Uptake of MicroRNAs from Exosome-Like Nanovesicles of Edible Plant Juice by Rat Enterocytes

MicroRNAs (miRNAs) are small RNAs present in extracellular vesicles (EVs) that, when transferred to a target cell, affect its biological functions. Plant miRNAs regulate the expression of certain mammalian genes. Here, we characterized EVs in fruit and vegetable juice, and their miRNA cargo, and investigated whether such miRNA-containing EVs could be taken up by mammalian enterocytes in vitro. Using filtration and ultra-centrifugation methods, EVs were purified from commercially available and manually squeezed plant juice. EV morphological features and subcellular localization were analyzed using the NanoSight tracking system and electron microscopy. Plant EV miRNA levels were evaluated using quantitative reverse transcription PCR. For the in vitro EV uptake experiments, rat intestinal epithelial cells (IEC6) were used. Plant EVs shared morphological features with mammalian EVs and contained miR156a-5p, miR166a-3p, and miR168a-5p. EVs were present in the cell sap-filled central vacuoles and were taken up by IEC6 cells. Edible plant cells produce EVs that contain various miRNAs and release them into the central vacuole. The exogenous plant EVs are taken up by mammalian enterocytes in vitro. These findings suggest the possibility that exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract.

ISOLATION OF CUCURBITACIN-B FROM CUCUMIS CALLOSUS AND ITS HYPOGLYCEMIC EFFECT IN ISOLATED RAT ENTEROCYTES

Objective: The pericarp of fruits of Cucumis callous (Rottl.) Cogn. (Cucurbitaceae) is traditionally used for curing diabetes, epilepsy, and diarrhea. It has an active compound include Cucurbitacin-B (CuB), which acts as a potent inducer of CYP450 of rat enterocytes. This study was conducted with the aim of elaborating and reconciling our previous finding on the glucose-lowering effect of Cucumis callosus (Rottl.) Cogn. fruits.Methods: In vivo hypoglycemic potential for methanolic pericarp extracts from C callosus (MPCC, 350 mg/kg b.w. p. o), methanolic seed extract of C callosus (MSCC, 250 mg/kg b.w. p. o) and CuB (80 µg/kg b.w. p. o) were studied in streptozotocin (STZ, 55 mg/kg b.w. i. p) induced diabetic rats. Metformin (25 mg/kg b.w. p. o) served as reference drug. Ex vivo model of intestinal tissue preparation of Swiss albino rats named Single Pass Intestinal Perfusion (SPIP) technique was performed for ex vivo hypoglycemic study. The glucose levels in the serosal fluid were determined by commercially available glucose oxidase kit and compared with the standard drug metformin (0.1 mg/kg).Results: In vivo results showed that administration of MPCC (350 mg/kg b.w. p. o) and Cucurbitacin-B (80 µg/kg b.w. p. o) produced the hypoglycemic effect. The MPCC (1.4 mg/kg) and CuB (0.4 µg/kg) produced hypoglycemic effect in ex vivo technique. These effects are due to induction of 0.53 mµmoles of CYP450 proteins with maximum absorption at 454 mµ in rat enterocytes.Conclusion: The present investigation gave evidence that bitter pericarp of C callosus fruit has a hypoglycemic effect due to the presence of Cucurbitacin B as phytoconstituent but seeds did not have such effects.

Parathyroid Hormone-Related Protein (1-40) Enhances Calcium Uptake in Rat Enterocytes Through PTHR1 Receptor and Protein Kinase Cα/β Signaling

Background/Aims: Parathyroid hormone-related protein (PTHrP) is implicated in regulating calcium homeostasis in vertebrates, including sea bream, chick, and mammals. However, the molecular mechanism underlying the function of PTHrP in regulating calcium transport is still not fully investigated. This study aimed to investigate the effect of PTHrP on the calcium uptake and its underlying molecular mechanism in rat enterocytes. Methods: The rat intestinal epithelial cell line (IEC-6) was used. Calcium uptake was determined by using the fluo-4 acetoxymethyl ester fluorescence method. The expression levels of RNAs and proteins was assessed by RT-PCR and Western-blot, respectively. Results: PTHrP (1-40) induced rapid calcium uptake in enterocytes in a dose-dependent manner. PTHrP (1-40) up-regulated parathyroid hormone 1 receptor (PTHR1) protein but not 1,25D3-MARRS receptor. Pre-treatment of anti- PTHR1 antibody abolished the PTHrP (1-40)-induced calcium uptake. PTHrP (1-40) significantly up-regulated four transcellular calcium transporter proteins, potential vanilloid member 6 (TRPV6), calbindin-D9k (CaBP-D9k), sodium-calcium exchanger 1 (NCX1) and plasma membrane calcium ATPase 1 (PMCA1), in a dose- and time-dependent manner. Pre-treatment with TRPV6 or CaBP-D9k antibodies or knockout of rat TRPV6 or CaBP-D9k markedly inhibited PTHrP (1-40)-induced calcium uptake, whereas inhibition of NCX or PMCA1 by antibodies or inhibitors had no effect on PTHrP(1-40)-induced calcium uptake. Furthermore, PTHrP (1-40) treatment up-regulated protein levels of protein kinase C (PKC α/β) and protein kinase A (PKA). Pretreatment of PKC α/β inhibitor (but not PKA inhibitor) inhibited PTHrP (1-40)-induced calcium uptake. Conclusion: PTHrP (1-40) stimulates calcium uptake via PTHR1 receptor and PKC α/β signaling pathway in rat enterocytes, and calcium transporters TRPV6 and CaBP-D9k are necessary for this stimulatory effect.

Infrared Spectroscopy as a Tool to Study the Antioxidant Activity of Polyphenolic Compounds in Isolated Rat Enterocytes

The protective effect of different polyphenols, catechin (Cat), quercetin (Qc) (flavonoids), gallic acid (GA), caffeic acid (CfA), chlorogenic acid (ChA) (phenolic acids), and capsaicin (Cap), against H2O2-induced oxidative stress was evaluated in rat enterocytes using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy and Fourier Transform Infrared Microspectroscopy (FTIRM), and results were compared to standard lipid peroxidation techniques: conjugated dienes (CD) and Thiobarbituric Acid Reactive Substances (TBARS). Analysis of ATR-FTIR and FTIRM spectral data allowed the simultaneous evaluation of the effects of H2O2and polyphenols on lipid and protein oxidation. All polyphenols showed a protective effect against H2O2-induced oxidative stress in enterocytes, when administered before or after H2O2. Cat and capsaicin showed the highest protective effect, while phenolic acids had weaker effects and Qc presented a mild prooxidative effect (IR spectral profile of biomolecules between control and H2O2-treated cells) according to FTIR analyses. These results demonstrated the viability to use infrared spectroscopy to evaluate the oxidant and antioxidant effect of molecules in cell systems assays.

NHE3 Regulatory Factor 1 (NHERF1) Modulates Intestinal Sodium-dependent Phosphate Transporter (NaPi-2b) Expression in Apical Microvilli

Pi uptake in the small intestine occurs predominantly through the NaPi-2b (SLC34a2) co-transporter. NaPi-2b is regulated by changes in dietary Pi but the mechanisms underlying this regulation are largely undetermined. Sequence analyses show NaPi-2b has a PDZ binding motif at its C terminus. Immunofluorescence imaging shows NaPi-2b and two PDZ domain containing proteins, NHERF1 and PDZK1, are expressed in the apical microvillar domain of rat small intestine enterocytes. Co-immunoprecipitation studies in rat enterocytes show that NHERF1 associates with NaPi-2b but not PDZK1. In HEK co-expression studies, GFP-NaPi-2b co-precipitates with FLAG-NHERF1. This interaction is markedly diminished when the C-terminal four amino acids are truncated from NaPi-2b. FLIM-FRET analyses using tagged proteins in CACO-2BBE cells show a distinct phasor shift between NaPi-2b and NHERF1 but not between NaPi-2b and the PDZK1 pair. This shift demonstrates that NaPi-2b and NHERF1 reside within 10 nm of each other. NHERF1−/− mice, but not PDZK1−/− mice, had a diminished adaptation of NaPi-2b expression in response to a low Pi diet. Together these studies demonstrate that NHERF1 associates with NaPi-2b in enterocytes and regulates NaPi-2b adaptation.