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  • br Materials and methods br


    Materials and methods
    Discussion Dex has been shown to induce apoptosis in osteoblastic cell lines (MC3T3-E1, UAMS-32 cell) [24], [25]. Consistent with previous reports, we find high-dose Dex (1 or 5 μM) can induce apoptosis of MC3T3-E1 ap5 within 24 h as evidenced by Annexin V/PI staining. This was also confirmed by the expression of apoptosis-related molecules. Dex treatment significantly increased bax expression while reduced bcl-2 expression. The proliferation of MC3T3-E1 cells was also reduced after Dex treatment. Although osteoblast apoptosis is commonly accepted as a key player in the pathogenesis of GIOP, the underlying mechanism remains poorly understood. Previous studies have shown that Dex induces osteoblast apoptosis by increasing E4BP4 expression or ER stress [26], [27], [28]. Autophagy activation also participates in Dex-induced apoptosis [28]. In the present study, we find that Dex significantly reduced HO-1 expression after incubation for 24 h with MC3T3-E1 cells. HO-1 is a stress-responsive enzyme that exerts potent cytoprotective effects in a wide range of cells and disease models. HO-1 deficiency results in increased apoptotic cells in mouse placentas [29]. Carbon monoxide, one of the by-products of HO-1, attenuates TNF-α–induced apoptosis of osteoblasts [30]. Based on previous studies, our findings suggest that HO-1 may participate in ap5 Dex-induced apoptosis of osteoblasts. Consistent with our results, Singh and Haldar [31] also observed that Dex represses HO-1 expression in peripheral blood mononuclear cells. However, Han et al [32] have reported that Dex increases HO-1 expression after incubation for 6 h with MC3T3-E1 cells. The difference may be caused by different culture conditions. HO-1 is responsible for ROS scavenging [33]. HO-1 deficiency results in increased ROS levels in visceral adipose precursor cells [34]. ROS induction leads to oxidative stress, a process that alters bone metabolism and contributes to the pathogenesis of osteoporosis [35]. As expected, we find a concomitant increase of ROS levels in MC3T3-E1 cells treated with Dex. Previous studies have reported that ROS is responsible for Dex- or homocysteine-induced apoptosis of osteoblasts [24], [36]. This result provides further evidence that HO-1 is involved in Dex-induced apoptosis of MC3T3-E1 cells. We also find that the antiapoptotic effects of HO-1 induction were associated with the activation of ERK1/2. The ERK1/2 pathway regulates cell apoptosis, proliferation, autophagy and differentiation. Previous studies have reported that ERK activation can attenuate osteoblast apoptosis induced by Dex or 17β-estradiol [37], [38]. In the present study, using U0126, an inhibitor of ERK activation, we find that inhibition of ERK activation significantly abrogated the protective effect of CoPP against Dex-induced apoptosis. Moreover, inhibition of ERK activation also reduced the expression of HO-1 in MC3T3-E1 cells. These results suggest that CoPP triggers the activation of ERK1/2 pathways, which induces an antiapoptotic effect in osteoblasts. Previous studies have shown that ERK kinase can phosphorylate Bad or Nrf2 [39], [40]. Bad is a Bcl-2 family protein, and its phosphorylation can prevent apoptosis. Nrf2 is a key transcription factor that mediates antioxidant and antiinflammatory responses. ERK/Nrf2 signalling pathway plays an important role in CoPP-induced antiapoptotic response. However, identifying the downstream target of ERK needs more investigation.
    Conflict of interest
    Introduction Nitric oxide (NO) exerts a number of protective effects to maintain normal vascular function, including modulating blood pressure, inhibiting adhesion of immune cells and platelets to the endothelium [[1], [2], [3]]. A reduction of NO bioavailability by excessive production of reactive oxygen species (ROS) is implicated in the initiation and progression of several cardiovascular diseases [1,3]. There is increasing evidence that NADPH oxidase expressed in endothelial cells is a major source of superoxide (O2−) [[4], [5], [6]]. Excess generations of O2− and derivative ROS promote expression of proinflammatory molecules, lipid peroxidation and foam cell formation in the vasculature. Mounting evidence indicates that the increased activation of NADPH oxidase has been linked to impairment of endothelial NO function in patients with coronary artery disease, and is involved in vascular oxidative stress and endothelial dysfunction in a variety of pathologic conditions such as hypertension, atherosclerosis, intimal hyperplasia and hyperlipidemia [[4], [5], [6], [7]].