Vitamin K2 (MK-7) Intercepts Keap-1/Nrf-2/HO-1 Pathway and Hinders Inflammatory/Apoptotic Signaling and Liver Aging in Naturally Aging Rat.

Aging is a naturally occurring physiological process with a deleterious impact on various body organs and humans' well-being. The aging population is increasing worldwide, which imposes the need for the exploration of nutritional options that can intercept the impact of the aging processed on various body organs. Vitamin K2 (VK2) is a fat-soluble vitamin with emerging evidence on its therapeutic merits. In the current study, natural aging induced a significant liver deterioration with a disrupted Keap-1/Nrf-2/HO-1 axis and increased COX-2, iNOS and TNF-α expression and apoptotic and fibrotic changes. VK2 administration, on the other hand, improved the biochemical indices of liver function (total protein, albumin, ALT and AST); the suppressed hepatic expression of Keap-1 and increased the hepatic expression of Nrf-2 with a parallel increase in the hepatic activity of HO-1. Subsequently, the liver content and hepatic expression of TNF-α, COX-2 and iNOS were significantly retracted. In context, the liver content and hepatic expression of the fibrotic biomarkers TGFß and TIMP significantly retracted as well. Moreover, the TUNEL assay confirmed the retraction of liver apoptotic changes. Of notice, electron transmission microscope examination confirmed the preservation of mitochondrial functions and preservation of the ultra-microscopical structures. In conclusion, the VK2-mediated interception of aging-induced Keap-1/Nrf-2/HO-1 signaling suppressed the hepatic contents of inflammatory and fibrotic biomarkers, as well as apoptotic changes with preservation of the hepatic architectural and functional status. VK2 can be presumed to be an effective nutritional supplement to the aging population to spare the liver, amongst other body organs, against aging-induced deleterious injury.

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.

Development of a simple and valid nutrition screening tool for pediatric hospitalized patients with acute illness.

Background: Nutritional screening, intervention and assessment in patients with undernutrition are key components of any nutritional care. The goal of any nutritional assessment is to determine the specific nutritional risk(s). Presently, there are no guidelines on any ideal screening tool to be used on admission for identification of children that are at risk of developing malnutrition during their hospital stay. The objective of the study was to develop a valid and simple nutritional screening tool which can be used on hospital admission to identify pediatric patients at risk of malnutrition .Methods: This study was cross sectional analytical that enrolled children (n:161) admitted with acute illness to the general wards at Cairo University Children Hospitals (CUCH). The answers to the developed questionnaire were compared to the Subjective Global Assessment (SGA), those with high accuracy (≥80%) were used for validity with anthropometric measures. Results: In the 'less than two years of age' group, the simple and valid nutritional screening tools were the following questions: (Is there a problem during breast-feeding?), (Is there scanty breast milk?), (Is there appetite loss?). The simple and valid nutritional screening tools during the 'early childhood' group were the following questions: (Is there appetite loss?), (Is there any skipping of meals?), (Are they watching TV, videotapes and/or playing computer games for more than two hours/day?). The simple and valid  nutritional screening tools during the 'late childhood' group were the following questions: (Is there appetite loss?), (Are they watching TV, videotapes and/or playing computer games for more than two hours/day?). Conclusion: The simple and valid nutritional screening tools differ according to age groups. The one which is valid in all ages is the question about the appetite loss.

Protective effect of metformin on rat diabetic retinopathy involves suppression of toll-like receptor 4/nuclear factor-k B expression and glutamate excitotoxicity.

Microvascular complications of diabetes mellitus are progressively significant reasons for mortality. Metformin (MET) is considered as the first-line therapy for type 2 diabetes patients, and may be especially beneficial in cases of diabetic retinopathy although the precise mechanisms of MET action are not fully elucidated. The current study was designed to inspect the antioxidant and modulatory actions of MET on DRET in streptozotocin-induced diabetic rats. The effect of MET on the toll-like receptor 4/nuclear factor kappa B (TLR4/NFkB), inflammatory burden and glutamate excitotoxicity was assessed. Twenty-four male rats were assigned to four experimental groups: (1) Vehicle group, (2) Diabetic control: developed diabetes by injection of streptozotocin (60 mg/kg, i.p.). (3&4) Diabetic + MET group: diabetic rats were left for 9 weeks without treatment and then received oral MET 100 and 200 mg/kg for 6 weeks. Retinal samples were utilized in biochemical, histological, immunohistochemical and electron microscopic studies. MET administration significantly decreased retinal level of insulin growth factor and significantly suppressed the diabetic induced increase of malondialdehyde, glutamate, tumor necrosis factor-α and vascular endothelial growth factor (VEGF). Further, MET decreased the retinal mRNA expression of NFkB, tumor necrosis factor-α and TLR4 in diabetic rats. The current findings shed the light on MET's efficacy as an adjuvant therapy to hinder the development of diabetic retinopathy, at least partly, via inhibition of oxidative stress-induced NFkB/TLR4 pathway and suppression of glutamate excitotoxicity.

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.

Renoprotective effect of calycosin in high fat diet-fed/STZ injected rats: Effect on IL-33/ST2 signaling, oxidative stress and fibrosis suppression.

Type 2 diabetes mellitus (T2DM) is a disease with a drastically growing worldwide prevalence. It is usually associated with numerous complications of which; diabetic nephropathy (DN); is a main complication of microvasculature and more seriously, a common cause of end-stage renal disease (ESRD). Unfortunately, both the lack of a definitive remedy alongside the economic and the social burden on DN patients enforces considerable impetus for developing alternative therapies. IL-33 is a newly discovered member of the IL-1 cytokine family. IL33/ST2 signaling plays a crucial role in acute and chronic kidney diseases. Calycosin is an isoflavone with reported IL33 signaling inhibitory activity. The present study aimed to investigate if calycosin possess renal protective effect in high-fat diet/STZ-induced T2DM model and to clarify the potential underlying mechanisms. HFD-STZ control rats showed functional and structural renal damage confirmed by increased serum creatinine, blood urea nitrogen and albuminuria associated with marked renal glomerulosclerosis and interstitial fibrosis. Initiation of inflammation, oxidative stress, and fibrosis was evident as depicted by elevated renal levels of IL33/ST2 mRNA as well as increased renal NF-κBp65, TNF-α, IL-1ß, MDA, and TGF-ß contents with suppressed Nrf2 and TAC. Calycosin treatment markedly improved the aforementioned makers of renal injury and dysfunction, modulated IL33/ST2 signaling, inflammatory cytokines, oxidative stress and fibrotic processes. This was accompanied by improvement of T2DM-induced renal ultramicroscopic and histopathological alterations.

Carbamazepine Alleviates Retinal and Optic Nerve Neural Degeneration in Diabetic Mice via Nerve Growth Factor-Induced PI3K/Akt/mTOR Activation.

Aim: Diabetic retinopathy causes loss of vision in adults at working-age. Few therapeutic options are available for treatment of diabetic retinopathy. Carbamazepine (CARB), a widely used antiepileptic drug, was recently accounted for its neuroprotective effect. Nerve growth factor (NGF) activates various cascades among which, PI3K/Akt/mTOR pathway has a vital action in NGF-mediated neuronal differentiation and survival. This study evaluated the effect of CARB in the treatment of diabetic retina and unveiled some of the underlying molecular mechanisms. Main Methods: Alloxan diabetes model was induced in 36 albino well-acclimatized mice. After establishment of the diabetic model in 9 weeks, mice were assigned to treatment groups: (1) saline, (2) alloxan-diabetic, (3 and 4) alloxan+CARB (25 or 50 mg per kg p.o) for 4 weeks. After completion of the therapeutic period, mice were sacrificed and eyeballs were enucleated. Retinal levels of NGF and PI3K/Akt were assessed using real-time polymerase chain reaction. Further, total and phosphorylated TrKA, PI3K, Akt, mTOR as well as Caspase-3 were measured by Western blot analysis. Key Findings: Histopathological examination demonstrated that CARB attenuated vacuolization and restored normal thickness and organization of retinal cell layers. In addition, CARB increased pTrKA/TrKA ratio and ameliorated diabetes-induced reduction of NGF mRNA and immunostaining in retina. Additionally, it augmented the mRNA expression of PI3K and Akt, as well as the protein level of the phosphorylated PI3/Akt/mTOR. Significance: Results highlighted, for the first time, the neuronal protective effect for CARB in diabetic retina, which is mediated, at least in part, by activation of the NGF/PI3K/Akt/mTOR pathway.

Crocin reverses unilateral renal ischemia reperfusion injury-induced augmentation of oxidative stress and toll like receptor-4 activity.

Renal Ischemia (RI) usually develops as a secondary manifestation of hypertension, various cardiovascular disorders and renal transplantation. It exerts hypoxic oxidative stress to kidneys, together with stimulation of several immune-mediated inflammatory cascades. Such events eventually damage renal tubules and glomeruli, driving acute kidney injury (AKI) and ultimately, renal failure. Crocin; the main bioactive constituent of Crocus sativus extract has been reported to demonstrate numerous pharmacological merits. In the current study, unilateral renal ischemia reperfusion injury (URIRI) was induced in rats by unilateral clamping of the left renal pedicle for 45 min followed by 24 h of reperfusion. Daily pre-treatment with crocin (20 mg/kg, orally) for 7 days, significantly improved all signs of renal injury. Biochemically, kidney functions; including serum creatinine (Sr Cr), blood urea nitrogen (BUN), proteinuria and creatinine clearance (Cr Cl) significantly improved. Inflammatory biomarkers; serum lactate dehydrogenase (LDH) and kidney nitric oxide (Nos) contents significantly declined. Oxidant/antioxidant balance was significantly restored; manifested in recovery of renal superoxide dismutase (SOD) activity, glutathione (GSH) concentration, malondialdehyde (MDA) content and restoration of serum catalase activity. Kidney contents of inflammatory cytokine interleukin-6 (IL6) and toll-like receptors 4 (TLR4) significantly declined as well. Histopathologically, crocin pretreatment resulted in signs of improvement with minimal renal lesions with significant decrease in renal inflammatory cells count. In conclusion, crocin induced restoration of normal kidney functions is mediated through multiple mechanisms including mainly attenuation of oxidative stress and inflammation via down-regulation of renal TLR4 and IL6 expression.

Correlation between testicular mast cell count and spermatogenic epithelium in non-obstructive azoospermia.

Although there is emerging evidence that mast cells are involved in infertility, their exact role has not been elucidated clearly. Here we carried out a retrospective case-control study to find out whether there is a correlation between mast cell (MC) count and proliferation (Ki67 index) of the spermatogenic epithelium as well as of the Sertoli cells (vimentin-positive) in non-obstructive azoospermia (NOA). We assessed MCs, Ki67 and vimentin expression in Sertoli cells in testicular biopsies of germ cell aplasia (GCA, n = 14) and maturation arrest (MA, n = 14) vs. normal spermatogenesis (n = 14) cases. There was a significant decrease in the spermatogonial Ki67 index (1.25 ± 0.91, 4.21 ± 1.81 vs. 39.57 ± 3.92) and Johnsen score (2.48 ± 0.65, 4.89 ± 1.05 vs. 9.75 ± 0.30) as well as a significant increase (P < 0.001) in MC count (29.00 ± 4.11, 7.57 ± 1.95 vs. 3.00 ± 1.30) in seminiferous tubules of infertile cases with GCA and MA vs. controls. On the other hand, the percentage of vimentin-expressing Sertoli cells was significantly decreased (P < 0.001) in biopsies of cases with MA (35.50 ± 15.62) compared to those of cases with GCA and controls (72.64 ± 10.67 and 98.57 ± 1.45 respectively). Additionally, a significant negative correlation was detected between MC count and Ki67 index as well as Johnsen score in the MA group which became more significant in the GCA group. The significant increase in MC count in the GCA group and to a lesser extent in the MA group indicates their possible role in NOA particularly at the spermatogonial proliferation level and this is supported by the significant negative correlation with the Ki67 index.

Crocin mediated amelioration of oxidative burden and inflammatory cascade suppresses diabetic nephropathy progression in diabetic rats.

Diabetic Nephropathy (DN) is one of the main complications associated with diabetes mellitus. Persistently elevated blood glucose level drives histopathological changes in renal tissues that hinder normal kidney functions. In the current study, crocin; the main bioactive constituent of Crocus sativus was investigated as a reno-protective agent against DN by virtue of its numerous pharmacological activities. Diabetes was induced in male Sprague-Dawely rats through intravenous injection of streptozocin (STZ) (50 mg/kg), DN was confirmed eight weeks post diabetes induction. Daily oral crocin for eight weeks (20 mg/kg) significantly reduced blood glucose level with a significant increase in insulin level. Moreover, crocin improved impaired kidney functions as manifested in reduction of serum creatinine levels, blood urea nitrogen and proteinuria with concomitant increase in urinary creatinine clearance. Furthermore, biomarkers of cell injury and tissue necrosis like LDH activity was significantly reduced, kidney content of NOS significantly declined likewise. In addition, renal antioxidants such as SOD, GSH and serum catalase activity significantly increased with concomitant reduction of kidney MDA; biomarker of oxidative load. Kidney content of toll-like receptors 4 and IL-6 significantly declined with simultaneous suppression of nuclear factor kappa-B (NF-κB/p65) protein expression and immuno-staining in rat renal cortex. Furthermore, crocin inhibited progression of renal fibrosis as seen with reduction of renal hydroxyproline and collagen content, TGF-ß immuno-staining and Masson's Trichrome positive tissue. Histopathologically, crocin pretreatment was associated with minimal renal damage with fewer fibrotic lesions. There was a concomitant restoration of renal tubules integrity with preservation of glomerular space area. In conclusion, crocin's ameliorative impact on DN may be attributed to its free radicals scavenging properties, its ability to enhance host antioxidant defense system and its ability to inhibit inflammatory and fibrotic cascades activation.

Ultrastructural changes and nestin expression accompanying compensatory renal growth after unilateral nephrectomy in adult rats.

BACKGROUND: Several renal disorders affect the glomerular podocytes. Compensatory structural and functional changes have been observed in animals that have undergone unilateral renal ablation. These changes occur as a pliant response to quench the increased functional demand to maintain homeostasis of fluid and solutes. Nestin is an intermediate filament protein present in the glomerular podocytes of the adult kidney and is linked with the maintenance of its foot process structure. Structural changes in the podocytes ultimately restructure the filtration barrier. Very few studies related to the ultrastructural and histopathologic changes of the podocytes are documented. The present study aimed to assess the histopathologic changes at the ultrastructural level in the adapted kidney at different time intervals following unilateral renal ablation in adult rats and its relation with nestin. METHODS: Forty-eight rats were divided into four groups (n=12 in each group). The animals of Group A were control naïve rats, while the group B, group C and group D animals underwent left unilateral nephrectomy and the remaining right kidney was removed on days 10, 20 and 30, respectively. Each group included four sham-operated rats, which were sacrificed at the same time as the naïve rats. Each nephrectomized sample was weighed and its sections were subjected to hematoxylin and eosin examination, transmission electron microscopic study as well as immunostaining using the intermediate filament protein nestin. RESULTS: No difference was found between the kidney sections from the control group and the sham-operated groups. A significant increase in the weight of the right kidneys was noted in groups B, C and D (P<0.001). The ultrastructural adaptive changes seen in the glomeruli of group B were subsequently reduced in groups C and D. This finding corresponded to a similar pattern of nestin expression in the podocytes, which showed significant increase in group B followed by reduced expression in groups C and D. Histopathologic and transmission electron microscopic evaluation of group B showed signs of kidney injury. On the other hand, group C animals showed markedly reduced renal adaptive changes and similar changes were also noted in group D. CONCLUSION: Correlation between nestin expression and the ultrastructural changes confirms that nestin has a role in increasing the mechanical stability of the podocytes in order to enhance their morphologic changes in response to the tensile glomerular capillary wall. However, further studies investigating more remote ultrastructural changes and their relation with nestin expression are needed to confirm this relationship.

Implication of Nrf2/HO-1 pathway in the coloprotective effect of coenzyme Q10 against experimentally induced ulcerative colitis.

PURPOSE: Given the increased incidence of ulcerative colitis worldwide, the current study was designed to investigate the coloprotective potential of CoQ10 against experimentally induced ulcerative colitis (UC) and specify the implicated mechanisms. METHODS: Ulcerative colitis was induced by intracolonic instillation of [2 ml, 3% v/v acetic acid (AA)]. Rats in the different experimental groups received CoQ10 (10 or 30 and 100 mg/kg, orally) for eight consecutive days, either in a protective or curative regimen. RESULTS: Intracolonic AA instillation significantly increased colon/body weight index, colon weight/colon length ratio, clinical evaluation and macroscopic scoring of UC, serum LDH, C-reactive protein and decreased the serum total antioxidant capacity. Colon MDA, TNF-α and calcium content significantly increased as well, with concomitant reduction in colon GSH, SOD, CAT, Nrf2 and HO-1 contents. Moreover, immunohistochemical staining of colon specimen revealed increased expression of caspase-3 with significant histopathological changes. Coenzyme Q10 suppressed the release of inflammatory biomarkers and restored oxidants/antioxidants hemostasis. In a dose-dependent manner, CoQ10 significantly decreased colon/body weight index, colon weight/colon length ratio, clinical evaluation and macroscopic scoring of UC, serum LDH, C-reactive protein, colon MDA, TNF-α, caspase-3 expression and increased the serum total antioxidant capacity. Colon GSH, SOD, CAT, Nrf2 and HO-1 contents significantly increased. Moreover, coenzyme Q10 significantly preserved tissue histopathological architecture. It appears that the coloprotective effect of CoQ10 was calcium-independent. CONCLUSION: Coenzyme Q10 dose-dependently protects against AA-induced UC mainly via modulation of Nrf2/HO-1 and caspase-3 pathways. Antioxidant, anti-inflammatory and anti-apoptotic properties of CoQ10 are implicated in its observed therapeutic benefit.

Sulforaphane protects against sodium valproate-induced acute liver injury.

Drug-induced hepatotoxicity is one of the most commonly encountered obstacles in the field of medical practice. Sodium valproate (VPA) is among many drugs with reported hepatotoxic effects. Sulforaphane (SFN) is a thiol compound found in wide abundance in cruciferous plants that has numerous reported therapeutic efficacies. The current investigation sheds light on the potential hepatoprotective effect of SFN against VPA-induced liver injury in rats. Twice daily VPA (700 mg/kg, i.p.) for 7 days induced significant biochemical alterations and hepatic histopathological damage. SFN (0.5 mg/kg, orally) for 7 days significantly boosted liver function biomarkers; it reduced serum alanine transaminase, aspartate aminotransferase, and alkaline phosphatase, and restored serum albumin concentration in a significant manner. Meanwhile, SFN significantly mitigated VPA-induced histopathological alterations. To highlight the mechanisms implicated in the observed hepatoprotective action, hepatic malondialdehyde and tumour necrosis factor α content significantly declined with concomitant increase in hepatic heme oxygenase-1 content and glutathione concentration with SFN treatment. In conclusion, SFN can significantly ameliorate VPA-induced hepatotoxicity and liver injury primarily by direct association between antioxidant and anti-inflammatory properties.

Increased Proliferative Activity Accompanies the Local Inflammatory Response of Gastric Mucosa After Intragastric Balloon Insertion.

BACKGROUND AND AIM: The use of BioEnterics intragastric balloon (BIB) is progressively increasing, owing to the fact that morbid obesity becomes a global epidemic together with risks of bariatric surgery. Yet, the possible local BIB effect on gastric mucosa is not clearly elucidated. The aim of the current study was to assess the histological changes occurring in the gastric mucosa post-BIB insertion. METHODS: Gastric mucosa biopsy was obtained from 87 cases of morbid obesity both pre-BIB and 6 months post-BIB insertion to compare the local changes by histological and immunohistochemical analysis. RESULTS: An inflammatory reaction was detected in the post-BIB mucosa which displayed a positive CD20, CD3 (p < 0.05), and the proliferation index (Ki67) increased significantly compared with that of the pre-BIB cases. The Ki67 index showed a significant positive correlation with CD20 and CD3 immunoexpression in the post-BIB gastric mucosae. CONCLUSION: Our results demonstrated a possible increase in the gastric proliferative activity after BIB insertion accompanied with a remarkable local inflammatory reaction. Although these findings may be reactive and transient, endoscopic follow-up is recommended for early detection of further pathological changes.