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。

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.

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.

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.

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.

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.

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.

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.

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.

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.

Influence of magnetic iron oxide nanoparticles on red blood cells and Caco-2 cells

The interactions of two types of cells (red blood cells, Caco-2 cells) with magnetic iron oxide nanoparticles (non-grafted, citrate-grafted, dendrimer-grafted) of 11 nm in size have been investigated. We focused on two important physiological parameters of the cells, the intracellular pH and the intracellular Ca2+ content. The results show that the nanoparticles do not have a significant influence on the pH and Ca2+ content of Caco-2 cells. The Ca2+ content of red blood cells is also not affected but the intracellular pH is slightly reduced.

Antioxidant Activity and Protective Effects of Alcalase-Hydrolyzed Soybean Hydrolysate in Human Intestinal Epithelial Caco-2 Cells

Soybeans are known as a promising source of bioactive peptides.However,knowledge on the antioxidant behaviors of soybean protein hydrolysate(SPH)in the human intestinal epithelium is limited.In this study,SPH was prepared with Alcalase and subsequently ultrafiltered into four peptide fractions as SPH-I(<3 kDa),SPH-II(3~5 k Da),SPH-III(5~10 k Da)and SPH-IV(>10 kDa).The antioxidant properties of SPH and membrane fractions were investigated using different chemical assays and their protective effects against oxidative stress were evaluated using H2 O2-stressed human intestinal Caco-2 cells.Results showed that SPH-I exhibited the strongest 2,2-diphenyl-1-picrylhydrazyl(DPPH)radical scavenging activity(IC50=2.56 mg/m L)and reducing capacity while SPH-III had the best metal ion-chelating activity(IC50=0.29 mg/m L).Both SPH and the peptide fractions dose-dependently suppressed intracellular reactive oxygen species(ROS)accumulation induced by H2O2 in Caco-2 cells,but the strongest inhibitory effect was observed for SPH-I.Amino acid(AA)results revealed that SPH-I was rich in hydrophobic and antioxidant AAs,which could contribute to its stronger antioxidant properties.Additionally,SPH-I protected Caco-2 cells from H2O2-induced oxidative stress via inhibiting lipid peroxidation and stimulating antioxidant enzyme activities.These results suggest that SPH-I and constitutive peptides can be beneficial ingredients with antioxidant properties and protective effects against ROS-mediated intestinal injury.

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。

黄芩苷-小檗碱复合物及纳米晶在Caco-2细胞单层模型的吸收特性研究

[目的]研究黄芩苷(BA)-小檗碱(BBR)复合物及纳米晶的吸收特性,探讨纳米晶技术对BA-BBR复合物吸收特性的改善。[方法]利用Cell Counting Kit-8(CCK-8)法筛选BA-BBR复合物及纳米晶对人结肠腺癌细胞系(Caco-2)细胞的给药浓度,构建Caco-2细胞单层模型探究BA-BBR复合物及纳米晶的跨膜转运方式,并且加入P-糖蛋白(P-gp)抑制剂维拉帕米研究对其累积转运量和表观渗透系数(Papp)的影响。[结果]复合物组中BA和BBR的Papp值均低于物理混合物组;与复合物组相比,纳米晶组中BA和BBR的累积转运量和Papp值均提高,Papp值为复合物组的1.3倍和2.3倍。加入维拉帕米后,复合物组BA和BBR的Papp值分别提高1.5倍和2.0倍,而纳米晶组Papp值无明显提高。[结论]BA-BBR复合物不同于物理混合物,具有低渗透性,同时易受到P-gp外排影响。纳米晶技术可以显著改善BA-BBR复合物的渗透性,并且减弱外排作用对其的影响。

Ponatinib and gossypol act in synergy to suppress colorectal cancer cells by modulating apoptosis/autophagy crosstalk and inhibiting the FGF19/FGFR4 axis

Objective:To evaluate the efficacy of ponatinib plus gossypol against colorectal cancer HCT-116 and Caco-2 cells.Methods:Cells were treated with ponatinib and/or gossypol at increasing concentrations to evaluate synergistic drug interactions by combination index.Cell viability,FGF19/FGFR4,and apoptotic and autophagic cell death were studied.Results:Ponatinib(1.25-40μM)and gossypol(2.5-80μM)monotherapy inhibited HCT-116 and Caco-2 cell viability in a doseand time-dependent manner.The combination of ponatinib and gossypol at a ratio of 1 to 2 significantly decreased cell viability(P<0.05),with a>2-and>4-fold reduction in IC50,respectively,after 24 h and 48 h,as compared to the IC50 of ponatinib.Lower combined concentrations showed greater synergism(combination index<1)with a higher ponatinib dose reduction index.Moreover,ponatinib plus gossypol induced morphological changes in HCT-116and Caco-2 cells,increased beclin-1 and caspase-3,and decreased FGF19,FGFR4,Bcl-2 and p-Akt as compared to treatment with drugs alone.Conclusions:Gossypol enhances ponatinib's anticancer effects against colorectal cancer cells through antiproliferative,apoptotic,and autophagic mechanisms.This may open the way for the future use of ponatinib at lower doses with gossypol as a potentially safer targeted strategy for colorectal cancer treatment.

莱柯霉素在Caco-2细胞单层模型中的转运特性研究

【目的】研究莱柯霉素在Caco-2细胞单层模型中的转运特性,为进一步深入了解莱柯霉素在体内的吸收过程提供试验依据。【方法】建立Caco-2细胞单层模型,通过测定跨膜电阻(TEER)值、低渗透性药物荧光素钠通透性试验的表观渗透率Papp(AP-BL)来验证Caco-2细胞单层模型的致密性并进行模型筛选。考察在Caco-2细胞单层模型中莱柯霉素从顶侧(apical side,AP)到基底侧(basolateral side,BL)及基底侧到顶侧的转运特性,分析药物浓度、pH及外排转运蛋白(P-gp、MRP2和BCRP)抑制剂3种因素对莱柯霉素转运的影响。用超高效液相色谱串联质谱(UPLC-MS/MS)法检测药物浓度,计算其表观渗透率和外排率。【结果】连续20 d的跨膜电阻值检测及荧光素钠通透性试验显示,随培养时间增加,Caco-2细胞单层模型的跨膜电阻值逐渐增加,在20 d时达到400Ω·cm 2左右;荧光素钠在0.1~6μg/mL内线性关系良好(R^(2)=0.991),计算荧光素钠AP侧向BL侧的表现渗透率(Papp(AP-BL))为(1.57×10^(-7)±9.92×10^(-9))cm/s,小于试验规定的0.5×10^(-6) cm/s,表明Caco-2细胞单层模型建立成功,可用于莱柯霉素的转运特性研究。莱柯霉素Papp(AP-BL)值随时间无明显变化且水平较低,而BL侧向AP侧的表现渗透率(Papp(BL-AP))值随时间递增,在孵育120 min时达到最高水平(2.0350×10^(-6) cm/s),表明肠道外排莱柯霉素的能力较强。随莱柯霉素浓度递增,Papp(BL-AP)值呈上升趋势,具有浓度依赖性。当溶液pH为8.0时,Papp(BL-AP)值(39.3871×10^(-6) cm/s)明显高于pH为6.5和7.4的溶液,表明碱性环境可加速莱柯霉素外排。维拉帕米、丙磺舒和GF1209183种转运蛋白抑制剂均能显著抑制莱柯霉素由BL侧向AP侧的转运,外排率均>1.5,可能有外排蛋白参与转运。【结论】莱柯霉素在体外Caco-2细胞单层模型上的转运较差,吸收率低,表现为低渗透性化合物;随着浓度和pH的升高,莱柯霉素外排增强;外排转运蛋白P-gp、MRP2及BCRP可能参与莱柯霉素转运过程。

细胞代次对t-BHP诱导Caco-2细胞构建氧化应激模型的影响

目的:探究细胞代次对叔丁基过氧化氢(t-BHP)诱导构建人克隆结肠腺癌细胞(Caco-2)氧化应激模型的影响。方法:以不同浓度t-BHP(1、2和3 mmol/L)处理第F15代Caco-2细胞1、2、3、4、5和6 h后测定不同处理下细胞丙二醛(MDA)和8-羟基脱氧鸟苷(8-OHdG)的含量,以确定构建Caco-2细胞氧化应激模型的基础条件。再以8-OHdG为分子标志物,探究细胞代次(F14、F15、F18、F19和F20)对Caco-2细胞氧化应激模型构建的影响。结果:采用1、2和3 mmol/L t-BHP处理F15代Caco-2细胞3~6 h均可稳定构建氧化应激模型。当以2.0 mmol/L t-BHP处理不同代次Caco-2细胞5 h后,F14、F15和F18代细胞的8-OHdG浓度均显著高于对照(P<0.05),而F19和F20代细胞的8-OHdG浓度与对照无显著差异。结论:Caco-2细胞代次会影响其氧化应激模型的构建,F19代之前的Caco-2细胞采用2 mmol/L t-BHP处理细胞5 h可稳定构建氧化应激模型。