Sirt3 regulates NLRP3 and participates in the effects of plantainoside D on acute lung injury sepsis

Proteomic analysis results

The changes of lung tissue proteins in septic ALI mice and control mice were analyzed by proteomics. The results showed that 38 differential proteins were identified. Among them, 18 proteins were up-regulated and 20 proteins were down-regulated (Figure 1B). Through bioinformatics technology, it was found that Sirt3 was the protein with the most obvious changes, and it was significantly down-regulated (Figure 1A). In order to further verify the expression of Sirt3, we detected their mRNA and protein levels by PCR and Western blot. The results showed that compared with control mice, the mRNA level of Sirt3 was significantly decreased (Figure 1C) and the protein levels of Sirt3 was significantly decreased (Figure 1D, 1E).

Figure 1. Proteomic analysis of protein differences in sepsis with acute lung injury. (A) Differential protein volcano map analysis; (B) Up and down regulation of protein; (C) mRNA levels of Sirt3; (D, E) protein levels of Sirt3. (N=6) All the data was presented as mean ± SD. Compared with control group: ^#P<0.05, ^##P<0.01.

Effect and mechanism of PHL on ALI in vivo and in vitro

In sepsis mice

The effects of PD on the w/d, ROS and MPO in sepsis mice. As shown in Figure 2A–2C, the levels of w/d, ROS and MPO were markedly increased in model group than control group. Compare with LPS group, Sirt3 KO significantly increased levels of w/d, ROS and MPO. While, compared with LPS and Sirt3 KO groups, PD significantly decreased the levels of w/d, ROS and MPO.

Figure 2. The effects of PD on ROS, w/d, MPO and oxidative stress. (A–C) ROS, w/d and MPO (n=6); (D–F) SOD, MDA and GSH-Px in serum (n=6); (G–I) SOD, MDA and GSH-Px in serum (n=6). All the data was presented as mean ± SD. Compared with control group: ^#P<0.05, ^##P<0.01. Compared with model group: ^*P<0.05, ^**P<0.01.

The effects of PD on oxidative stress, inflammation and lung histopathology in sepsis mice. As shown in Figure 2D–2I, the levels of MDA in serum and lung tissues were increased, SOD and GSH-px in serum and lung tissues were decreased compared to the control group.

Further, Compare with LPS group, Sirt3 KO significantly decreased SOD and GSH-px in serum and lung tissues, increased MDA in serum and lung tissues. Compare with LPS group, Sirt3 KO significantly decreased the levels of SOD, GSH-px and increased MDA. While, compared with LPS and Sirt3 KO groups, PD significantly decreased MDA and increased SOD and GSH-px in serum and lung tissues.

The histopathological test results of lung tissue after HE staining are shown in Figure 3A, which shown that the control group has regular lung tissue morphology, clear and complete alveolar structure, and no obvious bleeding or inflammatory cell infiltration, alveoli collapse, alveolar wall and alveolar septum thickening and inflammatory cell infiltration in lung tissue of ALI model group mice. Compare with LPS group, the above changes were more serious in Sirt3 KO group. While, compared with LPS and Sirt3 KO groups, PD significantly restored those changes.

Figure 3. The effects of PD on lung histopathology and cytokine in sepsis mice. (A) Representative heart tissue sections photomicrographs for hematoxylin-eosin (HE) staining. Original magnification: 200, scale bar: 50 μm. (B–D) The levels of TNF-α, IL-1β, IL-6 in serum. (E–G) The levels of IL-6, TNF-a and IL-1β. All the data was presented as mean ± SD. Compared with control group: ^#P<0.05, ^##P<0.01. Compared with model group: ^*P<0.05, ^**P<0.01.

In order to evaluate inflammatory reaction, cytokines (TNF-α, IL-1β and IL-6) were detected. As expected, the levels inflammatory cytokines TNF-α, IL-1β, IL-6 were increased in serum (Figure 3B–3D) and lung (Figure 3E–3G) in sepsis mice compared with control group. Compare with LPS group, Sirt3 KO significantly increased the levels of cytokines TNF-α, IL-1β, IL-6 in serum and lung, While, compared with LPS and Sirt3 KO groups, PD significantly decreased the levels of cytokines TNF-α, IL-1β, IL-6 in serum and lung.

The effects of PD on Sirt3/NLRP3 pathway associated protein in sepsis mice. As shown in Figure 4A–4F, compared with control group, the level of Sirt3 was significantly decreased, NLRP3, ASC, caspase-1 and IL-1β were increased. Compare with LPS group, Sirt3 KO significantly increased the levels of NLRP3, ASC, caspase-1, IL-1β. While, compared with LPS and Sirt3 KO groups, PD significantly increased the level of Sirt3, and decreased the levels of NLRP3, ASC, caspase-1 and IL-1β. The results of immunofluorescence were consistent with those of Western blot (Figure 4G).

Figure 4. The effects of PD on NOX4/ROS/NLRP3 pathway in sepsis mice. (A–F) The levels of NLRP3, ASC, Caspase-1 and IL-1β; (G) Immunofluorescence of NLRP3 in lung (n=3). Original magnification: 200, scale bar: 20 μm. All the data was presented as mean ± SD. Compared with control group: ^#P<0.05, ^##P<0.01. Compared with model group: ^*P<0.05, ^**P<0.01.

In MLE-12 cells

The effects of PD on the cell viability and ROS in LPS-induced MLE-12 cells. As shown in Figure 5A, 5B, the level of cell viability was decreased and ROS were markedly increased in model group than control group. Compare with LPS group, Sirt3 KO significantly increased levels of ROS and decreased cell viability. While, compared with LPS and Sirt3 KO groups, PD significantly decreased the levels of ROS and increased cell viability.

Figure 5. The effects of PD on oxidative stress and cytokine LPS-induced MLE-12 cells. (A, B) ROS and cell viability; (C–E) The level of SOD, MDA, GSH-Px; (F–H) The levels of TNF-a, IL-1β, IL-6. All the data was presented as mean ± SD. Compared with control group: ^#P<0.05, ^##P<0.01. Compared with model group: ^*P<0.05, ^**P<0.01.

The effects of PD on oxidative stress and inflammation in MLE-12 cells. As shown in Figure 5C–5E, the level of MDA in MLE-12 cells was increased, SOD and GSH-px in in MLE-12 cells were decreased compared to the control group. Further, Compare with LPS group, Sirt3 KO significantly decreased SOD and GSH-px in MLE-12 cells, increased MDA in in MLE-12 cells. While, compared with LPS and Sirt3 KO groups, PD significantly decreased MDA and increased SOD and GSH-px in MLE-12 cells.

As expected, the levels inflammatory cytokines TNF-α, IL-1β, IL-6 were increased in MLE-12 cells (Figure 5F–5H) compared with control group. Compare with LPS group, Sirt3 KO significantly increased the levels of cytokines TNF-α, IL-1β, IL-6 in MLE-12 cells, While, compared with LPS and Sirt3 KO groups, PD significantly decreased the levels of cytokines TNF-α, IL-1β, IL-6 in MLE-12 cells.

The effects of PD on Sirt3/NLRP3 pathway associated protein in LPS induced MLE-12 cells. As shown in Figure 6A–6F, compared with control group, the level of Sirt3 was significantly decreased, NLRP3, ASC, caspase-1 and IL-1β were increased. Compare with LPS group, Sirt3 KO significantly increased the levels of NLRP3, ASC, caspase-1 and IL-1β. While, compared with LPS and Sirt3 KO groups, PD significantly increased the levels of Sirt3 and decreased the levels of NLRP3, ASC, caspase-1, IL-1β.

Figure 6. The effects of PD on Sirt3/NLRP3 pathway in LPS-induced MLE-12 cells and molecules docking results of PD and Sirt3. (A–F) The levels of NLRP3, ASC, Caspase-1 and IL-1β; (G, H) molecules docking results of PD and Sirt3. All the data was presented as mean ± SD. Compared with control group: ^#P<0.05, ^##P<0.01. Compared with model group: ^*P<0.05, ^**P<0.01.

Molecules docking results of PD and Sirt3

Molecules Docking technology is a convenient and effective means to explore the interaction between small molecules and target targets. Here, we used Vina 1.1.2 software to study the docking of compound PD with Sirt3 protein. As shown in the Figure 6G, 6H, the interaction diagram between the molecule PD and Sirt3 protein. The result that PD forms hydrogen bonds with arg-87, leu-67, arg-89, ile-70, asp-75, ser-88 and tyr-64 on Sirt3 protein. In addition, it also forms hydrophobic interaction with glu-65, ile-70, glu-65 and tyr-64, further strengthening the binding effect. A negative number of binding affinities indicates the possibility of binding. Generally, a value less than -6 kcal/mol is considered to be more likely to bind. In this complex, the binding affinity score given by docking software was -6.3 kcal/mol, which means that PD has potential active effect on Sirt3.

Reagents

PD, specification: 20 mg, purity: > 98%, batch number: 2021-0576, produced by Sichuan Vicky biological company. Hematoxylin eosin (HE) staining solution was purchased from Solarbio Company, USA. Fetal blood serum, phosphate buffer (PBS) and DMEM medium were purchased from Hyclone Company, USA. Transfection reagent Transit-X2 was purchased from Shenzhen Minas Bio-Tech Co., Ltd., USA. Endotoxin-free plasmid small amount extraction kit was purchased from Qiagen Company of Germany. BCA protein quantitative kit was purchased from Beijing Pulilai Company, China and Western blot kit was purchased from Promega Company, USA. LPS was purchased from Sigma-Aldrich Company, USA. Diamino polyethylene glycol was purchased from Yarebio Company, China. All antibodies were purchased from Cell Signaling Technology Company, USA.

Animals

WT mice (ICR, male, 6 weeks, 16-18g), Sirt3 KO mice (ICR, male, Sirt3^-/-, 6 weeks, 16-18g) were obtained from the Cyagen Biosciences Inc. All animals’ operations were approved by the Research Council and Animal Care and Use Committee of Animal Center of Suzhou University.

Proteomics in ALI mice

Mice were divided into two groups, control mice and ALI mice. The ALI mice were administrated by intraperitoneal injection of lipopolysaccharide 10 mg/kg. The control group was given the same amount of saline in the same way. After 24 hours, the lung tissues of mice were collected for proteomic detection. Specific operation method according to previous reports [11].

Establishment of ALI mouse model

ALI model of mice was established by intraperitoneal injection of lipopolysaccharide (LPS, 10 mg/kg). The WT mice (control group) was given the same amount of saline in the same way. Mice were divided into the following groups: WT mice (control), LPS, Sirt3 KO + LPS, Sirt3 KO + LPS + PD (50 mg/kg). After 24 hours of intervention, PD (50 mg/kg) (intraperitoneal injection) was given to mice by gavage for 7 consecutive days.

In vitro model establishment

Lung epithelial cell line MLE-12 cells were seeded at 6-well-plate at a density of 1×10⁶ cells/well for 24 h. After that, the cells were divided into these groups: control, LPS (model, 50 μM), LPS + Sirt3 siRNA, LPS + Sirt3 siRNA + PD (5 μmol/L). The cells were treated with PD (5 μmol/L) for 24h, and 50 μM LPS were co-incubated for 24h, while the control group was challenged with an equal PBS. Cell supernatant and cells were collected for subsequent detection.

Cell transfection

The MLE-12 cells were inoculated in a 6-well plate with 5×10⁴ cells per well, and cultured in DMEM containing 10% fetal bovine serum in an incubator based on 37° C and 5% CO2. When the cells grew to 50%-70% fusion degree, various shRNA were transfected with Translipid HL Transfection Reagent with a final concentration of 80 nmol/L according to the instructions of Translipid HL Transfection Reagent. Before transfection, the cells were cultured in serum-free DMEM medium 2 mL for 2 h, and after transfection for 6h, the conventional DMEM medium containing 10% fetal bovine serum was changed for further culture. Six hours after transfection, the expression of green fluorescent protein in MLE-12 cells was observed under fluorescence microscope. Ten low-power fields were randomly selected, and the transfection efficiency was evaluated by counting the percentage of transfected cells in the total number of cells. Western blot was used to evaluate the success of cell transfection. The small interfering RNA (siRNA) targeted for Sirt3 (5’-A C C C A G T G G C A T T C C A G A C3’, 5’-G G C T T G G G G T T G T G A A A G A A G-3’ and GAPDH, 5’-G C A C C G T C A A G G C T G A G A C-3’, 5’-T G G T G A A G A C G C C A G T G G A-3’ both synthesized by Sigma-Aldrich Company, USA, was designed on the basis of the Thomas Tuschl protocol. Lyophilized single-stranded RNA oligonucleotides were resuspended in sterile RNase free water (100 μM), denatured (heated at 95° C for 5 min), aligned and slowly annealed with decreasing temperature, resulting in the formation of double-stranded siRNA at 50 μM.

Measurement of wet-dry ratio of lung (w/d)

The right lung of mice was removed. The trachea and esophagus were separated passively, the right lung lobe was obtained, and the wet weight was measured immediately. Subsequently, the lungs were dried at 60° C for 72 hours to remove all moisture, and then the dried lungs were weighed and the wet weight/dry weight ratio of the lungs was calculated.

ROS and MPO test

10 mg of lung tissue was added with 1:10 normal saline, centrifuged at 12000r/min for 10min, and the supernatant was collected and detected by MPO and ROS commercial kit. The operation process strictly followed the instructions of the kits.

Detection of oxidative stress

The levels of SOD, MDA and GSH-Px were detected by commercial kits, and the operation method was carried out according to the instructions.

Cytokine measurement

The concentrations of IL-6, IL-1β and TNF-α in serum, lung tissues and cell supernatant were analyzed by enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions.

Hematoxylin-eosin (HE) staining

The lung was fixed in 4% formalin solution, embedded in paraffin, cut into 4μm thick sections, and placed on a glass slide for HE staining.

Cell viability and ROS assay

MLE-12 cells used for cell viability, the medium was removed and 200 μL 0.5 mg/ml MTT was added to each well for 4 h. The supernatant was discarded and added 150 μl DMSO to each well. The absorbance value of each well was determined at 490 nm with a microplate spectrophotometer. For ROS assay, the detection was carried out according to the instructions of the kit.

Immunohistochemistry

Lung tissue slices were baked at 60° C for 1 h, then paraffin was removed by xylene, dehydrated by gradient ethanol and heated by sodium citrate buffer for antigen repair. After natural cooling to room temperature, it was cultured with 3% hydrogen peroxide for 10 min. Each section was sealed with 3% BSA at room temperature. After removing the blocking solution, the section was incubated with the primary antibody at 4° C overnight, the secondary antibody was incubated for 10 min, and PBS was washed three times, each time for 3 min, the third antibody was incubated for 10 min and washed with PBS for 3 min each time. Samples were stained with DAB and hematoxylin, dehydrated by gradient ethanol and xylene, dried, sealed with neutral resin, and the expression of related proteins in lung tissue was observed under light microscope.

Western blot analysis

Total protein was extracted from the lysate of lung tissue and MLE-12 cells, and the protein content was determined by BCA method. SDS-PAGE electrophoresis was performed on protein samples and transferred to PVDF membrane. The 5% skimmed milk powder was sealed at room temperature for 2 h, and incubated with related equal primary antibodies respectively. The membrane was rinsed with TBST and then reacted with horseradish peroxidase coupled secondary antibody. The membrane was rinsed with TBST and then developed with enhanced chemiluminescence (ECL) luminescent reagent. The optical density of the main band was measured by gray-scale imaging software (UVP, UK) to calculate the expression level of the above proteins in lung tissue.

PD and Sirt3 molecular docking

The structures of Sirt3 and PD were drawn. After the energy of MM2 was minimized, it was further processed by Autodock tools1.5.6, and the torsion was set as the default value. Finally, it was saved as a pdbqt file for Autodock Vina to conduct molecular docking.

Statistical analysis

All data were expressed as mean ± standards deviation (SD) and analyzed by one-way analysis of variance (ANOVA) followed by Tukey multiple comparison test using GraphPad prism 5 (GraphPad Software, USA). A value of p<0.05 was considered statistically significant.

Availability of data and material

Data can be requested by the corresponding author.