Anti-aging effect of nifuroxazide on skin changes of aged male rat models via modulating immunoreactivity of IL-6/NF-κB/Caspase-3.

PURPOSE: To evaluate nifuroxazide's (NIF's) anti-aging characteristics in a skin-aging rat model for the first time in order to create effective preventive measures and anti-aging skin therapies. MATERIALS AND METHODS: Thirty randomly selected aged male rats were assorted into three equal groups; aged control group, treated NIF I, aged rats were treated with NIF (10mg/kg, orally once daily for 14 consecutive days), and treated NIF II, aged rats were treated with NIF (20mg/kg, orally once daily for 14 consecutive days). Skin samples were obtained from the dorsal skin of the aged male rats and processed for tissue biochemical MDA, histological (Hx&E and Masson's Trichrome stains), and immunohistochemical (IL-6, NF-κB, and caspase-3) analysis. RESULTS: Group I aged male albino rat skin illustrated evident distorted epidermis and dermis, disorganization of collagen fibers with marked multiple spaces of collagen fibers loss in the dermis, marked reduction of total epidermal thickness and mean area percent of collagen fibers, elevated tissue MDA level and strong positive IL-6, NF-κB, and caspase-3 immune reaction. The anti-aging benefits of NIF on skin aging are demonstrated by a marked improvement in histological alterations in the form of a well-organized epidermis and dermis, most collagen fibers in the dermis appear closely packed, significant elevation of total epidermal thickness and mean area percent of collagen fibers, a significant decrease of tissue MDA level, and immunoexpression of the inflammatory markers, IL-6, and NF-κB, and the apoptotic marker caspase-3. CONCLUSIONS: This study found that group III, which received 20mg/kg of NIF, experienced more pronounced and noticeable improvements in skin aging than group II, which received 10mg/kg of NIF.

Targeting STAT3 prevents bile reflux-induced oncogenic molecular events linked to hypopharyngeal carcinogenesis.

The signal transducer and activator of transcription 3 (STAT3) oncogene is a transcription factor with a central role in head and neck cancer. Hypopharyngeal cells (HCs) exposed to acidic bile present aberrant activation of STAT3, possibly contributing to its oncogenic effect. We hypothesized that STAT3 contributes substantially to the bile reflux-induced molecular oncogenic profile, which can be suppressed by STAT3 silencing or pharmacological inhibition. To explore our hypothesis, we targeted the STAT3 pathway, by knocking down STAT3 (STAT3 siRNA), and inhibiting STAT3 phosphorylation (Nifuroxazide) or dimerization (SI3-201; STA-21), in acidic bile (pH 4.0)-exposed human HCs. Immunofluorescence, luciferase assay, Western blot, enzyme-linked immunosorbent assay and qPCR analyses revealed that STAT3 knockdown or pharmacologic inhibition significantly suppressed acidic bile-induced STAT3 activation and its transcriptional activity, Bcl-2 overexpression, transcriptional activation of IL6, TNF-α, BCL2, EGFR, STAT3, RELA(p65), REL and WNT5A, and cell survival. Our novel findings document the important role of STAT3 in bile reflux-related molecular oncogenic events, which can be dramatically prevented by STAT3 silencing. STA-21, SI3-201 or Nifuroxazide effectively inhibited STAT3 and cancer-related inflammatory phenotype, encouraging their single or combined application in preventive or therapeutic strategies of bile reflux-related hypopharyngeal carcinogenesis.

Nifuroxazide ameliorates pulmonary fibrosis by blocking myofibroblast genesis: a drug repurposing study.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a serious interstitial lung disease with a complex pathogenesis and high mortality. The development of new drugs is time-consuming and laborious; therefore, research on the new use of old drugs can save time and clinical costs and even avoid serious side effects. Nifuroxazide (NIF) was originally used to treat diarrhoea, but more recently, it has been found to have additional pharmacological effects, such as anti-tumour effects and inhibition of inflammatory diseases related to diabetic nephropathy. However, there are no reports regarding its role in pulmonary fibrosis. METHODS: The therapeutic effect of NIF on pulmonary fibrosis in vivo was measured by ELISA, hydroxyproline content, H&E and Masson staining, immunohistochemistry (IHC) and western blot. Immune cell content in lung tissue was also analysed by flow cytometry. NIF cytotoxicity was evaluated in NIH/3T3 cells, human pulmonary fibroblasts (HPFs), A549 cells and rat primary lung fibroblasts (RPLFs) using the MTT assay. Finally, an in vitro cell model created by transforming growth factor-ß1 (TGF-ß1) stimulation was assessed using different experiments (immunofluorescence, western blot and wound migration assay) to evaluate the effects of NIF on the activation of NIH/3T3 and HPF cells and the epithelial-mesenchymal transition (EMT) and migration of A549 cells. RESULTS: In vivo, intraperitoneal injection of NIF relieved and reversed pulmonary fibrosis caused by bleomycin (BLM) bronchial instillation. In addition, NIF inhibited the expression of a variety of cellular inflammatory factors and immune cells. Furthermore, NIF suppressed the activation of fibroblasts and EMT of epithelial cells induced by TGF-ß1. Most importantly, we used an analytical docking experiment and thermal shift assay to further verify that NIF functions in conjunction with signal transducer and activator of transcription 3 (Stat3). Moreover, NIF inhibited the TGF-ß/Smad pathway in vitro and decreased the expression of phosphorylated Stat3 in vitro and in vivo. CONCLUSION: Taken together, we conclude that NIF inhibits and reverses pulmonary fibrosis, and these results support NIF as a viable therapeutic option for IPF treatment.

Synthesis and in vitro antileishmanial efficacy of benzyl analogues of nifuroxazide.

Leishmaniasis is a vector-borne parasitic disease that mostly affects populations in tropical and subtropical countries. There is currently no vaccine to protect against and only a handful of drugs are available to treat this disease. Leishmaniasis is curable, but its eradication and elimination are hindered by the emergence of multidrug resistant strains of the causative pathogens, accentuating the need for new and effective antileishmanial drugs. In search for such agents, nifuroxazide, a clinical antibiotic, was evaluated through investigation of its benzyl analogues for in vitro antileishmanial efficacy against promastigotes of various Leishmania (L.) strains. The monobenzylated analogues 1 and 2 were the most potent of all, possessing nanomolar activities up to 10-fold higher than the parent drug nifuroxazide against all three tested Leishmania strains. Both analogues stand as antipromastigote hits for further lead investigation into their potential to act as new antileishmanial agents.

Nifuroxazide Mitigates Angiogenesis in Ehlrich's Solid Carcinoma: Molecular Docking, Bioinformatic and Experimental Studies on Inhibition of Il-6/Jak2/Stat3 Signaling.

Nifuroxazide is an antidiarrheal medication that has promising anticancer activity against diverse types of tumors. The present study tested the anticancer activity of nifuroxazide against Ehrlich's mammary carcinoma grown in vivo. Furthermore, we investigated the effect of nifuroxazide on IL-6/jak2/STAT3 signaling and the possible impact on tumor angiogenesis. The biological study was supported by molecular docking and bioinformatic predictions for the possible effect of nifuroxazide on this signaling pathway. Female albino mice were injected with Ehrlich carcinoma cells to produce Ehrlich's solid tumors (ESTs). The experimental groups were as follows: EST control, EST + nifuroxazide (5 mg/kg), and EST + nifuroxazide (10 mg/kg). Nifuroxazide was found to reduce tumor masses (730.83 ± 73.19 and 381.42 ± 109.69 mg vs. 1099.5 ± 310.83) and lessen tumor pathologies. Furthermore, nifuroxazide downregulated IL-6, TNF-α, NFk-ß, angiostatin, and Jak2 proteins, and it also reduced tumoral VEGF, as indicated by ELISA and immunohistochemical analysis. Furthermore, nifuroxazide dose-dependently downregulated STAT3 phosphorylation (60% and 30% reductions, respectively). Collectively, the current experiment shed light on the antitumor activity of nifuroxazide against mammary solid carcinoma grown in vivo. The antitumor activity was at least partly mediated by inhibition of IL-6/Jak2/STAT3 signaling that affected angiogenesis (low VEGF and high angiostatin) in the EST. Therefore, nifuroxazide might be a promising antitumor medication if appropriate human studies will be conducted.

Combination of attenuated Salmonella carrying PD-1 siRNA with nifuroxazide for colon cancer therapy.

Colon cancer is a member of malignant tumors in the digestive system. Traditional treatment strategies are ineffective and improving the treatment of colon cancer is an urgent need. Targeting programmed cell death-1 (PD-1) by monoclonal antibodies has shown some therapeutic effectiveness and has advantages. Additionally, the Stat3 inhibitor nifuroxazide was employed to promote the antitumor activity. Here, we hypothesized that combining nifuroxazide with PD-1 small interfering RNA carried by attenuated Salmonella would exert a synergistic antitumor effect on colon cancer. Indeed, treatment with this combination effectively inhibited the development of colon cancer in mice and improved the survival rate. These two novel anticancer agents worked synergistically to elicit potent antitumor immunity and achieve improved therapeutic efficacy. The underlying mechanisms are mainly involved with immune regulation and cell apoptosis. This study provides a previous framework for combining this Stat3 inhibitor with RNAi designed to block immune checkpoint signaling for cancer therapy.

Nifuroxazide induces apoptosis, inhibits cell migration and invasion in osteosarcoma.

Osteosarcoma is the most common primary malignancy of bone and characterized by an appendicular primary tumor with a high rate of metastasis to the lungs. Unfortunately, there is no effective strategy to treat osteosarcoma in current clinical practice. In this study, the anticancer effects and potential mechanisms of nifuroxazide, an oral nitrofuran antibiotic, on two osteosarcoma cell lines were investigated. The results of the antiproliferative activity in vitro showed that nifuroxazide inhibited cell proliferation of UMR106 and MG63 cells in a dose- and time-dependent manner. Interestingly, nifuroxazide showed low toxicity to non-tumor cells (HEK 293 T). In addition, ROS-mitochondrial mediated apoptosis was observed after treatment of nifuroxazide. Moreover, nifuroxazide could significantly inhibit osteosarcoma cells migration and invasion via p-Stat3, MMP-2 and MMP-9 mediated signaling pathway. Taken together, our results suggested that nifuroxazide could be a promising agent for osteosarcoma treatment by inhibiting cell proliferation, inducing cell apoptosis and impairing cell migration and invasion.

[Effect of Nifuroxazide on Proliferation, Migration, and Invasion of Thyroid Papillary Carcinoma Cells].

OBJECTIVE: To explore the effect of nifuroxazide on proliferation, migration, and invasion of thyroid papillary carcinoma cells. METHODS: BCPAP and TPC-1 cell lines treated with different concentration (0, 1.25, 2.5, 5, 10, 20 µmol/L) of nifuroxazide, respectively. Cell viability and proliferation of BCPAP and TPC-1 was evaluated by MTT and colony formation assay. Apoptosis analysis and cell nuclear changes were determined by staining with Hoechst 33258 and visualized by a fluorescence microscope after treatment with nifuroxazide. Western blot analysis was used to evaluate protein expressions of apoptosis and invasion of BCPAP cells treated (48 h) with nifuroxazide. Transwell assay was conducted to evaluate ability of cell migration and invasion. RESULTS: After being treated with nifuroxazide (0, 1.25, 2.5 µmol/L and 0, 1.25 µmol/L) for 24, 48, 72 h respectively, decreased proliferations of BCPAP and TPC-1 cell lines were not obvious ( P>0.05). However, treated BCPAP and TPC-1 cells with higher concentration respectively (5, 10, 20 µmol/L and 5, 10 µmol/L) of nifuroxazide for 24, 48, 72 h, the inhibitory effects were significantly obvious ( P<0.05), and the inhibitory effects were increased in a CM(155mm]concentration- and time-dependent manner. The inhibition in proliferation of TPC-1 cell with nifuroxazide (2.5, CM)]5 µmol/L) took effect from 72 h and 48 h ( P<0.05), respectively. Clone formations of BCPAP and TPC-1 cells were significantly inhibited after being exposed to nifuroxazide (2.5, 5 µmol/L) for 10 d ( P<0.05). Hoechst 33258 staining assay showed that nifuroxazide (10 µmol/L) treatment resulted in cell shrinking, nuclear fragmentation and formation of condensed nuclei with bright-blue fluorescence. After 48 h, the percentage of apoptotic cells of BCPAP and TPC-1 significantly increased respectively as the concentration of nifuroxazide with 10 µmol/L ( P<0.005). Pro-apoptotic protein CC-3 and Bax expression levels increased significantly ( P<0.05), and the expression of anti-apoptotic protein Bcl-2 decreased significantly ( P<0.05) in BCPAP cells after nifuroxazide-treatment (10 µmol/L) for 48 h. The percentage of migrations and invasions of BCPAP and TPC-1 significantly decreased ( P<0.05) in the presence of nifuroxazide (10 µmol/L, 48 h). Nifuroxazide (10 µmol/L) treatment significantly decreased the expressions of matrix metalloproteinase (MMP)-2 and MMP-9 in BCPAP cells ( P<0.05) . Expression of MMPs family inhibitor-tissue inhibitors of metalloproteinase (TIMP)-2 increased ( P<0.05). CONCLUSION: Nifuroxazide inhibits the proliferation of thyroid cancer cells BCPAP and TPC-1, induceds the cell apoptosis by up-regulating the expressions of CC-3 and Bax proteins in vitro, and blocks migration and invasion of cells in vitro by reducing protein expressions of MMP-2 and MMP-9.

Renoprotective effect of nifuroxazide in diabetes-induced nephropathy: impact on NFκB, oxidative stress, and apoptosis.

The prevalence of diabetes mellitus (DM) is drastically increased worldwide. Diabetic nephropathy (DN) is a microvascular complication of DM and a common cause of end stage renal disease (ESRD). DN has been recently reported as the most common cause among ESRD patients. Shortage of a definitive cure for DN and the social and economic burden of this disease provide considerable impetus for development of new therapies. In the present study, we evaluated the effect of nifuroxazide, a potent inhibitor of Janus kinase/signal transducers and activators of transcription (JAK2/STAT3), on nuclear factor kappa B (NFκB), oxidative stress, and apoptosis in diabetic kidney. Following induction of diabetes by single dose of streptozotocin (50 mg/kg), nifuroxazide was administrated to diabetic rats (25 mg/kg/day, orally) for 8 weeks. Our results showed that nifuroxazide treatment, attenuated diabetes-induced damage in renal structure, ameliorated oxidative stress, triggered antioxidant defense, reduced NFκB nuclear translocation and cleaved caspase-3 expression and down regulated the activity of apoptotic enzymes (caspase-3/caspase-8/caspase-9) in diabetic kidney. In conclusion, nifuroxazide exhibited renoprotective effect in diabetic kidney via dampening NFκB activation, oxidative stress, and apoptosis.

Nifuroxazide repurposing for protection from diabetes-induced retinal injury in rats: Implication of oxidative stress and JAK/STAT3 axis.

The prevalence of diabetes mellitus (DM) is alarmingly increasing worldwide. Diabetic retinopathy (DR) is a prevailing DM microvascular complication, representing the major cause of blindness in working-age population. Inflammation is a crucial player in DR pathogenesis. JAK/STAT3 axis is a pleotropic cascade that modulates diverse inflammatory events. Nifuroxazide (Nifu) is a commonly used oral antibiotic with reported JAK/STAT3 inhibition activity. The present study investigated the potential protective effect of Nifu against diabetes-induced retinal injury. Effect of Nifu on oxidative stress, JAK/STAT3 axis and downstream inflammatory mediators has been also studied. Diabetes was induced in Sprague Dawley rats by single intraperitoneal injection of streptozotocin (50 mg/kg). Animals were assigned into four groups: normal, Nifu control, DM, and DM + Nifu. Nifu was orally administrated at 25 mg/kg/day for 8 weeks. The effects of Nifu on oxidative stress, JAK/STAT3 axis proteins, inflammatory factors, tight junction proteins, histological, and ultrastructural alterations were evaluated using spectrophotometry, gene and protein analyses, and histological studies. Nifu administration to diabetic rats attenuated histopathological and signs of retinal injury. Additionally, Nifu attenuated retinal oxidative stress, inhibited JAK and STAT3 phosphorylation, augmented the expression of STAT3 signaling inhibitor SOCS3, dampened the expression of transcription factor of inflammation NF-κB, and inflammatory cytokine TNF-α. Collectively, the current study indicated that Nifu alleviated DR progression in diabetic rats, suggesting beneficial retino-protective effect. This can be attributed to blocking JAK/STAT3 axis in retinal tissues with subsequent amelioration of oxidative stress and inflammation.

Nifuroxazide Prevents Chikungunya Virus Infection Both In Vitro and In Vivo via Suppressing Viral Replication.

Chikungunya virus (CHIKV) is a reemerging arbovirus causing disease on a global scale, and the potential for its epidemics remains high. CHIKV has caused millions of cases and heavy economic burdens around the world, while there are no available approved antiviral therapies to date. In this study, nifuroxazide, an FDA-approved antibiotic for acute diarrhea or colitis, was found to significantly inhibit a variety of arboviruses, although its antiviral activity varied among different target cell types. Nifuroxazide exhibited relatively high inhibitory efficiency in yellow fever virus (YFV) infection of the hepatoma cell line Huh7, tick-borne encephalitis virus (TBEV) and west nile virus (WNV) infection of the vascular endothelial cell line HUVEC, and CHIKV infection of both Huh7 cells and HUVECs, while it barely affected the viral invasion of neurons. Further systematic studies on the action stage of nifuroxazide showed that nifuroxazide mainly inhibited in the viral replication stage. In vivo, nifuroxazide significantly reduced the viral load in muscles and protected mice from CHIKV-induced footpad swelling, an inflammation injury within the arthrosis of infected mice. These results suggest that nifuroxazide has a potential clinical application as an antiviral drug, such as in the treatment of CHIKV infection.

The enteroprotective effect of nifuroxazide against methotrexate-induced intestinal injury involves co-activation of PPAR-γ, SIRT1, Nrf2, and suppression of NF-κB and JAK1/STAT3 signals.

Methotrexate (MTX) has long manifested therapeutic efficacy in several neoplastic and autoimmune disorders. However, MTX-associated intestinal toxicity restricts the continuation of treatment. Nifuroxazide (NIF) is an oral antibiotic approved for gastrointestinal infections as an effective antidiarrheal agent with a high safety profile. The current study was designed to explore the potential efficacy of NIF in alleviating intestinal toxicity associated with MTX chemotherapy with the elucidation of the proposed molecular mechanisms. Rats were administered NIF (50 mg/kg; p.o.) for ten days. On day five, a single i.p. injection of MTX (20 mg/kg) was given to induce intestinal intoxication. At the end of the experiment, duodenal tissue samples were isolated for biochemical, Western blotting, immunohistochemical (IHC), and histopathological analysis via H&E, PSA, and Alcian blue stains. NIF showed antioxidant enteroprotective effects against MTX intestinal intoxication through enhanced expression of the redox-sensitive signals of PPAR-γ, SIRT1, and Nrf2 estimated by IHC. Moreover, NIF down-regulated the pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6), NF-κB protein expression, and the phosphorylation of JAK1/STAT3 proteins, leading to mitigation of intestinal inflammation. In accordance, the histological investigation revealed that NIF ameliorated the intestinal pathological changes, preserved the goblet cells, and reduced the inflammatory cells infiltration. Therefore, NIF could be a promising candidate for adjunctive therapy with MTX to mitigate the associated intestinal injury and increase its tolerability.

Alfalfa biomass as a green source for the synthesis of N,S-CDs via microwave treatment. Application as a nano sensor for nifuroxazide in formulations and gastric juice.

BACKGROUND: Researchers have investigated different techniques for synthesis of carbon dots. These techniques include Arc discharge, laser ablation, oxidation, water/solvothermal, and chemical vapor deposition. However, these techniques suffer from some limitations like the utilization of gaseous charged particles, high current, high temperature, potent oxidizing agents, non-environmentally friendly carbon sources, and the generation of uneven particle size. Therefore, there was a significant demand for the adoption of a new technology that combines the environmentally friendly aspects of both bio-based carbon sourcing and synthesis technique. RESULTS: Medicago sativa L (alfalfa)-derived N, S-CDs have been successfully synthesized via microwave irradiation. The N,S-CDs exhibit strong fluorescence (λex/em of 320/420 nm) with fluorescence quantum yield of 2.2 % and high-water solubility. The produced N,S-CDs were characterized using TEM, EDX, Zeta potential analysis, IR, UV-Visible, and fluorescence spectroscopy. The average diameter of the produced N, S-CDs was 4.01 ± 1.2 nm, and the Zeta potential was -24.5 ± 6.63 mv. The stability of the produced nano sensors was also confirmed over wide pH range, long time, and in presence of different ions. The synthesized N, S-CDs were employed to quantify the antibacterial drug, nifuroxazide (NFZ), by fluorescence quenching via inner filter effect mechanism. The method was linear with NFZ concentration ranging from 1.0 to 30.0 µM. LOD and LOQ were 0.16 and 0.49 µM, respectively. The method was applied to quantify NFZ in simulated gastric juice (SGJ) with % recovery 99.59 ± 1.4 in addition to pharmaceutical dosage forms with % recovery 98.75 ± 0.61 for Antinal Capsules® and 100.63 ± 1.54 for Antinal suspension®. The Method validation was performed in compliance with the criteria outlined by ICH. SIGNIFICANCE AND NOVELTY: The suggested approach primarily centers on the first-time use of alfalfa, an ecologically sustainable source of dopped-CDs, and a cost-effective synthesis technique via microwave irradiation, which is characterized by low energy consumption, minimized reaction time, and the ability to control the size of the produced CDs. This is in line with the growing global recognition of the implementation of green analytical chemistry principles.

ALDH1A3-acetaldehyde metabolism potentiates transcriptional heterogeneity in melanoma.

Cancer cellular heterogeneity and therapy resistance arise substantially from metabolic and transcriptional adaptations, but how these are interconnected is poorly understood. Here, we show that, in melanoma, the cancer stem cell marker aldehyde dehydrogenase 1A3 (ALDH1A3) forms an enzymatic partnership with acetyl-coenzyme A (CoA) synthetase 2 (ACSS2) in the nucleus to couple high glucose metabolic flux with acetyl-histone H3 modification of neural crest (NC) lineage and glucose metabolism genes. Importantly, we show that acetaldehyde is a metabolite source for acetyl-histone H3 modification in an ALDH1A3-dependent manner, providing a physiologic function for this highly volatile and toxic metabolite. In a zebrafish melanoma residual disease model, an ALDH1-high subpopulation emerges following BRAF inhibitor treatment, and targeting these with an ALDH1 suicide inhibitor, nifuroxazide, delays or prevents BRAF inhibitor drug-resistant relapse. Our work reveals that the ALDH1A3-ACSS2 couple directly coordinates nuclear acetaldehyde-acetyl-CoA metabolism with specific chromatin-based gene regulation and represents a potential therapeutic vulnerability in melanoma.

Utilizing machine learning to identify nifuroxazide as an inhibitor of ubiquitin-specific protease 21 in a drug repositioning strategy.

Ubiquitin-specific protease (USP), an enzyme catalyzing protein deubiquitination, is involved in biological processes related to metabolic disorders and cancer proliferation. We focused on constructing predictive models tailored to unveil compounds boasting USP21 inhibitory attributes. Six models, Extra Trees Classifier, Random Forest Classifier, LightGBM Classifier, XGBoost Classifier, Bagging Classifier, and a convolutional neural network harnessed from empirical data were selected for the screening process. These models guided our selection of 26 compounds from the FDA-approved drug library for further evaluation. Notably, nifuroxazide emerged as the most potent inhibitor, with a half-maximal inhibitory concentration of 14.9 ± 1.63 µM. The stability of protein-ligand complexes was confirmed using molecular modeling. Furthermore, nifuroxazide treatment of HepG2 cells not only inhibited USP21 and its established substrate ACLY but also elevated p-AMPKα, a downstream functional target of USP21. Intriguingly, we unveiled the previously unknown capacity of nifuroxazide to increase the levels of miR-4458, which was identified as downregulating USP21. This discovery was substantiated by manipulating miR-4458 levels in HepG2 cells, resulting in corresponding changes in USP21 protein levels in line with its predicted interaction with ACLY. Lastly, we confirmed the in vivo efficacy of nifuroxazide in inhibiting USP21 in mice livers, observing concurrent alterations in ACLY and p-AMPKα levels. Collectively, our study establishes nifuroxazide as a promising USP21 inhibitor with potential implications for addressing metabolic disorders and cancer proliferation. This multidimensional investigation sheds light on the intricate regulatory mechanisms involving USP21 and its downstream effects, paving the way for further exploration and therapeutic development.

5-Nitro-2-furfuriliden derivatives as potential anti-Trypanosoma cruzi agents: design, synthesis, bioactivity evaluation, cytotoxicity and exploratory data analysis.

The anti-Trypanosoma cruzi activity of 5-nitro-2-furfuriliden derivatives as well as the cytotoxicity of these compounds on J774 macrophages cell line and FN1 human fibroblast cells were investigated in this study. The most active compounds of series I and II were 4-butyl-[N'-(5-nitrofuran-2-yl) methylene] benzidrazide (3g; IC50=1.05µM±0.07) and 3-acetyl-5-(4-butylphenyl)-2-(5-nitrofuran-2-yl)-2,3-dihydro,1,3,4-oxadiazole (4g; IC50=8.27µM±0.42), respectively. Also, compound 3g was more active than the standard drugs, benznidazole (IC50=22.69µM±1.96) and nifurtimox (IC50=3.78µM±0.10). Regarding the cytotoxicity assay, the 3g compound presented IC50 value of 28.05µM (SI=26.71) against J774 cells. For the FN1 fibroblast assay, 3g showed IC50 value of 98µM (SI=93.33). On the other hand, compound 4g presented a cytotoxicity value on J774 cells higher than 400µM (SI >48), and for the FN1 cells its IC50 value was 186µM (SI=22.49). Moreover, an exploratory data analysis, which comprises hierarchical cluster (HCA) and principal component analysis (PCA), was carried out and the findings were complementary. The molecular properties that most influenced the compounds' grouping were ClogP and total dipole moment, pointing out the need of a lipophilic/hydrophilic balance in the designing of novel potential anti-T. cruzi molecules.

Pharmacological updates of nifuroxazide: Promising preclinical effects and the underlying molecular mechanisms.

Nifuroxazide (NFX) is a safe nitrofuran antibacterial drug used clinically to treat acute diarrhea and infectious traveler diarrhea or colitis. Recent studies revealed that NFX displays multiple pharmacological effects, including anticancer, antioxidant, and anti-inflammatory effects. NFX has potential roles in inhibiting thyroid, breast, lung, bladder, liver, and colon cancers and osteosarcoma, melanoma, and others mediated by suppressing STAT3 as well as ALDH1, MMP2, MMP9, Bcl2 and upregulating Bax. Moreover, it has promising effects against sepsis-induced organ injury, hepatic disorders, diabetic nephropathy, ulcerative colitis, and immune disorders. These promising effects appear to be mediated by suppressing STAT3 as well as NF-κB, TLR4, and ß-catenin expressions and effectively decreasing downstream cytokines TNF-α, IL-1ß, and IL-6. Our review summarizes the available studies on the molecular biological mechanisms of NFX in cancer and other diseases and it is recommended to translate the studies in experimental animals and cultured cells and repurpose NFX in various diseases for scientific evidence based on human studies.

IDO2-siRNA Carried by Salmonella Combined with Nifuroxazide Attenuates Melanoma Growth.

BACKGROUND: Melanoma, a highly malignant skin cancer, is a hot topic in oncology treatment research. Nowadays, tumor immunotherapy, especially immunotherapy combined with other therapies, has attracted more and more attention. Indoleamine 2,3-dioxygenase 2 (IDO2), a ratelimiting enzyme of the tryptophan metabolism pathway in the urine of dogs with immunosuppression, is highly expressed in melanoma tissue. Additionally, IDO2 significantly inhibits the anti-tumor immunity of the body and has become a novel target of melanoma treatment. Nifuroxazide, as an intestinal antibacterial agent, was found to be able to inhibit Stat3 expression and exert an anti-tumor effect. Therefore, the present study aimed to examine the therapeutic effect of a self-designed IDO2-small interfering RNA (siRNA) delivered by attenuated Salmonella combined with nifuroxazide on melanoma- bearing mice, as well as determine its underlying mechanism. METHODS: The effect of nifuroxazide on melanoma was detected by flow cytometry, CCK-8 and colony- forming ability assays, respectively, in vitro. The plasmid of siRNA-IDO2 was constructed, and the mice-bearing melanoma model was established. After the treatment, the tumor growth and survival rate were monitored, and the morphological changes of tumor tissue were detected by HE staining. The expression of related proteins was detected by Western blotting, and the expression of CD4 and CD8 positive T cells in tumor tissue was detected by IHC and IF, and the proportion of CD4 and CD8 positive T cells in spleen was detected by flow cytometry. RESULTS: The results demonstrated that the combination therapy effectively inhibited the phosphorylation of Stat3 and the expression level of IDO2 in melanoma cells, which effectively inhibited tumor growth and prolonged the survival time of tumor-bearing mice. The mechanistic study revealed that, compared with control groups and monotherapy groups, the combination treatment group reduced the atypia of tumor cells, increased the apoptotic rate, enhanced the infiltration of T lymphocytes in tumor tissue and increased the CD4+ and CD8+ T lymphocytes in the spleen, suggesting that the mechanism may be associated with the inhibition of tumor cell proliferation, the increase of apoptosis and the enhancement of the cellular immunity. CONCLUSION: In conclusion, IDO2-siRNA combined with nifuroxazide therapy could serve a significant role in the treatment of melanoma-bearing mice, enhance the tumor immunity and provide an experimental basis for identifying a novel combination method for the treatment of melanoma clinically.

Design, synthesis and biological evaluation of novel diarylacylhydrazones derivatives for the efficient treatment of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized with high mortality, unknown etiology, and lack of effective treatment. Many evidences validate that inhibiting the activation of STAT3 is an attractive therapeutic strategy for IPF. Herein, based on our previous findings that nifuroxazide (NIF) could effectively attenuate pulmonary fibrosis by inhibiting STAT3 activation, a series of diarylacylhydrazones derivatives have been designed and synthesized. Among them, compounds 44 and 52 could inhibit TGF-ß1-induced abnormal activation of NIH-3T3 and A549 cells, as well as migration and EMT of A549 cells. In a bleomycin-induced mouse pulmonary fibrosis model, the oral administration of 44 and 52 (bioavailability F = 31.75% and 42.08%) improved mouse lung function and slowed the progression of IPF. Moreover, 52 could reverse the pulmonary fibrosis in treatment model. Collectively, this work shows 44 and 52 could be a potential lead compound for the treatment of IPF, and it is worthy of further study.

Nifuroxazide mitigates doxorubicin-induced cardiovascular injury: Insight into oxidative/NLRP3/GSDMD-mediated pyroptotic signaling modulation.

Doxorubicin (DOX) is a widely used powerful anthracycline for treatment of many varieties of malignancies; however its cumulative and dose-dependent cardio-toxicity has been limited its clinical use. In the current study, in vivo and in vitro (neonatal rat's cardiomyocytes) experiments were conducted to identify the impact of nifuroxazide (NIFU) on DOX-induced cardiomyopathy, vascular injury, and hemato-toxcity and plot the underlying regulatory mechanisms. Cardiovascular injury was induced in vivo by I.P. injection of an overall dose of DOX (21 mg/kg) administered (3.5 mg/kg) twice weekly for 21 days. NIFU (10 and 30 mg/kg) was administered orally once daily for 21 days, 1 week after DOX injection initiation. In vivo experiments confirmed NIFU to restore blood cells counts and hemoglobin concentration. Moreover, NIFU normalized the myocardial functional status as confirmed by ECG examination and myocardial injury markers; CK-MB, LDH, and AST. NIFU restored the balance between TAC and both of ROS and MDA and down-regulated the protein expression of TLR4, NF-kB, TXNIP, NLR-family pyrin domain containing 3 (NLRP3), caspase-1, IL-1ß, and GSDMD-N terminal, with inhibition of the up-stream of NLRP3 and the down-stream DOX-induced pyroptosis. The in vitro assay confirmed well preserved cardiomyocytes' architecture, amelioration of NLRP3/IL-1 ß-mediated cell pyroptosis, enhanced cell viability, and improved spontaneous beating. Moreover, NIFU normalized the disturbed aortic oxidant-antioxidant balance; enhanced eNOS- mediated endothelial relaxation, and down regulated IL-1ß expression. Thus, NIFU may be proposed to serve as a cardioprotective agent to attenuate DOX-induced cardio-toxicity and vascular injury.