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Biochemical analysis and microRNA profiling in a high glucose In vitro model with resveratrol intervention
Author(s)
Tshivhase, Abegail Mukhethwa
Date Issued
2023
Type
Thesis
Publisher
Cape Peninsula University of Technology
Abstract
Background: The prevalence of diabetes has reached an alarming level worldwide.
Individuals with diabetes experience impaired glucose metabolism, which results in an
augmented inflammatory response and heightened oxidative stress, contributing to the
upregulation of inflammatory and pro-apoptotic genes. These effects ultimately exacerbate
complications associated with diabetes, which significantly compromise patients’ quality of life
and life expectancy. Thus, there is an urgent need to identify safe and effective drugs that
provide anti-diabetic benefits while protecting against complications of diabetes. Furthermore,
alternative biomarkers are required to facilitate early identification of complications and risk
management to improve the quality of life. Circulating miRNAs have emerged as potential
contributors to disease etiology and progression, including diabetes; hence, they can be of
significant use as novel markers with the potential for innovative diagnostic and therapeutic
tools. Additionally, the aberrant expression of miRNAs may be implicated in various pathways,
such as glucose metabolism, inflammation, oxidative stress, and apoptosis. The therapeutic
effects of natural compounds have been widely recognized for centuries. This study aimed to
investigate the effect of RES on oxidative stress, inflammation, apoptosis, and glucose
metabolism under high glucose-induced conditions as well as investigate the effect of high
glucose levels and evaluate the influence of RES on high glucose-induced miRNA
dysregulation. Methods: HepG2 liver cells were divided into six groups: control, High glucose (40 mM), Low
resveratrol (LR) (25 μM), High resveratrol (HR) (50 μM), HG+LR, and HG+HR. The
supernatant was collected after 48 and 72 hours of exposure; total RNA and miRNAs were
extracted according to the manufacturer's instructions. Total RNA was reverse transcribed into
cDNA and used for gene expression analyses. The extracted total miRNAs were used for
miRNA expression analyses using quantitative Polymerase Chain Reaction (qPCR). The
collected supernatant was utilized for ELISA, Bioplex, and lactate dehydrogenase (LDH)
assays. All statistical analyses were performed using GraphPad Prism version 8.0.0
(GraphPad Software, San Diego, California, USA). The Student’s t-test and one-way analysis
of variance (ANOVA) were used. All assays were performed in triplicate, and differences were
considered statistically significant at p<0.05.
Results: A significant reduction was observed in the expression levels of miR-126-3p, miR-
182-5p, and miR-30a-5p when HepG2 cells were exposed to high glucose conditions.
Intriguingly, resveratrol treatment reversed the reduction of miR-126-3p, miR-182-5p, and miR-
30a-5p caused by high glucose in HepG2 cells. Moreover, our research demonstrates that high glucose resulted in an increase in Neuronal Differentiation 1 (Neurod1) expression in
HepG2 cells. Conversely, the expression of Neurod1 was found to be reduced in response to
resveratrol. There was a significant increase in the mRNA expression of nuclear factor kappa
B (NF-kB), IkB kinase α (IKKα), and IkB-α when HepG2 cells were exposed to high glucose.
Resveratrol treatment markedly reduced NF-kB, IKKα, and IkB-α expression levels. A notable
increase in Sprouty-related EVH1 domain containing 1 (SPRED1) expression was observed
in cells treated with high glucose, leading to augmented expression levels of tumour necrosis
factor-alpha (TNF-α), Interleukin-6 (IL-6), Cyclooxygenase 2 (COX2), and Interleukin-1 beta
(IL-1β). Nevertheless, resveratrol treatments reduced the expression levels of SPRED1, TNF-
α, IL-6, COX2, and IL-1β in HepG2 cells. In cells treated with high glucose, there was a
significant increase in the expression of FOXO1. This increase subsequently led to an increase
in the expression of genes associated with gluconeogenesis, namely phosphoenolpyruvate
carboxykinase (PEPCK) and Glucose-6-phosphate (G6P). Simultaneously, there was a
concurrent reduction in the expression of glucokinase (GCK). In contrast, resveratrol treatment
reduced FOXO1, PEPCK, and G6P expression while increasing GCK expression. A significant
reduction in nuclear factor erythroid 2–related factor 2 (Nrf2) expression, (p<0.0001) and
antioxidant enzymes (SOD, Superoxide dismutase; GPx1, Glutathione peroxidase 1; CAT and
NQO1, NAD(P)H quinone oxidoreductase 1) were observed when HepG2 cells were exposed
to high glucose. Remarkably, resveratrol increased Nrf2 expression, subsequently triggering
an increase in genes associated with antioxidant enzymes (SOD, CAT, GPx1, and NQO1).
High glucose exposure notably decreased B-cell lymphoma 2 (BcL-2) gene expression,
whereas resveratrol treatment significantly increased BcL-2 expression. Prolonged exposure
of HepG2 cells to high glucose (72 h) increased LDH release. Intriguingly, resveratrol treatment
showed a noteworthy reduction in LDH release. High glucose exposure reduced Oxoguanine
glycosylase-1 (OGG1) expression, while resveratrol significantly increased OGG1 mRNA
levels (p<0.0001).
Conclusion: Data obtained from this study showed that high glucose levels influence miR-
126-3p, miR-182-5p, and miR-30a-5p in HepG2 liver cells. While resveratrol treatment
reversed high glucose-induced downregulation of miR-126-3p, miR-182-5p, and miR-30a-5p
in HepG2 cells. Thus, suggesting a promising role for resveratrol in regulating miRNA
expression patterns implicated in diabetes. Our findings demonstrated that high glucose
disrupts pathways (glucose metabolism, inflammation, oxidative stress, and apoptosis) related
to diabetes. Moreover, our findings demonstrated that resveratrol may ameliorate the
pathologic processes involved in DM complications by reducing inflammation and oxidative
stress, increasing anti-apoptotic and DNA-repair genes, and regulating glucose metabolism.
Individuals with diabetes experience impaired glucose metabolism, which results in an
augmented inflammatory response and heightened oxidative stress, contributing to the
upregulation of inflammatory and pro-apoptotic genes. These effects ultimately exacerbate
complications associated with diabetes, which significantly compromise patients’ quality of life
and life expectancy. Thus, there is an urgent need to identify safe and effective drugs that
provide anti-diabetic benefits while protecting against complications of diabetes. Furthermore,
alternative biomarkers are required to facilitate early identification of complications and risk
management to improve the quality of life. Circulating miRNAs have emerged as potential
contributors to disease etiology and progression, including diabetes; hence, they can be of
significant use as novel markers with the potential for innovative diagnostic and therapeutic
tools. Additionally, the aberrant expression of miRNAs may be implicated in various pathways,
such as glucose metabolism, inflammation, oxidative stress, and apoptosis. The therapeutic
effects of natural compounds have been widely recognized for centuries. This study aimed to
investigate the effect of RES on oxidative stress, inflammation, apoptosis, and glucose
metabolism under high glucose-induced conditions as well as investigate the effect of high
glucose levels and evaluate the influence of RES on high glucose-induced miRNA
dysregulation. Methods: HepG2 liver cells were divided into six groups: control, High glucose (40 mM), Low
resveratrol (LR) (25 μM), High resveratrol (HR) (50 μM), HG+LR, and HG+HR. The
supernatant was collected after 48 and 72 hours of exposure; total RNA and miRNAs were
extracted according to the manufacturer's instructions. Total RNA was reverse transcribed into
cDNA and used for gene expression analyses. The extracted total miRNAs were used for
miRNA expression analyses using quantitative Polymerase Chain Reaction (qPCR). The
collected supernatant was utilized for ELISA, Bioplex, and lactate dehydrogenase (LDH)
assays. All statistical analyses were performed using GraphPad Prism version 8.0.0
(GraphPad Software, San Diego, California, USA). The Student’s t-test and one-way analysis
of variance (ANOVA) were used. All assays were performed in triplicate, and differences were
considered statistically significant at p<0.05.
Results: A significant reduction was observed in the expression levels of miR-126-3p, miR-
182-5p, and miR-30a-5p when HepG2 cells were exposed to high glucose conditions.
Intriguingly, resveratrol treatment reversed the reduction of miR-126-3p, miR-182-5p, and miR-
30a-5p caused by high glucose in HepG2 cells. Moreover, our research demonstrates that high glucose resulted in an increase in Neuronal Differentiation 1 (Neurod1) expression in
HepG2 cells. Conversely, the expression of Neurod1 was found to be reduced in response to
resveratrol. There was a significant increase in the mRNA expression of nuclear factor kappa
B (NF-kB), IkB kinase α (IKKα), and IkB-α when HepG2 cells were exposed to high glucose.
Resveratrol treatment markedly reduced NF-kB, IKKα, and IkB-α expression levels. A notable
increase in Sprouty-related EVH1 domain containing 1 (SPRED1) expression was observed
in cells treated with high glucose, leading to augmented expression levels of tumour necrosis
factor-alpha (TNF-α), Interleukin-6 (IL-6), Cyclooxygenase 2 (COX2), and Interleukin-1 beta
(IL-1β). Nevertheless, resveratrol treatments reduced the expression levels of SPRED1, TNF-
α, IL-6, COX2, and IL-1β in HepG2 cells. In cells treated with high glucose, there was a
significant increase in the expression of FOXO1. This increase subsequently led to an increase
in the expression of genes associated with gluconeogenesis, namely phosphoenolpyruvate
carboxykinase (PEPCK) and Glucose-6-phosphate (G6P). Simultaneously, there was a
concurrent reduction in the expression of glucokinase (GCK). In contrast, resveratrol treatment
reduced FOXO1, PEPCK, and G6P expression while increasing GCK expression. A significant
reduction in nuclear factor erythroid 2–related factor 2 (Nrf2) expression, (p<0.0001) and
antioxidant enzymes (SOD, Superoxide dismutase; GPx1, Glutathione peroxidase 1; CAT and
NQO1, NAD(P)H quinone oxidoreductase 1) were observed when HepG2 cells were exposed
to high glucose. Remarkably, resveratrol increased Nrf2 expression, subsequently triggering
an increase in genes associated with antioxidant enzymes (SOD, CAT, GPx1, and NQO1).
High glucose exposure notably decreased B-cell lymphoma 2 (BcL-2) gene expression,
whereas resveratrol treatment significantly increased BcL-2 expression. Prolonged exposure
of HepG2 cells to high glucose (72 h) increased LDH release. Intriguingly, resveratrol treatment
showed a noteworthy reduction in LDH release. High glucose exposure reduced Oxoguanine
glycosylase-1 (OGG1) expression, while resveratrol significantly increased OGG1 mRNA
levels (p<0.0001).
Conclusion: Data obtained from this study showed that high glucose levels influence miR-
126-3p, miR-182-5p, and miR-30a-5p in HepG2 liver cells. While resveratrol treatment
reversed high glucose-induced downregulation of miR-126-3p, miR-182-5p, and miR-30a-5p
in HepG2 cells. Thus, suggesting a promising role for resveratrol in regulating miRNA
expression patterns implicated in diabetes. Our findings demonstrated that high glucose
disrupts pathways (glucose metabolism, inflammation, oxidative stress, and apoptosis) related
to diabetes. Moreover, our findings demonstrated that resveratrol may ameliorate the
pathologic processes involved in DM complications by reducing inflammation and oxidative
stress, increasing anti-apoptotic and DNA-repair genes, and regulating glucose metabolism.
Additional information
Thesis (DPhil (Biomedical Sciences))--Cape Peninsula University of Technology, 2023
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