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. 2024 Apr 5;14(1):8043.
doi: 10.1038/s41598-024-58545-2.

Centella asiatica mitigates the detrimental effects of Bisphenol-A (BPA) on pancreatic islets

Oly Banerjee  1   2 , Siddhartha Singh  1 , Tiyesh Paul  1 , Bithin Kumar Maji  1 , Sandip Mukherjee  3
Affiliations

Centella asiatica mitigates the detrimental effects of Bisphenol-A (BPA) on pancreatic islets

Oly Banerjee et al. Sci Rep. .

Abstract

Bisphenol-A (BPA) is widely used in food packaging and household products, leading to daily human exposure and potential health risks including metabolic diseases like type 2 diabetes mellitus (T2DM). Understanding BPA's mechanisms and developing intervention strategies is urgent. Centella asiatica, a traditional herbal medicine containing pentacyclic triterpenoids, shows promise due to its antioxidant and anti-inflammatory properties, utilized for centuries in Ayurvedic therapy. We investigated the effect of Centella asiatica (CA) ethanol extract on BPA-induced pancreatic islet toxicity in male Swiss albino mice. BPA administration (10 and 100 μg/kg body weight, twice daily) for 21 days caused glucose homeostasis disturbances, insulin resistance, and islet dysfunction, which were partially mitigated by CA supplementation (200 and 400 mg/kg body weight). Additionally, heightened oxidative stress, elevated levels of proinflammatory cytokines, loss of mitochondrial membrane potential (MMP), abnormal cell cycle, and increased apoptosis were implicated in the detrimental impact of BPA on the endocrine pancreas which were effectively counteracted by CA supplementation. In summary, CA demonstrated a significant ability to mitigate BPA-induced apoptosis, modulate redox homeostasis, alleviate inflammation, preserve MMP, and regulate the cell cycle. As a result, CA emerged as a potent agent in neutralizing the diabetogenic effects of BPA to a considerable extent.

Keywords: Centella asiatica; Apoptosis; Bisphenol A (BPA); Inflammation; Insulin resistance; Oxidative stress.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Protective efficacy of CA against BPA-mediated impaired fasting blood glucose (FBG) level and oral glucose tolerance test (OGTT). (a) FBG, (b) OGTT and (c) area under the curve (AUC) for OGTT in BPA (10 and 100 µg/kg body weight for 21 days) treated mice with or without supplementation of CA (200 and 400 mg/kg body weight/day for 21 days). Data were presented as mean ± SEM (n = 6). Normality of data was tested by Shapiro–Wilk test. Significance level based on one-way ANOVA (for FBG and AUC), p < 0.05, or two-way ANOVA (for OGTT), p < 0.05. Significance level based on Tukey’s post hoc test *p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001, ns—not significant. (d) Significance level based on Tukey’s multiple comparison test for blood glucose level during OGTT at 120 min.
Figure 2
Figure 2
CA supplementation extends protection against BPA-induced pancreatic islet cell dysfunction and insulin resistance. Changes in plasma insulin and c-peptide level, HOMA-IR and HOMA-β index and insulin expression in pancreatic islets on exposure to BPA (10 and 100 µg/kg body weight for 21 days) with or without supplementation of CA (200 and 400 mg/kg body weight/day for 21 days): (a) fasting plasma insulin level, (b) fasting C-peptide level, (c) HOMA-IR index and (d) HOMA-β index. Data were presented as mean ± SEM (n = 6). Normality of data was tested by Shapiro–Wilk test. Significance level based on one-way ANOVA, p < 0.05. Significance level based on Tukey’s post hoc test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant. (e) Representative photomicrograph of immunohistochemical analysis of insulin in pancreatic islets of BPA (10 and 100 µg/kg body weight for 21 days) treated mice with or without supplementation of CA (200 and 400 mg/kg body weight/day for 21 days). Results are representative of six mice. Magnification ×200 and scale bar: 50 µm for all panels. (f) Quantification of immunohistochemical images presented in (e) by Image J software and expressed as integrated density (pixel density). Data were presented as mean ± SEM (n = 6). Normality of data was tested by Shapiro–Wilk test. Significance level based on one-way ANOVA, p < 0.05. Significance level based on Tukey’s post hoc test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant.
Figure 3
Figure 3
Beneficial impact of CA supplementation on BPA-induced change in pancreatic islet cell morphology. Representative photomicrograph of haematoxylene-eosine stained pancreas sections of BPA (10 and 100 µg/kg body weight for 21 days)-treated mice with or without supplementation of ethanol extract of Centella asiatica (CA: 200 and 400 mg/kg body weight/day for 21 days). (a) Control, (b) CA supplemented (200 mg/kg body weight/day for 21 days), (c) CA supplemented (400 mg/kg body weight/day for 21 days), (d) BPA treated (10 µg/kg body weight for 21 days), (e) BPA treated (100 µg/kg body weight for 21 days), (f) BPA10 + CA supplemented (200 mg/kg body weight/day for 21 days), (g) BPA10 + CA supplemented (400 mg/kg body weight/day for 21 days), (h) BPA100 + CA supplemented (200 mg/kg body weight/day for 21 days) and (i) BPA 100 + CA supplemented (400 mg/kg body weight/day for 21 days). Magnification ×400 and scale bar: 50 µm for all panels. (j) Quantification of cytoplasmic vacuolization of pancreatic islets. Vacuolization score was calculated using the following: > 2 vacuoles/100 μm islet diameter—1, > 4 vacuoles/100 μm islet diameter—2, > 8 vacuoles/100 μm islet diameter—3 (n = 6; from each animal, 3 islets were considered for scoring). (k) Median islet diameter (µm). Data of median islet diameter were presented as mean ± SEM (n = 6). Normality of data was tested by Shapiro–Wilk test. Significance level based on one way ANOVA, P < 0.001. Significance level based on Tukey’s post hoc test *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant.
Figure 4
Figure 4
Adverse effect of BPA on lipid profile parameters of mice: protective role of CA. Serum lipid profiles of BPA (10 and 100 µg/kg body weight for 21 days)-treated mice with or without CA supplementation (200 and 400 mg/kg body weight/day for 21 days). (a) Total cholesterol (mg/dl), (b) triglycerides (mg/dl), (c) HDL (mg/dl), (d) LDL (mg/dl) and (e) VLDL (mg/dl). Data were presented as mean ± SEM (n = 6). Normality of data was tested by Shapiro–Wilk test. Significance level based on one way ANOVA, p < 0.05. Significance level based on Tukey’s post hoc test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant.
Figure 5
Figure 5
CA regulates oxidative stress in pancreatic islets, enhancing its protective effects against the toxicity induced by BPA. Effect of BPA (10 and 100 µg/kg body weight for 21 days) on oxidative stress parameters with or without supplementation of ethanol extract of CA (200 and 400 mg/kg body weight/day for 21 days): (a) NO level, (b) MDA level, (c) intracellular ROS generation: isolated islet cells undergo staining with H2DCFDA and subsequent analysis via flow cytometry (higher dose of BPA was considered only). The values displayed in the representative flow cytometry data represent the intensity of DCF fluorescence for the entire cell population. (d) The accompanying bar graph illustrates quantitative information regarding the mean DCFDA. Data were presented as Mean ± SEM (n = 3, for each sample isolated islets were pooled from two animals). CA (200 and 400 mg/kg body weight/day for 21 days) supplementation further harmonize BPA (10 and 100 µg/kg body weight for 21 days)-induced alteration in antioxidant parameters: (e) SOD activity (f), CAT activity (g), GSH level, and (h) glutathione peroxidise (GPx) activity. Except intracellular ROS, all data were presented as Mean ± SEM (n = 6). Normality of data was tested by Shapiro–Wilk test. Significance level based on one-way ANOVA, p < 0.05. Significance level based on Tukey’s post hoc test *p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant.
Figure 6
Figure 6
Impact of CA supplementation on heightened inflammation, loss of mitochondrial membrane potential (MMP) and impaired cell cycle caused by BPA. Serum level of (a) IL-6, (b) TNF-α and (c) c-reactive protein (CRP) in BPA (10 and 100 µg/kg body weight for 21 days) treated mice with or without supplementation of CA (200 and 400 mg/kg body weight/day for 21 days). Data were presented as mean ± SEM (n = 6). Effect of CA supplementation (200 and 400 mg/kg body weight/day for 21 days) on BPA (100 µg/kg body weight for 21 days)-mediated MMP loss and impaired cell cycle. (d) i) MMP loss measured by flow cytometry using the DiOC6 fluorescent probe ii) mean fluorescence intensity (as fold of control) (mean ± SEM, n = 3, for each sample isolated islets were pooled from two animals) of traces shown in (d) i). (e) i) flow cytometry analysis of cell cycle progression determined using propidium iodide (PI), ii) and iii) % of cells in G0/G1 and S, respectively (mean ± SEM, n = 3, for each sample isolated islets were pooled from two animals). Normality of data was tested by Shapiro–Wilk test. Significance level based on one-way ANOVA, p < 0.05. Significance level based on Tukey’s post hoc test *p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant.
Figure 7
Figure 7
Protective role of CA against BPA-induced apoptosis of pancreatic islet cell and altered expression of Bcl2, Bax, cleaved caspase-3 and caspase-9. Effect of BPA (100 µg/kg body weight for 21 days) on apoptosis of pancreatic islet cells and expression of pro-apoptotic and anti-apoptotic markers with or without supplementation of ethanol extract of CA (200 and 400 mg/kg body weight/day for 21 days). (a) i) flow cytometry analysis of apoptosis determined using Annexin V/PI, ii) Apoptotic cells (mean ± SEM, n = 3, for each sample isolated islets were pooled from two animals) of traces shown in (a) i). (b) i) Representative photomicrograph of immunohischemical analysis of Bcl2 and Bax in pancreatic islets. Results are representative of six mice. Magnification ×200 and scale bar: 50 µm for all panels. (c) i) Western blot of cleaved caspase-3 and caspase 9, ii) and iii) Quantification of cleaved caspase-3 (17 kDa) and caspase 9 (mean ± SEM, n = 3, for each sample isolated islets were pooled from two animals). Normality of data was tested by Shapiro–Wilk test. Significance level based on one-way ANOVA, p < 0.05. Significance level based on Tukey’s post hoc test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant.
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