In vivo and in vitro data supporting the involvement of oxidative stress in aging-related and metabolic diseases

Background: Oxidative stress (OxS) is caused by the derangement of the balance between production of oxidants, mainly represented by reactive oxygen species (ROS), and the antioxidant’s ability to detoxify them. The consequent biomolecular oxidative damage tends to accumulate in aged biological systems and it is widely believed to act as main trigger of aging- and metabolism- related diseases such as late onset Alzheimer’s disease (LOAD), postmenopausal osteoporosis (PO) and metabolic syndrome (MetS). The involvement of OxS in these pathologies has been nicely demonstrated in vitro and animal models. In contrast, the data from human studies are scarce and highly controversial. Similar controversies feature the body of population-based studies on the effects of antioxidant supplements or dietary interventions on OxS and related diseases. Objectives: To address these issues we have conducted three research projects with the following aims: Project 1: to evaluate a possible cross-sectional and/or longitudinal association between systemic markers of oxidative stress and the most common dementia-related diseases (Alzheimer’s disease and vascular dementia). Projects 2: to evaluate a possible involvement of OxS in the development of PO. Project 3: to evaluate in vitro the effects of flavonoids, in particular (-)-epicatechin (EC) and its metabolites (ECM), on the regulation of expression and activity of NADPH oxidase in hepatic cells (HepG2) treated with palmitate (Pal). Material and methods: Project 1: The patients enrolled (n=476 subjects) were divided, in accordance to the diagnosis of dementia, in: 105 late onset Alzheimer disease (LOAD), 54 vascular dementia (VaD), 199 mild cognitive impairment (MCI), which is consider as an intermediate state between normal aging and dementia, and 118 healthy controls. Among the MCI patients, a subgroup of 111 patients were follow-up for an average of 2 years (2.0 ± 0.6 years). The serum markers of OxS assessed in each subjects were: hydroperoxides (HY), advance oxidation protein products (AOPP), total antioxidant power (TAP), residual antioxidant power (RAP), uric acid (UA), and thiols (TH) Project 2: 290 women (age spanning from 21 to 65 years) were enrolled and divided in different groups according to menopausal status (reproductive age, peri- and post-menopause). In these women, serum markers of OxS (HY, AOPP, 8-isoprostane-F2-iso, 8-hydroxy-2'-deoxyguanosine-8-OH-dg, TAP, RAP, UA and TH) and serum bone markers (Bone-specific Alkaline Phosphatase-BAP and C-terminal telopeptide of type I collage-CTX-1) were assessed. Furthermore the areal bone density at lumbar spine, hip and total body by was assessed by dual energy X-ray absorptiometry (DXA) scanner. Project 3: HepG2 cells were incubated for 24 h with Pal (0.25 mM), in the absence/presence of EC (0.25-1 μM) and ECM (1 μM). By Real-Time PCR (RT-PCR) and western blot, mRNA and protein expression levels of NADPH oxidase subunits (NOX3, p22phox, p47phox) were assessed. Moreover, protein expression levels of insulin resistance pathway (IKK and JNK) were assessed by western blot as well. NADPH oxidase activity and cell oxidants were measured respectively with chemiluminescence assay and oxidant-sensitive probe 5-(and-6)-carboxy-2,7 dichlorodihydrofluorescein diacetate (DCF). Lipid accumulation was assessed by Red Oil O staining. Results: Project 1: Analysis of covariance showed that an oxidative derangement (high HY and/ low antioxidants) were present in MCI, LOAD and, though less pronounced in VaD. Multivariate logistic regression analysis showed that, compared with controls, high levels (over median value) of serum hydroperoxides were independently associated with an increase in the likelihood of having MCI (Odd Ratio: 2.59, 95% Confidence Interval: 1.08-6.21) or LOAD (OR: 4.09, 95%CI: 1.36-11.81). Moreover, low levels of residual antioxidant power were associated with increased risk of having MCI (OR: 3.97, 95% CI: 1.62- 9.72), but not LOAD (OR: 2.31, 95%CI: 0.83-6.63). No differences in either of these two OxS markers were found by comparing MCI patients who converted (n = 29) or not converted (n = 82) to LOAD. Project 2: Pearson’s correlation showed that increased serum levels of a lipid peroxidation marker, HY, were negatively and independently associated with decreased bone mineral density (BMD) in total body (r=-0.192, p<0.05), lumbar spine (r=-0.282, p<0.01) and total hip (r=-0.282, p<0.05), as well as with increased bone resorption rate (r=0.233, p<0.05), as assessed by the serum concentration of CTX-1. Importantly, multiple regression analysis revealed that HY is a determinant and independent factor for the association between lumbar spine BMD and CTX-1 levels. Project 3: Data obtained in vitro showed that Pal increases the production of oxidants in HepG2 through an up-regulation of protein levels and mRNA expression of NOX3. Pal was able to “mimic” an insulin resistance condition through an up-regulation of JNK and IKK expression. Moreover, our results demonstrated that EC and ECM decrease the activity of NADPH oxidase and the ROS production; furthermore, both EC and ECM decreased the expression of JNK and IKK, improving the insulin resistance condition. The Oil Red O staining showed that EC and ECM cannot significantly prevent Pal-induced lipid accumulation. Conclusion: Systemic OxS might be a significant feature of LOAD, PO and MetS. Moreover, our results suggest that rich-flavonoids food consumption may be an important way of prevention of age- and metabolism-related diseases. Abbreviations: OxS, oxidative stress; MCI, mild cognitive impairment; LOAD, late onset Alzheimer’s disease; VaD, vascular dementia; HY, hydroperoxides; AOPP, advanced oxidation protein products; TAP, total antioxidant power; RAP, residual antioxidant power; UA, uric acid; TH, thiol; BMD, bone mineral density; OR, Odds Ratio; EC, (-)-epicatechin; ECM, (-)-epicatechin metabolites; Pal, palmitate.