Susi Zara

Ibuprofen and Lipoic Acid Diamides as Potential Codrugs with Neuroprotective Activity

Current evidences support the hypothesis that non‐steroidal anti‐inflammatory drugs (NSAIDs) and antioxidant therapy might protect against the development of Alzheimers disease (AD). In the present work, our attention was focused on ibuprofen (IBU) used in clinical trails to prevent Alzheimers disease, and (R)‐α‐lipoic acid (LA) considered as a potential neuroprotective agent in AD therapy. In particular, we investigated a series of lipophilic molecular combinations obtained by joining (R)‐α‐lipoic acid and ibuprofen via an amide bond. These new entities might allow targeted delivery of the parent drugs to neurons, where cellular oxidative stress and inflammation seem related to Alzheimers disease. Our study included the synthesis of conjugates 1–3 and the evaluation of their physicochemical and in‐vitro antioxidant properties. The new compounds are extremely stable in aqueous buffer solutions (pH = 1.3 and 7.4), and in rat and human plasma they showed a slow bioconversion to ibuprofen and (R)‐α‐lipoic acid. Codrugs 1–3 displayed in vitro free radical scavenging activity and were hydrolyzed more rapidly in brain tissue than in rat serum indicating that these new entities might allow targeted delivery of the parent drugs to neurons. The immunohistochemical analysis of Aβ (1‐40) protein showed that Aβ‐injected cerebral cortices treated with ibuprofen or compound 1 showed few plaques within capillary vessels and, in particular, Aβ (1‐40) protein was less expressed in codrug‐1‐treated than in ibuprofen‐treated cerebral cortex.

Ibuprofen and Glutathione Conjugate as a Potential Therapeutic Agent for Treating Alzheimer's Disease

Non‐steroidal anti‐inflammatory drugs (NSAIDs) and antioxidant therapy might protect against the development of Alzheimers disease (AD). In the present work, we synthesized a molecular combination of glutathione (GSH) and ibuprofen (IBU) via an amide bond and investigated its potential for targeted delivery of the parent drugs to neurons, where cellular oxidative stress and inflammation are related to AD. Evaluation of its physicochemical and in‐vitro antioxidant properties indicated that compound 1 exhibits good stability toward human plasma enzymatic activity, and, like GSH, displays in‐vitro free radical scavenging activity in a time and concentration‐dependent manner. The new compound was also assessed by infusion in a rat model for Alzheimers disease for its potential to antagonize the deleterious structural and cognitive effects of β‐amyloid(1‐40). In behavioral tests of long‐term spatial memory, animals treated with codrug 1 performed significantly better than those treated with β‐amyloid (Aβ) peptide. Histochemical findings confirmed the behavioral data, revealing that Aβ protein was less expressed in cerebral cortex treated with 1 than that treated with IBU. Taken together, the present findings suggest that conjugate 1 treatment may protect against the oxidative stress generated by reactive oxygen species (ROS) and the cognitive dysfunction induced by intracerebroventricular (i.c.v.) infusion of Aβ(1‐40) in rats, and thus that codrug 1 could prove useful as a tool for controlling AD induced cerebral amyloid deposits and behavioral deterioration.

2‐Hydroxyethyl methacrylate inflammatory effects in human gingival fibroblasts

AIM To investigate the inflammatory response in human gingival fibroblasts (HGFs) treated with a relatively low 2-hydroxyethyl methacrylate (HEMA) concentration by studying reactive oxygen species (ROS) production, cyclooxygenase-2 (COX-2) and tumour necrosis factor-alpha (TNF-α) gene expression, and prostaglandin E2 (PGE₂) release. METHODOLOGY Cultured HGFs were exposed to 3 mmol L⁻¹ HEMA for 0, 24 or 96 h. ROS production was investigated by flow cytometry; TNF-α and COX-2 gene expression was determined by RT-PCR, and prostaglandin E2 production was detected by an enzyme immunoassay. RESULTS After 24- or 96-h HEMA incubation, ROS levels were approximately eightfold and elevenfold higher than controls, whilst COX-2 gene expression was approximately twofold or fourfold higher than controls, respectively. Twenty-four-hour exposure enhanced TNF-α mRNA levels by approximately 66%, whilst after 96-h incubation, TNF-α gene expression was fivefold higher than controls. Ninety-six-hour HEMA treatment increased PGE₂ concentration in the culture medium by around 17% compared with controls. CONCLUSIONS 2-Hydroxyethyl methacrylate treatment (3 mmol L⁻¹) induced an inflammatory response in HGFs modulated by ROS production, as well as by the increase in TNF-α and COX-2 gene expression and by PGE₂ release.

Ionizing Radiation Induces Apoptotic Signal Through Protein Kinase Cδ (delta) and Survival Signal Through Akt and Cyclic-Nucleotide Response Element-Binding Protein (CREB) in Jurkat T Cells

Although ionizing radiation induces a loss of proliferative capacity as well as cell death by apoptosis and necrosis, cells can oppose the damaging effects by activating survival signal pathways. Here we report the effect of 1.5- and 6-Gy doses of ionizing radiation on apoptotic protein kinase Cδ (PKCδ) and survival cyclic-nucleotide response element-binding protein (CREB) signal in Jurkat T cells. Cell cycle analysis, performed by flow cytometry, showed a significant G2M arrest 24 h after exposure to 6 Gy. This arrest was accompanied by dead cells, which increased in number up to 7 days, when cell viability was further reduced. The response was apparently promoted by caspase-3-mediated PKCδ activation, and thus apoptosis. Moreover, the presence of viable cells up to 7 days in samples exposed to 6 Gy is explained by Akt activation, which may influence the nuclear transcription factor CREB, leading to resistance to ionizing radiation. Thus, the knowledge of apoptotic and survival pathways activated in tumor cells may help in establishing specific therapies by combining selective inhibitors or stimulators of key signaling proteins with conventional chemotherapy, hormone therapy, and radiotherapy.

Ibuprofen and Lipoic Acid Diamide as Co-Drug with Neuroprotective Activity: Pharmacological Properties and Effects in β-Amyloid (1–40) Infused Alzheimer's Disease Rat Model

Both oxidative stress and inflammation are elevated in brains of Alzheimers disease patients, but their pathogenic significance still remains unclear. Current evidence support the hypothesis that non-steroidal anti-inflammatory drugs (NSAIDs) and antioxidant therapy might protect against the development of Alzheimers disease, and ibuprofen has the strongest epidemiological support. In the present work our attention was focused on (R)-α-lipoic acid considered as a potential neuroprotective agent in Alzheimers disease therapy. In particular, we investigated a new co-drug (1) obtained by joining (R)-α-lipoic acid and ibuprofen via a diamide bond, for evaluating its potential to antagonize the deleterious structural and cognitive effects of β-amyloid (1–40) in an infused Alzheimers disease rat model. Our results indicated that infusion of β-amyloid (1–40) impairs memory performance through a progressive cognitive deterioration; however, ibuprofen and co-drug 1 seemed to protect against behavioural detriment induced by simultaneous administration of β-amyloid (1–40) protein. The obtained data were supported by the histochemical findings of the present study: β-amyloid protein was less expressed in 1-treated than in ibuprofen and (R)-α-lipoic acid alone-treated cerebral cortex. Taken together, the present findings suggest that co-drug 1 treatment may protect against the cognitive dysfunction induced by intracerebroventricular infusion of β-amyloid (1–40) in rats. Thus, co-drug 1 could prove useful as a tool for controlling Alzheimers disease-induced cerebral amyloid deposits and behavioural deterioration.

Streptococcus mitis/human gingival fibroblasts co-culture: the best natural association in answer to the 2-hydroxyethyl methacrylate release

Di Giulio M, D’Ercole S, Zara S, Cataldi A, Cellini L. Streptococcus mitis/human gingival fibroblasts co‐culture: the best natural association in answer to the 2‐hydroxyethyl methacrylate release. APMIS 2011.

Biologic and clinical aspects of integration of different bone substitutes in oral surgery: a literature review

Many bone substitutes have been proposed for bone regeneration, and researchers have focused on the interactions occurring between grafts and host tissue, as the biologic response of host tissue is related to the origin of the biomaterial. Bone substitutes used in oral and maxillofacial surgery could be categorized according to their biologic origin and source as autologous bone graft when obtained from the same individual receiving the graft; homologous bone graft, or allograft, when harvested from an individual other than the one receiving the graft; animal-derived heterologous bone graft, or xenograft, when derived from a species other than human; and alloplastic graft, made of bone substitute of synthetic origin. The aim of this review is to describe the most commonly used bone substitutes, according to their origin, and to focus on the biologic events that ultimately lead to the integration of a biomaterial with the host tissue.

Ibuprofen and lipoic acid conjugate neuroprotective activity is mediated by Ngb/Akt intracellular signaling pathway in Alzheimer's disease rat model.

Background: Alzheimers disease (AD) is a frequent form of senile dementia. Neuroglobin (Ngb) has a neuroprotective role and decreases Aβ peptide levels. Ngb, promoting Akt phosphorylation, activates cell survival involving cyclic-nucleotide response element-binding protein (CREB). A new molecule (IBU-LA) was synthetized and administered to an AD rat model to counteract AD progression. Objective: The aim of this study was to investigate the IBU-LA-mediated induction of Ngb neuroprotective and antiapoptotic activities. Methods: Brain morphology was analyzed through Bielschowsky staining, Aβ(1-40) and Ngb expression by immunohistochemistry. Akt, p-Akt, CREB and p-CREB expression was evaluated by Western blot, apoptosis through cytochrome C/Apaf 1 immunocomplex formation, and TUNEL analysis. Results: Bielschowsky staining and Aβ(1-40) expression show few nerve connections and Aβ(1-40) expression in an Aβ sample, preserved neuronal cells and Aβ(1-40) expression lowering in an IBU sample, mostly in IBU-LA. The Ngb level decreases in Aβ samples, compared to control and IBU-LA samples. p-Akt/Akt and p-CREB/CREB ratios reveal a reduction in Aβ sample, going back to the basal level in control and IBU-LA samples. Cytochrome C/Apaf 1 co-immunoprecipitate occurs and TUNEL-positive nuclei percentage decreases in Aβ sample. Probe test performance shows an increased spatial reference memory in the IBU-LA compared to the Aβ sample; no significant differences were seen between the IBU-LA and IBU samples. Conclusion: This evidence reveals that IBU-LA administration has the capability to maintain a high Ngb level allowing Ngb to perform a neuroprotective and antiapoptotic role, representing a valid tool in the therapeutic strategy of AD progression.

PKC-δ signalling pathway is involved in H9c2 cells differentiation

H9c2 are rat heart embryonic myoblasts, with skeletal muscle properties, which terminally differentiate by fusing and forming multinucleated myotubes. Here we investigated the possible involvement of Protein Kinases C (PKCs) in H9c2 cell differentiation and explored the interplay of these enzymes both with reactive oxygen species (ROS), upstream physiological mediators of cell differentiation, and with nitric oxide (NO), downstream target of PKC activation, known for being involved in apoptosis induction in differentiated myoblasts. Cells were induced to differentiate (6 days) under low serum culture conditions and assayed for the expression of cell cycle (cyclin A) and differentiation markers (morphology and myogenin). Both ROS and in vivo production of NO were found increased after 6 days of differentiation, when the activation of PKC-δ isoform was 14-fold increased compared with the undifferentiated control cells. The parallel analysis of apoptotic features demonstrated a small increase in Annexin-V+ cells and a concomitant increase in PARP cleavage and Bax expression. Interestingly, a reduced percentage of differentiated cells was obtained both in the presence of Rottlerin, a highly selective PKC-δ pharmacologic inhibitor, and, moreover, with the use of PKC-δ siRNA technology, further supporting the involvement of PKC-δ in switching on the events related to skeletal muscle myoblast differentiation.

Maxillary sinus augmentation procedures through equine-derived biomaterial or calvaria autologous bone: immunohistochemical evaluation of OPG/RANKL in humans

Autologous bone is considered the gold standard for bone regeneration, even if different heterologous bone substitutes have been proposed to overcome the limits related to its use. The aim of this study was to analyze and to compare the molecular events switched on by autologous or heterologous bone graft insertion, focusing on TGFβ1 expression and OPG/RANKL ratio, to analyze resorption process, and estimating graft vascularization, new bone tissue deposition and its mineralization, through VEGF, BSP and SPARC expression evaluation, respectively. Patients needing pre-prosthetic rehabilitation of the posterior maxilla were treated using an equine-derived biomaterial (Group 1) or calvaria autologous bone (Group 2), according to the morphology of the bone defect. Bone graft integration was evaluated on bone samples obtained from the treated areas at the moment of dental implant insertion, by morphological and immunohistochemical analyses for TGFβ1, OPG, RANKL, VEGF, BSP, and SPARC expression. Morpho - logical analysis shows the presence of biomaterial residual granules in Group 1, in parallel to a good integration between graft and host tissue. Moderate TGFβ1 expression is seen in both Group 1 and Group 2. OPG/RANKL ratio appears higher in Group 1; VEGF expression appears very strong in Group 1 and strong in Group 2, while BSP and SPARC expression results weak in Group 1 and moderate in Group 2. Our results reveal the good integration between both types of graft and the host tissue, even though autologous graft seems to produce a faster regenerative process, as evidenced by the different expression of the investigated molecules. According to these observations, the clinical use of heterologous particulate equine-derived biomaterial may ensure long-term predictability of implantprosthetic rehabilitation, comparable to that obtained with autologous bone graft.