An Investigation into the Therapeutic Modulation of Aquaporin Five Expression in Salivary Gland Cells Treated with Natural Substances

Abstract

Hyposalivation is an excessive, objectively determinable reduction in salivation which affects primarily menopausal women and geriatric patients above 65 years of age. Ageing per se as a cause of hyposalivation has been controversial. Strong support for ageing as a cause of hyposalivation exists. For instance, Yamamura and co-workers observed an age-related drop in salivation in ageing C57BL/6 female mice. The present study aims to determine whether the age-related hyposalivation observed in ageing mice can be reversed by upregulating the water channel molecule Aquaporin 5 (AQP5) with edible bird’s nest (EBN) and ginger extracts (6-shogaol, 6-gingerol, 8-gingerol, and 10-gingerol). To evaluate whether the treatments upregulate AQP5 expression, a cell line assay had to be developed. Another aim of the present study was to determine the suitability of the human salivary gland (HSG) and A-253 cell lines for a cell line assay that studies the modulation of AQP5 expression. 26 female, C57BL/6 mice were arranged into four groups (8 control mice, 6 Decitabine-treated mice, 6 6-shogaol-treated mice, and 6 EBN- treated mice). Stimulated saliva was collected from the mice from weeks 6 to 16 to observe whether there was an age-related hyposalivation. The mice were treated from weeks 17 to 19, and their saliva collected to determine whether the treatments were able to reverse the age-related hyposalivation. HSG, A-253, and MCF7 cells were cultured and studied for endogenous AQP5 mRNA and protein expression. RNA was extracted from the cells, and reverse transcription polymerase chain reaction (RT-PCR) was conducted to identify whether these cells expressed AQP5 mRNA. Protein was extracted from the cells, and western blot was conducted to identify whether they expressed AQP5 protein. A-253 and MCF7 cells were treated with the natural substances. To evaluate whether their expressions were downregulated or upregulated from expression levels in the untreated cells, reverse transcription quantitative polymerase chain reaction (RT-QPCR) was conducted. An age-related hyposalivation was observed in the mice as they aged from weeks 6 to 16, validating the age-related hyposalivation observed by Yamamura and co-workers, and lending further support to the position that ageing per se is a cause of hyposalivation. EBN was unable to reverse this age-related drop in salivation, but 6-shogaol was able to, indicating that 6-shogaol is a potential therapeutic agent for age-related hyposalivation. Furthermore, the success of the mouse experiments indicate that the mouse experimental design is suitable for the screening of future natural substances for hyposalivation therapy. Neither HSG nor A-253 cells met the criteria required for the cell line assay, as neither expressed a basal level of AQP5 mRNA and protein. MCF7, due to its reliability in expressing AQP5, was proposed as a proxy cell line until a suitable cell line is discovered. QPCR results from the cell treatment studies indicate that the gingerols and EBN have an upregulation effect on AQP5 mRNA at the earlier durations of treatment (48 hours); however, these findings must be taken with a grain of salt, as A-253 cells are not suitably ideal for the type of cell line required.