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. 2020 Oct 13:14:4251-4261.
doi: 10.2147/DDDT.S270345. eCollection 2020.

EGCG Upregulates UCP3 Levels to Protect MIN6 Pancreatic Islet Cells from Interleukin-1β-Induced Apoptosis

Xu Jia #  1 , Ziren Luo #  2 , Ying Gao  3 , Hua Liu  3 , Xinghai Liu  3 , Wenli Mai  3 , Hong Liu  3 , Qian Zheng  3
Affiliations

EGCG Upregulates UCP3 Levels to Protect MIN6 Pancreatic Islet Cells from Interleukin-1β-Induced Apoptosis

Xu Jia et al. Drug Des Devel Ther. .

Abstract

Objective: The protective effects of epigallocatechin gallate (EGCG) on interleukin-1β (IL-1β)-induced apoptosis were investigated in murine MIN6 pancreatic β-cells. The role of uncoupling protein-3 (UCP3) signaling in this process was also explored.

Methods: After treatment with IL-1β and EGCG, cells were collected and analyzed. Cell viability was measured using the CCK8 assay and the function of β-cells was evaluated by analyzing insulin secretion. Detection of mitochondrial function in cells was performed by measuring mitochondrial membrane potential, the concentration of ATP and activity of ROS. Apoptosis was analyzed by Hochest33258 staining and flow cytometry. Expression levels of UCP3 were interrogated using immunohistochemistry, RT-PCR and Western blotting.

Results: Compared with the control group, IL-1β treatment (20nM) for 24 h significantly decreased cell viability and insulin secretion, damaged mitochondrial function and increased ROS activity. Results also showed increased apoptosis and a decrease in UCP3 expression levels (p<0.01). However, treatment with low (1mM) or high (5mM) concentrations of EGCG significantly decreased IL-1β-induced apoptosis (p<0.01), restored mitochondrial function and subsequently increased UCP3 levels in IL-1β-induced β-cells (p<0.01).

Conclusion: These results suggest that EGCG protects against IL-1β-induced mitochondrial injury and apoptosis in β-cells through the up-regulation of UCP3.

Keywords: EGCG; UCP3; apoptosis; pancreatic β-cells.

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Conflict of interest statement

The authors do not have potential financial conflicts of interest related to this manuscript. The authors report no conflicts of interest for this work.

Figures

Figure 1
Figure 1
EGCG treatment significantly improved the activity and function of IL-1β-stimulated MIN6 cells. MIN6 cells were stimulated with IL-1β followed by co-cultivation with low (1mM) or high (5mM) EGCG for 24 h. (A) Cell viability was assessed in each group using the CCK-8 assay. Mitochondrial membrane potential of MIN6 cells in each group was determined using flow cytometry. Basal insulin (B) and high glucose stimulated insulin (C) levels for group in the cell supernatant were determined using an ELISA kit. Data are representative of 2 independent experiments and presented as mean ±SD; *p < 0.05, **p < 0.01. Significant differences were evaluated using one-way ANOVA with a post hoc test (Fisher’s least significant difference).
Figure 2
Figure 2
EGCG treatment recovered mitochondrial function and reduced oxidative stress response in IL-1β-stimulated MIN6 cells. MIN6 cells were stimulated with IL-1β followed by co-cultivation with low (1mM) or high (5mM) EGCG for 24 h. (A) Mitochondrial membrane potential of MIN6 cells in was determined for each group using flow cytometry. (B) The bar chart show a summary of statistical results in (A). The activity of ATP (C) and content of ROS (D) in MIN6 cells for each group. Data are representative of 2 independent experiments and presented as mean ±SD; *p < 0.05, **p < 0.01. Significant differences were evaluated using one-way ANOVA with a post hoc test (Fisher’s least significant difference).
Figure 3
Figure 3
EGCG treatment decreased cell apoptosis in IL-1β-stimulated MIN6 cells. MIN6 cells were stimulated with IL-1β followed by co-cultivation with low (1mM) or high (5mM) EGCG for 24 h. Morphological analysis of the nuclei were stained using Hoechst 33258. Red arrows indicate apoptotic MIN6 cells. Representative images for each group (n=6 per group) are shown (magnification×400).
Figure 4
Figure 4
EGCG treatment decreased cell apoptosis in IL-1β-stimulated MIN6 cells. (A) Representative flow cytometry plots show percentages of early apoptotic cells (Annexin V-FITC+PI−), necrotic cells (Annexin V-FITC−PI+), late apoptotic cells and necrotic cells (Annexin V-FITC+PI+) and living cells (Annexin V-FITC−PI−) for each group of MIN6 cells receiving specified treatment (n ≥ 6 per group). (B) The bar charts show a summary of the statistical results in (A). Data are representative of 2 independent experiments and presented as mean ±SD; *p < 0.05, **p < 0.01. Significant differences were evaluated using one-way ANOVA with a post hoc test (Fisher’s least significant difference).
Figure 5
Figure 5
UCP3 was mainly expressed in the cytoplasm of MIN6 cells. MIN6 cells were stimulated with IL-1β followed by co-cultivation with low (1mM) or high (5mM) EGCG for 24 h. UCP3 protein levels were examined using immunohistochemistry and were indicated by red arrows. Representative histology images for each group (n=6 per group) are shown (magnification×400).
Figure 6
Figure 6
EGCG significantly increased the gene and protein expression levels of UCP3 in IL-1β-stimulated MIN6 cells. MIN6 cells were stimulated with IL-1β followed by co-cultivation with low (1mM) or high (5mM) EGCG for 24 h. (A) Summary of the immunohistochemical scores (IHS) for UCP3 protein levels for each group. (B) The gene expression levels of UCP3 in each group measured by qRT-PCR. (C) The protein levels of UCP3 in each group assessed by Western blotting and normalized to actin. (D) Bar chart showing a summary statistical results in (C). Data are representative of 2 independent experiments and presented as mean ±SD; *p < 0.05, **p < 0.01, ***p < 0.001. Significant differences were evaluated using one-way ANOVA with a post hoc test (Fisher’s least significant difference).
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