BV2 microglial cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. HT-22 neuronal cells were maintained in high-glucose dulbecco’s modified eagle medium (DMEM) containing 10% FBS. Subculture protocol: After aspirating the spent medium, cells were gently washed twice with 37 °C pre-warmed PBS, followed by digestion with 0.25% Trypsin-EDTA (45 seconds for BV2; 2.5 minutes for HT-22). Upon observation of intercellular gap widening under a microscope, serum-containing medium was added to neutralize trypsin. Cells were collected by centrifugation at 1000 rpm for 5 minutes and subcultured at a 1:3 ratio. Complete medium replacement was performed every 2 days, with cell density maintained between 30% and 80%. BV2 and HT-22 cells were purchased from Jiangsu KeyGENE Technology Co., Ltd. (Nanjing, China). All cell lines were validated by short tandem repeat (STR) profiling and tested negative for mycoplasma.

The Sparc coding DNA sequence (CDS) region sequence was searched through the national center for biotechnology information (NCBI) online website (https://www.ncbi.nlm.nih.gov/), and the CDS region sequence was imported into pcDNA3.1 expression plasmid to construct the Sparc overexpression recombinant plasmid. Sparc overexpression plasmid and Uba52 small interfering RNA (si-Uba52) sequences were synthesized by KeyGEN BioTECH Co., Ltd. (KeyGEN Biotech, Nanjing, China). When the fusion degree of BV2 cells reached about 70%, the cells were divided into 3 groups: control, lipopolysaccharides (LPS), and LPS + Sparc. Cells in the LPS group were treated with 200 ng/mL LPS (Sigma, St. Louis, MO, USA) for 24 hours. LPS + Sparc cells were transfected with Sparc overexpression plasmid by Lipofectamine 3000 (Thermo Fisher, Waltham, MA, USA), cultured for 24 hours and then treated with 200 ng/mL LPS for 24 hours. Cells in the control group were cultured normally for 24 hours.

In addition, the cells were divided into three groups again: LPS, LPS + Sparc, and LPS + Sparc + si-Uba52. Cells in the LPS and LPS + Sparc groups were treated as above. Cells in the LPS + Sparc + si-Uba52 group were co-transfected with the Sparc overexpression plasmid and si-Uba52 by Lipofectamine 3000. After 24 hours of culture, cells were treated with 200 ng/mL LPS for 24 hours.

Lysis conditions: 1 $\times$ 10⁷ cells were collected and lysed in pre-cooled radioimmunoprecipitation assay (RIPA) buffer (containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitors) on ice for 30 minutes, with vortexing for 5 seconds every 10 minutes. After centrifugation at 14,000 $\times$g for 15 minutes, the supernatant was collected, and protein concentration was quantified using the bicinchoninic acid (BCA) assay.

Immunoprecipitation: 1 mg total protein was incubated with Sparc antibody overnight at 4 °C with rotation, followed by 4 hours of incubation with Protein G agarose beads. Beads were washed 5 times with lysis buffer, and bound proteins were eluted using low-pH elution buffer. Target bands excised from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels were trypsin-digested at 37 °C for 16 hours for subsequent mass spectrometry analysis.

The transfected cells and untransfected cells were digested, counted, and prepared into cell a suspension with a concentration of 1 $\times$ 10⁵ cells/mL, and inoculated into a six-well plate. The next day, after the cells were attached, the corresponding medium containing 200 ng/mL LPS was added according to the group setting, and a negative control group was set up. After 24 hours of drug treatment, the cells were digested with 0.25% trypsin (without EDTA) and collected. The cells were washed once with PBS (centrifuged at 1000 rpm for 5 minutes) and the cell concentration was adjusted to 1 $\times$ 10⁶/mL. The cells were evenly suspended with 500 µL of prepared JC-1 working solution and incubated in an incubator at 37 °C with 5% CO₂ for 15–20 minutes. Cells were collected by centrifugation at room temperature (1000 rpm for 5 minutes) and washed twice with 1$\times$ Incubation Buffer. The cells were resuspended by absorbing 500 µL of 1$\times$ Incubation Buffer. Test on the machine.

The transfected cells and untransfected cells were digested, counted, and prepared into a cell suspension with a concentration of 1 $\times$ 10⁵ cells/mL, and inoculated into a six-well plate. The next day, after the cells were attached, the corresponding medium containing 200 ng/mL LPS was added according to the group setting, and a negative control group was set up. After 24 hours of drug treatment, the cells were digested with 0.25% trypsin (without EDTA) and collected. The cells were washed once with PBS (centrifuged at 1000 rpm for 5 minutes) and the cell concentration was adjusted to 1 $\times$ 10⁶/mL. 2^′,7^′-Dichlorofluorescin diacetate (DCFH-DA) was diluted in serum-free medium at 1:1000 to a final concentration of 10 µM. The cells were collected and suspended in the diluted DCFH-DA and incubated in a cell incubator at 37 °C for 20 minutes. The probe was mixed reversely every 3–5 minutes to make full contact with the cells. The cells were washed 3 times with serum-free cell culture medium to fully remove DCFH-DA that did not enter the cells. Flow cytometry was used to detect (Ex = 488 nm; Em = 525 nm).

After centrifugation, the colorless supernatant water was drawn and transferred to another centrifuge tube. After centrifugation, an equal volume of isopropanol and 70% ethanol was added. After centrifugation, RNase-free water was added to dissolve the RNA precipitation. After the concentration and purity of RNA were determined, 0.2 mL polymerase chain reaction (PCR) tubes that had been sterilized and had no nuclease were taken, and RNA (2 µg), 5$\times$ PrimeScript RT Master Mix (Takara Bio Inc., Kyoto, Japan, RR036B) were added in turn, and the products were obtained by centrifugation. The cDNA samples were diluted and TB Green® Premix Ex Taq™ The II (Tli RNaseH Plus) reagent (Takara, Japan, RR820A) contains 2$\times$ Real-time PCR Master Mix, SYBR Green, template, and primer Mix were successively added to 0.1 mL PCR tubes.

Cells were collected, lysates were added, protein samples were extracted, protein concentration was calculated according to the standard curve, 10% SDS-PAGE gel was prepared, electrophoresed, transferred to polyvinylidene fluoride (PVDF) membrane, blocked with 1% bovine serum albumin (BSA) for 1 hour, added primary antibody, incubated at 4 °C overnight, wash with PBS, add secondary antibody and incubate at room temperature for 2 hours. The obtained bands were imaged by ChemiDoc MP Imaging System 3.0.1 version (Bio-Rad, Hercules, CA, USA), and the gray level was analyzed by the Gel-Pro32 software 6.0 version (Media Cybernetics, Inc., Rockville, MD, USA).

The kits (Tumor necrosis factor-$\alpha$ (TNF-$\alpha$) kit: Mlbio, Shanghai, China, YJ002095; Interleukin-6 (IL-6) kit: Mlbio, Shanghai, China, YJ063159; Transforming growth factor-$\beta$ (TGF-$\beta$) kit: Mlbio, Shanghai, China, YJ057830; Interleukin-10 (IL-10) kit: Mlbio, Shanghai, China, YJ037873) were operated according to the instructions of each kit. The original standard substance was diluted in an EP tube, and through the process of adding sample, adding enzyme, incubation, mixing, washing, and color development, the absorbance (OD) value of each well was measured at 450 nm wavelength 15 minutes after adding the termination solution.

Co-culture of BV2 cells and HT-22 cells was performed using Transwell chambers. BV2 cells were seeded in the upper chamber of Transwell, and HT-22 cells were seeded in 24-well plates. The co-culture cells were divided into 4 groups: Control/HT-22, LPS/HT-22, LPS + Sparc/HT-22, LPS + Sparc + si-Uba52/HT-22. BV2 cells in each group were treated as above and co-cultured with HT-22 cells for 24 hours.

24 hours after transfection, BV2 cells were digested, counted, and prepared into a cell suspension with a concentration of 1 $\times$ 10⁴ cells/mL. A total of 200 µL cell suspension was added to each well of the 24-well cell co-culture upper chamber, and HT-22 cells in logarithmic growth phase were digested, counted, and prepared into a cell suspension with a concentration of 1 $\times$ 10⁴ cells/mL. An amount of 800 µL of HT-22 cell suspension was added to each well in the lower chamber. After cell attachment, the co-cultured cells were treated with 200 ng/mL LPS for an additional 24 hours. The lower layer of HT-22 cells was removed, the medium was discarded, and the cells were washed twice with PBS. EdU (medium preparation 50 µM), 500 µL cell fixative (i.e., PBS containing 4% paraformaldehyde), 200 µL 2 mg/mL glycine, 500 µL PBS, 1$\times$ Apollo® staining reaction solution, and 500 µL osmotic agent (0.5% TritonX-100 in PBS) were added sequentially to cells in each well and 500 µL of 1$\times$ Hoechst 33342 reaction solution was prepared prior. After washing with PBS, the plates were sealed, and the pictures were taken under a fluorescence microscope (200$\times$) (Olympus, Tokyo, Japan).

The co-cultured HT-22 cells were collected by 0.25% trypsin (without EDTA) digestion. Cells were washed twice with PBS (centrifuged at 1000 rpm for 5 minutes) to collect 1 $\times$ 10⁶ cells. A total of 500 µL Binding Buffer was added to suspend the cells. After 5 µL Annexin V-APC was added to the mix, 5 µL Propidium Iodide was added to the mix. The reaction time was 5–15 minutes at room temperature in the dark. Cell apoptosis was detected by flow cytometry.

The lower layer of HT-22 cells was removed, the medium was discarded, and the cell samples were allowed to dry naturally. The cells were immersed in 4% paraformaldehyde fixator for 30 min or overnight to improve cell permeability, and the cells were immersed in PBS for 3 minutes $\times$ 3. The cells were covered with 3% H₂O₂–methanol solution and blocked at room temperature (15–25 °C) for 10 minutes. After soaking with PBS three times, 100 µL of ready-to-use goat serum was added and incubated at room temperature for 20 minutes. Then 100 µL of rabbit anti-III-tubulin (Abclonal, Wuhan, China, A17913, 1:50), FITC-labeled goat anti-rabbit IgG-HRP (KeyGEN Biotech, Nanjing, China, KGC6214-0.1, 1:5000), and prepared Hoechst staining solution were added in turn, placed in a dark place at room temperature for 5 minutes, and washed 3 times with PBS. A total of 1 mL PBS was added and the expression was observed under a confocal microscope (Olympus, Tokyo, Japan). Three highly expressed regions were taken and photographed, and stored (600$\times$).

For dual-colour immunofluorescence co-localisation of Uba52 and Sparc, BV2 cells seeded on glass coverslips were fixed with 4% paraformaldehyde for 30 minutes, permeabilised with 0.2% Triton X-100, and blocked with 3% BSA for 30 minutes. Sequential tyramide signal amplification (TSA) labelling was performed on the same slide. First, rabbit anti-Uba52 (Abclonal, Wuhan, China, A20876, 1:500) was applied overnight at 4 °C, followed by goat anti-rabbit IgG-HRP (KeyGEN Biotech, Nanjing, China, KGC6202, 1:5000) and Alexa Fluor 488-TSA. After stringent antibody elution (37 °C, 15–30 minutes in citrate-based buffer), the second cycle was executed with rabbit anti-Sparc (Proteintech, Wuhan, China, 15274-1-AP, 1:1000), identical HRP-secondary and Alexa Fluor 594-TSA. Nuclei were counterstained with 4^′,6-Diamidino-2^′-phenylindole (DAPI). Coverslips were mounted with anti-fade medium and imaged on a confocal laser-scanning microscope (Olympus, Tokyo, Japan).

All experimental data are presented as mean $\pm$ standard deviation. One-way analysis of variance (ANOVA) was used for comparison among multiple groups, and the LSD method was used for post hoc comparison. Statistical software SPSS (ver. 23.0, IBM, Beijing, China), the error line represents the standard deviation of three independent means, *p 0.05; **p 0.01; ***p 0.001.

Mitochondrial dysfunction during neuroinflammation exacerbates inflammatory progression by reprogramming microglial metabolism. In LPS-induced BV2 cells. Sparc overexpression significantly restored mitochondrial membrane potential (Fig. 1A), attenuated ROS production (Fig. 1B), and upregulated mitochondrial oxidative phosphorylation (mt-OXPHOS)-related proteins voltage-dependent anion channel 1 (VDAC1), cytochrome c oxidase subunit 1 (COX1), and ATP synthase $\alpha$ subunit (ATP5A) (Fig. 1C). Besides, LPS induction markedly increased pro-inflammatory cytokines TNF-$\alpha$ and IL-6 while suppressing anti-inflammatory mediators IL-10 and TGF-$\beta$, which were reversed by Sparc overexpression (Fig. 1D).

Fig. 1.

Sparc overexpression suppresses pro-inflammatory responses and regulates mitochondrial homeostasis in lipopolysaccharides (LPS)-induced BV2 cells. (A) JC-1 method for detecting mitochondrial membrane potential. (B) 2^′,7^′-Dichlorofluorescin diacetate (DCFH-DA) probe for detecting reactive oxygen species (ROS) levels. (C) Western blot detection of mitochondrial functional proteins (voltage-dependent anion channel 1 (VDAC1), cytochrome c oxidase subunit 1 (COX1), ATP synthase $\alpha$ subunit (ATP5A)). (D) Enzyme-linked immunosorbent assay (ELISA) detection of cytokine levels (TNF-$\alpha$, IL-6, TGF-$\beta$, IL-10). *p 0.05, **p 0.01, and ***p 0.001. TNF-$\alpha$, tumor necrosis factor-$\alpha$; IL-6, interleukin-6; TGF-$\beta$, transforming growth factor-$\beta$.

Immunoprecipitation combined with mass spectrometry confirmed potential binding between Sparc and Uba52 (Fig. 2A,B). Subsequently, the interaction of the Sparc-Uba52 was confirmed through co-localization of co-immunoprecipitation and immunofluorescence (Fig. 2C,D). LPS stimulation suppressed both Uba52 mRNA and protein levels, whereas Sparc overexpression rescued its expression (Fig. 2E,F).

Fig. 2.

Sparc interacted with Uba52 and promoted Uba52 expression. (A–C) The interaction between Sparc and Uba52 protein was confirmed through co-immunoprecipitation coupled mass spectrometry. Input: 10% total lysate; Ig G: negative control; IP: anti-Sparc immunoprecipitated. (D) Co-localization analysis of the Sparc and Uba52 was performed using immunofluorescence. The white arrow represents the co localization area, where yellow (red and green overlapping) indicates spatial overlap between Sparc and Uba52. Scale bar: 10 µm. (E,F) Changes in expression levels of the Sparc and Uba52 genes and proteins in BV2 cells under the intervention of Sparc overexpression. (G) Uba52 knockdown validation was conducted using RT-qPCR. (H,I) Expression changes of the Sparc and Uba52 genes and proteins after transfection of si-Uba52 in BV2 cells. **p 0.01, ***p 0.001, and ns: p $>$ 0.05.

To knockdown Uba52 in BV2 cells, three si-Uba52 were designed. The verification results of RT-qPCR showed that si-Uba52#02 had the best inhibition rate on the expression of the Uba52 gene (Fig. 2G). Therefore, si-Uba52#02 was selected for the Uba52 knockdown experiment. Uba52 knockdown reduced the Uba52 gene and protein without altering the Sparc gene and protein expressions (Fig. 2F,H,I), establishing Uba52 as a downstream effector of Sparc.

Sparc overexpression reversed LPS-induced mitochondrial depolarization (Fig. 3A) and restored VDAC1, COX1, and ATP5A expressions (Fig. 3C), effects abolished by Uba52 knockdown. Sparc suppressed ROS accumulation (Fig. 3B), whereas Uba52 knockdown exacerbated it. Sparc overexpression inhibited TNF-$\alpha$ and IL-6 levels and enhanced IL-10 and TGF-$\beta$ expression, while Uba52 knockdown neutralized these anti-inflammatory effects (Fig. 3D).

Fig. 3.

Sparc interacts with Uba52 to regulate mitochondrial respiration and inhibit LPS-induced BV2 cell inflammatory responses. (A) Mitochondrial membrane potential measured by JC-1 assay. (B) Intracellular ROS levels detected by DCFH-DA probe. (C) Western blot analysis of mitochondrial respiratory chain proteins (VDAC1, COX1, ATP5A). (D) mRNA levels of inflammatory factors by qPCR. **p 0.01, ***p 0.001.

Transwell co-culture assays demonstrated that pro-inflammatory BV2 supernatants strongly inhibited HT-22 neuronal proliferation (Fig. 4A,B). Sparc overexpression rescued neuronal proliferation (Fig. 4B), whereas si-Uba52 intensified suppression. Apoptosis analysis revealed that Sparc attenuated LPS-induced neuronal apoptosis (Fig. 4C), while Uba52 silencing amplified cell death (Fig. 4C). III-tubulin expression patterns corroborated these findings: Sparc restored neurite outgrowth capacity, whereas si-Uba52 diminished it (Fig. 4D).

Fig. 4.

Sparc interacts with Uba52 to regulate pro-inflammatory microglia to promote neuronal cell axonal outgrowth. (A) Schematic diagram of Transwell co-culture system. Created by Adobe Illustrator 2022. (B) Quantification of HT-22 neuronal proliferation under different treatments. Left panels: Representative images of EdU staining (red, proliferating cells) and Hoechst 33342 (blue, nuclei). Scale bar: 50 µm. (C) Annexin V-APC/Propidium Iodide (PI) dual staining was used to detect apoptosis. (D) Quantification of axonal outgrowth in HT-22 neurons by III-tubulin immunofluorescence. Left panels: Representative confocal images of III-tubulin (green) and nuclei (blue, Hoechst). Scale bar: 10 µm. *p 0.05, **p 0.01, and ***p 0.001.