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. 2025 Oct 17;30:946–955. doi: 10.1016/j.reth.2025.09.004

Sevoflurane suppresses LPS-induced microglia injury by regulating USP11-mediated UHRF1 deubiquitination

Dongzhi Liu 1,⁎, Chengliang Sun 1, Xiuli Zhang 1, Shunheng Gao 1
PMCID: PMC12555821  PMID: 41159065

Abstract

Background

Spinal cord injury (SCI) is a devastating neurological and pathological condition that leads to severe motor, sensory, and autonomic dysfunctions. The neuroprotective effect of Sevoflurane (Sevo) in the rat model of SCI has been reported. However, the mechanism of Sevo is still elusive.

Methods

24 rats were divided into four groups: sham group, sham + Sevo group, SCI group, and SCI + Sevo group. On days 0, 14, 28, and 42 post-SCI, functional recovery was evaluated using the BBB test. The histological changes in the spinal cord were observed by HE staining. Western blotting, ELISA, and corresponding kits were used to detect the effects of Sevo on apoptosis, inflammatory factors, and oxidative stress. Human microglia HMC3 were induced by lipopolysaccharide (LPS) to mimic the in vitro environment of SCI. Ubiquitin-specific peptidase 11 (USP11) and ubiquitin-like, containing PHD and RING finger domains 1 (UHRF1), B-cell lymphoma-2 (BCL-2), and Bcl-2 related X protein (BAX) protein levels were determined using Western blot. Cell apoptosis, Interleukin-1β (IL-1β), and IL-6 levels were assessed using flow cytometry and ELISA. malondialdehyde (MDA), glutathione (GSH), and Reactive oxygen species (ROS) products were examined using special assay kits. After Ubibrowser prediction, GO enrichment, and protein-protein interaction (PPI) networks, the interaction between USP11 and UHRF1 was verified using Co-immunoprecipitation (CoIP) assay.

Results

Sevo treatment improved spinal cord functional recovery in rats, as evidenced by enhanced BBB locomotor rating scale and neuron death in vivo. Meanwhile, Sevo also reduced inflammation and oxidative damage in rats following SCI. Sevo exposure decreased USP11 and UHRF1 expression in LPS-treated HMC3 cells. Sevo repressed LPS-triggered HMC3 cell apoptosis, inflammatory response, and oxidative stress promotion by regulating USP11. At the molecular level, USP11 interacted with UHRF1 and maintained its stabilization by removing ubiquitin.

Conclusion

Sevo could protect LPS-induced HMC3 cell apoptosis, inflammation, and oxidative stress by regulating the USP11/UHRF1 axis, which might provide a novel therapeutic mechanism for Sevo in SCI.

Keywords: Spinal cord injury, Sevoflurane, Microglia, USP11, UHRF1, And inflammation

1. Introduction

As a destructive neurological and pathological state arising from direct or indirect spinal cord damage, Spinal cord injury (SCI) causes major motor, sensory, and autonomic dysfunctions and poses a significant global health concern [1,2]. At present, the pathophysiology of SCI involves primary and secondary injury cascades. The former is caused by mechanical compression, while the latter occurs after the primary damage and implicates a complex series of molecular events, such as oxidative stress, immune and inflammatory response, apoptotic pathway, ischemia, and locomotor dysfunctions [[3], [4], [5]]. As time passes, the lesion remodels and regeneration is severely inhibited by the development of an astroglial-fibrous scar surrounding coalesced cystic cavities, mainly comprising microglia, astrocytes, and NG2+-glia [6]. Of note, glial cells are a herd of cells that actively partake in nerve repair and regeneration after SCI, containing microglia, astrocytes, and their progenitors [7]. It has been reported that microglia exert a fundamental role in proliferation, differentiation, and synaptic hemi-channel growth in neurons and help in tissue repair and breakdown in the central nervous system (CNS) and SCI recovery [8,9]. As the resident immune cells of CNS, microglia respond first to spinal nerve injury signals by initiating widespread inflammatory cascades and release, such inflammatory factors as IL-6, IL-1β, and TNF-α [10]. Meanwhile, inflammation could enhance the production of ROS, thereby promoting oxidative stress [11]. At present, there are no effective drugs or therapeutics against SCI for successfully initiating functional recovery [12]. Therefore, understanding and identifying the specific mechanisms responsible for secondary injury is critical to exploring novel drugs to mitigate secondary injury.

Numerous studies have suggested that Sevoflurane (Sevo) is a drug widely used for volatile anesthetic agents during surgeries and the management of neuropathic pain [13,14]. Compared with currently available agents, Sevo is an excellent agent for inhalation inductions in patients of all ages due to the lower blood-gas solubility, safe potency, high efficiency, and rapid [15]. Interestingly, increasing evidence is pointing towards the neuroprotective properties of Sevo by directly acting on neurons or intra-neural glial cells [16,17]. For example, Sevo preconditioning could protect experimental ischemic stroke by promoting anti-inflammatory microglia phenotype polarization via the GSK-3β/Nrf2 pathway [18]. Furthermore, a recent study has indicated that preconditioning with 2.4 % Sevo could ameliorate spinal cord ischemia/reperfusion injury in rats by regulating the expression of miRNAs [19]. However, whether Sevo could exhibit protective effects in SCI is still unclear.

Recently, mounting evidence has consistently indicated that the maintenance of protein homeostasis is a central player in proper cell function [20]. Furthermore, an imbalance in proteostasis often leads to misfolding and accumulation of abnormal proteins, which is strongly correlated with neuroprotection after SCI [21]. Currently, the ubiquitin-proteasome system has been considered the major mechanism responsible for maintaining protein homeostasis [22,23]. Ubiquitination is a protein modification process that is closely associated with multiple tumor pathways [24]. Deubiquitination is a pivotal post-translational modification in the protein reaction of the human body. As a critical regulator of deubiquitination, Deubiquitinating enzyme (DUB) could remove ubiquitin moieties from diverse ubiquitinated substrates and maintain protein stability [25]. Notably, ubiquitin-specific peptidases (USPs) constitute the largest DUB family and present an important modulatory role in SCI by the mediation of deubiquitination. For example, USP18 could bind, deubiquitinate, and stabilize SOX9 protein, thereby positively regulating reactive astrogliosis [26]. Beyond that, USP7 could remove the ubiquitination from its substrate NRF1 to maintain NRF1 protein stability, thus alleviating neuronal inflammation and apoptosis in SCI [27]. Of interest, USP11 is a DUB that is highly expressed in the human brain and could control cortical neurogenesis and neuronal migration by stabilizing SOX11 [28]. Moreover, a previous study has suggested that USP11 could accelerate autophagy-dependent ferroptosis after spinal cord ischemia-reperfusion injury through deubiquitinating and stabilizing Beclin 1 [29]. Nevertheless, whether USP11 can mediate the neuroprotective effects of Sevo in SCI is unknown. Herein, we found that USP11 expression was reduced in LPS-stimulated microglial cells (HMC3) after Sevo treatment. Furthermore, ubibrowser and PPI network analysis predicted that USP11 may be a DUB regulating ubiquitin-like, containing PHD and RING finger domains 1 (UHRF1). Therefore, we inferred that Sevo could affect LPS-induced microglia injury by regulating the USP11/UHRF1 axis.

2. Materials and methods

2.1. SCI model

Under standard conditions at temperature of 25 °C, relative air humidity of 50 %, and a programmed 12 h light/12 h dark cycle, Sprague Dawley male rats (weight, 200–250 g; age, 9–11 weeks, Slaike Jingda Laboratory, Hunan, China) were randomly assigned into four groups (n = 6 per group): sham group (rats underwent surgery with ligation) sham + Sevo group (rats underwent surgery with ligation and were then given 20 mg/kg/day), SCI group (rats were given normal saline at 0.2 mL/d through nasal feeding after modeling), and SCI + Sevo group (rats were given 20 mg/kg/day through nasal feeding after modeling).

All rats were provided with normal free access to sterilized standard food and water before the operation. Process of modeling: rats were intraperitoneally injected with 50 mg/kg pentobarbital (2.5 mL/kg, Sigma-Aldrich, St. Louis, MO, USA) for about 30 min. Subsequently, the limbs of the rats were first bound and fixed on the operating table. After depilation, the skin at the surgical site was routinely disinfected using 10 % iodophor and 75 % alcohol, and sterile operation sheets were laid. In a sterile environment, 2 cm midline incision was made in the midline of the skin and subcutaneous tissue along the eighth thoracic vertebral segment (T8). The spinous processes and vertebral plates of the T8 vertebral segment were removed (laminectomy). After that, a 10 g rod was thrown over the spinal cord from a distance of 5 cm, and the rod was allowed to remain at the site of the lesion for 3 min. When the spinal cord injury is completed, the skin, muscle, and other tissues of rats are gradually sutured, followed by intraperitoneal injection with saline. For the sham group, only T8 laminectomy was performed, and the corresponding site was marked in the same way and sutured. All mice were raised in single cages after the operation and were given 200,000 U of penicillin intramuscularly for three days. Meanwhile, manual urination was conducted twice a day until urinary function was restored. Animal experiments were approved by the Animal Ethics Committee of the First People's Hospital of Lianyungang.

For behavioral analysis, the basso-beattie-bresnahan (BBB) score was performed to evaluate the spontaneous recovery of locomotor function after SCI. According to the BBB rating scale [30], these rats were used for this test on days 0, 7, 14, 21, and 28 after spinal injury. The rats were free to walk around, and the movements of the hind limbs were observed, scored, and averaged.

For HE staining, all rats were sacrificed after day 28 of the spinal cord injury model. Then, the collected spinal cord tissue of each group was fixed with 4 % paraformaldehyde for 48 h, followed by embedding in paraffin. After consecutive slices were made from the center of the injured spinal cord with a slice thickness of 5 μm, these sections were stained with hematoxylin and eosin (Solarbio, Beijing, China) according to the standard protocol, followed by observation using a light microscope (Olympus, Tokyo, Japan). Finally, the spinal cord injury area was quantified using Image J.

2.2. Cell culture and treatment

In this study, human microglial clone 3 cell line HMC3 (CL-0620; Procell, Wuhan, China) was cultured in special medium (CM-0620; Procell) at 37 °C with 5 % CO2. To imitate the process of SCI cell model injury in vitro, transfected cells were added with 5 μg/mL LPS (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. Then, the culture plates were put into an airtight container with a gas mixture, whose inlets were linked to an outlet of an anesthesia gas detector at a concentration of 3.3 %. After that, the container was placed in a 37 °C incubator for 1h, and the cells were put into a 37 °C 5 % CO2 incubator and eluted for 10 min.

2.3. Cell transfection

For USP11 knockdown, HMC3 cells were transfected with either 20 nM of siRNA against USP11 (si-USP11) and non-targeting siRNA (si-con, a negative control, GenePharma, Shanghai, China). For the overexpression system, cDNA sequences of USP11 and UHRF1 were respectively amplified and introduced into the pcDNA vector (GenePharma), termed as pcDNA-USP11/UHRF1 (oe-USP11/UHRF1). After that, 50 ng of plasmids were transfected into HMC3 cells. In this assay, Lipofectamine 3000 reagent (Invitrogen, Paisley, Scotland, UK) was used for each cell transfection. At last, these harvested cells were treated with 5 μg/mL LPS and 3.3 % Sevo.

2.4. Western blot assay

Briefly, total proteins were extracted from spinal cord tissue and HMC3 cells using RIPA buffer (Keygen, Nanjing, China), followed by quantification with BCA method. After mixing with loading buffer and incubated at 100 °C for 5 min, 40 μg protein extracts were subjected to 10 % SDS-PAGE gel, transferred to PVDF membrane, and 5 % fat-free milk sealing, followed by reacting with corresponding primary antibodies at 4 °C overnight: USP11 (sc-365528, 1:1000, Santa Cruz Biotech, Delaware Avenue, CA, USA), UHRF1 (sc-136264, 1:1000, Santa Cruz Biotech), BCL2 (ab194583, 1:500, Abcam, Cambridge, MA, USA), BAX (ab32503, 1:1000, Abcam), and β-actin (ab7817, 1:1000, Abcam). After incubation with secondary antibody at 37 °C for 2 h, protein bands were visualized using ECL (Solarbio). Three biological replicates were performed for each Western blot.

2.5. Cell apoptosis

In short, 1 × 106 HMC3 cells were trypsinized and washed, followed by dual staining with 5 μL Annexin V-FITC and 5 μL PI (BD Biosciences, Heidelberg, Germany) in binding buffer. 15 min later in the dark, apoptotic cells were analyzed by flow cytometry.

2.6. Enzyme-linked immunosorbent assay (ELISA)

Briefly, the inflammatory cytokines IL-1β and IL-6 in spinal cord tissue or HMC3 cell supernatant of every group were assessed using commercial ELISA kits (PI303, PI328, PI330, PI305, Beyotime). Furthermore, the levels of Malondialdehyde (MDA) and Glutathione (GSH) in spinal cord tissue or HMC3 cells were measured using MDA Assay Kit (88-3909-22, eBioscience, San Diego, CA, USA) and GSH Assay Kit (88-50600-22, eBioscience).

2.7. Reactive oxygen species (ROS) detection

The levels of ROS in the spinal cord and HMC3 cells of each group were determined using the ROS assay kit (CA1410, Solarbio). Concisely, the spinal cord was added to the pancreatic enzyme solution for 2 min, followed by filtration of the homogenate through a 200-mesh sieve to produce the cell suspension. After that, the collected tissue cell suspensions and HMC3 cell suspension were incubated with 10 μM 2′,7′-dichlorofluorescin diacetate (DCFH-DA), followed by incubation for 30 min in the dark. After completely covering the probe with the cells, the samples of each group were imaged under a microscope (Olympus, Tokyo, Japan) and analyzed using Image J.

2.8. Gene ontology (GO) enrichment analysis

GO pathway enrichment analysis explored the possible function of the first 50 substrates of USP11. Functional enrichment analysis was carried out using the R package clusterProfiler, and the enrichment with statistical significance (P < 0.05) was obtained.

2.9. Construction of PPI networks and analysis of pivotal genes

In brief, these key genes in the inflammatory response, neuronal apoptosis-related signaling pathway, and ubiquitination-related signaling pathway from GO enrichment analysis were used to construct a protein-protein interaction (PPI) network. Subsequently, Cytoscape v3.8.0. CytoHubba and MCODE plug-ins were applied to screen the Hub genes and modules in the target genes. After that, the PPI network was topologized to identify pivotal genes using the Maximum Clip Centrality (MCC) algorithm in the Cytohubba plugin.

2.10. Co-immunoprecipitation assay (Co-IP)

According to Ubibrowser database prediction, the interaction between USP11 and UHRF1 was further verified using Co-IP assay in HMC3 cells with Pierce Crosslink Magnetic CoIP Kit (Invitrogen). Briefly, HMC3 cells were suspended in IP Lysis buffer. Then, the whole cell lysates were collected and centrifuged, and 10 % cleared lysates served as input control. Meanwhile, the remaining was incubated with anti-USP11, anti-UHRF1, and anti-IgG, followed by mixing with protein-A/G magnetic beads (Millipore). After washing and elution, samples were determined using Western blot.

2.11. Ubiquitylation assay

In short, si-con or si-USP11-transfected HMC3 cells were washed with PBS and lysed for immunoprecipitation assay using anti-UHRF1 antibody. Then, the UHRF1 ubiquitination was detected by Western blot assay using anti-Ub.

2.12. Statistical analysis

Data in this research were analyzed using GraphPad Prism7 and exhibited in the form of mean ± standard deviation (SD). Statistical significance was indicated by P < 0.05. Student's t-test or one-way ANOVA with Tukey's tests was applied for group comparison.

3. Results

3.1. Sevo treatment relieved the damage of SCI rats

To investigate the functional role of Sevo treatment in vivo, SCI rat models were established. At first, the evaluation of locomotor function verified that the BBB scores of the SCI group were lower than those of the sham group throughout the experiment, while BBB scores of rats treated with 2.7 % Sevo were obviously increased compared with those of the SCI rats (Fig. 1A), suggesting that Sevo improved locomotor function recovery after SCI. To further assess tissue damage and recovery, rat spinal cord tissue sections were assessed using HE staining. As shown in Fig. 1B, the sham rats had more neurons and regular cell arrangement, whereas the SCI group was atrophied and irregularly shaped. Meanwhile, compared with the SCI group, the SCI + Sevo group could clearly alleviate these pathological changes. These data suggested that Sevo had the ability to protect neurons in SCI rats. It has been reported that Bcl-2 is an anti-apoptotic protein, whose increased expression suggests inhibition of apoptosis, while BAX is a pro-apoptotic protein, whose increased expression suggests enhanced apoptosis. Then, Western blot analysis presented an increase in apoptosis in the SCI group, while Sevo stimulation could partly overturn these effects, as described by reduced BAX and enhanced BCL2 at the lesion site (Fig. 1C). In terms of neuro-inflammatory response, secretions of pro-inflammatory cytokines IL-1β and IL-6 at the lesion site after SCI were highly induced, which were partly abolished after Sevo exposure (Fig. 1D and E). MDA is the end product of lipid peroxidation, and its elevated level reflects the aggravation of oxidative damage, whereas GSH is an important antioxidant substance, and its elevated level suggests the enhancement of cellular antioxidant capacity. Herein, increased oxidative stress in the SCI group was also remarkably attenuated by Sevo treatment, accompanied by lower MDA and ROS levels (Fig. 1F and H), and higher GSH level (Fig. 1G). Together, these data suggested the neuro-protective effect of Sevo on SCI rats in vivo.

Fig. 1.

Fig. 1

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Effects of Sevo treatment on the damage of SCI rats. The mice were divided into four groups (6 rats in each group): 1) the sham group; 2) the sham group + Sevo; 3) the SCI group; 4) the SCI + Sevo group. (A) BBB score was determined in different groups. (B) Representative images of hematoxylin-eosin (HE) in each group, % of preserved tissue” = (area of undamaged tissue/total spinal cord cross-sectional area) × 100 %. (C) BCL2 and BAX protein levels were measured using Western blot in each group. (D and E) The secretions of IL-1β and IL-6 were analyzed using ELISA in different groups. (F–H) Corresponding kits assessed MDA, GSH, and ROS levels in different groups. ∗∗P < 0.01, ∗∗∗∗P < 0.0001.

3.2. Downregulation of USP11 attenuated LPS-triggered HMC3 cell damage

Furthermore, it has been reported that USP11 is associated with spinal cord ischemia-reperfusion injury. Therefore, we further explored the effects of USP11 in LPS-induced HMC3 microglia injury. As shown in Fig. 2A, the USP11 protein level was remarkably upregulated in LPS-treated HMC3 cells, which was partially overturned after si-USP11 transfection. After that, Western blot assay displayed that LPS treatment led to an apparent increase in BCL2 and a substantial decrease in BAX level in HMC3 cells, while these effects were abrogated by USP11 deficiency (Fig. 2B). Consistently, LPS-mediated HMC3 cell apoptosis promotion was markedly ameliorated by USP11 downregulation (Fig. 2C). Besides, USP11 silencing could strikingly mitigate LPS-caused inflammation and oxidative stress promotion in HMC3 cells, as evidenced by reduced IL-1β and IL-6 levels (Fig. 2D and E), lower MDA and ROS levels (Fig. 2F and H), and higher GSH level (Fig. 2G). Overall, these data indicated that USP11 knockdown protected HMC3 cells against LPS-triggered damage by inhibiting apoptosis, inflammation, and oxidative stress.

Fig. 2.

Fig. 2

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Effects of USP11 knockdown on LPS-induced HMC3 injury. HMC3 cells were treated with 0 μg/mL LPS (con), 5 μg/mL LPS, 5 μg/mL LPS + si-con, or 5 μg/mL LPS + si-USP11. (A) USP11 protein level was detected using Western blot. (B) BCL2 and BAX protein levels were determined using Western blot. (C) Cell apoptosis rate was assessed using flow cytometry. (D and E) The concentrations of IL-1β and IL-6 were detected using ELISA kits. (F–H) MDA, GSH, and ROS levels were examined to evaluate oxidative stress. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

3.3. Sevo exposure prevented LPS-induced microglia apoptosis, inflammation, and oxidative stress by regulating USP11

Next, to further explore whether the influence of Sevo in LPS-induced microglia damage was correlated with USP11, in vitro gain-of-function analyses were performed in HMC3 cells. As displayed in Fig. 3A, Sevo exposure could evidently decrease USP11 protein level in LPS-treated HMC3 cells, which were partly ameliorated after oe-USP11 transfection. Subsequently, functional analysis exhibited that USP11 upregulation could clearly weaken the promotion of Sevo on cell apoptosis in LPS-treated HMC3 cells, as described by enhanced BCL2 and reduced BAX (Fig. 3B and C). Moreover, LPS-induced inflammatory response was prominently alleviated by Sevo treatment, and this protection was partially abolished by USP11 overexpression, as evidenced by higher IL-1β and IL-6 (Fig. 3D and E). In parallel, the forced expression of USP11 also remarkably abated the repression of Sevo exposure on oxidative stress in LPS-treated HMC3 cells, accompanied by declined MDA and ROS levels (Fig. 3F and H), and elevated GSH level (Fig. 3G). Collectively, these results indicated that Sevo treatment repressed LPS-caused HMC3 cell injury by regulating USP11.

Fig. 3.

Fig. 3

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Sevo/USP11 regulated LPS-triggered HMC3 cell injury. HMC3 cells were treated with 0 μg/mL LPS (con), 5 μg/mL LPS, 5 μg/mL LPS + Sevo, or 5 μg/mL LPS + Sevo + oe-USP11. (A) Western blot assay was used to measure USP11 protein level. (B) Western blot analysis of BCL2 and BAX protein levels. (C) Flow cytometry analysis of cell apoptosis rate. (D and E) The concentrations of IL-1β and IL-6 were assessed using ELISA kits. (F–H) Corresponding kits identified MDA, GSH, and ROS levels in HMC3 cells. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

3.4. USP11 interacted with and stabilized UHRF1

Based on ubibrowser database analysis, many substrates of USP11 were found in this study. Then, the top 50 predicted substrates were assessed using GO enrichment analysis. As shown in Fig. 4A, the predicted substrates were enriched in the regulation of inflammatory response, regulation of neuron apoptotic process, histone ubiquitination, and regulation of neuron apoptotic process. Then, these key genes were selected by the PPI network (Fig. 4B). Among them, UHRF1 was associated with Spinal cord injury. Therefore, UHRF1 was selected for further research. Furthermore, a prominent increase level of ubiquitinated UHRF1 was observed in HMC3 cells with low-expressed USP11 (Fig. 4C). Meanwhile, CoIP assay also confirmed the ability of endogenous USP11 and UHRF1 to bind in HMC3 cells (Fig. 4D). Overall, these results suggested that USP11 could mediate deubiquitination and stabilization of UHRF1 in HMC3 cells.

Fig. 4.

Fig. 4

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USP11 regulated UHRF1 ubiquitination. (A) Database ubibrowser predicted the top 50 substrates of USP11, followed by GO analysis showing the enrichment of these 50 substrate genes. (B) Key genes were selected by combining pivotal genes in the protein-protein interaction (PPI) network with characterized genes identified. (C) Analysis of UHRF1 protein and ubiquitination levels by Western blot in HMC3 cells transfected with si-USP11 or si-con. (D) The binding association between UHRF1 and USP11 in HMC3 cells using Co-IP assay. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

3.5. USP11 inhibition relieved LPS-induced HMC3 cell injury by regulating UHRF1

Given the regulatory role of USP11 on UHRF1 expression in HMC3 cells, we further determined whether the effects of USP11 on LPS-induced HMC3 cell damage were correlated with UHRF1. At first, Western blot analysis exhibited that USP11 silencing could significantly hinder UHRF1 protein levels in LPS-treated HMC3 cells, which were partly overturned after oe-UHRF1 co-transfection (Fig. 5A). After that, USP11 downregulation diminished BAX protein level and enhanced BCL2 level in LPS-treated HMC3 cells, whereas these effects were partly abolished by UHRF1 overexpression (Fig. 5B). Similarly, the results of flow cytometry presented that UHRF1 upregulation could prominently abolish the repression of USP11 deficiency on HMC3 cell apoptosis (Fig. 5C). In addition, LPS-evoked inflammatory response and oxidative stress in HMC3 cells were markedly blocked by USP11 knockdown, while these influences were effectively reversed through UHRF1 overexpression, as depicted by enhanced IL-1β and IL-6 secretions (Fig. 5D and E), increased MDA and ROS levels (Fig. 5F and H), and reduced GSH level (Fig. 5G). All these results demonstrated that USP11 downregulation could ameliorate LPS-caused HMC3 cell damage by interacting with UHRF1.

Fig. 5.

Fig. 5

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USP11/UHRF1 affected LPS-induced HMC3 cell injury. HMC3 cells were treated with 0 μg/mL LPS (con), 5 μg/mL LPS, 5 μg/mL LPS + si-USP11, or 5 μg/mL LPS + si-USP11+UHRF1. (A) UHRF1 protein level was examined using Western blot. (B) BCL2 and BAX protein levels were detected using Western blot. (C) Cell apoptosis rate was assessed using flow cytometry. (D and E) IL-1β and IL-6 levels were determined using ELISA kits. (F–H) MDA, GSH, and ROS levels were measured using corresponding kits. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

3.6. Validation of Sevo/USP11/UHRF1 regulatory axis in LPS-treated HMC3 cells

Based on the above finding, we speculated that Sevo could exert its neuro-protective role partially by the USP11/UHRF1 regulation pathway. To confirm the assumption, we further investigated whether Sevo could affect the expression of UHRF1 by USP11. Western blot results demonstrated that Sevo exposure could strikingly restrain UHRF1 protein level in LPS-treated HMC3 cells, which was partly reversed by USP11 upregulation (Fig. 6A and B). In addition, Western blot results displayed that Sevo exposure could obviously decrease USP11 and UHRF1 protein levels in the SCI rat group (Fig. S1). Taken together, these results suggested that Sevo treatment regulated UHRF1 expression by targeting USP11.

Fig. 6.

Fig. 6

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Sevo repressed UHRF1 expression by modulating USP11 in LPS-treated HMC3 cells. (A and B) UHRF1 protein level was detected in HMC3 cells treated with 0 μg/mL LPS (con), 5 μg/mL LPS, 5 μg/mL LPS + Sevo, or 5 μg/mL LPS + Sevo + oe-USP11 using Western blot. ∗∗P < 0.01, ∗∗∗P < 0.001.

4. Discussion

Nowadays, the clinical application and neuroprotective mechanism of Sevo, an important volatile anesthetic, in some human diseases have received increasing attention due to the rapid recovery time with little emergence agitation [[31], [32], [33]]. For example, it has been reported that Sevo exposure could decrease the inflammation response and neuronal apoptosis in rats and mice with cerebral ischemia/reperfusion [34,35]. Beyond that, Sevo also could ameliorate neuronal deficits by repressing microglia MMP-9 expression after spinal cord ischemia/reperfusion in rats [36]. In addition, numerous literatures have demonstrated the protective effect of Sevo preconditioning in spinal cord ischemia/reperfusion injury and acute traumatic SCI by different mechanisms [19,]. However, whether Sevo treatment exhibits a neuroprotective effect in SCI is far from being addressed. Herein, our data verified that Sevo treatment could improve locomotor function recovery and neuron loss after SCI. Meanwhile, Sevo exposure also could efficiently ameliorate the developing secondary damage after SCI in rats by reducing apoptosis, inflammatory response, and oxidative stress. In total, these findings and evidence make it plausible to indicate that Sevo confers neuro-protection against SCI in rats.

Accumulating evidence has highlighted that the deubiquitinating enzyme USP11 exerts a key role in the modulation of neurodevelopment disorders [28], apoptosis regulation [37], oxidative stress, and inflammation [38]. Furthermore, it has been reported that USP11 upregulation in mice led to poor functional recovery after Spinal cord ischemia-reperfusion injury [29]. To elucidate whether USP11 could partake in the regulation of SCI, human microglia HMC3 were treated with LPS to mimic the in vitro environment of SCI. As a result, we found that USP11 silencing could relieve LPS-induced HMC3 injury through inhibiting apoptosis, inflammation, and oxidative stress. Of note, previous studies have suggested that the interaction between Sevo and USPs could affect cognitive function [39], but the regulatory role of this interaction on SCI remains unknown. In order to further explore whether Sevo mitigated SCI by regulating USP11 expression, we overexpressed USP11 and subjected LPS-treated HMC3 cells to Sevo treatment. Subsequently, our results displayed that Sevo treatment could obviously decrease USP11 protein level in LPS-triggered HMC3 cells. Functional analysis presented that overexpressing USP11 could partly abolish the inhibitory role of Sevo on LPS-caused HMC3 cell apoptosis, inflammation, and oxidative stress. These results confirmed that the protective mechanism of Sevo can be mediated by targeting USP11, providing an experimental and theoretical basis for the clinical anesthesia of SCI.

Regarding the molecular mechanism, DUBs could influence protein function and degradation by removing the ubiquitin of target protein substrates [40,41]. Herein, ubibrowser database, GO enrichment, and PPI network analysis found that UHRF1 was a key substrate of USP11. Then, our results confirmed that USP11 could stabilize UHRF1 protein by preventing its protein degradation. It has been reported that UHRF1 expression was upregulated in SCI [42]. Furthermore, a recent study has indicated that UHRF1 knockdown could improve motor function in mice with SCI [43]. Herein, our data validated that UHRF1 upregulation could partially reverse the repression of USP11 deficiency on LPS-induced HMC3 cell injury. Further, rescue experiments verified that overexpressing USP11 could partly overturn Sevo-mediated UHRF1 expression inhibition in LPS-treated HMC3 cells, supporting that Sevo could exert the protective effect in LPS-induced HMC3 cell damage by regulating the USP11/UHRF1 modulatory mechanism.

5. Conclusion

In summary, our study revealed that Sevo treatment could attenuate LPS-caused HMC3 cell apoptosis, inflammation, and oxidative stress by altering the USP11/UHRF1 (Fig. 7). These results provided an available preclinical basis for researching the application of Sevo for SCI.

Fig. 7.

Fig. 7

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Sevo could relieve LPS-induced microglia apoptosis, inflammation, and oxidative stress by regulating USP11-mediated UHRF1 deubiquitination.

Authors’ contributions

Xiuli Zhang and Shunheng Gao designed and performed the research; Dongzhi Liu analyzed the data; Dongzhi Liu and Chengliang Sun wrote the manuscript. All authors read and approved the final manuscript.

Availability of data and materials

The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.

Animal studies

Animal studies were performed in compliance with the ARRIVE guidelines and the Basel Declaration. All animals received humane care according to the National Institutes of Health (USA) guidelines.

Funding

None.

Declaration of competing interest

The authors declare that they have no conflicts of interest.

Acknowledgement

None.

Footnotes

Peer review under responsibility of the Japanese Society for Regenerative Medicine.

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.reth.2025.09.004.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Fig. S1.

Fig. S1

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Effects of Sevo treatment on UHRF1 and USP11 expression in SCI rats. The mice were divided into four groups (6 rats in each group): 1) the sham group; 2) the sham group + Sevo; 3) the SCI group; 4) the SCI + Sevo group. (A and B) UHRF1 and USP11 protein levels were determined using western blot. ∗∗∗P < 0.001.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.

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