Introduction
Sepsis-associated encephalopathy (SAE), a common and severe complication in patients with sepsis, is a diffuse brain dysfunction caused by sepsis, mainly manifests as long-term cognitive impairments and psychiatric diseases, and is closely associated with increased morbidity and mortality [
1,
2]. Cognitive deficits are found in about 70% of survivors who recovered from severe systemic infections [
3]. Although the pathogenesis of SAE is not fully elucidated, sepsis-induced neuroinflammation is thought to be an essential contributor to cognitive dysfunction [
4].
Microglia are the central innate immune cells and the primary producers of pro-inflammatory cytokines in the brain. Therefore, they are the focus of research when investigating neuroinflammation. Microglia are rapidly activated in response to various stimuli, including infectious, pathological stimuli, or Aβ peptides. Activated microglia can secrete substantial pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-1β. This amplified neuroinflammation in the brain exacerbates neuronal damage or even death, resulting in behavioral and psychological symptoms of SAE [
5].
Activation of the NLRP3 inflammasome and the occurrence of pyroptosis in microglia are implicated in the pathogenesis of SAE [
6,
7]. Microglia are the main cells where pyroptosis occurs in the central nervous system (CNS) [
8]. NLRP3 inflammasome, the most well-characterized inflammasome, is a multi-protein complex composed of Nod-like receptors (NLRs), apoptosis-associated speck-like proteins containing a caspase recruitment domain (ASC), and pro-Caspase-1 [
9]. NLRs recognize multiple stimuli to form complexes to cleave pro-Caspase-1 into activated Caspase-1. Activated Caspase-1 then cleaves the pore-forming protein gasdermin D (GSDMD), pro-IL-1β, and pro-IL-18, leading to pyroptosis and IL-1β, IL-18 secretion. Pyroptosis is a pro-inflammatory programmed cell death mediated by GSDMD-N (N-terminal fragment of GSDMD) cleaved by Caspase-1. GSDMD-N binds to membranes to form membrane pores and promotes the release of inflammatory factors, notably IL-1β [
10,
11]. Pyroptosis is closely related to neuroinflammatory diseases, such as subarachnoid hemorrhage (SAH), cerebral venous sinus thrombosis (CVST), and spinal cord injury (SCI). TREM-1 exacerbates neuroinflammatory injury via microglia-mediated pyroptosis in SAH [
12]. NLRP3 inflammasome activation and pyroptosis in microglial modulated by the cGAS–STING pathway are involved in the pathogenesis of CVST [
13]. CD73 alleviates GSDMD‐mediated microglia pyroptosis in SCI through PI3K/AKT/Foxo1 signaling [
14]. However, the mechanisms by which it occurs are not well-understood in SAE.
Recent evidence has suggested that endoplasmic reticulum (ER) stress is involved in NLRP3 inflammasome activation [
15]. ER is responsible for protein synthesis and processing and cellular Ca
2+ homeostasis. Several stimuli, such as ischemia, hypoxia, and bacterial infections, can induce the accumulation of misfolded proteins and result in ER stress [
16]. Then ER initiates three signaling pathways mediated by ER-resident protein folding sensors inositol-requiring enzyme 1 alpha (IRE1α), PKR-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) to restore ER homeostasis [
17]. Hyperactivation of ER stress leads to Ca
2+ release and oxidative stress, which triggers a downstream cascade leading to inflammation [
18]. IRE1α is the most conserved ER stress sensor and has both kinase and RNase activity. IRE1α autophosphorylates, activating RNase activity, and splices X-box-binding protein 1 (XBP1) to form spliced XBP1 (XBP1s) under ER stress. Plentiful studies have proved that IRE1α signaling could activate NLRP3 inflammasome and pyroptosis in multiple diseases such as mellitus [
19], hypoxic-ischemic brain injury [
20], and nonalcoholic fatty liver disease [
21]. Nevertheless, whether it is involved in the pathogenesis of SAE remains unknown.
Erbin, first discovered as an interacting protein of ErbB2, is a member of the epidermal growth factor receptor family. Recently, the role of Erbin in various diseases and signaling pathways has been extensively studied. In most cases, Erbin is involved in regulating multiple cell signaling as a negative regulator, including MAP kinase pathway [
22], NF-κB [
23], Ras–Raf–ERK signaling pathway [
24], and transforming growth factor-beta-signaling pathways [
25]. Erbin deficiency could negatively regulate NOD2-mediated NF-κB activation and overexpressed Erbin in mouse embryonic fibroblasts, significantly inhibiting the production of pro-inflammatory cytokines induced by MDP [
26]. Shen et al. [
27] found that in the DSS-induced mouse colitis model, the expression of Erbin in colon tissue was reduced considerably, and Erbin-deficient mice were more prone to small intestinal inflammation. The above studies show that Erbin plays an essential role in inflammatory diseases. However, whether Erbin regulates the microglia-mediated neuroinflammation and its role in SAE has not yet been elucidated.
We hypothesized that Erbin could attenuate NLRP3 inflammasome activation and inhibit pyroptosis in microglia, reducing neuroinflammation in SAE. In the present study, we first validated the role of Erbin in SAE by behavioral tests. Then, we found that Erbin suppressed microglia pyroptosis by restricting the flow of Ca2+ from ER to the cytoplasm. Finally, we confirmed that Erbin inhibits neuroinflammation and improves cognitive function by negatively regulating IRE1α/Xbp1s pathways.
Materials and methods
Animals
Male C57BL/6 J aged 6–8 weeks (weighing 20–25 g) were enrolled in this study. WT mice were obtained from Hubei Province Center for Animal Experiments, and Erbin-knockout (Erbin−/−) mice were purchased from Wuhan Xianran Biotechnology Co., Ltd. (Contract Number: Mouse-2018-9-25-WJB-3, China). Mice were housed at 22 °C with a relative humidity of 50–60% and a 12 h of light/dark cycle; food and water were available ad libitum. All animal care and experiments were performed according to the ethical regulations set by the Animal Experimentation Committee of Wuhan University (WQ20210298).
Sepsis model and drug treatment
Laparotomy was performed to isolate the cecum after intraperitoneally anesthetized with pentobarbital sodium (50 mg/kg). The cecum was ligated using 3.0 silk and then punctured twice with a 21 G needle. Next, the abdominal incision was sutured, and the animals received 1 ml of 0.9% normal saline as resuscitation subcutaneously. Mice in the Sham group underwent the same operation without the ligation and puncture of the cecum. For further studies on the effect of STF083010 on septic mice, intranasal administration was performed according to the previously used protocol [
28]. 1 h after cecal ligation and puncture (CLP), the mice were anesthetized with a 2% isoflurane and positioned supine. A total volume of 5 μl of STF083010 (2.5 mg/kg, MedChemExpress) was instilled into the nasal cavity of mice. Repeated dosing once daily for three consecutive days. The Sham group was given the same volume of DMSO at the same time.
Behavioral tests
Open field test (OFT)
In this study, an OFT was performed to evaluate the locomotor activity of mice. Each mouse was gently placed in the center of the box (40 × 40 cm) and allowed to explore the apparatus for 5 min. The movement of mice was recorded, and the total traveled distance was analyzed.
Novel object recognition test (NORT)
The NORT was carried out to evaluate recognition memory based on a previous study reported [
29]. Before the experiment, each mouse was gently placed in a field arena (30 × 30 cm) for 3 min to acclimate to the environment. Then, the mice were randomly placed in the center of the arena to explore two identical objects for 5 min each. 24 h post the training, one of the familiar objects was replaced with a novel object, and each mouse was allowed to explore for 5 min. The preference index was calculated as [time spent exploring the novel object/time spent exploring the two objects] × 100%.
Morris Water Maze (MWM) test
The MWM test was implemented 10 days after the operation to assess the spatial learning and memory of the mice. Briefly, the mice were trained for four consecutive days, followed by a probe trial on the 5th day. The MWM consisted of a round steel pool (diameter of 1.2 m, height of 0.6 m) filled with water maintained at 23°C and a hidden platform (diameter of 0.1 m) located in the southwest quadrant of the pool approximately 1 cm below the water surface. Titanium dioxide was used to make the water opaque. Each mouse was randomly placed into each pool quadrant every day during the training period. Mice were allowed to find the platform for 60 s, and the latency to the platform was recorded in each trial. If the mouse failed to find the platform within 60 s, it was guided to the platform to rest for 10 s. On the 5th day, removed the platform from the swimming pool and released the mice into the water from the northeast quadrant. Each mouse was allowed to swim freely for 60 s, and the number of platform crossings and the seconds of search time in the target quadrant were recorded.
Nissl staining
Nissl staining was performed to evaluate neuronal damage and loss. After paraffin embedding and sectioning (4 μm), brain tissues were stained with a 1% toluidine blue solution (Boster Biotechnology, China).
Immunofluorescent staining
For primary microglia, after indicated treatment, cells were fixed with 4% paraformaldehyde for 30 min and then permeabilized by 0.5% Triton X-100 for 30 min. Following blocking with 5% BSA for 1 h, cells were incubated with indicated primary antibodies overnight at 4°C. After three washes with PBS, cells were stained with DayLight 488/CY3-conjugated secondary antibodies (1:400, Abbkine) for 1 h. DAPI was used to stain nuclei. Images were recorded by Leica confocal microscopy. For brain tissue, double immunofluorescence staining was performed on formalin-fixed, paraffin-embedded brain sections. After blocking with 5% BSA, 4 μm thick paraffin brain slices were incubated with rabbit anti-Caspase-1 (1:200, Abclonal), rabbit anti-GSDMD (1:200, Abclonal), or mouse anti-Iba-1 (1:200, Abcam) antibodies overnight at 4°C. Then, the slices were incubated with CY3-conjugated duck anti-rabbit or DayLight 488-conjugated duck anti-mouse secondary antibodies (1:400, Abbkine) for 1 h at 37 °C. After washing with PBS, DAPI was used to label the nuclei. The number of double-positive cells was detected under a fluorescence microscope.
Cell culture and treatment
Primary mouse microglia were obtained from newborn WT and
Erbin−/− mice at postnatal days 1–2 as previously reported [
30]. Briefly, the pups were decapitated, removed the meninges, and cut cortices into small pieces. After trypsin enzymatic digestion, cells were filtered by passing through a cell strainer (70 μM pores, Corning, USA), then resuspended in complete DMEM-F12 (GIBCO, USA) with 10% fetal bovine serum (FBS) (GIBCO, USA) and 1%penicillin/streptomycin (Biosharp, China) and seeded on poly-
d-lysine-coated T75 flasks. Then, the cells were cultured in an incubator (37 °C, 5% CO
2), and the medium was changed every 3–4 days. About 2 weeks later, cells were shaken using a horizontal orbital shaker at 200 rpm for 2 h at a constant temperature (37˚C) to isolate the matured microglia.
The mouse microglia BV2 cell line and mouse hippocampal neuron HT22 cell line were purchased from the Procell Life Science&Technology Co., Ltd (Wuhan, China) and cultured in DMEM (GIBCO, USA) supplemented with 10% FBS and 1% penicillin/streptomycin at 37˚C in a humid air atmosphere containing 5% CO2. LPS (E. coli 0111:B4, L2630, Sigma) and nigericin (HY-100381, MedChemExpress) were used to establish the activation of the NLRP3 inflammasome cell model. For NLRP3 inflammasome activation treatment, primary microglia and BV2 cells were treated with LPS (500 ng/ml) for 4 h and then treated with nigericin (10 µΜ) for 45 min. For intervention experiments, primary microglia and BV2 cells were pretreated with 30 µΜ STF083010 (dissolved in DMSO) (HY-15845, MedChemExpress) for 2 h, followed by treatment with LPS and nigericin. DMSO was designed as vehicle control for treatment conditions.
Establishment of microglia-conditioned medium (CM)
Primary microglia were plated onto 6-well plates and then treated with DMSO, STF083010, or LPS/nigericin at the appointed times. 45 min after nigericin was added, replaced the old medium with fresh medium, and continued to culture the microglia for 24 h. Then, the supernatant was collected and centrifuged at 1000 rpm for 5 min. Next, the HT22 cells’ medium was replaced with the collected CM and cultured for 12 h.
Plasmids and siRNA transfection
Small interfering RNA against Erbin (siErbin) was purchased from Tsingke Biotechnology Co., Ltd (Nanning, China) and transfected into BV2 cells using jetPRIME (Polyplus transfection reagent, France) according to the manufacturer's instructions. After 48 h, the cells were used for further experiments. The following sequences were used: siErbin sense: 5'-GCAAGCGGUGUCCUUGUUATT-3', negative control siRNA (siNC) sense: 5'-UUCUCCGAACGUGUCACGUTT-3'.
The ER-targeted plasmid CMV-ER-LAR-GECO1 (#61,244, Addgene), a sensitive single-wavelength Ca
2+ indicator for detecting Ca
2+ dynamics within the ER of mammalian cells [
31], was a gift from Han song. Moreover, the changes in Ca
2+ concentration in ER were observed by a confocal microscope or a flow cytometer.
Cell viability
Primary microglia were seeded into a 96-well plate (1 × 104 cells/well), and LPS was added to the cells for 4 h and then stimulated with 10 μM nigericin for 45 min. HT22 cells were plated in 96-well plates at a density of 103 cells per well, cultured for 24 h, then replaced the media with microglia-CM, and cultured for another 12 h. Finally, 10 µL CCK-8 reagents (Biosharp, China) were added per well. The absorbance at 450 nm was measured to calculate the cell viability using a microplate reader.
LDH assay
LDH is an indicator of cell membrane integrity and is also used to indirectly indicate the onset of pyroptosis [
32]. The culture medium was collected and centrifuged at 500×
g, 4 °C for 10 min. Then, the supernatant was transferred into 1.5 mL EP tubes. LDH release was measured with an LDH assay kit (Beyotime, China) according to the manufacturer’s manual.
Reverse transcription real-time quantitative polymerase chain reaction (RT q-PCR)
Total RNA was extracted from BV2 cells with TRIZOL Reagent (Invitrogen, USA). RNA was reverse-transcribed to complementary DNA using the RT-qPCR Fast Master Mix (Vazyme, China). Real-time fluorescence quantitative PCR detection was performed according to the manufacturer's instructions. The GAPDH gene was used as an internal control for IL-1β, TNF-α, and IL-6 mRNA expressions analysis. The gene expression was analyzed using the 2 − ΔΔCt method for quantification. All gene primer sequences were used as follows: IL-1β, forward 5′-GAAATGCCACCTTTTGACAGTG-3′, and reverse 5′-TGGATGCTCTCATCAGGA.
CAG-3′; TNF-α, forward 5′-GGCATGGATCTCA AAGACAAC-3′ and reverse 5′-TGGATGCTCTCATCAGGACAG-3′; IL-6, forward 5′-CATGTTCTCTGGGAAAT.
CGTGG-3′ and reverse 5′-GTACTCCAGGTAGCTATGGTAC-3′; GAPDH, 5′-AGGT.
CGGTGTGAACGGATTTG-3′ and reverse 5′-TGTAGACCATGTAGTTGAG GTA-3′.
Flow cytometry
Fluo4-AM (1 μM, Beyotime, China) was used to indicate the cytoplasmic Ca2+ concentration. Cells were incubated with Fluo4-AM solution for 30 min in a cell incubator and washed three times using PBS. CMV-ER-LAR-GECO1 was used to indicate ER Ca2+. 48 h after transfection of plasmids. Primary microglia were treated with DMSO, STF083010, or LPS/nigericin according to the appointed time. Then discarded medium and washed three times with PBS. The green (intracytoplasmic Ca2+, FITC) and red (ER Ca2+, PE) fluorescence were measured by flow cytometry.
Transmission electron microscopy (TEM)
After transcardial perfusion with 50 mL of saline and followed by 50 mL of 4% paraformaldehyde, about 1 mm3 hippocampal CA1 tissues were collected and fixed in 2.5% glutaraldehyde, dehydrated through a grade ethanol series and propylene oxide, and then embedded in Epon. The ultrathin sections (70 nm) were placed onto 200 mesh copper grids and then stained with 4% uranyl acetate and 0.04% lead citrate. The stained sections were observed under a transmission electron microscope (Hitachi H-600, Hitachi, Tokyo, Japan). The thickness of the postsynaptic density (PSD) was assessed as the length of the vertical line from the postsynaptic membrane to the most convex part of the synaptic complex. The width of the synaptic cleft was evaluated by measuring the narrowest and widest parts of the synapse and then taking the average of these values.
Western blot analysis
The total protein content was extracted from hippocampal tissue or cultured cells using RIPA lysis buffer (Beyotime, China) containing PMSF (BioSharp, China) and phosphatase inhibitor (Beyotime, China). The protein concentration in the supernatant was quantified by a BCA protein assay (Beyotime, China). Equal amounts of protein samples were then separated by SDS–PAGE and transferred to PVDF (MilliPole, UK). PVDF membranes were then blocked with 5% BSA for 2 h at room temperature and incubated with primary antibodies (Erbin (NBP2-56,104, Novus), NLRP3 (A5652, Abclonal), GSDMD (A10164, Abclonal), Caspase-1 (AG-20B-0042, adipogenic), Iba-1 (ab178846, Abcam), IRE1α (3294, CST), p-IRE1α (ab48187, Abcam), Xbp1s (24,868–1-AP, proteintech), PSD95 (3409, CST), Synaptophysin (AF0257, affinity), Synapsin-1 (5297, CST), β-actin (HRP-60008, proteintech)) antibody overnight at 4 °C. After incubation, membranes were washed with TBST and then incubated with the corresponding HRP-conjugated secondary antibodies for 2 h. Then, washed the membrane with TBST. Finally, immunoreactive bands were detected with an enhanced chemiluminescence detection reagent. Band intensities were quantified by spot densitometric analysis using ImageJ software, and results were normalized to β-actin levels and reported as relative intensities to controls.
ELISA
The levels of IL-1β, IL-18, TNF-α, and IL-6 in the hippocampus and medium of primary microglia were determined using an ELISA kit (Beijing 4A Biotech Co., Ltd) according to the manufacturer's instructions. The absorbance of the samples at 450 nm wavelength was measured with a BioTek microplate reader.
Statistical analysis
GraphPad Prism 8.3.0 was used to analyze data and construct the graphs. All data were expressed as mean ± SEM. MWM training experiments were analyzed using two-way ANOVA for repeated measures followed by Bonferroni correction for multiple testing. Multiple groups were analyzed by two-way ANOVA followed by the Bonferroni post-tests. P < 0.05 was considered statistically significant.
Discussion
Erbin is involved in inhibiting various cell signaling pathways as a scaffold protein. Erbin plays a cardioprotective role in cardiovascular disease, and the down-regulation of Erbin is associated with cardiac hypertrophy and heart failure in both mice and humans [
40]. A few research reports showed that Erbin exerted a protective effect against inflammatory bowel disease by suppressing autophagic cell death, and
Erbin−/− mice were more prone to small intestinal inflammation [
27]. Though Erbin is highly expressed in the brain, its role in CNS in SAE mice has not been explored. In this study, we discovered the neuroprotective effects of Erbin in SAE by regulating IRE1α/Xbp1s-Ca
2+ axis and consequently inhibiting microglia pyroptosis.
SAE is a common and severe complication of sepsis and is closely associated with mortality and morbidity. Several studies have shown an association between brain injury and long-term psychological or cognitive impairment in SAE [
41,
42]. Consistent with previous studies, septic mice exhibited severe learning and memory impairment 7 days after CLP assessed by behavioral tests. Lack of Erbin exacerbated cognitive impairment and accelerated death in septic mice. More importantly, Erbin was down-regulated in septic mice. Although we cannot conclude that this downregulation is a cause of SAE rather than its consequence, it is a reasonable hypothesis based on our results in mice. Thus, for the first time, this study shows the role of Erbin in SAE and suggests that it is crucial in attenuating cognitive dysfunction induced by CLP.
The hippocampus is one of the brain structures that is more sensitive to the cognitive dysfunction caused by sepsis [
43]. Damage to hippocampal CA1 and CA3 regions, critical brain regions for learning and memory, may lead to cognitive impairment [
44]. Our study observed more severe neuronal damage in the CA1 and CA3 areas in
Erbin−/− septic mice. Dysregulation of synaptic ultrastructure is associated with cognitive impairment [
45,
46]. Aberrant alterations in the morphological structure of postsynaptic densities may result in long-term synaptic plasticity impairment and synaptic transmission dysfunction [
47]. Subsequently, the TEM result showed that the synaptic structure was deteriorated with thinner postsynaptic densities and broader synaptic clefts in
Erbin−/− septic mice. Synapse-related proteins, including SYP, SYN1, and PSD95, play crucial roles in synaptic plasticity and memory formation [
48]. SYP is present in neuroendocrine cells and implicated in synaptic transmission. Low expression of SYP led to behavioral changes, such as difficulty in object novelty recognition and reduction in spatial learning [
49]. SYN1 exists in the nerve terminal of axons and is involved in the regulation of synaptogenesis and neurotransmitter release. PSD95, a protein density, is almost completely attached to the postsynaptic membrane, participates in anchoring synaptic proteins, regulating synaptic plasticity, and plays an important role in long-term potentiation [
50]. Here,
Erbin deletion in septic animals further downregulated synapse-related protein levels compared with the CLP group. Therefore, it is speculative that Erbin ameliorates cognitive dysfunction by reducing neuronal damage, improving the synaptic structure, and increasing synapse-associated protein expressions.
Systemic inflammation during sepsis could activate microglia in the brain, then activated microglia, and following secreted pro-inflammatory cytokines are detrimental to neuronal survival, growth, synaptogenesis, and phagocytosis [
51]. Aberrant activation of NLRP3 inflammasome and pyroptosis has been described as dominant factors in microglia-mediated neuroinflammation in SAE development [
7]. Once NLRP3 inflammasome activated, pro-Caspase-1 was cleaved into active Caspase-1, then activated Caspase-1 cleaved the GSDMD, pro-IL-1β and pro-IL-18, leading to pyroptosis and IL-1β and IL-18 secretion. IL-1β should be the main perpetrator of neurogenesis. Studies have shown that neurons highly express IL-1 receptors, and excessive IL-1β can significantly inhibit the proliferation and growth of neurons [
52]. Overexpression of IL-1β in the hippocampus may affect neuronal synaptic plasticity and impairs hippocampal-dependent memory [
53]. Treated with MCC950 (the NLRP3 inhibitor) [
7] or Ac-YVAD-CMK (the Caspase-1 inhibitor) [
6] prevented sepsis-induced neuronal damage and cognitive deficits. To investigate whether the effect of Erbin is related to pyroptosis-mediated neuroinflammation in microglia, we systematically examined the activation of microglia and the expression of pyroptosis-associated genes and found that Iba-1 and pyroptosis-associated proteins were all overexpressed in hippocampus tissue after CLP. What is more, the increase in the above indicators was more pronounced in
Erbin−/− septic mice, as well as increased pro-inflammatory cytokine levels. We also observed intense Caspase-1 and GSDMD immunostaining in Iba-1-labeled microglia and remarkable growth in hippocampal lesions in
Erbin−/− mice. Collectively, the present results define that the pyroptosis occurring in microglia can be inhibited by Erbin in SAE. We further verified the conclusion in primary mouse microglia and BV2 cells. Afterward, the viability of HT22 cells cultured with CM of LPS/nigericin-treated
Erbin-knockout microglia was obviously reduced. This suggested that
Erbin's deletion exacerbated neuronal damage and cognitive dysfunction due to the overactivation of NLRP3 inflammasome and pyroptosis.
Ca
2+ mobilization is an essential upstream event for NLRP3 inflammasome activation. It occurs by opening plasma membrane channels or releasing ER-linked intracellular Ca
2+ stores to allow Ca
2+ to flow into the cytosol [
54]. Inhibition of Ca
2+ mobilization has been reported to reduce the activation of the NLRP3 inflammasome [
37,
55]. Our study detected a pronounced accumulation of intracytoplasmic Ca
2+ in response to LPS/nigericin stimulation, and a further increase in intracytoplasmic Ca
2+ was observed when
Erbin was knockout or knockdown, indicating that Erbin might have a negative effect on the regulation of Ca
2+ in the cytoplasm. Given that ER is the most important intracellular Ca
2+ store and increased intracellular Ca
2+ influx from the ER is an endogenous signal which drives cell death and immune responses [
56], we continued to explore whether the increased cytosolic Ca
2+ came, at least in part, from the ER. Results of flow cytometry and confocal microscopy both showed that Ca
2+ in ER was significantly reduced after LPS/nigericin administration. ER stress is closely linked to dysregulated Ca
2+ homeostasis, and three ER stress signaling is involved in regulating ER Ca
2+ release [
38,
39]. We then found only IRE1α was phosphorylated upon LPS stimulation, accompanied by increased splicing of downstream XBP1. In agreement with previous works, toll-like receptors (TLR), which primarily recognize microbial ligands like LPS, selectively stimulate IRE1α, but not PERK or ATF6 [
57]. Also, IRE1α/XBP1 activated by TLR is essential for optimal and sustained production of pro-inflammatory cytokines in macrophages [
57]. Therefore, we speculate that the activation of the IRE1α/Xbp1s signaling may be related to the increase of intracytoplasmic Ca
2+ and the decrease of ER Ca
2+. Furthermore,
Erbin depletion increased the expression of p-IRE1α and Xbp1s, indicating that Erbin negatively modulates IRE1α/Xbp1s pathways in LPS/nigericin-treated microglia and CLP-induced SAE mice.
The movement of Ca
2+ across the ER membrane is mediated by Ca
2+ release channels, including inositol-1,4,5-triphosphate receptors (InsP3Rs) and ryanodine receptors (RyRs). Some studies have demonstrated that IRE1α regulates the flow of Ca
2+ between ER and mitochondria by determining the distribution of InsP3Rs located in ER membrane in mouse liver [
58]. Also, Xbp1s could induce the expression of InsP3Rs, facilitating Ca
2+ release from ER [
59]. However, whether IRE1α or Xbp1s can regulate ER Ca
2+ movement in microglia in SAE is unknown. To verify this, we blocked the IRE1α/Xbp1s pathway using STF083010, a specific inhibitor of IRE1α RNase activity inhibitor. Just as we envisioned, Ca
2+ in the cytoplasm decreased, and Ca
2+ in ER increased in LPS/nigericin-treated
Erbin-silenced primary microglia or BV2 cells after being given STF083010. This suggests that Erbin can limit the efflux of Ca
2+ from ER to cytoplasm by inhibiting IRE1α/Xbp1s pathway activity. Meanwhile, pyroptosis-associated proteins were all reduced both in
Erbin-deficient primary microglia and BV2 cells after blocking IRE1α/Xbp1s signaling. The number of Caspase-1/Iba-1 and GSDMD/Iba-1 double-positive cells in the
Erbin−/− + CLP + STF083010 group was less than that in the
Erbin−/− + CLP group, as well as descending inflammatory cytokines levels. Moreover, the addition of STF083010 did not affect Erbin's expression. Combining the previous results, we can conclude that Erbin negatively regulates IRE1α/Xbp1s pathway in LPS/nigericin-treated microglia or CLP-induced SAE mice. When LPS added or sepsis occurs, the expression of Erbin decreases and the activity of the IRE1α/Xbp1s pathway is enhanced, mobilizing Ca
2+ to flow from ER to cytoplasm, resulting in Ca
2+ accumulation in the cytoplasm, thereby inducing inflammasome activation and pyroptosis, release inflammatory factors.
Finally, we assessed neurological impairment and behavioral performance in SAE mice followed by the administration of STF083010. As expected, STF083010 significantly attenuated neuronal damage in hippocampal increased pyroptosis-associated proteins expressions and improved cognitive dysfunction in Erbin−/− septic mice. These results further support the idea that Erbin exerts neuroprotective effects by inhibiting the IRE1α/Xbp1s pathway in SAE mice.
ER stress-related TXNIP/NLRP3 inflammasome activation is involved in the pathophysiology of SAH [
60], neonatal hypoxic-ischemic brain injury [
20], type 2 diabetes [
61], and many other diseases. Hyperactivated IRE1α activates the NLRP3 inflammasome through elevated TXNIP protein expression, resulting in caspase-1 cleavage and IL-1β secretion. TXNIP knockout reduces pyroptosis during ER stress. Small molecule IRE1α RNase inhibitors STF083010 inhibit TXNIP production from blocking IL-1β secretion. In our study, we also detected TNXIP expression in the hippocampus and microglia and found that LPS treatment resulted in decreased TXNIP expression (see Additional file
1: Fig. S4). Nevertheless, we did not find that IRE1α RNase signaling promotes inflammasome formation by increasing TXNIP expression.
However, some limitations in our study cannot be ignored. Firstly, the increased Ca2+ in the cytoplasm may come from the efflux of Ca2+ in intracellular organelles or from the influx of extracellular Ca2+. We did not further explore whether other organelles such as mitochondria and lysosomes have Ca2+ efflux into the cytoplasm to lead to NLRP3 inflammasome activation, nor did we completely rule out the effect of extracellular Ca2+ influx on NLRP3 inflammasome and pyroptosis. Secondly, we have not yet elucidated the exact mechanism of how the IRE1α/Xbp1s pathway regulates ER Ca2+ efflux and whether IRE1α/Xbp1s regulates ER Ca2+ efflux by affecting the Ca2+ channel InsP3Rs on the ER. Therefore, although the present study is helpful in understanding the neuroprotective role of Erbin in SAE, further experimental verification is still needed.