Caco-2 Cell Monolayer Model Research Articles

Gastrointestinal metabolism of Astragalus membranaceus polysaccharides and its related hypoglycemic mechanism based on gut microbial transformation

Astragalus membranaceus polysaccharides (AMP) was reported to exhibit hypoglycemic potential in diabetic host. However, the metabolic fate of AMP in gastrointestinal tract and its underlying hypoglycemic mechanisms remained unclear. Our current study aimed to reveal the structure alteration of AMP in gastrointestinal tract and its hypoglycemic mechanism from the perspective of microbial transformation. Caco-2 monolayer cell model revealed that AMP exhibited poor intestinal absorption. The in-vitro digestion and fermentation study revealed that AMP remained intact after gastrointestinal digestion while it could be degraded and utilized by gut microbiota with increased SCFA formation and decreased levels of all the monosaccharides in AMP except for mannose. Additionally, Diversity of gut microbiota was improved with the increased abundance of Dubosiella and Monoglobus and decreased abundance of Escherichia-Shigella and Acinetobacter after fermentation of AMP. Further hypoglycemic mechanism study for the first time revealed that both AMP and its potential microbial metabolites, SCFA salt mixture, could enhance intestinal integrity significantly on LPS induced Caco-2 cell model, while only SCFA salt mixture rather than AMP could significantly stimulate GLP-1 secretion in NCI-H716 cell model possibly via promoting GPCR43 expression. Such findings provided insights into the hypoglycemic mechanism of AMP from the perspective of microbial transformation.

Bio-Calcium from Skipjack Tuna Frame Attenuates Bone Loss in Ovariectomy-Induced Osteoporosis Rats.

Bio-calcium derived from fish frames may offer several advantages for osteoporosis prevention. This study aimed to evaluate the effects of bio-calcium derived from skipjack tuna frames on bone loss in ovariectomized rats. Tuna bio-calcium was prepared through enzymatic hydrolysis, defatting, bleaching, and grinding processes. The bioavailability of calcium was tested using the Caco-2 cell monolayer model, showing that 13% of tuna bio-calcium was absorbed, compared to 10% for calcium carbonate. Rats were divided into the five following groups: (1) OVX, (2) sham-operated, (3), OVX + estrogen-treated (4) OVX + calcium carbonate-treated, and (5) OVX + tuna bio-calcium-treated. All groups were raised for eight weeks. Tuna bio-calcium was able to increase BV/TV by 26% in the femur and 29% in the tibia, compared to 13% and 17% in the OVX group, respectively. Trabecular thickness in the femur upsurged to 360 µm in the tuna group, while a thickness of 290 µm was observed in the control. Additionally, osteoclast numbers were reduced to 5 N.Oc/mm in the femur and 6 N.Oc/mm in the tibia in the tuna group, compared to 35 and 45 N.Oc/mm in the control. Overall, tuna bio-calcium effectively prevented bone loss and can serve as a promising natural alternative for managing osteoporosis.

Bee pollen from bracatinga (Mimosa scabrella): Effects of gastrointestinal digestion and epithelial transport in vitro on phenolic profile and bioactivities

The main objective of the present work was to assess the phenolic profile of bracatinga (Mimosa scabrella) bee pollen, and its antioxidant and anti-inflammatory activities after gastrointestinal digestion in vitro and epithelial transport in a Caco-2 cell monolayer model. The botanical origin of bee pollen was confirmed by optical microscopy and scanning electron microscopy. As major results, 34 phenolic compounds (13 phenylamides, 14 flavonols, and 7 flavanones) were tentatively identified in the extract of bracatinga bee pollen by HPLC-ESI-QTOF-MS. The aglycone forms of quercetin and p-coumaric acid were identified only after digestion, indicating the breakage of flavonols and phenylamides, respectively. These compounds may have contributed to the decrease in NF-κΒ activation up to 54% and in the release of TNF-α and CXCL2/MIP-2 by 26% and 21%, respectively, in raw 264.7 murine macrophages activated with microbial lipopolysaccharide and treated with the digested fraction. Among all tentatively identified phenolic compounds, five of them were found in the basolateral fraction. These compounds, represented by four aglycone flavonoids (quercetin, kaempferol, naringenin, and herbacetin methyl ether) and a phenolic acid (p-coumaric acid) may be responsible for its outstanding antioxidant activity in Caco-2 cells, as well as for its remaining capacity in mitigating CXCL2/MIP-2 release after transport through the Caco-2 cell monolayer, as an intestinal barrier model. Therefore, our work sheds light on the phenolic profile and bioactivities of an interesting functional food produced by bees throughout a simulated gastrointestinal system.

Studies on absorption mechanism and pharmacokinetic properties of albendazole-bile acid conjugate: In vivo and in vitro

PurposeTo improve the oral bioavailability of albendazole (ABZ), a series of albendazole-bile acid conjugates (ABCs) were synthesized. ABC's transmembrane transport mechanism and in vivo pharmacokinetic properties were preliminarily studied. MethodsThe transmembrane transport mechanism of ABCs was studied using the Caco-2 monolayer cell model and intestinal perfusion model. The concentration of ABCs and ABZ were evaluated using High-Performance Liquid Chromatography (HPLC) and HPLC-Mass Spectrometry (HPLC-MS/MS). ResultsCompared to ABZ, better permeability was observed for different types and concentrations of ABCs using the Caco-2 monolayer cell model, with ABC-C8 showing the highest permeability. The transmembrane transport of ABCs was affected by ASBT inhibitors, indicating an ASBT-mediated active transport mechanism. Additionally, introducing cholic acid resulted in ABZ no longer being a substrate for P-gp, MRP2, and BCRP, effectively reversing ABZ efflux. In vivo unidirectional intestinal perfusion results in rats showed that ABCs altered the absorption site of ABZ from the jejunum to the ileum. The absorption efficiency of ABCs in each intestinal segment was higher than that of ABZ, and the transmembrane transport efficiency decreased with increasing concentrations of ASBT inhibitors. This further confirmed the presence of both passive diffusion and ASBT-mediated active transport mechanisms in the transport of ABCs. The solubility of ABCs in gastric juice and pharmacokinetics in rats showed that ABZ-C4 exhibited enhanced solubility. Moreover, ABCs significantly increased oral bioavailability compared to ABZ, with ABC-C4 showing an approximately 31-fold increase in bioavailability. ConclusionThe transmembrane transport mechanism of ABCs involves a combination of ASBT-mediated active transport and passive diffusion. Moreover, the incorporation of BAs successfully reverses the efflux of ABZ by efflux proteins. Among the synthesized conjugates, ABC-C4 demonstrated superior dissolution behavior both in vitro and in vivo.

Deer Skin Collagen Peptides Bound to Calcium: In Vitro Gastrointestinal Simulation of Digestion, Cellular Uptake and Analysis of Antioxidant Activity.

The by-product of deer skin, which has mostly been used as a decorative material, is rich in collagen and amino acids that could bind to Ca2+. Therefore, the preparation process, stability, antioxidant activity and calcium transport capacity of deer skin collagen peptide calcium chelate (Ca-DSCP) were investigated. In addition, the structure of the new chelate was characterized. The preparation process of Ca-DSCP was optimized using one-way experiments and response surface methodology. The ideal conditions were pH 9, 48 °C, and a peptide-to-calcium mass ratio of 5:1. The chelation rate was (60.73 ± 1.54)%. Zeta potential, XRD, UV-vis and FTIR analyses yielded that deer skin collagen peptides (DSCP) underwent a chelating reaction with calcium ions to form new structures. The stability of Ca-DSCP and the fraction of bioavailability of calcium ions were determined using in vitro gastrointestinal digestion and a Caco-2 cell monolayer model. The results showed that fraction of bioavailability and stability of DSCP were improved by influencing the structural characterization. The antioxidant activities of DSCP and Ca-DSCP were evaluated by measuring relevant oxidative stress indicators, DPPH radical scavenging capacity and hydroxyl radical scavenging capacity. Finally, bioinformatics and molecular docking techniques were utilized to screen and study the antioxidant mechanism of DSCP.

Comparison of intestinal absorption of soybean protein isolate-, glutenin- and peanut protein isolate-bound Nε-(carboxymethyl) lysine after in vitro gastrointestinal digestion

Advanced glycation end products (AGEs), a heterogeneous compound existed in processed foods, are related to chronic diseases when they are accumulated excessively in human organs. Protein-bound Nε-(carboxymethyl) lysine (CML) as a typical AGE, is widely determined to evaluate AGEs level in foods and in vivo. This study investigated the intestinal absorption of three protein-bound CML originated from main food raw materials (soybean, wheat and peanut). After in vitro gastrointestinal digestion, the three protein-bound CML digests were ultrafiltered and divided into four fractions: less than 1 kDa, between 1 and 3 kDa, between 3 and 5 kDa, greater than 5 kDa. Caco-2 cell monolayer model was further used to evaluate the intestinal absorption of these components. Results showed that the absorption rates of soybean protein isolate (SPI)-, glutenin (Glu)-, peanut protein isolate (PPI)-bound CML were 30.18%, 31.57% and 29.5%, respectively. The absorption rates of components with MW less than 5 kDa accounted for 19.91% (SPI-bound CML), 22.59% (Glu-bound CML), 23.64% (PPI-bound CML), respectively, and these samples were absorbed by paracellular route, transcytosis route and active route via PepT-1. Taken together, these findings demonstrated that all three protein-bound CML digests with different MW can be absorbed in diverse absorption pathways by Caco-2 cell monolayer model. This research provided a theoretical basis for scientific evaluation of digestion and absorption of AGEs in food.

Uncovering the antiinflammatory potential of Lactiplantibacillus Plantarum fermented Cannabis Sativa L seeds

Inflammation acts as a dual role in disease initiation and progression, while Cannabis sativa L. (hemp) seeds, known for their abundance of anti-inflammatory phytochemicals, present a promising food source. Additionally, fermentation may optimize the food matrix, thereby augmenting its developmental prospects. This study explores the anti-inflammatory potential of hemp seeds fermented with 10 different probiotic strains. Among these, Lactiplantibacillus plantarum fermented hemp seeds (FHS) demonstrated a significant anti-inflammatory ability, accompanied by a reduction in the expression of critical inflammatory markers such as TLR4, NF-κBp65, and iNOS. Moreover, there is a noteworthy dose-dependent inhibition of inflammatory cytokines TNF-α, IL-6, IL-1β, and NO within a concentration range of 50 to 500 µg/mL. Subsequently, metabolomics analysis using UHPLC-QTOF-MS highlighted significant metabolic alterations in FHS compared to raw hemp seeds (RHS). Through multivariate, univariate, and correlation analyses, indolelactic acid (IA) and homovanillic acid (HVA) emerged as the main anti-inflammatory metabolites in FHS. Validation via HPLC confirmed the concentration of IA and HVA in RHS and FHS and both organic acids demonstrated lower IC50 values for TNF-α, IL-1β, IL-6, IL-18, and NO inhibition, showcasing their potent anti-inflammatory abilities. Furthermore, in vitro gastro-intestinal digestion coupled with the Caco-2 cell monolayer model validates the uptake and bioaccessibility of FHS, further affirming IA and HVA as major anti-inflammatory compounds. Overall, this research sets the stage for the development of novel hemp seed-based products targeting inflammation-associated disorders.

Stability, Digestion, and Cellular Transport of Soy Isoflavones Nanoparticles Stabilized by Polymerized Goat Milk Whey Protein.

Soy isoflavones (SIF) are bioactive compounds with low bioavailability due to their poor water solubility. In this study, we utilized polymerized goat milk whey protein (PGWP) as a carrier to encapsulate SIF with encapsulation efficiency of 89%, particle size of 135.53 nm, and zeta potential of -35.16 mV. The PGWP-SIF nanoparticles were evaluated for their stability and in vitro digestion properties, and their ability to transport SIF was assessed using a Caco-2 cell monolayer model. The nanoparticles were resistant to aggregation when subjected to pH changes (pH 2.0 to 8.0), sodium chloride addition (0-200 mM), temperature fluctuations (4 °C, 25 °C, and 37 °C), and long-term storage (4 °C, 25 °C, and 37 °C for 30 days), which was mainly attributed to the repulsion generated by steric hindrance effects. During gastric digestion, only 5.93% of encapsulated SIF was released, highlighting the nanoparticles' resistance to enzymatic digestion in the stomach. However, a significant increase in SIF release to 56.61% was observed during intestinal digestion, indicating the efficient transport of SIF into the small intestine for absorption. Cytotoxicity assessments via the MTT assay showed no adverse effects on Caco-2 cell lines after encapsulation. The PGWP-stabilized SIF nanoparticles improved the apparent permeability coefficient (Papp) of Caco-2 cells for SIF by 11.8-fold. The results indicated that using PGWP to encapsulate SIF was an effective approach for delivering SIF, while enhancing its bioavailability and transcellular transport.

Optimization of Conditions of Zanthoxylum Alkylamides Liposomes by Response Surface Methodology and the Absorption Characteristics of Liposomes in the Caco-2 Cell Monolayer Model.

Zanthoxylum alkylamides, as a numbing substance in Zanthoxylum bungeanum has many physiological effects. However, the numbing taste and unstable properties limited its application. This study aimed to optimize the preparation process of Zanthoxylum alkylamides liposomes by response surface methodology (RSM) and to investigate the in vitro absorption characteristics of the liposomes through the Caco-2 cell monolayer model. The process parameters of liposomes were as follows: Zanthoxylum alkylamides was 15 mg, phospholipid-feedstock ratio was 6.14, phospholipid-cholesterol ratio was 8.51, sodium cholate was 33.80 mg, isopropyl myristate was 29.49 mg, and the theoretical encapsulation efficiency of the prepared liposomes could reach 90.23%. Further, the particle size of the liposomes was 155.47 ± 3.16 nm, and the ζ-potential was -34.11 ± 4.34 mV. Meanwhile, the liposomes could be preserved for 14 days under the condition that the content of Zanthoxylum alkylamides was less than 2 mg/mL and the preservation temperature was lower than 25 °C. Moreover, the uptake characteristics of the Zanthoxylum alkylamides liposomes in the Caco-2 cell monolayer model were also investigated. The results showed that the Zanthoxylum alkylamides liposomes could be taken up and absorbed by Caco-2 cells. Also, the Zanthoxylum alkylamides liposomes had a better uptake performance than the unembedded Zanthoxylum alkylamides and conformed to the passive uptake.

Structural characterization and calcium absorption-promoting effect of sucrose-calcium chelate in Caco-2 monolayer cells and mice.

Sucrose emerges as a chelating agent to form a stable sucrose-metal-ion chelate that can potentially improve metal-ion absorption. This study aimed to analyze the structure of sucrose-calcium chelate and its potential to promote calcium absorption in both Caco-2 monolayer cells and mice. The characterization results showed that calcium ions mainly chelated with hydroxyl groups in sucrose to produce sucrose-calcium chelate, altering the crystal structure of sucrose (forming polymer particles) and improving its thermal stability. Sucrose-calcium chelate dose dependently increased the amount of calcium uptake, retention, and transport in the Caco-2 monolayer cell model. Compared to CaCl2 , there was a significant improvement in the proportion of absorbed calcium utilized for transport but not retention (93.13±1.75% vs. 67.67±7.55%). Further treatment of calcium channel inhibitors demonstrated the active transport of sucrose-calcium chelate through Cav1.3. Cellular thermal shift assay and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays indicated that the ability of sucrose-calcium chelate to promote calcium transport was attributed to its superior ability to bind with PMCA1b, a calcium transporter located on the basement membrane, and stimulate its gene expression compared to CaCl2 . Pharmacokinetic analysis of mice confirmed the calcium absorption-promoting effect of sucrose-calcium chelate, as evident by the higher serum calcium level (44.12±1.90mg/L vs. 37.42±1.88mmol/L) and intestinal PMCA1b gene expression than CaCl2 . These findings offer a new understanding of how sucrose-calcium chelate enhances intestinal calcium absorption and could be used as an ingredient in functional foods to treat calcium deficiency. PRACTICAL APPLICATION: The development of high-quality calcium supplements is crucial for addressing the various adverse symptoms associated with calcium deficiency. This study aimed to prepare a sucrose-calcium chelate and analyze its structure, as well as its potential to enhance calcium absorption in Caco-2 monolayer cells and mice. The results demonstrated that the sucrose-calcium chelate effectively promoted calcium absorption. Notably, its ability to enhance calcium transport was linked to its strong binding with PMCA1b, a calcium transporter located on the basement membrane, and its capacity to stimulate PMCA1b gene expression. These findings contribute to a deeper understanding of how the sucrose-calcium chelate enhances intestinal calcium absorption and suggest its potential use as an ingredient in functional foods for treating calcium deficiency.

Investigation of selenium and selenium species in Cardamine violifolia using in vitro digestion coupled with a Caco-2 cell monolayer model

Inadequate Se intake can enhance vulnerability to certain health risks, with supplementation lessening these risks. This study investigated the bioavailability of Se and Se species in five Se compounds and in Se-rich Cardamine violifolia using in vitro digestion coupled with a Caco-2 cell monolayer model, which enabled the study of Se transport and uptake. Translocation results showed that SeCys2 and MeSeCys had high translocation rates in C. violifolia leaves (CVLs). The uptake rate of organic Se increased with time, and MeSeCys exhibited a higher uptake rate than that for SeCys2 and SeMet. The translocation mechanisms of SeMet, Se(IV), and Se(VI) were passive transport, whereas those of SeCys2 and MeSeCys were active transport. The bioavailability of organic Se was higher than that of inorganic Se, with a total Se bioavailability in CVLs of 49.11 %. This study would provide a theoretical basis for the application of C. violifolia in the functional food.

Inhibition of α-glucosidase activity and intestinal glucose transport to assess the in vivo anti-hyperglycemic potential of dodecyl-acylated phlorizin and polydatin derivatives.

A diet containing natural active compounds that can inhibit the hydrolytic activity of α-glucosidase on carbohydrates and intestinal glucose absorption is an effective means of controlling postprandial hyperglycemia. Phlorizin and polydatin as phenolic glycosides have a high affinity for the catalytic site of α-glucosidase, but exhibited unsatisfactory competitive inhibitory capacity, with an IC50 of 0.97 and >2 mM, respectively. However, dodecyl-acylated derivatives of phlorizin and polydatin exerted α-glucosidase inhibitory capacity, with an IC50 of 55.10 and 70.95 μM, respectively, which were greatly enhanced and much stronger than that of acarbose with an IC50 of 2.46 mM. The SPR assay suggested the high affinity of dodecyl phlorizin and dodecyl polydatin to α-glucosidase with equilibrium dissociation constant (KD) values of 12.0 and 7.9 μM, respectively. Both dodecyl phlorizin and dodecyl polydatin reduced the catalytic ability of α-glucosidase by reversible noncompetitive and uncompetitive mixed inhibition, which bind noncovalently to the allosteric site 2 through hydrogen bonds and hydrophobic interactions, thereby inducing the secondary structure unfolding and intrinsic fluorescence quenching of α-glucosidase. Confocal microscopy detection visually showed significant inhibitory effects on FITC-labeled glucose uptake in intestinal Caco-2 cells by phlorizin, polydatin, dodecyl phlorizin and dodecyl polydatin. In addition, based on the differentiated Caco-2 cell monolayer model, dodecyl phlorizin and dodecyl polydatin suppressed intestinal glucose transport more effectively than phlorizin and polydatin, suggesting that they were promising in vivo hypoglycemic active compounds.

Study on the transport and internalisation mechanism of dietary supplement nattokinase in the small intestine using animal and Caco-2 cell monolayer models

Maintaining proper blood flow is critical to promoting good health. Nattokinase is a serine protease from Bacillus subtilis that has significant in vitro thrombolytic activity, but its mechanism as a dietary supplement to prevent thrombosis through intestinal absorption and transport is still unclear. The purpose of this study is to study the transport and internalisation mechanism of NK in the small intestine using animal models and Caco-2 cell monolayer models. This study first evaluated the preventive effect of supplementing low dose (4000 FU (Fibrin Unit)/kg, n = 6), medium dose (8000 FU/kg, n = 6), and high dose (12000 FU/kg, n = 6) of nattokinase on carrageenan induced thrombosis in mice. Subsequently, we used the rat gut sac model, ligated intestinal loop model, and Caco-2 cell uptake model to study the intestinal transport mechanism of NK. Results indicate that NK is a moderately absorbed biomolecule whose transport through enterocytes is energy- and time-dependent. Chlorpromazine, nystatin and EIPA all inhibited the endocytosis of NK to varying degrees, indicating that the endocytosis of NK in Caco-2 cells involves macropinocytosis, clathrin-mediated and caveolae-mediated pathway. These findings offer a theoretical basis for investigating the mechanism of oral NK supplementation in greater depth.

Novel Ca-Chelating Peptides from Protein Hydrolysate of Antarctic Krill (Euphausia superba): Preparation, Characterization, and Calcium Absorption Efficiency in Caco-2 Cell Monolayer Model.

Antarctic krill (Euphausia superba) is the world's largest resource of animal proteins and is thought to be a high-quality resource for future marine healthy foods and functional products. Therefore, Antarctic krill was degreased and separately hydrolyzed using flavourzyme, pepsin, papain, and alcalase. Protein hydrolysate (AKH) of Antarctic krill prepared by trypsin showed the highest Ca-chelating rate under the optimized chelating conditions: a pH of 8.0, reaction time of 50 min, temperature of 50 °C, and material/calcium ratio of 1:15. Subsequently, fourteen Ca-chelating peptides were isolated from APK by ultrafiltration and a series of chromatographic methods and identified as AK, EAR, AEA, VERG, VAS, GPK, SP, GPKG, APRGH, GVPG, LEPGP, LEKGA, FPPGR, and GEPG with molecular weights of 217.27, 374.40, 289.29, 459.50, 275.30, 300.36, 202.21, 357.41, 536.59, 328.37, 511.58, 516.60, 572.66, and 358.35 Da, respectively. Among fourteen Ca-chelating peptides, VERG presented the highest Ca-chelating ability. Ultraviolet spectrum (UV), Fourier Transform Infrared (FTIR), and scanning electron microscope (SEM) analysis indicated that the VERG-Ca chelate had a dense granular structure because the N-H, C=O and -COOH groups of VERG combined with Ca2+. Moreover, the VERG-Ca chelate is stable in gastrointestinal digestion and can significantly improve Ca transport in Caco-2 cell monolayer experiments, but phytate could significantly reduce the absorption of Ca derived from the VERG-Ca chelate. Therefore, Ca-chelating peptides from protein hydrolysate of Antarctic krill possess the potential to serve as a Ca supplement in developing healthy foods.

Bioavailability and Antioxidant Activity of Rambutan (Nephelium lappaceum) Peel Polyphenols during in Vitro Simulated Gastrointestinal Digestion, Caco-2 Monolayer Cell Model Application, and Colonic Fermentation.

The bioavailability of rambutan peel polyphenols (RPPs) was studied via in vitro simulated digestion, a Caco-2 monolayer cell model, and colonic fermentation. Total phenolic content of RPPs decreased with the progress of the simulated digestion. A total of 38 phenolic compounds were identified during the digestion and colonic fermentation, of which 12 new metabolites were found during colonic fermentation. The possible biotransformation pathways were inferred. Geraniin was transformed into corilagin, ellagic acid, and gallic acid during the digestion and colonic fermentation. Ellagic acid could be further transformed into urolithin under the action of intestinal microbiota. The transformation of ellagitannins could be beneficial to transport on Caco-2 monolayer cell. The antioxidant capacity of RPPs increased with the progress of gastrointestinal digestion. Furthermore, RPPs could increase the yield of short-chain fatty acids, decrease the pH value, promote the growth of beneficial bacteria, and inhibit the growth of pathogenic Escherichia coli/Shigella during colonic fermentation.

Regulation and function of adiponectin in the intestinal epithelial cells in response to Trichinella spiralis infection

Besides metabolic homeostasis regulation, adipokines are recently emerged as important players in regulating immunity and inflammation. Helminth infection has known to modulate circulating adipokine secretion; however, the regulation and function of adipokines in response to helminth infection is still unclear. Here, we investigated the regulation and function of adiponectin during T. spiralis infection. While there was no change in circulating level of adiponectin, we found an increased adiponectin, but not leptin expression in the small intestine. Interestingly, the intestinal adiponectin expression was strongly associated with the expression of epithelial cell-derived cytokines IL-25, IL-33, and TSLP following infection. Indeed, mice deficiency of IL-25 receptor exhibited no intestinal adiponectin induction upon helminth infection. Interestingly, IL-25-induced adiponectin modulated intestinal epithelial cell responses by enhancing occludin and CCL17 expression. Using LPS-induced intestinal epithelial barrier dysfunctions in a Caco-2 cell monolayer model, adiponectin pretreatment enhanced a Transepithelial electrical resistance (TEER) and occludin expression. More importantly, adiponectin pretreatment of Caco2 cells prevented T. spiralis larval invasion in vitro and its administration during infection enhanced intestinal IL-13 secretion and worm expulsion in vivo. Altogether, our data suggest that intestinal adiponectin expression induced by helminth infection through the regulation of IL-25 promotes worm clearance and intestinal barrier function.

Structural Characterization of Zinc-Sucrose Complex and Its Ability to Promote Zinc Absorption in Caco-2 Monolayer Cells and Mice.

Sucrose emerges as a metal-ion chelating agent with excellent stability that may increase metal-ion absorption. This study aimed to characterize the structure of zinc-sucrose complex and investigate its ability to promote zinc absorption in Caco-2 monolayer cells and mice. Based on the results of the inductively coupled plasma emission spectrometer (ICP-ES), scanning electron microscopy-energy-dispersive X-ray spectroscopy (SEM-EDX), and Fourier transform infrared spectroscopy (FT-IR), it can be inferred that zinc and sucrose were chelated at a 1:1 ratio, with the hydroxyl groups playing a significant role. The Caco-2 monolayer cell model revealed that zinc-sucrose complex increased the amount of zinc uptake, retention, and transport in a dose- and time-dependent manner. Through the upregulation of genes and proteins for ZIP4, MT1, and DMT1, treatment with zinc-sucrose complex improved the proportion of absorbed zinc utilized for transport compared to ZnCl2 (26.21 ± 4.96 versus 8.50 ± 1.51%). Pharmacokinetic analysis of mice confirmed the zinc absorption-promoting effect of zinc-sucrose complex, as indicated by the considerably higher serum zinc level (4.16 ± 0.53 versus 2.56 ± 0.45 mg/L) and intestinal ZIP4, MT1, and DMT1 gene expression than ZnCl2. Further treatment of different zinc channel inhibitors and CETSA demonstrated the direct interaction of zinc-sucrose complex with ZIP4 protein and ZIP4-mediated cellular transport of zinc-sucrose complex. These findings provide a novel insight into the zinc absorption-promoting mechanism of zinc-sucrose complex, which could be used as an ingredient in functional foods to treat zinc deficiency.

Liposome-Micelle-Hybrid (LMH) Carriers for Controlled Co-Delivery of 5-FU and Paclitaxel as Chemotherapeutics.

Paclitaxel (PTX) and 5-fluorouracil (5-FU) are clinically relevant chemotherapeutics, but both suffer a range of biopharmaceutical challenges (e.g., either low solubility or permeability and limited controlled release from nanocarriers), which reduces their effectiveness in new medicines. Anticancer drugs have several major limitations, which include non-specificity, wide biological distribution, a short half-life, and systemic toxicity. Here, we investigate the potential of liposome-micelle-hybrid (LMH) carriers (i.e., drug-loaded micelles encapsulated within drug-loaded liposomes) to enhance the co-formulation and delivery of PTX and 5-FU, facilitating new delivery opportunities with enhanced chemotherapeutic performance. We focus on the combination of liposomes and micelles for co-delivery of PTX and 5_FU to investigate increased drug loading, improved solubility, and transport/permeability to enhance chemotherapeutic potential. Furthermore, combination chemotherapy (i.e., containing two or more drugs in a single formulation) may offer improved pharmacological performance. Compared with individual liposome and micelle formulations, the optimized PTX-5FU-LMH carriers demonstrated increased drug loading and solubility, temperature-sensitive release, enhanced permeability in a Caco-2 cell monolayer model, and cancer cell eradication. LMH has significant potential for cancer drug delivery and as a next-generation chemotherapeutic.

Arabinogalactan ameliorates benzo[a]pyrene-induced intestinal epithelial barrier dysfunction via AhR/MAPK signaling pathway

Benzo[a]pyrene (B[a]P), a kind of pollutant, can disrupt the gut microbiota, but its effects on the function of intestinal epithelial barrier (IEB) is still unclear. Arabinogalactan (AG), a natural polysaccharide, can protect intestinal tract. Thus, the purpose of this study was to evaluate the effect of B[a]P on IEB function and the mitigation effect of AG on the IEB dysfunction induced by B[a]P using a Caco-2 cell monolayer model. We found B[a]P could damage the IEB integrity by inducing cell cytotoxicity, increasing lactate dehydrogenase leakage, decreasing the transepithelial electrical resistance, and increasing fluorescein isothiocyanate-dextran flux. The mechanism of B[a]P-induced IEB damage may through induction of oxidative stress, including increasing reactive oxygen species levels, decreasing glutathione levels, reducing the activity of superoxide dismutase, and increasing malonaldehyde levels. Moreover, it can be due to increasing secretion of pro-inflammatory cytokines (interleukin [IL]-1β, IL-6, and tumor necrosis factor [TNF]-α), down-regulated expression of tight junction (TJ) proteins (claudin-1, zonula occludens [ZO]-1, and occludin), and induced activation of aryl hydrocarbon receptor (AhR)/mitogen activated protein kinase (MAPK) signaling pathway. Remarkably, AG ameliorated B[a]P-induced IEB dysfunction through inhibited oxidative stress and pro-inflammatory factor secretion. Our study demonstrated B[a]P could damage the IEB and AG could alleviate this damage.

Screening quality markers (Q-markers) of Xiaoer Chaige Tuire Oral Liquid by in vitro sequential metabolism and in vivo biopharmaceutical analysis

BackgroundXiaoer Chaige Tuire Oral Liquid (XCT) is a preparation composed of 7 traditional Chinese medicines including Bupleuri Radix, Puerariae Lobatae Radix, Scutellariae Radix, Gypsum Fibrosum, Artemisiae Annuae Herba, Paeoniae Radix Alba and Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle in proportion. According to traditional Chinese medicine theory, it has the function of dispelling wind evil and relieving exterior syndrome, clearing summer heat and dampness, and reducing internal heat. So, it is indicated for pediatric upper respiratory tract infection caused by exogenous wind-heat. Modern pharmacological studies have indicated that XCT has a variety of activities such as anti-inflammation and antivirus. PurposeTo screen potential quality markers (Q-markers) of XCT by tracking in vivo bioactive compounds concomitantly using in vitro sequential metabolism and in vivo biopharmaceutical analysis. MethodsIn vitro metabolic models including artificial gastric juice, intestinal juice, intestinal microbiota, Caco-2 cell monolayer and liver S9 were employed to simulate metabolism of main compounds of XCT in the body. High performance liquid chromatography with diode-array detection (HPLC-DAD) was used to quantitatively determine main components of XCT preparation and its sequential metabolism samples. Ultra performance liquid chromatography with QExactive Orbitrap tandem mass spectrometry (UPLC-QExactive-HF-x-Orbitrap-MS) was used to qualitatively determine in vivo components of XCT preparation in rat plasma and metabolites obtained with liver S9 fraction of rats. ResultsTwenty-five compounds were identified from the preparation of XCT. Sequential in vitro metabolism studies indicated that most of these compounds except baicalin and baicalein were stable in artificial gastric juice, albiflorin, glycyrrhizic acid, gallic acid and baicalein were unstable in artificial intestinal juice, daidzin, liquiritin and genistin were hydrolyzed into their aglycones daidzein, liquiritigenin and genistein by intestinal microbiota, and 7 compounds thereout including benzoic acid, puerarin, 3′-methoxypuerarin, paeoniflorin, scopoletin, daidzein and liquiritigenin were shown to be well absorbed with Caco-2 cell monolayer model. These 7 compounds were demonstrated to be metabolized via hydroxylation and glycosylation by liver S9 system. Ten components of XCT preparation including puerarin, baicalin, wogonoside, benzoic acid, daidzein, baicalein, wogonin, oroxylin A, isoscopoletin and isoliquiritigenin were identified from rat plasma by in vivo biopharmaceutical analysis. Most of the compounds screened with both in vitro and in vivo metabolic studies were shown to be active against inflammation and influenza virus. ConclusionsA screening strategy for potential quality markers (Q-markers) of XCT preparation based on tracking in vivo bioactive compounds using the combination of in vitro sequential metabolism and in vivo biopharmaceutical analysis was established. With this strategy, a total of 12 compounds including puerarin, daidzein, benzoic acid, baicalin, baicalein, wogonoside, wogonin, oroxylin A, 3′-methoxypuerarin, paeoniflorin, scopoletin and liquiritigenin were screened to be potential Q-markers of XCT, which provides a material basis for quality control and development of XCT.