Caecal ligation and puncture (CLP)-induced sepsis model

Animal experiments in this assay were ethically approved by the Third Xiangya Hospital of Central South University. Eighteen C57BL/6 mice (male, 23–25 g, 7 weeks old) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). The mice were kept at 25 °C with 12 h light each day in a single cage and supplied with regular diet and water. The abovementioned mice were randomly divided into three groups: (1) Sham group, (2) CLP group, and (3) CLP + Rg3 group (n = 6). The CLP-induced sepsis model was established in accordance with previous reports [23, 24]. Mice in the sham group were subjected to laparotomy without ligation and perforation. After intraperitoneal inoculation with Rg3 (20 mg/kg) (Sigma–Aldrich, St. Louis, MO, USA) for 1 h, mice in the CLP + Rg3 group underwent CLP. At 6 h post CLP treatment, all animals were sacrificed, and livers were resected for subsequent assays.

Haematoxylin-eosin(H&E) staining

This assay was implemented to detect liver tissue injury. After fixation with 10% formaldehyde for 2 days, live tissues were embedded in paraffin. Generated paraffin sections (4 μm) were dyed with an H&E staining kit (Solarbio, Beijing, China) according to the manufacturer’s instructions and then observed under a light microscope (magnification: 100 ×).

Cell culture and LPS treatment

Human primary hepatocytes (Corning Inc., Corning, NY, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; HyClone, Logan, UT, USA) supplemented with 10% FBS (HyClone) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA).

LPS (1 μg/mL, Sigma–Aldrich) was added to treat cells for 12 h until 80% confluence, and the cells were recorded as LPS group. 100 mM stock solution of Rg3 was prepared with dimethyl sulfoxide (DMSO) and diluted to the indicated 25 μM concentration with culture medium before treatment. For cells in the LPS + Rg3 group, cells pretreated with 25 μM Rg3 for 6 h were exposed to 1 μg/mL LPS.

Cell transfection

Small hairpin RNA (shRNA) targeting lncRNA TUG1 (sh-TUG1) was transfected into human primary hepatocytes to silence its expression, with sh-NC as a negative control. The total sequence of TUG1 was inserted into the pcDNA3.1 vector to generate a TUG1-overexpressing vector (pcDNA3.1-TUG1), and pcDNA3.1-NC was used as a negative control. miR-200a-3p mimics were introduced into human primary hepatocytes to upregulate miR-200a-3p expression, with miR-NC as a negative control. The above oligonucleotides and plasmids were obtained from GenePharma (Shanghai, China). Cell transfection assays were conducted using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s directions. After 48 h, transfection efficiency was determined by qRT–PCR assays.

Isolation of mitochondria

Mitochondria derived from human primary hepatocytes were extracted using the Mitochondria Isolation Kit (Cat. NO. 89874, Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the user’s manual and then kept on ice before downstream processing.

Western blot analysis

Radioimmunoprecipitation assay (RIPA) buffer (CWBIO, Beijing, China) was utilized to prepare protein samples according to the manufacturer’s instructions. After determining the concentration of samples with a bicinchoninic acid assay (BCA) kit (Sigma–Aldrich), 30 μg protein samples were separated through 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and then transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) by a wet electrophoretic transfer approach. After blockage with 5% bovine serum albumin (BSA) (Sigma–Aldrich) at room temperature for 1 h, membranes were incubated with primary mouse antibodies against mitochondrial respiratory chain-related proteins Complex I (ab110245, 1:1000 dilution, Abcam, Shanghai, China), Complex II (ab110410, 1:1000 dilution, Abcam), and OPA1 Mitochondrial Dynamin Like GTPase (OPA1) (ab119685, 1:2000 dilution, Abcam), autophagy-related proteins light chain 3 (LC3) (ab243506, 1:3000 dilution, Abcam), P62 (ab56416, 1:3000 dilution, Abcam), and Beclin-1 (ab118148, 1:2000 dilution, Abcam), mitochondrial biogenesis related transcription factors peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1-α) (ab77210, 1:3000 dilution, Abcam), Nuclear Respiratory Factor 1 (NRF-1) (ab55744, 1:2000 dilution, Abcam), and Transcription Factor A (Tfam) (ab119684, 1:1000 dilution, Abcam), SIRT1 (ab110304, 1:3000 dilution, Abcam), AMPK (ab80039, 1:3000 dilution, Abcam), Acetyl-CoA Carboxylase (ACC) (sc-137,104, 1:2000 dilution, Santa Cruz), p-ACC (sc-271,965, 1:1500 dilution, Santa Cruz), Voltage Dependent Anion Channel 1 (VDAC1) (ab186321, 1:5000 dilution, Abcam), β-Actin (ab8226, 1:5000 dilution, Abcam), or primary rabbit antibody against p-AMPK (Cat. NO. PA5–110151, 1:2000 dilution, Invitrogen) at 4 °C overnight and then probed with goat anti-mouse immunoglobulin G (IgG) H&L (HRP) (ab205719, 1:8000 dilution, Abcam) or goat anti-rabbit IgG H&L (HRP) (ab205718, 1:8000 dilution, Abcam) secondary antibodies at room temperature for 2 h. Subsequently, protein signals were generated by using an enhanced chemiluminescence (ECL) Western blot detection system (Millipore) and analysed by ImageJ software (NIH, Bethesda, MD, USA). Anti-VDAC1 or anti-β-Actin acted as internal controls for mitochondria and cytoplasm, respectively.

Quantitative real-time polymerase chain reaction (qRT–PCR)

Live tissues or treated human primary hepatocytes were exposed to TRIzol reagent (Invitrogen) for extraction of total RNA. To detect lncRNA TUG1 and SIRT1 expression, total RNA was subjected to complementary DNA (cDNA) synthesis utilizing M-MLV Reverse Transcriptase (Invitrogen), and then qRT–PCR assay was performed with SYBR Master Mix (Applied Biosystems, Foster City, CA, USA). For the miR-200a-3p expression assay, cDNA was generated using a TaqMan miRNA Reverse Transcription Kit (Applied Biosystems), followed by qRT–PCR assay utilizing a miRNA-specific TaqMan MiRNA Assay Kit (Applied Biosystems). All primers involved in the qRT–PCR assay were listed in Table 1. The relative expression was determined via 2-ΔΔCt method. GAPDH and U6 were used as the internal parameters for genes (TUG1 and SIRT1) and miR-200a-3p, respectively.

Table 1 Primers used for qRT-PCR assayCell counting Kit-8 (CCK-8) assay

After treatment with LPS or LPS + Rg3, human primary hepatocytes were plated in 96-well plates (5 × 103 cells/per well). Then, 10 μL CCK-8 solution (Sigma–Aldrich) was added to each well. After 1 h of incubation with CCK-8 solution, cell viability was evaluated by measuring the absorbance at 450 nm using a microplate reader.

Reactive oxygen species (ROS) measurement

MitoSOX™ Red reagent (Cat. NO. M36008, Thermo Fisher Scientific) was applied to detect mitochondrial ROS level in human primary hepatocytes based on the protocol supplied by the manufacturer. Human primary hepatocytes after treatment and/or transfection were washed by PBS twice, then incubated with l μM MitoSOX Red for 20 min. Subsequently, cells were trypsinized and suspended in PBS. Later, a fluorescence spectrophotometer was used to assess fluorescent intensity (Ex/Em = 510/580 nm).

Detection of mitochondrial transmembrane potential (MTP)

JC-1 (CBIC2) dye (ab141387, Abcam) was employed to measure the MTP of human primary hepatocytes. JC-1 (10 μM) in DMEM was added to human primary hepatocytes at 37 °C for 20 min, followed by measurement of JC-1 aggregate fluorescence (red) (Ex/Em = 488/583 nm) and JC-1 monomer fluorescence (green) (Ex/Em = 488/525 nm) under a fluorescence microscope.

Bioinformatic analysis

In the present study, the putative binding site between miR-200a-3p and TUG1 or SIRT1 was analysed using the starBase online database (http://starbase.sysu.edu.cn/).

Dual-luciferase reporter assay

For the dual-luciferase reporter assay, wild-type luciferase reporter plasmids (TUG1-WT and SIRT1-WT) containing predicted binding sites with miR-200a-3p, as well as mutant plasmids (TUG1-MUT and SIRT1-MUT) containing mutant binding sites, were established by RIBOBIO (Guangzhou, China). Subsequently, human primary hepatocytes were cotransfected with 100 ng luciferase reporter plasmid and 40 nM miR-NC or miR-200a-3p mimic. At 48 h post transfection, the relative luciferase density was evaluated utilising a Dual-Luciferase Reporter detection System (Promega, Shanghai, China) and normalised to Renilla luciferase activity.

RNA immunoprecipitation (RIP) assay

RIP assay was carried out to validate the binding efficiency between lncRNA TUG1 and miR-200a-3p using the Magna RIP Kit (Millipore) following the recommended instructions. Human primary hepatocytes were subjected to lysis in specific lysis buffer. Generated cell lysate was mixed with magnetic beads conjugated with anti-Argonaute-2 (Ago2) antibody or anti-immunoglobulin G (IgG) antibody overnight on a shaker. Afterwards, proteinase K was added to purify RNA. Enrichment of lncRNA TUG1 and miR-200a-3p was measured by qRT–PCR assays.

Statistical analysis

Quantitative data derived from at least 3 independent experiments were presented as the mean ± standard deviation (SD). Statistical significance in groups was determined by the Mann–Whitney U test (for 2 groups) or Kruskal–Wallis-Wallis test (for 3 or more groups). A P value < 0.05 was defined as statistically significant.