Fig 1.
Effects of baicalein on PA-induced cytotoxicity and ER stress.
INS-1 cells were treated with various concentrations of PA (0–1000 μM) for different times (0–48 h) (A). INS-1 cells were treated with different concentrations of baicalein (0–50 μM) for 3 h and then PA (500 μM) was added and incubation continued for a further 8 h or 24 h (B). Cell viabilities were assessed using a MTT assay, and the results are shown as % of control (2% BSA treatment at 0 h). Apoptotic DNA fragmentation was assessed using a Cell Death Detection ELISA Plus kit, and the results are shown as OD at 405 nm (C). INS-1 cells were pretreated with baicalein (50 μM) for 3 h and then PA (500 μM) was added and incubation continued for a further 8 h (for qPCR) or 24 h (for western blot). The mRNA expressions (D) and protein (E) levels of ER stress markers were assessed by qPCR or western blotting. The results shown are representative of three independent experiments and are expressed as means ± SDs. #P<0.05 vs. controls; *P<0.05 vs. PA alone.
Fig 2.
Effects of baicalein on HO-1 expression.
INS-1 cells were treated with different concentrations of baicalein (0–100 μM) for 11 h (A, for qPCR) or 27 h (B, for western blot). HO-1 mRNA expression and protein levels were assessed by qPCR or western blotting, respectively. The results shown are representative of three independent experiments and are expressed as means ± SDs. *P<0.05 vs. controls.
Fig 3.
Involvement of HO-1 upregulation in the protective effect of baicalein on PA-mediated lipotoxicity.
INS-1 cells were pretreated with or without ZnPP (10 μM) for 30 min, followed by baicalein (50 μM) for 3 h and then PA (500 μM) for a further 8 h (for qPCR) or 24 h (for western blot). The mRNA expressions (A and C) and protein (B and D) levels of HO-1 or ER stress markers were assessed by qPCR or western blotting, respectively. Apoptotic DNA fragmentation was assessed using a Cell Death Detection ELISA Plus kit (E). INS-1 cells were pretreated with CoPP (10 μM) for 1 h, followed by PA (500 μM) for a further 8 h, and mRNA expressions were determined by qPCR (F). The results shown are representative of three independent experiments and are expressed as means ± SDs. #P<0.05 vs. controls; *P<0.05 vs. PA alone; &P<0.05 vs. PA plus baicalein treatment.
Fig 4.
Effects of baicalein on PA-induced lipotoxicity in isolated pancreatic islets.
Pancreatic islets isolated from C57BL/6J mouse (9 weeks old, male) were pretreated with or without ZnPP (10 μM) for 30 min, followed by baicalein (50 μM) for 3 h and then PA (500 μM) for a further 8 h. The mRNA expressions of HO-1 (B) or ER stress (A) and proinflammatory markers (C) were assessed by qPCR. The results shown are representative of three independent experiments and are expressed as means ± SDs. #P<0.05 vs. controls; *P<0.05 vs. PA alone; &P<0.05 vs. PA plus baicalein treatment.
Fig 5.
Effects of ERK activation on baicalein-induced HO-1 expression.
INS-1 cells were pretreated with baicalein (50 μM) for 3 h in the absence or presence of ZnPP (10 μM), or PD98059 (10 μM) and then PA (500 μM) was added and incubation continued for a further 8 h (for qPCR) or 24 h (for western blot). The mRNA expression of HO-1 (A) and protein levels of HO-1, pERK1/2 and ERK1/2 (B) were assessed by qPCR or western blotting, respectively. Results are representative of three independent experiments and are presented as means ± SDs. #P<0.05 vs. controls; *P<0.05 vs. PA alone; &P<0.05 vs. PA plus baicalein treatment.
Fig 6.
Effects of baicalein on PA-induced inflammation and reduced insulin secretion.
INS-1 cells were pretreated with either ZnPP (10 μM) or PD98059 (10 μM) for 30 min, followed by baicalein (50 μM) for 3 hr. And then PA (500 μM) was added and further incubated for 24 h. To examine glucose-stimulated insulin secretion, culture medium was replaced with medium containing 25 mM glucose for 1 h, and then insulin secretion to medium was measured by ELISA (A). INS-1 cells were pretreated with baicalein (50 μM) for 3 h in the absence or presence of ZnPP (10 μM), or PD98059 (10 μM) and then PA (500 μM) was added and incubation continued for a further 8 h (for qPCR) or 24 h (for western blot). Levels of TNF-α and IL-6 were assessed by ELISA (B). The protein levels of ER stress markers were determined by Western blotting (C). The results shown are representative of three independent experiments and are expressed as means ± SDs. #P<0.05 vs. controls; *P<0.05 vs. PA alone; &P<0.05 vs. PA plus baicalein treatment.
Fig 7.
Attenuation of the protective effects of baicalein by HO-1 knockdown.
INS-1 cells were transfected with 20 nM HO-1 siRNA or control siRNA for 36 h, and the reduction of HO-1 mRNA expression was determined (A left). HO-1 siRNA or control siRNA transfected cells were treated with baicalein (50 μM) for 3 h, and then HO-1 mRNA expression was determined by qPCR (A right). After HO-1siRNA transfection, cells were treated with baicalein (50 μM) for 3 h and PA was then added and incubation continued for 8 h (for qPCR) or 24 h (for western blot or ELISA). The expression levels of ER stress makers (B) were assessed by qPCR. Protein levels of ER stress markers and phosphorylated ERKs were determined by Western blotting (C). Insulin secretion (D) and secreted TNF-α and IL-6 (E) levels were assessed by ELISA. The results shown are representative of three independent experiments and are expressed as means ± SDs. #P<0.05 vs. controls; *P<0.05 vs PA alone; &P<0.05 vs. PA plus baicalein with control siRNA transfection.
Fig 8.
Effects of RuCO (a CO donor) on PA-mediated lipotoxicity.
INS-1 cells were treated with RuCO (150 μM) for 1 h and then incubated with PA (500 μM) for 8 h. The mRNA expression levels of ER stress markers were detected by qPCR. The results shown are representative of three independent experiments and are expressed as means ± SDs. #P<0.05 vs. controls; *P<0.05 vs PA alone.