Piper nigrum (black pepper), an important spice, were evaluated for their neuroprotective potential in hydrogen peroxide-induced oxidative stress in human neuroblastoma cells (SH-SY5Y). The extracts were also subjected to GC/MS to identify the important bioactives present. The extracts exhibited neuroprotection by significantly decreasing the oxidative stress and restoring the mitochondrial membrane potential in the cells. Additionally, the extracts displayed potent anti-glycation and significant anti-Aβ fibrilization activities. The extracts were competitive inhibitors of AChE. The multitarget neuroprotective mechanism displayed by Piper nigrum indicates it as a potential candidate in the treatment of NDs.Neurodegenerative diseases (NDs) are a family of disorders that cause progressive structural and functional degeneration of neurons. Among all the organs in the body, the brain is the one that is the most affected by the production and accumulation of ROS. Various studies have shown that an increase in oxidative stress is a common pathophysiology for almost all NDs, which further affects various other pathways. The available drugs lack the wide spectrum necessary to confront these complexities altogether. Hence, a safe therapeutic approach to target multiple pathways is highly desirable. In the present study, the hexane and ethyl acetate extracts of  Neurodegenerative diseases (NDs) are a family of disorders  that lead to the progressive structural and/or functional degeneration of neurons. ND pathophysiology involves neuronal malfunction, synaptic dysfunction, and aggregation of specific proteins in the brain . The proPiper nigrum L.; Piperaceae family) is a flowering vine native to South Asia, crowned as the “King of Spices” due to its significant place culinarily for over 2000 years, and was the most valuable spice  traded from India to different parts of the world. The peppercorns were used as currency by ancient Greeks and European countries between 500 and 1500 AD [Black pepper  inhibition, and advanced glycation end product (AGE) inhibition.Therefore, based on the above therapeutic importance of black pepper, we explored the role of the hexane and ethyl acetate extracts of black pepper (dried fruit) on HThe chemicals 6,6′-dinitro-3,3′-dithiodibenzoic acid, bis(3-carboxy-4-nitrophenyl) disulfide (DTNB), acetyl thiocholine chloride, galantamine, gallic acid, ascorbic acid, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tripyridyl-s-triazine (TPTZ), Folin–Ciocalteu reagent (FCR), 2′,7′-dichlorofluorescin diacetate (DCFDA), tetramethylrhodamine, ethyl ester (TMRE), hydrogen peroxide, thioflavin T (ThT), bovine serum albumin (BSA), sodium azide, aminoguanidine, dextrose, and acetylcholinesterase  were bought from Sigma-Aldrich . Aβ1-42 (Aggresure™) was acquired from AnaSpec . The WST-8 kit was obtained from Roche Diagnostics GmbH . All organic solvents of HPLC grade were purchased from Sigma-Aldrich.The dried black pepper fruits were procured from the Expat Mart . The samples were weighed and powdered using a pestle mortar. The powder was extracted sequentially in n-hexane and ethyl acetate. The extracted fractions were dried, weighed, and stored at 4 °C until further experiments. The sample was analyzed on a fused-silica capillary column  installed on a GCMS-QP2020 . The oven temperature was programmed at 60 °C for 2 min, 100 °C at 4 °C/min, 290 °C at 10 °C/min, and finally to isothermic for 10 min. The split injection mode (1:10) was used and hexane and ethyl acetate fractions  were injected into the GC/MS via an auto-injector. The carrier gas was helium at a constant flow mode rate of 1 mL/min. The injection port, ion source, and interface temperatures were: 280, 280, and 150 °C, respectively. The energy of ionization was 70 eV. The mass spectra were obtained in full scan mode (40–700 AMU). The Folin−Ciocalteu method , with moThe total flavonoids were estimated following the method of Ribarova et al. , with moThe method described previously , with miThe free radical scavenging capacity of extracts was measured by the method described earlier . ABTS ra4·7H2O at various concentrations (1 mM). For the assay, the extract was incubated with 300 µL of FRAP reagent, and the reduction of ferric tripyridyltriazine to a ferrous complex by the extract was monitored at 593 nm  after 30 min of incubation at RT. FRAP values of the sample were expressed as µM Fe2+/g. The FRAP assay was carried out to evaluate the metal-chelating ability of the extracts by modifying a previously described method . The wor50 values were determined by GraphPad Prism 9.5. The AChE activity was monitored by slight modifications to Ellman’s method . The extA previously reported method was followed with slight modification . The assThe aggregation inhibition was calculated as: 50 values were determined by GraphPad Prism 9.5.The glycation reaction was carried out as described  by incub2, and a 95% humidified atmosphere in the incubator. The cells were passaged twice per week and the experiments were performed at 80–90% cell confluency.Human neuroblastoma SH-SY5Y cells  were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (FBS), 1% kanamycin, and 1% penicillin  at 37 °C with 5% CO4 cells/well) in sterile 96-well plates and subjected to various concentrations of extracts for 24 h. The extracts were removed and the cells were washed twice with 1X PBS and incubated in the fresh medium with 10% WST-8 reagent  for 2 h. The absorbance was measured at 450 nm in a multi-plate reader . The percent cytotoxicity was calculated as:For the cell viability assay, cells were seeded (1 × 1050).The plot of percent cytotoxicity versus sample concentration was used to calculate the extract concentration that killed 50% of the cells (IC2O2-induced oxidative stress in SH-SY5Y was performed as previously described [4 cells/well) were seeded in a 96-well plate and incubated for 18–24 h. After stabilization, the cells were pre-treated with the extracts for 24 h. The extracts were then removed and treated with H2O2 (100 μM) for 6 h. A solvent control, H2O2 alone, and extract alone treatments were also included. After incubation, the % cell viability was determined using WST-8 reagent in triplicate experiments. The neuroprotective effect of extracts on Hescribed . The cel4 cells/well) were seeded in a 96-well plate and incubated for 18–24 h, after which they were pre-treated with the extract for 12 h. The extracts were then removed, followed by a 4 h treatment with H2O2 (100 μM). Then, 25 μM H2DCFDA was added and the cells were incubated for another 2 h in the dark at 37 °C. The fluorescence intensity  was measured by a microplate reader . The ROS was calculated as a percentage of the untreated control cells (100%) in triplicate measurements.The ROS was measured using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) as previously described . The cel4 cells/well) were seeded in a 96-well plate and incubated for 18–24 h; after which, they were pre-treated with the extract for 12 h. The extracts were then removed, followed by a 2 h treatment with H2O2 (200 μM). The cells were incubated for 1 h with 1 μM TMRE at 37 °C. The fluorescence  was read in a microplate reader . The ΔΨm was calculated as a percentage of the untreated control cells (100%) in triplicate measurements.The mitochondrial membrane potential was measured using the tetramethylrhodamine, methyl ester (TMRE) staining method . The celp < 0.001, ##, ** represents p < 0.01, and #, * represents p < 0.05. The symbol # indicates significance compared to the H2O2 control while * indicates significance compared to the untreated control. The IC50 values were determined using non-linear regression. The Michaelis–Menten plot was drawn using a non-linear plot by GraphPad Prism 9.5, and the Vmax and Km were calculated from it. Lineweaver–Burk plots were drawn using linear regression analysis by GraphPad Prism 9.5. Statistical analysis was established by a one-way ANOVA followed by Dunnett’s post-hoc test. Data are registered as the mean ± SD of at least three experiments. The symbols ###, *** represents Piper nigrum  extracts were recorded to identify their bioactive compounds. The GC–MS chromatogram of Pep-EA was much clearer and a single major peak (52.4%) of piperidine, 1--1-oxo-2,4-pentadienyl)-, and - {syn. chavicine} was recorded , 2,4-Decadienamide, N-isobutyl-, - {syn. pellitorine} (7.75%), (E)-9-Octadecenoic acid ethyl ester (5.65%) {syn. ethyl elaidate}, (E)-5--1-(piperidine) {syn. dihydropiperine} (9.59%), -N-Isobutylhexadeca-2,4,10-trienamide {syn. pipercide} (11.40%), Piperidine, 1--1-oxo-2,4-pentadienyl)-, - {syn. chavicine} (8.71%), (E)-7--1-(piperidine-1-yl)hept-6-en-1-one) {syn. piperolein A} (6.94%), -9--1-(piperidin-1-yl)nona-2,4,8-trien-1-one) {syn. dehydropipernonaline} (7.69%), and (E)-9--1-(piperidin-1-yl)hept-6-en-1-one) {syn. piperolein B} (10.03%) B.P. retrofractum acted as anti-obesity agents [The key bicyclic sesquiterpene contributing to the piquancy of Pep-H is β-Caryophyllene (BCP), which is also the first “dietary cannabinoid” with GRAS  status and certified for food use by the FDA . Apart fy agents .The total phenolic content (TPC) and flavonoid content (TFC) were estimated in the extracts using colorimetric assays. Phenols and flavonoids are secondary metabolites with an important role in the growth, communication, and defense of plants. Pep-EA had a higher phenol and flavonoid content compared to Pep-H. The phenolic content in the Pep-H and Pep-EA extracts was calculated to be 14.78 ± 1.99 mg GAE/g and 24.10 ± 0.67 mg GAE/g, respectively, while the flavonoid content was 20.15 ± 0.78 mg QE/g (Pep-H) and 41.86 ± 0.69 mg QE/g (Pep-EA).Previously, the methanolic extract of black pepper had a TPC of 6.71 ± 0.34 mg GAE/g and TFC of 63.11 ± 3.16 mg QE/g . However+ radical scavenging activity and electron transfer (FRAP) were better in the case of Pep-EA (22.91 ± 1.19% and 41.65 ± 0.53 µM Fe2+/g) compared to Pep-H (14.79 ± 0.58% and 6.41 ± 0.01 µM Fe2+/g) at 50 μg/mL. In a previous study, the water extract of black pepper exhibited 26.67% (DPPH assay), 74.87% (ABTS assay), and 20.42% (FRAP assay) activity at 500 μg/mL [The antioxidant potential of the extracts was also evaluated by different assays. The percent of radical scavenging activity observed in the DPPH assay was lower in the case of Pep-H 34.39 ± 0.24%) compared to Pep-EA (54.8 ± 0.39%). However, the ABTS00 μg/mL . The hyd00 μg/mL . The los00 μg/mL . The hig00 μg/mL . In GC–M ± 0.24% 00 μg/mL . HoweverGlycation is a non-enzymatic reaction that results in the formation of AGEs which are the cross-linked structures formed between proteins and reducing sugars, eventually leading to inflammation and oxidative stress in addition to affecting cell signaling. Chronic stress accelerates the formation and aggregation of AGEs in the body, which in turn fuels oxidative stress . The AGE50 value of 35.6 μg/mL, much lower than Aminoguanidine, the positive control (IC50 91.42 μg/mL). In a previous report, a similar IC50 value of 91.2 μg/mL was reported for Aminoguanidine [50 119.9 μg/mL) as the control  content. In addition, piperine forms a stable albumin-piperine complex by interacting with its subdomain IIA, which could be another possible mechanism for the anti-glycation action [It has been known that spices are a rich source of polyphenols and could inhibit AGE formation considerably  through 50 values  was conducted. The IC50 value of Pep-H was 41.72 μg/mL and that of Pep-EA was 28.59 μg/mL. In previous studies, the methanolic pepper extract had shown an IC50 value of 11.13 μg/mL [50 value (5.9 μg/mL) [50 value of the positive control, Galantamine, was calculated as 0.94 μg/mL, like the previously reported value of 1.45 μg/mL [50 values of the extracts and the positive control have been shown in To evaluate the neuroprotective effect of black pepper extracts, we also studied their anti-acetylcholine esterase  activity. The higher AChE induces apoptosis and affects synaptic integrity neurodevelopment  therefor13 μg/mL  whereas 9 μg/mL) . The IC545 μg/mL . The IC5The Lineweaver-Burk plot was used to elucidate the mechanism of AChE inhibition. Competitive inhibition was observed for both Pep-H and Pep-EA  and the Similar Vmax  values were obtained for the no-inhibitor (1.375 μmole/min/mg) with Pep-H (1.364 μmole/min/mg at 50 μg/mL and 1.372 μmole/min/mg 100 μg/mL) and Pep-EA (1.333 μmole/min/mg at 50 μg/mL and 1.360 μmole/min/mg 100 μg/mL), while an increase in the Km  values was observed: 7.37 mM (no-inhibitor), Pep-H , and Pep-EA  at 50 μg/mL and 100 μg/mL, respectively . The incP. nigrum components (such as piperine), facilitate hydrophobic connections (five in the case of piperine) with amino acids present in the proteins, resulting in enhanced enzyme inhibition [P. longum extract using T. californica AChE suggested a possible hydrogen bonding with Tyr70 [114, Trp126, Trp351, and Phe392 of the T. castaneum AChE enzyme [297 and Trp286 residues in AChE from Electrophorus [P. nigrum also formed a hydrogen bond with Ser200 and His440 at the catalytic site of T. californica AChE [50 value of Pep-EA compared to Pep-H could be due to the higher content of chavicine compared to Pep-H. However, the Pep-H also displayed commendable AChE inhibition which could be the result of the synergistic action of bio-active components present in the extract as reported in the case of P. longum [An in silico study proposed that the presence of several functional groups  in the benzodioxol moiety of hibition ,55. The th Tyr70 . In a prE enzyme . In addirophorus . The molica AChE . The rea. longum . In the p < 0.001) at 50 μM, comparable to the previously described value [p < 0.05) inhibition of 31.84 ± 7.76% compared to the control . Phenol red exhibited a 67.49 ± 3.73% inhibition  improved memory in an aluminum chloride-induced neurotoxicity mice model by significantly modulating the expression of amyloid-producing isoforms (APP770 and APP695) in the brain. The extract decreased the expression of the amyloidogenic APP770 isoform with a concomitant improvement in the expression of the APP695 (non-amyloidogenic) isoform in the hippocampus, amygdala, and cortex. Chavicine was identified as the main pharmacologically active component of the extract responsible for neuroprotection [Aβ fibrilization is the vital component of amyloid plaques in AD pathology which contribute to oxidative stress and neuroinflammation. It is speculated that the main component of Pep-EA, chavicine, might be interacting with the β sheet of the protein through π-stacking or hydrophobic interaction to exert anti-fibrilization activity . Our resotection .In a previous study, black pepper oil exhibited weak fibrilization inhibition (33.17 ± 6.67%) at 100 μg/mL . Howeverp < 0.01) at 50 μg/mL (The cellular viability in the neuroblastoma cell line (SH-SY5Y) was analyzed using WST-8 dye after 24 h of treatment with different concentrations of the extracts . No cytotoxicity was observed up to 50 μg/mL for Pep-H, however, for Pep-EA, the cell viability decreased to 76.57%  for 12 h.The neuroprotective effects of the extracts were assessed by Hp < 0.05) increased the cell viability at 1 μg/mL and ## p < 0.01 at 3–30 μg/mL compared to H2O2 control. Pep-H significantly increased the cell viability at 3 μg/mL (# p < 0.05) and at 10 and 30 μg/mL (## p < 0.01) as compared to the H2O2 control. However, the lower concentrations (0.1–1 μg/mL) were ineffective for Pep-H  exposure for 4 h. The fluorescent dye (H2DCFDA) was used to monitor ROS production. H2O2 treatment generated 156.9 ± 5.0% ROS compared to the untreated cells (100%). Pre-treatment of the cells with extracts resulted in a dose-dependent decrease in ROS production in Pep-H. The Pep-EA was slightly more effective and significant at 1 μg/mL  and 10 μg/mL  compared to Pep-H  is disturbed due to oxidative damage to the cell which affects membrane permeability and the release of Cytochrome C and/or pro-apoptotic factors in the cytoplasm. Therefore, the loss of MMP is regarded as an early marker of apoptosis and a major contributor to NDs ,66.2O2 concentration and time of induction were optimized using a fluorescent dye (TMRE) which has an affinity for active mitochondria. In our experiment, H2O2 at 200 μM concentration reduced the MMP to ~50% of the untreated cells, hence, this condition was used for further examination. H2O2 treatment depolarized the mitochondria resulting in a decreased membrane potential. The cells pre-treated with Pep-H for 12 h followed by H2O2 (200 μM) treatment for 2 h (after removing the extracts) displayed a significant dose-dependent increase in MMP at 1 μg/mL  and 10 μg/mL , after which it declined significantly . The effect of Pep-EA was milder than Pep-H, showing improvement in MMP at 1 μg/mL  and 10 μg/mL  which decreased afterward  at 25 μg/mL  has a favorable pharmacokinetics profile with a high affinity towards brain tissue (98.4–98.5%), plasma protein (96.2–97.8%), and a brain distribution volume of 36.32 ± 1.40 mL/g [max (the maximum drug concentration observed in the sampled blood or plasma) was reported as 3.77 μg/mL and 6.59 μg/mL, respectively, in healthy volunteers [The present study was conducted to explore the neuroprotective mechanism exerted by .40 mL/g . Additiolunteers . In cont


This study aimed to verify the optimal pH of HMP for DCD livers. Rat livers were retrieved 30 min post-cardiac arrest and subjected to 3-h cold storage (CS) in UW solution (CS group) or HMP with UW-gluconate solution (machine perfusion [MP] group) of pH 7.4 , 7.6, 7.8, and 8.0  at 7–10 °C. The livers were subjected to normothermic perfusion to simulate reperfusion after HMP. All HMP groups showed greater graft protection compared to the CS group due to the lower levels of liver enzymes in the former. The MP-pH 7.8 group showed significant protection, evidenced by bile production, diminished tissue injury, and reduced flavin mononucleotide leakage, and further analysis by scanning electron microscopy revealed a well-preserved structure of the mitochondrial cristae. Therefore, the optimum pH of 7.8 enhanced the protective effect of HMP by preserving the structure and function of the mitochondria, leading to reduced reperfusion injury in the DCD liver.Ex vivo hypothermic machine perfusion (HMP) is a strategy for controlling ischemia-reperfusion injury in donation after circulatory death (DCD) liver transplantation. The pH of blood increases with a decrease in temperature and water dissociation, leading to a decrease in [H Expanding donor sources in liver transplantation is a global challenge . As a so®), which is used in liver  = PVP (cmH2O)/portal flow (mL/min/g liver). Hepatic enzyme release (IU/g liver) = [Ct × V]/LW (g). Where Ct is the enzyme activity (mIU/mL) in the perfusion medium after t min of reperfusion, V (mL) is the perfusate volume (300 mL), and LW (g) is the liver weight before CS or HMP.PVR [cmH2/min/g liver) = (Cin − Cout) × portal flow (mL/min/g liver), where Cin and Cout are the O2 concentrations in the inflow and outflow, respectively. O2 concentration (µmol O2/mL) = pO2 (kPa)·SO2 (37 °C) (µmol O2/mL/kPa), where SO2 (37 °C) is the oxygen solubility in water at 37 °C. SO2 (37 °C) = 0.01056 (µmol O2/mL/kPa).OCR  staining using the In Situ Apoptosis Detection Kit , according to the manufacturer’s instructions. Nuclear counterstaining was performed using hematoxylin. The apoptotic index was calculated as the ratio of TUNEL-positive hepatocytes per cell number in high-power fields (HPFs). The average of three HPFs represented each sample.4), washed in a phosphate buffer, immersed in 25 and 50% dimethyl sulfoxide, and frozen on a flat aluminum block. Then the specimens were immersed again in 0.1% OsO4 for 96 h at 20 °C to remove cytoplasmic proteins. The specimens were post-fixed in 1% OsO4, stained with tannic acid and 1% OsO4, dehydrated, lyophilized in an VFD-21S freeze-dryer , mounted onto a metal plate, and coated with osmium using an HPC-1SW plasma device  for evaluation using a Regulus 8100 scanning electron microscope (Hitachi).Osmium maceration was performed as previously described . Brieflyg and 4 °C. The first pellet contained the nuclear fraction. The supernatant was centrifuged for 10 min at 15,000× g and 4 °C. The pellet contained a mitochondrial fraction. The resulting supernatant contained the microsomal (cytoplasmic) fraction. The protein concentration of each fraction was determined using a bicinchoninic acid assay .The frozen tissues were minced and homogenized with a glass-Teflon homogenizer and subjected to nuclear, mitochondrial, and microsomal (cytoplasmic) fractionation using a LysoPure™ Nuclear and Cytoplasmic Extractor Kit with an inhibitor cocktail of proteases (1%) and phosphatases (1%). Briefly, the homogenate was centrifuged for 10 min at 600× ® TGX Stain-Free™ Protein Gels , transferred onto a low fluorescent polyvinylidene difluoride (PVDF) membrane using Trans-Blot® Turbo™ (Bio-Rad), and incubated with diluted primary antibodies (1:1000), pan-AMPK-alpha, phospho-AMPK-alpha, pan-SQSTM-1, phospho-SQSTM-1, ATG5, PARKIN, PINK-1, pan-JNK, and phospho-JNK overnight at 4 °C. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit (1:5000) or anti-mouse (1:1000) IgG secondary antibodies. Protein bands were detected using a ChemiDoc XRS® chemiluminescent detector (Bio-Rad) and normalized using the total protein normalization method.Every 12 micrograms of the cytosolic or mitochondrial proteins were subjected to standard SDS polyacrylamide gel electrophoresis (SDS-PAGE) with 7.5% Mini-PROTEANv/v), where the acetic acid buffer was a mixture of 4 M sodium acetate (20 mL) and 50% acetic acid (10 mL) in 1 L of deionized water; flow (0.7 mL/min), and injection volume (25 µL). FMN, FAD, and RF contents were expressed as mmol/L.Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and riboflavin (RF) in the perfusate of HMP were measured using a high-performance liquid chromatography (HPLC) method. The sample (perfusate) was deproteinized by mixing it with pure methanol, followed by centrifugation and filtration. An aliquot of the resulting deproteinized supernatant was directly injected into the HPLC system. The HPLC system consisted of a GASTORR BG-42 degasser , L-7100 pump , model 234 autoinjector , ATC-10 column oven , L-7400 UV detector (Hitachi), NOD-10 UV detector (Eicom), and F-1050 fluorescence spectrophotometer (Hitachi) with C18 column for reverse phase HPLC analysis InertSustain AQ-C18 (5 µm) and a guard column E . The HPLC conditions were as follows: ODS column , column oven (40 °C), UV–Vis detector (254 nm), fluorescence detector , mobile phase . We used one-way ANOVA and Dunnett’s tests to evaluate statistical significance compared with the CS or MP groups using JMP® 15 . Statistical significance was set at p < 0.05.Data were examined, excluding the control group, and were expressed as the mean ± SD . The CS group showed a value of 8.0 to 9.0 (cmH2O/mL × min × g liver), whereas the MP and MP-pH groups showed values of 9.0 to 10.0 (cmH2O/mL × min × g liver) with no significant differences.  in the CT group, increased to 35.6 ± 14.7 (×10−1 IU/g liver) in the CS group, and significantly reduced to 21.4 ± 8.1 (×10−1 IU/g liver) in the MP group. AST activities were significantly reduced in all the MP-pH groups compared to those in the CS and MP groups: MP-pH 7.6, 14.4 ± 2.3, MP-pH 7.8, 11.6 ± 2.9, MP-pH 8.0, and 17.5 ± 4.2 (×10−1 IU/g liver). Only the MP-pH 7.8 group appeared to show a significantly lower AST activity than the other MP groups.The AST activity of the perfusate at 90 min after reperfusion was 10.5 ± 2.3 (×10−1 IU/g liver) in the CT group, increased to 14.0 ± 6.9 (×10−1 IU/g liver) in the CS group, and significantly reduced to 6.2 ± 3.7 (×10−1 IU/g liver) in MP group. ALT activities were significantly reduced in all the MP-pH groups compared to those in the CS and MP groups as follows: MP-pH 7.6, 3.7 ± 1.8, MP-pH 7.8, 3.0 ± 0.7, MP-pH 8.0, and 5.4 ± 2.5 (×10−1 IU/g liver).The ALT activity of the perfusate at 90 min after reperfusion was 1.8 ± 1.2 (×10−1 IU/g liver) in the CT group, increased to 58.10 ± 26.67 (×10−1 IU/g liver) in the CS group, and significantly reduced to 22.77 ± 14.08 (×10−1 IU/g liver) in the MP group. LDH activity was significantly reduced in all the MP-pH groups compared to those in the CS and MP groups ).The LDH activity of the perfusate at 90 min after reperfusion was 1.57 ± 0.06 (×102/min/g liver) in the CT group, 0.82 ± 0.21 in CS group, 0.72 ± 0.12 in the MP group, 0.81 ± 0.12 in the MP-pH 7.6 group, 0.78 ± 0.06 in the MP-pH 7.8 group, and 0.88 ± 0.19 (µmol O2/min/g liver) in the MP-pH 8.0 group, with no significant differences.Mitochondrial respiratory function was determined using OCR d. The OCIntegrated liver function during IPRL was evaluated based on bile production. Bile production was the highest at 72.0 ± 17.6 (µL/g) in the CT group. It was significantly reduced to 11.76 ± 3.21 (µL/g) in the CS group, whereas it was significantly higher in all the MP-pH groups as compared to that of the CS group as follows: 18.3 ± 4.9 in the MP-pH 7.6 group, 23.0 ± 5.7 in MP-pH 7.8 group, and 19.8 ± 3.3 (µL/g) in MP-pH 8.0 group. Bile production in the MP group (17.3 ± 2.8 µL/g) was not statistically different from that of the CS group. In addition, bile production did not differ in the CS and MP-pH groups e.Liver histological examination 90 min after reperfusion showed an almost normal appearance in the CT group. Severe vacuolization and condensed or swollen nuclei with heterogeneous staining were observed in the CS and MP groups. In contrast, these findings were suppressed in the MP-pH groups, especially at pH 7.8 a.TUNEL-positive cells were not observed in the CT group. The TUNEL-positive cell ratio increased in the CS and MP groups, whereas it decreased in the MP-pH 7.8 group with no significant differences b.In the osmium-maceration SEM 90 min after reperfusion, the cell morphology of the CT group was well preserved, while in the CS group, there were several cells with numerous vacuolization, enormous mitochondria, and coarse cristae structures. The MP and MP-pH groups preserved the general structure of the cell under low magnification. However, there were differences in the density and architecture of the cristae between the groups when focused on the mitochondria. The mitochondria in the MP group had pendulous, flat structures, fewer rod-like structures, and low cristae density. Some of these cristae structures were observed in the MP-pH 7.6 and pH 8.0 groups. In contrast, rod-like structures and dense cristae were preserved in the pH 7.8 group .Macro- and microautophagy were evaluated through measurements of cytosolic AMPK-α, SQSTM-1, ATG5, mitochondrial PARKIN, and PINK-1 using western blotting. AMPK-α and SQSTM-1 are denoted as phospho/pan, which indicates the ratio of the activated signal. ATG5, PARKIN, and PINK-1 represent normalized band intensities. There were no differences in any of the signals between the groups. Acute stress responses were evaluated by western blotting of the cytosolic and mitochondrial MKK4-JNK pathways. JNK is denoted by phospho/pan, as described above. There were no significant differences among the groups .FMN was measured in the effluent using HPLC as a marker of mitochondrial damage. FMN showed an increasing trend over time, with the highest value in the MP group and lower and similar values in the MP-pH 7.6, 7.8, and 8.0 groups. Perfusate FMN in the MP group was significantly higher than that in the MP-pH 7.8 group at the end of the 180 min of HMP a. Other In the current study, we investigated the optimal pH for liver HMP by adjusting the pH of the UW-gluconate solution. HMP for DCD liver is more effective than CS in reducing transaminase levels . In addiThe use of DCD livers as a solution to donor shortages in liver transplantation is attracting attention, and research has been conducted to suppress the progression of reperfusion injury associated with warm ischemia. A warm ischemic period, even for a short duration, profoundly affects the liver endothelial phenotype, making it dysfunctional, and leads to an immediate increase in hepatic vascular tone, inflammation, polymorphonuclear cell infiltration, and cell death in hepatocytes . These r2 species, FAD, FMN, and RF [The increase in FMN in the perfusate of HMP was suppressed in the pH-regulated HMP compared to that in the pH-unregulated one. In particular, significant suppression was observed at pH 7.8, which was the most effective HMP in this experiment, suggesting that the protective effect of HMP on mitochondrial electron transport chain complex I was enhanced by avoiding acidosis at 7 °C. Previous studies have reported that FMN release in HMP is associated with ischemia-reperfusion injury and is a marker of mitochondrial injury. However, in these studies, the fluorescence intensity was measured without separating the vitamin B, and RF ,30,31. IIn the osmium-maceration SEM findings, differences in mitochondrial size and cristae, the mitochondrial inner membrane structure, were observed among the groups. The morphology of the mitochondrial inner membrane is important for the formation of proton gradients across the inner membrane for ATP synthesis, and increasing the density of the inner mitochondrial membrane alters the ATP production capacity of mitochondria ,33. ExteWe explored mitochondrial repair or mitophagy based on scanning electron microscopy findings as a mechanism to suppress reperfusion injury. In damaged or dysfunctional mitochondria, PINK-1 accumulates in the outer mitochondrial membrane. PARKIN in the cytosol then migrates into the mitochondrial outer membrane and is activated, inducing mitophagy . In thisA limitation of this study was the method used to control the pH of the UW-gluconate solution, into which NaOH, a strong base, was intermittently added. The pH adjustment method is complex. Thus, to minimize the fluctuation of perfusate pH during HMP, further investigation of buffer components, pH monitoring, and adjustment methods are necessary for the future development of MP. Another limitation is that the method of evaluation was an IPRL, which simulates reperfusion without blood components only for 90 min. It is necessary to evaluate this hypothesis in liver transplantation in both small and large animals.In conclusion, we found that the most effective pH was 7.8 for HMP at 7 °C in DCD rat liver. At this pH, mitochondrial ultrastructure and function of the electron transport chain complex were well preserved, leading to reduced hepatic ischemia and reperfusion injury.


O6 position of guanine and eventually forming dG-dC interstrand cross-links (ICLs). However, the clinical application of BCNU is hindered to some extent by the absence of tumor selectivity, poor stability and O6-alkylguanine-DNA alkyltransferase (AGT) mediated drug resistance. In recent years, tumor microenvironment has been widely utilized for advanced drug delivery. In the light of the features of tumor microenvironment, we constructed a multifunctional hypoxia/esterase-degradable nanomicelle with AGT inhibitory activity named HACB NPs for tumor-targeting BCNU delivery and tumor sensitization. HACB NPs was self-assembled from hyaluronic acid azobenzene AGT inhibitor conjugates, in which O6-BG analog acted as an AGT inhibitor, azobenzene acted as a hypoxia-responsive linker and carboxylate ester bond acted as both an esterase-sensitive switch and a connector with hyaluronic acid (HA). The obtained HACB NPs possessed good stability, favorable biosafety and hypoxia/esterase-responsive drug-releasing ability. BCNU-loaded HACB/BCNU NPs exhibited superior cytotoxicity and apoptosis-inducing ability toward the human uterine cervix carcinoma HeLa cells compared with traditional combined medication of BCNU plus O6-BG. In vivo studies further demonstrated that after a selective accumulation in the tumor site, the micelles could respond to hypoxic tumor tissue for rapid drug release to an effective therapeutic dosage. Thus, this multifunctional stimulus-responsive nanocarrier could be a new promising strategy to enhance the anticancer efficacy and reduce the side effects of BCNU and other CENUs.Carmustine (BCNU), a vital type of chloroethylnitrosourea (CENU), inhibits tumor cells growth by inducing DNA damage at The online version contains supplementary material available at 10.1186/s12951-023-02062-3. O6 position followed by covalently connecting with the complementary cytosine to form interstrand cross-links (ICLs) [O6-Alkylguanine-DNA alkyltransferase (AGT), a DNA-repair enzyme, can restore the BCNU-induced DNA damage by transferring the alkyl groups from the O6-position of guanine to the cysteine 145 (Cys145) residue in the active site [O6-alkylated guanine blocks the formation of ICLs, resulting in resistance of cells to BCNU, especially in the tumor tissues with high expression levels of AGT [As a classical type of chloroethylnitrosoureas (CENUs), carmustine (BCNU) has been demonstrated to be efficient against various tumors including brain tumors, bronchogenic carcinoma and carcinoma of the female genitalia –3. It exs (ICLs) , 5. The s (ICLs) . Besidesive site , 9. Thiss of AGT .O6-Benzylguanine (O6-BG) is an effective AGT inactivator, which is often used as chemotherapeutic adjuvants to improve the therapeutic efficacy of BCNU. It acts as a pseudosubstrate to transfers its benzyl group to the Cys145 residue at the active site of AGT, resulting in the ubiquitination and degradation of AGT enzyme. A large amount of research has revealed that O6-BG administration could effectively reverse the resistance of cancer cells to BCNU both in vitro and in vivo [O6-BG analog moiety in one molecule [ in vivo –14 In remolecule –18. Nevemolecule . Thus, nTumor hypoxia is a unique feature of most solid tumors stemming from the rapid proliferation of tumor cells, abnormal vasculatures, and high interstitial pressures of solid tumors , 21. DueO6-BG conjugates for the construction of a hypoxia-responsive delivery system for targeted delivery of BCNU. However, a critical issue faced by hypoxia-responsive delivery systems is that because of the heterogeneity of hypoxia inside tumors, the loaded chemotherapy cannot be rapidly released in oxygen-rich tumor cells nearby blood vasculatures, which reduces the sensitivity of the nanocarrier to tumor environment and thereby decreases drug efficacy [Nanomicelles constructed by the self-assembly behavior of polymer-drug conjugates possess advantages of simple process, high drug-loading and controllable release, displaying great potential in tumor-targeted delivery . Hyaluroefficacy , 31. As efficacy , 33. Theefficacy , 35. Su efficacy  developeO6-BG derivative (BG-AZO-COOH) through an ester bond, forming HA-AZO-COO-BG (abbreviated as HACB). In an aqueous solution, BCNU was encapsulated within the synthesized HACB to form micelles (HACB/BCNU NPs) via self-assembly behavior. In the micelles, BCNU itself was not modified, but rather hosted within the hydrophobic core of the stimuli-responsive drug-delivery system. As displayed in Scheme O6-(3-amino-benzyl) guanine acting as an AGT inhibitor is yielded via the reduction of the azo bond to aniline, and BCNU is fully released simultaneously, thus achieving an enhanced anticancer therapeutic efficacy. In vitro and in vivo investigations were carried out to confirm the dual responsive release behavior and the anticancer activity of HACB/BCNU NPs. This study is expected to contribute to the development of novel tumor-targeting nitrosourea chemotherapies with enhanced efficacy and reduced side effect.In this study, we successfully constructed an amphiphilic conjugate by covalently linking HA and the azobenzene-modified N-Hydroxysuccinimide (NHS) and 1-ethyl-3-(3-(dimethylamino) propyl) carbodiimide (EDC) were acquired from Aladdin Reagent Co., Ltd. . Reduced nicotinamide adenine dinucleotide phosphate oxidase (NADPH) and coumarin-6 (Cou6) were got from Sigma-Aldrich . BCNU was brought from Tokyo Chemical Industry Co., Ltd. . Esterase from porcine liver was provided by MACKLIN Co., Ltd. . Fetal bovine serum (FBS) was bought from Zhejiang Biological technology stock Co., Ltd. . Rat liver microsomes were obtained from PrimeTox . Roswell Park Memorial Institute 1640 Medium (1640) and penicillin–streptomycin solution were purchased from HyClone . 3--2,5-diphenyl-2-H-tetrazolium bromide (MTT) was provided by Beyotime Biotechnology Co., Ltd . Hoechst33342/PI apoptosis detection kit, Annexin V-FITC/PI apoptosis detection kit and Alamar blue were acquired from Solarbio Life Science . All other reagents were purchased from Beijing Chemicals Co.  and J&K Scientific Ltd. .3-Aminobenzylalcohol, phenol and sodium hydride were purchased from Energy Chemical . 1-Methylpyrrolidine and 6-chloropurine were obtained from Bidepharm . Hyaluronic acid  was brought from HEOWNS Co., Ltd. . 2 incubator . Hypoxic conditions in cell culture were generated by a hypoxia anaerobic incubator chamber  supplemented with an appropriate humidified gas mixture containing 1% O2 and 5% CO2 balanced with N2.The human uterine cervix carcinoma cells (HeLa), the human non-small cell lung cancer cells (A549), the hepatocellular carcinoma cells (SMMC-7721) and mouse brain endothelial cells (b End. 3) were purchased from Peking Union Medical College . All cells were cultured in RPMI-1640 medium containing 10% FBS, 100 U/mL of penicillin and 100 µg/mL of streptomycin at 37 ℃ using a humidified 5% CO7 HeLa cells (100 µL) into the left side of each mouse near the armpit. Tumor volume (V) was determined by measuring the length (L) and the width (W), and was calculated according to Eq.  were purchased from Vital River Laboratory Animal Technology Co. Ltd . All animal experiments were conducted under a protocol approved by Beijing University of Technology Institutional Animal Care and Use Committee. Subcutaneous tumor models were planted by injecting 1 × 10µL deionized water. Then, 800 µL hydrochloric acid and 1 mL cold sodium nitrite solution (2 mmol/L) were dropwise added to the above solution stirring for 20 min at 0–4 ℃. After that, 96 µL phenol was added to the solution stirring for 24 h at 0–4 ℃. Then the solution was extracted with distilled water/ethyl acetate (1:1). The organic layers were collected, dehydrated with sodium sulfate and evaporated to get a bronzing solid . 1H NMR  δ: 10.26 , 7.69–7.83 , 7.43–7.53 , 6.94–6.96 , 5.35–5.38 , 4.60–4.61 . 13C NMR  δ: 161.38, 152.56, 145.72, 144.47, 129.48, 128.85, 125.25, 121.69, 119.68, 116.41, 63.00. IR (KBr) υ: 3479.41, 3184.31 (OH), 2881.61 (CH2), 1593 (C=C), 1506.40 (N=N), 1145.05 (C–N), 1145.05 (C–O), 838.95 (=C–H), 694.11 (C–H).3-Aminobenzylalcohol (123 mg) was dissolved in 800 b was synthesized according to the method described by Keppler et al. with modification [N, N-dimethylformamide (DMF). Then, 1-methylpyrrolidine (3 mL) was added into the DMF solution to react under stirring for 20 h at room temperature. Subsequently, acetone (3 mL) was added to the above solution for precipitation. Then, the precipitates were filtered and washed twice by ether to get a white solid . 1H NMR  δ: 13.45 , 8.35 , 7.12 , 4.63–4.57 , 4.01–3.95 , 3.66 , 2.34–2.20 , 2.11–2.00 . 13C NMR  δ: 161.30, 159.52, 157.63, 150.63, 141.81, 117.07, 65.23, 51.50, 22.37. IR (KBr) υ: 3290.57 (NH2), 3350.92 (NH), 3184.60 (=C–H), 2897.59 (CH2), 1631.80 (C=N), 1564.10 (C=C), 1366.87 (C–C), 1293.28 (C–N), 816.40 (=C–H), 789.91 (N–H), 625.06 (C–H).Compound fication  Briefly,a (580 mg) was dissolved in 10 mL DMF, to which 600 mg potassium tert-butoxide and 300 mg compound b were added. Then, the mixture was stirred under a nitrogen atmosphere for 6 h at room temperature. Subsequently, 1 mL of deionized water containing 100 µL glacial acetic acid was added to the above solution. Then, the mixture was extracted with ethyl acetate/deionized water (1:1). The organic layers were dried with anhydrous sodium sulfate and concentrated under a vacuum. Finally, the crude product was purified by column chromatography with dichloromethane/methanol (50:1–10:1) as an eluent to obtain a yellow solid . 1H NMR  δ: 12.48 , 10.37 , 7.93 , 7.84–7.79 , 7.64–7.57, 6.96–6.94 , 6.36 , 5.60 . 13C NMR  δ: 161.57, 160.13, 152.67, 145.67, 138.73, 138.41, 130.64, 129.90, 125.39, 122.26, 122.05, 116.43, 66.73. IR (KBr) υ: 3683.45 (OH), 3487.84 (NH2), 3340.02 (NH), 2802.47 (CH), 1587.03 (C=C), 1501.92 (N=N), 1355.59 (C–C), 1283.92 (C–O–C), 1146.54 (C–N), 837.46 (=C–H), 786.69 (N–H), 628.41 (C–H).Compound c was added to the above solution and stirred at 40 ℃ for 48 h. The obtained crude product was then dialyzed by a dialysis bag (MWCO 3500 Da) against an excess amount of water/DMSO (1:3 to 1:1) for 1 day and against distilled water for another 2 days. The solution was filtered through a microporous membrane with 0.45 µm pores followed by freezing and lyophilizing to obtain a yellowish fluffy powder, named HACB.An aliquot of HA  was dissolved in distilled water (7 mg/mL) followed by addition of 15 mg EDC and 9 mg NHS, and the mixture was stirred at room temperature for 0.5 h to activate the carboxyl groups of HA. Then, 243 mg of compound µm pores followed by lyophilizing and stored at − 20 ℃ until further use. For visualization, Cou6-loaded micelles (HACB/Cou6 NPs) were prepared using Cou6 instead of BCNU.HACB/BCNU NPs were prepared by solvent volatilization method according to the literature with some modifications . Briefly2O, PBS (pH 7.4) or DMEM with 10% FBS was measured at different time points .Dynamic light scattering (DLS) measurements were performed to examine the hydrodynamic size, zeta-potential and poly-dispersity (PDI) of HACB NPs and HACB/BCNU NPs using a laser diffraction particle sizer . The optical absorption was determined with a UV–vis spectrophotometer . The morphology of the micelles was monitored using transmission electron microscopy . To investigate the stability of HACB NPs, the mean particle size of the micelles dissolved in Hµm membrane filters. The obtained filtrate was used for the determination of the total BCNU concentration in the solution. All the samples were analyzed by HPLC and the ultraviolet absorption was monitored at 230 nm for BCNU. The values of EE and DL of HACB/BCNU NPs were calculated according to Eqs. (The encapsulation efficiency (EE) and drug loading (DL) of HACB/BCNU NPs were determined by measuring the concentration of the loaded drugs using HPLC  with a C18 column . Briefly, the total dispersoid was filtered with an Amicon Ultra 10 kD molecular weight centrifugal filter  to obtain filtrate, which contained the unencapsulated BCNU. Then, the total mixture of the drug was dissolved in ten-fold volume of methanol followed by filtering through 0.22 2S2O4 was added into the aqueous solution with HACB NPs (1 mg/mL) and the mixture was stirred continuously at 37 ℃. The change of the absorption peak of the azobenzene group in the reaction was recorded by a UV–vis spectrophotometer. Moreover, HACB NPs was incubated with or without Na2S2O4 for 1 h at 37 ℃, and then the hydrodynamic diameter distributions and the morphology were observed by DLS and TEM, respectively.To simulate the hypoxic reductive environment, 1 mM NaHACB NPs dispersion at a concentration of 1 mg/mL was incubated with or without 30 U/mL porcine liver esterase at 37 ℃ under continuous shaking (100 rpm) for 1 h. After that, the hydrodynamic diameter distributions of the micelles were monitored by DLS, and the morphology was observed by TEM.µg/mL rat liver microsomes and 100 µM NADPH. Air and Nitrogen gas were bubbled through the reaction mixture to create normoxic and hypoxic conditions, respectively. Then, each group was gently shaken at 37 ℃ at a speed of 100 rpm. At the predetermined time point, 0.5 mL of the mixture containing the released Cou6 was drawn for measurement and replaced with fresh medium to maintain a constant volume. Three independent repeats were performed for each sample (n = 3).On account of the high instability of BCNU in solution, a Cou6 model was used to examine the in vitro release profile of the micelles. In brief, 2 mL HACB/Cou6 NPs dispersion (containing 2 mg Cou6) was placed into a dialysis bag (MWCO of 1 kDa), and then immersed in 40 mL release media containing 0.5% w/v sodium dodecyl sulfate, porcine liver esterase (0 or 30 U/mL), 790 5/well and cultured for 24 h at 37 ℃. Then, cells were treated with Cou6 or HACB/Cou6 NPs (Cou6 concentration of 10 ng/mL) for 30 min and 2 h under normoxia and hypoxia. To demonstrate the assisted targeting effect of HA, the cells were pretreated with 5 mg/mL HA for 2 h before exposure to HACB/Cou6 NPs. After treatment, the cells were washed twice with PBS, stained by Hoechst 33342 (10 µg/mL) for 15 min and observed by an IX-51 inverted fluorescence microscope . To further reveal the intracellular distribution of HACB/Cou6 NPs, the intracellular fluorescence was observed under a confocal laser scanning microscope .Hela cells were seeded in 12-well plates at a density of 1.5 × 103/well and cultured for 24 h at 37 ℃. Then, the cells were treated with BCNU and HACB/BCNU NPs  under normoxic and hypoxic conditions. For the combination-treated groups, the cells were pretreated with 40 µM O6-BG for 2 h before exposure to BCNU. Moreover, HeLa cells and a normal cell line b End.3 cells were treated with HACB NPs  to evaluate the cytotoxicity of blank micelles. After a 24-h treatment, 10 µL MTT solutions (5 mg/mL) were added in each well, and the cells were incubated in the dark for 4 h. Subsequently, the medium was removed and formazan crystals were dissolved in 150 µL DMSO. Finally, the absorbance at 560 nm was determined by a Thermo Scientific Multiskan FC . The cell viability was calculated according to Eq. .HeLa cells were seeded in 48-well plates at 8 × 10O6-BG or HACB/BCNU NPs (containing 0.05 mM BCNU) under normoxia or hypoxia. The dose of BCNU in each group was 0.05 mM. After treatment for 24 h, the medium was replaced with fresh medium followed by incubation for another 8 days. For the BCNU + O6-BG group, the cells were pretreated by 40 µM O6-BG for 2 h before exposure to BCNU. The cell clones were stained with crystal violet for 10 min followed by washing three times with PBS for observation.HeLa cells were seeded in 6-well plates at 800 cells per well and maintained for 24 h. Thereafter, the medium was replaced with fresh medium containing BCNU, BCNU + 5 cells per well and incubated for 24 h. Later, the cells were exposed to BCNU or HACB/BCNU NPs (containing 0.1 mM or 0.3 mM BCNU) under normoxic or hypoxic conditions for 24 h. The combination-treated groups were pretreated by 40 µM O6-BG for 2 h before BCNU exposure. Then, the cells were digested with trypsin without EDTA, centrifuged and washed twice with pre-cooled PBS. The cells were resuspended in a 1 × binding buffer and co-stained with Annexin V-FITC/PI kit for detection by FACS Calibur flow cytometer .The apoptosis induced by HACB/BCNU NPs was evaluated by Annexin V-FITC/PI apoptosis detection kit. HeLa cells were seeded in 6-well plates at 4 × 105 cells per well and cultured till approximately 90% confluence. Afterward, the medium was removed and a 10 µL pipette tip was used to generate a wound area by scraping the cell monolayer. The cells were treated with BCNU, BCNU + O6-BG or HACB/BCNU NPs under normoxia or hypoxia for 24 h. The concentration of BCNU in each group was 0.1 mM. For the BCNU + O6-BG group, the cells were pretreated with 40 µM O6-BG for 2 h before exposure to BCNU. The wound width was observed and recorded by a fluorescent microscope . The cell migration rate was calculated according to Eq. . The cell-cultured samples were fixed by 2.5% Karnovsky’s fixative overnight at 4 ℃ and dehydrated in a graded series of ethanol . After that, the samples were critical-point dried and sputter-coated with gold before imaging with a SU3500 SEM  [Three-dimensional (3D) tumor spheroids were established to mimic the in vivo hypoxic tumor microenvironment by culturing HeLa cells in a chitosan-hyaluronic acid (C-HA) scaffold, which was prepared according to the method described by Florczyk et al. . HeLa ceO6-BG, HACB/BCNU NPs with proposed concentrations  in normoxia or hypoxia for 24 h. As described above, cells were exposed to 40 µM O6-BG for 2 h before BCNU exposure for the combination-treated groups. The cells were washed with PBS followed by adding 500 µL medium containing 10% Alamar blue. After incubation for 2 h, 100 µL Alamar blue solution was transferred to a 96-well plate followed by measuring the absorbance at 570 nm and 630 nm. Ultimately, the cell viability (%) was calculated according to Eq.  via the tail vein 1 h before being treated with HACB/BCNU NPs. At the prearranged time points after injection, NIR fluorescent images were captured by an in vivo imaging system . Furthermore, major organs  and tumors of each mouse were collected and imaged via the in vivo imaging system.In order to determine the bio-distribution of HACB/BCNU NPs, DiR-labelled HACB NPs were injected into the HeLa tumor xenograft mice via the tail vein followed by observation via a non-invasive near-infrared (NIR) optical imaging technique. When the tumor reached about 300 mm3, the mice were randomly divided into five groups (five mice per group): (I) control group (PBS); (II) BCNU group at 15 mg BCNU/kg; (III) BCNU + O6-BG group ; (IV) HACB/BCNU NPs-low dose group at 5 mg BCNU/kg; (V) HACB/BCNU NPs-high dose group at 15 mg BCNU/kg. The body weights and the tumor volumes were recorded every 2 days. At day 15, the mice were sacrificed, and the tumors were collected and weighed. All tumors and major organs were fixed in 10% neutral buffered formalin for histological examination. TdT-mediated dUTP nick-end labeling (TUNEL) assay and hematoxylin and eosin (HE) assay were performed to further evaluate the antitumor effect of different formulations. Meanwhile, the major organs were also analyzed by the HE staining at the end of the treatment to determine the biocompatibility and adverse effect of formulations.Tumor growth inhibition of HACB/BCNU NPs was evaluated on HeLa tumor xenograft models. When the tumor reached about 100 mmt-test or one-way ANOVA. The values between groups were compared using Student’s t-test. A p-value of less than 0.05 was considered statistically significant.The data are given as mean ± S. D.. The statistical significance was tested by a two-tailed Student’s a was synthesized via diazotization between 3-aminobenzylalcohol and phenol. Then, compound c was formed by the condensation between compound a and compound b under the nitrogen atmosphere in presence of potassium tert-butoxide. Due to its azobenzene group, which undergoes azo bond cleavage and releases AGT inhibitor through electrons reduction under tumor hypoxic conditions, compound c is considered to be a hypoxia-degradable AGT inhibitor derivative [c acts as the hydrophobic terminal of HACB conjugate. Finally, amphiphilic conjugates HACB were synthesized by linking -COOH of HA and -OH of compound c with the esterification reaction. The chemical structure of the HACB conjugate was characterized and verified by 1H NMR and FT-IR. As shown in Fig. 3 of HA and aromatic groups of compound c, respectively. Compared to the FT-IR spectrum of HA, the peak of HACB observed at 1730 cm−1 was assigned to stretching vibration of the carbonyl group from the newly formed ester  in an aqueous solution. As shown in Fig. The particle size determines the tumor targeting ability and internalization efficiency of vehicles –45. Henc2S2O4 and HACB NPs in aqueous solution at 37 ℃ [2S2O4 treatment. The above data confirmed that HACB NPs possessed high hypoxia sensitivity and could be disintegrated via the breakage of azo bonds under hypoxia conditions, resulting in the changes of morphology and hydrodynamic size of the micelles.To verify the responsiveness of HACB NPs to hypoxia, the hypoxic bioreductive environment was stimulated by the incubation of Na at 37 ℃ . As showO6-BG and acts as an esterase-sensitive switch to control the drug release locating at the core of the micelles. To evaluate the esterase responsiveness of HACB NPs, we recorded the hydrodynamic size and morphology of the micelles after incubation with esterase by DLS and TEM, respectively. As shown in Fig. The phenyl hydroxyl ester structure in HACB NPs, which is a typical moiety undergoing rapid response to esterase , combinep < 0.05) than that of the group with esterase addition under normoxia. These results confirmed that the HACB/BCNU NPs were stable under normoxic physiological conditions, but the micelles could be disintegrated under high esterase content and hypoxic environment achieving rapid BCNU release in tumor tissues.Cou6 is a classic model for the exploration of drug-releasing characteristics. Considering the poor stability of BCNU in aqueous solution, Cou6-loaded HACB NPs was prepared as an alternative of HACB/BCNU NPs. The release behavior of HACB/Cou6 NPs was analyzed by detecting the absorbance of Cou6 at 457 nm via HPLC. As shown in Fig. The cellular uptake behavior of HACB/Cou6 NPs was observed by the fluorescent microscopic. Hoechst33342, a blue dye, was used to stain the nucleus for locating HeLa cells. Cou6 and HACB/Cou6 NPs were shown as green fluorescent. As illustrated in Fig. The intracellular localization of nano-micelles was further observed by CLSM Fig. B. HACB/CO6-BG showed a lower survival rate compared with BCNU treatment. The IC50 values for the BCNU + O6-BG-treated groups were 213.2 µM and 257.3 µM in normoxia and hypoxia, respectively, which was lower than 489.5 µM (normoxia) and 512.5 µM (hypoxia) of the BCNU-treated groups   and 2.4-fold (p < 0.001) higher than that of HAB/BCNU NPs-group, respectively  and 1.44-fold (p < 0.01) higher than those of BCNU + O6-BG-treated group, respectively. These results confirmed that HACB/BCNU NPs could efficiently release BCNU and produce AGT inhibitors to induce apoptosis of HeLa cells under hypoxia.BCNU exerts antitumor activity by inducing the cancer cells to form the dG-dC ICLs, which causes cellular apoptosis by blocking the opening of DNA double strands in replication and transcription , 50. Basely Fig. C and D. O6-BG under normoxic and hypoxic conditions. On the contrary, HACB/BCNU NPs exhibited superior hypoxia-responsive inhibition in HeLa cell migration. The cell migration ratio of HACB/BCNU NPs was 5.2% under hypoxia, obviously lower than that of BCNU (29.7%) and BCNU + O6-BG (14.4%).To further evaluate the antitumor efficiency of HACB/BCNU NPs, the wound healing assay was carried out to investigate the inhibition of HACB/BCNU NPs on HeLa cell migration. As shown in Fig. 50 of HACB/BCNU NPs in normoxia and hypoxia were 185.2 µM and 175.2 µM, respectively, which were remarkably lower (p < 0.001) than those treated with BCNU (800.8 µM in normoxia and 805.8 µM in hypoxia) and BCNU + O6-BG (442.4 µM in normoxia and 392.4 µM in hypoxia)  tumor models possess similar characteristics to solid tumors, such as oxygen-deficient microenvironment, enhanced resistance and abnormal enzyme level . In this50 of BCNU and BCNU + O6-BG in HeLa tumor spheroids were higher than those of 2D adherent cells . As illustrated in Fig. O6-BG, and HACB/BCNU NPs. As shown in Fig. O6-BG showed an enhanced inhibitory effect (p < 0.05), but was still insufficient to completely eliminate HeLa tumors. It is noticeable that a higher inhibitory effect on tumor growth was achieved in the low-dose HACB/BCNU NPs group compared with BCNU + O6-BG group, which was attributed to the effective accumulation and degradability of the micelles on tumor tissue. Besides, the high-dose HACB/BCNU NPs group showed the strongest tumor inhibition potency. On day 15, the tumor weight of the high-dose HACB/BCNU NPs group was only 14.5% and 24.9% of the weight of BCNU and BCNU + O6-BG groups, respectively  of the mice were excised for pathological examination to evaluate the biological toxicity of HACB/BCNU NPs. As showed in Fig. In this study, a multifunctional hypoxia/esterase-sensitive nanomicelle was fabricated for the application of BCNU responsive delivery and tumor sensitization. The poor solubility and low stability of free BCNU in physiological buffers were improved by packaging BCNU in a spherical micelle with a hydrophilic surface. HACB/BCNU NPs could selectively disintegrate by azo bond cleavage under hypoxic conditions, leading to the emergence of AGT inhibitors and the release of BCNU. In vitro characterization experiment verified the good stability, high loading capacity of BCNU, HA-mediated high cell uptake and effective hypoxia/esterase responsiveness. By cytotoxicity assays, apoptosis analysis, wound-healing assays and in vivo antitumor assays, we observed that HACB/BCNU NPs exhibited significantly potentiated anticancer effect compared with conventional BCNU chemotherapeutics. Moreover, in vivo fluorescence imaging indicated that HACB NPs could specifically target tumor tissue. HACB/BCNU NPs showed favorable biocompatibility and biosafety in vivo. This high-performance stimulus-responsive nanocarrier paves a promising way for enhancing the anticancer efficacy and reducing the side effects of BCNU and other CENUs.Additional file 1.Fig. S1 Chemical structural formula (S1.1) and 1H NMR (S1.2) and 13C NMR (S1.3) characterization of 4-((3-(hydroxymethyl) phenyl) diazenyl) phenol (a). Fig. S2 Chemical structural formula (S2.1) and 1H NMR (S2.2) and 13C NMR (S2.3) characterization of 1-(2-amino-9H-purin-6-yl)-1-methylpyrrolidin-1-ium chloride (b). Fig. S3 Chemical structural formula (S3.1) and 1H NMR (S3.2) and 13C NMR (S3.3) characterization of 4-((3-(((2-amino-9H-purin-6-yl) oxy) methyl) phenyl) diazenyl) phenol (c). Fig. S4 The zeta pontential of HACB NPs after esterase incubation. Table S1 Encapsulation efficiency and drug loading of HACB/BCNU NPs for different mass ratios of BCNU and HACB (w/w). Table S2 The inhibitory concentration of each cell line after treatment. Table S3 The inhibitory concentration of HeLa spheroids after treatment.


The mean reduction of grain yield was only 26.66% at N50 ranging from 15.0% to 34.2%. The significant finding of the study is the identification of flag leaf chlorophyll fluorescence traits  and Ci associated with grain yield under N50, which can be used to screen N use efficient genotypes in rice under reduced N application. Out of nine NUE indices assessed, NUpE, NUtE, and NUEyield were able to delineate the high-yielding genotypes at N50 and were useful to screen rice under reduced N conditions. Birupa emerged as one of the high yielders under N50, even though it is a moderate yielder at N100 and infers the possibility of cultivating some of the released rice varieties under reduced N inputs. The study indicates the possibility of the existence of promising genetic variability for grain yield under reduced N, the potential of flag leaf chlorophyll fluorescence, and gas exchange traits as physiological markers and best suitable NUE indices to be deployed in rice breeding programs.Higher grain yield in high-yielding rice varieties is mostly driven by nitrogen (N) fertilizer applied in abundant amounts leading to increased production cost and environmental pollution. This has fueled the studies on nitrogen use efficiency (NUE) to decrease the N fertilizer application in rice to the possible extent. NUE is a complex physiological trait controlled by multiple genes, but yet to be completely deciphered in rice. With an objective of identifying the promising physiological traits associated with NUE in rice, the performance of 14 rice genotypes was assessed at N0, N50, N100, and N150 for four (two wet and two dry) seasons using agro-morphological, grain yield, flag leaf traits, photosynthetic pigment content, flag leaf gas exchange traits, and chlorophyll fluorescence traits. Furthermore, the data were used to derive various NUE indices to identify the most appropriate indices useful to screen rice genotypes at N50. Results indicate that with the increase in N application, cumulative grain yield increased significantly up to N100 (5.02 t ha Rice, a grain crop, is the prime source of food for more than half of the global population . Owing t2, which is 75% higher than the world average. However, the NUE is only 28%–35%, which is 15%–20% lower than that of the global average NUE  and photosynthetic nitrogen use efficiency (PNUE), which is the ratio of Pn to leaf N content, are the two primary attributes affecting the photosynthesis and nutrient utilization by plant leaves . FurtherIn rice, the relationship between leaf traits and NUE is yet to be characterized . Similarviz., Anjali, Birupa, Daya, Heera, Indira, Nidhi, N22, Tella Hamsa, VL Dhan 209, Vasumati, IR64, GQ25, Varadhan, and MTU 1010, were selected for characterization of their physiological traits. Details of rice genotypes used in the study are given in Based on studies conducted earlier under NEWS project, genotypes with varying yield potential, −1. During dry season 2021, mean maximum temperature was 32.4°C while mean minimum temperature was 16.1°C. The mean relative humidity was 87.5%, with a total rainfall of 16.8 mm and mean bright sunshine hours was 7.9 h day−1. During wet season 2021, mean maximum temperature was 30.7°C while mean minimum temperature was 22.6°C. The mean relative humidity was 93.9%, with a total rainfall of 823.8 mm and mean bright sunshine hours was 5.0 h day−1. During dry season 2022, mean maximum temperature was 32.5°C while mean minimum temperature was 17.3°C. The mean relative humidity was 84.4%, a total rainfall of 14.0 mm was received, and mean bright sunshine hours was 7.5 h day−1.Important weather parameters recorded during the crop growing period is given in 2 . Four graded levels of N, viz., N150 [150% recommended dose of N (RDN)—150 kg N ha−1], N100 (100% RDN—100 kg N ha−1), N50 (50% RDN—50 kg N ha−1) and N0 (0% RDN—0 kg N ha−1), were used.The experimental plot soil was clay in texture, slightly alkaline (pH 8.25), non-saline (EC - 0.76 dS/m), and medium in organic carbon content (0.53%). Soil available nitrogen was low (213 kg/ha) with high available phosphorus (92 kg/ha) and potassium (641 kg/ha). Experiments were arranged in a split-plot design with nitrogen application rates as the main plot and genotypes as the subplot with three replications. The size of each plot was 15 mThe number of days taken for 50% of plants to flower in each genotype and each treatment was noted as days to 50% flowering and was expressed in days. The number of days taken from sowing to physiological maturity was recorded and was expressed in days. The flag leaf traits along with SLA and SLW were measured from five randomly selected leaves per plot in three replications during 50% flowering stage . Flag le2 g−1. Specific leaf weight (SLW) was determined by dividing leaf dry weight with leaf area, using the formula of −2.Flag leaf thickness is measured using a digital caliper and expressed in millimeters (mm). The flag leaves were oven dried after measuring length, width, and thickness for 3 days at 80°C and flag leaf dry weight was recorded using an electronic balance . Specific leaf area (SLA) was calculated by dividing leaf area with leaf dry weight, employing the formula of 2 area were harvested and threshed grain weight was determined after drying to 14% moisture content and converted to t ha−1 and straw weight was also recorded for the same . The content of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids were calculated as per the formulas given by For the quantitative determination of leaf chlorophyll content, at 50% flowering stage, five plants were randomly chosen in each plot and the flag leaf was labeled to investigate gas exchange traits and photosynthetic pigment content. For the determination of pigment content, leaf tissue of each sample was cut into small pieces with a sharp razor blade and 25 mg of cut leaf pieces was placed into 10-mL tubes containing 10 mL of 80% acetone and stored in the dark for 48 h to ensure complete extraction of leaf chlorophyll pigment. The absorbance of the chlorophyll solution was measured by using a UV-VIS double beam spectrophotometer . Chlorophyll a, chlorophyll b, and carotenoids were measured at 663.2, 646.8, and 470.0 nm, respectively, and expressed in mg gs), transpiration rate (E), and internal CO2 concentration (Ci) were recorded at 50% flowering stage by using the Infra-Red Gas Analyzer portable photosynthesis measurement system  attached to a leaf chamber fluorometer, which was used as the light source. During measurements, the photosynthetically active radiation (PAR) was kept at 1,200 µmol m−2 s−1. The CO2 concentration was maintained at 387 ± 6 ppm. These measurements were made between 10:00 a.m. and 12.00 noon at all the sampling dates. Pn was expressed in μmol (CO2) m−2 s−1, gs was expressed in mol (H2O) m−2 s−1, E was expressed in mmol (H2O) m−2 s−1, and Ci was expressed in ppm. PNUE was calculated as given by Gas exchange traits in flag leaf such as photosynthetic rate (Pn), stomatal conductance (gv/Fm), actual quantum yield of PSII (ΦPSII), electron transport rate (ETR), coefficient of photochemical quenching (qP), and coefficient of non-photochemical quenching (qN)  during 50% flowering stage. The instrument was connected to a desktop PC with WinControl-3 software. The flag leaves were dark-adapted for 30 min before recording fluorescence traits and the following fluorescence traits were calculated: the maximum quantum yield of PSII (Fing (qN) .2 area at harvest. Samples were dried under shade and then in hot air oven at 60°C. Oven-dried samples were ground to fine powder using a grinder and stored in butter paper covers for estimating N concentration. The samples were digested in sulfuric acid (H2SO4) using block digestion unit and analyzed for their total N content by the micro Kjeldahl distillation method using automatic N analyzer  with steam distillation and the N content was expressed as percentage. NUE indices such as nitrogen uptake efficiency (NUpE), nitrogen utilization efficiency (NUtE), nitrogen use efficiencyyield (NUEyield), agronomic efficiency (AE), physiology efficiency (PE), partial factor productivity (PFP), apparent nitrogen recovery efficiency (ANRE), and nitrogen harvest index (NHI) were calculated as per formulas given in Flag leaf samples collected at 50% flowering stage were used to determine flag leaf N content, and grain and straw samples were collected from 1 mf is the amount of N in a fertilized plant, Plant N0 is the amount of N in a non-fertilized plant, Yieldf is the grain yield of a fertilized plant, and Yield0 is the grain yield of a non-fertilized plant.where Plant N is the amount of N in a plant, Yield N is the amount of grain N in a plant, Plant NTwo-way analysis of variance (ANOVA) was performed using an open source software R  with Agrv/Fm. Season × treatment was significant for Pn, gs, E, days to 50% flowering, days to physiological maturity, chlorophyll a, total chlorophyll, carotenoids, PNUE, total dry matter, grain N uptake, straw N uptake, and total N uptake. Season × treatment × genotypes was significant for gs, E, days to physiological maturity, and photosynthetic pigments. Among the NUE indices, ANOVA showed significant effect of treatment and variation among genotypes and interaction between treatment and genotypes. Three indices  noted significant interaction for season × treatment and six indices  noted significant interaction for season × genotypes. Significant interaction was not observed in any of the indices for season × treatment × genotypes.ANOVA indicates that 29 morpho-physiological traits including grain yield and N uptake traits noted significant variation with treatment and among the genotypes Table 3.Table 3ANOVA for morpho-physiological parameters, grain yield and nitrogen use efficiency indices.−1 and total dry matter significantly increased from 7.43 to 10.89 t ha−1 with the increase in N fertilizer application among the treatments . Among the genotypes, Vasumati at N50 and MTU 1010 at N100 recorded the highest grain yield (4.22 and 5.84 t ha−1), while N22 recorded the lowest (2.46 and 3.28 t ha−1) at N50 and N100. The highest total dry matter was recorded in Varadhan (9.87 and 12.08 t ha−1) whereas N22 recorded the lowest (6.29 and 7.37 t ha−1) at N50 and N100. With increased N application from N0 to N150, mean days to 50% flowering and physiological maturity significantly increased from 96 to 101 days and 125 to 130 days. Among the genotypes, days to 50% flowering ranged from 83  to 111 days (Birupa) at N50 and 84  to 112 days (Birupa) at N100. Days to maturity ranged from 114  to 137 days (Daya) at N50 and 114  to 138 days (Birupa) at N100.The genotype-wise values of all the measured traits of the study are presented in 2, flag leaf thickness increased from 0.281 to 0.374 mm, and flag leaf dry weight increased from 0.121 to 0.170 g. Among the genotypes, Indira exhibited the highest flag leaf length (34.8 and 37.2 cm), area (39.2 and 43.0 cm2), and dry weight (0.177 and 0.185 g), while the lowest flag leaf length (24.6 and 27.8 cm), area (24.3 and 28.3 cm2), and dry weight (0.111 and 0.123 g) were observed in IR64, at N50 and N100. The highest flag leaf width (1.52 and 1.60 cm) was noticed in Heera at N50 and N100, whereas MTU 1010 exhibited the lowest values (1.28 and 1.35 cm). Flag leaf thickness was the highest (0.347 mm) in Birupa and the lowest in GQ25 (0.280 mm) at N50, and the highest (0.371 mm) in N22 and the lowest (0.312 mm) in Varadhan at N100. SLA increased significantly with increased application of N whereas significant reduction in SLW is observed. From N0 to N150, mean values of SLA increased from 209.0 to 241.1 cm2 g−1 and SLW decreased from 4.80 to 4.15 mg cm−2. Among the genotypes, SLA ranged from 213.5 (Heera) to 223.5 cm2 g−1 (Tella Hamsa) at N50, and from 220.7 (N22) to 236.0 cm2 g−1 (Nidhi) at N100. SLW ranged from 4.48 (Tella Hamsa) to 4.69 mg cm−2 (Heera) at N50, and from 4.25 (Nidhi) to 4.54 mg cm−2 (N22) at N100.Flag leaf length, width, area, thickness, and dry weight increased significantly with increased application of N. From N0 to N150, mean values of flag leaf length increased from 26.3 to 35.2 cm, flag leaf width increased from 1.28 to 1.55 cm, flag leaf area increased from 25.4 to 41.1 cm−1 fw, chlorophyll b increased significantly from 0.478 to 0.921 mg g−1 fw, total chlorophyll increased significantly from 2.19 to 3.83 mg g−1 fw, and carotenoid increased significantly from 0.510 to 0.802 mg g−1 fw with the increase in N application from N0 to N150. Among the genotypes, the highest chlorophyll a content (2.34 and 3.03 mg g−1 fw) was recorded in Heera at N50 and Varadhan at N100 while the lowest content (1.81 and 2.32 mg g−1 fw) was recorded in N22 at N50 and N100. IR64 at N50 and Varadhan at N100 recorded the highest contents of chlorophyll b (0.756 and 0.939 mg g−1 fw),whereas N22 at N50 and Heera at N100 recorded the lowest contents (0.512 and 0.675 mg g−1 fw). Total chlorophyll content was the lowest (2.33 and 3.01 mg g−1 fw) in N22 at N50 and N100 and the highest (3.03 and 3.97 mg g−1 fw) in MTU 1010 at N50 and Varadhan at N100. Carotenoid content was the highest (0.710 and 0.834 mg g−1 fw) in Varadhan at N50 and Heera at N100 and the lowest (0.554 and 0.673 mg g−1 fw) in N22 at N50 and N100.Among the treatments, mean contents of chlorophyll a increased significantly from 1.71 to 2.91 mg g2) m−2 s−1, gs increased significantly from 0.281 to 0.706 mol (H2O) m−2 s−1, and E increased significantly from 4.94 to 8.42 mmol (H2O) m−2 s−1 while Ci decreased significantly from 279.6 to 256.5 ppm with an increase in N application from N0 to N150. MTU 1010 exhibited the highest Pn [1.3 and 26.0 µmol (CO2) m−2 s−1], gs [0.550 and 0.750 mol (H2O) m−2 s−1], and E [7.24 and 8.47 mmol (H2O) m−2 s−1], and the highest Ci (296.5 and 287.7 ppm) was observed in Daya at N50 and Tella Hamsa at N100, while the lowest Pn [17.9 and 22.0 µmol (CO2) m−2 s−1] was observed in Tella Hamsa at N50 and N22 at N100, the lowest gs [0.370 and 0.581 mol (H2O) m−2 s−1] was recorded in Daya at N50 and Tella Hamsa at N100, the lowest E [5.29 and 7.16 mmol (H2O) m−2 s−1] was noticed in N22 at N50 and Nidhi at N100, and the lowest Ci (242.4 and 238.1 ppm) was recorded in Nidhi at N50 and Daya at N100. Among the treatments, mean flag leaf N content increased significantly from 2.11% to 2.86% and mean PNUE increased significantly from 15.8 to 21.6 μmol (CO2) g−1 N s−1 with an increase in N application from N0 to N150. Flag leaf N content was the highest (2.68% and 2.96%) in Tella Hamsa at N50 and IR64 at N100 and the lowest (2.23% and 2.43%) in Daya at N50 and N100. MTU 1010 exhibited the highest PNUE [19.4 and 22.6 μmol (CO2) g−1 N s−1] at N50 and N100 whereas Anjali exhibited the lowest [14.9 and 18.0 μmol (CO2) g−1 N s−1].Mean Pn increased significantly from 15.7 to 25.4 µmol  at N50 and N100, whereas Daya at N50 and N22 at N100 recorded the lowest (0.792 and 0.805). ΦPSII was the highest (0.354 and 0.388) in MTU 1010 at N50 and Tella Hamsa at N100 and the lowest (0.297 and 0.335) in Nidhi at N50 and N100. MTU 1010 at N50 and Tella Hamsa at N100 recorded the highest ETR (25.1 and 27.4) whereas Nidhi at N50 and Birupa at N100 recorded the lowest (21.8 and 25.3). qP was the highest (0.564 and 0.622) in VL Dhan 209 at N50 and MTU 1010 at N100 and the lowest (0.474 and 0.553) in Nidhi at N50 and N100. GQ25 exhibited the highest qN (0.414 and 0.396) at N50 and N100 whereas Indira exhibited the lowest qN (0.361 and 0.341).F−1, mean straw N uptake from 24.5 to 41.2 kg N ha−1, and as total plant from 55.1 to 110.3 kg N ha−1. Grain N uptake ranged from 28.8 (N22) to 52.3 kg N ha−1 (MTU 1010) at N50, and from 41.8 (N22) to 80.5 kg N ha−1 (MTU 1010) at N100. Straw N uptake ranged from 22.5 (N22) to 37.6 kg N ha−1 (MTU 1010) at N50, and from 26.5 (N22) to 42.6 kg N ha−1 (VL Dhan 209) at N100. Total N uptake ranged from 51.4 (N22) to 89.8 kg N ha−1 (MTU 1010) at N50, and from 68.3 (N22) to 120.5 kg N ha−1 (MTU 1010) at N100.Increased N application from N0 to N150 resulted in a significant increase in mean grain N uptake from 30.6 to 69.1 kg N hav/Fm, ΦPSII, ETR, qP, and Ci with grain yield were only significant at N50. Furthermore, ΦPSII, ETR, and qP showed a significant negative correlation and qN noted a non-significant positive correlation with flag leaf nitrogen (FLN) at N100. In contrast, ΦPSII, ETR, qP, and qN noted a significant positive correlation with FLN at N50.Multiple correlation analysis 2 of morR2 > 0.8) correlated with grain yield at all the N levels and seasons (R2 ≥ 0.5) or highly significant (R2 ≥ 0.7) positive correlation with grain yield at all the N levels and seasons (R2 ≥ 0.5) or non-significant (R2< 0.5) positive correlation with grain yield at all the N levels and seasons  with grain yield at all N levels and seasons , Birupa (2), MTU 1010 (14), Varadhan (13), and Indira (5) have exhibited maximum NUEyield and grain yield at N50. At N100 and N150, MTU 1010 (14), Vasumati (10), Varadhan (13), Indira (5), and VL Dhan 209 (9) have shown higher grain yield and NUEyield. N22 (7) and Tella Hamsa (8) have shown the least NUEyield and grain yield at all N levels. AE showed significantly positive correlation with grain yield at all N levels and seasons except for wet season 2021 at N0 (R2 = 1) with grain yield at all N levels and seasons  is an essential nutrient for the growth, development, and maintenance of rice . As soilUnder N50, a reduction of 26.99% of grain yield in comparison with N100 was observed while 43.88% reduction was observed under N0. However, only marginal increment in grain yield (1.31%) was observed from N100 to N150 Figure 7indica rice cultivars  have shown a significant increase and specific leaf weight (SLW) has shown a significant decrease with increased N application in the present study. Earlier studies have also reported a significant increase in length and width of flag leaf , an incrultivars  and 2.9%ultivars , was repultivars .indica hybrid rice , Pn, gs, and E were significantly higher under medium (120 kg N ha−1) and high N (180 kg N ha−1) levels (2) m−2 s−1 at 150% of RDN was noted compared with a Pn of 17.41 µmol (CO2) m−2 s−1 at 0% of RDN  levels . A signi% of RDN . Flag le% of RDN . Leaf N % of RDN . PNUE is% of RDN . Leaf N % of RDN .v/Fm, ΦPSII, ETR, and qP showed an increasing trend, while qN decreased as the N rate increased as reported in hybrid rice  and Ci among the gas exchange traits were significantly correlated with grain yield at N50. As these traits were measured using flag leaf, the correlation of these traits with FLN revealed significant differences between N50 and N100. Hence, flag leaf at 50% flowering can be a good source to assess chlorophyll fluorescence traits under reduced N conditions and can differentiate rice genotypes varying in yield and NUE. In photosystem II of light reaction, Fv/Fm explains the maximum photochemical conversion (quantum yield), ΦPSII explains the effective photochemical conversion, ETR explains the amount of electron transfer at the reaction center in PSII, and qP represents the functional or open proportion of PSII involved in photochemical conversion. The available literature indicates that N-deficient conditions like N50 can result in improper formation and function of PSII affecting photochemical conversion followed by yield  to screen genotypes under N50 with known low-yielding and high-yielding genotypes as checks. As optimum N can show the highest quantum efficiency followed by yield and genotype-specific optimum N requirement is unknown, Birupa, a moderate yielder at N100, emerged as one of the top five yielders at N50 and vice versa in the case of VL Dhan 209. It indicates differential response of the genotypes for yield potential with varied levels of N.Among the chlorophyll fluorescence traits, Frid rice . The apprid rice . Among tby yield  and reduby yield , whereasby yield . Hence, −1 and decreased with further uplift in N application in some recently released Egyptian rice varieties , NUEyield increased up to 210 kg N ha−1 and then decreased  grain yielders at N100 were identified as promising genotypes for efficient use of N by NUpE, NUtE, and NUEyield indices at N50. Moreover, NUEyield is the product of NUpE and NUtE. Hence, among the nine indices, these three  can be further used to identify promising genotypes at N50.Screening of 14 genotypes under four graded N levels across four seasons revealed wide genotypic variation in their response in terms of grain yield. An increase of agro-morphological traits, photosynthetic pigments, and flag leaf traits (except SLW) was observed with an increase of N fertilizer application. At N50, Fv/Fm, ΦPSII, ETR, and qP) and Ci were identified to be associated with grain yield under N50, which could be deployed in the breeding for NUE in rice. Among the tested genotypes, Birupa, which is a relative moderate yielder at N100, emerged as a high yielder under N50, which indicates the potential of the moderate-yielding genotypes at N100 to produce better grain yield at N50. Therefore, this study recommends the evaluation of the released rice varieties at N50 to determine their suitability under low N input conditions. Among the nine NUE indices studied, NUpE, NUtE, and NUEyield are useful to identify promising genotypes at N50.The present study has clearly demonstrated the existence of genetic variability among the rice genotypes through N response under graded N levels. The grain yield penalty ranged only from 15% to 34.2% at N50 across the 14 genotypes in comparison with N100, suggesting the possibility of reduction of N fertilizer application. Most importantly, through the evaluation of flag leaf physiological traits at the flowering stage, chlorophyll fluorescence traits (FThe original contributions presented in the study are included in the article/BS: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft. DS: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing. DSR: Data curation, Software, Visualization, Writing – review & editing. SR: Methodology, Writing – review & editing. KaS: Software, Writing – review & editing. PR: Resources, Writing – review & editing. KuS: Methodology, Writing – review & editing. RS: Resources, Writing – review & editing. CN: Conceptualization, Supervision, Writing – review & editing.


Plasmodium falciparum Kelch 13 (PfK13) protein are associated with artemisinin resistance, but the unique or common mechanism which results in this resistance is unclear.The emergence and spread of artemisinin resistance threaten global malaria control and elimination goals, and encourage research on the mechanisms of drug resistance in malaria parasites. Mutations in PfK13 mutation on the transcriptome and proteome of P. falciparum at different developmental stages. Additionally, the number of merozoites, hemozoin amount, and growth of P. falciparum 3D7C580Y and P. falciparum 3D7WT were compared. The impact of iron supplementation on the number of merozoites of P. falciparum 3D7C580Y was also examined.We analyzed the effects of the PfK13 mutation did not significantly change glycolysis, TCA, pentose phosphate pathway, or oxidative phosphorylation, but did reduce the expression of reproduction- and DNA synthesis-related genes. The reduced number of merozoites, decreased level of hemozoin, and slowed growth of P. falciparum 3D7C580Y were consistent with these changes. Furthermore, adding iron supply could increase the number of the merozoites of P. falciparum 3D7C580Y.We found that the PfK13 mutation reduced hemoglobin ingestion, leading to artemisinin resistance, likely by decreasing the parasites' requirement for haem and iron. This study helps elucidate the mechanism of artemisinin resistance due to PfK13 mutations.These results revealed that the The online version contains supplementary material available at 10.1186/s13071-023-06024-4. Plasmodium falciparum Kelch 13 (PfK13) protein have been associated with artemisinin resistance [PfK13 include C580Y, R539T, I543, and Y493Y, with the C580Y mutation becoming increasingly dominant in the Greater Mekong subregion [PfK13 is located in the cytostome (cell mouth), through which the parasite takes up hemoglobin [PfK13 mutation likely confers artemisinin resistance by dampening hemoglobin endocytosis and artemisinin activation [PfK13 mutations and relevant genetic and physiological backgrounds [PfK13 mutation [PfK13 mutations were still parasitaemic on day 3 after treatment with artemisinin derivatives [PfK13 mutations that reduced hemoglobin endocytosis and led to decreased artemisinin activation were not the sole causes of artemisinin resistance. Malaria parasites that survive with PfK13 mutations show reduced hemoglobin endocytosis and have lower haem and iron (HI) requirements. We recently showed that malaria parasites depend on HI, and that artemisinin likely kills malaria parasites by disturbing their utilization of HI [ Plasmodium parasites may result in their high sensitivity to artemisinin [Plasmodium with PfK13 mutations. However, regardless of which explanation is better supported, it is essential to clarify how PfK13 mutations affect malaria parasites.The emergence and spread of artemisinin resistance endanger worldwide malaria control and elimination goals –4. Althosistance –8. Frequmoglobin , the uptmoglobin . PfK13 mtivation . Althougkgrounds , 13, notmutation . Some paivatives , thus, ton of HI . The higemisinin , 20; thuPlasmodium falciparum 3D7WT and 3D7C580Y were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with 25 mmol/L HEPES, 0.5% AlbuMAX, 0.2% sodium bicarbonate, 0.2 mmol/L htpoxanthine, and 20 μg/mL gentamicin sulfate at 37 °C in an incubator supplied with 5% CO2 and 5% O2. Fresh O positive human red blood cells from healthy human donors were used to maintain parasitaemia; the parasites were maintained at 2% hematocrit, based on a previous report [s report . The par g for 3 min and preserved in TRIzol reagent . RNA was extracted using phenol and isopropanol precipitation. RNA quantification and quality determination were performed using Nanodrop 2000 .The cultures were tightly synchronized to obtain 0- to 3-h ring stages. Parasites were harvested at 0, 6, 12, 18, 24, 30, 36, and 42 h post-infection (hpi) for transcriptome analysis. Samples at different stages were collected by centrifugation at 1000RNA integrity was assessed using an Agilent 2100 Bioanalyzer . Libraries were constructed using the TruSeq Stranded mRNA LT Sample Prep Kit  according to the manufacturer’s instructions. Transcriptome sequencing and analysis were conducted by LC Sciences . The libraries were sequenced on an Illumina HiSeq X Ten platform, and 150 base pair paired-end reads were generated. About 49.10 million raw reads for each sample were generated. Raw data in FASTQ format were first processed using Trimmomatic, and low-quality reads were removed to obtain the clean reads . About 4P. falciparum genome by using HISAT2 [https://bioconductor.org/packages/release/bioc/html/edgeR.html). The threshold for significant differential expression was set at P- < 0.05, fold change > 2, or fold change < 0.5, and the minimum number of reads for at least one sample had to exceed 100. Gene Ontology (GO) enrichment analysis was performed using R or David software (https://david.ncifcrf.gov). Differentially expressed genes were functionally annotated using PlasmoDB and UniProt.Clean reads were mapped to the g HISAT2 . The frag HISAT2 , 25, andg HISAT2 . DifferePlasmodium falciparum 3D7WT and P. falciparum 3D7C580Y were treated to obtain parasites 0–3 h after invasion, as described above, and samples were harvested at 12, 24, and 30 h. Extraction, purification and mass spectrometry analysis of the protein were performed by LC-Bio Technology  through liquid chromatography–tandem mass spectrometry. During the differentially expressed protein analysis, fold change > 2 or fold change < 0.5 served as the screening criterion. The selected differentially expressed proteins were subjected to GO enrichment analysis.P. falciparum 3D7WT and P. falciparum 3D7C580Y was isolated using TRIzol reagent. Following DNase treatment, total RNA (800 ng) was reverse transcribed into complementary DNA using the ChamQ Universal SYBR qPCR master mix  according to the manufacturer’s instructions on a 7500 Real-Time PCR System (Applied Biosystems). We selected genes such as merozoite structure-related genes, merozoite surface protein MSA180 gene, rhoptry protein genes, DNA replication-related genes, DNA replication licensing factor MCM5 gene, and proliferating cell nuclear antigen 1 gene, which showed differential expression in the transcriptomes. Seryl-transfer RNA synthetase served as the internal control, and its expression was measured by detecting the fluorescence from SYBR green dye incorporated into the amplifying target. RNA expression was normalized to the corresponding internal control genes, and relative changes were calculated using the 2−Ct method [https://www.ncbi.nlm.nih.gov/tools/primer-blast/). Primers used for quantitative real-time polymerase chain reacion (qPCR) are listed in Additional file Total RNA from t method , 28. ThePlasmodium falciparum 3D7WT and P. falciparum 3D7C580Y were synchronized several times to obtain 0- to 3-h ring stages. The initial infection rate was maintained at 0.3% with hematocrit at 2%, after which normal culture conditions were used. The parasites were cultured for 144 h, and infected red blood cells were collected every 48 h. These were then incubated with Hoechst 33342  in the dark at 37 °C for 10 min. After two washes with RPMI 1640, positive cells were sorted using the BD FACS AriaII cell sorter based on a 355-nm (Hoechst 33342) fluorescence signal. Uninfected red blood cells were used as controls. All data were processed using FlowJo software (10.8.1).Mature schizonts were synchronized using a 40–70% Percoll gradient and cultured at 37 °C for 3 h with shaking to reduce multiple infections. After synchronization with 5% sorbitol, the parasite was cultured for another 42–45 h. The number of merozoites in the schizonts was counted under light microscopy after Giemsa staining. Data were collected through repeated experiments conducted by three operators.Plasmodium falciparum 3D7WT and P. falciparum 3D7C580Y were purified using a 40–70% Percoll gradient and treated with 5% sorbitol to obtain 6-h ring stages. Then, P. falciparum 3D7WT and P. falciparum 3D7C580Y were cultured for 36–42 h with parasitaemia adjusted to 0.5–1% by using normal erythrocytes while maintaining 2% hematocrit. Approximately 10 ml of the culture medium was centrifuged at 1000 g for 3 min, and then treated with 0.15% saponin solution on ice for 10 min to lyse the cells. After centrifugation at 12,000 g for 10 min, the supernatant was discarded, and the sediment was washed with 25 mmol/L Tris (pH 7.8) containing 2.5% sodium dodecyl sulphate until the supernatant was clear. The sediment was dissolved in 250 µL of 2.5% sodium dodecyl sulphate buffer and 20 µL of 2.5 mol/L NaOH. The absorbance of hemozoin at 400 nm was measured using a Nanodrop 2000 spectrophotometer to quantify its content [ content .Plasmodium falciparum 3D7WT and P. falciparum 3D7C580Y were tightly synchronized to obtain the 0- to 3-h ring stages. Synchronized ring-stage parasites were incubated with 50 µmol/L iron sucrose, and the control group was incubated with an equal volume of RPMI 1640 complete medium. After 90–96 h of incubation, the merozoites were counted under a light microscope following Giemsa staining. Data were obtained from experiments repeated at least three times.Plasmodium falciparum 3D7WT at 1.5–2.5% parasitaemia was treated with desferrioxamine (DFO)  at a final concentration of 500 nmol/L for 24 h. A portion of the mixture was centrifuged at 1000 g for 5 min to remove the supernatant. After the addition of cells suspended in PBS and Hoechst 33342 (1:100) (Biyuntian), the mixture was incubated at 37 °C for 10 min under continuous mixing, centrifuged at 1000 g for 5 min, and washed three times with phosphate-buffered saline. The positive cell rate was determined using a BD FACS AriaII cell sorter. Infected red blood cells were identified by using 355-nm fluorescence spectroscopy, with uninfected cells serving as controls. All data were processed using FlowJo software (10.8.1).t-test (unpaired) was performed to compare the changes in parasitaemia in the groups. P < 0.05 was considered to indicate statistical significance.Graphpad Prism 8.0.2 software was used to perform the statistical analysis. Student's P. falciparum 3D7WT and P. falciparum 3D7C580Y were analyzed at 0, 6, 12, 18, 24, 30, 36, and 42 hpi  accounted for 96.05% and 95.12% of the differentially expressed genes, respectively  accounted for 61.20%, 69.18%, and 56.94%, respectively , oxidative phosphorylation, reproduction, DNA replication, pyrimidine metabolism, fatty acid metabolism, amino acid metabolism, purine metabolism, and redox pathways, including the glutathione and thioredoxin systems   Fig. A, B.Fig.PfK13 mutation on HI utilization, we compared the hemozoin content of an equivalent number of P. falciparum 3D7C580Y and P. falciparum 3D7WT at 36 hpi. The hemozoin content of P. falciparum 3D7WT was 16.02 ± 2.44 nmol and that of P. falciparum 3D7C580Y 10.72 ± 2.13 nmol. The hemozoin content of P. falciparum 3D7C580Y was significantly lower than that of P. falciparum 3D7WT (P < 0.01)  (P < 0.01). This suggested that the growth and reproductive capability of P. falciparum 3D7C580Y was lower than that of P. falciparum 3D7WT   Fig. C, D, whi7WT Fig. D.P. falciparum 3D7C580Y. To further elucidate the role of iron in Plasmodium proliferation and development, we treated P. falciparum 3D7 with DFO for 24 h and observed its effect on the parasites. The parasitaemia of P. falciparum 3D7 decreased from 3.21% ± 0.55% to 1.03% ± 0.56% after treatment with DFO (P < 0.01)  at different stages, B fatty acid metabolism, C amino acid metabolism, D purine metabolism, and E redox processes.Additional file 2: Table S1. Primer sequences of key target genes.Additional file 3: Table S2. Analysis of differentially expressed genes at different stages of the parasite.Additional file 4: Table S3. Analysis of differentially expressed proteins at 12, 24, and 30 h post-infection.