Korean J Physiol Pharmacol 2022; 26(3): 165-174
Published online May 1, 2022 https://doi.org/10.4196/kjpp.2022.26.3.165
Copyright © Korean J Physiol Pharmacol.
Jingying Gao1,2,*, Lixia Xia1, and Yuanyuan Wei1,2
1Department of Pediatrics, Shanxi Medical University, 2Pediatric Internal Medicine, Children’s Hospital of Shanxi Province, Shanxi Medical University, Taiyuan 030001, China
Correspondence to:Jingying Gao
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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 speck-like 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.
Keywords: Heme oxygenase-1, Nuclear factor (erythroid-derived 2)-like 2 protein, Nuclear factor kappa B, Oxymatrine, Pyroptosis
The main pathobiology of diabetes involves the decreased number and dysfunction of islet cells [1,2]. Inflammatory damage mediated by cytokines affects insulin secretion and the survival of islet cells [3,4]. Abnormal activation of Nod-like receptor family pyrin domain containing 3 (NLRP3) induces cell pyroptosis .
Pyroptosis is a programmed cell death mediated by various injury stimuli
Studies have found that high glucose, high fat, reactive oxygen species (ROS), and nuclear factor kappa B (NF-κB) can activate NLRP3 inflammasome and impair islet function [8-10]. ROS can damage islet β cells and inhibit insulin synthesis and secretion . Nuclear factor (erythroid-derived 2)-like 2 protein (Nrf2) is a redox-sensitive transcription factor. Activated Nrf2 regulates downstream gene transcription to enhance the production of proteins, such as NAD (P)H: quinone oxidoreductase 1, glutamate-cysteine ligase, heme oxygenase-1 (HO-1), and superoxide dismutase, which reduce ROS production and ameliorate cell function disorders . Nrf2 mainly mediates Keleh-like ECH-associated protein (Keap1)-Nrf2/antioxidant response element (ARE) signaling pathway. Under normal physiological conditions, Nrf2 is primarily negatively regulated by Keap1. Nrf2 bound to Keap1 remains in the cytoplasm, maintaining low transcriptional activity . Under oxidative stress, Nrf2 is released from Keap1, translocates to the nucleus, and binds to the ARE to activate target gene transcription and produce antioxidant enzymes . The number and activity of β cells have been found to be significantly lower in
Oxymatrine is the main component of a traditional Chinese herb,
The INS-1 cell line was obtained from AiYan Biological technology Co., Ltd. (Shanghai, China). The cells were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with streptomycin (100 μg/ml; Sigma-Aldrich, St. Louis, MO, USA), penicillin (100 U/ml; Sigma-Aldrich), β-mercaptoethanol (50 μM; Gibco), sodium pyruvate (0.11 g/L; Sangon Biotech Co., Ltd., Shanghai, China), and fetal bovine serum (10%; Gibco) at 37°C in a humidified atmosphere containing 5% CO2. The INS-1 cells were treated as follows: a control group: no treatment; HG + PA group: high glucose (30 mM glucose [Sangon Biotech Co., Ltd.]) + high fat (400 μM palmitic acid sodium [Sigma-Aldrich]); HG + PA + oxymatrine (1 μM) group: high glucose (30 mM glucose) + high fat (400 μM palmitic acid sodium) + oxymatrine (1 μM [Sigma-Aldrich]); HG + PA + oxymatrine (10 μM) group: high glucose (30 mM glucose) + high fat (400 μM palmitic acid sodium) + oxymatrine (10 μM). The concentrations of oxymatrine were based on the results of our previous work .
The FAM-FLICA Caspase-1 Assay Kit (Immuno Chemistry Technologies, Bloomington, MN, USA) was employed for cell pyroptosis detection. The INS-1 cells were seeded in 6-well plates at a density of 4 × 105 cells/ml. After treatment for 24 h, the cells were collected, washed, and centrifuged (1,000 rpm, 5 min). The supernatant was discarded, and the pellets were mixed with FAM-FLICA caspase-1 (10 μl:290 μl) and incubated at 37°C for 1 h. After the medium was removed by centrifugation and the samples washed three times with 1 × Apoptosis wash buffer, a working solution of propidium iodide (100 μg/ml) was added to the cell suspension (1 μl:100 μl). The cells were incubated at room temperature for 15 min and detected using a Beckman DxFLEX flow cytometer (Beckman Coulter, Inc., Indianapolis, IN, USA).
A Reactive Oxygen Species Assay Kit (Beyotime Biotechnology, Shanghai, China) was used to measure ROS production. After treatment for 24 h, the INS-1 cells were digested with trypsin without EDTA (Gibco), centrifuged (1,500 rpm, 5 min) at 4°C, and collected. Each sample was incubated with 1.5 ml of a working solution of dichlorodihydrofluorescein diacetate (5 μM) at 37°C in the dark for 20 min. Subsequently, the cells were centrifuged (1,500 rpm, 10 min), and the staining solution was carefully discarded. After washing two times with phosphate-buffered saline (PBS), the cells were resuspended with PBS (200 μl/well). Beckman DxFLEX flow cytometer was used to detect the fluorescence signals of 10,000 cells within half an hour to obtain a curve.
The release of LDH in the supernatants was measured by using a commercially available LDH assay kit (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. The INS-1 cells were treated as follows: control; HG + PA; HG + PA + oxymatrine (1 μM); HG + PA + oxymatrine (10 μM). The cells were cultured in a 24-well plate for 24 h. The absorbance was determined using a multifunctional microplate reader (Berthold Technologies, Bad Wildbad, Germany) at 450 nm.
The INS-1 cells were treated as follows: control; HG + PA; HG + PA + oxymatrine (10 μM). After the cells were digested and centrifuged twice (1,000 rpm, 5 min, and 1,600 rpm, 5 min), the supernatant was removed and the cell masses collected. The samples were fixed (2.5% glutaraldehyde and 1% osmic acid), dehydrated (ethanol and acetone), embedded in epoxy resin, sectioned using a microtome to a thickness of 50 nm, and stained (uranium acetate and lead citrate). Finally, the specimens were observed and images acquired using a transmission electron microscope (TEM; Hitachi, Tokyo, Japan).
IL-1β and IL-18 concentrations were measured using an IL-1β ELISA kit (R&D Systems, Minneapolis, MN, USA) and an IL-18 ELISA kit (Huijia Biological Technology Co., Ltd., Xiamen, China), respectively. The INS-1 cells were treated as follows: control; HG + PA; HG + PA + oxymatrine (10 μM). The cells were seeded in 96-well plates (5 × 103 cells/well) for 24 h. After the cells were digested and centrifuged (2,000 rpm, 20 min), the supernatant was collected, and the assay performed according to the manufacturer’s instructions. The optical density was determined at 450 nm by using the multifunctional microplate reader.
Caspase-1 activity was assessed by using a caspase-1 activity assay kit (Beyotime Biotechnology) according to the manufacturer’s instructions. The INS-1 cells were treated as follows: control; HG + PA; HG + PA + oxymatrine (10 μM). The cells were incubated in 96-well plates (5 × 103 cells/well) for 24 h. After the cells were digested and centrifuged (700 rpm, 5 min, 4°C), the supernatant was discarded. Subsequently, the cells were resuspended with pyrolysis buffer (100 μl pyrolysis buffer for 2 × 106 cells), lysed in an ice bath for 15 min, and centrifuged (3,800 rpm, 10 min, 4°C). The supernatant was mixed with precooled Ac-YVAD-pNA (2 mM) and incubated at 37°C for 60 min. The absorbance was measured at a wavelength of 405 nm using the multifunctional microplate reader.
The protein levels were analyzed by western blotting. The INS-1 cells were treated as follows: control; HG + PA; HG + PA + oxymatrine (1 μM); HG + PA + oxymatrine (10 μM). The cells were cultured in 6-well plates (4 × 105 cells/ml) for 24 h. In brief, the cell lysates were electrophoresed and transferred onto polyvinylidene fluoride membranes. Then, the membranes were incubated with the following primary antibodies (diluted in Tris-buffered saline with 0.1% Tween-20 [TBST] buffer): anti-NLRP3 antibody (ab214185, 1:1,000), anti-IL-1β antibody (ab205924, 1:1,000), anti-NF-κB p65 antibody (ab16502, 1:2,000), anti-Nrf2 antibody (ab89443, 1:500), anti-HO-1 antibody (ab13243, 1:2,000), anti-Keap1 antibody (ab119403, 1:1,000), anti-ASC antibody (ab180799, 1:2,000), and β-actin antibody (ab8226, 1:1,000) from Abcam (Cambridge, UK); anti-Gsdmd antibody (93709, 1:1,000) and anti-Histone H3 antibody (4499, 1:2,000) from Cell Signaling Technology (Boston, MA, USA); and anti-caspase-1 antibody (NBP1-45433, 1:1,000) from Novus Biologicals (Littleton, CO, USA). After washing, the membranes were incubated with the appropriate secondary antibody (diluted in TBST buffer) from Abcam (1:5,000, Cambridge, UK). Image Pro Plus 6.0 software (Media Cybernetics, Houston, TX, USA) was used to determine the intensity of the protein bands.
A firefly luciferase reporter gene assay kit was obtained from Beyotime Biotechnology. The NF-κB-luc reporter plasmid and Nrf2-luc reporter plasmid were purchased from Genomeditech (Shanghai, China). The INS-1 cells were treated as follows: control; HG + PA; HG + PA + oxymatrine (10 μM). After 36‒48 h of co-transfection with the plasmids, the cells in each well were treated with 100 μl of pyrolysis buffer and centrifuged (3,700 rpm, 3 min). Then, the supernatant was collected and mixed with luciferase test reagent (100 μl sample:100 μl luciferase test reagent) to measure the relative luciferase activity using the multifunctional microplate reader. The measurement time was 10 sec and the interval was 2 sec.
The data were expressed as mean ± standard deviation (SD). Sigmaplot (Systat Software, San Jose, CA, USA) was used to statistical analysis. Statistical differences were tested by one-way analysis of variance (ANOVA) and Tukey’s test. p < 0.05 was considered significant.
Flow cytometry, the LDH release assay, and TEM were employed to determine the effects of oxymatrine on pyroptosis in INS-1 cells. As shown in Fig. 1, mitochondrial cristae and abundant organelles were visible in the control cells; no mitochondrial swelling or vacuolar degeneration was observed. The number of autophagosomal vesicles was higher in the HG + PA cells, which also showed mitochondrial swelling, vacuolar degeneration, irregular nuclear morphology, local nuclear membrane depression, decreased organelles, and a large number of intracellular vacuoles (Fig. 1A). The HG + PA cells showed an increase in caspase-1 activity to 18.70% ± 1.48% at 24 h (p < 0.01
After the cells were treated under different conditions, the levels of NLRP3, Gsdmd-N, caspase-1, ASC, and IL-1β were detected by Western blotting, while IL-1β and IL-18 secretion was measured using ELISA. Caspase-1 activity was examined by spectrophotometry. The levels of NLRP3, Gsdmd-N, caspase-1, IL-1β, and ASC (p < 0.01
As indicated in Fig. 3, ROS production was higher in HG + PA cells than in the control cells (p < 0.01
Treatment of INS-1 cells under different conditions for 0.5 h, 1 h, or 2 h altered NF-κB (p65) protein levels. In HG + PA cells, the nuclear p65 protein levels increased over time (0.5 h: 2.32 ± 0.22; 1 h: 3.64 ± 0.32; 2 h: 3.99 ± 0.35) (p < 0.01
As shown in Fig. 5, the levels of HO-1 and nuclear Nrf2 proteins increased in HG + PA cells compared with those in the control cells (p < 0.01
The DNA-binding activity of NF-κB (p65) and Nrf2 were assessed in INS-1 cells after 24 h treatment under different conditions. Compared with the control cells, the transcriptional activity of p65 and Nrf2 was activated in HG + PA cells (p < 0.01
Previous reports have indicated that high glucose and high fat concentrations can induce cell pyroptosis. Li
Hyperglycemia reduces the binding affinity of the NF-κB (p65) subunit to IκB alpha, causing increased nuclear translocation of p65  and transcription of target genes involved in inflammatory responses . Saturated fatty acids may also directly activate the toll-like-4 receptor, resulting in the activation of the downstream c-Jun NH 2-terminal kinase and inhibitor kappa B kinase β/NF-κB cascade [34,35]. Lipotoxicity, glucotoxicity, and glucolipotoxicity induce metabolic stress, which manifests as increased oxidative stress and ROS production [36-38]. As mentioned above, the activation of NLRP3 is affected by the NF-κB (p65) pathway and ROS production. Under oxidative stress, the transcriptional activity of Nrf2 was increased, and Nrf2 reduced ROS production by mediating the transcription and expression of a series of antioxidant stress proteins such as HO-1. Thus, both NF-κB and Nrf2 regulate NLRP3 by different mechanisms. Oxymatrine has been reported to regulate NF-κB pathway and Nrf2 expression. The NF-κB pathway is suppressed by oxymatrine in colon cancer cells and fibroblast-like synoviocytes [39,40]. By increasing the levels of Nrf2 and HO-1, oxymatrine reduces renal ischemia-reperfusion injury, cerebral ischemia-reperfusion injury, arsenic trioxide (As2O3)-affected liver injury, and lipopolysaccharide/D-galactosamine-induced acute liver failure [41-44]. Therefore, to explore the mechanism of oxymatrine-inhibited pyroptosis in INS-1 cells, we analyzed the effects of oxymatrine on these factors. The results showed that high glucose and high fat increased the DNA-binding activity of NF-κB (p65) and Nrf2. Treatment with oxymatrine suppressed the transcriptional activity of NF-κB and reduced the entry of P65 into the nucleus. In contrast, oxymatrine promoted
In summary, oxymatrine inhibits NLRP3-mediated pyroptosis in INS-1 cells, which may be related to its inhibition of the NF-κB pathway and activation of the Nrf2 pathway. These results contribute to understanding the mechanism of oxymatrine-regulated islet cell function and provide a theoretical basis for the clinical application of oxymatrine for the treatment of diabetes.
This work was supported by grants from Youth Science Foundation of Shanxi Province (201801D221395), Startup Foundation for Doctors of Shanxi Medical University (BS03201640).
The authors declare no conflicts of interest.
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