Ozone at low concentration modulates microglial activity in vitro: A multimodal microscopy and biomolecular study

Abstract

Oxygen-ozone (O 2 -O 3 ) therapy is an adjuvant/complementary treatment based on the activation of antioxidant and cytoprotective pathways driven by the nuclear factor erythroid 2-related factor 2 (Nrf2). Many drugs, including dimethyl fumarate (DMF), that are used to reduce inflammation in oxidative-stress-related neurodegenerative diseases, act through the Nrf2-pathway. The scope of the present investigation was to get a deeper insight into the mechanisms responsible for the beneficial result of O 2 -O 3 treatment in some neurodegenerative diseases. To do this, we used an integrated approach of multimodal microscopy (bright-field and fluorescence microscopy, transmission and scanning electron microscopy) and biomolecular techniques to investigate the effects of the low O 3 concentrations currently used in clinical practice in lipopolysaccharide (LPS)-activated microglial cells human microglial clone 3 (HMC3) and in DMF-treated LPS-activated (LPS + DMF) HMC3 cells. The results at light and electron microscopy showed that LPSactivation induced morphological modifications of HMC3 cells from elongated/ branched to larger roundish shape, cytoplasmic accumulation of lipid droplets, decreased electron density of the cytoplasm and mitochondria, decreased amount of Nrf2 and increased migration rate, while biomolecular data demonstrated that Heme oxygenase 1 gene expression and the secretion of the pro-inflammatory cytokines, Interleukin-6, and tumor necrosis factor-α augmented. O 3 treatment did not affect cell viability, proliferation, and morphological features of both LPS-activated and LPS + DMF cells, whereas the cell motility and the secretion of proinflammatory cytokines were significantly decreased. This evidence suggests that modulation of microglia activity may contribute to the beneficial effects of the O 2 -O 3 therapy in patients with neurodegenerative disorders characterized by chronic inflammation.

Mitochondrial Features of Mouse Myoblasts Are Finely Tuned by Low Doses of Ozone: The Evidence In Vitro

“…Consistently with similar findings obtained in other cell lines [4,18,28], low doses of O 3 did not affect the percentage of C2C12 cells in the S-phase, indicating that the decreased viability observed in these cells was not due to O 3 -mediated alteration in cell proliferation.…”

“…Ten µg O 3 was safe for all time points, consistent with our previous data on other cell types [9,18,[28][29][30]. Conversely, 20 µg O 3 induced a significant decrease in cell viability at both 24 h and 48 h after treatment.…”

“…O 3 was used at the concentrations currently administered in the clinical practice for intramuscular injections, i.e., 10 and 20 µg O 3 /mL O 2 . In addition, these concentrations proved to be non-toxic for various cultured cells and tissues [4,8,9,18,28,29]. Pure O 2 was administered to cells to discern the effect elicited by O 3 from O 2 , as gas treatment is dispensed as a mixture of the two.…”

“…The Nrf2 protein stability is tightly regulated: under basal conditions, Keap1 targets Nrf2 for continuous ubiquitination and proteasomal degradation in the cytoplasm [10][11][12]. Keap1 is a critical sensor of oxidative stress [13], and its chemical modification [14,15] or the direct disruption of the Keap1-Nrf2 binding interface [16,17] prevents the degradation of Nrf2 that translocates to the nucleus [18], where it promotes the transcription of ARE-driven genes [7,19]. Several lines of evidence showed that, among its cytoprotective activities, Nrf2 mediates the maintenance of cell redox homeostasis by regulating and sustaining the structural and functional integrity of the mitochondria [20].…”

Modifications of Blood Molecular Components after Treatment with Low Ozone Concentrations

“…It has been reported that the treatment of blood with therapeutic doses of O 3 , after causing an initial slight and transient decrease in the antioxidant capacity of the plasma (fully reconstituted within a few minutes) [5], gives rise to a prompt plasmatic and cellular antioxidant response [4,18] that is likely responsible for the increased antioxidant capacity in our O 3 -treated samples. In particular, it has already been experimentally demonstrated that an O 3 concentration of 10 µg is able to stimulate an antioxidant cytoprotective response through the activation of the Keap1-dependent nuclear factor erythroid 2-related factor 2 (Nrf2) pathway [27][28][29][30][31][32][33][34][35]. Interestingly, in the present study, we found that also the very low concentration of 5 µg O 3 is able to induce a significant increase in the antioxidant capacity.…”