Blood samples collecting

Between January 2019 and December 2021, 23 septic patients and 13 healthy volunteers were admitted to the Emergency Department at Xijing Hospital. The initial group, consisting of healthy controls (n = 3) and Sepsis patients (n = 3), was utilized for scRNA-seq. The subsequent group, comprising healthy controls (n = 10) and Sepsis patients (n = 20), were employed for cytology analysis (refer to Table S2 and S3). Inclusion criteria included: (1) diagnosis of sepsis upon emergency department admission; (2) adherence to Sepsis 3.0 criteria (infection with a SOFA score ≥ 2); (3) between 18 and 60 years old, and (4) consent to participate, provided personally or via legal representatives. Exclusion criteria encompassed: (1) length of stay < 1 day; (2) acute and chronic infectious diseases; (3) chemoradiotherapy within 3 months; (4) fatal trauma. All participants gave informed consent, and received ethical approval from the Ethics Committee of the First Affiliated Hospital of Fourth Military Medical University.

Single cell collection and single-cell RNA-seq

PBMCs were extracted from whole blood using Ficoll-Paque (TBD, Tianjin, China). The viability of the cells, exceeding 95%, was assessed using flow cytometry. A 10X Genomics GemCode Single-cell instrument was used to load cellular suspensions, resulting in single-cell Gel Bead-In-Emulsion (GEMs). Libraries were generated and sequenced from the cDNAs with Chromium Next GEM Single Cell 5’ Reagent Kits v3.1.

Bioinformatic analysis

Data quality control and genome alignment. In summary, reads with poor-quality barcodes and UMIs were excluded and subsequently aligned to the reference genome. Reads that were uniquely aligned to the transcriptome and overlapped an exon by at least 50% were taken into account for UMI counting. Prior to quantification, UMI sequences underwent error correction, and valid barcodes were identified using the EmptyDrops method. Cell by gene matrices were generated by counting UMIs and calling cell barcodes. Batch effects were corrected using the Harmony algorithm.

Cell clustering

Each sample’s cell by gene matrix was separately imported into Seurat version.

For downstream analysis

To analyze differentially expressed genes that are upregulated, the expression values of each gene within a specific cluster were compared to those in other cells using the Wilcoxon rank sum test. Various criteria were used to identify significantly upregulated genes. Initially, genes had to exhibit a minimum 1.28-fold overexpression in the target cluster. Secondly, they needed to be expressed in more than 25% of the cells in the target cluster. Finally, the p value should be less than 0.05. Gene Ontology (GO) is a globally recognized system for classifying gene functions, providing an evolving controlled vocabulary and precise definitions to thoroughly describe the characteristics of genes and their products across all organisms.

Primary human T cell isolation and lentiviral transduction

5 mL peripheral blood was extracted from each patient and healthy volunteer. PBMCs were isolated from the buffy coats samples using Lymphoprep density separation Invitrogen (Ficol, Kewei) density gradient centrifugation according to the manufacturer’s protocol, then isolated using a pan T cell isolation kit. T cells were resuspend in RPMI-1640 medium supplemented with 10% FBS. CD3/CD28-coated Dynabeads (Gibco) were used to activate cells in complete RPMI medium containing 100 IU/ml IL-2 for 2 days. Cells were then transduced with a concentrated lentiviral cocktail, infected 10 times per virus, and incubated for 1 h at 1800 g, 32℃ on RetroNectin (Takara)-coated plates.

Cell culture

EL4 cells were obtained from the American Type Culture Collection (ATCC). They were cultured in complete Dulbecco’s modified Eagle medium (Invitrogen, Carlsbad, CA, USA) with an addition of 10% fetal bovine serum (Sigma Chemical Co., St. Louis, MO, USA) and 1 × antibiotic/antimycotic (Invitrogen). Cells were grown in a humidified atmosphere containing 5% CO2 at 37 ℃. Once the cells covered 65% of the culture dish, they were exposed to LPS at a concentration of 10 µg/mL. RNA and protein were collected 48 h post-treatment. Afterward, the cell supernatants were gathered and preserved at − 70℃ for cytokine analysis.

Cell transfection

In order to knockdown the expression of SNHG5, miR-324-5p and CDK16, 50 nM of si-SNHG5 or si-NC/ad-SNHG5 or ad-NC, along with 100 nM of miR-324-5p inhibitor or inhibitor NC and ad-CDK16 or ad-NC, were transfected into cells, with all materials provided by HANBIO Technology Co. Ltd., Shanghai). EL4 cells were exposed to adenoviruses for 8 h, after which the media was replaced with fresh media. The cells were then collected 48 h later for further experiments.

Assessment of cell viability and proliferation

To assess EL4 cell viability and proliferation, the Cell Counting Kit-8 (CCK-8, Beyotime, Shanghai, China) was utilized. In short, 100 μL of EL4 cell suspension (1 × 105 cells/mL) was placed in 96-well plates and incubated at 37 °C with 5% CO2 for 24 h. Then, 10 μL of the CCK-8 solution was added to the plates for 1-h incubation periods at 24, 36, 48, and 60 h, respectively. Cell proliferation was estimated by measuring the OD value at a 450 nm excitation wavelength using a microplate reader (Bio-Rad 680, Hercules, CA, USA).

Animal experiments

The Animal Care Committee of Fourth Military Medical University (Xi’an, China) approved the animal experiments, which were carried out following their strict guidelines. The university’s animal center supplied 60 male BALB/c mice, aged 6 to 8 weeks, which were housed under standard conditions. Out of these, 30 mice were randomly divided into three experimental groups, with 10 mice in each group: sham control, CLP model, and CLP + sh-SNHG5. A total of 30 mice were used to track survival over a 48-h timeframe, and survival curves were created. An additional 30 mice were randomly split into three groups, with each group containing 10 mice: sham control, CLP model, and CLP + sh-SNHG5. As previously described, sepsis was triggered using CLP surgery, and treatments were given right after the operation (Shen et al. 2023, 2025). Lung tissues, serum, and bronchoalveolar lavage fluid (BALF) were gathered 24 h after CLP to assess the treatment effects.

Flow cytometry

Flow cytometry was conducted using the CytoFLEX system from Beckman Coulter, USA. The data were analyzed with FlowJo V10 software. The antibodies employed in the flow cytometry process included: human CD4 (1:20, 300,528, BioLegend), human CD8 (1:40, 344,706, BioLegend). 1 × 106 cells stained for 30 min at 4 °C in the dark. Additionally, Annexin Ⅴ (1:40, 640,951, BioLegend) and its binding buffer (Cat# 422,201, BioLegend), 7-AAD (1:20, Cat# 420,404, BioLegend) was used for apoptosis detection. To stain for apoptosis, cells were gathered, rinsed three times with PBS, and then incubated with Annexin V in its binding buffer (5 μL Annexin V in 100 μL binding buffer) for 15 min at room temperature. For intracellular staining, cells were fixed and permeabilized using the Cyto-Fast™ Fix/Perm Buffer Set as per the manufacturer’s guidelines (Cat. No.426803, BioLegend).

ROS and 8OHdG activity of T cells

The intracellular ROS levels in lymphocytes were assessed by oxidizing 7-dichlorodihydrofluorescein diacetate (DCFH-DA) to 2,7-dichlorodihydrofluorescein, using a ROS detection kit from Boster, China, following the manufacturer’s instructions. 8OHdG immunofluorescence staining using an anti-8-oxo-dG Monoclonal antibody (Abcam, USA) was employed to detect DNA damage, adhering to the suggested protocols. The mean fluorescent intensity (MFI) was used to measure intracellular ROS levels.

RT-qPCR

RT-qPCR was used to determine the expression patterns of Bax, Bcl-2, lncRNA SNHG5, miR-324-5p, and CDK16 in mouse PBMCs or EL4 cells. The complete RNA was isolated from the PBMCs or cells following the TRIzol reagent (Invitrogen) instructions precisely. First, mRNA was converted into cDNA with the PrimeScript RT Master Mix (Yeasen, Shanghai, China). Then, miRNA was reverse transcribed into cDNA using the PrimeScript ll 1 st Strand cDNA Synthesis Kit. The SYBR Premix Ex Taq kit from Yeasen was used for real-time PCR on the ABI 7500 system by Applied Biosystems, with actin serving as the internal control. Using the 2 − ΔΔCt method, the gene’s relative expression was determined, and the RT-qPCR primers are shown in the Table S1.

Western blot analysis

The Caspase3, Cle-Caspase3, Bcl-2, Bax, and CKD16 protein levels were determined by Western blotting. The cells were lysed with radioimmunoprecipitation and lysis solution (AccuRef Scientic, China) containing protease inhibitors, and then the protein concentration was measured with the BCA protein assay kit (AccuRef Scientic, China). The separation of proteins (30 μg per well) was achieved through sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by their transfer to polyvinylidene fluoride membranes(Thermo Fischer Scientific), followed by blocking with BSA for 1 h at 4 °C. Following a rinse, the membranes were treated with the primary antibodies: Caspase3(at a dilution ratio of 1: 200, TU409364, Abmart), Cle-Caspase3(at a dilution ratio of 1: 2000, #9664, CST), Bcl-2(at a dilution ratio of 1: 200, TA6139, Abmart), Bax(at a dilution ratio of 1: 200, T40051, Abmart), and CKD16 (at a dilution ratio of 1: 200, PHT2648, Abmart) and Anti-beta Actin (at a dilution ratio of 1: 1000, ab8227). Subsequently, the membranes were incubated with horseradish peroxidase-linked IgG (diluted 1:2000, ab6721, Abcam) at 24 °C for one hour. The blots were visualized using the Pierce ECL Plus Substrate (AccuRef Scientific, China). The imaging analysis system (Bio-Rad) was used to capture digital images. ImageJ software was then employed to analyze the bands, with their intensity being normalized to β-actin.

Dual-luciferase reporter gene assay

The interactions between lncRNA SNHG5 and miR-324-5p, as well as between miR-324-5p and CKD16, were confirmed through a dual-luciferase reporter gene assay. Fragments of SNHG5 and CKD16, both wild type (WT) and mutant type (MUT), containing miR-324-5p binding sites, were cloned into the pMIR-reporter vector (HANBIO Technology Co. Ltd., Shanghai). The dual-luciferase reporter plasmids that were constructed were co-transfected with either mimic-NC or miR-324-5p-mimic into HTR-8/SVneo trophoblasts. After 48 h, the cells were collected, lysed, and analyzed using the luciferase assay system for analysis of the luciferase activity.

Statistical analysis

Excel 2019 was used for data collation and SPSS 25.0 software was applied for statistical analysis while the GraphPad Prism 8.0 software was applied for graphing. The experimental data were expressed as cases and percentages. Data following a normal distribution were shown as the mean and standard deviation (SD), and group differences were assessed using a t-test. For data that were not normally distributed, the median and interquartile range (IQR) were used, and differences between groups were assessed using non-parametric tests. The adoption rate or constituent ratio was used to describe categorical variables, and the χ2 test assessed group differences. Statistical significance was set at P < 0.05.