Evaluating the Effect of Cinnarizine on Promastigotes and Amastigotes forms of Leishmania major

: As an important global disease, cutaneous leishmaniasis is associated with complications such as secondary infections and atrophic scars. The first line treatment with antimonials is expensive and reported to have serious side effects and enhance resistance development. The main objective of this study was to evaluate the effect of Cinnarizine on standard strains of Leishmania major because of paucity of information on this subject. Methods: In this experimental study, four concentrations of the drug (5, 10, 15 and 20 μg/ml) were added to Leishmania major cultures at 24, 48 and 72 hours intervals. MTT assays were performed to determine parasite viability and drug toxicity. Leishmania major promastigotes were augmented to the in vitro cultured macrophages (J774 cells) and then incubated for 72 hours. Half maximal inhibitory concentration (IC50) was ascertained by counting parasites. The inhibitory effect of the drug was compared with that of Glucantime. Flow-cytometry was performed to investigate apoptosis. Each test was repeated thrice. Results: The IC50 values of Cinnarizine after 72 hours were calculated to be 34.76 μg/ml and 23.73 μg/ml for promastigotes and amastigotes, respectively. The results of MTT assays showed 48 % promastigote viability after 72 hour-exposure to Cinnarizine at 20 μg/ml concentration. Programmed cell death in promastigote- and amastigote-infected macrophages was quantified to be 13.66 % and 98.7 %, respectively. Flow- cytometry analysis indicated that Cinnarizine induced early and late apoptosis in parasites. All treatments produced results which differed significantly from control group (P<0.05). Conclusion: Cinnarizine showed low toxicity with anti-leishmanial and apoptosis effects on both promastigote and intracellular amastigote forms. Therefore, we may suggest further assessment on animal models of this drug as candidates for cutaneous leishmaniasis therapy.

Antileishmanial Activity and Influence on Mitochondria of the Essential Oil from Tagetes lucida Cav. and Its Main Component

Current antileishmanial drugs are toxic, expensive, and resistance to them has emerged. Several studies have focused on natural products as alternatives. In the present work, the chemical composition, in vitro antileishmanial activity, cytotoxicity effects, and the influence on mitochondrial function of the essential oil from Tagetes lucida Cav. was determined, as well its main compound estragole. Forty-nine compounds were detected in the oil by gas chromatography-mass spectrometry (GC-MS), of which estragole was the main constituent (97%). The oil showed inhibition of the promastigotes of L. tarentolae and L. amazonensis (IC50 = 61.4 and 118.8 µg/mL, respectively), decreased oxygen consumption of L. tarentolae, disrupted mitochondrial membrane potential in L. amazonensis, inhibitory activity on the intracellular amastigote of L. amazonensis (IC50 = 14.2 ± 1.6 µg/mL), and cytotoxicity values ranging from 80.8 to 156 µg/mL against murine macrophages and J774 cells. Estragole displayed higher activity on promastigotes (IC50 = 28.5 and 25.5 µg/mL, respectively), amastigotes (IC50 = 1.4 ± 0.1 µg/mL), and cytotoxicity values ranging from 20.6 to 14.5 µg/mL, respectively, while on mitochondria, it caused a decrease of the membrane potential but did not inhibit oxygen consumption. The potential antileishmanial activity of the essential oil from T. lucida and estragole makes these compounds favorable candidates for exploration in further studies.

Acyclic Triterpenoid Isolated from Alpinia katsumadai Alleviates Formalin-Induced Chronic Mouse Paw Inflammation by Inhibiting the Phosphorylation of ERK and NF-κB

Chronic and excessive inflammation can destroy host organs and cause inflammatory diseases such as inflammatory bowel disease, asthma, and rheumatoid arthritis. In this study, we investigated the anti-inflammatory effects of Alpinia katsumadai seed-derived 2,3,5,22,23-pentahydroxy-2,6,10,15,19,23-hexamethyl-tetracosa-6,10,14,18-tetraene (PHT) using lipopolysaccharide (LPS)-stimulated J774 cells and a formalin-induced chronic paw inflammation mouse model. The in vitro results showed that PHT exhibited no cytotoxicity and decreased LPS-induced NO secretion. Additionally, PHT inhibited LPS-induced inducible NO synthase (iNOS) and cyclooxygenase 2 (COX2) protein expression. The quantitative real-time PCR results showed that PHT downregulated the gene expression of the proinflammatory cytokines interleukin-1β (IL-1β) and interleukin-6 (IL-6) but not tumor necrosis factor α (TNF-α). PHT inhibited the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK) and nuclear factor kappa light chain enhancer of activated B cells (NF-κB). In a mouse model, oral administration of 50 mg/kg PHT significantly alleviated both mouse paw thickness and volume. These results indicate that PHT has potential anti-inflammatory effects and should be considered a possible functional material.

Antibiotic Resistance and Azithromycin Resistance Mechanism of Legionella pneumophila Serogroup 1 in China

ABSTRACT Legionnaires’ disease, caused by Legionella pneumophila, has been treated primarily with antibiotics. However, few reports have been published on antibiotic-resistant Legionella in China. Our aim was to determine the azithromycin resistance mechanism of L. pneumophila serogroup 1 in China. The sensitivities of 149 L. pneumophila serogroup 1 strains, isolated from clinical cases or environmental water in China from 2002 to 2016, to five antibiotics, including erythromycin, azithromycin, levofloxacin, moxifloxacin, and rifampin, were evaluated. The mechanisms of the resistance of L. pneumophila serogroup 1 to azithromycin were studied. The expression levels of efflux pump gene lpeAB and the MIC of azithromycin-resistant strains in the presence and absence of the efflux pump inhibitor carbonyl cyanide-chlorophenylhydrazone (CCCP) were determined. All 149 strains were sensitive to erythromycin, levofloxacin, moxifloxacin, and rifampin, among which 25 of the strains exhibited azithromycin resistance. These 25 strains, including strains of sequence type 1 (ST1), ST144, ST150, ST154, and ST629, were screened. Expression of lpeAB was responsible for the reduced azithromycin susceptibility in all 25 of these strains. The phenotypes of 25 strains with virulence were linked by evaluating the intracellular growth ability in mouse macrophage J774 cells. Among the 25 strains, 60% were more virulent than the ATCC 33152 reference strain. The results determined in our study represent data supporting the further study of the antibiotic sensitivity of L. pneumophila strains in China.

Attenuation of Atherosclerosis by Protocatechuic Acid via Inhibition of M1 and Promotion of M2 Macrophage Polarization (P04-063-19)

Objectives Macrophage polarization has a vital impact on the progression of atherosclerosis (AS). Protocatechuic acid (PCA), a flavonol, displays notable atheroprotective effects, but its mechanisms have not been clearly defined. Therefore, we investigated whether PCA attenuated AS by regulating macrophage polarization. Methods Male apolipoprotein E-deficient (ApoE−/−) mice fed a normal chow diet (NCD), a high-cholesterol diet (HCD) or a high-cholesterol diet with PCA (PCA, 15 mg/kg body weight) for 14 weeks. In in vitro studies, mouse-macrophage cell line (J774 cells) and mouse-bone-marrow-derived macrophages (BMDMs) were stimulated with interferon γ (IFN γ) plus lipopolysaccharide (LPS) or interleukin 4 (IL-4) with or without pretreated with PCA for 24 h. Flow cytometry analysis, immunofluorescence, real-time quantitative reverse transcriptase and western blotting were performed for the next measurement. Results PCA consumption inhibited HCD-induced plaque formation (17.84% and 8.21% in the HCD and HCD with PCA groups, respectively; P < 0.05) and inflammatory responses in apolipoprotein E-deficient (ApoE−/−) mice (Fig. 1). Moreover, PCA suppressed the classically activated macrophage (M1) polarization, which decreased the secretion of synthesis of nitric oxide synthase (54.63% and 32.86% in the HCD and HCD with PCA group, respectively; P < 0.05) and proinflammatory factors (Fig. 2). PCA promoted alternatively activated macrophage (M2) activation, which increased the expression of arginine I (6.97% and 26.19% in the HCD and HCD + PCA group, respectively; P < 0.001) and anti-inflammatory factors (Fig. 2). PCA also regulated M1-M2 polarization in J774 cells and mouse-bone-marrow-derived macrophages. Finally, PCA reduced PI3K-Akt-mediated nuclear-factor-κB activation, thereby suppressing M1 polarization, and provoked signal-transducers-and-activators-of-transcription-6 phosphorylation and peroxisome-proliferator-activated-receptor-γ activation, leading to enhanced M2 activation. Conclusions Our data revealed that PCA alleviated AS progression by suppressing M1 polarization and promoting M2 activation. Funding Sources This work was received funding from the Major Projects of Guangzhou Health Collaborative Innovation, the State Key Program of National Natural Science Foundation of China and the Guangdong Science and Technology Project. Supporting Tables, Images and/or Graphs

Reduction of Real-Time Imaging of M1 Macrophage Chemotaxis toward Damaged Muscle Cells is PI3K-Dependent

Macrophages migrate and invade into damaged muscle rapidly and are important for muscle repair and subsequent regeneration. The exact cellular and biological events that cause macrophage migration toward injured muscle are not completely understood. In this study, the effect of macrophage differentiation on the chemotactic capability to invade local damaged muscle was investigated using an in vitro model of muscle injury. We used C2C12 cell myoblasts and J774 cell macrophages, and the “killed-C2C12” cells were combined with live C2C12 cells as a partially damaged muscle model. The cultured J774 cells, with or without lipopolysaccharide (LPS), were treated with Ly294002 (Ly), which is an inhibitor of phosphoinositide 3-kinase (PI3K). In order to evaluate the polarization effect of LPS stimulation on J774 cells, expression of cell surface Toll-like receptor 4 (TLR4), CD11c and CCR2, and expression of F-actin intensity, were analyzed by flow cytometry. The real-time horizontal chemotaxis assay of J774 cells was tested using the TAXIScan device. The expressions of TLR4, CD11c, and F-actin intensity in LPS-treated cells were significantly higher than those in Ctrl cells. In LPS-treated cells, the chemotactic activity toward damaged muscle cells completely disappeared. Moreover, the reduced chemotaxis depended far more on directionality than velocity. However, Ly treatment reversed the reduced chemotactic activity of the LPS-treated cells. In addition, cell-adhesion and F-actin intensity, but not CCR2 expression, in LPS-treated cells, was significantly reduced by Ly treatment. Taken together, our results suggest that the PI3K/Akt activation state drives migration behavior towards damaged muscle cells.

Microbial co-infection alters macrophage polarization, phagosomal escape, and microbial killing

Macrophages are important innate immune cells that respond to microbial insults. In response to multi-bacterial infection, the macrophage activation state may change upon exposure to nascent mediators, which results in different bacterial killing mechanism(s). In this study, we utilized two respiratory bacterial pathogens, Mycobacterium bovis (Bacillus Calmette Guẻrin, BCG) and Francisella tularensis live vaccine strain (LVS) with different phagocyte evasion mechanisms, as model microbes to assess the influence of initial bacterial infection on the macrophage response to secondary infection. Non-activated (M0) macrophages or activated M2-polarized cells (J774 cells transfected with the mouse IL-4 gene) were first infected with BCG for 24–48 h, subsequently challenged with LVS, and the results of inhibition of LVS replication in the macrophages was assessed. BCG infection in M0 macrophages activated TLR2-MyD88 and Mincle-CARD9 signaling pathways, stimulating nitric oxide (NO) production and enhanced killing of LVS. BCG infection had little effect on LVS escape from phagosomes into the cytosol in M0 macrophages. In contrast, M2-polarized macrophages exhibited enhanced endosomal acidification, as well as inhibiting LVS replication. Pre-infection with BCG did not induce NO production and thus did not further reduce LVS replication. This study provides a model for studies of the complexity of macrophage activation in response to multi-bacterial infection.