Role of PEPT1in the transport of cinnabar in Caco-2 cells

Cinnabar, a mercury-containing mineral medicine, has been used as an ingredient in Traditional Chinese Medicines for treatment of various diseases for thousands of years and is still widely used today. The toxicity of cinnabar is much less than other mercury-containing compounds. This study aimed to evaluate the possible role of oligopeptide transporter1 (PEPT1) in intestinal uptake of cinnabar. Thus, the Caco-2 cell model was employed to investigate the differential transport levels and the probable transporter involved in the transport of cinnabar, mercury sulfide (HgS) and mercury chloride (HgCl₂). Cells were incubated with the same molar concentration of cinnabar, HgS or HgCl₂ and then the inorganic mercury content of apical (AP), cellular and basolateral (BL) side of the cell was measured by ultra-high liquid chromatography-inductively coupled plasma mass spectrometry (UPLC-ICP/MS) after the treatment, respectively. Their transportation levels were also investigated when pH was changed to 5.5 in AP side to define the role of the H⁺ dependent transporter. Effects of cinnabar, HgS or HgCl₂ on transporter mRNA and protein expression levels were assayed by RT-PCR and Western-blot method, respectively. The possible transporter involved in the transport was examined by siRNA silencing and chemical inhibition. The results showed that the levels of inorganic mercury in the BL side for cinnabar and HgS were 49.39% and 30.41% of that in HgCl₂ group. The transport levels of cinnabar and HgCl₂ were significantly increased when the pH was changed to 5.5 on the AP side as compared with the control group (pH 7.4). Cinnabar significantly decreased the mRNA and protein expression of PEPT1. Transport levels of cinnabar were significantly decreased by PEPT1-siRNA and chemical inhibition of PEPT1. The present study demonstrates that PEPT1 may be an important transporter in the entry of cinnabar into the intestinal epithelium, and intestinal transport levels of cinnabar and HgS was lower than that of HgCl₂.