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.

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.

Anti-inflammatory and neuroprotective activity of boswellic acids in rotenone parkinsonian rats.

There is evidence that inflammation and oxidative stress contribute to the neurodegenerative changes observed in Parkinson's disease. Unfortunately, there is a lack of curative treatment for this debilitating movement disorder. Boswellic acids (BAs) are pentacyclic triterpene molecules of plant origin that have been utilized for treating many inflammatory conditions. The current study was conducted to explore the protective role of BAs against rotenone-induced experimental parkinsonism. Twenty-four rats were assigned to one of four treatment groups. The first two groups were a vehicle group (no rotenone) and a rotenone control group in which rats received rotenone (1 mg/kg) every 48 h. The next 2 groups received rotenone (1 mg/kg every 48 h) plus protective oral doses of BAs (125 or 250 mg/kg daily). Rats in the rotenone group showed motor dysfunction when tested in the open-field arena and cylinder and rotarod tests. Moreover, inflammatory markers increased, whereas the dopamine level was lower in the striata of rats in the rotenone group versus those in the vehicle group. BAs taken by rats with rotenone-induced parkinsonism showed enhanced general motor performance, reduced inflammatory markers, and increased striatal dopamine level and nigral tyrosine hydroxylase immunostaining. In conclusion, BAs are promising agents in slowing the progression of Parkinson's disease if appropriate data become available about their safety and efficacy in humans.

Protective effects of evening primrose oil on behavioral activities, nigral microglia and histopathological changes in a rat model of rotenone-induced parkinsonism.

Parkinson's disease (PD) is a neurodegenerative illness described as damage to dopaminergic neurons. There is increasing evidence that neuroinflammatory activity mediated by microglia is extensively involved in the initiation and development of PD. This study assessed the protective effect of evening primrose oil [EPO] as an anti-inflammatory mediator in rotenone-induced Parkinsonism in rats. Forty-eight adult male albino rats were distributed into four groups. Group I: control. Group II: rotenone [1.5 mg/kg/48 h] was administered subcutaneously to the rats. Groups III and IV: the rats had rotenone plus daily oral [EPO] 5 and 10 mg/kg respectively. After 24 days, motor behaviour was assessed by the open field and rotarod tests. The brain striata were isolated and tested for tumor necrosis factor (TNF)-α, interleukin 6, NF-B [nuclear factor-kappa B], and dopamine levels. The mid-brain tissues were processed for light and electron microscopy examinations, and immunohistochemical staining for tyrosine hydroxylase [TH], and microglia cells' markers: [CD68 and IBA1]. Results revealed that rotenone-treated rats had poor motor function, a significantly increased striatal level of inflammatory markers, markedly shrunken neurons, degeneration, pyknotic neuroglia, neuropil vacuolation, markedly destructed swollen mitochondria with loss of their cristae, and dilated rough endoplasmic reticulum, as well as decreased TH and increased CD68 and IBA1-positive cells. Treatment with EPO ameliorates all the neuropathological changes of rotenone in the rat brain. In conclusion, EPO enhanced the motor performance, reduced the inflammatory marker levels, restored dopamine levels, and ameliorated the neurohistopathological lesions of rats with experimental parkinsonism, suggesting its neuroprotective and anti-inflammatory effects.

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.

Protective Effect of Topiramate against Diabetic Retinopathy and Computational Approach Recognizing the Role of NLRP3/IL-1ß/TNF-α Signaling.

The possible impact of topiramate against diabetic retinopathy (DREN) and its molecular mechanisms in relation to the nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome has not been studied before. Thus, in the present study, we aimed to utilize a computational approach to investigate the possible protective effect of topiramate on experimental DREN and explore its impact on NLRP3/interlukin-1ß signaling and brain-derived neurotrophic factor (BDNF) expression. Male albino mice were distributed to four experimental groups and assigned the following categorizations: (i) saline, (ii) diabetic, (iii) diabetic + topiramate 10 mg/kg and (iv) diabetic + topiramate 30 mg/kg. We observed shrinkage of total retinal thickness and elevation in retinal glutamate, malondialdehyde, NLRP3 and interlukin-1ß but decreased glutathione (GSH) levels in the diabetic mice. Additionally, retinal ultra-structures in the diabetic group showed abnormalities and vacuolations in the pigmented epithelium, the photoreceptor segment, the outer nuclear layer, the inner nuclear layer and the ganglion cell layer (GCL). Mice treated with topiramate 10 or 30 mg/kg showed downregulation in retinal malondialdehyde, NLRP3 and interlukin-1ß levels; improvements in the retinal pathologies; enhanced immunostaining for BDNF and improved ultra-structures in different retinal layers. Overall, the current results suggest topiramate as a neuroprotective agent for DREN, and future studies are warranted to further elucidate the mechanism of its protective action.

Autophagy modulation by irbesartan mitigates the pulmonary fibrotic alterations in bleomycin challenged rats: Comparative study with rapamycin.

AIMS: In pulmonary fibrosis, autophagy handles the maintenance of alveolar epithelial cells, prevents epithelial-mesenchymal transition (EMT), and controls collagen turnover. The mammalian target of rapamycin (mTOR) and its translational-dependent proteins are essential regulators of autophagy. Irbesartan (IRB) has earlier ameliorative effects in experimental pulmonary fibrosis. The current study aimed to explore therapeutic autophagy-modulated pulmonary fibrotic changes by IRB versus rapamycin (RAPA) in bleomycin (BLM)-challenged rats. MATERIALS AND METHODS: A single intratracheal BLM dose at day (0), IRB in different doses (10, 20, and 40 mg/kg) or RAPA (2.5 mg/kg) was given daily for 14 continuous days. KEY FINDINGS: IRB significantly diminished the fibrotic lung scores. Pulmonary levels of transforming growth factor (TGF)-ß1 and hydroxyproline exhibited marked attenuation in IRB (40 mg/kg)-treated rats compared to other treated groups. IRB (40 mg/kg) was not significantly different from RAPA. It downregulated the fibrotic lung phosphorylated mammalian target of rapamycin (p-mTOR) levels and augmented lung Unc-51-like autophagy activating kinase 1 (ULK1), LC3-I and LC3-II more than IRB (10 and 20 mg/kg)-treated fibrotic groups. SIGNIFICANCE: Autophagic effects via the mTOR signalling pathway may play a role in IRB's antifibrotic effects. Consideration of IRB as a therapeutic antifibrotic agent in pulmonary fibrosis needs further experimental and clinical long-term validation, especially in comorbid with primary hypertension, heart failure, and diabetic renal insults.

Long Non-Coding RNAs Gene Variants as Molecular Markers for Diabetic Retinopathy Risk and Response to Anti-VEGF Therapy.

BACKGROUND: Long non-coding RNAs (lncRNAs) play essential roles in molecular diagnosis and therapeutic response in several diseases. PURPOSE: For the first time, we aimed to evaluate the association of four lncRNAs TUG1 (rs7284767G/A), MIAT (rs1061540T/C), MALAT1 (rs3200401C/T), and SENCR (rs12420823C/T) variants with susceptibility to diabetic retinopathy (DR), disease severity, and early therapeutic response to intravitreous anti-vascular endothelial growth factor aflibercept therapy. PATIENTS AND METHODS: This case-control study enrolled 126 adult patients with type 2 diabetes. TaqMan assays using Real-Time PCR were run for genotyping. Multivariable regression analyses were applied to assess the role of each polymorphism after the adjustment of covariates. RESULTS: Carriers of TUG1 A/G and MIAT T/C and C/C genotypes were more likely to develop DR [OR=3.15 (95% CI=1.15-8.64), and OR=4.31 (95% CI=1.78-10.47)], while MALAT1 T/C conferred protection (OR=0.40, 95% CI=0.16-0.99). For TUG1, MALAT1, MIAT, and SENCR genotype combinations, GTCT and GCCC had a higher disease risk (P=0.012). For disease severity, MIAT T/T homozygosity was associated with higher DR grade [33.3% (T/T) vs 10% (C/C) and 4.2% (C/T) carriers, P=0.012]. Otherwise, patients with the SENCR T variant exhibited better pre-treatment best-corrected visual acuity level (p=0.021). Following aflibercept administration, carrying the TUG1 A or MIAT T/C was associated with a poor therapeutic response (OR=5.02, 95% CI=1.60-15.76, and OR=10.23, 95% CI=1.51-69.15, respectively). CONCLUSION: The lncRNAs TUG1 (rs7284767G/A) and MIAT (rs1061540T/C) were associated with increased DR susceptibility and poor response to aflibercept treatment, while MALAT1 (rs3200401C/T) conferred protection to DR. These genetic determinants could be useful in DR risk stratification and pharmacogenetics after validation in large-scale studies.

Clopidogrel or prasugrel reduces mortality and lessens cardiovascular damage from acute myocardial infarction in hypercholesterolemic male rats.

AIMS: Hypercholesterolemia is a hazard for increasing susceptibility of the heart to myocardial infarction (MI) by inducing platelet hyperaggregability. Clopidogrel and prasugrel have documented cardioprotective effects in clinical studies. Herein, we investigated whether clopidogrel and prasugrel could protect against isoproterenol-induced acute MI (A-MI) under hypercholesterolemic conditions in rats. MAIN METHODS: Dietary hypercholesterolemic rats were subjected to acute doses of isoproterenol. Serum lipids, inflammatory markers, aortic endothelin1 and endothelial nitric oxide synthase (eNOS) mRNAs expression and immunexpression of BCL2 were determined. KEY FINDINGS: Hypercholesterolemic rats showed infiltration of inflammatory cells and reduction in aortic wall thickness, deposition of fibrous tissue between cardiac muscle fibers. Protective doses of prasugrel or clopidogrel for 28 days before A-MI increased survival, amended the ECG parameters -including ST segment elevation- and improved the histopathological picture in hypercholesterolemic rats. This was coupled with reductions in platelet aggregation, creatine kinase-MB activity, endothelin 1, systemic inflammation and cardiac lipid peroxidation and increment in aortic eNOS expression. Clopidogrel and prasugrel groups showed enhanced BCL2 expression in cardiac fibers and aortic wall. SIGNIFICANCE: Prasugrel and clopidogrel protected against A-MI via anti-aggregatory and anti-inflammatory effects. These results add to the value of these drugs in correcting cardiovascular dysfunction in patients vulnerable to A-MI after confirmation by appropriate human studies.

Supplement With Calcium or Alendronate Suppresses Osteopenia Due to Long Term Rabeprazole Treatment in Female Mice: Influence on Bone TRAP and Osteopontin Levels.

BACKGROUND AND PURPOSE: Rabeprazole, a proton pump inhibitor (PPIs) is much endorsed to patients with increased gastric acidity. PPIs were accused to have osteoporotic effects on patients who chronically use them. The point of the current investigation was to decide the impact of rabeprazole on osteoporosis and to explore the modulatory effects of dietary calcium or alendronate on this side effect. METHODS: 80 female mice were alienated into four groups maintained for 18 weeks: [1] Vehicle group: given distilled water in 12 ml/kg, P.O. [2] Rabeprazole control group: given rabeprazole in a dose equals 10 mg/kg every 48 h, P.O. [3] Rabeprazole + calcium: given rabeprazole (10 mg/kg every 48 h) along with calcium supplement. [4] Rabeprazole + alendronate: given rabeprazole (10 mg/kg every 48 h) and alendronate (1 mg/kg per week, i.p.). Serum calcium, phosphorus and parathyroid hormone were measured. Both femurs were kept in paraformaldehyde, and then the right one was used for X-ray examination with analysis by Digora software and the left one for histopathological examination (H&E) and immunohistochemical stains for osteopontin and tartrate resistant acid phosphatase (TRAP). RESULTS: Calcium supplementation or administration of alendronate along with rabeprazole significantly restored the mean bone density as shown by X-ray analysis. Femurs from mice received rabeprazole showed widely separated, thin-walled bone trabeculae and increased number of osteoclasts. Calcium or alendronate with rabeprazole showed thick bone trabeculae without full recovery from rabeprazole induced damage. Adding calcium supplementation to rabeprazole did not affect the histological abnormalities related to osteoclasts meanwhile alendronate produced inactivation of osteoclasts. Both calcium and alendronate decreased the rabeprazole-induced increment in the femur osteopontin level. CONCLUSION: Calcium or alendronate can be recommended for female patients on PPI therapy who are at risk of osteopenia.

Neuroprotective effect of levetiracetam in mouse diabetic retinopathy: Effect on glucose transporter-1 and GAP43 expression.

AIMS: Retinopathy is a neurodegenerative complication associating diabetes mellitus. Diabetic retinopathy (DR) is the primary reason of visual loss during early adulthood. DR has a complicated multifactorial pathophysiology initiated by hyperglycaemia-induced ischaemic neurodegenerative retinal changes, followed by vision-threatening consequences. The main therapeutic modalities for DR involve invasive delivery of intravitreal antiangiogenic agents as well as surgical interventions. The current work aimed to explore the potential anti-inflammatory and retinal neuroprotective effects of levetiracetam. MAIN METHODS: This study was performed on alloxan-induced diabetes in mice (n: 21). After 10 weeks, a group of diabetic animals (n: 7) was treated with levetiracetam (25 mg/kg) for six weeks. Retinal tissues were dissected and paraffin-fixed for examination using (1) morphometric analysis with haematoxylin and eosin (HE), (2) immunohistochemistry (GLUT1, GFAP and GAP43), and (3) RT-PCR-detected expression of retinal inflammatory and apoptotic mediators (TNF-α, IL6, iNOS, NF-κB and Tp53). KEY FINDINGS: Diabetic mice developed disorganized and debilitated retinal layers with upregulation of the gliosis marker GFAP and downregulation of the neuronal plasticity marker GAP43. Additionally, diabetic retinae showed increased transcription of NF-κB, TNF-α, IL6, iNOS and Tp53. Levetiracetam-treated mice showed downregulation of retinal GLUT1 with relief and regression of retinal inflammation and improved retinal structural organization. SIGNIFICANCE: Levetiracetam may represent a potential neuroprotective agent in DR. The data presented herein supported an anti-inflammatory role of levetiracetam. However, further clinical studies may be warranted to confirm the effectiveness and safety of levetiracetam in DR patients.

Pharmacological Action of a Pregnane Glycoside, Russelioside B, in Dietary Obese Rats: Impact on Weight Gain and Energy Expenditure.

Background and purpose: Russelioside B (RB) is a pregnane glycoside obtained from Caralluma quadrangula; a herb with antidiabetic, anti-inflammatory, and antihyperlipidemic activities. The present experiment tested the possible role of RB in controlling weight gain in rats fed on high fat (HF) diet. Methods: RB was separated from the n-butanol fraction of the crude methanolic extract by chromatographic separation on a Si gel column according to the procedures described previously. The experiment of the biological assessment of RB used 32 male Wistar rats (4 groups, n = 8). Group 1 rats were fed with a palatable normal diet. Group 2, 3, and 4 were fed on HF diet for 16 weeks. Group 2 served as the HF diet control group while Group 3 and 4 received daily oral doses of RB (25 and 50 mg/kg) during the last four weeks. Animals' parameters like weight gain, fasting level of blood sugar, serum lipids, and serum liver enzyme activities were measured. Liver or adipose tissue weight was divided by the rat's body weight and multiplied by 100 to obtain the liver or adipose tissue index, respectively. Adipose tissues were processed for histopathological examination, measurement of mRNA expression of visfatin, leptin, adiponectin, uncoupling protein-1 (UCP-1), and carnitine palmitoyl transferase-1 (CPT-1). Furthermore, serum levels of insulin, interleukin-6 (IL-6), IL-1ß, tumor necrosis factor-α (TNF-α), leptin, resistin, and adiponectin were assessed using ELISA kits. Results: Rats fed with the HF diet exhibited significant body weight gain, abnormal liver function, disturbed lipid profile, and greater serum level of pro-inflammatory cytokines in addition to greater insulin resistance, adipose tissue and liver indices. Further, rats fed with the HF diet displayed upregulations in the expression of visfatin and leptin with downregulations in the expression of adiponectin, UCP-1, and CPT-1 compared to normal rats. Interestingly, RB (25 or 50 mg/kg) favorably modulated the measured parameters. Conclusion: Data from this study documented the beneficial role of RB in diminishing weight gain, improving the inflammatory perturbations and energy expenditure in HF diet fed rats. Therefore, RB might be a promising candidate for obesity.

Renoprotective effect of the isoflavonoid biochanin A against cisplatin induced acute kidney injury in mice: Effect on inflammatory burden and p53 apoptosis.

Cisplatin is a potent widely-used chemotherapeutics; however, its clinical use is associated with nephrotoxicity. Renoprotective approaches are being discovered to halt the tubular cell death due to inflammatory and apoptotic burdens. In the present study, the renoprotective effects of different doses of biochanin A (10, 20 or 40 mg/kg) in mice treated with a single injection of cisplatin (10 mg/kg) were reported. Cisplatin administration resulted in marked increases in serum creatinine and blood urea nitrogen. Further, renal homogenates showed increased level of inflammatory cytokines and upregulation of the expression of p53 up-regulated modulator of apoptosis (PUMA), p53 and caspase 3 but downregulation in Nrf2 expression. Furthermore, cisplatin group showed marked necrosis and degenerated tubular lining epithelial cells with frequently detected apoptotic bodies. Mice treated with biochanin A (10, 20 or 40 mg/kg) for 14 days prior to cisplatin abrogated cisplatin-mediated damage. Furthermore, the elevated serum creatinine and urea levels were lessened by some doses of biochanin A, indicating protection against renal injury. Similarly, the changes in apoptosis and inflammatory markers have ameliorated to significant levels (P < 0.05). The results suggest biochanin A as a nephroprotective agent against cisplatin toxicity. Overall, this nephroprotective effect of biochanin A involved anti-inflammatory and antiapoptotic activities.

Chemopreventive effect of leflunomide against Ehrlich's solid tumor grown in mice: Effect on EGF and EGFR expression and tumor proliferation.

UNLABELLED: i) AIMS: The current study aimed to examine the effect of leflunomide on tumoral expression of epidermal growth factor and its receptor (EGFR) in Ehrlich's ascites carcinoma (EAC) grown in mice. ii) MATERIALS AND METHODS: Mice were injected subcutaneously with EAC cells and allocated into four groups; Group i: EAC control group. Groups ii-iv: mice treated with leflunomide (3, 10 or 30mg/kg/day, p.o.), respectively. Pharmacologic treatments were initiated at day 8 and continued for 14days. iii) KEY FINDINGS: Treatment with leflunomide evoked antitumor properties as indicated by reduction in tumor mass, histopathological score, number of intratumoral PCNA immunopositive nuclei. Leflunomide (3, 10 or 30mg/kg) exerted an anti-inflammatory effect as indicated by the reduction in serum tumor necrosis factor-α. Furthermore, leflunomide demonstrated anti-angiogenic activity which was expressed as a decline in serum vascular endothelial growth factor and down-regulation of intratumoral EGF protein and mRNA expression as well as EGFR expression in addition to suppression of immunostaining for the endothelial marker, CD31. iv) SIGNIFICANCE: Taken together, the present results demonstrated that leflunomide possessed anti-angiogenic and anti-proliferative activity against EAC solid tumors that might be correlated to down regulation of EGF and EGFR. Further, the current data indicated that leflunomide may have utility in the management of human cancer.