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.

Influence of Surface-Modification via PEGylation or Chitosanization of Lipidic Nanocarriers on In Vivo Pharmacokinetic/Pharmacodynamic Profiles of Apixaban.

Nanostructured lipid carriers (NLCs) have been proven to significantly improve the bioavailability and efficacy of many drugs; however, they still have many limitations. These limitations could hinder their potential for enhancing the bioavailability of poorly water-soluble drugs and, therefore, require further amendments. From this perspective, we have investigated how the chitosanization and PEGylation of NLCs affected their ability to function as a delivery system for apixaban (APX). These surface modifications could enhance the ability of NLCs to improve the bioavailability and pharmacodynamic activity of the loaded drug. In vitro and in vivo studies were carried out to examine APX-loaded NLCs, chitosan-modified NLCs, and PEGylated NLCs. The three nanoarchitectures displayed a Higuchi-diffusion release pattern in vitro, in addition to having their vesicular outline proven via electron microscopy. PEGylated and chitosanized NLCs retained good stability over 3 months, versus the nonPEGylated and nonchitosanized NLCs. Interestingly, APX-loaded chitosan-modified NLCs displayed better stability than the APX-loaded PEGylated NLCs, in terms of mean vesicle size after 90 days. On the other hand, the absorption profile of APX (AUC0-inf) in rats pretreated with APX-loaded PEGylated NLCs (108.59 µg·mL-1·h-1) was significantly higher than the AUC0-inf of APX in rats pretreated with APX-loaded chitosan-modified NLCs (93.397 µg·mL-1·h-1), and both were also significantly higher than AUC0-inf of APX-Loaded NLCs (55.435 µg·mL-1·h-1). Chitosan-coated NLCs enhanced APX anticoagulant activity with increased prothrombin time and activated partial thromboplastin time by 1.6- and 1.55-folds, respectively, compared to unmodified NLCs, and by 1.23- and 1.37-folds, respectively, compared to PEGylated NLCs. The PEGylation and chitosanization of NLCs enhanced the bioavailability and anticoagulant activity of APX over the nonmodified NLCs; this highlighted the importance of both approaches.

Memantine mitigates ROS/TXNIP/NLRP3 signaling and protects against mouse diabetic retinopathy: Histopathologic, ultrastructural and bioinformatic studies.

Diabetic retinopathy (DRET) triggers vision loss in adults, however, little therapeutic options are existing. Memantine is an anti-Alzheimer drug that antagonizes the activity of glutamate at N-methyl-D-aspartate (NMDA) receptors. Glutamate and thioredoxin-interacting protein (TXNIP) are known to be overexpressed in diabetic retinas and can produce activation of NOD-like receptor protein 3 (NLRP3) with subsequent secretion of interlukin-1ß. This study repurposed memantine for its neuroprotective effect in experimental DRET and tested its impact on ROS/TXNIP/NLRP3. In addition, KEGG pathway database and STRING database identified the protein-protein interaction between glutamate receptors and TXNIP/NLRP3. Male Swiss albino mice received alloxan (180 mg/kg) to induce DRET. After 9 weeks, we divided the mice into groups: (a) saline, (ii) DRET, (iii and iv) DRET + oral memantine (5 or 10 mg per kg) for 28 days. Then, mice were euthanized, and eyeballs were removed. Retinal samples were utilized for biochemical, histopathological, and electron microscopy studies. Retinal levels of glutamate, TXNIP, NLRP3 and interlukin-1ß were estimated using ELISA technique as well as retinal malondialdehyde. Histopathological and ultrastructural examination demonstrated that oral memantine attenuated vacuolization and restored normal retinal cell layers. Moreover, memantine reduced TXNIP, NLRP3, interleukin-1ß and MDA concentrations. These results provide evidence demonstrating memantine' efficacy in alleviating DRET via suppressing reactive oxygen species/TXNIP/NLRP3 signaling cascade. Therefore, memantine might serve as a potential therapy for retinopathy after adequate clinical research.

Metformin suppresses LRG1 and TGFß1/ALK1-induced angiogenesis and protects against ultrastructural changes in rat diabetic nephropathy.

Diabetic nephropathy (DN) has high prevalence and poor prognosis which make it a research priority for scientists. Since metformin, a hypoglycaemic drug, has been found to prolong the survival of mice with DN. This study aims at investigating the molecular mechanisms leading to DN in rats and to explore the role of leucine-rich α-2-glycoprotein-1 (LRG1), activin-like kinase1 (ALK1), and transforming growth factor-ß (TGFß1) in the pathologic alterations seen in DN. The aim was also extended to explore the protective action of metformin against DN in rats and its influence on LRG1and ALK1/TGFß1 induced renal angiogenesis. 24 male rats were used. Rats were assigned as, the vehicle group, the diabetic control group and diabetic + metformin (100 and 200 mg/kg) groups. Kidney samples were processed for histopathology, immunohistochemistry and biochemical analysis. Bioinformatic analysis of studied proteins was done to determine protein-protein interactions. Metformin reduced serum urea and creatinine significantly, decreased the inflammatory cytokine levels and reduced LRG1, TGFß1, ALK1 and vascular endothelial growth factor (VEGF) proteins in rat kidneys. Bioinformatic analysis revealed interactions between the studied proteins. Metformin alleviated the histopathological changes observed in the diabetic rats such as the glomerular surface area and increased Bowman's space diameter. Metformin groups showed decreased VEGF immunostaining compared to diabetic group. Metformin shows promising renoprotective effects in diabetic model that was at least partly mediated by downregulation of LRG1 and TGFß1/ALK1-induced renal angiogenesis. These results further explain the molecular mechanism of metformin in DN management.

Serum and Urinary Interferon-Gamma-Inducible Protein 10 in Lupus Nephritis.

BACKGROUND: Lupus nephritis is a serious manifestation of systemic lupus erythematosus (SLE). The objective of this study was to identify the sensitivity, specificity, and cut-off values of IP-10 in the serum and urine of patients with lupus nephritis compared to renal biopsy, albumin/creatinine ratio, and serum anti-dsDNA. METHODS: Thirty female SLE patients were included. SLEDAI was calculated and blood and urine samples were collected. Patients were divided into 10 SLE patients with renal involvement (six active and four inactive), 10 active SLE, and 10 inactive SLE patients. Ten age-matched healthy (control) were included. Serum and urinary levels of IP-10 were measured by ELISA. Anti-dsDNA, urine albumin/creatinine ratio were performed. RESULTS: Serum and urinary IP-10 in active SLE patients had significantly increased levels as compared to inactive SLE patients (P = 0.015, P = 0.033, respectively). However, there was no difference in serum and urinary levels between active renal and active non-renal patients. Albumin/creatinine ratio is a better marker in differentiating between lupus nephritis and SLE with no renal involvement. Any of serum and urinary IP-10, albumin/creatinine ratio, and anti-dsDNA did not correlate with the class of lupus nephritis in renal biopsy. CONCLUSION: Urinary and serum IP-10 are useful markers of lupus activity, but not indicative of renal activity. Albumin/creatinine ratio is superior in identifying lupus nephritis and renal activity.