Cytotoxicity In RAW Research Articles

Green extraction of marine phospholipids from Conger eel by-product using SC-CO2: Lipidomic profiles and biological activities

This paper presents an eco-friendly approach to recovering phospholipids (PLs) from the Conger myriaster head (CMH) via supercritical carbon dioxide (SC–CO2) extraction using ethanol as a cosolvent. The lipidomic profile and biological activities of the extracted PLs were investigated. Purity assessments, PL composition analysis, and fatty acid profiling were performed to characterize the PLs. Biological activity was determined through antioxidant, antimicrobial, antihypertensive, anti-inflammatory, and anticancer evaluations. The results showed that the yields of the SC–CO2 extracted CMH PLs (SCHP) and organic solvent extracted CMH PLs (OCHP) were 3.26% ± 0.20 % (76.18% ± 0.09% purity) and 9.02% ± 0.21% (purity = 18.29% ± 0.04%), respectively. The 31P NMR analysis revealed a diverse PL composition in both extracts, with the SCHP showing higher PC content and selective extraction characteristics. The SCHP exhibited significantly superior antioxidant, antimicrobial, antihypertensive, and anti-dementia activities compared to the OCHP. Both extracts inhibited nitric oxide production in RAW 264.7 cells, suggesting anti-inflammatory activity. Selective anticancer activity was observed, with high toxicity in SK-N-SH cells but no cytotoxicity in RAW 264.7 and HaCaT cells. The findings highlight the potential of SC-CO2 extraction using ethanol to obtain bioactive PLs from marine by-products, with promising applications in the pharmaceutical, cosmetic, and food industries.

Cytoprotective and Anti-inflammatory Effects of Derma Genie™-H002 against Paraphenylenediamine-induced Cytotoxicity and Inflammation in RAW 264.7 Cells

Purpose: This study aimed to establish an <i>in vitro</i> model to examine paraphenylenediamine (PPD)-induced cytotoxicity and inflammation within RAW 264.7 cells. Additionally, we investigated the cytoprotective and anti-inflammatory effects of Derma Genie™-H002 derived from extracts of five medicinal herbs. Methods: Various biochemical analyses, including water-soluble tetrazolium salt, crystal violet staining, and lactate dehydrogenase leakage assays, were employed to evaluate the impact of Derma Genie™-H002 on PPD-induced cytotoxicity and inflammation. Furthermore, apoptosis- and inflammation-related gene expression was analyzed using reverse transcription-polymerase chain reaction and Western blotting. Results: Derma Genie™-H002 exhibited no significant cytotoxicity in RAW 264.7 cells, whereas PPD induced notable cytotoxicity at concentrations >25 μM. Cotreatment with PPD and Derma Genie™-H002 not only improved cell proliferation rates but also alleviated cell viability in PPD-induced RAW 264.7 cells. Furthermore, findings demonstrated that Derma Genie™-H002 mitigates the cytotoxicity of PPD-induced RAW 264.7 cells by reducing the expression of the proapoptotic protein Bax and increasing the expression of the antiapoptotic protein Bcl-2. Moreover, cotreatment with PPD and Derma Genie™-H002 downregulated the mRNA expression of inflammasome-related genes, inflammatory cytokines, and allergic contact dermatitis-related genes, all elevated by PPD. The increased expression of nuclear factor-κB protein induced by PPD was also reduced upon cotreatment with Derma Genie™-H002, providing compelling evidence of its anti-inflammatory efficacy in the PPD-induced inflammation model. Conclusion: This study demonstrated that Derma Genie™-H002 effectively alleviates PPD-induced cytotoxicity and inflammation in RAW 264.7 cells. Therefore, the potential application of Derma Genie™-H002 as a naturally derived functional anti-inflammatory material has been validated.

Nanostructured Lipid Carriers for oral treatment of leishmaniasis: Design and preclinical evaluation

Pentavalent antimonials are the primary treatment for leishmaniasis due to their proven efficacy and cost-effectiveness. However, they present poor oral absorption, parenteral administration, and serious adverse effects due to prolonged use. Nanostructured Lipid Carriers (NLC) offer a promising solution, providing prolonged release, enhanced intestinal permeability, and oral bioavailability. This study aimed to address these issues by developing Nanostructured Lipid Carrier loaded with meglumine antimoniate (NLC-MA) for oral leishmaniasis treatment. We used design of experiments (DoE) for optimization. The chosen NLC-MA was characterized assessing size, polydispersity index (PdI), zeta potential (ZP), entrapment efficiency (EE), pH, and by analytical techniques such as DSC, FTIR, XRD, and TEM. Stability under simulated gastrointestinal conditions, in vitro release kinetics, cytotoxicity in RAW 264.7 macrophages, and leishmanicidal activity were assessed. NLC-MA presented 41.13 nm, PdI 0.227, ZP −27.9 mV, EE of 62.43%, and pH 5.23. Characterization techniques confirmed drug incorporation and nanoparticles with reduced crystallinity. TEM images showed spherical morphology. NLC remained stable in gastrointestinal pH and under refrigeration storage, while lyophilized formulations retained their initial properties. MA release from NLC followed the Peppas-Sahlin model, an anomalous transport mechanism. Importantly, MA showed no cytotoxicity in macrophages, but the NLCs exhibited cytotoxicity beyond 50 µg/mL. NLC-MA displayed improved efficacy against Leishmania infantum. Overall, these results highlight the potential of utilizing a lipid nanocarrier incorporating meglumine antimoniate as an innovative drug delivery platform for oral treatment against leishmaniasis.

Yanang water extract exhibits a protective effect against methomyl-induced cytotoxicity in RAW 264.7 cells via suppression of apoptosis and cell cycle arrest

Methomyl, an extremely hazardous substance, is widely used for controlling insects and pests in agricultural production. This compound can cause several illnesses in humans. In this research, we determined the alleviation potential of Yanang water extract (YWE) against methomyl-induced cytotoxicity in RAW 264.7 macrophage cells and examined the underlying mechanisms of action. The YWE (2.5 – 10 μg/mL) exhibited no toxicity against RAW 264.7 cells, whereas methomyl significantly reduced RAW 264.7 cell growth, resulting in the progression of apoptosis and cell cycle arrest. Supplementation of YWE on methomyl treated RAW 264.7 cells revealed a pronounced protective effect via the suppression of caspase-9 and caspase-3 mRNA expression levels. Proteomics studies were used to assess the effect of methomyl on RAW 264.7 cells, revealing an increase of proteins associated with apoptosis and cell cycle arrest, whereas in the YWE co-treatment condition these proteins were suppressed. Moreover, an increase in proteins involved with cell cycle progression and anti-apoptosis was found in YWE co-treatment. Our findings suggest that YWE is potentially involved in mitigating methomyl-induced cytotoxicity, via the suppression of apoptosis and cell cycle arrest.

Time maters: Exploring the dynamics of bioactive compounds content, bioaccessibility and antioxidant activity during Lupinus angustifolius germination

Germination is a process that enhances the content of health-promoting secondary metabolites. However, the bioaccessibility of these compounds depends on their stability and solubility throughout the gastrointestinal tract. The study aimed to explore how germination time influences the content and bioaccessibility of γ-aminobutyric acid and polyphenols and antioxidant capacity of lupin (Lupinus angustifolius L.) sprouts during simulated gastrointestinal digestion. Gamma-aminobutyric acid showed a decrease following gastrointestinal digestion (GID) whereas phenolic acids and flavonoids exhibited bioaccessibilities of up to 82.56 and 114.20%, respectively. Although the digestion process affected the profile of phenolic acids and flavonoids, certain isoflavonoids identified in 7-day sprouts (G7) showed resistance to GID. Germination not only favored antioxidant activity but also resulted in germinated samples exhibiting greater antioxidant properties than ungerminated counter parts after GID. Intestinal digests from G7 did not show cytotoxicity in RAW 264.7 macrophages, and notably, they showed an outstanding ability to inhibit the production of reactive oxygen species. This suggests potential benefit in mitigating oxidative stress. These findings contribute to understand the dynamic interplay between bioprocessing and digestion in modulating the bioaccessibility of bioactive compounds in lupin, thereby impacting health.

A comprehensive investigation of the medicinal efficacy of antimicrobial fusion peptides expressed in probiotic bacteria for the treatment of pan drug-resistant (PDR) infections.

The present work aimed to examine the intracellular antibacterial efficacy of Recombinant Lactobacillus acidophilus/antimicrobial peptides (AMPs) Melittin and Alyteserin-1a, specifically targeting Gram-negative bacteria. The first assessment was to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Recombinant L. acidophilus/AMPs versus Gram-negative and Gram-positive bacteria. In addition, the researchers examined the in vitro viability and safety of AMPs generated by L. acidophilus. The experiments included exposing the AMPs to elevated temperatures, proteases, cationic salts at physiological levels, and specific pH settings. The safety aspect was evaluated using hemolytic analysis utilizing sheep erythrocytes; cytotoxicity assays employing cell lines, and experiments on beneficial gut lactobacilli. An experiment was done using a time-kill method to assess the intracellular antibacterial efficacy of Recombinant L. acidophilus/AMPs compared to pathogenic varieties in HEp-2 cells. Previous investigations have shown that the MBC levels of recombinant L. acidophilus/AMPs were consistently two to four times higher than the equivalent MIC values when evaluated versus Gram-negative bacteria. Furthermore, the stability of the Recombinant L. acidophilus/AMPs showed variability when exposed to elevated temperatures (70 and 90℃), treated with protease enzymes (proteinase K, lysozyme), exposed to higher concentrations of physiological salts (150mM NaCl and 2mM MgCl2), and varying pH levels (ranging from 4.0 to 9.0). The recombinant L. acidophilus/AMPs are non-hemolytic towards sheep erythrocytes, exhibit little cytotoxicity in RAW 264.7 and HEp-2 cells, and are considered safe when compared to beneficial gut lactobacilli. The research examined the intracellular bacteriostatic effects of recombinant L. acidophilus/AMPs on Gram-negative bacteria inside HEp-2 cells. Nevertheless, no notable bactericidal impact was seen on Gram-positive bacteria (P > 0.05). The research shows that recombinant L. acidophilus/AMPs, namely (L. acidophilus/melittin/Alyteserin-1a) as the focus of the investigation, effectively eliminate Gram-negative bacteria. Therefore, more investigation is necessary to elaborate on these discoveries.

Innate Immune-Enhancing Effect of Pinus densiflora Pollen Extract via NF-κB Pathway Activation.

Considering the emergence of various infectious diseases, including the coronavirus disease 2019 (COVID-19), people's attention has shifted towards immune health. Consequently, immune-enhancing functional foods have been increasingly consumed. Hence, developing new immune-enhancing functional food products is needed. Pinus densiflora pollen can be collected from the male red pine tree, which is commonly found in Korea. P. densiflora pollen extract (PDE), obtained by water extraction, contained polyphenols (216.29 ± 0.22 mg GAE/100 g) and flavonoids (35.14 ± 0.04 mg CE/100 g). PDE significantly increased the production of nitric oxide (NO) and reactive oxygen species (ROS) but, did not exhibit cytotoxicity in RAW 264.7 cells. Western blot results indicated that PDE induced the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. PDE also significantly increased the mRNA and protein levels of cytokines and the phosphorylation of IKKα/β and p65, as well as the activation and degradation of IκBα. Additionally, western blot analysis of cytosolic and nuclear fractions and immunofluorescence assay confirmed that the translocation of p65 to the nucleus after PDE treatment. These results confirmed that PDE increases the production of cytokines, NO, and ROS by activating NF-κB. Therefore, PDE is a promising nutraceutical candidate for immune-enhancing functional foods.

Lignin nanoparticles as a novel carrier for efficacious delivery of toll like receptor 7/8 agonist: Physicochemical and in-vitro evaluation

Imidazoquinolinone (IMDQ) derivatives are known TLR7/8 agonists and are approved by the FDA for antiviral and skin cancer treatment. Their use as innate immune system activating molecules has been limited by poor pharmacokinetic properties and toxicities associated with systemic administration. In the present study, an IMDQ derivative, 1-(3-(aminomethyl) benzyl)-2-butyl-1H-imidazo[4,5-c] quinolin-4-amine (meta-aminomethyl BBIQ, 4), was encapsulated in biopolymer lignin nanoparticles (LNPs) to develop slow-release delivery system and to enhance its immune activating properties. A co-precipitation method was used to synthesize LNPs of alkali lignin. Characterization studies demonstrated the formation of spherical shaped nanoparticles of ∼150 nm size. The encapsulation efficiency and loading capacity for meta-aminomethyl BBIQ in LNPs was found to be 99% and 70%, respectively. In-vitro release studies showed 78% release over 24 h at pH 7.4 followed by sustained release. Kinetic modelling studies showed the release profile followed Weibull order kinetics with β value ≤ 0.75 corresponding to Fickian diffusion. Moreover, blank as well as meta-aminomethyl BBIQ-loaded LNPs were non-hemolytic and did not show significant cytotoxicity in RAW 264.7 and MDA-MB-23 cells at all tested concentrations. This study confirms the potential to use LNPs as a drug delivery system for immune modulators or vaccine adjuvants.

Chemical Characterization and Leishmanicidal Activity In Vitro and In Silico of Natural Products Obtained from Leaves of Vernonanthura brasiliana (L.) H. Rob (Asteraceae).

Vernonanthura brasiliana (L.) H. Rob is a medicinal plant used for the treatment of several infections. This study aimed to evaluate the antileishmanial activity of V. brasiliana leaves using in vitro and in silico approaches. The chemical composition of V. brasiliana leaf extract was determined through liquid chromatography-mass spectrometry (LC-MS). The inhibitory activity against Leishmania amazonensis promastigote was evaluated by the MTT method. In silico analysis was performed using Lanosterol 14alpha-demethylase (CYP51) as the target. The toxicity analysis was performed in RAW 264.7 cells and Tenebrio molitor larvae. LC-MS revealed the presence of 14 compounds in V. brasiliana crude extract, including flavonoids, flavones, sesquiterpene lactones, and quinic acids. Eriodictol (ΔGbind = -9.0), luteolin (ΔGbind = -8.7), and apigenin (ΔGbind = -8.6) obtained greater strength of molecular interaction with lanosterol demethylase in the molecular docking study. The hexane fraction of V. brasiliana showed the best leishmanicidal activity against L. amazonensis in vitro (IC50 12.44 ± 0.875 µg·mL-1) and low cytotoxicity in RAW 264.7 cells (CC50 314.89 µg·mL-1, SI = 25.30) and T. molitor larvae. However, the hexane fraction and Amphotericin-B had antagonistic interaction (FICI index ≥ 4.0). This study revealed that V. brasiliana and its metabolites are potential sources of lead compounds for drugs for leishmaniasis treatment.

Pomegranate juice fermented by tannin acyl hydrolase and Lactobacillus vespulae DCY75 enhance estrogen receptor expression and anti-inflammatory effect

Phenolics are phytochemicals in plants, fruits, and vegetables have potential health-promoting efficacies. However, mostly available as a complex form. So, to increase the contents and nutritional value of the phenolic compounds, fermentation is most readily used in the food industry. Especially, the hydrolyzable tannins present in the pomegranate that can be liberated into monomolecular substances, which enhances biological activity. Thus, this study aims to convert hydrolyzable tannins to ellagic acid by fermentation using Tannin acyl hydrolase (TAH) and a novel bacteria strain Lactobacillus vespulae DCY75, respectively to investigate its effect on Estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) mRNA expression along with inflammation inhibition. As a result, the fermentation enhanced the ellagic acid content up to 70% by the synergetic effect of TAH and DCY75. Furthermore, fermented pomegranate (PG-F) increased cellular proliferation as well as upregulated the gene expression of estrogen regulators such as ERα, ERβ, and pS2 in breast cancer cell line (MCF-7), which commonly used to evaluate estrogenic activity. Moreover, to study the inflammation associated with low estrogen in menopause, we have analyzed the inhibition of nitric oxide (NO)/inducible nitric oxide synthase (iNOS) in RAW 264.7 cells. The PG-F juice did not exert any cytotoxicity in RAW 264.7 cells and inhibited NO production along with the downregulation of a major pro-inflammatory cytokine iNOS which indicates the anti-inflammatory potential of it. To sum it up, the fermented commercial pomegranate juice using a novel bacteria strain increased the amount of ellagic acid that the value added bioactive of pomegranate and it has significantly increased the estrogenic activity via upregulating estrogen related biomarkers expression and reduced the risk of related inflammation via NO/iNOS inhibition. This study could be a preliminary study to use fermented pomegranate as a potential health functional food after further evaluation.

Talaromyces purpurogenus from a marine-polluted environment inhibits pro-inflammatory markers in LPS-stimulated RAW 264.7 cells and in carrageenan-induced paw edema

ObjectivesTalaromyces purpurogenus is a fungus species widely found in the marine environment. When in a hostile environment, it produces a series of secondary metabolites with important biotechnological potential, such as meroterpenoids. In our previous report, we demonstrated that the culture broth ethyl acetate extract from T. purpurogenus reduced the cellular infiltration of lymphoid organs and circulating levels of TNF-α. To elucidate this anti-inflammatory effect, this work aimed to study T. purpurogenus isolated from a marine-polluted environment regarding the chemical composition of its culture broth and mycelial ethyl acetate extract (CBEAE and MEAE respectively), as well as its action on pro-inflammatory markers in LPS-stimulated RAW 264.7 cells and carrageenan-induced mice paw edema. MethodsChemical characterization was performed by HPLC-DAD-UV. Cytotoxicity in vitro and endotoxin quantification in extracts were preformed prior the experiments. In vitro inhibitory effect of T. purpurogenus extracts were evaluated by cytokines and nitrite quantification in cell supernatant of RAW 264.7 cells. The effect of T. purpurogenus in murine acute inflammation model of carrageenan-induced paw edema was verified by thickness kinetic and histology evaluation. ResultsThe CBEAE demonstrated a richer chemical composition than MEAE, presenting nine meroterpenoid compounds, while MEAE displayed only two meroterpenoid and one phenolic compound. Cytotoxicity in RAW 264.7 macrophages was not observed at any concentration treatment for both extracts. Endotoxin quantification also demonstrated absence of endotoxins in both extracts. At 500 μg/mL, the CBEAE and MEAE extracts inhibited nitrite (p = 0.0450 and p = 0.0497), TNF-α (p = 0.0076 and p = 0.0001) and IL-1β (p = 0.0042 and p = 0.0031), but only CBEAE inhibited IL-6 levels in LPS-stimulated cells (p = 0.0234). CBEAE, but not MEAE, also inhibited nitrite (p = 0.0353) and IL-1β (p = 0.0386) in non-stimulated cells. In addition, CBEAE at 250 and 500 mg/kg inhibited edema thickness and mast cells (p = 0.0303 and p = 0.0130) in λ-carrageenan-induced mice paw edema. ConclusionsTalaromyces purpurogenus isolated from marine-polluted environment inhibited pro-inflammatory markers in LPS-stimulated RAW 264.7 cells and in murine model of paw edema. The promising anti-inflammatory activity in vitro and in vivo, especially the culture broth ethyl acetate extract, demonstrates its potential for further studies to elucidate its mechanism of action.

Low-Level Laser Irradiation Stimulates RANKL-Induced Osteoclastogenesis via the MAPK Pathway in RAW 264.7 Cells

Low-level laser therapy (LLLT) is recognized as an effective medical tool for the treatment of various conditions requiring tissue repair, pain relief, inflammation treatment, and restoration of tissue dysfunction, and its development and research are growing rapidly. However, studies that analyze molecular biology by applying LLLT to osteoclasts are still insufficient to understand the mechanism. In order for LLLT to be suggested as an appropriate treatment method for the treatment of various bone diseases, it is necessary to elucidate the effect and mechanism of LLLT on osteoclast differentiation. In this study, we investigated the effect of LLLT on osteoclast differentiation using murine macrophage (RAW 264.7) cells by means of a Ga-As-Al laser (λ = 810, 80 mW). Our results indicate that LLLT did not induce cytotoxicity in RAW 264.7 cells. When LLLT was applied for 15 s to osteoclasts exposed to RANKL, the expression of NF-κB, ERK, p38, and c-Fos, which are associated with expression of NFATc1, was increased. The RT-PCR results also demonstrated significantly increased expression of osteoclast-specific genes, including NFATc1, TRAP, the calcitonin receptor, and cathepsin K, compared with the control. Taken together, we concluded that low-level laser irradiation induces osteoclastogenesis by enhancing the expression of NF-κB, MAPKs (ERK, p38), c-Fos, and NFATc1 in RAW 264.7 cells. These findings indicate that low-level laser irradiation could be considered a potential treatment option in various metabolic bone diseases that require osteoclastic activity and bone formation.

Phytochemical profile and antioxidant activity of extracts of the peruvian peppertree Schinus areira L. from Chile.

The Andean tree Schinus areira L. has multiple traditional uses, from the treatment of bronchitis and rheumatic diseases to menstrual cycle regulation and wound healing. With reported hypotensive, analgesic, antitumoral and anti-inflammatory properties, it acts predominantly against diseases related to oxidative stress. This study focuses on the antioxidant activity and phytochemical profile of the extracts of Schinus areira L.Serial extraction of the fruits was performed both by maceration and by Soxhlet. Total phenols and flavonoids were measured using the Folin-Ciocalteu method and AlCl3, respectively. In vitro antioxidant activity was determined by FRAP and DPPH.Results were similar for both extraction methods. Primary metabolites detected included carbohydrates, proteins and amino acids; secondary metabolites included tannins, flavonoids, saponins, steroids and triterpenes. Antioxidant activity was confirmed for ethyl acetate, methanolic and aqueous extracts. The methanolic extract had both the highest polyphenol content (>195 mg GAE/ g dry weight) and the highest antioxidant activity [EC50 > 476 μg/mL; >273 mg AA/g dry weight (DPPH); >301 mg AA/ g dry weight (FRAP)]. The extract does not produce macrophage cytotoxicity in RAW 264.7, which is indicated by an average cytotoxicity of 2% over 24 h.Our study serves as a starting point for future research on the pharmacological properties of Schinus areira L.

Antimicrobial efficacy of Cecropin A (1–7)- Melittin and Lactoferricin (17–30) against multi-drug resistant Salmonella Enteritidis

The present study evaluated intracellular antibacterial efficacy of two short-chain cationic antimicrobial peptides (AMPs) namely, Cecropin A (1–7)-Melittin and lactoferricin (17–30) against three field strains of multi-drug resistant Salmonella Enteritidis. Initially, antimicrobial ability of both the AMPs was evaluated for their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against multi-drug resistant S. Enteritidis strains. Besides, the AMPs were evaluated for its in vitro stability (high-end temperatures, proteases, physiological concentrations of cationic salts and pH) and safety (haemolytic assay in sheep erythrocytes; cytotoxicity assay in murine macrophage RAW 264.7 cell line and human epithelioma HEp-2 cell line and beneficial gut lactobacilli). Later, a time-kill assay was performed to assess the intracellular antibacterial activity of Cecropin A (1–7)-Melittin and lactoferricin (17–30) against multi-drug resistant S. Enteritidis in RAW 264.7 and HEp-2 cells. The observed MBC values of Cecropin A (1–7)-Melittin and lactoferricin (17–30) against multi-drug resistant S. Enteritidis (128 μM; 256 μM) were generally twice or four-fold greater than the MIC values (64 μM). Further, both the AMPs were found variably stable after exposure at high-end temperatures (70 °C and 90 °C), protease treatment (trypsin, proteinase K, lysozyme), higher concentration of physiological salts (150 mM NaCl and 2 mM MgCl2) and hydrogen ion concentrations (pH 4.0 to 8.0). Both the AMPs were found non-haemolytic on sheep erythrocytes, revealed minimal cytotoxicity in RAW 264.7 and HEp-2 cells, and was tested safe against beneficial gut lactobacilli (L. acidophilus and L. rhamnosus). Intracellular bacteriostatic effect with both cationic AMPs against multi-drug resistant S. Enteritidis was evident in RAW 264.7 cells; however, in both the cell lines, the significant bactericidal effect was not observed (P > 0.05) with both cationic AMPs understudy against multi-drug resistant S. Enteritidis. Based on the results of the present study, both the cationic AMPs understudy may not be useful for the intracellular elimination of multi-drug resistant S. Enteritidis; hence, further studies such as conjugation of these AMPs with either cell-penetrating peptides (CPP) and/or nanoparticles (NPs) are warranted.

Cuminum cyminum Prevents Lipotoxicity and Apoptosis but Cuminaldehyde Fails to Do So: A Study on Mouse Macrophage (RAW 264.7) Cells

Aim:The present study aims to compare the cytoprotective effect of Cuminum cyminum L. (CC) extract and cuminaldehyde (CA) against lipotoxicity induced by oxidized low density lipoprotein (Ox-LDL) in mouse macrophage (RAW 264.7) cells.Objective:To assess comparative Cytoprotective potential of CC and CA against Ox-LDL induced cytotoxicity. ii) To study efficacy of CC and CA in preventing Ox-LDL induced apoptosisMethods:Protective effect of CC extract and CA aganist Ox-LDL induced cytotoxicity in RAW 264.7 cells was assessed by MTT assay. DCFDA stain was used to check the generation of ROS followed by analysis of apoptotic genes by quantitative RT-PCR.Results:CC extract was found to be non-toxic up to 300 μg/ml but CA showed significant toxicity from 50 to 300 μg/ml. Cells treated with Ox-LDL recorded 80 % decrement in cell viability as compared to the control cells. But Ox-LDL+CC treated group accounted for improved cell viability (88 %) which was comparable to that of control. However, Ox-LDL+CA treated cells did not record any improvement in cell viability (19 %). DCF-DA staining revealed that the presence of CC could minimize intracellular oxidative stress but similarly this was persistent in CA supplemented group. Furthermore, mRNA expression of apoptotic genes revealed that Ox-LDL induced upregulation of Bax and downregulation of Bcl-2 genes were not recorded in Ox-LDL+CC treated group.Conclusion:It can be concluded that CC extract efficiently prevented Ox-LDL induced lipotoxicity and apoptosis and has an anti-atherosclerotic potential. The failure of CA emphasizes the importance of naturally occurring polyherbal formulations over pure compounds in imparting bioactivity and for therapeutic applications.

Multimodality labeling strategies for the investigation of nanocrystalline cellulose biodistribution in a mouse model of breast cancer

MethodsWe have developed a nuclear and fluorescence labeling strategy for nanocrystalline cellulose (CNC), an emerging biomaterial with versatile chemistry and facile preparation from renewable sources. We modified CNC through 1,1′-carbonyldiimidazole (CDI) activation with radiometal chelators desferrioxamine B and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), allowing for the labeling with zirconium-89 (t½ = 78.41 h) and copper-64 (t½ = 12.70 h), respectively, for non-invasive positron emission tomography (PET) imaging. The far-red fluorescent dye Cy5 was added for ex vivo optical imaging, microscopy and flow cytometry. The multimodal CNC were evaluated in the syngeneic orthotopic 4T1 tumor model of human stage IV breast cancer. ResultsModified CNC exhibited low cytotoxicity in RAW 264.7 macrophages over 96 h, and high radiolabel stability in vitro. After systemic administration, radiolabeled CNC were rapidly sequestered to the organs of the reticulo-endothelial system (RES), indicating immune recognition and no passive tumor targeting by the enhanced permeability and retention (EPR) effect. Modification with NOTA was a more favorable strategy in terms of radiolabeling yield, specific radioactivity, and both the radiolabel and dispersion stability in physiological conditions. Flow cytometry analysis of Cy5-positive immune cells from the spleen and tumor corroborated the uptake of CNC to phagocytic cells. ConclusionsFuture studies on the in vivo behavior of CNC should be concentrated on improving the nanomaterial stability and circulation half-life under physiological conditions and optimizing further the labeling yields for the multimodality imaging strategy presented. Advances in knowledgeOur studies constitute one of the first accounts of a multimodality nuclear and fluorescent probe for the evaluation of CNC biodistribution in vivo and outline the pitfalls in radiometal labeling strategies for future evaluation of targeted CNC-based drug delivery systems. Implications for patient careQuantitative and sensitive molecular imaging methods provide information on the structure–activity relationships of the nanomaterial and guide the translation from in vitro models to clinically relevant animal models.

Effect of Lactobacillus Fermentation on the Anti-Inflammatory Potential of Turmeric.

Curcumin, the major bioactive constituent of turmeric, has been reported to have a wide range of pharmacological benefits; however, the low solubility in water has restricted its systemic bioavailability and therapeutic potential. Therefore, in the current study, we aimed to investigate the effect of turmeric fermentation on its curcumin content and anti-inflammatory activity by using several lactic acid bacteria. Fermentation with Lactobacillus fermentum significantly increased the curcumin content by 9.76% while showing no cytotoxicity in RAW 246.7 cells, as compared to the unfermented turmeric, regardless of the concentration of L. fermentum-fermented turmeric. The L. fermentum-fermented turmeric also promoted cells survival; a significantly higher number of viable cells in lipopolysaccharide (LPS)-induced RAW 264.7 cells were observed as compared to those treated with unfermented turmeric. It also displayed promising DPPH scavenging activity (7.88 ± 3.36%) and anti-inflammatory activity by significantly reducing the nitrite level and suppressing the expression of the pro-apoptotic tumor necrosis factor-alpha (TNF-α) and Toll-like receptor-4 (TLR4) in LPS-induced RAW 264.7 cells. Western blot analysis further revealed that the anti-inflammatory activity of the fermented turmeric was exerted through suppression of the c-Jun N-terminal kinase (JNK) signal pathway, but not in unfermented turmeric. Taken together, the results suggested that fermentation with lactic acid bacteria increases the curcumin content of turmeric without increasing its cytotoxicity, while strengthening the specific pharmacological activity, thus, highlighting its potential application as a functional food ingredient.

Effects of mineral trioxide aggregate and bioceramics on macrophage differentiation and polarization in vitro

Mineral trioxide aggregate (Pro-Root MTA, PR-MTA) and bioceramics (iRoot® SP Injectable Root Canal Sealer, iR-BC) are used for making apical plugs used in apexification, repairing root perforations during root canal therapy, and treating internal root resorption. The purpose of the present invitro study was to compare the biological effects of PR-MTA- and iR-BC-based dental sealers in the mouse macrophage cell line RAW 264.7. Cytotoxicity and cell proliferation were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell hemocytometer, respectively. Protein expression of biomarkers of cell proliferation, autophagy, and osteoclast differentiation was determined by western blotting. Pro-inflammatory gene expression was examined using quantitative reverse transcription-PCR. PR-MTA induced cytotoxicity in RAW 264.7cells in a dose-dependent manner, and iR-BC was more cytotoxic than PR-MTA. Low-dose and short-term treatments of both PR-MTA and iR-BC induced RAW 264.7cell proliferation. PR-MTA induced autophagy, whereas iR-BC did not. Neither PR-MTA nor iR-BC induced osteoclastogenesis. Pro-inflammatory genes were activated by both materials. However, the expression of inducible nitric oxide synthase (iNOS) mRNA was upregulated by iR-BC treatment, but not by PR-MTA treatment. Overall, dental PR-MTA and iR-BC induced pro-inflammatory genes but did not induce osteoclastogenesis in macrophages. PR-MTA and iR-BC induced M2 and M1 polarization, respectively, of RAW 264.7cells.

Investigating the Antioxidant Characteristics of Saccharina Latissima and Its Ability to Reduce Inflammation in RAW 264.7 Cells (P06-091-19)

ObjectivesThe goal of this study is to understand the anti-inflammatory characteristics of Saccharina latissima (sugar kelp). Sugar kelp is a brown macroalga that grows readily off the coast of Maine. Other seaweeds are known to contain bioactive chemicals that reduce inflammation—a contributing factor to metabolic syndrome and type 2 diabetes. It is hypothesized that injuring farmed sugar kelp by trimming the kelp may increase the beneficial bioactives and promote anti-inflammatory effects. The specific aims are (1) to produce extracts of Maine-grown sugar kelp through accelerated solvent extraction (ASE), (2) to measure the samples’ antioxidant capacity, and (3) to investigate the samples’ anti-inflammatory effect in vitro. MethodsSugar kelp samples were freeze-dried and extracts were produced using ASE. Total phenolic content was measured using the Folin-Ciocalteau assay, and radical scavenging capacity was measured with a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The sugar kelp extracts were tested for cytotoxicity in RAW 264.7 macrophages using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Lipopolysaccharide (LPS) was used to induce inflammation in the cells, and the attenuation effect of extract doses ranging from 12.5–50 μg total phenolics (TP)/mL was observed by measuring nitric oxide (NO) secretion with the Griess assay. ResultsPreliminary data shows that sugar kelp contains polyphenols (8.4–16.0 mg gallic acid equivalent/g dried biomass) and radical scavenging capacity (IC50 = 61.8–79.5 μg/mL). The data did not demonstrate the trimming the kelp has a beneficial effect to increase phenolic compounds. The polyphenolic contents of the trimmed samples were lower than the untrimmed (9.5 vs. 13.1 mg gallic acid equivalent/g dried biomass). The radical scavenging capacity decreased when the samples were trimmed (14.7%). The greatest decrease in NO production was at a concentration of 50 μg TP/mL for a trimmed sample (up to 78.5%), which supports the hypothesis. ConclusionsThis data suggests that sugar kelp may be a good source of antioxidants and trimmed samples may contain additional bioactives to help decrease inflammation. Further investigation is needed to confirm the hypothesis. Funding SourcesThe University of MaineThe University of New England

Zn Supplement-Antagonized Cadmium-Induced Cytotoxicity in Macrophages In Vitro: Involvement of Cadmium Bioaccumulation and Metallothioneins Regulation.

Cadmium (Cd) is a toxic metal leading to multiple forms of organ damage. Zinc (Zn) was reported as a potential antagonist against Cd toxicity. The present study investigates the antagonistic effect of Zn (20 μM) on Cd (20 or 50 μM) cytotoxicity in macrophages in vitro. The results shows that Cd exposure caused dose-dependent morphologic and ultrastructural alterations in RAW 264.7 macrophages. Zn supplement significantly inhibited Cd cytotoxicity in RAW 264.7 or HD-11 macrophages by mitigating cell apoptosis, excessive ROS output, and mitochondrial membrane depolarization. Notably, Zn supplement for 12 h remarkably prevented intracellular Cd2+ accumulation in 20 μM (95.99 ± 9.93 vs 29.64 ± 5.08 ng/106 cells; P = 0.0008) or 50 μM Cd (179.78 ± 28.66 vs 141.62 ± 22.15 ng/106 cells; P = 0.003) exposed RAW 264.7 cells. Further investigation found that Cd promoted metallothioneins (MTs) and metal regulatory transcription factor 1 (MTF-1) expression in RAW 264.7 macrophages. Twenty μM Zn supplement dramatically enhanced MTs and MTF-1 levels in Cd-exposed RAW 264.7 macrophages. Intracellular Zn2+ chelation or MTF-1 gene silencing inhibited MTs synthesis in Cd-exposed RAW 264.7 macrophages, which was accompanied by the declined expression of MTF-1, indicating that regulation of Zn on MTs was partially achieved by MTF-1 mobilization. In conclusion, this study demonstrates the antagonism of Zn against Cd cytotoxicity in macrophages and reveals its antagonistic mechanism by preventing Cd2+ bioaccumulation and promoting MTs expression.