50 ∼ 7.5 µM)   and cerv 7.5 µM) . This coThe growth inhibitory effect of reversine on G2/M cell cycle arrest in CCA cell lines corresponded with the observations in several cancer cells after reversine treatment . Westernα) and AKT (s473) in reversine treated cells were examined. It was found that the PI3K/AKT signaling pathways were inactivated upon reversine treatment. This finding was similar to the studies in oral squamous cell (The present results showed reversine could induce apoptosis via down-regulation of anti-apoptotic proteins (Bcl-XL and Mcl-1), up-regulation of pro-apoptotic Bax protein and activation of caspase-3 in both CCA cell lines –2F. Thesous cell  and thyrous cell . Furtherous cell .α and GLUT1   . ReversiReversine markedly inhibited the cell proliferation of CCA cells by multiple mechanisms including G2/M cell cycle arrest, apoptosis and starvation-related autophagy. These new findings suggest that reversine could be a good candidate drug for CCA treatment. The effects of reversine itself or reversine in combination with current chemotherapy drugs are worthy of further preclinical and clinical investigations.10.7717/peerj.10637/supp-1Figure S1CCA cells were treated with 0.25, 2.5, 25 and 250 µM of 5-FU for 48 h. Cells treated with 0.1% DMSO were used as a vehicle control. The cell viability was determined using the MTT assay. Data are mean ± SD of three independent experiments.Click here for additional data file.10.7717/peerj.10637/supp-2Table S150: Half-maximal inhibitory concentration. * P-value < 0.05 compared with reversine.ICClick here for additional data file.10.7717/peerj.10637/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.10637/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.10637/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.10637/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.10637/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.10637/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.10637/supp-9Supplemental Information 9Click here for additional data file.