All compounds exhibittwo structural phase transitions, and the lowest temperature phasesof IMPbBr3 and IM3PbBr5 are noncentrosymmetric, as confirmed by measurementsof second-harmonic generation (SHG) activity. X-ray diffraction andthermal and Raman studies demonstrate that the phase transitions featurean order–disorder mechanism. The only exception is the isostructural P1̅ to P1̅ phase transitionat 141 K in IM2PbBr4, which is of a displacivetype. Dielectric studies reveal that IMPbBr3 is a switchabledielectric material, whereas IM3PbBr5 is animproper ferroelectric. All compounds exhibit broadband, highly shiftedStokes emissions. Features of these emissions, i.e., band gap and excitonic absorption, are discussed in relation tothe different structures of each composition.Hybrid organic–inorganiclead halide perovskites have emergedas promising materials for various applications, including solar cells,light-emitting devices, dielectrics, and optical switches. In thiswork, we report the synthesis, crystal structures, and linear andnonlinear optical as well as dielectric properties of three imidazoliumlead bromides, IMPbBr Three imidazolium lead bromides of variouschemical compositionsand crystal structures display broadband photoluminescence that canbe tuned from bluish-green to orange. All compounds exhibit two structuralphase transitions, which lead to interesting optical and electricalproperties such SHG activity, ferroelectricity, or dielectric switching. Three-dimensional (3D) lead halide perovskiteswere reported for only four organic cations: methylammonium (MA+),8 formamidinium(FA+),9 methylhydrazinium(MHy+),12 and aziridinium.13 Themost well-known MAPbI3 and FAPbI3 exhibit convenientsolution processability as well as great optical and electrical propertiesfor solar cell applications like narrow band gaps, high carrier mobility,and strong light absorption.9 On the other hand, recentlydiscovered 3D MHyPbBr3 exhibits four kinds of functionalproperties such as photoluminescence (PL), second-harmonic generation(SHG), two-photon excited PL, and switchable dielectric property,whereas MHyPbCl3 shows PL and quadratic nonlinear optical(NLO) switching between two SHG-on states.11In recent years, the field of hybrid organic–inorganicperovskiteshas become one of the most promising research directions for a varietyof optoelectronic applications like solar cells,16Since the 3D lead halide perovskites can be constructed foronlya few organic cations, a lot of attention has been paid to the low-dimensionalanalogues, which also exhibit unique optoelectronic parameters, butsurpass their 3D counterparts in terms of resistance to moisture andirradiation stresses.19 and ferroelectric20 materials.Furthermore, the desirability of lead bromide perovskites also comesfrom their interesting emission properties. For light-emitting devices,diverse distribution of emitting wavelengths and intense PL is required.The extant literature describes plenty of ways of creating wavelength-tunablematerials.16 One possibility of tuningthe emission wavelength is to synthesize lead halide perovskites comprisingtwo different halides, like in MAPbIx3–Brx or MHyPbBrx3–Clx.22 Another way is to decrease the dimensionality to 2D, one-dimensional(1D), or zero-dimensional (0D)24 or manipulate the thicknessof the inorganic slabs in 2D perovskites by changing the number ofinorganic bromide layers (n), like in (RNH3)2(MA)n−1PbnBrn+13 (RNH3 = long organic ammonium cation).25 It is worth mentioning that the type and energy of emissions bothdepend on the crystal structure. In this respect, three differenttypes of PL are recognized in lead halide perovskites, i.e., broadband PL with a large Stokes shift assigned to self-trappedexcitons (STEs), relatively narrow PL with a small Stokes shift dueto bound excitons (BEs), and a narrow PL explained by free excitons(FEs).28 Narrow PL attributed to FE and BE states is typically observed for3D perovskites and 2D analogues with the crystallographic orientation⟨100⟩,30 while broadband STE-related PLoften occurs in corrugated 2D structures (⟨110⟩ and⟨111⟩),30 as well as in 1D and 0D structures.33 However, PL related to STE states can also be observedfor some ⟨100⟩-oriented perovskites. For instance, bothFE- and STE-type PL was reported for (PMA)2PbBr4 (PMA = phenylmethylammonium) and MHy2PbBr4.34There is no denying that for photovoltaic applicationsiodide-basedperovskites are more prospective than bromide analogues mostly becauseof their narrow band gaps. Nevertheless, bromide-based perovskites,both 3D and two-dimensional (2D), were found to be attractive NLO37 Small IM+ cations can also be doped intoa 3D perovskite structure, enhancing the efficiency of solar cells.38 Regarding imidazolium lead bromides, only onecompound was reported in the literature with the chemical compositionIMPbBr3.39 Its crystal structure,solved at 173 K, is orthorhombic, space group Pnma, and it adopts a distorted 4H perovskite-like structure with face-and corner-sharing octahedra.39It is worth mentioning that due tothe hydrophobic properties ofimidazolium derivatives, they are used for improving the hydrophobicproperties of lead halide perovskites and thus their resistance tomoisture.3 andits optical and electrical properties are unknown, we decidedto conduct comprehensive thermal, single-crystal X-ray diffraction(SCXRD), Raman, optical, PL, and dielectric studies of this perovskite.We will show that IMPbBr3 undergoes two structural phasetransitions below room temperature (RT) and that this compound exhibitsintense red PL as well as switchable dielectric properties. We alsosuccessfully grew large crystals of previously unreported IMI2PbBr4 and IM3PbBr5 with uniquecrystal structures, which undergo structural phase transitions andexhibit broadband PL. Furthermore, IM3PbBr5 isa rare example of a 1D perovskite exhibiting polar order at low temperatures.Sincetemperature-dependent structural changes of IMPbBr2 98%, imidazolium99%, HBr 48 wt % in H2O) used for the synthesis were commerciallypurchased from Sigma-Aldrich and used without further purification.To grow single crystals of IMPbBr3, a solution was preparedby dissolving 5 mmol of imidazole and 5 mmol of PbBr2 inhydrobromic acid. The clear solution was obtained after stirring for20 min and then left for crystallization at room temperature (RT).The same method was used to crystalize IM2PbBr4 and IM3PbBr5 but with imidazolium/PbBr2 ratios of 2:1 and 4:1, respectively. The crystals were harvestedafter one week, decantated, and then dried at RT . The comparison of their powder X-ray diffraction(XRD) patterns with the calculated ones based on the single-crystaldata attests the phase purity of bulk samples .Allreagents (PbBr1 radiation (λ = 1.54056 Å).PowderXRD patterns were measuredin the reflection mode on an X’Pert PRO X-ray diffraction systemequipped with a PIXcel ultrafast line detector and Soller slits forCu Kα–1. Sample weights were 29.0, 31.3, and 26.8 mg forIMPbBr3, IM2PbBr4, and IM3PbBr5, respectively. The excess heat capacity associatedwith the phase transition was evaluated by subtracting from the datathe baseline representing the variation in the absence of the phasetransitions.Heat flow wasmeasured using a Mettler Toledo DSC-1 calorimeter with a high resolutionof 0.4 μW. The cooling and heating speed rate was 5 K min40 using SHELXT41 andrefined with SHELXL.42 For all structuresmeasured at 295 K or less, H-atom parameters were constrained, whilefor measurements at 415 K (IM2PbBr4) and 400K (IM3PbBr5), H atoms were not inserted dueto the dynamic disorder of IM+. The main experimental detailsfor all of the reported compounds are shown in Tables S1–S3, together with selected geometric (Tables S4–S6) and hydrogen-bonding (Tables S8–S9) parameters. The concisestructure details, i.e., symmetry, unit cell parameters,and refinement factors, are as follows.Single-crystal X-raydiffraction (SCXRD) experiments were carried out using an Xcaliburfour-circle diffractometer (Oxford Diffraction) with an Atlas CCDdetector and graphite-monochromated Mo Kα radiation. Absorptionwas corrected by multiscan methods using CrysAlis PRO 1.171.41.93a. Empirical absorption correctionusing spherical harmonics, implemented in the SCALE3 ABSPACK scalingalgorithm, was applied. Crystal structures were solved in Olex2 1.53 : hexagonal, P63/mmc, a = b = 9.0871(5)Å, c = 13.8501(7) Å, V = 990.45(12) Å3, Z = 4, R1 = 0.03, wR2 = 0.06, S = 1.08; : orthorhombic, P212121, a = 9.0432(8) Å, b = 13.7661(7) Å, c = 31.053(3) Å, V = 3865.8(5) Å3, Z = 16, R1 = 0.13, wR2 = 0.38, S = 1.05; : orthorhombic, Pna21, a = 14.2042(3) Å, b = 8.8719(2) Å, c = 14.9262(2), V = 1880.97(6) Å3, Z = 8, R1 =0.03, wR2 = 0.06, S = 1.09.IMPbBr2PbBr4 : orthorhombic, Cmmm, a = 9.2682(8) Å, b = 27.049(4) Å, c = 6.1301(5) Å, V = 1536.8(3) Å3, Z = 4, R1 =0.05, wR2 = 0.12, S = 1.05; : triclinic, P1̅, a =6.0676(2) Å, b = 9.3485(4) Å, c = 13.7370(7) Å, α = 74.336(4)°, β = 87.025(3)°,γ = 88.612(3)°, V = 749.21(6) Å3, Z = 2, R1 =0.03, wR2 = 0.08, S = 1.08; : triclinic, P1̅, a = 6.0086(2) Å, b = 9.4176(3) Å, c = 13.4597(5) Å,α = 73.804(3)°, β = 86.516(2)°, γ = 87.157(2)°, V = 729.64(4) Å3, Z = 2, R1 = 0.02, wR2 = 0.06, S = 1.07.IM3PbBr5 : orthorhombic, Cmmm, a = 9.2708(18) Å, b = 18.346(4)Å, c = 6.1547(8) Å, V =1046.8(3) Å3, Z = 2, R1 = 0.05, wR2 = 0.13, S = 1.03; : triclinic, P1̅, a =6.0690(2) Å, b = 9.3749(5) Å, c = 9.7338(6) Å, α = 66.98(1)°, β = 86.87(1)°,γ = 88.62(1)°, V = 508.97(5) Å3, Z = 1, R1 =0.03, wR2 = 0.07, S = 1.04; : triclinic, P1, a = 5.9724(3) Å, b = 9.3996(4) Å, c = 9.4745(5) Å, α= 67.42(1)°, β = 85.47(1)°, γ = 86.93(1)°, V = 489.42(4) Å3, Z = 1, R1 = 0.03, wR2 = 0.05, S = 1.05.IM–1.Temperature-dependent Raman spectrawere measured using a Renishaw inVia Raman spectrometer, equippedwith a confocal DM2500 Leica optical microscope, a thermoelectricallycooled CCD as a detector, and a diode laser operating at 830 nm. Thetemperature was controlled using a THMS600 stage, and the spectralresolution was 2 cmThe dielectric measurements wereperformed using a Novocontrol Alpha impedance analyzer. The temperaturewas controlled by the Novo-Control Quattro system using a nitrogengas cryostat. All dielectric measurements were taken every 1 K duringthe cooling cycle. Low temperatures were achieved using liquid nitrogen.The gas flow cryostat ensures temperature stability better than 0.1K. Since the obtained single crystals were not large enough to performsingle-crystal dielectric measurements, pellets made of well-driedsamples were measured instead. The silver paste was deposited on thepellet surface to ensure good electrical contact. The AC voltage withamplitude 1 V and frequency in the range 1 Hz to 1 MHz was appliedacross the sample.RT absorptionspectra of the powdered samples were measured using a Varian Cary5E UV–vis–near-infrared (NIR) spectrophotometer. Emissionspectra at various temperatures under 266 or 375 nm excitation froma diode laser were measured with the Hamamatsu photonic multichannelanalyzer PMA-12 equipped with a BT-CCD linear image sensor. The temperatureof the single-crystal sample was controlled using a Linkam THMS600heating/freezing stage. The quantum efficiency was measured on a HamamatsuAbsolute PL quantum yields (PLQYs) measurement system C9920-02G.3) or 1500 nm (IM2PbBr4 and IM3PbBr5) and was used unfocused.Laser fluence values of samples were equal to 0.28 and 0.32 mJ cm–2 at 1400 and 1500 nm, respectively.Temperature-resolved SHG studies were performedusing a laser system employing a wavelength-tunable Topaz Prime vis–NIRoptical parametric amplifier (OPA) pumped by the Coherent AstrellaTi:Sapphire regenerative amplifier providing femtosecond laser pulses at a 1 kHz repetition rate. The output of OPA wasset to 1400 .Temperature control of the sample was performed (dT/dt = 5 K min–1) using a LinkamLTS420 heating/freezing stage. Temperature stability was equal to0.1 K.The singlecrystals of IMPbBr3 showtwo anomalies at T1 = 243/240 and T2 = 221/200 K upon heating/cooling, confirmingthe existence of two reversible phase transitions (S3). The symmetric shapes of these anomalies indicate thatthese phase transitions are of the first-order type. The associatedchanges in entropy (ΔS) and enthalpy (ΔH) were estimated to be ∼4.47 J mol–1 K–1 and ∼1.05 kJ mol–1 for the phase transition at T1 and ∼7.41J mol–1 K–1 and ∼1.43 kJmol–1 for the phase transition at T2. Based on the Boltzmann equation, ΔS = R ln(N), where R is the gas constant and N denotes theratio of the number of distinguishable orientations, the value of N was calculated as 1.71 (2.43) for the phase transitionat T1 (T2).These values indicate that the phase transitions are of an order–disordertype.The DSC measurementsfor IMPbBrnsitions 1a and S32PbBr4 at T1 = 407/401 and T2 = 185/178 K. Theestimated average values of ΔS, ΔH, and N are ∼20.41 J mol–1, ∼6.72 kJ mol–1 K–1,and 11.59 for the phase transition at T1 and ∼5.31 J mol–1 K–1, ∼1.04 J mol–1, and 1.89 for the phasetransition at T2, respectively. As canbe noticed, the N values are very large for IM2PbBr4 and IM3PbBr5 at T1, indicating a very pronounced disorder ofthe HT phases I, which decreases significantly in the intermediatephases II. The anomalies at T2 are much weaker, implying either weaker order–disorder contributionor the displacive character of the phase transitions to phases III.Two anomalies are alsoobserved for IM85/178 K1b. The c43/141 K 1c and S33, solved at 173 K, hasalready been reported.39 It is a 3D derivativeof the perovskite structure builtof face-sharing PbBr6 octahedra, which additionally sharethe free corners to form a three-dimensional network. This so-called4H-hexagonal polytype develops channels propagating along the c-direction, which are occupied by protonated amines. Elliotet al. reported the low-temperature (LT) structure of the orthorhombic Pnma symmetry.39 Due to the factthat this compound exhibits two phase transitions at 200 and 240 K,we decided to follow the thermal evolution of its structure. Below,we summarize the results.The crystal structure of IMPbBr3 crystallizesin the hexagonal P63/mmc symmetry, which is characteristic of 4H-hexagonal perovskites.43 This high, hexagonal symmetry implies the disorder of IM+ amines, which is illustrated in + in all directions, and thesecond one allows only in-plane movements. The lack of hydrogen bonds(HBs) leaves the PbBr6 octahedra almost undisturbed, withnegligible octahedral distortion and Pb–Br distances changingbetween 3.01 and 3.02 Å; see At RT, IMPbBrao = ah, bo = ch, co = 2(2bh – ah). Due to the twinning, the only reasonable model ofthe structure could be solved in the P212121 symmetry, which has not been evidencedin the SHG results (see the next paragraph). However, still, thisnonperfect refinement may shed light on the atom rearrangement inthe intermediate phase. Figure S4 showsthe asymmetric unit, which consists of four independent lead ions,12 Br– ligands, and four ordered IM+ counterions.The large displacement parameters may originate from the poor qualityof the twinned data set, as well as from the nonresolved disorderassociated with small-angle librations of the amines. As the DSC analysisimplies the order–disorder mechanism of the first phase transition,the presented model seems reasonable. Additionally, the chiral P212121 intermediate phasehas been found in two DMA 4H perovskite derivatives (DMA+ = dimethylammonium cation).43Thephase transition at 240 K leads to symmetry reduction to theorthorhombic structure, with the lattice parameters being the 2-foldsuperstructure of the ortho-hexagonal C-centered phase with the followingrelationships between the lattice parameters: 3, and the symmetry is noncentrosymmetric Pna21 (which is confirmed by the SHG). The phasetransition is triggered by the alternating arrangement of the IM+, which couples to the antiphase rotation of the octahedraaround the b-axis. Although the view of the structure,displayed in Pnma phase,39 the lack of the symmetry center has an irrefutableinfluence on the physical properties of this phase. The dipoles assignedto the IM+ cations give rise to uncompensated spontaneouspolarization. Examining the octahedral distortions, collected in Withfurther cooling, the second phase transition occurs, and itis associated with the translational and space group symmetry change.The volume of the unit cell is reduced twice from 3866 down to 1881Å2PbBr4 crystallizes in thetriclinic P1̅ symmetry and possesses two additionalpolymorphic forms depending on temperature, i.e.,a disordered, high-temperature (HT) orthorhombic Cmmm phase above 400 K and another triclinic P1̅isomorphic phase below 178 K. The lead bromine substructure is similarto that found in C4N2H14PbBr4.44 In both compounds, the 1D inorganicpart consists of [PbBr42–]∞ double chains which are separated by protonated amines. The chainsare built of edge-sharing bioctahedra, which in turn share apicalbromines and form a polymeric structure expanding along the a-direction; see 46IM+, N–H···Brhydrogen bonding, and octahedral distortions of PbBr6 units.The HT orthorhombic phase is characterized by disordered IM+ cations, which may freely perform in-plane rotations. Symmetry reductionat 401 K to the triclinic, P1̅ arises due tothe ordering of IM+. In this phase, the cations are anchored via weak N–H···Br and C–H···BrHBs; however, the hydrogen-bonded network is not complete, some NHgroups do not have the hydrogen acceptor in this phase. Moreover,quite large displacement parameters of all atoms, as well as insignificantchanges in octahedral distortion parameters Å in II up to 3.070(1) Å in III, whereas the Pb1–Br1–Pb1 angle decreases from 178to 172°. The unusual elongation of a bond at LT is also observedfor Pb1–Br3 (from 2.994(1) Å up to 3.058(1) Å) beinga product of the increase in the strength of N10–H···Br3HB (the donor-to-acceptor distance decreases from 3.56(1) Å in II to 3.365(50) Å in III). As a result ofchanges in the configuration of HBs, there is a continuous increasein the shortest distance between the chains, which refers to the b-lattice parameter in II and III. Thus, the negative thermal expansion develops in the LT phases ofIM2PbBr4 along the b-direction.The phase transitions appear as a result of the interplaybetweenthe temperature-activated rotations of IMters see 1, imply 3PbBr5 is a representative of the 1D perovskites built on [PbBr5]n3– chains arrangedfrom the single, corner-sharing PbBr6 octahedra. The motifof linear chains of corner-sharing PbX6 octahedra is quiterare for A3PbX5 compounds. A few examples maybe found for A3PbI5 crystallizing with guanidinium,47 iodoformamidinium,48 protonated thiourea,49 mixed methylammonium/dimethylsulfoxide (DMSO),50 A3PbCl5 representatives with protonated methyltiourea,51 and melamine.52 Tothe best of our knowledge, IM3PbBr5 is the firstexample of lead bromine with the linear chains of corner-sharing PbBr6 octahedra.The last polymorph IM+ moieties, stackingalong the b-direction and along the chains (in the a-direction). As in the previous compounds, three phasesinduced by temperature can be distinguished in this material. In theHT phase of the orthorhombic Cmmm symmetry, whichis stabilized above 395 K, all IM+ cations are disorderedand perform in-plane and out-of-plane rotations. The transition tothe RT phase is triggered by the reduction of the degrees of freedomof IM+, which are placed in the stacks along the triclinic b-direction. A temporary hydrogen-bonded network is formed,in which each ordered cation is hydrogen-bonded to one inorganic chain.The second phase transformation is driven by the further IM+ ordering and rearrangement of the existing HBs. In III, which is triclinic P1, the complete 3D supramolecularstructure is formed, as each IM+ interacts with two neighboringchains. Figure S5 shows thermally inducedchanges in the crystal structure. The lack of the symmetry centerimplies polar properties of this phase originating from noncompensateddipoles of IM+.The RT structure is shown in b-lattice parameter,increases with the temperature lowering from 9.27 Å (I) to 9.375 Å (II) and finally to 9.400 Å (III), implying negative thermal expansion along this direction.The strong N–H···BrHBs influence the packingof the crystal structure. The shortest distance between the chains,which actually refers to the 3PbBr5 polymorphs presented here,the octahedral distortion parameters  and 3D  perovskites reportedby us earlier.22In all IMII and III of IM2PbBr4 and IM3PbBr5 feature acentric structural order or not. The screening ofSHG activity was performed using 1500 nm ultrafast laser pulses forall crystal phases that were detected using DSC and crystallography(vide supra). Indeed, in our SHG studies of leadhalide perovskites, we generally employ long-wavelength femtosecondlaser excitations (1300–1500 nm), for several reasons. Oneis that lead halide perovskites, with their optical band gaps mainlylocated in the visible spectral range, are very good two-photon absorbersat corresponding two-photon resonances at doubled wavelengths;17 accordingly, the irradiation of perovskite sampleswith, e.g., 800 nm laser pulses, typically resultsin giant two-photon excited luminescence that largely obscures theSHG response.18 The other factor is that intenseexcitation through resonant nonlinear absorption processes can leadto the photochemical generation of defects and associated with thismodification of the optical properties such as luminescence quenchingor emergence of defect-derived SHG response. Thus, to minimize convolutingphenomena, it is preferred to shift away the laser excitation deepinto the NIR region, where multiphoton absorption cross sections aremuch lower in intensity so that the upconverted emissions are correspondinglyweaker.The following section is devoted to theNLO spectroscopic verification of whether phases 3 and IM3PbBr5 has been realized through thetemperature-resolved irradiation experiments of the size-graded sampleswith 1400 and 1500 nm femtosecond laser pulses, respectively. Figure S6.Screening of the SHG activity for IMPbBr3 demonstratethat out ofthree crystal phases only the LT phase III produces SHG.Indeed, upon cooling, the SHG signal at 700 nm could be detected aslow as at 175 K and is partially overlapped with weak multiphotonexcited luminescence (MPEL) ; upon the heating run, SHG is no longer detected at about 185 K. Thus, plots of integral SHGintensities for cooling and heating runs are separated by ca. 10 K-wide thermal hysteresis (Collected results for IMPbBre (MPEL) S6a; uponII of IM3PbBr5 displays broad, moderateMPEL , but no SHG response is present. This confirms the centrosymmetriccharacter of this crystal phase. However, one sees that upon coolingbelow 120 K the SHG signal at 750 nm starts to emerge and sits atthe shoulder of the broad MPEL emission , which also increases its intensity due to decreased thermalquenching. Overall, it is clear that only the LT phase III of IM3PbBr5 is noncentrosymmetric.It is apparent that HT phase 2PbBr4. In this case, only MPEL signals of various intensities havebeen registered, but no trace of SHG at 750 nm could be noticed . Combined with crystallographic results,it led us to the conclusion that all three crystal phases of IM2PbBr4 feature centrosymmetric structures.Underidentical irradiation conditions (1500 nm), the screeningof the SHG activity was also performed for IM53 This fact motivated us to perform comprehensive temperature-dependentdielectric measurements of IMPbBr3, IM2PbBr4, and IMI3PbBr5. To depict the structuralchanges associated with the changes in the internal dipole moment,the temperature-dependent complex dielectric permittivity ε*(ε* = ε′ – iε″) spectra werecollected (M* = 1/ε*) was also used  being onthe order of 6 for 1 MHz , slightly obscured by some conductivity process. The well-depictedsteplike tendency and a bell-shaped curve of real and imaginary parts,respectively, of both ε and M, indicate thedipolar relaxation process in this compound . The complex dielectric spectra for the IM2PbBr4 compound collected in the frequency domain revealed the characteristicrelaxation process just on the electric modulus . This behavior suggests that the observed relaxationprocess is related to conductivity. In the case of IM3PbBr5, in the studied frequency range, two dipole relaxation processesare visible only below the phase transition temperature. The secondrelaxation process, observed only in the electric modulus spectra,is related to thermally induced conductivity .To quantify the observed relaxation processes,an analysis of spectrain the frequency domain was performed. M, the data were parameterized using a single Havriliak–Negami(H–N) function: ∞ is the high-frequency contribution, and parameters α and βdescribe symmetrical and asymmetrical broadening of the relaxationpeak. The relaxation times were calculated from the peak maximum frequencyas τmax = (2πfmax)−1. 3 and IM3PbBr5 and the electricmodulus for IM2PbBr4. To depict the structuralrelaxation dynamics, we have determined the temperature-dependentbehavior of the dielectric relaxation times obtained from both dielectricpermittivity and electric modulus data τmax and scaledlog τmax as a function of 1000/T . Thesechanges are consistent with the increase in the crystal symmetry whengoing from phases III to phases II. Interestingly,the phase transitions from phases II to phases I do not lead to any apparent change in the number of Ramanbands despite clear symmetry changes revealed by the X-ray diffractionmethod. This behavior can be most likely attributed to the fact thatIM+ cations in phases II and III of the studied compounds have very similar structures.3, IM2PbBr4, and IM3PbBr5, respectively. The energy band gaps (Eg) of these compounds, calculated using theKubelka–Munk formula, are 3.24, 3.58, and 3.52 eV, respectively. These band gaps are significantlylarger than reported for 3D MAPbBr3, FAPbBr3, and MHyPbBr3 hybrid perovskites (2.18–2.48 eV).59 According to literature data, a decrease in dimensionality and changein PbBr6 connectivity from corner-shared to edge- or face-sharedlead to an increase in the band gap and a blue shift of the excitonicabsorption. For instance, band gaps near above 3 eV were reportedfor many 2D layered corner-shared bromide perovskites,62 as well as 0D and 1D analogues containing corner-,edge-, or face-shared PbBr6 octahedra.64 Thus, large band gaps of the studied compounds areconsistent with their low dimensionality. Since IMPbBr3 is isostructural to DMAPbBr3,43 it is worth comparing band gaps and excitonic absorptions of thesecompounds. Using our data reported previously for DMAPbBr3,65 we estimate the excitonic absorptionand the band gap of this compound as 388 nm (3.20 eV) and 3.34 eV,respectively. Accordingly, IMPbBr3 shows more red-shiftedexcitonic absorption and a narrower band gap compared to DMAPbBr3. Taking into account that the band gap and energy of excitonicabsorption increase with increasing distortion of the inorganic network,66 the observed differences between IMPbBr3 and DMAPbBr3 indicate a smaller octahedral distortion in the former case.Diffuse reflectance spectraregistered for the investigated perovskites consist of absorptionin the UV range and the excitonic bands centered at 397 (3.12), 376(3.30), and 378 (3.28) nm (eV) for IMPbBr3 recorded at 80 K shows anintense and very broad band (FWHM = 168 nm) centered at 668 nm compared to the PL of isostructural IMPbBr3. Figure S16a shows that the intensity of PL ofthe IMPbBr3 sample increases on heating up to 120 K, followedby typical luminescence quenching beyond 120 K with Ea of 148 meV .The PL quantum yield (PLQY) of IMPbBr3 reaches 0.9% atRT. Anomalous enhancement of PL on heating is often observed due tothe presence of phase transitions. For instance, such behavior wasreported for MAPbBr3 and explained as resulting from reducednonradiative recombination due to increased dielectric screening andthe reduction of defects at the order–disorder phase transition.68 Since IMPbBr3 does not exhibit anyphase transition near 120 K and the dielectric data do not show anyincrease of the dielectric permittivity near 120 K, this explanationcannot be adopted. We suppose, therefore, that the observed behaviorcan be explained in the same way as proposed for a few other leadbromide perovskites exhibiting anomalous temperature dependence ofthe PL intensity, i.e., this behavior can be attributedto thermally activated trapping–detrapping of excitons.69The PL spectrum of IMPbBrt 668 nm 12a. The 2PbBr4 also exhibits very broad PL (FWHM= 124 nm), which can be attributed to the intrinsic STEs or excitonstrapped at the defects . The PL quantumyield (PLQY) of IM2PbBr4 reaches 1.9% at RT.IM defects 12b. Howe3PbBr5 . The PL quantumyield (PLQY) of IMI3PbBr5 reaches 1.5% at RT.Broadband PL (FWHM = 140 nm) centered at 574 nm is also observedfor IMI3PbBr5 12c. Thisi.e., it changes from 668 nm for IMPbBr3 to 574 nm for IM3PbBr5 and 501 nm  forIM2PbBr4. d and Br–Pb–Br angles are comparable for allcompounds. Octahedral angle variance (σ2) is thelargest for IMPbBr3 but small and similar for IM2PbBr4 and IM3PbBr5. Thus, the differencein the distortion parameters does not explain a very large differencein the Stokes shift observed for the studied compounds. We concludetherefore that the different behavior of each composition can be attributedmainly to the different connectivity of PbBr6 octahedralunits in the studied compounds, i.e., whereas inIMPbBr3, each PbBr6 octahedron is linked tofour nearest neighbors (one by face-sharing and three by corner-sharing),in IM3PbBr5, all PbBr6 octahedraare corner-sharing, forming 1D chains, while in IM2PbBr4, the PbBr6 octahedra form edge-shared dimers,which then join by corners forming extending chains. The shift inthe registered PL bands leads to a change in the emission color fromorange for IMPbBr3, yellow for IM3PbBr5, and bluish-green for IM2PbBr4  depends strongly on thechemical composition, 3 and two novel IM2PbBr4 and IM3PbBr5 compounds. These compounds havebeen studied using various experimental methods to monitor their crystalstructures, mechanism of phase transitions, and dielectric and opticalproperties.We have synthesized three imidazolium lead bromides:previouslyreported IMPbBr3 crystallizesin the 4H perovskite structure with face- and corner-shared PbBr6 octahedra (space group P63/mmc), with disordered IM+ cations. On cooling,it exhibits phase transitions at 240 and 200 K associated with anordering of IM+ cations. Most interestingly, the LT phaseis polar, Pna21. The noncentrosymmetricnature of this phase was confirmed by its SHG activity. IM2PbBr4 adopts a 1D double-chain structure with edge-sharedoctahedra. At high temperatures, IM+ cations are disorderedand the symmetry is orthorhombic (Cmcm). On cooling,a phase transition to the ordered P1̅ phaseoccurs at 401 K. Another isostructural and displacive-type phase transitionoccurs at 178 K. IM3PbBr5 crystallizes in the1D single-chain structure with corner-shared PbBr6 octahedra.The crystal structure of the HT phase is orthorhombic (space group Cmmm), and IM+ cations are disordered. Thesecations exhibit partial ordering at 395 K, and the symmetry changesto P1̅. On further cooling, IM3PbBr5 transforms at 141 K to the ordered polar phase of the P1 symmetry, which is SHG active.Single-crystal X-ray diffraction revealed that crystalstructureschange strongly with the chemical composition. IMPbBr3 exhibitsa significant steplike dielectric anomaly at 200 K, allowing us toclassify this bromide as a switchable dielectric. Furthermore, theobserved change in dielectric permittivity of IM3PbBr5 near 145 K indicated that this compound is an improper ferroelectric.Dielectric studiesrevealed the presence of dielectric relaxationsin all compounds. Most interestingly, IMPbBr3, IM2PbBr4, and IM3PbBr5, respectively. The band gaps are 3.24, 3.58, and 3.52 eV. All compoundsexhibit broadband highly Stokes-shifted PL attributed either to theintrinsic STEx states or to excitons trapped at the defects . We attributedthe different behavior of each composition to the different connectivityof PbBr6 octahedral units in the studied compounds.Linear optical studies revealed that the excitonic bands are centeredat 397 (3.12), 376 (3.30), and 378 (3.28) nm (eV) for IMPbBr


XRD analysis confirms that low-content Mn-ion insertion at the lattice sites of CBTNF does not distort the pseudo-tetragonal phase lattice of CBTNF:xMn ceramics, but enhances the functional behavior of the ceramic system, specifically at x = 0.15 wt%Mn. Compared to pure CBT and CBTNF ceramics, CBTNF:0.15Mn has demonstrated a highly dense relative density (~96%), a saturated polarization (SP) of 15.89 µC/cm2, a storage energy density (STW) of ~1.82 J/cm3, an energy-conversion efficiency (ƞ) of ~51% and an upgraded piezoelectric behavior (33d) of 27.1 pC/N at room temperature. Sharp temperature-dependent dielectric constant (rε) peaks display the solid ferroelectric behavior of the CBTNF:0.15Mn sample with a Curie temperature (CT) of 766 °C. The thermally stable piezoelectric performance of the CBTNF:0.15Mn ceramic was observed at 600 °C, with just a 0.8% 33d loss (25 pC/N). The achieved results signify that multi-valence Mn ions have effectively intercalated at the lattice sites of the pseudo-tetragonal phased CBTNF counterpart and enhanced the multifunctional properties of the ceramic system, proving it to be a durable contender for utilization in energy-storage applications and stable high-temperature piezoelectric applications.In the present era of advanced technology, the surge for suitable multifunctional materials capable of operating above 300 °C has increased for the utilization of high-temperature piezoelectric devices. For this purpose, a pseudo-tetragonal phased CaBi Recently, Wu et al. reported the fabrication of Gd/Mn co-coped CaBi4Ti4O15 ceramics, where a high dc conduction activation energy value of 1.87 eV was reported [0.85 (LiCe)0.075Bi4Tix4−ZnxO15 ceramic system and reported a high 33d of 20 pC/N [1−x x/2Bi4Ti4O15 ceramic system via a conventional solid-state reaction method, where a high resistivity of 4.51 × 1011 Ω cm was reported with an improved CT of 814 °C [4Tix4−(Nb1/2Fe1/2)xO15 ceramic system with a low dielectric loss (tanδ) of 5.9% at 400 °C [The materials of the Aurivillius-type family, also known as bismuth-layered structure ferroelectrics (BLSFs) with a  ions, along with the various allotropic forms, have made MnO2 a strong candidate for the improvement of dielectric, ferroelectric and capacitive properties as a dopant/additive of the materials [0.5 (K0.2Na0.8)0.5TiO3 as a ceramic with improved ferroelectric, piezoelectric and dielectric properties [3 film capacitor, with a remanent polarization of 66 μC/cm2 [0.60 (Li0.5Bi0.5)0.40Bi2Nb2O9:xwt%MnO2 complex ceramic scheme, with an improvement in the ferroelectric and piezoelectric properties of the ceramic system [4Tix4- (Nb0.5Fe0.5)0.02O15 (CBTNF) usually shows a reduced dielectric loss at high temperatures with better piezoelectric properties, the combination of it with multi-valence additive MnO2 as CaBi4Tix4- (Nb0.5Fe0.5)0.02O15:xwt%MnO2 (CBTNF:xMn) with its new scheme needs to be explored. Here, it is expected that, during the high- temperature fabrication of CBTNF, the diffusion of Ca/Bi ions from the A site will be replaced by Mn2+/Mn3+ ions, while the evaporation of Ti ions will be occupied by Mn4+ ions. Such a diverse or unique approach can be helpful in exploring BLSFs as a step forward in practical applications. Moreover, the capacitive performance of the CBT-based ceramic is measured for the first time.MnOtoxicity ,21. Moreaterials . Recentloperties . Yang et6 μC/cm2 . Similarc system . Keeping2 as an additive is analyzed in a pseudo-tetragonal phased CBTNF system through the composition of CBTNF:xMn with x = 0–0.2 to explore the structural, morphological, ferroelectric and piezoelectric measurements. For CBTNF:0.15Mn ceramic, the existence of a pseudo-tetragonal region, along with the influence of multi-valance Mn ions at the A site of CBTNF, have caused a strong multifunctional character with a high rP of ~9.8 µC/cm2, high 33d of 27.1 pC/N and impedance of ~109 Ω, which are much better than pure CBT or pure CBTNF ceramics.Herein, the influence of MnOxMn (x = 0–0.20) ceramics are presented in A21am space group), without the presence of impurities. The existence of a strong (119) diffraction peak at angle 30.8° for all compositions is the characteristic peak of the CBT-based ceramics [m + 1) diffraction peak of BLSFs [xMn (x = 0–0.20 wt%) compositions. The pattern of the pure CBT ceramic is labeled PDF # 52–1640 [xMn (x = 0–0.20) ceramics was observed. As high-temperature sintering was performed for the fabrication of ceramics, the A site vacancies varied due to the diffusion of Ca and Bi ions, causing the diminution of lattice parameters a and c, while b decreased. In a/b ratio. The tetragonality increased with the variation of vacancies at the A site due to the evaporation of Bi at high-sintering temperatures, which has already been reported in articles belonging to the complex pseudo-tetragonal phase [xMn ceramics as well. All CBTNF:xMn (x = 0–0.20) ceramics maintained their pseudo-tetragonal identity, verifying that Mn ions have efficaciously intercalated at the vacancies of parent CBTNF material, without damaging the structure as the content of additive Mn ions was kept very low.The XRD study of pure single-phase CBT ceramic for comparison with pseudo-tetragonal phased CBTNF:ceramics . XRD patof BLSFs ,26. An i 52–1640 . The 200al phase . In thisx = 0.1, 0.15 and 0.20) ceramics were performed by employing the general diffraction-analysis software FULLPROF. Orthogonal symmetry with space group 21amA was the best-fitting reference structure between the observed and calculated intensities. From 2 refined factors were achieved, confirming the better agreement between the observed and calculated patterns. Obviously, there existed no large change in the lattice parameters , which is confirmation that Mn did not influence the pseudo-tetragonal phase of the CBTNF counterpart and maintained its identity 2+ layer, due to surrounding environment [3/2 was located at a binding energy value of 458.6 eV, while the peak for Ti 2p1/2 was located at a binding energy value of 464.4 eV. These peak positions of Ti 2p3/2 and Ti 2p1/2 are the characteristic features of standard anatase phase TiO2 [3/2 and Ti 2p1/2 (ΔBE = Ti 2p3–2 − Ti 2p1/2) was equal to 5.8 eV, which is associated with typical Ti4+–O bonding in TiO2 [3/2 at a binding energy of 206.8 eV and Nb 3d5/2 at a binding energy of 210.2 eV  was equal to 11.8 eV. The XPS spectra for Mn 2p of CBTNF:0.15Mn ceramic is shown in 3/2 and Mn 2p1/2 peaks were located at the binding energies of 642.11 eV and 653.73 eV, respectively. The difference in binding energies between Mn 2p3/2 and Mn 2p1/2 (ΔBE = Mn 2p3–2 − Mn 2p1/2) was equal to 11.6 eV. Moreover, the Mn 2p3/2 and Mn 2p1/2 were fitted to visualize the multi-valence states of added Mn ions. From the simulation of the Mn 2p3/2 peak, three fitting peaks were located at binding energies 642.1, 642.75 and 644.6 eV, which were associated with Mn2+, Mn3+ and Mn4+, respectively. From 2+ is much lesser than that of the other two peaks, confirming that a low amount of Mn2+ ions were generated compared to the Mn3+ and Mn4+ ions [XPS analysis for the binding energy range of 0 to 750 eV was performed for the comparison study of pure CBTNF and CBTNF:0.15Mn ceramics. Additionally, the main course of the study was to analyze the presence of multi-valance manganese ions in the CBTNF:0.15Mn ceramic sample. Photoemissions of Ca-2p, Bi-4f, Ti-2p, Nb 3d and Fe-2p core levels are analyzed in nfluence a. These ironment . It is aase TiO2 . Moreove in TiO2 . No obvi210.2 eV d. The pe210.2 eV b,d of pu723.5 eV . The difn4+ ions ,36. Durin4+ ions . Mn ionsP-E loops) of the pure CBT and CBTNF:xMn (x = 0–0.20) ceramics is measured and presented in SP) of ~15.8 µC/cm2 was measured for CBTNF:0.15Mn. The measured SP value for CBTNF:0.15Mn was much improved compared to the pure CBT (SP ~13.8 µC/cm2) ceramic and pure CBTNF (SP ~10.8 µC/cm2) ceramic. A high electric field (120 kV/cm) was employed to all samples for these measurements. From Pr increased with the variation of additive Mn-ion content until the CBTNF:0.15Mn ceramic, which can be attributed to the presence of multi-valence Mn additives which acted as a soft agent for the CBTNF component, causing the improvement in the domain wall mobility, which allowed better broad switching within the ceramic [SP ~14.7 µC/cm2 started to decrease , stored energy density (stW) and energy-conversion efficiency (ƞ) calculations are achieved from the measured P-E loops, and the equations for the calculations are as follows [maxP, rP and E are achievable from the measured P-E loops. recW, stW and ƞ versus the x values of Mn addition in CBTNF at 120 kV/cm. The values of stored energy density (stW) of all the samples were above 1.67 J/cm3 (1.67–1.82 J/cm3) and the energy-conversion efficiency (ƞ) was above 44% (44–63%). The highest STW of 1.82 J/cm3 and ƞ of 51% were calculated for CBTNF:0.15Mn ceramic.Recently, the dielectric capacitor behavior of ceramics has achieved substantial attention in the electronic field of instant energy-storage applications, owing to their rapid charge–discharge time, high power density and exceptional chemical constancy. Recoverable energy density ( follows :(1)Wrec=33d) values of pure CBT and CBTNF:xMn (x = 0–0.20) ceramics are presented in 33d ~27.1 pC/N, much more improved than pure CBT (~ 8 pC/N) ceramic, with a 70% improvement and pure CBTNF (20 pC/N) ceramic with a 24.8% improvement. The thermal stability of the piezoelectric performance of the synthesized material under the influence of high temperatures was observed: the degradation of the piezoelectric properties in ceramic is observed and illustrated in 33d value of pure CBTNF remained much stronger (11 pC/N) at 700 °C than pure CBT. However, 33d of CBTNF:0.15Mn showed the most stable character with 25 pC/N at 600 °C (0.8% loss of 33d) and 21 pC/N at 700 °C (23% loss of 33d). The applied electric field is the main reason for the polarization of the ceramics: this electric field creates the configuration of the ferroelectric domains within the CBTNF counterpart, and a liberated eccentric recompense of charges occurs at the CBTNF/CBTNF grain boundaries. During the annealing process, there exists a natural phenomenon within ceramics when materials transfer from an aligned domain configuration to a non-aligned domain configuration. This phenomenon is called back-switching at the diffusion temperature. The addition of Mn ions due to its multi-valence nature is capable of occupying the unbound misfits, which results in the development of electronic-charge dispersal at the grain boundaries, which reduce back-switching occurrence even at high-temperature annealing [33d value decreased to 25.2 pC/N (at room temperature) as the solubility limit of the Mn ions at vacant A and B sites in the CBTNF counterpart attained its maximum limit (percolation threshold). Later, ions will gather at the CBTNF/CBTNF grain boundaries, affecting the piezoelectric properties [rε) plots of the fully poled CBT and CBTNF:xMn (x = 0–0.20) samples are depicted in rε of CBTNF:0.15Mn ceramic is at its maximum (rε ~418) at a frequency of 100 Hz, which is better than all other compositions. For instance, all compositions of the ceramics were poled, causing the creation of dielectric anomalies along the thickness of the ceramic at certain frequencies (~212/225 kHz). Poled ceramics possess the novel capability to create deformation in their piezoelectric properties due to the influence of an applied 0.5 AC voltage, resulting in the formation of dielectric anomalies [9 Ω) and CBTNF (~3.05 × 109 Ω) ceramics were higher than all other compositions at a frequency 100 Hz. Mn ions caused the reduction in the impedance values of the materials, but this is a minute decrement (~1.2 × 109 Ω for CBTNF:0.15Mn ceramic). Furthermore, the piezoelectric anomalies along the thickness of the impedance curves are consistent with the specific frequency (εr vs. f plot) detected because of piezoelectric (33d) influence [The piezoelectric co-efficient  of the materials, where they not only lose their piezoelectric behavior, but also transform from the ferroelectric to the paraelectric phase. CT of the synthesized pure CBT and CBTNF:xMn (x = 0–0.20) ceramics was taken at 100 kHz by temperature-dependent dielectric constant plots for a temperature range of 0 to 850 °C. Plots are presented in CT ~789 °C, consistent with previous reports. The CT values of pure CBTNF, CBTNF:0.1Mn, CBTNF:0.15Mn and CBTNF:0.2Mn ceramics were 779 °C, 770 °C, 766 °C and 753 °C, respectively. The attained values demonstrate the prominence of the fabricated material for utilization in various purpose devices. Frequency-dependent dielectric loss (tanδ) curves of the CBT and CBTNF:xMn (x = 0–0.20) samples are presented in The thermal stability of 4Ti4O15 and CaBi4Tix4- (Nb0.5Fe0.5)0.02O15 ceramics were synthesized by a rapid liquid-sintering technique with a rapid increase and decrease in temperature > 20 °C/min to avoid the excessive diffusion of Bi ions and oxygen during the high-temperature sintering process. The high-purity raw powders of CaCO3 , Bi2O3 , TiO2 , Fe2O3  and Nb2O5  were weighed according to the desired stoichiometric ratio of CBT and CBTNF, mixed in a milling machine for 12 h in ethanol. Later on, the solution was dried at 100 °C overnight, calcined at 900 °C for 4 h, milled again for 12 h and dried at 100 °C. The dried powders of CBT and CBTNF were pelletized with a diameter of 7–11 mm under a hydrostatic pressure of 160 MPa and sintered at 1100 °C for 2–4 h. MnO2  powder was stoichiometrically added in as a fabricated single-phased CBTNF powder, with a variation from 0.00 wt% to 0.20 wt% and a composition of CaBi4Tix4- (Nb0.5Fe0.5)0.02O15:xwt%MnO2 (CBTNF:xMn). Powders were milled for 12 h and dried, and then polyvinyl butyral (PVB) was added as a binder to make the powder more dense. Lastly, the powders were pressed into 7–11 mm diameter disk ceramics under a hydrostatic pressure of 160 Mpa and sintered at 1100 °C for 4 h through a 20/50 °C/min heating/cooling rate.Composition CaBiKα 1: λ = 1.54056 Å, step: 0.02°). The morphological and elemental mapping of the fabricated ceramics were observed by a field-emission scanning electron microscope . Later on, the fabricated ceramics were polished to a thickness of ~0.7–0.9 mm for the ferroelectric, electric, piezoelectric and dielectric measurements. Silver paste was coated on both surfaces of the disks to work as an electrode. The polarization versus the electric field loops (P-E loops) were recorded by an aixACC TF Analyzer 1000 (ferroelectric tester) at the fix frequency (1 Hz) at room temperature. Ceramic samples of all the compositions were fully poled at a temperature of 150 °C, with a high dc electric field of 140 kV/cm, followed by the measurement of the piezoelectric coefficient (33d) at 60 Hz by IAAS ZJ-30, Institute of Acoustics of CAS, Beijing, China (piezoelectric tester) at room temperature. The thermal depoling of the ceramics was checked by annealing the poled ceramics at a variable 20–825 °C temperature range for 30 min, and then the 33d values of the annealed samples were measured at room temperature. The dielectric constant (rε), dielectric loss and impedance versus frequency curves were analyzed using an HP4284A precision impedance analyzer . The dielectric constant (rε) versus the temperature curves were measured using an LCR analyzer  attached to a computer with a furnace. Structural analysis, purity and phase transformation were analyzed by X-ray diffraction 0.02O15:xwt%MnO2 (CBTNF:xMn), with x = 0–0.20, ceramics are engineered to inspect the ferroelectric, electric, dielectric and temperature-dependent piezoelectric response. CBTNF:0.15Mn ceramic has shown the best performance among all compositions, with the highest SP value of 15.89 µC/cm2, stW of ~1.82 J/cm3, ƞ of ~51%, improved 33d of 27.1 pC/N and impedance of ~ 109 Ω at room temperature. A high CT of ~766 °C is the main reason for the thermally stable piezoelectric performance of CBTNF:0.15Mn ceramic, even at 600 °C, with just 0.8% 33d loss (25 pC/N). A low dielectric loss < 4%, even at a high temperature of 700 °C, confirms the successful intercalation of multi-valence Mn ions at the A and B vacant lattice sites of CBTNF, resulting in an improvement in the multifunctional properties. The achieved outcomes confirm the ability of the engineered ceramic to be employed in energy-storage and high-temperature piezoelectric applications.Pseudo-tetragonal boundary phased CaBi


Rhodiola rosea L. We established a corticosterone (CORT)-induced or lipopolysaccharide (LPS)-induced mice in vivo, and CORT, or nigericin (NLRP3 agonist)-induced PC12 cells in vitro. Our findings demonstrated that Sal profoundly mediated CORT or LPS-induced depressive behavior and improved synaptic plasticity by upregulating the expression of brain-derived neurotrophic factor (BDNF) gene. The data showed upregulation of proteins associated with NLRP3-mediated pyroptosis, including NLRP3, cleaved Caspase-1, IL-1β, IL-18, and cleaved GSDMD. The molecular docking simulation predicted that Sal would interact with P2X7 of the P2X7/NF-κB/NLRP3 signaling pathway. In addition, our findings showed that the NLRP3-mediated pyroptosis was regulated by P2X7/NF-κB/NLRP3 signaling pathway. Interestingly, Sal was shown to ameliorate depression via suppression of the P2X7/NF-κB/NLRP3 mediated pyroptosis, and rescued nigericin-induced pyroptosis in the PC12 cells. Besides, knock down of the NLRP3 gene by siRNA markedly increased the inhibitory effects of Sal on pyroptosis and proinflammatory responses. Taken together, our findings demonstrated that pyroptosis plays a crucial role in depression, and Sal ameliorates depression by suppressing the P2X7/NF-κB/NLRP3-mediated pyroptosis. Thus, our study provides new insights into the potential treatment options for depression.Depression is a common and serious mental disorder. Data on its pathogenesis remain unclear and the options of drug treatments are limited. Here, we explored the role of pyroptosis, a novel pro-inflammatory programmed cell death process, in depression as well as the anti-depression effects and mechanisms of salidroside , a bioactive extract from  Depression is a widespread mental health disorder, which is associated with disability and high mortality rates . TypicalAlthough the pathogenesis of depression is unclear, it is affected by various genetic, psychological, and physiological factors. Previous data showed that factors such as structural changes within the hippocampus, loss of BDNF, and excessive neuroinflammation are important in the development of depression . Recent Previous data has shown that activation of the NLRP3 inflammasome can trigger caspase-1 associated pyroptosis, also referred to as “caspase-1 dependent programmed cell death”. Pyroptosis executor gasdermin D (GSDMD) is cleaved by activated caspase family proteins and releases the N-terminal, which creates holes in the cell membrane, leading to enhanced release of inflammatory products, cell rupture and death . Recent The P2X7 receptor is distributed on cell membranes and is a P2X subtype closely associated with depression. After exposure to stressors, large amounts of ATP is released, which leads to activation of the P2X7R and NLRP3-mediated inflammatory responses . The relRhodiola rosea L., has various pharmacological effects, including anti-inflammatory, antioxidant, and regulation of cognitive functions , a kind of phenylpropanoid glycoside found in the herb unctions . Recent unctions . Data haunctions . Althoug® 80 (T118633) were purchased from Aladdin . Lipopolysaccharide  was purchased from Sigma Aldrich . Salidroside  was purchased from Meilunbio . Nigericin  was purchased from MedChemExpress . The primary antibodies against P2X7, ASC, IL-18, IL-1β were purchased from Proteintech Group . All chemicals and reagents were summarized in Corticosterone , Fluoxetine  and TWEENThe male C57BL/6 mice  were acquired from the Qinglongshan Farm . To avoid the influence of female hormones on depressive behaviors, male mice were used in our study . The experimental animals were housed in cages with free access to food and water at the Laboratory Animal Research Center of China Pharmaceutical University. The laboratory temperature was controlled at 20–22°C with 50%–60% relative humidity, with a 12-h light-dark cycle. All animals are acclimatized to the laboratory environment for 1 week before the experiment. The protocol of experiments was supported by the Ethics Committee of China Pharmaceutical University.n = 10 per group): 1) the control group, 2) the CORT-injection group, 3) the CORT + fluoxetine treatment group (CORT + FLU), 4) the CORT +20 mg/kg Sal [CORT + Sal (20 mg/kg)] group, 5) the CORT +40 mg/kg Sal [CORT + Sal (40 mg/kg)] group. CORT was suspended in physiological saline containing 0.1% DMSO and 0.1% Tween-80, and the suspension was administered through subcutaneous injection (20 mg/kg) at a volume of 10 ml/kg once daily for 21 days to induce depression-like symptoms : 1) the control group, 2) the LPS treated group, 3) the LPS +20 mg/kg FLU (LPS + FLU) group, 4) the LPS +20 mg/kg Sal [LPS + Sal (20 mg/kg)] group, 5) the LPS + 40 mg/kg Sal [LPS + Sal (40 mg/kg)] group. LPS (1 mg/kg) was dissolved in saline and injected intraperitoneally (10 ml/kg) for 5 days to induce acute depressive symptoms , followed by the sucrose preference test (SPT) and the last one was the forced swim test (FST).Based on previous studies, moderately modified OFT was used to assess the locomotor capacity of mice . BrieflyThe absence of the ability to feel pleasure from pleasant activities is a classic symptom of human depression. On the other hand, depressed animals showed a decreased rate of preference for sucrose solution intake. The sucrose preference test was conducted using two bottles of either containing 1% sucrose solution or water respectively. Mice were acclimated to sucrose solution for 24 h. For the next 24 h, 1% sugar water was replaced by water. After the mice were starved of food and water for 24 h, the animals housed solely and had free access to the two bottles containing either 1% sucrose solution or water during the formal experiments. After 24 h, the ratio of sucrose consumption and total fluid intake was calculated as a sucrose preference ratio .Mice were subjected to FST as noted in previous studies . Mice wehttp://www.pdb.org) in PDB format. The 3D structure of Sal and A804598  was downloaded from https://pubchem.ncbi.nlm.nih.gov/. The P2X7 receptor was pretreated with pyMOL software to remove small molecule ligands, dehydrate, and hydrogenate. Then, both the small molecule and protein receptor formats are converted to PDBQT format. Finally, AutoDock Vina 1.1.2 was employed for molecular docking  structure was downloaded from the RCSB website  for 24 h.The PC12 cells were cultured in DMEM  with 10% FBS, and placed in an incubator at 5% COTo determine whether NLRP3 can activate CORT-induced pyroptosis, the NLRP3 agonist nigericin (10 μM) was used in this study. The PC12 cells were divided into six groups for different treatments: 1) the Control group; 2) the 200 μM CORT (CORT) group; 3) the 10 μM nigericin (Nig group); 4) the 200 μM CORT +50 μM Sal  group; 5) the 10 μM Nig +50 μM Sal  group; 6) the 200 μM CORT +50 μM Sal +10 μM nigericin  group. The cells were treated with Sal (50 μM) for 2 h, and then incubated with CORT (200 μM) or Nig (10 μM) for 24 h.NLRP3 siRNA and NC (negative control) siRNA provided by RiboBio  were used in our study. The nucleotide sequences of siRNAs are listed in test–Ablank)/(Acontrol–Ablank)*100%. The absorbance of different samples represented by Atest, absorbance of unprocessed cells is expressed as Acontrol, and Ablank is the absorbance of the well that is not inoculated with cells.The cell viability was quantified by the CCK-8 kit according to the manufacturer’s protocol. In brief, 10 µl of CCK-8 solution was added to each well of the 96-well plate and cultivated at 37°C for 1 h without light. The absorbance at 450 nm was detected by an enzyme marker. The cell viability of each well was computed according to the formula : Cell ViLDH, mainly located in the cytoplasm, can be released into the extracellular when cell membranes are disrupted . The relThe cell membrane integrity was detected by Calcein/PI Cell Viability/Cytotoxicity Assay Kit  according to the manufacturer’s instructions. Briefly, the cells were seeded in 24-well plates, then stained with Calcein-AM and PI mixed solution at 37°C for 30 min. The images were obtained using a fluorescent microscope  (living cells appeared green and dead cells displayed red fluorescence) and the percentage of PI-positive cells was quantified by ImageJ software.ELISA kits were used to determine the levels of BDNF and inflammatory cytokines (IL-18 and IL-1β) in our study according to the protocol’s guidelines.Brain samples were fixed in 4% paraformaldehyde and embedded in paraffin for histopathological analysis . Serial Immunohistochemistry was carried out as previously reported with minor modifications . Brain tThe expressions of P2X7, NLRP3 and Cleaved caspase-1 in PC12 cells were evaluated via immunofluorescence assay. Immunofluorescence was carried out as previously reported, with slight modifications . In brieThe hippocampus tissues and cells were homogenized in cold RIPA buffer containing various protease inhibitors. The equal amounts of protein were loaded and separated by 12% SDS-PAGE gels. After being transferred to PVDF and blocked by 5% skim milk powder for 2 h, and the membranes were incubated with antibodies targeting BDNF , P2X7 , ASC , NF-κB , P-NF-κB , NLRP3 , IL-18 , IL-1β , Cleaved GSDMD , Cleaved caspase-1 , β-actin  at 4°C overnight. After washing with TBST, the protein bands were incubated with the HRP-binding secondary antibody (1:1000) for 1 h at room temperature. Finally, blots were displayed with ECL reagent and quantified with ImageJ software.p < 0.05 means that the difference is statistically significant. All statistical analyses were analyzed by GraphPad Prism eight software. Statistical significance of various groups was analyzed by one-way ANOVA followed by Tukey’s test.Results are expressed as mean ± SEM. p < 0.01], elevated distance  and time  in the central zone, as well as increased average velocity    = 7.636,  < 0.05) . In cont 0.0003] . Howevep < 0.05; CA3: F  = 14.98, p < 0.05; DG: F  = 17.01, p < 0.05]  = 8.891,  < 0.05] .p < 0.05]. In addition, our findings showed that repeated CORT injections resulted in decreased BDNF protein levels in the hippocampus   = 27.17, p < 0.05], DG:  ] . Togethep < 0.0001] and IL-1β  in the hippocampus of the CORT group compared to the control mice  = 8.986, p < 0.01] and cleaved GSDMD , which were all reversed by 40 mg/kg Sal and FLU. Collectively, our data illustrated that CORT triggers pyroptosis, which can be relieved by Sal.We next evaluated the effect of Sal on pyroptosis-associated proteins using Western blot and ELISA. Our ELISA results showed significantly increased expression of IL-18 , P-NF-κB , NLRP3 , ASC , Cleaved caspase-1  in the CORT treated mice. However, Sal and FLU notably down-regulated the expression of the P2X7/NF-κB/NLRP3 signaling pathway-related proteins (p < 0.01), CA3:  = 8.742, p < 0.01), DG:  = 11.22, p < 0.001)), NLRP3  = 13.23, p < 0.001), CA3:  = 15.61, p < 0.001), DG:  = 9.262, p < 0.01)) and cleaved Caspase-1 , changes that were reversed by Sal and FLU . Overall, our results demonstrated the correlation between pyroptosis and the P2X7/NF-κB/NLRP3 signaling pathway, and that Sal exerts its effect by suppressing the expression of proteins related with the signaling pathway.We performed Western blot and immunohistochemical analyses to study the effect of Sal on the P2X7/NF-κB/NLRP3 signaling pathway in CORT-induced mice. Western blot assay showed increased expression of P2X7 , the time spent in the central zone , average velocity , as well as the total distance  were markedly decreased in mice administered with LPS than those in the normal group  = 10.71, p < 0.0001] in contrast with the control group  = 7.419, ol group , phenomep < 0.01]. Furthermore, Western blot analyses demonstrated that LPS decreased hippocampus BDNF levels, while Sal treatment remarkably enhanced the production of hippocampus BDNF  (p < 0.05), CA3:  = 9.655, p < 0.05), DG:  = 10.97, p < 0.05)] ] . These rp < 0.05] and IL-18   = 8.367, p < 0.05], and the cleaved GSDMD  induced by LPS  = 26.07,  < 0.05] . Similard by LPS . Our datp < 0.01], P-NF-κB , NLRP3 , ASC , and Cleaved caspase-1  in the LPS group. Sal markedly decreased the expression of proteins in the P2X7/NF-κB/NLRP3 signaling pathway (p < 0.05); CA3:  = 11.53, p < 0.01); DG:  = 8.022, p < 0.05)], NLRP3  = 10.96, p < 0.05); CA3:  and Cleaved caspase-1  were notably up-regulated in the mice exposed to LPS. Conversely, administration of Sal and FLU effectively rescued the LPS-induced changes in the P2X7/NF-κB/NLRP3 cascade .Mechanisms of the effects of Sal on pyroptosis in LPS-induced mice remain undefined. Western blot and immunohistochemical analyses of LPS-injected mice demonstrated increased expression of proteins such as P2X7  . 200 μM CORT reduced the cell viability to 56% and was then chosen in subsequent experiments. Compared with the model group, the cell survival rates of 10 μM and 50 μM Sal were 65% and 70%, respectively, which were significantly increased  (p < 0.001] (p < 0.05)  = 11.69, p < 0.05]  . Further < 0.05] . These fp < 0.001] and IL-18  in the PC12 cells as shown in . On the other hand, the expression of pyroptosis-related proteins was measured by Western blot assays  = 10.92, p < 0.01], IL-18  and cleaved GSDMD  proteins were upgraded in the PC12 cells, which were all significantly reversed by Sal. The cellular membrane integrality was evaluated by Calcein-AM/PI staining and LDH release. These results also indicated that CORT-induced pyroptosis in PC12 cells. Taken together, these findings demonstrated that the protective effects of Sal against CORT-induced cell injury was mediated through inhibiting of pyroptosis.GSDMD-dependent membrane pore formation is an ultimate event of pyroptosis, which promotes the release of pro-inflammatory mediators, such as IL-18 and IL-1β . We emplt assays . In syncp < 0.01], P- NF-κB , NLRP3 , ASC , and Cleaved caspase-1  were significantly higher in the CORT-damaged PC12 cells, and the proteins were obviously down-regulated by Sal  = 16.85, p < 0.01] and Cleaved caspase-1  in the PC12 cells  = 9.217, d by Sal . Our imm12 cells . These rp < 0.001], ASC , Cleaved caspase-1 , IL-1β , IL-18  and Cleaved GSDMD  in vitro. Nonetheless, these effects were all abolished by the Sal (p < 0.05). Nig weakened the inhibitory effect of Sal on pyroptosis. Thus, these results demonstrated that the anti-pyroptosis effect of Sal is associated with NLRP3.Based on the essentiality of NLRP3 in pyroptosis, we assessed the effect of nigericin (an NLRP3 agonist). Our Western blot assay showed that treatment with CORT and nigericin increased the expression of NLRP3   = 14.67, p < 0.01], IL-1β , IL-18 , and cleaved GSDMD  in the PC12 cells treated with CORT.Next, to investigate the impact of NLRP3 on Sal-specific anti-pyroptosis effects, the PC12 cells were transfected with Si-NLRP3. NLRP3 was successfully knocked down by Si-RNA, as demonstrated by Western blot data . We sele < 0.05] . In addi < 0.05] . Our res < 0.05] . As illuRecent studies have reported that Sal exerts various pharmacological effects such as anti-inflammatory, antioxidant activities . Other sin vivo results showed that CORT administration significantly decreased sucrose preference rate, increased the time of immobility in FST, and reduced motility in OFT. However, Sal administration profoundly improved the behavioral defects in SPT, OFT and FST, implying that Sal alleviates depression-like behavior in mice. Furthermore, our data showed that CORT induced damage in the PC12 cells, release of LDH and increased PI staining, which are reversed by Sal. In a previous study, fluoxetine, a typical selective serotonin reuptake inhibitor (SSRI), was shown to effectively reverse anxiety/depression-like behaviors (The pathogenesis of depression is very complex, and the proposed “neurotropism and plasticity” theory is gradually gaining attention in recent years. According to this theory, depression is caused by decreased neurotrophic factors, impaired synaptic plasticity and reduced neural plasticity . BDNF isehaviors . In agreNeuroinflammation is an essential occurrence in the pathogenesis of psychiatric diseases, which is associated with the pathogenesis of depression in vivo and in vitro results indicated that Sal treatment significantly decreased pyroptosis-related protein levels including cleaved GSDMD, cleaved caspase-1, IL-1β, and IL-18. Therefore, the protective effect of Sal against depression-like behavior may be associated with the inhibition of pyroptosis. Pyroptosis is often accompanied by inflammation, which performs an essential role in the fight against infections. The activated NLRP3 inflammasome is one of the most important mediators of pyroptosis, which is involved in various inflammatory and immune diseases (in vitro. In addition, knockdown of the NLRP3 with siRNA significantly inhibited cell pyroptosis and improved cell viability. These results demonstrated that NLRP3 silencing and Sal treatment could prevent cell damage and ameliorate pyroptosis in PC12 cells. Our findings implied that NLRP3 may be a potential therapeutic target for depression and Sal can improve depressive symptoms by inhibiting the NLRP3-mediated pyroptosis.The NLRP3 inflammasome, an important component of the immune system, is one of the most intensively studied inflammasomes, and consists of NLRP3, apoptosis speck-like protein and pro-caspase-1. The NLRP3 inflammasome is present in microglia and astrocytes in the CNS . An incrdiseases . Interes+ ionophores (It has been reported that NLRP3 inflammasome is activated by various stimuli, including ATP and Knophores . The P2Xnophores . P2X7 renophores . In our nophores . NF-κB, nophores . In addinophores . In our Previous studies demonstrated that LPS stimulation leads to pyroptosis and secretion of active IL-1β . In addiIn conclusion, our findings indicated that NLRP3-mediated pyroptosis exerted an essential effect on depressive symptoms. Precisely, Sal can ameliorate pyroptosis via suppressing the P2X7/NF-κB/NLRP3 pathway. Furthermore, we revealed that Sal provides novel therapeutic strategies for the treatment of depression.


Escherichia coli (E. coli) in young chicks. Amongst the different combinations of innate immune stimulants, the in ovo administration of CpG ODN 10 µg in combination with 15 µg of poly I:C was the most effective combination, offering 100% protection from YSI. It is expected that the introduction of these reagents to management practices at the hatchery level may serve as a potential replacement for antibiotics for the reduction of early chick mortality (ECM) due to YSI/colibacillosis.Innate immune stimulants, especially toll-like receptor (TLR) ligands and agonists, are the main players in the initiation of innate immunity and have been widely studied as alternatives to antibiotics to control infection. In the present study, we characterized the dosage levels of various innate immune stimulants, including unmethylated cytosine-phosphate-guanosine dinucleotide -containing oligodeoxynucleotides (CpG ODN), polyinosinic-polycytidylic acid (poly I:C), cyclic polyphosphazene 75B (CPZ75B), avian beta-defensin 2 (ABD2), and combinations of these reagents given in ovo. Data derived from a series of animal experiments demonstrated that the in ovo administration of 10–50 µg CpG ODN/embryo (on embryonic day 18) is an effective formulation for control of yolk sac infection (YSI) due to avian pathogenic  In the last few decades, the poultry industry has witnessed extraordinary production gains. For instance, in 1925, a 1.1 kg chicken was produced in 112 days, whereas, in 1950, the same weight was attainable in 70 days. In 2015, the production of a 2.27 kg chicken was possible in less than 50 days . HoweverClostridial infection (causing necrotic enteritis), and Salmonellosis or asymptomatic colonization of Salmonella and Campylobacter play a critical role in profitability, food security, and food safety issues associated with poultry production [E. coli and Salmonella infections in Canadian human populations [E. coli and Salmonella infections in humans [E. coli strains isolated from broilers in the Netherlands were resistant to cefotaxime  in 2007. However, this resistance prevalence decreased sharply and reached a level of 2.9% in 2014 after the ban on the use of ceftiofur in hatcheries in 2010 [E. coli isolated from retail chicken meat samples and human clinical samples shared approximately 39% similarity, implying that ESBL genes could be transmitted from poultry to humans through the food chain [E. coli in Sweden, as only 0.09% of the population might be expected to carry poultry-associated isolates [E. coli between the Netherlands and Sweden [Bacterial infections, including yolk sac infection (YSI) and colibacillosis ), clinical and subclinical oduction ,7,8,9. Foduction . For insulations . Interesn humans . Similar in 2010 . Anotherod chain . Howeverd Sweden . Thus, iE. coli (APEC) [APEC. It is expected that the introduction of these reagents at the hatchery level will be helpful in reducing ECM, enhancing profitability, and reducing biological, environmental, and ecological hazards associated with the use of antibiotics in young chicks. Omphalitis, or yolk sac infection (YSI), in young chicks, is the major cause of ECM, with most death occurring within 24 h of hatching and peaking at 5 to 7 days . These ii (APEC) . In ovo i (APEC) . In thisAll animal trials were conducted according to animal protocols (AUP 20160079) approved by the Animal Care Committee of the University of Saskatchewan.E. coli, yolk, liver, and heart tissue samples were taken from 5 randomly selected embryos on embryonic day (ED) 18 prior to the administration of innate immune stimulants and cultured on blood agar  and MacConkey agar  plates. Eighteen-day-old embryonated eggs were injected with 100 µL of test formulations or sham (PBS) solutions in ovo into the amniotic sac. Right after hatching, the chicks were transferred to the animal care unit of the Vaccine and Infectious Disease Organization (VIDO), University of Saskatchewan, for the challenge studies. All hatched birds were administered antibiotic-free feed and water ad libitum. In all experiments, specific-pathogen-free (SPF) eggs  were incubated at the Poultry Science hatchery of the University of Saskatchewan. To ensure that the embryos were free of In this animal trial, multiple dosage levels of select innate immune stimulants were characterized in ovo. The treatment groups (n = 20 embryos/group) included CpG ODN at 50, 20, 10, 5, and 1 µg per embryo; cyclic polyphosphazene 75B (CPZ75B) (developed at VIDO) at 10 µg and 20 µg per embryo; and poly I:C at 5, 10, and 20 µg per embryo. The control groups received 100 µL PBS/embryo. This trial was conducted to evaluate formulations prepared from stand-alone or combinations of CpG ODN, poly I:C, avian beta-defensin 2 , and CPZ75B. All formulations and controls were administered in ovo in a volume of 100 µL/embryo. Briefly, on ED18, a total of 350 viable SPF chicken embryos were divided into seven groups designated as A to G comprised of 50 embryos (n = 50) per group. Stand-alone immune stimulants, including CpG ODN 20 µg/embryo, poly I:C 20 µg/embryo, and ABD2 10 µg/embryo, were administered to groups A, B, and C, respectively. Formulations containing combinations of immune stimulants included (Group D) CpG ODN 10µg + poly I:C 15 µg, (Group E) poly I:C 15 µg + ABD2 10 µg, and (Group F) CpG ODN 10µg + poly I:C 15 µg + ABD2 10 µg per embryo. Group G was injected with 100 µL of sterile PBS as a sham control.In this experiment, additional combinations of innate immune stimulants were tested. On ED18, 120 viable SPF chicken embryos were randomly divided into 4 equal groups (n = 30/group) designated as A to D. Groups A, B, and C were given formulations including (i) CpG ODN 20 µg + CPZ75B 10 µg, (ii) poly I:C 10 µg + CPZ75B 10 µg, or (iii) CpG ODN 20 µg + poly I:C 10µg + CPZ75B 10 µg/embryo, whereas group D was treated with 100 µL sterile PBS. E. coli strain EC317. E. coli EC317 was originally isolated from a case of septicemia in a turkey. This APEC strain was found to be highly pathogenic and causes both sepsis and cellulitis in chickens. E. coli EC317 belongs to serotype O2, has a K1 capsule and Type-1 pili, produces aerobactin, and is serum-resistant [E. coli. The daily survival rate was determined as the ratio of the number of alive birds in a specific group on that day to the number of challenged birds in that group.The efficacy of each formulation was evaluated through the reduction in ECM for 7 days following a severe challenge using avian pathogenic esistant . Birds wp < 0.05.The survival curves from the treatment and control (PBS) groups were statistically analyzed using GraphPad PRISMTM 8 software . The data were normalized by log ranking. Statistical differences were determined by one-way analysis of variance  and Chi-square tests (Gehan–Breslow–Wilcoxon). The differences were considered statistically significant at APEC) in trial 1. For CpG ODN, our data showed survival rates of 76%, 81%, 77%, 60%, and 44% on the seventh day post-challenge following the in ovo administration of 50 µg, 20 µg, 10 µg, 5 µg, and 1 µg, respectively. There were no significant differences between the dosage levels of 50 µg, 20 µg, and 10 µg of CpG ODN. Survival rates at dosage levels of 5 µg and 1 µg were not different from the controls  following the in ovo administration of CpG ODN 20 µg/embryo, poly I:C 20 µg/embryo, and ABD2 10 µg/embryo, respectively . These sMore combinations of immune stimulants, including (group A) CpG ODN 20 µg in combination with CPZ75B 10 µg/embryo, (group B) poly I:C 10 µg plus CPZ75B 10 µg/embryo, and (group C) a combination of three immune stimulants consisting of CpG ODN 20 µg plus poly I:C 10 µg plus CPZ75B 10 µg/embryo, were tested in trial 3. In these studies, groups A and C showed significantly higher survival rates  compared with the control group at 62%, whereas group B showed survival at 60% which was not statistically different from that of the control group at 7 days PC .Mycobacterium tuberculosis, Listeria, and Francisella species, following the administration of CpG ODN in mice [E. coli infection [The use of antibiotics in food animal production is one of the major sources of the emergence of antimicrobial resistance (AMR) in animals and humans. AMR causes ~700,000 deaths per year worldwide ,21. Anti in mice ,32,33,34 in mice ,36. Simi in mice ,38,39,40nfection . Yersinia enterocolitica. Lanteir et al. (2014) showed that protection from Cryptosporidium and Salmonella Typhimurium infection following the administration of poly I:C in neonatal mice critically depends on IL-12p40, IFN-α, and IFN-β induction mediated through the activation of MDA5 and TRIF via TLR3 signaling [APEC-based YSI through the in ovo administration of 10–50 µg CpG ODN; meanwhile, using a dose of 5 µg/embryo or lower exhibited similar levels of protection to the PBS group. No differences in survival were found among groups treated with CpG ODN at 10, 20, and 50 µg/embryo, suggesting that the cost-effective dosage levels for the stand-alone use of CpG ODN delivered in ovo are between 10 and 20 µg/embryo . Additionally, birds receiving 20 µg of poly I:C or 10 µg of ABD2/embryo alone had survival rates comparable to those of birds treated with 20 µg of CpG ODN . Among the three innate immune stimulants , CpG ODN is more expensive than poly I:C or ABD2. Hence, in order to apply these immunostimulants in the commercial poultry sector, the co-administration of CpG and poly I:C or ABD2 using a half-dose each may be beneficial in lowering the cost and enhancing APEC protection in young birds. Poly I:C, another innate immune stimulant investigated in these studies, mediates innate immune activation through its binding to multiple dsRNA sensors, including retinoic acid-inducible gene I (RIG1), melanoma differentiation-associated protein 5 (MDA5), and TLR3 . TLR3 anignaling . Similarignaling . In conjignaling . Data frSeveral reports have shown that the co-stimulation of multiple TLRs with selective synthetic TLR ligands may activate different TLR-associated adaptor proteins and signaling cascade-modulating factors leading to the augmentation or suppression of immune responses ,48. For Innate immune stimulants, such as CpG ODN and poly I:C, are not only potential anti-infection reagents, but also are good adjuvants to enhance immune responses for their incorporated vaccines. These immunostimulatory reagents have been intensively studied as adjuvants in poultry for improving both humoral and cellular immune responses. For instance, the in ovo administration of encapsulated CpG ODNs 2007 with herpesvirus of turkey (HVT) vaccine could moderately enhance HVT efficacy and reduce tumor incidence . CpG ODNWe have demonstrated that the in ovo co-administration of CpG 10 µg/embryo and poly I:C 15 µg/embryo provided the highest degree of protection against YSI  in young birds. In addition, stand-alone use via the in ovo route of CpG ODN 10–50 µg/embryo, poly I:C 20 µg/embryo, or ABD2 10 µg/embryo could significantly reduce ECM due to severe YSI, with survival ranging from 76 to 84%. Similarly, the synergetic effects of various combinations of innate immune stimulants, including: (i) CpG ODN 20 µg + poly I:C 10 µg + CPZ75B 10 µg , (ii) CpG ODN 20 µg + CPZ75B 10 µg , or (iii) poly I:C 15 µg + ABD2 10 µg  showed significant improvements in the survival rate following severe YSI. Further characterization of these formulations is warranted to investigate their effectiveness against other bacterial infections in young chicks.


Takifugu obscurus) was characterized by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Fourier-transform infrared spectroscopy (FTIR), and fibril-forming tests. The results indicate that extraction at 12 °C can effectively improve the extraction efficiency of natural collagen compared with extraction at 4 °C. However, extraction at 20 °C results in a decrease in molecular integrity, thus, inducing the resultant collagen to degrade or even lose fibril-forming ability. Transmission electron microscope (TEM) images revealed that ASC-4, PSC-4, ASC-12, and PSC-12 can assemble into fibrils with D-periodicities, and ASC-20 associated into molecular aggregates alongside partial D-banded fibrils, while no well-defined fibrils were observed in PSC-20. Scanning electron microscope (SEM) analysis confirmed the well-defined fibril morphologies of ASC-4, PSC-4, ASC-12, and PSC-12 with imino acid contents between 190.0 and 197.8 residues/1000 residues. The denaturation temperature of ASC-4, PSC-4, ASC-12 and PSC-12 was 30.0, 27.6, 25.9 and 22.7 °C, respectively. This study indicates that ASC and PSC extracted at 4 °C and 12 °C could be alternatives to terrestrial collagens for industrial applications.Acid-solubilized (ASC) and pepsin-solubilized collagen (PSC) extracted at 4 °C (ASC-4 and PSC-4), 12 °C (ASC-12 and PSC-12), and 20 °C (ASC-20 and PSC-20) from the skin of farmed pufferfish ( Collagen is the most abundant extracellular protein found in skin, bone, tendon, ligament, and corneal and other connective tissues . The hieTakifugu obscurus), a fish species of economic importance, belongs to the family Tetradontidae of teleost fish. Pufferfish are famous for their puffing behavior and have become increasingly popular among consumers for their non-toxicity, desirable flavor, and high nutritional value [Pufferfish  [Saurida tumbil) [Chlorurus sordidus) [Huso huso) [Reinhardtius hippoglossoides) [Oncorhynchus mykiss) [So far, skin collagen has been extracted from many fish species, including barramundi (carifer) , lizardf tumbil) , parrotfordidus) , sturgeoso huso) , Greenlassoides)  and rain mykiss) . Traditi mykiss) . However mykiss) ,20. Limi mykiss) . Optimum mykiss) . Thus, tTakifugu rubripes) (89.31%) [The proximate composition of pufferfish skin is moisture (72.15 ± 2.55%), ash (2.84 ± 0.11%), protein (24.12 ± 0.86%), and fat (0.51 ± 0.05%). The protein content in pufferfish skin is higher than that of Spanish mackerel (17.17%) and Nile perch (21.6%) . In addip < 0.05), indicating that pepsin treatment can efficiently cleave the cross-linked collagen molecules at telopeptide regions and induce extraction with higher yield. Extraction temperature also had significant influence on the extraction efficiency. The recovery rate of ASC-12 (52.21%) and PSC-12 (63.41%) was markedly increased when compared with that of ASC-4 (40.52%) and PSC-4 (46.42%) after 20 h of extraction (p < 0.05). However, with extraction at a higher temperature (20 °C), the recovery rate of PSC-20 (57.42%) was lower than that of ASC-20 (65.11%) after 20 h of extraction (p < 0.05) and then decreased continuously as extraction time proceeded. This result might be attributed to the excessive pepsin digestion at the high temperature, resulting in an increase in lower-MW (molecular weight) protein fragments. Thus, pepsin treatment and extraction temperature are two important parameters that influence the recovery rate and extraction efficiency. Samples acquired under the condition that produced a high recovery rate at each extraction temperature  were prepared for the subsequent experiments.The recovery rate of extracted collagen of all treatments is shown in T. obscurus skin might be type I collagen. The subunit composition of ASC-4, PSC-4, ASC-12, PSC-12 and ASC-20 was similar, suggesting that the extraction temperature applied does not significantly induce collagen molecule chains to degrade. However, PSC-20 contained reduced proportions of β-chain as well as low-MW fragments (<97.4 kDa), indicating that high-Mw chains are partially degraded by excessive pepsin digestion at higher temperature.The SDS-PAGE analysis showed that all extracted samples were composed of one β chain and at least two α chains (α1 and α2) as their major subunits . The mol−1) is related to N–H stretch coupled with hydrogen bonds and moves to a lower frequency  when N–H groups are involved in the formation of hydrogen bonds [−1, respectively. This result indicates that fewer N–H groups in collagen extracted at higher temperature are involved in hydrogen bond formation, suggesting the triple-helical structure is less intact.FTIR spectra exhibited four amides  identified as the major peaks of extracted collagens . Amide Aen bonds . The ami− and is the important factor in investigating the secondary structure of proteins [−1, respectively; meanwhile, the amide II bands were detected at 1550, 1548, 1548, 1545, 1542 and 1541 cm−1, respectively. This result represents that the degree of molecular order of ASC-20 and PSC-20 is relatively lower than that of ASC-4, PSC-4, ASC-12 and PSC-12. The amide III band is associated with C–N stretching and N–H deformation from amide linkages as well as the absorption caused by the wagging vibration of the CH2 groups of the glycine backbone and the proline side chains [−1. Based on SDA-PAGE and FTIR spectra data, extraction at higher temperature with pepsin digestion results in a decrease in molecule order in extracted collagen.The amide I band is associated with the stretching vibrations of the carbonyl group (C=O) or hydrogen bond coupled with COOproteins . The amiproteins ,29. The e chains . The amiIn vivo, collagen molecules can undergo self-assembly into highly ordered supramolecular structures and further intertwine into fibrils which can form even larger bundles . PreviouAs shown in Therefore, it was speculated that higher extraction temperature with pepsin treatment could impair the structural integrity in whole or in part, thus, causing a decrease in fibril formation ability. It can also be seen in The TEM images of the resulting fibrils are shown in D-periodical banding serves as an indication for the reconstruction of native-like fibrils and is important in maintaining the mechanical stability and biological functions of tissues . It has Combined with the turbidity–time curves and fibril-forming degree, the results revealed that pepsin treatment influences the fibril-forming rate of the extracted collagens, and collagen lacking telopeptides might form a looser fibrillary structure ,39, whilThe SEM images of resulting fibrils are shown in Loligo vulgaris squid mantle collagen (585–591 residues/1000 residues) [Amino acid compositions of four extracted collagens expressed as residues per 1000 total amino acid residues are shown in esidues) . Hydrophesidues) . This reThe imino acid (proline + hydroxyproline) contents of ASC-4, PSC-4, ASC-12 and PSC-12 were 192.6, 197.8, 191.6, and 190.0 residues/1000 residues, which are similar to those of unicorn leatherjacket (187–190 residues/1000 residues) , higher dT values of ASC-4, PSC-4, ASC-12 and PSC-12 were 30.0, 27.6, 25.9 and 22.7 °C, respectively  was higher than that of collagens from the skin of Spanish mackerel (14.7–15.1 °C) and rainbow trout (21.4–21.5 °C) [dT of ASC-4 and PSC-4 was higher than that of several invertebrates, including Cyanea nozakii jellyfish (23.8 °C) and Loligo vulgaris squid (21–22 °C) [The PSC-4 27.–30.0 °C 21.5 °C) , but low21.5 °C) . This re21.5 °C) . Moreove1–22 °C) . TherefoThe effect of pH on the solubility of extracted collagens is shown in The solubility values of extracted collagens decreased sharply at a high NaCl concentration above 2% B. The prAcid-solubilized and pepsin-solubilized collagen (type I) was prepared from the skin of pufferfish at different temperatures. Results showed that an extraction temperature of 12 °C markedly increases the yield and extraction efficiency compared to an extraction temperature of 4 °C. However, extraction at 20 °C results in a degradation of subunit integrity, which leads to the formation of poorly banded and not well-ordered fibrillary aggregates. Moreover, the triple-helical structure of collagens extracted at 4 °C and 12 °C is maintained, as observed under FTIR spectra, turbidity assay, and TEM analyses. In conclusion, ASC-4, PSC-4, ASC-12 and PSC-12 exhibit better fibril formation abilities and have the potential to be explored further as an alternative collagen source.Acetic acid, sodium chloride, sodium dodecyl sulfate (SDS), ammonium persulfate, and Coomassie Brilliant Blue R-250 were purchased from Sinopharm Chemical Reagent Co., Ltd. . High-MW marker was purchased from Solarbio Biotechnology Co., Ltd. . Pepsin (EC 3.4.23.1) was purchased from Sigma-Aldrich Chemical Company . All other reagents used were of analytical grade.Moisture, ash, crude protein, and fat of raw materials were measured according to the procedures of AOAC (2003)  method nT. obscurus) skin was obtained from Shenshi Aquatic Product Co., Ltd. . Fresh skin was kept in ice at a ratio of 1:2 (w/w) and transported to the laboratory within 24 h. Upon arrival, fish skin was washed with iced tap water (0–4 °C) and cut into small pieces (approximately 0.5 × 0.5 cm2). To remove non-collagenous protein and pigment, skin pieces were soaked in 0.1 M NaOH with a skin/solution ratio of 1:20 (w/v) for 12 h. The mixture was continuously stirred, and the alkaline solution was changed every 4 h. Alkali-treated skin was washed with distilled water until pH was neutral, homogenized, and then lyophilized [Pufferfish (philized .w/v). Extraction procedures were conducted at different temperatures  for 0–50 h with continuous stirring following the method of Li et al. [g for 30 min at 4 °C. Then, ASC in the supernatant was precipitated by adding NaCl to a final concentration of 0.9 M and collected by centrifuging at 10,000× g for 30 min at 4 °C. The precipitates were re-dissolved in a minimum volume of 0.5 M acetic acid, dialyzed at 4 °C against 0.1 M acetic acid for 12 h and distilled water for 48 h, and then lyophilized. The obtained ASCs extracted at 4, 12 and 20 °C were referred to as ASC-4, ASC-12 and ASC-20, respectively.Pretreated skin was soaked in 0.5 M acetic acid with a skin/solvent ratio of 1:50  containing 1% (w/w) pepsin. Extraction procedures were conducted at different temperatures  for 0–50 h with continuous stirring following the method of Li et al. [g for 30 min at 4 °C. Precipitate collagen was re-dissolved in 0.5 M acetic acid, dialyzed against 0.02 M Na2HPO4 solution for 12 h, 0.1 M acetic acid for 12 h, and distilled water for 48 h, and then lyophilized. The obtained PSCs extracted at 4, 12 and 20 °C were referred to as PSC-4, PSC-12 and PSC-20, respectively.Pretreated skin was soaked with 0.5 M acetic acid in a ratio of 1:50  concentration at 4 °C. The obtained solution was mixed with 0.1 M phosphate buffer (pH 7.4), and the final pH was adjusted to neutral (pH 7.0 ± 0.2). The solution was kept at 25 °C to reconstruct the fibrils. Fibril formation rate was obtained by measuring the turbidity changes by the increase in absorbance at 310 nm using a UV–visible spectrophotometer . After the fibril formation experiment, the mixture was centrifuged at 8000× g for 30 min. Fibril formation degree was calculated as follows:Fibril formation ability was measured following the method of Pal et al.  with sliw/v) phosphotungstic acid. TEM images were observed using a transmission electron microscope .The TEM observations of collagen fibrils were obtained following the method of Li and Douglas . Samplesv/v) glutaraldehyde for 12 h, then rinsed with 0.1 M phosphate buffer (pH 7.2). After dehydration in a graded series of ethanol, fibril samples were dried in a critical point dryer . A scanning electron microscope  was applied to observe the SEM morphology at 10,000× and 40,000× magnification.The SEM observations of fibril samples were obtained according to the method of Zhang et al.  with sliAmino acid compositions were analyzed following the method of Li et al. . CollagedT) were determined following the method of Yan et al. [The denaturation temperatures  − ηsp(minimum))/(ηsp(maximum) − ηsp(minimum)), where ηsp is the specific viscosity. dT was determined as the temperature at which the change in viscosity was 50% decreased.Fractional viscosity = . The relative solubility was calculated as a percentage of that obtained at the pH giving the highest solubility.The solubility of extracted collagens, as influenced by pH and NaCl concentration, was determined by the methods of Shen et al. . Samplesg for 30 min. Protein content in the supernatant was determined as described above. The relative solubility was calculated in comparison to the total collagen in 0.5 M acetic acid.Samples were redissolved in 0.5 M acetic acid at 4 °C for 12 h to a concentration of 6 mg/mL. An 8 mL amount of sample solution was mixed with an equal volume of NaCl in 0.5 M acetic acid solution to obtain final concentrations of 1, 2, 3, 4, 5 and 6%. The mixtures were stirred for 20 min at 4 °C, then centrifuged at 10,000× p < 0.05.An analysis of variance (ANOVA), followed by Duncan’s multiple-range test, was used for comparisons by SPSS 25.0 software . Experiments were performed in triplicate. Mean values with standard deviations (SD) were reported. Difference was considered to be significant if