Caco-2 Cell Viability Research Articles

Effects of Peroxisome Proliferator-Activated Receptor γ on Modulating Angiopoietin-Like Protein 4 Synthesis in Caco-2 Cells Exposed to <i>Clostridium butyricum</i>

Angiopoietin-like protein 4 (ANGPTL4) is considered to be one of the important circulating mediators linking intestinal microorganisms and host lipid metabolism. The objective of this study was to assess the effects of peroxisome proliferator-activated receptor γ (PPARγ) on modulating ANGPTL4 synthesis in Caco-2 cells exposed to Clostridium butyricum. The viability of Caco-2 cells and the expression of PPARγ and ANGPTL4 in Caco-2 cells were detected after the Caco-2 cells were co-cultured with C. butyricum at the concentration of 1 × 106, 1 × 107 and 1 × 108 CFU/mL. The results showed that cell viability was enhanced by C. butyricum. Besides, PPARγ and ANGPTL4 expression and secretion in Caco-2 cells was significantly increased by 1 × 107 and 1 × 108 CFU/mL of C. butyricum. Furthermore, the effects of PPARγ on modulating ANGPTL4 synthesis in Caco-2 cells regulated by 1 × 108 CFU/mL of C. butyricum was also be expounded in PPARγ activation/inhibition model based on Caco-2 cells and via ChIP technique. It was found that C. butyricum promoted the binding of PPARγ to the PPAR binding site (chr19: 8362157-8362357, located upstream of the transcriptional start site of angptl4) of the angptl4 gene in Caco-2 cells. However, the PPARγ was not the only way for C. butyricum to stimulate ANGPTL4 production. Taken together, PPARγ played a role in the regulation of ANGPTL4 synthesis by C. butyricum in Caco-2 cells.

Encapsulation of St. John's Wort extract in β-Cyclodextrin carrier: In-vitro antioxidant, antimicrobial and Caco-2 cell viability studies

St. John's wort (SJW) is a plant flowering species, and its extract shows antidepressant, anticancer, wound-healing, antibiotic, antiviral, antioxidant, anxiolytic and anti-inflammatory activities. However, SJW extract has low aqueous solubility and biological efficacy; it might be due to the presence of lipophilic components such as hyperforin and hypericin. To increase the aqueous solubility of SJW extract, in the present report an attempt has been made to prepare SJW extract/β-cyclodextrin inclusion complex (IC). The LCMS analysis of SJW extract showed the presence of hyperforin and hypericin components. Morphological investigation depicted the spherical shaped mono-disperse particles of 30 nm size and DLS confirmed hydrodynamic diameter around 100 nm. The encapsulation efficiency of IC was 76%, and 92% SJW components were released from IC in 13.3 h. Caco-2 cell viability study pointed towards improved safety profile of SJW extract after β-CD complex formation. Furthermore, synthesized IC showed higher antioxidant activity than native SJW extract. The prepared IC showed moderate antimicrobial activity for Escherichia coli (E. coli), Enterococcus faecalis (E. faecalis) and Bacillus cereus (B. cereus. The work would help to enhance the biological efficacy of SJW extract.

Modified Gegen Qinlian decoction ameliorated ulcerative colitis by attenuating inflammation and oxidative stress and enhancing intestinal barrier function in vivo and in vitro

Ethnopharmacological relevanceModified Gegen Qinlian decoction (MGQD), which was first documented in Treatise on Febrile Disease, is recognized as a classic prescription to treat ulcerative colitis (UC). However, its protective mechanism against UC remains to be fully elucidated. Aim of the studyTo explore the impact and the potential molecular mechanism of MGQD on dextran sodium sulfate (DSS)-induced UC mice and tumor necrosis factor alpha (TNF-α)-induced Caco-2 cell monolayer model of intestinal barrier. Materials and methodsThe chemical components of MGQD and MGQD drug containing serum (MGQD-DS) were characterized by LC-MS/MS. The therapeutic effect of MGQD on DSS-induced UC was evaluated based on body weight, disease activity index (DAI), colon length, colonic histopathological injury, inflammatory cytokines, oxidative stress response and intestinal barrier function. Cell Counting Kit (CCK)-8 assay was applied to detect the effect of MGQD-DS on the viability of Caco-2 cells. Additionally, TNF-α-induced Caco-2 cell monolayer model of intestinal barrier was established in vitro. The Caco-2 cell monolayers were administered blank serum or MGQD-DS to observe the effects of MGQD-DS on transepithelial electrical resistance (TEER), permeability of fluorescein isothiocyanate (FITC)-dextran, inflammatory cytokines, oxidative stress indicators and intestinal epithelial barrier (IEB). ResultsMGQD significantly improved symptoms and pathological damage in UC mice by downregulating the expression of interleukin (IL)-1β and malondialdehyde (MDA), attenuating the loss of goblet cells and the destruction of intestinal epithelial ultrastructure, and upregulating the expression of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), zonula occludens-1 (ZO-1), Occludin, Claudin-1 and E-cadherin. In vitro, MGQD-DS significantly reduced the flux of FITC-dextran, increased the TEER, inhibited the expression of IL-21, IL-17A and MDA, and promoted the expression of IL-4, IL-10, transforming growth factor-β (TGF-β), SOD, CAT, GSH, Occludin and E-cadherin in TNF-α-induced Caco-2 cell monolayer model of intestinal barrier. ConclusionMGQD can ameliorate DSS-induced UC mice and TNF-α-induced Caco-2 cell monolayer model of intestinal barrier, and the protective effect is related to its inhibition of inflammation, alleviation of oxidative stress, and repair of intestinal barrier damage.

2'-Fucosyllactose and 3-Fucosyllactose Alleviates Interleukin-6-Induced Barrier Dysfunction and Dextran Sodium Sulfate-Induced Colitis by Improving Intestinal Barrier Function and Modulating the Intestinal Microbiome.

Ulcerative colitis is an inflammatory bowel disease (IBD) with relapsing and remitting patterns, and it is caused by varied factors, such as the intestinal inflammation extent and duration. We examined the preventative effects of human milk oligosaccharides (HMOs) on epithelial barrier integrity and intestinal inflammation in an interleukin (IL)-6-induced cell model and dextran sodium sulfate (DSS)-induced acute mouse colitis model. HMOs including 2'-fucosyllactose (FL) and 3-FL and positive controls including fructooligosaccharide (FOS) and 5-acetylsalicylic acid (5-ASA) were orally administrated once per day to C57BL/6J mice with colitis induced by 5% DSS in the administered drinking water. 2'-FL and 3-FL did not affect the cell viability in Caco-2 cells. Meanwhile, these agents reversed IL-6-reduced intestinal barrier function in Caco-2 cells. Furthermore, 2'-FL and 3-FL reversed the body weight loss and the remarkably short colon lengths in DSS-induced acute colitis mice. Moreover, 2'-FL and 3-FL obviously protected the decreasing expression of zonula occluden-1 and occludin in colon tissue relative to the findings in the DSS-treated control group. 2'-FL and 3-FL significantly reduced IL-6 and tumor necrosis factor-α levels in serum relative to the control findings. The summary of these results shows that HMOs prevent colitis mainly by enhancing intestinal barrier function and advancing anti-inflammatory responses. Therefore, HMOs might suppress inflammatory responses and represent candidate treatments for IBD that protect intestinal integrity.

Delayed Release HPMC Capsules for Efficient Delivery of Cholecalciferol Solid Dispersion

Abstract: Introduction: The deficiency of Vitamin D is associated with an increased risk of various diseases and deficiency of this Vitamin is recognized in individuals all over the world. Therefore, the intake of Vitamin D has become essential. Objectives: Cholecalciferol (Vitamin D3) is a poorly soluble molecule and it is very sensitive to degradation under environmental factors such as light, temperature, and oxygen. Its stability is also affected adversely under acidic conditions. Therefore, solid dispersion-based formulation for cholecalciferol was developed and encapsulated in delayed release hydroxypropylmethyl cellulose (HPMC) capsules. Materials and Methods: Cholecalciferol solid dispersion was developed and characterized by Fourier transform–infra red spectroscopy (FTIR), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and X-ray diffraction analysis. The effect of various concentrations of cholecalciferol formulations on the viability of Caco-2 cells was determined by using MTT assay. Dissolution profile and stability study of the developed product was also evaluated. Results: The results demonstrated improved solubility of cholecalciferol in solid dispersion-based formulation. The drug content of solid dispersions was in the order of 91±2.3% and various studies showed the amorphous form of cholecalciferol in the solid dispersion. The cell viability assay in Caco-2 cells demonstrated that the surfactant used in the solid dispersion formulation of cholecalciferol had no adverse effect on intestinal cells. Further, dissolution profile of HPMC capsule encapsulated solid dispersion showed improved dissolution of cholecalciferol. Moreover, the stability study indicated no significant changes in the cholecalciferol content in the developed formulation under storage at experimental conditions. Conclusion: The solid dispersion-based formulation of cholecalciferol exhibited improved solubility and found to be compatible with Caco-2 cells. The delayed release HPMC capsule encapsulated solid dispersion of cholecalciferol (DRHCap-SD) showed improved dissolution and acceptable stability profile and this represent a potential delivery system for oral administration of cholecalciferol. Keywords: Solid dispersion, Cholecalciferol, HPMC capsule, Caco-2 cells, Oral delivery, Dissolution.

Combined Effects of Acrylamide and Ochratoxin A on the Intestinal Barrier in Caco-2 Cells.

Acrylamide (AA) and ochratoxin A (OTA) are contaminants that co-exist in the same foods, and may create a serious threat to human health. However, the combined effects of AA and OTA on intestinal epithelial cells remain unclear. The purpose of this research was to investigate the effects of AA and OTA individually and collectively on Caco-2 cells. The results showed that AA and OTA significantly inhibited Caco-2 cell viability in a concentration- and time-dependent manner, decreased transepithelial electrical resistance (TEER) values, and increased the lucifer yellow (LY) permeabilization, lactate dehydrogenase (LDH) release and reactive oxygen species (ROS) levels. In addition, the levels of IL-1β, IL-6, and TNF-α increased, while the levels of IL-10 decreased after AA and OTA treatment. Western blot analysis revealed that AA and OTA damaged the intestinal barrier by reducing the expression of the tight junction (TJ) protein. The collective effects of AA and OTA exhibited enhanced toxicity compared to either single compound and, for most of the intestinal barrier function indicators, AA and OTA combined exposure tended to produce synergistic toxicity to Caco-2 cells. Overall, this research suggests the possibility of toxic reactions arising from the interaction of toxic substances present in foodstuffs with those produced during processing.

New sulfa drug derivatives and their zinc(II) complexes: synthesis, spectroscopic properties and in vitro cytotoxic activities

Synthesis of four sulfa drug derivatives (L1–L4 ) and Zn(II) complexes derived from sulfonamide group antibiotic substances was carried out using the hydrothermal technique (HT) and their structures of the obtained compounds were explained using elemental analysis (EA), FT-IR and NMR (1H- and 13C-). Cytotoxic activities of four novel sulfa drug based-Schiff base compounds and their Zn(II) complexes were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay using MCF-7 (human breast cancer), Caco-2 (human colorectal adenocarcinoma), A2780 (human ovarian cancer) and LNCaP (human prostate adenocarcinoma) cell lines. LogIC50 values of all obtained compounds were computed with the Graphpad Prism 6 program after 24 h of treatment for MCF-7, Caco-2, A2780 and LNCaP cells. Comet assay experiments were performed using LogIC50 concentrations of all compounds to determine DNA damage. Based on the data obtained, all compounds significantly decreased MCF-7, Caco-2, A2780 and LNCaP cell viability compared to the control groups (p < 0.05).

Characterization and Protective Properties of Lactic Acid Bacteria Intended to Be Used in Probiotic Preparation for Honeybees (Apis mellifera L.)-An In Vitro Study.

Lactic acid bacteria (LAB) are widely used probiotics and offer promising prospects for increasing the viability of honeybees. Thus, the probiotic potential of 10 LAB strains was determined, which in our previous studies showed the most potent protective abilities. In the current study, we investigated various properties of probiotic candidates. The tested LAB strains varied in susceptibility to tested antibiotics. Isolates showed high viability in sugar syrups and gastrointestinal conditions. None of the LAB strains exhibited β-hemolytic activity, mutual antagonism, mucin degradation, hydrogen peroxide production capacity, or bile salt hydrolase (BSH) activity. Additionally, the cytotoxicity of LAB cell-free supernatants (CFS) was assessed, as well as the effect of CFS from P. pentosaceus 14/1 on the cytotoxicity of coumaphos and chlorpyrifos in the Caco-2 cell line. The viability of Caco-2 cells reached up to 89.81% in the presence of the highest concentration of CFS. Furthermore, LAB metabolites decreased the cytotoxicity of insecticides (up to 19.32%) thus demonstrating cytoprotective activity. All tested LAB strains produced lactic, acetic, and malonic acids. This research allowed the selection of the most effective LAB strains, in terms of probiosis, for future in vivo studies aimed at developing an ecologically protective biopreparation for honeybees.

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 (1.25-40 μM) and gossypol (2.5-80 μM) monotherapy inhibited HCT-116 and Caco-2 cell viability in a dose- and time-dependent manner. The combination of ponatinib and gossypol at a ratio of 1 to 2 significantly decreased cell viability (P&lt;0.05), with a &gt; 2- and &gt; 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&lt;1) with a higher ponatinib dose reduction index. Moreover, ponatinib plus gossypol induced morphological changes in HCT-116 and 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.

Growth Inhibitory Effects of Lactobacillus acidophilus, Lactobacillus reuteri, and Bifidobacterium bifidum Supernatant on Blastocystis Subtypes 1 and 3 Via the TLR4 Axis

Background: Blastocystis is a common protozoan parasite found in the human intestinal tract and is associated with several gastrointestinal symptoms as well as inflammatory conditions in the bowel. Probiotics are groups of beneficial microorganisms with a substantial impact on overall human health. Objectives: This study aimed to investigate the growth inhibitory effects of Lactobacillus acidophilus, Lactobacillus reuteri, and Bifidobacterium bifidum on Blastocystis subtypes (ST) 1 and 3 through the evaluation of expression changes in toll-like receptor 4 (TLR4) in Caco2 cell culture. Materials and Methods: The parasite and Caco2 cells were cultured and maintained, and the supernatant of bacteria was prepared. The viability of parasites and cell cultures exposed to supernatants was measured separately by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, in comparison with metronidazole. In addition, TLR4 expression changes in cell cultures and co-cultures exposed to the supernatants were measured using real-time polymerase chain reaction (PCR). Results: The mean viability of Caco2 cells exposed to the highest and lowest (100 vs. 10 µg/mL) concentrations of supernatants was in a similar range. The survival of Blastocystis ST1 and ST3 exposed to the 15, 25, and 35 µg/mL of probiotics supernatants significantly decreased during 24, 48, and 72 hours. Although not statistically significant, the findings indicated a decrease in TLR4 expression in Caco2 cells and an increase in gene expression in co-cultures exposed to the probiotic supernatants (15, 25, and 35 µg/mL). Conclusion: This novel therapeutic field of study using probiotics compounds deserves further exploration to find unprecedented therapies against Blastocystis infection.

Amelioration of cadmium cytotoxicity to human cells by nutrient cation contents and the building of a biotic ligand model

A variety of important major and trace elements may competitively inhibit cadmium (Cd) absorption in human cells and reduce Cd toxicity. However, the impact of essential elements on the cytotoxicity of metals can be difficult to quantify and anticipate. Cd acute toxicity to Caco-2 cell viability was studied in culture solutions and modeled by a biotic ligand model (BLM). The individual effects of the cations potassium (K+), calcium (Ca2+), magnesium (Mg2+), ferrous ion(Fe2+), zinc (Zn2+) and manganese (Mn2+) on Cd toxicity were also investigated. The results indicated that the toxicity of Cd in culture solutions to cell viability declined with increasing concentrations of Zn2+ and Mn2+ in the solutions, while K+, Ca2 +, Mg2 + and Fe2+ had no significant effect. Using the BLM, the stability constants for the binding of Cd2 +, Zn2+, and Mn2+ to biotic ligands were determined to be logKCdBL = 5.76, logKZnBL = 4.39 and logKMnBL = 5.31, respectively. Moreover, it was calculated that 51% occupancy of the biotic ligand sites for Cd by Cd was required to cause a 50% reduction in Caco-2 cell viability. A BLM was successfully established using the estimated constants to predict the Cd cytotoxicity to Caco-2 cell viability as a function of solution characteristics, so that the effective concentrations that reduced cell viability by 50% (EC50) could be predicted by the BLM within 1.6 fold changes of the observed EC50. The application's viability and precision for foretelling Cd toxicity in Caco-2 cells are discussed.

Prevention of Cell Proliferation Signaling Via RNA Interference of c-myc Gene Expression in Colon Cancer In-vitro

Colon cancer is a cancer begins in the large intestine (colon). Its aggression is due to late diagnosis, so poor prognosis and higher mortality rates are reported. Colon cancer has become a thorny research area that requires more examination of cellular pathways involved in its emergency. Based on this, we sought to check the biological role of cMyc in colon cancer cell proliferation through targeting its expression by a respective siRNA using CaCo-2 cell lines. Notably, the cytotoxic potential of cMyc knockdown was monitored by cell imaging using an inverted microscope, the production of lactate dehydrogenase (LDH) and viability of the transfected cells using MTT assay. The relative gene expression of c-myc and other related factors such as Raf-1 was investigated using quantitative real-time PCR (qRT-PCR). ELISA assay has been used to achieve the production of pro and anti-inflammatory cytokines released from transfected cells. Interestingly, the transfection with a new designed siRNA antagonist cMyc markedly decreased CaCo-2 cell viability rate in a dose-dependent manner compared with the transfection with specific siRNA antagonist Luciferase gene, which served as control. Likewise, number of survived cells significantly reduced following transfection with siRNA against c-myc, while the relative production of LDH significantly increased. Importantly, the cytotoxic effect of the transfection protocol showed neglected influence in normal colon epithelial cells (NCM-460 cell lines) indicating the selective regulation of colon cancer cell proliferation by transfection with siRNA targeting c-myc. Noteworthy, the knockdown efficiency of c-myc showed more than 80% inhibition of its expression in CaCo-2 cells transfected with the siRNA antagonist c-myc indicated by qRT-PCR. The relative gene expression of Raf-1 significantly decreased in c-myc knockdown cells, while the relative expression of both P53 and Caspase 3, as cell death indicators, significantly upregulated compared with control-transfected cells. Furthermore, the transfection with siRNA targeting c-myc markedly increased the production of interleukin 1 alpha (IL-1α ) and IL-1β, as pro-inflammatory cytokines, while decreased the production of IL- 4 and IL-10, as anti-inflammatory cytokines. These data provide evidence for the involvement of c-myc gene expression in colon cancer cell proliferation and immune response during cancer development.

Silybin Showed Higher Cytotoxic, Antiproliferative, and Anti-Inflammatory Activities in the CaCo Cancer Cell Line while Retaining Viability and Proliferation in Normal Intestinal IPEC-1 Cells.

The anticancer potential of silymarin is well known, including its anti-inflammatory as well as antiproliferative effect mediated by influencing the cell cycle, suppression of apoptosis, and inhibition of cell-survival kinases. However, less is known about silybin, the main component of the silymarin complex, where studies indicate its dual effect on the proliferation and immune response of various cell types in a dose-dependent manner. Moreover, there is a lack of studies comparing the effect of silybin on the same type of healthy and tumor cells, especially intestinal ones. Therefore, our study aimed to investigate the concentration-dependent effect of silybin on the normal intestinal porcine epithelial cell line-1 (IPEC-1) and the human epithelial colorectal adenocarcinoma cell line (CaCo-2). The metabolic viability, cell cycle, mitochondrial membrane potential, apoptosis, and the relative gene expression for pro- and anti-inflammatory cytokines were monitored in cells treated with silybin. Silybin stimulates metabolic viability as well as proliferation in IPEC-1 cells, protects the mitochondrial membrane, and thus exerts a cytoprotective effect, and has only a minimal effect on the gene expression of pro-inflammatory cytokines but significantly increases the expression of anti-inflammatory TGF-β. In contrast, it inhibits metabolic viability in tumor intestinal CaCo-2 cells, has an antiproliferative effect accompanied by increased apoptosis, and significantly reduces the expression of genes for pro-inflammatory interleukins as well as TGF-β. The antiproliferative and anti-inflammatory effect of silybin on tumor intestinal cells without a negative effect on healthy cells is a prerequisite for its potential use in the adjuvant therapy of colon cancer; however, further studies are necessary.

Effects of Peroxisome Proliferator-Activated Receptor γ on Modulating Angiopoietin-Like Protein 4 Synthesis in Caco-2 Cells Exposed to Clostridium butyricum

Angiopoietin-like protein 4 (ANGPTL4) is considered to be one of the important circulating mediators linking intestinal microorganisms and host lipid metabolism. The objective of this study was to assess the effects of peroxisome proliferator-activated receptor у (PPARγ) on modulating ANGPTL4 synthesis in Caco-2 cells exposed to Clostridium butyricum. The viability of Caco-2 cells and the expression of PPARγ and ANGPTL4 in Caco-2 cells were detected after the Caco-2 cells were co-cultured with C. butyricum at the concentration of 1 x 10^(6), 1 x 10^(7) and 1 x 10^(8) CFU/mL. The results showed that cell viability was enhanced by C. butyricum. Besides, PPARγ and ANGPTL4 expression and secretion in Caco-2 cells was significantly increased by 1 x 10^(7) and 1 x 10^(8) CFU/mL of C. butyricum. Furthermore, the effects of PPARγ on modulating ANGPTL4 synthesis in Caco-2 cells regulated by 1 x 10^(8) CFU/mL of C. butyricum was also be expounded in PPARγ activation/inhibition model based on Caco-2 cells and via ChIP technique. It was found that C. butyricum promoted the binding of PPARγ to the PPAR binding site (chr19: 8362157-8362357, located upstream of the transcriptional start site of angptl4) of the angptl4 gene in Caco-2 cells. However, the PPARγ was not the only way for C. butyricum to stimulate ANGPTL4 production. Taken together, PPARγ played a role in the regulation of ANGPTL4 synthesis by C. butyricum in Caco-2 cells.

Ethyl Protocatechuate Encapsulation in Solid Lipid Nanoparticles: Assessment of Pharmacotechnical Parameters and Preliminary In Vitro Evaluation for Colorectal Cancer Treatment.

Colorectal cancer is one of the most diffused tumoral diseases. Since most medicaments employed for its treatment are debilitating, the use of naturally derived products, which can be effective against the mutated cells and, in addition, can reduce most inflammatory-related effects, could be extremely beneficial for the continued treatment of this disease. In this research, ethyl protocatechuate (PCAEE), a protocatechuic acid prodrug, was encapsulated in solid lipid nanoparticles (SLN) (prepared without and with Tween 80), which were characterized in terms of size, polydispersity index (PDI), zeta potential and thermotropic behavior. Encapsulation efficiency, release profile and interaction with a model of biomembrane were also assessed. The nanoparticles were tested in vitro on both healthy cells and on a model of tumoral cells. SLN prepared with Tween 80 was promising in terms of physicochemical properties (z-average of 190 nm, PDI 0.150 and zeta potential around -20 mV) and encapsulation efficiency (56%); they showed a desirable release profile, demonstrated an ability to penetrate and release the encapsulated PCAEE into a biomembrane model and were nontoxic on healthy cells. In addition, they caused a greater dose-dependent decrease in the viability of CaCo-2 cells than PCAEE alone. In conclusion, the formulation could be proposed for further studies to assess its suitability for the treatment of colorectal cancer.

Protective role of circRNA CCND1 in ulcerative colitis via miR-142-5p/NCOA3 axis

BackgroundIncreasing research indicates that circular RNAs (circRNAs) play critical roles in the development of ulcerative colitis (UC). This study aimed to determine the role of circRNA CCND1 in UC bio-progression, which has been shown to be downregulated in UC tissues.MethodsReverse transcription quantitative polymerase chain reaction was used to determine the levels of circRNA CCND1, miR-142-5p, and nuclear receptor coactivator-3 (NCOA3) in UC tissues and in lipopolysaccharide (LPS)-induced Caco-2 cells. Target sites of circRNA CCND1 and miR-142-5p were predicted using StarBase, and TargetScan to forecast potential linkage points of NCOA3 and miR-142-5p, which were confirmed by a double luciferase reporter-gene assay. Cell Counting Kit 8 and flow cytometry assays were performed to assess Caco-2 cell viability and apoptosis. TNF-α, IL-1β, IL-6, and IL-8 were detected using Enzyme-Linked Immunosorbent Assay kits.ResultsCircRNA CCND1 was downregulated in UC clinical samples and LPS-induced Caco-2 cells. In addition, circRNA CCND1 overexpression suppressed LPS-induced apoptosis and inflammatory responses in Caco-2 cells. Dual-luciferase reporter-gene assays showed that miR-142-5p could be linked to circRNA CCND1. Moreover, miR-142-5p was found to be highly expressed in UC, and its silencing inhibited LPS-stimulated Caco-2 cell apoptosis and inflammatory responses. Importantly, NCOA3 was found downstream of miR-142-5p. Overexpression of miR-142-5p reversed the inhibitory effect of circRNA CCND1-plasmid on LPS-stimulated Caco-2 cells, and the effects of miR-142-5p inhibitor were reversed by si-NCOA3.ConclusionCircRNA CCND1 is involved in UC development by dampening miR-142-5p function, and may represent a novel approach for treating UC patients.

Plasma etching effect on the molecular structure of chitosan-based hydrogels and its biological properties

To reasonably use hydrogels in healthcare field, this study four kinds of chitosan (CTS)-based hydrogels with different molecular structures. With plasma etching, the morphology, chemical groups' proportion, and hydrophilicity of the hydrogel surface were changed. At 40 min of modification, the ratios of CO and NH2 on the CTS40-based hydrogel surface increased and reached their maximum values of 40.31 % and 89.17 %, respectively. Combined with the changes in hydrophilic chemical groups and the hydrogel's network structure, the hydrogel surface's wettability changed after plasma etching. From the results, CTS40-based hydrogel showed the lowest contact angle (77.40 ± 3.89°) with 80 min modification due to its dense network structure of CTS and appropriate ratio of hydrophilic groups on the surface. With these molecular structural changes, the antibacterial properties of CTS-based hydrogels against Staphylococcus aureus were improved. Moreover, the functional components delivery system coating with these CTS-based hydrogels showed colon-site controlled-release property. The hydrogels also facilitated the growth of Caco2 and Hic cells, which had 72.74 %–453.27 % cell viability of Caco2 cells on the surface. Therefore, the antibacterial property and biocompatibility of plasma modified CTS-based hydrogels have been demonstrated. The mechanism between molecular structure changes of CTS with plasma etching and its properties was discussed, which would provide a promising carrier material for utilizing healthcare field.

Micro-sized polyethylene particles affect cell viability and oxidative stress responses in human colorectal adenocarcinoma Caco-2 and HT-29 cells

Plastic is a widely utilized material and polyethylene is one of the most used plastic types. Microplastics are plastic particles (size <5 mm) which are primarily a micro-size range or results from degeneration of larger plastic pieces in the environment. Drinking water and food are two main human exposure sources for microplastics and consequently effects of microplastics in gastrointestinal tract are considered important. Still, only little is known how microplastics and plastic associated chemicals affect the human health. The aim of our study was to evaluate the ability of micro-sized polyethylene to cause harmful effects in human intestinal cells. Raw ultra-high molecular-weight polyethylene (size 5–60 μm) was used. In addition, polyethylene particles were extracted with ethanol to determine the effect of extraction process on toxicity of the particles. In the experiments, human colorectal adenocarcinoma Caco-2 and HT-29 cells were exposed to polyethylene (0.25–1.0 mg/ml) or extracts for 48 h. After exposure, cell viability and cytotoxicity were assessed with MTT and lactate dehydrogenase assay. Reactive oxygen species (ROS) production was measured with dichlorofluorescin diacetate and cytoplasmic production of superoxide with dihydroethidium and mitochondrial superoxide production with MitoSOX. The 48-h exposure to polyethylene decreased dose-dependently cell viability and increased oxidative stress, especially mitochondrial superoxide production, in both cell lines. Effects on ROS or cytosolic superoxide production were not observed. Also, exposure to extracts decreased cell viability and increased oxidative stress in cell cultures, but there were differences between cell lines. These effects were most probably caused by the remaining particles rather than the compounds released from the plastic during the extraction. In conclusion, our study shows that micro-sized polyethylene and ethanol-extracted polyethylene in high concentrations decreased cell viability and increased oxidative stress responses in intestinal cells. These results contribute to the existing evidence on potential adverse human health effects of microplastics.

Plantaricin BM-1 decreases viability of SW480 human colorectal cancer cells by inducing caspase-dependent apoptosis.

Plantaricin BM-1 is a class IIa bacteriocin produced by Lactobacillus plantarum BM-1 that has significant antimicrobial activity against food-borne bacteria. In this study, a cell proliferation assay and scanning electron microscopy were used to detect changes in the viability of SW480, Caco-2, and HCT-116 colorectal cancer cells treated with plantaricin BM-1. We found that plantaricin BM-1 significantly reduced the viability of all colorectal cancer cell lines tested, especially that of the SW480 cells. Scanning electron microscopy showed that plantaricin BM-1 treatment reduced the number of microvilli and slightly collapsed the morphology of SW480 cells. Fluorescence microscopy and flow cytometry demonstrated that plantaricin BM-1 induced apoptosis of SW480 cells in a concentration-dependent manner. Western blotting further showed that plantaricin BM-1-induced apoptosis of SW480 cells was mediated by the caspase pathway. Finally, transcriptomic analysis showed that 69 genes were differentially expressed after plantaricin BM-1 treatment (p < 0.05), of which 65 were downregulated and four were upregulated. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that expression levels of genes involved in the TNF, NF-κB, and MAPK signaling pathways, as well as functional categories such as microRNAs in cancer and transcriptional misregulation in cancer, were affected in SW480 cells following the treatment with plantaricin BM-1. In conclusion, plantaricin BM-1 induced death in SW480 cells via the caspase-dependent apoptosis pathway. Our study provides important information for further development of plantaricin BM-1 for potential applications in anti-colorectal cancer.

In vitro safety assessment of electrohydrodynamically encapsulated Lactiplantibacillus plantarum CRD7 and Lacticaseibacillus rhamnosus CRD11 for probiotics use

The current study aimed to validate the safety of electrohydrodynamically encapsulated Lactiplantibacillus plantarum CRD7 and Lacticaseibacillus rhamnosus CRD11 in accordance with guidelines of FAO/WHO and ICMR/DBT. In vitro assays such as mucin degradation, hemolysis of blood cells, antimicrobial susceptibility pattern, possession of virulence factors, biogenic amine, and ammonia production were assessed. In results, the cross-streak and co-culture techniques revealed that CRD7 and CRD11 were compatible in vitro. Upon visual inspection through scanning electron and fluorescence microscopy, the integrity of bacterial cell membrane was confirmed even after the encapsulation process. CRD7 and CRD11 were non-hemolytic and showed negative responses to gelatinase, urease, and DNase activities. Non-mucinolytic activity of CRD7 and CRD11 was verified by measuring cell growth rate (p < 0.05) in different modified media followed by SDS-PAGE. High-performance liquid chromatography analysis revealed that both the strains did not produce biogenic amines (putrescine, cadaverine, histamine, and tyramine). Neither of the Lactobacillus strains produced ammonia after growing in BHI broth for 5 days at 37 °C. L-lactate production was highest (p < 0.05) in CRD11 (8.83 g/L), followed by CRD7 (8.16 g/L), whereas the lowest (p < 0.05) D-lactate was registered for encapsulated CRD11 (0.33 g/L) and CRD7 (0.49 g/L). The antibiogram profile determined through minimum inhibitory concentration showed that CRD7 and CRD11 were sensitive to key antibiotics suggested by EFSA except for gentamycin and kanamycin. PCR data on virulence genes demonstrated that both strains were safe for probiotic use. Moreover, CRD7 and CRD11 strains caused insignificant (p > 0.05) changes in the cell viability of Caco-2 cells as estimated by MTT (98.94–99.50%) and NR uptake (95.42–97.03%) assays and showed sensitivity to human serum. According to the results of these evaluated attributes, it is concluded that L. plantarum CRD7 and L. rhamnosus CRD11 are safe, non-toxic to human epithelial cells, and thus may be potentially suitable for various food/feed applications.