Bergenin inhibits palmitic acid-induced pancreatic β-cell inflammatory death via regulating NLRP3 inflammasome activation

Abstract

Background: Bergenin, an active constituent of plants of the genus Bergenia, has been reported to have antidiabetic properties. This study investigated whether bergenin is beneficial for treating type 2 diabetes mellitus (T2DM) via regulating NOD-like receptor family-pyrin domain containing 3 (NLRP3) inflammasome.

Methods: Two pancreatic β-cell lines, INS-1 and MIN6, were treated with 1, 3, or 10 µM bergenin in the absence or presence of palmitic acid (PA). Cell Counting Kit (CCK)-8, flow cytometry, quantitative reverse transcription-polymerase chain reaction, western blotting, and immunofluorescent staining were performed.

Results: Bergenin with concentrations of 1, 3, and 10 µM had no cytotoxicity in INS-1 and MIN6 cells. However, bergenin dose-dependently relieved PA-induced pancreatic β‑cell loss and apoptosis. Bergenin dose-dependently inhibited NLRP3 inflammasome-related inflammation, as observed by the downregulation of NLRP3, apoptosis associated speck like protein (ASC), cleaved caspase-1, and gasdermin-D (GSDMD)-N, as well as the decreased release of cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1β, and IL-18. The NOD-like receptor signaling pathway was predicted to be a downstream signaling pathway regulated by bergenin. Autodock Vina software docked bergenin with NLRP3. The binding energy of interaction was -5.101 kcal/mol and the root-mean-square deviation (RMSD) score was 1.5901A. Treating pancreatic β‑cells with bergenin accelerated the degeneration of NLRP3. Furthermore, restoration of NLRP3 expression using plasmid transfection reversed the protective effects of bergenin on pancreatic β-cells.

Conclusions: Our data suggests that bergenin is a potential agent for treating T2DM through preventing NLRP3 inflammasome-related inflammation in pancreatic β-cells.

Keywords: Diabetes mellitus; NOD-like receptor family-pyrin domain containing 3 (NLRP3); bergenin; palmitic acid; pancreatic β-cell.

Figure 1

Bergenin inhibits PA-induced pancreatic β-cell apoptosis. (A) INS-1 and MIN6 cells were treated with 1, 3, or 10 µM bergenin for 48 h and cell survival was measured by CCK-8 kits; (B,C) INS-1 (B) and MIN6 cells (C) were treated with 1, 3, or 10 µM bergenin in the absence or presence of 400 µM PA for 0–72 h. Cell survival was analyzed using CCK-8 kits. (D,E) INS-1 (D) and MIN6 cells (E) were treated with 1, 3, or 10 µM bergenin in the absence or presence of 400 µM PA for 48 h. The apoptosis rate was measured by flow cytometry. ***P<0.001 vs. control group; n.s., no significance, ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. PA group. PA, palmitic acid.

Figure 2

Bergenin inhibits PA-induced pancreatic β-cell inflammation. (A-J) INS-1 and MIN6 cells were treated with 1, 3, or 10 µM bergenin in the absence or presence of 400 µM PA for 48 h. The mRNA levels of IL-6 (A,E), TNF-α (B,F), IL-1β (C,G), and IL-18 (D,H) were measured by qRT-PCR. Protein levels of these cytokines were measured by western blot (I,J). ***P<0.001 vs. control group; n.s., no significance, ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. PA group. PA, palmitic acid.

Figure 3

Bergenin inhibits PA-induced NLRP3 inflammasome activation in pancreatic β-cells. (A-D) INS-1 and MIN6 cells were treated with 1, 3, or 10 µM bergenin in the absence or presence of 400 µM PA for 48 h. Protein levels of NLRP3, ASC, cleaved caspase-1, and GSDMD-N were measured by western blot (A,B). Immunofluorescent staining of NLRP3-labelled cells (C,D). Scale bar =50 µm. PA, palmitic acid; NLRP3, NOD-like receptor family-pyrin domain containing 3; ASC, apoptosis associated speck like protein; GSDMD-N, gasdermin-D N.

Figure 4

Bergenin inhibits NLRP3 expression via regulating its protein stability. (A,B) SwissTargetPrediction and CTD databases screened 4 signaling pathways (A) and identified NOD-like receptor, TNF, HIF-1, and IL-17 signaling pathways (B). (C,D) Autodock Vina software docked bergenin with NLRP3 protein. Molecular docking results in 2D (C) and 3D view (D). (E) MIN6 cells were treated with 100 µM CHX for the indicated time, and the degradation of NLRP3 was analyzed. *P<0.05 and ***P<0.001 vs. control group. NLRP3, NOD-like receptor family-pyrin domain containing 3; CTD, Comparative Toxicogenomics Database; CHX, cycloheximide.

Figure 5

Bergenin inhibits PA-induced MIN6 cell apoptosis via regulating NLRP3. (A) The protein levels of NLRP3 in MIN6 cells transfected with NLRP3 overexpression plasmids or the empty vector were measured by western blot. (B-D) MIN6 cells were transfected with NLRP3 overexpression plasmids or the empty vector and then treated with 10 µM bergenin in the absence or presence of 400 µM PA for 48 h. NLRP3 protein levels were analyzed by western blotting (B). Cell survival and apoptosis were analyzed by the CCK-8 assay (C) and flow cytometry (D), respectively. ***P<0.001 vs. control group; ^###P<0.001 vs. PA group; ^&P<0.05 and ^&&P<0.01 vs. bergenin + vector group. NLRP3, NOD-like receptor family-pyrin domain containing 3.

Figure 6

Bergenin inhibits PA-induced MIN6 cell inflammation via regulating NLRP3. (A-E) MIN6 cells were transfected with NLRP3 overexpression plasmids or the empty vector and then treated with 10 μM bergenin in the absence or presence of 400 µM PA for 48 h. The mRNA levels of IL-6 (A), TNF-α (B), IL-1β (C), and IL-18 (D) were measured by qRT-PCR. Protein levels of these cytokines were measured by western blot (E). ***P<0.001 vs. control group; ^###P<0.001 vs. PA group; ^&P<0.05 and ^&&P<0.01 vs. bergenin + vector group. NLRP3, NLR family pyrin domain containing