Protective role of antioxidants in bone and bowel pathological alterations related to oxidative stress

Free radicals are common outcome of normal aerobic cellular metabolism and endogenous antioxidant system plays its decisive role in prevention of excessive amount of free radicals. However, imbalanced defence mechanism of antioxidants, overproduction or incorporation of free radicals from environment to living system leads to various pathological states. In particular, given the involvement of oxidative stress and antioxidants in bone remodelling alterations and inflammatory bowel diseases, the aim of this thesis was to study: 1) the role and molecular mechanisms of the physiologically predominant estrogen, 17β-Estradiol (17β-E2), and natural antioxidants on stress induced apoptosis in osteocytes; 2) the antioxidant ability of resveratrol (RE) and of its benzoselenophene derivatives, VD0, VD1 and VD2, in restoring the physiological redox state in both osteocytes and intestinal myofibroblasts; 3) the role of oxidative stress and TNFα on the expression and activation of TNF-α converting enzyme (TACE) involved in chronic intestinal inflammation. Estrogen deficiency causes bone loss as a result of microdamage, oxidative stress and osteocyte apoptosis. A relationship between oxidative stress-induced apoptosis, c-Jun N-terminal kinase (JNK) activation, and expression of factors involved in bone remodelling has been demonstrated in osteocytes. However, the molecular regulation of these events in osteocytes treated with 17β-E2 remains unexplored. The MLO-Y4 murine osteocyte-like cell line was used as a model to study starvation-induced apoptosis and ROS production during 17β-E2 treatment. Expression of glutathione S-transferase P1-1 (GSTP1-1), receptor activator kB ligand (RANKL), osteoprotegerin (OPG), sclerostin, and kinases activation were measured by western blot. In addition, the GSTP1-1/JNK association was assessed by immunoprecipitation, and GSTP1-1 involvement in the osteocyte response to 17β-E2 was detected by specific siRNA transfection. 17β-E2 prevents starvation-induced apoptosis (DNA fragmentation and caspase-3 activation), the increase in sclerostin expression and the RANKL/OPG ratio, which are all related to osteoclastogenic and JNK activation due to oxidative stress in osteocytes. Estrogen prevention occurs through GSTP1-1 overexpression, which can inhibit JNK activation by formation of a GSTP1-1/JNK complex. No early antioxidant effect of 17β-E2 has been found, however, the estrogen effect results similar to N-acetylcysteine which, by decreasing the intracellular oxidative state, maintains JNK bound to GSTP1-1. Thus, the antiapoptotic and osteogenic effect of 17β-E2 in MLO-Y4 occurs by a redox- independent process involving GSTP1-1/JNK association. Moreover, the role of blueberry juice (BJ) obtained from Vaccinium myrtillus berries, native in Italian Apennines, on ROS production, apoptosis and expression of osteoclastogenic factors in MLO-Y4 osteocytes, was investigated and compared to that of dry extracts of blueberry (BE), green tea (GTE) and Hypericum perforatum (HE), being the last two known for their powerful antioxidant properties and beneficial effects in reducing the bone mass loss. The results show that both fresh juice, and blueberry dry extract reduce the apoptosis and the expression of osteoclastogenic factors induced by oxidative stress in starved MLO-Y4 osteocytes in the same manner as GTE and HE containing equal amount of total soluble polyphenols. However, blueberry antiapoptotic effect is not closely related to its antioxidant effect in MLO-Y4 cells, differently to that observed in GTE- and HE-treated MLO-Y4 cells. This suggests that the effect of blueberries on osteocyte apoptosis and bone turnover can be due to their antioxidant properties but also to other factors, not strictly redox regulated, which remain to be elucidated in further studies. RE, a polyphenolic compound present in some food and plants, is characterized by anti-inflammatory and antioxidant properties. However, it is quickly metabolized with consequent loss of its efficacy. The antioxidant effect of 2-phenyl-benzoselenophene derivatives of RE (VD0, VD1 and VD2) was detected in human colon mucosa myofibroblasts, CCD-18Co (18Co), and in MLO-Y4 cells in which the oxidative stress was induced by buthionine sulfoximine (BSO), inhibitor of GSH synthesis, or serum starvation, respectively. The results show that RE derivatives have major antioxidant power in reducing and/or restoring radical oxygen species to control values respect to RE in both cell types. Moreover, derivatives have different antioxidant capacity in 18Co and in MLO-Y4 and this can be due to different degree of oxidative stress and structural characteristics of these compounds. Some of the synthesized RE analogues have shown antibacterial role in IBD and anti-resorptive activity in bone pathologies related to inflammatory and osteoporotic processes. Thus, it has been speculated the use of benzoselenophene derivatives as an alternative therapy and/or therapeutic support in intestinal inflammatory disease and osteoporosis. Finally, the role of oxidative stress and TNFα on the expression and activation of TACE has been investigated in 18Co cells treated with BSO and/or stimulated with TNFα. Previously, in these cells it has been shown that oxidative stress and TNFα are both related to an increased expression of ICAM-1 and release of sICAM-1 by a TACE proteolytic cleavage. In literature no data are reported on the expression, activation and regulation of this enzyme in relationship to the intracellular oxidative state in intestinal myofibroblasts. The data obtained indicate a strong relationship between TACE expression and activation and intracellular ROS levels. In fact, GSH depletion by BSO and TNFα, both increase ROS levels and TACE expression and activation, whereas N-acetylcysteine (NAC), precursor of GSH synthesis, and diphenyleneiodonium (DPI), inhibitor of NADPH oxidase, restore these events to control values, by reducing ROS levels. Given that TACE is involved in the cleavage of adhesion molecules and pro-inflammatory cytokines, which increase in condition of oxidative stress, antioxidants may have beneficial role in reducing the activation of inflammatory cascade and preventing the progression of chronic inflammatory disease.