Pyrroloquinoline quinone ameliorates diabetic cardiomyopathy by inhibiting the pyroptosis signaling pathway in C57BL/6 mice and AC16 cells

Fig. 1. PQQ ameliorated diabetes-induced myocardial hypertrophy. a Representative H&E staining in the study groups. Chromatin and nucleic acids are stained blue and the cytoplasm and extracellular matrix are stained red; magnification, × 400; n = 5 per group. b Comparison of cardiomyocyte area in the mice from each group; n = 5. Relative mRNA expression levels of c ANP, d BNP, and e β-MHC were measured by real-time RT-PCR; n = 4 per group. Data are presented as mean ± SD, *P < 0.05, ***P < 0.001 vs. Ctl, #P < 0.05, ###P < 0.001 vs. DCM. DCM diabetic cardiomyopathy group, DCM + PQQL diabetic mice treated with 10 mg/kg BW/day PQQ, DCM + PQQM diabetic mice treated with 20 mg/kg BW/day PQQ, DCM + PQQH diabetic mice treated with 40 mg/kg BW/day PQQ

Fig. 2. PQQ ameliorated diabetes-induced cardiac fibrosis. a Representative Masson’s trichrome staining of interstitial fibrosis in the study groups; magnification, × 400; n = 5 per group. b Representative Masson’s trichrome staining of perivascular fibrosis in the study groups; magnification, × 400; n = 5. Collagen is stained blue and the myocardium is stained red. c Analysis of cardiac fibrosis area; n = 5 per group. Data are presented as mean ± SD, ***P < 0.001 vs. Ctl, ###P < 0.001 vs. DCM. DCM. DCM diabetic cardiomyopathy group, DCM + PQQL diabetic mice treated with 10 mg/kg BW/day PQQ, DCM + PQQM diabetic mice treated with 20 mg/kg BW/day PQQ, DCM + PQQH diabetic mice treated with 40 mg/kg BW/day PQQ

Fig. 3. PQQ ameliorated cardiac fibrosis-related mRNA and protein levels. The relative mRNA expression levels of a Col I (n = 5), b Col III (n = 5), c TGF-β1 (n = 4), and d CTGF (n = 5) were measured by real-time RT-PCR. Protein expression levels of e COL I, f COL III, and g TGF-β1 were determined by western blotting analysis in the study groups, n = 4 per group. Data are presented as mean ± SD, *P < 0.05, **P < 0.01 vs. Ctl, #P < 0.05, ##P < 0.01 vs. DCM. DCM. DCM diabetic cardiomyopathy group, DCM + PQQL diabetic mice treated with 10 mg/kg BW/day PQQ, DCM + PQQM diabetic mice treated with 20 mg/kg BW/day PQQ, DCM + PQQH diabetic mice treated with 40 mg/kg BW/day PQQ

Fig. 4. PQQ ameliorated diabetes-induced inflammation. Levels of the inflammatory cytokines a IL-1β, b IL-6, and c TNF-α were measured by ELISA; n = 4 per group. Data are presented as mean ± SD, *P < 0.05, **P < 0.01 vs. Ctl, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. DCM. DCM. DCM diabetic cardiomyopathy group, DCM + PQQL diabetic mice treated with 10 mg/kg BW/day PQQ, DCM + PQQM diabetic mice treated with 20 mg/kg BW/day PQQ, DCM + PQQH diabetic mice treated with 40 mg/kg BW/day PQQ

Fig. 5. PQQ protected the heart from diabetes-induced oxidative stress. The activity or quantity of the myocardial damage-related indicators, a LDH and b CK-MB, and the oxidative stress-related indicators, c MDA, d SOD, e GSH-px, and f CAT were measured using commercial kits; n = 4 per group. Data are presented as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 vs. Ctl, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. DCM. DCM. DCM diabetic cardiomyopathy group, DCM + PQQL diabetic mice treated with 10 mg/kg BW/day PQQ, DCM + PQQM diabetic mice treated with 20 mg/kg BW/day PQQ, DCM + PQQH diabetic mice treated with 40 mg/kg BW/day PQQ

Fig. 6. PQQ ameliorated the diabetes-induced pyroptosis of cardiomyocytes. The relative mRNA expression levels of a NLRP3, and b caspase-1 were measured by real-time RT-PCR; n = 5. The protein levels of c NLRP3 (n = 4), d caspase-1 (n = 4), e activated-caspase-1 (n = 4), f IL-1β (n = 4), and g IL-18 (n = 3) were determined by western blotting analysis. Data are presented as mean ± SD, *P < 0.05, **P < 0.001 vs. Ctl, #P < 0.05, ##P < 0.01 vs. DCM. DCM. DCM diabetic cardiomyopathy group, DCM + PQQL diabetic mice treated with 10 mg/kg BW/day PQQ, DCM + PQQM diabetic mice treated with 20 mg/kg BW/day PQQ, DCM + PQQH diabetic mice treated with 40 mg/kg BW/day PQQ

Fig. 7. The effects of PQQ on cell viability and pyroptosis signaling in AC16 cells under HG conditions. a & b AC16 cells were treated with different concentrations of PQQ (1, 10, 100, 1,000, 5,000, 10,000, and 10,0000 nmol/L) in basic DMEM (5.5 mmol/L glucose) or HG (35 mmol/L glucose) media for 24 h. Cell viability was measured by MTT assay, n = 6. The protein levels of c NLRP3, d cleaved-caspase-1, e caspase-1, f IL-1β, and g IL-18 were analyzed by western blotting, n = 4 per group. Mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 vs. Ctl, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. HG. HG high glucose; HG + 1 nmol/L PQQ, high glucose with 1 nmol/L PQQ; HG + 10 nmol/L PQQ, high glucose with 10 nmol/L PQQ

Fig. 8. PQQ ameliorated NF-κB activation and ROS generation in AC16 cells under HG conditions. a Protein levels of phosphorylated and total NF-κB p65 were determined by western blotting analysis, n = 4. b Protein levels of phosphorylated IκB and total IκB were determined by western blotting analysis, n = 4; c Intracellular ROS levels were determined using a DCFH-DA assay kit, n = 3. d Mitochondrial membrane potential was measured in AC16 cells using a JC-1 mitochondrial membrane potential assay kit, n = 3. Mean ± SD, *P < 0.05 vs. Ctl, #P < 0.05, ##P < 0.01 vs. HG. HG high glucose; HG + 1 nmol/L PQQ, high glucose with 1 nmol/L PQQ; HG + 10 nmol/L PQQ, high glucose with 10 nmol/L PQQ