Neurologic damage often leads to neuropathic pain, for which there are no effective treatments owing to its complex pathogenesis. The purinergic receptor P2X4 is closely associated with neuropathic pain. Astragalin (AST), a compound that is used in traditional Chinese medicine, has protective effects against allergic dermatitis and neuronal injury, but its mechanism of action is not well understood. The present study investigated whether AST can alleviate neuropathic pain in a rat model established by chronic constriction injury (CCI) to the sciatic nerve. The model rats exhibited pain behavior and showed increased expression of P2X4 and the activated satellite glial cell (SGC) marker glial fibrillary acidic protein in dorsal root ganglia (DRG). AST treatment partly abrogated the upregulation of P2X4, inhibited SGC activation, and alleviated pain behavior in CCI rats; it also suppressed ATP-activated currents in HEK293 cells overexpressing P2X4. These data demonstrate that AST relieves neuropathic pain by inhibiting P2X4 and SGC activation in DRG, highlighting its therapeutic potential for clinical pain management. More than 100 million people worldwide suffer from pain, which severely affects their quality of life and work productivity and imposes a considerable financial burden on their families and society . NeuroloThe purinergic receptor (P2 receptor) family is divided into P2X (ionic) and P2Y (G protein-coupled) receptors . P2X2/3,P2X4 expressed by satellite glial cells (SGC) of the dorsal root ganglia (DRG) has been shown to be involved in neuropathic pain . Large aIn Vivo Experiments.Male Sprague–Dawley rats weighing 200 ± 20 g were purchased from Changsha Tianqin Biological Co.  and allowed to acclimate to the laboratory environment for 7 days before they were used in experiments. The rats were housed in a ventilated room with the temperature controlled at 23°C on a 12:12 h light/dark cycle with free access to water and food. All animal procedures were approved by the Animal Protection and Use Committee of the Medical School of Nanchang University. The experiments in this study followed International Association for the Study of Pain ethics guidelines and were carried out according to the guidelines of Animal Research: Reporting of 21H20O11, MW 448.38, >98% purity) was purchased from Nanjing Herb Source Biotechnology Co.  and dissolved in 0.5% dimethyl sulfoxide (DMSO). The P2X4 antagonist 5-(3-bromophenyl)-1H-benzofurodiazepin-2(3H)-one (5-BDBD) was purchased from Bio-Techne . All other chemicals were of analytic grade and obtained from standard commercial suppliers.Astragalin (Cn = 8): control (Ctrl), sham operation (Sham), rats with sciatic nerve CCI (CCI), Ctrl rats treated with AST (Ctrl + AST), CCI rats treated with AST (CCI + AST), and DMSO-treated CCI rats (CCI + DMSO). The control rats were designed as a blank control without any drugs. The CCI + AST and Ctrl + AST groups were intragastrically administered with AST  once a day for 2 weeks starting on day 2 after CCI. Rats in the CCI + DMSO group were orally administered by gavage with the same volume of DMSO  daily for 2 weeks. In order to evaluate the role of P2X4 receptors and the involvement of extracellular signal-regulated kinase (ERK) signaling in the effects of P2X4, the P2X4 antagonist 5-BDBD was added in the experiment. 5-BDBD (dissolved in 0.5% DMSO) was prepared to be 100 μM. After fixation under anesthesia, each rat in the CCI + 5-BDBD group was intravenously injected with 10 μl of 5-BDBD through the tail vein, while the CCI + DMSO group rat was injected with 10 μl of 0.5% DMSO once daily from days 7 to 13 after CCI operation. A new microsyringe was changed every day for each rat. There were no unexpected, new, and/or significant hazards or risks associated with this work.In order to determine the concentration of AST, we carried out a preliminary experiment and designed three concentrations  of AST to observe the analgesic effect of AST by intragastric administration. The dose of AST was selected based on the results of our pilot experiments and previous literature . Rats weWe used CCI of the rat sciatic nerve as a neuropathic pain model. Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (3 ml/kg). After immobilizing the rat in a prone position, the right hind leg was disinfected with 75% ethanol solution. A skin incision was made along the inside of the femur, and the sciatic nerve was completely exposed after separating the subcutaneous tissue and muscle. The sciatic nerve was ligated four times using a 4-0 chromic gut suture starting 7 mm distal to the sciatic nerve trunk, with each suture separated by a distance of 1 mm. Sham rats underwent the same operation as CCI rats but without nerve ligation.Pain behavior was measured on day 0 (before the experiment) and days 1, 3, 5, 7, 9, 11, 13, and 15 after CCI. Rats were allowed to adapt to the surrounding environment before behavioral testing. The mechanical withdrawal threshold (MWT) was determined by observing the withdrawal response induced by mechanical stimulation using a BME-404 electronic mechanical pain detector . The ratThermal withdrawal latency (TWL) was assessed with a BME-410C Thermal Paw Stimulation System  . The rat1 and the Python scripts ligand4.py and prepare receptor4.py. The target protein binding pocket was identified with the ADT molecular observer. The parameters were saved as system default values. Finally, MGLTools2 and PyMOL software3 were used to view the output file.Using human (h) P2X4 as the target protein and AST as the ligand, molecular docking simulations were carried out using the AutoDock Tools (ADT) of AutoDock 4.2 softwareL4–L6 DRG were removed from four rats in each group anesthetized with chloral hydrate on day 15 after CCI and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 1 day , followe–ΔΔCT method).L4–L6 DRG isolated from rats anesthetized with chloral hydrate on day 15 after CCI were rinsed with ice-cold PBS, and total RNA was extracted using the Total RNA Extraction kit  according to the manufacturer’s instructions. The RNA was reverse transcribed into cDNA using the cDNA Synthesis kit . RNA samples were analyzed by qPCR with SYBR Green MasterMix on an ABI Prism 7500 instrument  using the following forward and reverse primers : P2X4, 5′-CCCTTTGCCTGCCCAGATAT-3′ and 5′-CCGTACGCCTTGGTGAGTGT-3′; and β-actin, 5′-AAGATCCTGACCGAGCGTGG-3′ and 5′-CAGCACTGTGTTGGCATAGAGG-3′. The threshold cycle (CT) was used to calculate the expression level of target genes in each sample normalized to that of β-actin , 0.2% bromophenol blue, and 20% glycerol] and heating at 95°C for 10 min, samples containing equal amounts of protein (20 μg) were separated by SDS–polyacrylamide gel electrophoresis  on a 12% acrylamide gel, and the proteins were transferred to a polyvinylidene difluoride membrane. Non-specific antigens were blocked by incubating the membrane for 2 h at room temperature in 5% skim milk; this was followed by overnight incubation at 4°C with the following primary antibodies: rabbit anti-P2X4 , rabbit anti-ERK1/2 , and anti-phosphorylated (p)ERK1/2  ; and rabbit anti-tumor necrosis factor receptor (TNF-R)1 (1:800) and mouse anti-GFAP (1:800) . After incubation with horseradish peroxidase-conjugated secondary antibody for 2 h, protein bands were visualized with a chemiluminescent substrate . The membrane was stripped and reprobed with anti-β-actin antibody , which was used to normalize target protein expression level. Quantification of protein level expressed as integrated optical density was measured using Image Pro-Plus software .On day 15 after CCI, L4–L6 DRG was isolated from rats anesthetized with chloral hydrate and immediately washed with precooled PBS. DRG tissue was homogenized on ice by trituration in lysis buffer  and incubated on ice for 40 min. The homogenate was centrifuged at 6,000 × 2 humidified incubator. Full-length cDNA encoding rat P2X4  was inserted into the pcDNA3.1(+)/EGFP/IRES vector (ampicillin resistance) containing an enhanced green fluorescent protein (EGFP) reporter to generate pcDNA3.1(+)/EGFP/IRES-P2RX4. Cloning site was NheI/XbaI. The plasmid was constructed by Shanghai Nuovo Biological Technology Co.  and was transiently transfected into cells using Lipofectamine 2000 reagent  according to the manufacturer’s instructions. When the cells reached 70–80% confluence, the culture medium was replaced with OptiMEM for 2 h. The transfection medium was prepared as follows: (a) Dilute 4 μg of plasmid with OptiEME to 250 μl, (b) dilute 10 μl of Lipo2000 with OptiEME to 250 μl, and (c) mix the above two solutions and incubate at room temperature for 20 min. Then, 500 μl of cDNA/lipofectamine solution was added to each well followed by incubation for 6 h at 37°C and 5% CO2. Control cells were transfected with the empty pcDNA3.1(+)/EGFP/IRES vector. After incubation, the cells were washed in minimal essential medium containing 10% fetal bovine serum and cultured for 24–48 h. GFP fluorescence was evaluated to determine the transfection efficiency. Whole-cell patch clamp recordings were carried out 1–2 days after transfection.HEK293 cells were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 1% penicillin, and streptomycin in a 37°C, 5% CO2 (0.1), and Na3GTP (0.3). The bath was continuously perfused with extracellular solution composed of (mM) KCl (2.5), NaCl (126), glucose (10), CaCl2 (2.4), MgCl2 (1.2), and NaHCO3 (18). The osmotic pressure of the extracellular and internal solutions was adjusted to 340 mOsm with sucrose. The pH of the extracellular solution was adjusted to 7.4 with NaOH and that of the internal solution was adjusted to 7.3 with KOH. The resistance of the recording electrode was 2–6 MΩ. The holding potential was −70 mV. ATP  and the P2X4 antagonist 5-BDBD were dissolved in extracellular solution; AST was dissolved in 0.5% DMSO and diluted to different concentrations with extracellular solution. The drugs were rapidly administered through a manifold consisting of 10 capillaries made of fused silica coated with polyimide with an inner diameter of 200 μm. The distance from the tubule mouth to the examined cell was approximately 100 μm. The solution was transported from a separate container by gravity flow. One barrel was used to apply the drug-free extracellular solution to rapidly terminate drug application. The drug was applied every 4 min for 2 s, and the effect was reproducible. The data were low-pass filtered at 2 Hz, digitized at 5 kHz, and stored on a laboratory computer using the Digidata 1200 system and pClamp 10.0 software (Axon Instruments/Molecular Devices).Electrophysiologic recordings were carried using a whole-cell patch clamp amplifier . HEK293 cells with green fluorescence  were selected for recording. Microelectrodes were filled with an internal solution composed of (mM) K gluconate (145), MgATP (2), EGTA (0.75), HEPES (10), CaClpost hoc test. Pain behavior data were evaluated by two-way ANOVA. p < 0.05 was considered statistically significant.Data were analyzed using SPSS software  and are presented as mean ± SEM. Multiple comparisons for western blotting, qPCR, immunofluorescence, and electrophysiology data were performed by one-way analysis of variance (ANOVA) followed by Fisher’s p > 0.05). On days 1, 3, 5, 7, 9, 11, 13, and 15 post-CCI, MWT (p < 0.001) and were increased in the CCI + AST group from day 5 to day 15. On the final day of the experiment, MWT (p > 0.05), and no difference between CCI and CCI + DMSO groups (p > 0.05). By day 15, TWL was decreased in CCI (13.118 ± 1.103) and CCI + DMSO (14.13 ± 1.105) groups relative to the Ctrl group (25.181 ± 0.62) but increased in CCI + AST rats (26.975 ± 0.993) (p > 0.05), or between CCI and CCI + DMSO (p > 0.05) groups. 5-BDBD increased MWT and TWL from day 9 to day 13 compared with CCI rats  . There wCCI rats .We performed molecular docking to predict whether AST and P2X4 interact. The optimal docking configuration between AST and hP2X4 revealed that AST strongly interacts with Tyr299 in the A and B chains of hP2X4 protein. The binding energy for the interaction was calculated as −7.3 kcal/mol .p < 0.01) but was reduced in CCI + AST rats (0.96 ± 0.06) relative to CCI rats (p < 0.001) , and Ctrl + AST (0.94 ± 0.13) groups or between CCI + DMSO (1.57 ± 0.11) and CCI (p > 0.05) groups. P2X4 protein level was also higher in CCI rats (1.23 ± 0.11) than in Ctrl rats (0.32 ± 0.02) (p < 0.001), and lower in the CCI + AST group (0.34 ± 0.01) than in the CCI group (p < 0.001) (p > 0.05). In order to observe whether blocking the activation of P2X4 receptor affects the expression of P2X4 receptor, we also detected the expression of P2X4 receptor in CCI rats treated with 5-BDBD. The results were as follows: Ctrl (0.17 ± 0.03), Sham (0.188 ± 0.04), CCI (0.85 ± 0.06), and CCI + 5-BDBD (0.21 ± 0.05)  relative to the Ctrl group (< 0.001) . There w< 0.001) . There w ± 0.05) . P2X4 prp < 0.001) and less colocalization in the CCI + AST group (1.13 ± 0.04) than in CCI rats (p > 0.05).We examined whether P2X4 is expressed in activated SGCs by double immunofluorescence labeling of DRG with antibodies against P2X4 and GFAP. The results showed that P2X4 colocalized with GFAP in SGCs , with grCCI rats . There wp < 0.001). Meanwhile, no differences were observed between Sham (1.22 ± 0.17), Ctrl, and Ctrl + AST (1.07 ± 0.14) groups (p > 0.05). GFAP level was lower in CCI + AST rats (1.13 ± 0.20) than in CCI rats (p < 0.001). There was no difference in GFAP protein level between the CCI + DMSO (2.74 ± 0.28) and CCI groups (p > 0.05).To determine the degree of SGC activation, we also evaluated GFAP protein expression in DRG by western blotting . GFAP exp < 0.001; p < 0.001); however, there were no differences between the Ctrl, Sham (0.33 ± 0.04), and Ctrl + AST (0.43 ± 0.03) groups or between the CCI + DMSO (1.12 ± 0.13) and CCI groups (p > 0.05).TNF-α released from the activated glial cells acts on TNF-R1 expressed by DRG neurons. Upregulation of TNF-R1 expression in DRG neurons enhances their sensitivity to mechanical and thermal stimuli following peripheral nerve injury. Western blotting analysis revealed increased TNF-R1 expression in the CCI group (1.11 ± 0.03) compared to the Ctrl group (0.33 ± 0.04) . There were no differences in pERK1/2 between the Ctrl, Sham (0.65 ± 0.1), and Ctrl + AST (0.53 ± 0.09) groups or between the CCI + DMSO (2.82 ± 0.42) and CCI groups (p > 0.05).To determine whether the ERK signaling pathway mediates the effects of P2X4, we examined the expression levels of ERK and phosphorylated extracellular-regulated protein kinase (pERK) in DRG. Total ERK level did not differ significantly across groups (ATP-activated currents in HEK293 cells transfected with pcDNA3.1(+)/EGFP/IRES-P2RX4 plasmid and control cells transfected with the empty pcDNA3.1(+)/EGFP/IRES vector were recorded by whole-cell patch clamping. Currents were generated in HEK293 cells overexpressing P2X4 but not in control cells by application of ATP (100 μM). AST (50 μM) treatment blocked the ATP current generated in P2X4-overexpressing cells, but to a lesser degree than the P2X4 antagonist 5-BDBD (10 μM). DMSO has no inhibition effect on ATP-activated current in cells expressing P2X4 receptors. AST inhibited ATP activated current in a manner of concentration–response .Neuropathic pain is a type of chronic pain caused by nerve injury . The resP2X4 expression is upregulated in peripheral nerve injury, and P2X4 activation in DRG is a key factor in the development and maintenance of neuropathic pain . Using aPrimary sensory ganglia contain sensory neurons and surrounding SGCs , which hNeuropathic pain involves communication between SGCs and neurons in sensory ganglia . ActivatThe ERK signaling pathway in glial cells and neurons is important for the transmission of pain information in the CCI model . P2X4 isIn order to observe whether AST could specifically act on the P2X4 receptor, HEK293 cells transfected with P2X4 receptor plasmid were used. Our results showed that AST significantly inhibited the ATP-activated currents in HEK293 cells transfected with P2X4 receptor. The electrophysiological results combined with downregulation of P2X4 mRNA and protein after AST treatment confirmed that AST inhibited the transmission of nociceptive signaling by acting on the P2X4 receptor. Some literatures have shown that the expression of P2X4 is increased in inflammatory state . After CIt has been reported that there is interaction between neurons and SGCs in the sensory ganglia , and purIn conclusion, the upregulated P2X4 receptor in SGCs of dorsal root ganglion is involved in the process of CCI-induced neuropathic pain. AST reduces the upregulated expression and function of P2X4 receptor and then inhibits the activation of SGC, thus alleviating the pain behaviors of CCI rats. These results provide evidence that AST has therapeutic potential for the effective clinical management of neuropathic pain.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Animal Protection and Use Committee of the Medical School of Nanchang University.SDL and SML designed the study. MW, YW, and XC prepared the DRG tissue samples and performed immunofluorescence analysis, microscopy, and data analysis, and wrote the manuscript. MW, SZL, NW, and RS performed the pain behavior experiment. MW and SZL performed western blotting and qPCR experiments and data analysis. RS and JX assisted with animal breeding and maintenance. SML supervised the study and contributed to manuscript writing. 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.


Intervertebral disc degeneration (IDD) is a major cause of lower back pain. This study aimed at exploring the effects of histone deacetylase 4 (HDAC4) and its upstream and downstream signaling molecules on IDD development.A murine IDD model was established by inducing a needle puncture injury to the vertebrate, whereupon we isolated and transfected of nucleus pulposus (NP) cells. Disc height index (DHI) of the mice was determined by X-ray tomography, while the pain experienced by the IDD mice was evaluated by mechanical and thermal sensitivity tests. Next, the interaction between GSK3β and HDAC4 as well as that between HDAC4 and KLF5 acetylation was assessed by co-immunoprecipitation, while the promoter region binding was assessed identified by chromatin immunoprecipitation. By staining methods with TUNEL, Safranin O fast green, and hematoxylin and eosin, the NP cell apoptosis, degradation of extracellular matrix, and morphology of intervertebral disc tissues were measured. Furthermore, mRNA and protein expressions of GSK3β, HDAC4, KLF5, and ASK1, as well as the extent of HDAC4 phosphorylation, were determined by RT-qPCR and Western blotting.GSK3β was identified to be downregulated in the intervertebral disc tissues obtained from IDD mice, while HDAC4, KLF5, and ASK1 were upregulated. HDAC4 silencing alleviated IDD symptoms. It was also found that GSK3β promoted the phosphorylation of HDAC4 to increase its degradation, while HDAC4 promoted ASK1 expression through upregulating the expression of KLF5. In IDD mice, GSK3β overexpression resulted in increased DHI, inhibition of NP cell apoptosis, alleviation of disc degeneration, and promoted mechanical and thermal pain thresholds. However, HDAC4 overexpression reversed these effects by promoting ASK1 expression.Based on the key findings of the current study, we conclude that GSK3β can promote degradation of HDAC4, which lead to an overall downregulation of the downstream KLF5/ASK1 axis, thereby alleviating the development of IDD.The online version contains supplementary material available at 10.1186/s13148-021-01005-9. Intervertebral disc degeneration (IDD) is one of the most common causes of lower back pain and related disability , 2. BackHistone deacetylase 4 (HDAC4) is regarded as an important mediator of disease processes due to its essential function on the transcriptional regulation and cell cycle progression . In addiInterestingly, as previously reported, glycogen synthase kinase-3β (GSK3β) could promote HDAC4 degradation by phosphorylating HDAC4 , while iAs previously reported, HDAC4 promotes the development of IDD . With thAfter that, the expression of HDAC4 in the mouse model was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting, which revealed that HDAC4 mRNA and protein expressions were increased in NP tissues of IDD mice when compared to control or sham mice, while there was no difference in the mRNA and protein expressions of HDAC4 between control and sham mice Fig. c, d. NP Moreover, apoptosis in NP cells was found to be significantly increased in IDD mice that were normalized by HDAC4 silencing Fig. l. MoreovA previous study revealed that HDAC4 can remove the acetylation of KLF5 protein through its deacetylase function and promote its transcription function . At the After establishing the promoting effect of HDAC4 on ASK1 expression, we further investigated the effect of HDAC4 on IDD by regulating ASK1. First, we detected the silencing efficiency of ASK1 expression using RT-qPCR, which showed that both ASK1 silencing sequences significantly inhibited the expression of ASK1 Fig. a. sh2-ASGSK3β is capable of promoting HDAC4 degradation through its phosphorylation . MeanwhiThen we proceeded to elucidate the mechanism by which GSK3β-mediated HDAC4 degradation affects IDD. GSK3β overexpression increased DHI in IDD mice, while further HDAC4 overexpression reversed the trend Fig. a. MoreovFinally, we aimed to determine whether GSK3β can arrest the IDD through ASK1. GSK3β overexpression increased DHI in IDD mice significantly, which was inhibited secondary to ASK1 overexpression Fig. a. H&E stThe present study revealed several key findings. Firstly, HDAC4 expression was increased in IDD. HDAC4 silencing alleviated IDD by increasing DHI, improving morphology of NP tissue, decreasing NP cell apoptosis, and reducing pain. Next, HDAC4 was found to increase the expression of KLF5, resulting in an increase in ASK1 expression. HDAC4 also promoted IDD, as indicated by the decrease in DHI, and increase in NP cell apoptosis and pain. These effects of HDAC4 were reversed secondary to ASK1 silencing. In addition, IDD presented with reduced GSK3β expression. GSK3β overexpression degraded HDAC4 and hence improved IDD; however, this effect was reversed by either HDAC4 overexpression or ASK1 overexpression. Collectively, these results suggest that GSK3β degraded HDAC4 to alleviate IDD through KLF5 and ASK1. Therefore, our findings showed that GSK3β could potentially serve as a novel therapeutic target for slowing the progression of IDD and back pain and this study paves the way for further investigations on the topic.The thorough understanding of the mechanism underlying the occurrence of IDD can aid the development of a novel treatment for lower back pain, as IDD is a major cause of this condition , 3. In tAnother important finding was that GSK3β degrades HDAC4 in IDD, suggesting GSK3β may be an upstream regulator of HDAC4. This result was also consistent with a previous study, suggesting that GSK3β promoted HDAC4 degradation by HDAC4 phosphorylation . MoreoveIn addition to upstream, we also found KLF5 as a downstream mediator of HDAC4-mediated IDD. HDAC4, as a histone deacetylase, increased KLF5 expression by removing the acetylation of KLF5 . KLF5 haFurthermore, we found that KLF5 promoted the expression of ASK1, which was consistent with a previous study . MoreoveThere are a few notable limitations in this study. First, the study utilized traumatic injury model of IDD. Although this is a valid IDD model, other pathologies, such as chronic stress, can also lead to the development of IDD. Therefore, further investigations with other animal models of IDD are required for verification of our findings. Second, we used HDAC4 and GSK3β overexpression to determine their roles on IDD. We also used ASK1 overexpression to study its role on GSK3β-mediated improvement of IVD. However, the role of KLF5 remains unclear. Therefore, a more detailed study involving KLF5 overexpression and its role in IDD is encouraged.In conclusion, GSK3β could serve as a negative regulator of HDAC4, resulting in the alleviation of IDD, including pain Fig. . This beThe current study was approved by the Animal Ethics Committee in Guangxi University of Chinese Medicine, Guangxi International Zhuang Medicine Hospital. Extensive efforts were made to ensure minimal suffering of the animals used in the study.n = 130, 8-week old, 20–25 g, Experimental Animal Center of Guangxi University of Chinese Medicine, Guangxi International Zhuang Medicine Hospital) were used to establish an IDD (n = 112) model and the remaining 18 mice served as controls. In addition, in the control group, 9 were naïve and 9 were in sham operation. Mice were anesthetized with 3% sodium pentobarbital . Mice were placed in a prone position, and a 20-gauge needle was used to puncture three intervertebral discs  under a microscope [n = 108 out of 112). IDD mice were randomly divided into 12 groups (9 mice per group). After successful modeling, a positive control group was set. The mice in this group received treatment with the Food and Drug Administration (FDA)-approved tasquinimod  dissolved in drinking water at a daily dose of about 25 mg/kg of body weight. Lentiviral vector LV5-GFP was used for overexpression , and pSIH1-H1-copGFP was used for gene silencing . All plasmids used were made by Gene Pharma . 293 T cells were used for lentivirus packaging. 293 T cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 complete medium containing 10% fetal bovine serum (FBS) and passaged every other day. A small incision was made in the left side of the mice to expose the previously punctured IDD. An adenoviral vector (1 × 109 pfu/100 μL) was slowly injected into the punctured disc with a 33-gauge needle . Injections were repeated 4 and 8 weeks later. NP tissues were collected for further experiments.Male C57BL/6 J mice  and passaged. The third to fifth passages of NP cells were used [5/mL, the cells were inoculated to a 6-well plate for 24 h. When cell fusion reached about 75%, cells were transfected with following plasmids: sh-negative control (NC), sh1-GSK3β, sh2-GSK3β, oe-NC, oe-GSK3β, sh1-HDAC4, sh2-HDAC4, using Lipofectamine 2000 reagents .Mouse NP tissue samples were isolated, cut into small pieces, and treated with 0.25% streptomycin  for 30 min. After that, the tissues were treated with 0.1% type II collagenase  at 37 °C for 4 h . Then, tere used . When in−ΔΔCt method. Each sample was tested in triplicate.TRIzol reagents  were used to extract the total RNA from cells in each group. The RNA concentration, purity, and integrity were determined by Nano-Drop ND-1000 spectrophotometry and 1% agarose gel electrophoresis. Reverse transcription reaction was performed on a PCR amplification equipment to synthesize a complementary DNA (cDNA) template using a reverse transcription kit . Reverse transcription system (20 μL) was performed according to the instructions of EasyScript First-Strand cDNA Synthesis SuperMix . Reaction solution was collected for real-time quantitative PCR by SYBR® Premix Ex TaqTM II Kit  using ABI 7500 system. Primer sequences are shown in Table Total tissue protein was extracted by a radioimmunoprecipitation assay (RIPA) kit . Protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit . Protein (40 µg) from each sample was loaded and separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane and blocked with Tris-buffered saline Tween-20 (TBST) solution containing 5% bovine serum albumin (BSA) at room temperature. Membranes underwent incubation with primary antibodies against GSK3β , phosphorylated (p)-HDAC4, ASK1 , HDAC4 , KLF5 , cleaved caspase-3 , Bcl-2 , Bax , and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)  at 4 °C overnight. Membranes were subsequently incubated with secondary antibody goat anti-rabbit immunoglobulin G (IgG) antibody  or goat anti-mouse IgG antibody  and at room temperature. Protein bands were developed using enhanced chemiluminescence reagents. Gray intensity in each protein band was analyzed by ImageJ software and normalized to internal reference protein GAPDH. Each sample was tested in triplicate.ChIP detection was performed using an EZ-Magna ChIP kit . In brief, cells were fixed with 4% paraformaldehyde and incubated with glycine for 10 min to produce DNA–protein crosslinks. Then, the cells were lysed using cell lysis buffer and nuclear lysis buffer, which were subsequently sonicated to produce 200–300 bp chromatin fragments. Next, the cell lysates were immunoprecipitated with magnetic protein A beads bound to the antibodies: HDAC4 , acetyl-histone H3 , KLF5 , and ASK1 . Meanwhile, Rabbit IgG  was used as a negative control. Finally, the KLF5 and ASK1 promoters in the precipitated DNA were analyzed and quantified by RT-qPCR.Transfected cells were treated in a lysis buffer , and cell debris was removed by centrifugation. The clear cell lysate was incubated with 1 μG HDAC4 antibody  or anti-GSK3β  and 15 μL protein A/G beads  for 2 h. After washing, beads were boiled at 100 °C for 5 min. Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane , and then immunoblotted. To detect endogenous protein interactions, cells were lysed in ice-cold lysis buffer. The cleared cell lysate underwent incubation with 5 µg of anti-HDAC4 antibody and 20 μL of protein A/G beads at 4 °C overnight. Anti-HDAC4 antibody or anti-GSK3β antibody was used to detect endogenous levels of HDAC4 or GSK3β, respectively.NP cells with HADC4 overexpression were transfected with the plasmid expressing Flag-KLF5 and cultured for 48 h, and the IP was purified by Flag antibody. The precipitate was washed with pre-lysis buffer and analyzed by Western blotting. Antibodies used in immunoblotting and co-immunoprecipitation were HADC4 , anti-acetyl-histone H3 .2O2 and 0.1% Triton X-100 for 10 min and washed 3 times with phosphate-buffered saline (PBS). Cells were then fixed with 4% paraformaldehyde in situ following the instructions in an apoptosis detection kit  and stained with 4′,6-diamidino-2-phenylindole (DAPI). Apoptosis was observed under a light microscope . The experiment was repeated three times.TUNEL staining was used to detect apoptotic DNA fragments. Degenerative disk NP cells were cultured with 3% HMouse intervertebral disc tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm sections. Tissue sections were then stained with Alcian blue  and Safranin O fast green . In H&E staining, tissue sections were dewaxed with xylene I for 10 min and xylene II for 5 min, followed by rehydration with anhydrous alcohol for 1 min, 95% alcohol for 1 min, and 85% alcohol for 1 min. After that, tissue sections were stained with hematoxylin  for 5 min and then with eosin  for 2 min. Tissues were then dehydrated with 85% alcohol dehydration for 20 s, 95% alcohol for 1 min, absolute alcohol I for 2 min, anhydrous alcohol II for 2 min, xylene I for 2 min, and xylene II for 2 min, and sealed with neutral or Canadian balsam resin. Finally, the sections were observed under a light microscope .Mouse intervertebral disc tissue sections were dewaxed and hydrated as above and stained for 3 min with hematoxylin. Sections were differentiated with 1% hydrochloric acid alcohol for 5 s and stained with 0.02% fast green solution for 10 min . After wMice were anesthetized, and their limbs and tails were secured so that their tail muscles were relaxed vertically. The Faxitron Cabinet X-ray system  was used to scan the mice on week 4 and 8 after modeling. Disc height was obtained and DHI was calculated . The chaMice were habituated to the test environment for at least 2 days. Mechanical sensitivity was tested by the von Frey Hair  test. Prior to testing, mice were placed in a metal mesh box for 30 min. A series of von Frey hairs with logarithmically increasing stiffness were used to vertically stimulate the plantar surface of each hind paw, and the paw withdrawal observed. Each mouse was tested 3 times, and the average of the thresholds was calculated.To demonstrate thermal hyperalgesia, withdrawal latency of mouse feet to thermal stimulation was measured. An analgesic device  was used to provide a heat source, and animals were placed in boxes with smooth and temperature-controlled glass floors. The heat was transmitted through the heat source to the hind paw. When the hind paw was retracted, the stimulus was turned off (or removed after 20 s to prevent tissue damage). In all experiments, the intensity of the thermal stimulus remained constant. In control animals, withdrawal latency was 9–14 s. Thermal stimuli were delivered to each hind paw three times at 5–6-min intervals [t test. Data from multiple groups were compared by one-way analysis of variance (ANOVA) and post hoc Tukey’s test. Data comparison between groups at different time points was made using repeated-measures ANOVA and post hoc Bonferroni test. Differences were considered significant when p < 0.05.Data analysis was performed by SPSS 21.0 statistical software . Data are expressed as mean ± standard deviation (SD). Data from two groups were compared by unpaired Additional file 1. Figure S1 H&E staining of disc tissues in each group (200 ×). A: HDAC4 promotes IDD by elevating ASK1 expression. B: GSK3β alleviates IDD by inhibiting HDAC4 expression. C: GSK3β alleviates IDD by inhibiting ASK1 expression.Additional file 2. Figure S2 Safranin O fast green staining of disc tissues in each group (200 ×). A: HDAC4 promotes IDD by elevating ASK1 expression. B: GSK3β alleviates IDD by inhibiting HDAC4 expression. C: GSK3β alleviates IDD by inhibiting ASK1 expression.


Herein, we hypothesized that sugarcane plants supplied with more NO3− as a source of N would produce more NO under water deficit. Such NO would reduce oxidative damage and favor photosynthetic metabolism and growth under water limiting conditions. Sugarcane plants were grown in nutrient solution and received the same amount of nitrogen, with varying nitrate:ammonium ratios (100:0 and 70:30). Plants were then grown under well-watered or water deficit conditions. Under water deficit, plants exhibited higher root [NO3−] and [NO2−] when supplied with 100% NO3−. Accordingly, the same plants also showed higher root NR activity and root NO production. We also found higher photosynthetic rates and stomatal conductance in plants supplied with more NO3−, which was associated with increased root growth. ROS accumulation was reduced due to increases in the activity of catalase in leaves and superoxide dismutase and ascorbate peroxidase in roots of plants supplied with 100% NO3− and facing water deficit. Such positive responses to water deficit were offset when a NO scavenger was supplied to the plants, thus confirming that increases in leaf gas exchange and plant growth were induced by NO. Concluding, NO3− supply is an interesting strategy for alleviating the negative effects of water deficit on sugarcane plants, increasing drought tolerance through enhanced NO production. Our data also provide insights on how plant nutrition could improve crop tolerance against abiotic stresses, such as drought.Nitric oxide (NO) is an important signaling molecule associated with many biochemical and physiological processes in plants under stressful conditions. Nitrate reductase (NR) not only mediates the reduction of NO Nitric oxide (NO) is a diatomic radical gas and important signaling molecule in animals , fungi , bacteriNO synthesis is increased in plants under drought and its role in promoting acclimation responses to cope with water deficit has been suggested . NO and 2•-) and enhancement of the antioxidant system in sugarcane plants under drought , which oxidizes e and NO . Althouge and NO . NO prode and NO .3− reduction to NO2−, which is then reduced to NH4+ during the N assimilatory pathway (3− supply (Physcomitrella patens occurred when it received a NR inhibitor (3− reduction (3− supply in plants under water deficit remain unknown.One of the most important pathways for NO production in land plants is through nitrate reductase (NR) , a multi pathway . Arasimo− supply . Furthernhibitor . Althougnhibitor , some aueduction . However3−), ammonium (NH4+), and urea (CO(NH2)2) are the main forms of fertilizers and, thus, are the main sources of N for crops  and NH4+ (30%). As a consequence of NO production, oxidative damage will be reduced under water deficit, favoring photosynthetic metabolism and plant growth.The literature concerning NOSaccharum spp.) cv. IACSP95-5000 developed by the Sugarcane Breeding Program of the Agronomic Institute  were used. Six-week-old plants were transferred to plastic boxes (4 L) containing nutrient solution modified from −1 N (nitrate 90% + ammonium 10%); 9 mmol L−1 Ca; 0.5 mmol L−1 Mg; 1.2 mmol L−1 P; 1.2 mmol L−1 S; 24 µmol L−1 B; 16 µmol L−1 Fe; 9 µmol L−1 Mn; 3.5 µmol L−1 Zn; 1 µmol L−1 Cu; and 0.1 µmol L−1 Mo. Plants received this solution for 2 weeks until the establishment of treatments and the nutrient solution was renewed every 3 days throughout the experimental period.Pre-sprouted sugarcane plants  and a portable pH meter . The nutrient solution volume was also checked daily and completed with water when necessary. The nutrient solution was aerated continuously by using an air compressor .Electrical conductivity of nutrient solution was maintained between 1.8 and 2.0 mS cm−2 s−1.The experiment was carried in a growth chamber , with a 12 h photoperiod, air temperature of 30/20°C (day/night), air relative humidity of 80% and photosynthetic photon flux density (PPFD) about 800 µmol m4+ in nutrient solution without compromising their photosynthesis and growth ; and −0.75 MPa , totaling four treatments (2 NO3−:NH4+ ratios × 2 water treatments) with four biological replicates. The water deficit was induced by adding polyethylene glycol  to the nutrient solution, seven days after imposing NO3−:NH4+ ratios. To prevent osmotic shock, PEG-8000 was gradually added to the nutrient solution, reducing the osmotic potential of the solution by −0.20 MPa per day, i.e., −0.75 MPa was reached after three days (3th day of the experiment). Plants were allowed to recover from water deficit after returning them to control conditions on the 7th day of the experiment. They remained for four days under such conditions, when the experiment ended. For the biochemical analyzes, there were four biological replicates for both leaves and roots and samples were taken at the maximum water deficit (7th day) and at the end of the rehydration period (11th day). Samples were collected, immediately immersed in liquid nitrogen and then stored at −80°C. Biochemical analyzes were performed in technical triplicates.Our previous study revealed that sugarcane plants can be supplied with 30% NHd growth . Thus, tg for 5 min, the supernatants were collected, and chloroform and deionized water were added to them. The mixture was shaken vigorously and then centrifuged for 3 min at 2,000 g for phase separation. The upper aqueous phase was collected and maintained in a water bath at 37°C to remove traces of chloroform, and then the extracts were stored at −20°C (Fresh plant tissue samples (500 mg) were ground in liquid nitrogen and extraction medium containing methanol/chloroform/water (12:5:3 v/v). After centrifugation at 2,000 at −20°C .2SO4). After 20 min, 2 N NaOH was added and the solution stirred. After cooling to room temperature, the absorbance was read in a spectrophotometer at 410 nm and the nitrate content calculated from a standard curve using KNO3 (100–1000 nmol) (4)2SO4 was used to estimate the ammonium content. Concentrations were corrected to sample water content and expressed on dry weight basis.For nitrate determination, an aliquot of the extract was pipetted into test tubes containing reaction medium  . For nit00 nmol) . For amm00 nmol) . A stand2−) production , deionized water, and 0.5 mM PMSF. Then, the crude extracts were centrifuged at 10,000 g for 10 min at 4°C, and the supernatant was collected and maintained on ice. The extract was added to the assay medium containing 100 mM Tris-HCl buffer (pH 7.5), 10 mM KNO3, 0.05 mM NADH, and triton 1% X-100 (v/v), mixed and incubated at 30°C for 10 min. The reaction was quenched by adding 1% sulfanilamide solution in 1 M HCl and 0.01% N-naphthyl ethylenediamine. Nitrite production was determined by absorbance at 540 nm using a standard curve with KNO2. The NR activity was expressed as µmol NO2− min−1 mg−1 protein.Leaf and root nitrate reductase  activity was estimated as the rate of nitrite (NOoduction . The enzg and 4°C. The enzyme extract was added in to the assay medium (20 mM HEPES buffer pH 8.0 and 1.8 mM NADH) at 25°C, and maintained in the dark. The reaction was started by adding 4 mM GSNO (−1 cm−1 at 340 nm) and expressed as nmol NADH min−1 mg−1 protein.Leaf and root S-nitrosogluthatione reductase  activity was determined spectrophotometrically at 25°C by monitoring the oxidation of NADH at 340 nm, based on  mM GSNO  and the −1). This condition allowed the detection of free NO released from the S-nitrosothiols present in the leaf and root protein homogenate. The samples were run in triplicate, and the calibration curve was obtained with newly prepared GSNO solutions. The data were compared with the standard curve obtained and normalized against fresh weight. The SNO content was expressed as nmol NO mg−1 protein.The total leaf and root proteins were extracted in deionized water and the resulting homogenate was used to estimate the S-nitrosothiol content through an amperometer, as described by 2, pH 6.15), at room temperature for 15 min. Then, these segments were incubated in solution of 10 µM DAF2-DA, mixing for 40 min in the dark at room temperature (NO was assayed in leaf and root segments according to perature . The sam2•−) was determined in 50 mg of fresh tissue incubated in an extraction medium consisting of 100 μM EDTA, 20 μM NADH, and 20 mM sodium phosphate buffer, pH 7.8. The reaction was initiated by adding 25.2 mM epinephrine in 0.1 N HCl. The samples were incubated at 28°C under stirring for 5 min and the absorbance was read at 480 nm over a further 5 min (2•− production was assessed by the accumulation of adrenochrome using a molar extinction coefficient of 4.0×103 M−1 cm−1 (2•− concentration was expressed as µmol O2•− g−1 dry weight.The concentration of the superoxide anion (Oer 5 min . O2•− prM−1 cm−1 . O2•− co2O2) was performed following g and 4°C for 15 min. The reaction medium consisted of 1 mM KI, 0.1 M potassium phosphate buffer (pH 7.5) and crude extract. The microtubes were left on ice in the dark for 1 h. After this period, the absorbance was read at 390 nm. A standard curve was obtained with H2O2, and the results were expressed as µmol H2O2 g−1 dry weight.The quantification of hydrogen peroxide (Hg for 15 min. The supernatant was added to 0.5% thiobarbituric acid (w/v) in 20% TCA (w/v), and the mixture incubated at 95°C for 20 min  was measured and used as a proxy of lipid peroxidation. 200 mg of fresh tissue were macerated in extraction medium containing 0.1% TCA (w/v) and centrifuged at 10,000 r 20 min . After tM−1 cm−1  and resug for 15 min at 4°C. The specific medium for CAT and SOD consisted of 0.1 M potassium phosphate buffer (pH 6.8), 0.1 mM EDTA, 1 mM PMSF and 1% PVPP, according to The crude enzymatic extracts for the determination of superoxide dismutase activity (SOD), catalase (CAT) and ascorbate peroxidase (APX) were obtained from 100 mg of plant tissue in specific medium, followed by centrifugation at 12,000 −1 mg−1 of protein.Superoxide dismutase  activity was determined according to 2O2. The reaction was carried out in a water bath at 25°C for 2 min, and CAT activity was assessed by the decrease in absorbance at 240 nm, using the molar extinction coefficient of 36 M−1 cm−1 and expressed activity as nmol min−1 mg−1 of protein.Catalase  activity was quantified following the procedure described by 2O2. The reaction was carried out at 25°C for 2 min and APX activity quantified by the decrease in absorbance at 290 nm, using the molar extinction coefficient of 2.8 M−1 cm−1 and expressing activity as μmol min−1 mg−1 of protein.Ascorbate peroxidase  activity was evaluated as described by The protein levels were determined by the Bradford method , using b2 assimilation rate (An), stomatal conductance (gs) and the effective quantum efficiency of photosystem II (ϕPSII) were measured under PPFD of 2000 µmol m−2 s−1 and air CO2 concentration of 400 µmol mol−1. The measurements were performed between 10:30 and 12:30 h, as carried out previously  equipped with a modulated fluorometer . Net COeviously . The vap2, 5 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 10 µM leupeptin. The resulting solution was centrifuged at 14,000 g for 5 min at 4°C. An aliquot of leaf extract was incubated with the reaction medium containing 100 mM bicine-NaOH (pH 8.0) 10 mM NaHCO3, 20 mM MgCl2, 3.5 mM ATP, 5 mM phosphocreatine, 0.25 mM NADH, 80 nkat glyceraldehyde-3-phosphate dehydrogenase, 80 nkat 3-phosphoglyceric phosphokinase, and 80 nkat creatine phosphokinase, for 10 min at 25°C. The oxidation of NADH was initiated by adding 0.5 mM ribulose-1,5-bisphosphate (RuBP) and total Rubisco activity was measured. The reduction of absorbance at 340 nm was monitored for 3 min  was quantified in approximately 200 mg of leaves, which were macerated and homogenized in 100 mM bicine-NaOH buffer (pH 7.8), 1 mM ethylenediaminetetraacetic (EDTA), 5 mM MgClor 3 min .g for 25 min at 4°C. The supernatant was collected, and the reaction medium for PEPC activity contained 50 mM Tris-HCl buffer (pH 7.8), 5 mM MgCl2, 5 mM glucose 6-phosphate, 10 mM NaHCO3, 33 nkat malic dehydrogenase, and 0.3 mM NADH. The reaction was initiated by adding 4 mM phosphoenolpyruvate at 30°C. The oxidation of NADH was monitored a 340 nm for 1 min  was also evaluated in approximately 200 mg of leaves, which were macerated and homogenized in 100 mM potassium phosphate buffer (pH 7), 1 mM EDTA, 1 mM PMSF, and centrifuged at 14,000 or 1 min .Proteins were extracted from leaf samples with extraction buffer composed of 100 mM Tris, 1 mM EDTA, 5 mM DTT, 1 mM PMSF, and separated by SDS-PAGE . The firth day), and four days after returning plants to the control condition .A chlorophyll meter  was used to assess the relative chlorophyll content (Chl). The relative water content was calculated using the fresh (FW), turgid (TW) and dry (DW) weights of leaf discs, according to Leaf and root dry masses were quantified after drying samples in an oven (60°C) with forced-air circulation until constant weight. Leaf area of each plant was evaluated with a portable leaf area meter .3– and subjected to water deficit were in fact caused by NO. We used a NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). cPTIO is a stable organic radical developed by 2. In plants supplied with only NO3- as N source, the following treatments were evaluated: (a) well-watered condition, with an osmotic potential of the nutrient solution of −0.15 MPa; (b) water deficit, with an osmotic potential of nutrient solution of −0.75 MPa; and (c) same as b with 100 µM cPTIO.An additional experiment was performed to verify whether the benefits found in plants supplied with only NO2 assimilation rate (An) and stomatal conductance (gs) as described previously.First, plants were moved and roots placed in a moist chamber, where they were sprayed with cPTIO and remained in the dark for 1 hour. After this treatment, the plants were returned to the boxes with the original nutrient solution. This procedure was performed for four consecutive days from the moment the water deficit (−0.75 MPa) was installed. We also evaluated the production of intracellular NO, plant biomass, net COhttps://jasp-stats.org/). When significant differences were detected, the mean values (n=4) were compared using Bayes Factor (BF10). Our interpretation of Bayes Factor as evidence for alternative hypothesis (H1) was based on 10 < 3, there is a weak support to H1; 3 < BF10 < 20 indicates positive support to H1; and BF10 > 20 indicates strong support to the alternative hypothesis.The experiment was carried out in a completely randomized design and two causes of variation were analyzed: water availability and nitrogen source. Data were analyzed using Bayesian statistics and we used the JASP software  activity was higher in plants supplied with 100% NO3– exhibited higher NO production than those supplied with 70% NO3–  and leaf catalase (CAT) activities under water deficit  activity due to changes in NO2 assimilation rate (An), however, plants supplied with more NO3– exhibited higher photosynthetic rates than those under NO3–:NH4+ 70:30 (An after re-watering when compared to ones receiving 70% NO3– (3– supply (3– supply (data not shown).Low water availability caused a large reduction in net CO3– under low water availability. In addition, the root dry mass of plants supplied with 70% NO3– was significantly reduced under water deficit , which in turn can lead to the formation of NO2− and the hydroxyl radical. ONOO− can also add a nitro group to tyrosine residues—a process known as tyrosine nitration , S-nitrosylation increased the activity of cytosolic ascorbate peroxidase -4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO)—an endogenous NO scavenger —to check3− as nitrogen source were more tolerant to water deficit, and this response was associated with increased NO production and high nitrate reductase activity in roots. Herein, increasing NO3− supply was enough to stimulate NO synthesis and alleviate the effects of water deficit on sugarcane plants by increasing the activity of antioxidant enzymes, photosynthesis, stomatal conductance, and root growth. From a broad perspective, our data show that supplying more NO3− during nitrogen fertilization may improve sugarcane tolerance and be beneficial to field-grown sugarcane.Sugarcane plants grown in nutrient solution containing only NOAll datasets generated for this study are included in the article/S-nitrosoglutathione. AS and MTP performed the analysis of S-nitrosothiols and nitrite. EM, LS, and RR edited and revised the manuscript.MDP executed the experiments, analyzed the data, and drafted the manuscript. NS assisted in the execution of biochemical analyzes, data analysis, and revised the manuscript. PP assisted in the analysis of antioxidant metabolism, nitrate reductase, and This work was supported by the São Paulo Research Foundation ; the Coordination for the Improvement of Higher Education Personnel ; and the National Council for Scientific and Technological Development .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.


Fusarium oxysporum f. sp. cubense race 4 (Foc4) causes Fusarium wilt that affects banana plants, and hence, the molecular mechanisms of its virulence need to be investigated. We purified an exo-polygalacturonase (exo-PG), Pgc4, from Foc4. Pgc4 has an apparent molecular weight of 50.87 kDa based on sodium dodecyl sulphate–polyacrylamide gel electrophoresis. We further performed its sequence analysis and biochemical characterization. The two pgc4 genes encoding Pgc4 from Foc4 and Foc1 were 1434 bp in length and encoded 477 amino acids with differences, due to some nucleotide differences between the two. The Km and Vmax values of Pgc4 purified from Foc4 were determined to be 0.45 mg/mL and 105.26 Units·mg·protein−1 ·min−1, respectively. The recombinant proteins, r-Foc1-Pgc4 and r-Foc4-Pgc4, were expressed and purified from Pichia pastoris and showed optimal Pgc4 activity at 55 °C and pH 4.0; both could induce tissue maceration and necrosis in the “Guangfen-1” and “Baxi” varieties of banana but to a different extent. Phenotypic assays and complementation analyses revealed that, compared to the wild-type, the generated Foc4Δpgc4 mutant strain showed a lower aerial hyphal growth, grew slower, and had a reduced virulence. Therefore, our results demonstrate the function of Pgc4 as a pathogenicity factor of Foc4. Fusarium oxysporum can induce the vascular wilt disease in many important cash crops and is one of the most common and important Fusarium species worldwide [F. oxysporum f. sp. cubense is a serious threat to global banana (Musa spp.) production [Fusarium oxysporum f. sp. cubense (Foc) were divided into three races based on the banana cultivars they infect: race 1 (Foc1) infects the “Gros Michel” (AAA) and “Silk” (AAB) cultivars, race 2 (Foc2) causes disease only in the “Bluggoe” (ABB) cultivar, and race 4 (Foc4) infects nearly all banana cultivars [orldwide . The Fusoduction . The banultivars . Foc1 anultivars .Although many efforts have been made to improve the resistance of banana to Foc, the number of available varieties of banana that are resistant to Fusarium wilt disease are still limited. There are no banana varieties that are completely resistant to Foc4, and there are no suitable management strategies for Fusarium wilt plants infected by Foc4. Thus, learning about the molecular pathogenesis of Fusarium wilt may lead to the discovery of efficient ways to control the banana Fusarium wilt disease. Only some of the pathogenic genes associated with Foc4 have been reported ,7,8. HowCarbohydrate-active enzymes (CAZymes) play an important role in host invasion by fungal pathogens. The plant cell wall is the first chemical and physical barrier against pathogen invasions. It is mainly composed of pectin, cellulose, and hemicellulose. Some CAZymes secreted by Foc4 have been proved to be important pathogenicity factors during infection ,10. ManyPichia pastoris [Polygalacturonases (PGs) are a type of pectinase, speculated to be related to the pathogenicity and toxicity of fungi . They arpastoris ,10. BothP. pastoris. We performed their biochemical characterization and performed assays for tissue maceration and necrosis. Furthermore, we generated the Foc4Δpgc4 mutant strain and evaluated the phenotypic differences between the wild-type (WT) and mutant strains. We also compared the difference in virulence between the WT and Foc4Δpgc4 mutant strains against banana plants.In this study, we purified an exo-PG from Foc4 and named it as Pgc4. The objective was to explore the functions of Pgc4 in Foc4. We compared the Pgc4 from Foc1 and that from Foc4 and cloned and expressed both in Polygalacturonase activity in the Foc4 culture supernatant could be detected when grown on potato dextrose broth (PDB) supplemented with 1% citrus pectin. The PGs were purified from Foc4 by ultrafiltration, gel filtration, and then cation exchange chromatography. A significant peak (collection tube nos. 9–23) of PG activity could be observed after the cation exchange chromatography. Several proteins could be seen on the sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) upon combining all the collection fractions associated with the PG peak. When we concentrated each eluate collection tube, a single protein band could be seen on the SDS–PAGE gel for collection tubes No. 9 (Pgc4), No. 12 (Pgc3), No. 19 (Pgc1), and No. 20 (Pgc2), respectively. Besides the two known PGs (Pgc2 and Pgc3), a novel PG, Pgc4, was purified and had an enzyme activity of 3.08 Units (U). The specific activity of Pgc4 increased from 3.59 U/mg in a crude culture supernatant to 25.67 U/mg for the purified protein. The total protein amounts in the purified and crude culture supernatant were 0.15 mg and 36.6 mg, respectively ; this amF. oxysporum as the organism; the top matches with 100% similarity were a probable exo-polygalacturonase from F. oxysporum (SCO91513.1) and an uncharacterized protein FOIG_10423 from Foc tropical race 4 54006 (XP_031059473.1).The SDS–PAGE analysis showed that the molecular weight of Pgc4 was approximately 47 kDa, which is close to that of Pgc3 (45 kDa) but lower than that of Pgc2 (63 kDa) purified previously . N-termipgc4 was isolated by a rapid amplification of the cDNA ends (RACE) polymerase chain reaction (PCR); then, specific primer pairs were designed to amplify pgc4 from Foc1. A full-length of a 1728 bp DNA sequence was isolated and sequenced; the gene structure has six introns of size 51, 47, 53, 48, 47, and 50 bp. The open reading frame (ORF) of pgc4 was 1434 bp, encoding a protein consisting of 477 amino acids. The SignalP prediction showed that a putative N-terminal signal peptide sequence of 20 amino acids would be cleaved (with a probability of 0.9330) to produce a mature protein of 457 amino acids. The molecular weight and isoelectric point (pI) of the mature protein were predicted to be 50.87 kDa and pI 5.62, respectively. Two potential N-glycosylation sites, 225 NGSI and 403 NITV, could be detected in Foc4 pgc4 based on the analysis from the NetNGlyc 1.0 online server. The protein sequence of Foc4 pgc4 shared a 58% and 30% identity with those of Foc4 pgc2 and Foc4 pgc3, respectively.Based on the N-terminal sequence of Pgc4, the coding sequence of Foc4 pgc4 and Foc1 pgc4 were 217, 649, 857, 1140, 1231, and 1261  after methanol induction, the media supernatants were harvested, and the expression products were visualized by SDS–PAGE  by purified Pgc4 from Foc4 was analyzed; galacturonic acid (GA) was the only degradation product detected, and the hydrolysis of 3% of the substrate leading to a 21.5% reduction in viscosity demonstrated that Pgc4 is an exo-PG. The Km and Vmax values of Pgc4 from Foc4 were determined to be 0.45 mg/mL and 105.26 U/mg·min, respectively.The optimal pH and temperature for the PG activity of the recombinant Pgc4 proteins were investigated; both enzymes exhibited the highest activity at pH 4.0 A and at To estimate the pH stability of recombinant Pgc4, samples were incubated in buffers of different pH values at 4 °C for 24 h, and the remaining PG activity was assayed. Both recombinant Pgc4 retained more than 60% activity at pH 3.0–7.0 C.To investigate the thermostability of recombinant Pgc4, both enzymes were incubated at different temperatures in 100 mM of a potassium phosphate buffer, with a pH of 4.0 for 2 h, and then the residual activity was determined. Both recombinant Pgc4 retained more than 50% activity at 20–50 °C D.Purified r-Foc1-Pgc4 and r-Foc4-Pgc4 proteins were inoculated into banana tissues to examine their ability to macerate tissue. Sterilized banana tissues were inoculated with 1 U of r-Foc1-Pgc4 or r-Foc4-Pgc4 mixed with 1 mL of 50 mM sodium acetate buffer pH 4.0; the maceration was evaluated after 48 h. The maceration activity of r-Foc1-Pgc4 on “Guangfen-1” (Musa AAB cv. “Guangfen-1”) was higher than that of r-Foc4-Pgc4, while the maceration activity of r-Foc1-Pgc4 on “Baxi” (Cavendish banana) was lower than that of r-Foc4-Pgc4. Both exo-PGs showed a higher maceration activity on “Guangfen-1” than “Baxi” . In the stems of Cavendish cv. “Baxi” plants injected with 1 U of r-Foc1-Pgc4 or r-Foc4-Pgc4, the vascular tissues showed partial necrosis after 5 days. The r-Foc1-Pgc4 induced less necrosis as compared to that by r-Foc4-Pgc4. It seems that r-Foc1-Pgc4 had a lower activity than that of r-Foc4-Pgc4. Sterile double distilled water and 50 mM sodium acetate buffer pH 4.0, used as controls, did not cause any symptoms when injected into the stems of bananas .pgc4, the upstream and downstream sequences of the pgc4 gene were amplified and fused together by PCR. The complete pgc4 gene was replaced by the fused fragment through homologous recombination. The complementation strain, Foc4Δpgc4+pgc4, was generated by cloning the complete pgc4 gene under the control of the native promoter. The knockout and complementation strains were selected in the medium containing the appropriate antibiotics and validated by PCR and Southern blot analyses. On the potato dextrose agar (PDA) plates, the mutant and the complementation strains showed a similar morphology, with their colonies being smaller and more compact than those of the WT. Furthermore, the mutants produced fewer and shorter aerial hyphae, while the WT produced abundant aerial hyphae on PDA plates. The hyphal growth rate of the mutant and complementation strains was lower than that of the WT .Foc4 was obtained from Guangzhou, China, while Foc1 was obtained from Nanning, China. The pathogenicity of the isolate was periodically confirmed by plant assays. The wild-type and mutant strains were cultured and maintained in a PDA culture medium at 25 °C. For pectinolytic enzyme production and RNA extraction, fungal strains were grown in Synthetic Medium (SM)  supplemew/v), 50 mM sodium acetate buffer (pH 4.5), and various amounts of enzyme solution at 50 °C for 30 min. The number of reducing groups, expressed in terms of galacturonic acid (GA), released by enzymatic action was determined according to standard protocols [The PG activity was assayed in a mixture  containing 0.5% PGA  , which contained a cut-off membrane with a molecular weight of 10 kDa. The concentrated filtrate was passed through a gel filtration column  and eluted with 50 mM sodium acetate buffer (pH 4.5) at a flow rate of 1 mL/min. The active fraction containing PG was collected, passed through a cation exchange column , equilibrated with 20 mM sodium acetate buffer (pH 4.5), and eluted with an NaCl gradient (0–0.7 M) at a flow rate of 4 mL/min. The fractions were gathered, passed through another cation exchange column  balanced with 20 mM sodium acetate buffer (pH 4.5), and then eluted with an NaCl gradient (0–0.7 M) at a flow rate of 2 mL/min.The purified Pgc4 was run on SDS–PAGE, transferred onto a polyvinylidene difluoride  membrane, and then submitted for the N-terminal analysis by automated Edman degradation .pgc4-P1 (5′-ACNYTNCCNGCNGGNGT-3′) and pgc4-P2 (5′-GAYATHWSNGARTTYMGN-3′), along with Oligo (dT) 20 primers. The amplification conditions were as follows: 1 cycle of 3 min at 94 °C; 35 cycles of 30 s at 94 °C, 30 s at 52 °C, and 90 s at 72 °C; then 10 min at 72 °C. The amplified fragments were purified and sequenced.For RACE PCR, Foc4 cDNA was synthesized from RNA by reverse transcription-PCR (RT-PCR) with murine leukemia-virus reverse transcriptase . Two primers were designed based on the N-terminal amino acid sequence of Pgc4. The 3′-RACE PCR was conducted using the primers pgc4-F1 (5′-ATGCGCTCCTTGCAAATTATTT-3′) and pgc4-R1 (5′-CTACTGTGGATGGAAAGCGCCC-3′). The amplification conditions were as follows: 1 cycle of 3 min at 94 °C; 35 cycles of 30 s at 94 °C, 30 s at 52 °C, and 90 s at 72 °C; then 10 min at 72 °C.Based on the sequence results of the RACE PCR, the gene sequences and coding sequences of Foc1 and Foc4 were obtained by RT-PCR using the primers pgc4 from Foc4 and Foc1 have been deposited in the GenBank database under the accession numbers MT385837 and MT385838.The nucleotide sequences of pgc4-Myc-His6 was constructed as follows: the pgc4 cDNA was amplified using the forward primer, pgc4-F2 (5′-GAATTCATGCGCTCCTTGCAAATTATTT-3′), and the reverse primer, pgc4-R2 (5′-TCTAGACTGTGGATGGAAAGCGCCC-3′), containing the EcoRI and XbaI restriction sites, respectively. The PCR product was then cloned into the pPICZαA vector  in fusion with a C-terminal Myc and His6 tag. The correct sequence was verified by sequencing. A yeast transformation was performed. SacI was used to digest and linearize the recombinant plasmids, pPICZαA-Foc1-pgc4 and pPICZαA-Foc4-pgc4. The linearized plasmid was transformed into a P. pastoris strain SMD1168 by electroporation. The transformed P. pastoris was grown on a yeast extract peptone dextrose (YPD) plate containing 1% yeast extract, 2% dextrose, 2% peptone, and 100 µg/mL Zeocin  at 28 °C for 48 h.The r-Pgc4 expression vector pPICZαA--pgc4 and pPICZαA-Foc4-pgc4 were inoculated in 10 mL BMGY medium  at 28 °C for 24 h, and then inoculated in 200 mL BMMY . Samples were collected every 12 h. To each sample, 1 mL of 100% methanol was added every 24 h to make sure that the final concentration of methanol was 0.5%, assuming that the methanol would get completely utilized in 24 h. The culture supernatant was analyzed by 10% (w/v) SDS–PAGE, followed by silver staining.The transformants containing pPICZαA-Foc1w/v) SDS–PAGE. The proteins were transferred to a PVDF membrane for a Western blot analysis. The r-Pgc4 protein was detected using an anti-Myc-HRP antibody . The membrane was developed using the chemiluminescent substrate HRP-DAB kit  according to the manufacturer’s instructions.The recombinant protein was extracted from the culture supernatant and purified using a Ni-NTA His Bind resin column following the manufacturer’s instructions . All steps were carried out at 4 °C. The protein concentrations were determined using the Bradford assay and analyzed by 10% (w/v) PGA in 50 mM sodium acetate buffer (pH 4.5), incubated at 50 °C for 10, 20, 30, 40, 50, and 60 min, and subsequently used for a PG activity assay. The hydrolysis of 3% of the substrate by endo-PGs can lead to a 50% reduction in viscosity, while the exo-PGs need to hydrolyze 20% of the substrate for the same reduction in viscosity.To analyze the hydrolysis products, the samples (0.02 U enzyme in 0.5 mL water) were added to 1 mL of 0.5% (Km) and Vmax values were determined from Lineweaver–Burk plots of enzyme activity measured with PGA as substrate at concentrations between 0.25% and 1.25%, at an optimum pH and temperature.The Michaelis constant (w/v) PGA as the substrate. The consequence of temperature on the PG activity was determined in 100 mM potassium phosphate buffer at pH 4.5, between 10 and 90 °C. Three replicates were tested per treatment and negative control.To determine the optimal pH, the PG activity was assayed using 100 mM potassium phosphate buffer with a pH between 3 and 10 at 50 °C and using 0.5%  were taken from the healthy stems of the four-leaf stage banana and placed in test tubes. A mixture of 1 U purified enzyme with 1 mL of 50 mM sodium acetate buffer, pH 5.0, was inoculated with the sterilized banana tissue; the control tubes contained the same buffer without enzyme. The maceration was evaluated after 48 h at 45 °C. The released reducing sugar was calculated from the standards of GalA after incubation for 48 h at 45 °C.To evaluate banana tissue maceration, the cultivars tested comprised For assaying the tissue necrosis, 1 U enzyme was injected into the stems of healthy banana plants. Sterile double distilled water and 50 mM sodium acetate buffer pH 5.0 were used as controls. Five days after each treatment, stems were cut into vertical sections to observe vascular necrosis; ten replicates were used for each treatment.pgc4, we created the vector pTHPRΔpgc4. About 800 bp upstream and 800 bp downstream, flanking sequences of the Foc4 pgc4 gene were amplified by PCR, using genomic DNA as a template, with primer pairs pgc4-upF (5′-TCTAGAGCTTGGGGCACTGTTTCTCA-3′)/pgc4-upR (5′-CCGCGGTGGTACCAGGCATTCTCTTG-3′) and pgc4-downF (5′-CCGCGGCCCCAAGGATGTCACGAACGA-3′)/pgc4-downR (5′-AAGCTTCACTATCATACGGCACGCAATCAA-3′), respectively. The two PCR fragments were linked by overlap the extension PCR using primers, pgc4-upF/pgc4-downR, and the amplified products were cloned into pMD19-T simple vectors to form the plasmid pMD19Δpgc4. Both the pMD19Δpgc4 and pTHPR1 vectors were digested by XbaI and HindIII enzymes. The recombinant vector, pTHPRΔpgc4, was obtained by ligating the digested fragments with T4 DNA ligase. Positive plasmid was confirmed by PCR using the primers, vec-F (5′-CTAACGCCGCCATCCAGTGTC-3′) and vec-R (5′-CGGCGGCGCTCTTGTTCA-3′), followed by sequencing. The positive strain was named as Foc4Δpgc4 and confirmed by Southern blot analysis.For the deletion of Foc4 pgc4 strain with pgc4, a vector pTHPR1-Zeocin was first developed. The pTHPR1-Zeocin vector contained a 1200 bp Zeocin resistance gene fragment in the pPICZαA vector. Foc4Δpgc4 was plated onto the PDA plates with different Zeocin concentrations and cultured at 25 °C. Foc4Δpgc4 could not grow on PDA plates with Zeocin concentrations of 100 and 150 μg/mL. Using the primers pgc4-F3 (5′-GTTTAAACATGCGCTCCTTGCAAATTATTT-3′)/pgc4-R3 (5′-TCTAGACTACTGTGGATGGAAAGCGCCC-3′) and Foc4 genomic DNA as a template, pgc4 was amplified and cloned for complementation. PmeI and XbaI were used for double enzyme digestion. The DNA fragment and pTHPR1-Zeocin, a functional complementation vector, were used for double enzyme digestion and ligated by T4 DNA ligase. Foc4Δpgc4+pgc4 was obtained by PCR confirmation, using the primers pgc4-F3/pgc4-R3.For the complementation of the Foc4Δ6 Foc4 conidia/mL were inoculated in a PDB medium at 22 °C for 3 days. The mycelia were collected, cleaned, and resuspended in 20 mL of phosphate buffer supplemented with 7 mg/mL of lysing enzyme . The protoplastization was performed at 30 °C under agitation at 80 rpm for 4 h. A solution containing the inactivation cassette constructs and 50 μL of a polyethylene glycol solution  was added to 200 μL of the suspension containing 107 protoplasts/mL. The mixture was incubated at 0 °C for 20 min. An additional 500 μL of the PEG solution was added, followed by an incubation at 25 °C for 20 min. The protoplasts were plated on PDA containing 0.56 M saccharose and incubated at 22 °C for 48 h. After protoplast regeneration, 5 mL of a semi-solid PDA medium containing 60 μg/mL hygromycin  was added. Later, the hygromycin-resistant transformants were selected and purified using monosporic isolation.Approximately 106 conidia/mL of the wild-type and mutant strains, maintained in Petri dishes containing paper disks moistened with sterile distilled water, and incubated for 7 days at 22 °C under a photoperiod of 16 h light and 8 h dark. The disease symptoms were assessed after 60 days. To verify the reproducibility of the results, inoculation was performed in triplicate.To test for pathogenicity, detached common bean leaves were inoculated with 10P. pastoris protein expression system. Both could induce tissue maceration and necrosis in the “Guangfen-1” and “Baxi” varieties of banana. The phenotypic assays and complementation analyses revealed that, compared to the wild-type, the generated Foc4Δpgc4 mutant strain showed a lower aerial hyphal growth, grew slower, and had a reduced virulence. Our results demonstrate the function of Pgc4 as a pathogenicity factor of Foc4 and will provide new ideas for the prevention and control of banana wilt.The present study provides the basic characterization of a newly purified Pgc4 protein. Pgc4 from both Foc4 and Foc1 can be produced as a fully functional PG by using the


All these four compounds exerted better inhibitory activities than the positive control . The fluorescence quenching study and kinetic analysis revealed that all these compounds directly bind to α-glycosidase and belonged to the noncompetitive α-glycosidase inhibitors. Then, the binding modes of these four compounds were carefully investigated. Significantly, these four compounds showed nontoxicity (IC50 > 100 μM) toward the human normal hepatocyte cell line (LO2), which indicated the potential of developing into novel candidates for type 2 diabetes treatment.α-Glycosidase inhibitors could inhibit the digestion of carbohydrates into glucose and promote glucose conversion, which have been used for the treatment of type 2 diabetes. In the present study, 52 candidates of α-glycosidase inhibitors were selected from commercial Specs compound library based on molecular docking–based virtual screening. Four different scaffold compounds  were identified as α-glycosidase inhibitors with IC Diabetes is a metabolic disorder that causes high blood sugar and could directly increase the risk of other deadly diseases, such as cancer, stroke, and cardiovascular diseases . AccordiControlling blood glucose levels is thought to be the main strategy for treating diabetes and reducing diabetes complications . α-GlucoVirtual screening has been proven to be a very effective tool capable of providing drug hits or leads with structural diversity and makes drug discovery faster and more efficient . In thisThe protein coordinates in the α-glycosidase crystal complex structure (PDB code 3W37) were prepared by the Protein Preparation Wizard panel inserted in the Maestro with the default settings. Residues within 15 Å centered on acarbose were defined as compound-binding sites in which the docking grid was generated by the Receptor Grid Generation panel. The default settings were adopted for the cutoff, neutralization, etc. The docked compounds in Specs database were prepared with LigPrep panel. Then, the prepared compounds were docked to the aforementioned docking gird with extra precision (XP) mode. “Clustering Molecules” protocol inserted in Pipeline Pilot 7.5 was employed to achieve the cluster analysis. The top ranked compounds assessed by XP GScore were clustered into 30 clusters. To increase the diversity of selected compounds, at least one candidate was selected in each cluster. In addition, we gave priority to the compounds with simple structure and/or small molecular weight.50 values were estimated with six different concentrations, and each sample was measured three times in parallel experiments.The α-glucosidase inhibitory evaluation of the purchased 52 compounds was performed according to the previously described protocol . α-GlucoAccording to the previously reported method , all flu50 values. The inhibitory kinetics of the investigated compounds on α-glucosidase was analyzed using the Lineweaver–Burk plot of the substrate concentration and velocity.The inhibition type of the inhibitors against α-glucosidase activities was evaluated based on a described method . Increas2 at 37°C. Cell suspensions were plated in 96-well plates at a density of 2 × 104 cells/cm3. Compounds were solubilized in DMSO at six different concentrations. After incubation for 24 h, the cells were treated with various concentrations of tested substances for 48 h and then incubated with 100 μL of MTT at 37°C for 2 h. The formazan dye product was measured by the absorbance at 490 nm on a Tecan Spark multimode microplate reader (Switzerland).The LO2 cell line was cultured in a proper medium supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% COAs the crystal structure of α-glycosidase–acarbose complex has been determined (PDB code 3W37) , molecul50 values of these four compounds were further determined. As shown in 50 values of 17.36 ± 1.32, 35.19 ± 2.14, 31.34 ± 3.11, and 9.99 ± 0.43 μM, respectively. All of them showed better activity than the positive reference control 1-deoxynojirimycin , and compound 44 exhibited the most potent activity.The selected 52 candidates were initially evaluated for their inhibitory ratios against α-glycosidase at 100 μM with 1-deoxynojirimycin as positive reference. The α-glycosidase enzymatic inhibition bioassay results indicated that four compounds, namely, 7, 22, 37, and 44 with representing totally different scaffolds, exhibited an inhibition ratio above 50% at 100 μM . Then, tThe interactions of 7, 22, 37, and 44 with α-glycosidase were explored through the fluorescence quenching experiments. As displayed in After treated by compounds 7, 22, 37, and 44 with increasing concentration , the fluKi values of 24.18, 11.34, 11.27, and 15.39 μM, respectively.To explore the mechanism of the interaction modes of compounds 7, 22, 37, and 44 with the enzyme, kinetic assay was conducted to study their inhibition types using Lineweaver–Burk plot analysis . The resThe interaction mechanisms of compounds 7, 22, 37, and 44 with α-glycosidase were carefully analyzed with the molecular docking results, as shown in 50 values more than 100 μM toward LO2 cells, suggesting they are nontoxic toward liver cells. Thus, further structural optimization and biological evaluation for 7, 22, 37, and 44 deserved further investigation.Since most of the drugs are metabolized in the liver, there is a great focus on the hepatic safety of new medicines. Thus, the cytotoxicity of inhibitors 7, 22, 37, and 44 was evaluated in human normal hepatocyte (LO2) cells using the MTT method . The resin vitro evaluation. Among them, compound 44 had the best α-glycosidase inhibitory activity with IC50 and Ki values of 9.99 ± 0.43 and 15.39 μM, respectively. The fluorescence quenching experiment indicated all these compounds could directly bind to α-glycosidase, and the kinetic study revealed a noncompetitive α-glycosidase inhibitory mechanism of these compounds toward α-glycosidase. In addition, binding mode analysis provided the detailed binding mechanism of these four α-glycosidase inhibitors, which made further structural optimization feasible. Moreover, the in vitro cytotoxicity bioassay demonstrated these α-glycosidase inhibitors were nontoxic toward LO2 cells. Based on these results, these compounds can serve as promising hit compounds for further bioactivity optimization and anti–type 2 diabetes study.In this study, four novel α-glycosidase inhibitors 7, 22, 37, and 44 with distinct structural features were identified through virtual screening and


Iron-containing particulate catalysts of 0.1–1 µm size were prepared by wet and ball-milling procedures from common salts and characterized by FTIR, TGA, UV-Vis, PXRD, FEG-SEM, and XPS analyses. It was found that when the wet method was used, semi-spherical magnetic nanoparticles were formed, whereas the mechanochemical method resulted in the formation of nonmagnetic microscale needles and rectangles. Catalytic activity of the prepared materials in the oxidation of 1-phenylethanol to acetophenone was assessed under conventional heating, microwave (MW) irradiation, ultrasound (US), and oscillating magnetic field of high frequency (induction heating). In general, the catalysts obtained by wet methods exhibit lower activities, whereas the materials prepared by ball milling afford better acetophenone yields (up to 83%). A significant increase in yield (up to 4 times) was observed under the induction heating if compared to conventional heating. The study demonstrated that MW, US irradiations, and induction heating may have great potential as alternative ways to activate the catalytic system for alcohol oxidation. The possibility of the synthesized material to be magnetically recoverable has been also verified. Alcohols and related compounds, e.g., diols or benzoins, are among the most abundant and used starting synthetic materials, and their partial oxidation can provide a significant added value to a number of chemical processes, since many aldehydes, ketones, and carboxylic acids are important by themselves and also can be used for further processing ,2,3. GenMany transition-based catalysts are currently used for the mild partial alcohol oxidation , and nonTherefore, in this work, we performed the preparation of new iron-containing dispersed materials by simple one-pot wet, dry, and combined methods, and studied their activity and reusability in the oxidation of secondary alcohols to the corresponding carbonyl-containing compounds under various energy inputs. 1-Phenylethanol was selected as a model substrate, while the conventional and induction heating, MW- and US-irradiations were used to activate the proposed catalytic system.1, “dry”  for 3 and 4, and combined (for 2) methods as described in Experimental section and The catalytic materials were prepared by “wet” (solvent-based) for 1 and 2 (peaks in the 3300–3400 and 1600–1700 cm−1 ranges), while peaks at 500–600 cm−1 can be attributed to the stretching vibration of the Fe-O bond  were recorded in the wavelength range of 200–800 nm using an ISR 2600 plus integration sphere in diffuse reflectance mode using BaSO4 as reference. Spectra were recorded at room temperature, and the data were transformed through the Kubelka–Munk function. Thermogravimetric analyses were carried out with a Perkin–Elmer instrument system  at a heating rate of 10 °C·min−1 under a dinitrogen atmosphere, in the range from room temperature to ca. 800 °C. Diffraction  were collected in a D8 Advance Bruker AXS θ-2θ diffractometer, equipped with a LYNXEYE XE detector and a copper radiation source , operated at 40 kV and 30 mA. A planetary ball mill PM100/200 Retsch GmbH was equipped with a 250 mL grinding bowl, 10 stainless steel balls of 10 mm size and used for the ball-milling preparation of catalysts. FTIR spectra were obtained as diffuse reflectance (DRIFT) measurements on a Nicolet 6700 in the 400–4000 cmMorphology and distribution of metal composites were characterized using a scanning electron microscope  JEOL 7001F  with Oxford light elements EDX detector and EBSD detector X-ray photoelectron spectroscopy (XPS) characterization was performed using a dual anode XSAM800 spectrometer . Spectra were obtained using non-monochromatic Al Kα X-radiation (hν = 1486.6 eV) or Mg Kα X-radiation (hν = 1253.6 eV). Other operating conditions were described elsewhere . For cha−1 to 200 °C and held at this temperature for 1 min. Helium was used as the carrier gas.An Anton Paar Monowave 300 microwave reactor fitted with a rotational system and an IR temperature detector was used for the catalytic studies with a sealed cylindrical Pyrex reaction vessel . The ultrasonic experiments were performed using an ATU ultrasonic thermoregulated bath. For inductive heating, a commercial portable induction hob (model: MX-PIP2195) has been used. Ferromagnetic flat plate (140 × 140 × 1 mm) with square shape and round hole Ø 25 cm in the center has been placed on top of the hob to initiate it. The reactor has been positioned in the center of the hole with stirring from above. GC analysis was undertaken by using a Fisons Instruments GC 8000 series gas chromatograph with a DB-624 (J&W) capillary column , FID detector, and the Jasco–Borwin v.1.50 software. The temperature of injection was 240 °C. The initial temperature was maintained at 120 °C for 1 min, then raised from 10 °C·minFor comparison purposes, the preparation of catalysts  was carr1: In 100 mL round bottom flask, 0.7843 g, 2 mmol of (NH4)2Fe(SO4)2·6H2O and 1.0812 g, 4 mmol of FeCl3·6H2O were added to 80 mL of deionized water and the solution was magnetically stirred (600 rpm) at 50 °C. Total of 1 M NaOH was added dropwise to the solution until pH 10–12. Then the reaction was stopped, the formed precipitate was washed with water and ethanol and dried overnight at 80 °C.2: Total of 0.39 g (1 mmol) (NH4)2Fe(SO4)2·6H2O and 0.54 g (2 mmol) FeCl3·6H2O were treated in ball mill  at room temperature for 10 min, with rotational inversions every 5 min. The thus obtained orange solid was transferred to a glass vial and 2 mL of deionized water were added. Then 1 M NaOH was added until pH 10–12 and the suspension was stirred for 30 min. After that, the formed black precipitate was filtered off, washed with water and ethanol, and dried overnight at 80 °C.3: Total of 3.14 g (8 mmol) (NH4)2Fe(SO4)2·6H2O) and 2.61 g (16 mmol) FeCl3 were mixed and ball milled  at room temperature for 30 min, with rotational inversions every 5 min. The thus formed reddish-brown powder was dried overnight at 80 °C.4: Total of 0.25 g (6.2 mmol) NaOH, 3.32 g (8 mmol) (NH4)2Fe(SO4)2·6H2O) and 2.61 g (16 mmol) FeCl3 were treated in ball mill as indicated above. The formed brown powder was dried overnight at 80 °C.t-BuOOH were added in this order. The tube was then sealed and kept for 1 h at 80 °C under microwave irradiation (5–10 W). Finally, 5 mL of MeCN (to extract substrate and products from the reaction mixture) and 300 µL of benzaldehyde  were added. The obtained mixture was stirred for 5 min and then a sample (1 µL) was taken from the organic phase and analyzed by GC using benzaldehyde as an internal standard method.The microwave-assisted oxidations were performed in a cylindric Pyrex tube to which 5 mmol 1-phenylethanol, 50–500 μmol catalyst and 5 mmol of 70% aq. solution of t-BuOOH were added to a 10 mL Pyrex reactor and the reaction proceeded under US irradiation at 80 °C for 1h. Induction heating was performed as follows: the reaction mixture was added to a 10 mL glass vial as indicated above and the vial was placed on the center of the induction hob covered with a steel plate with a hole. The device was turned on with an induction power of 200 W for 1 h. The temperature was measured at the end of the reaction being ca. 70 °C. For reactions under conventional heating, a 10 mL glass reactor with the reaction mixture analogous to the ones described above was placed into an oil bath and heated for 1 h at 80 °C. At the end the product was extracted with MeCN and analyzed by GC with benzaldehyde used as an internal standard.Under the optimized conditions, several energy inputs were tested. A total of 2.5 mmol 1-phenylethanol, 0.33 mmol catalyst, and 2.5 mmol of 70% aq. solution of 4)2Fe(SO4)2·6H2O and FeCl3·6H2O) catalytically behave differently; the dry mechanochemical preparation, can increase the catalytic activity more than six-fold. It is also demonstrated that the activation of catalysts by ultrasounds and an oscillating magnetic field of high frequency can significantly increase the overall effectiveness of a catalytic system. Finally, the prepared catalysts can be magnetically recovered and reused at least three times.Wet, dry, and combined synthetic methods were used to prepare iron materials with different compositions, particle sizes (0.1–1 µm), shapes , and magnetic susceptibilities (paramagnetic or diamagnetic). Moreover, the materials prepared by wet, dry, and combined methods from the same starting materials (namely (NH


Epithelial-mesenchymal transition (EMT) plays an important role in fibrosis, chronic inflammation, tumor metastasis, etc. Glycyrrhizin, an active component extracted from licorice plant, has been reported to treat a variety of inflammatory reactions through inhibiting high-mobility group box1 (HMGB1), which has been suggested to be a significant mediator in EMT process. However, whether glycyrrhizin affects the EMT process or not remains unclear.β1 to induce EMT. Elisa was used to detect HMGB1 concentrations in cell supernatant. RNA interference and lentivirus infection experiments were performed to investigate the involvement of HMGB1 in EMT process. Cell Counting Kit-8 (CCK-8) was used to detect the viability of A549 and BEAS-2B cells treated with glycyrrhizin. Finally, the effects of glycyrrhizin on EMT changes, as well as the underlying mechanisms, were evaluated via Western blot, immunofluorescence and transwell assays.Human alveolar epithelial cell line A549 and normal human bronchial epithelial cell line BEAS-2B were treated with extrinsic TGF-β1, and knockdown of HMGB1 expression reversed TGF-β1-induced EMT in A549 and BEAS-2B cells. Ectopic HMGB1 expression or TGF-β1 treatment caused a significant increase in HMGB1 release. Notably, we found that glycyrrhizin treatment effectively suppressed TGF-β1-induced EMT process by inhibiting HMGB1. Also, glycyrrhizin significantly inhibited the migration of both A549 and BEAS-2B cells promoted by TGF-β1. Mechanistically, HMGB1 overexpression could activate Smad2/3 signaling in A549 and BEAS-2B cells. Glycyrrhizin significantly blocked the phosphorylation of Smad2/3 stimulated either by TGF-β1 or by ectopic HMGB1 in A549 and BEAS-2B cells.Our results showed that HMGB1 expression was increased by TGF-β1 in lung epithelial cells. Importantly, glycyrrhizin can effectively block Smad2/3 signaling pathway through inhibiting HMGB1, thereby suppressing the EMT progress.HMGB1 is a vital mediator of EMT changes induced by TGF- In the ant role .High-mobility group box1 (HMGB1), a highly conserved nonhistone chromosomal protein in the nucleus, mainly stabilizes the structure of nucleosomes and regulates gene transcription, which can be released when stressed . ExtraceGlycyrrhizin, the main active component obtained from licorice roots, has many pharmacological and biological functions such as protecting liver cells, anti-inflammation, anti-virus, immunomodulation, has been widely applied in the treatment of clinically related hepatic diseases . Glycyrrβ1/Smad2/3 pathway is involved in the EMT process in lung epithelial cells. Our findings indicated that glycyrrhizin suppresses EMT by inhibiting HMGB1 and blocking the downstream Smad2/3 signaling pathway.In this study, we found the crosstalk between HMGB1 and TGF-2. Cells were passaged no more than 15 times after thawing. TGF-β1  was prepared with a buffer containing 0.1% bovine serum albumin (BSA) and the final concentration used was 5ng/ml (β1(5 ng/ml), with different concentration of glycyrrhizin pretreatment for 2 h.Human lung alveolar epithelial cell line A549 and normal bronchial epithelial cell line BEAS-2B were obtained from the American Type Culture Collection . All cell lines were maintained in 1,640 medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin), at 37 °C in a humidified incubator with 5% COs 5ng/ml . Glycyrr3 cells per well) in triplicate and treated with glycyrrhizin (ranged from 25 to 400 µM) or vehicle for 24 or 48 h. Cell Counting Kit-8  was used to detected the cell proliferation rate of each group and 10 µl WST-8 was added into each well. After incubation for 1 h, the optical density (OD) of each hole was measured at 450 nm using an enzyme standard instrument.Cells were seeded into 96-well plate  according to the manufacturer’s instruction. All the samples were analyzed in triplicate. The concentrations of HMGB1 in the samples were determined from standard curve.Cells were seeded into 6-well plate with a density of 2.0 × 104 per well. Cells were transfected with siRNAs  using Lipofectamine 2000  or infected with prepared HMGB1 virus  at different MOI respectively in accordance with the manufacturers’ instructions. The transfection or infection effects were detected by RT-PCR and Western blot. The siRNA sequences are listed as follows: HMGB1-siRNA-1: sense 5′-GGAGAGAUGUGGAAUAACAtt-3′and antisense 5′-UGUUAUUCCACAUCUCUCCtt-3′; HMGB1-siRNA-2: sense 5′-CCAUUGGUGAUGUUGCGAAtt-3′and antisense 5′-UUCGCAACAUCACCAAUGG-3′.Cells were seeded in a 6-well plate with a density of 1.0 × 10β-actin expression. The transcription primers are shown as follows:Total RNA from cultured A549 and BEAS-2B cells was extracted using TRIzol reagent  according to the manufacturer’s instructions. 1 mg RNA samples were used for cDNA synthesis. Amplification was conducted using a SYBR Green I PCR kit . The cycling conditions were as follows: 95 °C for 30s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 20s. The relative expression levels of HMGB1 mRNA were normalized to human HMGB1: forward, 5′-GAGAGGCAAAATGTCATCAT-3′, reverse, 5′- GGGATCCTTGAACTTCTTTT-3′;β-actin: forward, 5′-AGTTGCGTTACACCCTTTCTTG-3′, reverse, 5′- CACCTTCACCGTTCCAGTTTT-3′.β1, p-Smad3 . Gray values for each protein band were analyzed by Image J analysis software.Collected cells were lysed in radioimmunoprecipitation (RIPA) buffer  containing protease inhibitors to extract the protein effectively. Protein concentrations were detected by BCA assay kit . A 40 µg of protein samples were electrophoresed on 10% SDS polyacrylamide gel and resolved to PVDF membrane. Non-specific binding sites were blocked at room temperature for 1 h by 5% non-fat milk dissolved in Tris buffered saline (TBS) containing 0.1% Tween20 or blocking solution containing 1% BSA. Then the membranes were incubated overnight at 4 °C with the primary antibodies including GAPDH , E-cadherin, p-Smad2 , vimentin, HMGB1, TGF-3 cells/ml, followed the incubation as above. Wash the slides with phosphate-buffered saline (PBS), fixed the cells with 4% paraformaldehyde at room temperature for 30 min, and then permeabilized them in 0.1% Triton X-100 for 10min. After being blocked with the goat serum for 1 h, cells were incubated with primary antibody (1:150) overnight at 4 °C. The next day, the slides were washed for three times and incubated with secondary antibody (1:200) for 1 h at room temperature in dark. PBST (PBS containing 0.1% Teen20) were used to washed away the remaining secondary antibodies and then the slides were mounted in antifade solution with DAPI . Images were captured with an Olympus BX51 micro-scope (Japan).Cells were seeded onto glass slides at a density of 8 × 104cells/well) were plated in Matrigel-coated Transwell chambers  with 8 µm pore size polycarbonate microporous membranes. 200 µL serum-free medium was added into the upper chamber while for 600 µL medium with 10% FBS was added into the lower chamber. After incubation for 24 h, the cells migrated the lower surface were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% Hematoxylin for 15 min. Then, the invading cells were photographed by the fluorescence microscope and counted by Image J software.The incubated cells  and Graphpad Prism 7  and were shown as mean ± standard deviation (SD). Difference of P < 0.05 was considered statistically significant.All results were performed at least three separate experiments. Data were analyzed using two-tailed unpaired β1 in A549 and BEAS-2B cells, HMGB1 gene was knocked down by siHMGB1(siRNA-1 and siRNA-2) transfection. The mRNA level of HMGB1 was reduced  for 24 h, with or without different concentrations of glycyrrhizin pretreatment for 2 h. We detected the high level of HMGB1 in cell supernatant in TGF-β1 treated group with Elisa. It indicated that TGF-β1 triggered HMGB1 release to supernatant, while glycyrrhizin treatment could reduce it  has been shown to promote EMT through multiple signaling pathways (β1 can activate the tetrameric complex of T beta R I (TGFR1) and T beta R II(TGFR2), then the post-receptor sensors Smad2 and Smad3 are phosphorylated and combined with Smad4 to form a relatively stable trimeric complex, which can regulate the EMT process through synergistic action with the related transcription factors -dependently (β1-induced EMT of FaDu hypopharyngeal carcinoma cells through activation of RAGE (β1 was suppressed. We had detected the elevated expression of TGF-β1, p-Smad2 and p-Smad3 in the HMGB1 overexpression cells, indicating that HMGB1 overexpression promoted the phosphorylation of Samd2 and Smad3, thereby activating the TGF-β1/Smad2/3 signaling pathway. Moreover, TGF-β1 treatment upregulated the HMGB1 expression and promoted the HMGB1 release into cell supernatant, further confirming that there was a crosstalk between HMGB1 and TGF-β1 signaling in EMT.HMGB1 can be involved in the process, in which TGF-pathways . TGF- β1 factors . It has endently , regulatendently . HMGB1 c of RAGE . In our β1-mediated pathways, including Smad2, Smad3 as an active ingredient in astragalus saponins (β, Snail, and E-cadherin Kinases (β1-induced EMT progress in the two cell lines were suppressed, along with the obstruction of the phosphorylation of Smad2 and Smad3.In recent years, studies on glycyrrhizin are not only limited to its anti-inflammatory and antiviral effects, but also its therapeutic effects on fibrosis. Glycyrrhizin inhibited pro-fibrotic cytokine TGF-saponins , changedsaponins . Glycyrrsaponins . The lat Kinases . To testin vitro. Clinical trials will be carried out if approved, notably, the adverse reactions of glycyrrhizin and the possible interactions with other drugs taken concurrently should be explored.EMT has been proved to be significantly associated with pulmonary fibrosis and lung cancers, providing a new target for clinical drug treatment work. But for now, there are still no specific drugs that can slow down or even reverse the EMT progress. Most previous studies on EMT have focused on the development and metastasis of tumors, however the key role of EMT in the occurrence and development of chronic airway disease should not be ignored. Glycyrrhizin is expected to be put into clinical trials in the adjuvant treatment of airway diseases for those therapeutic effects above. Our current work is at the cellular level, and we chose the appropriate concentrations of glycyrrhizin that did not affect cell viability. We need further animal experiments to verify the results of experiments β1/Smad2/3 pathway related to EMT progress. In the alveolar epithelial cell line A549 and the human bronchial epithelial cell line BEAS-2B, knockdown of HMGB1 expression restrained the EMT process mediated by TGF-β1, while HMGB1 overexpression activated the TGF-β1/Smad2/3 pathway involved in EMT. As a pleiotropic inhibitor of HMGB1, we found glycyrrhizin can inhibit the EMT induced by HMGB1 overexpression. Further study showed that by inhibiting HMGB1 release, glycyrrhizin suppressed TGF-β1-induced EMT progress, blocked the TGF-β1/Smad2/3 pathway and inhibited the cells migration. These findings may provide certain guidance for clinical treatment of EMT-related chronic airway diseases.Our research indicated that there was a crosstalk between HMGB1 and TGF-10.7717/peerj.8514/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.8514/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.8514/supp-3Supplemental Information 3n = 3.Click here for additional data file.10.7717/peerj.8514/supp-4Supplemental Information 4n = 3.Click here for additional data file.