Previous studies show that stressful events after ovulation in sows significantly impaired the embryo cleavage with a significant elevation of blood cortisol. However, the effects of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and cortisol on fertilization and embryo development remain to be specified, and whether they damage pig embryos directly or indirectly is unclear. This study demonstrated that embryo development was unaffected when pig parthenotes were cultured with different concentrations of CRH/ACTH/cortisol. However, embryo development was significantly impaired when the embryos were cocultured with pig oviductal epithelial cells (OECs) in the presence of CRH/cortisol or cultured in medium that was conditioned with CRH/cortisol-pretreated OECs (CRH/cortisol-CM). Fertilization in CRH/cortisol-CM significantly increased the rates of polyspermy. CRH and cortisol induced apoptosis of OECs through FAS and TNFα signaling. The apoptotic OECs produced less growth factors but more FASL and TNFα, which induced apoptosis in embryos. Pig embryos were not sensitive to CRH because they expressed no CRH receptor but the CRH-binding protein, and they were tolerant to cortisol because they expressed more 11-beta hydroxysteroid dehydrogenase 2 (HSD11B2) than HSD11B1. When used at a stress-induced physiological concentration, while culture with either CRH or cortisol alone showed no effect, culture with both significantly increased apoptosis in OECs. In conclusion, CRH and cortisol impair pig fertilization and preimplantation embryo development indirectly by inducing OEC apoptosis via the activation of the FAS and TNFα systems. ACTH did not show any detrimental effect on pig embryos, nor OECs. Although it is known that stress can influence female reproduction, how stress affects embryo development is not very clear. Because there are reports that the early stages of pregnancy are more sensitive to stress than the later stages are ,2, the pStresses are commonly associated with the enhanced activity of the hypothalamo-pituitary-adrenal (HPA) axis. The activation of the HPA axis leads to secretion of corticotrophin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and glucocorticoids. For instance, restraint of female mice increased their CRH concentration in both serum and ovaries . An immeIt is known that reproduction in female pigs is sensitive to various stressors, which can activate the HPA axis and facilitate the secretion of CRH, ACTH and cortisol ,15. FurtThus, the major objectives of this study were to specify the effects of CRH, ACTH and cortisol on pig embryo development; to clarify whether these hormones would damage pig embryos directly or indirectly by changing the oviductal environment; and to explore whether they would affect fertilization in the oviducts.The procedures for animal care and handling were performed according to guidelines approved by the Animal Care and Use Committee of the Shandong Agricultural University P. R. China (Permit number: SDAUA-2001-001). Unless pointed out otherwise, all chemicals and reagents used in this study were bought from Sigma-Aldrich Corp. .Pig ovaries and oviducts were obtained from the Feicheng slaughterhouse of Yinbao Food Corporation Ltd. . Only ovaries and oviducts at the follicular stage were collected from gilts, approximately 6 months after birth (approximately 100 kg bodyweight). While the ovaries were transported in a thermos bottle with sterile saline containing 100 IU/mL penicillin and 0.05 mg/mL streptomycin, maintained at 30–35 °C, the oviducts were transported in physiological saline solution, maintained at 4 °C, to the laboratory within 3 h after slaughtering.2·2H2O, 0.49 mM MgCl2·6H2O, 0.1% polyvinyl alcohol, 0.03 mM phenol red, 50 IU/mL penicillin and 50 µg/mL streptomycin. The COCs recovered were washed three times in D-PBS, and those showing a uniform ooplasm and compact cumulus were selected for in vitro maturation. Approximately 80 ovaries were obtained on each experimental day, and the oocytes recovered from them were pooled and divided into different treatments.To collect cumulus-oocyte complexes (COCs), 3–6 mm follicles were aspirated using a syringe containing Dulbecco’s phosphate-buffered saline  supplemented with 0.88 mM CaClg for 30 min, we collected the supernatants and filtered them sequentially through 0.22 μm syringe filters. The prepared follicular fluid was stored at −20 °C until use. We placed the maturation medium in culture wells of a 96-well plate (150 μL per well), and performed a pre-equilibration at 38.5 °C in an atmosphere of 5% CO2 in humidified air for 3 h before the introduction of the oocytes. We then washed the COCs three times in D-PBS and once in the maturation medium, and then, placed them in the wells (approximately 25 per well) covered with mineral oil before culture for 44 h at 38.5 °C under 5% CO2 in humidified air.The culture medium used for oocyte maturation was TCM-199  containing 10% porcine follicle fluid, 0.1% PVP, 0.91 mM sodium pyruvate, 3.05 mM glucose, 0.05 IU/mL FSH, 10 ng/mL EGF, 0.05 IU/mL LH, 0.57 mM cysteine, 50 μg/mL streptomycin and 100 IU/mL penicillin. We aspirated porcine follicular fluid from 3–6 mm follicles of the follicular stage ovaries. After centrifugation at 1600× After the maturation culture, the cumulus cells were mechanically removed from oocytes by repeatedly pipetting in D-PBS with 0.1% hyaluronidase using a small-bore pipette. For activation treatment, the cumulus-free oocytes were first cultured for 5 min in D-PBS containing 5 µM ionomycin, and then, they were incubated for 5 h in PZM-3 medium with 2 mM 6-DMAP after being washed three times in PZM-3 medium.v/v) fetal calf serum (Gibco) and 0.5% (v/v) penicillin/streptomycin solution (Gibco) and washed them twice by centrifugation . We added the final suspension (1~2 × 105 cells/mL) to wells (500 μL per well) of a 24-well culture plate and cultured them at 38.5 °C in a humidified atmosphere of 5% CO2 in air. At 24 h after seeding, when most OECs had attached, the medium was renewed for the first time, and then, the medium was renewed every 48 h. Mouse OECs were prepared as reported previously [One oviduct was used on each experimental day. We dissected the oviducts free from surrounding tissues, ligated them at both ends, and washed them three times in Hanks’ balanced salt solution at 4 °C. After being closed off at one end with a clip, the oviduct was filled with 0.25% trypsin solution and closed off at the other end. Then, the oviduct was incubated at 37 °C for 45 min before being squeezed with tweezers to obtain the contents. We resuspended the recovered pellets in Dulbecco’s Modified Eagles Medium/Ham’s F12  containing 10%  and Cy3-conjugated goat anti-rabbit IgG , and nuclei were stained with Hoechst 33324. Then, the sample was mounted, sealed and visualized under a fluorescent microscope. Each treatment contained three coverslips and three fields were observed on each coverslip. Approximately 200 cells were counted in each field to calculate rates of positive cells. The results showed that the cytokeratin 18-positive rates were 96.4 ± 2.0% and 98.2 ± 1.2% in OECs cultured to 20% and 90% of confluence, respectively.g to collect CM. We froze and stored the CM in aliquots at −20 °C before use.To prepare CM, when the cultured OECs reached 60–70% of confluence, we replaced the spent DMEM/F12 with fresh serum-free DMEM/F12 without (control) or with CRH, ACTH or cortisol, and we cultured the growing OECs further for 48 h. Then, we replaced the medium with fresh PZM-3 medium without hormones and cultured cells for 24 h. After the culture, we collected the supernatants and centrifuged them for 10 min at 1000× v/v) and 0.02% (v/v), respectively.According to the experimental design, CRH, ACTH and cortisol and their antagonists were added to PZM-3 medium or to serum-free DMEM/F12 to observe their effects on the embryos and OECs, respectively. To prepare stock solutions, we dissolved CRH (10 mM) and ACTH (10 mM) in water, cortisol (10 mM) and RU486 (10 mM) in ethanol and antalarmin (10 mM) in dimethylsulfoxide (DMSO). We stored the stock solutions at −20 °C before use. When the OECs or embryos were treated with cortisol, RU486 or antalarmin, the control OECs/embryos were cultured with the same amount of ethanol or DMSO. The final concentrations of ethanol and DMSO in culture medium were 0.02–0.1%  and observed under a fluorescence microscope. For embryo culture, each treatment was repeated four times on four different experimental days with each replicate containing approximately 25 oocytes.2, 2 mM caffeine and 0.6% BSA. To prepare mTBM-CM, when the cultured OECs grew to 60–70% confluence, they were changed into serum-free DMEM/F12 without (control) or with CRH or cortisol and cultured further for 48 h. Then, the cells were cultured for 24 h in mTBM without hormones. At the end of the culture, we collected supernatants and centrifuged them for 10 min at 1000× g to obtain mTBM-CM. We froze and stored the mTBM-CM in aliquots at −20 °C before use. The freshly prepared mTBM or mTBM-CM was kept in 5% CO2 incubator for 10 h to stabilize its pH at 7.2 to 7.4.Modified Tris-buffered medium (mTBM) was used for the fertilization medium, which contained 113.1 mM NaCl, 3.0 mM KCl, 20.0 mM Tris, 11.0 mM glucose, 5.0 mM Na-pyruvate, 7.5 mM CaClg for 4 min. After the washing procedure, we resuspended the sperm pellets in 1 mL of mTBM or mTBM-CM and incubated them in a CO2 incubator at 38.5 °C for 1 h for capacitation. After capacitation and appropriate dilution with mTBM or mTBM-CM, we added 30 μL of the sperm suspension to 50 μL of the medium containing oocytes, producing a final concentration of 2.5 × 105 sperm/mL. Then, the oocytes and spermatozoa were co-incubated for 6 h at 38.5 °C in an atmosphere of 5% CO2 in air.After the maturation culture, we removed cumulus cells as described above, and oocytes showing the first polar bodies were washed three times in mTBM or mTBM-CM. After washing, 15 oocytes were placed in 50 μL mTBM or mTBM-CM under mineral oil in a well of a 96-well plate. The plate was kept in an incubator until spermatozoa were added for fertilization. Freshly-collected semen (1 mL) was washed once in D-PBS and once in mTBM or mTBM-CM by centrifugation at 200× 2 in air. Then, the zona pellucida was removed by treating oocytes with 0.5% pronase for 2 min. The zona-free oocytes were fixed in 4% paraformaldehyde for 2 min and stained with 10 μg/mL Hoechst 33342 for 5 min before being mounted on a slide and examined for fertilization under a fluorescence microscope at a magnification of ×400. Oocytes with one or no pronucleus, oocytes with two pronuclei and those with three or more pronuclei were considered unfertilized, normally fertilized and polyspermic, respectively. Each treatment was repeated three times on three experimental days with each replicate including approximately 15 oocytes.After the co-incubation, oocytes were washed twice with pre-equilibrated PZM-3 medium and cultured for 10 h in PZM-3 (15 oocytes/150 μL medium) at 38.5 °C in an atmosphere of 5% COCultured OECs were stained with Hoechst 33342 in situ in wells of a 24-well culture plate. Briefly, at the end of culture, OECs were washed three times with PBS, and then, stained for 5 min in situ using 10 μg/mL Hoechst in the dark. Then, the stained cells were observed under a Leica DMLB fluorescence microscope at a magnification of 200×. The heterochromatin was heavily stained and was identified by its characteristic bright fluorescence. While the apoptotic cells showed pyknotic nuclei full of heterochromatin, healthy cells showed normal nuclei with sparse heterochromatin spots. Three to five fields were randomly observed in each well, and percentages of apoptotic cells were calculated from 200–250 cells observed in each field. For each experimental series, all images were acquired with identical settings. Cell numbers were counted on raw images by using the Image J software . Briefly, the image was first converted into an 8-bit black and white format (image-Type-8-bit). The brightness threshold was then adjusted to eliminate the background brightness. Then, the threshold value for all the cells and for positive cells were set to 90–255 and 145–255, respectively. Finally, the range of nuclear area detected was set to 100 pixels. Each treatment was repeated three times on three different experimental days with each replicate including cells from one well of a 12-well culture plate.The OECs were first stained with Annexin V-FITC/propidium iodide (PI) staining  and then measured by flow cytometry. Briefly, the OECs were washed in PBS and digested at 37 °C with 0.25% trypsin for 3–5 min. After that, a DMEM/F12 medium containing 10% fetal bovine serum was added to neutralize the residual trypsin. We resuspended the cells of each sample in 100 μL binding buffer, containing 5 μL Annexin V-FITC, and incubated them for 10 min at room temperature in the dark. Finally, we added 5 μL PI to the OECs and analyzed the fluorescence of 10,000 cells per sample using a LSRFortessa (BD). Each treatment was repeated three times on three different experimental days with each replicate including cells from one well of a 24-well culture plate.We first prepared a radio immuno-precipitation assay (RIPA) buffer containing 150 mM NaCl, 1 mM phenylmethyl sulfonyl fluoride, 50 mM Tris (pH 8), 1.0% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS. We then washed the OECs in cooled PBS, and lysed them in 100 μL RIPA buffer. After that, the total protein concentration was determined using a BCA Protein Assay Kit  and was adjusted to 1 μg/μL. Then, 20 μL of total protein was placed in a 0.5-mL microfuge tube and frozen at −80 °C before use. To extract proteins, we added 5 μL of 5× sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer to each tube and heated the tubes to 100 °C for 5 min. We ran SDS-PAGE on a 10% polyacrylamide gel. We transferred the proteins electrophoretically onto polyvinylidene fluoride membranes. After that, we washed the membranes in TBST , blocked them with TBST containing 3% BSA at 37 °C for 2 h and incubated them at 4 °C overnight with primary antibodies. We then washed the membranes in TBST, and incubated them for 1.5 h at 37 °C with secondary antibodies. Finally, we detected signals using a BeyoECL Plus kit . Each treatment was repeated three times on three different experimental days with each replicate including OECs from one well of a 6-well plate.The primary antibodies we used are as follows: rabbit anti-activated caspase-3 antibodies , goat anti-CRHR1 polyclonal antibodies , rabbit anti-GR antibodies , rabbit anti-Fas , rabbit anti-TNFR , mouse anti-ACTB monoclonal antibodies , and mouse anti-GAPDH monoclonal antibody . The secondary antibodies are: HRP-conjugated rabbit anti-goat IgG , goat anti-rabbit IgG  and goat anti-mouse IgG .2O) and digested with RNase-free DNase I . Spectroscopical quantification of RNA was performed at 260 nm. The RNA purity and integrity were assessed by determining the A260:A280 ratio (1.8–2.0) and performing electrophoresis in 1% agarose.RNA was isolated by treating the OECs with 1 mL TRIzol reagent, resuspended in diethyl pyrocarbonate-treated MilliQ water  we incubated the mixture at 42 °C for 2 min in a PCR instrument ; (c) we added 4 μL 5× PrimeScript Buffer 2, 1 μL PrimeScript RT Enzyme Mix I, 1 μL RT Primer Mix and 4 μL RNase Free dH2O to the reaction tube; and (d) we incubated the mixture at 37 °C for 15 min, at 85 °C for 5 s before store at −20 °C until use. We conducted reverse transcription using a total volume of 20 μL and PrimeScript RT Reagent Kit (Takara RR047A): (a) we mixed 2 μL RNA sample, 2 μL 5× gDNA Eraser Buffer, 1 μL gDNA Eraser and 5 μL DEPC-dHIGF1, 60 °C for 34 s for BDNF, 58 °C for 34 s for TGFB1, 59 °C for 34 s for CRHBP, 63 °C for 34 s for 11-beta hydroxysteroid dehydrogenase 1 (HSD11B1) and HSD11B2. To determine reaction specificity, PCR products were analyzed by sequencing, dissociation-curve analysis and gel electrophoresis. Gene expression was normalized to internal control (GAPDH), and the values were expressed relative to calibrator samples by using the 2−(ΔΔCT) method. For the OECs, each treatment was repeated three times on three different experimental days with each replicate including cells from one well of a 24-well plate, and for embryos, each treatment was repeated three times with each replicate including 250 2- or 4-cell embryos.A Mx3005P Real-Time PCR System  was used to carry out the mRNA quantification, which was performed in a 10-μL reaction volume, containing 1 μL of cDNA, 5 μL of 2× TB Green Premix Ex Taq , 0.2 μL ROX Reference Dye II, 3.4 μL of RNase-free water and 0.2 μL each of forward and reverse gene-specific primers . The following cycle amplification conditions were used: (a) an initial denaturation step at 95 °C for 5 min; (b) 40 cycles at 95 °C for 5 s; and (c) 59 °C for 34 s for https://www.moma.dk/normfinder-software/, accessed on 30 May 2022), an algorithm-based tool, to identify the most stable genes among the six commonly used reference genes in samples derived from 10 different primary pOECs cultures. The ranking of expression stability calculated by Normfinder in the genes analyzed was GAPDH = HMBS > ACTB > HPRT1 > RPL13A > YWHAZ, and thus, Gapdh could be used as a stable internal reference gene in our experiments.We used the Normfinder . Total RNA was extracted from OECs as described above. Approximately 300 2-cell and 4-cell stage embryos or 50 blastocysts were used to isolate total RNA using a RNAqueous-Micro Total RNA Isolation Kit . Reverse transcription was conducted as mentioned above. Amplification reactions were performed in a 15 μL reaction volume containing 0.6 μL of cDNA, 1.5 μL of 10× PCR buffer, 0.97 μL dNTP, 0.06 μL Taq , 11.27 μL RNase-free water and 0.3 μL each of forward and reverse gene-specific primers 10 μM, . Amplifi2O, 1 μL template, 5 μL 2× One Step TB Green RT-PCR Buffer, 0.2 μL PrimeScript PLUS RTase Mix, 0.6 μL Takara Ex Taq HS Mix, and 0.2 μL ROX Reference Dye II). We analyzed the relative gene expression using the 2−∆∆CT method following normalization against sus scrofa actin beta (ACTB). We conducted mRNA quantification with the Mx3005P real-time PCR instrument . Each treatment was repeated three times with each replicate including 10 blastocysts.To extract RNA, 10 blastocysts were lysed with a commercial cell lysis kit . The RNA obtained was analyzed by real-time PCR using One Step TB Green PrimeScript PLUS RT-PCR Kit  and primers for Bcl2, Bax and Actb . We usedg, and added 100 μL of supernatant or standards in the coated wells. We then added 50 μL of conjugate to each well before incubation for 1 h at 37 °C. After we washed the coated wells with washing solution and dried them with paper towels, we added 50 μL of substrates A and B to each well before incubation for 15 min at 37 °C. Lastly, we terminated the reaction with 50 μL of stop solution, and measured the optical density within 15 min at 450 nm using a plate reader . We calculated the concentrations of FASL or TNFα against respective standard curves. Each treatment was repeated three times on three different experimental days with each replicate containing CM from one well of a 24-well plate.We carried out ELISA for FASL and TNFα in CM using Porcine Factor Related Apoptosis Ligand (FASL) Elisa kit  and Porcine tumor necrosis factor α (TNF-α) Elisa kit , respectively. We centrifuged CM for 10 min at 1000× 4, pH 7); (b) permeabilization with 0.25% TritonX-100 to observe GR; (c) block for 1 h in PBS with 3% bovine serum albumin (BSA); (d) overnight incubation at 4 °C with primary antibodies in PBS with 3% BSA; (e) 1-h incubation with secondary antibodies in PBS with 3% BSA; (f) 10-min incubation in PBS with 10 μg/mL of Hoechst 33342; and (g) observation under a Leica laser-scanning confocal microscope . We washed the embryos in PBS between treatments. We detected fluorescence using band pass emission filters . We also processed negative control samples with the primary antibody omitted to verify the specificity of the secondary antibodies.Immunofluorescence microscopy was performed to localize CRHR1, glucocorticoid receptor (GR), TNFR and FAS in embryos. We removed zona pellucida from the 2-cell, 4-cell and blastocyst embryos with 0.5% pronase in PBS at 37 °C. Then, the embryos were treated as follows: (a) fixation for 30 min in 4% paraformaldehyde in PHEM buffer  for CRHR1; rabbit anti-GR polyclonal antibodies  for GR; rabbit anti-TNFR1 polyclonal antibodies  for TNFR; and rabbit anti-FAS  for FAS. Cy3-conjugated AffiniPure goat anti-rabbit IgG  were used as secondary antibodies for GR, TNFR1 and FAS and Cy3-conjugated donkey anti-goat IgG  were used for CRHR1 secondary antibodies.FASL gene were FL siRNA-1 (5-CAA TCT ACC CTC TGA GAAA-3), FL siRNA-2 (5-GTC AGT ACT GCA ACA ACCA-3), and FL siRNA-3 (5-GAT GAA CTA TTG CAC TACT-3); those targeting the TNFα gene were TN siRNA-1 (5-CTC AGA TCA TCG TCT CAAA-3), TN siRNA-2 (5-CGA CTA TCT GGA CTT TGCT-3) and TN siRNA-3 (5-CCT ACC AGA CCA AGG TCAA-3); and we used siR-RiboTM for negative control. We performed transfection with 100 nM siRNAs using the lipofectamine RNAiMAX reagent . When OECs grew to approximately 60% of confluence, we replaced the spent medium in the wells with 450 μL fresh DMEM/F-12 medium and transfected the cells using the forward transfection method. To prepare the transfection complex, we diluted 1 μL of a 20 μM solution of each siRNA in 50-μL Opti-MEM medium (Invitrogen), and mixed it with 3 μL of Lipofectamine RNAiMAX reagent (Invitrogen) diluted in 50 μL Opti-MEM medium. Following incubation for 5 min at room temperature, we added 50 μL of the transfection complex to the wells before incubation for 6 h at 38.5 °C in a humidified 5% CO2 atmosphere. The transfected cells were further cultured for 48 h in serum-free DMEM/F12 with or without CRH or cortisol before recovery of CM or cells for ELISA measurement of FASL or TNFα or for flow cytometry analysis of apoptosis, respectively.The RiboBio  designed and synthesized the siRNAs targeting mRNAs and the negative control siRNA. The sense strands of siRNAs targeting the t-test to analyze data when each measure contained only two groups, and used ANOVA when each measure had more than two groups. We carried out a Duncan multiple comparison test to locate differences during ANOVA. We expressed data as means ± SEM, and considered differences significant when p < 0.05.Each treatment contained at least three replicates, unless otherwise specified. We used the software of Statistics Package for Social Sciences  to perform data analysis. We arc sine-transformed the percentage data before analysis. We used independent −8 M, 3 × 10−11 M and 4 × 10−7 M, respectively, our results suggest that none of the three hormones impaired development of pig preimplantation embryos even at levels much higher than the physiological concentrations.In vitro matured (MII) pig oocytes showing the first polar body (PB1) were chemically activated and cultured in the presence of different concentrations of CRH, ACTH or cortisol for 7 days before examination for embryo development. The results revealed no significant effects of any concentration of any hormone tested on any stage of embryo development . Because−6 M and 1 × 10−6 M, respectively  or cortisol (4 × 10−7 M) alone or both before examination for apoptosis by flow cytometry. The results show that although culture with either 1 × 10−8 M CRH or 4 × 10−7 M cortisol alone did not increase apoptosis, culture with both 1 × 10−8 M CRH and 4 × 10−7 M cortisol significantly increased early and late apoptosis of pig OECs , ACTH (1 × 10−8 M) or cortisol (1 × 10−6 M) for 7 days before observation for embryo development. Control oocytes were cocultured with OECs without stress hormones. Although culture with ACTH showed no effect, the presence of CRH or cortisol significantly decreased the blastocyst rates and the cell number per blastocyst in pig embryos cocultured with OECs , or with OECs pretreated with either CRH (CRH-CM) or cortisol (Cort-CM) before examination for fertilization. Oocytes with one or no pronucleus, oocytes with two pronuclei and those with three or more pronuclei were considered unfertilized, monospermic and polyspermic, respectively A. Althou−6 M of CRH or 1 × 10−6 M of cortisol for 48 h before RT-PCR assays. Treatment with either CRH or cortisol significantly decreased the expression of BDNF, IGF1, TGFB1 and OVGP1 mRNAs  higher than that in CM from the untreated control OECs (27.83±2.66 pg/mL), the TNFα concentration in CM from corticosterone-treated mouse OECs (33.00 ± 6.15 pg/mL) was significantly (p < 0.05) lower than that in CM from control OECs (69.59 ± 7.10 pg/mL). The results confirmed the species difference in the effect of glucocorticoids on TNFα expression in OECs.Pig OECs were treated with 2 × 10and TNFα E,F. Beca−6 M of CRH or 1 × 10−6 M of cortisol for 48 h before the Western blotting analysis. While treatment with CRH significantly increased CRHR1 expression, treatment with cortisol decreased GR expression significantly , FASL or TNFα siRNAs were cultured in DMEM/F12 without hormones for 48 h before recovery of CM for ELISA measurement for FASL or TNFα. The FASL and TNFα levels decreased significantly after transfection with NC siRNA A,B. To se of CRH C,E or coe of CRH D,E. The −8 M CRH and 4 × 10−7 M cortisol with OECs that had been transfected with NC siRNA or co-transfected with both FASL siRNA-1 and TNFα siRNA-3. Co-transfection of OECs with FASL and TNFα siRNAs significantly (p < 0.05) increased both the blastocyst rates and the cell number per blastocyst in comparison with those following transfection with NC siRNA . Our previous study [−8 M CRH or 1 mg/mL cortisol did not affect pig oocyte development into blastocysts, culture with both significantly decreased blastocyst rates. The present results showed that although culture with neither stress-induced concentration of CRH (1 × 10−8 M) nor that of cortisol (4 × 10−7 M) alone increased apoptosis, culture with both 1 × 10−8 M CRH and 4 × 10−7 M cortisol significantly increased early and late apoptosis of pig OECs. Thus, the results suggest that in vivo, stresses might increase the release of multiple hormones that work together to induce a symptom at lower concentrations than in vitro in the presence of only a single hormone.In this study, the effective concentrations of CRH and cortisol that caused the significant apoptosis of pig OECs were higher than peak serum concentrations reported in stressed pigs. Du et al. ,54 obserus study  demonstrIn summary, this study shows that although ACTH showed no effect on pig embryo development, CRH and cortisol impaired preimplantation embryo development and increased polyspermy indirectly by inducing the apoptosis of OECs. Thus, after the treatment of OECs with CRH or cortisol, while the expression level of GR decreased, that of CRHR1, FAS and TNFR1 increased significantly, which would lead to apoptosis . The apoThe results of this study demonstrate that CRH and cortisol impair pig fertilization and preimplantation embryo development indirectly by inducing OEC apoptosis via the activation of the FAS and TNFα systems. However, ACTH did not show any detrimental effect on pig embryos, nor on OECs. The data are important for us to understand the mechanisms by which stress impairs reproduction and to take measures to ameliorate the harmful effects of stress on fertilization and embryo development.


Bifidobacterium longum BB68S (BB68S). Moreover, we examined the water content, water activity, enzyme activities, and scanning electron microscope of BB68S to explore mechanisms underpinning the effect of temperature on viability. Our analyses revealed the highest survival rates of BB68S collected after pre-freezing and sublimation drying at −40 °C (94.9 ± 2.2%) and −10 °C (65.4 ± 3.8%), respectively. Additionally, response surface methodology demonstrated that the optimum conditions for freeze-drying of BB68S were pre-freezing temperature at −45.52 °C and sublimation temperature at −6.58 °C, and the verification test showed that survival rates of BB68S could reach 69.2 ± 3.8%. Most of the vitality loss occurred during the sublimation drying phase. Further studies showed that different sublimation temperatures affected water content and activity, β-galactosidase, lactate dehydrogenase, Na+-K+-ATP and Ca2+-Mg2+-ATP activities. In conclusion, the temperature during freeze-drying, especially sublimation temperature, is a key factor affecting the survival rate of BB68S, and the vitality loss during freeze-drying process might be due to compromised cell membrane integrity and permeability.Maintaining optimum temperature during freeze-drying is crucial to ensuring the viability of strains. In this study, we evaluated the effect of pre-freezing, sublimation and desorption temperatures on the viability of  Probiotics play a key role in the food and dairy industries, and probiotics have been shown to exert positive effects on health status of the host . PowdereLactobacillus plantarum strains. Aragón-Rojas et al. [Lactobacillus fermentum K73. The temperature during different freeze-drying stages might affect the survival rates of probiotic strains. It is crucial to optimize pre-freezing, sublimation and desorption temperatures for the improvement of survival rates. However, the mechanisms underpinning the effect of freeze-drying temperatures on the viability of bacterial strains have remained unexplored.The freeze-drying process has three stages: pre-freezing, sublimation drying and desorption drying . The wats et al.  observedBifidobacterium are used as probiotics, among which Bifidobacterium longum is widely used and has a history of safe use [Bifidobacterium longum BB68S  was isolated from healthy centenarians in Bama longevity villages of Guangxi province in China. The aim of this investigation was to optimize pre-freezing, sublimation and desorption temperatures during the freeze-drying process of BB68S. Furthermore, we explore the putative mechanisms through which freeze-drying temperatures affect bacterial survival rate to provide the rationale behind the selection of temperatures during the freeze-drying of probiotics.At present, numerous species of safe use . Bifidobv/v).BB68S was cultured in Man–Rogosa–Sharpe broth at 37 °C for approximately 12 h. The biomass fermentation was performed in a 5 L fermenter  with 3 L working volume, at 80 rpm for 12 h. The medium composition was 1.5% yeast peptone, 1.0% yeast extract powder, 0.5% beef extract powder, and 4.0% glucose. The microbial inoculation rate was 2% . Sublimation drying survival rates of BB68S were calculated after 2 h of pre-freezing (−40 °C) and 20 h of sublimating at different temperatures . Desorption drying survival rates of BB68S were calculated after 2 h of pre-freezing (−40 °C), 20 h of sublimating (−10 °C), and 15 h of treating with different desorption temperatures .2 was applied to optimize pre-freezing temperature  and sublimation temperature  during the freeze-drying process (1) and sublimation temperature (X2). The relationship between response (Y) and factors (X) was described using the following equation:Design Expert Version 12.0  software was applied for RSM design and statistical analysis. According to RSM, an experimental design 3 process . The desi and xj represent independent coded variables ; β0, βi, βii and βij represent the coefficients for intercept, linear, quadratic and interaction, respectively.In this equation, Y represents the response variable ; xViable cell counts were detected using the plate-counting method. The powder was rehydrated with PBS and incubated on MRS plates at 37 °C for 48 h for counting. The survival rates (%) after pre-freezing, after drying and after freeze-drying were calculated as follows: N1/N0 × 100%, N2/N0 × 100% and N3/N0 × 100%, where N1 represents the viable counts after pre-freezing, N2 represents the viable counts after drying, N3 represents the viable counts after freeze-drying, and N0 represents the viable counts before freeze-drying .w was measured using a water activity meter  at room temperature. The freeze-dried samples were oven dried at 105 °C until constant weight. The weight loss was measured to estimate water content.The ag for 10 min at 4 °C The supernatant was used to measure extracellular enzyme activity. For collecting cell-free extracts, 10 mg/mL lysozyme was added, and the mixture was kept at 37 °C for 15 min. Then, 4 mol/L sodium chloride solution was added, and incubated at 37 °C for 50 min. Finally, the mixture was centrifuged at 10,000× g for 10 min.After rehydration, the bacterial cells were centrifuged at 10,000× +-K+-ATP and Ca2+-Mg2+-ATP were analyzed using commercial kits  according to the instructions. One unit of β-galactosidase activity is defined as the amount of enzyme that releases 1 nmol o-nitrophenol per hour at 37 °C. The amount of released o-nitrophenol was measured at 400 nm. One unit of lactate dehydrogenase activity is defined as the amount of enzyme that releases 1 nmol pyruvate per min at 37 °C. The amount of pyruvate was measured at 450 nm. One unit of Na+-K+-ATPase and Ca2+-Mg2+-ATPase are both expressed as 1 μmol of inorganic phosphate released per milligram of protein per hour, and the sample was measured at 660 nm.The activities of β-galactosidase, lactate dehydrogenase, NaLeyophilized powder was fixed on a glass slide usingconductive tape and sputtered with gold at 10 mA for 2 min. The morphology of gold-clad samples was evaluated with a Hitachi SU8020 field emission scanning electron microscope (Japan) at 5 KV.p < 0.05 was used as a threshold for statistically significant differences.All experiments were conducted with three biological replicates, and the data are expressed as the mean ± standard deviation (SD). Statistical analyses were performed using SPSS . Significant differences among the data were detected using ANOVA. p < 0.05) (94.9 ± 2.2%) compared to those after pre-freezing at −20 °C (82.1 ± 2.2%) and −60 °C (92.3 ± 3.8%) (p < 0.05) (65.4 ± 3.8%) compared to those collected after sublimation drying at −20 °C (42.3 ± 3.8%) and 0 °C (56.4 ± 2.2%)  A. The su ± 2.2%) B. No dif ± 2.2%) C. Overal1) and sublimation temperatures (X2) on the survival rate of BB68S was evaluated by the application of RSM, and predicted values were similar to experimental values , and the p-value for lack of fit was not significant (p = 0.837), indicating a good fit. The coefficient of determination (R2) was higher than 90% (R2 = 0.9899), and adjusted R2 was 0.9827, indicating that the model could explain 90% of the response variation. In decreasing order of F values, the factors affecting the survival rates of BB68S were X12  > X22  > X2  > X1  > X1* X2 . As shown in w of BB68S under different sublimation temperatures are shown in w of BB68S were obtained under the condition of sublimation temperature of −10 °C (p < 0.05).The water content and a+-K+-ATPase and Ca2+-Mg2+-ATPase activities were significantly higher at the sublimation temperature of −10 °C than those of at 0 °C and −20 °C (p < 0.05). As for extracellular β-galactosidase and lactate dehydrogenase, activities revealed that those of the powders obtained at a sublimation temperature of −10 °C were the lowest (p < 0.05).As shown in The SEM of BB68S obtained under different sublimation temperatures are shown in w. However, desorption temperature did not seem to affect the survival rates of BB68S in the current study.In the current study, we evaluated the effect of pre-freezing and sublimation temperatures on the survival rate of BB68S. Previous research showed that pre-freezing temperatures could affect pre-freezing rates, under the same pre-freezing time. The pre-freezing rate showed a trend to reduce with the increase of pre-freezing temperature . On the RSM is a collection of mathematical and statistical methods, and RSM is often used for the development, improvement and optimization of process parameters . Recentlw are crucial to the storage of probiotics products [w of each dried product was in the range of 0.012–0.020, and water content was in the range of 2.76–3.27%, which were equal to or lower than previously reported values, thus the samples were considered sufficiently dehydrated under drying conditions used in this study [w and water content of obtained probiotics after freeze-drying are in the optimal range, water may interact with functional groups and block reaction sites, thus avoiding interactions with oxygen and oxidation reactions that lead to lipid and protein degradation in cells [Similar to previous reports, water content was associated with sublimation temperatures during the freeze-drying process . Water cproducts ,23,24. Tis study ,25. When+-K+-ATPase and Ca2+-Mg2+-ATPase membrane-bound enzymes, and ATPase are crucial to cell functions and intracellular ions gradients [+-K+-ATPase and Ca2+-Mg2+-ATPase activities of bacterial cells are considered to be related to the enhancement of cell membrane integrity and tolerance to freeze-drying [ATP-hydrolyzing enzymes such as Naradients ,27. The e-drying . β-galace-drying . On the e-drying . The free-drying . TherefoDuring the dehydration of strains, most of the water inside the cells was expelled through membrane channels. During the freeze-drying process, the cells could not fully recover due to the destruction of cell structure by dehydration, thus reducing cell viability . In the Freeze-drying is an excellent method for preserving probiotics; however, negative effects on the survival rates of strains are inevitable. This experiment showed that pre-freezing temperatures and sublimation temperatures during freeze-drying affects the survival rates of BB68S, and the optimization of pre-freezing and sublimation temperatures could increase the viability of BB68S. Additionally, survival rates affected by different temperatures might be related to cell membrane integrity and permeability. These results provide more strategy for the improvement of probiotics survival rates. Furthermore, this study provides theoretical support for selecting pre-freezing and sublimation temperatures during the freeze-drying process.


D) and detonation pressure (P) of NTZF were calculated. NTZF is insensitive to impact and friction  and has higher detonation velocity and pressure  compared to TNT . NTZF has appropriate sensitivity and detonation performance, so it can be used as a low explosive and gas generant.The synthesis mechanism of 3-nitro-4-(tetrazol-5-yl)furazan (NTZF) was calculated by Gaussian 09 for the first time, and NTZF was successfully synthesized based on the theoretical design. Its ionic salts (RbNTZF and CsNTZF) were synthesized and studied by single-crystal X-ray diffraction firstly. The thermal stability of NTZF was investigated by TG-DSC and the kinetic data of thermal decomposition were calculated. The sensitivity of NTZF was measured. The formation heat, detonation velocity ( The synthesis mechanism of 3-nitro-4-(tetrazol-5-yl)furazan (NTZF) was calculated by Gaussian 09 for the first time, and NTZF was successfully synthesized based on the theoretical design. PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>N, C–N. Some energetic nitrogen-rich compounds have been synthesized for application in the field of military chemistry and explosion safety, such as high-energy insensitive explosives,1,2 small propulsion system solid fuels,3,4 smokeless pyrotechnics,5,6 gas generators,7,8 flame low temperature fire extinguishing agents9,10 and so on.As a new type of energetic materials, nitrogen-rich compounds contain a high enthalpy of formation and high energy density. Their molecular structures include a large number of chemical bonds N–N, C–N, N3N7HO3. The nitrogen content of it is 53.55%. The structure of it is very stable because of the π-conjugate formed by the heterocyclic system. Thus, the NTZF is a stable energetic compound which has good application prospects in the field of explosives. NTZF was synthesized for the first time by Wang11 with ATZF as the raw material. However, the synthesis mechanism of NTZF hasn't been studied to date.The molecular formula of NTZF is C1H NMR, 13C NMR and EA. Single crystals of its ionic salts were cultivated firstly. The thermal stability was investigated by TG-DSC and friction sensitivity was measured. Additionally, its formation heat, detonation pressure and detonation velocity were calculated.In this study, the synthesis mechanism of NTZF was studied by theoretical calculations for the first time, and its structure was characterized by IR, 2.2.1All chemical used reagents and solvents were analytically pure, and purchased commercially.In this paper, 3-amino-4-(tetrazol-5-yl)furazan (ATZF) was synthesized according to the method in 2SO4 (4 mL) and ATZF  were added to a three-necked flask at room temperature. The reaction mixture was stirred until the solid had dissolved. Na2WO4  was added to 30% H2O2 (24 mL) and the mixture was stirred gently at −10 °C, until the solid had completely dissolved. The solution was added dropwise to the three-necked flask where a vigorous exothermic reaction occurred. The temperature of the reaction was adjusted to 35 °C and keep 3 h. The resulting solution was filtered and extracted with acetate (20 mL × 3). The yellow-green product of NTZF were washed with saturated brine, dried and evaporated. Yield 77.50%. C3N7HO3: calcd C 20.04, N 52.12, H 0.87%; found: C 19.68, N 53.55, H 0.55%; IR : 1566.77, 1328.01 (–NO2), 3095.65, 1144.77, 1114.42 (Tetrazole ring), 1471.58, 1397.51, 996.10 (Furan ring). 1H NMR : 12.17 . 13C NMR : 160.02, 147.37, 141.34.Concentrated HRbNTZF and CsNTZF: the synthesis of RbNTZF/CsNTZF was carried out from NTZF by reaction with rubidium/cesium. 1.83 g (10 mmol) NTZF was added in a three-necked flask with 20 mL of water at 40 °C. After stirred fully to dissolve, the mixture was added 1.15 g (5 mmol) rubidium carbonate/1.63 g (5 mmol) cesium carbonate. The reaction produced a large number of bubbles. After fully responsed to clarification, the reaction liquid was filtered and extracted. The filtrate was mixed with toluene by certain proportion. The product was steamed at 60 °C and dried backspin steamed.2.2A portion of the solution extracted during the above reaction was removed. The sample was placed in a clean beaker after filtering. The beaker was placed in a thermostatic incubator for 3 days until the colorless crystal which can be used for single crystal test appear.2.3Infrared spectra (IR) was recorded with a Nicolet FT-IR6700. Elemental analysis was measured with a Vario EL elemental analyzer. The NMR spectrum was carried out with a DRX500 Bruker NMR spectrometer. Single crystal structure characterization was determined by Rigaku 742+ single crystals diffractometer.−1 flux over the temperature ramp of 50–400 °C, at a heating rate of 2, 5, 10, 20 °C min−1.Differential scanning calorimeter (DSC) and thermogravimetric analysis (TG) were carried out with a STARe system and a CDR-4P differential scanning calorimeter and thermogravimetric analyzer. TG-DSC of ATZF was measured under nitrogen with a 50 mL minThe impact and the friction sensitivities were tested according to BAM standard methods. The flame sensitivity was determined by HGY-1 flame sensitivity instrument. The sample mass used for each test was 20 ± 2 mg. The sample was compressed into a fire cap at 58.8 MPa and the cap was ignited by the standard black powder column. The 50% ignition height indicated the friction sensitivity.3.3.13.1.114 The geometric structure of the NTZF molecule is shown in †The structure of NTZF was optimized with B3LYP/6-311 ++ G **  using thBased on the principle of minimum energy, the lone pair electrons on one oxygen atom of the nitro group repulsively opposes the lone pair of nitrogen atoms in tetrazole. The furazan ring, tetrazole ring and nitro in NTZF are not coplanar. The bond length of furazan ring is between 1.30–1.43 Å and the bond angles are similar to each other. Therefore, it tends to form a stable five-membered ring structure. The repulsion between the lone pair of the oxygen atom and nitrogen atoms makes the bond angles of the N5–O1–N4 in the furazan ring become larger. What's more, the bond length in the tetrazole ring is between 1.30–1.36 Å and the bond angles are similar. It is also a stable five-membered ring structure. In summary, the optimized molecular structure of NTZF is stable.3.1.234 system is larger than that in π-conjugated tetrazole, therefore, the positive charge carried by C3 is much larger than C2. The conjugate system of the tetrazole ring accepts the electron the hydrogen atom was supplied with, so that the hydrogen with the positive charge is more easily ionized.Natural bond orbital (NBO) analysis was performed on the geometry of the optimized NTZF at B3LYP/6-311 ++ G ** level. The NBO charge distribution of each atom in NTZF was obtained . The hea3.1.33 isosurfaces of electron density elaluated at the B3LYP/6-311++G** level. The colors range from −3.00 × 10−2 to +5.00 × 10−2 eV with dark blue denoting extremely electron-deficient regions [V(r) ≥ 5.00 × 10−2] and red denoting electron-rich regions [V(r) ≤ −3.00 × 10−2].As shown in 3.1.4E = 5.42 eV) is the energy required for the electrons in the molecule to excite from the ground state to the excited state. Above all, the molecule is relatively stable.3.22O2 is reflected in its own heterogeneous ability. As is shown in 2O2 move to the oxygen atom and undergo isomerization, forming a water molecule with one active oxygen atom. The active oxygen atom is the key to oxidation. The amino-group (–NH2) in ATZF is oxidized by H2O2 to form nitroso-group (–NO), which is further oxidized by H2O2 to form a nitro group .The relevant electron energy parameters of NTZF are obtained by analyzing the optimized NTZF geometry results at the B3LYP/6-311 ++ G ** level . By subs4.(1) Based on the density functional theory (DFT), the molecular structure of NTZF is designed and optimized. The synthesis mechanism of NTZF is explored by the transition state theory for the first time.(2) NTZF was successfully synthesized based on the theoretical design. Its ionicsalts (RbNTZF and CsNTZF) were synthesized and studied by single-crystal X-ray diffraction firstly.−1, 7.838 km s−1 and 27.32 GPa.(3) The stability and detonation performance of NTZF were studied. It has good thermal stability (the exothermic decomposition peak temperature is at 176.83–248.50 °C). NTZF is insensitive toward impact and friction . The formation heat, detonation velocity and detonation pressure is 256.32 kJ molThese indicate that NTZF is an energetic heterocyclic compound with low sensitivity which has broad application prospects in low explosives and gas generant.There are no conflicts to declare.RA-008-C8RA02682C-s001RA-008-C8RA02682C-s002


Structural analysis revealed two distinct patterns influenced by the counter ions where L acts as a tridentate chelating ligand. The in vitro antitumor activity of L and L′ ) diacetate) as well as their metal complexes, was tested by the measurement of their cytostatic and cytotoxic properties towards the blood cancer mastocytoma cell line P815. We have also investigated their interactions with the antioxidant enzyme system. As a result, [MnL′Cl2] (1′) exhibited the strongest activity compared to reference cis-platin with no cytotoxicity towards normal cells PBMCs . On the other hand, the antioxidant enzyme activity showed that the efficiency of metal complex 1′ against P815 tumor cells was via the rise in the SOD activity and inhibition of CAT enzyme activity. This proof of concept study allows disclosure of a new class of molecules in cancer therapeutics.The complexes: [CoL 2](ClO4)2 (1), [FeL2](ClO4)2 (2), [NiL2](ClO4)2 (3) and [MnLCl2] (4), with L = diethyl-1,1′-bis(5-methyl-1H-pyrazole-3-carboxylate), were synthesized and fully characterized.The complexes: [CoL The situation is particularly worrying in Europe, with 23% of reported cancers and 20% of deaths while in the United States, it is considered as the second leading cause of death, after heart disease.2 Therefore, developing an urgent effective treatment remains one of the greatest challenges in clinical oncology. Treatment options for cancer depend on the type of islet cell cancer, the extent of metastasis, and the general health of a patient. Standard treatment options for cancer include surgery, chemotherapy, hormone therapy, radiotherapy or the introduction of biological drugs. The emergence of resistant tumours, however, considerably limits the effectiveness of conventional chemotherapies. This is why it is becoming interesting to explore various therapeutic avenues, including the development of new drugs that are active on resistant cancers and prevent the formation of metastases. Heterocycles represent a majority of active components of cancer agents,3 some of them being coordinated to metal complexes. For example, nitrogen-donor chelating ligands have been widely used in the architectural design of metal complexes with biological applications, including cytotoxic activities.4–7 For instance, Singh et al. synthesized a series of 5-iodouracil complexes with Mn(ii), Co(ii), Cu(ii), Zn(ii) and Cd(ii) ions, some of which were active against Sarcoma-180 and L929 tumor cells.8 Later, the effects of 5-bromouracil complexes with Cr(iii), Fe(iii) and Al(iii) were reported on P815 murine mastocytoma.9 We recall that P815 is a mastocytoma cell line commonly used as an experimental tumor model due to its several advantages for in vivo experimentation. It is derived by methylcholanthrene treatment of a male DBA/2 mouse.10 Other examples include transition metal complexes used as pro-drugs where a cytotoxic agent bound to the metal ion can be released during treatment.11According to the World Health Organization (WHO), cancer remains one of the world's most damaging diseases, with 18.1 million new cases of cancer and not less than 9.6 million deaths in 2018.12 It is also involved in the synthesis and activation of numerous enzymes and in the regulation of glucose and lipid metabolism in humans.13 In addition to its physiological role, manganese may have beneficial therapeutic and preventive effects on infectious diseases and, compared to other metal-based drugs, Mn(ii) complexes generally exert lower toxicity and have less side effects.However, such complexes are limited by severe side effects, general toxicity, and drug resistance. To limit these side effects, manganese is among the most promising metals that is essential to human physiology. In the human body, manganese is one of the components required for Mn superoxide dismutase which is primarily responsible for trapping reactive oxygen species in mitochondrial oxidative stress.14,15 The pyrazole scaffold is a privileged pharmacophore encountered in many chemical compounds confirmed to be associated with various biological assets such as anti-inflammatory,16 antimicrobial and anticancer properties.17,18 Furthermore, many compounds bearing a pyridine moiety have been identified to be biologically active19–22 and therapeutically relevant in medicinal chemistry and to display versatile biological activities including antibacterial and anticancer activities.18,23 In view of their biological significant activity, the combination of pyrazole and pyridine moieties may lead to the synthesis of a new class of therapeutical active compounds, and is thus very much attractive.24,25 In particular, because such materials are rarely cited for their individual anticancer activities, despite their significant activities,26,27 which could be attributed to the electronic interactions between the metal centre and π electrons in their aromatic rings.28,29Heterocyclic compounds containing pyrazole and pyridine were reported to show remarkable biological properties.in vitro individual anticancer activities with particular emphasis on their complexes of Co, Fe, Ni, and Mn. This led us to prepare diethyl 2,2′-) diacetate (L′)30 and diethyl 1,1′- bis(5-methyl-1H-pyrazole-3-carboxylate) (L) (2](ClO4)2 (1), [FeL2](ClO4)2 (2), [NiL2](ClO4)2 (3), [MnLCl2] (4), [MnL′Cl2] (1′) and [CdL′Cl2] (2′) were also investigated. A structure–activity relationship study taking into account the nature of ligands and coordination metals is discussed.To this end, we have designed novel derivatives of nitrogen-chelating pyrazolyl–pyridine ligands with CC (L′) and CN (L) junctions, to study their ate) (L) . Metal c2.2.1.−1. 1H and 13C NMR spectra were acquired on a Bruker AC 300 MHz instrument. Chemical shifts (δ) are given in ppm referring to the signal centre using the solvent peaks as reference: CDCl3 7.26 ppm/77.36 ppm. High-resolution mass spectrometry HRMS data were obtained with a Q Exactive Thermofisher Scientific ion trap spectrometer using ESI ionization. UV-visible spectra were recorder using a Shimadzu 3600 plus spectrometer equipped with Harrick praying mantis modulus which allows direct analysis of powders in reflectance mode. Melting points were measured using a Koffler bench. A 57Fe Mössbauer spectrum was recorded in transmission geometry mode with a constant acceleration mode conventional spectrometer equipped with a 50 mCi 57Co(Rh) source and a Reuter Stokes proportional counter. The powdered sample was sealed in aluminium foil, and the spectrum was recorded at room temperature. The spectrum was fitted using Recoil 1.05 Mössbauer Analysis software.31 The isomer shift values are given with respect to α-Fe at 298 K. Thermogravimetric Analyses (TGA) were carried out on a Mettler Toledo TGA/SDTA 851e analyser by loading 3–4 mg of sample, and the mass loss was monitored under nitrogen on warming from room temperature to 900 °C at 10 °C min−1. Magnetic susceptibilities were measured on a quantum design MPMS-5s SQUID magnetometer. The magnetic data were corrected for the sample holder and diamagnetic contributions. The crystal sample was quickly loaded into a gelatin capsule and immediately inserted within the SQUID cavity.All solvents and chemicals were obtained from commercial sources and used without purification. The progress of reactions and the elution of products were followed by TLC (silica gel). Infrared spectra were recorded on a PerkinElmer 1310 Spectrometer FT-IR in the region 4000–400 cm2.1.12(g) flow. The data were integrated with the CrysAlisPro software.32 The structures were solved by direct methods using the SHELXT program33 and refined by full matrix least squares on |F2| using SHELXL2014/7.33 The summary of data collection and crystallographic parameters of 1–4 are listed in Table S1.2073690–2073693 contain the supplementary crystallographic data for this paper.†Single-crystal X-ray diffraction data of 1–4 were collected on a MAR345 image plate detector using Mo Kα radiation at either ambient temperature or flash cooled to 150 K in a gaseous N2.1.2l-glutamine. Incubation was performed at 37 °C in humidified atmosphere containing 5% CO2.Murin mastocytoma cell line P815 was cultured in RPMI 1640 medium supplemented with 5% heat-inactivated fetal bovine serum, 1% penicillin G-streptomycin, and 0.2% of 2.1.334 Briefly, the target cells (P815 murine mastocytoma) were washed twice with phosphate-buffered saline (PBS) and seeded on 96-well microtiter plates at the density of 104 cells per well. Then, 100 μL of the culture medium supplemented with different concentrations (0–200 μM) of the compounds were added. Control cells were treated with DMSO alone. In all cases, the final concentration of DMSO never exceeded 0.2%. Following 48 h of incubation in humidified atmosphere at 37 °C and 5% CO2, 20 μL of MTT solution (5 mg mL−1 in PBS) was added to each well and incubated under the same conditions. After 4 h, 100 μL of medium was carefully removed from each well and replaced with 100 μL of acid–isopropanol (0.04 N HCl in isopropanol). The cell viability was measured in a spectrophotometer (MultisKan EX apparatus (Labsystem)) at λ = 540 nm and expressed as the percentage of control cells, using the following formula:A0 and Ai are the optical density of control cells and treated cells, respectively. The cytotoxic activity of the compounds was compared by calculating the IC50 values (concentration leading to 50% of cell inhibition).Cellular viability was performed using the 3--2,5-diphenyltetrazolium bromide (MTT) assay.2.1.4This test was realized in order to evaluate the effect of the ligands and their active metal complexes against non-cancerous cells using the MTT colorimetric assay described below. To isolate the human PBMCs, blood samples in sterile heparinized tubes were collected under medical and ethical committee control from healthy volunteer donors. PBMCs were isolated using standard Ficoll-Hypaque density centrifugation. The interface lymphocytes were washed twice with phosphate buffer solution (PBS). The cytotoxic effect was measured in the same conditions and concentrations as detailed above for the tumor cells.2.1.5g for 10 min. The pellet was suspended in 500 μL of lysis buffer composed of 50 mM Tris–HCl, 1 mM phenylmethanesulfonyl (PMSF), 0.1% (v/v) Triton X-100, in 1.5 mL Eppendorf tubes and maintained in constant agitation at 4 °C for 30 minutes. The homogenate was then centrifuged  at 4 °C. The supernatant (enzyme extract solution) was kept at −80 °C or used for the determination of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glutathione reductase (GR) activities.P815 tumor cells were treated with the ligands and their active complexes for 48 h. Then, after washing once with PBS, the cells were harvested and centrifuged 12 000et al.35 with some modifications. Briefly, the reaction mixture was composed of 0.05 M phosphate buffer, pH, 7.5, 10 mM methionine, 0.1 μM EDTA, 2 μM riboflavin, 75 μM Nitro Blue Tetrazolium (NBT) and the enzyme extract. The SOD activity was measured at 560 nm. One unit of SOD activity was defined as the quantity of SOD required to obtain a 50% inhibition of the reduction of NBT. The activity was expressed as units per mg of protein content.SOD activity was assayed according to the method of Sun 36 with some modifications. The final reaction volume of 0.5 mL included 0.1 M phosphate buffer, pH 7.0, 15 mM H2O2, and enzyme extract. The decomposition of H2O2 was monitored at 240 nm for 2 min at 25 °C. The extinction coefficient (43.6 M cm−1) was used to calculate the catalase activity, expressed as μmol of H2O2 decomposed per min per mg protein.The catalase activity was measured by the method of Aebi37 Briefly, the reaction mixture contained 0.1 M phosphate buffer, pH 7.6, 1 mM GSSG and 0.2 mM NADPH. The contents were incubated at 25 °C for 3 min and the reaction was initiated by adding enzyme extract. GR activity was expressed as nmol of NADPH oxidized per min per mg of protein by using the extinction coefficient of 6.2 mM−1 cm−1.The GR activity was determined by the oxidation of NADPH at 340 nm as described by Carlberg and Mannervik.38 with some modifications. The reaction mixture contained 0.1 M potassium phosphate, pH 7.0, 1 mM EDTA, 1 mM sodium azide, 1 mM GSH, GR (10 μg mL−1), 0.25 mM NADPH and enzyme extract. The mixture was incubated at 25 °C for 3 min and completed by adding 0.25 Mm of H2O2. The rate of NADPH oxidation was monitored at 340 nm for 5 min. GPx activity was calculated and expressed as μmol of NADPH oxidized per min per mg of protein by using the extinction coefficient of 6.2 mM−1 cm−1.GPx activity was carried out according to the method described by Lawrence & Burk39 using Bovine Serum Albumin (BSA) as a protein standard.The total soluble protein content of the enzyme extracts was determined following the method of Bradford,2.2.H-pyrazole-3-carboxylate (Lpyz), which is carried out in two steps, as reported previously by our group  and potassium tert-butoxide  were both dissolved under Ar(g) at r.t. in dry and degassed DMSO 99.8% (20 mL) to give a dark red solution. The mixture was stirred for 30 min, then dibromopyridine  solubilized in DMSO 99.8% (10 mL) was added. The mixture was kept under Ar(g) for 24 h at 140 °C. After the reaction time has elapsed, it was cooled to r.t. and filtered. A white precipitated was formed by adding water (50 mL), which was filtered and washed with water (3 × 20 mL). Yield 42% (4.82 g). Mp = 125(1) °C. FT-IR/cm−1: 2985 (w), 1724 (s), 1601 (m), 1427 (s), 1243 (s), 810 (m). 1H NMR  δ (ppm) = 2.62 ; 2.62 ; 4.43 ; 6.74 ; 7.93 . 13C NMR  δ (ppm) = 13.97 , 14.79 (CH3–CH2), 61.18 (CH2–O), 111.06 (CHpyrazolyl), 117.35–142.63 (pyridine), N PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>Cpyz(150.89). MS (ESI), m/z: 406.1483 [MNa+].The synthesis of L was carried out adapting a literature method.2.2.24)2·6H2O  and L  in MeOH (10 mL) was stirred for 15 min. The solution was left at room temperature. The obtained powder was recrystallized by slow diffusion of diethyl ether (30 mL) into the methanolic solution. Pink needle crystals of 1 suitable for X-ray measurements were filtered off after 10 days. Yield 23% (12 mg). FT-IR/cm−1: 3134 (m), 2989 (w), 1623 (m), 1235 (s), 856 (w). HRMS (ESI): m/z: 924.1997 [L235ClO459Co].A solution of Co(ClO2.2.34)2·6H2O  in acetone (10 mL) was added to a solution of 10 mL of L  in chloroform (20 mL), and the mixture was stirred for 15 min. An orange/yellow solution was formed. A yellow powder was obtained by slow addition of diethyl ether (40 mL) into this solution. After 30 min stirring, the solution was filtered and the residue was dissolved in nitromethane (6 mL), and vapour diffused with Et2O. Diffusion was complete in about 2 weeks and X-ray quality yellow crystals were collected by filtration. Yield 31% (16 mg). FT-IR/cm−1 3134 (w), 2994 (w), 1635 (m), 1235 (s), 856 (w). HRMS (ESI): m/z: 921.2016 [L235ClO456Fe].A solution of Fe(ClO2.2.44)2·6H2O  in methanol (7 mL) was added to a solution of L  in nitromethane (10 mL) and the mixture was stirred for 10 min. To this blue solution, diethyl ether (30 mL) was slowly diffused to obtain blue needle crystals suitable for X-ray diffraction. Yield: 24% (12.1 mg). FT-IR/cm−1 2985 (w), 1731 (m), 1596 (m), 1240 (s), 856 (w). HRMS (ESI): m/z: 923.2020 [L235ClO458Ni].A solution of Ni and L  in a ratio 1 : 2 was stirred at r.t. for 16 h then concentrated. The obtained powder was solubilised in nitromethane (6 mL) and recrystallized by slow diffusion of diethyl ether (30 mL) at r.t. After 10 days, white single crystals were obtained. Yield: 38% (9.5 mg) FT-IR/cm−1 3150 (w), 2964 (w), 1737 (s), 1627 (m), 1442 (s), 856 (w). HRMS (ESI): m/z: 473.0655 [L35Cl55Mn].15 mL of acetonitrile solution of MnCl3.3.1.41L has been prepared by nucleophilic aromatic substitution reaction of pyrazolic anions with 2,6-dibromopyridine . Given t41 shown in The most common impurity, when unsymmetrically substituted pyrazole is used, is the monosubstituted intermediate 2′,(g) and dimethyl sulfoxide extra dry as solvent at very high temperature. We point out that the use of 3(5) pyrazole substituted asymmetrical pyrazole possessing an ester function could, by its particular electronic effects, induces the orientation of the alkylation on the α-nitrogen.42 Furthermore, the use of potassium tert-butoxide as a base, acts as a strong base and weak nucleophile, thus leading to the stabilization of β-nitrogen pyrazolate anion through the K+ cation (compound A), and to the decrease of its free energy relative to that of α-nitrogen pyrazolate anion (compound B) .Therefore, we have abandoned these classic processes and we carried out this reaction under an inert atmosphere, using Arpound B) . This im3.2.mer coordination with an overall planar skeleton , Fe(ii) and Ni(ii) complexes the presence of weakly coordinating ClO4− anions, do not play a big role in stabilizing the crystal packing as they were mostly found to be disordered. Nevertheless, the double interaction between a carbonyl oxygen and the twisted pyrazolyl ring centroids  also displays this double interaction.Single-crystal X-ray analysis revealed that 1–4 are mononuclear coordination complexes  and 2, Ls Fig. S3 leads inmer coordination mode of the tridentate ligands necessarily places the pyridyl nitrogen atoms in axial position, with four nitrogen of four pyrazolyl groups from two ligands L in the equatorial plane, to form an octahedral geometry around the central metal atom. The ligands are therefore located on opposite sides of the metal centre with a dihedral angle between the ligand planes  of 67.09°, 59.97°, and 75.75° for 1, 2, and 3 respectively. Table S2The overall molecular structure of complexes 1–3 is quite similar with a maximal root-mean-square deviation (RMSD) of 0.89 Å, taking all atoms in account  complex followed by 3.857 Å in the Co(ii) and 3.500 Å in the Fe(ii) complex  complex for which no disorder was observed during structure refinement.A large torsion angle is observed between one pyrazolyl ring and the central pyridyl ring in complexes 1, 2 and 3. Both angles being 23.4(3)°, 19.8(3)° and 21.9(4)°, 20.6(4) ° in 1 and 2, respectively and 24.5(2)° in 3 which is in line with the values from complexes 1 and 2, and 9.8(2), which is significantly less. Furthermore, the torsion angle bends both pyrazolyl rings of both ligands towards each other, bringing both rings over each other, with a longer inter-centroid distance 4.112 Å in the Ni and Ni(ii) complexes with bpp ligands.45–47 Iron complex 2 expected to be high-spin (HS) at room temperature, as earlier noticed for Fe(ii) complexes with NNN tridentate ligands with narrow bites like bpp or terpyridine.48,49 Indeed, the Fe–N bond length of 2.18 Å (Table S2D2d symmetry. This angular distortion involves a decrease in N(pyridine)–Fe–N(pyridine) angle from its optimal value 180° to 157.6(1)° and twisting of the two planes defined by the tridentate ligands away from the perpendicular 90° decreasing to 59.97°, which is characteristic of an angular Jahn–Teller distortion. Two parameters are discussed: Σ and Θ which defines more specifically the degree of trigonal distortion of the coordination geometry from an octahedron towards a trigonal prism -three-fold axis.50 An ideal octahedron would afford zero distortion parameters. Herein, we compare Θ and Σ values in other HS and LS iron(ii) bis (pyrazolyl)pyridine (bpp) complexes  ions show less variation than HS complexes, as a consequence of their more regular geometry. Iron centre 2 remains HS on cooling, which is consistent with our magnetic study (vide infra).It is clear that there is a strong correlation between distortion parameters 54 revealed by the contraction of the Fe–N bonds required for the LS state as observed in earlier examples.55This behaviour is due to the rigid lattice and to steric hindrance of 2 which prevents any SCO to occur, since a rigid lattice cannot accommodate the resulting structural changes,2] (4) (ii) ion is penta-coordinated by one trigonal pincer ligand L using two pyrazole nitrogen and one pyridine N atom, the two remaining sites are occupied by two monodentate chloride counter anions.When chloride is used instead of perchlorate, another geometry is obtained [MnLCl2] (4) . The Mn(τ5 = (α − β)/60, where α and β are the two largest coordination angles56 (τ = 0.00 for square pyramid (SP) and 1.00 for trigonal bipyramidal (TP)). In our case, the Addison distortion index τ5 = 0.07; indicates that the Mn complex has a nearly perfect square pyramid geometry, with one Cl atom in the axial position. Table S3ii) complexes with τ5 = 0.046.57 Furthermore, complex 4 is located on a 2-fold axis, running though the pyridyl ring and the Mn atom, with the asymmetric unit being half a formula unit in space group R3̄. During structure refinement the pyridyl ring was found slightly off the 2-fold axis and disorder was modelled for the central ring of the ligand. Also, here the ethyl groups are disordered and refined over two sites. In the crystal packing all disordered ethyl groups are pointing towards the crystallographic 3-fold axis, forming a hydrophobic channel of alkyl chains , Fe(ii), Ni(ii) and Mn(ii) complexes suggesting that coordination of the metal ions hardly alters the intrinsic electronic properties of the ligand.60 Additional bands are observed in the UV region for compounds 1–4, which can be assigned to metal-to-ligand charge transfer (MLCT) processes. However, weak bands in the visible region at λ = 563 nm, 473 nm and 589 nm are observed respectively in cobalt, iron and nickel complexes corresponding to d–d transition. In 1, a broad band is observed, which presumably originates from the association of molecules though intermolecular hydrogen bonding.61Solid-state UV/vis spectra of L and its coordination complexes 1–4 are displayed in 3.5.2(g) atmosphere. Complexes 1, 2 and 3 were not studied since their structures include perchlorate ions, which are potentially explosive.62 The thermogravimetric analysis plot of the ligand L (see ESI19H24N4O4: 96.2%), thus demonstrating its relative high thermal stability. The thermal decomposition of complex 4 takes place in three steps (see ESI7H12O2 units. The second step occurs within the temperature range 380–660 °C and represents the volatilization of two Cl anions and four pyrazole's nitrogen with mass loss of 23.7% (theo. of 24%). The last step above 660 °C was assigned to pyridine pyrolysis.Powder X-ray diffraction (XRD) patterns of 1–4 at room temperature well match the diffractograms simulated from the single-crystal X-ray diffraction data, thus confirming a phase purity of the bulk samples see ESI. The theL see ESI shows ons see ESI. The fir3.6.δ = 1.02(1) mm s−1 and quadrupole splitting ΔEQ = 1.87(3) mm s−1. These parameters are characteristic of HS iron(ii) bpp complexes. The half-width at half-maximum was Γ/2 = 0.17(2) mm s−1. The observed asymmetry of the lines is due to a texture effect, while measuring single crystals. Such asymmetry was not taken into account in the current fit. While doing so, a fraction of Fe(iii) species were detected as a result of the long acquisition in air of the spectrum.63 No colour change was observed on cooling to liquid nitrogen suggesting that 2 remains in the HS ground state. This will be confirmed in the next section by recording temperature dependent magnetic susceptibility data.A Mössbauer spectrum of 2 as single crystals recorded at room temperature is shown in 3.7.χMT, χM being the molar magnetic susceptibility for 2, was investigated over the range 4–400 K  cm3 K mol−1, in agreement with the HS state of iron, as detected by Mössbauer spectroscopy , [CdL′Cl2] (2′). The in vitro antitumor activity was determined by the measurement of their cytostatic and cytotoxic properties towards the tumor cell line P815 using the MTT assay.We have investigated the biological activity of L and its coordination complexes 1–4 as well as the one of L′,  (CisPt) as a positive control with IC50 = 1.42 ± 0.035 μM. L′ and 1′ exhibit a potent cytotoxic activity with IC50 = 35.12 μΜ and 34.77 μM, respectively. However, L, 1 and 4 display a moderate cytotoxic activity, whereas 2′, 2 and 3 show a lower cytotoxic activity with IC50 values higher than 200 μM. Given together, the eight compounds can be classified on the basis of their cytotoxic level against the P815 cell line in the following order: 1′ ≥ L′ > 1 > L > 4 > 2 > 2′ > 3.64 Bioactive conformation is one of the important pharmacophore which is defined by the IUPAC as “an ensemble of steric and electronic features that is necessary to ensure the optimal supramolecular interactions with a specific biological target and to trigger (or block) its biological response”.65,66 On the other hand, the improvement in activity is also due to the complexation metal. Interestingly the cytotoxicity activity of L′ was maintained in 1′ but decrease considerably when substituting Mn(ii) by Cd(ii) for 2′. The manganese complexes (1′ and 4) have considerably better activity than the other complexes, in their respective series, especially the Mn complex formed with the C–C type ligand (L′). This is not surprising given that tumor-targeting manganese complexes induce reactive oxygen species (ROS)-mediated apoptotic and autophagic cancer cell death.67Thus, the cytotoxicity of L′ is much higher than the one of L. Such activity is most likely due to the conformation of C–C pyridine–pyrazole bond which is rigid, stable and likely bioactive. In contrast to the C–N pyridine–pyrazole bond, found in L, which exhibits greater flexibility suggesting that the ligand passes through different conformations. It is known that conformational stability is a determinant of drug cytotoxicity.3.8.268 In this study, we have tested our compounds towards tumor cells, against human PBMCs from healthy donors in order to determine their effects against normal cells. Interestingly, ligands and their active metal complexes did not show a significant cytotoxicity towards human normal PBMCs  are the first normal cell populations that come into contact with antitumor drugs used in conventional chemotherapy and that collapses from the first week of intravenous treatment of patients resulting in significant immune deficiency and increased side effects.al PBMCs . These f3.8.369 However, the elevation of ROS to a certain level may be lethal for tumor cells themselves. As a normal strategy from cells to face the oxidative stress, they increase the levels of antioxidant enzyme's activity, where SOD is recognized as the primary defense barrier against ROS by catalyzing the dismutation of superoxide anion radicals (O2˙−) to hydrogen peroxide (H2O2). H2O2 generated by the activity of SOD is eliminated by its conversion to H2O in subsequent reactions by CAT and GPx.69 Accumulation of H2O2 in the cell was reported to be implicated in the induction of apoptosis.70 The intracellular antioxidant capacity is mainly also conferred by glutathione (GSH) dependent systems. In fact, the important enzymes implicated in the regulation of redox homeostasis such as glutathione reductase are limited to the pool of GSH as a source of reducing equivalents.71The main objective of this study is to better understand the mechanisms of our compounds in the protection of P815 cell line against stress. Many studies have reported that cancer cells have an elevated level of ROS compared to normal cells.In the current study, we show that P815 tumor cells exposure to studied compounds differentially affects the SOD, CAT, GPx and GR activities. According to 2O2 by its ability to inhibit the catalase activity. The other compounds seem to modulate the pool of GSH by decreasing the activity of GR.Taken together, these findings suggest that 1′ acts through the induction of intracellular oxidative stress. In addition, 1′ may induce the accumulation of H4.2+ derivatives belonging to C–N junction, bearing sterically substituents with Co(ii), Fe(ii), Ni(ii) and Mn(ii). Our results have shown mononuclear structures promoted by tridentate ligand via two pyrazolyl N-atoms and one N-pyridine donor atom to set up distorted octahedra for Co2+, Fe2+, Ni2+ and a distorted square-pyramidal geometry for Mn2+. Moreover, L′ (C–C junction) and its Mn(ii) 1′ complex exhibited better antitumor activity compared to the other compounds, also all cytotoxic products are safe on normal cells PBMCs. 1′ stimulate SOD and inhibit CAT, this inhibition induces the accumulation of H2O2 in the cell, that could implicate the induction of apoptosis and consequently cell death. As a conclusion, this study highlighted the strong correlation between pyridine–pyrazolyl rings junction and anticancer activity, however further investigation is required in this important research area.This study has investigated synthesis, structural chemistry, cytotoxic and antioxidant activity of a series of [M(bispyrazolyl pyridine)]There are no conflicts to declare.RA-011-D1RA05918A-s001RA-011-D1RA05918A-s002


In this work, graphitic carbon nitride (g-C3N4) photocatalysts were synthesized using mixtures of low-cost, environment-friendly urea and thiourea as precursors by varying calcination temperatures ranging from 500 to 650 °C for 3 h in an air medium. Different analytical methods were used to characterize prepared g-C3N4 samples. The effects of different calcination temperatures on the structural, morphological, optical, and physiochemical properties of g-C3N4 photocatalysts were investigated. The results showed that rhodamine B (RhB) dye removal efficiency of g-C3N4 prepared at a calcination temperature of 600 °C exhibited 94.83% within 180 min visible LED light irradiation. Photocatalytic activity of g-C3N4 was enhanced by calcination at higher temperatures, possibly by increasing crystallinity that ameliorated the separation of photoinduced charge carriers. Thus, controlling the type of precursors and calcination temperatures has a great impact on the photocatalytic performance of g-C3N4 towards the photodegradation of RhB dye. This investigation provides useful information about the synthesis of novel polymeric g-C3N4 photocatalysts using a mixture of two different environmentally benign precursors at high calcination temperatures for the photodegradation of organic pollutants.Recently, polymeric graphitic carbon nitride (g-C Nevertheless, the substantial electron-hole pair recombination in these photocatalysts significantly decreases their photocatalytic efficiency. Furthermore, ZnO, TiO2, and SnO2 can absorb solar energy exclusively in the UV region because of their wide energy bandgap, which accounts for just 4% of the total solar energy irradiated. Nowadays, because of its fascinating physicochemical properties, g-C3N4 is a superior alternative for visible light-assisted photocatalytic wastewater treatment than other photocatalysts such as TiO2, ZnO, SnO2, and so on [Nowadays, growing industrial activities have resulted in the generation of huge amounts of hazardous pollutants that are released into environmental water bodies ,2. The mnd so on .The majority of photocatalysis research has used xenon lamps ,20, halo3N4 has piqued the interest of researchers as a visible-light-driven semiconductor photocatalyst [3N4 is an extensively explored n-type semiconductor for the removal of organic pollutants due to its inexpensiveness, non-toxicity, eco-friendliness, simple synthesis, mid-energy bandgap, and robust physicochemical stability [3N4 can be synthesized by thermal calcination of various nitrogen-rich precursors, such as cyanamide [Since its discovery by Wang’s group in 2009, g-Ccatalyst . Polymertability . In geneyanamide , melaminyanamide , dicyandyanamide , thioureyanamide , and ureyanamide .3N4 suffers from a low photocatalytic performance due to the rapid recombination of photogenerated charge carriers, limited range absorption of visible light, and low specific surface area [3N4, such as elemental doping [3N4-based heterojunctions [3N4 for photocatalytic elimination of pollutants [Nevertheless, pristine g-Cace area . Therefol doping ,34,35,36l doping , and conunctions ,39,40. Mllutants ,24,41, pllutants ,43,44, wllutants ,45, and 3N4 photocatalysts. Enumerating some of the recent outcomes, Zhang et al. fabricated g-C3N4 by pyrolysis using an aggregate of urea and thiourea as precursors to enhance the specific surface area and separation of photo-induced electron-hole pairs, consequently improving photocatalytic activity of g-C3N4 photocatalysts [3N4 nanosheets by heating low-cost urea and thiourea precursors at 520 °C for 2 h, and g-C3N4 showed the best photocatalytic efficiency for the elimination of cationic MB dye when exposed to visible light [3N4 materials by simply calcining mixtures of urea and thiourea precursors at 550 °C for photocatalytic degradation of RhB upon irradiation of visible light [3N4 by polycondensation of melamine and urea at 550 °C for 3 h. For a melamine-to-urea molar ratio of 1:1, the photocatalytic degradation rate for MB was about 75% after 4 h of light exposure [Recently, many researchers have reported the impact of different types of precursors on the structural, optical, morphological, and physicochemical properties as well as the efficiency of g-Catalysts . Similarle light . In addile light . Guo et exposure .3N4 photocatalyst. As a result, the synthesis of g-C3N4 by varying calcination temperature allows improvements in the surface area, crystallinity, extended absorption range of visible light, and overall enhanced photocatalytic activity [3N4 photocatalytic performance. Enumerating a few of the outcomes, Fang et al. prepared porous g-C3N4 through direct pyrolysis of low-cost urea by controlling the condensation temperature in the range between 400 and 650 °C. In addition, the synthesized U-g-C3N4 had better photoactivity and long-term photocatalytic stability [3N4 by calcining urea at temperatures between 350 and 750 °C. It was discovered that increasing the calcination temperature to 550 °C enhanced the photocatalytic activity of g-C3N4 for the degradation of MB dye, whereas increasing the temperature beyond the optimum value decreased the photoactivity [3N4 for the decomposition of rhodamine B (RhB) and methyl orange (MO) via thermal polycondensation of melamine at different calcination temperatures ranging from 450–700 °C. The g-C3N4 prepared at a calcination temperature of 700 °C revealed robust photocatalytic activity and stability against RhB and MO decomposition [3N4 using urea and thiourea were generally considered to be in the range of 520–550 °C and 450–650 °C, respectively [Similarly, calcination temperature control is one of the most common reaction parameters affecting the performance of the g-Cactivity ,20,32. Tposition . In addiectively .3N4 photocatalyst using mixtures of stochiometric amounts of urea and thiourea at different calcination temperatures, especially in wastewater treatment using visible LED light irradiation, has not been reported in the previous literatures. Herein, in this work, we designed a novel single-step synthesis route to fabricate g-C3N4 nanomaterials by controlling various calcination temperatures of a mixture of stochiometric amounts of low-cost, environment-friendly urea and thiourea for complete photocatalytic degradation of RhB dye. Furthermore, the influence of varying calcination temperatures on the structural, optical, morphological, and physicochemical properties of g-C3N4 photocatalysts was carefully examined. Subsequently, the properties of the prepared g-C3N4 samples were characterized using several analytical techniques, and the photocatalytic activities of g-C3N4 samples were assessed by measuring the removal of rhodamine B (RhB) after irradiation of visible LED light.Synthesis of the g-C4N2O, 99.5%), and thiourea  were bought from Merck, Mumbai, India. Rhodamine B (99.9%) was obtained from Loba Chemie Pvt. Ltd., Mumbai, India. All reagents were used without further purification as received.Urea  with Cu-Kα radiation (λ = 1.5406 Å), operating at 40 kV, and 15 mA. Diffraction intensity was continuously recorded over a 2θ range from 10° to 70° with a sweep step width of 0.01° and a scan rate of 10°/min. Fourier transform infrared (FTIR) spectroscopy  was used to probe functional groups in the range 400–4000 cm−1. The optical properties of the materials were evaluated with a UV-Vis diffuse reflectance spectrometer  in the range 200–800 nm using barium sulfate (BaSO4) as background. Photoluminescence (PL) spectral data were obtained using a PL spectrophotometer  at an excitation wavelength of 360 nm. The width of the excitation and emission slits was 10 nm, and the thermal stability of each g-C3N4 sample was obtained by TGA . The morphology of the g-C3N4-600 °C sample was investigated by FE-SEM and HR-TEM.X-ray diffraction patterns of g-C3N4 photocatalysts with different calcination temperatures was evaluated by decomposing RhB as a model pollutant under visible-LED-light irradiation. In a typical experiment, 50 mg of g-C3N4 photocatalyst was dispersed in 100 mL of RhB solution (10 mg/L of RhB in 100 mL of distilled water). The suspension was stirred vigorously for 60 min in the dark before irradiation to ensure proper dispersion and establish adsorption–desorption equilibrium between the photocatalysts and RhB dye. The initial concentration (C0) of suspensions was obtained at adsorption–desorption equilibrium. A visible light source of  LED lamp  was used to initiate the photocatalytic reaction. At predetermined intervals, 3 mL of the solution was taken and centrifuged to separate the photocatalyst and to obtain the supernatant. Subsequently, the concentration of RhB in the supernatant was measured with a UV-Vis NIR spectrophotometer  at its maximum absorption wavelength of The photocatalytic activity of g-C0 represents the concentration of RhB dye at adsorption–desorption equilibrium (mg L−1), and C is the concentration of RhB at reaction time t; similarly, A and A0 represent corresponding values.The intensity change of the major absorption peak was used to assess dye degradation. The following equation was used to calculate photocatalytic degradation efficiency :(1) Degr0 is the concentration of RhB dye at adsorption–desorption equilibrium (mgL−1), C is the concentration of RhB at reaction time t, k is the apparent reaction rate constant (min−1), and t is the irradiation time (min). Furthermore, the photolysis experiment was carried out following the same procedure without the addition of a catalyst.To solve the apparent degradation rate constant (k) of the RhB dye, a simplified Langmuir–Hinshelwood pseudo-first-order kinetic model was used :(2) lnC03N4 prepared at various temperatures, as shown in 3N4 samples generated at calcination temperatures of 500, 550, 600, and 650 °C revealed two main diffraction peaks at around 13.1° and 27.2°. The characteristic peak at 27.2° represents interplanar graphitic stacking, while the peak at 13.1° represents inplanar structural packing, demonstrating g-C3N4 formation in all prepared g-C3N4 samples [3N4 samples becomes sharper, while its width becomes narrower. As a result, an increase in peak sharpness indicated a higher degree of crystallinity of a photocatalyst [X-ray diffraction patterns were used to identify the crystal structure of g-C samples ,50. As tcatalyst . Therefocatalyst .3N4 completely decomposed into gases including CO2, H2S, NH3, and water. Therefore, at calcination temperatures above 650 °C, g-C3N4-prepared samples become thermally unstable, and optimal calcination could be between 600–650 °C [3N4 samples prepared at calcination temperatures of 500, 550, 600, and 650 °C is 27.25, 27.08, 27.30, 27.19, and 27.25°, respectively.No solid sample was produced when the calcination temperature reached 700 °C during calcination, indicating that g-C0–650 °C . In addi3N4 samples was determined using the Debye–Scherrer formula [hkl) can be calculated as follows:Moreover, the crystallite size of synthesized g-C formula :(3) D=kλ3N4 samples at various temperatures corresponding to the (002) plane.3N4 samples. A sharp intense peak at high calcination temperatures implies that the s-triazine layer arrangement is more regular and that there is a significant degree of aggregation [regation .3N4 samples.Photoluminescence (PL), UV-Vis diffuse reflectance spectra (DRS), and FTIR spectra were used to investigate the optical properties of the prepared g-C3N4 samples prepared at various calcination temperatures were obtained at 360 nm excitation wavelength. As shown in 3N4 samples exhibited an intense and broad photoluminescence peak between 437–440 nm and showed a slight red-shift at a temperature of 550 °C. A slight shift of PL peak was attributable to differences in the bandgap energy of each sample [3N4 samples increased as the calcination temperature increased. This indicated that g-C3N4 prepared at 550 °C could have the lowest recombination rate of photoinduced electron-hole pairs [Photoluminescence spectra are useful for understanding the separation and recombination of electron-hole pairs, which is an important mechanism in photocatalysis . Lowerinh sample . Except le pairs . In addile pairs .3N4 samples was observed with changing calcination temperatures, as shown in −/h+ pairs [3N4 is a well-known indirect bandgap semiconductor material [3N4 samples were calculated by extrapolating the linear portion of the 1/2 versus 3N4 samples prepared at different calcination temperatures of 500, 550, 600, and 650 °C was estimated to be 2.78, 2.75, 2.82, and 2.89 eV, respectively [Diffuse reflectance spectroscopy was used to study the optical properties of all prepared photocatalysts at different calcination temperatures, as shown in h+ pairs . The banh+ pairs :(5) FRhvmaterial . Therefoectively ,47. In cectively .3N4 samples synthesized at various calcination temperatures. As shown in −1, which corresponds to the stretching mode of conjugated heterocycles [3N4 samples seen at wavenumber about 1458, 1560, and 1630 cm−1. However, the C=O stretching mode at wavenumber 1740 cm−1 was observed in the g-C3N4 samples prepared at calcination temperatures of 500, 600, and 650 °C. FTIR peaks at wavenumber around 1230, 1319, and 1400 cm−1 belong to the aromatic C-N stretching mode, and also the peaks at wavenumber of 805 and 885 cm−1 resulted from the distinctive breathing mode of tri-s-triazine ring units. Moreover, the absorbed H2O molecules cause the occurrence of N-H and O-H stretching modes at broad bands situated in the wavenumber range of 3074–3320 cm−1 [3N4.FTIR spectroscopy is used to analyze the chemical compositions and functional groups of g-C320 cm−1 ,58,59. I3N4 samples was performed by heating them from ambient temperature to 700 °C in an air atmosphere. 3N4 photocatalysts synthesized at various calcination temperatures. The weight loss is less at temperatures below 450 °C but increases as calcination temperatures decrease from 650 to 500 °C, which may be caused by the evaporation of water molecules that have been adsorbed on the surface of catalysts [3N4-650 °C sample did not lose weight significantly at a temperature below 450 °C, which is an indication of its great thermal stability. In addition, the significant weight loss of all prepared samples between temperatures 450 °C and 700 °C is attributed to the decomposition and condensation of g-C3N4 photocatalysts [3N4 is non-volatile up to 600 °C and will be practically entirely degraded when the temperature climbs to 700 °C [Thermal analysis of g-Catalysts . Meanwhiatalysts ,61. A tho 700 °C .3N4 synthesized at a calcination temperature of 600 °C. 3N4 prepared at 600 °C consists of large nanoflakes [3N4 prepared at 600 °C, and it was almost transparent, which was due to its thin structure. The results indicated that g-C3N4 prepared at 600 °C was fluffy [3N4 photocatalysts calcined at 600 °C.noflakes . In addinoflakes . Figure s fluffy . Therefo3N4 samples was shown by plots of C/C0 vs. irradiation time (min), as shown in 3N4, the degradation rate of RhB was found to be almost insignificant, showing that the photolysis of RhB itself was negligible. Therefore, in the absence of a g-C3N4 photocatalyst, the rhodamine B dye solution was stable under visible LED light irradiation. The degradation of RhB was greatly improved when photocatalysts were introduced. As a result, the degradation of rhodamine B dye was caused by the presence of g-C3N4 in the solution.The photocatalytic activity of rhodamine B for all the synthesized g-C3N4 photocatalysts prepared at 500, 550, 600, and 650 °C were about 1.85%, 0.66%, 3.5%, and 1.55% for RhB dye solution, respectively. The g-C3N4 sample prepared at a calcination temperature of 600 °C displayed the highest adsorption ability. Meanwhile, the photodegradation efficiency of g-C3N4-500 °C, g-C3N4-550 °C, g-C3N4-600 °C, and g-C3N4-650 °C was 18.34%, 22.2%, 94.83%, and 94.80%, after 180 min irradiation, as shown in 3N4 at various temperatures towards RhB dye removal are shown in The adsorption efficiencies of g-C3N4 photocatalysts was obtained as calcination temperatures vary between 600 and 650 °C. Furthermore, the crystallinity of the as-prepared samples increased at higher calcination temperatures.Generally, 3N4-600 °C with other previously reported photocatalysts. As shown, g-C3N4-600 °C was a very potential photocatalyst and was extremely attractive for the actual treatment of industrial wastewater containing organic pollutants.3N4 samples. The presence of a typical g-C3N4 pattern was demonstrated by the XRD and FTIR spectra of all prepared g-C3N4 samples at various calcination temperatures. The influence of calcining temperatures on photocatalytic activities of g-C3N4 samples was investigated by photodegradation of RhB when exposed to visible LED light. The best photocatalytic activity of g-C3N4 photocatalyst was achieved at a temperature of 600 °C, due to its efficient separation of photoinduced charge carriers, high crystallinity, and enhanced visible light absorption range. Therefore, single-step synthesis of g-C3N4 using a mixture of urea and thiourea at high calcination temperature leads to the highest photocatalytic activity for the degradation of RhB dyes. This work can give a good insight into the synthesis of a cost-effective and efficient g-C3N4 photocatalyst at high calcination temperature for the treatment of industrial wastewater containing organic pollutants.A visible LED light-driven graphitic carbon nitride photocatalyst was fabricated by single-step calcination of mixtures of urea and thiourea at different temperatures. Meanwhile, various analytical tools were used to study the structural, morphological, optical, and physicochemical properties of all prepared g-C