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Gigantol is a phenolic component of precious Chinese medicine Dendrobii Caulis, which has many pharmacological activities such as prevent tumor and diabetic cataract. This paper aimed to investigate the molecular mechanism of gigantol in transmembrane transport in human lens epithelial cells(HLECs). Immortalized HLECs were cultured in vitro and inoculated in the laser scanning confocal microscopy(LSCM) medium at 5 000 cells/mL. The fluorescence distribution and intensity of gigantol marked by fluorescence in HLECs were observed by LSCM, and the absorption and distribution of gigantol were expressed as fluorescence intensity. The transmembrane transport process of gigantol in HLECs were monitored. The effects of time, temperature, concentration, transport inhibitors, and different cell lines on the transmembrane absorption and transport of gigantol were compared. HLECs were inoculated on climbing plates of 6-well culture plates, and the ultrastructure of HLECs was detected by atomic force microscopy(AFM) during the transmembrane absorption of non-fluorescent labeled gigantol. The results showed that the transmembrane absorption of gigantol was in time and concentration-dependent manners, which was also able to specifically target HLECs. Energy and carrier transport inhibitors reduced gigantol absorption by HLECs. During transmembrane process of gigantol, the membrane surface of HLECs became rougher and presented different degrees of pits, indicating that the transmembrane transport of gigantol was achieved by active absorption of energy and carrier-mediated endocytosis.
Subject(s)
Humans , Lens, Crystalline/pathology , Cataract/prevention & control , Bibenzyls/pharmacology , Epithelial Cells , Cells, Cultured , ApoptosisABSTRACT
Oral nanoparticles （NPs） has gradually become a approach to improve oral bioavailability of biopharmaceutics classification system （BCS） Ⅱ， Ⅲ， Ⅳ drugs， and the transmembrane transport mechanism in the gastrointestinal tract largely depends on physicochemical characteristics of NPs. It would be beneficial to design the NPs with high transport efficiency and effectively improve the oral bioavailability of drugs by adopting a reasonable research model to analyze the transmembrane mechanism of the oral NPs and exactly reveal the relationship between the physicochemical properties and the transport mechanism of NPs. This review focused on summarizing the transmembrane approaches of oral NPs， comparing the advantages and disadvantages of the common cell models， concluding the potential interaction between the physicochemical properties and transmembrane process of NPs， and proposing the research strategy of transport mechanism based on in situ intestinal perfusion， with the purpose of discovering a suitable research model for studying the transport mechanism of different NPs， providing a basis for regulating the transport performance of the NPs to improve the oral bioavailability， and expanding the application of oral NPs in the development of new drugs.
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Plants produce a series of secondary metabolites in cells. These secondary metabolites will be transported to specific organelles for storage or secreted extracellularly. Transporters are a class of membrane proteins that mediate transmembrane transport of chemicals and intracellular signal exchange, and also play an important role in transmembrane transport of plant secondary metabolites. Identification of the function of the secondary metabolite transporters of medicinal plants will help to elucidate the biosynthetic pathways of secondary metabolites in medicinal plants and the molecular mechanism of the transport process of bioactive compounds. In this review, the structure and classification of plant transporters are described in detail. The research progress of plant secondary metabolic transporters and the methods for functional verification of transporter, which have been summarized in this article, will provide a basis for elucidating the biosynthetic pathways and utilization of secondary metabolites in medicinal plants.
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Objective:To explore the absorption and transport properties of flavanomarein in the Madin-Darby canine kidney(MDCK) monolayer cell model. Method:Methyl thiazolyl tetrazolium(MTT) assay was used to investigate the toxicity of flavanomarein in MDCK cells. The resistance value of MDCK monolayer cell model was detected by Millicell-ERS-2 cell resistometer. The effects of mass concentration of flavanomarein,administration time,sodium-glucose cotransporter(SGLTs) inhibitor and glucose transporter 2(GLUT2) inhibitor on the transmembrane transport of flavanomarein were investigated. The concentration of flavanomarein was determined by UPLC-MS/MS, and the apparent permeability coefficient(Papp) and the efflux ratio(ER) were calculated. Result:When the concentration of flavanomarein was 5.625-120 mg·L-1, there was no significant toxic effect on MDCK cells. The transport of flavanomarein in MDCK monolayer cell model was time-dependent and concentration-dependent. The Papp values of flavanomarein were basically between 1×10-6 cm·s-1 to 10×10-6 cm·s-1. Compared with the blank group, the phlorizin group significantly reduced the transport of flavanomarein on the MDCK monolayer cell model at 60 min and 90 min. Conclusion:Flavanomarein is a moderately absorbed drug in the intestine, its transmembrane transport mechanism is dominated by passive transport along with active transport. SGLTs may be involved in mediating the transport of flavanomarein on the MDCK monolayer cell model.
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OBJECTIVE To study transmembrane transport and methylation metabolism of quercetin based on the human intestinal absorption model. METHODS Caco-2 cell monolayer was used as the human intestinal absorption model. The incubation concentration of quercetin was 9.0 and 18.0 mg-L'1. Samples were collected at 30, 60, 90, 120 and 150 min, and the contents of quercetin, isorhamnetin and tamarixetin on the loading side and receiver side were determined by high performance liquid chromatography-mass spectrometry (LC-MS) method. Bupropion was used as the internal standard, and the injection volume was 20 pL. The specific inhibitors of P-glycoprotein (P-pg) or multidrug resistant protein 2 (MRP2), cyclosporins (CysA) 10 mmol-L-1 or MK571 1 mmol • L-1 (final concentration), were added to the apical side. After 15 min incubation, quercetin 9.0 or 18.0 mg-L-1 (final concentration) was added to the apical side, respectively. The quercetin content on the receiver side was determined by the same method. RESULTS During bi-directional transport, the dynamic change of quercetin residues on the loading side showed a continuous decline within 150 min (P<0.05 between adjacent time points), while the amount of quercetin on the receiver side tended to increase before decreasing, reaching the peak at 120 min and falling at 150 min (P<0.05 between adjacent time points). Isorhamnetin and tamarixetin could be detected on both the loading side and receiver side. But the difference was that when quercetin was loaded on the apical side or the basolateral side, there was much more isorhamnetin and tamarixetin on the apical side than on the basolateral side after 30-60 min. Analysis of the percentage of quercetin, isorhamnetin and tamarixetin on the loading side and receiver side found that when incubated for 30 min, the residual quercetin on the loading side was less than 20%-25%, and quercetin on the receiver side was only about 1% of the loading amount. At 150 min, the residual quercetin decreased to <10%, while quercetin in the receiver side was only 6%-7% of the loading amount, and isorhamnetin and tamarixetinin on both sides were only 0.1%-0.3% of loading quercetin. Compared with the quercetin control group, the addition of CysA or MK571 in advance significantly increased the transport of quercetin from the apical side to the basolateral side (P<0.05). CONCLUSION The transport of quercetin on the Caco-2 monolayer cell model shows a trend of rise and fall, accompanied by methylation metabolism. The efflux of P-pg and MRP2 may have an effect on the transmembrane transport of quercetin.
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Objective:To investigate the transport mechanism of punicalagin in MDCK monolayer model .Methods:The safe con-centration of punicalagin in MDCK cells was determined by CCK8 assay.Millicell -ERS was used to measure cell monolayer TEER value to determine the integrity of the cell monolayer .The effects of direction , drug concentration , time, P-gp inhibitor and EDTA-Na2 on the absorption and transport of punicalagin were studied systematically .And then the drug concentration was analyzed by HPLC to calculate the apparent permeability coefficient (Papp) and efflux ratio(ER).Results: Punicalagin transport in MDCK cells was time and concentration dependent .Punicalagin showed poor absorption in MDCK cells .Papp from apical to basolateral side ( AP-BL) within the concentration range of 100-300μg· ml-1 was (6.13 ±0.12) ×10 -7 cm· s-1 , (6.96 ±0.26) ×10 -7 cm· s-1 and (5.94 ±0.10) ×10 -7 cm· s-1 , respectively .P-gp inhibitor and EDTA-Na2 could significantly increase the transport of punicalagin in AP-BL direc-tion, while the transport decreased at 4℃.Conclusion:The transport mechanism of punicalagin might be passive diffusion as the dom-inating process involving active transportation .Punicalagin is one of P-gp substrates with exocytosis and absorbed via the paracellular route.
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Objective To study the inhibition of berberine on organ anion transporters (OATs) and its bidirectional trans-membrane transport.Method The transgene cell lines of the organ anion transporters including OAT1,OAT2,OAT3,OAT4,OAT7,and URAT1 were constructed and selected by animal cell transgenic method mediated by transporter Lipo 3000.Wild type (WT) cells were used as control group,and activity of OATs was verified by adding their radiolabeled substrates and inhibitors.The inhibition of 100 μmol/L berberine on the transporters was investigated in vitro.The IC50 of berberine on URAT1 was also determined.The bidirectional transport of berberine was studied through the Caco-2 model.Result The results showed that 100 μmol/L berberine inhibited the activity of OAT1,OAT2,OAT3,OAT4,OAT7 and URAT1 to (70.48±4.23)％,(69.13±1.28)％,(72.12±3.28)％,(79.77±6.49)％,(69.51 ±5.99)％ and (38.4 ± 2.67)％ respectively,the IC50 of berberine to URAT 1 was 13.19 μmol/L,the Papp (A-B) of 50 μmol/L and 100 μmol/L berberine were separately 0.28 × 10-6 and 0.40 × 10-6 cm/s,and the effiux rates were separately 3.18 and 3.15.Conclusion Berberine shows a stronger inhibition to URAT1 compared to OAT1,OAT2,OAT3,OAT4 and OAT7.Berberine may be the substrate of some effiux transporters.This study provides theoretical basis for explaining the low bioavailability ofberberine and forecasting the possible drug-drug interaction.
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Objective:To summarize the single nucleotide polymorphisms ( SNPs) in ABCC2 and the effect on clinical drug appli-cation. Methods:According to the related articles published in domestic and abroad, the correlation between the single nucleotide pol-ymorphisms in ABCC2 and drug responses was classified and summarized. Results:ABCC2 translocator played an important role in the transmembrane transport of many physiological compounds and exogenous compounds. Numerous studies have demonstrated that the single nucleotide polymorphisms in ABCC2 possibly affected the expression or activity of ABCC2, which leading to the variation in the absorption, distribution and excretion of certain drugs and toxicants. However, the limitation and controversy were still emerged in the results. Conclusion:The influence of ABCC2 single nucleotide polymorphisms on clinical drug application shows significantly referen-tial value for the guidance of medication and the evaluation of efficacy, however, it can not be used as the only indicator yet.
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Aim To study the transport of geniposide and geniposide in Zhizi Bopi Decoction in MDCK cell membrane model. Methods The safety concentration of geniposide and Zhizi Bopi Decoction in MDCK cells were determined by MTT assay. Then the MDCK cell membrane model was used to investigate the transport of drugs. Firstly, the effects of time, drug concentra-tion, P-gp inhibitor and EDTA on the absorption and transport of geniposide were studied systematically. Secondly, the differences were compared between the transport of the same concentration of geniposide as single compound and that in Zhizi Bopi Decoction in MDCK cell model. The drug concentration was deter-mined by high performance liquid chromatography ( HPLC) to calculate the apparent permeability coeffi-cient (Papp). Results Geniposide transport in MDCK cell monolayer was time and concentration dependent. P-gp inhibitors had no significant effect on its transport and the transport of geniposide was enhanced by ED-TA. The absorption Papp of different concentrations of geniposide in Zhizi Bopi Decoction were ( 8. 96 ± 0. 35 ) × 10 -7 cm · s-1 , ( 8. 95 ± 0. 38 ) × 10 -7 cm · s-1 and (9. 16 ± 0. 30) × 10 -7 cm·s-1, significantly higher than the absorption Papp of geniposide as single compound(5. 85 ± 0. 44) × 10 -7 cm·s-1, (6. 88 ± 0. 38) × 10 -7 cm·s-1 and (6. 31 ± 0. 19) × 10 -7 cm ·s-1 ( P<0. 05 ) . Conclusion The transport of ge-niposide in MDCK cell membrane model is passive transport and is not affected by P-gp. Geniposide may transport via the paracellular route. The Zhizi Bopi De-coction can increase the absorption of geniposide.
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Objective To study the expression of tight junction factors in human placental tissues derived from assisted reproductive technology (ART) and natural pregnancy and its role in placental barrier.Methods Ten placental samples were collected from the women who had undergone ART treatment and 11 placenta were collected from control group.Transmission electron microscope (TEM) examination was utilized to detect the morphology of placental tight junctions.The mRNA of claudin (CLDN) 1,CLDN4,CLDN5,CLDN8,zonula occudens (ZO) 1 was detected by real-time PCR and the protein of CLDN4,CLDN8 and occludin (OCLN) were measured by western blot.Results TEM microscopy results showed that placenta samples derived both ART and control placenta had normal microscopic histological features of tight junctions,localized in the apical part of the syncytium and also between the cell-cell contacts of fetal blood vessel endothelial.The expression level of CLDN4 mRNA were 0.87 ±0.17 in ART group and 1.18 ± 0.30 in control group,respectively.The expression level of CLDN8 mRNA were 3.25 ± 2.32 in ART group and 1.08±0.41 in control group,respectively.The mRNA level of CLDN4 and CLDN8 were significantly differentially expressed in ART derived placenta when compared with control groups.The expression level of CLDN1,CLDN5,OCLN and ZO1 mRNA were 0.49 ± 0.44,0.80 ± 0.20,0.92 ± 0.18 in ART group and 1.09±0.82,1.21 ±0.78,0.80± 0.27 in control group,respectively,in which there were no significant differences between two groups.Western Blot analysis showed the protein levels of tight junctions CLDN4,CLDN8 and OCLN did not differ between groups.Conclusions Tight junction factors were expressed in human placental tissues.Tight junction derived from ATR platenta might have mild dysfunction.
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OBJECTIVE: To investigate the mechanism of transmembrane transport of mycophenolate mofetil (MMF) and its active metabolite mycophenolic acid (MPA). METHODS: Caco-2 cell monolayer model was developed for MMF and MPA transport experiment, and the LC-MS/MS method was used for the determination of MMF and MPA concentration in transport medium. The apparent permeability coefficient (Papp) and efflux ratio (Pratio) were calculated and used for the evaluation of the ability of drug transport and drug efflux across Caco-2 cell monolayer. RESULTS: The Papp of MPA in two-way transport experiment (Papp,ab and Papp,ba) at 10, 50 and 100 μmol · L-1 were(9.70 ± 0.40) × 106 cm · s-1 [(13.52 ± 0.28) × 106 cm · s-1], (9.35 ± 0.62) × 106 cm · s-1 [(11.38 ± 0.59) × 106 cm · s-1], (8.69 ± 0.69) × 106 cm · s-1 [(10.53 ± 0.64) × 106 cm · s-1], respectively. Pratio were 1.39, 1.22 and 1.22, respectively. Pratio of MPA was reduced by verapamil, a well-known P-glycoprotein inhibitor. MMF was mostly hydrolyzed to MPA during the transport process across the Caco-2 cell monolayer. The amount of MPA transported increased with time, but the amount of MMF transported changed not significantly over time and no significant effect of verapamil was observed. CONCLUSION: The transmembrane transport of MPA is affected by active transport efflux mediated by transporter with P-gp involved. No effect of P-gp is found in MMF transmembrane transport, which is passive transport.
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Objective To label the amino-glucomannan(AGM) with fluram and to explore the properties of the compound of fluram-AGM in transmembrane transport.Methods After AGM was labeled with fluram,the peripheral blood mononuclear cells(PBMC) and HepG2 were respectively cultured with Fluram-AGM,then observed under fluorescence microscope with violet light as exciting light.Results Both PBMC and HepG2 showed intracellular indigotic fluorescence.Conclusion AGM can be transported into cells across cell membrane.