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. 2021 Apr;24(4):460-468.
doi: 10.22038/ijbms.2021.52449.11850.

Voltage-gated potassium channels are involved in oxymatrine-regulated islet function in rat islet β cells and INS-1 cells

Jingying Gao  1   2 , Lixia Xia  1 , Yuanyuan Wei  1   2
Affiliations

Voltage-gated potassium channels are involved in oxymatrine-regulated islet function in rat islet β cells and INS-1 cells

Jingying Gao et al. Iran J Basic Med Sci. 2021 Apr.

Abstract

Objectives: Oxymatrine can regulate glucose metabolism. But the underlying mechanisms remain unclear. We investigated the relationship of oxymatrine and voltage-gated potassium (Kv) channel in rat islet β cells and INS-1 cells.

Materials and methods: Insulin secretion and Kv channel currents were tested by radioimmunoassay and patch-clamp technique, respectively. The INS-1 cell viability was detected using cell counting kit-8 experiments. Flowcytometry analysis and western blot were employed for cell apoptosis and protein levels, respectively. INS-1 cell proliferation was assessed by the 5-Ethynyl-2'- deoxyuridine method.

Results: Oxymatrine potentiated insulin secretion at high glucose (P<0.01 vs 11.1 G, P<0.01 vs 16.7 G) and inhibited KV currents at 40 mV (45.73±15.34 pA/pF for oxymatrine, 73.80±19.23 pA/pF for control, P<0.05). After the INS-1 cells were treated with oxymatrine for 12 and 24 hr, KV2.1 channel protein was up-regulated (P<0.01 vs Control). At the same time, compared with the high glucose and high fat group, cell viability and proliferation ability were increased (P<0.01). The cell apoptotic rate was reduced, reaching 17.30%±1.00% at 12 hr and 10.35%±1.52% at 24 hr (P<0.01). These protective effects of oxymatrine were reversed by using Stromatoxin-1, a kv channel inhibitor.

Conclusion: The results indicate that oxymatrine can stimulate insulin secretion and decrease kv channel currents in islet β cells. Besides, oxymatrine also increases cell viability, proliferation, and reduces cell apoptosis in INS-1 cells. The effects of oxymatrine are related to kv channels. This finding provides new insight into the mechanisms of oxymatrine-regulated islet function.

Keywords: Apoptosis; Diabetes mellitus; Insulin secretion; Oxymatrine; Potassium channel; Voltage-gated.

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Figures

Figure 1.
Figure 1.
Oxymatrine promotes glucose-stimulated insulin secretion in rat pancreatic islets. Rat islets were incubated in Krebs-Ringer bicarbonate-HEPES buffer containing 2.8 mmol/l glucose (2.8 G), 11.1 mmol/l glucose (11.1 G), or 16.7 mmol/l glucose (16.7 G) in the absence or presence of oxymatrine as indicated. Each tube contains 6 islets, each group contains 7 tubes.**P<0.01
Figure 2.
Figure 2.
Oxymatrine inhibits kv channels in rat islet β cells. The cells were treated with or without oxymatrine (1 μmol/L,10 μmol/l, and 100 μmol/l). kv currents were recorded 10 min after different treatments from a holding potential of -70 mV to various test pulses (-70 to 80 mV) in 10 mV steps. (A) Current-voltage curves of Kv channel currents under different treatments. (B) Summary of the mean current density of Kv channel currents at 40 mV (each group represents different cells, n=6 β cells/group), *P<0.05, **P<0.01
Figure 3
Figure 3
Oxymatrine increases the expression level of kv2.1 channel protein in INS-1 cells. The INS-1 cells were treated with or without oxymatrine (1 μmol/l,10 μmol/l, and 100 μmol/l). The expression level of kv2.1 channel protein was detected by Western blot assay, (A) Representative Western blot bands of kv2.1 channel protein were shown. (B) Analysis of the amount of kv2.1 channel protein (normalized to control). The experiments were repeated three times. Data represent an average of three experiments, 2×105 cells/well. *P<0.05 vs Control, **P<0.01 vs Control, #P<0.05 vs Oxymatrine (1 μmol/l), ##P<0.01 vs Oxymatrine (1 μmol/l)
Figure 4
Figure 4
Oxymatrine promotes cell viability in INS-1 cells under high glucose and high-fat conditions. The INS-1 cells were treated with or without oxymatrine (10 μmol/l) for 12 hr and 24 hr under different conditions. Data are normalized to control. INS-1 cells were preincubated with stromatoxin-1 for 30 min before the addition of oxymatrine, high glucose, and palmitic acid sodium. Then, the INS-1 cells were treated together with all corresponding drugs for 12 or 24 hr. The experiments were repeated three times. Data represent an average of three experiments, 8×103 cells/well. Control: not treated; HG: high glucose (30 mmol/l glucose); PA: palmitic acid sodium (400 μmol/l); stromatoxin-1 (a kv channel inhibitor, 100 nmol/l). *P<0.05, **P<0.01
Figure 5
Figure 5
Oxymatrine increases the proliferation capacity of INS-1 cells in high glucose and high-fat. The proliferating nuclei were stained red with EdU and the nuclei of all cells were stained blue with Hoechst 33342. (A) Representative pictures from confocal microscopy were shown. (B) Analysis of the numbers of EdU positive and Hoechst 33342 positive cells. INS-1 cells were preincubated with stromatoxin-1 for 30 min before the addition of oxymatrine, high glucose, and palmitic acid sodium. Then, the INS-1 cells were treated together with all corresponding drugs for 24 hr. The experiments were repeated three times. Data represent an average of three experiments, 8×103 cells/well. Control: not treated; HG: high glucose (30 mmol/l glucose); PA: palmitic acid sodium (400 μmol/l); stromatoxin-1 (100 nmol/l); 5-Ethynyl-2’-deoxyuridine: Edu, **P<0.01
Figure 6
Figure 6
Oxymatrine reduces high glucose and high fat-induced INS-1 cell apoptosis. (A) Representative pictures from flowcytometery analysis were shown. (B) The apoptotic rate analysis of different groups. INS-1 cells were preincubated with stromatoxin-1 for 30 min before the addition of oxymatrine, high glucose, and palmitic acid sodium. Then, the INS-1 cells were treated together with all corresponding drugs for 12 or 24 hr. The experiments were repeated three times. Data represent an average of three experiments, 1×106 cells/ml. Control: not treated; HG: high glucose (30 mmol/l glucose); PA: palmitic acid sodium (400 μmol/l); stromatoxin-1 (100 nmol/l); *P<0.05, **P<0.01
Figure 7
Figure 7
Oxymatrine affects the protein expressions of Bax, Bcl-2, and Caspase-3 in INS-1 cells under high glucose and high-fat conditions. The INS-1 cells were treated with or without oxymatrine (10 μmol/l) for 12 hr and 24 hr under different conditions. Data are normalized to Control. INS-1 cells were preincubated with stromatoxin-1 for 30 min before the addition of oxymatrine, high glucose, and palmitic acid sodium. Then, the INS-1 cells were treated together with all corresponding drugs for 12 or 24 hr. The experiments were repeated three times. Data represent an average of three experiments, 2×105 cells/well. Control: not treated; HG: high glucose (30 mmol/l glucose); PA: palmitic acid sodium (400 μmol/l); stromatoxin-1 (100 nmol/l); (A) Representative Western blot bands of Bax, Bcl-2 and Caspase-3 were shown. (B, E) Analysis of the amount of Bax protein. (C, F) Analysis of the amount of Bcl-2 protein. (D, G) Analysis of the amount of Caspase-3 protein. *P<0.05, **P<0.01
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