Major Outer Membrane Protein from Legionella pneumophila Inhibits Phagocytosis but Enhances Chemotaxis of RAW 264.7 Macrophages by Regulating the FOXO1/Coronin-1 Axis

Legionella pneumophila is an intracellular pathogen that can cause Legionnaire’s disease by invading alveolar epithelial cells and macrophages. The major outer membrane protein (MOMP) plays an important role in the interaction between bacteria and host cells. However, the role of MOMP in the process of L. pneumophila invasion of macrophages and its working mechanism remain unknown. We aimed to explore the effects of MOMP on phagocytosis and chemotaxis of RAW 264.7 macrophages. The chemotactic activity, toxicity, and phagocytosis of RAW 264.7 cocultured with different concentrations of MOMP were determined by Transwell, CCK-8, and neutral red uptake assays, respectively. Target genes were detected by double-luciferase and pull down assays. qRT-PCR and Western blot were performed to analyze the expression of several important proteins involved in the immune response pathway, including coronin-1, interleukins (IL-10), forkhead transcription factor 1 (FOXO1), nucleotide-binding oligomerization domain protein (NOD) 1, NOD2, and receptor-interacting protein (RIP) 2. After coculturing with MOMP, cytological observation indicated a decrease of phagocytosis and a marked increase of chemotaxis in RAW 264.7 macrophages. The phagocytosis degree of RAW 264.7 macrophage varied with the concentration gradient of MOMP in a time-dependent manner. MOMP could increase the expression levels of MCP-1, IL-10, NOD2, and RIP2 and decrease the expression levels of FOXO1 and coronin-1 in cell culture supernatants. In addition, we found that FOXO1 could promote its transcription by binding to the promoter of coronin-1. The results of the present study suggested that MOMP could inhibit phagocytosis and facilitate chemotaxis of RAW 264.7 macrophage, which might be associated with the FOXO1/coronin-1 axis.

Thymol and Piperine-Loaded Poly(D,L-lactic-co-glycolic acid) Nanoparticles Modulate Inflammatory Mediators and Apoptosis in Murine Macrophages

This study aimed at preparing and characterizing thymol, eugenol, and piperine-loaded poly(D,L-lactic-co-glycolic acid) nanoparticles and evaluating the effect on inflammatory mediators secretion and apoptosis in Raw 264.7 macrophage cells. Nanoparticles were produced by the solvent evaporation technique. Dynamic light scattering and scanning electron microscopy were used to study the physicochemical characteristics. Raw 264.7 macrophage cells were used as a model for in vitro assays. The 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium assay was used to determine the cytotoxicity of the formulated nanoparticles. An annexin V apoptosis detection kit was used to assess apoptosis. Nitric oxide production was determined using the Griess reagent, and the inflammatory mediators level was evaluated with Th1/Th2 cytokine and fluorometric cyclooxygenase kits. The loaded nanoparticles showed a particle size around 190 nm with a low polydispersity between 0.069 and 0.104 and a zeta potential between–1.2 and–9.5 mV. Reduced cytotoxicity of nanoparticles compared to free molecules against Raw 264.7 macrophage cells was observed and seemed to occur through a mechanism associated with apoptosis. A decrease in cyclooxygenase enzyme activity with an increasing concentration was observed. Both free molecules and nanoparticles showed their capacity to modulate the inflammatory process mostly by inhibiting the investigated inflammatory cytokines. The data presented in this study indicate that thymol and piperine-loaded poly(D,L-lactic-co-glycolic acid nanoparticles could serve as a novel anti-inflammatory colloidal drug delivery system with reduced toxicity. However, further study should be considered to optimize the formulation’s loading capacity and thereby probably enhance their bioactivity in treating inflammatory diseases.

Integrated Phytochemical Analysis Based on UPLC–MS/MS and Network Pharmacology Approaches to Explore the Effect of Odontites vulgaris Moench on Rheumatoid Arthritis

Odontites vulgaris Moench has the effect of clearing away heat, detoxification, dispelling wind, and clearing dampness. In this study, the potential anti-inflammatory compounds of O. vulgaris were investigated using ultra-high-performance liquid chromatography–mass spectrometry (UPLC–MS/MS) combined with the network pharmacology approach and further confirmed on an LPS-activated RAW 264.7 macrophage model. Monomer compounds were prepared from the active fraction using modern advanced separation and purification methods. UPLC–Q-Exactive HRMS was used to identify the chemical compounds in the active fractions of O. vulgaris. D-mannitol, geniposidic acid, salidroside, shanzhiside methyl ester, eleutheroside B, geniposide, 7,8-dihydroxycoumarin, gardoside methyl ester, arenarioside, vanillic acid, p-hydroxy-cinnamic acid, melampyroside, syringaresinol, tricin, and diosmetin were isolated from O. vulgaris for the first time. A compound database of O. vulgaris was established based on the existing literature to predict the mechanism of O. vulgaris in the treatment of rheumatoid arthritis. The results suggest that the PI3K-Akt pathway mediates O. vulgaris and deserves more attention in the treatment of RA. Finally, the anti-rheumatoid arthritis effects of the four target compounds were validated with the decreased levels of NO, TNF-α, IL-6 and IL-1β in RAW 264.7 macrophage cells treated with LPS. The present study explored the potential targets and signaling pathways of O. vulgaris in the treatment of RA, which may help to illustrate the mechanisms involved in the action of O. vulgaris and may provide a better understanding of the relationship between O. vulgaris and RA. This study provides novel insights into the development of new drugs and utilization of Mongolian traditional Chinese medicine resources.

In vitro Cytotoxicity and Antioxidant Study of Rhodomyrtus tomentosa (Aiton) Hassk. Ethanolic Leaf Extract on LPS-induced RAW 264.7 Macrophage Cells

Aims: Ethanol extract of Rhodomyrtus tomentosa leaves found specifically on Malaysian soil was used to further investigate the antioxidant properties and cytotoxicity against RAW 264.7 macrophage cells in the search for a safer and effective natural antioxidant agent. Study Design: Antioxidant potential of R. tomentosa were analyzed through series of spectrometric assays and cell-based bioassays model. Place and Duration of Study: This study was carried out at Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 43200, Selangor, Malaysia from the year of 2019 to 2021. Methodology: R. tomentosa leaves were subjected to extraction with 95% ethanol. The extracts were then denoted as ethanolic leaves extract of R. tomentosa or EtRT extract. EtRT extract were then screen for its antioxidant activity (AOA) and total antioxidant capacity (TAC) through DPPH radical scavenging assay and ferric reducing antioxidant power (FRAP) assay. After that, EtRT extract were brought to observe its toxicity against RAW 264.7 macrophage cells in MTT assay. Once their toxicity was obtained, EtRT extracts were finally tested for their ability to inhibit intracellular reactive oxygen species (ROS) and nitric oxide (NO) inhibition in RAW 264.7 macrophage cells to further analyze their antioxidant properties. Results: In this study, EtRT extracts dose dependently showed the ability to scavenge DPPH radicals and reduce ferric ions during DPPH radicals scavenging activity assay and ferric reducing antioxidant power assay (FRAP), respectively. In DPPH radical scavenging activity assay, EtRT extracts showed EC50 value at 12.37 ± 1.73 µg/mL with ARP value of 0.08 almost as near as ascorbic acid’s ARP value which is 0.09. Further into the study, EtRT extract were not cytotoxic to RAW 264.7 macrophage cells at concentrations 3.91 µg/mL and lower which showed more than 86.4% cell viability with IC50 value at 204.70 ± 5.30 µg/mL. EtRT extract possessed the ability to inhibit ROS production on LPS-induced RAW 264.7 macrophage cells at 7.813 µg/mL and lower, with the highest concentration can reduce up to 30.20% ± 1.01 out of the total ROS produced by the induced cells. Furthermore, EtRT extract also have evidenced that it is able to significantly inhibit NO production by the LPS-induced RAW 264.7 macrophage cells at 7 µg/mL and lower being the highest at 56.73% ± 0.11 inhibition of the highest concentration tested. Conclusions: This study suggests that EtRT extracts have the potential to reduce LPS-induced oxidative stress due to the antioxidant activities of phenolic compounds in the extracts, and that at low doses, EtRT extracts had low to no cytotoxicity on RAW 264.7 macrophage cells. As a result, EtRT extract could be a promising natural medicinal agent for the treatment of oxidative stress.

Antifungal Activity of Isolated Compounds from the Leaves of Combretum erythrophyllum (Burch.) Sond. and Withania somnifera (L.) Dunal against Fusarium Pathogens

Crop diseases caused by Fusarium pathogens, among other microorganisms, threaten crop production in both commercial and smallholder farming. There are increasing concerns about the use of conventional synthetic fungicides due to fungal resistance and the associated negative effects of these chemicals on human health, livestock and the environment. This leads to the search for alternative fungicides from nature, especially from plants. The objectives of this study were to characterize isolated compounds from Combretum erythrophyllum (Burch.) Sond. and Withania somnifera (L.) Dunal leaf extracts, evaluate their antifungal activity against Fusarium pathogens, their phytotoxicity on maize seed germination and their cytotoxicity effect on Raw 264.7 macrophage cells. The investigation led to the isolation of antifungal compounds characterized as 5-hydroxy-7,4′-dimethoxyflavone, maslinic acid (21-hydroxy-3-oxo-olean-12-en-28-oic acid) and withaferin A (4β,27-dihydroxy-1-oxo-5β,6β-epoxywitha-2-24-dienolide). The structural elucidation of the isolated compounds was established using nuclear magnetic resonance (NMR) spectroscopy, mass spectroscopy (MS) and, in comparison, with the available published data. These compounds showed good antifungal activity with minimum inhibitory concentrations (MIC) less than 1.0 mg/mL against one or more of the tested Fusarium pathogens (F. oxysporum, F. verticilloides, F. subglutinans, F. proliferatum, F. solani, F. graminearum, F. chlamydosporum and F. semitectum). The findings from this study indicate that medicinal plants are a good source of natural antifungals. Furthermore, the isolated antifungal compounds did not show any phytotoxic effects on maize seed germination. The toxicity of the compounds A (5-hydroxy-7,4′-dimethoxyflavone) and AI (4β,27-dihydroxy-1-oxo-5β,6β-epoxywitha-2-24-dienolide) was dose-dependent, while compound B (21-hydroxy-3-oxo-olean-12-en-28-oic acid) showed no toxicity effect against Raw 264.7 macrophage cells.