Novel In Vitro Assay of the Effects of Kampo Medicines against Intra/Extracellular Advanced Glycation End-Products in Oral, Esophageal, and Gastric Epithelial Cells

Abstract

Kampo medicines are Japanese traditional medicines developed from Chinese traditional medicines. The action mechanisms of the numerous known compounds have been studied for approximately 100 years; however, many remain unclear. While components are normally affected through digestion, absorption, and metabolism, in vitro oral, esophageal, and gastric epithelial cell models avoid these influences and, thus, represent superior assay systems for Kampo medicines. We focused on two areas of the strong performance of this assay system: intracellular and extracellular advanced glycation end-products (AGEs). AGEs are generated from glucose, fructose, and their metabolites, and promote lifestyle-related diseases such as diabetes and cancer. While current technology cannot analyze whole intracellular AGEs in cells in some organs, some AGEs can be generated for 1-2 days, and the turnover time of oral and gastric epithelial cells is 7-14 days. Therefore, we hypothesized that we could detect these rapidly generated intracellular AGEs in such cells. Extracellular AEGs (e.g., dietary or in the saliva) bind to the receptor for AGEs (RAGE) and the toll-like receptor 4 (TLR4) on the surface of the epithelial cells and can induce cytotoxicity such as inflammation. The analysis of Kampo medicine effects against intra/extracellular AGEs in vitro is a novel model.

Keywords: Kampo medicines; absorption; advanced glycation end-products; digestion; esophageal epithelial cells; gastric epithelial cells; in vitro; metabolism; oral epithelial cells.

Figure 1

Schematic diagram of progression of Kampo medicines used as oral administration in a mouse model. Medicines are digested in the stomach, absorbed in the small intestine, metabolized in the liver, and passed into the blood.

Figure 2

Schematic diagram of the novel in vitro assay for analysis of the effects of Kampo medicines on the complete organism (mouse), esophagus, and/or stomach. In this assay, the steps of absorption and metabolism can be removed, and preparation of hydrolyzed samples allows assessment of stomach-digested medicine. Blue triangles indicate medicine or digestate transportation pathways that can be closed.

Figure 3

Schematic diagram of application of the novel in vitro assay for assessing the effects of Kampo medicines on intracellular advanced glycation end-products (AGEs) rapidly generated in oral and gastric epithelial cells. RAGE: receptor for AEGs. TLR4: toll-like receptor 4.

Figure 4

Schematic diagram of application of the novel in vitro assay for assessing the effects of Kampo medicines against extracellular advanced glycation end-products (AGEs) that can bind to receptor for AEGs (RAGE) and/or toll-like receptor 4 (TLR4) in oral, esophageal, and gastric epithelial cells. Yellow AGEs: AGEs released from organs. Pink AGEs: dietary AGEs.