A sequence analysis revealed that PsGR is a protein consisting of 451 amino acids, and homology modeling demonstrated that PsGR has fewer hydrogen bonds and salt bridges, which might lead to improved conformational flexibility at low temperatures. PsGR possesses the flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) binding motifs. Recombinant PsGR (rPsGR) was purified using Ni-NTA affinity chromatography and was found to have a molecular mass of approximately 53.5 kDa. rPsGR was found to be optimally active at 25 °C and a pH of 7.5. It was found to be a cold-adapted enzyme, with approximately 42% of its optimal activity remaining at 0 °C. Moreover, rPsGR was most active in 1.0 M NaCl and 62.5% of its full activity remained in 3.0 M NaCl, demonstrating its high salt tolerance. Furthermore, rPsGR was found to have a higher substrate affinity for NADPH than for GSSG (oxidized glutathione). rPsGR provided protection against peroxide (H2O2)-induced oxidative stress in recombinant cells, and displayed potential application as an antioxidant protein. The results of the present study provide a sound basis for the study of the structural characteristics and catalytic characterization of cold-adapted GR.A new glutathione reductase gene ( E. coli has been carried out [Streptococcus pneumoniae (S. pneumoniae) was overexpressed and purified and its crystal structure was determined [S. pneumoniae has a dimeric structure that includes an antiparallel beta-sheet at the dimer’s interface [In low temperature, psychrophilic bacteria experience a lot of cold-induced stress. Low temperatures impose strict physical and chemical constraints on membrane fluidity, enzyme kinetics, and macromolecular interactions . As a reried out . GR fromtermined . Regarditermined ,12. GR fnterface . Two fornterface . ComparePsychrobacter sp. strain ANT206 is a psychrotrophic bacterium that has been isolated from Antarctic sea-ice; the thiol-dependent antioxidant protein peroxiredoxin from this strain also exhibited special catalytic properties and has the function of protecting super-coiled DNA from oxidative damage [psgr from an Antarctic psychrophilic bacterium. Then, we analyzed psgr by its expression in E. coli, and the recombinant GR was studied in terms of its molecular and biochemical properties and oxidative resistance.In recent years, the Antarctic has attracted attention due to its special climate ,15. The e damage . Here, wpsgr gene revealed an open reading frame (ORF) finder of 1356 bp and psgr encoded a protein consisting of 451 amino acids. The GR from E. coli, Colwellia psychrerythraea (C. psychrerythraea), Sphingomonas sp. (S. sp.), Pseudomonas aeruginosa (P. aeruginosa) encoded 450, 454 [Psychrobacter arcticus GR (90.69%), followed by the E. coli GR (75.17%) and the Streptococcus thermophilus (S. thermophilus) GR (67.18%). In addition, alignment of the amino acid sequence revealed that it has one FAD binding motif as well as one NADPH binding motif (17E) and the NADPH binding motif (GXGYIAX18RX5R) that are typically present in the other GR homologs, such as the GR from Psychrobacter sp. YGAH215 and E. coli.Sequence analysis of the 450, 454 , and 451450, 454  amino acng motif . FurtherE.coli GR, 1GET) were compared and found to superimpose well  and E. coli were found to be 634-fold and 45% [E. faecalis GR (34.43 μmol/min/mg) [Rhodospirillum rubrum (R. rubrum) GR (102 μmol/min/mg) [rPsGR with an approximately molecular mass of 53.5 kDa was found to expressed in oli BL21 , Lane 3,t of ~50  and 48.7t of ~50 , respect and 45%  and 58.3 and 45% . The spe/min/mg)  and Rhod/min/mg) .Chlamydomonas sp. [S. sp. exhibited optimum activity at 60 °C [Phaeodactylum tricornutum [Pennisetum glaucum [Chlamydomonas sp. 23.5% of the highest activity remained at 0 °C [C. psychrerythraea GR were 30 and 40 min [E. coli GR was found to be 2 min at 37 °C [Chlamydomonas sp. approximately 60% of its initial activity remained after incubation at 55 °C for 30 min [E. coli was quite heat stable, denaturing significantly only after incubation for 10 min at 70 °C [S. sp. GR was denatured after incubation at 80 °C for 1 h [onas sp. . Unexpecat 60 °C . The optcornutum  and Penn glaucum  were 32  at 0 °C , suggestat 37 °C . In addir 30 min . rPsGR cat 70 °C . S. sp.  for 1 h . These rS. sp. GR [E. coli GR [Chromatium vinosum (C. vinosum) GR (pH 7.0) [E. coli S33 was stable at pH values ranging from 7.5 to 9.5 [Chlamydomonas sp. GR only lost 10% of its highest activity at a pH of 5.0 after incubation for 30 min [Phaeodactylum tricornutum was found to decrease significantly above a pH of 8.5 [E. coli S33 GR activity was noticed at pH values below 7.5 [E. coli GR showed almost complete inactivation at pH values below 5.5 and over 7.5 [On the basis of the above results, the optimal pH for rPsGR activity was analyzed at 25 °C c. The in. sp. GR  and E. c coli GR . Differe(pH 7.0) . After i5 to 9.5 . rPsGR mr 30 min . rPsGR aH of 8.5 . Moreoveelow 7.5 , and theover 7.5 .Setaria cervi bovine filarial worms were found to exhibit maximum activity between 0 and 0.2 M NaCl [Based on the above described single-factor experimental results, we used the optimal enzyme activity assay system (with the standard method) to study the effects of various factors on enzyme activity. Enzyme activity was affected by the presence of various NaCl concentrations e. The ma2 M NaCl , which i2 M NaCl .2. This increase in activity suggests that Mg2+ is important to the activity, similar results were obtained for the GR from Chlamydomonas sp. [2 (0.25 mM) slightly inhibited rPsGR activity. K+ had also been found to inhibit Setaria cervi GR activity [2, CuCl2, and HgCl2 strongly inhibited the activity of rPsGR. The addition of 0.25 mM CuCl2 to C. vinosum GR inhibited its activity by 33% [2 to rPsGR and C. vinosum GR inhibited their activity by approximately 67% and 10% [2. Pb2+ and Cu2+ were found to have significant inhibitory effects on rPsGR. These two metal ions were also found to inhibit the activity of GR from Chlamydomonas sp. [The effect of different additives on rPsGR activity were measured . For theonas sp. . Furtheractivity . Additioy by 33% . The add and 10% , respectonas sp. .Vm for GSSG was 172.41 mmol/min/mg , mesophilic baker’s yeast (3.9 μM) [E. coli K-12 (4.8 μM) [Km for NADPH from S. pneumoniae (23.2 μM) [E. coli GR (25 μM) [Xanthomonas campestris (X. campestris) (52.6 μM) [Km for GSSG was 68.96 μM, which is lower than GR from C. vinosum (7 × 103 μM) [Km for NADPH (16.95 μM) was lower than the value of GSSG (68.96 μM). This result indicates that the rPsGR had a higher substrate affinity for NADPH than for GSSG, which is consistent with the general trend [kcat is a fundamental kinetic parameter that characterizes an enzymatic reaction and the enzymatic reaction rate is given by the catalytic constant kcat [kcat for rPsGR at 25 °C was 94.97 1/s. A kcat for E. faecalis of 145 1/s [C. psychrerythraea of 700 1/s have also been reported [The kinetic parameters of rPsGR were determined with GSSG and NADPH as substrates at 25 °C . The Vm l/min/mg , and Vm nditions ,34. At 2(4.8 μM) . The Km  (25 μM) , and Xan 103 μM) . Further 103 μM) . The Km ant kcat . The kcaeumoniae .2 μM [E.G is, the more stable the enzyme is. The ΔG values of the NADPH substrate increased from 60.07 to 61.80 kJ/mol as the temperature increased from 0 to 25 °C, suggesting that the thermostability of the rPsGR increased as the temperature increased. This trend was also observed in other cold-adapted enzymes [H, showed a downward trend from 0 to 25 °C. The ΔH of GR from Red Spruce was found to be 54 and 43 KJ/mol at 10 and 35 °C, respectively [H, which allows them to increase kcat at low temperatures [H as a cold-adapted xylanase [kcat value at 0 °C that was approximately 4.98 times lower than that obtained at 25 °C, and kcat value of GR from Red Spruce at 10 °C was found to be eight times lower than that at 35 °C [Thermodynamic parameters of the rPsGR were calculated at different temperatures . The fre enzymes ,38. Addiectively , withouteratures . It has at 35 °C . Therefo2O2 were performed by a disk diffusion assay  were significantly smaller than those of the controls (on the plates with a diameter of 4.0 cm). These results demonstrate that rPsGR had an antioxidant effect and overcame the oxidative stress induced by H2O2. Thus, rPsGR might have application as a potential source of antioxidant proteins in the pharmaceutical and food industries. Similarly, GR from the oxygen-tolerant Streptococcus mutans was also shown to protect cells from oxidative stress induced by H2O2 [To determine the oxidation resistance of GR in recombination strains, the sensitivities of BL21/pET-28a(+) and BL21/pET-28a(+)-rPsGR to Hon assay . After t by H2O2 .The strains used in this study are listed in Psychrobacter sp. ANT 206 (GenBank accession numbers MK968312), isolated from a sample of Antarctic sea-ice , was cultured in 2216E sea water medium  and 1 g of yeast extract powder  per 1 L of solution, pH 7.5) at 12 °C with shaking at 200 rpm and then frozen in glycerol at −80 °C for long term storage. Psychrobacter sp. ANT206 was used as a source of chromosomal DNA. All strains were stored in 16% glycerol at −20 °C, BL21/pET-28a(+) and E. coli BL21 were cultured in Luria Bertani (LB) medium , 5 g of yeast extract powder, and 10 g of NaCl  per 1 L of solution, pH 7.0) at 37 °C.psgr coding sequence by PCR: A forward primer with the BamH I site 5′-ATCGGATCCATGACAAAACATTATG-3’ and a reverse primer with the Xho I site 5′-TAACTCGAGAGCGCATCGTCACAAA-3′. PCR was performed with Taq DNA polymerase and consisted of initial denaturation at 94 °C for 5 min, and then 30 cycles of denaturation at 94, 59.1, and 72 °C for 1 min, with a final extension at 72 °C for 10 min. The reaction was performed in 20 µL of solution containing 13.8 µL of H2O, 2 µL of 10 × PCR buffer (Mg2+ plus), 0.125 mM dNTP mixture, 0.5 µM of each primer, 1 µL of template DNA, and 1 U of Taq DNA polymerase. All reagents used in PCR were obtained by TaKaRa Bio . The complete amino acid sequence and the multiple sequence alignments of PsGR were obtained by ORF and the BioEdit and EScript tools , respectively.According to the genomic sequence and the annotation of ANT206 (manuscript in preparation), genomic DNA was used as a template and the following primers were used to amplify the E. coli capable of encoding 450 amino acids was selected as a template for a 3D model, and the quality of the model was evaluated by a Ramachandran plot analysis. The 3D molecular viewer PyMOL  was used to generate homology model images. Meanwhile, ESBRI  was used to investigate salt bridges, and protein electrostatic interactions were predicted by the Protein Interactions Calculator program . The 3D structures of the NADPH and GSSG ligands were obtained from the Zinc database  and Open Babel was used to change sdf files to pdb files. To study the interactions between proteins and ligands, AutoDock Vina [The 3D structure of the PsGR was obtained using a SWISS-MODEL server. GR  in ock Vina  by the dBamH I  and Xho I  to insert the psgr gene, as well as to create a nick in the expression vector, T4 DNA ligase  was used to connect the psgr gene and pET-28a(+) for 12 h at 16 °C. Then, the ligation system was added to BL21 competent cells  and blown evenly. After that, ice bath for 30 min, heat at 42 °C for 90 s, and then placed in an ice bath for 2 to 3 min. Then, it was transferred to 1 mL of LB medium and incubated at 37 °C for 1.5 h. The bacterial solution (200 μL) was applied to a kanamycin (100 µg/mL)-resistant LB solid plate and cultured overnight at 37 °C. Then, the recombinant bacterium was cultured in LB medium at 37 °C until the optical density at 600 nm (OD600) was 0.4–0.6. Expression was induced by addition of 0.5 mM isopropyl β-D-thiogalactoside  and the induction was continued for an additional 24 h at 25 °C. After the cells were induced, they were centrifuged to obtain inclusion bodies. Then, the inclusion bodies were washed twice with 20 mM potassium phosphate buffer (pH 8.0), containing 1 mM dithiothreitol, 1mM EDTA, and 10 μM FAD, and then treated with 8 M urea for 1 h and centrifuged  for 15 min, and the supernatant containing crude extract of recombinant PsGR (rPsGR) was recovered. The resulting supernatant was loaded into a Ni-NTA affinity chromatography  column equilibrated with a buffer , and the proteins were eluted with an elution buffer . Then, the proteins were separated by SDS-PAGE (12.5% polyacrylamide), and the purity and the molecular weight of the rPsGR were estimated.The PCR product and the pET-28a(+) vector were digested using the restriction enzyme GR activity was measured by monitoring the oxidation of NADPH in the reaction mixture (200 μL) for 2 min at 340 nm and 25 °C. The reaction mixture contained 100 mM potassium phosphate  buffer (pH 7.5), 0.1 mM NADPH , 1.2 mM GSSG , and 12.8 µg purified rPsGR. One unit of enzyme activity was defined as the amount of GR that oxidized 1 μmol NADPH per min. The remaining activity was detected using an ultraviolet spectrophotometer  and a quartz cuvette  with a path length of 0.1 cm.2HPO4/NaH2PO4 (pH 6.0–8.5) buffer. As for its pH stability, rPsGR was incubated at 25 °C for 30 min in buffers with various pHs, and the remaining activity was tested. To assay its salt tolerance, rPsGR was incubated in 0–3.0 M NaCl at 25 °C for 30 min, and the residual activity was determined. Using the standard method, the effect of adding various reagents of 0.25 and 1 mM  was examined. First, KCl, CoCl2, MgCl2, CaCl2, ZnCl2, FeCl2, CuCl2, HgCl2, CrCl2, CdCl2, Pb(NO3)2, BaCl2, and EDTA were mixed with purified rPsGR at 25 °C for 30 min, respectively, and then 100 mM potassium phosphate buffer, 0.1 mM NADPH, and 1.2 mM GSSG were added. The above experiment was carried out three times. Relative activity was expressed as a percentage of the control enzyme’s activity. Furthermore, all temperatures were controlled using a temperature control system JASCO PTC-343 . All chemicals were used without further purification.The purified rPsGR (10.6 µg) was used in a reaction system and the enzymatic properties were investigated. rPsGR activity was determined at different temperatures (0–45 °C) and the optimal temperature of rPsGR was obtained using a standard method described above. The temperature stability of rPsGR was analyzed by pre-incubating the recombinant protein for 60 min at 25 to 55 °C, and the residual activity was detected by the standard method. In order to investigate the optimal pH of rPsGR, two buffers were used for enzyme activity by running the standard assay at 25 °C, and they were the NaAc/HAc buffer (pH 4.5–6.0) and Nakcat values were obtained at 0–25 °C, and then based on previously described methods [H, ΔS, and ΔG) for rPsGR using NADPH as a substrate were determined based on the kcat values. The experiment was carried out three times.The kinetic parameters of the purified rPsGR (12.5 μg) were determined from the secondary plots of intercepts versus reciprocal concentrations of GSSG  and NADPH  at 0–25 °C. Different  methods , the theE. coli BL21 with pET-28a(+) (BL21/pET-28a(+)) and E. coli BL21 with pET-28a(+) containing GR (BL21/pET-28a(+)-rPsGR), a disk diffusion analysis was conducted. The IPTG and low temperature (25 °C) induced bacterial cultures were spread on top of LB agar plates. After placing two disks (with a diameter of 3 mm) of Whatman filter paper  on the plate, 1.5 and 3 μL 30% of H2O2 were added onto the disks. Furthermore, after the plates were treated for 12 h at 37 °C, the cleared zones that reflected the oxidation resistance were measured. The experiment was carried out three times.In order to compare the survival rates of psgr gene of Psychrobacter sp. ANT206, overexpression of the protein in E. coli, and characterization of the rPsGR. The analysis of the homology modeling showed that PsGR exhibited a decreased number of hydrogen bonds and salt bridges. Therefore, rPsGR showed maximum activity at 25 °C, which proves it to be a cold-adapted enzyme. It was shown to have a high salt-tolerance (1.0 M NaCl). Thus, rPsGR is resistant to low temperatures and high concentrations of NaCl. In addition, the GR in recombinant strains might provide protection to these strains against H2O2-oxidative stress. Taken together, our results indicate that rPsGR is a novel cold-adapted GR and further reveals its structural characteristics and catalytic properties.The present study described the cloning of the


Cordyceps cicadae (CC) has been used as a traditional medicine for improving renal function via its anti-inflammatory effects. N6-(2-hydroxyethyl)adenosine (HEA), a physiologically active compound, has been reported from CC mycelia (CCM) with anti-inflammatory effects. We hypothesize that HEA could protect human proximal tubular cells (HK–2) from NSAID-mediated effects on differential gene expression at the mRNA and protein levels. To verify this, we first isolated HEA from CCM using Sephadex® LH–20 column chromatography. The MTT assay revealed HEA to be nontoxic up to 100 µM toward HK–2 cells. The HK–2 cells were pretreated with HEA (10–20 µM) and then insulted with the NSAIDs diclofenac  and meloxicam  for 24 h. HEA (20 µM) effectively prevented ER stress by attenuating ROS production (p < 0.001) and gene expression of ATF–6, PERK, IRE1α, CDCFHOP, IL1β, and NFκB within 24 h. Moreover, HEA reversed the increase of GRP78 and CHOP protein expression levels induced by DCF and MXC, and restored the ER homeostasis. These results demonstrated that HEA treatments effectively protect against DCF- and MXC-induced ER stress damage in human proximal tubular cells through regulation of the GRP78/ATF6/PERK/IRE1α/CHOP pathway.Nonsteroidal anti-inflammatory drugs (NSAIDs) belong to a class of universally and commonly used anti-inflammatory analgesics worldwide. A diversity of drawbacks of NSAIDs have been reported including cellular oxidative stress, which in turn triggers the accumulation of unfolded proteins, enhancing endoplasmic reticulum stress, and finally resulting in renal cell damage.  Although NSAIDs have long been considered as safe medication practices in the treatment of inflammatory diseases, NSAIDs have received much special attention in the past few years due to the adverse effects in the inhibition of prostaglandin synthesis . ProstagAs one of the critical organelles, endoplasmic reticulum (ER) carries out fundamental biological functions including protein folding, protein modification, lipid synthesis and calcium signaling . Under sDCF-induced acute renal kidney through induction genomic DNA fragmentation and necrotic cell death of proximal and distal convoluted tubules . SimilarCordyceps cicadae (CC) contains abundant biologically active compounds including polysaccharides [6-(2-hydroxyethyl)-adenosine (HEA) [charides ,13, adencharides , N6-(2-hne (HEA) , and ergne (HEA) . It has ne (HEA) . In vivone (HEA) . CC polyne (HEA) . CCP signe (HEA) . Prelimine (HEA) .2+ antagonist and an anti-inflammatory agent [HEA is a Cary agent ,21,22. Iry agent . Recentlry agent ,23,24,25ry agent . Controvry agent , triggerry agent , while ary agent .The CCAAT/enhancer-binding proteins (C/EBP) are a family of transcription factors strongly implicated in the control of genes involved in intermediary metabolism. The C/EBP homologous protein (CHOP) is considered a key event for endoplasmic reticulum (ER) stress-mediated ,30. WithWhether HEA can protect the kidneys from ER stress resulting from treatment of DCF and MXC has never been documented. We propose that HEA could be beneficial to the prevention of renal damage via suppressing the CHOP-related ER stress, and this work is conducted to uncover such a mechanism of action, to point out the shortcomings of current NSAIDs therapy and comment the possible alternative treatments.w/w, denoted hereafter as CC-M2) crude extract. After the HPLC analysis, the crude extract prepared from CC mycelia was shown with a major compound of HEA . At 24 h, AFT6, IRE1α, PERK were stimulated by 250-fold, 1.5-fold and 8.5-fold, respectively, which were ameliorated by HEA down to ≤3-fold, 1.0-fold, and ≤2.5-fold, respectively (p < 0.001). In contrast, at 24 h, the levels of CHOP and IL–1β were upregulated to 20-fold and 400-fold, respectively. While CHOP was suppressed by HEA 20 µM to 17-fold, and to less than 2-fold by HEA 20 µM (p < 0.001) (p < 0.001)  , panel h< 0.001) .p < 0.001), 680-fold, and 180-fold respectively compared to the control (p < 0.001) (p < 0.001). After 24 h, ATF and PERK were completely attenuated by HEA (p < 0.001). CHOP expression was not completely inhibited until the concentration of HEA reached at 20 µM. Instead, IL–1β once upregulated by 9.3-folds was attenuated to 2.4-fold and normal value after being treated with HEA 5 µM and 20 µM, respectively (p < 0.001).In contrast, MXC behaved quite differently. At 2 h of treatment, ATF6, PERK, and CHOP were all highly upregulated after being treated with MXC 400 µM, reaching 27-fold (< 0.001) , panel bectively , panel gAs described above, most of the genes’ expressions, including AFT6, IRE1α, PERK and IL–1β, were upregulated by eliciting MXC 400 µM and DCF 200 µM; only the CHOP gene expression remained the same in the MXC 400 µM treatment at hour 24 . We inveC. cicadae mycelia extract, CC-M2) and a purity of 95.40%   C.Cyclooxygenases (COXs) occur in two forms: the constitutive (COX–1) and the inducible COX–2) isoforms. COX–1 could provide cytoprotective effects, whereas COX–2 is both inducible and the major isoform for inflammatory responses  isoforms. At low ER stress was associated with some deleterious events including ROS overproduction and apoptosis . We obseIn the cells, the major source of reactive oxygen species (ROS) is the microsomal monooxygenase (MMO) system, which is composed of cytochrome P450 (P450), NADPH-dependent cytochrome P450 reductase (NPR) and phospholipids ,36. ElecNSAIDs have been shown to induce reactive oxygen species (ROS) in different cell types including gastric cancer cells ,38. DCF Apparently HEA (10–20 µM) alone was a good antioxidant, rather than a prooxidant. Pretreatment with HEA (20 µM) abrogated the ROS production stimulated by these two medicines to only 1.7-fold that of the control HEA , indicatWhen an ER stress occurs, the cell initiates an adaptive response called the unfolded protein response (UPR), the role of which is to maintain protein homeostasis by decreasing the load of unfolded proteins and increasing the protein folding capacity ,40. ER sIn response to endoplasmic reticulum (ER) stress, the REDD1 (regulated in development and DNA damage responses) is transcriptionally upregulated through a mechanism involving the activation of PERK, phosphorylation of eIF2α, and increased ATF4 expression , promotiCHOP plays an important role in the induction of apoptosis. As mentioned, without ER stress, CHOP expression is low. Abrupt and remarkable expression can be induced in response to ER stress through IRE1-, PERK- and ATF6-dependent transcriptional inductions , evidencThe overexpression of CHOP promotes apoptosis in several cell lines, whereas CHOP-deficient cells are resistant to ER stress-induced apoptosis ,30, poinTaken together the results from The IL–1β gene is involved in the proliferation, differentiation and apoptosis of cells. We showed that the IL–1β gene was upregulated by DCF, while HEA downregulated its expression .iNOS and stimulating production of NO. Fattori et al. raised the first report demonstrating that DCF induced kidney cell apoptosis, upregulated proinflammatory cytokines, and induced the activation of NF-κB in renal tissue, resulting in acute kidney injury [−7–1 × 10−6 molL−1) decreased both endogenous and TNFα-induced NFκB activation [iNOS) gene expression to produce NO, which protected hepatocytes from TNF-α- and Fas-mediated apoptosis [According to a previous report, the upregulation of IL–1β with interleukin–1 receptor-associated kinase–2 (IRAK2) is responsible for triggering ER stress ,47 and ly injury  also demtivation . NFκB stpoptosis ,51. Combpoptosis .To summarize, HEA was beneficial in protecting from renal damages caused by NASIDs, DCF and MXC. Pretreatment with HEA could maintain or restore the ER homeostasis via suppressing ER stress-related elements including ATF6, PERK, IRE–1α, CHOP, ROS, IL–1β, and NFκB.Antibody against HRP (horseradish peroxidase)-conjugated goat anti(rabbit IgG) antibody (ab97051) was purchased from Abcam . β-Actin (tcba13655), GRP78 (tcea19663) and CHOP (tcba1658) were purchased from Taiclone . Diclofenac (DCF) and meloxicam (MXC) were obtained from Novatis  and Boehringer Ingelheim Pharma GmbH & Co. KG , respectively. MTT and DCFH-DA were purchased from Sigma-Aldrich .C. cicadae (BCRC MU30106) was gifted by the Grape King Biotech Co. . HK–2 (ATCC®CRL–2190TM) (human kidney–2) cell line, a proximal tubular cell (PTC) line derived from normal kidneys, was purchased from the Biosource Collection and Research Center .The fungal strain C. cicadae (BCRC MU30106) was grown on PDA, transferred to 500 mL flask containing basal medium 200 mL, and incubated at 25 °C with continuous agitation at 120 rpm for three days. The culture was transferred into a 100 L fermenter maintained at 25 °C for four days while continuously stirred at 150 rpm. The fermentation broth was centrifuged and the supernatant was filtered under pressure through a filtering press. The filtrate was lyophilized to obtain the crude mycelial powder [l powder .2) under 5000 psi at 60 °C for 1 h, then subjected to solvent extraction with boiling water (1:10 w/v) for 2 h, filtered through 0.22 µm microporous membrane to obtain the aqueous extract which was evaporated under vacuum to one tenth original volume (designated as CCM–1). The filtrate was concentrated under vacuum on a rotary evaporator and freeze-dried (CCM–2). The obtained sample (CCM–2) was then subjected to column separation using the Sephadex® LH–20 column (id × ℓ = 1.5 × 30 cm) and eluted with double distilled water at a flow rate 1 mL/min. The eluent was collected, each fraction from 3 min, until a total of 40 fractions. The OD at 260 nm was used to detect the absorbance of each tube. The fractions from fractions 33–35 were pooled and subjected to further HPLC-ESI-(+)-MS/MS analysis to identify the purified HEA, and then lyophilized (1.07 ± 0.04 mg/g dry weight). The obtained fraction was used for biochemical analyses of HK–2 cells.The mycelial powder was defatted using supercritical extraction carbon dioxide  and attached to a UV detector (Hitachi L–7455) monitored at 260 nm. The injection volume was 20 µL, the column was maintained at temperature 35 °C, and the flow rate was 0.3 mL/min. The mobile phase was composed of two reagents: phases A and B. Mobile phase A was 10 mM ammonium formate . Mobile phase B was acetonitrile containing 0.1% formic acid. The programmed elution condition was: at min 0, A:B = 100:0; at min 3, A:B =100:0. At min 10, A:B = 85:15; at min 15, A:B = 5:95; at min 25, A:B = 5:95; at min 30, A:B =100:0, and at min 50, A:B = 100:0. The compounds having been eluted and separated were further identified with triple quadruple (QQQ) mass spectrometer  in positive ionization mode with the operating parameters as follows: nitrogen used both as a drying gas at a flow rate of 10 L/min and as a nebulising gas at a pressure of 30 psi, drying gas temperature 325 °C, and a potential of 3500 V applied across the capillary, fragmentor voltage 90 V, and the collision voltage 15 V. Quadrupole 2 was applied for the scanning ions produced by nitrogen collision of ionized compounds in the range 100–800 2 at 37 °C. The culture medium comprised of Dulbecco’s Modified Eagle Medium: Nutrient Mixture F–12 medium (DMEM/F–12) containing 10% of fetal bovine serum and 1% penicillin/streptomycin, which were all purchased from Gibco/Thermo Fisher Scientific, Inc. .HK–2 cells were incubated in a humidified atmosphere with 5% CO3 cells/well were seeded onto a 96 well-plate and incubated for 24 h. To each well medium containing different concentration of extracts with DCF (100–500 µM), MXC (100–500 µM), and/or HEA (1–1000 µM) was added as indicated. MTT  were separately added at hour 24, 48, 72, and 96 and left to stand for 2 h to facilitate the reaction. After the supernatant was sucked off, DMSO was added to dissolve the product formazan crystals. The optical density was read at 570 nm with ELISA Reader .Method of Mosmann et al.  was foll4 cells/mL and incubated for 24 h. The cells were pretreated with HEA at dose 5, 10, and 20 µM, respectively, for 30 min. Drugs DCF (200 µM) or MXC (400 µM) was added as indicated and incubated for additional 4 h. The used culture medium was sucked off, replaced with fresh medium containing 10 µM DCFH-DA, and further incubated at 37 °C for 30 min to facilitate the reaction. The medium was sucked off. The cells were rinsed twice with cold PBS and observed under an invert Olympus IX71 microscope.HK–2 cells were seeded onto a 12-well plate at a density of 1 × 104 cells/mL were seeded onto a 6 cm petri dish for 24 h. Cells were pretreated with HEA at 10 and 20 µM for 2 h. DCF (200 µM) and/or MXC (400 µM) was added, and the incubation was continued for an additional 24 h. The medium was sucked off and the cells were rinsed with 2 mL PBS. We added 0.05% trypsin 0.02% EDTA to detach the cells, which were collected and centrifuged at 4 °C under 1000× g for 5 min. The supernatant was discarded. The sediment cells, rinsed with 3 mL ice cooled PBS, were suspended in 1 mL ice cooled PBS and stained with a mixture containing 100 µL 1× Binding buffer, 5 µL Annexin-V and 5 µL propidium iodide. The cells were dispersed homogeneously, left to stand at 4 °C for 30 min avoiding direct sunlight, and immediately immersed in ice and subjected to flowcytometric analysis using the BD Accuri™ C6 Flow Cytometer . The excitation and the emission wavelengths used were λex = 488 nm; and λem = 610 nm, respectively.HK–2 cells at a density 5 × 105 cells/mL were seeded onto a 6 cm dish and incubated for 24 h. Cells were pretreated with HEA at 10 and 20 µM for 2 h, respectively. DCF (200 µM) and MXC (400 µM) were, respectively, added and incubated for 24 h. The used culture medium was sucked off and the cells were rinsed twice with ice-cooled PBS. TRIzol® Reagent  (1 mL) was added. The culture was transferred into an Eppendorf vial, 200 µL chloroform was added and vortexed for 10 min, left to stand for 15 min and centrifuged at 13,000× g for 15 min. The supernatant (400 µL) was transferred into an Eppendorf vial, 600 µL isopropanol was added, shaken up and down, then left to stand for 30 min, centrifuged at 4 °C under 13,000× g for 15 min, and the supernatant was discarded. The sediment was rinsed with 500 µL ethanol (70%), centrifuged at 4 °C under 13,000× g for 20 min. The supernatant was discarded. The sediment was redissolved in diethylpyrocarbonate (DEPC) (ThermoFisher) water and stored at −80 °C for use.The HK–2 cells at a density 4 × 10TM RT Reagent Kit  . The operation protocol was carried out by following the instructions given by Takara PrimeScriptCA, USA) . The fin® FAST (KAPA biosystems). The 20 μL reaction mixture contained 9.2 μL of cDNA, 0.4 μL of 10 μM forward and reverse primers, 10 μL of KAPA SYBR FAST qPCR Master Mix (2×). Amplification was performed in an StepOnePlus™ Real-Time PCR System . The DNA fragments were amplified for 40 cycles . The expression of β-actin was determined as the internal control. Relative expression level was calculated using the 2Ct−∆∆ method.An equal amount of cDNA was used for the subsequent qPCR performed with the SYBRg for 5 min. The supernatant was separated and the protein content was determined and frozen at −30 °C until use. An aliquot of the supernatant, containing 20–30 µg of protein, was measured and mixed with 1/5 × Laemmli sample buffer. The protein samples were then separated on a 10% SDS-PAGE and electroblotted to the nitrocellulose membranes. After blocking with TBS buffer containing 5% nonfat milk, the membrane was incubated overnight at 4 °C with various specific antibodies including GRP78 , CHOP  and β-actin , followed by treatment with horseradish peroxidase-conjugated antimouse IgG. The results were visualized with the ECL chemiluminescent detection kit  and quantified by with the Image J gel analysis software.Following our previous report , the expp < 0.05 was considered to be statistically significant.Data acquired in this study were presented in a mean ± SD manner and further analyzed by soft GraphPad Prism Program . One way analysis of variance (ANOVA) was used for analysis of variations in each group. Tukey’s post hoc test was used for analysis of the significance of difference among the means. A confidence level ® LH–20 column chromatography was successfully applied to purify HEA from CC mycelia crude extract for the first time. HEA has been revealed with promising protective capability against NSAIDs-induced ER stress in kidney cells. The pathway, as shown in In this report, an efficient, simple, and cost-effective Sephadex


Acinetobacter junii LH-1-1 and Klebsiella pneumoniae BPBA052 during deltamethrin and 3-PBA degradation were established, respectively. When the inoculum proportion of the strains LH-1-1 and BPBA052 was 7.5:2.5, and LH-1-1 was inoculated 24 h before inoculation of strain BPBA052, 94.25% deltamethrin was degraded and 9.16 mg/L of 3-PBA remained within 72 h, which was 20.36% higher and 10.25 mg/L lesser than that in monoculture of LH-1-1, respectively. And the half-life of deltamethrin was shortened from 38.40 h to 24.58 h. Based on gas chromatography–mass spectrometry, 3-phenoxybenzaldehyde, 1,2-benzenedicarboxylic butyl dacyl ester, and phenol were identified as metabolites during deltamethrin degradation in co-culture. This is the first time that a co-culture degradation pathway of deltamethrin has been proposed based on these identified metabolites. Bioremediation of deltamethrin-contaminated soils with co-culture of strains LH-1-1 and BPBA052 significantly enhanced deltamethrin degradation and 3-PBA removal. This study provides a platform for further studies on deltamethrin and 3-PBA biodegradation mechanism in co-culture, and it also proposes a promising approach for efficient bioremediation of environment contaminated by pyrethroid pesticides and their associated metabolites.Deltamethrin and its major metabolite 3‐phenoxybenzoic acid (3‐PBA) have caused serious threat to the environment as well as human health, yet little is known about their degradation pathways by bacterial co-cultures. In this study, the growth and degradation kinetics of  Co-culture was used to degrade both deltamethrin and its toxic intermediate metabolite 3-PBA.Co-culture was more efficient in degradation of deltamethrin and 3-PBA than the monoculture.A degradation pathway in co-degradation of deltamethrin and 3-PBA was first reported.Deltamethrin-contaminated soils could be efficiently bioremediated by co-culture.Pyrethroid pesticides (PPs) are a type of synthetic insecticides, which are widely used in agriculture, forestry, horticulture, public health, and indoor places to control pests, cockroaches, mosquitoes, and livestock parasites because of their broad spectrum, high efficiency, and low mammalian toxicity as compared to that of organophosphate pesticides  was 75.60% in 72 h and the content of 3-PBA was 10.31 mg/L. As compared with the degradation by strain B-1 alone, the degradation of cypermethrin increased by 24.52% and 3-PBA content reduced by 12.5 mg/L in case of degradation of cypermethrin in co-culture. However, co-culture pathways for degradation of deltamethrin and its intermediate metabolites have remained largely unexplored.Currently, biodegradation has become a mainstream technology for removal of pesticide residues or pesticide metabolites, as it is eco-friendly, cost-effective, convenient, and possesses excellent degradation properties  was purchased from Ronch Chemicals , 3-PBA standard (98.0%) and chromatographic-grade acetonitrile were obtained from TCI  and Adamas-Beta , respectively. Other chemicals were analytical grade and commercially available.2PO4, 1.5 g/L K2HPO4, 1.5 g/L (NH4)2SO4, 0.2 g/L MgSO4, 0.5 g/L NaCl, 0.01 g/L FeCl3, and supplemented with 3 g/L tryptone as extra carbon source, the pH was adjusted to 7.0. At each stage, pre-cultures were incubated in Luria–Bertani , containing 0.5 g/L KHA. junii LH-1-1  was isolated from strawberry rhizosphere soil and could degrade 82.36% of deltamethrin (75 mg/L) at 30 °C in OMSM after incubation for 96 h. Klebsiella pneumoniae BPBA052  was obtained from the soybean rhizosphere soil, and it could degrade 96.37% of 3-PBA (100 mg/L) within 72 h in mineral salt medium , Xm represents the maximum cell concentration (OD600), t represents the culture time (h), and μm represents the maximum specific growth rate (h−1). C0 is the initial substrates concentration (mg/L), Ct is the substrates concentration at time t (mg/L), t is the degradation time (h), k is the degradation rate constant (h−1), and t1/2 is the half-life of substrates. Inoculum (5.0% v/v) was added to 100 mL OMSM containing 75 mg/L substrates in a 250 mL Erlenmeyer flask, OD600 and substrates concentration were measured at regular intervals. Considering that a growth and degradation kinetics model can predict the status of degrading-strain and residue of pollutants in the future , strains LH-1-1 and BPBA052 were mixed at a proportion of 10:0, 7.5:2.5, 6.7:3.3, 5:5, 3.3:6.7, 2.5:7.5, and 0:10. Each inoculum was inoculated to 100 mL OMSM with 75 mg/L deltamethrin for 72 h to measure the residues of deltamethrin and 3-PBA, and non-inoculated cultures served as controls.Under the optimal inoculum proportion of strains LH-1-1 and BPBA052, three different inoculation sequences of strains LH-1-1 and BPBA052 were investigated. Sequence 1: LH-1-1 was first inoculated and cultured in OMSM for 24 h before inoculation of strain BPBA052. Sequence 2: strains LH-1-1 and BPBA052 were inoculated in the OMSM, simultaneously. Sequence 3: BPBA052 was first inoculated and cultured in OMSM for 24 h before inoculation of strain LH-1-1. Each sequence was inoculated to 100 mL OMSM with 75 mg/L deltamethrin at an interval of 12 h to measure the residues of deltamethrin and 3-PBA, and non-inoculated cultures served as controls.Three sets of experiments  were set up to investigate the degradation of deltamethrin and its metabolic products in the co-culture. In experiment L, 5% (v/v) inoculum of strain LH-1-1 was inoculated in 200 mL OMSM containing 75 mg/L of deltamethrin as a positive control. In experiment LP, under the optimal inoculation proportion and inoculation sequence, co-culture of strains LH-1-1 and BPBA052 ) were inoculated in OMSM. In experiment C, non-inoculated cultures served as negative control. The metabolites of deltamethrin at an interval of 12 h were extracted and identified by gas chromatography-mass spectrometry (GC–MS) as described by Tang et al. .8 CFU/g, pH: 6.38, organic matter: 18.40 g/kg, total nitrogen: 2.52 g/kg, total phosphorus: 0.43 g/kg, water content: 34.52% (w/w). After the soil was air-dried and sieved, deltamethrin was added to the soil to make the final concentration 20 mg/kg, and then kept overnight for further studies.Soil samples were collected from top 0–10 cm of a field in Xihua University, Sichuan, China, which had never been contaminated by pyrethroid insecticides. The characteristics of the collected soil samples were as follows: total bacteria: 4.7 × 10Both sterile and nonsterile soils were used to investigate the co-culture effect of strains LH-1-1 and BPBA052 on degradation of deltamethrin and its metabolite, 3-PBA. Each soil sample  was prepared by using 500 g of nonsterile soil in 500 mL Erlenmeyer flask. In sample L, only strain LH-1-1 was added in the nonsterile soil. In sample LP, under the optimal inoculation proportion and inoculation sequence, co-culture of strains LH-1-1 and BPBA052 was added in the nonsterile soil. And non-inoculated, nonsterile soil served as sample C (control). The mentioned above experiments  were also performed once again in sterile soil. All the experiments were performed in triplicate. The water content of these soil samples was maintained at 35.0 ± 5.0% and the temperature was maintained at 30 ± 5 °C. The soil samples were collected and the content of deltamethrin and 3-PBA was measured every day.600) of strains were nonlinearly fitted in the growth kinetics model. The OD600 of strain LH-1-1 rose from 0.06 to 0.74 and the growth kinetics parameters were μm = 0.12346 h−1, X0 = 0.06183, Xm = 0.74619 and substituting (Eq. XLH-1-1 = 0.06183et0.12346/(1–0.082861(1–e0.12346t)), R2 = 0.986. The results of growth kinetics of BPBA052 in OMSM containing 75 mg/L of 3-PBA were as follows: XBPBA052 = 0.0918et0.09419/(1–0.080152(1–e0.09419t)), where μm = 0.09419 h−1, X0 = 0.0918, Xm = 1.14532, R2 = 0.985.As shown in Fig. CLH-1-1 = 74.02463et−0.01805, k = 0.01805, t1/2 = 38.40 h, R2 = 0.996. The formation of 3-PBA was accompanied with the degradation of deltamethrin, reaching a maximum concentration of 22.40 mg/L in 96 h. It was noteworthy that the OD600 of strain LH-1-1 was 0.74, which was far lower than that of the strain BPBA052 (1.04). It may be because the toxic intermediate 3-PBA has antibacterial activity and inhibits microbial growth  was degraded within 96 h in OMSM. The degradation rate was slightly slower in OMSM than in mineral salt medium, this inhibitory effect may be related to metabolism of additional carbon sources, which was also observed in the case of strain ZS-19  by LH-1-1 or BPBA052, respectively.The RA. junii LH-1-1 and K. pneumoniae BPBA052, the co-culture of these two strains was used to improve the degradation of deltamethrin and decrease the content of metabolite 3-PBA during deltamethrin degradation process. As shown in Fig. A. junii LH-1-1 inoculum and the increase in the amount of inoculum of K. pneumoniae BPBA052, the degradation rate of deltamethrin first increased and then decreased, while the content of 3-PBA gradually decreased within 72 h. When only strain LH-1-1 (10:0) was inoculated, the degradation rate of deltamethrin was 73.90% and a large amount of 3-PBA was accumulated (up to 19.41 mg/L), suggesting that the strain LH-1-1 could not effectively degrade 3-PBA, which could have also inhibited further degradation of deltamethrin. When only strain BPBA052 (0:10) was inoculated, the residue of 3-PBA was the least, but the degradation rate of deltamethrin was only 38.03%, because BPBA052 is an efficient 3-PBA degrading strain with poor deltamethrin-degrading ability. However, when the inoculation proportion of LH-1-1 and BPBA052 was 7.5:2.5, the degradation rate of deltamethrin reached the highest—89.67%, and only 9.30 mg/L 3-PBA remained at 72 h. Therefore, in our study the most appropriate proportion of strains LH-1-1: BPBA052 was 7.5:2.5, which used for subsequent studies.Based on the degradation of different substrates by CLH-1-1+BPBA052 = 75.411et−0.0282, k = 0.0282, t1/2 = 24.58 h, R2 = 0.974. The degradation rate of deltamethrin was 20.35% higher than that in monoculture A. junii LH-1-1, and the half-life was shortened from 38.40 h to 24.58 h. The results showed that deltamethrin could be efficiently co-degraded by A. junii LH-1-1 and K. pneumoniae BPBA052.Based on the optimal inoculum proportion of the strains LH-1-1 and BPBA052, the optimal inoculation sequence of the strains for preparation of the co-culture was investigated. As shown in Fig. A. junii LH-1-1 and K. pneumoniae BPBA052, samples were detected and characterized by GC–MS , three compounds were detected Fig. a within A. junii LH-1-1 and K. pneumoniae BPBA052 was proposed , 1,2-benzenedicarboxylic butyl dacyl ester (C), and phenol (D) within 72 h. A new peak with retention time 6.525 min was observed and was identified as phenol, which was not detected in experiment L (monoculture strain LH-1-1). The formation of phenol may be due to the oxidization and cleavage of diaryl ether of 3-PBA by the strain BPBA052 , co-culture strains A. junii LH-1-1 and K. pneumoniae BPBA052 (LP sample), and the control (C sample) are shown in Fig. The content of deltamethrin and 3-PBA in the sterile soil and nonsterile soils after degradation by monoculture strain Bacillus licheniformis B-1 and Sphingomonas sp. SC-1 to degrade cypermethrin and its metabolite was 3.3:6.7, this signifies that the optimal inoculum proportion in case of different strains and substrates in a co-culture is different. But the inoculation sequence was confirmed the observations made by Liu et al. benzoic acid. But all these 3-PBA-degrading strains could not metabolize PPs. Fortunately, in our current study co-culture of strains LH-1-1 and BPBA052 not only achieved the detoxification of deltamethrin by ester bond hydrolysis, but further transformed its toxic metabolite (3-PBA) by cleavage of the diaryl bond, achieving completely mineralized pathway. That is a small amount of hydrolyzed 3-phenoxybenzaldehyde (B) was metabolized with diaryl cleavage to form 1,2-benzenedicarboxylic butyl dacyl ester by strain LH-1-1, and through the cleavage of aromatic rings to achieve complete metabolism. While a large amount of transient 3-phenoxybenzaldehyde (B) was rapidly degraded with oxidation and cleavage of diaryl ether by BPBA052 to generate phenol releasing the toxicity inhibition of 3-PBA and achieving completely mineralized pathway.Although metabolic mechanism is the basis of application, reports on co-culture biodegradation pathway and mechanism are still rare (Chen et al. o et al. , whereino et al.  and Toppo et al.  reportedn et al.  reportedThe potential of the co-culture strains LH-1-1 and BPBA052 to remediate deltamethrin- contaminated soil has been confirmed. Whether in sterilized soils or in unsterilized soils, the degradation rate of deltamethrin and 3-PBA concentration in co-cultured strains LH-1-1 and BPBA052 is much higher and lower than that in monoculture strain LH-1-1. And both in case of degradation by monoculture of strain LH-1-1 or co-culture of strains LH-1-1 and BPBA052, the degradation rate of deltamethrin in nonsterile soil was higher than that in sterile soil, and the content of 3-PBA in nonsterile soil was lower than that in sterile soil. These results confirmed the observations made by Chen et al.  and ZhanIn summary, the inoculum proportion and the inoculation sequence played an important role in effective degradation of deltamethrin in co-culture of strains LH-1-1 and BPBA052. And the co-culture degraded deltamethrin via a novel metabolic pathway with the formation of 3-phenoxybenzaldehyde by ester hydrolysis reaction by strain LH-1-1, which was further transformed by strain LH-1-1 and BPBA052 to form 1,2-benzenedicarboxylic butyl dacyl ester and phenol without any persistent accumulative product, indicating that the co-culture harbors a complete metabolic pathway for degradation and mineralization of deltamethrin. Simultaneously, the study revealed that use of co-culture is a potential and efficient strategy for bioremediation of environments contaminated with pyrethroid and their toxic intermediate metabolites.


HBNs1–9 as alpha-phenyl-N-tert-butylnitrone (PBN) analogues for stroke therapy. In vitro neuroprotection studies of HBNs1–9 against Oligomycin A/Rotenone and in an oxygen-glucose-deprivation model of ischemia in human neuroblastoma cell cultures, indicate that -1,1′-bis(N-benzylmethanimine oxide) (HBN6) is a potent neuroprotective agent that prevents the decrease in neuronal metabolic activity (EC50 = 1.24 ± 0.39 μM) as well as necrotic and apoptotic cell death. HBN6 shows strong hydroxyl radical scavenger power (81%), and capacity to decrease superoxide production in human neuroblastoma cell cultures , values significantly superior to the neuroprotective and antioxidant properties of the parent PBN. The higher neuroprotective ability of HBN6 has been rationalized by means of Density Functional Theory calculations. Calculated physicochemical and ADME properties confirmed HBN6 as a hit-agent showing suitable drug-like properties. Finally, the contribution of HBN6 to brain damage prevention was confirmed in a permanent MCAO setting by assessing infarct volume outcome 48 h after stroke in drug administered experimental animals, which provides evidence of a significant reduction of the brain lesion size and strongly suggests that HBN6 is a potential neuroprotective agent against stroke.We herein report the synthesis, antioxidant power and neuroprotective properties of nine homo-bis-nitrones  W-AZN  to brain tissues7. Bis-nitrones are well-known antioxidant and neuroprotective agents showing high clinical potential. For instance, bis-nitrone AZN Fig. , an azul8. For instance, hydroperoxides formed in the reaction of ROS with unsaturated fatty acids are very reactive, and constitute the origin of extensive cell death9. Nowadays, it is widely accepted that the formation of ROS is one of the main biological events involved in the etiology of stroke10. For this reason, there exists a growing interest in the search for new and more efficient ROS scavengers as potential therapeutic agents for stroke9.Despite ROS play key roles in physiological functions at low concentrations, they can also be very toxic in highly oxidative stress dysregulated conditions11, we have already investigated nitrones derived from (hetero)aromatic aldehydes13, quinolylnitrones17, and cholesteronitrones18. More recently, we have designed bis-nitrones derived from alpha-phenyl-N-tert-butylnitrone (PBN)  1–9 result from the incorporation of a second identical nitrone moiety at the para (p-HBNs 1–3), meta (m-HBNs 4–6) and ortho (o-HBNs 7–9) positions and bearing methyl, tert-butyl or benzyl substituents, respectively, as the N-alkyl groups attached to the nitrone motif. Among these nitrones, only HBNs 121, 222, 323, 425, and 526 have been previously described in the literature, but in studies not related to their antioxidant properties and/or potential use for stroke therapy. In fact, the present work is the first study aimed at exploring the neuroprotective and antioxidant properties of bis-nitrones, analogues of the parent PBN. The hypothesis behind the present design is that two “nitrone” scavenging motifs in the same scaffold should afford a higher antioxidant power than only one. Indeed, as it will be shown later on, we have identified -1,1′-bis(N-benzylmethanimine oxide) (HBN6) as a potent neuroprotective ligand (EC50 = 1.24 ± 0.39 µM), whose neuroprotective and antioxidant capacities are higher than those of PBN.In our current program targeted to identify new nitrones for the therapy of strokeHBNs 1–9 hydroxylamine hydrochlorides (Methods and Supplementary Information). All compounds were isolated as pure Z isomers at the double PhC=N(O)R bond, and exhibited analytical and spectroscopic data in agreement to those previously reported26.The synthesis of 27, we selected the appropriate experimental conditions and tested the neuroprotective effect of HBNs 1–9 at different concentrations , added 10 min before the administration of O10 μM /R30 μM (O/R), and using PBN, at the same concentrations , as a reference compound28.One of the first events taking place in the initial stages of stroke is the collapse of the mitochondrial electron transport chain (ETC), which leads to extended cell death and brain damage due to the formation of ROS. In order to mimic this event into suitable experiments, we tested the effect of the bis-nitrones on cell death induced by Oligomycin A and Rotenone (O/R), inhibitors of mitochondrial complexes V and I, respectively. To this end, we used the XTT cell viability test, a colorimetric assay that detects the cellular metabolic activities. Based on a previous work from our laboratoryPBN and HBNs 1–9 for 24 h in a concentration-dependent manner  (EC50 = 5.16 ± 1.60 μM). From the structure–activity relationship (SAR) point of view, note that, among the bis-nitrones of the same group, HBNs having a benzyl or tert-butyl group at the nitrogen atom of the nitrone motif systematically afforded a higher neuroprotection.As the highest neuroprotective activity  was similar in all cases, we can conclude, by regarding the ECHBNs 1–9 was evaluated in an in vitro oxygen glucose deprivation (OGD) model, followed by ischemic reperfusion (IR)29. Tested compound concentrations ranged from 0.01 to 1,000 μM, after IR. After OGD (I) (4 h), a loss of metabolic activity between 50–80% was observed, showing a small cell recovery after 24 h reperfusion (IR) of 38% to 61% . HBNs 1–9 , this order was as follows: HBN3 < < HBN6 ≤ HBN 5 ≤ HBN 9. Then, given that HBN3, despite having the lowest EC50, has a low maximal activity, we could conclude that the neuroprotective capacity of HBN3 is similar to that of HBN9, a compound that shows a high maximal neuroprotective activity at a higher EC50, and that both HBNs have lower overall neuroprotective capacity than HBN6 and HBN5, both with a low EC50 and a high maximal neuroprotective capacity.Based on these encouraging results, we have determined the ECHBNs 3, 6 and 9 bear a benzyl group at the nitrogen atom of the nitrone motif, (2) HBN9 bears the two nitrone motifs in an ortho arrangement at the aromatic ring, and (3) the meta relative position of nitrones, present in HBN5 and HBN6, is the preferred arrangement to provide an effective neuroprotection. Moreover, the neuroprotection afforded by HBN5 and HBN6 is very similar to that of NAC (EC50 = 2.58 ± 0.91 μM).From the SAR point of view, note that: (1) the best neuroprotective 30. Under these circumstances, lactate dehydrogenase (LDH), a soluble cytosolic enzyme, easily crosses the damaged membrane, and for this reason, it is possible to determine the extent of the cell necrosis taking place in the OGD experiment by comparing its extracellular to its intracellular activity. As shown in Fig. HBNs 1–9 at 1–500 µM concentrations (PBN and NAC as the reference compounds), we concluded that all HBNs, with the exception of HBN3, PBN and NAC, significantly decreased the release of LDH, reaching 100% of the LDH activity inhibition , the cells were lysated, DEVD-AMC was added, and the fluorescence measured. As shown in Fig. para-HBNs, HBN3 was the best agent, as the caspase-3 activity was reduced at 10 μM dose. HBN5, HBN6, HBN8 and HBN9 showed also potent antiapoptotic activity, being ortho-HBN8 and HBN9, which bear tert-Bu and Bn substituents, respectively, more potent than the corresponding meta-HBN5 and HBN6. Both, the antiapoptotic and antinecrotic effects of the most potent HBNs , were very similar to those found for NAC. However, the antiapoptotic effect of HBNs 1–9 was greater than that of PBN, despite the fact that they have a similar anti-necrotic effect to this base nitrone.Next, and in order to evaluate the extent of cell death by apoptosis, we determined the caspase-3 activity, by using DEVD-AMC as a substrate, which affords fluorescent AMC upon hydrolysis. So, after OGD (4 h), and adding HBNs 4–6 , HBN4 , and HBN1 , were neurotoxic.Due to the observed decrease of the neuroprotective effect by 4–6 Fig.  or the e2·−). O2·− detection was carried out by using dihydroethidium (DHE), after OGD (3 h) and IR (3 h), with or without HBNs 1–9, including PBN and NAC as standards. Compound concentrations from 0.1 to 1,000 μM were tested, after IR. As shown in Fig. P < 0.05, one way Anova test) than ROS production after OGD alone . As expected, HBNs 1–9 were able to partially or totally reverse the increase in ROS levels induced by IR, in a concentration-dependent manner  was lower for HBNs 1–4 and higher forHBNs 5–6 and HBN8–9, we conclude that, regarding the antioxidant capacity against IR-induced superoxide production, m-HBNs 4–6 exhibit the best antioxidant properties followed by p-HBNs 1–3, whose effect is very similar to that of PBN. However, the fact that o-HBN8 and o-HBN9 have higher maximal activity than the other HBNs, despite their higher EC50, makes them highly antioxidant nitrones with a similar activity to those of HBNs 1–3, which, despite their lower EC50, exhibit the lowest maximal activity.The results shown in the previous sections prompted us to investigate whether the observed neuroprotection was a consequence of their capacity to act as antioxidants and ROS scavengers, particularly of superoxide radical anion  ranging from 0.807 to 0.983, and with a statistical significance > 0.001 in the case of nitrones with tert-Bu and Bn substituents and < 0.01 or < 0.05 for HBNs with Me substituent and PBN (data not shown).Finally, to examine whether the antioxidant effect of wer Fig. B versus wer Fig. C, as shoHBN5 and HBN6 are the best and most balanced bis-nitrones of the studied series in terms of neuroprotection  and antioxidant power. In addition, the antioxidant power of HBN5 and HBN6 is very similar to the antioxidant power of NAC (EC50 = 3.23 ± 0.35 μM).To sum up, it becomes clear that the results of the ROS trapping experiment are in good agreement with the neuroprotection analyses and firmly confirm that HBNs 5–9 on diverse antioxidant tests, using PBN, NDGA and Trolox as standards for comparative purposes.Based on the neuroprotection results, we have also investigated the antioxidant power analysis of HBN9 was able to inhibit 80% lipid peroxidation (LP), in the same range as Trolox (88%), in the same experiment, as well as lipoxygenase (LOX) (85 µM) and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS+.) (23%), albeit in a poorer extent than NDGA (0.45 µM), and Trolox (91%), respectively. Note also that HBN6 was the most potent hydroxyl radical scavenger (81%), overcoming HBN5 and HBN9, and in the same range that Trolox (83%). Finally, compared to PBN, HBN5, HBN6 and HBN9 showed more potent balanced antioxidant capacity, in good agreement with the higher calculated ClogP values, which are also consistent with the neuroprotection results (see above).As shown in Table 31. Permanent middle cerebral artery occlusion (pMCAO) is a commonly used stroke model in mice32. Using the pMCAO procedure33, we analyzed the in vivo contribution of HBN6 to brain damage prevention. As expected, animals in the sham operated group showed no infarct (not shown). In striking contrast, groups subjected to pMCAO showed, 48 h after the occlusion procedure, infarcted regions which included exclusively the cerebral cortex  in vehicle treated animals, a value significantly greater than that for the HBN6 treated group, 0.37 ± 0.15 mm3 (media ± SEM)  , this area represents 0.92 ± 0.05% and 0.088 ± 0.03% of the total brain volume, for vehicle and HBN6-treated animals, respectively .Permanent ischemia models reflect the most frequent variants of stroke in patients who are outside of therapeutic windows, or are non-responders to recombinant tissue plasminogen activator, or surgical thrombectomy. In addition, permanent ischemia (no reperfusion) has also been associated with substantial accumulation of ROSHBN6 as compared to the parent nitrone PBN. To this end, we first explored the reaction between the oxygen-centered radical HO· and HBN6. Three different pathways were envisaged  calculations were carried out at the dispersion corrected B3LYP-D3/def2-SVP level  to gain more insight into the higher neuroprotective response of see Fig. , namely From the data in Fig. HBN6 and PBN. Data for HBN5 are also included to enable a direct comparison (i.e. PBN and HBN5 possess a tert-Bu group as a nitrone substituent). As clearly seen from the computed energy values, all the possible pathways are less favored for the processes involving the parent PBN, which is fully consistent with the lower neuroprotective response observed for this nitrone. Except for the hydrogen abstraction reaction, the addition of OH· is only slightly favored for HBN6 as compared to its t-Bu substituted counterpart HBN5, which again is also consistent with the slightly higher response of the HBN6 as compared to HBN5 (see above). Therefore, it can be concluded that the neuroprotective ability of the considered bis-nitrones may be directly related to the exergonicity of their initial reaction (mainly addition reaction) with the corresponding ROS.Table  HBNs 1–9 and PBN as reference compound. We have used the QikProp software , and results are summarized in Table 34 violation and three nitrones showed one violation  value should be ≤ 5. For nitrones bearing the N-benzyl moiety  QPlogPo/w values range from 6.048 to 6.247. The number of hydrogen bond donors (NdonorHB) and hydrogen bond acceptors (NaccptHB) for all the nitrones were in agreement with the drug-likeness requirements of the Lipinski’s rule of five34 . The predicted central nervous system (CNS) activity with a score range from − 2 (inactive) to + 2 (active) indicated that bis-nitrones HBNs 1–9 had lower activity in the CNS  than PBN . Molecular volume is another crucial factor for binding at the active site. It was found that all bis-nitrones have molecular volumes between 667.298 and 1,188.993 Å3 . The aqueous solubility (QPlogS) of a compound significantly affects its absorption and distribution characteristics. Typically, a low solubility goes along with a bad absorption. Only HBN3 (QPlogS: − 6.81) and HBN6 (QPlogS: − 6.87) presented solubility values out of the limits (− 6.5 to 0.5). This is the reason why these two nitrones showed one violation of the rule-of-three (ROT)36. QPPCaco, which predicts permeability of a molecule for the gut-blood barrier through passive transport, is also one important factor to be considered. Compounds with high Caco-2 permeability (QPPCaco) are easy to absorb. Caco-2 cell permeability prediction of the tested bis-nitrones indicated excellent results, predicting good intestinal absorption. The prediction of Blood Brain Barrier (BBB) permeability, determined by the logBB was also assessed. Compounds with logBB below − 1 are poorly distributed to the brain and are improbable to operate as effective CNS drugs. All bis-nitrones displayed logBB > − 1, pointing towards potential BBB permeability. The number of likely metabolic reactions (metab) is necessary for determining the level of accessibility of compounds to their target sites after entering into the blood stream. The predicted average number of possible metabolic reactions indicated that all bis-nitrones possessed metab values in the recommended range (1–8). All bis-nitrones also exhibited an excellent predicted percentage of oral absorption, 100%. The optimum value polar surface area (7–200 Å) holds a great importance on the oral bioavailability of the molecules; in the present study, bis-nitrones were exhibited 18.99–45.83 Å value of PSA, indicating good bioavailability by oral route.Drug-like properties were determined forHBN6 and PBN, the more significant observed data were the following: (1) Total Solvent Accessible Surface Area (SASA), in square Å, using a probe with a 1.4 Å radius: 686.317 and 429.378, respectively ; (2) Estimated number of hydrogen bonds (donorHB) that would be accepted by the solute: 0 for both compounds (limits: 0.0–6.0); (3) Predicted octanol/water partition coefficient (QPlogPo/w): 6.244 and 3.450, respectively (limits − 2.0 to 6.5); (4) Predicted aqueous solubility. S, in mol/dm3, is the concentration of the solute’s saturated solution that is in equilibrium with crystalline solid (QPlogS): − 6.873 and − 3.342, respectively (limits − 6.5 to 0.5); (5) Predicted brain/blood partition coefficient (QPlog BB): − 0.601 and 0.197, respectively (limits − 3.0 to 1.2); (6) Van der Waals surface area of polar nitrogen and oxygen atoms (PSA): 43.368 and 18.991, respectively (limits 7.0–200.0); (7) Number of violations of Lipinski's Rule Of Five (ROF): 1 and 0, respectively; number of violations of Jorgensen's rule of three (ROT): 1 and 0, respectively. To sum up, the predicted results indicate that the ADME properties for HBN6 are quite similar to those of PBN, except that HBN6 must be less soluble in water due to its highest lipophilicity (QPlogPo/w > 5). Nevertheless, an orally active drug should have no more than one violation of the Lipinski's Rule, as it is the case of agent HBN6, and consequently, no problems with its bioavailability should be observed.In particular, for HNBs 1–9 derived from PBN for the potential treatment of stroke. The biological evaluation of the bis-nitrones included neuroprotection against Oligomycin A/Rotenone, and in an ischemia in vitro model under OGD conditions in human neuroblastoma SH-SY5Y cells, and diverse antioxidant tests. Our design, supported on literature precedents, was based on the hypothesis that two nitrone motifs installed in the same scaffold should afford higher neuroprotective power than only one nitrone group. For our approach, we have used PBN -1,1′-bis(N-benzylmethanimine oxide) (HBN6), bearing the two nitrone motifs in meta position, and two N-benzyl groups at the nitrone scaffold, as a potent neuroprotective agent (EC50 = 1.24 ± 0.39 µM) with strong hydroxyl radical scavenger power (81%), in the same range as Trolox (83%), and a strong capacity to decrease superoxide production in human neuroblastomas cell cultures, which exceeds the neuroprotective and antioxidant capacities of the parent PBN. This may be related to the exergonicity of the addition of the oxygen-centered radical to the carbon atom of the nitrone moiety. In silico results allowed us to conclude that HBN6 is predicted to be a potential drug candidate, via oral administration, due to its relevant drug-likeness profile, bioavailability, excellent liposolubility and adequate pharmacokinetics, including CNS permeability, although with low water solubility. To sum up, all these observations confirmed that our initial design hypothesis was correct (“twobetterthanone”), and could be used as a guideline to design new and more efficient neuroprotective agents for the therapy of stroke.In this work we have described the design, synthesis and biological evaluation of nine bis-nitrones 3 or DMSO-d6 using TMS as internal standard for 1H NMR spectra. In 13C NMR spectra, CDCl3 central signal (77.0 ppm) and DMSO-d (39.5 ppm) were used as references. 1H-NMR and 13C-NMR spectra were obtained in Bruker Avance 300 (300 MHz) and Bruker Avance 400 III HD (400 Hz) spectrometers. Chemical shifts (δ) are given in ppm. Coupling constants (J) are given in Hz. Signal multiplicity is abbreviated as: singlet (s), doublet (d), triplet (t), quartet (c), doublet of doublets (dd), triplet of doublets (td), or multiplet (m). IR spectra were recorded on a Perkin-Elmer Spectrum One B spectrometer. Units are cm−1. Low resolution mass spectra were recorded on an Agilent HP 1,100 LC/MS Spectrometer, whereas High Resolution mass spectrometry (Exact Mass) was performed in an AGILENT 6,520 Accurate-Mass QTOF LC/MS Spectrometer. Elemental analyses were performed in an Elementary Chemical Analyzer LECO CHNS-932.Compound purification was performed by column chromatography with Merck Silica Gel (40–63 µm) or by flash chromatography (Biotage Isolera One equipment) and the adequate eluyent for each case. Reaction course was monitored by thin layer cromatography (t.l.c.), revealing with UV light (λ = 254 nm) and ethanolic solution of vanillin or ninhydrin. Melting points were determined using a Reichert Thermo Galen Kofler block and are uncorrected. Samples were dissolved in CDCl3 (3 equiv), Na2SO4 (4 equiv) and the corresponding N-alkylhydroxylamine hydrochloride (3 equiv) were added. The mixture was irradiated at 90 °C, and 15 bar, for the time indicated in each case. Then, the mixture was cooled, the solvent removed, and the crude purified by column chromatography. Method B: As in Method A, but at room temperature (rt). Method C: As in Method A, but in dry THF as solvent.Method A: To a suspension of the bis(tris)carbaldehyde (1 mmol) in dry EtOH (20 mL), anhydrous NaHCO3 , Na2SO4  and N-methylhydroxylamine hydrochloride , in EtOH (20 mL), for 1 h, after work-up and purification by column chromatography eluting with MeOH:CH2Cl2 at 3%, gave HBN1 : mp > 220 °C; IR (KBr) ν 3,423, 1591, 1,418, 1,129 cm-1; 1H NMR  δ 8.22 , 7.85 , 3.77 ; 13C NMR  δ 133.9 , 132.3 , 127.9 , 54.6 ; MS (ESI) m/z: 193 [M + 1]+, 215 [M + Na]+, 404 [2 M + Na]+. Anal. Calcd for C10H12N2O2: C, 62.49; H, 6.29; N, 14.57. Found: 62.48; H, 6.28; N, 14.57.Following the general Method A, the reaction of terephthalaldehyde  with NaHCO3 , Na2SO4  and N-tert-butylhydroxylamine hydrochloride , in EtOH (20 mL), for 3,5 h, after work-up and purification by column chromatography eluting with AcOEt: hexane (2:3), gave HBN2 : mp > 220 °C; IR (KBr) ν 3,434, 1569, 1,361, 1,125 cm−1; 1H NMR  δ 8.34 , 7.86 , 3.31 ; 13C NMR  δ 132.6 , 128.9 , 128.3 , 71.0 , 28.3 3]; MS (ESI) m/z: 277 [M + 1]+, 299 [M + Na]+. Anal. Calcd for C16H24N2O2: C, 69.53; H, 8.75; N, 10.14. Found: C, 69.31; H, 8.69; N, 10.13.Following the general Method A, the reaction of terephthalaldehyde  with NaHCO3 , Na2SO4  and N-benzylhydroxylamine hydrochloride , in EtOH (20 mL), for 2,5 h, after work-up and purification by column chromatography eluting with AcOEt, gave HBN3 : mp > 220 °C; IR (KBr) ν 3,435, 1,570, 1,459, 1,152 cm−1; 1H NMR  δ 8.24 , 8.12 , 7.54–7.31 , 5.08 ; 13C NMR  δ 135.1 (C6H5), 133.4 , 132.3 , 129.4, 128.9, 128.8 (C6H5), 128.2 , 70.6 ; MS (ES) m/z (%): 350 [M + 1]+, 372 [M + Na]+. Anal. Calcd for C22H20N2O2 2/7 H2O: C, 75.59; H, 5.93; N, 8.01. Found: C, 75.68; H, 6.22; N, 7.88.Following the general Method A, the reaction of terephthalaldehyde  with NaHCO3 , Na2SO4  and N-methylhydroxylamine hydrochloride , in EtOH (20 mL), for 1 h, after work-up and purification by column chromatography eluting with MeOH:CH2Cl2 al 4%, gave HBN4 : mp 148–150 °C; IR (KBr) ν 3,419, 1584, 1,415, 1,172, 1,157 cm−1; 1H NMR  δ 8.92 , 8.34 , 7.90 , 7.49 , 3.80 ; 13C NMR  δ 134.0 , 131.6 , 129.1 , 128.9 (C-3), 127.6 (C-6), 54.6 ; MS (ESI) m/z: 193 [M + 1]+, 215 [M + Na]+, 404 [2 M + Na]+. Anal. Calcd for C10H12N2O2: C, 62.49; H, 6.29; N, 14.57. Found: 62.21; H, 6.23; N, 14.58.Following the general Method A, the reaction of isophthalaldehyde  with NaHCO3 , Na2SO4 , and N-tert-butylhydroxylamine hydrochloride , in EtOH (20 mL), for 3,5 h, after work-up and purification by column chromatography eluting with AcOEt: hexane (3:2), gave HBN5 : mp 147–9 °C; IR (KBr) ν 3,435, 2,976, 1573, 1,361, 1,180 cm−1; 1H NMR  δ 9.22 , 8.39 , 7.82 , 7.44 , 1.50 ; 13C NMR  δ 131.88 , 129.59 , 128.92 , 128.50 (C-5), 70.95 , 28.28 3]; MS (ESI) m/z: 277 [M + 1]+, 299 [M + Na]+. Anal. Calcd for C16H24N2O2: C, 69.53; H, 8.75; N, 10.14. Encontrado: C, 69.26; H, 8.75; N, 9.87.Following the general Method A, the reaction of isophthalaldehyde  with NaHCO3 , Na2SO4  and N-benzylhydroxylamine hyrochloride , in EtOH (20 mL), for 2.5 h, after work-up and purification by column chromatography eluting with AcOEt: hexane (3:2), gave HBN6 : mp 185–187 °C; IR (KBr) ν 3,435,1582, 1,458, 1,175 cm−1; 1H NMR  δ 8.98 , 8.29 , 8.12 , 7.51–7.32 , 5.06 ; 13C NMR  δ 135.1 (C6H5), 133.4 , 131.5 , 129.4 , 129.4 (C-5), 129.0, 128.8, 128.7 (C6H5), 128.0 (C-2), 70.5 ; MS (ES) m/z (%): 350 [M + 1]+, 372 [M + Na]+. Anal. Calcd for C22H20N2O2: C, 76.72; H, 5.85; N, 8.13. Found: C, 76.59; H, 6.02; N, 8.28.Following the general Method A, the reaction of isophthalaldehyde  with NaHCO3 , Na2SO4  and N-methylhydroxylamine hydrochloride , in EtOH (20 mL), for 3 d, after work-up and purification by column chromatography eluting with MeOH:CH2Cl2 al 6%, gave HBN7 : mp 153–155 °C; IR (KBr) ν 3,413, 1592, 1,415, 1,170 cm−1; 1H NMR  δ 8.99 , 8.02 , 7.44 , 3.84 ; 13C NMR  δ 130.6 , 129.5 , 128.8 , 127.4 , 55.1 ; MS (ES) m/z (%): 193 [M + 1]+, 215 [M + Na] +. Anal. Calcd for C10H12N2O2: C, 62.49; H, 6.29; N, 14.57. Found: C, 62.20; H, 6.23; N, 14.57.Following the general Method B, the reaction of phthalaldehyde  with NaHCO3 , Na2SO4  and N-tert-butylhydroxylamine hydrochloride , in EtOH (20 mL), for 5 d , after work-up and purification by column chromatography eluting with AcOEt, gave HBN8 : mp 130–132 °C; IR (KBr) ν 2,977, 1554, 1,360, 1,195, 1,164, 1,116 cm−1; 1H NMR  δ 8.55 , 7.77 , 7.43 , 1.52 ; 13C NMR  δ 130.0 , 129.1 , 128.2 , 127.5 , 71.1 , 28.2 ; MS (ES) m/z (%): 277 [M + 1]+, 299 [M + Na]+. Anal. Calcd for C16H24N2O2: C, 69.53; H, 8.75; N, 10.14. Found: C, 69.51; H, 8.75; N, 10.21.Following the general Method B, the reaction of phthalaldehyde  with NaHCO3 , Na2SO4  and N-benzylhydroxylamine hydrochloride  in EtOH (20 mL), for 1 d, after work-up and purification by column chromatography eluting with AcOEt: hexane (3:2), gave HBN9 : mp 152–154 °C; IR (KBr) ν 3,422, 1564, 1,456, 1,149 cm−1; 1H NMR  δ 8.91 , 8.27 , 7.53–7.50 , 7.42–7.35 , 5.10 ; 13C NMR  δ 135.1 , 130.2 , 129.8 , 129.4 , 128.9 , 128.8 , 127.5 , 71.0 ; MS (ES) m/z (%): 350 [M + 1] +, 372 [M + Na]+. Anal. Calcd for C22H20N2O2: C, 76.72; H, 5.85; N, 8.13. Found: C, 76.46; H, 5.94; N, 8.11.Following the general Method B, the reaction of phthalaldehyde  with NaHCO27. Cultures were seeded into flasks containing supplemented medium and maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Culture media were changed every 2 d. Cells were sub-cultured after partial digestion with 0.25% trypsin–EDTA. For assays, SHSY5Y cells were subcultured in 96 or 48-well plates at a seeding density of 0.50–1 or 2–2.5 × 105 or cells per well, respectively. When the SHSY5Y cells reached 80% confluence, the medium was replaced with fresh medium containing 0.01–1,000 μM compound concentrations or PBS in the controls, as indicated in each assay.The human neuroblastomas cell line SH-SY5Y were cultured in Dulbecco's: Ham's F12, 1:1 [vol/vol] containing 3.15 mg/mL glucose, 2.5 mM Glutamax and 0.5 mM sodium pyruvate DMEM/F-12, GlutaMAX™; GIBCO, Life Technologies, Madrid (Spain), 1% Antibiotique-Antimitotic  , 1% gentamicine 15 mg/mL  and 10% Foetal Calf Serum (FCS)  as described2/5% CO2 for 30 min) and maintained in an anaerobic chamber containing a gas mixture of 95% N2/5% CO2 and humidified at 37 °C at a constant pressure of 0.15 bar. Cells were exposed to OGD for a period of 4 h (OGD 4 h), as indicated. At the end of the OGD period, culture medium was replaced with oxygenated serum-free medium, and cells were placed and maintained in the normoxic incubator for 24 h to recovery (R24h). In the neuroprotection experiments, HBNs 1–9 and PBN (0.01 μM − 1 mM) were added at the beginning of the recovery period (see below). Control cultures in Dulbecco’s medium containing glucose were kept in the normoxic incubator for the same period of time as the OGD (C4h), and then culture medium was replaced with fresh medium and cells were returned to the normoxic incubator until the end of the recovery period (C24h). In each experiment a series of different controls were performed containing the same final % of dimethyl sulfoxide (DMSO) as the samples with the tested compounds . Cultured cells were washed and placed in glucose-free Dulbecco’s medium  were incubated with the XTT solution (Cell Proliferation Kit II (XTT), Sigma, Aldrich, Madrid) at 0.3 mg/ml final concentration for 2 h in a humidified incubator at 37 °C with 5% CO2 and 95% air (v/v) and the soluble orange formazan dye formed was spectrophotometrically quantified, using a Biotek Power-Wave XS spectrophotometer microplate-reader at 450 nm (reference 650 nm). All XTT assays were performed in triplicate in cells of at least three different cell batches. Control cells treated with DMEM alone were regarded as 100% viability. Controls containing different DMSO concentrations (0.001–1% DMSO) were performed in all assays.Measurements of cell viability in human SHSY5Y neuroblastoma cells were carried out into 96-well culture plates as described38. Data are given as the percentage of LDH release with respect to the total LDH content (LDH in the culture medium and LDH inside the cells).For these assays, cultured neuroblastoma cells grown in 96-well culture dishes at a density of 1.5 × 105 cells/well were used. LDH activity was measured as the rate of decrease of the absorbance at 340 nm, resulting from the oxidation of NADH to NAD + as described5 cells/well, were used. After OGD treatment, cells were treated with different nitrones or indicated positive controls at 1 − 500 μM concentrations and subjected to 24 h reperfusion. Attached cells were lysed at 4 °C in a lysis medium containing 5 mM Tris/HCl (pH 8.0), 20 mM ethylenediaminetetraacetic acid, and 0.5% Triton X-100 and centrifuged at 13.000g for 10 min. The activity of caspase-3 was measured using the fluorogenic substrate peptide DEVD-amc , as described29. Proteins were measured by the Bradford assay. Results were expressed as arbitrary fluorescence units [(AFU)/μg protein/h].For these assays, cultured neuroblastoma cells grown in 48-well culture dishes, at a density of 2.5 × 105 cells/well) were exposed to OGD for a period of 4 h (OGD4h). At the end of the OGD period, the culture medium was replaced with oxygenated Dulbecco’s modified Eagle’s medium containing glucose and 10% fetal calf serum. Cells were treated in the absence (controls) or presence of indicated concentrations of nitrones or different known neuroprotective agents and maintained at 37 °C in a normoxic incubator for 3 h for recovery. At the end of this period, 20 μM DHE  was added and fluorescence was recorded every 15 − 30 s during a 15 min period, using an excitation filter of 535 nm and an emission filter of 635 nm in a spectrofluorimeter (Bio-Tek FL 600) as previously described34. Linear regression of fluorescence data [expressed as arbitrary fluorescence units (AFU)] was calculated for each condition, and the slopes (a) of the best fitting lines (y = ax) were considered as an index of O2·− production. SNP was used as a positive control of superoxide production37.SHSY5Y human neuroblastoma cells /(1 + (x/EC50)^(-Hillslope), by using the program SigmaPlot v.11 . Lineal correlation analysis  and statistical significances of regression line equations) were performed by the Pearson Product Moment Correlation Test, carried out with SigmaPlot v.11.Data were expressed as mean ± SEM of results obtained from at least three independent experiments from different cultures, each of which was performed in triplicate. Statistical comparisons between the different experimental conditions were performed using one-way analysis of variance (ANOVA), followed by Holm–Sidak’s post-test when the analysis of variance was significant. A P value < 0.05 was considered statistically significant. Concentration–response curves for the estimation of ECHBN6 on stroke recovery, administration of the vehicle and HBN6 was performed intraperitoneally, to 8weekold male C57BL/6 J mice (Harlan) weighing 25–30 g. All procedures with animals were carried out under a protocol approved by the Ethical Committee of the Spanish National Research Council (CSIC), and recommendations of the European Council. A special effort was made to keep to a minimum necessary the number of animals to achieve adequate significance. For surgery, anaesthesia induction was carried out with 3% isoflurane , followed by 2% isoflurane for maintenance during stroke procedure. Rectal temperature was maintained at 36.5 °C with the use of a heating pad. The frontal branch of the MCA was, after craniotomy, exposed and occluded permanently by suture ligation as previously reported, with modifications33. The permanent occlusion involved exclusively the frontal branch of the middle cerebral artery, the stem of this artery remaining untie. This procedure yielded a smaller infarct size than that determined by ligature of the arterial stem, allowing a better assessment of final infarct volume among vehicle control and HBN6 treated groups, and also reducing the sample (n) size. To ensure a complete artery occlusion during surgery, cortical blood flow was monitored by non-invasive laser Doppler flowmetry, as a quality control, with the aid of a Perimed equipment . The study was exclusively performed in animals that showed post-ligature a drop of blood flow of at least 65%. Animals subjected to surgery for longer than 15 min were excluded of the study. Physiological parameters were maintained as previously reported33. Experiments were performed in each of the following groups: (a) sham operated ; (b) pMCAO vehicle control group  (n = 6), and (c) pMCAO HBN6 treated group (100 mg/kg HBN6 dissolved in vehicle) (n = 6). Drug administration was 15 min after arterial ligature. Determination of infarct size in vehicle and HBN6 treated mice was performed by means of the 2,3,5-triphenyltetrazolium chloride (TTC) staining procedure of sequential coronal 1 mm-thick brain slices obtained from the operated animals with the aid of a Brain Matrix  as reported previously33. The experiments compared infarct volume outcome between group’s b and c. Sham operated control group (a) showed with certainty that stroke was not due to the surgical pre-occlusive procedure. Infarct volumes, shown in mm3, were obtained integrating infarcted areas by counting pixels contained within the regions of interest. Each side of the coronal sections was sampled. Images were taken with the aid of a digital camera (Pentax Optio S7) that provided good resolution of infarct boundaries. With the use of the free software ImageJ 1.33u software , acquired images were analyzed. Student two-sample t-test was carried out to determine the statistical significance of differences of infarct values between the vehicle and the HBN6 treated mice. P value < 0.05 was considered significant.To study of the effects of P values (BioByte Home Page. Available online: https://www.biobyte.com).Bioloom of Biobyte Corp was used for the theoretical calculation of lipophilicity as Clog NDGA), Trolox, 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS)Soybean LOX linoleic acid sodium salt were purchased from the Aldrich Chemical Co. Milwaukee, WI, (USA). Phosphate buffer (0.1 M and pH 7.4) was prepared mixing an aqueous KH2PO4 solution , and an aqueous of NaOH solution ; the pH (7.4) was adjusted by adding a solution of KH2PO4 or NaOH). For the in vitro tests a Lambda 20  UV–Vis double beam spectrophotometer was used.Nordihydroguaiaretic acid  dihydrochloride (AAPH) is used. The final solution in the UV cuvette consisted of ten microliters of the 16 mM linoleate sodium dispersion 0.93 mL of 0.05 M phosphate buffer, pH 7.4, thermostatted at 37 °C. 50 μL of 40 mM AAPH solution was added as a free radical initiator at 37 °C under air and 10 μL of the tested compounds.14. The tested compounds  dissolved in DMSO were incubated at room temperature with sodium linoleate (0.1 mM) and 0.2 mL of enzyme solution . The conversion of sodium linoleate to 13-hydroperoxylinoleic acid at 234 nm was recorded and compared with the appropriate standard inhibitor NDGA (IC50 0.45 μM and 93% at 100 μM).The oxidation of linoleic acid sodium salt results in a conjugated diene hydroperoxide. The reaction is monitored at 234 nm. Soybean lipoxygenase inhibition study in vitro. In vitro study was evaluated as reported previously3+/ascorbic acid system. EDTA (0.1 mM), Fe 3 + (167 μM), DMSO (33 mM) in phosphate buffer , the tested compounds (0.1 mM) and ascorbic acid (10 mM) were mixed in test tubes. The solutions were incubated at 37 °C for 30 min. The reaction was stopped by CCl3COOH (17% w/v) and the % scavenging activity of the tested compounds for hydroxyl radicals was given.The hydroxyl radicals were produced by the FeSupplementary information


A highly sensitive electrocatalytic sensor was designed and fabricated by the incorporation of NiO dope Pt nanostructure hybrid (NiO–Pt–H) as conductive mediator, bis   nickel(II) hexafluorophosphate , and electrocatalyst into carbon paste electrode (CPE) matrix for the determination of cysteamine. The NiO–Pt–H was synthesized by one-pot synthesis strategy and characterized by XRD, elemental mapping analysis (MAP), and FESEM methods. The characterization data, which confirmed good purity and spherical shape with a diameter of ⁓ 30.64 nm for the synthesized NiO–Pt–H. NiO–Pt–H/B,1,10, P,1,10, PDNiPF6/CPE, showed an excellent catalytic activity and was used as a powerful tool for the determination of cysteamine in the presence of serotonin. The NiO–Pt–H/B,1,10, P,1,10, PDNiPF6/CPE was able to solve the overlap problem of the two drug signals and was used for the determination of cysteamine and serotonin in concentration ranges of 0.003–200 µM and 0.5–260 µM with detection limits of 0.5 nM and 0.1 µM, using square wave voltammetric method, respectively. The NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE showed a high-performance ability for the determination of cysteamine and serotonin in the drug and pharmaceutical serum samples with the recovery data of 98.1–103.06%. Cysteamine is prescribed as a radiation-protective agent drug to treat radiation sickness2. Overdose of cysteamine brings about many side effects such as dizziness, rash, skin odor, headache, and tiredness. Therefore, the analysis of cysteamine should be monitored for administrated patients. On the other hand, taking cysteamine is effective in the serotonin neurotransmission level in body3. Also, the use of cysteamine in the treatment of patients can change tissue serotonin in duodenal ulceration4. Therefore, the simultaneous measurement of these two compounds is pivotal in the patients treated with cysteamine. Due to the important analysis of these compounds, many researchers focused on the fabrication of analytical sensors for their determination in biological and clinical samples9. Although there have been no reports on the simultaneous measurement of cysteamine and serotonin, various analytical methods such as chromatography, spectroscopy, and electrochemical sensors have been separately reported for each of these compounds13. Due to simple operations and low-cost analysis systems, electrochemical sensors have been widely used for the analysis of pharmaceutical and biological compounds21. Near over-potential oxidation of cysteamine and serotonin on the surface of a bare electrode is one of the most important problems in the simultaneous analysis of cysteamine and serotonin, using electrochemical methods22. Therefore, the application of electro-catalyst is necessary for resolving this issue in the simultaneous analysis30. EC/ electrochemical mechanism is a suitable strategy for the determination of two analytes with near over-potential in electrochemical analysis systems37. The interaction of one analyte with a suitable electrocatalyst and remaining of another analyte at its potential is a very interesting strategy based on electrocatalytic systems in the simultaneous analysis41. Selecting a suitable catalyst with high selectivity is one of the most important steps in the design of electrocatalytic sensors46. According to previous reports, inorganic complexes, especially complexes with nickel central atom can be used as a suitable electrocatalyst in the analysis of important pharmaceutical and biological constituents52. Although nanomaterials such as carbon nanotubes and graphene with high-conductivity have been proposed for the preparation of electrochemical sensors, their high cost, hard synthesis methods, and high capacitive charging current are some of the most important criteria for their use in electrochemical sensors61. Accordingly, the use of metal oxides has been used as a suitable alternative to this category of materials66. Recent research suggests that modifying metal oxides with metal nanoparticles such as platinum could increase the electrical conductivity of the sensors and create the right conditions for the design of high-sensitivity sensors67.Cysteamine is a simple aminothiol with a variety of medicinal applications used to treat various diseases such as cystinosis and hypothyroidismTherefore, in this study, we fabricated NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE as a first and highly sensitive electroanalytical sensor for simultaneous determination of cysteamine and serotonin. The NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE was successfully used for the determination of cysteamine and serotonin in real samples.Electrochemical signals were recorded by Potentiostat/Galvanostat Electrochemical Instrument PGSTAT-302N (Netherlands). The Field Emission SEM machine model Mira 3-XMU was used for morphological and EDS analysis of NiO dope Pt nanostructure hybrid. The structural analysis of NiO dope Pt nanostructure hybrid was characterized by XRD machine model X’Pert Pro. The Ag/AgCl/KClsat and electrochemical cell were purchased from Azar Electrode Company.Cysteamine hydrochloride and serotonin hydrochloride were purchased from Sigma-Aldrich Company. The 0.01 M stock solution of cysteamine hydrochloride and serotonin hydrochloride was prepared by dissolving 0.113 g and 0.212 g compounds in 100 mL phosphate buffer solution (PBS) pH 7.0.Nickel nitrate hexahydrate, sodium hydroxide, and platinum (II) chloride were purchased from Merck Company and used for one-pot synthesis of NiO dope Pt nanostructure hybrid. Phosphoric acid was purchased from Acros Company and used for the preparation of PBS.The 100-mL solution containing 16 mg platinum (II) chloride and 0.5 M nickel nitrate hexahydrate were stirred for 30 min at room temperature. The 100-mL sodium hydroxide 1.0 M was added to the previous solution over a 1.5 h period. The green precipitated sample was filtered and then dried at 15 h at 100 °C. The green powder was calcined at 400° C in a furnace for 3 h.The NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE was designed and made by using slurry composed of 0.95 gr of graphite powder 0.04 g + NiO–Pt–H and 0.05 g of B,1,10,P,1,10,PDNiPF6 using 13 drops of paraffin oil as binders into mortar and pestle in the presence of 10 mL ethanol. After evaporation of the ethanol solvent, the mixture was hand-mixed for 90 min to obtain a homogeneous paste.Cysteamine capsules (150 mg) were purchased from a local pharmacy and after opening 10 capsules, the powder was dissolved into a 100-mL solution containing ethanol/PBS 1:1 and stirred for 1 h. Dilution was performed using phosphate buffer solution.Electrochemical behavior of cysteamine was investigated by recording cyclic voltammograms of 500 µM cysteamine on the surface of NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE, B,1,10,P,1,10,PDNiPF6/CPE, NiO–Pt–H/CPE and CPE at pH 7.0 and scan rate 10 mV/s. Then, the recorded cyclic voltammograms were compared together for the investigation catalytic effect of the fabricated sensors.–6 g of cysteamine in solution. In the next step and after the addition of interference with a maximum acceptable value of 1,000 fold (w/w) into an electrochemical cell, the signal of the solution containing cysteamine and interference was recorded. The 5% error in current was acceptable and showed that interference did not have a significant effect on the analysis signal.Interference study was investigated by recording square wave voltammogram (SWV) of 20.0 µM cysteamine on the surface of NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE. The 20.0 µM in the electrochemical cell containing 10 mL buffer solution was equal to 2.2 × 10Stability of NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE as an electroanalytical sensor for determination of cysteamine was investigated by recording SWV of 100.0 µM cysteamine over a course of 60 days on the surface of the fabricated sensor. The drug signal was recorded every ten days and the current obtained was compared with the initial current of the drug.The purity and particle distribution of NiO–Pt–H were characterized by MAP analysis. The results are presented in Fig. 2+/Ni3+ in the absence of cysteamine. After the addition of 500 µM cysteamine on the surface of NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE, the oxidation current of B,1,10,P,1,10,PDNiPF6 increased and the reduction signal of mediator was removed (EOxidation ~ 120 mV). This phenomenon exhibits a kind of electrocatalytic behavior between the intermediate  and the cysteamine , a linear relation was observed between the oxidation signal of cysteamine and ν1/2 on the surface of NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE  as a kinetic parameter, containing useful information about the rate-determining step, was calculated by the Tafel plot Fig. . The sloThe Electrochemical impedance spectroscopy (EIS) technique was used to confirm the electro-catalytic process between B,1,10,P,1,10,PDNiPF6 and cysteamine on the surface of NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE  and catalytic rate constant, kh were determined using chronoamperogram recording of NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE in the absence of Fig. B. The sl–5 cm2/s.The mean value of D for cysteamine was determined as ~ 2.45 × 10The catalytic rate constant between B,1,10,P,1,10,PDNiPF6 and cysteamine can be determined according to the method of Galus equation Eq. :2\documeC/IL  vs. t1/2 and Eq.  was also used for the examination of electrode processes at NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE Fig. D. Forwarpa = 0.0385 Ccysteamine + 0.9602 (R2 = 0.9978) and a linear range between 0.5 and 260 µM with equation Ipa = 0.0238 CSerotonin + 0.4139 (R2 = 0.9969) for the determination of serotonin on the surface of NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE. The detection limit (LOD) was 0.5 nM cysteamine and 0.1 µM serotonin, according to the definition of LOD = 3sb/m.The square wave voltammograms (SWV) of cysteamine and serotonin were recorded separately on the surface of NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE. The results showed a linear relation between oxidation current of cysteamine and its concentration in the range of 0.003–200 µM with equation IOn the other hand, the square wave voltammograms of NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE in the solution containing different concentrations of cysteamine and serotonin was recorded and the obtained signals were presented in Fig. The reproducibility and stability of NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE were investigated by recording SWV of 100.0 µM cysteamine at pH 7.0. We detected a relative standard deviation of (RSD%) 2.1% for five successive recorded signals that confirmed good repeatability for NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE as an electroanalytical sensor. Moreover, the stability of the NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE was examined by the storage of the sensor in the lab. Then, NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE was used for the determination of 100.0 µM cysteamine using SWV. The recorded signal showed 93.2% of its initial response relative to 100.0 µM cysteamine after 60 days, using NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE, indicating good stability for the suggested sensor. To study the reproducibility of NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE in determination of 500 µM cysteamine, five modified electrodes were prepared in the same condition and the oxidation signal of cysteamine was recorded on the surface of NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE with a scan rate of 10 mV/s. A relative standard deviation of (RSD%) 3.33% was detected for the determination of cysteamine on the surface of these electrodes, confirming good reproducibility for the fabrication of NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE (Fig. The selectivity of NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE as an electroanalytical sensor was checked in the solution containing 20.0 µM cysteamine with an acceptable error of 5% in oxidation current. The results are presented in Table In the final step, we check the ability of NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE as a powerful electroanalytical sensor for determination of cysteamine and serotonin in capsule and pharmaceutical serum samples. The recovery data between 98.5–103.06% for analysis of cysteamine and 98.1–101.68% for analysis of serotonin were observed on the surface of NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE Table , confirmIn this study, a novel and highly sensitive electroanalytical sensor was fabricated for the determination of cysteamine by modification of CPE with NiO–Pt–H and B,1,10,P,1,10,PDNiPF6 as two mediators. The NiO–Pt–H was synthesized by an one-pot procedure resulting in a spherical shape with diameter of 30.64 nm. The NiO–Pt–H/B,1,10,P,1,10,PDNiPF6/CPE was used as the first electrochemical sensor for the simultaneous analysis of cysteamine and serotonin with detection limits of 0.5 nM and 0.1 µM, respectively. In addition, NiO–Pt–H and B,1,10,P,1,10,PDNiPF6 showed a powerful ability for the determination of cysteamine and serotonin in the drug and pharmaceutical serum samples with recovery data of 98.1–103.06%.