Aims: Lactococcus lactis is a non-colonizing, generally-regarded as safe (GRAS) lactic acid bacteria that has been frequently studied as a potential vector for bactofection. To mediate bactofection, a series of interaction between the bacteria and the host cell needs to occur. This study aims to investigate the in vitro bacterial-cell interaction between a locally-isolated L. lactis M4 strain with human colorectal cancer line, Caco-2. Methodology and results: Bacterial interaction was evaluated via adherence and internalisation assays. A 250:1 ratio of bacteria to cancer cell was selected as the optimum multiplicity of infection for all assays. After 2 h, L. lactis M4 was able to adhere to and internalise into Caco-2 cells at comparable rates to commercial strains L. lactis NZ9000 and MG1363. Conclusion, significance and impact of study Findings from this study showed that this strain has similar interaction properties with the commercial strains and would make a promising candidate for future bactofection studies and development of bacteria-mediated DNA vaccination against various diseases.
Lactococcus lactis ; Colorectal Neoplasms ; Caco-2 Cells

Nanocrystals self-stabilized Pickering emulsion(NSSPE) is a new kind of emulsion where only nanocrystals of poorly soluble drugs are used as stabilizers. Our previous study showed that NSSPE with Ligusticum chuanxiong oil as the main oil phase can significantly promote oral absorption of puerarin. The present study aimed to explore its absorption mechanism in oral administration. The in vitro dissolution test was carried out to study the effect of NSSPE on release of puerarin. The effects and mechanism of NSSPE on uptake and transport of puerarin across Caco-2 cell were investigated. The results showed that the drug release rate of NSSPE was similar to that of nanocrystals, with their cumulative dissolution of puerarin not affected by pH of releasing mediums, both significantly higher than that of crude material. The uptake of puerarin in NSSPE was concentration-dependent and significantly higher than that of solution or surfactant stabilized emulsion. Genistein and indomethacin, inhibitors of lipid rafts/caveolin, could significantly reduce the uptake of puerarin in NSSPE. Compared with solution, NSSPE and surfactants stabilized emulsion obviously increased transport rate K_a and apparent permeability coefficient P_(app) of puerarin in AP → BL direction, but there was no significant difference in BL → AP direction. It could be inferred that there were both passive and active transport mechanisms, as well as lipid raft/caveolin mediated endocytosis for absorption of NSSPE. The promoted oral absorption of puerarin in NSSPE was mainly related to the existing nanocrystal form which could promote dissolution, puerarin as well as Ligusticum chuanxiong oil which could promote drug transmembrane transport and inhibit drug efflux. It is the unique structure and composition of the compound NSSPE that promoted the oral absorption of puerarin.
Caco-2 Cells ; Drugs, Chinese Herbal ; Emulsions ; Humans ; Isoflavones ; Nanoparticles

OBJECTIVETo establish and assess the Caco-2 cell in vitro absorption model.

METHODSCaco-2 cells were cultured on the millipore filters fixed in Snapwell transport chamber. The cell morphology, transepithelial electrical resistance, mannitol efflux rate and alkaline phosphatase activities were monitored during culture.

RESULTSAfter 21 days of in vitro culture, formation of tight junction was observed between the cells. The transepithelial electrical resistance reach a relatively stable value of 620-/+47 Omega.cm(2), the mannitol efflux rate was lower than 0.3%.h(-1).cm(-2), and the alkaline phosphatase activity in the apical side was significantly higher than that in the basolateral side.

CONCLUSIONThe established Caco-2 cell model shows similar morphology to intestinal epithelial cells with formation of polarity, and can be used as an in vitro model for absorption studies.

Caco-2 Cells ; Cell Culture Techniques ; methods ; Epithelial Cells ; cytology ; metabolism ; Humans ; Intestinal Absorption ; Intestines ; cytology

OBJECTIVETo investigate the role of cyclic adenosine monophosphate(cAMP) in the regulation of intestinal epithelial barrier function under hypoxia.

METHODSIntestinal epithelial barrier was established by Caco-2 monolayers. Cells were divided into four groups: normoxia (Nx), normoxia plus Forskolin(Nx+FSK), hypoxia(Hx), hypoxia plus SQ22536(Hx+SQ22536). cAMP concentrations of different groups were assessed by cAMP enzyme immunoassay kit. RT-PCR and Western blotting were used to detect the mRNA and protein expressions of claudin-1 and occludin under normoxic and hypoxic condition. Caco-2 monolayers were grown on Millicell filters, and transepithelial electrical resistance(TER) was measured using a Millipore electric resistance system.

RESULTSThe concentration of cAMP under hypoxic conditions(Hx group) was higher compared with Nx group [(6.30±0.50) pmol/L vs. (2.38±0.18) pmol/L, P<0.01]. At the same time, both mRNA and protein expressions of claudin-1 and occluding were lower in Hx group than those in Nx group(all P<0.05). TER decreased by 76.30±0.64(P<0.01). When the monolayers were exposed to hypoxia plus SQ22536 (Hx+SQ22536 group), the concentration of cAMP was(2.12±0.23) pmol/L, which was lower than that under hypoxic conditions(Hx group, P<0.01). Both mRNA and protein expressions of claudin-1 and occludin were higher compared to Hx group (all P<0.01). TER increased by 32.96±2.16 (P<0.05).

CONCLUSIONWhen Caco-2 cells are exposed to hypoxia, barrier function, claudin-1 and occludin expression are diminished in parallel with a high level of intracellular cAMP compared with the normoxic condition. Inhibition of the intracellular cAMP level under hypoxia can maintain the intestinal epithelial function through regulating the claudin-1 and occludin expression and attenuate the permeability of intestinal mucosa.

Adenosine Monophosphate ; Caco-2 Cells ; Claudin-1 ; metabolism ; Humans ; Intestinal Mucosa ; metabolism ; Intestines ; Occludin ; metabolism

Although the essential oil of Xiangfu Siwu decoction (XFSWD) has strong pharmacological activity, its special physical and chemical properties restrict the clinical application and curative effect. In this paper, Xiangfu Siwu decoction essential oil (XFS-WO) was prepared by forming inclusion complex with β-cyclodextrin (β-CD). The present study is to investigate the effect of β-CD inclusion complex on the transport of major components of XFSWO using Caco-2 cell monolayer model, thus to research the effect of this formation on the absorption of drugs with low solubility and high permeability, which belong to class 2 in biopharmaceutics classification system. A sensitive and rapid UPLC-MS/MS method was developed for simultaneous quantification of senkyunolide A, 3-n-butylphthalide, Z-ligustilide, dehydrocostus lactone and α-cyperone, which are active compounds in XFSWO. The transport parameters were analyzed and compared in free oil and its β-CD inclusion complex. The result revealed that the formation of XFSWO/β-CD inclusion complex has significantly increased the transportation and absorption of major active ingredients than free oil. Accordingly, it can be speculated that cyclodextrin inclusion complex can improve bioavailability of poorly water-soluble drugs. Above all these mentioned researches, it provided foundation and basis for physiological disposition and pharmaceutical study of XFSWD.
Biological Transport ; Caco-2 Cells ; Drugs, Chinese Herbal ; analysis ; Humans ; Oils, Volatile ; analysis ; beta-Cyclodextrins ; pharmacology

OBJECTIVETo investigate the intestinal absorption mechanism of swertiamain metabolite {(Z)-5-ethylidene-8-hydroxy-3,4,5,6,7,8-hexahydro-1H-pyrano [3,4-c]pyridine-1-one, EHPO}.

METHODThe time depended hi-directional transport of EHPO in Caco-2 monolayer model was investigated with the factors of concentration, pH and verapamil. EHPO concentration was measured by HPLC assay and the apparent permeability coefficients (Papp) were calculated.

RESULTIn the this cell model, EHPO could be absorbed through Caco-2 soon. The P(app AP-BL) was equal to the P(app BL-AP), and Papp keep almost constant with the selected concentration investigated. But the Papp could be influenced by pH and verapamil (100 mg x L(-1)).

CONCLUSIONThe absorption of EHPO in Caco-2 cell model is a passive one. And absorption of EHPO in the intestines is quite good.

Biological Transport ; Caco-2 Cells ; Cell Membrane Permeability ; Drugs, Chinese Herbal ; chemistry ; pharmacokinetics ; Humans ; Models, Biological

OBJECTIVETo determine the concentration of ginsenoside Rb(2) and study the absorption characteristics of ginsenoside Rb(2) in Caco-2 cell monolayer.

METHODSLC-MS-MS was used to determine the concentration of ginsenoside Rb(2), and the apparent permeability coefficient (P(app)) of ginsenoside Rb(2) was calculated.

RESULTSP(app(AP-BL)) was 3.27 x 10(-7) cm.s(-1), P(app(BL-AP)) was 3.16 x 10(-6) cm.s(-1), and the efflux ratio (P(app(BL-AP))/P(app(AP-BL))) was 9.63.

CONCLUSIONThe absorption characteristics of ginsenoside Rb(2) in Caco-2 cell model have been demonstrated.

Caco-2 Cells ; Gas Chromatography-Mass Spectrometry ; methods ; Ginsenosides ; pharmacokinetics ; Humans ; Intestinal Absorption

OBJECTIVETo study the absorption mechanism of icariin by using Caco-2 monolayer model.

METHODCaco-2 cell monolayer model was used to study the bi-direction transport of icariin. The effects of time, drug concentration and inhibitor on the absorption of icariin were studied. The concentration of icariin in cell culture medium was measured by UPLC and the apparent permeability coefficients (Papp) was calculated.

RESULTThe amount of icariin which was transported increased linearly with the time (14 hr). The ratio of PBA/PAB was larger than 4. Verapamil, the P-glycoprotein inhibitor, could cause significantly effect on transport of icariin, PAB increased, the ratio of PBA/PAB decreased.

CONCLUSIONThe reason for low absorption of icariin in Caco-2 cell model may be the secretion of the P-gp transporter.

Absorption ; Caco-2 Cells ; Epimedium ; chemistry ; Flavonoids ; pharmacokinetics ; Humans ; Models, Biological

Shuxiong prescription (Notoginseng Radix et Rhizoma, Chuanxiong Rhizome and Carthami Flos) has the function of activating blood circulation to dissipate blood stasis, activating meridians to stop pain. This paper was mainly aimed to discuss the transport characteristics of Shuxiong prescription across Caco-2 cell monolayer. Safe concentration range of Shuxiong prescription against Caco-2 cell monolayer model was determined by MTT assay. The mechanism of Shuxiong prescription bidirectional transport was investigated by Caco-2 cell monolayer model. The apparent permeability coefficient Papp of digoxin was determined by high performance liquid chromatography (HPLC). The test results showed that the Papp of extract from Notoginseng Radix et Rhizoma, Chuanxiong Rhizome, Carthami Flos, Chuanxiong Rhizome+Carthami Flos and Shuxiong prescription transport from apical (AP) side to basolateral (BL) side was (3.12±0.73)×10⁻⁶, (2.58±0.41)×10⁻⁶, (4.97±0.64)×10⁻⁶, (4.63±0.57)×10⁻⁶, (5.79±0.68)×10⁻⁶ cm·s⁻¹, respectively, indicating that the transport of digoxin across Caco-2 cell monolayer model was active absorption, and the P-gp protein took part in the process. Chuanxiong Rhizome could significantly decrease the transport of digoxin from BL→AP(<0.01) and increase its transport from AP→BL(<0.05) significantiy. After the addition of Shuxiong prescription, the transport of digoxin from BL→AP was significantly inhibited(<0.01). The results suggested that the extract of safflower had no effect on P-gp transport, nor on the independence diffusion of digoxin. The transport of digoxin could be degraded by the extract of Chuanxiong Rhizome and the extract of Shuxiong prescription from BL→AP(<0.01), significantly; pseudo-ginseng had no effect on the independence diffusion of digoxin; the extract of safflower+Chuanxiong Rhizome had the same experimental result as Chuanxiong Rhizome extract.
Biological Transport ; Caco-2 Cells ; Chromatography, High Pressure Liquid ; Digoxin ; pharmacokinetics ; Drugs, Chinese Herbal ; pharmacokinetics ; Humans

To investigate the effect and mechanism of metformin on intestinal epithelial barrier injury in ulcerative colitis. A cell model of colitis was established by co-culture of human colon cancer cell line Caco-2 and human monocyte cell line THP-1. The colitis model cells were treated with metformin at concentration of for Flow cytometry was used to detect Caco-2 cell apoptosis, and Western blotting was used to detect the protein expression of tight junction proteins and endoplasmic reticulum stress-related proteins. After metformin treatment, the apoptosis rate of Caco-2 cells was decreased from (14.22±2.34)% to 0.61)% (=3.119, <0.05), and the expression levels of tight junction protein-1 and claudin-1 increased (=5.172 and 3.546, both <0.05). In addition, the expression levels of endoplasmic reticulum-related proteins glucose regulated protein (GRP) 78, C/EBP homologous protein (CHOP) and caspase-12, as well as the phosphorylation level of PRKR-like endoplasmic reticulum kinase (PERK) and eukaryotic translation initiation factor 2α (eIF2α) decreased (all <0.05). Metformin may alleviate the intestinal epithelial barrier damage in colitis by reducing intestinal epithelial cell apoptosis and increasing the expression of tight junction proteins, which may be associated with the inhibition of endoplasmic reticulum stress-induced apoptotic pathway.
Apoptosis ; Caco-2 Cells ; Colitis, Ulcerative ; Endoplasmic Reticulum Stress ; Humans ; Metformin/pharmacology*