Simvastatin (Sigma-Aldrich; Merck KGaA) was dissolved in DMSO to make a 50 mM stock solution, which was maintained at 4°C before use. The MTT and 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) assays were purchased from Sigma-Aldrich (Merck KGaA) and Nanjing KeyGen Biotech Co., Ltd., respectively. Antibodies targeted against Beclin 1, LC3 and GAPDH were purchased from Abcam. RPMI-1640 and FBS were purchased from Gibco (Thermo Fisher Scientific, Inc.). ELISA kits were purchased from Hangzhou Multi Sciences (Lianke) Biotech Co., Ltd.

Peripheral venous blood samples (10 ml) were obtained from six healthy volunteers (HP) and six patients with gram-negative SP, who were admitted to the ICU of the Department of Anesthesiology of The First Affiliated Hospital of Soochow University (Suzhou, China) between January 2019 and December 2019. All study participants or their relatives (age, ≥18 years) provided written informed consent in accordance with the Declaration of Helsinki. The present study was approved by the Institutional Review Board and Ethics committee of The First Affiliated Hospital of Soochow University (approval no. 138/2018). The inclusion criteria were as follows: i) Age, ≥18 years; and ii) diagnosed with SP based on the third international consensus definitions for sepsis and septic shock (Sepsis-3) (15). Furthermore, the exclusion criteria were as follows: i) HIV infection; ii) organ transplantation; and iii) pregnancy. The mean age of the participants was 55.0±10.9 years for the SP group and 57.8±10.5 years for the HP group. There were three males and three females in both the SP and HP groups. Venous blood samples were collected within 1 h after enrollment. Human neutrophils were isolated by discontinuous density gradient centrifugation as previously described (16). Briefly, the leukocyte-enriched pellets were collected by centrifugation at 250 × g for 6 min at room temperature. Subsequently, in a centrifuge tube, 3 ml 65% Percoll solution (Pharmacia Biotech) was layered onto 3 ml 75% Percoll solution, followed by the layering of 4 ml of peripheral blood. The tube was then centrifuged at 330 × g for 25 min at room temperature and the interface between the 75 and 65% layers was carefully aspirated. After hypotonic lysis of erythrocytes by RBC Lysis Buffer (Roche Diagnostics), neutrophils were isolated to reach 98% purity as determined by Giemsa staining. After isolation, 1×106 cells were fixed in methanol for 5 min at room temperature and stained with Giemsa solution for 1 min at room temperature and were subsequently imaged using light microscopy. The viability of neutrophils was >95% as indicated by Trypan blue staining. Briefly, neutrophil suspensions were added to an equal volume of 0.4% trypan blue solution in a microtube and mixed for 2 min on ice. Neutrophil viability was analyzed using a Neubauer chamber and an optic microscope.

Neutrophils were cultured in a humidified atmosphere containing 5% CO₂ at 37°C. Cells were adjusted to ~1×10⁶ cells/ml in RPMI-l640 medium supplemented with 10% FBS and 2 mM L-glutamine, and then plated in 96-well culture plates. Cells were treated with vehicle (DMSO) or simvastatin (5 µM) for 24 h. For inhibitory assays, cells were pre-incubated with the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenyleneiodonium (DPI; 20 µM) or the autophagy inhibitor 3-methyladenine (3-MA; 5 nM) for 1 h at 37°C before each experiment as previously described (17,18). 3-MA and DPI were both dissolved in DMSO and diluted to the working concentrations. DMSO was used as the negative control. Neutrophil media were centrifuged at 400 × g for 5 min at 4°C, and the supernatants were used for quantification of cytokines (TNF-α and IL-6).

Cell viability was detected using the MTT assay. After treatment for 24 h, MTT solution was added to the medium and incubated at 37°C for 4 h. Subsequently, the MTT solution was replaced with DMSO (200 µl) for 30 min at 37°C. The optical density was assessed using a microplate reader (Bio-Rad Laboratories, Inc.). Neutrophil viability was analyzed at different doses of SIM (0, 1, 5, 10 or 15 µM) for 24 h to assess the lethal concentration of SIM. The mean absorbance values were recorded and normalized to the values obtained for cells cultured with vehicle.

For intracellular ROS assessment, cells were seeded into plates (1×10⁵ cells/well). After treatment, cells were incubated with DCFH-DA (50 µM; Nanjing KeyGen Biotech Co., Ltd.) at 37°C for 20 min. DCF fluorescence was determined and analyzed using a flow cytometer (FACSVerse; BD Biosciences) and FlowJo version 10 (Tree Star, Inc.).

Cells were lysed with lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 1 mM EDTA and protease inhibitor cocktail). After centrifugation at 12,000 × g for 10 min at 4°C, the supernatants were collected. For western blotting, 30 µg of protein/lane was separated by SDS-PAGE on a 15% gel and then transferred to a PVDF membrane. After blocking with 5% non-fat milk in TBST (10 mM Tris, 150 mM NaCl and 0.1% Tween-20) for 1 h at 37°C, the membranes were probed with primary antibodies targeted against rabbit anti-Beclin-1 (1:2,000; cat. no. ab210498; Abcam), rabbit anti-LC3 (1:1,000; cat. no. ab192890; Abcam) or rabbit anti-GAPDH (1:1,000; cat. no. ab181602; Abcam) with gentle agitation overnight at 4°C. After washing with TBST, the membranes were further incubated with HRP-conjugated goat anti-rabbit secondary antibodies (1:5,000; cat. no. ab205718; Abcam) in blocking buffer for 1 h at room temperature. Specific protein bands were visualized using the ECL detection kit (Amersham; Cytiva). The blots were scanned, and the intensity of the obtained blot bands was semi-quantified and analyzed using Science Lab Image Gauge software (version 4.0; FUJIFILM Wako Pure Chemical Corporation).

The concentrations of TNF-α and IL-6 in the supernatants were determined using Human TNF-α Quantikine ELISA Kit (cat. no. DTA00D; R&D Systems, Inc.) and Human IL-6 Quantikine ELISA Kit (cat. no. D6050; R&D Systems, Inc.) according to the manufacturer's protocols. Absorbance was determined at a wavelength of 450 nm using a VersaMax microplate reader (Molecular Devices, LLC). The concentrations were calculated according to calibration curves.

Statistical analyses were performed using SPSS software (version 22.0; IBM Corp.). Data are presented as the mean ± SD from three replicates. Comparisons among multiple groups were analyzed using one-way or two-way ANOVA followed by Bonferroni's post hoc test. The unpaired Student's t-test was used for comparisons between two groups. P<0.05 was considered to indicate a statistically significant difference.

To investigate the viability of neutrophils in SP, an MTT assay was performed. The results demonstrated that the viability of neutrophils was significantly decreased in the SP group compared with that in the HP group (Fig. 1A).

Next, whether simvastatin could protect neutrophils from SP was investigated. To obtain a non-toxic dose of the simvastatin, a viability test was performed, in which the neutrophils from the HP group were treated with different doses of simvastatin (0, 1, 5, 10 or 15 µM) for 24 h to assess the lethal concentration. A significant decrease in neutrophil viability was observed following treatment with 10 or 15 µM simvastatin compared with that in untreated neutrophils (Fig. 1B). Moreover, there was no significant difference in viability between the 0 and 5 µM simvastatin groups, thus 5 µM was considered as the optimal concentration in the present study. Subsequently, neutrophils from the SP and HP groups were treated with simvastatin (5 µM), and the results indicated that the viability of neutrophils was 98.33% in the HP group, but 42.00% in the SP group (Fig. 2A). Moreover, simvastatin significantly inhibited the decreased viability of neutrophils in the SP group (42.00 vs. 71.33%, P<0.01).

Subsequently, ROS production in neutrophils was assessed. The results demonstrated that the neutrophils from the SP group displayed a significantly higher production of ROS compared with neutrophils from the HP group. In addition, the elevated level of ROS was abrogated with treatment by DPI, an inhibitor of NADPH oxidase (Figs. 1C and S1). Following treatment with simvastatin, neutrophils in the SP group displayed significantly decreased levels of ROS (223.33±30.86%) compared with those from the untreated SP group (606.67±38.24%, P<0.001; Fig. 2B). Furthermore, simvastatin did not affect ROS production in neutrophils from the HP group.

To investigate whether simvastatin had an effect on the inflammatory response in neutrophils, the concentrations of TNF-α and IL-6 in the supernatants of neutrophil-conditioned media were assessed. The results demonstrated that the concentrations of TNF-α and IL-6 were significantly higher in the SP neutrophil-conditioned media compared with those in the HP neutrophil-conditioned media (Fig. 3A and B). However, TNF-α and IL-6 concentrations were significantly reduced after treatment with simvastatin in septic neutrophils (SP vs. SP + Sim TNF-α, 124.67±5.51 vs. 89.33±8.14 pg/ml, P=0.003; SP vs. SP + Sim, IL-6, 142.67±4.04 vs. 115.33±4.73 pg/ml, P=0.002).

Subsequently, whether simvastatin affected autophagy in septic neutrophils was investigated. The conversion of LC3I to LC3II was significantly increased in neutrophils from the SP group compared with that in the HP group (Fig. 4A). Moreover, SP neutrophils exhibited a significant increase in Beclin 1 protein expression (another autophagy-related molecule) compared with HP neutrophils (P<0.001; Fig. 4B). Notably, treatment with simvastatin promoted the conversion of LC3I to LC3II and elevated the expression level of Beclin1 in SP neutrophils compared with the untreated SP group, and these effects were abrogated by treatment with 3-MA, a well-known autophagy inhibitor (Fig. 4C and D). These results indicated that simvastatin enhanced autophagy in neutrophils from the SP group.

To investigate the importance of autophagy activation in the protective effect of simvastatin, neutrophils were treated with 3-MA. The results demonstrated that 3-MA administration completely blocked the protective effect of simvastatin, as evidenced by the decreased viability of neutrophils compared with that in the SP group treated with simvastatin alone (SP + Sim vs. SP + Sim + 3-MA, 72.33±6.03 vs. 41.67±3.51%, P=0.002; Fig. 5A). Furthermore, the inhibition of ROS production by simvastatin was reversed by treatment with 3-MA in septic neutrophils (SP + Sim vs. SP + Sim + 3-MA, 217.67±18.23 vs. 566.67±31.34, P<0.001; Fig. 5B). Subsequently, the role of autophagy in the inflammatory regulation of simvastatin was examined. It was found that the suppression of autophagy using 3-MA eliminated the inflammatory inhibitory effect of simvastatin in septic neutrophils, as evidenced by the increased concentrations of IL-6 (SP + Sim vs. SP + Sim + 3-MA, 109.00±6.00 vs. 143.67±5.69 pg/ml, P=0.002) and TNF-α (SP + Sim vs. SP + Sim + 3-MA, 81.67±3.06 vs. 114.00±6.24 pg/ml, P=0.001; Fig. 5C and D).