Therefore, we designed and synthesized a ratiometric fluorescent Zn2+ probe ER-Zn targeting ER stress. The probe displayed a specific Zn2+ induced blue shift at the spectral maximum values of excitation (80 nm) and emission (30 nm). The ratio imaging capability of Zn2+ under dual excitation mode can be applied not only to quantitative and reversible detection of exogenous Zn2+, but also the observation of the Zn2+ level change under ER stress, elucidating the different behaviors of Zn2+ release in ER stimulated by tunicamycin and thapsigargin. Additionally, the NIR imaging capability of ER-Zn provides an important basis for further research on animal models and is expected to realize the visualization and treatment of ER stress-related diseases through the regulation of ER stress by Zn2+. We envision that this probe can be applied to screen drugs for diseases related to ER stress regulation.The endoplasmic reticulum (ER) is the main storage site of Zn For one thing, the styrene group was introduced into the α site of BODIPY fluorophore, which not only increased its excitation wavelength, but also extended the emission wavelength of the fluorophore to the NIR region for reducing the interference of spontaneous fluorescence in organisms and damage to cells during imaging. For another, N, N, N′-tri (pyridin-2-ylmethyl) ethane-1,2-diamine (TPEA) was introduced at the α position as the Zn2+ chelating group. Finally, p-toluenesulfonamide was introduced at the meso position of the fluorophore as the ER targeting group to construct a highly selective NIR Zn2+ fluorescent probe targeting ER, ER-Zn. Cell imaging experiments showed that ER-Zn had good cytocompatibility, which not only realized reversibly ratiometric imaging of exogenous Zn2+, but also quantitatively monitored the fluctuation of Zn2+ level in ER under ER stress. ER-Zn provides a new method to study the relationship between ER stress regulation and Zn2+ level.In this paper, due to the lack of research on near-infrared (NIR) ratiometric Zn4, MgCl2·6H2O, CaCl2, Zn(NO3)2·7H2O, NaCl, KCl, FeCl2, CoCl2·6H2O, and NiCl2·6H2O were dissolved in ultrapure water to prepare corresponding concentrations of metal ion stock solution. The stock solution of Zn2+ was prepared by dissolving ZnCl2 in ultrapure water.The commonly used chemicals  and general solvents  were all from the domestic reagents purchased by School of Chemistry and Chemical Engineering of Nanjing University, and they were not further purified before use. In the spectral characterization, the solvents such as DMSO and DMF were both spectrally pure reagents purchased from Aldrich. The water used was ultrapure from Millipore. CuSO1H and 13C NMR were determined by Bruker DRX-500 and Bruker DRX-400 NMR spectrometers using standard pulse sequence with tetramethylsilane (TMS) as internal standard (298 K). The absorption spectra were measured by PerkinElmer Lambda 35 UV-vis spectrometer. Fluorescence emission spectra were recorded by FluoroMax-4 fluorescence spectrometer (HORIBA Jvon Inc.). The pH assay was determined by SevenCompact S210 pH meter (Mettle Toledo). Cell imaging was performed on Olympus FV10-ASW laser confocal fluorescence microscope and Leica SP8 STED 3X confocal microscope. The excitation wavelength was 570 and 650 nm, and the collection wavelength was 660–720 nm.Mass spectrometry was determined by LCQ electrospray ionization mass spectrometry . High-resolution mass spectrometry (HRMS) was determined by Thermo scientific quadrupole orbit trap tandem high-resolution mass spectrometer. The synthesis steps of ER-Zn are shown in 1H NMR  δ 7.65 , 7.25–7.17 , 7.13 , 5.96 , 2.53 , 2.38 , 1.25 . 13C NMR  δ 155.71, 144.16, 142.77, 140.52, 137.52, 135.27, 132.08, 131.38, 129.59, 129.12, 127.42, 122.31, 121.32, 21.55, 14.58, and 14.45.At 0°C, 3  and 4-methylbenzenesulfonyl chloride  were dissolved in dichloromethane (20 ml), and then several drops of pyridine were added to the solution. After stirring for 2 h, the solvent was removed by rotary evaporation. The target product in orange solid form  was obtained by using dichloromethane/petroleum ether (v:v = 2:1) preparative chromatography on silica gel. 2SO4. After the solid was removed by filtration, the crude product in the filtrate was collected by rotary evaporation. The target product in blue-black solid form  was obtained by preparative chromatography with dichloromethane/methanol  system on silica gel. 1H NMR  δ 8.57 , 7.66–7.63 , 7.61 , 7.42 , 7.38 , 7.24–7.20 , 7.20–7.16 , 7.12 , 6.55 , 6.52 , 5.93 , 3.93 , 3.20 , 2.91 , 2.56 , 2.37 , 2.11 , 1.28 , and 1.24 . 13C NMR  δ 174.77, 158.46, 155.07, 152.89, 149.77, 149.26, 148.83, 143.97, 142.53, 140.30, 140.03, 138.28, 137.63, 137.56, 137.25, 136.92, 135.43, 133.06, 132.34, 131.08, 129.52, 129.47, 127.40, 125.20, 123.52, 122.46, 122.20, 120.46, 117.82, 113.97, 112.62, 106.55, 60.04, 52.66, 40.91, 21.54, 21.01, 14.79, 14.58, and 14.33. HRMS : Calcd: 913.39896, found: 913.39630 for [M + H]+ and Calcd: 935.38090, found: 935.37921 for [M + Na]+.ER-Zn: 3  and TPEA-CHO  were weighed and added into a 50 ml three-necked flask. Then the activated molecular sieve was added into the bottle with a water separator. Pumped the pressure of the system to vacuum. Under the protection of nitrogen, 10 ml toluene, a drop of acetic acid and a drop of pyridine were added and refluxed overnight. The reaction process was monitored by TLC until the reaction finished. The reaction was further processed after cooling to room temperature. The saturated NaCl solution was used to extract for three times, and the organic phase was selected to remove water by Na3, pH = 7.2).7.78 mg ER-Zn was accurately weighed and then dissolved with 8.52 ml DMSO to prepare 1 mM ER-Zn storage solution. No special instructions, the test system is 50 mM HEPES solution with 60% DMSO (containing 100 mM KNO2 (3 mM) was equivalently dropped into the test system to record the absorption titration spectra, fluorescence titration spectra, and excitation titration spectra. The absorption band was collected from 480 to 750 nm, the excitation spectra test parameters are as follows: the emission at 700 nm and excitation collection band from 475 to 685 nm, the slit is 4 * 4 nm.10 μM ER-Zn solution was prepared with 3 ml buffer solution, and its absorption and fluorescence spectra were collected. Then, 1 μl ZnCl2+, K+, Ca2+ and Na+, and 10 μM Ni2+, Co2+, Al3+, Cr3+, Cu2+, Fe3+, Mn2+, Pb2+ solutions, respectively, were dripped to the probe solution before collecting their excitation spectra. Then, 10 μl ZnCl2 solution (3 mM) was added into the test system containing different ions to determine their excitation spectra.Selectivity test: The different metal ions 2 mM Mg3, pH = 7.2) was adjusted to pH range of 2–10, then 30 μl ER-Zn storage solution (1 mM) was added to the solutions to make the final concentration as 10 μM. Finally, 10 μl ZnCl2 storage solution (3 mM) was dropped into the above solutions, and the fluorescence spectra were performed.pH-dependent experiment: Using NaOH and HCl, the buffer solution  containing 10% fetal bovine serum (FBS), penicillin (100 units/ml), and streptomycin (100 mg/ml). There is no special indication that HeLa cells are used for cell imaging.The HeLa cells used in the experiment were cultured in the air with 5% CO2 incubator. After cell adherent growth for 12 h, the original culture medium was washed away, and 200 μl ER-BDP with different concentrations were added to each well, and then put back into the incubator for 24 h. Then 30 μl MTT solution was added to each well. After incubation in the incubator for 12 h, the supernatant was carefully removed, and 200 μl DMSO was added to each well to fully dissolve the purple precipitate. Finally, the absorbance of each well at 490 nm was measured by a microplate reader. Each group of experiments was measured three times in parallel.MTT -2,5-diphenyltetrazolium bromide) method was used to determine the number of living cells by using MTT to produce purple precipitate formazan with the mitochondrial succinate dehydrogenase of living cells. HeLa cells in the exponential growth phase were collected and inoculated into 96-well plates. About 5,000 cells and 100 μl medium containing 10% FBS were added to each plate. HeLa cells in 96-well plates were placed in a 37°C, 5% COTwo groups of cells were prepared, and the co-localization experiments between the nucleus, ER and ER-Zn were carried out respectively. Two groups of cells were treated with 100 nM ER-Tracker™ Blue-White DPX dye for 30 min and 100 nM DAPI for 10 min, respectively. The cells were taken out and washed three times with PBS buffer, then incubated with 5 μM ER-Zn solution for 30 min and washed three times with PBS buffer.The excitation wavelength and emission channel of ER-Blue Tracker dye were as follows: the excitation wavelength was 405 nm, and the emission channel was 415–450 nm; the excitation wavelength of the probe ER-Zn was 570 nm, and the emission channel was 660–720 nm. For DAPI, the excitation wavelength was 405 nm, and the emission channel was 430–500 nm; the excitation wavelength of ER-Zn was 633 nm, and the emission channel was 645–700 nm.2 and 5 mM 2-mercaptopyidine-N-oxide aqueous solution (diluted to a specific concentration by DMEM). After incubation for 20 min, the imaging was performed with a laser confocal fluorescence microscope. After imaging, the cells were washed three times with PBS without metal ions and then treated with 50 μM TPEN (diluted by the TPEN storage solution through DMEM medium) for 20 min, and then washed once with PBS.The cultured cells were washed three times with metal-free PBS solution and then incubated with 5 μM ER-BDP solution at room temperature for 2 h. Remove the probe solution, wash the cells three times with PBS solution without metal ions, and then observe them with a laser confocal fluorescence microscope. The introduction of exogenous zinc in cells was carried out by incubation in a 1:1 mixture of 5 mM ZnClTime-course imaging: The cells were incubated with 5 μM ER-Zn for 2 h and then washed with PBS buffer three times. Then, the cells were incubated with 25 μg/ml thapsigargin (TG) and collected images with time.Cell imaging under different stimuli: Three groups of cells were stimulated by TM and TG to induce ER stress, respectively. The cells were treated with medium, 25 μg/ml tunicamycin (TM), and 25 μg/ml TG for 12 h, respectively. After incubation with 5 μM ER-Zn solution for 2 h, the cells were washed three times with PBS buffer. The cells were washed with PBS solution three times, and the cells were imaged.2+ fluorescent probes has encountered the following problems so far: 1) Sensitivity and selectivity. Since DPA (Di (2-methylpyridine) amine) was first linked with fluorescein, the chelating group has become the most commonly used recognition group for the construction of Zn2+ sensors  Orgahe probe . 3) Image, ER-Zn . Its syn3, pH = 7.2). Absorption and fluorescence titration were performed by adding different concentrations of Zn2+. As can be seen from 2+ concentration, the absorbance at 610 nm decreases continuously until it disappears, while a new absorption peak at 575 nm appears and keeps increasing. Therefore, when Zn2+ was added, the absorption peak was blue-shifted from 610 to 575 nm with an isosbestic point appearing at 588 nm, and the solution color changed from cyan to blue. The nitrogen atom in the chelating group of the free probe had a strong electron donating ability. However, Zn2+ binding TPEA weakened the electron-donating ability of nitrogen atom, which diminished the ICT effect of the structure, inducing a hypochromatic shift of absorption peak.The probe molecule ER-Zn was prepared as a 10 μM solution in HEPES buffer  reached equilibrium when Zn2+ was added to 1 eq. It indicated that ER-Zn binds with Zn2+ in the manner of 1:1. In addition, the detection limit (3σ/slope) of this probe was estimated to be 31.8 nM  ranges from 0.1 to 1. As shown in 2+ is 1:1. Notably, the dissociation constant of the ER-Zn-Zn2+ complex was calculated as 3.65 nM  as an exogenous Zn2+ supplement and TPEN as an intracellular Zn2+ chelator. When the cells were incubated with ER-Zn for 2 h, excitation ratiometric imaging was performed, and the probe showed dual-channel imaging in the cells. Then the cells were incubated with 5 μM ZnPT for 10 min for cell imaging. As shown in 2+ in cells. When the cells were incubated with 50 μM TPEN for 20 min, the image ratio was significantly decreased. It is proved from the result that ER-Zn can be used for quantitative imaging of exogenous Zn2+ in cells, which provides a basis for quantitative and dynamic imaging of endogenous Zn2+ in ER.The ER targeting ability of ER-Zn has been confirmed via a colocalization experiment. To further study the sensing ability of ER-Zn on Zn2+ in the ER, we set up cell imaging of endogenous Zn2+ by ER-Zn under ER stress. TG can affect ER calcium (Ca2+) homeostasis by specific inhibition of ER Ca2+-ATPase and induce ER stress  on ZnR stress . Therefo2+ level in ER. We speculate that Zn2+ homeostasis, like Ca2+ homeostasis, is controlled by IP3Rs, which may allow Zn2+ to enter the cytoplasm and pump Zn2+ from the cytoplasm into ER through TG sensitive ATPase activity (2+ will be released from the ER into the cytoplasm, resulting in the decrease of Zn2+ in the ER.In addition, TM is also commonly used to induce ER stress . We stimactivity . When ER2+, good selectivity, and anti-interference ability. In addition, ER-Zn not only possesses double excitation ratio detection of exogenous Zn2+ but made clear the relationship between ER stress regulation by different stimuli and the endogenous Zn2+ level in the ER. ER-Zn with the ability of NIR imaging provides a reliable technical basis and design strategy for the further development of quantitative imaging of Zn2+ probes in vivo. We envision that this probe can be applied to screen drugs for diseases related to ER stress regulation.In summary, based on the ICT mechanism, we designed and synthesized a novel NIR ratiometric fluorescent probe targeting ER, ER-Zn, which has a good ratio response to Zn


Microglia play an important role in the pathology of various central nervous system disorders, including multiple sclerosis (MS). While different methods exist to evaluate the extent of microglia activation, comparative studies investigating the sensitivity of these methods are missing for most models. In this study, we systematically evaluated which of the three commonly used histological methods  immunohistochemical stains) is the most sensitive method to detect subtle changes in the microglia activation status in the context of MS. To this end, we used the toxin-induced cuprizone model which allows the experimental induction of a highly reproducible demyelination in several central nervous system regions, paralleled by early microglia activation. In this study, we showed that after 3 weeks of cuprizone intoxication, all methods reveal a significant microglia activation in the white matter corpus callosum. In contrast, in the affected neocortical grey matter, the evaluation of anti-IBA1 cell morphologies was the most sensitive method to detect subtle changes of microglial activation. The results of this study provide a useful guide for future immunohistochemical evaluations in the cuprizone and other neurodegenerative models. Microglia represent the resident innate immune cells of the central nervous system (CNS), originating from the progenitor cells of the embryonic yolk sac . These iMS is a chronic inflammatory demyelinating CNS disease causing disability in more than 2.5 million people worldwide. On the histopathological level, the brains of MS patients show perivascular inflammation, peripheral immune cell recruitment, demyelination, axonal injury, as well as astrocyte and microglia activation. Of note, demyelination is not restricted to the white matter but can as well be found in the cortical and subcortical grey matter areas ,16. MicrTo determine the role of microglia for CNS disease development and progression, it is essential to visualize their activation status in histological sections. Several methods exist to (semi-)quantify the extent of microglia activation. First, one can estimate microglia numbers within a predefined region of interest (ROI) by either assessing the number of microglia applying stereological methods  or by deIn this study, we systematically evaluated which of these histological methods  is most sensitive to detect subtle changes in the microglia activation status in the context of MS. To this end, we used the toxin-induced cuprizone model which allows the experimental induction of a highly reproducible demyelination in several CNS regions, paralleled by robust microglia activation in the course of cuprizone intoxication ,30.n = 20, 19–23 g) were ordered from Janvier Labs  as part of a large cohort study. Female mice were only used in this study. They were housed in cages which were changed once per week with a maximum of five mice per cage with ad libitum water and food. Further, the animals were housed under standard laboratory conditions, including 12 h light/dark cycle and controlled temperature 22 ± 2 °C. As suggested by the Federation of European Laboratory Animal Science Associations (FELASA), we implemented microbiological testing. The acclimatization phase of mice lasted for seven days previous to the start of cuprizone intoxication. The assignment of animals to the experimental groups was random. The Review Boards for the Care of Animal Subjects of the district government Mecklenburg–Western Pomerania permitted the experiments (reference number 7221.3-1-001/19).C57BL6/J mice .For tissue preparation, animals were injected intraperitoneally with ketamine (100 mg/kg) and xylazine (10 mg/kg). Afterward, mice were transcardially perfused with phosphate-buffered saline (PBS) and 3.7% paraformaldehyde solution (pH 7.4). At Bregma −2.3 (corresponding to Franklin and Paxinos atlas ), we mad2O2) in PBS for 30 min. After washing in PBS, the slides were incubated with the EnVision System for 1 h . The antigenic sites were detected by a reaction with 3,3′-diaminobenzidine  and H2O2, yielding a brownish deposit.Previously established protocols were used to conduct immunohistochemistry ,36. In b+ cells were counted within the ROI by two evaluators (MW and YB/AS) blinded to the treatment groups. Results are shown as the number of cells per mm2.To quantify microglia cell numbers, sections were digitalized using a Nikon ECLIPSE E200 microscope : 0.4) equipped with a camera (Basler acA1920-40uc). For densitometrical  and morphological analyses  of microglia cells, the Leica DM6 B automated microscope  equipped with a DMC6200 camera was used and the images were taken using constant illumination settings. To quantify the number of microglia, the program ViewPoint  was used. The area of the ROI was outlined manually, and IBA1For staining intensity measurement, we performed a semi-automated densitometrical analysis with the software ImageJ . The pictures were transformed to grayscale pictures and a threshold was set to divide every pixel of all pictures in black or white . Moreover, the threshold analysis functions by setting a value cutoff in which every pixel larger in comparison to the cutoff value refers to one class and every pixel smaller in comparison to the cutoff value refers to the other class. After setting a threshold, relative staining intensities were semi-quantified in binary converted images and the results were transformed into percentage area values. Results are shown as staining intensity in (%) area of the entire ROI.Z-stack-multifocus images were generated with a 0.2 μm step size using the Leica DM6 B automated microscope (see c) and the projection area (Ap) [c, the anti-IBA1-positive area of one individual cell was outlined using the polygon tool in ImageJ. To measure Ap, a Convex Hull Analysis was performed which measures the size of the cellular field by interpreting a branched structure as a solid object controlling a given amount of physical space. Resting microglia are characterized by long and thin ramified processes with a relatively large Ap and a relatively small Ac. Activated microglia show a hypertrophic cell body with hypertrophic and retracted cell processes. During microglial activation, the ramification index approaches a value close to 1.To analyze the microglial morphology, cope see D–F. Accorea (Ap) . To measp-values of ≤ 0.05 were considered to be statistically significant. The following symbols are used to indicate the level of significance: * p  ≤  0.05, ** p  ≤  0.01, *** p  ≤  0.001, n.s. indicates “not significant”. Significant outliers were excluded from the densitometrical analyses using the Grubbs’ test . To analyze differences between groups, the non-parametric Kruskal–Wallis test followed by Dunn’s multiple comparison test was applied.All data are given as arithmetic means ± SD. Differences between groups were statistically tested using the software GraphPad Prism  with confidence intervals of 0.05. + cell numbers, (ii) densitometrical analyses of IBA1 staining intensities, and (iii) morphological changes of IBA1+ cells by determining the cell ramification index (2 vs. 1 week (1 w) 196.97 ± 51.14 cells/mm2, n.s.; staining intensity: (Ctrl) 1.89 ± 0.96% vs. (1 w) 4.84 ± 0.84%, n.s.; ramification indices: (Ctrl) 0.66 ± 0.04 vs. (1 w) 0.81 ± 0.06, n.s.). During the course of cuprizone-induced demyelination, all three values steadily increased. At week 3, cell numbers were (Ctrl) 87.79 ± 20.52 cells/mm2 vs. 3 weeks (w) 803.14 ± 264.54 cells/mm2, p = 0.006; staining intensities were (Ctrl) 1.89 ± 0.96% vs. (3 w) 28.41 ± 17.31%, p = 0.004; and the cell ramification indices were (Ctrl) 0.66 ± 0.04 vs. (3 w) 0.87 ± 0.03, p = 0.008. At week 5, cell numbers were (Ctrl) 87.79 ± 20.52 cells/mm2 vs. (5 w) 945.12 ± 370.25 cells/mm2, p = 0.001; staining intensities were (Ctrl) 1.89 ± 0.96% vs. (5 w) 37.57 ± 16.1%, p = 0.002; and the cell ramification indices were (Ctrl) 0.66 ± 0.04 vs. (5 w) 0.88 ± 0.04, p = 0.002.In the cuprizone model, various regions of the brain are affected such as the corpus callosum which is an integral commissural fiber tract connecting the left and right cerebral hemispheres, enabling communication between them. In a first step, we analyzed the progression of microglia activation in the medial corpus callosum using the (i) quantification of anti-ionized calcium-binding adaptor protein-1 (IBA1)on index . None of2 vs. (1 w) 87.27 ± 17.79 cells/mm2, n.s.; staining intensity: 2.34 ± 1.07% vs. 1.97 ± 0.13%, n.s.; ramification indices: 0.44 ± 0.04 vs. 0.49 ± 0.04, n.s.). Comparable to what we observed in the corpus callosum, during the course of cuprizone-induced demyelination, all three values increased, although in a delayed time course. At week 3, cell numbers were (Ctrl): 92.31 ± 13.46 cells/mm2 vs. (3 w) 99.13 ± 14.54 cells/mm2, n.s; staining intensities were (Ctrl) 2.34 ± 1.07% vs. (3 w) 4.39 ± 1.18%, n.s.; and the cell ramification indices were (Ctrl) 0.44 ± 0.04 vs. (3 w) 0.55 ± 0.04, p = 0.02. At week 5, cell numbers were (Ctrl) 92.31 ± 13.46 cells/mm2 vs. (5 w) 207.32 ± 39.2 cells/mm2, p = 0.03; staining intensities were (Ctrl) 2.34 ± 1.07% vs. (5 w) 6.52 ± 3.12%, p = 0.01; and the cell ramification indices were (Ctrl) 0.44 ± 0.04 vs. (5 w) 0.56 ± 0.03, p = 0.005. To conclude, just the ramification index was able to detect significant microglia activation at week 3.During the course of cuprizone intoxication, besides the white matter tract corpus callosum, various grey matter areas, such as the neocortex, are affected as well. Due to the a priori lower myelin content in the grey matter compared to the white matter, the extent of microglia activation during the course of cuprizone-induced demyelination is less severe . In a ne2 vs. (1 w) 107.88 ± 10.77 cells/mm2, n.s.; staining intensity: (Ctrl) 1.17 ± 0.89% vs. (1 w) 5.94 ± 4.37% n.s.; ramification indices: (Ctrl) 0.53 ± 0.04 vs. (1 w) 0.6 ± 0.04, n.s.). Again, all three parameters increased during the course of cuprizone intoxication, but a plateau was reached at week 3 with no further increase until week 5. At week 3, cell numbers were (Ctrl) 85.42 ± 8.89 cells/mm2 vs. (3 w) 195.61 ± 27.98 cells/mm2, p = 0.0013; staining intensities were (Ctrl) 1.17 ± 0.89% vs. (3 w) 5.3 ± 1.26%, p = 0.014; and the cell ramification indices were (Ctrl) 0.53 ± 0.04 vs. (3 w) 0.64 ± 0.03, p = 0.003. At week 5, cell numbers were (Ctrl) 85.42 ± 8.89 cells/mm2 vs. (5 w) 180.56 ± 9.9 cells/mm2, p = 0.007; staining intensities were (Ctrl) 1.17 ± 0.89% vs. (5 w) 4.8 ± 0.99%, p = 0.05; and the cell ramification indices were (Ctrl) 0.53 ± 0.04 vs. (5 w) 0.62 ± 0.03, n.s.Finally, we tested by which method microglia activation in the hippocampus can be optimally analyzed . No methIn this study, we were able to demonstrate that all of the tested methods to characterize the extent of microglia cell activation in the cuprizone model of demyelination, id est, the estimation of cell numbers, the analyses of anti-IBA1 staining intensity, and the quantification of the cellular morphology by determining the cell ramification index, can be used and provide comparable results.Cxcl10, Ccl2, and Ccl3. Some of these, for example, CXCL10, appear to be functionally involved in the complex process of microglia activation in this animal model [In the cuprizone model, the kinetics of cellular alterations are relatively well characterized. Days after the initiation of the cuprizone intoxication, apoptotic oligodendrocytes can be histologically observed ,39,40,41al model ,45,46,47Microglia activation is a simple term describing a rather complex cellular process which includes changes in cell numbers, cellular morphology, gene expression, cytokine release, and the potential to present antigens, among others. While the necessity to apply reliable methods to quantify the extent of microglia activation is undisputed, the characterization of “activated” microglia is a challenging task for several reasons. For example, firstly, commonly used markers to label microglia cells in most instances also label peripheral monocytes that have invaded the brain parenchyma. Thus, a clear differentiation between microglial cells and peripheral macrophages is challenging. Secondly, while marker proteins have been identified which label microglia but not peripheral monocytes, for example, the transmembrane protein TMEM119  or the pIn this study, we used three commonly accepted methods to evaluate the extent of microglia cell activation, and each of the three methods has its weaknesses and strengths. Firstly, we used the parameter “number of cells”. One requirement for this method is to reliably identify the cell bodies to be able to estimate cellular numbers. This identification was challenging in the corpus callosum at week 3 and 5 of the cuprizone intoxication because of pronounced microgliosis. Further, it is worth mentioning that following the described approach, one does not evaluate cell numbers but cell densities (cells per area). To obtain entire cell numbers in a given region of interest, design-based stereology methods have to be applied as published in several models, including MS animal models ,55. The Secondly, we used the parameter “staining intensity”. The advantage of this method is that it is objective, reproducible, and can be used in areas of intense microglia activation where other methods are not precise. For example, after 5 weeks of cuprizone intoxication, the midline of the corpus callosum displays such an intense anti-IBA1 staining that cell counting and the estimation of microglia cell morphologies is complicated compare . A limitp and a relatively small Ac. In contrast, in activated microglia cells (or macrophages), the ramification indices value approaches 1 due to a hypertrophy of the cell body paralleled by a retraction of the cell processes [Finally, we determined the degree of the cellular ramification to estimate the extent of the microglia activation in anti-IBA1-stained sections. It has been demonstrated that IBA-1 regulates actin-crosslinking involved in membrane ruffling in microglia . Becauserocesses . At leasrocesses , the detTo recommend an evaluation method of choice during the course of cuprizone intoxication, areas with intense microglia activation have to be distinguished from areas with subtle microglia changes. The cell number evaluation and the morphological method should be preferred in areas with bland microglia activation, whereas in areas with a high degree of microglia activation, densitometrical analysis appears to have a higher validity.


Second-generation antipsychotics (SGAs), in particular, olanzapine and clozapine, have been associated with the development of type 2 diabetes mellitus (T2D) and metabolic syndrome in individuals with schizophrenia. In this context, beta cell dysfunction is a plausible mechanism by which SGAs cause T2D. Herein, we analyzed the direct effects of olanzapine, a commonly prescribed SGA with diabetogenic properties, on the INS-1 (821/13) beta cell line and isolated pancreatic islets. Treatment of INS-1 beta cells with non-toxic concentrations of olanzapine (3–6 μM) during 4 h activated endoplasmic reticulum (ER) stress-mediated signaling by increasing PERK/eIF2α phosphorylation, IRE-1 phosphorylation and XBP-1 splicing. Moreover, glucose-stimulated insulin secretion (GSIS) was inhibited when olanzapine was present for 16 h. The insulin secretory function of INS-1 cells was restored by inhibiting olanzapine-induced ER stress with tauroursodeoxycholic acid (TUDCA). Similar effects of olanzapine with or without TUDCA on ER-stress-mediated signaling and GSIS were found in pancreatic islets from female mice. Our results indicate that early activation of ER stress in pancreatic beta cells is a potential mechanism behind the alterations in glucose homeostasis induced by olanzapine. Second-generation antipsychotics (SGAs) are the main line of treatment for schizophrenia, and other severe mental illness (SMI), due to their higher clinical efficacy compared to first-generation antipsychotics (FGA) . HoweverThe different SGAs vary in their relative diabetogenic effects. In this regard, clozapine and olanzapine induce the greatest weight gain and confer a higher risk for metabolic disturbances compared with other antipsychotics . HoweverDespite numerous investigations, controversy remains over the effects of SGAs on insulin secretion and beta cell function, effects that seem to be dependent on the antipsychotic binding to different receptors, and on the dose and treatment time . In thisBeta cells are extremely dependent on the endoplasmic reticulum (ER) to sustain insulin production. In situations of insulin resistance, beta cells increase the production of insulin to compensate for the higher peripheral insulin demand, thereby enhancing ER function. However, exceeding the functional capacity of the ER can lead to accumulation of misfolded proteins and activation of the unfolded protein response (UPR) . The UPRPrevious studies have illustrated that the most diabetogenic SGAs, including olanzapine, clozapine and risperidone, induce ER stress in pancreatic beta cells , liver ,13 and hBased on the concentrations used in previously reported in vitro studies with olanzapine in beta cells lines ,17, we eA previous study reported that olanzapine may activate UPR in beta cells through effects in proinsulin folding . Therefop < 0.001) (p < 0.05)  c. Moreov < 0.05) d.†† p < 0.01, ††† p < 0.001 vs control cells) and eIF2α (†††p < 0.001 vs control cells) in INS-1 beta cells. Likewise, the combination of olanzapine (6 μM) and TUDCA counteracted XBP1 splicing  expression and serotonin production in pancreatic beta cells, causing beta cell dysfunction by interfering with the regulation of the serotoninergic system and also by inhibiting Ca2+ entry . Howeverculation .db/db mice in parallel to the alterations in glucose homeostasis [Interestingly, in our recent study we found that inhibition of insulin secretion in female mice receiving a chronic treatment with olanzapine seemed to be, at least in part, due to defective insulin granule maturation . Amorphieostasis .Herein, we show activation of ER-stress-mediated signaling in INS-1 beta cells and pancreatic islets by olanzapine, particularly PERK/eIF2α and IRE1α phosphorylation, and XBP1 splicing, in line with the results reported by Ozasa et al. . ImportaFetal bovine serum (FBS) and culture media RPMI-1640 containing 11.1 mM glucose were purchased from Sigma-Aldrich . RPMI-1640 without glucose, L-glutamine, sodium pyruvate and penicillin/streptomycin were purchased from Gibco . Olanzapine was obtained from Glentham Life Sciences (UK) and stocked at 100 mM in DMSO at −20 °C. Tauroursodeoxycholic acid (TUDCA) was purchased from Sigma-Aldrich . All other chemicals were purchased form Sigma-Aldrich unless otherwise noted.2. Cells were passaged twice a week by trypsin incubation. INS-1 cells were seeded in 6-well tissue plates at a density of 6 × 105 cells/well, and after 24 h cells were treated with olanzapine at concentrations of 1–6 μM, 250 μM TUDCA or vehicle (0.01% DMSO) for 3–4 or 24 h. For all compounds prepared in DMSO, the final concentration of this solvent in the culture medium was kept at less than 0.01%.The rat insulinoma INS-1 832/13 cell line (referred as INS-1) was kindly provided by Dr. Hindrik Mulder  . Mycopla5 cells/well in a 12-well plate, and the day after treated with some concentration (1–100 μM) of olanzapine. For assessment of cell viability using crystal violet, individual wells were fixed with 4% paraformaldehyde (PFA) for 15 min and washed with PBS. After, wells were incubated with a 0.1% crystal violet solution  for 20 min. Then, wells were washed with water and air dried for several days. A solution of 10% acetic acid was used to dissolve the crystal violet and absorbance was measured in this supernatant at 590 nm. The MTT -2,5-diphenyltetrazolium bromide) tetrazolium reduction assay was also used to measure cell viability . After 24 h of treatment, 50 µL of MTT solution (5 mg/mL) was added to each well and the plate was incubated at 37 °C for 3 h. Then, 150 µL of MTT solvent was added to the wells and the plate was incubated for 15 min. Absorbance was read at 590 nm.INS-1 cells were seeded at a density of 3 × 103, 1.1 MgCl2, 1.2 NaH2CO4, 2.5 CaCl2, 25 HEPES and 0.25% bovine serum albumin (BSA) was prepared with the addition of olanzapine with or without 250 μM TUDCA. Briefly, INS-1 cells were preincubated with 1 mL of KRB supplemented with 2.8 mM glucose for 30 min at 37 °C in an atmosphere of 5% CO2. Afterwards, the KRB solution was replaced with 1 mL of fresh KRB solution supplemented with 2.8 mM glucose, and INS-1 cells were then incubated for 1 h at 37 °C in an atmosphere of 5% CO2. The supernatant was collected into a 1.5 mL tube, and cells were incubated again with 1 mL of KRB solution supplemented with 16.7 mM glucose for 1 h at 37 °C in an atmosphere of 5% CO2. The supernatant was also collected into a 1.5 mL tube. Both tubes were centrifuged at 5600 rcf for 10 min at 4 °C, and the supernatants were collected and stored at −20 °C until analysis. Insulin was measured by ELISA .INS-1 cells were seeded in 6-well plates, and when 90% confluence was reached, they were treated for 24 h with olanzapine at 1–6 μM. Krebs–Ringer buffer (KRB) solution containing (in mM) 2.8 glucose, 115 NaCl, 5 KCl, 1.2 NaHCOAfter GSIS, 500 µL of glycine/NP-40 lysis buffer  was added to the wells. INS-1 cells were scrapped, and the contents were transferred into a 1.5 mL tube. The samples were freeze–thawed in liquid nitrogen and a 37 °C water bath and then centrifuged for 15 min at 15,700 rcf. These supernatants were stored at −20 °C until analysis of insulin content. Values were normalized to protein content.Clostridium histolyticum  mixed with Hank’s balanced salt solution (HBSS) (1:14) . Islets were recovered in a 35 mm culture plate overnight at 37 °C and 5% CO2 in complete RPMI-1640 medium  FBS) containing 5.6 mM glucose. The next day, islets were pooled, randomized and incubated with olanzapine at the indicated concentrations in the absence or presence of TUDCA (250 μM). Control islets were treated with 0.01% (vol/vol) DMSO.Animal experiments were approved by the Animal Ethics Committees of the Spanish National Research Council and Comunidad de Madrid  in accordance with Spanish (RD 53/2013) and European Union (63/2010/EU) legislation. Twelve-week-old female mice were sacrificed by cervical dislocation, and the pancreas was excised after infusing the animal in the common bile duct (CBD) with 3–4 mL of collagenase P solution (13.5 U/mL) derived from 3, 1.1 MgCl2, 1.2 NaH2CO4, 2.5 CaCl2, 25 HEPES, 2.8 glucose and 0.25% BSA, pH 7.4, for 1 h at 37 °C and 5% CO2. Thereafter, batches of 3 islets per condition matched by size were transferred into a 96-well plate containing 200 μL KRB per well with the addition of 2.8 and 16.7 mM glucose , and the plate was incubated at 37 °C and 5% CO2 for 1 h. After, 100 μL of the supernatant was collected and stored at −20 °C until the analysis. Experiments were performed with 4–6 replicates per condition and animal. Insulin concentration was determined with a Mouse Insulin ELISA kit following manufacturer’s instructions . Values were normalized per islet number.Pancreatic islets, obtained as detailed above, were incubated in KRB containing (in mM) 115 NaCl, 5 KCl, 10 NaHCO® reagent , and reverse transcription was performed using a High-Capacity cDNA Reverse Transcription Kit  using random primers and Superscript III enzyme  according to the manufacturer’s instructions. RT-PCR was used for the detection of XBP1 mRNA splicing with forward (5′-CCTTGTAATTGAGAACCAGG-3′) and reverse (5′-CCAAAAGGATATCAGACTCGG-3′) primers as described [Total RNA from INS-1 cells was extracted with TrizolINS-1 cells were lysed in lysis buffer containing 10 mM Tris pH 7.5, 5 mM EDTA, 50 mM HCl, 30 μM sodium pyrophosphate, 50 mM NaF, 100 mM o-vanadate sodium, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 10 µg/mL of protease inhibitors, pH 7.4–7.6. Pancreatic islets were lysed in Cell Lysis Buffer  supplemented with 1 mM PMSF and 10 µg/mL of protease inhibitors. Protein extracts were quantified using the Bio-Rad Protein Assay solution , and 15–30 µg was loaded in an 8–12% SDS polyacrylamide gel for electrophoresis (SDS-PAGE). Gels were transferred to PVDF membranes . Then, membranes were incubated in blocking solution  for 1 h at room temperature (RT), followed by o/n incubation (4 °C) with primary antibodies. Antibodies used were phospho-eIF2α , eIF2α , phospho-IRE1α , IRE1α , phospho-PERK , PERK , vinculin , GAPDH  and α-tubulin . Blots were incubated with secondary anti-mouse  or anti-rabbit  antibody. Membranes were developed with chemiluminescent substrate , and different exposure times were used for each primary antibody with radiographic films in a radiology cassette (AGFA), or they were developed in a ChemiDoc imager (Bio-Rad). Densitometric analysis of the bands was performed using the Image J software . Samples from each experiment were analyzed individually. Details of proteins detected in the same gel and the number of independent experiments and replicates are included in the corresponding figure legends.† p < 0.05, **, †† p < 0.01, ***, ††† p < 0.001.Statistical analysis was performed using GraphPad Prism software version 8 . A Shapiro–Wilk normality test was run to determine if the samples followed a parametric or non-parametric distribution. In parametric distributions, student’s T-test was used to compare mean differences between 2 groups, whereas one-way ANOVA with Bonferroni post-hoc test was used to compare mean differences between 3 or more groups. In non-parametric distributions, the Mann–Whitney U test was used to compare mean differences between 2 groups, and the Kruskal–Wallis test to compare mean differences between 3 or more groups. Two-way ANOVA was employed to compare 2 different categorical independent variables. Use of statistical analysis other than those described above is indicated in the figure legends. The data are presented as the mean ± SEM (standard error of the mean). Statistical significance was set to *,


To understand its electrophysiological fundamental, whole-cell patch-clamp was used to record the L-type calcium current  and potassium currents (IK and IK1) in the ventricular myocytes in rats. Oral administration of PDQ exerted obvious antiarrhythmic effects, as indicated by the decreased incidence rate, lower number of occurrences, and shorter duration time of ventricular tachycardia and ventricular tachycardia, decreased mortality rate and increased survival time. ICa,L and IK were inhibited by PDQ treatment while IK1 was not affected. To conclude, PDQ may have an anti-arrhythmia effect through inhibiting ICa,L and IK.Pseudo-ginsengenin DQ (PDQ) is the product of the oxidative cyclization of protopanaxadiol. PDQ exhibits various bioactivities, including reversal of multidrug resistance in cancer, renal protective effects against acute nephrotoxicity and attenuating myocardial ischemia injury induced by isoproterenol or ligation of coronary arterials, but its effect on arrhythmias has not been clear until now. Because of the complicated effects of ginseng on the cardiovascular system, it is necessary to investigate whether PDQ affects arrhythmias, which are always concomitant with other cardiac diseases. Aconitine was used to induce arrhythmia  2+ and K+ currents in ventricular myocytes.Pseudo-ginsengenin DQ (PDQ) is the product of the oxidative cyclization of protopanaxadiol. PDQ could ameliorate aconitine-induced arrhythmias by influencing Ca Ocotillol-type ginsenosides were reported to have multiple biological activities such as neuroprotective, anti-inflammatory, anti-bacterial, and anti-tumor effects.11–14 Therefore, some ocotillol-type saponins have been semi-synthesized by modifying dammarane-type saponins. Among them, Pseudo-ginsengenin DQ (PDQ) can be prepared by the oxidation and cyclization of the side-chain of protopanaxadiol with a yield of 75% (ZL 200510016774.4).15 PDQ has not only shown chemo-reversal ability in cancer via overcoming multidrug resistance,16 but also renal protective effects against CDDP-induced acute nephrotoxicity through the caspase and Sirt1/NF-κB signaling pathway.17 In addition, PDQ can attenuate isoproterenol-induced myocardial ischemia injury through suppressing the activity of glutathione peroxidase, reducing the superoxide dismutase and increasing the malondialdehyde content.18 The development of myocardial ischemia injury, along with the structural heart diseases and genetic deficits, has frequently been associated with arrhythmias.19 Thus, whether PDQ has anti-arrhythmic activity is necessary to be investigated.Arrhythmias, as major types of heart malfunctions, often occur due to production or conduction of cardiac rhythm, which can cause hemodynamic changes, cardiac dilation, and ventricular filling pressure overload.etc.20 Its electrophysiological mechanism involves mainly in multiple ion channels in myocyte membrane, such as activation of sodium channels to accelerate Na+ influx, depolarize cell membranes, and enhance the autonomy of fast-acting cells such as atrial conduction tissue and atrioventricular bundle-Purkinje fiber system, increasing Ca2+ inflow and inhibiting outward K+ current to prolong repolarization time, increase the incidence of post depolarization, and cause reentry excitation.Aconitine is a fatal toxin isolated from aconitum plant, which is the commonly used chemical to induce different kinds of arrhythmias, including ventricular premature beat (VP), ventricular tachycardia (VT), ventricular fibrillation (VF) and cardiac arrest, in vitro.In the present study, PDQ pills were chosen as the test drug. PDQ pills were the dosage form with PDQ as drug substance and PEG 4000 (Polyethylene Glycol 4000) as excipients with a ratio of 1 : 3 in mass. We evaluated the anti-arrhythmic effects of PDQ pills on aconitine-induced arrhythmia in rats and investigated its electrophysiological mechanism of action in the cardiomyocytes of rat/guinea pigs 2.2.1PDQ (purity > 98.0%) was provided by the School of Pharmaceutical Sciences of Jilin University . PDQ pills (the dosage form) were prepared by Jilin Shengya Pharmaceutical Technology Co., Ltd. . PEG 4000 was bought from Hunan Erkang Pharmaceutical Co., Ltd. . Chloral hydrate was purchased from Sinopharm Chemical Reagent Co., Ltd. Aconitine was bought from Hainan Sinochem United Pharmaceutical Industry Co., Ltd. Diltiazem hydrochloride tablets  were purchased from Tianjin Tianbian Pharmaceutical Co., Ltd.2.22.2.1In vivo experiment was carried out in rats and the rat ventricular myocytes were separated to investigate ion channel.All the animal experiments were conducted based on the guide for the administration of laboratory animals  and were approved by the Review Committee of Animal Care and Use of Jilin University. Male SPF Wistar rats weighing between 200 g and 220 g were purchased from the Animal Center of Norman Bethune Medical College of Jilin University (production certificate no. SCXK (JI) 2007-0003, SCXK (JI) 2013-0001; using certificate no. SCXK (JI) 2007-0011, SCXK (JI) 2013-0005). All rats had free access to standard laboratory food and water. They were maintained under standardized laboratory conditions . Guinea pigs weighing between 300 g and 350 g were purchased from the Animal Center of Norman Bethune Medical College of Jilin University (using certificate no. SCXK (JI) 2013-0005). Ventricular myocytes were separated to investigate action potentials.2.2.2n = 10 per group): (1) normal control group, (2) PEG 4000 group (240 mg kg−1), (3) aconitine-induced arrhythmic model group, (4) H-PDQ , (5) M-PDQ , (6) L-PDQ , (7) positive control group . The rats of normal and model groups were administrated 0.9% NaCl aqueous solution (5 mL kg−1). The rats of PEG 4000 group were administrated PEG 4000 (48.0 mg mL−1). PDQ groups were administered with PDQ pills aqueous solution , which were grinded and suspended in 0.9% NaCl aqueous solution. The rats of positive drug group were administered with diltiazem hydrochloride aqueous solution (1.2 mg mL−1).After a week of acclimatization, the rats were randomly divided into seven groups  was administrated via sublingual vein within 5 seconds, while the rats of normal group were injected with physiological saline.21 Electrocardiogram was recorded using a Power Lab Multi-channel physiological recorder .The rats in treatment groups were intragastrically administered with PEG 4000, PDQ or diltiazem hydrochloride 4 h before the experiment. Then, rats were anesthetized using 10% chloral hydrate. Aconitine .Normal perfusion condition: the cells were divided into DMSO (0.1%) perfused control group and different concentrations of PDQ (0.2 μg mL2.42+ Tyrode's solution contained (in mM): NaCl 143.0, KCl 5.4, CaCl2·2H2O 1.8, MgCl2·6H2O 1.8, NaH2PO4·2H2O 0.25 and HEPES 5.0 (adjusted to pH 7.4 with NaOH). The Ca2+-free Tyrode's solution was prepared by removing CaCl2 from the Tyrode's solution. The extracellular solution contained (in mM): NaCl 115.0, KCl 4.7, MgSO4 1.2, CaCl2·2H2O 2.0, NaHCO3 2.5, KH2PO4 1.2, glucose 10.0, tetrodotoxin (TTX) 20.0, 4-aminopyridine (4-AP) 5.0 (pH 7.4 adjusted with NaOH). The pipette solution for recording ICa,L and IK1 (the inward rectifier potassium current) contained (in mM): cesium aspartate 120.0, CsCl 30.0, KH2PO4 10.0, MgSO4 3.0, NaCl 5.0, HEPES 5.0, K2ATP 5.0, EGTA 0.1 (pH 7.3 adjusted with CsOH). The pipette solution for recording IK (outward delay rectifier potassium current) contained (in mM): potassium aspartate 80.0, KCl 42.0, KH2PO4 10.0, Mg2+-ATP 10.0, phosphocreatin 3.0, K+-EGTA 5.0 (pH 7.2–7.4 adjusted with KOH). The Kreb's solution used for cell storage contained (in mM): KOH 70.0, l-glutamic acid 50.0, KCl 40.0, taurine 20.0, KH2PO4 20.0, MgCl2·6H2O 3.0, glucose 10.0, HEPES 10.0, EGTA 0.5 (pH 7.4 adjusted with KOH).Ca−1.PDQ was dissolved in 0.1% DMSO, the final concentration was 200 μg mL2.5−1, intraperitoneally), and the heart was excised and fixed in a Langendorff apparatus. The Ca2+ Tyrode's solution was used to retrograde perfusion in the heart for the first 2 min, then the Ca2+-free Tyrode's solution for 5 min, and at last the enzymatic solution (15 mg collagenase in 100 mL Ca2+-free Tyrode's solution) for 10 min. Afterwards, the ventricular muscle were cut into small pieces (1 mm3) and lightly oscillated for 10 min at 37 °C. Ventricular myocytes were filtered through nylon mesh and stored in Krebs solution at 4 °C. The perfusate was bubbled with 95% O2 + 5% CO2 at a flow of 8 mL min−1 at 37 °C. Ventricular myocytes from Wistar rats were used to measure the L-type calcium currents, while the myocytes of adult guinea pigs were used to measure the action potential of ventricular myocytes and the potassium currents.Wistar rats/adult guinea pigs were anesthetized with sodium phenobarbital  and linked to a 12 bit analog to digital converter (AD/DA) card  at 3 KHz. pCLAMP 6.03 software (Axon Instruments) was applied to generate voltage clamp protocols and retrieve data. Clampfit 6.02 software  was used to analyze the current traces.2.7ICa,L and IK, a molecular docking study was carried out to measure the relative binding energies and localized binding sites in the active pocket. The study was performed using GLIDE (Grid-based Ligand Docking with Energetics)  software developed by Schrödinger. Maestro Elements (2015-2) was used for all the steps involving protein and ligand preparation, receptor grid generation, and docking. The X-ray crystal structure of calcium channel [Protein Data Bank (PDB) code: 4TMF] and potassium channel (PDB code: 2A79) were retrieved from the PDB database (http://www.rcsb.org/pdb) according to previous studies.22,23 The Protein Preparation Wizard in the GLIDE software was used to prepare the receptors. The structures of proteins were optimized after a series of processes, including assigning bond orders and water orientations, removing water, adding hydrogen, and creating zero-order bonds to metals and di-sulphide bonds.24 Two-dimensional structure of PDQ (ligand) were drawn with Maestro Elements . LigPrep module (2015-2) of Schrödinger Suite was used to generate the three-dimensional structure of PDQ by assigning the bond orders and angles. In addition, the ligand was subjected to minimization using the OPLS3 force field. For GLIDE docking, the prepared structure of calcium channel, potassium channel, and ligand (PDQ) were imported into the workspace using GLIDE v6.7 from Schrödinger Suite.25–27 Extra precision (XP) docking was carried out, and the parameters of scaling factor and partial charge cutoff were set at the default values 0.80 and 0.15, respectively.28 Figures of the docking results were subsequently prepared using PyMOL (Schrödinger).To clarify the mode of action of PDQ on 3.p < 0.05 was considered statistically significant.All data are expressed as the mean ± standard deviation. GraphPad Prism 7.0 software was applied to analyze the data. Data analyses were performed using one way ANOVA. 4.4.1p < 0.01). As for both of the positive control group and H-PDQ group, the survival time were 600 s, which showed significantly difference compared with model group (p < 0.01), which was similar to the positive group  was 40%, while the mortality rates of low-, medium- and high-doses of PDQ  were 20%, 10%, and 0% respectively . In addiel group . Moreoveve group . PDQ red4.250) between PDQ treated groups and normal group. However, 2.0 μg mL−1 (p < 0.05) and 20.0 μg mL−1 PDQ (p < 0.01) significantly prolonged the APD at 90% repolarization (APD90). In addition, 20.0 μg mL−1 PDQ remarkably reduced the action potential amplitude (APA) (p < 0.001) versus the control group. The absolute values of resting potential (RP) were significantly reduced by 2.0 μg mL−1 PDQ and 20.0 μg mL−1 PDQ treatment. The results were shown in PDQ pre-treatment has the tendency of prolonging action potential duration (APD) with the increase of dosage. There is no significant difference for APD at 50% repolarisation  in ventricular cells. Flip potential was at +50 mV to +60 mV, the maximum ICa,L  was −3.64 ± 0.35 pA pF−1. The current was sensitive to verapamil, indicating that it was L-type Ca2+ current. The average maximum ICa,L of 0.2, 2.0 and 20.0 μg mL−1 PDQ were −3.64 ± 0.34 pA pF−1, −3.03 ± 0.27 pA pF−1 (p < 0.05) and −2.91 ± 0.24 pA pF−1 (p < 0.01) vs. −3.64 ± 0.35 pA pF−1 in control group  in ventricular cells within 400 ms. In the present study, IK was activated at −30 mV in the normal group, and the maximum peak outward current was 16.67 ± 1.78 pA pF−1 at +50 mV. After adding tetraethylammonium (TEA), the potential amplitude was inhibited, indicating it was IK current. After perfusion for 3 min using different concentrations of PDQ, the peak current at +50 mV of 0.2, 2.0 and 20.0 μg mL−1 PDQ were 15.63 ± 0.36 pA pF−1, 14.63 ± 0.92 pA pF−1 (p < 0.05) and 14.15 ± 1.12 pA pF−1 (p < 0.05) vs. 16.67 ± 1.78 pA pF−1 in control group. Current–voltage curve moved downward, suggesting that PDQ can inhibit IK in a dose-dependent pattern  in 10 mV increments in ventricular cells. Then it reached a steady state, and didn't change over time, showing inward rectification characteristics. Flip potential was about −40 mV, and the maximum IK1 was −23.12 ± 3.55 pA pF−1 at −100 mV. The IK1 of 0.2, 2.0 and 20.0 μg mL−1 PDQ were −22.35 ± 3.58 pA pF−1, −20.55 ± 3.94 pA pF−1 and −19.71 ± 2.80 pA pF−1, which showed no significant difference compared with the normal group, indicating PDQ had no effect on IK1 . PDQ formed one hydrogen bond at a distance of 1.9 Å with the residue (GLU-226), and 2.4 Å with the residue (THR-221). Docking calculation predicted three hydrogen bonds forming between PDQ and ASN-158 and ARG-189 of the potassium channel  and Key Technologies Research and Development Program (No. 2017YFC1702105).The authors declare that they have no conflict of interest concerning this article.RA-010-D0RA01683G-s001RA-010-D0RA01683G-s002RA-010-D0RA01683G-s003RA-010-D0RA01683G-s004


In addition, mitochondrial production of free radicals, membrane depolarization, decreased basal respiration, and ATP production were inhibited by dutasteride, while finasteride had no effect. In conclusion, the present results indicate that dutasteride treatment prevents enteric neuronal damages in the MPTP mouse model, at least in part through anti-inflammatory and mitochondrial effects. This suggests that drug repurposing of dutasteride might be a promising avenue to treat enteric neuroinflammation in early PD.Gastrointestinal disorders in Parkinson’s disease (PD) have been associated with neuronal alteration in the plexus of the gut. We previously demonstrated the immunomodulatory effect of female hormones to treat enteric neurodegeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. This study made the hypothesis of obtaining similar neuroprotection as with hormone treatments by affecting steroidogenesis with two 5α-reductase inhibitors, finasteride and dutasteride. These drugs are approved to treat benign prostatic hyperplasia and alopecia and display mitochondrial effects. In MPTP-treated mice, the dopaminergic and vasoactive intestinal peptide (VIP) neurons alteration was prevented by finasteride and dutasteride, while the increase in proinflammatory macrophages density was inhibited by dutasteride treatment but not finasteride. NF-κB response, oxidative stress, and nitric oxide and proinflammatory cytokines production  Several studies have demonstrated the bidirectional connection between brain and gut . Hence, Inflammation also plays a major role in the degeneration of dopamine (DA) neurons in the development of PD . An incr+) inhibits complex I of the mitochondrial respiration chain, reducing ATP production and increasing the presence of oxidative stress , many studies have been performed to understand the sexual differences in this disease . It has in vitro.It is well known that hormonal therapies induce various undesirable side effects  . In ordeTen weeks old C57BL/6 male mice were purchased from Charles River Canada . Animals were housed in cages under standard laboratory conditions, had access to food and water, and were acclimatized to a controlled temperature environment maintained under a 12-h light/dark cycle. Mice were handled in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, using a protocol approved by the Laval University Animal Care Committee. All efforts were made to minimize animal suffering and to reduce the number of mice used.Mice were divided into six groups comprising 10 mice per group. Mice were treated once daily (s.i.d.) with vehicle , finasteride , or dutasteride  for 10 days. On Day 5, mice received four injections of MPTP  at 2-h intervals, whereas the control groups received a saline solution. On Day 11, mice were anesthetized with isoflurane and killed to collect the brain and gut. The effects of finasteride and dutasteride treatment were previously reported to be neuroprotective against MPTP in the brain of these mice .Guts collection and preparation method was previously described . BrieflyAs previously reported, DA neurons and macrophages of the myenteric plexus were identified on free-floating sections following immunohistochemistry . BrieflyVIP intensity and proinflammatory macrophage density were measured using an immunofluorescence-based approach. Free-floating tissues were incubated overnight at room temperature with a monoclonal rat antibody for the polyclonal rabbit VIP antibody  or the major histocompatibility complex (MHC) class II molecule . Myenteric plexus were stained with a donkey anti-rat secondary antibody conjugated to Alexa Fluor 488  the next day. Nuclei were counterstained with 0.022% DAPI (Invitrogen). Images were taken using a confocal laser-scanning microscope  with a two-frame Kalman filter and several focal plans were acquired with a ×20 objective lens to generate maximum intensity projections.+ and Iba-1+ cell bodies were counted only if they did not cross the forbidden contours. Densities were calculated as the number of cells divided by the traced area (in mm2). MHCII and VIP quantifications were performed using the NIH ImageJ software . Section contours were also traced as a virtual overlay on images with the freehand selection option. VIP immunofluorescent staining quantification was executed on gray scale values images and measured by the mean pixel intensity. MHCII+ cell count was performed using the embedded cell counter plugin and densities were calculated as the number of cells per area unit (in mm2). All figures were assembled using Adobe Illustrator CS3.Stereological analyses were performed with the Stereo Investigator software 6.0  operated by an observer (blind to treatments), as previously described . The con2 in RPMI 1640 (Sigma) supplemented with 10% (v/v) heat-inactivated fetal bovine serum , 100 μg/ml Normocin (InvivoGen), 100 units/ml penicillin/streptomycin (Sigma), and 200 μg/ml of Zeocin (InvivoGen). Cell density was maintained <2 × 106 cells/ml and used before passage 20.Three different immortal cell types were used in this study: human neuroblastoma SH-SY5Y cells , human monocytic THP-1 cells (ATCC), and THP1-XBlue cells . These cell lines were grown at 37°C in a humidified incubator of 5% COin vitro treatments were performed as described previously  to induce their differentiation. Cell medium was changed before incorporating finasteride (1 µM) or dutasteride (1 µM) to the cells for 24 h at 37°C; the doses of finasteride and dutasteride were as used by +; 500 µM; Sigma-Aldrich Chemical) was added to cell media for 18 h. Supernatants were used for nuclear factor-kappa B (NF-κB), IL-1β, IL-6, and nitrite assays, whereas cells were used for the MTT viability test, cellular oxidative stress, mitochondrial free radicals production, mitochondrial membrane potential measurements, and seahorse analysis.Experiments with eviously . Cells w2 at 37°C, allowing them to form the blue formazan product. After the removal of 75 µl of cell supernatant, 50 μl of 100% DMSO was added and plates were then gently swirled for 10 min at room temperature to dissolve the precipitate. Optical density (OD540) was monitored using a spectrophotometer .Cell viability test was performed by adding 100 μl of a 12-mM sterile filtered 3--2,5-diphenyl-tetrazolium bromide  solution to each well. Cells were further incubated for 4 h in 5% CO+ incubation, THP-1 supernatants were added with equal volumes of sulfanilamide solution (1% sulfanilamide in 5% H3PO4) for 10 min, and then, 0.1% of N-(1-napthyl) ethylenediamine dihydrochloride in water for an additional 10 min. Subsequently, optical density (OD535) was measured using a spectrophotometer (Biotek).Griess reagent  was used to determine nitric oxide (NO) contractions in the supernatants. Following treatments and MPPTHP1-XBlue cells (InvivoGen) are THP-1 cells stably transfected with an NF-κB/AP-1 inducible reporter construct expressing a secreted embryonic alkaline phosphatase (SEAP) for monitoring NF-κB and AP-1 activation. After treatments and incubation, NF-κB activity was determined by adding QUANTI-Blue (InvivoGen) as a substrate of SEAP in the supernatants. Further incubation was pursued for 8 h at 37°C. The absorbance was then measured at 633 nm by spectrophotometry.Levels of IL-1β and IL-6 proinflammatory cytokines were detected with enzyme-linked immunosorbent assays (ELISAs). The cytokines concentrations of treated and untreated THP-1 supernatants were measured in triplicate using the Human Quantikine ELISA kit . IL-1β and IL-6 levels were measured using a spectrophotometer. Data at 540 nm were subtracted from the data at 450 nm, according to the manufacturer’s instructions.+ as described previously . Cells were then washed twice and resuspended in ice-cold PBS for flow cytometry analysis (488 nm excitation wavelength with 585/42 nm bandpass filter).Cellular oxidative stress was quantified by flow cytometry with CellROX Orange reagent (Thermo Fisher Scientific), a membrane-permeable reactive oxygen species (ROS)-sensitive probe. After plating THP-1 or SH-SY5Y cells, they were incubated with PMA and treated with finasteride, dutasteride, MPPeviously . Then, 5+ (500 µM) with 5 µM MitoSox Red diluted in Dulbecco’s phosphate-buffered saline (D-PBS) was added to the cell media for 6 h (37°C in the dark). This combination avoids the MitoSOX Red saturation due to continuous ROS accumulation in the mitochondria. The incubation time of 6 h was determined by a time course experiment. At least 1 × 106 cells/treatment were mechanically removed from the plate and were collected by centrifugation (500g for 5 min). Cells were then washed twice and resuspended in ice-cold PBS for flow cytometry analysis (488 nm excitation wavelength with 585/42 nm bandpass filter).Mitochondrial free radicals were measured using MitoSOX Red (Thermo Fisher Scientific), a fluorescent probe specific for mitochondrial superoxide. THP-1 cells were plated and incubated with PMA for 48 h as described in + was added to cell media for 6 h. This time point was determined by a time course experiment. Cells were incubated in the dark at 37°C with JC-1 dye (5 μg/ml) 30 min before the experiment, according to the manufacturer’s instructions. At least 1 × 106 cells/treatment were mechanically removed from the plate and were collected by centrifugation (500g for 5 min). THP-1 cells were then washed twice and resuspended in ice-cold PBS for flow cytometry analysis (488 nm excitation wavelength with 530/30 nm and 585/42 nm bandpass filters).Flow cytometry was also used to analyze mitochondrial membrane potential with the fluorescent probe JC-1 (Thermo Fisher Scientific). This lipophilic cationic dye accumulates in mitochondria in a membrane potential-dependent manner. In cells with polarized mitochondria membrane, JC-1 selectively enters the mitochondria and forms orange-red aggregates. These punctate orange-red fluorescent staining are replaced by diffuse green monomers fluorescence in the presence of depolarized mitochondrial membrane. The red/green (FL2/FL1) ratio of the two fluorescence intensities allows the comparative measurement of mitochondrial membrane potential. THP-1 cells were plated and incubated with PMA for 48 h as described in All analyses were performed with BD FACSCantoII system  and the fluorescence properties of 10,000 cells were recorded for each sample. Data were analyzed using FlowJo version 8.7 . Results are expressed as number of positive cells or as mean fluorescence intensity (MFI) of 10,000 cells.4 THP-1 and 4.5 × 104 SH-SY5Y cells/well) and incubated with PMA for 48 h as described in + (125 µM on THP-1 and 50 µM on SH-SY5Y cells) was added to cell media for overnight incubation. Optimization of MPP+ doses was determined to identify the half maximal inhibitory concentration (IC50) and to choose the best concentration. Cells were washed and incubated in XF basal medium supplemented by 2 mM glutamine, 10 mM glucose, and 1 mM sodium pyruvate. Measurements of mitochondrial basal respiration and ATP production, following sequential injections of 1 µM oligomycin and 1 µM/0.1 µM rotenone/antimycin A, were performed as described previously  were performed using an XFe-96 Extracellular Flux Analyzer (Agilent/Seahorse Bioscience). THP-1 or SH-SY5Y cells were seeded to confluence in XF cell plates  followed by a Bonferroni post-test . All treatment comparisons were performed with GraphPad Prism 7.0 . Data are presented as group mean ± SEM in all histograms. Statistical significance was determined with a threshold value of p < 0.05.Statistical analyses of in vivo experiments were performed using a one-way analysis of variance (ANOVA) followed by a Bonferroni post-test , while + neurons density immunochemistry staining (brown DAB staining) were measured in the myenteric plexus of distal ileum of MPTP lesioned mice to seek their possible neuroprotection by 5α-reductase inhibitors. + neuron density  = 8.267, p < 0.0001) (+ neuron density. Finasteride treatment at the higher dose (investigated at 12.5 mg/kg) but not at the lower dose (5 mg/kg) prevented the MPTP-induced loss of TH+ neurons , the TH+ neuron density was neither different from controls nor vehicle-treated MPTP-lesioned mice  and TH 0.0001) . The MPT neurons . Dutastened mice .F = 6.607; p < 0.0001)  and dutasteride treatment at 5 mg/kg in MPTP mice  . As for PTP mice . Also, aPTP mice . MoreovePTP mice .F = 7.065; p < 0.0001) (MPTP-lesioned mice exhibit an activation of the innate immune response due to infiltration of macrophages in the myenteric plexus . We prev 0.0001) . The MPT 0.0001) . Dutaste 0.0001) .F = 11.09; p < 0.0001)  . Similar 0.0001) . Dutaste 0.0001) .in vitro with the THP1-XBlue cell line with a NF-κB reporter gene. We previously showed that the MPTP metabolite, MPP+, induces an inflammatory response that upregulates the NF-κB response  = 0.5591, p = 0.4604; effect of the 5α-reductase inhibitors treatments: F = 0.7128, p = 0.4984; their interaction: F = 0.1846, p = 0.8324)  = 10.46, p = 0.0030; effect of the 5α-reductase inhibitors treatments: F = 15.01, p < 0.0001; interaction: F = 8.912, p = 0.0009) (+ (+ (+ mice left the NF-κB response at a level similar to the control mice (The anti-inflammatory activity of the 5α-reductase inhibitors was next investigated response . NF-κB a(+ toxin . THP1-XB 0.8324) . However 0.0009) . The NF-0009) (+ . Finaste9)  = 0.8832, p = 0.3658; effect of 5α-reductase inhibitors: F = 1.759, p = 0.2139; their interaction: F = 0.01573, p = 0.9844)  = 781.1, p < 0.0001; effect of 5α-reductase inhibitors: F = 168.7, p < 0.0001; their interaction: F = 195.3, p < 0.0001)  = 58.74, p < 0.0001; effect of 5α-reductase inhibitors: F = 19.55, p < 0.0001; their interaction: F = 9.506, p = 0.0006)  = 60.55, p < 0.0001; effect of 5α-reductase inhibitors: F = 18.89, p < 0.0001; their interaction: F = 8.109, p = 0.0015)  = 119.8, p < 0.0001; effect of 5α-reductase inhibitors: F = 73.44, p < 0.0001; their interaction: F = 19.38, p = 0.0002) , interleukin-1β (IL-1β), and interleukin-6 (IL-6) productions were measured in the supernatant of THP-1 monocytic cells treated with finasteride or dutasteride in the presence or the absence of MPP+ . THP-1 m 0.9844) . However 0.0001) , IL-1β c 0.0006) , IL-6 co 0.0015) , and the 0.0002) . NO accu 0.0002) , IL-1β c 0.0002) , IL-6 co 0.0002) , and the0002)  was quantified by Seahorse assays. Dose–response of MPP+ in THP-1 and SH-SY5Y cells are shown in +, the middle panel reported fluorescence intensity in the presence of finasteride showing a change compared to MPP+, and the right panel shows that dutasteride prevented the MPP+-induced fluorescence shift. The MPP+ effect on this fluorescence corresponds to an increase in mitochondrial oxidative stress production that was opposed by dutasteride but not by finasteride. + and finasteride + MPP+ treatments, led to a different pattern of depolarized membranes compared to the controls. By contrast, control, dutasteride, and dutasteride + MPP+ treatments showed similar patterns indicating that dutasteride opposed the effect of MPP+ and kept membranes more polarized. Histograms in + and finasteride and dutasteride treatments  = 236.2, p < 0.0001; effect of 5α-reductase inhibitors: F = 95.78, p < 0.0001; their interaction: F = 92.64, p < 0.0001; ATP production: effect of MPP+: F = 289.4 p < 0.0001; effect of 5α-reductase inhibitors: F = 113.7, p < 0.0001; their interaction: F = 151.7 p < 0.0001). MPP+ significantly reduced basal respiration and ATP production of THP-1 cells. Finasteride or dutasteride treatments alone left these parameters at control values. Finasteride did not oppose the effect of MPP+, whereas dutasteride completely prevented the MPP+-induced reduction of basal respiration and ATP production.Three parameters of mitochondrial function were evaluated in THP-1 cells exposed or not to MPP+ and the finasteride or dutasteride treatments  = 351.2, p < 0.0001; effect of 5α-reductase inhibitors: F = 18.57, p < 0.0002; their interaction: F = 1.694 p = 0.2249; +: F = 170.5 p < 0.0001; effect of 5α-reductase inhibitors: F = 32.88, p < 0.0001; their interaction: F = 3.684 p = 0.0734; and +: F = 192.4 p < 0.0001; effect of 5α-reductase inhibitors: F = 34.53 p = 0.0001; their interaction: F = 6.389 p = 0.0220). The percentage of positive SH-SY5Y cells to the oxidative stress marker CellROX were increased in the presence of MPP+  and dutasteride (at the lower dose tested) can prevent neuronal damage in the gut. Total and proinflammatory enteric macrophages density increased by MPTP were prevented by the dutasteride treatment but not by finasteride. The effects of these 5α-reductase inhibitors were then investigated in two immortalized human cell lines. In the human monocytic cell line (THP1-XBlue cells containing NF-κB reporter gene), the MPP50  in the MPTP mouse model . In the MPTP reduced the androgens testosterone and dihydrotestosterone plasma levels of these mice . This re+ and VIP+ enteric neurons and a decrease in these enteric VIP+ neurons in MPTP mice (VIP is an inhibitory neuropeptide inducing colonic relaxation by secretory effects on the colonic epithelium and is well documented to have anti-inflammatory activities . In PD pPTP mice . In the High levels of proinflammatory markers have been reported many times in PD patients and in the MPTP model . We prev+ to model the mitochondrial respiratory dysfunctions in PD. Interestingly, we showed that the MPP+-induced mitochondrial impairment was prevented by dutasteride but not finasteride treatment in monocytic cells and not in human neuroblastoma SH-SY5Y cells.Defective mitochondrial activity was reported in PD, including an impairment of the mitochondrial respiratory chain . AccumulThe calcium-dependent formation of the mitochondrial permeability transition pore is considered to initiate the apoptotic pathway of various neurodegenerative conditions . MoreoveIn conclusion, this study showed that dutasteride treatment prevented enteric neuronal damages in the MPTP mouse model with a better efficiency than finasteride. A shorter half-life of finasteride compared to dutasteride could have contributed to its lower neuroprotective activity. We observed in this study that the 5α-reductase inhibitor could have anti-inflammatory effects by targeting the mitochondria of immune cells. This suggests that drug repurposing of dutasteride might be a promising prophylactic avenue in familial forms of PD to treat enteric neuroinflammation to delay disease progression. For future studies, finasteride and dutasteride should be also tested in synucleinopathy models of PD to better assess the therapeutic potential of these drugs in the etiopathogenesis of PD.


Fusarium culmorum is a major pathogen of grain crops. Infected plants accumulate deoxynivalenol (DON), 3-acetyl-deoxynivalenol (3-ADON), or nivalenol (NIV), which are mycotoxins of the trichothecene B group. These toxins are also produced by F. graminearum species complex. New trichothecenes structurally similar to trichothecenes B but lacking the carbonyl group on C-8, designated NX toxins, were recently discovered in atypical isolates of F. graminearum from North America. Only these isolates and a few strains of a yet to be characterized Fusarium species from South Africa are known to produce NX-2 and other NX toxins. Here, we report that among 20 F. culmorum strains isolated from maize, wheat, and oat in Europe and Asia over a period of 70 years, 18 strains produced NX-2 simultaneously with 3-ADON and DON or NIV. Rice cultures of strains producing 3-ADON accumulated NX-2 in amounts corresponding to 2–8% of 3-ADON (1.2–36 mg/kg). A strain producing NIV accumulated NX-2 and NIV at comparable amounts . In F. graminearum, producers of NX-2 possess a special variant of cytochrome P450 monooxygenase encoded by TRI1 that is unable to oxidize C-8. In F. culmorum, producers and nonproducers of NX-2 possess identical TRI1; the reason for the production of NX-2 is unknown. Our results indicate that the production of NX-2 simultaneously with trichothecenes B is a common feature of F. culmorum. Fusarium head blight (FHB) is a cosmopolitan disease of small-grain cereals caused by several Fusarium species with a high economic impact TRI1 in aF. graminearum that facilitated the discovery of NX toxins was motivated by a failure to detect any trichothecene in a group of atypical strains of F. graminearum from the USA [F. graminearum accumulating NX-2 simultaneously accumulated 15-ADON. A search for new trichothecenes in  the USA ,33. Thes the USA  reportedF. culmorum strains in our study accumulated 3-ADON (most strains) or NIV (a single strain). In cultures of the 3-ADON chemotype, NX-2 accumulated at amounts corresponding to 2–8% of 3-ADON, and the content of NX-2 and trichothecenes B was tightly correlated . Reports . Fusarium strains producing DON, and substantial amounts of DON will be found in cultures of all strains producing NIV. We anticipate that with the widespread use of sensitive analytical methods, small amounts of NIV will often be found in cultures of Fusarium culmorum produce NX-2 toxin simultaneously with deoxynivalenol, 3-acetyldeoxynivalenol, or nivalenol. Strains producing NX-2 do not possess a specific variant of the TRI1 gene known from NX-2-producing strains of F. graminearum. Most strains of F. culmorum are listed in The strains of TEF-1α and RPB2 were amplified, and the PCR products subjected to high-resolution melting curve (HRM) analysis as described previously [TRI1 gene was amplified for sequencing as four overlapping fragments  adjusted to a 2 mM final MgCl2 concentration containing 100 µM of each deoxyribonucleoside triphosphate , 0.3 µM of each primer, 0.62 U HotStart-polymerase , and 1 µL of template DNA solution diluted 100 times. The thermocycling conditions are specified in Fungal DNA was extracted using a CTAB-based protocol  from 10 eviously . The TRIragments , which wTRI1 genes in Fusarium spp. were investigated using maximum-likelihood analysis using MEGA X under the assumption of Tamura’s and Nei’s substitution model [The sequences were quality-trimmed with Chromas version 2.6.6  and assembled to full-length gene sequences; the accession numbers are listed in on model .v/v/v)) overnight. Extracts were dried in a vacuum concentrator  and residues re-dissolved in methanol/water (20:80 (v/v)) as described previously [v/v), and phase B was methanol with 0.1% formic acid (v/v). The gradient was as follows: 0 to 0.2 min, 5% B; 0.2 to 8 min, 5% to 35% B; 8 to 8.5 min, 35% to 98% B; 8.5 to 12 min, 98% B; 12 to 12.5 min, 98% to 5% B; 12.5 to 16 min, 5% B. The calibration curve included 11 concentrations from 0.48 to 500 μg/L. A blank was analyzed after every 7th sample and a quality control standard after every 15th sample. The metabolites were detected in a multiple reaction monitoring (MRM) mode. The acquisition parameters and the limits of detection (LOD) and quantification (LOQ) are listed in Rice cultures were prepared in 50-mL Falcon tubes  by autoclaving 3 g of dry polished rice with 5 mL of tap water. The tubes were inoculated with plugs of PDA (0.5 cm diameter) overgrown with 5-day-old mycelium. Rice medium incubated with agar plugs without mycelium served as a control. The cultures were incubated for 21 days at 25 °C in the dark. Fungal metabolites were extracted by shaking with 30 mL acetonitrile/water/acetic acid  from 5-day-old fungal cultures. Rice cultures were incubated at 25 °C for 3 weeks in the dark. NX-2 was extracted with a 5-time excess of methanol/water/acetic acid (90:9:1) by shaking overnight. The extract was concentrated to approximately 15% of its original volume using a rotary evaporator R-100 . The concentrated extract was partitioned with the same volume of ethyl acetate (EtOAc) three times. Combined EtOAc fractions were dried in a rotary evaporator. The residue was dissolved in methanol/water (30:70) and cleared by centrifugation. The supernatant was subjected to flash chromatography  equipped with a binary pump (modules C-601/C-605) and a UV detector (module C-635). Metabolites were separated on a reverse-phase column . Mobile phase A was water with 0.2% acetic acid; phase B was methanol with 0.2% acetic acid. The gradient elution program was as follows: 0–2 min, 5% B; 2–42 min, from 5% to 50% B; 42–45 min, from 50% to 98% B; 45–55 min, 98% B; 55–58 min, from 98% to 5% B; 58–66 min, 5% B. The flow rate was 50 mL min−1. The separation was monitored at 254 nm. Fractions containing NX-2 were collected at 33 min and the solvent was evaporated in a rotary evaporator. The residue was re-dissolved in methanol/water (30:70) and subjected to a preparative-HPLC  equipped with a preparative reverse-phase column  and coupled to a UV/VIS detector . NX-2 was eluted by a gradient of water with 0.2% acetic acid (v/v) (solvent A) and methanol with 0.2% acetic acid (solvent B) as follows: 0–5 min, 5% B; 5–50 min, from 5% to 50% B; 50–55 min, from 50% to 98% B; 55–70 min, 98% B; 70–75 min, from 98% to 5% B; 75–90 min, 5% B. The injection volume was 3 mL, and the flow rate was 14 mL min−1. The separation was monitored at 254 nm. NX-2 was collected at 43 min and solvent was evaporated in a vacuum concentrator . NX-2 was further polished on Sephadex LH-20  in a 460 × 15 mm column eluted isocratically with methanol at a flow rate of 1 mL min−1. The eluent was monitored at 254 nm. 1H and 13C) NMR spectra were obtained from Bruker Avance 500 Hz and 2D  NMR spectra were obtained from an Agilent DD2 400 MHz system. The spectra were recorded at 500/400 MHz (1H) and 126/101 MHz (13C). Chemical shifts were referenced to internal TMS  or MeOH-d4 . The data are provided as 1D (


Mr, of 2.0~3.0 kG, and relatively large ferromagnetic resonance (FMR) linewidths, ΔFMRH, of 0.8~2 kOe. These properties lead to the development of high-performance, practical devices. Herein, crystallographically textured Ba-hexaferrite samples, of the composition Ba0.8La0.2Fe11.8Cu0.2O19, having excellent functional properties, are proposed. These materials exhibit strong anisotropy fields, Ha, of ~14.6 kOe, high remanent magnetization, 4πMr, of 3.96 kGs, and a low ΔFMRH of 401 Oe at zero-bias field at the Q-band. Concomitantly, the broadband millimeter-wave transmittance was utilized to determine the complex permeability, μ*, and permittivity, ε*, of textured hexaferrites. Based on Schlöemann’s theory of complex permeability, μ*, the remanent magnetization, 4πMr, anisotropy field, Ha, and effective linewidth, ΔeffH, were estimated; these values agree well with measured values.Excellent gyromagnetic properties of textured, bulk Ba-hexaferrite samples are required for low-loss, self-biased applications for microwave and millimeter-wave (MMW) devices. However, conventionally processed bulk Ba-hexaferrite ceramics typically demonstrate low remanent magnetization values, 4π Specifically, M-type Ba-hexaferrites  have attracted much attention for applications in monolithic-microwave-integrated circuits (MMIC) due to their strong and adjustable Ha, that acts as an effective internal field [Ferrite materials provide the necessary magnetic media to break time-reversal symmetry, allowing non-reciprocal behavior to passive control elements, such as circulators and isolators ,2,3. Desal field ,5,6,7.rM, and narrow ferromagnetic resonance linewidths, ΔFMRH, allow for the reduction in resonance losses [aH. These properties allow for the RF engineer to design the operational frequency  and device performance based on material properties. rM, and FMR linewidth, ΔFMRH, of bulk Ba-hexaferrites [Mr of 1.5~3.5 kGs and broad ΔFMRH of 0.5 to 2 kOe. Herein, we demonstrate crystallographically textured hexaferrites with concomitant high 4πrM and low ΔFMRH.Broadband, low-loss properties are prerequisites for high-performing, high-frequency, and self-biased MMW devices based on crystallographically textured Ba-hexaferrites . Improvee losses  and optiferrites ,15,16,17μ*, and dielectric permittivity, ε*, are two fundamental parameters that determine the response of ferrites to high-frequency electromagnetic fields. We employed high-Q resonant measurements to determine dielectric and magnetic properties at microwave frequencies [Furthermore, dynamic magnetic permeability, quencies . The accquencies .μ*, complex permittivity, ε*, and effective linewidth, ΔHeff, are determined by the magneto-optical measurements.Basic operational principles of MMW devices require the thickness of crystallographically textured ferrites often exceeding 100 μm , making 3+ ions are mainly substituted Ba2+ ions, and Cu2+ ions reside on the 2b and 4f2 sites  to produce a crystallographically textured Ba-hexaferrite cylindrical green compact, with the c-axes of Ba-hexaferrites particles aligned mostly perpendicular to the plane of the cylinder. The specimens were then sintered at 950~1050 °C for 2 h.Previous calculations  of densiites see b. IntrodKα radiation source. The cross-section morphology was observed using a field emission scanning electron microscope . The bulk density, d, was measured using a Micromeritics AccuPyc 1330 pycnometer. Room-temperature magnetic properties were determined by vibrating sample magnetometry  with a maximum applied magnetic field of 20 kOe. The absorbed power spectra were characterized by the TE10 rectangular transmission cavity perturbation at Q-band (30~50 GHz) [The crystallographic structure was determined by X-ray diffraction  using a Cu-~50 GHz) . BroadbaP63/mmc. The peaks perpendicular to the sample plane, corresponding to the c-axes reflections of (006), (008), and (0014), have the highest intensity. However, other weaker peaks are also present, namely (107), (206), and (220). As a reliable way to evaluate the degree of crystalline orientation, the Lotgering factor, fL, was calculated [I(00l)/∑I(hkl) is calculated based on the experimental data for crystallographically textured samples, and ∑I0(00l)/∑I0(hkl) is based on the XRD data from the JCPDF card. The evaluated degree of crystalline orientation, fL, for Ba0.8La0.2Fe11.8Cu0.2O19 samples reaches 83%, indicating the degree of the c-axis-preferred orientation.06), and 0. As a r0.8La0.2Fe11.8Cu0.2O19 sample demonstrates a microstructure consisting of hexagonal particles compactly aligned parallel to the c-axis. It is crucial to control the grain size during processing of textured samples in order to attain high remanent magnetization, 4πrM. We measure grain sizes to range from 1 to 2 μm, with an average grain size of 1.2 μm. This value lies within the estimated single domain critical size range of 1.0–1.5 μm [d, of the as-sintered Ba0.8La0.2Fe11.8Cu0.2O19 specimen was measured to be 97.5% of the theoretical density, ~5.28 g/cm3 [2O3 and CuO, establish a liquid channel at the grain boundaries. It is speculated that this structure promotes densification and grain alignment during sintering. Yet, some pores are still visible in The cross-sectional morphology of crystallographically textured Ba-hexaferrites is presented in 0–1.5 μm . Additio28 g/cm3 . As indi28 g/cm3 ,26, bismM versus H) of a crystallographic and magnetically textured Ba0.8La0.2Fe11.8Cu0.2O19 sample. The kink in the out-of-plane loop evolves slowly, thus indicating the strong c-axis orientation in the textured Ba0.8La0.2Fe11.8Cu0.2O19 sample. Because of a preferred orientation grain in the sample, the hysteresis loops between the in-plane and out-of-plane loops are obviously different. The reason can be explained that the magnetization prefers the direction of the c-axis. Thus, the magnetization of the sample with an external field applied in c-axis direction is larger than perpendicular to c-axis under same magnetic field. To describe obvious divergences between the in-plane and out-of-plane loops, the anisotropy field, aH, was obtained by the singular point detection (SPD) method [2M/dH2) of initial magnetization curve measured perpendicular to the direction of easy magnetization [K1 was calculated from the basic equation aH = 2K1/Ms. Representative magnetic properties are summarized in 0.8La0.2Fe11.8Cu0.2O19 samples show a strong uniaxial magnetic anisotropy with aH values of ~14.6 kOe and anisotropy constant K1 values of 2.52 × 105 J/m3. The measured 4πMr of 3.96 kGs is superior to previously reported results for bulk BaM  method , which wtization . Subsequ BaM see a. This iith Ref. . Such a  magnets ,15.H, ranging from 0 to 1 kOe, applied parallel to the cylinder axes. As depicted in f denotes the half width at half maximum (HWHM), and resf is the ferromagnetic resonance frequency. symL and asymD are symmetric and asymmetric contributions to the frequency, respectively. The corresponding ΔFMRH of the textured Ba0.8La0.2Fe11.8Cu0.2O19 sample was evaluated according to the equation ΔFMRH = 2 × Δf/γ and listed in H = 0 and 1 kOe, respectively. According to the Kittel formula [aH, obtained from FMR measurements, was extrapolated to be 15.1 ± 0.2 kOe, which is in close agreement with the value measured from the SPD method, 14.6 kOe. It is noteworthy that the crystallographically textured Ba0.8La0.2Fe11.8Cu0.2O19 sample exhibited a narrow ΔFMRH of 401 Oe at zero-bias field, an improvement of over 20% compared to the previous values for bulk BaM :P corresponds to porosity. This evaluation indicates 80~85% of the total FMR linewidth is determined by the porosity contribution. In other words, a relatively high density of 97.5% could allow for an even lower FMR linewidth in textured Ba0.8La0.2Fe11.8Cu0.2O19 samples.In order to probe the sources of the FMR linewidth, Δibutions .(3)ΔHFMm et al.  speculatchlömann ,32, base0.8La0.2Fe11.8Cu0.2O19 samples is presented as T, R, n, k, μ*, and ε* are the transmittance, reflectance, refractive index, absorption index, complex permeability, and complex dielectric permittivity, respectively. The dielectric permittivity, ε*, typically exhibits a very weak frequency dependence in the millimeter range, and therefore was reasonably assumed to be constant. By fitting the transmission spectrum far from the absorption region, it was deduced that the textured Ba0.8La0.2Fe11.8Cu0.2O19 sample had a real permittivity ε’ of 23, and an imaginary permittivity ε” of 0.29. The complex magnetic permeability was estimated based on the magnetocrystalline anisotropy, aH, and remanent magnetization, 4rπM [af* = (γ/2π)aH + jfG, and fM4πr = 2γ(4πrM) with γ being the gyromagnetic ratio, and G being the damping parameter. The fitting of the experimentally observed spectrum was performed by using Equation (6). The significant discrepancy between the experimental and simulated results for the resonance absorption region is attributed to the considerable contribution of “non-intrinsic” relaxation. The best-fit dissipation leads to aH = 15.4 kOe, 4πrM = 4.02 kGs, and G = 0.0011 far from the resonance. These values of aH and 4πrM agree well with those obtained from VSM and FMR measurements. According to the equation ΔeffH = 2πG·f/γ [effH, equals 108 Oe. This narrow ΔeffH is beneficial for the design of MMW devices that operate far from the FMR frequency.The millimeter-wave transmission spectrum of textured Baon, 4πMr :(6)μ∗/μ0 2πG·f/γ , the eff0.8La0.2Fe11.8Cu0.2O19 ferrites having both strong crystallographic and magnetic texture were successfully fabricated by employing conventional ceramic processes. These ferrites exhibit a strong crystallographic c-axis alignment, high sintering density, d, of 5.15g/cm3, strong anisotropy field, aH, of 14.6 kOe, and remanent magnetization, 4πrM, values up to 3.96 kGs. The corresponding FMR linewidth, ΔFMRH, with biasing fields, H, were measured to range from 0 and 1 kOe at the Q-band to 401 Oe and 379 Oe, respectively. Moreover, magneto-optical measurements allowed for the determination of the complex dielectric permittivity, ε*, permeability, μ*, and effective linewidth, ΔeffH. While the permeability, μ*, is strongly frequency-dependent, the real (ε’) and imaginary parts (ε”) of the dielectric permittivity, ε*, and effective linewidth, ΔeffH, are 23, 0.29, and 108 Oe away from resonance, respectively. These results indicate that Ba0.8La0.2Fe11.8Cu0.2O19 ferrites demonstrating both strong crystallographic and magnetic texture could be employed in self-biased and low-loss MMW devices, such as circulators or isolators.In summary, Ba