Raoultella ornithinolytica is an Enterobacteriaceae bacterium that can infect both humans and animals, while luteolin-7-O-glucoside (IOG) is a flavonoid that has broad effects on the intestinal microbiota of healthy animals. However, current studies lack sufficient data on intestinal microbiota dysbiosis and drug resistance transmission caused by R. ornithinolytica and the possible role of IOG. In this study, BALB/c mice were infected with R. ornithinolytica carrying blaNDM-1 gene and treated with IOG (3 mg/kg·d and 6 mg/kg·d) to analyze the diversity of intestinal microbiota and the transfer of blaNDM-1 between bacteria. The findings indicated that R. ornithinolytica B1645-1 exhibited a significant ability to enhance the Firmicutes/Bacteroidota ratio and increase the relative abundance of Lactobacillus and Bacillus after 48 h, where as 6 mg/kg·d IOG had an opposite effect. Moreover, R. ornithinolytica B1645-1 facilitated the emergence of drug-resistant bacteria and promoted blaNDM-1 gene transfer in Enterococcus, Escherichia, Klebsiella, Acinetobacter, Bacillus, Brevibacterium, and Lactobacillus. Enterococcus was the predominant genus at 48 h. Surprisingly, 6 mg/kg·d IOG significantly inhibited the production of drug-resistant bacteria and promoted blaNDM-1 gene transfer from Enterococcus to Lactobacillus at 144 h. However, the role of Lactobacillus as a recipient for drug-resistant genes should be of more concern. Lonicera japonica Thunb, Herbataraxaci, Forsythia, Polygonum cuspidatum, Ginger, and other plants  viagastric intubation for 24 h. Subsequently, the IFL3 and IFL6 groups received IOG at doses of 3 mg/kg·d and 6 mg/kg·d (200 µL) for five consecutive days, respectively. The Con was given an equivalent volume of 0.9% normal saline under identical conditions. At both the 24 h and 120 h time points after administration of IOG, the mice were euthanized. Fresh transverse colon contents (approximately 0.1 g) measuring approximately 1 cm in length were dissected and flash-frozen in liquid nitrogen for 30 s before being stored at −80 °C until DNA extraction for microbial diversity and blaNDM-1 gene analysis.All experiments using bacteria, IPM, IOG, and mice were performed at the Medical Center of Hubei University of Medicine (Licence No.: SYXK 2017-0093). The animal experiments were conducted upon approval from the Ethical Committee for Vertebrate Experiments of Hubei University of Medicine .Under ice-water bath conditions, 0.1 g of the sample was immersed in 1 mL of sterile phosphate-buffered saline (PBS) , vigorously vortexed, and then centrifuged at 200 rpm for 5 min to remove any remaining coarse particles. After this procedure was repeated three times, the combined supernatant was used to precipitate bacteria at 10,000–12,000 rpm for 10 min.® soil DNA kit (Omega) was utilized to extract DNA from the contents of mouse transverse colon. The V3–V4 regions of 16S rDNA were amplified with primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTA AT-3′). The amplification reaction was as follows: 95 °C pre-degeneration for 3 min, 95 °C degeneration for 30 s, 55 °C annealing for 30 s, and 72 °C extension for 30 s, for a total of 27 cycles, and then extension at 72 °C for 10 min. The raw sequence data were submitted to the NCBI’s Short Read Archive (SRA) (BioProject ID: PRJNA898665).The E.Z.N.A.Alpha diversity was used to assess species richness within group based on the Chao, ACE, Shannon, and Simpson indices. Beta diversity was performed to measure differences in abundance between groups with non-metric multidimensional scaling (NMDS) at the ASV level [p < 0.05 considered significant [The sequence data were processed using Quantitative Insights Into Microbial Ecology  . Paired-SV level . Linear nificant .blaNDM-1-positive colonies.Based on the operation flow shown in blaNDM-1-positive-colony screening results, PCR was used to detect the blaNDM-1 gene. Amplification was conducted with the primer set F(5′-CTCGCACCGAATGTCTGGC-3′) and R(5′-GGGGCGTAGTGCTCAGTGTC-3′). The PCR amplification reactions included 25 µL of 2 × NovoStar Green PCR Mix, 2 µL each of upstream and downstream primers (1 mM), 1 µL of DNA template, and ddH2O added to a total volume of 50 µL. The PCR conditions were as follows: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min, for a total of 34 cycles, and then extension at 72 °C for 5 min. The amplified products were separated via 1% agarose gel electrophoresis  and sequenced. The gene sequence was compared with the GenBank database to determine the blaNDM-1 gene, and the bacterial strains carrying blaNDM-1 were identified using mass spectrometry method [To determine the accuracy of p < 0.05 was considered to be statistically significant [One-way ANOVA with Tukey’s multiple comparison test and Kruskal–Wallis H test were used to compare the data among groups, and nificant . MetaPhlnificant . GraphPaA total of 3,829,368 sequences (>202 bp) were derived from 32 samples. After DADA2 noise reduction, 1,541,391 effective sequences were retained and 10,585 ASV sequences were clustered according to the minimum number of sample sequences   . The rarp < 0.05) (6) (p > 0.05).The Chao and ACE indices were used to determine species richness, while the Simpson and Shannon indices were used to estimate bacterial diversity . At 48 h < 0.05) A, and th.05) (6) B. The an.05) (6) C,D. No s.05) (6) E,F or di.05) (6) G,H was op = 0.009). There were significant differences in community composition between groups (p < 0.05), indicating that all four groups were effective , indicating that IOG had no further impact on the community   in the IF, IFL3, and IFL6 groups, while the relative abundance of Bacteroidota increased significantly . The Firmicutes/Bacteroidota ratio in the Con, IF, IFL3, and IFL6 groups were 1.16, 0.68, 0.57, and 1.26-fold, respectively   E. At 144 < 0.05) B, the reectively D. There  > 0.05) F.Norank_f__Muribaculaceae and Lactobacillus were clearly the dominant genera in the Con group (25.46% and 16.68%). At 48 h (Lactobacillus (p < 0.05). Although there was a decreasing trend in the relative abundance of norank_f__Muribaculaceae in the IF group, this difference did not reach statistical significance (p > 0.05). Following the administration of IOG, the relative abundance of Lactobacillus significantly decreased from 55.72% in the IFL3 group to 31.13% in the IFL6 group, and when the dosage was 6 mg/kg·d (IFL6), the relative abundance of Lactobacillus recovered to a level with no significant difference compared to the Con group (16.68%). This result suggested that IOG possesses the potential to reduce the increase in Lactobacillus count due to infection with R. ornithinolytica B1645-1. The relative abundance of Lactobacillus, Helicobacter, unclassified_f__Rikenellaceae, Candidatus_Saccharimonas, norank_f__Lachnospiraceae, and Skermanella exhibited significant differences between groups (p < 0.05)  C. The LE6 groups A.unclassified_f__Lachnospiraceae in the IF, IFL3, and IFL6 groups increased gradually, while that of Lactobacillus significantly decreased to 7.93%, 1.7%, and 5.66%, respectively (15.79% in Con). Prevotellaceae_UCG-001, Eubacterium_siraeum_group, Streptococcus, and Lachnospiraceae_UCG-004 were significantly different species (p < 0.05). The LEfSe analysis also showed that Prevotellaceae_UCG-001species and Streptococcus species had the greatest impact in the IFL3 and IFL6 groups, respectively . 3, and IFL6 groups increased significantly at 144 h compared to at 48 h, which were 5.03 ± 0.61 × 108 CFU/mL, 3.86 ± 1.32 × 108 CFU/mL, and 1.30 ± 0.26 × 107 CFU/mL, respectively. The bacterial count in the IFL6 group was still significantly lower than the counts in the IF and IFL3 groups (p < 0.001 and p < 0.01). On anaerobic agar medium, the number of anaerobic bacteria in the IF, IFL3, and IFL6 groups increased significantly at 48 h, reaching 1.17 ± 0.25 × 104 CFU/mL, 4.47 ± 0.85 × 102 CFU/mL, and 1.67 ± 0.51 × 104 CFU/mL, respectively. In addition, we also saw that at 144 h, the number of anaerobic bacteria in the IF, IFL3, and IFL6 groups was 3.53 ± 0.3 × 103 CFU/mL, 6.4 ± 0.48 × 103 CFU/mL, and 1.44 ± 0.03 × 104 CFU/mL, respectively. Combined with the number of colonies in the two culture media, the number of bacteria in the IF, IFL3, and IFL6 groups at 48 h was 1.53 ± 0.3 × 103 CFU/mL, 4.33 ± 0.3 × 103 CFU/mL, and 1.67 ± 0.3 × 103 CFU/mL, respectively. The bacterial numbers at 144 h were 5.03 ± 0.61 × 108 CFU/mL, 3.86 ± 1.32 × 108 CFU/mL, and 1.30 ± 0.26 × 107 CFU/mL, respectively.Drug-resistant strains were screened using Luria–Bertani agar medium and anaerobic agar medium supplemented with IPM at a concentration of 4 μg/mL. On Luria–Bertani agar medium, 6 group remained consistently lower than that in the IF and IFL3 groups, indicating IOG’s inhibitory effect on the production of drug-resistant bacteria.In conclusion, with the extension of infection time, the number of aerobic and anaerobic resistant bacteria significantly increased. However, the number in the IFLblaNDM-1-positive bacteria were successfully isolated from the IF, IFL3, and IFL6 groups, including Lactobacillus johnsonii, Enterococcus faecalis, Enterococcus gallinarum, Klebsiella pneumoniae, Escherichia coli, Brevibacterium linens, Acinetobacter baumannii,and Bacillus pumilus, of which Enterococcus was the dominant genus. However, at 144 h, there were still blaNDM-1-positive bacteria in five genera  in the IF, IFL3, and IFL6 groups  than in the Con group (1.1:1). Additionally, at 48 h, there was a notable increase in Lactobacillus abundance from 17% in the Con group to 60% in the IF group. During the initial phase (48 h), total colonic bacterial numbers (ASVs) decreased significantly while Lactobacillus increased, which may be attributed to pathogenic bacterial colonization and subsequent elimination of Bacteroidota [Firmicutes/Bacteroidota ratio recovered to 0.68:1 at 144 h and Lactobacillus was no longer the factor that affected the difference , which are strictly anaerobic bacteria, maintain a relatively low level of facultative anaerobes (such as Enterobacteriaceae and Enterococci), while Lactobacilli are abundant in rats and mice [Firmicutes/Bacteroidota ratio was significantly lower in the IFL6 group (2.91:1) compared to the IFL3 (7.46:1) and IF (5.52:1) groups, indicating that 6 mg/kg·d IOG had a stronger regulatory effect on intestinal microbiota recovery in infected mice than in other groups. Contrary to previous reports suggesting that IOG promotes an increase in Lactobacillus within the intestinal flora of healthy rodents [Lactobacillus numbers from 50% in the IFL3 group to 30% in the IFL6 group at 48 h. This regulatory effect was even more significant at 144 h, indicating that IOG primarily targets Lactobacillus as one of the main influencers on intestinal microbiota.The major groups of mammalian intestinal microbiota include  species . In animand mice ,15. IOG  rodents , our stuBacteroidota and Firmicutes) and genera , which are associated with the transmission of drug-resistant genes within intestinal microbiota. After 48 h of infection, the blaNDM-1 gene carried by R. ornithinolytica B1645-1 was detected in various intestinal bacteria, including E. faecalis, E. gallinarum, E. coli, K. pneumoniae, A. baumannii, B. pumilus, B.linens, and Lactobacillus johnsonii, with Enterococcus being the predominant genus. However, after a 120 h treatment with IOG, intestinal bacteria carrying the blaNDM-1 gene underwent significant changes and Lactobacillus  became the dominant carrier of the blaNDM-1 gene. This finding indicated that the blaNDM-1 gene had been transferred from pathogenic bacteria (R. ornithinolytica B1645-1) to Enterococcus within 48 h, and subsequently to Lactobacillus within 144 h, either through natural recovery in the IF group or after treatment with IOG in the IFL3 and IFL6 groups. Although IOG exhibited efficacy in reducing the number of bacterial genera or species carrying drug-resistant genes and the abundance of drug-resistant bacteria -producing enterobacteria (CPE) clones among patients and the transfer of carbapenemase-encoding genes between enterobacteria in individual patients’ intestinal microbiota have shaped the epidemiological characteristics of CPE ,37. Howebacteria , Lactobavantages , has becR. ornithinolytica B1645-1 induced a shift in the microbial composition, leading to ecological disorder. This finding has significant implications for comprehending the biological function of R. ornithinolytica and for clinical prevention and treatment. IOG rectified this interference and facilitated the translocation of the blaNDM-1 gene from Enterococcus to Lactobacillus, providing strong support for its clinical application. However, as a recipient for resistant genes, Lactobacillus warrants further investigation.R. ornithinolytica administered orally is sufficient to perturb the composition of intestinal microbiota and facilitate the transfer of the blaNDM-1 gene in intestinal microbiota. Luteolin-7-O-glucoside can modulate this disordered intestinal microbiota, inhibit the production of drug-resistant bacteria, and promote the transfer of the blaNDM-1 gene from the genus Enterococcus to Lactobacillus. However, Lactobacillus should be paid more attention as the recipient of drug-resistant genes because it may be an inevitable donor.


Clostridium perfringens is a widely distributed pathogen associated with several animal and human diseases, that produces many exotoxins, including the phospholipase C (CpPLC), the main virulence factor in gas gangrene. During this disease, CpPLC generates the formation of neutrophil/platelet aggregates within the vasculature, favoring an anaerobic environment for C. perfringens growth. This work demonstrates that CpPLC induces NETosis in human neutrophils. Antibodies against CpPLC completely abrogate the NETosis-inducing activity of recombinant CpPLC and C. perfringens secretome. CpPLC induces suicidal NETosis through a mechanism that requires calcium release from inositol trisphosphate receptor (IP3) sensitive stores, activation of protein kinase C (PKC), and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathways, as well as the production of reactive oxygen species (ROS) by the metabolism of arachidonic acid. Proteomic analysis of the C. perfringens secretome identified 40 proteins, including a DNAse and two 5´-nucleotidases homologous to virulence factors that could be relevant in evading NETs. We suggested that in gas gangrene this pathogen benefits from having access to the metabolic resources of the tissue injured by a dysregulated intravascular NETosis and then escapes and spreads to deeper tissues. Understanding the role of NETs in gas gangrene could help develop novel therapeutic strategies to reduce mortality, improve muscle regeneration, and prevent deleterious patient outcomes.Neutrophil extracellular traps (NETs) are networks of DNA and various microbicidal proteins released to kill invading microorganisms and prevent their dissemination. However, a NETs excess is detrimental to the host and involved in the pathogenesis of various inflammatory and immunothrombotic diseases.  Neutrophils play a critical role in the innate immune response against pathogens through chemokines and cytokines secretion, degranulation, phagocytosis, ROS generation, and the release of NETs . NETs arNETosis is an ancient and evolutionary conserved defense mechanism induced by microorganisms such as parasites, fungi, and viruses . In addiNETosis is classified into suicidal and vital NETosis . In bothC. perfringens is a Gram-positive, anaerobic, sporulated, nonmotile bacillus widely distributed in the environment and the gastrointestinal tract of humans and animals , necrotic enteritis toxin B (NetB), a hemolysin, and neuraminidase  using an automated workstation (Intavis). The resulting peptides were dried, redissolved in water with 0.1% formic acid, and submitted to nESI-MS/MS on a Q-Exactive Plus® mass spectrometer (Thermo-Fisher) as previously described , washed with 0.1% formic acid, and separated at 200 nl/min with a 3 μm particle, 15 cm × 75 μm C18 Easy-spray® analytical column using a nano-Easy® 1200 chromatograph (Thermo-Fisher). A gradient from 0.1% formic acid (solution A) to 80% acetonitrile with 0.1% formic acid (solution B) was developed: 1–5% B in 1 min, 5–26% B in 25 min, 26–79% B in 4 min, 79–99% B in 1 min, and 99% B in 4 min, for a total time of 35 min. MS spectra were acquired in positive mode at 1.9 kV, with a capillary temperature of 200°C, using 1 μscan at 400–1600 m/z, maximum injection time of 100 msec, AGC target of 3×106, and orbitrap resolution of 70,000. The top 10 ions with 2–5 positive charges were fragmented with AGC target of 1×105, maximum injection time of 110 ms, resolution of 17,500, loop count of 10, isolation window of 1.4 m/z, and a dynamic exclusion time of 5 s. MS/MS spectra were processed for peptide matching against C. perfringens protein sequences contained in the UniProt database or against the sequences of seven clostridial nucleases in a private database using Peaks X® (Bioinformatics Solutions) (The secreted proteins from the escribed . Each trlutions) . Cystein®-1119 . After centrifugation for 20 min at 400 x g without braking, the layer containing the granulocytes was recovered and washed with 10 ml of sterile phosphate buffer solution (PBS). Afterward, the cells were centrifuged at 400 x g for 10 min and the supernatant was discarded. Erythrocyte remnants were removed using 5 ml of lysis buffer  for 25 s. Then, 10 ml of sterile PBS was added to stop lysis, and the cell suspension was centrifuged at 400 x g for 10 min. The cell pellet was resuspended in 1 ml of sterile Roswell Park Memorial Institute (RPMI) medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (inactivated at 56°C) and 1% penicillin-streptomycin. All centrifugation steps were carried out at room temperature (r.t).Human neutrophils were purified from healthy volunteers, as described previously  with som5 per well in 500 μl of RPMI medium and incubated for 1 h at 37°C in a humid atmosphere with 7% CO2 to allow neutrophil adhesion. Cells were then treated with 100 μl of the proteins secreted by JIR325 (6.3 mg/ml). Phorbol 12-myristate 13-acetate (PMA) (100 nM) was added as a positive control, and untreated neutrophils were used as a negative control in each experiment. In some experiments, the neutrophils were exposed to proteins secreted by JIR325 pre-incubated for 1 h with the DNAse inhibitor CuSO4 (0.01 M). These cells were incubated at 37°C in a humid atmosphere with 7% CO2. After the incubation, the cells were fixed with 4% paraformaldehyde, washed three times with PBS for 5 min, and then permeabilized with 0.5% Triton X-100 for 1 min at r.t. After 3 washes with PBS, blocking was performed with Protein Block Serum-  in a humid chamber for 30 min at 37°C. Neutrophils were then incubated with the anti-neutrophil NE rabbit polyclonal antibody , diluted 1:100 in Tris-NaCl blocking buffer (TNB) for 1 h in a humid chamber at 37°C. After 3-5 -min washes with PBS, the coverslips were incubated for 1 h in a humid chamber at 37°C with a goat anti-rabbit IgG-FITC conjugate , diluted 1:50 in TNB buffer. Finally, the coverslips were washed 3 times with PBS for 5 min and mounted on glass slides, using Mounting Medium with DAPI-Aqueous Fluoroshield . Three assays were performed with three replicate samples .NETs induction of human neutrophils was carried out as previously described . A 24-wehttps://cellprofiler.org/) to analyze the fluorescence microscopy images. This protocol included two main steps according to the fluorescence channels: identifying total neutrophils  and identifying NE from NETs . Briefly, two independent steps with a function to identify primary objects were implemented to identify whole cells (size from 10 to 40 pixels) and NETs (size from 50 to 2000 pixels). Subsequently, a filtering step was performed to preserve only the cells that were releasing NETs or NETotic neutrophils using the mask functions in the program. The protocol was standardized with 10 sets of images and automatically applied to all images, including a quantification step. The performance was verified manually with the images generated from the analysis in all cases.Images were captured using a Lionheart TM LX Automated Microscope . An automatic protocol was implemented in the CellProfiler program  and 10X DNAse buffer  for 60 min at 37°C. Moreover, an experiment was carried out involving the incubation of supernatant with CuSO4 (0.01 M). The reaction was stopped by adding EDTA (50 mM) at 65°C for 10 min. Sterile RCM was used as a negative control. To visualize DNA degradation, 25 µl of the sample was mixed with 5 µl of gelRed (1:100 with loading buffer), and 20 µl of each sample was run on a 1.5% agarose gel.To measure nuclease activity, secreted proteins of C. perfringens culture at OD 600 = 0.6 was incubated with freshly isolated human neutrophils (4x105 cells) exposed to C. perfringens secreted proteins or sterile RCM (negative control) for 30, 60, and 120 min at 37°C, 5% CO2 atmosphere under aerobic conditions. Serial dilutions of each treatment were performed after cell resuspension, and a volume of 100 µl per dilution was pipetted onto each RCM agar plate. Plates were incubated in an anaerobic atmosphere for 48-72 h, and the CFU was counted. Three assays were performed with three replicated samples.4 cells were analyzed per replicate).Neutrophils were treated with recombinant CpPLC (25.12 ng/ml) for 30 min, and cell viability was measured using the cell-permeable stain 4′,6-diamidino-2-phenylindole (DAPI) and the cell impermeable stain propidium iodide (PI). After incubation, cells were analyzed by immunofluorescence using a Lionheart automated microscope. Three assays were performed with three replicate samples .ROS generation in neutrophils was determined by incubating the cells with recombinant CpPLC and the ROS-sensitive probe dichlorodihydrofluorescein diacetate (DCFDA) (5 mM) for 30 min. After 3 washes with PBS, the percentage of fluorescent cells was determined using a Lionheart automated microscope. Three assays were performed with three replicate samples  for 1 h at 37°C. Several pharmacological inhibitors were used to test the dependence of CpPLC NETs or ROS induction on different mediators. The substances and concentrations used were as follows: the glutathione precursor N-acetyl-L-cysteine (NAC) (1-6 mg/ml); the free radical scavengers 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron) (1-4 mg/ml), and pyrocatechol (50-200 µM); the mitochondrial-derived ROS scavenger MitoTEMPO (100-400 µM); the mitochondrial uncoupler 2,4-dinitrophenol (DNP) (200-800 µM); the NOX inhibitors diphenyleneiodonium chloride (DPI) (25-100 µM) and acetovanillone (0.1-0.4 mg/ml); the xanthine oxidase (XO) inhibitor allopurinol (4-16 mM); the inhibitors of arachidonic acid metabolism arachidonyl trifluoromethyl ketone (AACOCF3) (2.5-10 µM), and 5,8,11,14-eicosatetraenoic acid (ETYA) (12.5-50 µM): the noncompetitive antagonist of IP3 receptors 2-aminoethoxydiphenyl borate (2-APB) (50-400 µM); the scavenger of MPO-derived HClO luminol (50-200 µM); the PKC inhibitors bisindolylmaleimide I (GF109203X) (5-20 µM), safingol (7.5-30 µM) and hispidin (0.5-2 µM); the inhibitor of the MEK/ERK pathway U0126 (1.5-6 µg/ml). These drugs were not cytotoxic at the concentrations used because viability was greater than 95% in cells exposed only to the drugs compared to untreated cells.The capacity of the purified antibodies against CpPLC to neutralize the NETosis-inducing effect of the 4 per well and incubated for 1 h with each inhibitor at 37°C and 5% CO2 atmosphere. Then, cells were exposed to recombinant CpPLC for 1 h as previously described and fixed with 4% paraformaldehyde. Cells were permeabilized with 0.5% Triton X-100 for 1 min at r.t. After 1-2 min washes with PBS, cells were stained with Höechst 33342 (31.25 µg/ml) for 10 min and washed twice with PBS for 1 min each. Finally, NETotic neutrophil quantification was done using the automatic protocol implemented in the CellProfiler program. Identification of total neutrophils (size from 10 to 40 pixels) and NETs (size from 50 to 2000 pixels) was differentiated with the size delimited for each object, followed by manual verification. Three assays were performed with three replicate samples .A 96-well plate was coated with 0.001% poly-L-lysine for 1 h. Freshly isolated human neutrophils were seeded at a concentration of 7x10Results are expressed as mean +/- standard error of the mean (SEM) of at least three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison post-test. P values <0.01 and <0.05 were considered statistically significant.C. perfringens toxinotype A strain JIR325 were separated by SDS-PAGE (https://hdl.handle.net/10669/89483) were matched against the C. perfringens protein sequences in the UniProt database. The results allowed us to identify 39 proteins from the parental strain 13 , twelve cytosolic metabolic enzymes , two chaperones (P26823 and P26821), one folding enzyme (Q8XHK0), three membrane transporters , one adhesion membrane protein (Q93M90), two membrane enzymes (Q8XIW4 and Q8XN18), one secreted (Q8XIL2) and two periplasmic proteins (Q8XIN7 and Q8XLL1) of unknown functions, three cell wall-bound enzymes , and two cell wall-anchored enzymes (Q8XNP3 and Q8XIF9).The identified proteins include two major exotoxins: the CpPLC (Uniprot Accession number P0C216.1) and the thiol-dependent cytolysin, perfringolysin O (P0C2E9). Furthermore, C. perfringens Q8XIF9 bifunctional enzyme shows significant amino acid sequence identity to the virulence-associated S. pyogenes S5nA (Uniprot accession number A0A5S4TI37) (18.7%), the A0A3Q8B6L4 Streptococcus suis AdsA (19.4%) and the A3CKJ7 Nt5e Streptococcus sanguinis (24.0%) cell anchored 5´nucleotidases  and an 81.5 kDa bifunctional metallophosphatase/5’-nucleotidase (Q8XIF9). Amino acid sequence alignment analysis  incubated with human neutrophils exposed or not to C. perfringens secreted proteins was measured at 30, 60, and 120 min  (19.3%), the Q6X5T7 S. suis ssnA (20.8%) and the A3CPM7 S. sanguinis (20.6%) cell anchored nucleases  is produced. The results show that neutrophils became fluorescent and NETotic after exposure to CpPLC Table 2.3 receptors antagonist effectively prevents NETs formation . These three C. perfringens 5´nucleotidases share 36.7-66.0% sequence identity. They are homologous to enzymes relevant for pathogenicity in different streptococcal species  can be found in the article/The studies involving human samples were approved by Institutional Ethics Committee, University of Costa Rica. The studies were conducted in accordance with the local legislation and institutional requirements. The human samples used in this study were acquired from blood provided by healthy volunteers. Written informed consent for participation was not required from the participants in accordance with the national legislation and institutional requirements.LB-V: Writing – original draft, Writing – review & editing, Data curation, Formal Analysis, Investigation, Methodology, Visualization. RP: Investigation, Data curation, Formal Analysis, Methodology, Visualization, Writing – review & editing. JM-M: Formal Analysis, Methodology, Software, Visualization, Writing – review & editing. AA-G: Writing – original draft, Writing – review & editing, Data curation, Formal Analysis, Methodology, Resources, Visualization. MF-D: Writing – original draft, Writing – review & editing, Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization.


The developmental toxicity was evaluated according to the parameters of mortality, hatching rate, autonomous movement and heartbeat of embryos, and body length of larvae. The 9 dpf (days postfertilization)-LC50  of embryonic mortality was 0.077, 0.174 or 0.088 mg/L, and the 9 dpf-EC50 (effective concentration of 50% embryos hatching) of hatching rate was 0.079 mg/L, 0.21 mg/L and 0.092 mg/L when the embryos were exposed to PEG@Cu NCs, Kocide® 3000 or Cu(CH3COO)2, respectively. Kocide® 3000 and Cu(CH3COO)2 obviously decreased the spontaneous movements, while PEG@Cu NCs had no adverse effects on that of embryos. The reduced heartbeat can return to normal after exposure to PEG@Cu NCs for 96 h, while it cannot recover from Kocide® 3000. In addition, Kocide® 3000 (≥0.2 mg/L), PEG@Cu NCs and Cu(CH3COO)2 with 0.05 mg/L or higher concentration exhibited obvious behavioral toxicity to zebrafish larvae according to the parameters of movement distance, average velocity, absolute sinuosity, absolute turn angle and absolute angular velocity.Copper-based fungicides have been used to control various plant diseases for more than one hundred years and play very important roles in agriculture. Accumulation of copper in freshwater and environment pose severe threats to human health and the environment. The current study evaluated the developmental and behavioral toxicity of PEG@Cu NCs (copper nanoclusters), Kocide Daphnia, etc. Copper affected the normal growth and survival of fish by interfering in the nervous system, innate immune system and antioxidant system [Daphnia species [Copper exists naturally in a variety of mineral forms, and is widely used in industry, agriculture, cosmetics and food processing, etc. . Copper t system ,8. It al species ,10. In a species ,12.Xanthomonas citri subsp. Citri), angular leaf spot of cucumber (Pseudomonas syringae pv. Lachrymans), and downy mildew disease of grape (Plasmopara viticola) and cucumber (Pseudoperonospora cubensis) all over the world, including China [Copper was one of the first elements used as a plant fungicide. It can be traced back to the famous origin of Bordeaux mixture, containing a mixture of copper sulfate and lime, which could effectively control the destructive diseases of downy mildew in France vineyards . At presng China ,15. Thesng China ,15. Withng China . Compareng China . Howeverng China ,18.® 3000 (copper hydroxide), Cu(CH3COO)2 (copper acetate), and copper nanoclusters of PEG@Cu NCs (copper nanoclusters) on the development and behavior of zebrafish in early-life stages were studied.Copper usually enters the environment with aqueous discharges, such as urban wastewater, industrial and mine effluent and agriculture runoff, etc. ,19. A to® 3000 (46% copper hydroxide water-dispersible granule) is a commercial pesticide produced by DuPont Company , and 98% copper acetate anhydrous was purchased from Shanghai Macklin Biochemical Co., Ltd . Reconstituted water of pH 7.5 ± 0.5 was prepared according to the guideline of ISO-7346-2 with minor adjustments, which mainly contained 294 mg/L CaCl2 · 2H2O, 106.5 mg/L NaHCO3, 60 mg/L MgSO4 and 6 mg/L KCl [PEG@Cu NCs, a copper nanocluster pesticide, was provided by Professor Kun Qian from Southwest University of China . Kocidemg/L KCl .® 3000 was measured by transmission electron microscopy (TEM)  [® 3000 was measured by a nanoparticle-size and zeta potential analyzer .The shape, particle size of PEG@Cu NCs and Kocide, Japan) . The hydAdult zebrafish  were purchased from Beijing Hongda Gaofeng Aquarium Department, China. The male and female zebrafish were separately raised in the circulating filtration system  under conditions of 26 ± 1 °C and photoperiod of 14 h/10 h (light/dark). Adult zebrafish were fed with live brine shrimp three times a day.The night before the test, the male and female parent fish in a ratio of 1:1 were put into the spawning box, which was separated by a baffle and covered with black cloth for dark treatment. The fertilized embryos were collected and washed twice in reconstituted water the next morning. The normal and healthy embryos within 2 h postfertilization (hpf) were selected for subsequent tests.3COO)2, and 0.25, 0.5, 1, and 2, 4 mg/L for Kocide® 3000.Acute toxicity testing of embryos was carried out according to the OECD guideline 236  with modA total of 15 healthy embryos within 2 hpf were selected and placed in 40 mL test solutions with different concentrations. The reconstituted water was used as control. Three replicates were set for each test concentration and control. Test embryos were raised in an incubator in conditions of 26 ± 1 °C and photoperiod of 14 h/10 h (light/dark) for 96 h. The dead embryos were recorded and removed every 24 h during test periods. All procedures in this test complied with Chinese legislation and were approved by the Independent Animal Ethics Committee of China Agricultural University.3COO)2 or Kocide® 3000. A total of 15 healthy embryos were selected and immersed in 50 mL test solution with different concentrations. The reconstituted water was used as control. Test embryos were raised in an incubator in conditions of 26 ± 1 °C and photoperiod of 14 h/10 h (light/dark) for 9 days. The parameters of mortality and hatching rate, autonomous movement, heartbeat, locomotor behavior, body length and deformity were observed and recorded at different times during the test periods. The methods for locomotor behavior are described in The experiment was conducted according to the OECD guideline 212  with modn = 24) for each concentration.At 7 days postfertilization (dpf), the larvae without morphological abnormalities were selected and transferred into 24-well plates with one larva in 2 mL solution per well. At 8 dpf, all larvae were removed from incubators and acclimated at room temperature for 2 h. Then, the movement behavior of individual larvae was continuously recorded for 10 min in dark conditions using a DanioVision system  after acclimation in an observation chamber for 10 min. The total distance traveled, average velocity, absolute sinuosity, absolute turn angle, and absolute angular velocity were analyzed using EthoVision XT15 software . Tests were carried out in triplicate .® 3000 particle is irregular, and the average particle size cannot be determined accurately  of PEG@Cu NCs and Cu(CH3COO)2 for embryos was 0.166 mg/L and 0.276 mg/L, respectively 2 to embryos was 0.077, 0.174 and 0.088 mg/L, respectively (3COO)2 displayed higher embryo (or larvae) toxicity to zebrafish in early-life stages than Kocide® 3000.The copper fungicides exhibited different toxicity to embryos (or larvae). The 9 dpf-LCectively . The PEG® 3000 and Cu(CH3COO)2 with concentration of 0.0125 mg/L. At a concentration of 0.05 mg/L, the embryo mortality treated with PEG@Cu NCs and Cu(CH3COO)2 was significantly higher than Kocide® 3000. However, almost all embryos died after exposure to PEG@Cu NCs, Kocide® 3000 and Cu(CH3COO)2 at concentration of 3.2 mg/L.The mortality of embryos increased with the enhancement of copper concentration A. The em50 (effective concentration of 50% embryos hatching) of PEG@Cu NCs, Kocide® 3000 and Cu(CH3COO)2 to embryos was 0.079, 0.21 and 0.092 mg/L, respectively 2 at 0.0125 mg/L. At concentrations of 0.05 and 0.2 mg/L, the hatching rate of embryos between the PEG@Cu NCs and Cu(CH3COO)2 had no difference, but the hatching rate of embryos exposed to PEG@Cu NCs was significantly lower than that of Kocide® 3000. The hatching rate of embryos treated with PEG@Cu NCs was obviously lower than Kocide® 3000, but significantly higher than Cu(CH3COO)2 at concentration of 0.8 mg/L. When the concentration of PEG@Cu NCs, Kocide® 3000 or Cu(CH3COO)2 increased to 3.2 mg/L, almost no embryos could hatch.For each copper fungicide, the hatching rate of embryos decreased with enhancement in copper concentration B. The ha® 3000 (0.0125–0.8 mg/L) and Cu(CH3COO)2 (0.05–0.2 mg/L) obviously decreased the spontaneous movements of embryos. Almost all the embryos died after exposure to 3.2 mg/L Kocide® 3000 or Cu(CH3COO)2 at 0.8 mg/L and 3.2 mg/L.The three copper fungicides exhibited different effects on the autonomous movements of embryos . The spo® 3000 or Cu(CH3COO)2 with different concentrations gradually returned with the extension of exposure time within 48–96 h. The heartbeat reduced about 1.5–12.6%, 19.4–21.6% and 25.3–29.3% after exposure to PEG@Cu NCs (0.0125–3.2 mg/L), Kocide® 3000 (0.0125–0.8 mg/L) and Cu(CH3COO)2 (0.0125–0.2 mg/L) for 48 h, respectively (The copper fungicides exhibited different effects on the heartbeat of zebrafish in early-life stages with change in exposure time and concentrations A–C. In gectively A.® 3000 or Cu(CH3COO)2 recovered gradually, and the number of heartbeats was about 93.9%, 91.6% and 95.6% of the control, respectively (3COO)2 (0.0125–0.05 mg/L) returned to normal. However, the heartbeat after treatment with 0.2 mg/L Cu(CH3COO)2 reduced greatly. In contrast, heartbeat after treatment with Kocide® 3000 (0.0125–0.8 mg/L) was still inhibited, the number of heartbeats was about 87.0–88.5% of control  at 0.0125–0.05 mg/L, while it returned to normal at 0.2 mg/L and 0.8 mg/L. In contrast, Cu(CH3COO)2 stimulated growth (2.3–3.4%) at 0.0125 mg/L and 0.05 mg/L, and the body length of zebrafish larvae returned to normal after exposure to 0.2 mg/L Cu(CH3COO)2.Collectively, the copper fungicides at low concentrations displayed obvious promotion effects on the growth of zebrafish larvae . The bod® 3000 or Cu(CH3COO)2 compared with that of control larvae. However, the total movement distance and average velocity of zebrafish larvae reduced significantly when the exposure concentration increased to 0.05 mg/L or above 2. However, the absolute turn angle of zebrafish larvae decreased obviously when the exposure concentration increased to 0.05 mg/L or above (except 0.05 mg/L Kocide® 3000). When treated with PEG@Cu NCs, Kocide® 3000 or Cu(CH3COO)2 at 0.05 mg/L and 0.2 mg/L, the absolute turn angle decreased about 39.0–52.8%, 8.5–20.0%, and 22.0–35.3%, respectively 2. The absolute sinuosity and absolute angular velocity of zebrafish larvae decreased significantly after exposure to PEG@Cu NCs or Cu(CH3COO)2 at concentrations of 0.05–0.2 mg/L. The decrease was 42.0–61.2% and 38.9–52.8% for PEG@Cu NCs, 22.2–35.1% and 21.9–35.3% for Cu(CH3COO)2. In contrast, Kocide® 3000 reduced the absolute sinuosity and absolute angular velocity of zebrafish larvae only at 0.2 mg/L and 0.8 mg/L 2, which are copper-based fungicides, on the development and behavior of zebrafish in early-life stages were evaluated.The early-life stages of zebrafish mainly included embryos and larvae, which were widely used for the studies on aquatic toxicity as experimental models due to sensitivity to environment pollutants, including pesticides. In this study, the adverse effects of PEG@Cu NCs, KocideThe mortality, hatching rate, autonomous movement, heartbeat, and body length of zebrafish in early-life stages were used for evaluating the development toxicity of three copper fungicides in this study.® 3000 and Cu(CH3COO)2 exhibited obvious adverse effects on the hatching rate of embryos. The hatching rate of embryos decreased significantly as the concentration of the copper fungicides increased to 0.05 mg/L, and almost all embryos died at 3.2 mg/L (2+ (CuSO4·H2O) for 72 h [2 nanopesticide (CNPE) (Kocide 3000) at 4 mg/L or higher concentration [3COO)2 and higher toxicity than Kocide® 3000 at concentrations of 0.05 mg/L and 0.2 mg/L according to the hatching rate of embryos in this study. This may be caused by the characteristics of copper nanoparticles. The diameter of the chorionic membrane is approximately 0.5–0.7 µm, so copper nanoparticles are more likely to enter the embryonic chorionic membrane [2+ delay or impaired hatching of fish embryos occurred after inhibiting hatching enzyme activity, inducing reactive oxygen species (ROX) and downregulating wingless-type MMTV integration site family (Wnt) signaling [Hatching is very important in the development of zebrafish embryos and is considered a critical point for assessing the effects of toxic substances on fish in early stages . In this3.2 mg/L . Similarfor 72 h . A decrentration . The mecntration ,28. In amembrane . Nanoparmembrane . In addiignaling ,28,32.® 3000 (0.0125–0.8 mg/L) and Cu(CH3COO)2 (0.05–0.2 mg/L) obviously decreased the spontaneous movements of embryos. This demonstrated that Kocide® 3000 and Cu(CH3COO)2 had neurotoxicity to the embryo. However, PEG@Cu NCs at concentrations of 0.0125–3.2 mg/L had no obvious effects on spontaneous movements compared with that of control. Other studies also confirmed that oxine copper and Kocide® 3000 nanopesticide reduced the spontaneous movement of embryos greatly [The frequency of embryonic voluntary movements is associated with neurodevelopment and is one of the important biomarkers in neurodevelopmental toxicity studies ,34. The  greatly ,34. This greatly ,36. Henc3COO)2 only at 0.0125 and 0.05 mg/L. However, the heartbeat could not recover to normal after exposure to Kocide® 3000 under any concentration (0.0125–0.8 mg/L). The adverse effects of copper on the heartbeat were confirmed in various experiments. For example, CuSO4·5H2O at concentration of 327 μg/L and 464 μg/L significantly increased heartbeat of embryos at 28 hpf [2 nanopesticide (CNPE) (Kocide 3000) (at 4 mg/L or higher), CuO NPs (1 mg/L or higher), and oxine copper (10 μg/L) significantly decreased heartbeat of zebrafish embryos [The heart is one of the first organs formed during the embryonic development of zebrafish. Hence, heartbeat is an important parameter for evaluating cardiac function and toxicity of pesticides to zebrafish embryos ,38. The  embryos ,34,37® 3000, and Cu(CH3COO)2 obviously promoted the growth of larvae at 0.0125 and 0.05 mg/L, and did not exhibit inhibition to body length of larvae at 0.2 mg/L or 0.8 mg/L , PEG@Cu NCs and Cu(CH3COO)2 at 0.05 mg/L or higher concentrations (4·5H2O) at 125 μg/L reduced the mean velocity, total distance traveled and absolute turn angle of larvae. The 50 mg/L copper oxide nanoparticles (CuO NPs) reduced obviously the total movement distance, velocity, and angular velocity of larvae [The results indicated that the parameters of total distance traveled, average velocity, absolute sinuosity, absolute turn angle and absolute angular velocity obviously reduced when the zebrafish were exposed to Kocidetrations . This suf larvae . Oxine cf larvae . The disf larvae ,41,43.® 3000, PEG@Cu NCs and Cu(CH3COO)2) exhibited developmental and behavioral toxicity to embryos or zebrafish in early-life stages. In contrast, the adverse effects of PEG@Cu NCs on the growth and development of zebrafish in early-life stages seems to be less than Kocide® 3000 and Cu(CH3COO)2 according to the parameters of spontaneous movements of embryo, heartbeat and body length. However, the mechanisms underlying the developmental and behavioral toxicity are not clear and need to be thoroughly investigated in subsequent experiments.The results in this study indicated that copper fungicides (Kocide


Padina australis, a brown macroalga, has been demonstrated to have various pharmacological properties such as anti-neuroinflammatory activity. However, the underlying mechanism mediating the anti-neuroinflammatory potential of P. australis remains poorly understood. We explored the use of Malaysian P. australis in attenuating lipopolysaccharide (LPS)-stimulated neuroinflammation in BV2 microglial cells. Neuroinflammation and microglial activation are pathological features in central nervous system disorders. Excess levels of reactive oxygen species (ROS) and pro-inflammatory cytokines have been implicated in exacerbation of neuronal damage during chronic activation of microglial cells. P. australis were freeze-dried and subjected to ethanol extraction. The ethanol extract (PAEE) was evaluated for its protective effects against 1 µg/ml LPS-stimulated neuroinflammation in BV2 microglial cells. Fresh specimens of 2 (PGE2), intracellular reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6). However, the neuroinflammatory response was reversed by 0.5–2.0 mg/ml PAEE in a dose-dependent manner. Analysis of liquid chromatography-mass spectrometry (LC-MS) of PAEE subfractions revealed five compounds; methyl α-eleostearate, ethyl α-eleostearate, niacinamide, stearamide, and linoleic acid. LPS reduced the viability of BV2 microglia cells and increased the levels of nitric oxide (NO), prostaglandin EP. australis in combating continuous neuroinflammation. Our findings support the use of P. australis as a possible therapeutic for neuroinflammatory and neurodegenerative diseases.The protective effects of PAEE against LPS-stimulated neuroinflammation in BV2 microglial cells were found to be mediated by the suppression of excess levels of intracellular ROS and pro-inflammatory mediators and cytokines, denoting the protective role of  Emerging evidence suggests that neuroinflammation plays an important role in the onset and progression of neurodegenerative diseases. The neuroinflammatory response is mediated by cytokines, chemokines, reactive oxygen species (ROS), and secondary messengers produced by microglia, astrocytes, endothelial cells, peripherally derived T cells, macrophages, and dendritic cells, leading to physiological, biochemical, and psychological consequences . MicroglLipopolysaccharide (LPS) is an endotoxin derived from the outer membrane of Gram-negative bacteria. It is primarily recognized by the CD14/Toll-like receptor 4 (TLR-4) receptor complex, which is expressed on microglia and astrocytes. TLR-4 signaling mediates autoimmune responses and neuroinflammation in neurodegenerative diseases . ActivatPadina australis Hauck 1887  on the regulation of pro-inflammatory mediators  and pro-inflammatory cytokines  in LPS-stimulated BV2 microglial cells. However, the mechanism of the anti-neuroinflammatory activity of Harvesting of P. australis and preparation of the ethanol extractP. australis were harvested from Cape Rachado located in Negeri Sembilan, West Coast of Peninsular Malaysia. Morphological identification was conducted using standard botanical approaches , dissolved in Minimum Essential Medium Eagle (MEM) , and filtered-sterilized through a 0.2 µm-rated nylon membrane filter   served as a positive control  is an immortalized cell line that demonstrates the functional and morphological characteristics of microglia. BV2 microglial cells were maintained in Minimum Essential Medium Eagle (MEM)  supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin-streptomycin at 37±2 °C in a 5% CO control .3--2,5-diphenyltetrazolium bromide (MTT) cell viability assay 4 cells per well and incubated for 24 hr at 37±2 °C in a 5% CO2-humidified incubator. The supernatant was discarded and replaced with fresh medium containing different concentrations of selected PAEE (0.25–8.0 mg/ml)  and incubated for 24 hr (designated as Experiment 2.3B).The BV2 cells were plated into a 96-well plate at a density of 6.25 × 100 mg/ml)  and incumicroplate reader  and expressed as a percentage of viable cells relative to the negative control.The protective effects of PAEE against LPS-stimulated cytotoxicity in BV2 cells were studied by pre-treating the cells with a selected range of concentrations from Experiment 2.3A (0.25–2.0 mg/ml) for 2 hr followed by 1 µg/ml LPS for 24 hr (designated as Experiment 2.3C). Ten microliter of 0.5 mg/ml MTT  was added to each well and incubated for 4 hr. Supernatant was discarded, and insoluble formazan was dissolved in 100 µl dimethyl sulfoxide (DMSO). The MTT reduction was determined spectrophotometrically at 570 nm with a reference wavelength of 630 nm in a UV-Vis spectrophotometer The most optimum concentration of PAEE determined based on Experiment 2.3C (0.25–2.0 mg/ml) and 1 µg/ml LPS were selected for subsequent assays of neuroinflammation.Measurement of nitric oxide productionet al. . The NO production was determined spectrophotometrically at 550 nm in a UV-Vis spectrophotometer microplate reader  and expressed as micromolar (µM) relative to the negative control.The production of NO was measured according to the method described by Oh et al.  with minMeasurement of prostaglandin E2 production× 104 cells per well and incubated for 24 hr at 37±2 °C in a 5% CO2-humidified incubator. The supernatant was discarded and pre-treated with fresh medium containing 0.5, 1.0, or 2.0 mg/ml PAEE for 2 hr before exposure to 1 µg/ml LPS for 24 hr. The supernatant was then subjected to PGE2 measurement by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol of Parameter™ Prostaglandin E2 Immunoassay . The PGE2 production was determined spectrophotometrically at 450 nm with a reference wavelength of 570 nm in a UV-Vis spectrophotometer microplate reader  and expressed as picogram per milliliter (pg/ml) relative to the negative control.The BV2 microglial cells were plated into a 96-well plate at a density of 6.25 Intracellular reactive oxygen species assayet al.  , further incubated for 30 min at 37±2 °C, and washed twice with phosphate buffer saline (PBS). The fluorescence intensity was determined spectrophotometrically at an excitation wavelength of 485 nm and emission wavelength of 535 nm in a UV-Vis spectrophotometer microplate reader  and expressed as a percentage relative to the negative control.The intracellular ROS level was determined according to protocols described by Subermaniam et al.  with minWestern blot analysiset al. (29) with minor modifications. The BV2 microglial cells were plated into a 96-well plate at a density of 6.25 × 104 cells per well and incubated for 24 hr at 37±2 °C in a 5% CO2-humidified incubator. The supernatant was discarded and pre-treated with fresh medium containing 0.5, 1.0, or 2.0 mg/ml PAEE for 2 hr before exposure to 1 µg/ml LPS for 24 hr. The lysate was diluted in lysis buffer containing protease inhibitor cocktail  and 1 mM phenylmethylsulfonyl fluoride . An equal amount of proteins (20 μg) was separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes . The membranes were blocked with 5% skim milk in TBST  buffer for 1 hr at room temperature and incubated with primary antibodies  in blocking buffer at 4 °C overnight in a humidity chamber.Western blot analysis was carried out according to the method described by Kim β-actin .After washing five times in TBST, the membranes were incubated with a secondary antibody (horseradish peroxidase (HRP)-conjugated) for 1 hr at room temperature. Proliferating cell nuclear antigen  and β-actin  were employed as the loading controls. The HRP signal was detected by chemiluminescence  and visualized using the LAS-3000 LuminoImage analyzer . Resultant bands were quantified with Image J densitometry  and normalized to Measurement of pro-inflammatory cytokine secretion5 cells per well and incubated for 24 hr at 37±2 °C in a 5% CO2-humidified incubator. The supernatant was discarded and pre-treated with fresh medium containing 0.5, 1.0, or 2.0 mg/ml PAEE for 2 hr before exposure to 1 µg/ml LPS for 24 hr. The supernatant was collected, and the production of TNF-α and IL-6 was measured using the Mouse TNF-α Quantikine ELISA Kit and Mouse IL-6 Quantikine ELISA Kit . The concentration of TNF-α or IL-6 was determined by measuring the absorbance at 450 nm and 570 nm as reference wavelengths in a UV-Vis spectrophotometer microplate reader and expressed as picogram per milliliter (pg/ml) relative to the negative control.The BV2 microglial cells were plated into a 96-well plate at a density of 6.25 × 10Isolation and identification of compounds from PAEEpolarity of the eluting solvent was gradually increased starting from chloroform:hexanes (1:1) to chloroform:methanol (99:1-85:15). Eluted fractions were monitored via thin-layer chromatography (TLC) and appropriate fractions were combined into 11 main fractions (A-K). The selected combined fractions  were further fractionated using preparative radial chromatography (PRC)  into five, nine, and seven subfractions, respectively. The solvent systems used in the PRC included chloroform:hexanes (1-2:1-3), diethyl ether:hexanes (3:1), and dichloromethane. The eluted subfractions  were concentrated and further analyzed by liquid chromatography-mass spectrometry (LC-MS). P. australis. PAEE was subjected to flash column chromatography . The The LC-MS analysis was performed on Agilent 1290 infinity liquid chromatograph , coupled to the Agilent 6520 Accurate-Mass Q-TOF mass spectrometer with dual electrospray ionization (ESI) source. A reverse-phase high-performance liquid chromatography (HPLC) column (Agilent Eclipse XBD-C18) with a length of 150 mm and an internal diameter of 2.1 mm in a narrow-bore scale format along with a particle size of 3.5 µm was used (30). Automated mass spectrometry - mass spectrometry (aMSMS) mode was employed. Data were processed using Agilent MassHunter Qualitative Analysis software (Version B.07.00) and the Molecular Feature Extraction (MFE) small molecule algorithm. Compound identification was performed using the MassHunter METLIN Metabolite Personal Compound Database and Library (PCDL) .Statistical analysispost hoc multiple comparison tests were used for equal variances assumed, whereas the Welch test followed by Games-Howell multiple comparison test were used for equal variances not assumed. For non-normally distributed data, Kruskal-Wallis one-way ANOVA (k samples) followed by pairwise multiple comparison test were used. A P-value of <0.05 was considered statistically significant.Data analysis was performed using the Statistical Package for Social Sciences . Data were expressed as mean ± standard deviation (SD) from three replicates. The distribution of variables  was determined using the Shapiro-Wilk test whereas Levene’s test was employed to assess the homogeneity of variance. For normally distributed data, one-way ANOVA followed by Bonferroni Effect of PAEE on the viability of BV2 microglial cells P<0.05). Considering the lower concentrations of 0.25 to 2.0 mg/ml PAEE showed no significant difference in viability compared with the negative control (P>0.05), the concentration range was selected for subsequent assays of viability and NO production.The effect of PAEE on the viability of BV2 microglial cells was evaluated preceding the investigation of the anti-neuroinflammatory activities of the extract to eliminate the possible cytotoxic and proliferative effects. As shown in Effect of LPS on the viability of BV2 microglial cellsP<0.05). Considering exposure to 1 µg/ml LPS caused the lowest percentage of viability at 60.78 ± 2.48, the concentration was selected for the subsequent assays of neuroinflammation.The vulnerability of BV2 microglial cells to LPS-stimulated toxicity was investigated through exposure to different concentrations of LPS in the range of 0.125 to 8.0 µg/ml. Effect of PAEE on the viability of BV2 microglial cells treated with LPS P<0.05). However, pretreatment with PAEE in the range of 0.25 to 2.0 mg/ml and L-NAME significantly increased the viability to 107.88 ± 4.98% (0.25 mg/ml), 111.60 ± 1.21% (0.5 mg/ml), 100.18 ± 5.75% (1.0 mg/ml), 91.47 ± 8.33% (2.0 mg/ml), and 97.75 ± 8.51% (L-NAME) or 1.2- to 1.5-fold higher compared with LPS (P<0.05). We did not observe cytotoxicity effect for any tested concentrations (0.25 to 2.0 mg/ml) (P>0.05). Pre-treatment with PAEE was observed to be comparable with the effect of L-NAME in increasing viability.The protective effect of PAEE against 1 μg/ml LPS-stimulated cytotoxicity was investigated by pretreating BV2 microglial cells with 0.25 to 2.0 mg/ml PAEE for 2 hr followed by 1 μg/ml LPS for 24 hr. As shown in Effect of PAEE on NO production in BV2 microglial cells treated with LPS P<0.05). However, pretreatment with PAEE in the range of 0.25 to 2.0 mg/ml and L-NAME significantly reduced NO production to 14.18 ± 0.94 µM (0.25 mg/ml), 7.60 ± 1.25 µM (0.5 mg/ml), 2.62 ± 0.84 µM (1.0 mg/ml), 0.23 ± 0.60 µM (2.0 mg/ml), and 17.40 ± 0.20 µM (L-NAME) or 1.6- to 121.9-fold lower compared with LPS (P<0.05).  We evaluated NO production in culture supernatants by detecting the level of nitrite using a Griess reaction.  As shown in P<0.05). Thus, higher concentrations of PAEE (0.5 to 2.0 mg/ml) were selected for subsequent assays of neuroinflammation. Interestingly, the tested concentrations of PAEE exhibited 1.2- to 75.7-fold higher inhibitory activity compared with L-NAME (P<0.05).Although 0.25 mg/ml PAEE suppressed NO production to 14.18 ± 0.94 µM or 2.0-fold lower compared with LPS, it exhibited a weaker inhibitory effect against NO production compared with 0.5 to 2.0 mg/ml PAEE . However, pretreatment with 0.5 and 1.0 mg/ml PAEE significantly suppressed the PGE2 production to 527.60 ± 154.30 and 509.67 ± 66.81 pg/ml or 1.6- and 1.7-fold lower compared with LPS (P<0.05), respectively. On the other hand, 2.0 mg/ml PAEE failed to inhibit PGE2 production and actually increased PGE2 levels to 1120.67 ± 61.20 pg/ml or 1.3-fold higher compared with LPS (P<0.05). COX-2 catalyzes the conversion of free arachidonic acid to prostaglandins in LPS-stimulated neuroinflammation. As shown in Effect of PAEE on intracellular ROS generation in BV2 microglial cells treated with LPSP<0.05). However, pretreatment with 0.5, 1.0, and 2.0 mg/ml PAEE significantly reduced ROS generation to 744.12 ± 47.58, 636.76 ± 19.89, and 557.35 ± 131.34% or 3.8- to 5.1-fold lower compared with LPS (P<0.05).ROS act as second messengers to amplify the inflammatory function of microglia, activate the anti-oxidant response elements, and restore redox homeostasis. As shown in Effect of PAEE on the expression of iNOS and COX-2 in BV2 microglial cells treated with LPSP<0.05) (P<0.05), respectively. Treatment with 1 µg/ml LPS also significantly increased COX-2 expression from 0.04 ± 0.01 to 0.98 ± 0.12 or 24.5-fold higher compared with the negative control (P<0.05) (P<0.05).P<0.05) . However(P<0.05) . HoweverEffect of PAEE on the secretion of TNF-α and IL-6 in BV2 microglial cells treated with LPSP<0.05). However, pretreatment with 0.5, 1.0, and 2.0 mg/ml PAEE suppressed the TNF-α secretion to 7679.76 ± 217.31, 6499.32 ± 183.12, and 2035.67 ± 79.34 pg/ml, or 7.80, 21.97 and 75.56% lower compared with LPS (P<0.05), respectively. As shown in P<0.05). However, pretreatment with 0.5, 1.0, and 2.0 mg/ml PAEE markedly suppressed the IL-6 secretion to 4755.91 ± 151.69, 1865.96 ± 108.54, and -885.57 ± 158.10 pg/ml, or 84.58, 93.95 and 102.87% lower compared with LPS (P<0.05), respectively.Activated BV2 microglial cells showed induced secretion of pro-inflammatory cytokines, including TNF-α and IL-6. As shown in Identification of the major compounds in PAEE fractions m/z) and calculated (MFG) mass] of ± 5 ppm. These compounds included two esters, and an amide (stearamide) (31) from subfraction C-1, a polyunsaturated fatty acid (PUFA) (linoleic acid) from subfraction F-9, and an amide  (32) from subfraction K-2 (Liquid chromatography-mass spectrometry (LC-MS/MS using aMSMS mode) analysis of subfractions C-1, F-9, and K-2 detected 93, 73, and 31 corresponding peaks in the positive ion mass spectra. A total of five major compounds were identified by searching the Metlin database using a molecular formula generator (MFG) mass score above 90% and a mass deviation [difference between observed , ethyl α-eleostearate (C20H34O2), and stearamide (C18H37NO). The ESIMS of subfraction F-9 shows molecular ion peaks at m/z 281.246, establishing the molecular formula of linoleic acid (C18H32O2). The ESIMS of subfraction K-2 shows molecular ion peaks at m/z 123.0531, establishing the molecular formula of niacinamide (C6H6N2O) (The ESIMS of subfraction C-1 shows molecular ion peaks at C6H6N2O) . The chrP. australis has been demonstrated to attenuate oxidative damage induced by high-dose corticosterone in PC-12 cells mimicking the effects of depression due to the presence of phytochemical compounds  also inhibited LPS-stimulated NO and PGE2 production in BV2 microglial cells. Moreover, we found that the PAEE treatment demonstrated a higher percentage of NO reduction compared with L-NAME, indicating PAEE is a potent inhibitor of NOS. A study by Gany et al.  was able to inhibit NO production to 73.63 ± 3.81% and 75.67 ± 0.21%, respectively, which was similar to our finding that 0.5 mg/ml PAEE decreased NO production to 72.90%. Moreover, the P. australis methanol extract was found to inhibit PGE2 production by 30%, which was 7% lower than that of 0.5 mg/ml PAEE at 37%.  We observed that 1 µg/ml LPS profoundly increased the production of NO and PGEfindings , 35-38. h et al.  showed ty et al.  observed Neuroinflammation and oxidative damage are undoubtedly hallmarks of neurodegeneration characterized by the generation and accumulation of excessive free radicals, including ROS and RNS, therefore contributing to disease progression , 40. Impresults are very similar to those reported by Kim et al.  attenuated 94.45 ± 1.92% and 92.07 ± 1.99% of TNF-α and IL-6, respectively. The discrepancy in these findings may be due to genetic variations or phenolic composition of P. australis from different geographical locations, extraction procedure, particle size, storage conditions and time, and the presence of interfering substances in the extracts (M. myagroides (S. serratifolium (28) have also been observed to decrease the protein level of TNF-F-α, IL-1β, and IL-6 in BV2 microglial cells upon exposure to LPS. We also observed that 1 µg/ml LPS increased the secretion of TNF-α and IL-6, similar to the findings of other studies , 45, 46.y et al.  demonstrextracts . Likewisagroides  and S. set al. (48) reported that ethyl ɑ-eleostearate can reduce viability and induce apoptosis in human colon cancer CaCo-2 cells.  The LC-MS data revealed five compounds including esters, polyunsaturated fatty acids, and amides. Among these compounds, ethyl ɑ-eleostearate, stearamide, linoleic acid, and niacinamide have been reported to possess therapeutic properties. Ethyl ɑ-eleostearate or 9Z,11E,13E-octadecatrienoic acid ethyl ester is a conjugated PUFA derivative with anticancer properties. Yasui Channa pleurophthalma fin waste was found to possess anti-inflammation, antipruritic, antifungal, and antimicrobial properties using the Prediction of Activity Spectra for Substances (PASS) online tool  polyunsaturated fatty acid (PUFA) that can be elongated and desaturated to form arachidonic acid, the precursor of various inflammatory cytokines  . Collectcroalgae . Althougammation , both ω-ammation . In anotammation , pretreaal cells . Moreoveal cells  observedP. australis were attributed to the presence of linoleic acid and niacinamide.  In addition, niacinamide has been observed to inhibit poly (ADP-ribose) polymerase 1 (PARP-1) that facilitates diverse inflammatory responses orchestrated by pro-inflammatory cytokines, chemokines, and adhesion molecules . The abiP. australis and its compounds possess protective effects against LPS-stimulated neuroinflammation in BV2 microglial cells. The proposed anti-neuroinflammatory mechanism of PAEE is presented in 2, respectively. Upon exposure to 1 μg/ml LPS, the anti-oxidant substances , and pro-inflammatory cytokines (TNF-α and IL-6). Taken together, our findings suggest that P. australis and its potential compounds possessed protective effects against LPS-stimulated neuroinflammation in BV2 microglial cells. Taken together, our findings suggest that bstances  and majoP. australis may lead to the development of novel therapeutic strategies for neurodegenerative diseases. Our current study demonstrated PAEE has protective effects against LPS-stimulated neuroinflammation in BV2 microglial cells by suppressing the excessive generation of intracellular ROS, and pro-inflammatory mediators and cytokines. These effects can be attributed to major compounds such as linoleic acid and niacinamide. The anti-neuroinflammatory and anti-oxidant properties of KS and KHW conceived the study; KS and SYL prepared the original draft; YYY, SHL, WSY, LWL, and KHW reviewed and edited the manuscript; KS, SYL, and LWL helped with visualization and illustration; YYY, SHL, and KHW supervised the study. All authors have agreed to the contents and approved the final version for publication.None.


Paeonia lactiflora Pall.) has been widely used as a landscaping and economical plant around the world. However, the lack of an efficient and stable regeneration system in P. lactiflora restricts its rapid propagation and large-scale production. By testing the key factors affecting callus formation, proliferation, adventitious bud induction and rooting, here, we developed an in vitro system for callus induction and regeneration in P. lactiflora. Our results show that callus formation was affected by explant types, culture environment, basal medium and plant growth regulators. Using cotyledons as explants, we established good conditions for P. lactiflora callus induction and callus proliferation. We effectively obtained adventitious buds differentiated from callus in Murashige and Skoog (MS) medium containing kinetin (KT) and thidiazuron (TDZ). Adventitious bud growth can be further promoted by adding gibberellin 3 (GA3), 1-naphthaleneacetic acid (NAA) and 6-benzyleaminopurine (6-BA) into the MS medium. A high percentage of rooting can be achieved by adding indolebutyric acid (IBA) and activated carbon (AC) to ½ MS medium. Overall, our system promotes callus induction and adventitious bud regeneration for P. lactiflora through improved culture conditions and plant growth regulators in the culture media, and lays a foundation for subsequent genetic engineering research.Owing to its high ornamental, medicinal and horticultural values, herbaceous peony ( Paeonia lactiflora Pall.) is documented as the oldest ornamental perennial herbaceous flower in the family Paeoniaceae. Due to its graceful appearance, rich color, broad application value, and various application forms, P. lactiflora has fascinated people over the years, and it has also enjoyed the reputation of “Mayflower God” [P. lactiflora roots have important medicinal values, with their therapeutical effects on cardiovascular and immune system diseases [Herbaceous peony . The callus induction rate ranged between 83.89% and 98.89%  + 1.0 mg·L−1 TDZ + 0.5 g·L−1 Casein hydrolyzed (CH) + 1.0 mg·L−1 PVP when the proliferative callus obtained from cotyledon was used as the material. Our experiments show that the induction rate of calluses was positively correlated with the concentration of PIC. For example, the induction rate of calluses at 2.0 mg·L−1 PIC (46.67%) was significantly higher than that at 0 mg·L−1 PIC (20.00%)   . In 0 mgsh-green A,B. The browning C,D. Howe(46.67%) E,F. The −1 TDZ + 0.5 mg·L−1 2,4-D + 0.5 mg·L−1 NAA was selected as the material. We tested the impacts of a combination of two auxins  with 0.5 mg·L−1 TDZ. Overall, the callus in the medium with NAA, 2,4-D and TDZ was vibrantly green with full and loose tissue after 20 days of growth . A greater browning degree was observed for calluses cultured with high concentrations of BA. Little or moderate browning was observed when TDZ or KT was applied at the tested concentrations  and the highest stem height (2.54 cm), as well as the best leaf expansion rate (62.9%) (One of the major difficulties faced by  (62.9%) . The effP. lactiflora flora in a medium composed of ½ MS + 3.0 g·L−1 AC. No rooting (0%) was obtained when no auxins were added to the medium. Our test indicate that after 10 days of shoot inoculation, the adventitious roots began to elongate, but the roots were weak. After 20 days, the roots grew thicker and some explants had multiple roots. IAA and IBA had different effects on the induction of adventitious roots. With the same concentration, the ability of IBA to induce roots was stronger than that of IAA. When the concentration of IBA was 1.0 mg·L−1, the induction rate of roots was the highest (38.89%)  . The adv(38.89%) A. When 0re short B. When tP. lactiflora is related to the explant type, culture conditions, basic media and plant growth regulators. There are great differences in the states of calluses formed from different explants [Rosa hybrida, lotus and Datura innoxia have high callus induction rates only in dark environments [Rosa hybrida and Clivia miniata, which also reported high callus quality under dark conditions [Moso Bamboo, Toona ciliata and Ethiopian mustard [Abies koreana [−1 PIC into the medium induces more callus, with light yellow particles on the surface, which is similar to the results for peony [The success of callus induction in explants . Studiesexplants ,32. Coinronments ,34,35. Onditions ,36. We d mustard ,38. Furt koreana . However koreana . Many st koreana ,42,43,44or peony . HoweverP. lactiflora is primarily influenced by plant growth regulators. Our study has revealed that the combination of 1.0 mg·L−1 NAA, 1.0 mg·L−1 2,4-D and 0.5 mg·L−1 TDZ increased the callus proliferation coefficient of P. lactiflora to 3.13, and reduced the browning rate to 10.33%. Previous research has also demonstrated that the combination of NAA, 2,4-D and TDZ is beneficial to the callus proliferation of peony and Dendrocalamus luodianense [Clivia miniata [The callus proliferation of dianense ,47. Inte miniata . In our P. lactiflora is a challenging process primarily influenced by plant growth regulators. Both cytokinin and auxin can promote the formation and proliferation of shoot buds [Pogostemon cablin, Cotoneaster wilsonii and Allium hirtifolium [Cotoneaster wilsonii, the effect of TDZ on adventitious bud differentiation was better than that of 6-BA [−1, the shoot buds differentiation rate of P. lactiflora was the best. When the concentration of TDZ increased to 1.0 mg·L−1, the differentiation of adventitious buds of P. lactiflora was very difficult, although it was found that a small amount of callus of P. lactiflora could differentiate into adventitious buds when 1.0 mg·L−1 TDZ was added [P. lactiflora is not only related to the combination and concentration of plant growth regulators, but also may be related to the number of callus subcultures. Multiple subculturing results in a reduction in meristematic activity, impeding the differentiation of calluses into organ primordia.The formation of adventitious shoot buds from callus of oot buds ,50,51,52tifolium ,54,55. Ias added ,25. The P. lactiflora, which is consistent with the findings of the study of Zhao et al. [P. lactiflora [P. lactiflora with IBA alone was the highest. Overall, IBA treatment had the best rooting effect for P. lactiflora, which is similar to the results derived for Petunia hybrida and Oryza sativa [IBA, NAA and IAA are commonly used as plant growth regulators for rooting in plant tissue culture systems ,57,58. Oo et al. . Neverthctiflora . The rooctiflora . Interesa sativa ,62.P. lactiflora regeneration system has long been challenging. Although we have made some progress on the regeneration of P. lactiflora, the rates of adventitious bud differentiation and rooting in P. lactiflora are still relatively low. Thus, the efficient establishment of a P. lactiflora regeneration system still has a long way to go, and further exploration is necessary to effectively achieve the regeneration of P. lactiflora via the callus pathway.The establishment of a P. lactiflora hybrid seeds were soaked in water for 48 h, and we then peeled off the seed coats. Experiments of embryo germination were performed according to the protocol of Duan et al. [w/v) HgCl2 for 5–6 min followed by washing 4 times with sterile distilled water. Mature zygotic embryos were cut from seeds and inoculated in MS medium supplemented with 0.5 mg·L−1 GA3 and 1.0 mg·L−1 6-BA. After 20 days of culturing, the produced hypocotyls and cotyledons were used as explants for subsequent experiments. All culture media were added with 30 g/L sucrose and 6 g/L agar with pH of 5.8 and autoclaved at 121 °C for 20 min. Except for those subjected to special instructions, all cultures were maintained at 25 ± 1 °C, in a 16/8 h (light/dark) photoperiod with 2000–3000 lx light intensity. All components of the culture media used in this article were purchased from Beijing Solarbio Science & Technology Co., LTD . All plant growth regulators (PGRs) were purchased from Shanghai Yien Chemical Technology Co., LTD .The intervarietal hybrid seeds “Fen Yunu” ♀ × “Fen Yulou” ♂ were used as materials, and were collected via pollination from the germplasm resources nursery in Shenyang Agricultural University, Liaoning province, China. The seeds that were collected 100 days after pollination in the current year were shade-dried, and stored in a refrigerator at 4 °C. n et al.  with som−1 BA combined with 2.0 mg·L−1 PIC, 1.0 mg·L−1 BA combined with 2.0 mg·L−1 2,4-D or 1.0 mg·L−1 BA combined with 1.0 mg·L−1 NAA, respectively, to induce calluses under dark conditions. After 45 days, the callus induction rates of the three explants on different media were observed.The zygotic embryos, cotyledons, and hypocotyls were cultured in MS medium supplemented with 1.0 mg·L−1 TDZ, 0.5 mg·L−1 2,4-D and NAA , respectively. After 30 days, the callus induction rate and browning rate were calculated, and the growth status of the callus in each medium was observed.Experiments on callus induction were performed according to the protocol of Duan et al.  with som−1 TDZ, 0.5 mg·L−1 2,4-D and 0.5 mg·L−1 NAA and cultured in the dark for 10, 20 and 30 days, respectively, while normal light culturing was used as the control treatment (16 h light/8 h dark photoperiod with 2000–3000 lx light intensity). After dark culturing, they were transferred to light culturing conditions. After 30 days, the callus induction of cotyledons under different dark culturing times was observed.The cotyledons were transferred to MS medium supplemented with 0.5 mg·L−1 TDZ + 1.0 mg·L−1 2,4-D + 0.5 g·L−1 CH + 1.0 mg·L−1 PVP for 30 days to produce calluses, and then transferred to ½ MS medium supplemented with 1.0 mg·L−1 TDZ + 1.0 mg·L−1 NAA + 0.5 g·L−1 CH + 1.0 mg·L−1 PVP for callus proliferation. After 30 days, the obtained calluses were transferred to ½ MS (Ca2+) medium supplemented with 1.0 mg·L−1 TDZ + 0.5 g·L−1 CH + 1.0 mg·L−1 PVP + PIC , and the calluses were induced under dark conditions. After 30 days, the induction of calluses was observed.Cotyledons were cultured in ½ MS medium supplemented with 1.0 mg·L−1 TDZ + 0.5 mg·L−1 2,4-D + 0.5 mg·L−1 NAA were selected as the material. Experiments on callus proliferation were performed according to the protocol of Duan et al. [−1), 2,4-D  and TDZ (0.5 mg·L−1) for callus proliferation. Each treatment had 20 explants and 3 replicates. After 30 days, the callus proliferation coefficient and browning rate were measured.The calluses induced in MS + 0.5 mg·Ln et al.  with som−1 NAA + 1.0 mg·L−1 2,4-D + 0.5 mg·L−1 TDZ was used as the material. Experiments on adventitious bud induction were performed according to the protocol of Sun et al. [−1 NAA combined with TDZ , 0.2 mg·L−1 NAA combined with 6-BA  or 0.2 mg·L−1 NAA combined with KT  for shoot bud differentiation. The medium was supplemented with 1.0 mg·L−1 PVP to prevent browning. After 45 days, the shoot bud differentiation in each medium was observed.The callus proliferated in ½ MS + 1.0 mg·Ln et al.  with som−1 GA3 and tested at different concentrations and ratios of NAA and BA . Each treatment had 20 explants and 3 replicates. After 30 days, the stem pumping rate, leaf expansion rate and shoot height of adventitious buds were recorded.Experiments on adventitious bud growth were performed according to the protocol of Liu et al.  with som−1 AC combined with different concentrations of IAA  and IBA  for root induction. The rooting was observed after 45 days.The shoots with 2–4 leaves and which were 1.5–2.5 cm in height were selected as materials. The shoots cultured for 30 d were transferred to ½ MS medium supplemented with 3.0 g·Lp < 0.05. The significant data involved in this paper were calculated as follows: induction rate =  × 100%; proliferation coefficient = fresh proliferation callus weight/inoculated callus weight; browning rate =  × 100%; differentiation rate =  × 100%; stem pumping rate:  × 100%; leaf expansion rate:  × 100%; rooting rate =  × 100%.Except as noted, each treatment had 30 explants and 3 replicates. Statistical significance was calculated using SPSS software (Version 26.0) with the one-way analysis of variance (ANOVA) method, and significant difference was defined by Duncan’s multiple range test at P. lactiflora, and that resulted in indirect adventitious bud regeneration and their elongation to obtain shoots that were rooted, allowing us to obtain complete plants. The results of our system provide a new method for tissue culturing and the subsequent regeneration of P. lactiflora. However, the bud differentiation rate and rooting rate were low, which is a potential research avenue to be addressed in the future.In summary, our study established an efficient callus induction method by manipulating plant growth regulators. We developed a method that allows us to obtain and proliferate calluses from cotyledons of


This phase I trial evaluated pharmacokinetics/pharmacodynamics (PK/PD) characteristics, preliminary safety and efficacy of pemigatinib in Chinese patients with advanced, solid tumors. Patients with unresectable advanced or metastatic solid tumors bearing FGF/FGFR1-3 alterations received oral pemigatinib at 13.5 mg once daily (QD) on a 2-weeks-on/1-week-off schedule. The primary endpoint was PK/PD characteristics; secondary endpoints were safety and efficacy. Twelve patients were enrolled . PK data demonstrated pemigatinib (13.5 mg QD) was rapidly absorbed with a geometric mean elimination half-life of 11.3 h. The geometric mean values of maximum serum concentration and area under the plasma concentration–time curve from 0 to 24 h at steady state were 215.1 nmol/L and 2636.9 h·nmol/L, respectively. The mean clearance adjusted by bioavailability at steady state was low (11.8 L/h), and the apparent oral volume of distribution was moderate (170.5 L). The PD marker, serum phosphate level, increased on days 8 and 15 of cycle 1  and decreased to baseline post 1 week off. Three (25.0%) patients experienced grade ≥ 3 treatment-emergent adverse events. Partial response was confirmed in one patient with FGFR1-mutant esophageal carcinoma and one with FGFR2-mutant cholagiocarcinoma. Pemigatinib had similar PK/PD characteristics to Western population and demonstrated an acceptable safety profile and potential anti-cancer benefit in Chinese patients with FGF/FGFR1-3 altered, advanced, solid tumor. The online version contains supplementary material available at 10.1007/s10637-023-01396-x. The fibroblast growth factor (FGF) signaling pathway is associated with oncogenesis of human malignancy . A varieIn recent years, a great deal of effort has been devoted to develop precise anti-cancer therapeutics by selectively targeting FGFRs: such as FGFR1-4 inhibitors of futibatinib TAS-120) , derazan0 , derazThere is still a large unmet medical need for precision therapy in cancer patients in China, however, none of the selective-FGFR inhibitors were approved in the country when this study was initiated. Although pemigatinib has been deeply investigated in Western population and demonstrated clinical benefit in CCA and RRMLN and antitumor activity signals in several types of solid tumor including pancreatic cancer, gallbladder cancer, UC, etc. with acceptable safety , 17, 18,This phase I, open-label study was conducted to investigate pemigatinib in Chinese patients with FGF/FGFR1-3 altered, advanced, metastatic or recurrent, solid tumors. The primary objective was to assess the pharmacokinetics/pharmacodynamics (PK/PD) of pemigatinib; secondary objectives included safety and efficacy.The trial protocol was approved by the ethics committees of two participating hospitals. The study was conducted in accordance with the protocol, Good Clinical Practice Guidelines, Declaration of Helsinki, and local applicable regulatory requirements. Written informed consents were obtained before enrollment.This was a single-arm, phase I, open-label study conducted at two participating hospitals in China , which aimed to evaluate PK/PD characteristics, and preliminary safety and antitumor activity of pemigatinib in Chinese patients with FGF/FGFR1-3 altered, advanced solid tumors.Patients were ≥ 18 years old with unresectable advanced, recurrent or metastatic solid tumor bearing documented FGF/FGFR alterations, and had failed to prior therapy or no standard therapy available. Patients had at least one measurable lesion, an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, and an anticipated life expectancy of ≥ 12 weeks. Patients who previously received the selective FGFR inhibitor were excluded. Other key exclusion criteria included history of calcium and phosphate hemostasis disorder, and ophthalmologically confirmed corneal/retinal disease .Patients received oral pemigatinib at 13.5 mg once daily every 3 weeks on an intermittent 2-weeks-on/1-week-off dosing schedule. Tumor response was assessed by investigator according to Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 every 9 weeks after first dosing. If the patients were considered benefiting from the treatment by the investigator, pemigatinib could be continued until disease progression, unacceptable toxicity, initiation of other anti-tumor therapy, or for a maximum of 2-year treatment.max), time to Cmax (tmax), clearance, volume of distribution (Vd), elimination half-life (t1/2), etc.The primary endpoint was PK/PD characteristics of pemigatinib. For assessment of PK characteristics, plasma samples were collected from all the patients at before each pemigatinib administration on days 1, 2, 8, 14, 15, and 16 during cycle 1, and immediately after, and 0.5 h ± 2 min, 1 h ± 15 min, 2 h ± 15 min, 4 h ± 15 min, 6 h ± 30 min, and 8 h ± 30 min after each pemigatinib administration on days 1 and 14 during cycle 1. Plasma pemigatinib concentrations were measured using the validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) method. LC–MS/MS analysis for pemigatinib was carried out with a Sciex API 4000 mass spectrometer, coupled with an HPLC pump and an autosampler. PK parameters for evaluation included maximum serum concentration  by the central laboratory .The secondary endpoints were safety and efficacy including objective response rate (ORR), disease control rate (DCR), progression-free survival . Tumor was assessed per RECIST v1.1 by investigator at baseline, and every 9 weeks after first dosing. Safety was monitored up to 30 days after discontinuation of pemigatinib treatment. Adverse events (AEs) were graded per CTCAE v5.0. Treatment-emergent AEs  are reported. Sponsor-defined clinically notable TEAEs included hyperphosphatemia, hypophosphatemia, nail toxicities, and serous retinal detachment.All statistical analyses were conducted using SAS version 9.4 (or higher). The safety and efficacy were assessed in those patients who received at least 1 dose of pemigatinib. The PK characteristics were established from the PK analysis set (patients who had received at least one dose of pemigatinib and provided at least one post-baseline PK samples as per protocol).max, Tmax, clearance (CL), Vd, and t1/2. The apparent oral clearance at steady state (CLss/F) was computed as Dose/AUCss,0-τ, where F is the (unobserved) absolute bioavailability. It was included in the above relationship to indicate that the reported value was an apparent value based on extravascular administration. The accumulation ratio was computed as the ratio of AUCss,0–24 (steady-state area under the curve from hour 0 to 24) to AUCC1D1,0–24 .Standard non-compartmental pharmacokinetic methods with PKanalix2020R1  were applied to estimate PK parameters, including AUC, CThe Clopper-Pearson method was used to calculate the 95% confidence interval (CI) of ORR and DCR. The Kaplan–Meier method and the Brookmeyer and Crowley method were used to estimate PFS.From February 20, 2020 to July 2, 2020, 25 patients were screened; a total of 12 patients were enrolled and received treatment , including 4 (33.3%) patients with CRC, 3 (25%) gastric/gastroesophageal junction carcinoma, 2 (16.7%) esophageal carcinoma, 2 (16.7%) CCA, and 1 (8.3%) breast cancer. Five  patients received ≥ 3 lines of prior systemic anti-cancer treatment. All patients harbored one or more types of FGF/FGFR1-3 alterations including FGFR1 amplification , FGFR2 point mutation , FGFR1 point mutation , FGFR2 amplification , FGFR2 fusion , FGF3 point mutation , FGF3 amplification , FGF4 amplification , and FGF19 amplification . The demographics and baseline characteristics were listed in Table As of the data cutoff date of March 8, 2021, all patients discontinued treatment due to disease progression. The median duration of treatment was 62 days (range: 35–246) with median treatment cycle of 3.0 (range: 2–12). All patients were in compliance with pemigatinib dosing regimen.Twelve participants who received pemigatinib 13.5 mg daily on the intermittent schedule were assessed for PK on Cycle 1, Day 1 (C1D1) and Cycle 1, Day 14 (C1D14), respectively. Figure max of approximately 1.52 h at C1D1 and 1.99 h at steady state. The geometric mean terminal elimination half-life was 11.3 h. The steady-state geometric mean Cmax and AUC were 215.1 nmol/L7 and 2636.9 h·nmol/L, respectively. The degree of drug accumulation was measured by the accumulation ratio, and its geometric mean value was 1.32.Following daily administration of 13.5 mg pemigatinib, it attained peak plasma concentrations after a median tPD characteristics were analyzed in 11 out of 12 patients (with 1 subject excluded due to lack of baseline serum phosphate data). The serum phosphate concentrations increased on days 8 and 15 of cycle 1  and decreased to baseline post 1 week intermission. During subsequent treatment, the serum phosphorus remained below or near the baseline levels.Overall, confirmed partial response (PR) was observed in two patients per investigator assessments, including one patient with FGFR1-mutant (p.A354V) esophageal carcinoma and one with FGFR2-mutant (p.F276C) CCA Fig. . AdditioAll patients experienced TEAEs Table , most frAll patients experienced at least one sponsor-defined clinically-notable TEAE, all being grade 1 or 2 in severity, including hyperphosphatemia , hypophosphatemia , nail toxicity , nail disorder  and nail discolouration .The safety and efficacy with pemigatinib has been previously reported in Chinese patients with CCA . This phmax,ss (geomean [CV%]) of 215.1 nM [64.8%] vs 236 nM [56.4%] and AUCss,0–24 (geomean [CV%]) of 2636.9 h·nM [53.8%] vs 2620 h·nM [54.1%]. Serum phosphate was assessed as the PD marker and its concentration increased at first cycle and decreased to and remained around baseline levels after 1-week intermission. The serum phosphate concentration change from baseline (geomean [CV%]) was 2.15 (32.9%) in this study and was 2.6 (40.4%) in Western population (INCB 54828–101) [The PK data indicated rapid absorption of pemigatinib in Chinese patients and consistent PK parameters between single and multiple dosing. Pemigatinib exposures at steady state in this study were generally consistent with previous data in Western population (INCB 54828–101) , with Cm828–101) . Those fPreliminary anti-tumor activity was observed with pemigatinib in this previously treated patient population: 3 out of 12 patients achieved SD and 2 of 12 patients achieved PR. The overall efficacy of pemigatinib in the Chinese patients was similar to Western patients as reported in the phase I/II Fight-101 study Supplementary file2 (DOCX 18 KB)Below is the link to the electronic supplementary material.