The MPC5 cell line was purchased from the China Center for Type Culture Collection. The basic culture medium for this cell line was composed of 89% RPMI-1640 Medium (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Wuhan Servicebio Technology Co., Ltd.). The MPC5 cell line was maintained at a temperature of 37°C with 5% CO₂.

Cells were added to the culture medium following mixing in Opti-MEM for 15 min at room temperature (Thermo Fisher Scientific, Inc.), followed by the addition of antibiotic-free culture medium. pcDNA3.1-HO-1 was then transfected into MPC5 cells using EZ Cell Transfection Reagent (Life-iLab Biotech Co., Ltd.) at a 3:1 (µl/µg) ratio of reagent to DNA; the concentration of nucleic acid was 1,000 ng/µl and pcDNA3.1 was used as the negative control. pcDNA3.1-HO-1 was synthesized by Tsingke Biotech Co., Ltd., with 5′ HindIII and 3′ EcoRV restriction sites.

Small interfering (si)RNA targeting HO-1 (siHO-1) was transfected into MPC5 cells using Lipofectamine™ RNAiMAX (Thermo Fisher Scientific, Inc.). The final amount of siHO-1 used per well was 25 pmol, while the amount of transfection reagent was 7.5 µl. After 4–6 h of transfection at 37°C, the medium containing the EZ Trans complex was replaced with fresh medium, the cells were further cultured for 12 h and then treated with or without 0.2 mmol PA (Merck Corp) for 24 h. When the drug treatment was complete, the cell samples were used for protein and total RNA extraction. The following sequences were used: siRNA-HO-1 forward, 5′-GUUCAAACAGCUCUAUCGUTT-3′ and reverse, 5′-ACGAUAGAGCUGUUUGAACTT-3′; and siRNA negative control forward, 5′-UUCUCCGACAGUGUCACGUTT-3′ and reverse, 5′-ACGUGACACUGUCGGAGAATT-3′.

Cells were cultured without penicillin/streptomycin in 6-well plates for 24 h, then the culture medium was changed by RPMI-1640 Medium. Added 2 µl MCC950 (MedChemExpress) for 2 h, then 2 µl dapagliflozin were added for 4 h. After 48 h, the cells were used for fluorescent staining.

Following treatment with PA (0, 0.1, 0.2, 0.3 and 0.4 mmol) for 24 h at 37°C, the cell culture was centrifuged at 1,000 × g at 4°C for 20 min. Cell-free supernatant was collected for NLRP3 detection. NLRP3 content in the supernatant was measured using an ELISA kit (ELK Biotechnology Co., Ltd.), according to the manufacturer's instructions. Each sample was assayed in more than three technical replicates.

Cell Counting Kit-8 (CCK-8; Biosharp Life Sciences) assay was used to measure cell viability. When the cells had grown to 80–90% confluence, digestion was performed with 0.25% Trysin-EDTA. A total of 1×10⁵ MPC5 cell suspension (100 µl) was transferred into 96-well plates followed by treatment with different concentrations of PA or dapagliflozin for 24 h at 37°C. Subsequently, 10 µl CCK-8 solution was added to each well for 2 h. For the controls, the same concentration of CCK-8, culture medium and PA were added to a 96-well plate without the cells (29). Optical density was measured at 450 nm using a microplate reader (Synergy 2; BioTek China).

Total protein was extracted from 2.5×10⁶ MPC5 cells using RIPA lysis buffer (MedChemExpress) supplemented with 1% protease and phosphatase inhibitors for 20 min on ice, followed by centrifugation at 15,520 × g for 10 min at 4°C (30). The protein concentration was quantified using a BCA protein assay kit (Biosharp Life Sciences) and 25 µg protein/lane was separated by SDS-PAGE on a 12% gel. The separated proteins were then transferred onto a PVDF membrane (MilliporeSigma) and blocked with 5% nonfat dry milk for 1 h at room temperature. The membrane was incubated with primary antibodies against NLRP3 (cat. no. A12694, ABclonal Biotech Co., Ltd.), caspase-1 (cat. no. A0964, ABclonal Biotech Co., Ltd.), IL-18 (cat. no. A16737, ABclonal Biotech Co., Ltd.), IL-1β (cat. no. A11396&A16288, ABclonal Biotech Co., Ltd.) and HO-1 (cat. no. A19062, ABclonal Biotech Co., Ltd.), as well as a β-actin antibody (GB11001-100, Wuhan Servicebio Technology Co., Ltd.) used as the internal reference, for 10 h at 4°C. All antibodies were diluted at 1:1,000 in western blot primary antibody dilution buffer (ABclonal Biotech Co., Ltd.). Following primary incubation, the PVDF membrane was incubated with anti-rabbit IgG secondary antibody (cat. no. BL003A; 1:10,000; Biosharp Life Sciences) at room temperature for 1 h and then developed using a Pierce™ ECL chemiluminescence kit (Thermo Fisher Scientific, Inc.). The protein bands were exposed and observed in a multifunctional digital gel imaging analysis system and images were obtained (Bio-Rad GelDoc™ XR and ChemiDoc™ XRS System; Bio-Rad Laboratories, Inc.). Finally, ImageJ v1.51 (National Institutes of Health) was used for target protein expression analysis.

The cell culture medium was removed and the 2.5×10⁶ cells from 6-well plates were washed with 2 ml of 4°C pre-cooled PBS. The PBS was then thoroughly removed and 1 ml RNA extraction solution (Wuhan Servicebio Technology Co., Ltd.) was added to each sample in the 6-well plate, the bottom cells were gently shaken off the plate to fully contact the RNA extraction solution and the RNA extraction solution was then transferred to a 1.5-ml Eppendorf tube at room temperature for 5 min.

cDNA was synthesized using a reverse transcription kit (Novoprotein Scientific, Inc.), according to the manufacturer's instructions. The 2X SYBR Green qPCR Master Mix Kit (Wuhan Servicebio Technology Co., Ltd.) was used to quantify the relative mRNA levels of ASC, NLRP3, IL-18 and IL-1β (31). qPCR was performed using the qPCR System (CFX Connect; Bio-Rad Laboratories, Inc.) for 40 cycles, with GAPDH serving as internal controls. The sequences of primer pairs used for qPCR were as follows: NLRP3 forward, 5′-GCAGCCTCACCTCACACAGCT-3′ and reverse, 5′-TTTCACCCAACTGTAGGCTCT-3′; ASC forward, 5′-CTGACTGAAGGACAGTACCAG-3′ and reverse, 5′-TCCTGACTTTGTATACACAAT-3′; IL-1β forward, 5′-CTCACAAGCAGAGCACAAGC-3′ and reverse, 5′-AGCTGTCTGCTCATTCACGA-3′; IL-18 forward, 5′-GACTCTTGCGTCAACTTCAAGG-3′ and reverse, 5′-CAGGCTGTCTTTTGTCAACGA-3′; HO-1 forward, 5′-AAGGGAGAATCTTGCCTGGCT-3′ and reverse, 5′-ACCCCTCAAAAGATAGCCCCA-3′; GAPDH forward, 5′-AAGAAGGTGGTGAAGCAGGC-3′ and reverse, 5′-TCCACCACCCTGTTGCTGTA-3′.

The cultured cells were washed twice with PBS, followed by the addition of 1 ml cell staining buffer in the Hoechst 33342/PI double stain kit (Solarbio Science & Technology Co., Ltd.). Subsequently, 5 µl Hoechst 33342 staining solution and 5 µl PI staining solution were mixed and kept at 4°C for 20 min (32). Following staining, the cells were washed twice with PBS and observed with a 10Χ objective lens using a fluorescent microscope (IX71; Olympus Corporation).

GraphPad Prism 9 software (GraphPad Software; Dotmatics) was used for drawing and statistical analysis. Differences between the control group and each other group in Fig. 1A and B were analyzed using one-way ANOVA followed by Dunnett's post hoc test. An unpaired t-test was used for comparisons between two groups in Fig. 1C and D. Differences between multiple groups were analyzed using one-way ANOVA followed by Tukey's post hoc test (Fig. 2, Fig. 3, Fig. 4). P<0.05 was considered to indicate a statistically significant difference (33).

The pathophysiology of diabetes is complicated and involves glucose and lipid metabolism disorders. Fat deposits in the kidneys have been suggested to be instrumental to DN, so different concentrations of PA were used to pretreat cells in lipid metabolism-related experiments (34–36).

According to the results of the microplate reader detection of cell-released NLRP3 inflammasome, the present study showed that, under the induction of PA, MPC5 cells released a large amount of NLRP3 inflammasome (Fig. 1B). The protein expression of NLRP3 was also increased in MPC5 cells (Fig. 1D). However, inflammasome release was not significantly increased with increasing PA content. When the PA concentration reached 0.3 mmol, the release amount of NLRP3 inflammasome decreased (Fig. 1A) and a large number of dead cells floated in the culture dish at this time, which indicated that a high PA concentration had a detrimental effect on cell viability. In brief, 0.2 mmol was selected as the PA concentration in this experiment. Based on various relevant studies, concentrations of 2, 5, 10 and 15 µmol dapagliflozin were selected to verify the most suitable treatment methods, following treatment with 0.2 mmol PA. The drug effect was examined through a CCK-8 assay, which showed that when the concentration of dapagliflozin was >5 µmol, the number of viable cells began to decrease. Thus, the CCK-8 assay results showed that 2 µmol dapagliflozin was appropriate as the protective concentration for subsequent experiments (Fig. 1C).

Western blot results suggested that the expression of pyroptosis-related proteins NLRP3 and caspase-1 was increased compared with that in the control group. The expression of inflammatory cytokines IL-18 and IL-1β was increased by PA treatment. Following the addition of dapagliflozin, the expression of pyroptosis-related proteins decreased. At the same time, the content of intracellular inflammatory factors was also decreased (Fig. 2A).

qPCR results indicated that the mRNA levels of NLRP3 inflammasome, ASC, and inflammatory factors IL-18 and IL-1β were increased following the treatment of MPC5 cells with PA. Following the addition of dapagliflozin, the mRNA levels of inflammatory and pyroptosis-related factors were significantly decreased (Fig. 2B).

Hoechst 33342/PI fluorescence staining results indicated that the uptake of PI dye by MPC5 cells was increased following PA treatment and significantly decreased following the addition of dapagliflozin (Fig. 2C and D).

MCC950 is an effective and selective NLRP3 inhibitor, which can specifically inhibit activation of NLRP3 and reduced IL-1β production in vivo. After MCC950 was added, the uptake of PI dye by MPC5 cells also decreased. When both MCC950 and dapagliflozin were added to the cells, the uptake of PI dye decreased (Fig. S1). This result indicated that cell membrane damage was attenuated when NLRP3 was inhibited and dapagliflozin might also reduce the membrane damage by inhibiting the activity of NLRP3.

The protein expression of HO-1 decreased following the treatment of MPC5 cells with PA, which was consistent with the qPCR results. However, following dapagliflozin treatment, HO-1 protein and mRNA expression levels were both increased (Fig. 3).

Western blot analysis indicated that the expression levels of pyroptosis-related proteins NLRP3 and caspase-1, as well as inflammatory factors IL-18 and IL-1β, decreased following HO-1 overexpression in the PA group (Fig. 4A). Meanwhile, NLRP3 inflammasome, IL-18 and IL-1β increased following transfection with siHO1. Moreover, when dapagliflozin was added, NLRP3 inflammasome, IL-18 and IL-1β decreased (Fig. S2A).

qPCR results indicated that the mRNA levels of NLRP3 inflammasome and ASC, as well as inflammatory factors IL-18 and IL-1β, significantly decreased following HO-1 overexpression in MPC5 cells (Fig. 4B).

According to Hoechst 33342/PI fluorescence staining, when HO-1 was overexpressed in the PA group, the PI uptake of MPC5 cells was significantly reduced (Fig. 4C and D). This result suggested that a large number of cell membranes were damaged following treatment with PA, while HO-1 overexpression reduced this damage.