Exploring the role of Müller cells-derived exosomes in diabetic retinopathy.

Exosomes are nanosized vesicles that have been reported as cargo-delivering vehicles between cells. Müller cells play a crucial role in the pathogenesis of diabetic retinopathy (DR). Activated Müller cells in the diabetic retina mediate disruption of barrier integrity and neovascularization. Endothelial cells constitute the inner blood-retinal barrier (BRB). Herein, we aim to evaluate the effect of Müller cell-derived exosomes on endothelial cell viability and barrier function under normal and hyperglycemic conditions. Müller cell-derived exosomes were isolated and characterized using Western blotting, nanoparticle tracking, and electron microscopy. The uptake of Müller cells-derived exosomes by the human retinal endothelial cells (HRECs) was monitored by labeling exosomes with PKH67. Endothelial cell vitality after treatment by exosomes under normo- and hypoglycemic conditions was checked by MTT assay and Western blot for apoptotic proteins. The barrier function of HRECs was evaluated by analysis of ZO-1 and transcellular electrical resistance (TER) using ECIS. Additionally, intracellular Ca+2 in HRECs was assessed by spectrofluorimetry. Analysis of the isolated exosomes showed a non-significant change in the number of exosomes isolated from both normal and hyperglycemic condition media, however, the average size of exosomes isolated from the hyperglycemic group showed a significant rise when compared to that of the normoglycemic group. Müller cells derived exosomes from hyperglycemic condition media markedly reduced HRECs cell count, increased caspase-3 and Annexin V, decreased ZO-1 levels and TER, and increased intracellular Ca+ when compared to other groups. However, treatment of HRECs under hyperglycemia with normo-glycemic Müller cells-derived exosomes significantly decreased cell death, preserved cellular integrity and barrier function, and reduced intracellular Ca+2. Collectively, Müller cell-derived exosomes play a remarkable role in the pathological changes associated with hyperglycemia-induced inner barrier dysfunction in DR. Further in vivo research will help in understanding the role of exosomes as therapeutic targets and/or delivery systems for DR.

Prophylactic Evidence of MSCs-Derived Exosomes in Doxorubicin/Trastuzumab-Induced Cardiotoxicity: Beyond Mechanistic Target of NRG-1/Erb Signaling Pathway.

Trastuzumab (Trz) is a humanized monoclonal antibody targeting epidermal growth factor receptor 2 (HER2; ErbB2). The combined administration of Trz and doxorubicin (DOX) has shown potent anti-cancer efficacy; however, this regimen may be accompanied by severe cardiac toxicity. Mesenchymal stem cells (MSCs)-derived exosomes are nanosized vesicles that play a crucial role in cell-cell communication and have shown efficacy in the treatment of various diseases. In this study, we aim to investigate the cardioprotective effects of MSCs-derived exosomes in a DOX/Trz- mediated cardiotoxicity model, and the possible mechanisms underlying these effects are elucidated. Forty-nine male rats were randomly assigned into four groups: Group I (control); Group II (Dox/Trz); Group III (protective group); and Group IV (curative group). Cardiac hemodynamic parameters, serum markers of cardiac injury, oxidative stress indices, and cardiac histopathology were investigated. Further, transcript profile of specific cardiac tissue injury markers, apoptotic markers, and fibrotic markers were analyzed using qRT-PCR, while the protein expressions of pAkt/Akt, pERK/ERK, pJNK/JNK, pJNK/JNK, and pSTAT3/STAT3 were evaluated by ELISA. Additionally, cardiac mirR-21 and miR-26a were assessed. A combined administration of DOX/Trz disrupted redox and Ca2+ homeostasis in cardiac tissue induced myocardial fibrosis and myofibril loss and triggered cardiac DNA damage and apoptosis. This cardiotoxicity was accompanied by decreased NRG-1 mRNA expression, HER2 protein expression, and suppressed AKT and ERK phosphorylation, while triggering JNK phosphorylation. Histological and ultra-structural examination of cardiac specimens revealed features typical of cardiac tissue injury. Moreover, a significant decline in cardiac function was observed through biochemical testing of serum cardiac markers and echocardiography. In contrast, the intraperitoneal administration of MSCs-derived exosomes alleviated cardiac injury in both protective and curative protocols; however, superior effects were observed in the protective protocol. The results of the current study indicate the ability of MSCs-derived exosomes to protect from and attenuate DOX/Trz-induced cardiotoxicity. The NRG-1/HER2, MAPK, PI3K/AKT, PJNK/JNK, and PSTAT/STAT signaling pathways play roles in mediating these effects.

Design, synthesis and pharmacological screening of novel renoprotective methionine-based peptidomimetics: Amelioration of cisplatin-induced nephrotoxicity.

Cisplatin (CP) is an effective chemotherapeutic agent for treatment of various types of cancer, however efforts are needed to reduce its toxic side effect. Previous studies revealed promising effect of peptides in decreasing CP induced nephrotoxicity. Herein, novel Met-based peptidomimetics were synthesized using N-acylbenzotriazole as acylating agent in high yield. Evaluation of renoprotective effect of the synthesized targets on CP treated kidney cell line (LLC-PK1) revealed that pretreatment with 1/3 IC50 of targets II, IIIa-g attenuated CP induced cell death where the IC50 of CP was raised from 3.28 µM to 9.25-41.1 µM. The most potent compounds IIIg, II and IIIb exhibited antioxidative stress in CP-treated LLC-PK1 cells as confirmed by raising GSH/GSSG ratio and SOD concentration as well as decreasing ROS and MDA. Additionally, in vivo experiments using Sprague Dawley rats showed renoprotective effect of IIIg against CP-induced nephrotoxicity as evidenced by improved results of renal function tests and attenuated CP-induced renal structural injury. Moreover, antioxidant activity of IIIg was demonstrated via its ability to reduce renal MDA level and up-regulate renal antioxidant element GSH level. Further, immunohistochemistry of renal specimens showed the ability of IIIg to restore CP-induced suppression of Nrf2. Interestingly, in vivo and in vitro studies demonstrated that IIIg had no effect on CP antiproliferative activity. An assessment of the ADMET properties revealed that targets IIIg, II and IIIb showed good drug-likeness in terms of their physicochemical, pharmacokinetic properties. The findings presented here showcase that IIIg is a promising renoprotective candidate with antioxidative stress potential.

Homocysteine and Age-Related Central Nervous System Diseases: Role of Inflammation.

Hyperhomocysteinemia (HHcy) is remarkably common among the aging population. The relation between HHcy and the development of neurodegenerative diseases, such as Alzheimer's disease (AD) and eye diseases, and age-related macular degeneration (AMD) and diabetic retinopathy (DR) in elderly people, has been established. Disruption of the blood barrier function of the brain and retina is one of the most important underlying mechanisms associated with HHcy-induced neurodegenerative and retinal disorders. Impairment of the barrier function triggers inflammatory events that worsen disease pathology. Studies have shown that AD patients also suffer from visual impairments. As an extension of the central nervous system, the retina has been suggested as a prominent site of AD pathology. This review highlights inflammation as a possible underlying mechanism of HHcy-induced barrier dysfunction and neurovascular injury in aging diseases accompanied by HHcy, focusing on AD.

Nanogold Particles Suppresses 5-Flurouracil-Induced Renal Injury: An Insight into the Modulation of Nrf-2 and Its Downstream Targets, HO-1 and γ-GCS.

The development of the field of nanotechnology has revolutionized various aspects in the fields of modern sciences. Nano-medicine is one of the primary fields for the application of nanotechnology techniques. The current study sheds light on the reno-protective impacts of gold nano-particles; nanogold (AuNPs) against 5-flurouracil (5-FU)-induced renal toxicity. Indeed, the use of 5-FU has been associated with kidney injury which greatly curbs its therapeutic application. In the current study, 5-FU injection was associated with a significant escalation in the indices of renal injury, i.e., creatinine and urea. Alongside this, histopathological and ultra-histopathological changes confirmed the onset of renal injury. Both gene and/or protein expression of nuclear factor erythroid 2-related factor 2 (Nrf-2) and downstream antioxidant enzymes revealed consistent paralleled anomalies. AuNPs administration induced a significant renal protection on functional, biochemical, and structural levels. Renal expression of the major sensor of the cellular oxidative status Nrf-2 escalated with a paralleled reduction in the renal expression of the other contributor to this axis, known as Kelch-like ECH-associated protein 1 (Keap-1). On the level of the effector downstream targets, heme oxygenase 1 (HO-1) and gamma-glutamylcysteine synthetase (γ-GCS) AuNPs significantly restored their gene and protein expression. Additionally, combination of AuNPs with 5-FU showed better cytotoxic effect on MCF-7 cells compared to monotreatments. Thus, it can be inferred that AuNPs conferred reno-protective impact against 5-FU with an evident modulatory impact on Nrf-2/Keap-1 and its downstream effectors, HO-1 and γ-GCS, suggesting its potential use in 5-FU regimens to improve its therapeutic outcomes and minimize its underlying nephrotoxicity.

The co-chaperone UNC45A is essential for the expression of mitotic kinase NEK7 and tumorigenesis.

Cumulative evidence suggests that the heat shock protein 90 (Hsp90) co-chaperone UNC-45 myosin chaperone A (UNC45A) contributes to tumorigenesis and that its expression in cancer cells correlates with proliferation and metastasis of solid tumors. However, the molecular mechanism by which UNC45A regulates cancer cell proliferation remains largely unknown. Here, using siRNA-mediated gene silencing and various human cells, we report that UNC45A is essential for breast cancer cell growth, but is dispensable for normal cell proliferation. Immunofluorescence microscopy, along with gene microarray and RT-quantitative PCR analyses, revealed that UNC45A localizes to the cancer cell nucleus, where it up-regulates the transcriptional activity of the glucocorticoid receptor and thereby promotes expression of the mitotic kinase NIMA-related kinase 7 (NEK7). We observed that UNC45A-deficient cancer cells exhibit extensive pericentrosomal material disorganization, as well as defects in centrosomal separation and mitotic chromosome alignment. Consequently, these cells stalled in metaphase and cytokinesis and ultimately underwent mitotic catastrophe, phenotypes that were rescued by heterologous NEK7 expression. Our results identify a key role for the co-chaperone UNC45A in cell proliferation and provide insight into the regulatory mechanism. We propose that UNC45A represents a promising new therapeutic target to inhibit cancer cell growth in solid tumor types.

Antineoplastic Activity of Chrysin against Human Hepatocellular Carcinoma: New Insight on GPC3/SULF2 Axis and lncRNA-AF085935 Expression.

The natural flavonoid chrysin possesses antiproliferative activity against various types of cancers, including hepatocellular carcinoma (HCC), which is a common malignancy. However, the exact mechanism of chrysin antiproliferative activity remains unclear. This research was executed to explore the impact of chrysin on glypican-3 (GPC3)/sulfatase-2 (SULF2) axis and lncRNA-AF085935 expression in HCC using HepG2 cells. Cisplatin (20, 50, 100 µg/mL), chrysin (15, 30, and 60 µg/mL) and the combination of 50 µg/mL cisplatin with different concentrations of chrysin were applied for 24/48 h. Cell viability was determined by MTT assay. Protein levels of GPC3 and SULF2 were measured by ELISA at 24/48 h. GPC3 immunoreactivity was detected by immunocytochemistry. Moreover, GPC3 and SULF2 mRNA expressions in addition to lncRNA-AF085935 expression were assessed by qPCR at 48 h. The GPC3 protein, immunostaining and mRNA levels, SULF2 protein and mRNA levels, as well as lncRNA-AF085935 expression, were decreased significantly with cisplatin and chrysin alone when compared with the control untreated HepG2 cells. However, the combination treatment exhibited a better chemopreventive effect in a dose- and time-dependent manner. This study demonstrated, for the first time, the antiproliferative activity of chrysin against HCC through the suppression of the GPC3/SULF2 axis along with the downregulation of lncRNA-AF085935 expression. Synergistic effect of chrysin with cisplatin could potentiate their antiproliferative action in a dose- and time-dependent manner.

Clinical significance of the TNF-α receptors, TNFRSF2 and TNFRSF9, on cell migration molecules Fascin-1 and Versican in acute leukemia.

In human hematologic malignancies, some of the TNF receptor family members are up-regulated and have the ability to evoke reactions favoring tumor progression. Moreover, cell migration molecules, Fascin-1 and Versican are involved in proliferation, migration and invasion of cancer cells. They are linked to many human cancers. Therefore, we conducted this study to evaluate both the plasma and leukocytes concentrations of tumor necrosis factor receptor super family 2 (TNFRSF2), TNFRSF9, Fascin-1 and Versican in patients with acute leukemia, as well as to correlate these values with clinical features and treatment outcome. Therefore, forty-eight patients with acute myeloid leukemia (AML), thirty-two patients with acute lymphoblastic leukemia (ALL) and fifteen control subjects were included. TNFRSF2, TNFRSF9, Fascin-1 and Versican were measured in plasma and leukocytes of all subjects by enzyme-linked immunosorbent assay. We found that plasma TNFRSF9 was highly elevated in ALL and AML as compared with the control group. In addition, AML patients who failed to achieve complete remission showed a significant increase in leukocytes TNFRSF9 level. TNFRSF2 is significantly increased in plasma and leukocytes of ALL patients when compared with the control group and AML patients. Fascin-1 significantly increased in AML, but not in ALL cases. Plasma and leukocytes levels of Versican significantly increased in AML compared to both control and ALL subjects. Plasma Versican correlated with poor response to induction of chemotherapy in AML cases. In conclusion, TNFRSF2 and TNFRSF9 could act as a possible prognostic biomarkers for the outcomes of ALL patients and TNFRSF9 could be a potential target in AML. Versican may be used as a diagnostic biomarker and as a predictor of the response to chemotherapy in AML. In addition, plasma Fascin-1 is a potential biomarker for AML.

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.

The role of IL-18 in type 1 diabetic nephropathy: The problem and future treatment.

Diabetic vascular complication is a leading cause of diabetic nephropathy, a progressive increase in urinary albumin excretion coupled with elevated blood pressure leading to declined glomerular filtration and eventually end stage renal failure. There is growing evidence that activated inflammation is contributing factor to the pathogenesis of diabetic nephropathy. Meanwhile, IL-18, a member of the IL-1 family of inflammatory cytokines, is involved in the development and progression of diabetic nephropathy. However, the benefits derived from the current therapeutics for diabetic nephropathy strategies still provide imperfect protection against renal progression. This imperfection points to the need for newer therapeutic agents that have potential to affect primary mechanisms contributing to the pathogenesis of diabetic nephropathy. Therefore, the recognition of IL-18 as significant pathogenic mediators in diabetic nephropathy leaves open the possibility of new potential therapeutic targets.

Renal protective effects of arjunolic acid in a cisplatin-induced nephrotoxicity model.

Cisplatin is the first platinum-containing anti-cancer drugs. Cisplatin notable side effect of nephrotoxicity limits its use in clinic. Meanwhile, arjunolic acid possesses anti-inflammatory properties and plays protective roles against chemically induced organ pathophysiology. This study was conducted to find out whether arjunolic acid could attenuate kidney damage in rats, and to elucidate its possible mechanism of action. Fifty rats were treated with cisplatin (10mg/kg) in the presence/absence of 100 or 250mg/kg arjunolic acid. Arjunolic acid is given 1h after cisplatin. Morphological changes were assessed in kidney sections stained with Hematoxylin/Eosin and Masson Trichrome. Kidney samples were used for measurements of transforming growth factor (TGF)-ß1 and its type 1 receptor (TGF-ßR1), tumor necrosis factor (TNF)-α and interleukin (IL)-1ß by ELISA. Gene expression NFκB was determined by real time-PCR. Kidney tissue apoptosis was assessed by measuring the activities of caspase-3/8/9. The renal protective effect of arjunolic acid was confirmed by approximately normal appearance of renal tissue and the relatively unaffected serum creatinine and urea levels. Furthermore, arjunolic acid showed dose dependent reduction in cisplatin-induced elevation in renal levels of TGF-ßR1, TGF-ß1, TNF-α, IL-1ß and caspases. These findings demonstrated that arjunolic acid attenuates cisplatin nephrotoxicity either indirectly by enhancing body antioxidant activity or directly through several mechanisms, including inhibition of pro-inflammatory cytokines, blocking activation of TGF-ß1, and anti-apoptotic effects.

Phenethyl isothiocyanate potentiates anti-tumour effect of doxorubicin through Akt-dependent pathway.

The present study aims to investigate the in vivo and in vitro anti-tumour properties of phenethyl isothiocyanate (PEITC) alone and in combination with doxorubicin (Dox). The anti-tumour activity was evaluated in vitro by MTT assay using cultured human breast cancer cell line (MCF-7) and human hepatoma cell line (HepG-2) cell lines. In vivo, Ehrlich solid tumour model was used. Tumour volume, weight and antioxidant parameters were determined. Immunohistochemistry analysis for active (cleaved) caspase-3 was also performed. We tested the effect of PEITC treatment on pAkt/Akt ratio, NF-κB p65 DNA binding activity and caspase-9 enzyme activity in both MCF-7 and HepG-2 cell lines. Effect of PEITC treatment on cell migration was assessed by wound healing assay. PEITC and/or Dox treatment significantly inhibited solid tumour volume and tumour weight when compared with control mice. PEITC treatment significantly reduced oxidative stress caused by Dox treatment as indicated by significant increase in total antioxidant capacity and decrease in malondialdehyde level. Microscopic examination of tumour tissues showed a significant increase in active (cleaved) caspase-3 expression in PEITC and/or Dox treated groups. PEITC showed a dose-dependent inhibition of MCF-7 and HepG-2 cellular viability. PEITC inhibited Akt and NF-κB activation and increased caspase-9 activity in a dose-dependent manner. PEITC treatment effectively inhibited both MCF-7 and HepG-2 cell migration. We can conclude that PEITC acts via multiple molecular targets to elicit anti-carcinogenic activity. PEITC/Dox combination therapy might be a potential novel strategy, which may benefit patients with breast and liver cancers.

Adenosine kinase inhibition protects the kidney against streptozotocin-induced diabetes through anti-inflammatory and anti-oxidant mechanisms.

Adenosine provides anti-inflammatory effects in cardiovascular disease via the activation of adenosine A2A receptors; however, the physiological effect of adenosine could be limited due to its phosphorylation by adenosine kinase. We hypothesized that inhibition of adenosine kinase exacerbates extracellular adenosine levels to reduce renal inflammation and injury in streptozotocin-induced diabetes. Diabetes was induced in male C57BL/6 mice by daily injection of streptozotocin (50mg/kg/day, i.p. for 5 days). Control and diabetic mice were then treated with the adenosine kinase inhibitor ABT702 (1.5mg/kg, i.p. two times a week for 8 weeks, n=7-8/group) or the vehicle (5% DMSO). ABT702 treatment reduced blood glucose level in diabetic mice (∼20%; P<0.05). ABT702 also reduced albuminuria and markers of glomerular injury, nephrinuria and podocalyxin excretion levels, in diabetic mice. Renal NADPH oxidase activity and urinary thiobarbituric acid reactive substances (TBARS) excretion, indices of oxidative stress, were also elevated in diabetic mice and ABT702 significantly reduced these changes. ABT702 increased renal endothelial nitric oxide synthase expression (eNOS) and nitrate/nitrite excretion levels in diabetic mice. In addition, the diabetic mice displayed an increase in renal macrophage infiltration, in association with increased renal NFκB activation. Importantly, treatment with ABT702 significantly reduced all these inflammatory parameters (P<0.05). Furthermore, ABT702 decreased glomerular permeability and inflammation and restored the decrease in glomerular occludin expression in vitro in high glucose treated human glomerular endothelial cells. Collectively, the results suggest that the reno-protective effects of ABT702 could be attributed to the reduction in renal inflammation and oxidative stress in diabetic mice.

Pramipexole protects against diabetic neuropathy: Effect on oxidative stress, TLR4/IRAK-1/TRAF-6/NF-κB and downstream inflammatory mediators.

BACKGROUND: Diabetic neuropathy (DN) is a serious microvascular complication and a major cause of morbidity and mortality in diabetes mellitus. It is characterized by neurodegeneration of terminal sensory nerve fibers with subsequent pain, loss of sensation, and paresthesia, thus compromising the quality of life of diabetic patients. It is considered the leading cause of non-traumatic amputations worldwide, reflecting the insufficiency of current therapies. Pramipexole (PPX) is a dopamine receptor agonist used for the treatment of Parkinson's disease. The current study aims to investigate the potential neuroprotective effect of PPX in an experimental model of DN. METHODS: Sprague Dawley rats were randomly assigned into five groups: normal control, Normal + PPX (1 mg/kg) group, STZ control, STZ + PPX (0.25 and 1 mg/kg/day for eight weeks). The neuroprotective effect of PPX in rats was evaluated in terms of sciatic nerve histological alterations, oxidative stress, and protein expression of TLR4/MyD88/IRAK-1/TRAF-6/NF-κB axis and downstream inflammatory mediators. RESULTS: PPX administration ameliorated histopathological signs of neuronal inflammation and apoptosis. Additionally, PPX attenuated STZ-induced sciatic nerve oxidative stress and downregulated neural tissue expression of TLR4, MyD88, IRAK-1, TRAF-6, NF-κB and downstream mediators (TNF-α, IL-1ß and ICAM-1). CONCLUSION: Collectively, the current study sheds light on PPX as a potential protective medication to alleviate neuropathy progression in diabetic patients. PPX neuroprotective effect can be attributed to modulating TLR4/ MyD88/IRAK-1/TRAF-6/ NF-κB axis signaling in nerve tissues with subsequent attenuation of oxidative stress and inflammation.

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.

Potential roles of adenosine deaminase-2 in diabetic retinopathy.

The early activation of microglia that induces retinal inflammation in DR may serve as a target for therapeutic intervention of DR. Our demonstration that retinal inflammation is attenuated via adenosine receptor A(2A)AR supports the hypothesis that a mechanism to maintain extracellular concentrations of adenosine important in normal physiology is impaired in DR. Extracellular concentrations of adenosine are regulated by the interplay of equiliberative nucleoside transporter (ENT)s with enzymes of adenosine metabolism including adenosine deaminase-1 (ADA1), adenosine kinase (AK) and CD73. In the vertebrates but not rodents, a macrophage-associated ADA2 is identified. The role of ADA2 is, therefore, understudied as the sequencing probes or antibodies to mouse ADA2 are not available. We identified increased ADA2 expression and activity in human and porcine retinas with diabetes, and in Amadori glycated albumin (AGA)- or hyperglycemia-treated porcine and human microglia. In rodent as well as porcine cells, modulation of TNF-α release is mediated by A(2A)AR. Quantitative analysis of normal and diabetic porcine retinas reveals that while the expression levels of ADA2, A2AAR, ENT1, TNF-α and MMP9 are increased, the levels of AK are reduced during inflammation as an endogenous protective mechanism. To determine the role of ADA2, we found that AGA induces ADA2 expression, ADA2 activity and TNF-α release, and that TNF-α release is blocked by ADA2-neutralizing antibody or ADA2 siRNA, but not by scrambled siRNA. These results suggest that retinal inflammation in DR is mediated by ADA2, and that the anti-inflammatory activity of A(2A)AR signaling is impaired in diabetes due to increased ADA2 activity.

Origanum majorana L. Extract Attenuated Benign Prostatic Hyperplasia in Rat Model: Effect on Oxidative Stress, Apoptosis, and Proliferation.

Benign prostatic hyperplasia (BPH) is a widespread androgenic illness influencing elderly men. It is distinguished by prostatic epithelial and stromal muscle cell proliferation. Inflammation, oxidative stress, and apoptosis have all been interrelated to the development of BPH. Marjoram (Origanum majorana L.) is a herb with reported antiproliferative, proapoptotic, and antioxidative properties, which have not yet been studied in relation to BPH. Consequently, in this work, an ethanolic extract of O. majorana was prepared in two doses (250 and 500 mg/kg/day) to be injected into castrated rats after induction of a testosterone-BPH model. Testosterone propionate (TP) was subcutaneously injected (0.5 mg/kg/day) for one week after castration to induce BPH. Forty adult Wistar male rats were randomly allocated into five groups: control, BPH model, high and low O. majorana doses (250, 500 mg/kg/day), and finasteride (FN) (0.8 mg/kg/day) as a positive control. Treatment was continued with drugs/normal saline for 28 days. Rat's body and prostate were weighed, prostate index (PI) and % of prostate growth inhibition were calculated, serum dihydrotestosterone (DHT), prostatic content of superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (TAC), and malondialdehyde (MDA), DN damage, histopathological changes, immune expression of proliferating cell nuclear antigen (PCNA), caspase-3, α-SMA, and TGF-ß1 were assessed. In addition, molecular quantitative PCR and ELISA analyses were performed to identify the expression of mRNAs and related proteins of both caspase-3 and TGF-ß1 in prostate tissue from O. majorana-treated and untreated groups. Rats with BPH had significantly higher prostate weights and PI, higher DHT, DNA damage (8-hydroxyguanine, 8-OH-dG), and MDA levels with prominent PCNA, α-SMA, and TGF-ß expression, but lower SOD, CAT, and TAC activity and caspase-3 expression. O. majorana (250 and 500 mg/kg/day)-treated groups revealed a decrease in prostate weights and PI, lower levels of DHT, suppressed oxidative stress, reduced tissue proliferation and fibrosis, and restored antioxidant and proapoptotic activity. Additionally, quantitative PCR and ELISA analysis showed that treatment with O. majorana significantly upregulated the expression of caspase-3 and downregulated the expression of TGF-ß in prostate tissues of BPH rats. The data were confirmed by the immunohistological reactivity of these targeted markers in the prostate tissues. These effects were more significant with O. majorana 500 mg/mL/rat. In conclusion, the current study indicates the efficient use of O. majorana in the treatment of testosterone-induced BPH through its antiproliferative, proapoptotic, and antioxidative mechanisms.

Glycyrrhizic acid ameliorates submandibular gland oxidative stress, autophagy and vascular dysfunction in rat model of type 1 diabetes.

The burden of diabetes mellitus (DM) and associated complications is increasing worldwide, affecting many organ functionalities including submandibular glands (SMG). The present study aims to investigate the potential ameliorative effect of glycyrrhizic acid (GA) on diabetes-induced SMG damage. Experimental evaluation of GA treatment was conducted on a rat model of type I diabetes. Animals were assigned to three groups; control, diabetic and GA treated diabetic groups. After 8 weeks, the SMG was processed for assessment of oxidative stress markers, autophagy related proteins; LC3, Beclin-1 and P62, vascular regulator ET-1, aquaporins (AQPs 1.4 and 5), SIRT1 protein expressions in addition to LC3 and AQP5 mRNA expressions. Also, parenchymal structures of the SMG were examined. GA alleviated the diabetes-induced SMG damage via restoring the SMG levels of oxidative stress markers and ET-1 almost near to the normal levels most probably via regulation of SIRT1, AQPs and accordingly LC-3, P62 and Beclin-1levels. GA could be a promising candidate for the treatment of diabetes-induced SMG damage via regulating oxidative stress, autophagy and angiogenesis.

Decoding Diabetes Biomarkers and Related Molecular Mechanisms by Using Machine Learning, Text Mining, and Gene Expression Analysis.

The molecular basis of diabetes mellitus is yet to be fully elucidated. We aimed to identify the most frequently reported and differential expressed genes (DEGs) in diabetes by using bioinformatics approaches. Text mining was used to screen 40,225 article abstracts from diabetes literature. These studies highlighted 5939 diabetes-related genes spread across 22 human chromosomes, with 112 genes mentioned in more than 50 studies. Among these genes, HNF4A, PPARA, VEGFA, TCF7L2, HLA-DRB1, PPARG, NOS3, KCNJ11, PRKAA2, and HNF1A were mentioned in more than 200 articles. These genes are correlated with the regulation of glycogen and polysaccharide, adipogenesis, AGE/RAGE, and macrophage differentiation. Three datasets (44 patients and 57 controls) were subjected to gene expression analysis. The analysis revealed 135 significant DEGs, of which CEACAM6, ENPP4, HDAC5, HPCAL1, PARVG, STYXL1, VPS28, ZBTB33, ZFP37 and CCDC58 were the top 10 DEGs. These genes were enriched in aerobic respiration, T-cell antigen receptor pathway, tricarboxylic acid metabolic process, vitamin D receptor pathway, toll-like receptor signaling, and endoplasmic reticulum (ER) unfolded protein response. The results of text mining and gene expression analyses used as attribute values for machine learning (ML) analysis. The decision tree, extra-tree regressor and random forest algorithms were used in ML analysis to identify unique markers that could be used as diabetes diagnosis tools. These algorithms produced prediction models with accuracy ranges from 0.6364 to 0.88 and overall confidence interval (CI) of 95%. There were 39 biomarkers that could distinguish diabetic and non-diabetic patients, 12 of which were repeated multiple times. The majority of these genes are associated with stress response, signalling regulation, locomotion, cell motility, growth, and muscle adaptation. Machine learning algorithms highlighted the use of the HLA-DQB1 gene as a biomarker for diabetes early detection. Our data mining and gene expression analysis have provided useful information about potential biomarkers in diabetes.

Dapagliflozin, Liraglutide, and Their Combination Attenuate Diabetes Mellitus-Associated Hepato-Renal Injury-Insight into Oxidative Injury/Inflammation/Apoptosis Modulation.

In this study, we aim to explore the beneficial therapeutic impacts of dapagliflozin (Dapa), a highly potent, reversible, and selective sodium-glucose cotransporter-2 inhibitor, and liraglutide (Lira), a glucagon-like peptide-1 (GLP-1) receptor agonist, as hypoglycaemic agents for the management of diabetes mellitus (DM), as well as their combination against DM-induced complications, including hepato-renal injury. Indeed, the progression of DM was found to be associated with significant hepatic and renal injury, as confirmed by the elevated biochemical indices of hepatic and renal functions, as well as histopathological examination. Dapa, Lira, and their combination effectively attenuated DM-induced hepatic and renal injury, as confirmed by the recovery of hepatic and renal functional biomarkers. The administration of both drugs significantly reduced the tissue contents of MDA and restored the contents of GSH and catalase activity. Moreover, NF-κB and TNF-α expression at the protein and gene levels was significantly reduced in the liver and the kidney. This was in parallel with the significant reduction in the caspase-3 content in the liver and the kidney, as well as suppressed cleaved caspase-3 expression in the hepatic and renal specimens, as confirmed by immune-histochemical analysis. Notably, the combined Dapa/Lira treatment demonstrated an additive superior hepato-renal protective impact compared with the use of either drug alone. Thus, it appears that Dapa and Lira, through the coordinated modulation of oxidative, inflammatory, and apoptotic signalling, confer a significant hepato-renal protective impact against DM-induced complications and tissue injury.