Options for Topical Treatment of Oxidative Eye Diseases with a Special Focus on Retinopathies.

Antioxidants, usually administered orally through the systemic route, are known to counteract the harmful effects of oxidative stress on retinal cells. The formulation of these antioxidants as eye drops might offer a new option in the treatment of oxidative retinopathies. In this review, we will focus on the use of some of the most potent antioxidants in treating retinal neuropathies. Melatonin, known for its neuroprotective qualities, may mitigate oxidative damage in the retina. N-acetyl-cysteine (NAC), a precursor to glutathione, enhances the endogenous antioxidant defense system, potentially reducing retinal oxidative stress. Idebenone, a synthetic analogue of coenzyme Q10, and edaravone, a free radical scavenger, contribute to cellular protection against oxidative injury. Epigallocatechin-3-gallate (EGCG), a polyphenol found in green tea, possesses anti-inflammatory and antioxidant effects that could be beneficial in cases of retinopathy. Formulating these antioxidants as eye drops presents a localized and targeted delivery method, ensuring effective concentrations reach the retina. This approach might minimize systemic side effects and enhance therapeutic efficacy. In this paper, we also introduce a relatively new strategy: the alkylation of two antioxidants, namely, edaravone and EGCG, to improve their insertion into the lipid bilayer of liposomes or even directly into cellular membranes, facilitating their crossing of epithelial barriers and targeting the posterior segment of the eye. The synergistic action of these antioxidants may offer a multifaceted defense against oxidative damage, holding potential for the treatment and management of oxidative retinopathies. Further research and clinical trials will be necessary to validate the safety and efficacy of these formulations, but the prospect of antioxidant-based eye drops represents a promising avenue for future ocular therapies.

Increased Heat Pain Tolerance but Hyperalgesia to Tonic Inflammatory Pain in the CRND8 Mouse Model of Alzheimer's Disease.

BACKGROUND: The effects of Alzheimer's disease (AD) pathology on the experience of pain are poorly understood. OBJECTIVE: To understand the pathophysiological mechanisms underlying pain sensory transmission in the transgenic mouse model of AD, CRND8. METHODS: We explored AD-related pathology in the spinal cord and dorsal root ganglia of 18-week-old female CRND8 mice. We assessed nociceptive responses to both acute heat stimuli and persistent inflammatory pain in CRND8 mice and non-transgenic (non-Tg) littermates. In addition, we searched for differences in biochemical correlates of inflammatory pain between CRND8 and non-Tg mice. Finally, we investigated the excitability of dorsal horn noc iceptive neurons in spinal cord slices from CRND8 and non-Tg mice. RESULTS: We demonstrated the presence of intracellular AD-like pathology in the spinal cord and in the dorsal root ganglia nociceptive sensory neurons of CRND8 mice. We found that CRND8 mice had a reduced susceptibility to acute noxious heat stimuli and an increased sensitivity to tonic inflammatory pain. Tonic inflammatory pain correlated with a lack of induction of pro-opiomelanocortin in the spinal cord of CRND8 mice as compared to non-Tg mice. Electrophysiological recording in acute spinal cord slice preparations indicated an increased probability of glutamate release at the membrane of dorsal horn nociceptive neurons in CRND8 mice. CONCLUSION: This study suggests that an increased thermal tolerance and a facilitation of nociception by peripheral inflammation can coexist in AD.

Molecular Connections between DNA Replication and Cell Death in ß-Amyloid-Treated Neurons.

BACKGROUND: Ectopic cell cycle reactivation in neurons is associated with neuronal death in Alzheimer's disease. In cultured rodent neurons, synthetic ß-amyloid (Aß) reproduces the neuronal cell cycle re-entry observed in the Alzheimer's brain, and blockade of the cycle prevents Aß-induced neurodegeneration. DNA polymerase-ß, whose expression is induced by Aß, is responsible for the DNA replication process that ultimately leads to neuronal death, but the molecular mechanism(s) linking DNA replication to neuronal apoptosis are presently unknown. AIM: To explore the role of a conserved checkpoint pathway started by DNA replication stress, namely the ATM-ATR/Claspin/Chk-1 pathway, in switching the neuronal response from DNA replication to apoptosis. METHODS: Experiments were carried out in cultured rat cortical neurons challenged with toxic oligomers of Aß protein. RESULTS: Small inhibitory molecules of ATM/ATR kinase or Chk-1 amplified Aß-induced neuronal DNA replication and apoptosis, as they were permissive to the DNA polymerase-ß activity triggered by Aß oligomers. Claspin, i.e., the adaptor protein between ATM/ATR kinase and the downstream Chk-1, was present on DNA replication forks of neurons early after Aß challenge, and decreased at times coinciding with neuronal apoptosis. The caspase-3/7 inhibitor I maintained overtime the amount of Claspin loaded on DNA replication forks and, concomitantly, reduced neuronal apoptosis by holding neurons in the S phase. Moreover, a short phosphopeptide mimicking the Chk-1-binding motif of Claspin was able to prevent Aß-challenged neurons from entering apoptosis. CONCLUSION: We speculate that, in the Alzheimer's brain, Claspin degradation by intervening factors may precipitate the death of neurons engaged into DNA replication.

Wild Artichoke (Cynara cardunculus subsp. sylvestris, Asteraceae) Leaf Extract: Phenolic Profile and Oxidative Stress Inhibitory Effects on HepG2 Cells.

Cynara cardunculus subsp. sylvestris (wild artichoke) is widespread in Sicily, where it has been used for food and medicinal purposes since ancient times; decoctions of the aerial parts of this plant have been traditionally employed as a remedy for different hepatic diseases. In this study, the phenolic profile and cell-free antioxidant properties of the leaf aqueous extract of wild artichokes grown in Sicily (Italy) were investigated. The crude extract was also tested in cells for its antioxidant characteristics and potential oxidative stress inhibitory effects. To resemble the features of the early stage of mild steatosis in humans, human HepG2 cells treated with free fatty acids at the concentration of 1.5 mM were used. HPLC-DAD analysis revealed the presence of several phenolic acids (caffeoylquinic acids) and flavonoids (luteolin and apigenin derivatives). At the same time, DPPH assay showed a promising antioxidant power (IC50 = 20.04 ± 2.52 µg/mL). Biological investigations showed the safety of the crude extract and its capacity to counteract the injury induced by FFA exposure by restoring cell viability and counteracting oxidative stress through inhibiting reactive oxygen species and lipid peroxidation and increasing thiol-group levels. In addition, the extract increased mRNA expression of some proteins implicated in the antioxidant defense (Nrf2, Gpx, and SOD1) and decreased mRNA levels of inflammatory cytokines (IL-6, TNF-α, and IL-1ß), which were modified by FFA treatment. Results suggest that the total phytocomplex contained in wild artichoke leaves effectively modulates FFA-induced hepatic oxidative stress.

Protocatechuic Acid, a Simple Plant Secondary Metabolite, Induced Apoptosis by Promoting Oxidative Stress through HO-1 Downregulation and p21 Upregulation in Colon Cancer Cells.

Gastrointestinal cancers, particularly colorectal cancer, are mainly influenced by the dietary factor. A diet rich in fruits and vegetables can help to reduce the incidence of colorectal cancer thanks to the phenolic compounds, which possess antimutagenic and anticarcinogenic properties. Polyphenols, alongside their well-known antioxidant properties, also show a pro-oxidative potential, which makes it possible to sensitize tumor cells to oxidative stress. HO-1 combined with antioxidant activity, when overexpressed in cancer cells, is involved in tumor progression, and its inhibition is considered a feasible therapeutic strategy in cancer treatment. In this study, the effects of protocatechuic acid (PCA) on the viability of colon cancer cells (CaCo-2), annexin V, LDH release, reactive oxygen species levels, total thiol content, HO-1, γ-glutamylcysteine synthetase, and p21 expression were evaluated. PCA induced, in a dose-dependent manner, a significantly reduced cell viability of CaCo-2 by oxidative/antioxidant imbalance. The phenolic acid induced modifications in levels of HO-1, non-proteic thiol groups, γ-glutamylcysteine synthetase, reactive oxygen species, and p21. PCA induced a pro-oxidant effect in cancer cells, and the in vitro pro-apoptotic effect on CaCo-2 cells is mediated by the modulation of redox balance and the inhibition of the HO-1 system that led to the activation of p21. Our results suggest that PCA may represent a useful tool in prevention and/or therapy of colon cancer.

ß-amyloid monomers drive up neuronal aerobic glycolysis in response to energy stressors.

Research on cerebral glucose metabolism has shown that the aging brain experiences a fall of aerobic glycolysis, and that the age-related loss of aerobic glycolysis may accelerate Alzheimer's disease pathology. In the healthy brain, aerobic glycolysis, namely the use of glucose outside oxidative phosphorylation, may cover energy demand and increase neuronal resilience to stressors at once. Currently, the drivers of aerobic glycolysis in neurons are unknown. We previously demonstrated that synthetic monomers of ß-amyloid protein (Aß) enhance glucose uptake in neurons, and that endogenous Aß is required for depolarization-induced glucose uptake in cultured neurons. In this work, we show that cultured cortical neurons increased aerobic glycolysis in response to the inhibition of oxidative phosphorylation by oligomycin or to a kainate pulse. Such an increase was prevented by blocking the endogenous Aß tone and re-established by the exogenous addition of synthetic Aß monomers. The activity of mitochondria-bound hexokinase-1 appeared to be necessary for monomers-stimulated aerobic glycolysis during oxidative phosphorylation blockade or kainate excitation. Our data suggest that, through Aß release, neurons coordinate glucose uptake with aerobic glycolysis in response to metabolic stressors. The implications of this new finding are that the age-related drop in aerobic glycolysis and the susceptibility to Alzheimer's disease could be linked to factors interfering with release and functions of Aß monomers.

Role for Metallothionein-3 in the Resistance of Human U87 Glioblastoma Cells to Temozolomide.

Metallothioneins (MTs) are metal-binding proteins that are overexpressed in various human cancers and are thought to be associated with resistance to cytotoxic drugs. The knowledge on MT expression, regulation, and function in human gliomas is limited. We found that MT3 mRNA was highly expressed in cell lines derived from grade IV gliomas (i.e., A172 and U87 cells), as compared to grade II astrocytoma cells (i.e., 1321N1). Different from 1321N1, U87 cells were partly resistant to the alkylating drug, temozolomide (TMZ) (100 µM for 96 h), which induced a massive accumulation of U87 into the S and G2 fractions of the cell cycle but not apoptotic death. Silencing of MT3 did not significantly affect U87 cell proliferation and survival, but it delayed G1/S transition and favored the occurrence of apoptosis in TMZ-treated cells. Accordingly, the combination of MT3 silencing and TMZ treatment increased the protein levels of checkpoint kinase-1, which was ultimately responsible for the lasting G1 arrest and death of double treated U87 cells.

Inflammation as the Common Biological Link Between Depression and Cardiovascular Diseases: Can Carnosine Exert a Protective Role?

Several epidemiological studies have clearly shown the high co-morbidity between depression and Cardiovascular Diseases (CVD). Different studies have been conducted to identify the common pathophysiological events of these diseases such as the overactivation of the hypothalamic- pituitary-adrenal axis and, most importantly, the dysregulation of immune system which causes a chronic pro-inflammatory status. The biological link between depression, inflammation, and CVD can be related to high levels of pro-inflammatory cytokines, such as IL-1ß, TNF-α, and IL-6, released by macrophages which play a central role in the pathophysiology of both depression and CVD. Pro-inflammatory cytokines interfere with many of the pathophysiological mechanisms relevant to depression by upregulating the rate-limiting enzymes in the metabolic pathway of tryptophan and altering serotonin metabolism. These cytokines also increase the risk to develop CVD, because activation of macrophages under this pro-inflammatory status is closely associated with endothelial dysfunction and oxidative stress, a preamble to atherosclerosis and atherothrombosis. Carnosine (ß-alanyl-L-histidine) is an endogenous dipeptide which exerts a strong antiinflammatory activity on macrophages by suppressing reactive species and pro-inflammatory cytokines production and altering pro-inflammatory/anti-inflammatory macrophage polarization. This dipeptide exhibits antioxidant properties scavenging reactive species and preventing oxidative stress-induced pathologies such as CVD. In the present review we will discuss the role of oxidative stress and chronic inflammation as common pathophysiological events both in depression and CVD and the preclinical and clinical evidence on the protective effect of carnosine in both diseases as well as the therapeutic potential of this dipeptide in depressed patients with a high co-morbidity of cardiovascular diseases.

Effects of an extract of Celtis aetnensis (Tornab.) Strobl twigs on human colon cancer cell cultures.

Cancers of the digestive tract, in particular colorectal cancer (CRC), are among those most responsive to dietary modification. Research has shown that approximately 75% of all sporadic cases of CRC are directly influenced by diet. Many natural compounds have been investigated for their potential usefulness as cancer chemopreventive agents as they have been thought to suppress carcinogenesis mainly during the initiation phase due to their radical scavenger activity. Since there is an increasing interest in the in vivo protective effects of natural compounds contained in plants against oxidative damage involved in several human diseases such as cancer, the aim of the present research was to test the effects of a Celtis aetnensis (Tornab.) Strobl twig extract on a human colon carcinoma cell line (Caco2). In order to elucidate the mechanisms of action of this extract, LDH release, GSH content, ROS levels, caspase-3 and γ-GCS expression were also evaluated. The results revealed that the Celtis aetnensis extract reduced the cell viability of the Caco2 cells inducing apoptosis at the lowest concentration and necrosis at higher dosages. In addition, this extract caused an increase in the levels of ROS, a decrease in RSH levels and in the expression of HO-1. The expression of γ-GCS was not modified in the Celtis aetnensis-treated Caco-2 cells. These results suggest an interference of this extract on the oxidant/antioxidant cell balance with consequent cell damage. The present study supports the growing body of data suggesting the bioactivities of Celtis aetnensis (Tornab.) Strobl and its potential impact on cancer therapy and on human health.

Effects of Tithonia diversifolia (Hemsl.) A. Gray extract on adipocyte differentiation of human mesenchymal stem cells.

Tithonia diversifolia (Hemsl.) A. Gray (Asteraceae) is widely used in traditional medicine. There is increasing interest on the in vivo protective effects of natural compounds contained in plants against oxidative damage caused from reactive oxygen species. In the present study the total phenolic and flavonoid contents of aqueous, methanol and dichloromethane extracts of leaves of Tithonia diversifolia (Hemsl.) A. Gray were determined; furthermore, free radical scavenging capacity of each extract and the ability of these extracts to inhibit in vitro plasma lipid peroxidation were also evaluated. Since oxidative stress may be involved in trasformation of pre-adipocytes into adipocytes, to test the hypothesis that Tithonia extract may also affect adipocyte differentiation, human mesenchymal stem cell cultures were treated with Tithonia diversifolia aqueous extract and cell viability, free radical levels, Oil-Red O staining and western bolt analysis for heme oxygenase and 5'-adenosine monophoshate-activated protein kinase were carried out. Results obtained in the present study provide evidence that Tithonia diversifolia (Hemsl.) A. Gray exhibits interesting health promoting properties, resulting both from its free radical scavenger capacity and also by induction of protective cellular systems involved in cellular stress defenses and in adipogenesis of mesenchymal cells.

Neuroprotective effects of a glutathione depletor in rat post-ischemic reperfusion brain damage.

The induction of heme oxygenase (HO), the rate-limiting enzyme in heme degradation, occurs as an adaptative response to oxidative stress and is consequent to decrease in cellular glutathione levels. Our previous studies demonstrated significant increase in survival rates of rats treated with glutathione depletors and submitted to transient cerebral ischemia. The aim of the present research was to test the effects of L-Buthionine sulfoximine (BSO), a glutathione depletor, during cerebral post-ischemic reperfusion. Cerebral ischemia was induced by bilateral clamping of common carotid arteries for 20 min. Each sample was used for glutathione ad lipid peroxidation level dosage and for evaluating the expression of heme oxygenase both after a single subcutaneous administration of BSO and without treatment. In the same experimental conditions, endothelial, inducible and neuronal Nitric Oxide Synthase (eNOS, iNOS and nNOS) and Dimethylarginine Dimethyl amine Hydrolases (DDAH-1 and DDAH-2) were also evaluated. Results obtained in the present study suggested that HO-1 over-expression may be implicated in the protective effect of BSO in post-ischemic reperfusion brain damage, although the involvement of other important stress mediators cannot be ruled out.

Antioxidant activity of extracts of Momordica foetida Schumach. et Thonn.

Momordica foetida Schumach. et Thonn. (Cucurbitaceae) is a perennial climbing herb with tendrils, found in swampy areas in Central Uganda. Antidiabetic and antilipogenic activities were reported for some Momordica species, however the mechanism of action is still unknown. Oxidative stress may represent an important pathogenic mechanism in obesity-associated metabolic syndrome. The present study evaluated free radical scavenging capacity of different concentrations of aqueous, methanolic and dichloromethane leaf extracts of Momordica foetida Schumach. et Thonn. and the ability of these extracts to inhibit in vitro plasma lipid peroxidation; in addition, healthy human adipose mesenchymal stem cell cultures were used in order to test the hypothesis that these extracts may affect adipocyte differentiation. Results obtained in this study suggested that aqueous extract might be useful in preventing metabolic syndrome.

Effect of Treatment with Cyanidin-3-O-ß-D-Glucoside on Rat Ischemic/Reperfusion Brain Damage.

This study investigated the effect of cyanidin-3-O-ß-glucoside on an experimental model of partial/transient cerebral ischemia in the rats in order to verify the effectiveness of both pre- and posttreatments. Cyanidin-3-O-ß-glucoside-pretreated rats were injected with 10 mg/Kg i.p. 1 h before the induction of cerebral ischemia; in posttreated rats, the same dosage was injected during reperfusion (30 min after restoring blood flow). Cerebral ischemia was induced by bilateral clamping of common carotid arteries for 20 min. Ischemic rats were sacrificed immediately after 20 min ischemia; postischemic reperfused animals were sacrificed after 3 or 24 h of restoring blood flow. Results showed that treatment with cyanidin increased the levels of nonproteic thiol groups after 24 h of postischemic reperfusion, significantly reduced the lipid hydroperoxides, and increased the expression of heme oxygenase and γ-glutamyl cysteine synthase; a significant reduction in the expression of neuronal and inducible nitric oxide synthases and the equally significant increase in the endothelial isoform were observed. Significant modifications were also detected in enzymes involved in metabolism of endogenous inhibitors of nitric oxide. Most of the effects were observed with both pre- and posttreatments with cyanidin-3-O-ß-glucoside suggesting a role of anthocyanin in both prevention and treatment of postischemic reperfusion brain damage.

Biochemical modifications in Pinus pinaster Ait. as a result of environmental pollution.

Exposure to chemical pollution can cause significant damage to plants by imposing conditions of oxidative stress. Plants combat oxidative stress by inducing antioxidant metabolites, enzymatic scavengers of activated oxygen and heat shock proteins. The accumulation of these proteins, in particular heat shock protein 70 and heme oxygenase, is correlated with the acquisition of thermal and chemical adaptations and protection against oxidative stress. In this study, we used Pinus pinaster Ait. collected in the areas of Priolo and Aci Castello representing sites with elevated pollution and reference conditions, respectively. The presence of heavy metals and the levels of markers of oxidative stress (lipid hydroperoxide levels, thiol groups, superoxide dismutase activity and expression of heat shock protein 70, heme oxygenase and superoxide dismutase) were evaluated, and we measured in field-collected needles the response to environmental pollution. P. pinaster Ait. collected from a site characterized by industrial pollution including heavy metals had elevated stress response as indicated by significantly elevated lipid hydroperoxide levels and decreased thiol groups. In particular, we observed that following a chronic chemical exposure, P. pinaster Ait. showed significantly increased expression of heat shock protein 70, heme oxygenase and superoxide dismutase. This increased expression may have protective effects against oxidative stress and represents an adaptative cellular defence mechanism. These results suggest that evaluation of heme oxygenase, heat shock protein 70 and superoxide dismutase expression in P. pinaster Ait. could represent a useful tool for monitoring environmental contamination of a region and to better understand mechanisms involved in plant defence and stress tolerance.

Antiproliferative effect of oleuropein in prostate cell lines.

Currently, there is increasing interest in the in vivo protective effects of natural antioxidants found in dietary plants against oxidative damage caused by free radical species. Oxidative stress has been invoked as a causative agent in cancer and epidemiological data suggest that the consumption of fruits and vegetables may be associated with a lower incidence of cancer. The fruit of the Olea europaea L. and olive oil contain hundreds of phytochemicals and its extracts have recently been shown to exhibit antioxidant properties, due to the action of oleuropein. In view of these considerations, in this study, we investigated the effects of oleuropein on LNCaP and DU145 prostate cancer cell lines and on BPH-1 non-malignant cells. Oleuropein reduces cell viability and induces thiol group modifications, γ-glutamylcysteine synthetase, reactive oxygen species, pAkt and heme oxygenase-1. Exposing cell cultures to oleuropein induces an antioxidant effect on BPH-1 cells and a pro-oxidant effect on cancer cells. Our results confirm the beneficial properties of olive oil and oleuropein, suggesting its possible use as an adjuvant agent in the treatment of prostatitis, in order to prevent the transformation of hypertrophic to cancerous cells.

The DDAH/NOS pathway in human prostatic cancer cell lines: antiangiogenic effect of L-NAME.

Benign prostate hypertrophy (BPH) and prostate cancer (PC) are prostate chronic diseases that require a long period for development from a small lesion to clinical manifestation. PC is the most common cancer in men in Europe and the Americas. Tumor growth and metastasis depend upon the development of neovasculature around the tumor. This process, called angiogenesis, may be regulated by NO, and thus modulation of NO production could play an important role in tumor progression. Recent studies report the involvement of DDAH, an enzyme which metabolizes the endogenous NOS inhibitor ADMA, in the development of tumor vasculature. The aim of the present study was to verify the involvement of the DDAH/NOS pathway in the progression of prostate cancer. The effect of the NOS inhibitor L-NAME was evaluated in the human prostate cancer cell line LnCap and in BPH-1 cells which represent benign prostatic hypertrophy. Higher DDAH-2, eNOS, iNOS and VEGF expression was found in LnCap cells compared to BPH-1 cells. L-NAME treatment of LnCap cells resulted in a reduction in VEGF, iNOS and eNOS expression. VEGF, iNOS and eNOS inhibition is a promising approach for targeting tumor vasculature and certain NOS inhibitors could potentially serve as experimental agents for treatment of certain chemoresistant tumors, including prostate tumors. Moreover, since in our experimental conditions L-NAME was unable to reduce DDAH activity and expression, it is plausible to hypothesize the development of a targeted polypharmacological approach by developing dual and specific inhibitors of DDAH and NOS to better control NO biosynthesis.