Animals

C57BL/6 mice (weight 17–22 g, 6–8 weeks old) were purchased from the Animal Lab Center of Southern Medical University (Guangzhou, China). All animals were maintained in temperature-controlled conditions (temperature 23 ◦C–25 ◦C; humidity 50% ± 5%) with 12 h rhythm and fed with sterile food and water. All animal experiments were approved by the National Institutional Animal Care and Ethical Committee of Southern Medical University.

CLP animal models

C57 mice were anesthetized with isoflurane inhalation and their limbs were fixed. An approximately 2 cm incision was made along the middle of the abdominal wall of the mouse. The cecum was found and gently pulled out to the mesenteric vessels. The proximal cecal stool was gently squeezed to fill the cecum. Mesenteric surface vessels were then isolated and ligated with a 5-gauge sterile silk thread at a distance of 1 cm from the distal cecum. The cecum wall at the midpoint between the ligation and the distal cecum was pierced with a 21G sterile needle, and a little stool was gently squeezed. The cecum was returned to the abdominal cavity and sutured layer by layer with sterile No.4 surgical suture, and the postoperative mice were resuscitated with normal saline 50 mL/kg. In the sham-operation group, the cecum was explored by laparotomy without ligation or puncture.

To evaluate the role of HSPA8 in septic lung injury, mice were randomly divided into Sham, CLP, CLP + natural saline (NS), and CLP + GGA groups, with 3 mice in each group. In the CLP + GGA group, 800 mg/Kg GGA was used to gavage mice 2 h in advance, CLP modeling was then performed. The samples were taken 24 h after modeling for Co- immunoprecipitation (Co-IP) and Western blot analysis.

To observe the effect of HSPA8 agonist and inhibitor on the survival rate of CLP mice, C57 mice were randomly divided into three groups: CLP + DMSO, CLP + GGA, and CLP + VER155008, with 9 mice in each group. The mice were treated with 800 mg/Kg GGA by gavage 2 h before treatment, and 20 mg/kg VER155008 was injected intraperitoneally. The control group were given 1%DMSO intraperitoneally. Finally, a CLP model was established in each group of mice, the time of death in each group was recorded, and the survival rate was calculated.

Construction and transfection of adeno-associated virus

AAV9-HSPA8 (NM_031165) and AAV9-SKP2 (NM_013787) overexpressing adeno-associated viruses were constructed and tested by Genechem (Shangha, China). Normal C57 mice were pretreated with 1*10^11 v.g for 14 days, and then the CLP model was established according to the group. Lung tissue, alveolar lavage fluid and peripheral serum were collected 24 h after operation for subsequent detection. The expression efficiency was detected by Western blot and immunofluorescence (Fig.S4C-D; Fig.S5B-C).

Cell culture

Murine Lung Epithelial-12(MLE12, purchased from ATCC) cells. MLE12 cells were cultured in Dulbecco Modified Eagle medium (DMEM, Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum and 1% streptomycin/penicillin in a 37 ° C incubator containing 5% CO2. MLE12 cells were stimulated with LPS (10 µg/mL) for 24 h, and 5mM ATP was added to the new medium for 1 h to establish the cell model.

Isolation of primary ACE II

Mice were sacrificed by cervical dislocation and immersed in 75% alcohol for 5 min. The abdominal cavity was opened under sterile conditions, the bronchi were intubated, and the lungs were whitened by perfusion of PBS through the right ventricle to remove blood from the lungs. The lung tissue was then removed as soon as possible, trachea, bronchi, blood vessels and other tissues were removed in precooled PBS containing 1% streptomycin/penicillin. The PBS was washed repeatedly until clear.

The lung tissue was cut into 1 mm*1 mm*1 mm pieces and transferred to a 15mL centrifuge tube with 1 g/L trypsin added. The tube was incubated at 37 °C for 15 min, the digested cell suspension was transferred out, and an equal volume of DMEM medium containing 1% streptomycin/penicillin and 10% FBS was added to abrogate digestion.

Digestion of the remaining lung tissue was continued with trypsin and then incubated at 37 ° C for 15 min to remove the digestive fluid and stop digestion. Type I collagenase 1 g/L (2mL/ mouse) was added to the remaining lung tissue and incubated at 37 ° C for 15 min. The digestion solution was removed and the digestion was stopped with DMEM medium. The cell suspensions collected above were pooled and filtered through a 70 μm filter, followed by centrifugation at 1200 rpm/min for 5 min. Cells were transferred to IgG-coated dishes and incubated at 37 ° C for 60 min.

The suspension was aspirated and inoculated into another IgG-coated petri dish. The above step was repeated, and the non-adherent cells were aspirated, centrifuged at 1200 rpm/min for 5 min, seeded with cells, and then changed 2 to 3 times for further testing.

Plasmid transfection

The plasmid of overexpression HSPA8 and Myc-SKP2 and its negative control plasmid vector (pcDNA3.1) were constructed by GenePharma (Shanghai, China). The constructed plasmids were confirmed by amplification and sequencing. MLE12 and primary ACE II were then transfected with Lipo3000 reagent according to the manufacturer’s instructions. After 8 h, cells were changed to fresh medium containing 10% FBS for 48 h and then stimulated with LPS for 24 h. The expression efficiency was detected by Western blot (Fig.S4A-B).

RNA interference assay

An active oligonucleotide siRNA against HSPA8 or SKP2 was used for knockdown of HSPA8 or SKP2, with a scramble siRNA used as a Negative control. siRNA was transfected into MLE12 cells or primary ACE II with siRNA-Mate transfection reagent as indicated for 48 h and stimulated with LPS for 24 h, and the expression efficiency was determined by Western blot (Fig.S5A). Supplement Table 1 shows sequences of siRNAs against HSPA8 and SKP2.

Western blot

Proteins (30 µg) were extracted from lung tissue, MLE12 cells, and primary ACE II and separated by electrophoresis on a 10–12% SDS-polyacrylamide gel. The separated proteins were then transferred to a PVDF membrane. Membranes were blocked with TBST containing 5% skim milk powder for 1 h at room temperature. The membranes were incubated with the corresponding primary antibody overnight at 4 ° C and with horseradish peroxidase-coupled (HRP) secondary antibody for 1 h at room temperature. Specific bands were detected using Pierce ECL western blotting substrate. All samples were normalized by GAPDH and analyzed using ImageJ software.

Co-IP analysis

For Co-IP, whole cell and lung tissue extracts were lysed in IP buffer containing 1% phosphatase inhibitors, protease inhibitors, and PMSF. After centrifugation at 12,000 g for 15 min, the supernatant was removed and incubated overnight at 4 ° C with the corresponding IP monoclonal antibody, followed by Protein A + G magnetic beads for 3 h. Magnetic beads bound to proteins and antibodies were washed three to five times repeatedly with IP lysates. IP was denatured by boiling elution of IP lysate containing 1%SDS loading buffer. The samples collected were stored in the − 80 °C or -20 °C refrigerator for subsequent Western blot analysis.

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from MLE12 cells and lung tissue by Trizol reagent. 500ng RNA was reverse transcribed into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (+ gDNA wiper) kit, where genomic DNA was removed by DNase. Real-time qPCR was performed using SYBR Green Master Real-time PCR on a 7500 Real-Time PCR System (Applied Biosystems). The Ct value of β-actin was used as an internal reference for NLRP3, IL-1β, Caspase1, Caspase11, HMGB1, IL-1α, TNF-α、IL-6 and IL-1β mRNA in each sample. The qPCR primer sequences are shown in supplement Table 2.

Histopathology

Fresh lung tissues were fixed with 4% paraformaldehyde, embedded in paraffin after 24 h, and 4 μm sections were taken for hematoxylin and eosin (H&E) staining. The sections were placed under a light microscope for imaging(Carl Zeiss, Germany). Lung injury score was performed with reference to previous literature [18]: 0, no injury; 1, 25% injury; 2, 50% injury; 3, 75% injury; and 4, 100% injury. Each injury was scored in ten randomly selected fields (200×) from each slide.

Immunohistochemistry (IHC)

For immunohistochemical staining of HSPA8 in lung tissue, lung tissue sections were deparaffinized and antigen repaired, and nonspecific binding sites and endogenous peroxidase were blocked with 5% BSA and 0.3% H2O2, respectively. Lung tissue sections were incubated with HSPA8 primary antibody (1:200) overnight at 4 ° C, with goat anti-rabbit IgG for 1 h at room temperature, incubated with DAB (Dako, K5007), counterstained with hematoxylin, and dehydrated in ethanol. Finally, the sections were observed under a light microscope (Carl Zeiss Germany).

Immunofluorescence staining

MLE12 cells and primary ACE II in confocal dishes were fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.1% Tritonx-100 for 15 min, and blocked with goat serum for 1 h at room temperature. Next, cells were incubated with respective primary antibodies overnight at 4 °C and stained with Alexa Flour 488 or Alexa Flour 555 conjugated secondary antibodies for 1 h at room temperature in the dark, followed by DAPI (1:100) staining for 10 min. The cells were washed three times with PBS buffer for 10 min between each step. Finally, the stained cells were observed under a laser microscope.

Elisa

Mouse alveolar lavage fluid and serum were collected, and mouse IL-1β, TNF-α and IL-6 were detected by ELISA kit (R&D, USA) according to the instructions.

Statistical analysis

All data were analyzed by GraphPad Prism 8.0 software and presented as the mean ± SEM. The differences between groups were evaluated by two-tailed unpaired Student’s t-test and one way analysis of variance with the least significant difference (LSD) test or Dunnett T3 test for post hoc comparisons. P-values less than 0.05 were considered statistically significant.