Investigation of in vitro diabetic inflammation and treatment with Piper excelsum tea extract

Abstract

Induction of inflammation through hyperglycaemia and adiposity has been shown to contribute to the pathogenicity of type 2 diabetes (T2D). The pro-inflammatory cytokine, tumour necrosis factor α (TNF-α) was the first cytokine found to bridge the connection between the immune system and T2D. This was found due to its increased production in the disease state. TNF-α has specifically been linked to the onset of insulin resistance in T2D models. Evidence has shown that heat shock protein 60 (HSP60) may also play a role as a danger signal for the immune system as it can result in the induction of inflammation. Currently, there are no pharmaceutical options for T2D patients which target inflammation. Tea made from the leaves of kawakawa (Piper excelsum) has been used for many generations by Māori as a traditional rākau rongoā (traditional medicine) to treat a range of ailments including inflammation. The aim of this project was to investigate the modulation of inflammation using a commercially available kawakawa tea in human cancer cells in vitro.

This project involved measuring the growth rate, cytotoxicity, TNF-α production and HSP60 expression upon exposure to three treatments: high glucose, lipopolysaccharide (LPS) or kawakawa tea extract (KTE).

This study showed that a high concentration of KTE (1000 µg/mL) inhibited cell growth in both HEPG2 and HeLa cell lines. Cells treated with this concentration also showed typical morphological signs of apoptosis. Cytotoxicity testing confirmed that there was a significant (p <0.05) decrease in mitochondrial dehydrogenase activity for this treatment which confirmed the cells were likely dying through apoptosis. Over a course of 24-hours three LPS treatments were found to be cytotoxic. These were the 10 ng/mL, 10 µg/mL, and 100 µg/mL concentrations of LPS. After 48-hours all treatments apart from 100 µg/mL KTE treatment were also found to be cytotoxic. The cytotoxicity for these treatments was either due to a reduction in mitochondrial dehydrogenase activity (infers mitochondrial stress) or increased lactate dehydrogenase (LDH) release from cells (infers necrotic cell death). These cytotoxicity experiments allowed for identification of which concentrations should be selected or removed for future experiments.

Cytotoxicity levels were taken into consideration and experiments were run investigating the induction of TNF-α using enzyme-linked immunosorbent assays (ELISA). Issues regarding negative readings from samples arose which resulted in protocol changes and extensive trouble shooting. It was found that extended exposure times were required to attenuate a positive result from the positive control, LPS.

HSP60 expression was also measured for human cells exposed to high glucose and KTE treatments. No change in HSP60 expression was detected. However, 24-hour LPS exposure resulted in a significant increase in HSP60 expression compared to the control for the 100 ng/mL LPS treatment. Two concentrations were investigated; 100 ng/mL LPS and 100 µg/mL LPS which resulted in 1.4- and 2.1-fold increases compared to the control, respectively.

Overall, no conclusion could be drawn regarding the modulation of inflammation by KTE. However, this study was able to identify the effect the tea has on human cancer cell growth and the nature of cytotoxicity the tea poses to the cells. It is recommended to continue this research exploring different kawakawa tea blends and using alternative in vitro cell lines and whole animal models.