Methyl Thiazol Tetrazolium Research Articles

Influence of permeability enhancers on the paracellular permeability of metformin hydrochloride and furosemide across Caco-2 cells.

Permeability enhancers can affect absorption of paracellularly transported drugs. This study aims to evaluate effects of permeability enhancers (chitosan, methyl-β -cyclodextrin, sodium caprate, sodium lauryl sulfate, etc.) on the permeability of paracellularly absorbed furosemide and metformin hydrochloride. Methyl thiazole tetrazolium bromide test was carried out to determine the drug concentrations in permeability study. Trans-epithelial electrical resistance (TEER) values determined to assess the integrity of tight junctions. Permeability enhancers were applied at different concentrations alone, in dual/triple combinations. Permeability was determined using human colorectal adenocarcinoma (Caco-2) cells (TEER>400Ω·cm2). Permeability enhancers have no significant effect (<2-fold; p>0.05) on the permeability of furosemide (1.80×10-5±4.55×10-7cm/s); however, metformin permeability (1.36×10-5±1.25×10-6cm/s) increased significantly (p<0.05) with 0.3% and 0.5% (w/v) chitosan (2.0- and 2.7-fold, respectively), 1% methyl-β -cyclodextrin (w/v) (3.5-fold), 10 and 20µmol/L sodium caprate (2.2- and 2.8-fold, respectively), and 0.012% sodium lauryl sulfate (w/v) (1.9-fold). Furosemide permeability increased significantly (p<0.05) with chitosan-sodium lauryl sulfate combination (1.7-fold), and all triple combinations (1.4- to 1.9-fold). Chitosan containing dual/triple combinations resulted in significant increase (p<0.05) in metformin permeability (1.7 to 2.8-fold). All results indicated that absorption of furosemide and metformin can be improved by the combination of permeability enhancers. Therefore, it can be evaluated for the formulation of development strategies containing furosemide and metformin by the pharmaceutical industry.

Triptolide Inhibits Th17 Response by Upregulating microRNA-204-5p and Suppressing STAT3 Phosphorylation in Psoriasis.

Psoriasis is an immune and inflammation-related skin disease. Triptolide with immunosuppressive and anti-inflammatory properties has been utilized for psoriasis treatment. However, the potential immunological mechanisms of triptolide have not been fully elucidated. Using an imiquimod (IMQ)-induced psoriatic mouse model, we detected the effects of triptolide on psoriasis-like lesions including scales, thickening, and erythema. Methyl thiazol tetrazolium (MTT) cytotoxicity assay was performed for evaluating the influence of triptolide on cell viability. Gene expression at mRNA and protein levels were examined by reverse transcription-quantitative polymerase chain reaction and Western blot analysis, respectively. The combination between microRNA-204-5p (miR-204-5p) and signal transduction and transcription activator-3 (STAT3) was confirmed by luciferase reporter assay. Enzyme-linked immunosorbent assay was conducted to examine interleukin (IL)-17 and interferon-γ (IFN-γ) levels using corresponding kits. Hematoxylin and eosin staining was used for the visualization of epidermal thickness. Flow cytometry analysis was employed for examining T helper (Th) 17 cells. Triptolide ameliorated IMQ-induced psoriatic skin lesions manifested by the decreased psoriasis area and severity indexes (PASI) scores. Triptolide inhibited Th17 cell differentiation from splenocytes. Additionally, triptolide elevated miR-204-5p expression, whereas it downregulated STAT3 expression levels both in vitro and in vivo. Moreover, miR-204-5p directly targeted STAT3 in HaCaT cells. Furthermore, triptolide repressed the expression of proinflammatory cytokines in IMQ-evoked psoriasis-like mice. Triptolide inhibits STAT3 phosphorylation via upregulating miR-204-5p and thus suppressing Th17 response in psoriasis.

Synthesis and characterization of ZnO–TiO2–chitosan–escin metallic nanocomposites: Evaluation of their antimicrobial and anticancer activities

Abstract This work intended to formulate bio-nanocomposites of zinc oxide (ZnO), titanium oxide (TiO2), chitosan, and escin, characterize their physical properties, and evaluate their antimicrobial and anticancer properties. X-ray diffractometers (XRD) and scanning and transmission electron microscopes were applied to characterize the morphology and ultrastructure of chemically synthesized bio-nanocomposites. To investigate the functional groups of bio-nanocomposites, we used Perkin–Elmer spectrometers for Fourier transform infrared (FTIR) analysis and photoluminescence (PL) spectroscopy for PL spectrum analysis. Antimicrobial activities against bacterial and fungal strains were tested with agar well diffusion. Bio-nanocomposites were tested for anticancer effects on a MOLT4 blood cancer cell line using morphological analysis, methyl thiazole tetrazolium assay, apoptosis by acridine orange/ethidium bromide, and mitochondrial membrane potential (ΔΨm). In XRD, FTIR, and PL, the active compounds of ZnO–TiO2, chitosan, and escin peaks were observed. Our bio-nanocomposites demonstrated antimicrobial activity against bacterial and fungal pathogens. The bio-nanocomposite was cytotoxic to MOLT4 cells at an IC50 concentration of 33.4 µg·mL−1. Bio-nanocomposites caused cytotoxicity, changes in cell morphology, and mitochondrial membrane potential degradation, all of which resulted in apoptotic cell death. MOLT4 cells were found to be responsive to bio-nanocomposites based on ZnO–TiO2–chitosan–escin.

Novel ginsenoside derivatives induce apoptosis in HepG-2 cells via the MDM2-p53 signaling pathway

In this study, 6-aryl-5-cyanopyrimidines and quinazolinones were introduced into panaxadiol (PD) to synthesize 25 new panaxadiol derivatives. The cytotoxicity of these compounds against three cancer cells and one normal cell was examined by methylthiazoletetrazolium (MTT) cytotoxicity experiment. The findings demonstrated that PD cyanopyrimidine derivatives have superior anti-proliferative effects over quinazoline derivatives. Among them, PM14 had the strongest inhibitory activity on the proliferation of human hepatoma cell lines (HepG-2), the IC50 value was 2.13 ± 0.20 μM. 4′,6-diamidino-2-phenyl-indole (DAPI) staining showed that PM14 made HepG-2 nucleus shrink and had obvious apoptosis. Mechanistic studies confirmed that the derivative PM14 activates p53 signaling pathway by reducing the expression of MDM2 protein, and further induces an increase in the intracellular Bax/Bcl-2 ratio, down-regulated the expression of Caspase-3, up-regulated Cl-Caspase-3 expression, eventually leading to cell apoptosis. This lays the foundation for subsequent development of derivatives with stronger anti-proliferative activity.

Long Non-Coding RNA CASC2 Functions as A Tumor Suppressor in Colorectal Cancer via Modulating The miR-18a-5p/BTG3 Pathway.

Reportedly, long non-coding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2) is involved in regulating colorectal cancer (CRC) progression. However, the function and detailed downstream mechanism of CASC2 in CRC progression are not fully elucidated. The aim of the study was to investigate the potential function and molecular mechanism of CASC2 in CRC progression. In this experimental study, quantitative real-time polymerase chain reaction (qRT-PCR) was adopted to probe CASC2, microRNA-18a-5p (miR-18a-5p) and B cell translocation gene 3 (BTG3) mRNA expression in CRC tissues and cell lines. After CASC2 was overexpressed in Colo-678 and HCT116 cell lines, methylthiazol tetrazolium (MTT) and 5-bromo-2'-deoxyuridine (BrdU) assays were employed to examine the proliferation of CRC cells. Transwell migration and invasion assays were executed to evaluate the metastatic potential of CRC cells. The targeting relationships among CASC2, miR-18a-5p and BTG3 were validated by dual luciferase reporter gene assay. Western blot assay was applied to examine the regulatory effects of CASC2 and miR-18a-5p on BTG3 protein expression. CASC2 was decreased in CRC tissues and cell lines, and its low expression in CRC tissues was associated with larger tumor size and lymph node metastasis. CASC2 overexpression restrained proliferative, migrative and invasive capabilities of CRC cells. CASC2 could function as a molecular sponge for miR-18a-5p and repress the expression of miR-18a-5p. Furthermore, the inhibitory effects of CASC2 on the malignant phenotypes of CRC cells was counteracted by miR-18a-5p mimics. Additionally, CASC2 could positively regulate BTG3 expression via suppressing miR-18a-5p. CASC2 inhibits CRC development by suppressing miR-18a-5p and raising BTG3 expression.

Effects of propranolol on biological function of human esophageal squamous cell carcinoma cells

Objective: To investigate the effects of propranolol on the subcutaneous tumorigenesis of esophageal squamous cell carcinoma (ESCC) cells and the proliferation, migration, cell cycle, apoptosis and autophagy of ESCC cells and its possible molecular mechanisms. Methods: The cell proliferation was detected by MTT (methyl thiazol tetrazolium) assay: ESCC Eca109, KYSE-450 and TE-1 cells were routinely cultured. PBS (Phosphate buffer saline) group (without propranolol) and treated groups (40, 60, 80, 100 μmol/L propranolol) were set up with 5 wells in each group. After treatment for 0, 24, 48, 72 h, 10 μl (5 mg/ml) of MTT was added to each well, and the absorbance was measured at 490 nm. The cell migration was tested by Transwell assay: ESCC Eca109, KYSE-450 and TE-1 cells were routinely cultured, and PBS group (without propranolol) and treated groups (40, 60 μmol/L) were set up with 2 wells in each group. Photos were taken 40 h later, and the experiment was repeated for three times before statistical analysis. The cell cycle and apoptosis were detected by flow cytometry assay: ESCC Eca109, KYSE-450 and TE-1 cells were routinely cultured. PBS group (without propranolol) and treated group (80 μmol/L) were set up, fixed, stained, and fluorescence at 488 nm was detected. The protein levels were detected by Western blot: ESCC Eca109 and KYSE-450 cells were routinely cultured. PBS group (without propranolol) and treated groups (60, 80 μmol/L) were set up followed by gel electrophoresis, wet membrane transfer, and ECL imaging. The experiment was repeated for three times and then analyzed statistically. Subcutaneous tumor formation experiment in nude mice: 10 nude mice were assigned PBS group (without propranolol) and treated group (with propranolol). Five mice in each group were inoculated with 5×106 cells/100 μl (Eca109) into the right underarm. The treated group was given a gavage of 0.4 ml/kg (6 mg/kg) every other day, and the tumor size was measured every other day for 3 weeks. After 20 days, the nude mice were dislocated and sacrificed to take tumor tissue. Result: The results showed that propranolol inhibited the proliferation of Eca109, KYSE-450 and TE-1 cells with IC50 of around 70 μmol/L for 48 h. Eca109, KYSE-450 and TE-1 cell migration was inhibited by propranolol in a dose-dependent manner (P＜0.05); Propranolol blocked the cell cycle of Eca109 in G2/M phase, blocked the cell cycle of KYSE-450 and TE-1 in G0/G1 phase, and promoted apoptosis of three kinds of cells (P＜0.05). The results of cell fluorescence showed that LC3 fluorescence intensity of TE-1 was increased after 12 h, 24 h and 36 h treatment with propranolol (P＜0.05). Western blot results showed that compared with PBS group, the protein expressions of p-mTOR, p-Akt and cyclin D1 were down-regulated, while cleaved caspase 9 level was up-regulated (P＜0.05). The results of subcutaneous tumor formation in nude mice showed that the tumor weight of PBS group was (0.91±0.05)g, and that of the experimental group was(0.65±0.12)g, the difference was statistically significant (P＜0.05). Conclusion: Propranolol inhibits the proliferation, migration and cell cycle,promotes apoptosis and autophagy of ESCC cells, and inhibits subcutaneous tumor growth in nude mice. The mechanism might be related to the inhibition of PI3K/AKT/mTOR signaling pathway.

Preparation, characterization and in vitro cytotoxicity evaluation of free and liposome-encapsulated tylosin

Liposomes are the most attractive and well-known nanoparticles due to their high biocompatibility and biodegradability features which make them display less toxic effects for employing in vivo. Encapsulation of antibiotics within liposomes can provide a promising strategy for the effective treatment of resistant bacterial pathogens. This research was performed to prepare, characterize and evaluate the in vitro cytotoxicity effect of free and liposomal tylosin. Liposomal tylosin was produced by the thin-film hydration method. The characterization was performed by dynamic light scattering and high resolution transmission electron microscopy. Methylthiazol tetrazolium (MTT) assay was applied to evaluate the cytotoxicity of free and liposome-encapsulated tylosin against the NIH3T3 and NHDF. The obtained findings indicated well-formed liposome with ideal characteristics that efficiently encapsulated tylosin. The cytotoxicity data revealed cell viability of above 70% for liposomal tylosin and below 70% for free tylosin towards both NIH3T3 and NHDF at concentration of 1024 µg/mL. Therefore, liposome is a promising vehicle for an antibiotic delivery system with appropriate physicochemical, drug loading, release characteristics, and ideal biocompatibility properties.

Methotrexate loaded in alginate beads for controlled drug release against breast cancer

Methotrexate (MTX), as a folate antagonist is used for breast cancer chemotherapy, but its application due to the adverse side effects was limited. In this study, MTX were encapsulated in magnetic alginate beads coated with glutaraldehyde to control its release in order to reduce the side effects and improve its stability. The complex was characterized by physicochemical studies. The encapsulation efficiency was 75 % and the complex showed acceptable controlled release behavior. The cell cytotoxicity assessed using methylthiazol tetrazolium (MTT) method showed that magnetic alginate beads-MTX, in lower dosage has higher anticancer effect compared to the free MTX. The real-time polymerase chain reaction (PCR) was used to evaluate apoptotic factors Bcl2 associated X gene (Bax), B-cell lymphoma 2 (Bcl-2), and neuroinflammatory marker tumor necrosis factor-alpha (TNF-α) genes expression level on the treated cells. The findings demonstrated the significant increase of expression of Bax and a significant decrease in the expressions of Bcl-2 and TNF-α in Michigan cancer foundation-7 (MCF-7) cells. These results indicated that the developed drug can overcome the side effects of MTX and offer a controlled drug release for a sustained period with the long-term treatment of breast cancer.

Protective Effect of Rutin on Triethylene Glycol Dimethacrylate-Induced Toxicity through the Inhibition of Caspase Activation and Reactive Oxygen Species Generation in Macrophages.

Rutin, also called quercetin-3-rhamnosyl glucoside, is a natural flavonol glycoside present in many plants. Rutin is used to treat various diseases, such as inflammation, diabetes, and cancer. For polymeric biomaterials, triethylene glycol dimethacrylate (TEGDMA) is the most commonly used monomer and serves as a restorative resin, a dentin bonding agent and sealant, and a bone cement component. Overall, TEGDMA induces various toxic effects in macrophages, including cytotoxicity, apoptosis, and genotoxicity. The aim of this study was to investigate the protective mechanism of rutin in alleviating TEGDMA-induced toxicity in RAW264.7 macrophages. After treatment with rutin, we assessed the cell viability and apoptosis of TEGDMA-induced RAW264.7 macrophages using an methylthiazol tetrazolium (MTT) assay and Annexin V-FITC/propidium iodide assay, respectively. Subsequently, we assessed the level of genotoxicity using comet and micronucleus assays, assessed the cysteinyla aspartate specific proteinases (caspases) and antioxidant enzyme (AOE) activity using commercial kits, and evaluated the generation of reactive oxygen species (ROS) using a dichlorodihydrofluorescein diacetate (DCFH-DA) assay. We evaluated the expression of heme oxygenase (HO)-1, the expression of nuclear factor erythroid 2 related factor (Nrf-2), and phosphorylation of AMP activated protein kinase (AMPK) using the Western blot assay. The results indicated that rutin substantially reduced the level of cytotoxicity, apoptosis, and genotoxicity of TEGDMA-induced RAW264.7 macrophages. Rutin also blocked the activity of caspase-3, caspase-8, and caspase-9 in TEGDMA-stimulated RAW264.7 macrophages. In addition, it decreased TEGDMA-induced ROS generation and AOE deactivation in macrophages. Finally, we found that TEGDMA-inhibited slightly the HO-1 expression, Nrf-2 expression, and AMPK phosphorylation would be revered by rutin. In addition, the HO-1 expression, Nrf-2 expression, and AMPK phosphorylation was enhanced by rutin. These findings indicate that rutin suppresses TEGDMA-induced caspase-mediated toxic effects through ROS generation and antioxidative system deactivation through the Nrf-2/AMPK pathway. Therefore, rutin has the potential to serve as a novel antitoxicity agent for TEGDMA in RAW264.7 macrophages.

Anti-proliferative Effects of Ocimum basilicum Leaf Aqueous Extract on Colon Cancer Cell Lines and the Expression of Apoptotic Genes

Background: Colon cancer is one of the most diagnosed cancers in the modern world. Chemotherapy is one of the most relevant treatments for colon cancer, but this method of treatment has severe and sometimes irreversible side effects. Therefore, alternative treatments with fewer side effects are highly considered. Natural and herbal medicine has been proven to be a great source for these alternative treatment methods. Objectives: This study aimed to investigate the anti-proliferative and cytotoxic effects of Ocimum basillicum (OB) leaf aqueous extract on human colon cancer cell lines LS174T and COLO205 and the expression of apoptotic genes. Methods: Methyl thiazol tetrazolium (MTT) assay was performed to assess the cytotoxic effects of our extract on the aforementioned cell lines. The changes in the expression of BAX and BAD were analyzed by qPCR following the treatment of cells with our extract. Results: The results of the MTT assay indicated that the extract had a cytotoxic effect and could reduce the population of live cancer cells, especially LS174T cells. The expression of BAX and BAD genes showed a significant increase in qPCR analysis in both cell lines, especially in the LS174T cells. Conclusions: Our results showed that the extract had cytotoxic, anti-proliferative, and pro-apoptotic characteristics on previously mentioned cell lines, especially on the LS 174T cells.

Potential therapeutic effects of curcumin coated silver nanoparticle in the treatment of cutaneous leishmaniasis due to Leishmania major in-vitro and in a murine model

BackgroundLeishmaniasis is a destructive protozoan parasitic and vector-borne infection characterized as a neglected tropical disease (NTD) that is currently endemic in more than 100 countries. Herbal compounds such as curcumin derived from turmeric with complex and extensive structures have a wide range of applications in the treatment of parasitic infections; however, its usage has a major challenge due to low water solubility and bioavailability. Besides, metal nanoparticles such as silver have been applied for parasitic infection treatment. Synthesis of curcumin and silver nanoparticles conjugate facilitates in enhancing curcumin solubility and bioavailability. Here curcumin-coated silver nanoparticles (Cur@AgNPs) were synthesized, characterized, and then their cell cytotoxicity and antileishmanial activities were investigated both in-vitro and in mouse models. Materials and methodsCurcumin coated silver nanoparticles (Cur@AgNPs) synthesized by a simple one-pot green chemistry technique. After confirming their stability, in vitro cell cytotoxicity and leishmanicidal activity against promastigotes and amastigotes of the protozoan parasite, Leishmania major (L. major) was evaluated by using colourimetric yellow methyl thiazole tetrazolium (MTT) assay. Furthermore, antiparasitic properties of Cur@AgNPs in the mouse model were studied through quantifying footpads (FPs) lesion size and determining parasite burden in lymph nodes (LNs) and FPs of infected BALAB/c mice with L. major. ResultsCur@AgNPs represent a plasmonic peak of silver nanoparticles at 420 nm in UV–Vis spectra. The size of synthesized NPs was 29.1 ± 5.6 nm based on the analyzing TEM micrographs. The hydrodynamic diameter of the synthesized particles is 32.4 ± 2.3 nm. The zeta potential of washed Cur@AgNPs (4 rounds of washing) was −19.8 ± 1.5 mV. The stability of Cur@AgNPs at different time intervals (up to two months) in PBS buffer media has been investigated thoroughly. We confirmed that Cur@AgNPs decreased significantly in both forms of promastigotes and amastigotes of Leishmania parasite in a single treatment. The nanoparticle elucidated potent antileishmanial activity with IC50 of 58.99 μg/ml for promastigotes and EC50 58.99 μg/ml for amastigotes at 48 h post-infection with no harmful negative toxicity on the mice macrophages. In the treated infected BALB/c mice using Cur@AgNPs, the cutaneous leishmania (CL) lesion size in the FPs and Leishmania burden in both LNs and FPs were decreased significantly. ConclusionThe results of this study revealed that Cur@AgNPs could be, novel, safe, and effective promising candidates as an antileishmanial agent in the treatment of CL infection.

Adaptation of the methyl thiazole tetrazolium (MTT) reduction assay to measure cell viability in Vibrio spp.

Pathogenic vibrios of farmed shrimp have acquired resistance to antibiotics commonly used in aquaculture, leading to a constant search for new, effective products to control them. In vitro methods currently used to evaluate possible solutions are expensive, tedious, and time-consuming. In contrast, the methyl thiazole tetrazolium (MTT) reduction assay, widely used in mammalian cell lines to measure the cytotoxic effect of drugs, is inexpensive and reproducible. The objective of this study was to standardize an in vitro method, based on the reduction of the MTT compound, to measure cell viability and to evaluate the anti-Vibrio effect of bioproducts. The reduction capacity of MTT was evaluated by the formation of formazan crystals in Vibrio harveyi, V. campbellii, V. parahaemolyticus and V. vulnificus. Subsequently, the method was validated using essential oil of Origanum vulgare (EOOv) at different concentrations to obtain a dose-response effect. Oxytetracycline was used as a positive control at microbicidal doses. All vibrios were able to reduce the MTT to formazan and the effect of EOOv on the ability to reduce MTT depended on the Vibrio strains. The antibiotic affected the viability of the four vibrios at all concentrations evaluated and is understandable due to its microbicidal effect. In conclusion, the MTT reduction test was successfully adapted to measure viability and constitutes a suitable alternative to evaluate antimicrobial products quickly and accurately.

Cytotoxicity of HA-TCP Scaffold on Human Umbilical Cord Mesenchymal Stem Cells using MTT Assay

Hydroxyapatite-tricalcium phosphate (HA-TCP) scaffold is a three-dimensional structure used to support bone regeneration. Ideally, the scaffold should be biocompatible, biodegradable, and non-toxic. The tissue engineering technique uses a combined stem cell and scaffold to repair the bone defect. To prove the non-toxic properties of the scaffold, a cytotoxicity test is needed for the human umbilical cord mesenchymal stem cell (HUCMSCs). In this study, 27 samples were divided into eight groups with a variantof scaffold HA-TCP doses ranging from 5–1000 μg. Each treatment group of scaffolds was covered in HUCMSCs. The samples were added by methyl-thiazol-tetrazolium (MTT) colorimetric assay, counted using an optical density (OD) formula, and observed by a microplate reader. The viability of the cells was observed by an inverted TMS microscope with 100x magnification. The MTT assay’s test shows that the HUCMSCs cell viability shed each variant of HA-TCP scaffold dose, did not show any toxic effect. The higher the OD value, the higher the viability. It has been found that there is no significant difference between the variable scaffold doses to the percentage viability of umbilical cord cells

Cytotoxicity of HA-TCP Scaffold on Human Umbilical Cord Mesenchymal Stem Cells using MTT Assay

Hydroxyapatite-tricalcium phosphate (HA-TCP) scaffold is a three-dimensional structure used to support bone regeneration. Ideally, the scaffold should be biocompatible, biodegradable, and non-toxic. The tissue engineering technique uses a combined stem cell and scaffold to repair the bone defect. To prove the non-toxic properties of the scaffold, a cytotoxicity test is needed for the human umbilical cord mesenchymal stem cell (HUCMSCs). In this study, 27 samples were divided into eight groups with a variantof scaffold HA-TCP doses ranging from 5–1000 μg. Each treatment group of scaffolds was covered in HUCMSCs. The samples were added by methyl-thiazol-tetrazolium (MTT) colorimetric assay, counted using an optical density (OD) formula, and observed by a microplate reader. The viability of the cells was observed by an inverted TMS microscope with 100x magnification. The MTT assay’s test shows that the HUCMSCs cell viability shed each variant of HA-TCP scaffold dose, did not show any toxic effect. The higher the OD value, the higher the viability. It has been found that there is no significant difference between the variable scaffold doses to the percentage viability of umbilical cord cells.

Chemical composition, in vitro assessment of antioxidant properties and cytotoxicity activity of ethanolic and aqueous extracts of Ajuga orientalis L. (Lamiaceae)

Context: Ajuga orientalis L. (Lamiaceae) is an aromatic herb used in traditional medicine in Jordan and neighboring countries. Aims: To evaluate the cytotoxic potential of ethanolic and aqueous extracts from the aerial parts of A. orientalis against three cell lines MCF-7, Caco-2, and HDFa. In addition to assessing the total phenolic and flavonoid contents, antioxidant activity. Further to analyze the phytochemical constituents. Methods: The phytochemical analysis was performed using gas chromatography/mass spectrometry. The total phenolic (TPC) and flavonoid (TFC) contents were assessed using colorimetric methods. The antioxidant properties of both extracts were assessed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and reducing power ability. Cytotoxicity was investigated using methyl thiazol tetrazolium (MTT) assay. Results: The most abundant compounds in the extract were 9-octadecenoic acid, methyl ester, (E)- (27.2%), hexadecanoic acid, methyl ester (12.8%), and methyl stearate (9.6%). The ethanolic extract demonstrated higher TPC and TFC than the aqueous extract. Also, the ethanolic extract showed higher antioxidant activity than aqueous extract using DPPH and the reducing power ability. Furthermore, the aqueous extract of A. orientalis has a potent cytotoxic effect against the Caco-2 cell line (IC50 = 2.059 ± 0.10 μg/mL). In contrast, the ethanolic extract demonstrated a cytotoxic effect against the MCF-7 cell line (IC50 = 59.32 ± 0.04 μg/mL). Both extracts did not exhibit a toxic effect against normal dermal fibroblast cell line (HDFa). Compared to non-cancerous cells, the ethanolic extract of A. orientalis demonstrated high selectivity against MCF-7 cells and limited selectivity against Caco-2 cells. In comparison, the aqueous extract was highly selective against both cancerous cells. Conclusions: A. orientalis demonstrated antioxidant properties and significant antiproliferative potential against breast and colon cancer. Therefore, additional investigations are needed to study the mechanism of the cytotoxicity for this plant.

Cytotoxic Effect of Modified Gutta Percha by Incorporating Bioactive Glass 45S5 and Chitosan Particles As Potential Root Canal Filling Material

Aim: To investigate the cytotoxic effects of modified bioactive and antimicrobial gutta percha onthe fibroblast cells.Methods: The cytotoxic effects of modified bioactive and antimicrobial gutta percha on thefibroblast cells were investigated using Methylthiazol tetrazolium (MTT) assays at different times24, 48 and 72 hours.Results: there is no significant difference between controle and new modified gutta percha at first24 hrs. while with time at 48 and 72 hrs. there were significant difference.Conclusion: Bioactive bioglass BG45S5 and chitosan showed in vitro cytotoxic effects whenmixing with gutta percha in fibroblast cells as demonstrated by MTT assays, although toxic effectwas observed mostly as the same to control gutta percha.

Gallic Acid Inhibits Mesaconitine-Activated TRPV1-Channel-Induced Cardiotoxicity.

Aconiti Kusnezoffii Radix (Caowu) is often combined or processed with Chebulae Fructus (Hezi) to achieve attenuation purposes in Mongolian medicine. Mesaconitine (MA), a main bioactive ingredient of Caowu, is also famous for its high cardiotoxicity while exerting good anti-inflammatory and analgesic properties. Gallic acid (GA), one of the leading chemical components in Hezi, possesses cardiac protection. This study aimed to clarify the detoxification effects of GA from Hezi on MA-induced cardiotoxicity and whether the detoxification mechanism is related to the TRPV1 channel. Cell viability was determined by methyl thiazol tetrazolium (MTT), and lactate dehydrogenase (LDH) leakage rate was determined by ELISA. Hoechst 33258, JC-1, DCFH-DA, and Fluo-3 AM staining were conducted to detect apoptosis, mitochondrial membrane potential, reactive oxygen species (ROS), and Ca2+ respectively; TRPV1 channel current was recorded by whole-cell patch-clamp technology to observe the effect of GA and MA alone or in combination on TRPV1 channel. The results showed that GA exhibited pronounced detoxification effects on MA-induced cardiotoxicity. GA significantly inhibited the MA-induced decrease in cell viability; suppressed the MA-induced LDH leakage rate, apoptosis, and the release of ROS and Ca2+; and alleviated the reduction of mitochondrial membrane potential. We found that MA-induced cardiotoxicity was significantly attenuated in H9c2 cells pretreated with the TRPV1 antagonist BCTC. In the whole-cell patch-clamp experiment, the TRPV1 channel current increase was caused by the GA and MA treatment, whereas it was reduced by the cotreatment of GA and MA. Our data demonstrate that GA in Hezi can reduce MA-induced cardiotoxicity by inhibiting intracellular Ca2+ influx, restoring mitochondrial membrane potential, and reducing apoptosis. The detoxification mechanism may be related to the desensitization of the TRPV1 channel by the combined application of MA and GA.

1,2,4-Trioxolane and 1,2,4,5-Tetraoxane Endoperoxides against Old-World Leishmania Parasites: In Vitro Activity and Mode of Action.

Leishmaniasis remains one of the ten Neglected Tropical Diseases with significant morbidity and mortality in humans. Current treatment of visceral leishmaniasis is difficult due to a lack of effective, non-toxic, and non-extensive medications. This study aimed to evaluate the selectivity of 12 synthetic endoperoxides (1,2,4-trioxolanes; 1,2,4,5-tetraoxanes) and uncover their biochemical effects on Leishmania parasites responsible for visceral leishmaniasis. The compounds were screened for in vitro activity against L. infantum and L. donovani and for cytotoxicity in two monocytic cell lines (J774A.1 and THP-1) using the methyl thiazol tetrazolium assay. Reactive oxygen species formation, apoptosis, and mitochondrial impairment were measured by flow cytometry. The compounds exhibited fair to moderate anti-proliferative activity against promastigotes of the 2 Leishmania species, with IC50 values ranging from 13.0 ± 1.7 µM to 793.0 ± 37.2 µM. Tetraoxanes LC132 and LC138 demonstrated good leishmanicidal activity on L. infantum amastigotes (IC50 13.2 ± 5.2 and 23.9 ± 2.7 µM) with low cytotoxicity in mammalian cells (SIs 22.1 and 118.6), indicating selectivity towards the parasite. Furthermore, LC138 was able to induce late apoptosis and dose-dependent oxidative stress without affecting mithocondria. Compounds LC132 and LC138 can be further explored as potential antileishmanial chemotypes.

Cytotoxic Effect of Nano Fast Cement and ProRoot Mineral Trioxide Aggregate on L-929 Fibroblast Cells: an in vitro Study.

Statement of the Problem: Endodontic materials that are placed in direct contact with living tissues should be biocompatible. The cytotoxicity of Nano Fast Cement (NFC) compared to ProRoot Mineral Trioxide Aggregate (ProRoot MTA) must be evaluated.Purpose: This In vitro study aimed to assess the cytotoxic effects of NFC in comparison to ProRoot MTA on L-929 mouse fibroblast cells.Materials and Method: In this animal study, L-929 mouse fibroblast cells were grown in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in an atmosphere of 5% co2/95% air at 37 C̊. A total of 104 cells from the fourth collection were plated in each well of a 96-well micro-titer plate. Materials were mixed according to the manufacturer’s instruction and placed into the related plastic molds with 5 mm diameter and 3 mm height. After 24 hours and a complete setting, the extracts of the tested materials were produced at six different concentrations and placed in the related wells. Cells in DMEM served as the negative control group. DMEM alone was used as the positive control group. Methyl-thiazoltetrazolium (MTT) colorimetric assay was conducted after 24, 48, and 72 hours. The absorbance values were measured by ELISA plate reader at 540 nm wavelength. Three-way analysis of variance, post-hoc Tukey, LSD, and independent t-test were used for the statistical analyses using SPSS software, version 16.0.Results: There was no statically significant difference between MTA and NFC in cell viability values at different concentrations and different time intervals (p= 0.649). Viability values were significantly decreased after 72 hours, but there was no significant difference between the first and second MTT assays (p= 0.987). Cytotoxicity significantly increased at concentrations higher than 6.25 µɡ/ml.Conclusion: Cytotoxicity depends on time, concentration, and cement composition. There was no statistically significant difference between NFC and MTA concerning their cytotoxic effects on L-929 mouse fibroblast cells.

Fumonisin B2 Induces Mitochondrial Stress and Mitophagy in Human Embryonic Kidney (Hek293) Cells-A Preliminary Study.

Ubiquitous soil fungi parasitise agricultural commodities and produce mycotoxins. Fumonisin B2 (FB2), the structural analogue of the commonly studied Fumonisin B1 (FB1), is a neglected mycotoxin produced by several Fusarium species. Mycotoxins are known for inducing toxicity via mitochondrial stress alluding to mitochondrial degradation (mitophagy). These processes involve inter-related pathways that are regulated by proteins related to SIRT3 and Nrf2. This study aimed to investigate mitochondrial stress responses in human kidney (Hek293) cells exposed to FB2 for 24 h. Cell viability was assessed via the methylthiazol tetrazolium (MTT) assay, and the half-maximal inhibitory concentration (IC50 = 317.4 µmol/L) was estimated using statistical software. Reactive oxygen species (ROS; H2DCFDA), mitochondrial membrane depolarisation (JC1-mitoscreen) and adenosine triphosphate (ATP; luminometry) levels were evaluated to assess mitochondrial integrity. The relative expression of mitochondrial stress response proteins (SIRT3, pNrf2, LONP1, PINK1, p62 and HSP60) was determined by Western blot. Transcript levels of SIRT3, PINK1 and miR-27b were assessed using quantitative PCR (qPCR). FB2 reduced ATP production (p = 0.0040), increased mitochondrial stress marker HSP60 (p = 0.0140) and suppressed upregulation of mitochondrial stress response proteins SIRT3 (p = 0.0026) and LONP1 (p = 0.5934). FB2 promoted mitophagy via upregulation of pNrf2 (p = 0.0008), PINK1 (p = 0.0014) and p62 (p < 0.0001) protein expression. FB2 also suppressed miR-27b expression (p < 0.0001), further promoting the occurrence of mitophagy. Overall, the findings suggest that FB2 increases mitochondrial stress and promotes mitophagy in Hek293 cells.