Kv2 voltage-gated potassium channels are modulated by amphoterin-induced gene and open reading frame (AMIGO) neuronal adhesion proteins. Here, we identify steps in the conductance activation pathway of Kv2.1 channels that are modulated by AMIGO1 using voltage-clamp recordings and spectroscopy of heterologously expressed Kv2.1 and AMIGO1 in mammalian cell lines. AMIGO1 speeds early voltage-sensor movements and shifts the gating charge-voltage relationship to more negative voltages. The gating charge-voltage relationship indicates that AMIGO1 exerts a larger energetic effect on voltage-sensor movement than is apparent from the midpoint of the conductance-voltage relationship. When voltage sensors are detained at rest by voltage-sensor toxins, AMIGO1 has a greater impact on the conductance-voltage relationship. Fluorescence measurements from voltage-sensor toxins bound to Kv2.1 indicate that with AMIGO1, the voltage sensors enter their earliest resting conformation, yet this conformation is less stable upon voltage stimulation. We conclude that AMIGO1 modulates the Kv2.1 conductance activation pathway by destabilizing the earliest resting state of the voltage sensors. Kv2 potassium channels activate a potassium conductance that shapes neuronal action potentials. The AMIGO family of adhesion proteins modulate activation of Kv2 conductances, yet, which activation steps are modified is unknown. This study finds that AMIGO1 destabilizes the earliest resting conformation of the Kv2.1 voltage sensors to promote activation of channel conductance.+ conduction  channels of the Kv2 family open following membrane depolarization and are critical regulators of neuronal electrical excitability. Mammals have two Kv2 pore-forming α subunits, Kv2.1 and Kv2.2, which function as homo- or heterotetramers . However, it is not known which steps in the conductance activation pathway are modulated by any of these forms of regulation.Kv2 channels are abundant in most mammalian central neurons . GeneticG–V by -5 to -15 mV  and GxTX Lys27Pra(JP)) were used to monitor the chemical environment surrounding GxTX when localized to the channel ) was used to selectively modulate Kv2.1 channel gating and to fluorescently identify surface-expressing Kv2.1 channels . Conjuga channel . All mod channel , 47, 48.2. Chinese hamster ovary (CHO) cell lines were a tetracycline-regulated expression variant  and were cultured as described previously , prepared in 70% ethanol, was added to Kv2.1–CHO cells to induce rKv2.1 channel expression for 1.5 h to minimize series-resistance-induced voltage errors in K+-current recordings or for 48 h to produce sufficient Kv2.1 density necessary for recording gating currents. 5 min prior to transfection, cells were plated at 40% confluency in unsupplemented culture media free of antibiotics, selection agents, and serum and allowed to settle at room temperature. For imaging studies (except concentration-response), cells were plated in 35 mm no. 1.5 glass-bottom dishes . For concentration-response time-lapse imaging, cells were plated onto 22 × 22 mm no. 1.5H cover glass (Deckglaser). For electrophysiological studies, cells were plated in 35 mm tissue-culture-treated polystyrene dishes . Transfections were achieved with Lipofectamine 2000 . Each transfection included 220 μL Opti-MEM , 1.1 μL Lipofectamine, and the specified amount of plasmid DNA. HEK293 cell experiments included 0.1 μg of mKv2.1 DNA and either 0.1 μg plasmid encoding enhanced green fluorescent protein (pEGFP), mAMIGO1-pIRES2-GFP DNA, or hSCN1β-pIRES2-GFP. The pIRES2-GFP vector has an encoded internal ribosome entry site that promotes continuous translation of two genes from a singular mRNA  with enhanced yellow fluorescent protein (eYFP). pCAG-ChroME-mRuby2-ST encodes an mRuby2-tagged channelrhodopsin with a Kv2.1 PRC trafficking sequence  with 10% fetal bovine serum  and 1% penicillin/streptomycin  in a humidified incubator at 37°C under 5% COeviously . The Kv2eviously  was stabRuby2-ST  plasmidssequence ,52. mKv22, 2 MgCl2, and 10 glucose, adjusted to pH 7.3 with KOH, 345 mOsm; HEK293 external (in mM) 5 KCl, 160 NaCl, 10 HEPES, 2 CaCl2, 2 MgCl2, 10 glucose, adjusted to pH 7.3 with NaOH, 345 mOsm, and 5 μM tetrodotoxin was added to the recording solution: liquid junction potential (LJP) was 3.9 mV and EK was -89.0 mV with HEK293 internal; Kv2.1–CHO internal (in mM) 70 KCl, 5 EGTA, 50 HEPES, 50 KF, and 35 KOH, adjusted to pH 7.4 with KOH, 310 mOsm; Kv2.1–CHO external (in mM) 3.5 KCl, 155 NaCl, 10 HEPES, 1.5 CaCl2, 1 MgCl2, adjusted to pH 7.4 with NaOH, 315 mOsm: LJP was 8.5 mV and EK was -97.4 mV with Kv2.1–CHO cell internal; high Mg2+ Kv2.1–CHO external (in mM) 3.5 KCl, 6.5 NaCl, 10 HEPES, 1.5 CaCl2, 100 MgCl2, adjusted to pH 7.4 with NaOH, 289 mOsm: LJP was 13.1 mV and EK was -97.4 mV with Kv2.1–CHO internal. Osmolality was measured with a vapor pressure osmometer , and a 5% difference between batches was tolerated. LJP values were tabulated using Patcher’s Power Tools version 2.15 , and corrected post hoc during analysis. Voltage protocols list command voltages prior to LJP correction. Kv2.1CHO cells were harvested by scraping in Versene  or TrypLE . HEK293 cells were dislodged by scraping. Cells were washed three times in a polypropylene tube in the external solution used in the recording chamber bath by pelleting at 1000 x g for 2 min and rotated at room temperature (22–24°C) until use. Cells were then pipetted into a 50 μL recording chamber  and allowed to settle for 5 or more minutes. After adhering to the bottom of the glass recording chamber, cells were rinsed with external solution using a gravity-driven perfusion system. Cells showing plasma-membrane-associated YFP, or intracellular GFP of intermediate intensity, were selected for patching. Thin-wall borosilicate glass recording pipettes  were pulled with blunt tips, coated with silicone elastomer , heat cured, and tip fire-polished to resistances less than 4 MΩ. A series resistance of 3–9 MΩ was estimated from the whole-cell parameters circuit. Series resistance compensation (of <90%) was used as needed to constrain voltage errors to less than 10 mV, and the lag was 10 μs. Cell capacitances were 4–15 pF. Capacitance and Ohmic leak were subtracted using a P/5 protocol. Output was low-pass filtered at 10 kHz using the amplifier’s built-in Bessel and digitized at 100 kHz. Traces were filtered at 2 kHz for presentation. The intersweep interval was 2 s. HEK293 cells with less than 65 pA/pF current at +85 mV were excluded to minimize the impact of endogenous K+ currents  was an amplitude between 90 and 100 ms post depolarization. Mean tail current was the current amplitude between 0.2 and 1.2 ms into the 0 mV step. 100 μL of 100 nM GxTX-594 was flowed over cells with a membrane resistance greater than 1 GΩ, pulses to 0 mV were gauged over the time course of binding, and the G–V protocol was run. Data with a predicted voltage error Verror ≥10 mV was excluded from analysis. Verror was tabulated using estimated series resistance post compensation :Voltage clamp was achieved with an Axopatch 200B patch-clamp amplifier  run by Patchmaster . Solutions included HEK293 internal (in mM) 160 KCl, 5 EGTA, 10 HEPES, 1 CaClcurrents . The aveG–V profiles, the cell membrane voltage (Vmembrane) was adjusted by Verror and LJP:For G–V relations were individually fit with a fourth power Boltzmann:f(V) is normalized conductance (G), A is maximum amplitude, x is the number of independent identical transitions required to reach full conductance , V1/2 is activation midpoint, z is the valence in units of elementary charge (e0), F is the Faraday constant, R is the ideal gas constant, and T is absolute temperature. The half-maximal voltage (VMid) for fourth-power functions isTail currents were normalized by the mean current from 50 to 80 mV. Fitting was carried out using Igor Pro software, version 7 or 8  that employs nonlinear least squares curve fitting via the Levenberg-Marquardt algorithm. To represent the four independent and identical voltage sensors that must all activate for channels to open, zi and V1/2 ± SD. The minimum Gibbs free energy (ΔGAMIGO1) that AMIGO1 imparts to conductance, was tabulated asReconstructed Boltzmann curves use average R = 0.00199 kcal/(K⋅mol), and T = 298 K. fKv2.1+AMIGO1 is the reconstructed relative conductance of Kv2.1 + AMIGO1 at Vi,Mid of Kv2.1-control cells  and sigmoidicity values (σ)  were from fitting 1 to 100 ms of the current decay during 0 mV tail step with an exponential functionActivation time constants (lues (σ)  were derτdeact was the average after steps to +10 to +120 mV or +50 to +120 mV in GxTX–594. Kv2.1 deactivation kinetics became progressively slower after the establishment of the whole-cell mode, similar to Shaker deactivation after patch excision  155 NaCl, 50 HEPES, 20 KOH, 2 CaCl2, 2 MgCl2, 0.1 EDTA, adjusted to pH 7.3 with HCl, 347 mOsm; and Kv2.1-CHO single channel external (in mM) 135 KCl, 50 HEPES, 20 KOH, 20 NaOH, 2 CaCl2, 2 MgCl2, 0.1 EDTA, adjusted to pH 7.3 with HCl, 346 mOsm: LJP -3.3 mV with Kv2.1-CHO single-channel internal. Thick-wall borosilicate glass  was pulled, Sylgard coated and fire polished, to resistances >10 MΩ. Analysis methods were the same as those previously used  90 NMDG, 1 NMDG-Cl, 50 HEPES, 5 EGTA, 50 NMDG-F, 0.01 CsCl, adjusted to pH 7.4 with methanesulfonic acid, 303 mOsm; and gating-current external (in mM) 150 TEA-Cl, 41 HEPES, 1 MgCl2 · 6 H2O, 1.5 CaCl2, adjusted to pH to 7.3 with NMDG, 311 mOsm: LJP -3.3 mV with gating-current internal. To avoid KCl contamination of the recording solution from the pH electrode, pH was determined in small aliquots that were discarded. Cells were resuspended in Kv2.1-CHO external and washed in the recording chamber with 10 mL gating-current external. Pipettes has resistances of 6–14 MΩ. Series resistances were 14–30 MΩ and were compensated 50%. Cell capacitances were 6–10 pF. Verror was negligible (<1 mV). P/5.9 leak pulses were from a -133 mV leak holding potential. An early component ON-gating-charge movement was quantified by integrating ON gating currents in a 3.5-ms window  following the end of fast capacitive artifacts created from the test voltage step . The slow tail of the ON-charge movement is difficult to accurately integrate in these cells, making the cutoff point arbitrary. This 3.5-ms integration window resulted in a more positive QON,fast–V midpoint than with a 10-ms window . Currents were baseline subtracted from 10 to 20 ms into the step. Gating-charge density fC/pF was normalized by cell capacitance. Q–V curves were normalized to an average of 100–120 mV. Q–V relations were individually fit with a first-power Boltzmann (x = 1). Time constants (τON) were determined from a double-exponential fit function:Methods were the same as those used for whole-cell Kreported . Currentoltzmann , x = 1. τrise was not used in analyses. Ig,OFF was not well fit by τOFF was not analyzed. The voltage dependence of the forward voltage-sensor-activation (α) rate was determined by fitting the average τON–V weighted by the standard error:G‡AMIGO1) wask = α0mV. Estimates of ΔGAMIGO1 from Q–V relations were with Reverse rates were not analyzed. The energy of AMIGO1’s impact on the activation rate of all four voltage sensors  as calculated from integration above and below QOFF–V relations using a trapezoidal rule  run by ZEN black version 2.1. Laser lines were 3.2 mW (488 nm), 1.2 mW (514 nm), 0.36 mW (543 nm), and 0.60 mW (594 nm). Images were acquired with a 1.4 numerical aperture (NA) 63x (Zeiss 420782-9900-799), 1.3 NA 40x (Zeiss 420462-9900-000), or 1.15 NA 63x objectives (Zeiss 421887-9970-000). Images were taken in either confocal or airy disk imaging mode. The imaging solution was Kv2.1-CHO external supplemented with 0.1% bovine serum albumin and 10 mM glucose. The temperature inside the microscope housing was 24–28°C. Representative images had brightness and contrast adjusted linearly.μL GxTX–594 dilutions were applied for 10 min then washed out by flushing 10 mL at a flow rate of ∼1 mL/10 s. 15 min after wash out, the next GxTX-594 concentration was added. Airy disk imaging had a 1.4 NA 63x objective (Zeiss 420782-9900-799), 0.13 μm pixels, 0.85 μs dwell, and a 5-s frame rate. YFP excitation had 488 nm 2% power and an emission of 495–550 nm. GxTX–594 excitation had 594 nm 2% power and an emission of 495–620 nm. Intensities were extracted using FIJI (Kd) fit with fluorescence intensity at 0 nM GxTX–594 was set to 0 withCells plated on coverslips were washed 3 times with imaging solution and then mounted on an imaging chamber  with vacuum grease. 100 μL 100 nM GxTX-594 in imaging external was applied for 10 min then diluted with 1 mL Kv2.1-CHO external for imaging. Airy disk imaging had a 1.15 NA 63x objective (Zeiss 421887-9970-000), 0.11 μm pixels, 0.85 μs dwell, 2x averaging, and a 1-s frame rate. GxTX-594 excitation had 594 nm 1% power and an emission of 605 nm longpass. Cells with obvious GxTX-594 labeling were whole-cell voltage clamped. Voltage-clamp fluorimetry internal included (in mM) 70 mM CsCl, 50 mM CsF, 35mM NaCl, 1 mM EGTA, 10 mM HEPES, adjusted to pH 7.4 with CsOH, 310 mOsm: LJP -5.3 mV with Kv2.1–CHO external. Pipettes from thin-wall glass were less than 3.0 MΩ. Cells were held at -100 mV for 30 images and stepped up to +35 mV until fluorescence changes appeared complete. Intensity data were extracted using Zen Blue from regions of interest drawn around an apparent surface-membrane-excluding pipette region. For presentation, fluorescence-intensity traces were normalized from minimum to maximum. The rate of GxTX-594 dissociation (kΔF) was fit with a monoexponential function  solution for 5–10 min then washed with imaging solution. Spectral confocal imaging had a 1.4 NA 63x objective, 0.24 μm pixels, 8.24 μs dwell, 2x averaging. YFP excitation was 514 nm. GxTX Ser13Pra(JP) excitation was 594 nm. GxTX Lys27Pra(JP) excitation was 543 nm. Fluorescence counts were extracted in Zen Blue. JP emission spectra were fit with two-component split pseudo-Voigt functions (https://fityk.nieto.pl/), which employed a Levenberg-Marquardt algorithm. Goodness of fit was determined by root-mean-squared deviation values, which are listed in Environment-sensitive fluorescence imaging was done with GxTX Ser13Pra(JP) and GxTX Lys27Pra(JP). Cells were incubated in 100 unctions  using thn) are individual cells pooled over multiple transfections. The n values from each transfection for each figure are listed in n values were pooled. Statistical tests were conducted with Prism 9 , and details are included in the figure legends.Independent replicates (G–V relation of mouse Kv2.1 by -5.7 ± 2.3 mV (standard error of the mean [SEM]) (+ currents (Voltage-clamp recordings from co-transfected HEK293 cells indicate that mouse AMIGO1 shifts the n [SEM]) . This shn [SEM])  and smaln [SEM]) . This smn [SEM]) ,60 and tn [SEM]) , and flucurrents .As expression systems can influence auxiliary protein interactions with ion channels , 65, 66,+-current recordings and the prolonged 48-h induction period used for gating-current recordings or imaging studies. COVs were compared against an uninduced control (0-h induction) and against an engineered protein, ChroME-mRuby2, which contains the Kv2.1 PRC trafficking sequence but lacks the Kv2.1 voltage-sensing and pore-forming domains . With a limited 1.5-h induction, GxTX-594 was difficult to detect at the glass-adhered membrane, so we moved the confocal imaging plane further from the cover glass to image Kv2.1 on apical cell surfaces where GxTX-594 labeling was more apparent. On these apical surfaces, PCC1.5h and PCC48h were greater than PCC0h (A), consistent with some co-localization of AMIGO1-YFP and Kv2.1. The weakly significant increase of the PCC1.5h compared with PCC0h is consistent with some co-localization. Disproportionate expression can skew PCC values  or with AMIGO1-YFP (Kv2.1 + AMIGO1 cells) and were identified for the whole-cell voltage clamp based on the presence of cytoplasmic GFP fluorescence or plasma-membrane-associated YFP fluorescence, respectively (A). Macroscopic ionic-current recordings were made in whole-cell voltage-clamp mode, and the K+ conductance was measured from tail currents (B and C). In expectation of small AMIGO1 effects relative to cell-to-cell variation, recordings from control cells and AMIGO1 cells were interleaved during each day of experiments, and cell identity was blinded during analysis. G–V relations were fit with a fourth-power Boltzmann function , and average midpoints of half-maximal conduction  and steepness equivalents (zi) were determined , consistent with prior reports of Vi,Mid ranging from -3 to +8 mV in CHO cells , yet the average Vi,Mid was negatively shifted by -5.7 ± 2.2 mV (SE), similar to ΔVi,Mid from mouse Kv2.1 in HEK293 cells  was fit with the power of an exponential function (σ), which quantifies the delay before current rise, and activation time constant (τact). σ was not significantly altered by AMIGO1 , suggesting that the Kv2.1 activation pathway retains a similar structure to AMIGO1 , consistent with results of Maverick and colleagues . At 0 mV, we expected >85% of all Kv2.1-control voltage sensors or >95% of all Kv2.1–AMIGO1 voltage sensors to activate in less than 2 ms, such that the majority of single-channel openings represent stochastic fluctuations between a closed and open conformation of the pore. Neither the single-channel current amplitude  nor the intrasweep open probability (F) were significantly impacted by AMIGO1. AMIGO1 did not significantly impact the single-channel open or closed dwell times (G–L). These results constrain any impact of AMIGO1 on Kv2.1 pore opening to be smaller than the variability in these single-channel measurements.To more directly assess whether the pore-opening step of the Kv2.1 activation pathway is modulated by AMIGO1, we analyzed pore openings of single Kv2.1 channels during 1-s-long recordings to 0 mV A and B.mplitude C, D, E bability F were sll times G–L. TheVi,Mid of -22.1 ± 4.8 (SEM) with GxTX-594 was distinct from the AMIGO1 ΔVi,Mid of -5.7 ± 2.2 mV (SEM) without GxTX-594 , indicating that GxTX-594 amplifies the impact of AMIGO1 on Kv2.1 conductance. We did not observe a significant effect of AMIGO1 on τact or σ in GxTX-594 (J–N). We calculated the impact of AMIGO1 on a pore-opening equilibrium constant (Keq) at the midpoint of the Kv2.1 G–V relation and found a 3.7-fold bias toward a conducting conformation in 100 nM GxTX-594 versus a 1.4-fold bias under control conditions , which correspond to movement of Kv2.1 voltage sensors across the transmembrane electric field. Kv2.1–CHO cells were patch clamped in whole-cell mode in the absence of K+ (A) and given voltage steps to elicit gating currents (B and C). The resolvable ON-gating currents  represent an early component of gating-charge movement, but not all of the total gating charge; the later charge movements, which include any charge associated with the pore opening, move too slowly for us to resolve accurately in ON measurements , indicating that AMIGO1 did not alter the total charge translocated during early conformational transitions. However, between -10 and +50 mV, Kv2.1-control cells did not move as much gating charge as Kv2.1 + AMIGO1 cells, indicating a shift in gating-current activation (F). The shift in voltage dependence was quantified by fitting QON,fast–V with a Boltzmann  yielding ΔVg,Mid,ON,fast of -12.8 ± 3.5 mV (SEM) (K) and a Δzg,ON,fast of 0.215 ± 0.058 e0 (SEM) (J)  K and a e0 (SEM) J (TableSEM) (J) . This reτON) from the decay phase of Ig,ON that occurs before 10 ms  . Above +30 mV, the mean τON for Kv2.1 + AMIGO1 cells was faster than the mean τON from Kv2.1-control cells (N). Fitting the τON–V with rate-theory equations indicated that AMIGO1 accelerates the forward rate of gating-charge movement by 1.7x at neutral voltage and decreases the voltage dependence of this rate by 13% (N). This result indicates that voltage sensors activate faster in the presence of AMIGO1, consistent with destabilization of the earliest resting conformation of the voltage sensors by AMIGO1.To determine whether AMIGO1 modulates the kinetics of early gating-charge movement, we extracted a time constant  , as in  . In Kv2.ol cells N. Fittie by 13% N. This Ig,OFF) at -140 mV after 100-ms voltage steps . The density of QOFF elicited by voltage steps above -10 mV was not significantly different between Kv2.1-control and Kv2.1 + AMIGO1 cells (Q), indicating that AMIGO1 did not alter the density of channels expressed nor the total gating charge per channel. However, between -25 and -10 mV, Kv2.1-control cells did not move as much gating charge as Kv2.1 + AMIGO1 cells, indicating a shift in voltage dependence (Q). Boltzmann fits , yielded ΔVg,Mid,OFF of -10.8 ± 2.4 mV (SEM) (V) and a Δzg,OFF of 0.43 ± 0.20 e0 (SEM) (U)  V and a e0 (SEM) U (TableSEM) (U) , indicatkΔF) accelerated from 0.073 ± 0.010 s-1 (SEM) in control cells to 0.115 ± 0.015 s-1 (SEM) in cells positive for AMIGO1-YFP fluorescence . Fitting shows that the emission peaks, 644 and 617 nm, respectively, were unchanged with or without AMIGO1-YFP, consistent with the local electrostatic environment surrounding the JP probes positioned on resting Kv2.1 voltage sensors not being altered by AMIGO1 expression. Previous work has shown that the GxTX Lys27Pra(JP) emission-peak wavelength is sensitive to conformational changes among early resting states of voltage sensors (Full emission spectra of JP fluorescence from Kv2.1–CHO cells transfected with AMIGO1-YFP and treated with GxTX Ser13Pra(JP) or GxTX Lys27Pra(JP) were fitted with 2-component split pseudo-Voigt functions C and F. sensors . The abs2+ to minimize, or screen, the impact of fixed negative charges near the voltage sensors . Kv2.1 requires more positive voltage steps to activate in high Mg2+ solutions  (H) but did not change zi (G) . Hence, Mg2+ altered Kv2.1 activation in a manner consistent with surface-charge screening, yet Mg2+ did not detectably abrogate the AMIGO1 effect. However, a surface-charge site could be insensitive to 100 mM Mg2+. It remains a possibility that other manipulations, such as altering AMIGO1 glycosylation, could reveal that AMIGO1 surface charge impacts Kv2.1 gating. While neither extracellular fluorescence measurements nor surface-charge screening detected an extracellular impact of AMIGO1, we are not able to rule out the possibility of an extracellular coupling to AMIGO1 that was not detected by these methods.We also tested whether AMIGO1 acts by a surface-charge mechanism with a classical charge-screening approach. Surface-charge interactions can be revealed by increasing the concentration of Mg sensors ,79. If Aolutions , consistolutions . In highmV (SEM) H but dihange zi G (Tablee zi (G) . When coWe asked whether AMIGO1 modulates Kv2.1 conductance by modulating conformational changes of pore opening or voltage-sensor activation. We found that AMIGO1 destabilizes the resting, inward conformation of Kv2.1 voltage sensors, causing channels to activate at more negative voltages. This conclusion is supported by three major results.M). When voltage-sensor movements were measured directly, gating-current recordings revealed an acceleration of the forward rate constant (τON) of gating-charge activation in cells with AMIGO1. Between 0 and 120 mV, pore opening is 10–30 times slower than Ig,ON decay (M and N), too slow to influence the first few ms of Ig,ON. When the change in the forward rate α0mV (N) was used to estimate the amount of energy AMIGO1 contributes to modulating Kv2.1 conformational bias, we found that AMIGO1 imparted -1.3 kcal/mol per channel .AMIGO1 expression accelerated conductance activation only at a subset of voltages where the activation kinetics are voltage sensitive M. When ay M and N, too sate α0mV N was us channel  to ΔG‡AMormation A.Q–V than the midpoint of the G–V. The ΔG for AMIGO1’s impact on voltage-sensor activation ranged from -1.9 to -3.1 kcal/mol depending on the calculation method . With GxTX-594, the AMIGO1 G–V shift widens to -22.3 ± 4.8 (SEM)  . This re the G–V . GxTX stV,Midi of G–Vs, we performed calculations with a voltage-sensor-shift model composed of simple gating equations. This voltage-sensor-shift model incorporates distinct V1/2 values assigned to independent voltage-sensor  and pore  transitions, all of which must activate to allow channel opening. Calculations incorporating a constant ΔVVSD,1/2 shift with no change in VPore,1/2 demonstrate that the ΔVi,Mid of G–V can be malleable. In these calculations, an AMIGO1 shift of ΔVVSD,1/2 = -22.4 mV resulted in ΔVi,Mid = -5.0 mV (B), similar to the empirical measurement ΔVi,Mid = -5.7 mV of Kv2.1 with AMIGO1 . While the gating model implied by these calculations is highly simplified and does not recapitulate all of our data, it does demonstrate a mechanism by which a fixed modulation of voltage sensors could result in varying ΔVi,Mid shifts. As the voltage dependence of Kv2.1 activation is dynamically modulated by many forms of cellular regulation and can vary dramatically (G–V shift when the pore transition occurs at more negative potentials than voltage-sensor activation (G–V midpoint, channel opening is limited by voltage sensors occupying an AMIGO1-sensitive resting conformation, then the G–V midpoint will be very sensitive to AMIGO1; however, if almost all voltage sensors have already exited resting conformations at the G–V midpoint, then AMIGO1 destabilization of resting conformations will have little impact. A malleable impact of AMIGO1 in response to regulation of voltage-sensor or pore transitions could perhaps explain why a larger Kv2.1 G–V shift was originally reported (To test the idea that AMIGO1 modulation of voltage sensors could result in different Δ -5.0 mV B, similh AMIGO1 . However-21.8 mV B. Whileatically , 85, 86,tivation  C. Whilereported .Q–V curve (2+-activated K+ channels (The voltage-sensor-shift mechanism we propose does not require changes in pore opening or voltage sensor-pore coupling. Maverick and colleagues  suggeste–V curve , similarchannels . As the Vi,Mid as low as 5 mV, and standard deviations for Vi,Mid of 4–9 mV (VMid standard-deviation values from the standard errors and n values in these studies, standard deviations ranged from 1 to 17 mV, on par with our own. We suspect these notable VMid deviations result from the many different types of regulation to which Kv2.1 channels are susceptible (More detailed investigation of the AMIGO1 impact on the Kv2.1 activation pathway was limited by the relatively small magnitude of AMIGO1-dependent effects versus the cell-to-cell variability, with Δf 4–9 mV , 94, 95.ceptible ,21. Tech+ currents or at high density for gating-current and imaging experiments. Auxiliary subunit interactions with pore α subunits can be influenced by many factors that can alter their assembly and functional impact on channel currents (+-current recordings were modulated less by AMIGO1, we would expect a decrease in Boltzmann slope of the fit, a bimodal G–V relation, or increased cell-to-cell variability with AMIGO1. We do not observe any of these with CHO cells. The similar impact of AMIGO1 on Kv2.1 conductance in two cell lines (+-conductance measurements. Thus, while incomplete complex assembly and other factors could in theory influence the magnitude of the AMIGO1 impact on Kv2.1 conductance, we do not see evidence that would negate our biophysical assessment of the mechanism through which AMIGO1 alters Kv2.1 conductance.Our interpretations assume that the AMIGO1 effect is similar whether Kv2.1 is expressed at low density to measure Kcurrents , 101. Holl lines  and consll lines  further The most parsimonious explanation for the effect AMIGO1 has on the Kv2.1 conduction-voltage relation seems to be a direct interaction with Kv2.1 voltage sensors. However, it also seems possible that AMIGO1 proteins could change cellular regulation of which in turn modulates Kv2.1. Even if AMIGO1 acts by an indirect mechanism, our mechanistic conclusions remain valid, as they are not predicated on a direct protein-protein interaction between AMIGO1 and Kv2.1.in vivo was not recapitulated in CHO cells. Changes in Kv2 current density can modulate neuronal firing (K+ conductance activates at more negative voltages than wild type (AMIGO1 knockout mice display schizophrenia-related features , and AMIl firing , 108 andl firing ,28,109, ild type . While w4 model of activation (Vi,Mid. When the AMIGO1 ΔVi,Mid of Kv2.1–CHO voltage sensors are applied to SCG neuron parameters, Kv2 conductance at the end of the mock action potential decreases by 89% (In mammalian neurons, Kv2 conductance can speed action-potential repolarization ,110, damtivation . This sus by 89% . AlthougTo shift the activation midpoint of Kv2.1 conductance to lower voltages, AMIGO1 destabilizes the earliest resting conformations of Kv2.1 voltage sensors relative to more activated conformations. While we cannot rule out a direct influence on pore dynamics, we saw no indication of such. We propose that AMIGO1 shifts the voltage dependence of Kv2.1 conduction to more negative voltages by modulating early voltage-sensor movements.+ conductance.We also propose that because AMIGO1 acts on early voltage-sensor movements, modulation of Kv2 gating can alter the impact of AMIGO1 on KR.J.S., conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, visualization, writing – original draft, and writing – review & editing; R.G.S., formal analysis and visualization; L.A.V., formal analysis and visualization; P.T., resources; Z.W., resources; B.E.C., conceptualization, funding acquisition, project administration, and writing – review & editing; J.T.S., conceptualization, funding acquisition, investigation, methodology, project administration, supervision, writing – original draft, and writing – review & editing.


In this study, we established a chronic obstructive pulmonary disease (COPD) model by stimulating mice with cigarette smoke, and observed the effects of dendritic cells (DCs) overexpressing SOCS1 on Th17, Treg and other related cytokines in peripheral blood, bronchoalveolar lavage fluid and lung tissues of COPD mice.6), DC-SOCS1 (2 × 106) and immature DCs (1 × 106) via tail vein on days 1 and 7 of COPD fumigation modeling. After day 28 of modeling, the peripheral blood, BALF and lung tissue samples were extracted from the mice, and the changes of DCs, Th17 and Treg cells and related cytokines were detected by immunohistochemistry, immunofluorescence, HE staining, flow cytometry and ELISA.After successfully transfecting DCs with overexpressing SOCS1 (DC-SOCS1), the mice were injected with DC-SOCS1 (1 × 106 DC-SOCS1) was better than low concentration (1 × 106 DC-SOCS1). Moreover, the intervention effect was significant on day 1 compared with day 7. In the mice injected with DC-SOCS1, the expression of CD83, IL-4, Foxp3, and CCR6 was increased on day 1 than those on day 7, while IL-17 and IFN-γ was decreased.The results showed that DC-SOCS1 was able to reduce the secretion of pro-inflammatory factors and increase the anti-inflammatory factors in the COPD mice, and the effect of high concentration (2 × 10Intervention of COPD mice with high concentrations of DCs-SOCS1 reduced pro-inflammatory factor secretion and attenuated the inflammatory response in COPD.Trial registration Not applicable.The online version contains supplementary material available at 10.1186/s12890-022-01931-1. Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease of the airways characterized by incomplete reversibility and progressive airflow limitation, associated with an enhanced chronic inflammatory response of the airways and lungs to toxic particles or gases , 2. The In addition, it has been found that COPD may be an immune disease caused by smoking and its pathogenesis may be related to the deregulation of the autoimmune system , 5. T lyTherefore, this study was conducted to observe the effects of DCs overexpressing SOCS1 on Th17, Treg and other related cytokines in peripheral blood, bronchoalveolar lavage fluid (BALF) and lung tissues of COPD mice, and provided a new perspective for COPD treatment.6/ml cells in 6-well plates and incubated at 37 ℃ in a 5% CO2 incubator. Cells were plastered for about 1–2 days (d) and maturation was induced by adding LPS (100 ng/μl) on day 7 of culture.A number of healthy 6–8 weeks old C57BL/6 mice, purchased from the Animal Experiment Center of the Third Military Medical University. Mice were anesthetized and disinfected with 75% ethanol, then the femur and tibia were taken under aseptic conditions and washed twice with phosphate buffered saline (PBS) solution. The epiphysis was removed to expose the bone marrow cavity, and the bone marrow was flushed with PBS until the bone marrow cavity turned white. The flushed bone marrow was made into single cell suspension and centrifuged at 1000 r/min for 5 min (min), and the supernatant was discarded. Resuspended the cell precipitate with PBS. The cell suspension was slowly added into a 15 ml centrifuge tube pre-filled with 5 ml lymphocyte separation solution, centrifuged at 2000 rpm for 20 min, and the solution was divided into 5 layers after centrifugation, and the intermediate bone marrow stromal cell layer was aspirated and washed 3 times with PBS. The cells were inoculated with DC cell growth medium containing 10% fetal bovine serum and penicillin at a concentration of 10All animal experiments were carried out in accordance with the principles of the guidelines described in the Care and Use of Animals, and had been approved by the animal experimentation committee of the Zunyi Medical University.6 cells) were added to each flow assay tube; 50 μl Staining Buffer was added to the blank or isotype control tube. In each tube, 50 μl cell suspension (approximately 106 cells) was added and gently mixed. After incubation for 30 min in an ice bath, the cells were incubated with Staining Buffer (2 ml per tube and 200 μl per well) and centrifuged at 1000 rpm for 5 min at 4 °C. The supernatant was discarded and washed three times. 100 μl of the cells were resuspended and detected by FCM.CD80 and CD83 antibodies were used to identify DCs. The cells were collected after 24 h of Lipopolysaccharide (LPS) induction, then centrifuged at 1000 rpm for 5 min to precipitate the cells, and the supernatant was discarded. Resuspended the cells. Then, 50 μl diluted antibodies  was searched on the NCBI website, and NM_001271603.1 transcript was chosen for sequence synthesis. The Kozak sequence and EcoRI digested site were added at the 5′ end, and the BamHI site was added at the 3′ end. The pLVX-IRES-ZsGreen1 vector . After 24 h, the medium was replaced with fresh medium and observed under fluorescence microscope after 48 h of infection. DCs with green fluorescence were those infected with lentivirus overexpressing on SOCS1 gene. Meanwhile, samples were collected for further study.qRT-PCR for detecting transfection effects after lentivirus infection of DCs. SYBR Green I is a fluorescent dye that binds to double-stranded DNA and emits light, allowing detection of the amount of double-stranded DNA present in the PCR system based on the fluorescent signal. The RNA was first reverse transcribed into cDNA using random primers, and then specific primers and SYBR Green I fluorescent dye were designed for fluorescent quantitative PCR detection. The primer sequences were: SOCS1 (ID: 12703), mSOCS1F: CGCCTGCGGCTTCTATTG, mSOCS1R: CCCGAAGCCATCTTCACG, m actin f: GAGACCTTCAACACCCCAGC, m actin r. ATGTCACGCACGATTTCCC.Western blot (WB) was used to detect the transfection effect after lentivirus infection of DCs. The cultured DCs infected with lentivirus were lysed with RIPA lysis buffer, centrifuged at 12,000 rpm, 4 °C for 15 min, and the supernatant was collected. Then the protein concentration was determined by BCA protein assay kit. The protein was separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequently transferred to polyvinylidene fluoride membranes (Bio-Rad), which were subsequently blocked with Tris Buffered saline Tween (TBST) solution containing 5% skim milk at room temperature for 2 h. The corresponding primary antibody  was diluted to a certain concentration (1:500) with the blocking solution, and the final concentration of the internal reference primary antibody was 1:1000, and then incubated overnight at 4 °C. After 3 washes with TBST, they were incubated with secondary antibodies  at room temperature for 1 h. Finally, samples were incubated with the ECL-enhanced chemiluminescence detection kit (Thermo Scientific Pierce™) and OD values were calculated with ImageJ pro plus.Thirty-eight 8-week-old male SPF-grade C57BL/6 mice, weighing 20–25 g  were obtained. The 35 mice were randomly assigned into 7 groups (B-H groups), and the 7 groups were housed in identical environments Table . The aniAfter 28 days of modeling, the peripheral blood was collected from the eyeballs for FCM assay. The trachea was exposed by cutting the skin in the middle of the neck, and the trachea and alveoli were irrigated with saline . The BALF was collected and centrifuged , and the supernatant was used for enzyme linked immunosorbent (ELISA) assay and the BALF precipitate was taken for FCM assay. The abdominal cavity was opened, and fresh liver, spleen and lung tissues were washed in cold PBS, and part of the lung tissues were taken for FCM assay, while the rest of the tissues were stored at − 80 °C for ELISA assay. The tissues were fixed in 4% paraformaldehyde for more than 24 h for immunohistochemistry, immunofluorescence and hematoxylin–eosin staining.Tissues fixed in 4% paraformaldehyde fixative for 24 h were paraffin embedded, sectioned, stained with hematoxylin stain and eosin stain, and sealed with neutral resin and observed under the microscope. The nucleus became blue, while the cytoplasm became red or pink.Tissues fixed in 4% paraformaldehyde fixative for 24 h were subjected to IHC experiments after paraffin embedding, sectioning, and antigen thermal repair, and the changes of IL-17, IL-4, CD83, CCR6, Foxp3 and INF-γ factor levels were detected in each group. The tissues were washed with PBS and then closed with goat serum at room temperature for 1 h. Samples were then incubated with primary antibodies overnight at 4 °C . Following PBS wash, (HRP-labeled) secondary antibodies working solution was incubated at room temperature for 2 h. After DAB color development at room temperature for 1–10 min (avoid light), distilled water wash, hematoxylin re-staining for 5 min. Then wash back to blue with tap water, gradient alcohol dehydration , xylene transparent 2 times for 3 min. Lastly, the sections were sealed with neutral resin and observed under the microscope.Paraffin sections of the samples were dewaxed, rehydrated, washed with double-distilled water, and washed with PBS, then incubated with DAPI  for 15 min protected from light. Finally, anti-fluorescence quenching agent was added to seal the slides and the changes of each group was observed under fluorescence microscope.For Th17, individual nucleated cells were resuspended in RPMI 1640 (10% heat-inactivated FBS) and counted by Tissue Blue staining; the cell concentration was adjusted to 2 × 106 cells/ml, 500 μl. Cells were treated with PMA/Ionomycin Mixture (250 ×) for 5–18 h in each group, along with protein transport inhibitor (BFA/Monensin Mixture (250 ×).For Treg, the 100 µl cell suspension was added to each tube, with a cell count of approximately 1 × 106 cells. The surface antigens were labeled according to the cell surface antigen staining method, and the appropriate amount of CD4 and CD25 antibodies were added according to the instructions and incubated for 30 min at room temperature without light.Then, the cells were washed with pre-chilled PBS and the supernatant was discarded after centrifugation. Then the cells were resuspended by adding 1 ml of Fixation/Permeabilization solution and incubated for 30 min at room temperature under dark conditions. Subsequently, 1 ml Permeabilization Buffer (diluted 1:9 in deionized water) was added, and supernatant was discarded after centrifugation. Cells were resuspended with 100ul Permeabilization Buffer, added IL-17A (for Th17)and Foxp3 (for Treg) antibody, and incubated for 30 min at room temperature without light. Finally, it was washed with flow staining buffer, centrifuged at 300 g for 5 min, resuspended to 500ul with flow staining buffer, and detect on the machine.6 cells. The surface antigens were labeled according to the cell surface antigen staining method, and the appropriate amount of CD80 and CD83 (CCR5 and CCR6 for imDCs) antibodies were added according to the instructions and incubated for 30 min at room temperature without light. The cells were washed with pre-chilled PBS and the supernatant was discarded after centrifugation. Then, the cells were washed with flow staining buffer, centrifuged at 300 g for 5 min, resuspended to 500ul with flow staining buffer, and detect on the machine.For DCs, the 100 µl cell suspension was added to each tube, with a cell count of approximately 1 × 10The ELISA kit was used to determine the levels of interleukin (IL)-17, IL-21, IL-23, IL-10, transforming growth factor β (TGF-β), interferon-gamma (IFN-γ), IL-12, IL-4 and IL-5 in BALF supernatant and lung tissue samples in COPD mice. The kit applied double antibody sandwich method for the determination. The microtiters were coated with the purified mouse IL-17/IL-21/IL-23/IL-12/IL-10/IFN-γ/IL-4/TGF-β/IL-5 antibodies to make solid phase antibodies, and IL-17/IL-21/IL-23/IL-12/IL-10/IFN-γ/IL-4/TGF-β/IL-5 were added sequentially to the microplates. Then, it was conjugated with HRP-labeled IL-17/IL-21/IL-23/IL-12/IL-10/IFN-γ/IL-4/TGF-β/IL-5 antibodies to form antibody-antigen-enzyme-labeled antibody complexes, which were thoroughly washed and color developed by adding the substrate TMB. TMB was converted to blue by HRP enzyme and finally to yellow by acid. In addition, the shade of color was positively correlated with IL-17/IL-21/IL-23/IL-12/IL-10/IFN-γ/IL-4/TGF-β/IL-5 in the samples. The optical density (OD) value was measured at 450 m wavelength by microplate reader, and the concentration of mouse IL-17/IL-21/IL-23/IL-12/IL-10/IFN-γ/IL-4/TGF-β/IL-5 in the samples was calculated by the standard curve.P < 0.05 was considered as statistically significant.Statistical analysis was performed using SPSS version 26.0, and the quantitative data were analyzed by one way ANOVA and shown as mean ± standard deviation. The growth status of DCs in culture for 1 days, 48 h, after fluid change, 5 days, 7 days and after LPS induction was shown in Fig. The states of DCs transfected with overexpressed SOCS1 gene (DCs-Ad-SOCS1) and with GFP lentivirus no-load for 5 days (DCs-Ad-GFP) was observed under the fluorescence microscope as shown in Fig. 6 reinfusion was greater than that of C and F groups that received DC-SOCS1 1 × 106 reinfusion. Similarly, the body mass increase of mice in E group, which received imDCs reinfusion 1 day after fumigation, was greater than that in H group, which received imDCs reinfusion 7 d after fumigation. In addition, by immunofluorescence observation of lung tissue DCs, we found that compared with A group, there was a significant increase in DCs in B, E and H groups, more growth in C and F groups, a slight increase in G group and no significant change in D group , imDCs (CCR5+CCR6+), Th17 (CD4+IL-17A+), and Treg (CD4+CD25+Foxp3+) in peripheral blood, lung tissue, and BALF sediment were determined using FCM and expressed as percentage (%) (Table The content of mDCs (CD80eg CD4+CD5+Foxp3+ P < 0.05); the mDCs in E group (11.37 ± 0.15) was higher than that in D, F (8.98 ± 0.19) and G groups with statistically significant differences (P < 0.05); the mDCs of F group was significantly higher than that of D and G groups (P < 0.05). In the BALF sediment, the content of mDCs in D group (1.98 ± 0.11) was lower than that of E (3.58 ± 0.09), F (3.32 ± 0.11) and H (4.52 ± 0.16) groups with significant difference (P < 0.05); the content of mDCs of G group (2.66 ± 0.16) was significantly decreased compared with E and H groups (P < 0.05). However, no significant differences were found in the content of mDCs in lung tissue between the groups  was significantly higher than that in D (3.29 ± 0.21) and G (5.13 ± 0.28) groups (P < 0.05), while no differences were observed in the content of imDCs in the peripheral blood among the other groups. In the BALF sediment, the content of imDCs of D group (12.92 ± 0.01) was remarkably increased compared with that of E (5.95 ± 0.03), F (6.22 ± 0.16), and H (5.29 ± 0.33) groups (P < 0.05); compared with B group (4.92 ± 0.18), the content of imDCs of D and F groups was significantly increased (P < 0.05). In addition, the content of imDCs in lung tissue of F group (1.24 ± 0.01) was higher than that of B (0.58 ± 0.00) and H (0.64 ± 0.01) groups (P < 0.05), and the content of imDCs in D group (3.39 ± 0.32) was significantly higher than that in H group (P < 0.05). No difference was observed in the content of imDCs in BALF sediment and lung tissue among the other groups.  was significantly higher than that of B (1.27 ± 0.09) group (P < 0.05). Compared with B group (1.08 ± 0.01) in lung tissue, the content of Th17 of D (0.11 ± 0.01), G (0.28 ± 0.03) and H (0.89 ± 0.01) groups was significantly lower (P < 0.05). Additionally, the content of H group in lung tissue was significantly higher than that of D and G (0.28 ± 0.03) groups (P < 0.05). No significant differences were observed in Th17 in the BALF sediment between the groups  in peripheral blood was lower compared with D group (2.05 ± 0.09) (P < 0.05), and the content of Treg in BALF sediment of G group (4.93 ± 0.06) was higher than that of B (3.80 ± 0.09) and H (4.16 ± 0.06) groups (P < 0.05). However, There was no statistically significant differences were observed in the content of Treg of each group in the lung tissue  was significantly higher than that of B (2.83 ± 1.03), E (6.26 ± 0.29), and H (6.26 ± 1.04) groups ; the concentration of IL-17 in E, F and H groups was significantly higher than that of D group (P < 0.05); the concentration of IL-17 in E group was significantly higher than in F and G groups, but lower than that in H group (P < 0.05); the concentration of IL-17 in F group was lower than in H group but higher than that in G group with statistically significance (P < 0.05); the concentration of IL-17 in G group was lower than in H group with statistically significance (P < 0.05)  in C, D, E, F, G, and H groups  were significantly lower compared with that of B group (78.87 ± 0.91) (P < 0.05); the concentration of IL-21 in C, D, F (307.43 ± 1.04) and G groups were significantly lower than in H group (378.59 ± 0.23) (P < 0.05); the concentration of IL-21 in C, E and F groups were increased than that of G group with statistically significance (P < 0.05); the concentration of IL-21 in C group was significantly higher than in D group but lower than in E group (P < 0.05)  in C (284.33 ± 2.98), D (181.49 ± 6.82), E (344.41 ± 4.25) and G (226.29 ± 2.47) groups were significantly lower compared with B group (420.44 ± 6.49) (P < 0.05); the concentration of IL-23 in C, D, F and G groups were significantly lower compared with H group (P < 0.05); the concentration of IL-23 in C, D groups were decreased in comparison with E and F groups, and the concentration of IL-23 in F and G groups was lower than that of E group (P < 0.05); the concentration of IL-23 in C group was increased than that of D groups with statistically significance (P < 0.05)  in C, D, E, F, G, and H groups  were significantly decreased than in B group (200.62 ± 0.24) (P < 0.05); the concentration of IL-10 in C group was obviously higher than that in H group but lower than that in D group (P < 0.05); the concentration of IL-10 in E, F (54.78 ± 014), and H (44.56 ± 0.15) groups were significantly lower than that of D group (P < 0.05); the concentration of IL-10 in F group was significantly increased compared with H group (P < 0.05)  in C (58.71 ± 0.77), D (70.12 ± 0.40), E (50.74 ± 0.98) and G (62.99 ± 1.59) groups were significantly higher compared with B group (39.46 ± 1.12) (P < 0.05); the concentration of TGF-β in D group was significantly higher than that of C, E, F, G, H groups (P < 0.05); the concentration of TGF-β in C group was significantly higher compared with G, H groups (P < 0.05); the concentration of TGF-β in E, H groups were significantly decreased compared with G group (P < 0.05); the concentration of TGF-β in F group was significantly higher than that of H group, but lower than that in G group (P < 0.05)  in C, D, E, F, G, and H groups  were significantly increased than in B group (24.98 ± 0.08) (P < 0.05); the concentration of IFN-γ in B group was significantly higher than that of D, E (328.18 ± 10.08), and G (193.80 ± 10.18) groups (P < 0.05); the concentration of IFN-γ in D group was significantly decreased compared with C, F, H groups (P < 0.05); the concentration of IFN-γ in G group was significantly lower than that of E, H groups (P < 0.05); the concentration of IFN-γ in H group was significantly higher than that of C, G groups (P < 0.05)  in B 472.45 ± 13.15), C (252.28 ± 3.08), D (124.43 ± 0.88), E (328.18 ± 10.08), F (276.23 ± 4.21) and H (395.25 ± 4.45) groups were significantly increased compared with A group (92.48 ± 0.88) ; the concentration of IL-12 in C, D, F, G groups were significantly lower than that of B group (P < 0.05); the concentration of IL-12 in E, F, H groups were significantly higher than in C, D groups (P < 0.05); the concentration of IL-12 in E group was significantly higher than that in F group; the concentration of IL-12 in H group was significantly increased in comparison with E, F, G groups (P < 0.05)  in B (40.02 ± 0.29), C 30.75 ± 0.05), D (25.67 ± 0.31), E (35.15 ± 0.01), F (32.59 ± 0.11), G (28.37 ± 0.35) and H (38.58 ± 0.04) groups were significantly higher than that of A group (19.71 ± 0.47) ; the concentration of IL-4 in C, D (304.94 ± 0.82), E, F, G, and H groups were significantly increased than that of B group (P < 0.05); the concentration of IL-4 in C, E, F, G, H groups were significantly lower than that of D group (P < 0.05); the concentration of IL-4 in C group was significantly higher than that of E, F, H groups but lower than that of G group (P < 0.05); the concentration of IL-4 in G group was significantly higher than that of E, F, H groups (P < 0.05); the concentration of IL-4 in E, H groups were significantly lower than in F group (P < 0.05)  in B (101.16 ± 1.43), C (238.90 ± 0.90), E (148.89 ± 3.33), F 202.95 ± 2.19), G (270.50 ± 0.07) and H (120.39 ± 0.62) groups were significantly lower compared with A group (333.47 ± 2.17) ; the concentration of IL-5 in B group was significantly decreased than that of C, D, E, F, G groups (P < 0.05); the concentration of IL-5 in H group was significantly lower compared with C, D, E, F, G groups (P < 0.05); the concentration of IL-5 in E group was significantly decreased than that of C, D, G groups (P < 0.05); the concentration of IL-5 in C, F groups were significantly lower than that of D group (P < 0.05)  in B, C, D, E, F, G, and H groups  were significantly decreased compared with A group (12.77 ± 0.08) (P < 0.05); the concentration of IL-17 in C group was significantly higher than that of D group but lower than that of E, H (37.58 ± 0.17) groups (P < 0.05); the concentration of IL-17 in D group was significantly lower than in E, F, H groups (P < 0.05); the concentration of IL-17 in E group was lower than in H group with statistically significance (P < 0.05); the concentration of IL-17 in E, H groups were significantly higher than that of F, G groups (P < 0.05)  were significantly lower compared with B group (52.24 ± 1.16) (P < 0.05); the concentration of IL-21 in C, E, F (136.34 ± 0.16), and H (167.70 ± 1.36) groups were significantly higher than in D group (P < 0.05); the concentration of IL-21 in E, F, H (167.70 ± 1.36) groups were increased than that of G group with statistically significance (P < 0.05); the concentration of IL-21 in F group was significantly lower than in H group; the concentration of IL-21 in C group was significantly lower than in E, H groups (P < 0.05) , D (70.21 ± 0.93), E (150.19 ± 0.06) and G (99.35 ± 2.21) groups were significantly lower compared with B group (200.62 ± 0.24) (P < 0.05); the concentration of IL-23 in C, D, F, G groups were significantly lower compared with H group (P < 0.05); the concentration of IL-23 in C, F groups were increased in comparison with G group with statistically significance (P < 0.05); the concentration of IL-23 in D group was lower than that of E (176.28 ± 3.92) and F groups (P < 0.05)  were significantly increased than of C (139.88 ± 0.16), D (92.21 ± 2.52), F (152.26 ± 1.10), G (126.30 ± 0.43), and H (195.42 ± 0.68) groups (P < 0.05); the concentration of IL-10 in C (54.05 ± 1.77), E, F, and H (40.00 ± 0.00) groups were significantly lower than that of D group (P < 0.05) , E 43.53 ± 0.31), F (48.18 ± 0.65), and G (66.53 ± 1.85) groups were significantly higher compared with B group (35.02 ± 0.54) ; the concentration of TGF-β in C group was significantly higher than that of E, H groups but lower than that of D, G groups (P < 0.05); the concentration of TGF-β in D group was significantly higher compared with E, F, G, H groups (P < 0.05); the concentration of TGF-β in E, F, G groups were significantly increased compared with H group (P < 0.05); the concentration of TGF-β in G group was significantly higher than that of E, F groups (P < 0.05); the concentration of TGF-β in E group was lower than that of F group with statistically significance (P < 0.05)  were significantly increased than in B group (23.49 ± 0.09) (P < 0.05); the concentration of IFN-γ in B group was significantly higher than that of C, D (104.03 ± 7.39), E, F, G, and H groups (P < 0.05); the concentration of IFN-γ in H group was significantly increased compared with C, D, E, F, G groups (P < 0.05); the concentration of IFN-γ in D group was significantly lower than that of E and F groups (P < 0.05); the concentration of IFN-γ in F group was significantly higher than that of G group (P < 0.05) , C 168.58 ± 5.69), E (217.03 ± 7.67), F (195.78 ± 3.50), G (131.78 ± 2.51) and H (306.95 ± 4.38) groups were significantly increased compared with A group (41.53 ± 3.22) ; the concentration of IL-12 in C, D, E, F, and G groups were significantly lower than that of B group (P < 0.05); the concentration of IL-12 in C, E, F, and G groups were significantly higher than in D group (P < 0.05); the concentration of IL-12 in C, F, G groups were significantly decreased in comparison with E group (P < 0.05); the concentration of IL-12 in F group was higher than that of C, G groups with statistically significance (P < 0.05); the concentration of IL-12 in C group was significantly higher compared with G group (P < 0.05)  were significantly higher than that of A group (19.43 ± 0.03) (P < 0.05); the concentration of IL-4 in C, D, E, F, and G groups were significantly increased than that of B group (P < 0.05); the concentration of IL-4 in H groups were significantly lower than that of C, D, F, G groups (P < 0.05); the concentration of IL-4 in D group was significantly higher than that of C, E and F groups (P < 0.05); the concentration of IL-4 in E group was significantly lower than that of F, G groups (P < 0.05); the concentration of IL-4 in F group was significantly lower compared with G group (P < 0.05)  were significantly lower compared with A group (326.85 ± 0.81) (P < 0.05); the concentration of IL-5 in B group was significantly decreased than that of F, G, and H groups (P < 0.05); the concentration of IL-5 in H group was significantly lower compared with C, F, G groups (P < 0.05); the concentration of IL-5 in C group was significantly increased compared with E group (P < 0.05); the concentration of IL-5 in G group was significantly increased compared with C, E, F groups (P < 0.05) , C (6.12 ± 0.04), E (3.36 ± 0.16), F (5.81 ± 0.04), G (8.45 ± 0.02), and H (2.44 ± 0.04) groups were significantly decreased compared with A group (14.42 ± 0.06) , while anti-inflammatory factors such as IL-10 and TGF-β increased. The concentrations of pro-inflammatory factors in DCs-SOCS1 injected on day 7 after fumigation were higher than those on day 1, and the concentrations of anti-inflammatory factors decreased. It was suggested that the expression level of inflammatory factors was related to the concentration of injected DCs-SOCS1 and the time of intervention. As IL-10 was involved in the production of regulatory DCs, and overexpression of SOCS1 could induce IL-10 expression on the surface of DCs, so DCs could be converted to regulatory DCs when they produced large amounts of IL-10 [Moreover, in this study, the content of pro-inflammatory factors such as IL-17 was found to be lower in the 2 × 10of IL-10 . TherefoIn summary, early intervention of COPD mice with high concentrations of SOCS1 lentiviral-transfected DCs can reduce the secretion of pro-inflammatory factors in COPD, attenuate the inflammatory response in COPD.Additional file 1. Cover letter.


The morphology and structure of β-CD/CMK-8 were characterized by scanning electron microscope (SEM) and X-ray diffraction (XRD). In addition, the dates from electrochemical impedance spectroscopy (EIS) and Cyclic voltammetry (CV) demonstrated that the β-CD/CMK-8 possessed a fast electronic transfer rate and large effective surface area. Besides this, the β-CD/CMK-8 composite displayed high enrichment ability toward Cu2+. As a result of these impressive features, the β-CD/CMK-8 modified electrode provided a wide linear response ranging from 0.1 ng·L−1 to 1.0 mg·L−1 with a low detection limit of 0.3 ng·L−1. Furthermore, the repeatability, reproducibility and selectivity of β-CD/CMK-8 towards Cu2+ were commendable. The sensor could be used to detect Cu2+ in real samples. All in all, this work proposes a simple and sensitive method for Cu2+ detection, which provides a reference for the subsequent detection of HMIs.In this work, β-cyclodextrin (β-CD)/mesoporous carbon (CMK-8) nanocomposite was synthesized and used as an electrochemical sensing platform for highly sensitive and selective detection of Cu Accordingly, the Rct value of bare GCE was 876.4 Ω, while, β-CD/GCE presented a larger semicircle with Rct at about 1067.8 Ω, which was attributed to the low conductivity of β-CD. CMK-8/GCE displayed a minuscule semicircle with a Rct value of 49.6 Ω, demonstrating the high conductivity of CMK-8. The Rct value of β-CD/CMK-8/GCE was 417.8 Ω, which was between that of β-CD and CMK-8, confirming that the β-CD/CMK-8 composite was successfully prepared.Electrochemical impedance spectroscopy (EIS) is an available tool for exploring the electron transfer behavior of different modified electrodes, which usually consists of a straight line of low-frequency and a semicircle of high-frequency. The diameter of the high-frequency semicircle represents electron transfer resistance (Rct) . Figure −1 Cu2+ at different modified electrodes were researched by DPASV , 0.1 M phosphate buffer solution  and 0.1 M HAc-NaAc buffer solution  were conducted, and the results are displayed in 2+ in 0.6 M KCl solution was almost invisible (curve a), while an enhanced peak current was found in 0.1 M PBS solution (curve b). More obviously, the peak current of Cu2+ in 0.1 M ABS solution (curve c) was the largest. Therefore, ABS was applied as the optimal electrolyte solution for the detection of Cu2+.The effect of the support electrolyte type was also studied. Cu2+ increased with the increased β-CD/CMK-8 suspension when the volume of the suspension was less than 3 μL. This could be attributed to the fact that the active site increased with the increase of modification amount. However, the peak current decreased rapidly when the volume of β-CD/CMK-8 suspension was more than 3 μL. This phenomenon was because a high amount of β-CD/CMK-8 on the electrode surface caused considerable resistance against electron transfer. Therefore, 3 μL was used as the optimal modified volume of β-CD/CMK-8 suspension.The modification amount of β-CD/CMK-8 on GCE was optimized C. The re2+ was explored . −1 Cu2+ at β-CD/CMK-8/GCE with the scan rate ranging from 25 to 300 mV·s−1. As shown, the redox currents increased with the increase of the scan rate. There were good linear relationships between the anodic and cathodic currents and between the scan rates and the linear regression equations of pa =I 39.57 + 1.04 2=ν (R 0.995) and pcI = −27.07 − 0.61 2 =ν (R 0.996), respectively (2+ on β-CD/CMK-8/GCE was an adsorption control process.The effect of scan rate on the redox of Cuectively B. The phpaE = 0.085 log ν − 0.14 (2R = 0.999) and pcE = −0.059 log ν + 0.083 (2R = 0.996), respectively. According to the Laviron formulae, Moreover, from −1, averaged.Consequently, electron transfer coefficient , where SD was the standard deviation of intercept and S was the sensitivity. The LOD value was far lower than the prescribed value in drinking water by the World Health Organization (2000 μg·L−1) [2+ detection  . The β-Cshown in  [34,35,3−1 Cu2+ was explored by using one modified electrode . As the calculated p-value was bigger than 0.05, there was no significant difference between the 10 sets of current data. This result demonstrates that the repeatability of β-CD/CMK-8/GCE was satisfactory.The repeatability of β-CD/CMK-8/GCE toward 1.0 mg·Llectrode A. After −1 Cu2+ with 10 individual modified electrodes. As shown in p-value was calculated to be 0.052, using SPSS 18.0 software, which was bigger than our considered significance level 0.05, indicating that there was no significant difference between the measured currents. The result showed that β-CD/CMK-8/GCE owns excellent reproducibility.The reproducibility of the β-CD/CMK-8/GCE was confirmed by detecting 1.0 mg·L0 · 100%. I and I0 stand for the DPASV responses of β-CD/CMK-8/GCE toward 1.0 mg·L-1 Cu2+ under 100-fold common ions  and 1.0 mg·L−1 Cu2+, respectively. As shown in 2+, which indicated the excellent selectivity of β-CD/CMK-8/GCE toward Cu2+.Selectivity is also an important factor in evaluating the performance of a sensor, which could be further evaluated by selectivity factor (SF). Here, SF = I / I2+ standard solutions were spiked separately into the tap water sample and tested using β-CD/CMK-8/GCE. The data are summarized in 2+ was from 99.38% to 104.0%, and the RSD value was less than 4%, indicating that the β-CD/CMK-8 /GCE was feasible for the detection of Cu2+ in the tap water sample.Tap water was used as the real sample to verify the practicability of β-CD/CMK-8/GCE in the analysis of real samples. The tap water sample was firstly filtrated with 0.45 μm filter, and the pH value was adjusted to 5.0. Subsequently, different concentrations of Cu3, H2SO4, NaOH, Cu(NO3)2·3H2O, and Cd(NO3)2·4H2O were supplied from Aladdin . Absolute ethanol was obtained from Yishi Chemical Co., Ltd. . The water used in this work was deionized water.Mesoporous carbon (CMK-8) was obtained from XF Nano Co., LTD . β-cyclodextrin (β-CD), N, N-Dimethylformamide (DMF), KOH, HCl, HNOScanning electron microscopy (SEM)  and X-ray diffraction (XRD)  were used for the material characterization. All electrochemical measurements were carried out in CHI 660E electrochemical workstation . A conventional three-electrode cell, consisting of a working electrode (bare or modified glassy carbon electrode (GCE)), a opposite electrode (platinum electrode) and a reference electrode , was employed in this work.The amounts of 6 mg β-CD and 3 mg CMK-8 were added to 3 mL distilled water and 3 mL DMF, respectively, which were then ultra-sounded for 30 min to obtain uniform dispersion. Finally, β-CD/CMK-8 was obtained by mixing 1 mL β-CD dispersion and 1 mL CMK-8 dispersion uniformly.2+ sensor are shown in GCE was polished with 0.05 μm alumina powder, and washed with ethanol and deionized water successively. Subsequently, 3 μL β-CD/CMK-8 suspension was dropped on bare GCE by pipette gun and dried under the electric blast drying oven. The synthetic route of β-CD/CMK-8/GCE and sensing strategy of the Cu2+ was developed, based on the synergistic effect of β-CD and CMK-8. The multiple adsorption sites of β-CD and the good conductivity of CMK-8 enabled β-CD/CMK-8 to exhibit excellent detection performance with a satisfactory detection limit of 0.3 ng·L−1 and linear range from 0.1 ng·L−1 to 1.0 mg·L−1. At the same time, the repeatability, reproducibility and selectivity of β-CD/CMK-8 /GCE toward Cu2+ were satisfactory. In addition, the novel sensing platform proved useful to detect Cu2+ in tap water, demonstrating good application prospects.In this work, an effective electrochemical sensor for Cu


P21 with two molecules of the receptor, and two NH4+ and two PF6− ions in the asymmetric unit. In each of the complexes, the ammonium ion resides in the cavity of the receptor molecule and is fixed in its position by three N—H⋯N bonds, while the remaining hydrogen atom of the cation acts as a bifurcated binding site for N—H⋯F bonding to the counter-anion.The title complex crystallizes in the monoclinic space group  H-indazol-1-yl)methyl]benzene with ammonium hexafluorophosphate, C39H42N6·NH4+·PF6−, crystallizes in the monoclinic space group P21 with two molecules of the receptor, two NH4+ and two PF6− ions in the asymmetric unit. In each of the complexes the ammonium ion resides in the cavity of the receptor molecule and is fixed in its position by three N—H⋯N bonds, while the remaining hydrogen atom of the cation acts as a bifurcated binding site for N—H⋯F bonding to the counter-anion. The crystal is composed of one-dimensional supramolecular aggregates extending along the a-axis direction.The complex of 2,4,6-triethyl-1,3,5-tris[(4-methyl-1 The remaining H atom of the ammonium acts as a bifurcated binding site for N—H⋯F bonding with the counter-ion. The conformational difference between the receptor molecules is seen in the orientation of the three ethyl groups, leading to an ab′ab′ab′ (complex I) and an ab′aa′ab′ arrangement (complex II)  2.33–2.52 Å; Table 1a-axis direction. The packing is shown in Fig. 3PLATON program methyl]-2,4,6-triethylbenzene methyl]-2,4,6-triethylbenzene methyl]-2,4,6-trimethylbenzene methyl]-2,4,6-trimethylbenzene methyl]-2,4,6-trimethylbenzene methyl]-2,4,6-triethylbenzene methyl]-2,4,6-triethylbenzene methyl]-2,4,6-triethylbenzene  in dimethylformamide (9.0 mL) sodium hydroxide  was added and the suspension was stirred at room temperature for 30 minutes. Then 1,3,5-tris(bromomethyl)-2,4,6-triethylbenzene  was added and the mixture stirred at 343 K for 24 h. After cooling to room temperature, the reaction mixture was poured into ice–water (55 mL). The precipitate was filtered off, washed with a little ice–water and dried under reduced pressure. The crude mixture, containing the desired product as well as two other triethylbenzene derivatives and the unreacted 4-methyl-1H-indazole, was separated chromatographically. The first flash chromatography  allowed the isolation of the by-products 2,4,6-triethyl-1,3-bis[(4-methyl-1H-indazol-1-yl)methyl]-5[(4-methyl-2H-indazol-2-yl)methyl]benzene and 2,4,6-triethyl-1,3-bis[(4-methyl-2H-indazol-2-yl)methyl]-5[ enabled the removal of 4-methyl-1H-indazole. After crystallization from hexane/ethyl acetate  the title compound was obtained as colourless crystals ; m.p. 427–429 K. 1H NMR  δ = 0.80 , 2.58 , 2.89 , 5.63 , 6.87 , 7.03 , 7.12 , 8.01 . 13C NMR  δ = 14.7, 18.6, 23.8, 47.6, 107.0, 120.5, 124.8, 126.5, 130.6, 131.4, 132.0, 139.6, 145.7.To a solution of 4-methyl-16.Uiso(H) = 1.2 or 1.5Ueq(C). The hydrogen atoms of the ammonium ions were located in a difference-Fourier map and the N—H bond lengths refined to a target value of 0.90 Å. The crystal studied was refined as a two-component twin.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989022006867/ex2059sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989022006867/ex2059Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989022006867/ex2059Isup3.cmlSupporting information file. DOI: 2184249CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Depression, one of the most common causes of disability, has a high prevalence rate in patients with metabolic syndrome. Type 2 diabetes patients are at an increased risk for depression. However, the molecular mechanism coupling diabetes to depressive disorder remains largely unknown. Here we found that the neuroinflammation, associated with high-fat diet (HFD)-induced diabetes and obesity, activated the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) in hippocampal neurons. This factor repressed brain-derived neurotrophic factor (BDNF) expression and caused depression-like behaviors in male mice. Besides, the loss of C/EBPβ expression in C/EBPβ heterozygous knockout male mice attenuated HFD-induced depression-like behaviors, whereas Thy1-C/EBPβ transgenic male mice (overexpressing C/EBPβ) showed depressive behaviors after a short-term HFD. Furthermore, HFD impaired synaptic plasticity and decreased surface expression of glutamate receptors in the hippocampus of wild-type (WT) mice, but not in C/EBPβ heterozygous knockout mice. Remarkably, the anti-inflammatory drug aspirin strongly alleviated HFD-elicited depression-like behaviors in neuronal C/EBPβ transgenic mice. Finally, the genetic delivery of BDNF or the pharmacological activation of the BDNF/TrkB signaling pathway by 7,8-dihydroxyflavone reversed anhedonia in a series of behavioral tests on HFD-fed C/EBPβ transgenic mice. Therefore, our findings aim to demonstrate that the inflammation-activated neuronal C/EBPβ promotes HFD-induced depression by diminishing BDNF expression. Depression is a common and severe medical illness. Major depressive disorder is a complicated disease manifested by emotional, motivational, cognitive, and physiological domain symptoms, placing a heavy burden on patients, families, and the whole society. Studies have shown that inflammation is one of the most important factors of depression pathophysiology . Type 2 CCAAT/enhancer-binding protein β (C/EBPβ), a member of the C/EBP family, is an inflammation-associated transcriptional factor involved in neurodegenerative diseases, such as Alzheimer’s disease , 2022, PA recent study revealed that C/EBPβ could transcriptionally downregulate brain-derived neurotrophic factor (BDNF) in the peripheral and central nervous system . BDNF, aThus, we hypothesized that inflammation-activated C/EBPβ could mediate HFD-induced depression by downregulating BDNF and promoting AMPARs internalization . We perfAnti-phospho-C/EBPβ, CST, 3084s; Anti-C/EBPβ Antibody (H-7), Santa Cruz, sc-7,962; Anti-α-Tubulin, Sigma-Aldrich, T6074; Anti-beta-actin, Abcam, ab8227; Anti-Glial Fibrillary Acidic Protein (GFAP) antibody, Sigma-Aldrich, G3893; Anti-Iba1, VWR, 019–19,741; Anti-BDNF, Abcam, ab72439; Anti-BDNF, Abcam, ab72439; Anti-NeuN, Abcam, ab177487; Anti-TrkB, R&D, MAB397; Anti-pTrkB, Santa Cruz, sc-135,645; Anti-PSD95, CST, 3450; Anti-IL-6, R&D, AF506-SP; Anti-GluA1, Merck Millipore, AB1504; Anti-GluA2, Merck Millipore, AB10529.d-glucose, RPI, G32040.4′,6-diamidino-2-phenylindole (DAPI), Sigma-Aldrich, D9542; Aspirin, Sigma-Aldrich, A2093; 7,8-dihydroxyflavone , Tokyo Chemical Industry Co., Ltd., D1916; Human insulin, Eli Lilly, 00002831501; IL-1 alpha Mouse ELISA Kit, Thermo Fisher, 88–5,019-22; IL-6 Mouse ELISA Kit, Thermo Fisher, BMS603HS; TNF alpha Mouse ELISA Kit, Thermo Fisher, BMS607-3; BDNF ELISA Kit, Abcam, ab212166; Advanced Glucose Meter Test Strips, CVS Health™, 968,577; Advanced Bluetooth Glucose Meter, CVS Health™, 968,574; Ultra Sensitive Mouse Insulin ELISA Kit, Crystal Chem, 90,080.Wild-type C57BL/6J (C/EBPβ+/+) mice and C/EBPβ heterozygous knockout (C/EBPβ+/−) mice were purchased from the Jackson Laboratory . Since some of the homozygous mutations are lethal on pure-strain backgrounds, C/EBPβ+/− mice were maintained as heterozygotes on a C57BL/6 strain background.via crossbreeding with wild-type mice. In the end, male F1 heterozygous mutant mice were confirmed as the final deliverables for this project.Thy1-human C/EBPβ mouse is a gift from Dr. Keqiang Ye. To generate Thy1-human C/EBPβ and ApoE4- human C/EBPβ transgenic mice of C57BL/6 J background, mouse genomic fragments containing homology arms (HAs) were amplified from a bacterial artificial chromosome (BAC) clone by using high-fidelity Taq, and were sequentially assembled into a targeting vector together with recombination sites and selection markers. After confirming the correctly targeted ES clones by Southern Blotting, we selected some clones for blastocyst microinjection, followed by founder production. Founders were confirmed as germline-transmitted The genotypes of transgenic mice were validated by polymerase chain reaction (PCR): Primer mix #1 (to distinguish Non-Tg and C/EBPβ-Tg): Forward: TGAAGCATTCCCTAATGAGCCAC, reverse: CTCGCCTCCTCCGGCCACTGCTAG. Primer mixes #2 : Forward1: AGAGTTGGTTGGTCCTCTCCT, reverse: GCCATTTAAGCCATGGGAAGTTAG, forward2: TGGACAGAGGAGCCATAACTGCAG.Only male animals were used for the experiments. All mice were group-housed and kept under specific pathogen-free (SPF) conditions with a 12 h light/12 h dark cycle and with free access to food and water. Mice were randomly assigned to each group by using a random number table. The sample size was determined by Power and Precision (Biostat). All animal experimental protocols were approved by the Laboratory Animal Welfare Ethical Committee (IACUC) of Renmin Hospital of Wuhan University (IACUC Issue No. WDRM 20210102B).Animal care and handling was performed according to the NIH animal care guidelines and Wuhan University guidelines. All procedures involving animals followed the ethical standards of Renmin Hospital of Wuhan University Institutional Animal Care and Use Committee.At first, to investigate whether the HFD induced depressive behavior, we fed C57 BL/6 J wild-type mice with an HFD or chow diet for 2, 8, or 12 weeks and evaluated T2DM-associated phenotypes and multiple depression-related behaviors. Then, to find the key molecular factor underlying these phenotypes, we subjected the C57 BL/6 J wild-type mice fed with HFD for 12 weeks to a social interaction test . We then classified the mice as susceptible  or resilient  subpopulation and used mice fed with a chow diet for 12 weeks as a control group. Next, to test the function of the key molecular factor C/EBPβ in HFD-induced depression-like behavior, we fed C/EBPβ+/−mice and C/EBPβ Tg mice with HFD or chow diet for 12 weeks and 2 weeks, respectively. Finally, to rescue depression-like behavior, C/EBPβ Tg mice were fed with an HFD or chow diet for 12 weeks and given treatment of anti-inflammatory drug aspirin, overexpression of BDNF in their hippocampus, and administration of small molecular BDNF mimetic compound at the eighth week. We only employ male mice in our study. All mice were 8–10 weeks old and had 1 week of adaptation to their surroundings before experiments.Mice were fed with a chow diet or HFD . We administered aspirin through their drinking water at pH 6.4 (120 mg/kg body weight/day) during the last 4 weeks on the HFD. The mice received vehicle or 7,8-DHF through their drinking water. To dissolve 7,8-DHF in water, 1 M NaOH was added drop wise to the water and stirred at room temperature overnight. The final concentration of 7,8-DHF was 22 mg/l (pH 7.6–7.8). Water (pH 7.6–7.8) was used as the vehicle control. Since the daily water intake of C57BL/6 J mice is about 7 ml/30 g body weight , the ora9 vector genomes per μL was placed into each site at a rate of 0.25 μl min−1 using a 10 μl glass syringe with a fixed needle. After the injection, we left the needle in place for 10 min and removed it slowly over 2 min. Mice were placed on a heating pad until they recovered from anesthesia. Four weeks after the stereotactic injection, we performed the multiple behavior test on the mice.The vectors of AAV8-human mature BDNF (AAV-BDNF) and AAV8-enhanced GFP (AAV-GFP) were generated, produced and purified by Virovek  reveals despair/depression-like behavior by recording the time suspended mice remain immobile. In brief, each mouse was individually suspended 20 cm above the floor with adhesive tape placed 1 cm from the tip of the tail. Animals were considered as immobile when they hung passively for 10 s without body movement. The time during which mice remained immobile was quantified in a total of 6 min. A longer immobility time than control mice indicated depression-like behaviors.The forced swim test (FST) evaluates depression-like behaviors by measuring the time swimming mice spend immobile. Briefly, mice were individually placed into a glass cylinder (35 cm in height and 15 cm in diameter) filled with 10 cm of warm water (25 ± 1°C). After each trial, we renewed the water. We forced the mice to swim for 6 min, and recorded the immobility time during the final 4 min. Immobility was defined as floating or remaining motionless, without movement, except those motions necessary to keep the head above the water. The observers were blind to the treatment of the mice. An increased immobility time compared with control mice indicated depression-like behaviors.The sucrose preference test (SPT) assesses anhedonia which is a key feature of depression. Anhedonia is defined as a percentage of sucrose preference below 65% . We usedTo assess mobility and anxiety, we performed an open-field test (OF) in a rectangular chamber (50 × 50 × 50 cm) made of opaque white plastic. The ground area of the box was divided into two parts: a 35 × 35 cm central zone and the surrounding zone. Mice were gently put in the corner and allowed to explore the chamber for 5 min on two consecutive days. A video camera and a 25 W red light bulb were placed 180 cm above the center of the apparatus and recorded the movements with an ANY-maze video motility system. This system recorded the time spent in the central zone, the number of entries into the central zone, the total distance traveled in the whole field, and the average speed. Anxiety-related behavior was evaluated by recording these parameters on the second day. Lower center time and distance values than control mice indicated anxiety-related behavior.We also evaluated anxiety through the elevated plus maze (EPM) test. Mice were tested for 5 min on an elevated plus maze apparatus on two consecutive days. The elevated plus maze, which was elevated 40 cm above the floor, consisted of a plus-shaped platform with two open arms (30 × 5 × 0.5 cm), two closed arms (30 × 5 × 15 cm), and a connecting central zone (5 × 5 cm). At the beginning of each test, mice were placed in the central zone, facing an open arm. The movements of the mice during a 5 min trial period were tracked by a video camera above the center of the maze and recorded with the ANY-maze software. To count as an entry into an arm, the center body of the mouse had to cross the border to the arm. We recorded and calculated the total distance traveled in the elevated plus maze apparatus, the number of entries into open or closed arms, and the time spent in open and closed arms. Next, we quantified mobility using the total distance traveled in the both arms and the total number of entries into any arm during the 5 min test period. Anxiety-like behavior was evaluated using the time spent in the open arms and the number of entries into the open arms on the second day. A shorter time spent in the open arms and longer time spent in the closed arms compared with the control mice indicated anxiety-related behaviors.We assessed hippocampal-dependent memory by performing a novel object recognition (NOR) test in an open-field apparatus (50 × 50 × 50 cm). Before the test, mice were allowed to freely explore the testing apparatus for 10 min. On the first day, mice were presented with two identical objects (familiar objects). We placed the objects at the left and right corners of the area, and allowed the mice to freely explore the objects for 5 min. Then, on the second day, we replaced one of two familiar objects with another object (novel object), and again allowed the mice to freely explored for 5 min. The time spent in exploring each object (familiar and novel objects) was recorded using a digital video camera and recorded with ANY-maze software. We defined “exploration” as touching the object (except with the tail) or sniffing the object (distance <2 cm). To analyze the recognition ability, we defined a discrimination index as the ratio of the time spent exploring the novel object over the time spent exploring the familiar and novel objects. A preference for the novel object indicated intact spatial recognition memory. Mice that did not explore any of the two objects during the pre-test were excluded from the analysis.Bilateral hippocampal tissues were grinded and lysed on ice for 30 min with 80 μl of ice-cold lysis buffer containing protease and phosphatase inhibitors. We then centrifuged the samples at maximum speed for 15 min at 4°C, and collected the supernatant, and quantified proteins with a Thermo BCA Protein Assay Kit (Cat#23227). Protein samples were boiled in SDS loading buffer for 10 min, and then were separated by 8–12% SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for 1 h at room temperature and incubated overnight at 4°C with the appropriate primary antibodies. After washed 4–6 times with TBS-T, the membrane was incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibodies  and anti-rabbit secondary antibodies . Then, after washed 6–8 times with TBS-T, the membrane was immersed in enhanced chemiluminescence reagents, and images were captured by ChemiDoc™ Touch Imaging System. ImageJ software was used to analyze the bands. β-actin or tubulin was used as the loading control and all the experiments were performed at least three times.3) was performed as described previously . Then the tissue was cross-linked with gentle agitation (30 min at 4°C) and this reaction was stopped by adding 100 mM glycine (15 min at 4°C). Next, we centrifuged the tissue, resuspended it in ice-cold lysis buffer , and centrifuged it at 12000 g for 15 min at 4°C. Finally, the supernatant fraction was aliquoted and stored at −80°C before use in the Western blotting.Surface receptor cross-linking with bis(sulfosuccinimidyl)suberate  according to the manufacturer’s instructions and was quantified by the Nanodrop apparatus. After that, 1 μg of total RNA was used for cDNA synthesis with an NG Script I cDNA Synthesis kit  according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was performed in a Bio-rad Real-Time PCR System. For qRT-PCR, samples were heated to 95°C for 10 min followed by 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. PCRs for CEBPB and BDNF were performed in triplicate. The relative quantification of gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels and calculated using the ΔΔCt method. Gene expression analyses were expressed as mRNA levels relative to controls. Real-time PCR probes were bought from TaqMan® (Thermo Fisher Scientific).Mice abdominal aortic blood samples were collected and centrifuged  and then the supernatant was collected and stored at −80°C until use. Quantitative determination of IL-1β, IL-6, and TNF-α were performed using commercially available high-sensitivity kits according to the manufacturer’s instructions.Peripheral blood mononuclear cells (PBMC) were isolated from fresh blood by density gradient centrifugation over Lympholyte® Mammal  according to the manufacturer’s instructions. Briefly, fresh blood was diluted in sterile diluent with a ratio of 1:1, layered onto the Lymphoprep solution, and centrifuged at 450 g at room temperature for 20 min. After that, the PBMC layer at the interface was carefully collected and transferred into a 15-mL tube, which washed twice with sterile phosphate-buffered saline  and then centrifuged at 250 g for 10 min. The harvested cell pellet was stored at −80°C until analysis.We used free-floating 20 μm brain sections for immunofluorescence staining. The brain sections were washed three times with PBS and blocked with 1% bovine serum albumin (w/v) and 0.3% Triton X-100 (v/v) for 30 min, and were incubated with primary antibodies at 4°C overnight. On the second day, after washed three times with PBS, the brain sections were incubated at room temperature with a mixture of labeled secondary antibodies for 2 h. Finally, DAPI  was used for staining nuclei for 5 min followed by washed three times with PBS. Images were acquired with a Leica Confocal Imaging System.Mice brains were fixed in 10% formalin (v/v) for 24 h and then immersed in 3% potassium bichromate (w/v) for 3 days in the dark. The solution was changed every day. The brains were transferred into a 2% silver nitrate (w/v) solution and incubated for 7 days in the dark. Vibratome sections were cut at 50 μm, air dried for 10 min, dehydrated through 95 and 100% ethanol, cleared in xylene and coverslipped. Spine numbers were counted in Image J software.2PO4, 6.0 mM MgCl2, 26 mM NaHCO3, 2.0 mM CaCl2, and 10 mM glucose. Hippocampi were dissected and cut into 400-mm thick transverse slices with a vibratome. After incubation at room temperature (23–24°C) in a-CSF for 60–90 min, slices were placed in a recording chamber  on the stage of an up-right microscope (Olympus CX-31) and perfused at a rate of 3 ml/min with a-CSF (containing 1 mM MgCl2) at 23–24°C. A 0.1 MU tungsten monopolar electrode was used to stimulate the Schaffer collaterals. The field excitatory post-synaptic potentials (fEPSPs) were recorded in the CA1 stratum radiatum by a glass microelectrode filled with a-CSF with a resistance of 3–4 MU. The stimulation output  was controlled by the trigger function of an EPC9 amplifier . fEPSPs were recorded under current-clamp mode. Data were filtered at 3 kHz and digitized at sampling rates of 20 kHz using Pulse software (HEKA Elektronik). The stimulus intensity  was set to evoke 40% of the maximum fEPSP and the test pulse was applied at a rate of 0.033 Hz. The long-term potentiation (LTP) of fEPSPs was induced with three theta-burst-stimulation . The magnitudes of LTP are expressed as the mean percentage of the baseline fEPSP initial slope.Mice were anaesthetized with isoflurane, decapitated, and their brains dropped in ice-cold a-CSF containing 124 mM NaCl, 3 mM KCl, 1.25 mM NaHin vitro experiment, cells were lysed in RIPA buffer  on ice for 30 min. The lysates were centrifuged for 10 min at 15,000 rpm. We quantified proteins in the supernatant using a Coomassie Brilliant Blue protein assay kit (Bio-Rad). Next, we boiled the supernatant mixed with the same amount of SDS loading buffer. After SDS- PAGE, the samples were transferred to a nitrocellulose membrane. The membranes were blocked in 5% non-fat milk for 1 h at room temperature, then incubated with primary antibody at 4°C overnight. Then the blots were incubated with IRDye 800CW-conjugated affinity-purified anti-mouse or anti-rabbit IgG secondary antibody (Rockland). Immunoreactive bands were visualized using an Odyssey Infrared Imaging System .Mouse brain tissue samples were lysed in lysis buffer  on ice for 30 min. For the d-glucose (2 g/kg body weight). Each mouse was weighed before the injection to determine the appropriate dose. Blood samples from the tail vein were collected at 0, 15, 30, 60, and 90 min post-injection.We performed a glucose tolerance test (GTT) on mice fasted for 16 h after a peritoneal injection of For the insulin tolerance test (ITT), the mice fasted for 6 h. Each mouse was weighted and administered a peritoneal insulin injection of insulin (0.75 U/kg body weight). Blood glucose level from the tail vein were collected at 0, 15, 30, 60, and 90 min post-injection .t-test with Welch’s correction. For multiple-group comparisons, one-way ANOVA and Bonferroni’s multiple comparison test was applied. The two-way ANOVA and Bonferroni’s post hoc test compared the differences between groups that have been split on two independent factors. Values of p < 0.05 indicated statistical significance.We analyzed all data using GraphPad Prism (GraphPad Software) and expressed the results as the mean ± standard error of the mean (SEM) from three or more independent experiments. All the statistical procedures can be found in the figure legends for each experiment, including the statistical tests used, number of mice used. Sample sizes were determined by Power and Precision (Biostat). We compared group pairs using an unpaired The data that support the findings of this study are available on request from the corresponding author.p > 0.05; Chow-8 61.91 ± 6.334 vs. HFD-8112.2 ± 11.1, p = 0.0026; Chow-12 74.85 ± 7.588 vs. HFD-12132.3 ± 12.86, p = 0.0003; p > 0.05; Chow-8 75.75 ± 8.184 vs. HFD-8125.2 ± 10.43, p = 0.0036; Chow-12 80.12 ± 6.851 vs. HFD-12138.8 ± 14.26, p = 0.0003; p > 0.05; Chow-8 71.34 ± 4.305 vs. HFD-8 60.28 ± 4.346, p > 0.05; Chow-12 67.91 ± 4.883 vs. HFD-12 45.99 ± 3.365, p = 0.0045; To confirm whether the HFD consumption induced depressive behavior, we first confirmed that we had successfully established an HFD-induced insulin resistance murine model. Next, we evaluated multiple depression-related behaviors in mice fed with an HFD or chow diet for 2, 8, or 12 weeks . The bodp > 0.05; Chow-8 8.929 ± 0.629 vs. HFD-8 5.213 ± 0.4137, p = 0.0005; Chow-12 8.607 ± 0.7214 vs. HFD-12 4.06 ± 0.3762, p < 0.0001; p > 0.05; Chow-8800.1 ± 98.47 vs. HFD-8558.7 ± 64.43, p > 0.05; Chow-12721.1 ± 78.87 vs. HFD-12406 ± 37.62, p = 0.0449; p > 0.05; Chow-8 56.08 ± 5.014 vs. HFD-8 40.07 ± 2.938, p > 0.05; Chow-12 54.5 ± 4.403 vs. HFD-12 34.25 ± 2.558, p = 0.0105; p > 0.05; Chow-8157.9 ± 5.014 vs. HFD-8175.2 ± 3.657, p > 0.05; Chow-12165.5 ± 4.403 vs. HFD-12185.8 ± 2.558, p = 0.0137; To test whether these mice also exhibited anxiety-related behaviors, we performed open field (OF) test and elevated plus maze (EPM) test. In the OF test, the 8-week HFD group spent less time in the center and traveled shorter distances than the chow diet group, and this effect was more prominent in the 12-week HFD group  test. We found that the 12-week HFD group spent less time exploring new objects than familiar ones. Meanwhile, there was no difference among groups in the pre-test, indicating that 12-week HFD mice showed memory impairment  levels than control and resilient mice, indicating the activation of C/EBPβ in the hippocampus [p < 0.0001; C/EBPβ+/−-Chow 0.5234 ± 0.04809 vs. C/EBPβ+/−-HFD 0.5377 ± 0.08152, p > 0.05; p = 0. 0463; C/EBPβ+/−-Chow 1.224 ± 0.1883 vs. C/EBPβ+/−-HFD 1.154 ± 0.1361, p > 0.05; To assess the impact of C/EBPβ on HFD-elicited depression-like behaviors, we bred WT(C/EBPβ+/+) and C/EBPβ heterozygous knockout (C/EBPβ+/−) mice and treated them with HFD or chow diet for 12 weeks, then evaluated a series of depression-related behaviors and molecular component levels . Since sp = 0. 0407; C/EBPβ+/−-Chow 1.184 ± 0.2467 vs. C/EBPβ+/−-HFD 1.347 ± 0.1695, p > 0.05; p = 0. 0029; C/EBPβ+/−-Chow 1.08 ± 0.196 vs. C/EBPβ+/−-HFD 1.554 ± 0.1508, p > 0.05; p < 0.0001; C/EBPβ+/−-Chow 0.8333 ± 0.1604 vs. C/EBPβ+/−-HFD 1.387 ± 0.3051, p > 0.05; p = 0. 0091; C/EBPβ+/−-Chow 1.096 ± 0.0941 vs. C/EBPβ+/−-HFD 0.9267 ± 0.1983, p > 0.05; p = 0.0265; C/EBPβ/Tubulin: WT-Chow 1 ± 0.03988 vs. WT-HFD 1.878 ± 0.1962, p < 0.0001; BDNF/Tubulin: WT-Chow 1 ± 0.05771 vs. WT-HFD 0.6226 ± 0.06939, p = 0.0029; p > 0.05; C/EBPβ/Tubulin: C/EBPβ+/−-Chow 0.5835 ± 0.04955 vs. C/EBPβ+/−-HFD 0.6714 ± 0.07576, p > 0.05; BDNF/Tubulin: C/EBPβ+/−-Chow 1.186 ± 0.07754 vs. C/EBPβ+/−-HFD 1.051 ± 0.1829, p > 0.05; Next, we quantified the hippocampal levels of the pro-inflammatory cytokines including TNF-α, IL-1β and IL-6 in mice hippocampus by ELISA and found that they were all significantly increased in WT-HFD mice versus WT-chow diet mice while there was no significant change in C/EBPβ+/− mice either fed with HFD or chow diet. [TNF-α: WT-Chow 1 ± 0.1301 vs. WT-HFD 1.59 ± 0.2418, p < 0.0001; C/EBPβ+/−-Chow 63.78 ± 7.404 vs. C/EBPβ+/−-HFD 74.31 ± 8.298, p > 0.05; p < 0.0001; C/EBPβ+/−-Chow 67.35 ± 7.515 vs. C/EBPβ+/−-HFD 75.64 ± 7.691, p > 0.05; p = 0.0008; C/EBPβ+/−-Chow 73.3 ± 6.928 vs. C/EBPβ+/−-HFD 71.35 ± 5.48, p > 0.05; In alignment with these discoveries, we found that depression-related behaviors induced by HFD were more obvious in WT-HFD mice versus WT-chow diet mice both in TST and FST  and their littermate WT mice with HFD or chow diet for only 2 weeks before performing depression associated behaviors and molecular tests . The neup = 0. 0478; IL-1β: WT-Chow 1 ± 0.136 vs. WT-HFD 1.045 ± 0.01989, p > 0.05; C/EBPβ Tg-Chow 1.35 ± 0.1382 vs. C/EBPβ Tg-HFD 3.113 ± 0.6486, p = 0.0089; IL-6: WT-Chow 1 ± 0.1724 vs. WT-HFD 1.238 ± 0.0274, p > 0.05; C/EBPβ Tg-Chow 1.787 ± 0.3637 vs. C/EBPβ Tg-HFD 4.293 ± 0.6079, p = 0.0006; p = 0.0018; p > 0.05; C/EBPβ Tg-Chow 0.8163 ± 0.03716 vs. C/EBPβ Tg-HFD 0.5484 ± 0.04668, p = 0.0049; p > 0.05; C/EBPβ Tg-Chow 0.7601 ± 0.1085 vs. C/EBPβ Tg-HFD 0.4026 ± 0.1044, p = 0.0383; Next, we quantified pro-inflammatory cytokines  in mice hippocampus and found that HFD C/EBPβ transgenic mice had notably higher IL-1β and IL-6 levels than their corresponding-chow diet mice while there was no significant change between WT mice groups after a short-time HFD treatment. Meanwhile, TNF-α was not significantly increased between mice fed with HFD or chow diet TNF-α was not significantly increased between corresponding diet mice but there was an increasing trend between WT-chow and C/EBPβ Tg-HFD mice  curves in the CA1 region of hippocampal slices. The input/output curves were markedly reduced in the WT-HFD mice but with no significant change in the C/EBPβ+/− HFD mice . In addi/O curvesp < 0.0001; WT-HFD 1.043 ± 0.0328 vs. C/EBPβ+/−-HFD 0.9396 ± 0.0609, p > 0.05; IL-6: WT-Chow 0.9944 ± 0.0469 vs. WT-HFD 2.747 ± 0.2132, p < 0.0001; WT-HFD 0.714 ± 0.1018 vs. C/EBPβ+/−-HFD 0.9728 ± 0.158, p > 0.05; p < 0.0001; WT-HFD 15.54 ± 0.7127 vs. C/EBPβ+/−-HFD 13.85 ± 0.775, p > 0.05; p = 0.0002; WT-HFD 5.846 ± 0.191 vs. C/EBPβ+/−-HFD 5.154 ± 0.4213, p > 0.05; In addition, immunostaining of hippocampus slices revealed that C/EBPβ+/− mice largely blocked the reduction of PSD95 compared to WT-HFD mice  80.5 ± 8.382 vs. C/EBPβ Tg-HFD (12 W) 159.4 ± 12.82, p < 0.0001; p < 0.0001; C/EBPβ Tg-Chow (12 W) 75.77 ± 5.797 vs. C/EBPβ Tg-HFD (12 W) 157.8 ± 12.72, p < 0.0001; p = 0.0013; C/EBPβ Tg-Chow (12 W) 64.25 ± 4.505 vs. C/EBPβ Tg-HFD (12 W) 27.46 ± 2.939, p < 0.0001; To better understand the role of C/EBPβ in HFD-induced depression, we treated WT or Thy1-C/EBPβ Tg male mice with HFD or chow diet for 12 weeks. We found that HFD mice gained more weight than their chow-diet littermates regardless of WT or Thy1-C/EBPβ Tg male mice . ITT shop > 0.05; p > 0.05; C/EBPβ Tg-HFD + Vehicle 0.5919 ± 0.1277 vs. C/EBPβ Tg-HFD + Aspirin 0.9871 ± 0.1332 p = 0.0358; To ascertain that neuroinflammation is the indeed initiating factor, we tested the influence of non-steroid anti-inflammatory drug (NSAID) Aspirin or Vehicle on HFD-induced depression-like behaviors in mice. WT and Thy1 C/EBPβ Tg male mice were fed with HFD for 12 weeks, and were treated Aspirin or vehicle for the last 4 weeks . After tp = 0.0456; C/EBPβ Tg-HFD + Vehicle 1.514 ± 0.0933 vs. C/EBPβ Tg-HFD + Aspirin 0.942 ± 0.0674, p = 0.0009; IL-1β: WT-HFD + Vehicle 1 ± 0.1082 vs. WT-HFD + Aspirin 0.8884 ± 0.0820, p > 0.05; C/EBPβ Tg-HFD + Vehicle 2.566 ± 0.3666 vs. C/EBPβ Tg-HFD + Aspirin 1.184 ± 0.0641 p = 0.0002; IL-6: WT-HFD + Vehicle 1 ± 0.1407 vs. WT-HFD + Aspirin 0.7748 ± 0.0880, p > 0.05; C/EBPβ Tg-HFD + Vehicle 4.72 ± 0.3398 vs. C/EBPβ Tg-HFD + Aspirin 1.42 ± 0.1313 p < 0.0001; p > 0.05; C/EBPβ Tg-HFD + Vehicle 2.463 ± 0.0435 vs. C/EBPβ Tg-HFD + Aspirin 1.272 ± 0.0680, p < 0.0001; IL-1β: WT-HFD + Vehicle 1 ± 0.1716 vs. WT-HFD + Aspirin 0.792 ± 0.0901, p > 0.05; C/EBPβ Tg-HFD + Vehicle 1.754 ± 0.0555 vs. C/EBPβ Tg-HFD + Aspirin 1.363 ± 0.0824 p = 0. 027; IL-6: WT-HFD + Vehicle 1 ± 0.171 vs. WT-HFD + Aspirin 0.8113 ± 0.097, p > 0.05; C/EBPβ Tg-HFD + Vehicle 1.793 ± 0.0853 vs. C/EBPβ Tg-HFD + Aspirin 1.321 ± 0.1023 p = 0.0337; p > 0.05; C/EBPβ Tg-HFD + Vehicle 0.6168 ± 0.0322 vs. C/EBPβ Tg-HFD + Aspirin 0.9345 ± 0.0513 p = 0.0224; p > 0.05; BDNF/Tubulin: WT-HFD + Vehicle 1 ± 0.1134 vs. WT-HFD + Aspirin 0.9874 ± 0.0547, p > 0.05; C/EBPβ Tg-HFD + Vehicle 0.4012 ± 0.0810 vs. C/EBPβ Tg-HFD + Aspirin 0.7935 ± 0.0699 p = 0.0187; Next, we measured pro-inflammatory cytokines in the hippocampus and serum by ELISA. In both tissues, Thy1-C/EBPβ Tg mice treated with aspirin had lower TNF-α, IL-1β, and IL-6 levels than their corresponding vehicle mice.  alleviated HFD-induced depression-like behaviors, while overexpression of C/EBPβ (C/EBPβ Tg) accelerated them.Since synaptic plasticity impairment participates in depression, we tested C/EBPβ-induced synaptic plasticity impairment in WT (C/EBPβ+/+) and C/EBPβ+/− mice. As expected, electrophysiological examination showed that the input/output curves were markedly reduced in the WT-HFD mice but with no significant change in the C/EBPβ+/− HFD mice. In addition, C/EBPβ+/− HFD mice showed a normal provocation and maintenance of LTP in Schaffer collateral-CA1 compared to WT mice. Moreover, the level of synapse associated protein (PSD95) as well as the number of dendritic spines in WT-HFD mice was decreased versus WT-chow diet mice and C/EBPβ+/− mice. In addition, two surface AMPARs associated subunits, surface GluA1 and GluA2, were also downregulated in WT-HFD mice compared to C/EBPβ+/− mice, which was in line with Li’s report . Finallyvia suppressing hypothalamic protein kinase A  to Z-HW, National Key Research Projects of China (No. 2021YFA1302400) to Z-HW, Hubei Province Special Project Supported by Central Funds Guiding the Local Science and Technology Development (No. 2016ZYYD002) to MF, and Wuhan University Specific Fund for Major School-level Internationalization Initiatives (No. WHU-GJZDZX-PT02) to XX.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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