Abstract

Objective:

To investigate whether (Ba,Ca)(Ti,Sn)O3-based piezoelectric ceramics (BCTS) provide neuroprotection by inhibiting the IL-6/JAK2/STAT3 signaling pathway in microglia.

Methods:

BCTS surface morphology and elemental distribution were analyzed using Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDX), phase composition was determined via X-ray diffraction (XRD), and hydrophilicity was measured through contact angle analysis. Immunofluorescence (IF), Western blot, and ELISA were employed to evaluate the expression of microglial markers and inflammatory factors in the BV2 injury model and in spinal cord injury rats. Behavioral tests were conducted to evaluate motor function recovery in spinal cord injury rats. PC12 cells were cultured with BCTS-CM (supernatant from BCTS-treated BV2 cells) to assess the IL-6/JAK2/STAT3 signaling pathway expression and its effects on LDH release, antioxidant enzyme activity, apoptotic proteins, and β-III-tubulin expression.

Results:

BCTS exhibited a pure perovskite phase, densely packed grains, and favorable hydrophilicity. It did not affect BV2 cell viability but inhibited LPS-induced M1 microglial activation, reducing the expression of TNF-α, IL-1β, and IL-6. Simultaneously, BCTS promoted M2 microglial polarization, upregulating IL-4, IL-10, and TGF-β1. In PC12 cells, BCTS-CM increased cell survival, antioxidant activities, Bcl-2, and β-III-tubulin expression, while decreasing LDH release, MDA content, BAX and Cleaved Caspase-3 expression. BCTS-induced neuroprotection is mediated by the suppression of the IL-6/JAK2/STAT3 signaling pathway, as evidenced by the fact that IL-6 supplementation counteracts this protection while AG490 treatment further reinforces it compared to BCTS-CM alone. In the spinal cord injury rat model, BCTS inhibited the expression of microglia and inflammatory factors at the injury site, while improving the BBB score and reducing the error rate in the grid walking test.

Conclusion:

(Ba,Ca)(Ti,Sn)O3-based piezoelectric ceramics exhibit neuroprotective effects by inhibiting IL-6 secretion from microglia, thereby preventing the activation of the IL-6/JAK2/STAT3 signaling pathway in neurons.

(Ba,Ca)(Ti,Sn)O3-based piezoelectric ceramics (BCTS) were fabricated via a solid-state reaction: BaCO3, CaCO3, TiO2, and SnO2 powders were mixed, ball-milled, calcined, pressed into discs, and sintered to obtain dense ceramics. The BCTS ceramics attenuate neuronal injury and exert neuroprotective effects by reducing IL-6 release from microglia, thereby suppressing the excessive activation of the IL-6/JAK2/STAT3 signaling pathway in neurons.

1 Introduction

Spinal cord injury (SCI) is a common yet devastating condition of the Central Nervous System (CNS), often caused by trauma, compression, or ischemia. It directly impairs sensory, motor, and autonomic functions, frequently resulting in irreversible neurological deficits (Li et al., 2022; Sunshine et al., 2023). These impairments significantly diminish patients’ quality of life and life expectancy, while also imposing substantial physical, psychological, and economic burdens on individuals, families, and society at large (Karamian et al., 2022; Ma et al., 2023). While immediate stabilization is standard, effective neuroprotective strategies that arrest secondary injury mechanisms and promote functional recovery remain a critical unmet clinical need.

Electrical stimulation techniques are well-recognized for their reparative effects on CNS injuries, as they enhance the CNS plasticity (Kathe et al., 2022; Ziesel et al., 2023). However, traditional electrical stimulation techniques are limited by the need for external power sources and wired connections. To overcome these limitations, piezoelectric materials offer a compelling solution by converting physiological mechanical forces—such as respiration and spinal movement—into therapeutic bioelectric signals, enabling self-powered, wireless neuromodulation (Xu et al., 2023; Yan et al., 2022).

Furthermore, the excellent biocompatibility of piezoelectric materials minimizes tissue irritation and immune rejection, making them highly suitable for biomedical applications (Fu et al., 2023). Among lead-free piezoelectric ceramics, (Ba,Ca)(Ti,Sn)O3-based piezoelectric ceramics (BCTS) offer an optimal balance of electromechanical performance and physiological stability. While polymers like polyvinylidene fluoride (PVDF) and traditional piezoceramics like ZnO are biocompatible, their relatively low piezoelectric coefficients limit the generation of sufficient electrical output (Kapat et al., 2020). Conversely, high-performance perovskites like (K,Na)NbO3 (KNN) and (Bi,Na)TiO3 (BNT) face challenges in vivo: KNN is prone to hydrolytic degradation and ionic leakage in aqueous physiological environments, while BNT raises safety concerns due to potential cytotoxicity and leakage currents (Dong et al., 2022; Xu et al., 2023). Compared to pure BaTiO3, BCTS engineered at the morphotropic phase boundary exhibits significantly enhanced piezoelectric responsiveness, ensuring higher voltage output from mechanical inputs (Liu and Ren, 2009). Consequently, given its superior phase stability, higher piezoelectric constants, and enhanced chemical stability and biocompatibility under physiological conditions (Tang et al., 2025), specifically prepared BCTS were selected to investigate their potential neuroprotective effects.

Despite increasing interest in piezoelectric neuromodulation, its immunomodulatory mechanisms in SCI remain poorly defined. In particular, whether piezoelectric-generated bioelectricity influences microglial polarization and subsequently modulates neuron-specific inflammatory signaling pathways has not been determined. The therapeutic efficacy of BCTS-driven stimulation is likely mediated by the modulation of the immune microenvironment. Microglia, the primary immune cells of the CNS and the most abundant type of glial cells in the nervous system, play a pivotal role in CNS injuries (Borst et al., 2021). They are essential for maintaining neural tissue homeostasis, regulating neuronal activity, and contributing to neural repair processes (Rao et al., 2021; Au and Ma, 2022). Research indicates that microglia exhibit distinct functional roles depending on their phenotypic state (Tao et al., 2021). According to the widely accepted binary classification, M1-type microglia release pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 (Wang G. et al., 2022; Xian et al., 2022; Liu et al., 2024). These cytokines initiate and exacerbate neuroinflammatory responses, leading to further neuronal and tissue damage (Hui et al., 2023; Yu et al., 2023). Conversely, M2-type microglia secrete neurotrophic factors and anti-inflammatory cytokines such as transforming growth factor-β1 (TGF-β1), interleukin-4 (IL-4) and interleukin-10 (IL-10) (Wang L. et al., 2024; Zhong et al., 2024; Zhu et al., 2024), which inhibit inflammation and promote neuronal regeneration and synapse formation, thereby mitigating neural damage and facilitating tissue repair (Xie et al., 2021; Zou et al., 2024). Therefore, shifting the microglial balance from the destructive M1 state to the reparative M2 state represents a primary mechanism by which piezoelectric stimulation could exert neuroprotection.

To define the precise molecular mechanism, we focused on the crosstalk between microglia and neurons. Among the cytokines released by M1 microglia, Interleukin-6 (IL-6) is a critical mediator of neuronal damage. Excessive IL-6 activates the JAK2/STAT3 signaling pathway in neurons, a cascade implicated in excitotoxicity and structural degradation (Liu et al., 2022; Zhao et al., 2022; Lee J. Y. et al., 2023). We hypothesize that BCTS-mediated bioelectric signaling modulates microglial polarization toward the M2 phenotype, thereby suppressing IL-6 release, limiting neuronal JAK2/STAT3 overactivation, and exerting neuroprotective effects.

In this study, we utilized BCTS to validate this hypothesis. Specifically, we investigated how BCTS-driven stimulation modulates microglial polarization to regulate the IL-6/JAK2/STAT3 signaling pathway in neurons. Our objective is to elucidate the precise neuroprotective mechanisms of BCTS, thereby providing a solid theoretical basis and novel therapeutic strategies for the treatment of spinal cord injury.

2 Materials and methods2.1 Materials

JSM-5900 Scanning Electron Microscope (Japan), Xpert-PRO X-ray Diffractometer (Netherlands), Water Contact Angle Goniometer JC2000D1 (Shanghai Zhongchen Digital Technology Equipment Co., Ltd., China), Quasi-static d33 Meter ZJ-3AN (Institute of Acoustics, Chinese Academy of Sciences, China), Ultrasound Stimulator (GV-CSI1.0) (Green Valley BrainTech (ShenZhen) Medical Technology Co., Ltd. (China), BV2, PC12 (Wuhan Pricella Biotechnology Co., Ltd., China), Rabbit Anti-iNOS (Thermo Fisher Scientific, Inc.), Rabbit Anti-Iba-1, Arg-1, NeuN, Goat Anti-Rabbit IgG H&L (Alexa Fluor 488), Goat Anti-Mouse IgG H&L (Alexa Fluor 594) (Abcam, UK), Rabbit Anti-CD86, CD206, β-actin, HRP-conjugated Goat Anti-Rabbit IgG (H + L) (Wuhan Proteintech Biotechnology Co., Ltd., China), Rabbit Anti-IL-6, TNF-α, IL-4, IL-10, TGF-β1 (ImmunoWay, United States), Rabbit Anti-IL-1β, JAK2, P-JAK2, STAT3, P-STAT3, BAX, Bcl-2, Cleaved Caspase-3 (Shenyang Wanlei Biotechnology Co., Ltd., China), BCA Protein Assay Kit, CCK8 Cell Viability Assay Kit, LDH Cytotoxicity Assay Kit, SOD Activity Assay Kit, CAT Activity Assay Kit, GSH-Px Activity Assay Kit, MDA Assay Kit, Dapi (Beijing Solarbio Science & Technology Co., Ltd., China), LPS (Sigma, Germany), IL-6, AG490 (MCE, Germany), Laser Confocal Microscope (Olympus Corporation, Japan), SDS-PAGE Electrophoresis System (BIO-RAD, USA).

2.2 The preparation of BCTS piezoelectric ceramics

The Ba0.94Ca0.06Ti0.92Sn0.08O3 (abbreviated as BCTS) piezoelectric ceramics were prepared using the traditional solid-state reaction method. The raw materials included barium carbonate (BaCO3, 99.8%), calcium carbonate (CaCO3, 99.5%), titanium dioxide (TiO2, 99.0%), and tin oxide (SnO2, 99.99%). After drying, these materials were weighed according to their stoichiometric ratios and mixed using ZrO2 balls and anhydrous ethanol as the milling medium for 12 h to ensure thorough blending of the powders. The mixed powders were then dried and sieved, followed by calcination at 1,200°C for 2 h to promote solid-state reactions. The calcined powders were subjected to an additional 12-h milling, drying, and sieving process. The resulting powders were mixed with paraffin, and then pressed into discs with a diameter of 12 mm and a thickness of 1.2 mm under 10 MPa pressure. These discs were sintered in air at 1,440°C for 2 h, with a hold at 600°C for 2 h to complete the removal of paraffin.

2.3 Characterization

The surface microstructure of the sintered ceramic samples was observed using a Scanning Electron Microscope (ZEISS GeminiSEM 300, Germany). Since the ceramic samples are insulating materials, they were pre-treated by gold sputtering for 80 s before observation to ensure successful imaging. Additionally, Energy Dispersive X-ray Spectroscopy (EDX) was utilized to track the uniformity of the elemental distribution. Phase analysis of the prepared ceramics was conducted using an Xpert-PRO X-ray Diffractometer under the following conditions: Cu-Kα target (λ = 1.5406 Å), scan speed of 1°/min, tube voltage of 45 kV, tube current of 40 mA, diffraction angle range of 10°–80°, with a step size of 0.039°. The wettability of the piezoelectric ceramics was measured using a water contact angle goniometer (JC2000D1, Shanghai Zhongchen Digital Technology Equipment Co., Ltd., China). To measure the electrical properties, silver paste was first applied to the top and bottom surfaces of the samples and then sintered at 750°C for 30 min to form the electrodes. For ceramics used in biological experiments, thin copper foils were clamped onto both sides of the ceramic samples to create the electrodes. Subsequently, the samples were polarized in a silicone oil bath at room temperature using a DC electric field of 2.5 kV/mm. After polarization, the samples were left for 24 h to release residual stress and charge, and then their piezoelectric constants were evaluated using a quasi-static d33 meter (ZJ-3AN, Institute of Acoustics, Chinese Academy of Sciences, China).

The variation of the dielectric constant (εr) with temperature, from 20 to 150°C, was analyzed at frequencies of 100 Hz, 1 kHz, 10 kHz, 100 kHz, and 1 MHz using an LCR meter (TH2618B, Guangzhou Zhuo Yue Electronic Instrument and Equipment Co., Ltd., China). Ferroelectric hysteresis loops (P-E) were recorded at voltage of 1 kV using a ferroelectric tester (RT66A; Radiant Technologies Inc., Albuquerque, NM). To ensure the reproducibility of these material properties, all characterizations were performed on at least three independent samples from different batches, and representative results are presented.

2.4 Experimental animals

Adult Sprague-Dawley rats (equal numbers of males and females, 200–250 g) were obtained from the Experimental Animal Center of Guizhou Medical University (Certificate No.: SCXK(Gui)2025-0001). All experimental procedures were approved by the Ethics Committee of Guizhou Medical University (Approval No.: 2100034) and conducted in accordance with the Guidelines for Ethical Review of Animal Welfare in Experiments (GB/T 35892–2018). Animal handling, surgical procedures, and all experimental manipulations adhered strictly to the Regulations on the Administration of Experimental Animals (China) and the institutional guidelines for laboratory animal welfare and management.

2.5 Spinal cord injury model and BCTS intervention

SD rats were anesthetized via intraperitoneal injection with 30 mg/kg of 2% sodium pentobarbital (Ying et al., 2025) and placed in the prone position, after which a midline dorsal incision was made to expose the paraspinal muscles and vertebrae, T9 laminectomy was performed with careful removal of the lateral edges of the T9 articular processes, and the exposed spinal cord was subsequently compressed with an aneurysm clip applying a closing force of 30 g for 10 s (Lee P. H. et al., 2023; Vahabi and Öztürk, 2023). Successful spinal cord injury was confirmed by narrowing of the dorsal vein, hindlimb twisting or twitching, tail curling, and loss of voluntary movement below the injury site, after which hemostasis was achieved with a gelatin sponge and the wound was rinsed with sterile saline. In the intervention group, sterilized BCTS material was applied to the injury site and secured with a small amount of sterile fibrin glue, and the muscles, fascia, and skin were then sutured in layers with the incision disinfected using povidone-iodine. Sham-operated animals underwent the same procedure without spinal cord compression. Postoperatively, rats were individually housed with free access to food and water. Manual bladder expression was performed twice daily until spontaneous urination resumed, and the perineal area was kept dry. Intramuscular penicillin (0.1 mL/day) was administered for three consecutive days to prevent infection.

SD rats were randomly assigned to each experimental group using a computer-generated randomization table to ensure equal numbers of rats in each group: (1) Sham group, in which only laminectomy was performed without inducing spinal cord injury. (2) SCI group, in which spinal cord injury was induced without any further intervention. (3) BCTS group, in which rats received BCTS treatment after spinal cord injury. To activate the piezoelectric effect of BCTS materials, BCTS group rats were anesthetized and placed in the prone position for ultrasound (US) treatment. Each rat received ultrasound stimulation for 15 min per day for 3 consecutive weeks. US stimulation was applied to BCTS using the following parameters: frequency 1 MHz, amplitude 37% of the maximum output, pulse width 500 μs, pulse repetition period 7 ms, stimulation duration 1 s, and burst interval 5 s, corresponding to an acoustic pressure of 1.224 MPa (Zhang et al., 2023).

2.6 Cell culture

In this study, we utilized the BV2 cell line as a substitute for microglia and the rat pheochromocytoma PC12 cell line as a substitute for neurons. The BCTS samples were sterilized by soaking in 75% alcohol for 60 min, followed by three 5 min PBS washes and 30 min of UV sterilization. The BV2 cell line was seeded onto the negative polarization side of BCTS at a density of 1 × 105 cells per well, with 3 ml of complete DMEM medium (10% FBS + 1% penicillin/streptomycin) per well, cultured in a 37°C, 5% CO2 incubator with medium changes every 2 days, and passaged upon reaching 80% confluence. In this experiment, 1 μg/ml (Ismail et al., 2020) of LPS was utilized to establish damage models for both the BV2 cell line and the PC12 cell line. The supernatant from BCTS-treated BV2 cells, termed BCTS cell-conditioned medium (BCTS-CM) (Jiang et al., 2023), was collected, centrifuged (5 min at 1,000 × g), and then used to culture PC12 cells (Guo et al., 2016; Guo et al., 2024). Additionally, to verify the effect of the IL-6/JAK2/STAT3 signaling pathway on PC12 cells, the PC12 were treated with IL-6 at a concentration of 10 ng/mL (Wang Y. et al., 2024) and AG490 at 5 μM (Yang B. et al., 2020) in the BCTS-CM. In the BCTS group, ultrasound stimulation was applied with a frequency of 19.876 MHz, amplitude of 100 mVpp, and burst period of 100 ms, with a measured acoustic pressure of 2.45 MPa. BV2 cells were exposed to ultrasound stimulation for 5 min per day for 3 consecutive days (Zhang et al., 2023).

In BV2 cells, they are divided into groups, ① Con group (LPS– and BCTS–), ② LPS group (LPS+ and BCTS–), ③ BCTS group (LPS+ and BCTS +). In PC12 cells, they are divided into groups, ① Con group (LPS– and BCTS-CM– and IL-6– and AG490–), ② LPS group (LPS+ and BCTS-CM– and IL-6– and AG490–), ③ BCTS-CM group (LPS+ and BCTS-CM+ and IL-6– and AG490–), ④ IL-6 group (LPS+ and BCTS-CM+ and IL-6+ and AG490–), ⑤AG490 group (LPS+ and BCTS-CM+ and IL-6– and AG490+).

2.7 Cell viability assay

Cell viability was assessed using the CCK8 assay kit following the manufacturer’s instructions. Cells were plated at 100 μL per well in a 96-well plate and pre-incubated for 24 h. Following this, 10 μL of CCK8 reagent was added to each well, and the plate was further incubated for 2 h before measuring the optical density (OD) at 450 nm using a microplate reader to assess cell viability. Cell viability was calculated according to the following formula: Cell viability (%) = (ODtreatment – ODblank)/(ODcontrol−ODblank) × 100%. The control cells were considered to have 100% viability.

2.8 Analysis of LDH release, antioxidant enzyme activity and MDA levels

To assess lactate dehydrogenase (LDH) levels in the culture medium, supernatants were collected and analyzed using an LDH assay kit according to the manufacturer’s instructions, with cell suspensions undergoing 25 s of ultrasonic treatment followed by centrifugation (4°C, 15 min at 8,000 × g), and the procedures and calculations were based on the manufacturer’s manual. The supernatants were collected, and assay kits were used to measure the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and the content of malondialdehyde (MDA).

2.9 Western blot analysis

At 21 days post-spinal cord injury, rats were anesthetized with 1% sodium pentobarbital, and a 0.5 cm spinal cord segment adjacent to the injury site was dissected. The tissue was homogenized and lysed in protein lysis buffer containing protease inhibitors (PMSF: 100:1, v/v). The mixture was vortexed and centrifuged at 12,000 × g for 15 min at 4°C, and the supernatant was collected for protein quantification. After treatment, BV2 and PC12 cells were washed with 1 × PBS, lysed on ice with RIPA buffer (containing protease and phosphatase inhibitors), and centrifuged at 12,000 × g for 10 min at 4°C. The total protein concentration in each sample was measured using a BCA protein assay kit (Thermo Fisher Scientific, United States). Subsequently, protein samples were mixed with loading buffer and boiled to denature the proteins. Proteins of varying molecular weights were separated by SDS-PAGE using polyacrylamide gels of appropriate concentrations, and then transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk for 90 min to prevent non-specific binding. After blocking, the membranes were incubated overnight at 4°C with primary antibodies specific to the target proteins. The next day, the membranes were incubated with secondary antibodies at room temperature for 120 min, washed three times with 1 × TBST. The specific dilution ratios of the antibodies used in the experiments are detailed in Table 1). Immersed in ECL detection reagent, and protein bands were visualized using a chemiluminescence detection system. β-actin was used as a loading control, and the bands were analyzed using ImageJ software.

Target proteinDilutionArg-1 (WL02825)

BAX (bs0127R)1:2,000

1:1,500Bcl-2(bs-0038R)

CD206 (18704-1-AP)

CD86 (13395-1-AP)1:1,000

1:2,000

1:2,000Cleaved Caspase-3 (WL01992)1:1,000Il-10 (WL03088)1:2,000IL-1β (WL02257)

IL-4 (WL05916)

IL-6 (WL02841)

iNOS (WL0992a)1:1,000

1:3,000

1:3,000

1:1,500JAK2 (WL02188)

P-JAK2(WL02997)1:1,000

1:1,000P-STAT3 (WL06214)1:1,000STAT3 (WL01836)1:1,000TGF-β1(26155-1-AP)1:3,000TNF-α (17590-1-AP)1:1,500β-actin (20536-1-AP)1:5,000HRP-conjugated Goat Anti-Rabbit IgG (SA00001-2)1:10,000

Information of primary and secondary antibodies for western blot.

2.10 Immunofluorescence analysis

At 21 days post-spinal cord injury, rats were anesthetized and perfused with PBS followed by 4% paraformaldehyde (PFA) for fixation. The spinal cord segments were then dehydrated using a sucrose gradient and embedded for sectioning. Routine frozen sections were prepared. The sections were washed 3 times with 1 × PBS for 5 min each, followed by fixation with 4% PFA at 4°C for 20 min, and two additional washes with PBS. For cell samples, cells were seeded onto coverslips and allowed to adhere. After sufficient attachment, the coverslips were washed twice with 1 × PBS, followed by fixation with 4% PFA at 4°C for 20 min and two additional PBS washes. Primary antibodies were added and incubated overnight at 4°C, followed by gentle washing with PBS. The coverslips were then incubated with fluorescence-labeled secondary antibodies at room temperature for 120 min, washed with PBS, and stained with DAPI for 20 min. The specific dilution ratios of the antibodies used in the experiments are detailed in Table 2. After rinsing twice with PBS, the coverslips were mounted and observed under a laser confocal microscope. Fluorescence intensity was analyzed using ImageJ software.

Target proteinDilutioniNOS (MA5-17139)1:250Dapi (10 μg/mL) (D3571)1:500CD206 (18704-1-AP)1:100β-III-tubulin (ab18207)1:200Iba-1 (ab153696)1:200NeuN (ab177487)1:250Dapi (10 μg/mL)1:500Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) (ab150077)1:500Goat Anti-Mouse IgG H&L (Alexa Fluor 594) (ab150116)1:500

Information of primary and secondary antibodies for Immunofluorescence.

2.11 ELISA analysis

Collect the cell culture supernatant and centrifuge (10 min at 1,000 × g) to remove cell debris, and the levels of inflammatory cytokines (TNF-α, IL-1β, IL-6, TGF-β1, IL-4, and IL-10) were quantified according to the ELISA kit instructions, with absorbance measured at 450 nm using a microplate reader.

2.12 Behavioral tests

Basso-Beattie-Bresnahan (BBB) scale: Hindlimb motor function was assessed using the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale at specific time points following spinal cord injury, as well as preoperatively and weekly up to nine weeks post-surgery, excluding intervention days. Evaluations were conducted in a 100 cm × 100 cm open-field arena, where rats were allowed to move freely for 4–5 min. The BBB scale ranges from 0 (no observable hindlimb movement) to 21 (normal locomotor function). Two trained and blinded observers independently scored each rat based on hindlimb joint movement, weight support, coordination, trunk stability, toe clearance, and tail position.

Grid walking test: The grid-walk test was used to assess hindlimb motor function in rats. Prior to the experiment, rats were allowed to explore the grid for 5 min to acclimate to the environment. A 1.5 m × 1.5 m metal grid with squares measuring 5 cm × 5 cm was used for the test. The rats were placed on the grid and allowed to walk freely. The experiment was conducted by two blinded observers: one observer recorded the total number of steps taken by the rats, while the other recorded the number of hindlimb foot drops. Foot drops were defined as instances when the rat’s hind paw dropped below the grid during stepping, failing to maintain stable support. A foot fault was recorded when the hind paw completely fell into a square. The total number of foot faults and the total number of steps were recorded to calculate the average foot fault rate. The above data were obtained by two independent investigators who were blinded to the treatment assignments.

2.13 Motor evoked potentials

At 21 days post-spinal cord injury, rats were anesthetized, and recording electrodes were inserted into different regions of the ipsilateral gastrocnemius muscle, with a ground electrode placed subcutaneously at the neck. A stimulating electrode was positioned 5 mm rostral to the injury site to evoke motor-evoked potentials, and both amplitude and latency were recorded. Electrical stimulation was delivered at 200 mV and 1 Hz. MEPs were monitored and recorded for each group using electrophysiological acquisition software.

2.14 Statistical analysis

Data analysis was performed using SPSS 22.0, and statistical graphs were generated using GraphPad Prism 8.0, with all data presented as mean ± standard error of the mean (SEM) and assessed for normality and homogeneity of variance before statistical testing. For comparisons among multiple groups, one-way ANOVA followed by Tukey’s post-hoc test was used. For comparisons between two groups, Student’s t-test was applied. All tests were performed on data that met the assumptions of the corresponding statistical methods.

3 Results3.1 BCTS characterization

The XRD pattern of BCTS is shown, where the peaks correspond to the BaTiO3 perovskite structure, with no additional peaks observed, indicating the absence of any secondary phases in the ceramic. Additionally, the inset in the figure shows a distinct peak splitting near 2θ = 45° (Figure 1A), indicating the characteristic of multiphase coexistence. The presence of multiple phases in piezoelectric ceramics is beneficial for the deflection of dipoles, thereby enhancing piezoelectric properties (Zhu et al., 2013). Furthermore, the measured d33 value of BCTS is 450 ± 12 pC/N (Figure 1B). The microstructure of BCTS (Figures 1C,D) reveals the tightly packed grain arrangement of the piezoelectric ceramic. Elemental mapping images (Figures 1E–J) demonstrate uniform distribution of O, Ca, Ti, Sn, and Ba, indicating that the doped Ca2+ and Sn4+ have successfully integrated into the BaTiO3 lattice. Figure 1K show that at all measured frequencies, the dielectric constant initially increases with temperature, reaches a maximum value around 81.7°C (corresponding to the Curie temperature, Tc) and then decreases. At 37°C (physiological temperature), the dielectric constant ranges from 3426 to 3897, exhibiting a favorable dielectric response at body temperature. Studies have shown that piezoelectric materials with good dielectric properties can enhance the electrical coupling effect at the cell–material interface and promote the functional expression of electroactive cells (Jacob et al., 2018). Furthermore, the Curie temperature (81.7°C) of this material is significantly higher than physiological temperature, ensuring its structural stability and electrical performance retention under in vivo application conditions. Figure 1L show that the remanent polarization (Pr) was measured to be 4.99 μC/cm2, confirming the presence of switchable polarization and retained charge storage capability in the material. This retained polarization indicates that BCTS piezoelectric ceramics are capable of maintaining surface charge, which may locally modulate the electrical microenvironment at the cell–material interface. Such charge accumulation could potentially influence the activity of membrane receptors on neuronal cells (Thrivikraman et al., 2018), thereby affecting intracellular signaling pathways and subsequent cellular behaviors

BCTS characterization. (A) XRD spectrum of BCTS. (B) The d33 constant of BCTS. (C,D) SEM images showing the appearance of BCTS at different magnifications. Scale Bar = 10 and 4 μm. (E–J) Elemental mapping EDS spectra for O, Ca, Ti, Sn, and Ba. Scale Bar = 100 μm. (K) Temperature-dependent dielectric constant (εr) of BCTS ceramics measured over a temperature range of 20–150°C at frequencies of 100 Hz, 1 kHz, 10 kHz, 100 kHz, and 1 MHz. (L) Ferroelectric hysteresis loop of the BCTS ceramics, which exhibits typical characteristics of a relaxor ferroelectric material.

3.2 BCTS displays excellent biocompatibility

The hydrophilic property of a material plays a vital role in maintaining cell viability after implantation, as it enhances cell-substrate interactions and promotes cellular metabolism. The wettability results of BCTS (Figures 2A–C) indicate that the material exhibits excellent hydrophilicity. The water contact angle (WCA) of unpoled BCTS was measured to be 81.14 ± 0.8°, while the WCA values for the anode and cathode surfaces of BCTS were 72.42 ± 2.5° and 68.82 ± 2.3°, respectively (Figure 2D). Based on these results, the cathode surfaces of BCTS were selected for subsequent cell seeding. Furthermore, the effects of BCTS on BV2 cell viability were evaluated using CCK8 and LDH assays. The results (Figures 2E,F) demonstrated that BCTS did not exert any adverse effects on BV2 cell viability or LDH release, indicating that BCTS possesses excellent biocompatibility.

BCTS displays excellent biocompatibility. (A–D) Representative water contact angle results for BCTS. Data are expressed as mean ± SEM, n = 3. (E) Statistical analysis of BV2 cell viability after BCTS intervention. Data are expressed as mean ± SEM, n = 5. (F) Statistical analysis of LDH release from BV2 cells following BCTS intervention. Data are expressed as mean ± SEM, n = 5 vs. Unpoled BCTS group, aP < 0.05. vs. Anode surface of BCTS group, bP<0.05. ns, no significance.

3.3 BCTS promoted LPS-induced polarization of BV2 microglial cells toward the anti-inflammatory M2 phenotype

To explore the impact of BCTS on the polarization of BV2 microglial cells (Figure 3I), we utilized IF staining, Western Blot to assess the expression of M1 and M2 markers in BV2 cells. IF staining results revealed that LPS treatment significantly increased the iNOS expression in BV2 cells, indicative of M1 polarization. However, BCTS intervention markedly reduced the iNOS expression compared to the LPS group (P = 0.0447). In contrast, BCTS intervention markedly increased the Arg-1 expression compared to the LPS group (P = 0.0434) (Figures 3A–C). The trend observed in Western Blot is consistent with the IF staining. Specifically, after BCTS intervention, the expression of iNOS (P = 0.0425) and CD86 (P = 0.0222) (Figures 3D–F) was lower than that in the LPS group, while the expression of Arg-1 (P = 0.0371) and CD206 (P = 0.0145) was higher than in the LPS group (Figures 3G,H). The schematic model is presented in Figure 3I. Collectively, these findings suggest that BCTS effectively inhibited the pro-inflammatory M1 polarization while promoting the anti-inflammatory M2 polarization of BV2 microglial cells.

BCTS promoted M2 polarization of LPS-induced BV2 microglia. (A–C) the immunofluorescence results and statistical analysis of the M1 marker iNOS and M2 marker CD206 in microglial cells. (D–F) Western blot results and statistical analysis of iNOS and CD86 expression. (G,H) Western blot results and statistical analysis of Arg-1 and CD206 expression. (I) Schematic of the experimental procedure: LPS-injured BV2 cells were treated with ultrasound-driven BCTS, and the resulting conditioned medium was used to culture LPS-injured PC12 cells. Data are expressed as mean ± SEM, n = 5. Scale bar = 20 μm vs. Con group, aP < 0.05, ns, no significance vs. LPS group, bP < 0.05.

3.4 BCTS attenuates the inflammatory response in the LPS-induced BV2 injury model

To investigate the effect of BCTS on inflammatory factors following microglial polarization, ELISA was performed to measure the levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, as well as anti-inflammatory cytokines TGF-β1, IL-4, and IL-10 in the supernatant of BV2 cells. The results showed that, compared to the LPS group, the concentrations of pro-inflammatory cytokines TNF-α (P = 0.0481), IL-1β (P = 0.0112), and IL-6 (P = 0.038) were reduced in the BCTS group (Figures 4A–C), while the concentrations of anti-inflammatory cytokines TGF-β1 (P = 0.0418), IL-4 (P = 0.0311), and IL-10 (P = 0.0101) were increased (Figures 4D–F). These findings suggest that BCTS suppresses the pro-inflammatory response and enhances the anti-inflammatory response, thereby modulating the inflammatory response following microglial polarization.

BCTS reduced inflammation in the LPS-induced BV2 injury model. (A–C) Statistical analysis of the expression of pro-inflammatory cytokines TNF-α, IL-1β and IL-6. (D–F) Statistical analysis of the expression of anti-inflammatory cytokines TGF-β1, IL-4 and IL-10. Data are expressed as mean ± SEM, n = 5 vs. Con group,aP < 0.05, bP < 0.01 vs. LPS group, cP < 0.05.

3.5 BCTS-CM alleviates PC12 cell injury by inhibiting the IL-6/JAK2/STAT3 signaling pathway

To investigate whether polarized BV2 cells could exert neuroprotective effects by inhibiting the IL-6/JAK2/STAT3 signaling pathway, the conditioned medium from BCTS-treated BV2 cells (BCTS-CM) was collected and used to culture PC12 cells subjected to LPS-induced injury. The expression of proteins related to the IL-6/JAK2/STAT3 signaling pathway were analyzed by Western blotting (Figure 5A). The results showed that, compared with the LPS group, the expression of P-JAK2 and P-STAT3 were decreased in the BCTS-CM group. Furthermore, the addition of IL-6 to the BCTS-CM group increased the expression of P-JAK2 (P = 0.0187) and P-STAT3 (P = 0.0215), while treatment with the JAK2-specific inhibitor AG490 further reduced their expression (P-JAK2, P = 0.012; P-STAT3, P = 0.0353) (Figures 5B,C). These findings suggest that BCTS-CM attenuates the activation of the IL-6/JAK2/STAT3 signaling pathway, thereby exerting neuroprotective effects.