Korean J Physiol Pharmacol.  2022 May;26(3):165-174. 10.4196/kjpp.2022.26.3.165.

Oxymatrine inhibits the pyroptosis in rat insulinoma cells by affecting nuclear factor kappa B and nuclear factor (erythroidderived 2)-like 2 protein/heme oxygenase-1 pathways

• Gao J
• Xia L
• Wei Y

• KMID: 2529399
• DOI: http://doi.org/10.4196/kjpp.2022.26.3.165

Abstract

As the mechanism underlying glucose metabolism regulation by oxymatrine is unclear, this study investigated the effects of oxymatrine on pyroptosis in INS-1 cells. Flow cytometry was employed to examine cell pyroptosis and reactive oxygen species (ROS) production. Cell pyroptosis was also investigated via transmission electron microscopy and lactate dehydrogenase (LDH) release. Protein levels were detected using western blotting and interleukin (IL)-1β and IL-18 secretion by enzyme-linked immunosorbent assay. The caspase-1 activity and DNA-binding activity of nuclear factor kappa B (NF-κB) and nuclear factor (erythroid-derived 2)-like 2 protein (Nrf2) were also assessed. In the high glucose and high fat-treated INS-1 cells (HG + PA), the caspase-1 activity and LDH content, as well as Nod-like receptor family pyrin domain containing 3, Gsdmd-N, caspase-1, apoptosis-associated specklike protein containing a CARD, IL-1β, and IL-18 levels were increased. Moreover, P65 protein levels increased in the nucleus but decreased in the cytoplasm. Oxymatrine attenuated these effects and suppressed high glucose and high fat-induced ROS production. The increased levels of nuclear Nrf2 and heme oxygenase-1 (HO-1) in the HG + PA cells were further elevated after oxymatrine treatment, whereas cytoplasmic Nrf2 and Keleh-like ECH-associated protein levels decreased. Additionally, the elevated transcriptional activity of p65 in HG + PA cells was reduced by oxymatrine, whereas that of Nrf2 increased. The results indicate that the inhibition of pyroptosis in INS-1 cells by oxymatrine, a key factor in its glucose metabolism regulation, involves the suppression of the NF-κB pathway and activation of the Nrf2/HO-1 pathway.

Keyword

Heme oxygenase-1; Nuclear factor (erythroid-derived 2)-like 2 protein; Nuclear factor kappa B; Oxymatrine; Pyroptosis

Figure

• 

  Fig. 1 Oxymatrine protected INS-1 cells from pyroptosis. (A) The electron microscopic analysis of different cells. Scale bar: 2 μm; Oxymatrine: 10 μM. (B) Representative graphics from flow cytometry analysis were showed; Oxymatrine: 10 μM. (C) The caspase-1 activity analysis of different cells; Oxymatrine: 10 μM. (D) The LDH release analysis of different cells. control: no treatment; HG: high glucose (30 mM glucose); PA: palmitic acid sodium (400 μM). Arrows: the autophagosomal vesicles. Data are presented as mean ± SD and represent an average of three experiments. LDH, lactate dehydrogenase. **p < 0.01 vs. control, ##p < 0.01 vs. HG + PA; the experiments were repeated three times.

• 

  Fig. 2 Oxymatrine decreased the levels of NLRP3, Gsdmd-N, caspase-1, ASC, IL-1β, and IL-18 in INS-1 cells under high glucose and high fat conditions. (A) Representative Western blotting bands of NLRP3, Gsdmd-N, caspase-1, and IL-1β proteins were showed. (B) Analysis of the amount of NLRP3 proteins. (C) Analysis of the amount of caspase-1 proteins. (D) Analysis of the amount of IL-1β proteins. (E) Analysis of the amount of Gsdmd-N proteins. (F) Representative western blotting bands of ASC proteins were showed; Oxymatrine: 10 μM. (G) Analysis of the amount of ASC proteins; Oxymatrine: 10 μM. (H) Analysis of the secretion of IL-1β and IL-18; Oxymatrine: 10 μM. (I) Analysis of the activity of caspase-1; Oxymatrine: 10 μM. control: no treatment; HG: high glucose (30 mM glucose); PA: palmitic acid sodium (400 μM). Data are presented as mean ± SD and represent an average of three experiments. NLRP3, Nod-like receptor family pyrin domain containing 3; Gsdmd, Gasdermin D; caspase-1, cysteine aspartic acid-specific proteinase-1; ASC, apoptosis-associated speck-like protein containing a CARD; IL, interleukin. Data from Western blotting are normalized to control. **p < 0.01 vs. control, #p < 0.05, ##p < 0.01 vs. HG + PA; the experiments were repeated three times.

• 

  Fig. 3 Oxymatrine inhibited high glucose and high fat-induced ROS production in INS-1 cells. (A) Representative graphics of ROS production were showed. (B) The ROS production from different cells. control: no treatment; HG: high glucose (30 mM glucose); PA: palmitic acid sodium (400 μM); Oxymatrine: 10 μM. Data are presented as mean ± SD and represent an average of three experiments. ROS, reactive oxygen species; DCFH-DA, Dichloro-dihydro-fluorescein diacetate; MFI, mean fluorescence intensity. Data are normalized to control. **p < 0.01 vs. control, #p < 0.05 vs. HG + PA; the experiments were repeated three times.

• 

  Fig. 4 Oxymatrine affected NF-κB (p65) levels in INS-1 cells at different times. (A) Representative Western blotting bands of p65 proteins in the nucleus or cytoplasm were showed. (B) Analysis of the amount of p65 proteins in the nucleus when the cells were incubated for 0.5 h. (C) Analysis of the amount of p65 proteins in the cytoplasm when the cells were incubated for 0.5 h. (D) Analysis of the amount of p65 proteins in the nucleus when the cells were incubated for 1 h. (E) Analysis of the amount of p65 proteins in the cytoplasm when the cells were incubated for 1 h. (F) Analysis of the amount of p65 proteins in the nucleus when the cells were incubated for 2 h. (G) Analysis of the amount of p65 proteins in the cytoplasm when the cells were incubated for 2 h. Control: no treatment; HG: high glucose (30 mM glucose); PA: palmitic acid sodium (400 μM); Oxymatrine: 10 μM. Data are presented as mean ± SD and represent an average of three experiments. NF-κB, nuclear factor kappa B. Data are normalized to control. **p < 0.01 vs. control, ##p < 0.01 vs. HG + PA; the experiments were repeated three times.

• 

  Fig. 5 Oxymatrine affected Nrf2, HO-1, and Keap1 levels in INS-1 cells. (A) Representative Western blotting bands of Nrf2, HO-1, and Keap1 proteins in the nucleus or cytoplasm were showed. (B) Analysis of the amount of Nrf2 proteins in the nucleus. (C) Analysis of the amount of Nrf2 proteins in the cytoplasm. (D) Analysis of the amount of Keap1 proteins. (E) Analysis of the amount of HO-1 proteins. Control: no treatment; HG: high glucose (30 mM glucose); PA: palmitic acid sodium (400 μM); Oxymatrine: 10 μM. Data are presented as mean ± SD and represent an average of three experiments. Nrf2, nuclear factor (erythroid-derived 2)-like 2 protein; HO-1, heme oxygenase-1; Keap1, Keleh-like ECH-associated protein. Data are normalized to control. *p < 0.05 vs. control, **p < 0.01 vs. control, #p < 0.05 vs. HG + PA. ##p < 0.01 vs. HG + PA; the experiments were repeated three times.

• 

  Fig. 6 Oxymatrine affected DNA-binding activity of NF-κB (p65) and Nrf2 in INS-1 cells. (A) Analysis of the DNA-binding activity of NF-κB (p65). (B) Analysis of the DNA-binding activity of Nrf2. Control: no treatment; HG: high glucose (30 mM glucose); PA: palmitic acid sodium (400 μM); Oxymatrine: 10 μM . Data are presented as mean ± SD and represent an average of three experiments. NF-κB, nuclear factor kappa B; Nrf2, nuclear factor (erythroid-derived 2)-like 2 protein. Data are normalized to control. **p < 0.01 vs. control, #p < 0.05, ## p < 0.01 vs. HG + PA; the experiments were repeated three times.

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