Effect of dephytinization on bioavailability of iron,calcium and zinc from infant cereals assessed in the Caco-2 cell model

AIM： To test the effect of the dephytinization of three different commercial infant cereals on iron, calcium, and zinc bioavailability by estimating the uptake, retention, and transport by Caco-2 cells. METHODS： Both dephytinized （by adding an exogenous phytase） and non-dephytinized infant cereals were digested using an in vitro digestion protocol adapted to the gastrointestinal conditions of infants younger than 6 too. Mineral cell retention, transport, and uptake from infant cereals were measured using the soluble fraction of the simulated digestion and the Caco-2 cells. RESULTS： Dephytinization of infant cereals significantly increased （P 〈 0.05） the cell uptake efficiency （from 0.66%-6.05% to 3.93%-13%）, retention （from 6.04%-16.68% to 14.75%-20.14%） and transport efficiency （from 0.14%-2.21% to 1.47%-6.02%）, of iron, and the uptake efficiency （from 5.0%-35.4% to 7.3%-41.6%） and retention （from 4.05%-20.53% to 14.45%-61.3%） of zinc, whereas calcium only cell uptake showed a significant increase （P 〈 0.05） after removing phytate from most of the samples analyzed. A positive relationship （P 〈 0.05） between mineral solubility and the cell uptake and transport efficiencies was observed. CONCLUSION： Removing phytate from infant cereals had a beneficial effect on iron and zinc bioavailability when infant cereals were reconstituted with water. Since in developing countries cereal-based complementary foods for infants are usually consumed mixed with water, exogenous phytase additions could improve the nutritional value of this weaning food.

Assessment of Iron Bioavailability in Ten Kinds of Chinese Wheat Flours Using an in vitro Digestion/Caco-2 cell Model

Abstract Objective To compare iron bioavailability （Fe BV） from ten selected kinds of Chinese wheat flours in order to provide scientific basis for further human trials and enable plant breeding programs to screen biofortified wheat cultivars. Methods An in vitro digestion/Caco-2 cell model was used to assess Fe BV of ten flour samples from six leading Chinese wheat cultivars and the stability of Fe BV in one cultivar was studied across three growing environments. Results Significant differences were observed in both Fe BV and Fe bioavailability per gram of food （Fe BVPG） among cultivars （P〈0.01） grown at the same location with the same flour extraction rate. Zhongyou 9507 and Jingdong 8 had Fe BV 37%-54% and Fe BVP（3 103%-154% higher than the reference control. In the Anyang environment, Zhongyou 9507 had a higher wheat flour-Fe level and Fe BVPG. Differences in Fe BV were detected in cultivars with different flour extraction rates. Conclusion Zhongyou 9507 and Jingdong 8 were identified as the most promising cultivars for further evaluation of efficacy by using human subjects. The growing environments had no effect on Fe BV, but did have a significant effect on Fe BVPG. Fe bioavailabilities in low-extraction （40%） flours were higher than those in high-extraction （78%） flours.

Tartary buckwheat(Fagopyrum tataricum(L.)Gaertn)protein-derived antioxidant peptides:mechanisms of action and structure-activity relationship in Caco-2 cell models

Excessive reactive oxygen species(ROS)can cause oxidative damage and lead to various metabolic disease.Tartary buckwheat(Fagopyrum tataricum(L.)Gaertn)is a new kind of protein-rich functional food,the protein in which has been proved to have good antioxidant capacity.In this study,in order to further explore the antioxidant mechanism of Tartary buckwheat protein,4 peptides(CR-8,LR-8,GK-10 and SR-12)were isolated and identified from it.H2 O2 was used to induce oxidative damage to Caco-2 cells to evaluate antioxidant capacity of these peptides.The results of superoxide dismutase(SOD),total antioxidant capacity(T-AOC)and mitochondrial membrane potential etc.showed that these peptides have superior antioxidant capacity.CR-8 has the best antioxidant capacity.In order to further clarify the antioxidant mechanism of CR-8,metabolomics was used to analyze related metabolites and metabolic pathways.The results showed that after CR-8 intervention,the content of metabolites such as L-acetyl carnitine has increased.This indicated that CR-8 can improve the antioxidant capacity of damaged cells by intervening in multiple metabolic pathways.This also revealed the anti-oxidant mechanism of tartary buckwheat protein.In conclusion,it provided a theoretical basis for further studying the activity of tartary buckwheat portein and utilizing buckwheat resources.

Anti-oxidative and anti-inflammatory effects of different lutein isomers in Caco-2 cell model

The purpose of this work was to investigate the differences in the anti-oxidative stress and anti-inflammatory effects between all-E(trans)-lutein and its 9Z(cis)-and 13Z-isomers using the human intestinal Caco-2 cell model.I-TiO_(2) catalyst was used to isomerize all-E-lutein to obtain 9Z-lutein and 13Z-lutein.In anti-oxidative stress studies,all three lutein isoforms could significantly reduce H2O_(2)-induced interleukin-8(IL-8)and tumor necrosis factor-α(TNF-α)secretion(p<0.05)by 50%or more at 4μM.At lower concentration(2μM),9Z-lutein and 13Z-lutein showed higher protection against oxidative stress than all-E-lutein by significantly reducing IL-8 secretion by 48%and 40%(p<0.05),respectively.In addition,three lutein isomers,especially 9Z-lutein could up-regulate the activity of antioxidant enzymes including catalase(CAT),glutathione peroxidase(GPx),gluta-thione reductase(GR),and the content of endogenous antioxidant glutathione(GSH)at 2μM.In the anti-inflammatory assay,9Z-lutein and all-E-lutein exhibited higher anti-inflammatory effects than 13Z-lutein in reducing the secretion and expression of IL-8 in TNF-αinduced inflammatory Caco-2 cells.Compared with the positive control,9Z-lutein and all-E-lutein could reduce the expression of the IL-8 gene by 49%and 52%,respectively at 4μM.This study provided a theoretical basis for the potential health benefits of Z-luteins.

Mechanistic studies of the transport of peimine in the Caco-2 cell model

Fritillaria thunbergii Miq. has been widely used in traditional Chinese medicine for its expectorant, antitussive, antiinflammatory and analgesic properties. Moreover, modern pharmacological studies have demonstrated that F. thunbergii Miq. has efficacy in the treatment of leukemia and cancers of the liver and cervix. Although the alkaloid, peimine, is largely responsible for these pharmacological effects, it has very low oral bioavailability. The aim of this study was to investigate the intestinal absorption of peimine in Caco-2 cell monolayers. Having demonstrated that peimine is non-toxic to Caco-2 cells at concentrations o200 μmol/L, the effect of peimine concentration, p H, temperature, efflux transport protein inhibitors and EDTA-Na_2 on peimine transport were studied. The results show that peimine transport is concentration-dependent; that at p H 6.0 and 7.4, the P_(app(AP-BL))of peimine is not significantly different but the Papp(BL-AP)) is; that both Papp(AP-BL)and P_(app(BL-AP))at 4 1C are significantly higher than their corresponding values at 37 1C; that the P-glycoprotein(P-gp) inhibitors, verapamil and cyclosporin A, increase absorption of peimine; and that EDTA-Na2 has no discernible effect. In summary,the results demonstrate that the intestinal absorption of peimine across Caco-2 cell monolayers involves active transport and that peimine is a substrate of P-gp.

Quantitative Comparison of Bile Acid Distribution and Intestinal Transport from Native Cow-bezoar and Artificial and in vitro Cultured Substitutes using Caco-2 Cell Monolayer Model

Objective This study was conducted to examine the absorption and translocation of conjugated bile acids(BAs)in Calculus bovis and its substitutes to detect differences in these materials.Methods A Caco-2 monolayer cell model was used to compare the apparent permeability coefficient(Papp)value and efflux ratio(ER)of BAs in natural cow-bezoar(NCB),artificial cow-bezoar(ACB),and in vitro cultured cow-bezoar(Ivt-CCB).Papp and ER values were determined by liquid chromatography-mass spectrometry.Samples were separated on an analytical column.Results The distribution of BAs in NCB was significantly different from that in ACB and Ivt-CCB.The percentages of conjugated BAs were significantly higher in NCB than in the two substitutes.The distribution differences of conjugated and unconjugated BAs can be used to distinguish costly NCB from relatively inexpensive substitutes.Conclusion The transport characteristics of BAs in Ivt-CCB were more consistent with NCB than with ACB,even when the proportions of BAs in Ivt-CCB were closer to those of ACB.

内皮细胞特异性骨形态发生蛋白2对血管新生的影响:生物信息学分析和实验验证

背景:血管新生是心血管疾病的主要干预靶点,骨形态发生蛋白2具有调控血管新生作用,但内皮细胞特异性骨形态发生蛋白2对血管新生的调控作用不清楚。目的:探讨内皮细胞特异性骨形态发生蛋白2对血管新生的影响。方法:(1)生物信息学分析:通过Panglao DB公共基因表达数据库单细胞转录组荟萃分析观察骨形态发生蛋白2细胞群表达丰度和定位。血管新生小鼠和内皮(心内膜)过表达骨形态发生蛋白2小鼠转录组测序数据集探索内皮细胞骨形态发生蛋白2对血管新生信号通路的调控作用。(2)体内实验验证:建立小鼠后肢缺血模型,对比模型小鼠患侧与健侧缺血后肢7,14和21 d血流灌注情况,免疫荧光和免疫组织化学染色评估小鼠骨形态发生蛋白2和CD31的表达定位情况。(3)体外实验验证:体外培养人脐静脉内皮细胞,分为对照组、缺氧组和骨形态发生蛋白2抑制剂(Noggin蛋白)干预组,培养24 h,观察各组内皮细胞血管新生情况。结果与结论:(1)内皮细胞是表达骨形态发生蛋白2的重要细胞亚群,在血管新生内皮细胞和骨形态发生蛋白2过表达内皮细胞转录组再分析均发现骨形态发生蛋白2表达明显升高,血管新生通路明显激活。(2)缺血7 d小鼠新生血管周围骨形态发生蛋白2阳性血管明显增加(P<0.05),缺血2周骨形态发生蛋白2阳性血管明显减少(P<0.001)。(3)体外培养人脐静脉内皮细胞,缺氧干预后,内皮细胞迁移能力和血管出芽明显增加,血管新生因子血管内皮生长因子和血小板衍生生长因子的表达明显升高,Noggin明显减少了缺氧诱导的内皮细胞血管新生(P<0.001),并下调血管内皮生长因子和血小板衍生生长因子的表达(P<0.01)。(4)结果证实,内皮细胞特异性骨形态发生蛋白2具有调控血管新生作用,靶向性内皮细胞骨形态发生蛋白2可望改善血管新生。

基于Caco-2细胞模型的辣椒素与槲皮素协同调节小肠糖代谢的分子机制

为探究辣椒素与槲皮素协同调节小肠糖代谢的分子机制,借助Caco-2细胞单层模型,研究辣椒素与槲皮素单一及联合处理对Caco-2细胞存活率、细胞膜通透性的影响。采用免疫印迹法测定跨膜蛋白Occludin、胞质蛋白ZO-1、表面生长因子受体EGFR和纤维状肌蛋白F-actin以及葡萄糖转运关键蛋白GLUT2、SGLT1、Na^(+)/K^(+)-ATPase的表达量。结果显示:各试验处理组Caco-2细胞的增殖率均大于75%,表明药物对细胞没有明显毒性,可进行后续试验。Caco-2细胞单层模型的跨膜电阻值为(315.70±26.65)Ω·cm^(2),加药后各试验组的跨膜电阻值均呈现上升趋势,表明膜通透性变小,细胞屏障功能增强,可延缓小肠对葡萄糖的吸收。同时与空白对照组相比,各试验组上调了跨膜蛋白Occludin(>0.65)、胞质蛋白ZO-1 (>0.51)、表面生长因子受体EGFR(>0.19)和纤维状肌蛋白F-actin(>0.04)的相对表达量,并且下调了葡萄糖转运关键蛋白GLUT2(>1.00)、SGLT1(>0.78)、Na^(+)/K^(+)-ATPase(>0.87)的相对表达水平。综合来看,辣椒素与槲皮素在调节小肠糖代谢方面具有协同效应,二者在高剂量水平以1∶3比例混合效果最佳,主要通过增强肠道黏膜屏障功能,下调葡萄糖转运关键蛋白水平,从而抑制小肠对葡萄糖的吸收来达到调节糖代谢的效果。

天冬氨酸和谷氨酸及其甘氨酸二肽调控Caco-2细胞钙离子吸收的研究

为了探究天冬氨酸和谷氨酸及其甘氨酸二肽对钙离子吸收的影响机理,本文利用等温滴定量热技术、电化学、Caco-2细胞模型结合量子化学计算(密度泛函理论)对天冬氨酸和谷氨酸及其甘氨酸二肽与钙离子的相互作用进行了探究。结果表明,天冬氨酸和谷氨酸通过焓和熵驱动的放热反应与钙离子形成复合物,而其形成的二肽则是通过熵驱动的吸热反应与钙离子结合。此外,二肽比单独的氨基酸表现出更强的钙离子结合能力。量子化学计算表明,氨基酸/二肽中的羧基为钙离子的主要结合位点,在碱性条件下,会导致钙离子结合位点转移,氨基也可以参与钙离子结合。Caco-2细胞吸收实验表明,天冬氨酸和谷氨酸及其二肽均可促进钙的吸收,其中甘氨酸-谷氨酸二肽(Gly-Glu)的促进效果最好,促钙吸收率为1.33±0.115。此外,促钙吸收能力与钙离子结合能力之间没有明确的相关性,与其电子亲和力的关系更为密切。研究结果解释了天冬氨酸、谷氨酸及其甘氨酸二肽与钙之间的相互作用,为进一步开发钙补充剂提供科学依据。

黄芪多糖脂质体的制备及其在Caco-2细胞模型的转运特性研究

【目的】研究黄芪多糖脂质体(Astragalus polysaccharide liposomes,APSL)制备及其在Caco-2细胞模型中的转运。【方法】采用薄膜分散法制备APSL,通过透射电镜观察APSL结构,鱼精蛋白法检测APSL的包封率及载药量;建立Caco-2单层细胞模型,通过透射电镜观察其结构,测量细胞电阻值及荧光素钠表观渗透系数(apparent permeability coefficient,Papp)来评价Caco-2细胞模型的致密性和完整性;分别进行肠腔侧(apical side,AP)到基底侧(basolateral side,BL)及BL到AP侧两个转运方向的跨膜转运试验,分析药物浓度及P-糖蛋白(P-gp)的抑制剂维拉帕米溶液(Verapamil,Ver)对转运的影响。【结果】APSL包封率为71.74%±4.87%,载药量为2.92%,透射电镜下可见球状双层结构,粒径为(126.6±0.283)nm(n=3),聚合物分散性指数(polymer dispersity index,PDI)为0.190±0.009;透射电镜下观察Caco-2细胞紧密结合,形成微绒毛结构,跨膜电阻值达到650Ω·cm^(2),碱性磷酸酶(alkaline phosphatase,ALP)比值达到3.05,荧光素钠Papp为1.96×10^(-7)cm/s,可满足转运试验的要求。在不同浓度下(75、150、300μg/mL),黄芪多糖(APS)的外排率(efflux ratio,ER)均>1.5,APSL的ER均<1.5。3个浓度上AP-BL吸收方向上APSL组Papp均极显著高于APS组(P<0.01),且ER均低于APS组。加入P-gp蛋白抑制剂后,与APS组相比,APS+Ver组AP-BL方向Papp极显著升高(P<0.01),ER降低;而APSL+Ver组两侧的Papp均没有明显变化(P>0.05)。与APS组ER(3.995)相比,APSL组的ER(1.005)降低。【结论】APS在体外Caco-2细胞单层模型上的转运较差,为低渗透性化合物,可能存在P-gp蛋白外排,而APSL能够增加APS的转运吸收,提高药物渗透性。

Effects of extracellular iron concentration on calcium absorption and relationship between Ca^(2+) and cell apoptosis in Caco-2 cells

AIM: To determine the method of growing small intestinal epithelial cells in short-term primary culture and to investigate the effect of extracellular iron concentration ([Fe3+]) on calcium absorption and the relationship between the rising intracellular calcium concentration ([Ca2+]i) and cell apoptosis in human intestinal epithelial Caco-2 cells. METHODS: Primary culture was used for growing small intestinal epithelial cells. [Ca2+]i was detected by a confocal laser scanning microscope. The changes in [Ca2+]i were represented by fluorescence intensity (FI). The apoptosis was evaluated by flow cytometry. RESULTS: Isolation of epithelial cells and preservation of its three-dimensional integrity were achieved using the digestion technique of a mixture of collagenase Ⅺ and dispase Ⅰ. Purification of the epithelial cells was facilitated by using a simple differential sedimentation method. The results showed that proliferation of normal gut epithelium in vitro was initially dependent upon the maintenance of structural integrity of the tissue. If 0.25% trypsin was used for digestion, the cells were severely damaged and very difficult to stick to the Petri dish for growing. The Fe3+ chelating agent desferrioxamine (100, 200 and 300 μmol/L) increased the FI of Caco-2 cells from 27.50±13.18 (control, n = 150) to 35.71±13.99 (n = 150, P<0.01), 72.19±35.40 (n = 150, P<0.01) and 211.34±29.03 (n = 150,P<0.01) in a concentration-dependent manner. There was a significant decrease in the FI of Caco-2 cells treated by ferric ammonium citrate (FAC, a Fe3+ donor; 10, 50 and 100 μmol/L). The FI value of Caco-2 cells treated by FAC was 185.85±33.77 (n = 150, P<0.01), 122.73±58.47 (n = 150, P<0.01), and 53.29±19.82 (n= 150,P<0.01), respectively, suggesting that calcium absorption was influenced by [Fe3+]. Calcium ionophore A23187(0.1,1.0 and 10 μmol/L) increased the FI of Caco-2 cells from 40.45±13.95 (control, n = 150) to 45.19±21.95 (n = 150, P<0.01), 89.87±43.29 (n = 150, P<0.01) and 104.64±51.07 (n = 150,P<0.01) in a concentration-dependent manner. The positive apoptotic cell number of the Caco-2 cells after being treated with A23187 increased from 0.32% to 0.69%, 0.90% and 1.10%, indicating that the increase in the positive apoptotic cell number was positively correlated with [Ca2+]i. CONCLUSION: Ca2+ absorbability is increased with the decrease of extracellular iron concentration Fe3+ and hindered with the increase of Fe3+ consistence out of them. Furthermore, increase of [Ca2+]i can induce apoptosis in Caco-2 cells.

Comparative permeability of three saikosaponins and corresponding saikogenins in Caco-2 model by a validated UHPLC-MS/MS method

Saikosaponins(SSs)are the main active components extracted from Bupleuri Radix(BR)which has been used as an important herbal drug in Asian countries for thousands of years.It has been reported that the intestinal bacteria plays an important role in the in vivo disposal of oral SSs.Although the deglycosylated derivatives(saikogenins,SGs)of SSs metabolized by the intestinal bacteria are speculated to be the main components absorbed into the blood after oral administration of SSs,no studies have been reported on the characteristics of SGs for their intestinal absorption,and those for SSs are also limited.Therefore,a rapid UHPLC-MS/MS method was developed to investigate and compare the apparent permeability of three common SSs(SSa,SSd,SSb2)and their corresponding SGs(SGF,SGG,SGD)through a bidirectional transport experiment on Caco-2 cell monolayer model.The method was validated according to the latest FDA guidelines and applied to quantify the six analytes in transport medium samples extracted via liquid-liquid extraction(LLE).The apparent permeability coefficient(Papp)determined in this study indicated that the permeability of SGs improved to the moderate class compared to the corresponding parent compounds,predicting a higher in vivo absorption.Moreover,the efflux ratio(ER)value demonstrated an active uptake of SSd and the three SGs,while a passive diffusion of SSa and SSb2.

Gastric digestion of pea ferritin and modulation of its iron bioavailability by ascorbic and phytic acids in caco-2 cells

AIM： To understand the digestive stability and mechanism of release and intestinal uptake of pea ferritin iron in caco-2 cell line model.METHODS： Pea seed ferritin was purified using salt fractionation followed by gel filtration chromatography.The bioavailability of ferritin iron was assessed using coupled in vitro digestion/Caco-2 cell model in the presence or absence of ascorbic acid and phytic acid.Caco-2 cell ferritin formation was used as a surrogate marker of iron uptake. Structural changes of pea ferritin under simulated gastric pH were characterized using electrophoresis, gel filtration and circular dichroism spectroscopy.RESULTS： The caco-2 cell ferritin formation was significantly increased （P 〈 0.001） with FeSO4 （19.3±9.8 ng/mg protein） and pea ferritin （13.9 ± 6.19 ng/mg protein） compared to the blank digest （3.7 ± 1.8 ng/mg protein）. Ascorbic acid enhanced while phytic acid decreased the pea ferritin iron bioavailability. However,either in the presence or absence of ascorbic acid, the ferritin content of caco-2 cells was significantly less with pea ferritin than with FeSO4. At gastric pH, no band corresponding to ferritin was observed in the presence of pepsin either on native PAGE or SDS-PAGE. Gel filtration chromatography and circular dichroism spectroscopy revealed a pH dependent loss of quaternary and secondary structure.CONCLUSION： Under gastric conditions, the iron core of pea ferritin is released into the digestive medium due to acid induced structural alterations and dissociation of protein. The released iron interacts with dietary factors leading to modulation of pea ferritin iron bioavailability,resembling the typical characteristics of non-heme iron.

Molecular analysis and anticancer properties of two identified isolates,Fusarium solani and Emericella nidulans isolated from Wady El-Natron soil in Egypt against Caco-2(ATCC) cell line

Objective:To characterize,identify and investigate the anticancer properties of two new soil fungal isolates,Emericella nidulansand Fusarium solani isolated from Wady El-Natron in Egypt against colon cancer Caco-2(ATCCj cell line.Methods:Soil sample was cultured and two strains were chosen for morphological and phenotypical characterization.Partial sequences of the 18s rRNA gene and the internal transcribed spacer region ITS of the two isolates were amplified by PCR.Phylogenetic tree construction and analysis of the resulted multiple sequences from the two fugal isolates were also carried out.In vitro anticancer activity of the two strains was done against colon Caco-2 cancer cell line.Reverse transcription — PCR was carried out to detect level of expression of p53 in Caco-2 cell line.Results:HF.I displayed morphological and genotypic characteristics most similar to that of Fusarium solani while HF.2 was most similar to Emericella nidulans with high similarity of 99%and 97%respectively.The multiple sequence alignment of the two fungal isolates showed that,the maximum identical conserved domains in the 18s rRNA genes were identified with the nucleotide regions of Slst to 399th base pairs,88th to 525th base pairs respectively.While those in the ITS genes were identified with the nucleotide regions of 88th to 463rd and Slst to 274th.The two isolates showed IC<sub>50 value with（6.24±5.21） and（9.84±0.36） μ g/mL) concentrations respectively at 28h.Reverse transcription- PCR indicated that these cells showed high level of expression for p53 mRNA.Conclusions:The morphology and molecular analysis identified HF.1 and HF.2 to be Fusarium solani and Emericella nidulans;new isolates of anticancer producing fungi from Wady El-Natroon city in Egypt.Treatment with the two isolates caused P53 expression in Caco-2 cell line.These two isolates can be used as an anticancer agents.

Transepithelial transport of putrescine across monolayers of the human intestinal epithelial cell line, Caco-2

AIM: To study the transepithelial transport characteristics of the polyamine putrescine in human intestinal Caco-2 cell monolayers to elucidate the mechanisms of the putrescine intestinal absorption. METHODS: The transepithelial transport and the cellular accumulation of putrescine was measured using Caco-2 cell monolayers grown on permeable filters. RESULTS: Transepithelial transport of putrescine in physiological concentrations (】 0.5 mM) from the apical to basolateral side was linear. Intracellular accumulation of putrescine was higher in confluent than in fully differentiated Caco-2 cells, but still negligible (less than 0.5%) of the overall transport across the monolayers in apical to basolateral direction.EGF enhanced putrescine accumulation in Caco-2 cells by four fold, as well as putrescine conversion to spermidine and spermine by enhancing the activity of S adenosylmethionine decarboxylase. However, EGF did not have any significant influence on putrescine flux across the Caco-2 cell monolayers. Excretion of putrescine from Caco-2 cells into the basolateral medium did not exceed 50 picomoles, while putrescine passive flux from the apical to the basolateral chamber, contributed hundreds of micromoles polyamines to the basolateral chamber. CONCLUSION :Transepithelial transport of putrescine across Caco2 cell monolayers occurs in passive diffusion, and is not influenced when epithelial cells are stimulated to proliferate by a potent mitogen such as EGF.

Simultaneous LC-MS/MS Determination of Danshensu and Paeoniflorin for Permeability Studies in Caco-2 Intestinal Absorption Model

A high sensitive method based on liquid chromatography tandem mass spectrometry（LC-MS/MS） was developed and validated for the study of permeability of danshensu（DS） and paeoniflorin（PF） in Caco-2 intestinal absorption model. The DS and PF were extracted from cell culture by vacuum-lyophilizing and then separated on a Zorbax Stable Bond C18 column with 0.1% acetic acid aqueous solution and methanol as mobile phase. Detection was carried out by negative electrospray ionization（ESI ） with selected reaction monitoring（SRM） mode. The apparent permeability coefficients（Papp） of DS and PF in Caco-2 cell medium were calculated and the effects of verapamil on the coefficients Papp of the two test compounds were also illustrated. The permeability of PF was much better than that of DS when the two compounds were administrated individually. Co-administration of DS and PF led to the decrease of the transport from apical side to basolateral side for both the compounds. However, the transport in the contrary direction were accelerated. It was also observed that verapamil could accelerate the transport of the test compounds from apical side to basolateral side. However, the absorption-enhanced effect of verapamil was attenuated when DS and PF were co-administrated. These observations suggest that both passive diffusion and active efflux involved in P-gp would effect the passage of DS and PF across Caco-2 cell monolayer. At the same time, the co-administration of DS and PF to an alteration of transport behavior, which suggests that the interaction must be taken into account when ‘n-in-one＇ samples were used in Caco-2 intestinal model.

Transport and uptake of clausenamide enantiomers in CYP3A4-transfected Caco-2 cells： an insight into the efflux-metabolism alliance

Aim The present study developed a CYP3A4-expressed Caco-2 monolayer model at which effects of the efflux-metabolism alliance on the transport and uptake of clausenamide（CLA） enantiomers as CYP3A4 substrates were investigated. The apparent permeability coefficients （Papp） of （ - ） and （ ＋ ）CLA were higher in the ab- sorptive direction than those in the secretory direction with efflux ratios（ER） of 0. 709 ± 0.411 and 0. 867± 0. 250 （ Х10^-6 -1 cm · s ）, respectively. Their bidirectional transports were significantly reduced by （75.6 ± 87.5）% af- ter treatment with verapamil （ a P-glycoprotein inhibitor） that increased the rate of metabolism by CYP3 A4, whereas the CYP3A4 inhibitor ketoconazole treatment markedly enhanced the basolateral to apical flux of （ - ） and （ ＋ ） CLA with ERs being 2. 934 ± 1. 432 and 1. 877 ± 0. 148 （ Х 10^-6 cm/s） respectively. These changes could be blocked by the duel CYP3A4/P-glycoprotein inhibitor cyclosporine A, consequently, Papp values for CLA enanti- omers in both directions were significantly greater than those obtained by using verapamil or ketoconazole, and their ERs were similar to those following （ - ） or （ ＋ ）-isomer treatment alone. Furthermore, the uptake of （ - ）CLA was more than that of （ ＋ ）CLA in the transfected cells. Incubation with ketoeonazole decreased the intracellular concentrations of the two enantiomers. This effect disappeared in the presence of a CYP3A4 inducer dexametha- sone. These results indicated that CYP3A4 could influence P-gp efflux, transport and uptake of CLA enantiomers as CYP3A4 substrates and that a duel inhibition to CYP3A4/ P-glycoprotein could enhance their absorption and bioavailability, which provides new insight into the efflux-metabolism alliance and will benefit the clinical pharma- cology of （？） CLA as a candidate drug for treatment of Alzheimer＇ s disease.

Overloading of differentiated Caco-2 cells during lipid transcytosis induces glycosylation mistakes in the Golgi complex

Overloading the intestine enterocytes with lipids induced alteration of the Golgi complex(GC;Sesorova et al.,2020)and could cause glycosylation errors.Here,using differentiated Caco-2 cells with the established 0[I]blood group phenotype(no expression of the blood antigens A and B[AgA,AgB]under normal conditions)as a model of human enterocytes we examined whether the overloading of these cells with lipids could cause errors in the Golgi-dependent glycosylation.We demonstrated that under these conditions,there were alterations of the GC and the appearance of lipid droplets in the cytoplasm.Rare cells produced AgA and AgB.This suggested that after overloading of enterocytes with lipids,AgA were mistakenly synthesized in individual enterocytes by the Golgi glycosyltransferases.These mistakes could explain why in the absence of AgA and AgB antibodies against them exist in the blood.

Upregulation of 25-hydroxyvitamin D_3-1α-hydroxylase by butyrate in Caco-2 cells

AIM： To investigate the possible involvement of 25-hydroxyvitamin D3-1cx-hydroxylase [1α-25（OH）2D3] in butyrate-induced differentiation in human intestinal cell line Caco-2 cells. METHODS： Caco-2 cells were incubated either with 3 mmol/L butyrate and 1 umol/L 25（OH）2D3 or with 1 umol/L 1α-25（OH）2D3 for various time intervals ranging from 0 to 72 h. Additionally, cells were co-incubated with butyrate and either 25（OH）2D3 or 1α-25（OH）2D3. 1α-25（OH）2D3 mRNA was determined semi-quantitatively using the fluorescent dye PicoGreen. Immunoblotting was used for the detection of 1α-25（OH）2D3 protein. Finally, enzymatic activity was measured by ELISA. RESULTS： Both butyrate and 1α-25（OH）2D3 stimulated differentiation of Caco-2 cells after a 48 h incubation period, while 25（OH）2D3 had no impact on cell differentiation. Synergistic effects on differentiation were observed when cells were co-incubated with butyrate and vitamin D metabolite. Butyrate transiently upregulated 1α-25（OH）2D3 mRNA followed by a timely delayed protein upregulation. Coincidently, enzymatic activity was enhanced significantly. The induction of the enzyme allowed for comparable differentiating effects of both vitamin D metabolites. CONCLUSION： Our experimental data provide a further mechanism for the involvement of the vitamin D signaling pathway in colonic epithelial cell differentiation by butyrate. The enhancement of 1α-25（OH）2D3 followed by antiproliferative effects of the vitamin D prohormone in the Caco-2 cell line suggest that 25（OH）2D3 in combination with butyrate may offer a new therapeutic approach forthe treatment of colon cancer.

A SARS-CoV-2 neutralizing antibody discovery by single cell sequencing and molecular modeling

Although vaccines have been developed,mutations of SARS-CoV-2,especially the dominant B.1.617.2(delta)and B.1.529(omicron)strains with more than 30 mutations on their spike protein,have caused a significant decline in prophylaxis,calling for the need for drug improvement.Antibodies are drugs preferentially used in infectious diseases and are easy to get from immunized organisms.The current study combined molecular modeling and single memory B cell sequencing to assess candidate sequences before experiments,providing a strategy for the fabrication of SARS-CoV-2 neutralizing antibodies.A total of 128 sequences were obtained after sequencing 196 memory B cells,and 42 sequences were left after merging extremely similar ones and discarding incomplete ones,followed by homology modeling of the antibody variable region.Thirteen candidate sequences were expressed,of which three were tested positive for receptor binding domain recognition but only one was confirmed as having broad neutralization against several SARS-CoV-2 variants.The current study successfully obtained a SARS-CoV-2 antibody with broad neutralizing abilities and provided a strategy for antibody development in emerging infectious diseases using single memory B cell BCR sequencing and computer assistance in antibody fabrication.