2.1 Clinical specimen collection and ethics statement
We included 20 patients who were hospitalized in the Department of Critical Care Medicine in Guangdong Provincial People's Hospital from August 2021 to December 2021. ICI was diagnosed according to the Chinese Expert Consensus on the Diagnosis and Management of Infections in Neurosurgical Patients with Serious Illness (2017). Ten patients without ICI were assigned to non-ICI group, and another ten patients with ICI were grouped into ICI group. Clinical specimens of CSF were collected from patients (Table 1). MiR-338-3p expression were examined using qRT-PCR and the expression of TNF-α and IL-1β was measured by ELISA according to manufacturer’s instructions.
All patients provided informed consent for participation. All procedures performed in this study were in accordance with the Ethics Committee of Guangdong Province’s General Hospital (KY-Q-2022-176-02).
2.2 Animals
Adult male SD rats (150-200g) obtained from the Guangdong Medical Laboratory Animal Center were used in this study. They were raised in plastrocytesic cages under a 12-h light-dark cycle with food and water ad libitum. The room temperature was controlled at 24°C ± 1°C, and relative humidity was maintained at 50%-60%. The male rats used in this study were randomly divided into four groups: (I) Control group: The rats in this group were injected into ICV with phosphate buffered saline (PBS)(10µL); (II) ICV-LPS group: In this group, rats were injected into ICV with LPS (50µg in 10 µL saline) taken from Escherichia coli O111:B4 (Sigma-Aldrich, L4391) for 24h; (III) LPS + ICV-AAV-NC group: In this group, LPS were injected into ICV at 14 days after Adeno Associated Virus negative control reagent (AAV-NC) (4.2×1012 v.g/ml,10µl) was administrated into ICV; The rats were sacrificed after being ICV injected with LPS for 24h. (IV) LPS + ICV-AAV-miR group: In this group, LPS were injected into ICV at 14 days after AAV miR-338-3p (AAV-miR) (4.2×1012v.g/ml,10µl) was administrated into ICV. The rats were sacrificed after being ICV injected with LPS for 24h.
ICV injection of drugs was performed as previously described. Briefly, rats were anesthetized with sodium pentobarbitone by intraperitoneal (i.p.) injection (45 mg/kg) and then fixed in a stereotactic frame (RWD Life Science, Shenzhen, China). Subsequently, the skin of the heads was incised along the midline for about 1 cm. Referring to brain atlas, the left lateral ventricle was used as the administration target (millimeters from bregma): anterior-posterior = 1.0 mm, mediolateral = 1.5 mm, and dorsoventral = − 3.8 mm. A microinjector was used to infuse the compounds (10 µL) with a rate of 1µL/min, and the syringe was held in place for 10 min after infusion. The was used for local disinfection. Then the skin was sutured, and the rats were returned to the breeding room for recovery.
The rats were sacrificed at 24 h after injecting PBS or LPS (n = 3 per group). For qRT-PCR and Western blot, the brain was then dissected with a craniotomy. The CC was immediately isolated and placed in a cryotube, then rapidly removed to liquid nitrogen and subsequently transferred to − 80°C until use. For immunofluorescence analysis, the rats were perfused with PBS (150 mL) followed by 4% paraformaldehyde (pH = 7.3, 200 mL). The brain tissues were fixed in 4% paraformaldehyde for 24 h. All animal experimental procedures were consented by Institutional Animal Care and Use Committee, Guangdong Province, China. All efforts were made to reduce the number of rats used for experimentation and their suffering.
2.3 Primary culture of Astrocyte
Primary cortical astrocyte cultures were prepared using 1-day-old neonatal SD rats (obtained from Guangdong Medical Laboratory Animal Center, Guangdong Province, China). Before the experiment, Poly-L-lysine (PLL, 0.1 mg/ml) was used for pre-cultured coating in a flask. The 1-day-old rats were disinfected with alcohol. After decapitation, the brain was quickly removed. The adherent meninges were removed with a pair of fine forceps. Mixed glial cells separated from the CC were plated on a 75 cm2 flask at a density of 1.2×106 cells/mL in Dulbecco’s modified Eagle’s medium/F12 medium (DMEM/F12, Gibco) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT). The mixed cells were cultured for 8–9 days at 37°C in a humidified 5% CO2 incubator, and half of the medium was replaced about every 3–4 days. After 9–10 days, the flasks were shaken at room temperature at 180 rpm for 1 h to remove the microglial cells. After this, the flasks were added fresh DMEM/F12/FBS and continued to shake at 200 rpm for 16–20 h to remove OPCs. Subsequently, purified astrocytes were divided into several groups by different treatments.
Group Ⅰ
To study that miR-338-3p could mitigate the production of TNF-α and IL-1β in the astrocytes, purified astrocytes were cultured in cell medium for 1 day at 5% CO2 and 95% air at 37°C. The astrocytes, which were digested and plated at the same number in cell medium, were subdivided into the control group, LPS group, LPS + mimic-NC group, LPS + mimic-miR-338-3p group. In control group the astrocytes were treated with PBS. In LPS group, astrocytes were treated with LPS (1µg/ml) for 24h. In LPS + mimic-NC group, astrocytes were first treated with 50nM mimic-NC for 24h and then LPS for 24h. In LPS + mimic-miR group, astrocytes were first treated with 50nM mimic-miR-338-3p for 24h, then administrated with LPS for 24h.
Group Ⅱ
To explore whether miR-338-3p inhibit expression of STAT1, TNF-α and IL-1β through sponging STAT1 mRNA, purified astrocytes were cultured in cell medium for 1 day at 5% CO2 and 95% air at 37°C. The astrocytes were subdivided into the control group, LPS group, the LPS + mimic-miR-338-3p group and LPS + mimic-miR-338-3p + plasmid-STAT1 group. In control group the astrocytes were treated with PBS. In LPS group, astrocytes were treated with LPS (1µg/ml) for 24h. In the LPS + mimic-miR-338-3p group, astrocytes were first treated with 50nM mimic-miR-338-3p for 24h, then administrated with LPS for 24h. In the LPS + mimic-miR-338-3p + plasmid-STAT1 group, astrocytes were first treated with 50nM mimic-miR-338-3p and 0.8µg plasmid-STAT1 for 24h, then administrated with LPS for 24h.
Group Ⅲ
To examine whether STAT1 would facilitate the generation of TNF-α and IL-1β in the astrocytes administrated with LPS, purified astrocytes were cultured in cell medium for 1 day at 5% CO2 and 95% air at 37°C. Then, the astrocytes were subdivided into the control group, LPS group, LPS + si-NC group, LPS + si-STAT1 group. The astrocytes in the control group were treated with PBS. In LPS group, astrocytes were treated with LPS (1µg/ml) for 24h. In the LPS + si-NC group, astrocytes were first treated with 50nM si-NC for 24h, then administrated with LPS for 24h. In the LPS + si-STAT1 group, astrocytes were first treated with 50nM short interfering RNA-STAT1(si-STAT1) for 24h, then administrated with LPS for 24h.
Group Ⅳ
To examine if miR338-3p expression is localized in astrocytes, purified astrocytes were cultured in cell medium for 1 day at 5% CO2 and 95% air at 37°C, then used for immunofluorescence-fluorescent in situ hybridization (IF-FISH).
2.4 BV2 cell culture
Due to the low transfection efficiency of primary microglia cells, we used immortalized microglia BV2 (Purchased from Wuhan University Cell Bank) for cell tranfection, which was cultured in Minimum Essential Medium (MEM, Gibco) supplemented with 10% FBS (Hyclone, Logan, UT) at 37°C under 95% air and 5% CO2. In the experiment, BV2 cells were randomly assigned into the different groups according to different treatments.
Group Ⅰ
To investigate that miR-338-3p could suppress the generation of inflammatory cytokines in BV2 cells, BV2 cells were divided into control group, LPS group, LPS + mimic-NC group, LPS + mimic-miR-338-3p group. In control group, the BV2 cells were treated with PBS. In LPS group, BV2 cells were treated with LPS (1µg/ml) for 6h. In LPS + mimic-NC group, BV2 cells were first treated with 50nM mimic-NC for 24h, then administrated with LPS for 6h. In LPS + mimic-miR-338-3p group, BV2 cells were first treated with 50nM mimic-miR-338-3p for 24h, then administrated with LPS for 6h.
Group Ⅱ
To study whether miR-338-3p inhibit expression of STAT1, TNF-α and IL-1β through sponging STAT1 mRNA, BV2 cells were divided into the control group, LPS group, LPS + mimic-miR-338-3p group and LPS + mimic-miR-338-3p + plasmid-STAT1 group. In control group, the BV2 cells were treated with PBS. In LPS group, BV2 cells were treated with LPS (1µg/ml) for 6h. In LPS + mimic-miR group, BV2 cells were first treated with 50nM mimic-miR-338-3p for 24h, then administrated with LPS for 6h. In the LPS + mimic-miR-338-3p + plasmid-STAT1 group, BV2 cells were first treated with 50nM mimic-miR-338-3p and 0.8µg plasmid-STAT1 for 24h, then administrated with LPS for 6h.
Group Ⅲ
To explore whether STAT1 would promote the production of inflammatory cytokines in BV2 cells treated with LPS, BV2 cells were divided into control group, LPS group, LPS + si-NC group, LPS + si-STAT1 group. In control group, the BV2 cells were treated with PBS. In LPS group, BV2 cells were treated with LPS (1µg/ml) for 6h. In LPS + si-NC group, BV2 cells were first treated with 50nM si-NC for 24h, then administrated with LPS for 6h. In LPS + si-STAT1 group, BV2 cells were first treated with 50nM si-STAT1 for 24h, then administrated with LPS for 6h.
Group Ⅳ
To examine if miR-338-3p expression is localized in BV2 cells, these cells were cultured in cell medium for 1 day at 5% CO2 and 95% air at 37°C, then used for IF-FISH.
2.5 293T cells culture
293T cells were seeded in a 96-well white-walled plates at a density of 1.0 × 104 cells/hole in DMEM supplemented with 10% FBS. The mixed cells were cultured for 24h at 37°C in a humidified incubator containing 5% CO2. We divided 293T cells into four groups: Group I: 293T cells transfected with mimic-miR-338-3p and putative miR-338-3p-STAT1 binding site; Group II: 293T cells transfected with mimic-miR-338-3p and STAT1 Mutant control (MUT); Group III: 293T cells transfected with mimic NC and putative miR-338-3p-STAT1 binding site; GroupIV: 293T cells transfected with mimic NC and STAT1 MUT. The transfection concentration of mimics was 50nM, and the amount of plasmid was 50ng/well. Each group was set with 3 double wells. At 48 hours after transfection, double luciferase experiment is carried out.
2.6 ELISA
The protein levels of IL-1β and TNF-α in the CSF of the patients were measured using ELISA kit. The collected CSF was stored at -80°C. CSF was left to thaw before use, and its supernatant was collected after centrifugation at 3000 rpm for 20 min at 4°C. Supernatant samples were analyzed using commercially available ELISA kits according to the manufacturer's protocol. Optical density analysis was performed in a multifunctional microplate reader.
2.7 Double Immunofluorescence
Rats from each group were deeply anesthetized with 4% pentobarbital. Under deep anesthesia, the rats were perfused transcardially first with PBS, followed 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were removed and further fixed in 4% paraformaldehyde in 0.1 M phosphate buffer overnight at 4°C. After this, the brains were kept in 30% sucrose until use. Frozen sections were cut at 10 µm thickness mounted on glass slides and stored at − 20°C in cryoprotectant. Brain tissue sections derived from control group, ICV-LPS group, ICV-LPS + AAV-NC group and ICV-LPS + AAV-miR group were randomly divided into five groups. The brain sections in group I were from CC of rats sacrificed at 24h after ICV-LPS injection and corresponding controls, and incubated with antibody directed against anti-IL-1β and anti-GFAP (Table 2). The brain sections in group II were from CC of rats sacrificed at 24h ICV-LPS injection and corresponding controls, and incubated with antibodies directed against IL-1β and anti-Iba1 (Table 2). The brain sections in group Ⅲ were from CC of rats in ICV-LPS + AAV-NC group and ICV-LPS + AAV-miR group, and incubated with anti-GFAP or anti-Iba1. The brain sections in group Ⅳ were from CC of rats in ICV-LPS + AAV-NC group and ICV-LPS + AAV-miR group, and incubated with anti-GFAP and anti-IL-1β. The brain sections in groupⅤ were from CC of rats in ICV-LPS + AAV-NC group and ICV-LPS + AAV-miR group, and incubated with anti-Iba1 and anti-IL-1β. Incubation with the primary antibodies was overnight at 4°C. After washing for three times with PBS, the sections were incubated with secondary antibodies: 1) Alexa Fluor594 goat anti-Rabbit IgG (H + L) (Table 2) for IL-1β, Alexa Fluor488 goat anti-mouse IgG (H + L) (Table 2) for GFAP or Iba1 in group Ⅰ-II (control group and LPS group) for 1h.2)Alexa Fluor647 goat anti-mouse IgG (H + L) (Table 2) for GFAP or Iba1 in group III (ICV-LPS + AAV-NC group and ICV-LPS + AAV-miR group) for 1h. 3) Alexa Fluor594 goat anti-Rabbit IgG (H + L) (Table 2) for IL-1β, Alexa Fluor647 goat anti-mouse IgG (H + L) (Table 2) for GFAP or Iba1 in group IV-V (ICV-LPS + AAV-NC group and ICV-LPS + AAV-miR group) for 1h. Incubation of sections for all groups was carried out at room temperature. Finally, all sections were counterstained with DAPI (Sigma-Aldrich, St. Louis, MO, USA, Cat. No. D9542) and then observed using a fluorescence microscope (Olympus System Microscope Model BX53, Olympus Company Pte, Tokyo, Japan) or confocal microscope (ZEISS LSM 900, Oberkochen, Germany) .
Primary astrocytes or BV2 cells in all groups were washed for three times with PBS, and the cells were fixed in 4% paraformaldehyde for 30 minutes and then blocked in 1% BSA for 1 hour. Primary astrocytes in all groups were incubated with anti-TNF-α antibody (Table 2) and anti-GFAP antibody (Table 2) overnight at 4 ° C. BV2 cells in all groups were incubated with anti-TNF-α antibody (Table 2) and anti-Iba1 antibody (Table 2) overnight at 4°C. On the following day, primary astrocytes and BV2 cells were immunocytochemically stained after three washes with PBS (10 min each) according to the following protocol. After washing three times with PBS, primary astrocytes or BV2 cells were incubated with secondary antibodies: Alexa Fluor594 Goat Anti-Rabbit IgG (H + L) (Table 2) for TNF-α, Alexa Fluor488 Goat Anti-Mouse IgG (H + L) (Table 2) for GFAP or Iba1 for 1h at room temperature, then washed for three times with PBS, and incubated with DAPI for 5 min. The cells were observed under a fluorescence microscope.
After staining, brain sections were examined and images were captured using a fluorescence microscope or confocal microscope. For cell counting, three frozen sections were taken from each group. For each section, four different microscopic fields of the CC were observed at 40x magnification. Positive cells were counted using Image J software. IL-1β+/ GFAP + and IL-1β+/ Iba1 + labeled cells were counted and recorded for statistical analysis.
2.8 Western Blot Analysis
A protein extraction kit (Best Bio, BB-3101-100T) was used to extract the proteins from primary cultured astrocytes, BV2 cells and fresh CC tissues. The standard protocol as previously described (Deng and Lu et al., 2008) was used to detect the protein concentrations by BCA Protein Assay Kit (Thermo Scientific, 23250). Samples of supernatants containing 30 µg of total protein were collected and heated at 100°C for 10 min. These samples were separated by SDS-PAGE and then transferred to a 0.22 µm polyvinylidene fluoride membranes (Bio-Rad, USA), which were subsequently incubated with the primary antibodies according to the manufacturer’s recommendations. The primary antibodies and dilution concentration used in western blot are listed in Table 2. Incubation with all the primary antibodies was carried out overnight at 4°C. After three washes in tris-buffered saline Tween (TBST) containing 0.1% Tween-20, the membranes were incubated with the appropriate secondary antibodies including anti-mouse IgG (1:3000, Cell Signaling Technology, 7076S) or anti-rabbit IgG (1:3000, Cell Signaling Technology, 7074S) for 1 h at room temperature.The protein bands were visualized by chemiluminescence kit (Millipore, WBKLS0500) and detected by ImageQuant LAS 500 Imager (GE Healthcare Bio-Sciences AB). The optical density of each protein bands was quantified with image J software (n = 3 per group).
2.9 quantificational Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from primary cultured astrocytes, BV2 cells and fresh CC tissues subjected to different treatments by using Trizol reagent (Thermo Fisher Scientific). Total RNA extraction and reverse transcription to cDNA were carried out with a PrimescriptTM RT reagent kit (RR037A, Takara, Japan) on an ABI Veriti PCR (Applied Biosystems, USA). The reaction of qRT-PCR for each gene was detected in triplicate using the SYBR Premix Ex TaqTM II kit (RR820A, Takara, Japan) on an ABI 7900 thermocycler (Applied Biosystems, USA) according to the manufacturer’s instructions. The specific primer sequences are listed in Table 2. The specific primers of U6 and miR-338-3p (bulge-loop RT and qRT-PCR primers) were designed by RiboBio (RiboBio Co., Ltd, Guangzhou, China). GAPDH and U6 were used as the internal control for mRNA and miR-338-3p, respectively. Reaction without any template was served as negative controls. Relative mRNA expression was calculated by the 2−ΔΔCt method.
2.10 Bioinformatics Analysis and Dual-Luciferase Reporter Gene Assay
TargetScan online prediction software (https://www.targetscan.org/vert_72/) was used to screen the potential mRNAs that could bind to miR-338-3p, and the binding site between STAT1 and miR-338-3p was predicted.
We performed a luciferase activity assay using a Dual-Luciferase Reporter Assay Kit (Promega, Wisconsin, USA) according to the manufacturer’s instructions. Briefly, the wild-type (WT) or MUT of STAT1 3ʹ-UTR luciferase reporter constructs were co-transfected into 293T cells with mimic miR-338-3p or mimic NC using Lipofectamine 3000. After 24 h of transfection, the cells were collected and examined with a dual-luciferase reporter assay system (Promega, Wisconsin, USA).
2.11 Immunofluorescence-Fluorescent in situ hybridization (IF-FISH)
IF-FISH assay was executed to observe the location of miR-338-3p in astrocytes and BV2 cells. Briefly, cells were hybridized with specific Cy3-labeled miR-338-3p probes (Bersin Bio, Guangzhou, China) at 37°C overnight, and then the cells were incubated with primary GFAP or Iba1 antibody at 4°C overnight. On the following day, after washing for three times with PBS, the cells were then incubated with Alexa Fluor488 goat anti-mouse IgG (H + L) (Table 2) for GFAP or Iba1 at room temperature for 1h. Finally, the cells were counterstained with DAPI and examined under a confocal microscope.
2.12 Statistical Analysis
Data are represented as mean ± standard deviation (SD). The statistical analyses were carried out by using GraphPad Prism 9.4 (GraphPad Software, Inc., La Jolla, USA) and SPSS 22.0 (SPSS Inc., Chicago, IL, USA). The most appropriate statistical methods were used for different types of data. The data in Fig. 1d, e were analyzed by Pearson Correlation Analysis. The data in Fig. 1a-c, Fig. 2, Fig. 3, Fig. 4b-c, Fig. 5b-c, Fig. 7b-c, Fig. 8b-c were analyzed by t-test. The univariate-factor data in Fig. 4f-i, Fig. 5f-i, Fig. 6e-j, Fig. 7f-i, Fig. 8f-i were analyzed using one-way ANOVA because the data were homogeneity of variance. The data in Fig. 6b were analyzed using two-way ANOVA. p < 0.05 was considered as statistical significance.