Multiple biological factors, including age, sex, and genetics, influence Alzheimer’s disease (AD) risk. Of the 6.2 million Americans living with Alzheimer’s dementia in 2021, 3.8 million are women and 2.4 million are men. The strongest genetic risk factor for sporadic AD is apolipoprotein E-e4 (APOE-e4). Female APOE-e4 carriers develop AD more frequently than age-matched males and have more brain atrophy and memory loss. Consequently, biomarkers that are sensitive to biological risk factors may improve AD diagnostics and may provide insight into underlying mechanistic changes that could drive disease progression. Here, we have assessed the effects of sex and APOE-e4 on the miRNA cargo of cerebrospinal fluid (CSF) extracellular vesicles (EVs) in AD. We used ultrafiltration (UF) combined with size exclusion chromatography (SEC) to enrich CSF EVs . CSF EVs were isolated from female and male AD or controls (CTLs) that were either APOE-e3,4 or -e3,3 positive . MiRNA expression levels were quantified using a custom TaqMan™ array that assayed 190 miRNAs previously found in CSF, including 25 miRNAs that we previously validated as candidate AD biomarkers. We identified changes in the EV miRNA cargo that were affected by both AD and sex. In total, four miRNAs  were significantly increased in AD vs. CTL, independent of sex and APOE-e4 status. Pathway analysis of the predicted gene targets of these four miRNAs with identified pathways was highly relevant to neurodegeneration . There were also three miRNAs  that were significantly increased in females vs. males, independent of disease state and APOE-e4 status. We then performed a statistical analysis to assess the effect of APOE genotype in AD within each sex and found that APOE-e4 status affects different subsets of CSF EV miRNAs in females vs. males. Together, this study demonstrates the complexity of the biological factors associated with AD risk and the impact on EV miRNAs, which may contribute to AD pathophysiology. Alzheimer’s disease (AD) is the most common form of dementia, the sixth leading cause of death in the United States, and the fifth leading cause of death for those age 65 and older . In 2021There are multiple biological factors, including age, sex, and genetics, that influence AD risk . Of the In our prior studies, we focused on the utility of extracellular miRNAs as biomarkers for AD in total CSF that was not fractionated to separate and enrich for EVs. We initially discovered a set of 36 miRNAs in CSF from living donors that could classify AD patients from healthy controls (CTLs) . Our valExtracellular miRNAs in CSF have multiple carrier types, including extracellular vesicles (EVs), RNA-binding proteins, and high-density lipoproteins . EVs arevia blebbing of the plasma membrane, while microvesicles (MVs) are ∼150–1,000 nm and are released via budding of the plasma membrane. In contrast, exosomes measure ∼40–150 nm and arise from the endosomal pathway, which forms intracellular multivesicular bodies (MVB) that are secreted as vesicles into the extracellular space. Two additional nanoparticle types, exomeres and supermeres, have also been recently identified in cultured cells  also impact EV miRNA expression. We first established a protocol using living donor’s CSF and a combined approach of ultrafiltration (UF) plus size exclusion chromatography (SEC) to enrich EVs positive for canonical markers, such as Flotillin, TSG101, and CD81, while also depleting a majority of proteins and lipoproteins not associated with EVs. We then used UF plus SEC to isolate EVs from females and males that were either AD or CTLs and were either APOE-e3,4 or APOE-e3,3 positive. We quantified miRNA expression levels in the isolated EVs using a custom TaqMans for AD . We idenFor experiments that characterized CSF EVs by immunoblot, tunable resistive pulse sensing (TRPS), transmission electron microscopy (TEM), and single vesicle flow cytometry (vFC) , we poolThe OADC and USCD ADRC both used a standardized CSF collection protocol corresponding to that of other AD centers engaged in biomarker research . Lumbar HhaI (restriction enzyme site GCG^C) and then subjected to electrophoresis on polyacrylamide gels; the gels were stained with ethidium bromide, and the digested fragments were visualized by ultraviolet illumination. A unique combination of HhaI fragment sizes enabled typing of all homozygotic and heterozygotic combinations: HhaI cleaves at GCGC encoding 112arg (e4) and 158arg  but does not cut at GTGC-encoding 112cys  and 158cys (e2).APOE-e4 genotypes at OHSU were determined by sequencing the amplicon of APOE exon 4 . Genomic DNA was isolated from blood and amplified by Touchdown PCR with 250 µM dNTPs, 1 unit Taq DNA polymerase, buffer, 1X Q-solution , and 0.5 µM forward (5′-GGCGCTGATGGACGAGACC-3′) and reverse (5′-CCTGGGCCCGCTCCTGTAG-3′) primers to amplify the APOE exon 4. A product size of 443 nucleotides identified on a 1% agarose 1X TBE gel was excised, cleaned with ExoSAP-IT reagent (Affymetrix), and sequenced on a model 377 automated fluorescence sequencer (Applied Biosystems). Chromatogram traces were examined, and nucleotide sequences were determined using FinchTV . APOE-e4 genotypes at UCSD were determined by PCR amplification and restriction fragment length polymorphism (RFLP) , as prevFor single vFC studies, we examined total CSF that was not concentrated as well as 1.0 ml of CSF that was concentrated by UF using Microcon®-100 kDa Centrifugal Filters  to a final volume of 40 μL (25x). We analyzed the CSF by single vFC using fluorescence to estimate vesicle size, concentration, and surface cargo with a commercial flow cytometer  and an assay kit . In brief, 5 µL each of the unconcentrated and 25x concentrated CSF were stained according to the manufactures recommendations with a membrane stain (vFRed) and pool of fluorescence-labeled antibodies against tetraspanins  for 1 h at room temperature. The stained samples were diluted 1:1,000 prior to reading on the flow cytometer and detected using fluorescence triggering . To demonstrate the vesicular nature of the vFRed and/or TS-positive (TS+) events, an aliquot of the stained, concentrated 25x CSF was detergent treated (0.05% Triton X-100) for 1 min prior to being assayed. Data analysis was performed using FCS Express . Events were gated with respect to time (to eliminate spurious background that occurs at the start of each sample), vFRed pulse shape (to eliminate short pulse width background events), and violet side scatter (VSSC) vs. vFRed fluorescence (to include events with characteristic membrane fluorescence and light scatter) . NanoRaiEVs were isolated from CSF by a combined approach of UF and SEC for downstream use in immunoblots, TRPS, TEM, and miRNA qPCR arrays according to Protein concentrations were measured using the Qubit protein assay kit and Qubit 4 fluorometer  for the concentrated UF-SEC pools using 5 μL of the void and Fxs 6–9, 1 μL of Fxs 10–13, and 1 μL of 1:10 diluted Fxs 14–17. The remaining volume of each pool was prepared for SDS-PAGE by diluting in 4x NuPAGE™ LDS sample buffer (Thermo Fisher Scientific). To assess the separation of EV associated proteins from vesicle-free proteins and lipoproteins, we performed a total protein stain and immunoblots for APOA1, APOE, and albumin by loading equal volumes (37 μL) of the void, Fxs 6–9, 10–13, and 14–17 on a NuPAGE™ 4–12% Bis-Tris 1.5 mm x 10 well gels (Thermo Fisher Scientific). To identify the SEC Fxs that contained an enrichment of EV markers, equal concentrations (0.1 μg) of void, Fxs 6–9, 10–13, 14–17, and neurologically normal human frontal cortex lysate (positive control) were run on 4–12% gels, as described above, and immunoblotted for CD9, CD63, CD81, flotillin, TSG101, annexin V (AnnV), synaptophysin (SYP), NCAM-1, GLAST, CD11b, and TMEM119 proteins. In addition, gels loaded with 1.0 µg of void, Fxs 6–9, 10–13, 14–17, and human frontal cortex were immunoblotted for SYP and CD11b. Following electrophoresis, proteins were transferred to PVDF membranes and kept overnight at 4°C (30 V constant current). Membranes were incubated for 1 h with a blocking buffer (5% non-fat milk in TBS with 0.05% Tween 20 (TBST)) and then incubated with primary antibody diluted with TBST overnight at 4°C, and for 1 h with secondary antibody  at room temperature. Membranes were developed using the West Dura, Pico, or Femto chemiluminescence kits (Thermo Fisher Scientific) and imaged using the ChemiDoc Touch imaging system . The following antibodies were used for immunoblotting: albumin 1:1,000 , APOA1 (12C8) 1:200 , APOE 1:2000 , AnnV 1:5,000 , CD9 (C-4) 1:200 , CD11b 1:1,000 , CD63 1:1,000 , CD81 (B-11) 1:100 , flotillin 1:10,000 , GLAST 1:500 , NCAM-1 1:125 , SYP 1:1,000 , TMEM119 , and TSG101 1:1,000 . Secondary antibodies were purchased from Jackson ImmunoResearch  and included horseradish peroxidase (HRP)–conjugated donkey anti-mouse (#715-035-150), donkey anti-rabbit (#711-035-152), and donkey anti-goat (#705-035-003).Pools of the void volume (Fxs 1–5) and Fxs 6–9 were analyzed for particle size and concentration by TRPS using a qNANO gold instrument fitted with a NP80 nanopore (IZON Science). Prior to TRPS, 800 µL SEC pools were subjected to UF using Microcon®-30 kDa centrifugal filters and then brought to a final volume of 50 µL in 2x PBS . TRPS mePools of CSF SEC fractions including the void, Fxs 6–9, 10–13, and 14–17 were concentrated by UF to a final volume of 30 μL and then stored on wet ice until processing for TEM within 6 h  by the OEVs were isolated from 500 μL of CSF that was first concentrated by UF to 150 μL then fractionated by SEC . Fxs 6–92) amplifications with a Cq ≤ 34 and an AmpScore <1.0 or a CqConf <0.8 were excluded from analysis. Based on these criteria, miRNAs with a Cq ≤ 34, AmpScore >1.0, and CqConf >0.8 were deemed considered for further analysis. Next, we excluded miRNAs that were not expressed in at least 20% of the samples (11 out of 56), which would enable analysis of miRNAs differentially detected in one out of the eight subgroups . We also performed qPCR reactions with water only (no RNA—no template control) as a control for spurious PCR amplifications  based on Applied Biosystems recommendations . Cq values were calculated using automatic baseline, and threshold values were determined by ExpressionSuite Software v.1.3 (Thermo Fisher Scientific). The Cq value for each well was reported along with the amplification score (AmpScore) and a Cq confidence (CqConf), which are metrics for the quality of each amplification, as we have previously reported . Prior tications . MiRNAs t-test with a Benjamini–Hochberg false discovery rate (B-H FDR) of 0.20, a cutoff that is in line with a hypothesis-generating study, and the preference for some false positive findings in exchange for a larger list of potentially interesting true findings  followed by a Tukey’s multiple comparisons post hoc tests (n = 7/group). Given the small number of participants in this study, a covariate analysis of disease by sex by APOE genotype was not conducted. Thus, conclusions drawn from the analysis assume no interaction between all three variables.Data were analyzed with GraphPad Prism software v9.3.1 . Data are shown as either a Volcano plot  vs. the Log2(Fold change)) or the ΔCq mean ± standard error mean (SEM). Where possible the biological replicates are displayed as individual symbols. To assess the effects of AD on miRNA levels, participants were categorized as either AD (n = 28) or CTL (n = 28) and analyzed by Welch’s unpaired findings . To asseWe used TargetScan 7.2  and miRDevtrack.org) knowledgebase , a position study from the International Society of Extracellular Vesicles (ISEV) . The vFCedgebase . = 139.8, p < 0.0001, but no significant interactions with disease state and sex and/or genotype.CTL CSF was pooled and used for vFC, immunoblot, TRPS, and TEM experiments, while individual CSF samples from CTL and AD participants were used for the miRNA qPCR assays. Characteristics for the 28 AD and 28 CTL participants in this study  are shown in To measure the size and concentration of EVs in total CSF, we performed single vFC, which uses sensitive measurements of light scatter from a fluorescent lipid probe (vFRed) and surface immunofluorescence from staining with a pool of phycoerythrin (PE)-conjugated TS antibodies . In our prior studies on plasma, we diluted the sample 150-fold prior to staining to enable detection of TS + membranous particles in the diameter range of ∼75–600 nm at mean concentration of 1.3e10/mL . HoweverTo isolate EVs from human CSF, we first performed a series of SEC protocol refinement experiments. Using CSF pooled from multiple CTL samples, we tested 35 and 70 nm SEC columns for EV enrichment as indexed by the presence of established EV protein markers  and deplWe further characterized CSF fractionated using the 35 nm SEC columns by TEM and immunoblots . Figure Considering that extracellular miRNAs are associated with exosomes, MVs, apoptotic bodies, RNA binding proteins, and high-density lipoproteins , we sougp < 0.05 but were not considered significant based on the FDR  predictto 0.813 . In addito 0.813 . Aside fto 0.813 . TogetheExtracellular miRNAs have multiple carrier types including exosomes, exomeres, supermeres, MVs, apoptotic bodies, RNA-binding proteins, and high-density lipoproteins . In AD, Prior studies that investigated the miRNA cargo of CSF EVs in AD used precipitation, ultracentrifugation, or density gradient ultracentrifugation-based methods to fractionate CSF . EV prec42 treatment , with five being unique to Fxs 6–9 . In comparison, we identified very few miRNAs that were unique to the fractions that contained a majority of the vesicle-free lipoproteins and proteins (Fxs 10–13 and 14–17). However, we did observe 14 miRNAs that were in common between all three pooled fractions, including five AD CSF biomarkers . Also, there were two AD biomarkers detected only in the vesicle-free lipoprotein and protein fractions (miR-19b-3p and 193a-5p). Note that some miRNAs included in the AD, sex, and APOE analyses were excluded from this analysis  because of differential detection in AD only and not expressed in at least two of the four CTL samples used in this experiment. Together, these data demonstrate that SEC fractions, which contain EVs and small nanoparticles, also contain a majority of the miRNAs that are informative about disease state. However, miRNAs carried by lipoproteins and RNA-binding proteins also have biomarker potential. Thus, more comprehensive studies are needed to fully elucidate the effects of AD on miRNA transport in CSF.Considering that extracellular RNAs can be transported by EVs, exomeres, supermeres, lipoproteins, and RNA-binding proteins  and thatach pool . The larOur analysis of CSF EV miRNA expression between AD and CTL participants identified four miRNAs that were significantly increased in AD . We had Our target prediction analysis  found thvia ligand bound nuclear hormone receptors that bind and recruit co-activators and–repressors to gene promoter elements. In rodents, 17β-estradiol differentially regulates miRNA expression in an age-dependent manner in the hippocampus (APOE-e4 has been shown to disrupt exosome biogenesis and decreases the production of brain EVs in aged non-AD human brain and a humanized APOE-e4 mouse model . Furtherpocampus . Furtherpocampus . While tpocampus , and we In summary, our studies identified that AD and two of its risk factors, sex and APOE-e4 status, impact CSF EV miRNA levels. Importantly, APOE-e4 status within each sex-altered different subsets of miRNAs. Furthermore, the miRNAs differentially expressed in AD CSF EVs predicted gene targets and pathways that are known to contribute to neurodegeneration in AD. Together, these data demonstrate the need to perform large scale studies that take into consideration key biological factors in addition to the disease state in order to more precisely define biomarker profiles for the at-risk population. Our data also demonstrate that EV miRNAs could facilitate the identification of new targets for the therapeutic treatment of AD and define how EV-derived miRNAs may render females and/or APOE-e4 carriers more prone to AD.


Chitin nanocrystals (ChNCs) and cellulose nanocrystals (CNCs) have been recently used to stabilize emulsions; however, they generally require significant amounts of salt, limiting their applicability in food products. In this study, we developed nanoconjugates by mixing positively charged ChNCs and negatively charged CNCs at various ChNC:CNC mass ratios , and utilized them in stabilizing soybean oil–water Pickering emulsions with minimal use of NaCl salt (20 mM) and nanoparticle (NP) concentrations below 1 wt%. The nanoconjugates stabilized the emulsions better than individual CNC or ChNC in terms of a reduced drop growth and less creaming. Oppositely charged CNC and ChNC neutralized each other when their mass ratio was 1:1, leading to significant flocculation in the absence of salt at pH 6. Raman spectroscopy provided evidence for electrostatic interactions between the ChNCs and CNCs, and generated maps suggesting an assembly of ChNC bundles of micron-scale lengths intercalated by similar-size areas predominantly composed of CNC. The previous measurements, in combination with contact angles on nanoparticle films, suggested that the conjugates preferentially exposed the hydrophobic crystalline planes of CNCs and ChNCs at a 1:1 mass ratio, which was also the best ratio at stabilizing soybean oil–water Pickering emulsions. Emulsions, thermodynamically unstable heterogeneous mixtures of immiscible liquids, are a form of advanced materials that require addition of a stabilizer to prevent a phase separation induced by the fusion of droplets during storage. Surfactants and nano- or micro-particles are commonly used as stabilizers to prepare these emulsions as they stabilize the liquid/liquid interface. Although both surfactants and nanoparticles (NPs) adsorb to the interface, the mechanisms by which they stabilize emulsions vary significantly; however, adsorption to the interface can be considered a prerequisite for stabilization ,2,3,4. ICellulose and chitin are both abundant biopolymers, which have gained interest in the last few decades to develop advanced materials due to their unique physical and chemical properties along with their associated low cost, and low toxicity to humans and the environment ,7,8,9. CChitin-based NPs have been extensively studied as Pickering emulsion stabilizers for food applications ,15,16,17Huan et al. studied the oil/water emulsion stability by combining positively charged nanochitin (NCh) and negatively charged cellulose nanofibers (CNF) to prepare stable emulsion systems  and founThere are few reports focusing on the combination of CNC and ChNC to produce stable emulsions. Though oppositely charged chitin and cellulose nanofibers (CNF) have been reported in two studies ,27, the Commercially available CNCs were purchased from CelluForce Inc.  as a 6 wt% aqueous dispersion (NCV100-NAL90). The manufacturer synthesizes CNCs from the wood pulp by sulfuric acid hydrolysis. Chitin powder (shrimp shell), hydrochloric acid, sodium hydroxide (10 N), soybean oil (>99.5 purity), and n-heptane (>99.9% purity) were purchased from Fisher Scientific  and were used as received.Chitin nanocrystals (ChNC) were synthesized using procedures described elsewhere ,32. BrieAtomic force microscopy (AFM) in tapping mode  was used to obtain the topographic images of CNC and ChNC using a Bruker Di Innova microscope from Bruker . A drop of diluted CNC or ChNC suspension (0.0002 wt%) was deposited onto a freshly cleaved mica surface  and dried overnight in a desiccator. The images were processed using Gwyddion software to measure the dimensions of both NPs. A minimum of 175 rod-shaped individual nanocrystals were measured to obtain the size distributions.Conductometric titrations were completed to estimate the charge on ChNC and CNC surfaces. In the case of CNCs, the nanoparticles were first dialyzed against hydrochloric acid (1.0 M) to remove sodium counterions and protonate the superficial sulfate groups. The surrounding medium was replaced daily until constant pH. The protonated CNCs were then dialyzed against DI water to remove excess acid, following the same method. The resulting CNC dispersion had a pH of 3.4; then, 50 mL of 0.2 wt% CNC was titrated with 0.05 M NaOH while measuring the pH and electrical conductivity ,34. In tThe ζ-potential and mean hydrodynamic diameter of ChNC and CNC were measured as a function of pH using a Malvern ZetaSizer Nano ZS  for diameters lower than 300 nm. Larger aggregate sizes were measured using a laser diffraction particle size analyzer, Horiba Partica LA-960V2 . The ζ-potential and size of ChNC-CNC nanoconjugates were also measured for ChNC:CNC mass ratios of 3:0, 2:1, 1:1, 1:2, and 0:3, where 3:0 represents pure chitin and 0:3 represents pure CNC, while keeping the total NP concentration and the pH constant at 0.2 wt% and 6.0, respectively.Powder X-ray diffractograms (XRD) were collected using Malvern Panalytical Empyrean Nano Edition . Individual ChNC and CNC along with their mixtures were freeze-dried, loaded in a sample holder, and exposed to X-ray scans from 2θ angles of 5° to 60° at a step time of 0.6 s/step (0.003°/step) and sample revolution of 4 rev/min at 25 °C with Cu Kα radiation (λ = 0.1540 nm).Raman spectra and images were obtained using Horiba LabRam HR-Evolution  equipped with 600 grooves/mm grating, 532 nm laser excitation, CCD camera detection, and an MPlan N 100×/0.9 objective from Olympus . Raman maps were obtained with a 10 nm step size. The spectra were collected using an acquisition time of 1 s and 500 accumulations to improve the signal-to-noise ratio. Individual ChNC, CNC, and ChNC-CNC conjugates dispersions in DI water were drop-casted on a freshly cleaved mica surface and dried under ambient conditions prior to obtaining their Raman spectra. The spectra of different ChNC-CNC nanoconjugates were examined for spectral signatures to examine probable interactions between ChNC and CNC.ow) between soybean oil and aqueous phases containing ChNC, CNC, and ChNC-CNC nanoconjugates was measured under ambient conditions using a Kruss Scientific DSA100S  optical tensiometer as a function of pH and NP mass ratio. A pendant droplet of a 20 mM NaCl solution was formed on the tip of the needle submerged in soybean oil, while measuring γow for 60 min. Contact angles were measured using the sessile drop method to describe the macroscopic wettability of NP films (drop-casted and air-dried) submerged in soybean oil. The static equilibrium contact angle was measured after allowing the sessile drop of a 20 mM NaCl solution to equilibrate on NP films for 200 s.The equilibrium interfacial tension . At high ζ-potential values (>±30 mV), one would expect a relatively small particle size; however, as evidenced by the cloudiness of the dispersion in −1 and a peak at 955 cm−1  corresponding to carbonyl vibration bands in ChNC, and 1050–1150 cm−1 (blue regions) from skeletal bending and stretching, as well as glycosidic O-C-O bond vibrations [A Raman signal map is shown in brations . The grellomorph ,45. Inteα-chitin ,47,48,49ow between soybean oil and a 20 mM NaCl solution with dispersed nanocrystals under ambient conditions. Additionally, the three-phase contact angles (θ) of 20 mM NaCl solutions were measured on NP films submerged in soybean oil. ow between soybean oil and the aqueous phase, suggesting that both nanoparticles used in the study showed surface-active properties. It should be noted, however, that while there was a decrease in γow between the oil and aqueous phases with increasing NP concentration, the reduction was minimal (~3 mN/m in the case of CNC and ~4 mN/m for ChNC), similar to other systems containing unmodified nanocelluloses, as described in the recent literature [ow, whereas increasing the CNC concentration up to 2 wt% resulted in a monotonous reduction. The more significant γow reduction for ChNCs at low concentration was likely caused by their higher hydrophobicity (compared to CNC) arising from their superficial acetamido groups [The surface properties of CNC, ChNC, and ChNC-CNC nanoconjugates were studied in terms of the γo groups ,14.ow was also measured at a constant total NP concentration of 0.2 wt% for different ChNC-CNC nanoconjugates , all mass ratios resulted in approximately equal γow—within the error of the measurement—indicating that the conjugates retained the enhanced hydrophobicity of the ChNCs. Nevertheless, the effect of nanoconjugate addition on γow was too small to have a significant effect on emulsion stability.The effect of different mass ratios of nanoparticles on the γnjugates b. IncreaA different stabilization mechanism is expected if the NP adsorption energy is high, which is typically evidenced by contact angles close to 90° ,4,5,50. e planes ,29,30, we planes ,52. If tof ChNCs ,52. FutuThe contact angle measurements in Cellulose and chitin nanocrystals were used to stabilize emulsions containing 20 wt% soybean oil. A preliminary screening of emulsion creaming over 24 h is shown in the A more detailed coalescence and creaming study was performed to establish the effect of ChNC:CNC mass ratio on emulsion stability . For thiFor the emulsion stability studies, the salt concentration was maintained at just 20 mM, and the total nanoconjugate concentration was 0.8 wt%. Such a low salt concentration is adequate for food-grade emulsions, while the amount of nanocrystals is maintained as low as possible to lower the potential impact on the cost of a commercial product. After two and four weeks, the emulsions formed using CNC alone (0:3) were clearly unstable to the eye, and with significant coalescence of the remaining droplets with sizes growing from 26.16 µm σ = 23.57 µm) to 180.19 µm (σ = 213.35 µm) over 4 weeks. Emulsions prepared using ChNC (3:0) alone were comparatively more stable to coalescence, albeit with significant creaming. In that case, the drop size increment was from 40.21 µm (σ = 28.45 µm) to 63.78 µm (σ = 34.63 µm) in the same timeframe. The highest stability was found for samples containing both CNCs and ChNCs. There was very little change in the droplet size of the emulsions prepared with ChNC-CNC nanoconjugates at 1:1, and 2:1 over a 4-week storage. The average size of their emulsion drops only grew from 55.20 µm (σ = 31.64 µm) to 65.72 µm (σ = 26.90 µm) and 62.12 µm (σ = 37.52 µm) to 74.71 µm (σ = 40.84 µm) respectively. However, the 1:2 sample did coalesce significantly, with the drops growing from 67.91 µm (σ = 61.70) to 113.43 µm (σ = 63.73), even though the emulsion appeared to remain stable after enough coalescence occurred, presumably following an arrested coalescence behavior. These results appeared to be consistent with the enhanced contact angle of the mixed samples compared to pure ChNC or CNC, as shown in 7 µm to 1The structural orientation of nanoparticles, at the surface of emulsion droplets, was studied using SEM images of 1:1 emulsion samples dried using critical-point drying (CPD), leaving behind ChNC:CNC crusts deposited on TEM grids . HoweverThe morphology, interactions, and ability to stabilize emulsions of rod-like ChNC and CNC nanoconjugates (ChNC/CNC) were studied at various chitin-to-CNC ratios. Measurements of ζ-potential showed that CNCs were negatively charged in a wide range of pH (3 to 10), while ChNCs had an isoelectric point at around pH 8, below which ChNCs were positively charged. At pH 6, the ChNC:CNC conjugates with a mass ratio of 1:1 were electroneutral, resulting in aggregation. Significant aggregation was also observed for the 2:1 and 1:2 mass ratios despite ζ-potentials of 21 ± 5 mV and −33.3 ± 1 mV, respectively.The interactions between ChNC and CNC at pH 6 were analyzed by Raman spectroscopy, providing direct evidence for the interaction between negatively charged sulfate groups on the CNC surface and the positive ammonium groups on the ChNCs. In addition, Raman signal maps of ChNC and CNC characteristic vibrations in a 1:1 mass ratio film revealed that both NPs distributed in alternated, micron-long regions, of irregular but elongated shape. Contact angle (θ) measurements on multiple ChNC/CNC films indicated a maximum θ of 80.6 ± 0.8° at a 1:1 mass ratio, providing evidence for a preferential exposure of the nanocrystals’ hydrophobic planes when both NPs were combined. Such a high contact angle suggested that the optimal mass ratio for stabilizing soybean oil–water Pickering emulsions was 1:1, which was further confirmed by emulsion stability studies.By mixing ChNC and CNC at a 1:1 mass ratio, it was possible to produce highly stable soybean oil-in-water emulsions at a salt concentration of only 20 mM NaCl and a total NP concentration of 0.8 wt%. At these conditions, neither the CNCs nor ChNCs on their own could provide long-term stability, yet the nanoconjugates provided highly stable emulsions with emulsion drop sizes growing only 10 μm over a period of 4 weeks. The work herein presented suggest that nanoconjugates of ChNC and CNC have significant potential to provide enhanced long-term stability to food-grade emulsions in a variety of products that may include mayonnaise, salad dressings, nutritional drinks, sauces, and more.


Humulus lupulus L. (hops) is an important medicinal plant widely used in the food, beverage and pharmaceutical industries due to its strong antioxidant ability. In this study, APP/PS1 mutated transgenic mice and Aβ-injured osteoblasts were used to evaluate the protective effects of hops extracts (HLE) on SOP. Mice learning and memory levels were assessed by the Morris water maze. Mice femurs were prepared for bone micro-structures and immunohistochemistry experiments. The deposition of Aβ in the hippocampus, cortex and femurs were determined by Congo red staining. Moreover, protein expressions related to antioxidant pathways were evaluated by Western blotting. It was found that HLE markedly improved learning abilities and ameliorated memory impairment of APP/PS1 mice, as well as regulated antioxidant enzymes and bone metabolism proteins in mice serum. Micro-CT tests indicated that HLE enhanced BMD and improved micro-architectural parameters of mice femur. More importantly, it was discovered that HLE significantly reduced Aβ deposition both in the brain and femur. Further in vitro results showed HLE increased the bone mineralization nodule and reduced the ROS level of Aβ-injured osteoblasts. Additionally, HLE increased the expression of antioxidant related proteins Nrf2, HO-1, NQO1, FoxO1 and SOD-2. These results indicated that Humulus lupulus L. extract could protect against senior osteoporosis through inhibiting Aβ deposition and oxidative stress, which provides a reference for the clinical application of hops in the prevention and treatment of SOP.As aging progresses,  However, these are all relatively unclear pathogeneses of SOP. Current clinical drug treatments for SOP mainly include estrogen therapy, bisphosphonates supplementation, as well as calcium and active vitamin D, which cause more side effects and lack a clear target. Therefore, there is a desperate need to elucidate the pathogenesis of SOP and to find appropriate alternative drugs for SOP with few adverse effects.Senior osteoporosis (SOP) is a type of metabolic disease characterized by osteopenia, bone micro-structure degeneration and fracture. Aging is a major pathogenic factor causing SOP. Along with aging, the body will induce oxidative stress through releasing excessive reactive oxygen species (ROS), further decreasing bone formation in osteoblasts and increasing bone resorption in osteoclasts, eventually leading to a bone homeostasis imbalance . AccordiHumulus lupulus L. (hops) is an important medicinal plant widely used in the food, beverage and pharmaceutical industries due to its strong antioxidant capacity [β-induced oxidative damage and bone loss in APP/PS1 mice and osteoblasts [β deposition and anti-SOP properties.capacity , and hascapacity . In addicapacity ,8. It hacapacity ,10,11. Ieoblasts ,13. Thesβ aggregation and deposition in the cerebrum is a significant pathological feature in AD patients. Aβ deposition can cause neurotoxicity and oxidative stress, which leads to wide neurodegeneration [β not only exists in the cerebrum, but also in the bone [β42 is often abnormally elevated in osteoporosis patients [β deposition in femurs, which affect the activities of osteoblasts and osteoclasts [β deposition accompanied with peroxidation injury, and antioxidants could improve the cognitive ability and bone loss caused by this Aβ deposition and oxidative damage [β deposition and oxidative damage.Aneration . More rethe bone , and Aβ4patients . In addieoclasts . More ime damage . It is tβ deposition, and probed its potential mechanism using Aβ-injured osteoblasts, which can provide more references for the prevention and treatment of SOP.In view of the strong antioxidant effect of hops, the present study employed APP/PS1 mice to investigate the effect of hops on memory deficit and bone loss induced by Ap < 0.001), and the mice in treatment groups had a lower latency to load upon the platform compared with that of APP/PS1 mice during the last two training days (p < 0.05). On the probe test day, the swimming time spent in the target zone is shown in p < 0.05). APP/PS1 mice swam for less time in the target zone than the wild-type mice, and the mice treated with HLE or N-acetyl-L-cysteine  spent an increased amount of time in the target zone. Moreover, as shown in p < 0.001), while treatment with HLE (2 g/kg) or NAC significantly increased the number of platform crossings (p < 0.05). The swimming tracking showed that APP/PS1 mice mostly swam in the quadrant far from the target quadrant, while mice in wild-type group and other treatment groups mostly swam near the platform (p > 0.05). These results suggested that HLE had a good effect on improving the learning and memory abilities and could prevent spatial memory deficit of APP/PS1 mice.The Morris water maze (MWM) task was conducted to estimate whether hops extract could improve the long-term spatial memory of APP/PS1 mice. After 2 months administration, 11-month-old mice were prepared for the MWM task. The MWM task included two parts: task acquisition (days 1–5) and probe trial (day 6). During the experiment, the tank was videotaped from above, and all the relevant data were recorded. Detailed experimental steps are shown in “platform E. Howeveβ deposition, we applied Congo red strain to observe the pathological changes in Aβ plaque in the brain and femur. The brownish red or orange precipitates were positive for Aβ . HLE or NAC markedly decreased the amount of Aβ plaque in hippocampi and cortexes of APP/PS1 mice (p < 0.001). Moreover, we observed more Aβ deposition in the femurs of APP/PS1 mice than in wild-type mice (p < 0.01), while HLE or NAC could ameliorate the accumulation of Aβ in femurs of APP/PS1 mice (p < 0.01) (β deposition both in the brains and bones of APP/PS1 mice.To determine the effect of HLE on Ae for Aβ A. In the < 0.01) B–D. Thesp < 0.01), while HLE or NAC could reverse this decrease and enhance the BMD (p < 0.05). The bone volume fraction (BVF) indicates the ratio of bone volume to total volume. p < 0.01), while HLE or NAC could markedly increase the femur BVF in APP/PS1 mice (p < 0.05). In addition, as shown in Micro-CT was conducted to observe the bone structural properties of trabecular bone and BMD in femurs. As shown in the micro-CT images A, there p < 0.001). Moreover, osteocalcin (OCN), which plays an important role in regulating bone metabolism, was activated less in APP/PS1 mice (4.237 ± 0.41 ng/mL) than that in wild-type mice (11.250 ± 0.43 ng/mL) (p < 0.001). HLE or NAC could increase the SOD and OCN levels of APP/PS1 mice, respectively (p < 0.001), and there were no significant differences between the two dose groups. Inflammatory cytokines interleukin-1β (IL-1β) and interleukin-6 (IL-6) levels in the serum of APP/PS1 mice significantly increased from 5.028 ± 0.46 ng/L and 35.51 ± 1.18 ng/L in wild-type mice to 19.42 ± 0.68 ng/L and 51.70 ± 2.27 ng/L (p < 0.001), respectively. After treatment, HLE or NAC reversed the high level of IL-1β and IL-6 in the serum of APP/PS1 mice to almost ordinary levels (p < 0.001).As shown in p < 0.01). High doses of HLE significantly improved this inhibition (p < 0.01), indicating that endogenous Aβ reduced OPG content, and HLE might play a bone protective role by promoting the expression of OPG. As shown in p < 0.01), while high doses of HLE could return it to a normal level (p < 0.01), suggesting that HLE might alleviate oxidative stress through regulating Nrf2 and FoxO1 pathways.The bone metabolism index osteoprotegerin (OPG) and oxidative stress indexes nuclear factor erythroid 2-related factor 2 (Nrf2), forkhead box O1 (FoxO1) and SOD-2 in mice femurs were measured by immunohistochemistry. As shown in β deposition, osteoblasts were injured by Aβ for in vitro study. Bone mineralization levels were measured by Alizarin red staining. As shown in β-injured osteoblasts, proving that hops could promote bone mineralization and bone formation. As shown in β significantly improved the ROS release in osteoblasts, while HLE markedly reduced the ROS level in a dose-dependent manner (p < 0.01). In addition, oxidative stress related Nrf2 and FoxO1 pathways were measured by Western blotting. As shown in β markedly reduced the expression of Nrf2, heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO1), FoxO1 and SOD-2 compared to the control group. After treatment, HLE or NAC significantly reversed the decreased expression of Nrf2, HO-1, NQO1, FoxO1 and SOD-2 in Aβ-injured osteoblasts, indicating that HLE could alleviate oxidative stress caused by Aβ deposition through activating Nrf2 and FoxO1 pathways.To further verify the effect of hops on alleviating oxidative stress and promoting bone formation through inhibiting Aβ deposition in both the brain and bone. In vivo and in vitro experiments revealed hops could relieve Aβ-induced oxidative stress and protect against SOP.Osteoporosis is a degenerative chronic disease closely related to aging. It is crucial to fully recognize the potential risks and benefits of diagnosing and treating osteoporosis in elderly people. The high consumption of beer all over the world leads us to focus on hops plants that are essential in beer brewing, and our previous studies have detailed the potent effects of hops on relieving oxidative stress and alleviating bone loss. However, details of the mechanism of hops on preventing senior osteoporosis have not been clarified. In this study, it was determined for the first time that hops improved the learning ability and alleviated bone loss of APP/PS1 mice through inhibiting Aβ deposition and the dysfunction of antioxidant system play an important role in the pathogenesis of SOP and AD [β plaques with earlier onset than the single transgenic sample [β directly or indirectly modulates mitochondrial function and induces oxidative stress, which in turn enhances Aβ synthesis and aggregation. In this study, APP/PS1 mutated transgenic mice were used to mimic the SOP condition, and we found that there was Aβ deposition not only in the brain but also in the femurs of APP/PS1 mice, which was an important trigger of memory impairment and osteoporosis. After treatment, it was discovered that hops could both alleviate memory impairment and attenuate bone loss. More importantly, the Aβ amount in mice hippocampi, cortexes and femurs were markedly decreased after treatment with HLE, proving that hops could alleviate senior osteoporosis and dementia in APP/PS1 mice through removing Aβ deposition.In agreement with the clinical and epidemiological evidence, it has been found that AP and AD . ExtraceP and AD . In micec sample . Aβ direβ deposition led to bone loss. Hops could improve the micro-architecture, enhance the BMD, and increase the trabecular parameters in the femurs of APP/PS1 mice, suggesting that hops was effective in both preserving bone mass and rescuing the deterioration of bone micro-architecture when damaged by Aβ deposition. During bone remodeling, osteoblastic bone formation and osteoclastic bone resorption are a synergistic action. OCN and OPG are important enzymes characterized by the capacity of osteoblastic bone formation [β and IL-6 in serum were associated with increased ROS and osteoclastic bone resorption [β and IL-6 not only directly stimulate osteoclastogenesis and bone resorption but also stimulate RANKL production in osteoblastic cells in a synergistic fashion. In this study, hops can significantly increase the OCN and OPG expression and inhibit the serum IL-1β and IL-6 levels in APP/PS1 mice, indicating the potential of hops in maintaining bone homeostasis.The histomorphometric parameters of the trabecular bone obtained from micro-CT analysis can predict osteopenia and deterioration of bone quality, and BMD is known as the golden indicator for diagnosing osteoporosis . The preormation , while tsorption . IL-1β aβ deposition could interfere with the mitochondria, which causes cells to stop breathing and subsequently oxidative stress [β protein plaque and overexpression of hyperphosphorylated tau protein in neurons [β plaque. Conversely, Mn-SOD was overexpressed in APP/PS1 mice and they exhibited an increased antioxidant defense capability in the brain and a reduced level of Aβ plaque [β-injured osteoblasts. Fortunately, hops could increase the SOD level and reduce active oxygen release, thus relieving oxidative stress.Ae stress ,26. Stude stress . On the  neurons , all of β plaque . In our β-injured osteoblasts, suggesting that hops might alleviate oxidative stress and SOP through regulating Nrf2 and FoxO1 pathways.As a key redox-sensitive transcription factor, Nrf2 is conducive to maintaining cellular redox homeostasis and improving oxidative injury. Expressions of antioxidant enzymes SOD and CAT are intimately related with Nrf2 signaling activation . The by-β-induced senior osteoporosis primarily by ameliorating memory impairment, enhancing BMD and trabecular bone structure and improving osteoblastic MC3T3-E1 cell activities in APP/PS1 mutated transgenic mice. As for its mechanism, removing Aβ deposition in the brain and bone and its inducing of oxidative stress plays a crucial role.Collectively, we have, for the first time, demonstrated that hops extract protects against AHumulus lupulus L. was obtained from the Anguo Traditional Chinese medicine market  and identified through microscope identification and HPLC to test its quality  kits for determination of OCN, IL-1w/v) chloral hydrate. Serums were centrifuged and stored at −80 °C for biochemical assay, and femurs were prepared for micro-CT and immunohistochemistry experiments. All studies were conducted in accordance with the NIH publication and approved by the Committee on Ethics of Medical Research Second Military Medical University.Male APP/PS1 mutated transgenic mice  were obtained from the Nanjing Biomedical Research Institute of Nanjing University , and housed four per cage, maintained under constant temperature (23 ± 1 °C) and humidity (60 ± 10%) under a 12 h light/dark cycle (light from 7:30 am to 7:30 pm). Mice were freely provided with water and food and divided equally into five groups of ten mice, namely CON, APP/PS1, APP/PS1 + NAC (100 mg/kg/d), APP/PS1 + HLE-L (1 g/kg/d) and APP/PS1 + HLE-H (2 g/kg/d). The selection of the drug doses in the experiment was based on our previous study [The Morris water maze task was employed to test spatial learning and memory ability of mice in this study . The tasβ plaque, as described previously [After mice were sacrificed, the whole brain and right femurs were picked and fixed in 4% paraformaldehyde, then placed in 30% sucrose solution until they sunk to the bottom. The tissue was continuously cut, and every slice  was stained with Congo red to determine the total amount of Aeviously . In the Mice femur micro-architecture was measured with a micro-CT scanner (GE eXplore Locus SP). BMD and trabecular bone parameters of BVF, Tb.N., Tb.Th. and Tb.Sp. were auto calculated by the computer.β and IL-6 levels were measured by commercially available assay kits in accordance with the manufacturer’s instructions.Mice serum was centrifuged at 3000 r/min for 10 min, and the supernatant was collected for the biochemical markers assay. The SOD, OCN, IL-1The femur section was separated for immunohistochemistry by xylene and hydrated in ethanol of graded concentrations. The cross-section was bathed in sodium citrate buffer (pH = 6.0) and heated. Then, the section was incubated in 3% hydrogen peroxide for 25 min and 3% BSA solution for 30 min, successively. After that, the sections were incubated with primary and secondary antibodies at 4 °C, successively. Finally, slides were incubated by a DAB staining kit and stained with hematoxylin-eosin, and the positive section was stained brown-yellow. The coverage of positive staining was calculated with Image-pro plus software.2 at 37 °C. When reaching 80% confluence, osteoblasts could be used for follow-up studies. According to the experiment design, osteoblasts were incubated overnight and then pretreated with different concentration of HLE  or NAC (2.5 mM) for 4 h. After that, the plate was removed from the incubator and 1 μL of 1 mM Aβ1–42 oligomer mother liquor was added to each well, resulting in a final concentration of 10 μM Aβ. Osteoblasts were incubated for another 44 h, and cell growth in the orifice without the treatment was used as the control group.Osteoblastic MC3T3-E1 cells were purchased from the typical Culture Committee Cell Library of the Chinese Academy of Sciences, Shanghai, China, and cultured in DMEM containing 10% FBS in a humidified atmosphere of 5% CO4 cells/well) and then cultured with osteogenic differentiation medium  for 18 days. Then, cells were cultured in HLE or NAC, which contained 10 μM Aβ, for another 2 days. Osteoblasts were fixed in ice-cold 4% paraformaldehyde for 10 min, and then dyed with 0.1% Alizarin red solution at 37 °C for 30 min. After washing, osteoblasts were completely dissolved in 10% cetylpyridinium chloride for 15 min, and then measured at 570 nm.The cultured osteoblasts were added to 24-well plates overnight  reagents and imaged using the Gel imaging system. For protein bands with similar KD values, we used the removal solution to remove the previous protein imprint, and then carried out another incubation for other antibodies.Osteoblasts were seeded in 6-well plates with a density of 1 × 10p < 0.05 and determined by one-way analysis of variance and the Student–Newman–Keuls test for multiple comparisons. GraphPad Prism (version 5.0) was used for statistical analysis.All data were expressed as mean ± standard error of mean (SEM). Statistical significance was set at


Zea mays L.) seed germination under lead (Pb) stress, we comparatively analyzed the effects of applying different concentrations of iminodisuccinic acid  and combined an addition of exogenous substances regulating reactive oxygen species production on maize seed germination, seedling growth, H2O2 content, NADPH oxidase activity, and antioxidant enzyme activities under Pb-stressed and Pb-free conditions. Iminodisuccinic acid (100 mmol·dm−3) significantly delayed seed germination under normal germination conditions and alleviated the inhibitory effects of Pb stress (20 mmol·dm−3) on seed germination. Under normal conditions (without Pb stress), the iminodisuccinic acid-induced inhibition of seed germination was enhanced by treatment with dimethylthiourea (a specific scavenger of reactive oxygen species) or diphenyleneiodonium chloride (a specific inhibitor of NADPH oxidase), but diminished by treatment with H2O2, CaCl2, diethyldithiocarbamic acid (a specific inhibitor of superoxide dismutase), or aminotriazole . Under Pb stress, iminodisuccinic acid partially eliminated the excessive H2O2 accumulation, improved superoxide dismutase and catalase activity, and weakened the high NADPH oxidase activity. In addition, Ca2+ chelation may be essential for maintaining the reactive oxygen species’ balance and improving seed germination and seedling growth by iminodisuccinic acid supplementation in maize under Pb stress. The proposed iminodisuccinic acid supplementation-based method improved maize seed germination in Pb-polluted soil.To explore the effects of iminodisuccinic acid (a chelating agent) on maize ( In recent decades, rapid increases in urbanization and industrialization have caused the excessive release of heavy metals in farmlands, which has led to ecosystem damage ,2,3. HeaZea mays L.) exhibits the advantages of high biomass production and metal tolerant capacity; thus, it has been widely adopted for the phytomanagement of low- and medium-grade metal-contaminated soils [Studies have shown that using high-accumulating crops , such as nonfood products and dedicated bioenergy crops, may be a feasible strategy for the phytoremediation of heavy metal-polluted soils ,20. Amoned soils ,22,23. Med soils ,25,26,27ed soils . Therefoed soils .Lead (Pb) is a highly toxic and commonly occurring heavy metal contaminant in the environment ,31. Of tDirect seeding technology is widely used in mechanized maize production ,47. PhytHence, the objective of this study was twofold: to analyze the effects of IDS on seed germination and seedling growth under Pb-stressed and Pb-free conditions and to explore the regulatory mechanism underlying the effects of IDS on the seed germination process in terms of ROS balance to provide theoretical and technical support to facilitate the efficient implementation of phytoremediation projects for contaminated soil treatment.−3 IDS significantly delayed seed germination. Compared with the germination rate (GR) of seeds in the distilled water (control) treatment group, the GR of seeds treated with 100 mmol·dm−3 IDS was approximately 58%, 62%, 52%, and 9% less at 1, 2, 3, and 4 d after sowing, respectively (p < 0.05). However, 5 d after sowing, significant differences were not observed in the final GR of seeds across the IDS treatment groups.In the absence of heavy metal stress , an incr−3 PbCl2), the IDS treatment effectively alleviated the inhibitory effect of Pb stress on seed germination in maize . Similar changes were observed for maize seed germination under Pb stress in groups treated with 5 and 20 mmol·dm−3 IDS (p < 0.05).Although the final GR of seeds was reduced to approximately 18% under heavy metal stress . By contrast, Pb stress-inhibited seedling growth was significantly alleviated by IDS. Compared with the sprout length of seedlings under Pb stress alone, that of seedlings treated with 5, 20, and 100 mmol·dm−3 IDS while under Pb stress was approximately 20%, 50%, and 67% higher, respectively (p < 0.05).Five days after sowing, we assessed the growth of maize seedlings treated with different concentrations of IDS under Pb-stressed and Pb-free conditions. Under normal incubation conditions, treatment with high concentrations of IDS slightly but significantly reduced the growth of maize seedlings. For instance, the seedling sprout length decreased by approximately 7% and 12% after treatment with 20 and 100 mmol·dm2O2) and dimethylthiourea (DMTU) . A contrasting effect was observed in seeds treated with DMTU. For instance, in seeds incubated with Pb and Pb+IDS for 24 h, treatment with DMTU increased the GR by approximately 80% and 22%, respectively (p < 0.05). However, the GR of IDS-treated seeds increased by 46% and decreased by 42% after treatment with H2O2 and DMTU, respectively (p < 0.05).The germination of maize seeds treated with Pb, IDS, or their combination was significantly affected by treatment with redox reagents, such as hydrogen peroxide (Ha (DMTU) . After t2 on maize seed germination in the distilled water (control), Pb, IDS, and Pb+IDS treatment groups during the first 24 h of incubation (2-free conditions (p < 0.05). However, after the addition of DPI, the GR was approximately 120% and 26% higher in the Pb and Pb+IDS treatment groups, respectively, than in the corresponding DPI- and CaCl2-free condition (p < 0.05). Furthermore, compared with that in the DPI- and CaCl2-free conditions, the addition of CaCl2 increased the GR by approximately 21% and 25% in the control and IDS treatment groups, and reduced the GR by approximately 43% and 22% in the Pb and Pb+IDS treatment groups, respectively (p < 0.05).We investigated the effects of diphenyleneiodonium chloride (DPI) (a specific inhibitor of NOX) and CaClcubation . In the p < 0.05). Next, we investigated the effects of the antioxidant enzyme inhibitors diethyldithiocarbamic acid (DDC) (a specific inhibitor of SOD) and aminotriazole (ATZ) (a specific inhibitor of CAT) on the germination of maize seeds treated with Pb, IDS, or Pb+IDS. Twenty-four hours after the addition of DDC and ATZ, the GR of distilled water (control)- and IDS-treated maize seeds was significantly increased by approximately 16% and 33% (DDC) and by 20% and 44% (ATZ), respectively . However2O2 accumulation in maize seeds after incubation for 1, 3, and 5 d , compared with that after incubation for 1 d. However, H2O2 accumulation was significantly affected by treatment with IDS and Pb. After incubation for 1 d, Pb and Pb+IDS increased the H2O2 content of germinating seeds by approximately 80% and 49%, respectively, compared with the distilled water (control) treatment (p < 0.05). After 1, 3, and 5 d of treatment with IDS, the H2O2 content of germinating seeds was approximately 29%, 22%, and 11% lower, respectively, than that in the distilled water (control) treatment group (p < 0.05).We determined the effects of Pb, IDS, and Pb+IDS on H and 5 d . Throughp < 0.05). However, IDS significantly decreased NOX activity by 33%, 25%, and 11% after germination for 1, 3, and 5 d, respectively, compared with that in the control group (p < 0.05).Germination can significantly enhance NOX activity in maize seeds after incubation. Compared with the NOX activity after incubation for 1 d, germination increased the NOX activity by 47% and 79% after incubation for 3 and 5 d, respectively . After ip < 0.05). After treatment with Pb and Pb+IDS, the SOD activity was increased and was approximately 20–30% and 40–56% higher, respectively, than that of the distilled water (control) treatment after incubation for 1–5 d (p < 0.05). Similar patterns of change were observed for CAT activity in maize seeds (p < 0.05). Compared with distilled water treatment, Pb and Pb+IDS treatment increased CAT activity in maize seeds incubated for 1–5 d, by approximately 10–17% and 18–41%, respectively (p < 0.05). However, IDS decreased SOD and CAT activities. Treatment with IDS reduced SOD and CAT activities by approximately 13–32% and 10–25%, respectively, in maize seeds after incubation for 1–5 d, compared with those in the control (p < 0.05).The effects of Pb, IDS, and Pb+IDS on the SOD and CAT activities in maize seeds after incubation for 1, 3, and 5 d are illustrated in To counter the significant increase in the Pb content of cultivated soils near industrial areas ,53, chel−3 IDS noticeably delayed the germination of maize seeds that were not subjected to Pb stress  [2O2 and aggravated by DMTU in Pb-free conditions, and the opposite results were observed in Pb-stressed conditions  and ROS-scavenging enzymes  . In this of ROS)  after innditions . Therefo2+ (a non-specific activator of NOX) [2 treatment. Under Pb-free conditions, however, the IDS-delayed seed germination was enhanced by DPI but reversed by CaCl2. This phenomenon suggests that NOX-mediated ROS play a key role in IDS-regulated seed germination under Pb-stressed or Pb-free conditions. In addition, treatment with DDC (an inhibitor of SOD) and ATZ (an inhibitor of CAT) [We also investigated the effects of DPI (a specific inhibitor of NOX)  and Ca2+ of NOX)  on seed  of CAT)  further 2O2 content of seeds after the incubation period. Pb stress induced H2O2 overproduction in germinating seeds. However, IDS significantly decreased the Pb-enhanced H2O2 levels and reduced H2O2 accumulation under Pb-free conditions  to improve seed germination under Pb stress. The single and combined treatment with IDS and Pb revealed that under Pb stress, IDS, as a chelating agent of metal ions, did not restrict the activities of metal-containing SOD and CAT by chelating metal ions, such as Fe, Cu, Mn, and Zn, which are required for the activation of these enzymes [2+ on the activity of NOX. Therefore, further investigation is required to elucidate the mechanism underlying the regulation of antioxidant enzymes by IDS. However, protecting antioxidant enzymes from Pb-induced overproduction of ROS or free radicals, which can inactivate SOD and CAT [The activities of ROS-scavenging enzymes (SOD and CAT) increased gradually during seed germination in maize. However, compared with those in the control, SOD and CAT activities decreased significantly in the IDS-treated groups after incubation for 1, 3, and 5 d . This ca enzymes , but pla and CAT ,64, may 2+ chelation, which restricts NOX activity and protects and promotes SOD and CAT activity by inhibiting ROS overproduction, may be important for maintaining the ROS balance and improving seed germination and seedling growth under Pb stress resulting from IDS treatment in maize. In addition, IDS slightly interferes with the activity of SOD and CAT through the chelation of metal ions such as Fe, Zn, Cu, and Mn.To better understand the aforementioned findings, we propose a hypothetical model based on ROS levels to illustrate the mechanisms underlying IDS-regulated seed germination in Pb-free and Pb-stressed conditions . This moAll chemical reagents used in this work were of analytical grade. DPI, DMTU, DDC, and ATZ were purchased from Sigma-Aldrich . Other reagents were purchased from Aladdin Biochemical Technology Co., Ltd. .The seeds of the hybrid maize variety ‘Zhengdan 958’ used in the experiments were purchased from DONEED Seed Industry Co., Ltd, Beijing, China. The seeds were harvested in October 2020 and used in this experimental study in April 2021. The seed purity was greater than 99%, and the GR was greater than 95%. Before sowing, the seeds were surface-sterilized with 0.1% NaClO for 5 min. Healthy and uniform seeds with an intact seed coat were selected and submerged in distilled water (25 °C) for 1 h. The swollen seeds were then sown in a plastic box containing quartz stones (diameter: approximately 2–3 mm) to a depth of 2 cm. The plastic box was covered with parafilm and placed in an incubator . After incubation for 1, 3, and 5 d, the germinated seeds were collected and stored at −80 °C for use in further analysis.−3 IDS solution. The GR and seedling growth were monitored after sowing.Group 1: The swollen seeds were sown in a plastic box containing quartz stones moistened with 0, 5, 20, or 100 mmol·dm−3 PbCl2 as the Pb stress treatment condition. The swollen seeds were sown in a plastic box containing quartz stones moistened with 20 mmol·dm−3 PbCl2 + 0 mmol·dm−3 IDS, 20 mmol·dm−3 PbCl2 + 5 mmol·dm−3 IDS, 20 mmol·dm−3 PbCl2 + 20 mmol·dm−3 IDS, or 20 mmol·dm−3 PbCl2 + 100 mmol·dm−3 IDS solution. GR and seedling growth were monitored after sowing.Group 2: In accordance with the results of our previous study , we used2 (20 mmol·dm−3), IDS (100 mmol·dm−3), or PbCl2 (20 mmol·dm−3) + IDS (100 mmol·dm−3) solution. Subsequently, DMTU , H2O2 (10 mmol·dm−3), DPI , CaCl2 (0.1 mmol·dm−3), DDC , and ATZ  were applied separately. One milliliter of each ROS-related reagent solution was uniformly added to the quartz stones moistened with the distilled water, PbCl2, IDS, or PbCl2+IDS solutions, respectively.Group 3: The swollen seeds were sown in a plastic box containing quartz stones moistened with distilled water (control), PbClThe experiment included three treatment groups  as follows:All assays were repeated at least five times to minimize experimental error, and each replicate contained 100 seeds. The treatment concentration used in this study was based on the results of our preliminary experiments.The maize seeds were considered to have germinated when radicle emergence from the seeds was ≥1 mm. The number of germinated seeds was counted three times per day. The final GR was calculated as the percentage of seeds that had germinated 5 d after sowing .2, IDS, or their combination) were washed with distilled water. Excess water was removed using bibulous paper, and the sprout length was measured using a vernier caliper.Five-day-old maize seedlings  were determined using colorimetric measurements following the method described by Zhang et al. [2O2 was extracted by homogenizing germinating maize seeds (~0.5 g) in 3 mL of cold acetone. The samples were homogenized using a tissue homogenizer  at 4 °C and immediately centrifuged at 12,000 rpm for 15 min. A 1 mL aliquot of the supernatant solution was mixed with 0.1 mL of 5% titanium sulfate in concentrated HCl, followed by the addition of 0.2 mL aqueous NH3 (25%) to precipitate the peroxide–titanium complex. The mixture was then centrifuged at 12,000 rpm for 15 min. The precipitate was solubilized in 3 mL of 2 mmol·dm−3 H2SO4, and the absorbance was measured at 415 nm. A standard response curve was prepared with a known concentration of H2O2 using the aforementioned method.The Hg et al. . H2O2 wa−3 Tris–HCl (pH 7.5), 0.23 mol dm−3 sucrose, 5% polyvinylpyrrolidone, 1 mmol·dm−3 EDTA, 10 mmol·dm−3 KCl, 10 mmol·dm−3 MgCl2, and 2.5 mmol·dm−3 ascorbic acid (added to the buffer just before use). After homogenization and centrifugation , the supernatants were used for the enzyme assays.Germinating maize seeds (~3 g) were ground to a powder in liquid nitrogen. The powder was then homogenized in 30 mL of an ice-cold extraction buffer, which consisted of 0.1 mol dm−3 methionine, 75 μmol dm−3 nitro blue tetrazolium, 100 μmol dm−3 EDTA, and 2 μmol dm−3 riboflavin) was mixed with different volumes of enzyme extract in 50 mmol·dm−3 phosphate buffer (pH 7.8). The mixture was then exposed to light for 15 min. An increase in absorbance at 560 nm was observed due to formazan formation. One unit of SOD activity was defined as the amount of enzyme that inhibited nitro blue tetrazolium photoreduction by 50% [To measure SOD activity, the reaction buffer (containing 13 mmol·dmn by 50% .−3 potassium phosphate buffer (pH 7.0), 15 mmol·dm−3 H2O2, and 50 μL enzyme extract. The reaction was initiated by adding 15 mmol·dm−3 H2O2. CAT activity was determined based on the consumption of H2O2 (E = 39.4 dm3·mmol−1·cm−1) at 240 nm for 3 min [To measure CAT activity, a reaction mixture was prepared in such way that 3 mL of it contained 150 mmol·dmor 3 min .The soluble protein content was determined using the Bradford method , with bo−3 NADPH to a mixture containing 100 mmol·dm−3 Tris–HCl and 1 mmol·dm−3 EDTA. NOX activity was calculated by monitoring the decrease in absorbance at 340 nm, and the molar extinction coefficient used for NADPH was 6.22 × 103 dm3·mmol−1·cm−1 at pH 8.0 [The NOX enzyme was extracted, and its activity was assayed using a Plant NOX assay kit , following the manufacturer’s instructions. The reaction was initiated by adding 0.2 mmol·dmt pH 8.0 .p < 0.05.All experiments included five replicates, and the values represent the mean ± standard deviation (SD) of the five replicates. Statistical analyses were performed using analysis of variance (ANOVA) with SPSS software . The differences between treatments were determined by Duncan’s new multiple range test and considered statistically significant at −3) markedly delayed maize seed germination in the absence of Pb stress. Second, the Pb-mediated inhibition of seed germination and seedling growth was significantly alleviated by IDS. Third, IDS significantly inhibited the activity of ROS-producing enzymes, reduced the level of H2O2 in maize seeds under normal conditions, and markedly weakened the activities of ROS-producing enzymes under Pb stress. In contrast, the Pb stress-induced increase in the activities of ROS-scavenging enzymes was significantly enhanced by IDS. Under Pb stress, NOX (activated by Ca2+), SOD, and CAT were involved in maintaining the IDS-regulated ROS balance and promoting seed germination. Thus, Ca2+ chelation and protection of antioxidant enzymes may be essential for the regulatory roles of IDS. Overall, our results suggest that IDS can be used as an inexpensive, biodegradable, and readily available reagent for widespread application in agriculture to improve seed germination in Pb-polluted soils.Several significant conclusions can be drawn from the results of this study. First, IDS (100 mmol·dm


Dose–responsetreatments revealed that 1–5 improvedcell survival at nanomolar concentrations through the restorationof mitochondrial membrane potential and the reduction of reactiveoxygen species. Their ability to prevent the mitochondrial permeabilitytransition pore opening was also assessed, finding that 4 and 5 inhibited the channel at 0.001 μM. Thisinhibition was accompanied by a decrease in the expression of cyclophilinD, the main regulator of the pore, which was also reduced by 1 and 2. However, the activation of ERK and GSK3β,upstream modulators of the channel, was not affected by compounds.Therefore, their ability to bind cyclophilin D was evaluated by surfaceplasmon resonance, observing that 2–5 presented equilibrium dissociation constants in the micromolar range.All compounds also showed affinity for cyclophilin A, being 1 selective toward this isoform, while 2 and 5 exhibited selectivity for cyclophilin D. When the effectson the intracellular expression of cyclophilins A–C were determined,it was found that only 1 decreased cyclophilin A, whilecyclophilins B and C were diminished by most compounds, displayingenhanced effects under oxidative stress conditions. Results indicatethat furanoditerpenes 1–5 have mitochondrial-mediatedneuroprotective properties through direct interaction with cyclophilinD. Due to the important role of this protein in oxidative stress andinflammation, compounds are promising drugs for new therapeutic strategiesagainst neurodegeneration.Neuroprotective properties of five previously describedfuranoditerpenes  ROS accumulation produces the openingof the mitochondrial permeability transition pore (mPTP). The poreis a large nonspecific channel that connects the mitochondrial matrixand the cytosol and allows the passage of solutes with a molecularweight of less than 1500 Da, provoking an increase in the permeabilityof the inner mitochondrial membrane. mPTP opening is reversible, providinga pathway to release ROS and calcium from mitochondria, but if itis massive and persistent, it can lead to mitochondrial swelling andcell death.6Prevention of mitochondrial dysfunctionhas been proposed as atherapeutic strategy against several diseases. Age-related illnessesare closely associated with this organelle dysfunction and, therefore,with an increase in the release of damaging reactive oxygen species(ROS). Since mitochondria are responsible for oxidative phosphorylation,they are the main ROS producers when the electronic transport chainbegins to fail.7 Although ANTis still considered a part of the pore, F-ATP synthase has attractedattention as the main component of mPTP.6 In any case, the best-accepted regulator of the pore is cyclophilinD (CypD). This protein is the only mitochondrial isoform of a familywith more than 15 members that present the peptidyl–prolylisomerase activity and share the capacity to bind to cyclosporineA (CsA). The best-known isoforms include cyclophilin A (CypA), involvedin the CsA immunosuppressant activity, and cyclophilins B (CypB) andC (CypC), implicated in endoplasmic reticulum homeostasis.8 These enzymes have been associated with aging,inflammatory processes, and ROS augmentation.9 In fact, the role of CypD in mitochondria goes beyond mPTP modulation;it has been described as a regulator of the organelle proteome andfunction.10The molecular nature of the pore isstill an open question. Fora long time, mPTP was considered to be composed of adenine nucleotidetranslocase (ANT) in the inner membrane and voltage-dependent anionchannel (VDAC) in the outer membrane, but their genetic ablation didnot prevent pore opening.11Due to the high dependenceon the energy consumption of neurons,these cells are very sensitive to mitochondrial malfunction and oxidativedamage. In this context, neurodegenerative diseases have been associatedwith these pathological mechanisms, and therefore, mPTP opening andCypD have been proposed as important targets for counteracting thesedevastating illnesses.1–5) (S1–S5) isolated from the sponge Spongia (Spongia) tubulifera,2 including epispongiadiol (4)13 and spongian diterpene 17 (5),14 were tested in SH-SY5Y human neuroblastomacells to determine theirneuroprotective activity. In previous studies, compounds 1 and 4 displayed weak cytotoxicity in human cancer celllines,2 and 4 presented antiviralactivity,12 but their neuroprotective effectshave not been tested so far.In this work, five furanoditerpenes-2,5-diphenyltetrazoliumbromide (MTT) test. Compounds 1–5 did not display toxicity at any of the doses tested (data not shown).Therefore, neuroprotective experiments were performed with S. tubulifera metabolites at 0.001, 0.01, 0.1, and1 μM. Oxidative damage was induced by adding 150 μM H2O2 for 6 h since this concentration has been previouslyshown to significantly reduce cell viability, affect mitochondrialfunction, and increase the ROS release, mimicking the pathologicaleffects observed in neurodegenerative illnesses.15Before the beginning of neuroprotection assays, cytotoxiceffects of diterpenes 1–5 to improve cell survival under oxidative stress conditionswas determined with the MTT assay (p < 0.05) was observed. Compound 1 was not able to protect neuroblastoma cells from this damage, similar to theeffect observed with the positive control at 25 μM  with the fluorescentdye tetramethylrhodamine methyl ester (TMRM). As 1–5 did not produce effects on ΔΨm when added alone to SH-SY5Y cells, but all of them were capableof recovering mitochondria from the depolarization induced by H2O2 .Compound 1 improved ΔΨm aftertreatment at 0.001 μM   3a,where 0.01 μM 3b–d. Comol cells 3e.S. tubulifera metabolites to decrease ROS intracellular levels was evaluated (2O2 augmentedthe levels of oxidant molecules up to 125.1 ± 2.4% (p < 0.01) and the known antioxidant Vit E, used as a positive control,induced a reduction in ROS release of 24% with respect to cells treatedonly with H2O2 (p < 0.05).Cotreatment with 1 produced a significant decrease inthe ROS content at all of the doses assayed   4b. With s tested 4c. Finalol cells 4d,e.S. tubulifera compounds, the levels of the most importantnonenzymatic antioxidantof cells, glutathione (GSH), were assessed. Treatment with 1 did not affected the GSH content neither with the compound alonenor under oxidative damage  (3 (4 and 5 presented interesting results(4 increased the GSH content at the lowest concentrations testeduntil control cell levels (101.3–105.0%), and 5 recovered the endogenous antioxidant at 0.001  and 0.1 μM  compared to H2O2 controlcells.To confirm the antioxidant properties of e damage 5a. Regar < 0.05) 5b. No ef0.05)  was used to open the pore. Cells were loadedwith calcein-AM, which accumulates in cytosol and cell compartmentsand with CoCl2 that quenches cytosolic fluorescence butnot the mitochondrial signal.17 Then, fluorescencewas analyzed by flow cytometry (p < 0.001)due to mPTP opening. Cells treated with compounds 4 and 5 at 0.001 μM presented an increase in the fluorescenceof 78.9 ± 6.3% (p < 0.05) and 90.3 ±5.3% (p < 0.01), respectively, confirming thecapacity of these metabolites to inhibit mPTP aperture. This augmentationwas even higher than the increase produced by the known inhibitorof the pore CsA at 0.2 μM .Inview of the results obtained in the oxidative stress model and, inparticular, in those related to mitochondrial functioning, the abilityof compounds to inhibit mPTP opening was determined. For this assay,the minimal effective concentration in our previous assays was chosen(0.001 μM). SH-SY5Y cells were treated for 15 min with ytometry 6a. The a7 In this context, the expressionof this protein was analyzed after treatment with compounds. CypDcytosolic levels were evaluated after treatment with 1–5 alone for 6 h and after the coaddition ofditerpenes and H2O2 to get a complete insightinto diterpene mitochondria-related effects. SH-SY5Y cells treatedwith 1, 2, 4, and 5 at 0.001 μM presented a significant reduction in CypD expression,with levels among 64.8–71.9% of control cells, similar to theresults obtained after the addition of 0.2 μM CsA  .Compounds 1, 4, and 5 decreasedthe protein expression up to percentages of about 54% of H2O2 control cells  6b. Underol cells 6c.10 thestudy was continued by evaluating if S. tubulifera metabolites were affecting the activation of two kinases involvedin the control of mPTP by CypD, ERK, and GSK3β  and 67.3 ± 9.4%(p < 0.01), respectively. CsA at 0.2 μMalso reduced ERK phosphorylation in both cases, an effect previouslydescribed.19 On the other hand, GSK3βinhibition results in a reduction in mPTP opening since the kinasebinds to CypD, decreasing its interaction with mPTP components.10 In the case of GSK3β, Ser9 phosphorylationwas determined, a residue implicated in kinase inhibition carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS)and 50 μg/mL of human CypD dissolved in 5 mM sodium acetate(pH 4.5) was added. The remaining active esters were inactivated with1 M ethanolamine, and two injections of regeneration buffer (2.5 mMNaOH) were performed .20 No fall on the signal was observed, so compoundinjections were started. All of the assays were carried out with phosphate-bufferedsaline (PBS)—0.05% Tween (pH 7.2) as the running buffer, witha flow rate of 20 μL/min at 24 °C. At first, CsA at increasingconcentrations was added to set the optimal conditions and to confirmthe functionality of the protein. As can be observed in KD) of 0.13 ± 0.0003 μM.This constant is lower than previous values reported by our groupdue to the higher sensitivity of this instrument. In fact, in ourprevious assays, 5 μM was the lowest concentration used andno binding was detected, while it is the highest dose tested in theseassays.21Due to the lack of effects of diterpenes on CypDupstream regulators,we proceed to evaluate if compounds were capable of directly bindingto the protein. With this objective, surface plasmon resonance (SPR)experiments were performed. CypD was immobilized in the sensor surfaceas a ligand, and erformed S6.20 No 1 up to 50 μM, whereas association curves for 2–5 were obtained , and compounds at severalconcentrations were added. The same conditions set for CypD were usedfor CypA experiments, with a flow rate of 20 μL/min and PBS-T(pH 7.2) as the running buffer. All assays were performed at 24 °C.Again, CsA was utilized as a positive control, being injected at concentrationsbetween 2.5 and 0.01 μM. Typical association curves were obtained,reaching a response of 10.8 mdeg when the drug was added at 2.5 μM, while 3 had a higher affinity to thisisoform (KD = 6.39 ± 0.09 μM).Finally, 4 showed responses between 0.2 and 10.3 mdeg,with a KD of 27.4 ± 3.68 μM,and 5 presented a maximum signal of 13.6 mdeg (KD = 4.34 ± 0.17 μM). Kinetic constantsfor CypA and compounds are presented in Table S2.Due to the affinity of furanoditerpenesby mitochondrial CypD, their ability to bind to CypA was determinedto evaluate their selectivity between both isoforms. The protein wasimmobilized on the sensor surface by amino coupling, as describedabove S7, and c1–5, their effects on the expression ofthis protein,as well as in the intracellular levels CypB and CypC, the most studiedisoforms of this immunophilin family, were evaluated. Before startingthe experiments, antibody specificity was determined due to the conservedstructures along Cyps. With this objective, 1 μg of human recombinantCypA, CypB, CypC, and CypD was subjected to electrophoresis and Westernblot was performed as described for cell lysates . No cross-reaction was detected for anti-CypA,anti-CypB, and anti-CypC antibodies, but anti-CypD detected both CypAand CypD. Due to the differences in the molecular weight of theseproteins , the discrimination betweenboth bands was performed in cell lysates without uncertainty. Therefore,the assays were carried out. Human neuroblastoma cells were treatedwith compounds at 0.001 μM for 6 h, and Cyps expression wasdetermined by Western blot  when cells weretreated with metabolites alone, a comparable effect to that producedby CsA  (p < 0.05) of H2O2 control. Finally, CypCintracellular levels were analyzed, finding a decrease in its expressionafter the addition of 3–5 alone  , which currently does not have effective approved medicamentsfor counteracting its progression.25In this work, the mitochondrial-mediatedneuroprotective effects of five furanoditerpenes obtained from 2–5 displayed neuroprotectiveantioxidant properties against oxidative stress by decreasing theROS release and recovering ΔΨm at nanomolarconcentrations. Interestingly, compounds displayed a biphasic response,showing better outcomes at the lowest doses tested. This hormeticresponse has been described for several natural products, such ascurcumin or resveratrol, which have neuroprotective properties atlow concentrations and proapoptotic effects at high doses.27 This neuroprotective effect is related to the ability of S. tubulifera metabolites to bind CypD, as demonstratedby SPR assays. In fact, compound 1 was the less potentone in this in vitro model, being the only diterpenethat did not show an affinity for CypD in biosensor experiments. RegardingCypD expression, a decrease was found in physiological conditionswith compounds 1, 2, 4, and 5. This diminution in CypD expression after treatment withfuranoditerpenes points to an enhancement of the protein degradationby compounds. The CypD complex with heat shock protein-90 preventsprotein degradation, and their dissociation promotes CypD ubiquitinationand proteasomal degradation.28 In thiscontext, the binding of furanoditerpenes to CypD could be avoidingits association with the heat shock protein-90, triggering its degradationin the proteasome.Furanoditerpenes 10 and even modulates extra-mitochondrialsignaling.29 In this context, the decreaseproduced by furanoditerpenes 1, 2, 4, and 5 could be affecting mitochondria. However,the analysis of ROS levels and ΔΨm did notreveal any significant effect on the organelle function. The effectsof furanoditerpene 1 on CypD intracellular levels pointto an upstream target that could be associated with the protectiveeffect produced by this compound since it did not present affinityto CypD. However, when ERK and GSK3β were evaluated, none ofthe compounds showed effects on these well-recognized CypD regulators.So, other enzymes that are known to modify the CypD activity suchas sirtuin 3 or peroxisome proliferator-activated receptor αmight be potential targets of 1.10 On the other hand, compound 3, with a KD of 7.0 μM, did not affect CypD expression. Thislack of effect could be related to the low dose used in the assay(0.001 μM); higher concentrations should be tested in futureexperiments.Although the best-characterized role of CypDin cells is the regulationof mPTP opening, its function goes beyond the pore, as has been recentlydiscovered. CypD performs scaffolding functions, affects oxidativephosphorylation,1, 2, 4, and 5 underoxidativestress is very remarkable, as H2O2 increasesthe protein expression, making cells more susceptible to mPTP opening.31 To the best of our knowledge, the exact mechanism through whichoxidative stress promotes the expression of CypD has not been describedand transcriptional regulators of this protein remain elusive. Onlya recent work has identified BMP/Smad signaling as a regulator ofCypD gene expression,32 but its relationshipto oxidative stress has not been explored.The reduction produced in CypD expression by diterpenes 16 All compounds decreased ROSrelease, and 2, 4, and 5 wereable to recover the GSH content. These effects are also related totheir ability to target CypD, as the protein deletion attenuates H2O2-induced mitochondrial dysfunction and reducesintracellular ROS accumulation.30 AlthoughmPTP allows the release of excessive ROS from mitochondria in normalconditions, under pathological circumstances, mPTP opening promotesROS generation by mitochondria, generating a vicious cycle that isbeing interrupted by S. tubulifera metabolites.Surprisingly, only diterpenes 4 and 5 wereable to inhibit mPTP opening after oxidative injury. This discrepancycan be associated with the differences in the incubation time in bothexperiments. CypD expression was evaluated after a cotreatment withcompounds and the oxidant for 6 h, while cells were pretreated for15 min with 1–5 followed by oxidativeinjury for 3 min for mPTP assessment.CypD has been proposedas a target for neurodegenerative diseases,in which ROS increase and mitochondrial dysfunction are central pathologicalmechanisms.33 Moreover, a connection between tau and CypD has been recentlyestablished, observing that tau deletion leads to CypD downregulationand mPTP inhibition in a mouse model.34 With all of this evidence, the central role of CypD and mPTP inneurodegeneration is clear, so the discovery of compounds capableof targeting CypD and improving mitochondrial function is an importantchallenge. Despite the fact that CsA prevents mPTP opening, its usefor the treatment of neurological pathologies has been discarded formany reasons, such as oxidative stress generation. CsA has immunosuppressantactivity, it is not able to efficiently cross the blood–brainbarrier, and it has shown neurotoxicity, nephrotoxicity, and hepatotoxicity.35CypD and mPTP involvementin neurodegeneration and more particularlyin AD, the most prevalent neurodegenerative pathology, has been extensivelystudied. Elevated CypD expression has been detected in AD patients,and CypD-deficient mice presented improved memory.1–5 showed different affinities for CypDand CypA. In all cases, bindingamong S. tubulifera metabolites andCyps was reversible, as the injection of running buffer to the sensorwas enough to dissociate the compounds from the proteins, which discardsa covalent binding. Compounds 3 and 4 presentedsimilar KD values in both isoforms, whereas 1 was selective toward CypA and 2 and 5 displayed selectivity toward the mitochondrial isoform. These resultssuggest that the lack of a hydroxyl group at C-19 is critical forthe selective affinity for CypA binding since it is the only differencebetween 1 and 4. Moreover, the presenceof a Δ1 double bond along with a hydroxyl group atC-2 and a ketone carbonyl functionality at C-3 seems to be responsiblefor the selective affinity toward CypD since all of these featuresare only present in 2 and 5. In this sense,these furanoditerpenes could be useful as lead compounds to analyzethe structural requirements for CypA or CypD affinity, as well asvery helpful to better understand the implications of each Cyp oncellular signaling. In previous works, we have described a familyof natural sponge diterpenes, the gracilins, with an affinity forCypA and CypD, which presented both neuroprotective and immunosuppressantproperties.36 Interestingly, a pharmacophore-directed retrosynthesis led to obtainingsimplified derivatives with selective mitochondrial-mediated neuroprotectiveactivities.37 In this context, compounds 1–5, bearing similar but more rigid structuresto those of natural gracilins, could be used as scaffolds for newsynthetic analogues, thus avoiding the supply problem of marine naturalproducts.38 Moreover, rigid structuresare very useful to obtain information about the shape of the targetsite and to determine the conformation adopted by a ligand when itbinds to it.Interestingly, furanoditerpenes 1–5 bindto CypA, only 1 reduced its cytosolic expression at 0.001μM. This compound presented a KD of 15.2 μM, showing lower affinity than 3 and 5 for CypA , but it was the only one with a selectiveaffinity toward the cytosolic isoform. This selectivity could explainthe effects of 1 at such a low concentration. Along withits function as a chaperone, the best-characterized role of CypA incellular signaling is its interaction with calcineurin, which leadsto the activation of calcineurin phosphatase activity and provokesinterleukin-2 release. The inhibition of the CypA–calcineurincomplex by CsA is responsible for the immunosuppressant propertiesof this drug.40 Despite the fact that 2–4 did not alter CypA intracellular levels inSH-SY5Y cells at 0.001 μM, these compounds bind to this isoform,so the effects of furanoditerpenes 1–5 on calcineurin pathway should be studied in the following worksto clarify their potential immunosuppressant properties at higherdoses. Anyway, their ability of binding CypA could also contributeto neuroprotection since this protein has also been linked to neurodegeneration.Although CypA is mainly located in the cytosol, it can also be releasedinto the extracellular space, exerting proinflammatory activities.Extracellular CypA is a potent leukocyte chemoattractant and inducesthe expression of matrix metalloproteinases and cytokines.41 CypA has been involved in the blood–brainbarrier dysfunction observed in AD patients, more particularly inapolipoprotein E4 carriers.43 Moreover, CypA colocalizeswith Parkinson’s disease-associated protein α-synuclein,45 and targeting extracellular CypA has shown promising results againstamyotrophic lateral sclerosis in a murine model.46Although furanoditerpenes 8 Its overexpressionin SH-SY5Y cells induced protective effects;47 however, CypB serum levels are elevated in metabolic syndrome patientsand its intracellular levels are increased in liver and adipose tissueof obese mice.48 These tissues are of greatimportance at the beginning of obesity and type 2 diabetes, two knownrisk factors for dementia.50 With respect to CypC,it is located in the endoplasmic reticulum and the Golgi apparatus.This isoform participates in the redox regulation of endoplasmic reticulum,but its function remains elusive.51 It has beenreported that CypC levels are augmented in microglial and neuronalcells after focal ischemia.52 Moreover,high serum CypC levels have been recently described in the acute coronaryartery disease54 and its intra- and extracellularlevels are elevated in activated human T lymphocytes.55 These recent results point to an important role of CypCin inflammation-mediated disorders such as neurodegenerative diseases.56The function of the other immunophilins analyzed,CypB and CypC,has been less studied. CypB is located in the cytosol, the endoplasmicreticulum, and the nucleus, being also released to the extracellularspace. Both pro- and anti-inflammatory activities of this isoformhave been reported.2–5 isolated from S. tubulifera presentedmitochondrial-mediated neuroprotective effects associated with theiraffinity to CypD. Specifically, compounds 4 and 5 were the most promising ones since they were capable ofinhibiting mPTP opening at nanomolar concentrations. Furthermore, 1–5 presented different affinities forCypD and CypA related to their chemical structures, which turn theminto potential scaffolds for the designing of new selective Cyps inhibitorsuseful for the treatment of disorders associated with inflammationand oxidative stress, such as neurodegenerative diseases.In conclusion, furanoditerpenes 33.12DCFDA), MitoProbe Transition Pore Assay Kit,Pierce Protease Inhibitor Mini Tablets, Pierce Phosphatase InhibitorMini Tablets, Supersignal West Pico Luminiscent Substrate, and SupersignalWest Femto Maximum Sensitivity Substrate were purchased from ThermoFisher Scientific . Human CypD and CypA full-length proteinsand CsA were from Abcam . Recombinant human CypBwas from Antibodies-online, and recombinant human CypC was obtainedfrom BioVendor . Compounds were dissolved indimethyl sulfoxide (DMSO), and serial dilutions were performed incell medium. Vehicle concentration was always kept under 0.5% in celltreatments, and control cells were treated with the highest concentrationused to test its effects. Other chemicals were reagent grade and purchasedfrom Sigma-Aldrich .TMRM, Thiol Tracker Violet,5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate high-performanceliquid chromatography (HPLC) separation (H2O/CH3OH mixtures) to yield 1–3. Fractionationof the CH2Cl2 fraction by solid-phase extraction,using an RP-18 cartridge eluted with a stepped gradient from H2O, CH3OH, and CH2Cl2, yieldedenriched terpene fractions. RP-HPLC separation of these fractions,using H2O/CH3OH mixtures, gave 4 and 5. Full details on the isolation, purity, and structuralelucidation of compounds have been published elsewhere.2Compounds were obtained fromthe sponge 3.32 and 95% air and dissociatedweekly using 0.05% trypsin/ethylenediaminetetraacetic acid (EDTA).All of the reagents were provided by Thermo Fischer Scientific.Human neuroblastomaSH-SY5Y cells were purchased from American Type Culture Collection(ATCC), number CRL2266. Cells were cultured in Dulbecco’s modifiedEagle’s medium: nutrient mix F-12 (DMEM/F-12) supplementedwith 10% fetal bovine serum (FBS), 1% glutamax, 100 U/mL penicillin,and 100 μg/mL streptomycin. Cells were maintained at 37 °Cin a humidified atmosphere of 5% CO3.44 cells per well. After 24 h, cells weretreated with the metabolites at nontoxic concentrations (0.001–1μM) and 150 μM H2O2 for 6 h. VitaminE at 25 μM was used as a positive control. All of the experimentswere performed as previously described.15To determine the neuroprotective effectsof compounds, SH-SY5Y cells were seeded in 384-well plates at a densityof 2.5 × 102O2 was evaluated with 3--2,5-diphenyltetrazoliumbromide (MTT) test. After treatment, SH-SY5Y cells were washed threetimes with Locke’s buffer piperazine-N′-ethanesulfonicacid (HEPES), pH 7.4). Then, cells were incubated with 500 μg/mLMTT for 1 h at 37 °C and 300 rpm. After this time, cells weresolubilized with 5% sodium dodecyl sulfate. The absorbance of formazancrystals was measured at 595 nm with a plate reader. Saponin at 40mg/mL was used as the death control, and its absorbance value wassubtracted from the other data.The ability of compounds to protect cell viability from Hm were analyzedwith the fluorescent dye TMRM. For this assay, cells were washed twicewith Locke’s solution and 1 μM TMRM was added to eachwell. After 30 min of incubation at 37 °C and 300 rpm, cellswere lysed with H2O and DMSO at 50%. The fluorescence wasread in a plate reader at 535 nm excitation and 590 nm emission. Allof the assays were performed in triplicate three independent times.The effects of compounds inΔΨ3.515For these experiments, cellswere seeded in 384-well plates and treated as described above. Allof the assays were carried out as previously described.2DCFDA) was used to evaluate ROS levels.After treatment with compounds and H2O2, cellswere washed twice with serum-free medium. Next, cells were loadedwith 20 μM carboxy-H2DCFDA dissolved in serum-freemedium, and the plate was incubated for 1 h at 37 °C and 300rpm. Then, 100 μL of PBS were added to each well for 30 minat 37 °C and 300 rpm. After this incubation, the fluorescencewas read at 527 nm excitation, with an emission wavelength of 495nm.The fluorescent dye 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluoresceindiacetate , washed with PBS, and resuspended in PBS bufferwith 0.6 mM CaCl2. Cells were loaded with 0.01 μMcalcein-AM and incubated at 37 °C for 15 min. Then, 0.4 mM CoCl2 and compounds at 0.001 μM were added and cells wereincubated for 15 min at 37 °C. After this incubation, cells werecentrifuged, resuspended in 100 μL of calcium-free PBS, andkept on ice. Finally, TBHP at 1 mM was added for 3 min to the cellsto induce the pore opening. Fluorescence was measured at 488 nm excitationand 517 nm emission wavelengths by flow cytometry using the ImageStreamMKII instrument . The fluorescenceof 10 000 events was analyzed with IDEAS Application vs 6.0 . Experiments wereperformed three independent times, and CsA at 0.2 μM was usedas a positive control.The inhibition of mPTP by compounds was determinedwith the MitoProbe Transition Pore Assay Kit, as previously described.3.76 cells per welland allowed to settle down for 24 h. Then, cells were treated withcompounds at 0.001 μM for 6 h and oxidative damage was inducedwith 150 μM H2O2.SH-SY5Y cells werecultured in 12-well plates at 1 × 102, containingphosphatase and protease inhibitor cocktail). Cells were incubatedfor 15 min on ice and centrifuged at 3000 rpm and 4 °C for 15min. The supernatant was collected as the cytosolic fraction, andprotein concentration was quantified with the Direct Detect instrument.15Cells were washedtwice with ice-cold PBS, and a hypotonic solution was added , containing 15 μgof cytosolic protein from each sample. The Trans-Blot semidry transfercell (Biorad) was used to transfer proteins to poly(vinylidene difluoride)(PVDF) membranes (Merck Millipore). Snap i.d. system (Merck Millipore)was used for membrane blocking and antibody incubation. CypD was detectedwith an anticyclophilin F primary antibody , phosphorylated ERK was quantified with antiphospho-ERK1/2  , and the total levels of the enzyme weredetected with anti-ERK 1/2 antibody . Antiphospho-GSK3β (Ser9)  was used to recognizephospho-GSK3β, and anti-GSK3β  was utilized to measure the total GSK3βexpression. CypA was detected with anti-CypA primary antibody , CypB wasrecognized with anti-CypB antibody , and CypC was quantified with anti-CypC antibody . Proteinband intensity was corrected using anti-β-actin . The activation of kinaseswas analyzed as the ratio between phosphorylated and total proteinlevels. Immunoreactive bands were detected with Supersignal West PicoLuminiscent Substrate and Supersignal West Femto Maximum SensitivitySubstrate. Diversity GeneSnap system and software  were used for protein band detection. Experiments were performedthree independent times in duplicate.3.8N-hydroxysuccinimide(NHS). After activation, 50 μg/mL of CypA or CypD dissolvedin 5 mM sodium acetate buffer (pH 4.5) were injected for immobilizationon the sensor surface. The remaining reactive esters were deactivatedby the addition of 1 M ethanolamine (pH 8.5).22 For binding measurements, analytes were added at concentrationsbetween 0.001 and 50 μM for 5 min. Then, regeneration was performedby adding 2.5 mM NaOH for 1 min. CsA was used as a positive controlin these experiments. Compound injections were performed in serialmode, and the reference channel signal was subtracted from the analytesensorgram. Data were processed with MP-SPR Navi Data Viewer (BioNavis),and KD values were obtained using the1:1 Langmuir model with mass transport considerations of TraceDrawersoftware . All of the experimentswere performed three independent times.SPR measurements were performed using the MP-SPR Navi 210A VASA instrumentwith SPR Navi Control software . All ofthe analyses were carried out in three-dimensional (3D) carboxymethyldextran hydrogel-coated (CMD-3DM) sensors (BioNavis). PBS, 0.05% Tween(pH 7.2) was used as the running buffer for sensor activation, proteinimmobilization, and analyte injection. All buffers were degassed andfiltered by 0.22 μm before use. The flow rate and temperaturewere set at 20 μL/min and 24 °C, respectively. A commercialAmine Coupling Kit was used for sensor activation and Cyps immobilization. The chip surfacewas preconditioned with 2 M NaCl and 0.01 M NaOH. Then, the surfacewas activated by the addition of 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC) and 0.05 M 3.9p <0.05, **p < 0.01, and ***p <0.001.Data are presentedas mean ± SEM. Differences were evaluated by one-way ANOVA andDunnett’s post hoc test with Graph Pad Prism 8.0 software.Statistical significance was considered at *