Fig 1.
Suppression of cytokine-induced beta cell damage by bergenin.
(A) The chemical structure of bergenin. (B) Cellular ATP levels of INS-1E cells treated for two days with bergenin in the absence of cytokine treatment. INS-1E cells were treated for two days with proinflammatory cytokines (IL-1β, INF-γ, and TNF-α) in the presence or absence of bergenin (0.25–10 μM). (C) Dose-dependent effects of bergenin on cellular ATP levels after 48 hr treatment with cytokines. (D) Dose-dependent effects of bergenin on caspase-3 activity after 48 hr of treatment with cytokines. Data are represented as the mean ± standard deviation of 12 independent wells for A-B. * indicates p <0.05, ‡<0.01, and §<0.001 relative to cytokine-treated cells.
Fig 2.
Inhibition of cellular nitrite production and restoration of glucose-stimulated insulin secretion.
INS-1E cells were treated for two days with proinflammatory cytokines (IL-1β, INF-γ, and TNF-α) in the presence or absence of bergenin (0.25–10 μM). (A) Dose-dependent effects of bergenin on cellular nitrite production after 48 hr treatment with cytokines. Data are represented as the mean ± standard deviation of 12 independent wells for nitrite production. (B) The glucose-stimulated insulin secretion was measured in “low glucose” (2 mM), and “high glucose” (16 mM) conditions in the presence of bergenin. Data are represented as the mean ± standard deviation of six independent wells for insulin secretion. * indicates p <0.05, ‡<0.01, and §<0.001 relative to cytokine-treated cells.
Fig 3.
Cellular effects of bergenin on mitochondrial physiological parameters of beta cells in the presence of cytokines.
Effects of bergenin on mitochondrial membrane potential (ΔΨm), intracellular ROS production, and mitochondrial dehydrogenase activity (MDA) after 48 hr treatment with IL-1β, INF-γ, and TNF-α. For dose-response studies, INS-1E cells were treated with various concentrations of bergenin (0.25–10 μM). The treated INS-1E cells were assessed for their ability to restore mitochondrial membrane potential (ΔΨm) (A); to reduce cellular ROS production (B); and to improve mitochondrial dehydrogenase activity (C). Data are represented as the mean ± standard deviation of 12 independent wells for A-F. * indicates p <0.05, ‡<0.01, and §<0.001 relative to cytokine-treated cells.
Fig 4.
Bergenin suppresses cytokine-induced apoptosis in beta cells.
INS-1E cells were treated with cytokines (IL-1β, INF-γ, and TNF-α) in the presence or absence bergenin (2–10 μM) for 48 hr, and were assessed for apoptosis inhibition. (A) Represents the flow cytometric analysis of INS-1E cells treated with bergenin in the presence of cytokines cocktail. The viable cell populations are in the lower left quadrant (Annexin V-/PI-); the cells at the early apoptosis are in the lower right quadrant (Annexin V+/PI-); and the ones at the late apoptosis are in the upper right quadrant (Annexin V+/PI+); and dead cells are in the upper left quadrant (Annexin V-/PI+). (B) Represents the live and apoptotic beta cell population after treatment with cytokines and bergenin. Data are represented the mean ± standard deviation of 2 independent experiments. * indicates p <0.05, ‡<0.01 and § <0.001 relative to cytokine-treated cells.
Fig 5.
Bergenin suppressed cytokine-induced beta cell apoptosis.
Exposure of beta cell to proinflammatory cytokines (IL-1β, INF-γ, and TNF-α) lead to apoptosis. However, the addition of bergenin suppressed deleterious effects of cytokine cocktail and prevented apoptosis in INS-1E cells.
Table 1.
Activity profile of bergenin against beta cell viability, mitochondrial physiological parameters, beta cell function and apoptosis.
Fig 6.
Potential mechanism of action of bergenin in suppressing cytokine-induced apoptosis in INS-1E cells.
Proinflammatory cytokines (IL-1β, INF-γ, and TNF-α) initiate a cascade of signaling pathways leading to beta cell apoptosis. These cytokines stimulate JAK-STAT, NFκB, and MAPK pathways, which later induce intrinsic apoptotic pathway in beta cells. IL-1β also induces nitric oxide (NO) production, which causes inhibition of electron transport chain, increase in ROS production, decrease in glucose oxidation rate resulting in reduced ATP generation, and insulin production. Bergenin protected pancreatic beta cells through multiple mechanisms simultaneously. Bergenin suppressed beta cell apoptosis by potentially influencing NF-κB, MAPK and JAK/STAT pathways (represented by blue dashed lines). This is evident from the inhibition of downstream effector molecules (caspse-3, NO, ROS, and apoptosis) targeted in cell-based assays (represented by red dashed lines). By inhibiting cytokine-induced NO production, bergenin was able to increase in cellular ATP levels, decreased ROS production, and increased insulin production.