Leflunomide-induced cardiac injury in adult male mice and bioinformatic approach identifying Nrf2/NF-κb signaling interplay.

Leflunomide (LFND) is an immunosuppressive and immunomodulatory disease-modifying antirheumatic drug (DMARD) that was approved for treating rheumatoid arthritis. LFND-induced cardiotoxicity was not fully investigated since its approval. We investigated the cardiac injury in male mice and identified the role of nuclear factor erythroid 2-related factor 2/nuclear factor-κ B (Nrf2/NF-κB) signaling. Male albino mice were assigned into five groups as control, vehicle, and LFND (2.5, 5, and 10 mg/kg). We investigated cardiac enzymes, histopathology, and the mRNA expression of Nrf2, NF-κB, BAX, and tumor necrosis factor-α (TNF-α). The bioinformatic study identified the interaction between LFND and Nrf2/NF-κB signaling; this was confirmed by amelioration in mRNA expression (0.5- to 0.34-fold decrease in Nrf2 and 2.6- to 4.61-fold increases in NF-κB genes) and increased (1.76- and 2.625-fold) serum creatine kinase (CK) and 1.38- and 2.33-fold increases in creatine kinase-MB (CK-MB). Histopathological results confirmed the dose-dependent effects of LFND on cardiac muscle structure in the form of cytoplasmic, nuclear, and vascular changes in addition to increased collagen deposits and apoptosis which were increased compared to controls especially with LFND 10 mg/kg. The current study elicits the dose-dependent cardiac injury induced by LFND administration and highlights, for the first time, dysregulation in Nrf2/NF-κB signaling.

Critical Antileishmanial in vitro Effects of Highly Examined Gold Nanoparticles.

INTRODUCTION: The current therapeutic armory for visceral leishmaniasis (VL) caused by Leishmania donovani complex is inadequate, coupled with serious limitations. Combination therapy has proved ineffective due to mounting resistance; however, the search for safe and effective drugs is desirable, in the absence of any vaccine. There is a growing interest in the application of nanoparticles for the therapeutic effectiveness of leishmaniasis. Aimed in this direction, we assessed the antileishmanial effect of gold nanoparticles (GNP) against L. donovani in vitro. METHODS: GNP were synthesized and characterized for particle size by dynamic light scattering (DLS) and atomic force microscopy (AFM) and for optical properties by UV-visible spectroscopy. Cytotoxicity of GNP was measured by the MTT proliferation assay. The antileishmanial activity of the nanoparticles was evaluated against L. donovani promastigotes and macrophage-infected amastigotes in vitro. RESULTS: GNP showed a strong SPR peak at 520 nm and mean particle size, polydispersity index (PDI), and zeta potential of 56.0 ± 10 nm, 0.3 ± 0.1 and -27.0 ± 3 mV, respectively. The GNPs were smooth and spherical with a mean particle diameter of 20 ± 5 nm. Nanoparticles [1.2-100 µM] did not reveal any cytotoxicity on RAW 264.7 murine macrophage cell line, but exerted significant activity against both promastigotes and amastigote stages of L. donovani with 50% inhibitory concentrations (IC50) of 18.4 ± 0.4 µM and 5.0 ± 0.3 µM, respectively. GNP showed significant antileishmanial activity with deformed morphology of parasites and the least number of surviving promastigotes after growth reversibility analysis. CONCLUSION: GNP may provide a platform to conjugate antileishmanial drugs onto the surface of nanoparticles to enhance their therapeutic effectiveness against VL. Further work is warranted, involving more in-depth mechanistic studies and in vivo investigations.

Antitumor Activity of Nitazoxanide against Colon Cancers: Molecular Docking and Experimental Studies Based on Wnt/ß-Catenin Signaling Inhibition.

In colon cancer, wingless (Wnt)/ß-catenin signaling is frequently upregulated; however, the creation of a molecular therapeutic agent targeting this pathway is still under investigation. This research aimed to study how nitazoxanide can affect Wnt/ß-catenin signaling in colon cancer cells (HCT-116) and a mouse colon cancer model. Our study included 2 experiments; the first was to test the cytotoxic activity of nitazoxanide in an in vitro study on a colon cancer cell line (HCT-116) versus normal colon cells (FHC) and to highlight the proapoptotic effect by MTT assay, flow cytometry and real-time polymerase chain reaction (RT-PCR). The second experiment tested the in vivo cytotoxic effect of nitazoxanide against 1,2-dimethylhydrazine (DMH) prompted cancer in mice. Mice were grouped as saline, DMH control and DMH + nitazoxanide [100 or 200 mg per kg]. Colon levels of Wnt and ß-catenin proteins were assessed by Western blotting while proliferation was measured via immunostaining for proliferating cell nuclear antigen (PCNA). Treating HCT-116 cells with nitazoxanide (inhibitory concentration 50 (IC50) = 11.07 µM) revealed that it has a more cytotoxic effect when compared to 5-flurouracil (IC50 = 11.36 µM). Moreover, it showed relatively high IC50 value (non-cytotoxic) against the normal colon cells. Nitazoxanide induced apoptosis by 15.86-fold compared to control and arrested the cell cycle. Furthermore, nitazoxanide upregulated proapoptotic proteins (P53 and BAX) and caspases but downregulated BCL-2. Nitazoxanide downregulated Wnt/ß-catenin/glycogen synthase kinase-3ß (GSK-3ß) signaling and PCNA staining in the current mouse model. Hence, our findings highlighted the cytotoxic effect of nitazoxanide and pointed out the effect on Wnt/ß-catenin/GSK-3ß signaling.

Leflunomide Induces Dose-Dependent Lung Injury in Mice via Stimulating Vimentin and NLRP3 Inflammasome Production.

Recently, the therapeutic importance of the anti-rheumatic drug, leflunomide, has been increased after the involvement of leflunomide in treating other autoimmune diseases and its promising role in retarding human malignancies. Few studies have focused on the safety in human or animals without clear outlining of the pathologic features on target organs. One clinical study related leflunomide with significant pulmonary complications in predisposed individuals. The current study examined the dose-dependent lung injury produced by leflunomide in healthy mice. Albino mice were allocated into four different groups. Group (1): Vehicle control group, Group (2-4): mice received leflunomide (2.5, 5 or 10 mg/kg), respectively, for 8 weeks and then lungs were dissected from the mice for histopathological examination and fibrosis evaluation (Masson's trichrome staining and α-smooth muscle actin immunohistochemistry). Enzyme linked immunosorbent assay was used to assess the vimentin and other inflammatory factors in the lung homogenate whereas Western blot analysis was employed to assess α-smooth muscle actin, vimentin and collagen 1. Results indicated that leflunomide induced dose-dependent pulmonary injury and the high dose and increased the vimentin, inflammatory markers (NLRP3 and interlukin-1ß). Histologic examination showed distorted architecture, marked inflammatory cells infiltrate and increase collagen content. The findings were supported by Western blotting and the immunohistochemical study which showed greater pulmonary α-smooth muscle actin and vimentin content. In conclusion, the current results highlighted that leflunomide produced dose-dependent pulmonary toxicities that requires further investigation of the nature of injury.

Perinatal Immunization With Vaccine-Grade Listeria monocytogenes Provides Protection Against Murine Th2 Airway Inflammation.

PURPOSE: Asthma is a chronic respiratory disorder that leads to inflammation and narrowing of the airways. Its global prevalence has attained epidemic levels and treatment options that reach beyond temporary relief of symptoms are urgently needed. Since the processes leading to clinically symptomatic asthma start early in life, we set out to systematically evaluate a neonatal immunotherapeutic based on Listeria monocytogenes (Lm) for the control of allergic sensitization. METHODS: We modified Lm to express the model allergen, ovalbumin (OVA), and tested the ability of neonatal immunization with this strain to control allergic sensitization in a mouse model of OVA-induced asthma. Mice were immunized as newborns with live or heat killed LmOVA or live Lm, followed 6 weeks later by allergic sensitization with OVA. In order to determine whether the TH1-polarizing effect of this vaccine vector inadvertently may exacerbate development of certain TH1-driven allergic diseases, mice immunized as newborns were assessed in a model of adult hypersensitivity pneumonitis (HP). RESULTS: Both LmOVA and Lm-control vaccines were highly effective in providing long-lasting protection from airway inflammation after only one immunization given perinatally. Serum antibody levels and lung cytokine production suggest that this prophylactic strategy is associated with an allergen specific TH1-dominated response. Specifically, LmOVA vaccinated mice displayed significantly elevated OVA-specific serum IgG2a, but no difference in anti-OVA IgE antibodies and only slightly decreased anti-OVA IgG1 antibodies. Importantly, Lm-based neonatal vaccination did not exacerbate Th1/Th17 driven HP, arguing against broad spectrum immune skewing. CONCLUSIONS: Our findings highlight the promise of early life Lm-based immunomodulatory interventions as a prophylactic strategy for allergic asthma.