Studies on the recombinant production and anticancer activity of thermostable L- asparaginase I from Pyrococcus abyssi / Estudos sobre a produção recombinante e atividade anticancerígena da L-asparaginase I termoestável de Pyrococcus abyssi

L-Asparaginase catalysing the breakdown of L-Asparagine to L-Aspartate and ammonia is an enzyme of therapeutic importance in the treatment of cancer, especially the lymphomas and leukaemia. The present study describes the recombinant production, properties and anticancer potential of enzyme from a hyperthermophilic archaeon Pyrococcus abyssi. There are two genes coding for asparaginase in the genome of this organism. A 918 bp gene encoding 305 amino acids was PCR amplified and cloned in BL21 (DE3) strain of E. coli using pET28a (+) plasmid. The production of recombinant enzyme was induced under 0.5mM IPTG, purified by selective heat denaturation and ion exchange chromatography. Purified enzyme was analyzed for kinetics, in silico structure and anticancer properties. The recombinant enzyme has shown a molecular weight of 33 kDa, specific activity of 1175 U/mg, KM value 2.05mM, optimum temperature and pH 80°C and 8 respectively. No detectable enzyme activity found when L-Glutamine was used as the substrate. In silico studies have shown that the enzyme exists as a homodimer having Arg11, Ala87, Thr110, His112, Gln142, Leu172, and Lys232 being the putative active site residues. The free energy change calculated by molecular docking studies of enzyme and substrate was found as ∆G ­ 4.5 kJ/mole indicating the affinity of enzyme with the substrate. IC50 values of 5U/mL to 7.5U/mL were determined for FB, caco2 cells and HepG2 cells. A calculated amount of enzyme (5U/mL) exhibited 78% to 55% growth inhibition of caco2 and HepG2 cells. In conclusion, the recombinant enzyme produced and characterized in the present study offers a good candidate for the treatment of cancer. The procedures adopted in the present study can be prolonged for in vivo studies.

A L-asparaginase, que catalisa a degradação da L-asparagina em L-aspartato e amônia, é uma enzima de importância terapêutica no tratamento do câncer, especialmente dos linfomas e da leucemia. O presente estudo descreve a produção recombinante, propriedades e potencial anticancerígeno da enzima de Pyrococcus abyssi, um archaeon hipertermofílico. Existem dois genes que codificam para a asparaginase no genoma desse organismo. Um gene de 918 bp, que codifica 305 aminoácidos, foi amplificado por PCR e clonado na cepa BL21 (DE3) de E. coli usando o plasmídeo pET28a (+). A produção da enzima recombinante foi induzida sob 0,5mM de IPTG, purificada por desnaturação seletiva por calor e cromatografia de troca iônica. A enzima purificada foi analisada quanto à cinética, estrutura in silico e propriedades anticancerígenas. A enzima recombinante apresentou peso molecular de 33 kDa, atividade específica de 1.175 U / mg, valor de KM 2,05 mM, temperatura ótima de 80º C e pH 8. Nenhuma atividade enzimática detectável foi encontrada quando a L-glutamina foi usada como substrato. Estudos in silico mostraram que a enzima existe como um homodímero, com Arg11, Ala87, Thr110, His112, Gln142, Leu172 e Lys232 sendo os resíduos do local ativo putativo. A mudança de energia livre calculada por estudos de docking molecular da enzima e do substrato foi encontrada como ∆G ­ 4,5 kJ / mol, indicando a afinidade da enzima com o substrato. Valores de IC50 de 5U / mL a 7,5U / mL foram determinados para células FB, células caco2 e células HepG2. Uma quantidade de enzima (5U / mL) apresentou inibição de crescimento de 78% a 55% das células caco2 e HepG2, respectivamente. Em conclusão, a enzima recombinante produzida e caracterizada no presente estudo é uma boa possibilidade para o tratamento do câncer. Os procedimentos adotados na presente pesquisa podem ser aplicados para estudos in vivo.

Transcriptomic analysis of the ΔPaLoc mutant of Clostridioides difficile and verification of its toxicity / 中华预防医学杂志

Objective: Comparative analyses of wild-type Clostridioides difficile 630 (Cd630) strain and pathogenicity locus (PaLoc) knockout mutant (ΔPaLoc) by using RNA-seq technology. Analysis of differential expression of Cd630 wild-type strain and ΔPaLoc mutant strain and measurement of its cellular virulence changes. Lay the foundation for the construction of an toxin-attenuated vaccine strain against Clostridioides difficile. Methods: Analysis of Cd630 and ΔPaLoc mutant strains using high-throughput sequencing (RNA-seq). Clustering differentially expressed genes and screening differentially expressed genes by DESeq software. Further analysis of differential genes using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. Finally, cytotoxicity assays of ΔPaLoc and Cd630 strains were performed in the African monkey kidney epithelial cell (Vero) and the human colonic cell (Caco-2) lines. Results: The transcriptome data showed that the ΔPaLoc mutant toxin genes tcdA and tcdB were not transcribed. Compared to the wild-type strain, CD630_36010, CD630_020910,CD630_02080 and cel genes upregulated 17.92,11.40,8.93 and 7.55 fold, respectively. Whereas the hom2 (high serine dehydrogenase), the CD630_15810 (spore-forming protein), CD630_23230 (zinc-binding dehydrogenase) and CD630_23240 (galactitol 1-phosphate 5-dehydrogenase) genes were down-regulated by 0.06, 0.075, 0.133 and 0.183 fold, respectively. The GO and KEGG enrichment analyses showed that the differentially transcribed genes in ΔPaLoc were enriched in the density-sensing system, ABC transport system, two-component system, phosphotransferase (PTS) system, and sugar metabolism pathway, as well as vancomycin resistance-related pathways. Cytotoxicity assays showed that the ΔPaLoc mutant strain lost its virulence to Vero and Caco-2 cells compared to the wild-type Cd630 strain. Conclusion: Transcriptional sequencing analysis of the Cd630 and ΔPaLoc mutant strains showed that the toxin genes were not transcribed. Those other differential genes could provide a reference for further studies on the physiological and biochemical properties of the ΔPaLoc mutant strain. Cytotoxicity assays confirmed that the ΔPaLoc mutant lost virulence to Vero and Caco-2 cells, thus laying the foundation for constructing an toxin-attenuated vaccine strain against C. difficile.

Construction of spvD gene deletion mutant and compensation strains in Salmonella enteritidis and its effects on Caco-2 cells / 中华预防医学杂志

Objective: To analyze the effects of spvD gene on invasion and intracellular proliferation of Caco-2 cells and in order to provide insight into the function of that gene and the underlying mechanism of Salmonella caused infection. Methods: Functional verification of spvD gene deletion mutant and compensation strain. The deletion mutant strain was constructed through a suicide plasmid-mediated homologous recombination. The compensation plasmid constructed by cloning the coding sequence of spvD by PCR into plasmid pBAD33 was mobilized into the deletion mutant by conjugation and the pBAD33 was introduced into wild strains and deleted mutant strains as control. The relative expression of spvD mRNA was detected by quantitative reverse transcription PCR. In order to analyze the virulence of spvD against Caco-2 cells, Caco-2 cells was cocultured with wild type Salmonella enteritidis carrying spvD gene, the deletion mutant strain and compensation strain respectively. The expression level of spvD mRNA and the the number of Salmonella enteritidis after Caco-2 cells intervention were compared between the three groups by LSD-t test, and the invasion rate was compared by χ2 test. Results: The expression level of spvD mRNA in wild type Salmonella enteritidis was set as unit "1", the deletion mutant strain was "0.00", and the compensation strain was "2.60" (LSD-twild, deleted=1.11, P=0.31; LSD-twild, compensation=-1.77, P=0.13; LSD-t deleted, compensation=-2.88, P=0.03), which confirmed the successful construction of the deletion mutant strain and the compensation strain. The invasion experiment results of the above three Salmonella enteritidis strains on Caco-2 cells showed that the invasion rate of wild strain was 0.23%, the invasion rate of deleted mutant strain was 0.16%, and the invasion rate of compensation strain was 0.16%, with no statistical significance (χ2=1.13, P=0.570). By comparing the number of Salmonella enteritidis at different time points after Caco-2 cells intervention, it was discovered that the number of Salmonella enteritidis in wild strains (6.50×106 CFU/ml) and compensation strains (7.25×106 CFU/ml) was significantly increased than that in deletion mutant strain (1.90×106 CFU/ml) after 16 h coculture (LSD-twild, deleted=7.95, P=0.00; LSD-twild, compensation=-1.27, P=0.25; LSD-t deleted, compensation=-9.22, P=0.00). Conclusion: It is not considered that spvD gene can affect the invasion of Salmonella enteritidis on Caco-2 cells, but the gene can promote the reproduction of Salmonella enteritidis in Caco-2 cells.

Defensin c-thionin from Capsicum chinense improves butyrate cytotoxicity on human colon adenocarcinoma cell line Caco-2

BACKGROUND: Butyrate is a histone deacetylase inhibitor that induces apoptosis and inhibits cell proliferation of colorectal cancer cells. To improve its anticancer activity, butyrate has been evaluated mixed with drugs and different molecules. Plant antimicrobial peptides are attractive anticancer alternative molecules because they show selective cytotoxic activity against different cancer cell lines. In this work, we explore if the plant defensin c-thionin (Capsicum chinense) can improve butyrate activity on Caco-2 cell line and we also determined the mechanism of death activated. RESULTS: The combined treatment of c-thionin (3.5 mM) and butyrate (50 mM) showed higher cytotoxicity on Caco-2 cells with respect to single treatments. Also, the combined treatment reduced cell proliferation and exhibited a higher rate of apoptosis than single treatments. Combined treatment induced caspases 8 and 9 activation to an extent comparable with that of butyrate while c-thionin did not activate caspases. Additionally, reactive oxygen species generation preceded the onset of apoptosis, and superoxide anion production was higher in cells treated with the combined treatment. CONCLUSIONS: The c-thionin from Habanero chili pepper improved the butyrate cytotoxicity on Caco-2 cells. This effect occurred through apoptosis induction associated with reactive oxygen species production. Therefore, the combination of butyrate with cytotoxic antimicrobial peptides could be an attractive strategy for cancer therapy.

Biopharmaceutical classification and transport mechanism of 4 alkaloids in Mongolian herbal medicine Sophorae Flavescentis Radix / 中国中药杂志

In this study, we studied the solubility and permeability of matrine, oxymatrine, sophoridine, and oxysophocarpine, four alkaloids in the Mongolian herbal medicine Sophorae Flavescentis Radix, and evaluated the absorption mechanism with the Caco-2 cell model, so as to provide a basis for the new drug development and efficacy evaluation of Sophorae Flavescentis Radix. The results showed that all the four alkaloids had high solubility and high permeability and can be well absorbed, belonging to the class-I drugs of Biopharmaceutical Classification System(BCS). The absorption(AP→BL) and excretion(BL→AP) of matrine and oxymatrine were not affected by the concentration while the absorption depended on P-gp protein. The absorption(AP→BL) and excretion(BL→AP) of sophoridine and oxysophocarpine were positively related to the concentration and time, and the absorption process was independent from P-gp protein. The results provide scientific reference and an experimental basis for the development of Mongolian medical prescriptions containing Sophorae Flavescentis Radix.

Metformin alleviates intestinal epithelial barrier damage by inhibiting endoplasmic reticulum stress-induced cell apoptosis in colitis cell model / 浙江大学学报·医学版

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.

Studies on absorption and transportation of coumarins in Angelica dahurica 'Yubaizhi' across human intestinal epithelial by using human Caco-2 cell monolayers / 中国中药杂志

The absorption is the key to the resulted efficacy of orally administered drugs and the small intestine is the main site to absorb the orally administered drug. In this paper, internationally recognized human colon adenocarcinoma cell line(Caco-2) monola-yer model which can simulate small intestinal epithelial cell was used to comparatively study the absorption and transportation diffe-rences of total coumarins and main individual coumarin in Angelica dahurica 'Yubaizhi' by separately using 6-and 12-well plates. It was found that apparent permeability coefficient(P_(app)) values of oxypeucedanin hydrate, byakangelicin and phellopterin were at the quantitative degree of 1 × 10~(-5) cm·s~(-1) when the individual administration was conducted independently, indicating that they were well-absorbed compounds. P_(app) ratio of their bi-directional transportation was close to 1, indicating that they can be absorbed across Caco-2 monolayer by passive diffusion mechanism without carrier mediation during the transportation. The similar trend of transportation was also observed for imperatorin, isoimperatorin and bergapten. The P_(app) values of oxypeucedanin hydrate, byakangelicin and bergapten were at quantitative degree of 1 × 10~(-5) cm·s~(-1) when the administration of total coumarins in Angelica dahurica 'Yubaizhi' was conducted, indicating that they were well-absorbed compounds. The results were consistent with those of independent administration of individual coumarins. Whereas, the P_(app) values of imperatorin, phellopterin and isoimperatorin in the total coumarins decreased, indicating that the interaction between compounds may exist although the P_(app) value ratio of bi-directional transportation was between 0.5 and 1.5. The results laid the foundation for intestinal absorption study of Angelica dahurica 'Yubaizhi' coumarins in compound Chinese medicine.

Study on oral absorption mechanisms of puerarin in nanocrystals self-stabilized pickering emulsion / 中国中药杂志

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.

Effects of HPMCAS MF on absorption of silybin from supersaturable self-nanoemulsifying drug delivery system / 中国中药杂志

To evaluate the effects of Hydroxypropyl methylcellulose acetate succinate(HPMCAS MF) on absorption of silybin(SLB) from supersaturable self-nanoemulsifying drug delivery system which was pre-prepared at the early stage experiment. The cell toxicity of self-emulsifying preparation was evaluated by the MTT method, and the in vitro membrane permeability and absorption promoting effect of the self-emulsifying preparation were evaluated by establishing a Caco-2 cell monolayer model. The in vivo and in vitro supersaturation correlation was evaluated via the blood concentration of SLB. The results of MTT showed that the concentration of the preparation below 2 mg·mL~(-1)(C_(SLB) 100 μg·mL~(-1)) was not toxic to Caco-2 cells, and the addition of polymer had no significant effect on Caco-2 cells viability. As compared with the solution group, the transport results showed that the P_(app)(AP→BL) of the self-emulsifying preparation had a very significant increase; the transport rate of silybin can be reduced by polymer in 0-30 min; however, there was no difference in supersaturated transport between supersaturated SLB self-nanoemulsion drug delivery system(SLB-SSNEDDS) and SLB self-nanoemulsion drug delivery system(SLB-SNEDDS) within 2 hours. As compared with SLB suspension, pharmacokinetic parameters showed that the blood concentration of both SLB-SNEDDS and SLB-SSNEDDS groups were significantly increased, and C_(max) was 5.25 times and 9.69 times respectively of that in SLB suspension group, with a relative bioavailability of 578.45% and 1 139.44% respectively. C_(max) and relative bioavailability of SLB-SSNEDDS were 1.85 times and 197% of those of SLB-SNEDDS, respectively. Therefore, on the one hand, SSNEDDS can increase the solubility of SLB in gastrointestinal tract by maintaining stability of SLB supersaturation state; on the other hand, the osmotic transport process of SLB was regulated through the composition of its preparations, and both of them could jointly promote the transport and absorption of SLB to improve the oral bioavailability of SLB.

Prediction of anti-liver fibrosis effect of Piperis Longi Fructus based on network pharmacology / 中国中药杂志

Network pharmacology and liver fibrosis(LF) model in vitro were used to analyze the underly mechanism of anti-liver fibrosis effect that induced by Piperis Longi Fructus and its major active compounds. TCMSP and TCMIP were used to search for the chemical constituents of Piperis Longi Fructus, as well as the oral bioavailability(OB), drug-likeness(DL), intercellular permeability of intestinal epithelial cells(Caco-2) and Drug-likeness grading were set as limiting conditions. The related target genes of Piperis Longi Fructus were queried by TCMSP database, while related targets of LF were screened by GeneCards databases. Interaction network was constructed using Cytoscape 3.7.1. These above data were imported into STRING database for PPI network analysis. Enrichment of gene ontology(GO) and pathway analysis(KEGG) within Bioconductor database were utilized to note functions of related targets of Piperis Longi Fructus. Finally, the core targets and pathways were preliminarily verified by in vitro experiments. The effects of piperlongumine(PL), the major active component of Piperis Longi Fructus, on proliferation of rat liver stellate cells(HSC-T6) and expression of α smooth muscle actin(α-SMA) and collagen Ⅰ were investigated. The major factors TNF-α of tumor necrosis factor(TNF) pathway and NF-κB p65, IL-6 protein expressions of LF process were examined. A total of 12 active compounds such as PL were obtained by analyzing the bioavailability and drug-like properties, which inferred to 48 targets. The functional enrichment analysis of GO obtained 1 240 GO items, mainly involving in process of biology and molecular function. A total of 99 signaling pathways were enriched in the KEGG pathway enrichment analysis, including TNF signaling pathway, cGMP-PKG signaling pathway, calcium signaling pathways. CCK-8 assay showed that PL inhibited proliferation of HSC-T6 induced by transforming growth factor-β1(TGF-β1). Western blot analysis found that treated with PL suppressed the protein expressions of α-SMA, collagen Ⅰ, TNF-α and p65 in HSC-T6. Enzyme linked immunosorbent assay(ELISA) showed that PL inhibited the expressions of TNF-α and IL-6 in the cluture supertant of HSC-T6 cells. In conclusion, PL could play an anti-liver fibrosis role by regulating TNF/NF-κB signaling pathway. This study provided the mechanism basis of anti-LF effects induced by Piperis Longi Fructus and its major active compounds, which might help for the further study of the mechanism and key targets of Piperis Longi Fructus.

Short-chain fatty acid butyrate acid attenuates atherosclerotic plaque formation in apolipoprotein E-knockout mice and the underlying mechanism / 生理学报

This study was designed to evaluate the role of short-chain fatty acid butyrate acid on intestinal morphology and function, and atherosclerotic plaque formation in apolipoprotein E-knockout (ApoE

Efeitos do extrato aquoso de guaraná (Paullinia cupana) em pó nos mecanismos envolvidos com a absorção de colesterol in vitro e em células Caco-2 / Effects of the aqueous extract of guarana (Paullinia cupana) powder on the mechanisms involved with the absorption of cholesterol in vitro and in Caco-2 cells

Introdução - O risco de doenças cardiovasculares, dentre elas a aterosclerose, está associado com a hipercolesterolemia, resultado de fatores de risco comportamentais, metabólicos e genéticos. Intervenções dietéticas como o uso do guaraná (Paullinia cupana), podem ser úteis, uma vez que é um alimento rico em compostos bioativos como as catequinas, que possuem elevada atividade antioxidante, inibem a oxidação da lipoproteína de densidade baixa (LDL) e a peroxidação lipídica, sendo capazes de reduzir a concentração plasmática de colesterol, por mecanismos ainda não totalmente elucidados. Objetivo - Investigar os efeitos do extrato aquoso de guaraná (Paullinia cupana) em pó sobre mecanismos envolvidos com a absorção de colesterol em modelos in vitro e em células Caco-2. Métodos - O extrato aquoso do guaraná em pó foi submetido à digestão in vitro, os fenólicos totais foram quantificados pelo método de Folin-Ciocalteu e a determinação do perfil de fenólicos foi realizada por cromatografia líquida de alta eficiência (HPLC), com detector UV/VIS a 210 nm. A atividade antioxidante foi determinada pela capacidade de absorbância de radical oxigênio (Oxygen Radical Absorbance Capacity - ORAC) e foram avaliadas a capacidade de inibição da lipase pancreática, de ligação com ácidos biliares, de interação com a fosfatidilcolina micelar e de inibição da solubilização micelar do colesterol de forma in vitro. Foi realizado ensaio de permeação do colesterol por um período de 2 h em células Caco-2 semeadas e diferenciadas em placas transwell de 24 poços com uma solução de micelas de colesterol e extrato de guaraná em diferentes concentrações. O teor de colesterol permeado foi determinado por HPLC/UV a 206 nm. Foi realizado o ensaio de Western blotting para determinar o conteúdo proteico dos transportadores de colesterol (NPC1L1, ABCG5, ABCG8) e LXR-alfa em células intestinais Caco-2 após a incubação com diferentes concentrações do extrato em 2 e 16 horas. Resultados: O conteúdo total de fenólicos do extrato de guaraná foi 104,36 (± 3,62) mg EAG/g de guaraná em pó. Após o processo de digestão, este valor foi de 48,62 (± 3,97) mg EAG/g de guaraná em pó, demonstrando a bioacessibilidade dos polifenóis. Foram identificados os compostos procianidina B1 (1,56 ± 0,04 mg/g) e B2 (2,34 ± 0,06 mg/g), catequina (24,26 ± 0,63 mg/g), epicatequina (20,39 ± 1,20 mg/g) e cafeína (34,93 ± 0,75 mg/g) no extrato não digerido. Após digestão in vitro, somente a catequina, epicatequina e cafeína foram bioacessíveis com 181,16 %, 136,78 % e 213,51 % de bioacessibilidade, respectivamente. A atividade antioxidante do extrato aquoso não digerido foi menor que do digerido, sendo os valores finais de ORAC de 2512 (± 399) e 4321 (± 778) µmol Eq Trolox/g de guaraná em pó, respectivamente. O extrato exibiu uma atividade inibitória da enzima lipase pancreática de forma dose-dependente, com IC50 de 1033 µg/mL. O extrato também demonstrou uma capacidade de ligação ao taurocolato de sódio de 45,63 (± 8,50) % e de 44,30 (± 8,88) % com concentrações de 1,0 mg/mL e 0,5 mg/mL, respectivamente, correspondendo a 71,78 (± 13,36) % e 93,11 (± 18,65) % da capacidade de ligação da colestiramina quando utilizada nas mesmas concentrações. Observou-se uma redução de 20,47 (± 10,24) % e 17,06 (± 12,88) % na concentração da fosfatidilcolina micelar quando o guaraná foi utilizado nas concentrações de 1,0 e 0,50 mg/mL, respectivamente, em comparação com a micela padrão sem amostra e uma inibição da solubilização micelar do colesterol de 10,14 % pelo guaraná a 1,0 mg/mL. Após o experimento de permeação, as concentrações de colesterol no compartimento basolateral foram de 197,89 (± 3,95), 151,76 (± 1,39) e 94,51 (± 9,49) µg/mL, quando 0,025, 0,050 ou 0,075 mg/mL de guaraná foram utilizados, respectivamente, demonstrando redução de 23,31 % e 52,24 % à medida que a concentração de guaraná utilizada aumentou. Observou-se uma redução do conteúdo proteico do NPC1L1 após 16 horas de incubação com guaraná nas concentrações de 0,050 e 0,075 mg/mL. Em relação ao ABCG5, observou-se um aumento do conteúdo nas concentrações de 0,025 e 0,075 mg/mL de guaraná após 02 horas. Não foram observadas diferenças importantes nos conteúdos de ABCG8 e do LXR-alfa. Conclusões: O extrato aquoso de guaraná em pó demonstrou uma redução da absorção de colesterol em células Caco-2. O extrato também demonstrou uma atividade inibitória da lipase pancreática dose-dependente, uma boa capacidade de ligação aos ácidos biliares e de interação com a fosfatidilcolina micelar, além de uma capacidade de inibição da solubilização micelar do colesterol de forma in vitro. O extrato também foi capaz de modular negativamente o conteúdo proteico do NPC1L1 e positivamente o conteúdo do ABCG5. A sua ingestão pode ser considerada uma importante fonte alimentar com potencial efeito hipocolesterolêmico, capaz de reduzir a absorção de lipídios no intestino, podendo ser utilizado como adjuvante na regulação da hipercolesterolemia, provavelmente devido à presença de polifenóis.

Introduction - The risk of cardiovascular diseases, such as atherosclerosis, is associated with hypercholesterolemia, a result of behavioral and metabolic risk factors. Dietary interventions with the use of guarana (Paullinia cupana) may be useful, since it is a food rich in bioactive compounds such as catechins, which have high antioxidant activity, inhibit oxidation of LDLc and lipid peroxidation, being able to lower blood cholesterol levels, by mechanisms not yet fully elucidated. Objective - To investigate the effects of guarana (Paullinia cupana) powder aqueous extract on mechanisms involved with the absorption of cholesterol in vitro and in Caco-2 cells. Methods - The guarana powder aqueous extract was subjected to in vitro digestion, the total phenolics content were quantified by the Folin-Ciocalteu method and the determination of the phenolic profile was performed by high performance liquid chromatography (HPLC) UV/VIS at 210 nm. The antioxidant activity was determined by Oxygen Radical Absorbance Capacity (ORAC) and the ability to inhibit pancreatic lipase, to binding with bile acids, the interaction with micellar phosphatidylcholine and the inhibition of micellar solubilization of cholesterol in vitro were evaluated. Cholesterol permeation assay was performed for a period of 2 hours in Caco-2 cells seeded and differentiated in 24- transwell plates with a solution of cholesterol micelles and guarana extract at different concentrations. The permeated cholesterol content was determined by HPLC/UV at 206 nm. The Western blotting assay was performed to determine the protein content of cholesterol transporters (NPC1L1, ABCG5, ABCG8) and LXR-alpha in Caco-2 cells after incubation with different concentrations of the extract in 2 and 16 hours. Results: The total phenolic content of the guarana extract was 104.36 (± 3.62) mg EAG/g of guarana powder. After the digestion process, this value was 48.62 (± 3.97) mg EAG/g of guarana powder, demonstrating the bioaccessibility of the polyphenols. Procyanidin B1 (1.56 ± 0.04 mg/g) and B2 (2.34 ± 0.06 mg/g), catechin (24.26 ± 0.63 mg/g), epicatechin (20.39 ± 1.20 mg/g) and caffeine (34.93 ± 0.75 mg/g) were identified in the undigested extract. After in vitro digestion, only catechin, epicatechin and caffeine were bioaccessible with 181.16 %, 136.78 % and 213.51 % of bioaccessibility, respectively. The antioxidant activity of the undigested aqueous extract was lower than that digested, with the final ORAC values of 2512 (± 399) and 4321 (± 778) µmol Eq Trolox/g of guarana powder. The extract exhibited an inhibitory activity of the pancreatic lipase enzyme in a dose-dependent manner, with an IC50 of 1033 µg/mL. The extract also demonstrated a sodium taurocholate binding capacity of 45.63 (± 8.50) % and 44.30 (± 8.88) % with concentrations of 1.0 mg/mL and 0.5 mg/mL, respectively, corresponding to 71.78 (± 13.36)% and 93.11 (± 18.65)% of the cholestyramine binding capacity when used at the same concentrations. A reduction of 20.47 (± 10.24) % and 17.06 (± 12.88) % was observed in the concentration of micellar phosphatidylcholine when guarana was used at concentrations of 1.0 and 0.50 mg/mL, respectively, compared to the standard micelle without sample and an inhibition of 10.14 % micellar cholesterol solubilization by guarana at 1.0 mg/mL. After the permeation experiment, the cholesterol concentrations in the basolateral compartment were 197.89 (± 3.95), 151.76 (± 1.39) and 94.51 (± 9.49) µg/mL, when 0.025, 0.050 or 0.075 mg/mL of guarana were used, respectively, showing a reduction of 23.31 % and 52.24 % as the concentration of guarana used increased. A reduction in the protein content of NPC1L1 was observed after 16 hours of incubation with guarana at concentrations of 0.050 and 0.075 mg/mL. After 02 hours, an increase in the content of ABCG5 at concentrations of 0.025 and 0.075 mg/mL of guarana was observed. No significant differences were observed in the contents of ABCG8 and LXR-α. Conclusions: The aqueous extract of guarana powder showed a reduction in the absorption of cholesterol in Caco-2 cells. The extract also demonstrated a dose-dependent pancreatic lipase inhibitory activity, a good ability to bind bile acids and to interact with micellar phosphatidylcholine, in addition to an ability to inhibit micellar solubilization of cholesterol in vitro. The extract was also able to negatively modulate the protein content of NPC1L1 and positively the content of ABCG5. Its intake can be considered an important dietary source with a potential hypocholesterolemic effect, capable of reducing the absorption of lipids in the intestine, and can be used as an adjunct in the control of hypercholesterolemia, probably due to the presence of polyphenols.

Mast cells-derived MiR-223 destroys intestinal barrier function by inhibition of CLDN8 expression in intestinal epithelial cells

BACKGROUND: Mast cells (MCs) have been found to play a critical role during development of inflammatory bowel disease (IBD) that characterized by dysregulation of inflammation and impaired intestinal barrier function. However, the function of MCs in IBD remains to be fully elucidated. RESULTS: In our study, we used exosomes isolated from human mast cells-1 (HMCs-1) to culture with NCM460, HT-29 or CaCO2 of intestinal epithelial cells (lECs) to investigate the communication between MCs and lECs. We found that MCs-derived exosomes significantly increased intestinal epithelial permeability and destroyed intestinal barrier function, which is attributed to exosome-mediated functional miRNAs were transferred from HMCs-1 into lECs, leading to inhibit tight junction-related proteins expression, including tight junction proteins 1 (TJP1, ZO-1), Occludin (OCLN), Claudin 8 (CLDN8). Microarray and bioinformatic analysis have further revealed that a panel of miRNAs target different tight junction-related proteins. Interestingly, miR-223 is enriched in mast cell-derived exosome, which inhibit CLDN8 expression in IECs, while treatment with miR-223 inhibitor in HT-29 cells significantly reversed the inhibitory effect of HMCs-1-derived exosomes on CLDN 8 expression. Most importantly, enrichment of MCs accumulation in intestinal mucosa of patients with IBD compared with those healthy control. CONCLUSIONS: These results indicated that enrichment of exosomal miR-223 from HMCs-1 inhibited CLDN8 expression, leading to destroy intestinal barrier function. These finding provided a novel insight of MCs as a new target for therapeutic treatment of IBD.

Study on difference of liver toxicity and its molecular mechanisms caused by Tripterygium wilfordii multiglycoside and equivalent amount of triptolid in rats / 中国中药杂志

Tripterygium wilfordii multiglycoside( GTW),an extract derived from T. wilfordii,has been used for rheumatoid arthritis and other immune diseases in China. However its potential hepatotoxicity has not been investigated completely. Firstly,the content of triptolid( TP) in GTW was 0. 008% confirmed by a LC method. Then after oral administration of GTW( 100,150 mg·kg-1) and TP( 12 μg·kg-1) in female Wistar rats for 24 h,it was found that 150 mg·kg-1 GTW showed more serious acute liver injury than 12 μg·kg-1 TP,with the significantly increased lever of serum ALT,AST,TBA,TBi L,TG and bile duct hyperplasia even hepatocyte apoptosis. The expression of mRNA and proteins of liver bile acid transporters such as BSEP,MRP2,NTCP and OATP were down-regulated significantly by GTW to inhibit bile acid excretion and absorption,resulting in cholestatic liver injury. Moreover,GTW was considered to be involved in hepatic oxidative stress injury,although it down-regulated SOD1 and GPX-1 mRNA expression without significant difference in MDA and GSH levels. In vitro,we found that TP was the main toxic component in GTW,which could inhibit cell viability up to 80% in Hep G2 and LO2 cells at the dose of 0. 1 μmol·L-1. Next a LC-MS/MS method was used to detect the concentration of triptolid in plasma from rats,interestingly,we found that the content of TP in GTW was always higher than in the same amount of TP,suggesting the other components in GTW may affect the TP metabolism. Finally,we screened the substrate of p-glycoprotein( p-gp) in Caco-2 cells treated with components except TP extrated from GTW,finding that wilforgine,wilforine and wilfordine was the substrate of p-gp. Thus,we speculated that wilforgine,wilforine and wilfordine may competitively inhibit the excretion of TP to bile through p-gp,leading to the enhanced hepatotoxity caused by GTW than the same amount of TP.

Inhibitory effects of petasin on human colon carcinoma cells mediated by inactivation of Akt/mTOR pathway / 中华医学杂志(英文版)

BACKGROUND@#Colorectal cancer is the third most common cancer worldwide and still lack of effective therapy so far. Petasin, a natural product found in plants of the genus Petasites, has been reported to possess anticancer activity. The present study aimed to investigate the anticolon cancer activity of petasin both in vitro and in vivo. The molecular mechanism of petasin was also further explored.@*METHODS@#Caco-2, LoVo, SW-620, and HT-29 cell lines were used to detect the inhibitory effect of petasin on colon cancer proliferation. Cell viability was determined using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cell apoptosis was analyzed by flow cytometry. Hoechst 33258 staining was used to visualize morphological changes. Cell migration was assessed using a wound-healing migration assay, and cell invasion was investigated using Transwell chambers. Western blotting assays were employed to evaluate the expression levels of proteins in the protein kinase B/mammalian target of rapamycin (Akt/mTOR) signaling pathway. Finally, in vivo activity of petasin was evaluated using the SW-620 subcutaneous tumor model established in Balb/c nude mice. Twelve rats were randomly divided into control group and 10 mg/kg petasin group. The tumor volume was calculated every 7 days for 28 days. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed to assess the apoptotic effect of petasin. Differences between two groups were assessed by analysis of independent-sample t tests.@*RESULTS@#Petasin significantly inhibited the proliferation of human colon carcinoma cell lines, induced apoptosis, and suppressed migration and invasion in SW-620 cells. Western blotting results showed that petasin decreased the phosphorylation of Akt (1.01 ± 0.16 vs. 0.74 ± 0.06, P = 0.042), mTOR (0.71 ± 0.12 vs. 0.32 ± 0.11, P = 0.013), and P70S6K (1.23 ± 0.21 vs. 0.85 ± 0.14, P = 0.008), elevated the expression of caspase-3 (0.41 ± 0.09 vs. 0.74 ± 0.12, P = 0.018) and caspase-9 (1.10 ± 0.27 vs. 1.98 ± 0.22, P = 0.009), decreased the Bcl-2 protein (2.75 ± 0.47 vs. 1.51 ± 0.36, P = 0.008), downregulated the expression of matrix metalloproteinase (MMP)-3 (1.51 ± 0.31 vs. 0.82 ± 0.11, P = 0.021) and MMP-9 (1.56 ± 0.32 vs. 0.94 ± 0.15, P = 0.039) in SW-620 cell. In vivo, 10 mg/kg petasin inhibited tumor growth in Balb/c nude mice (924.18 ± 101.23 vs. 577.67 ± 75.12 mm at day 28, P = 0.001) and induced apoptosis (3.6 ± 0.7% vs. 36.0 ± 4.9%, P = 0.001) in tumor tissues.@*CONCLUSIONS@#Petasin inhibits the proliferation of colon cancer SW-620 cells via inactivating the Akt/mTOR pathway. Our findings suggest petasin as a potential candidate for colon cancer therapy.

Absorptive characteristic of Bletilla striata extract in Caco-2 cell monolayer / 中国中药杂志

To investigate the active fraction from Bletilla striata in Caco-2 cell monolayer,so as to explore its absorption mechanism of oral administration preliminarily.Active fraction from B.striata in Caco-2 cell monolayer was analyzed by UPLC-Q-TOF and detected by UPLC-MS/MS,and the effects of different concentrations,pH and P-glycoprotein inhibitors on Caco-2 cells Monolayer were investigated.Six compounds were isolated from the active fraction of B.striata in Caco-2 cell monolayer by UPLC-Q-TOF,and identified as B6,B12,B14,B17,B19 and B23,with concentration dependence.Within the 0-180 min,the uptake of B12 and B14 had a time dependence,while B6,B17,B19 and B23 tended to saturate after 60 min.All of the components had a good absorption in an acidic environment.B6 had a good absorption at pH 6.0,while the other components B12,B14,B17,B19 and B23 had a good absorption at pH4.0.The absorption of the 6 main components of B.striata were not be affected by P-glycoprotein inhibitors(verapamil/cyclosporin A).Compared with the control group,there was no difference in the absorption of B6 and B12,and the absorption of B14,B17,B19 and B23 increased,but with no significant difference.The absorption characteristic of B.striata extract across the Caco-2 cell monolayer is probably passive diffusion,and the absorption process was not affected by P-glycoprotein.

Growth suppression of colorectal cancer expressing S492R EGFR by monoclonal antibody CH12 / 医学前沿

Colorectal cancer (CRC) is a common malignant tumor in the digestive tract, and 30%-85% of CRCs express epidermal growth factor receptors (EGFRs). Recently, treatments using cetuximab, also named C225, an anti-EGFR monoclonal antibody, for CRC have been demonstrated to cause an S492R mutation in EGFR. However, little is known about the biological function of S492R EGFR. Therefore, we attempted to elucidate its biological function in CRC cells and explore new treatment strategies for this mutant form. Our study indicated that EGFR and S492R EGFR accelerate the growth of CRC cells in vitro and in vivo and monoclonal antibody CH12, which specifically recognizes an EGFR tumor-specific epitope, can bind efficiently to S492R EGFR. Furthermore, mAb CH12 showed significantly stronger growth suppression activities and induced a more potent antibody-dependent cellular cytotoxicity effect on CRC cells bearing S492R EGFR than mAb C225. mAb CH12 obviously suppressed the growth of CRC xenografts with S492R EGFR mutations in vivo. Thus, mAb CH12 may be a promising therapeutic agent in treating patients with CRC bearing an S492R EGFR mutation.

The Krüppel-like factor (KLF5) as a predictive biomarker in preoperative chemoradiation therapy for rectal cancer

PURPOSE: Preoperative chemoradiation therapy (CRT) has become the standard treatment for patients with locally advanced rectal cancer, 15%–30% of patients still progress while being treated with CRT. The aim of this study was to identify as important biomarker of poor response and evaluate the mechanism associated with CRT resistance. METHODS: This study included 60 human colon tumour pre-irradiation specimens. Expressions of epidermal growth factor receptor (EGFR), p53, Krüppel-like factor 5 (KLF5), C-ern, Ki67 were assessed and correlated with tumor regression grades and complete remission. We added in vitro study with biomarker which has been identified as important biomarker of poor response to evaluate the mechanism associated with CRT resistance. RESULTS: Pathologic complete remission (pCR) was achieved by 9 patients (18%). EGFR and KLF5 were significantly associated with pCR (P = 0.048, P = 0.023, respectfully). And multivariate analysis showed high KLF5 intensity was worse factor for pCR (P = 0.012). In vitro study, radiation or chemotherapy therapy stabilized KLF5 protein levels in a time- and dose-depended manner in HCT116 and Caco-2 cells. KLF5 overexpression in HCT116 stable cell line showed significantly better cell viability by increasing cyclinD1 and b-catenin compared to control cells in MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, suggesting that KLF5 mediates cell survival. CONCLUSION: KLF5 was significantly associated with the presence of KRAS mutations, and KLF5 was an independent poor response predictor of CRT in rectal cancer. Our study is pilot study and more research will be needed in the future.

Establishment of a congenital chloride diarrhea-associated SLC26A3 c.392C>G (p.P131R) polymorphism-expressing cell model and a preliminary analysis of its mechanism of action / 中国当代儿科杂志

OBJECTIVE@#To establish a congenital chloride diarrhea (CCD)-associated SLC26A3 c.392C>G (p.P131R) polymorphism-expressing cell model, and to investigate its biological function.@*METHODS@#The sequence of the SLC26A3 gene in GenBank was used to design the upstream and downstream single-guide RNA (sgRNA) that could specifically recognize the 392 locus of the SLC26A3 gene, and the sgRNA was mixed with the pSpCas9-puro vector after enzyme digestion to construct an eukaryotic recombinant expression plasmid (pSpCas9-SLC26A3). Caco-2 cells were transfected with the recombinant plasmid and synthesized single-stranded DNA oligonucleotides (ssODNs), and Taqman genotyping assay and Sanger sequencing were used to identify the expression of SLC26A3 c.392C>G (p.P131R) in Caco-2 cells. Wild-type Caco-2 cells were selected as normal control group and the Caco-2 cells with successful expression of SLC26A3 c.392C>G (p.P131R) was selected as P131R group. Both groups were treated with 100 ng/mL tumor necrosis factor-α (TNF-α), and then the normal control group was named as TNF-α group, and the P131R group was named as TNF-α+P131R group. Electric cell-substrate impedance sensing (ECIS) assay was used to evaluate the change in the monolayer barrier function of intestinal epithelial cells in the above four groups, and Western blot was used to measure the change in the expression of SLC26A3 protein in the normal control group and the P131R group.@*RESULTS@#The eukaryotic recombinant expression plasmid (pSpCas9-SLC26A3) was successfully constructed. Both Taqman genotyping assay and Sanger sequencing confirmed the successful establishment of the Caco-2 cell model of SLC26A3 c.392C>G (p.P131R) expression. ECIS assay showed that compared with the normal control group, the P131R group had a significant increase in the monolayer permeability of intestinal epithelial cells (PG (p.P131R) can reduce the expression of SLC26A3 protein, increase the monolayer permeability of intestinal epithelial cells, and thus lead to diarrhea.

Effects of circular RNA circ-SOD2 on intestinal epithelial barrier and ulcerative colitis / 北京大学学报(医学版)

OBJECTIVE@#To explore the expression profiling of circRNAs in ulcerative colitis(UC) and then determine the significantly changed circRNA and its influences on intestinal epithelial barrier.@*METHODS@#In this study, we selected 5 pairs of inflamed and normal colorectal mucosa tissues from UC patients to perform circRNAs microarray and identified the differentially expressed circRNAs in the UC inflamed colorectal mucosa tissues, and quantitative real-time PCR was used to identify the expression change of circ-SOD2 in 30 UC patients' inflamed and normal colorectal mucosa tissues. We detected the expression of circ-SOD2 in Caco2 and NCM460 cells after being treated with inflammatory factors (LPS, TNF-α, IL1-β). Fluorescence in situ hybridization (FISH) was used to determine the cellular location of circ-SOD2 in the UC colorectal mucosal tissues. The circ-SOD2 overexpression vector was constructed and produced and then transfected into Caco2 cells to examine the cells' trans-epithelial electrical resistance (TEER), permeability of FITC-dextran and the alterations of epithelial barrier related molecules.@*RESULTS@#We found 264 circRNAs (111 increased and 153 decreased) differentially expressed in the inflamed colon mucosa compared with normal colon mucosa using a P-value <0.05 and a >1.5-fold change cutoff. To validate the circRNA microarray results, we selected some circRNAs to perform qRT-PCR based on the following criteria: (1) circRNAs raw data >100 in each sample, (2) fold-change >2, (3) P<0.05. We identified 10 dysregulated circRNA, among them, circ-SOD2 was upregulated with maximum fold-change in the UC inflamed colorectal mucosa tissues. Then we identified circ-SOD2 was upregulated significantly through quantitative real-time PCR (qRT-PCR) in expanded 30 paired colorectal mucosa tissues(P<0.001). After treatments with LPS, TNF-α and IL1-β, circ-SOD2 was upregulated in Caco2 and NCM460 cells at different points from 1 to 7 h. Fluorescence in situ hybridization (FISH) indicated that circ-SOD2 located in intestinal epithelium mostly and few in mesenchyme and inflammatory cells. The overexpression of circ-SOD2 in Caco2 cells resulted in a decrease of transepithelial electrical resistance (TEER), an increase of the FITC-dextran permeability and the downregulation of epithelial barrier related molecule CLDN-8 (P<0.05).@*CONCLUSION@#The dysregulation of circRNAs existed in UC inflamed colorectal mucosa, among which, the upregulated circ-SOD2 weakened the intestinal epithelial barrier and thus might promote the occurrence of ulcerative colitis.