Recombinant irisin prevents cell death and mineralization defects induced by random positioning machine exposure in primary cultures of human osteoblasts: A promising strategy for the osteoporosis treatment

Abstract

Spaceflight exposure, like prolonged skeletal unloading, is known to result in significant bone loss, but the molecular mechanisms responsible are still partly unknown. This impairment, characterizing both conditions, suggests the possibility of identifying common signalling pathways and developing innovative treatment strategies to counteract the bone loss typical of astronauts and osteoporotic patients. In this context, primary cell cultures of human osteoblasts derived from healthy subjects and osteoporotic patients were exposed to random positioning machine (RPM) to reproduce the absence of gravity and to exacerbate the pathological condition, respectively. The duration of exposure to RPM was 3 or 6 days, with the aim of determining whether a single administration of recombinant irisin (r-irisin) could prevent cell death and mineralizing capacity loss. In detail, cellular responses were assessed both in terms of death/survival, by MTS assay, analysis of oxidative stress and caspase activity, as well as the expression of survival and cell death proteins, and in terms of mineralizing capacity, by investigating the pentraxin 3 (PTX3) expression. Our results suggest that the effects of a single dose of r-irisin are maintained for a limited time, as demonstrated by complete protection after 3 days of RPM exposure and only partial protection when RPM exposure was for a longer time. Therefore, the use of r-irisin could be a valid strategy to counteract the bone mass loss induced by weightlessness and osteoporosis. Further studies are needed to determine an optimal treatment strategy based on the use of r-irisin that is fully protective even over very long periods of exposure and/or to identify further approaches to be used in a complementary manner.

Keywords: apoptosis; bone loss; cell viability; mineralization; random positioning machine; recombinant irisin; simulated microgravity.

FIGURE 1

Effects of random positioning machine (RPM) exposure on the survival of primary osteoblast cultures derived from healthy (HEALTHY) subjects. (a,b) MTS assay: cell viability was completely (a) or partially (b) preserved in cells treated with r-irisin and exposed to RPM. (c,d) Intracellular levels of reactive oxygen species (ROS): the increase in oxidative stress induced by exposure to RPM was counteracted by r-irisin treatment. (e,f) Caspase-3/7 activity: total (e) or partial (f) prevention of caspase activity induced by RPM exposure in r-irisin-treated cells. (g) Western blotting analysis for Akt/protein kinase B (PKB): r-irisin treatment fully (3 days) or partially (6 days) preserved Akt expression. (h) Western blotting analysis for B-cell lymphoma 2 (Bcl-2) and Bax: significant increase in the Bcl-2/Bax ratio after r-irisin treatment (3 days); alteration of the Bcl-2/Bax ratio after 6 days of RPM exposure and r-irisin treatment, in favor of Bax.

FIGURE 2

Effects of random positioning machine (RPM) exposure on the survival of primary osteoblast cultures derived from osteoporotic (OP) patients. (a,b) MTS assay: cell viability was completely (a) or partially (b) preserved in cells treated with r-irisin and exposed to RPM. (c,d) Intracellular levels of reactive oxygen species (ROS): the increase in oxidative stress induced by exposure to RPM was completely (c) or partially (d) counteracted by r-irisin treatment. (e,f) Caspase-3/7 activity: total (e) or partial (f) prevention of caspase activity induced by RPM exposure in r-irisin-treated cells. (g) Western blotting analysis for Akt/protein kinase B (PKB): r-irisin treatment completely (3 days) or partially (6 days) preserved Akt expression. (h) Western blotting analysis for B-cell lymphoma 2 (Bcl-2) and Bax: significant increase in Bcl-2/Bax ratio after r-irisin treatment (3 days); restoration of basal levels of Bcl-2 and Bax after 6 days of RPM exposure and r-irisin treatment.

FIGURE 3

Effects of random positioning machine (RPM) exposure on the mineralization process in primary osteoblast cultures derived from healthy (HEALTHY) subjects. (a–h) Immunocytochemical analysis for PTX3: (a–d) significant increase in PTX3 expression (arrows) in cells exposed to 3 days of RPM and treated with r-irisin; (e–h) PTX3 expression (arrows) was not preserved by r-irisin treatment after 6 days of RPM exposure. (i) Western blotting analysis for PTX3: increased PTX3 expression in r-irisin-treated cells (3 days); PTX3 expression was not preserved by r-irisin treatment in cells exposed to 6 days of RPM. Images were magnified × 20, scale bar represents 50 μm.

FIGURE 4

Effects of random positioning machine (RPM) exposure on the mineralization process in primary osteoblast cultures derived from osteoporotic (OP) patients. (a–h) Immunocytochemical analysis for PTX3: (a–d) significant increase in PTX3 expression (arrows) in cells exposed to 3 days of RPM and treated with r-irisin; (e–h) PTX3 expression (arrows) was not completely preserved by r-irisin treatment after 6 days of RPM exposure. (i) Western blotting analysis for PTX3: increased PTX3 expression in r-irisin-treated cells (3 days); PTX3 expression was not completely by r-irisin treatment in cells exposed to 6 days of RPM. Images were magnified × 20, scale bar represents 50 μm.