Amelioration of streptozotocin‑induced pancreatic β cell damage by morin: Involvement of the AMPK‑FOXO3‑catalase signaling pathway

Abstract

Pancreatic β cells are sensitive to oxidative stress, which is one of the predominant causes of cell damage and the emergence of diabetes. The identification of effective therapeutic strategies to protect pancreatic cells from oxidative stress has increased interest in the screening of antioxidants from natural products. The present study aimed to investigate the protective effects of morin against streptozotocin (STZ)‑induced cell damage in a rat insulinoma cell line (RINm5F pancreatic β cells) and to identify the underlying mechanisms. The results indicated that morin inhibited the increase in intracellular reactive oxygen species, attenuated the activity of poly (ADP‑ribose) polymerase, restored intracellular nicotinamide adenine dinucleotide levels and reduced the apoptotic cell death of STZ‑treated pancreatic β cells. Treatment with morin significantly upregulated catalase in pancreatic β cells, and ameliorated the STZ‑induced loss of catalase at the genetic, protein and enzymatic level. In further experiments, morin induced the phosphorylation of 5' adenosine monophosphate‑activated protein kinase (AMPK), which subsequently promoted the translocation of forkhead box O3 (FOXO3) to the nucleus. Specific small interfering RNAs (siRNAs) against AMPK and FOXO3 suppressed morin‑induced catalase expression. Furthermore, catalase‑specific siRNA abolished the protective effects of morin against STZ‑stimulated cell death. Taken together, these results indicated that morin protected RINm5F cells from STZ‑induced cell damage by triggering the phosphorylation of AMPK, thus resulting in subsequent activation of FOXO3 and induction of catalase.

Figure 1

Toxic effects of STZ on RINm5F cells. RINm5F cells were treated with various concentrations of STZ and cell viability was determined after 24 h by MTT assay. Data are presented as the means ± standard error of the means from three experiments. ^*P<0.05 vs. control cells. STZ, streptozotocin.

Figure 2

Effects of morin on the viability of STZ-treated RINm5F cells. (A) Cells were treated with morin at various concentrations. After 1 h, 6 mM STZ was added to the cells and cell viability was determined after 24 h by MTT assay. ^*P<0.05 vs. control cells; ^**P<0.05 vs. STZ-treated cells. (B) Cells were treated with various concentrations of morin. Following a 24 h incubation, cell viability was determined by MTT assay. STZ, streptozotocin.

Figure 3

Effects of morin on STZ-induced intracellular ROS production. Cells were treated with morin (25 µM) for 1 h, after which 6 mM STZ was added to the cells for an additional 12 h. Intracellular ROS generation was detected by (A) flow cytometry following 2′,7′-dichlorodihydrofluorescein diacetate treatment. Measurements were conducted in triplicate and the values are presented as the means ± standard error of the mean. ^*P<0.05 vs. control cells; ^**P<0.05 vs. STZ-treated cells. (B) Representative confocal images illustrating the increase in red fluorescence intensity produced by ROS in STZ-treated cells compared with in the control cells, and the reduced fluorescence intensity in STZ-treated cells pretreated with morin. ROS, reactive oxygen species; STZ, streptozotocin.

Figure 4

Effects of morin on STZ-induced increases in PARP activity and depletion of NAD⁺ levels. Cells were treated with 25 µM morin for 1 h and were then incubated with 6 mM STZ for 24 h. (A) Cell lysates were electrophoresed and the protein expression levels of PAR were examined by western blot analysis. (B) Intracellular NAD⁺ content was measured using the NAD⁺/NADH quantification kit. The measurements were made in triplicate and the values are presented as the means ± standard error of the mean. ^*P<0.05 vs. control cells; ^**P<0.05 vs. STZ-treated cells. NAD⁺, nicotinamide adenine dinucleotide; PARP, poly (ADP-ribose) polymerase; ROS, reactive oxygen species; STZ, streptozotocin.

Figure 5

Protective effects of morin on STZ-induced damage in RINm5F cells. (A) Cell viability was determined by MTT assay. (B) Apoptotic body formation was observed under a fluorescence microscope following Hoechst 33342 staining; apoptotic bodies are indicated by arrows. (C) Apoptotic sub-G₁ DNA content was detected by flow cytometry following propidium iodide staining. Measurements were made in triplicate and the values are presented as the means ± standard error of the mean. ^*P<0.05 vs. control cells; ^**P<0.05 vs. STZ-treated cells. STZ, streptozotocin.

Figure 6

Effects of morin on catalase mRNA transcription, protein expression and enzyme activity. (A) Cells were treated with 25 µM morin for a series of time periods, total RNA was extracted and catalase mRNA expression was analyzed by qPCR. β-actin was used as the internal reference. (B) Cells were treated with 25 µM morin for the indicated time periods. Cell lysates were electrophoresed and the expression of catalase protein was detected using a catalase-specific antibody. β-actin was used as a loading control. (C) Cells were treated with morin for the indicated time periods. Catalase activity was measured using a colorimetric assay kit. ^*P<0.05 vs. control cells. (D) Cells were treated with 25 µM morin for 1 h, after which 6 mM STZ was added for 12 h. Total RNA was extracted and catalase mRNA expression was analyzed by qPCR. (E) Cells were treated with 25 µM morin for 1 h, 6 mM STZ was then added for 24 h. Cell lysates were electrophoresed and the expression of catalase protein was detected by catalase-specific antibody. (F) Catalase activity was measured using a colorimetric assay kit. ^*P<0.05 vs. control cells; ^**P<0.05 vs. STZ-treated cells. qPCR, quantitative polymerase chain reaction; STZ, streptozotocin.

Figure 7

Effects of morin on AMPK activation and FOXO3a translocation. Cells were treated with 25 µM morin for the indicated time periods. (A) Cell lysates underwent western blotting with primary antibodies against p-AMPKα and AMPKα. (B) Nuclear fractions were prepared and FOXO3a protein expression levels were examined by western blot analysis. TBP was used as a loading control. AMPK, 5′ adenosine monophosphate-activated protein kinase; FOXO3, forkhead box O3; p-AMPK, phosphorylated-AMPK; TBP, TATA binding protein.

Figure 8

Induction of catalase by morin via the AMPK-FOXO3a signaling pathway. (A) Cells were transfected with 10–50 nM siControl or siAMPK. A total of 24 h post-transfection, cells were treated with 25 µM morin for 24 h. The protein expression levels of catalase were then evaluated in cell lysates by western blot analysis. (B) Cells were transfected with 10–50 nM siControl or siFOXO3a. A total of 24 h post-transfection, cells were treated with 25 µM morin for 24 h, and the protein expression levels of catalase were evaluated in cell lysates by western blot analysis. (C) Cells were transfected with 10–50 nM siControl or siCatalase, followed by 1 h treatment with morin and exposure to 6 mM STZ for 24 h. Cell viability was measured using the MTT assay. ^*P<0.05 vs. STZ + siControl-transfected cells; ^**P<0.05 vs. morin + STZ + siControl-transfected cells. AMPK, 5′ adenosine monophosphate-activated protein kinase; FOXO3, forkhead box O3; si/siRNA, small interfering RNA; STZ, streptozotocin.