Momordica foetida facilitates glucose uptake independent of AMPK2 and PI3K to attenuate hyperglycemia-induced oxidative stress via a JNK-STAT3 mediated pathway in HepG2 cells.

Abstract

Introduction: The exponential rise in the global prevalence and incidence of type 2 diabetes is concerning. Hyperglycemia is a hallmark of type 2 diabetes that induces oxidative stress, leading to impairment of vital liver metabolic pathways. Metformin is the first-line treatment for type 2 diabetes mellitus. However, Momordica foetida has been used in folk medicine for the treatment and management of diabetes mellitus in various parts of the world including South Africa. Aim: In the present study, the cytoprotective effects of M. foetida on liver impaired glucose metabolism and oxidative stress damage were investigated on high glucose induced HepG2 cells, with Metformin as a positive drug control. Methods: The M. foetida leaves were used to prepare an aqueous lyophilized extract. The HepG2 cells were serum starved for 1 hour, then exposed to hyperglycemic conditions (30mM D-glucose) for 24 hours. Cells were treated with various concentrations (125 - 1000 μg/ml) of the lyophilized M. foetida aqueous extract for 24 hours, and the 3-(4,5- dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) assay evaluated the effects of high glucose and M. foetida on the metabolic activity of HepG2 cells. Antioxidants and prooxidants were assessed and quantified using luminometry, thiobarbituric acid reactive substances (TBARS) and nitric oxide synthase (NOS) assays. Western blot and quantitative real-time (qPCR) were used to observe the effects high glucose and M. foetida on signaling pathways and antioxidant response. Results: Glucose uptake in hyperglycemic conditions was mediated by increased gene expression of adenosine monophosphate-activated protein kinase alpha 2 (AMPKα2) (p˂0.05) and phosphatidylinositol 3‑kinase (PI3K) (p˂0.05), but glucose transporter 2 (GLUT2), glucokinase (GK) and glycogen synthase (GS) were downregulated (p˂0.05). Interestingly, an opposing response was noted for Metformin and M. foetida treatments, where AMPKα2 (p˂0.05) and PI3K (p˂0.05) were downregulated, whereas GLUT2, GK and GS were upregulated (p˂0.05) compared to the hyperglycemic control. When compared to the hyperglycemic conditions control, M. foetida treatments and Metformin showed an increase in glucose uptake. Hyperglycemic conditions induced toxicity indicated by increased extracellular lactate dehydrogenase (LDH) and decreased adenosine triphosphate (ATP), but Metformin and M. foetida decreased LDH activity back to xvi normoglycemic levels, indicating reduced cytotoxicity. Increased mitochondrial membrane potential (m) in hyperglycemic conditions was accompanied by increased lipid peroxidation (p˂0.05) and reactive nitrogen species (RNS) (p˂0.05). The m was increased further by M. foetida, with minimal effect on reactive oxygen species (ROS) production but effectively increasing RNS (p˂0.05). Oxidative damage was reduced in the hyperglycemic control but was increased by Metformin and M. foetida treatments prompting the activation of p53 in these cells (p˂0.05). Effective oxidative stress response was mounted by NRF2 (p˂0.05) and antioxidants SOD2 (p˂0.05) and GSH, but GPx1 and CAT (p˂0.05) were decreased. Interestingly, Metformin and M. foetida induced CAT (p˂0.05) and GPx1 (p˂0.05) in the antioxidant response, consequently decreasing GSH. Metformin decreased NRF2 (p˂0.05) and SOD2, while M. foetida increased NRF2 significantly and had no effect on SOD2 relative to the hyperglycemic control. Hyperglycemic conditions downregulated the oxidative stress response by MAPK (p-p38, pJNK and pERK1/2) (p˂0.05). However, Metformin upregulated pJNK (p˂0.05) and pERK1/2 (p˂0.05), but p-p38 (p˂0.05) was downregulated. Interestingly, M. foetida upregulated pJNK (p˂0.05), downregulated pERK1/2 (p˂0.05) and had no effect on p-p38. Hyperglycemic conditions also increased pSTAT3, which was downregulated by Metformin and M. foetida treatments (p˂0.05). Conclusion: Taken together, the results demonstrated that M. foetida enhanced the metabolic activity and reduced cell cytotoxicity in HepG2 cells. Furthermore, M. foetida facilitated glucose uptake independent of AMPK2 and PI3K. The main source of oxidative stress was increased RNS, which was alleviated by an effective MAPK/JNK and antioxidant response involving CAT.