Pharmacologic Comparison of High-Dose Hesperetin and Quercetin on MDCK II Cell Viability, Tight Junction Integrity, and Cell Shape.

The modulation of tight junction (TJ) integrity with small molecules is important for drug delivery. High-dose baicalin (BLI), baicalein (BLE), quercetin (QUE), and hesperetin (HST) have been shown to open TJs in Madin-Darby canine kidney (MDCK) II cells, but the mechanisms for HST and QUE remain unclear. In this study, we compared the effects of HST and QUE on cell proliferation, morphological changes, and TJ integrity. HST and QUE were found to have opposing effects on the MDCK II cell viability, promotion, and suppression, respectively. Only QUE, but not HST, induced a morphological change in MDCK II into a slenderer cell shape. Both HST and QUE downregulated the subcellular localization of claudin (CLD)-2. However, only QUE, but not HST, downregulated CLD-2 expression. Conversely, only HST was shown to directly bind to the first PDZ domain of ZO-1, a key molecule to promote TJ biogenesis. The TGFß pathway partially contributed to the HST-induced cell proliferation, since SB431541 ameliorated the effect. In contrast, the MEK pathway was not involved by both the flavonoids, since U0126 did not revert their TJ-opening effect. The results offer insight for using HST or QUE as naturally occurring absorption enhancers through the paracellular route.

Opposing Effect of Naringenin and Quercetin on the Junctional Compartment of MDCK II Cells to Modulate the Tight Junction.

Maintaining tight junction (TJ) integrity is important for epithelial cell barriers. Previously, the enhancement of TJ integrity, induced by citrus-derived flavonoids, naringin (NRG) and hesperidin (HSD), was demonstrated, but the effects of their aglycones naringenin (NAR) and hesperetin (HST), and the mechanisms, have not been systematically investigated. Here we compared three series of flavonoids related to NAR, HST, quercetin (QUE) and their glycosides with the Madin-Darby canine kidney (MDCK) II cell monolayers. The effect of flavonoids on the protein expression level of claudin (CLD)-2 and its subcellular localization were investigated. NAR, NRG, and HSD increased the CLD-2 localization at the TJ compartment, and its protein expression level. QUE and HST showed TJ-mitigating activity. Narirutin (NRT), neohesperidin (NHD) and rutin (RUT) did not affect the TJ. In addition, NAR and QUE induced an increase or decrease of the transepithelial electrical resistance (TEER) values of the MDCK II monolayers. Two known signaling pathways, phosphatidyl-inositol-3 kinase (PI3K) and 5'-AMP-activated protein kinase (AMPK), were further compared with NAR. Two-dimensional polyacrylamide electrophoresis (2D PAGE) analysis of whole-cell proteins treated with NAR, AICA-riboside (AMPK activator) and LY294002 (PI3K inhibitor) showed in both a distinct pattern. This suggests the target of NAR's CLD-2 or zonula occludens-1 (ZO-1) modulation was unique.

High dose of baicalin or baicalein can reduce tight junction integrity by partly targeting the first PDZ domain of zonula occludens-1 (ZO-1).

The tight junction (TJ) is the apical-most intercellular junction complex, serving as a biological barrier of intercellular spaces between epithelial cells. The TJ's integrity is maintained by a key protein-protein interaction between C-terminal motifs of claudins (CLDs) and the postsynaptic density 95 (PSD-95)/discs large/zonula occludens 1 (ZO-1; PDZ) domains of ZO-1. Weak but direct interaction of baicalin and its aglycon, baicalein-which are pharmacologically active components of Chinese skullcap (Radix scutellariae)-with ZO-1(PDZ1) have been observed in NMR experiments. Next, we observed TJ-mitigating activity of these flavonoids against Madin-Darby canine kidney (MDCK) II cells with the downregulation of subcellular localization of CLD-2 at TJs. Meanwhile, baicalein-but not baicalin-induced a slender morphological change of MDCK cells' shape from their normal cobblestone-like shapes. Since baicalin and baicalein did not induce a localization change of occludin (OCLN), a "partial" epithelial-mesenchymal transition (EMT) induced by these flavonoids was considered. SB431542, an ALK-5 inhibitor, reversed the CLD-2 downregulation of both baicalin and baicalein, while SB431542 did not reverse the slender morphology. In contrast, the MEK/ERK inhibitor U0126 reversed the slender shape change. Thus, in addition to inhibition of the ZO-1-CLD interaction, activation of both transforming growth factor-ß (TGF-ß) and MEK/ERK signaling pathways have been suggested to be involved in TJ reduction by these flavonoids. Finally, we demonstrated that baicalin enhanced the permeability of fluorescence-labeled insulin via the paracellular pathway of the Caco-2 cell layer. We propose that baicalin, baicalein, and Radix scutellariae extract are useful as drug absorption enhancers.