In mammals, a multi-oscillator circadian system generates behavioural, metabolic and physiological ~24h rhythms, with tissue-specific physiological cues enabling local circadian phase adjustments. Emerging work has shown musculoskeletal tissue homeostasis and mechanical responses to be under circadian control. Nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of the antioxidant response, is a clock-controlled target in several peripheral tissues and also modifies circadian gene expression and rhythmicity. However, the role of NRF2 in mechanical loading-induced changes in musculoskeletal tissues has yet to be elucidated. Wild-type (WT) and Nrf2 KO mice of young (3-6m) or old age (18-20m) harbouring a PER2::luciferase clock reporter were subjected to acute mechanical joint loading of the right leg (peak load 9N, 40 cycles of 10sec) during light phase whilst the contralateral (left) leg served as a non-loaded control. Musculoskeletal tissues were collected for analysis 4 hrs later. Real-time bioluminescence imaging of clock gene reporter activity, protein and mRNA levels of target markers, NRF2/ARE transactivation and genome-wide RNAseq analyses were undertaken. We show that acute mechanical loading in WT mice led to a decrease in gene expression of key members in the negative and auxiliary feedback loops of the molecular clock, associated with the phase-resetting of PER2::luc protein oscillations in the skeletal muscle and a knee joint. This was accompanied by a significant increase in the markers of oxidative burden as well as gene expression and protein abundance levels of antioxidant enzymes. Moreover, acute mechanical loading induced a significant activation of the redox-sensitive energy sensor, AMP-activated kinase (AMPK), known to be involved in molecular clock resetting. We thus examined whether the above acute mechanical responses were dependent on NRF2 activity. Nrf2 KO mice showed an altered response to acute mechanical loading, characterized by blunted circadian resetting and antioxidant responses, and altered AMPK activation. Furthermore, dampened responses to acute mechanical signals were found in ageing WT mouse musculoskeletal tissues, whilst AMPK activator treatments in WT mice induced circadian resetting and antioxidant responses in an NRF2-dependent manner. In conclusion, these data demonstrate that AMPK/NRF2 axis is required for relaying acute mechanical signals to the musculoskeletal system by controlling redox-mediated phase resetting of musculoskeletal clocks and antioxidant protection, which have important implications in understanding biomechanical mechanisms involved in musculoskeletal tissue maintenance in health and with ageing.

Imatinib, a tyrosine kinase inhibitor (TKI) is used as a standard treatment in chronic myeloid leukemia (CML) patients. Increased levels of BCR-ABL1 expression in CML cells are associated with oxidative stress induction due to overproduction of reactive oxygen species (ROS) or by deficient antioxidant system, disease progression, and imatinib resistance. Current scientific research confirms that oxidative stress is involved in CML pathogenesis and response to TKI treatment. Moreover, recent findings suggest that the antioxidant properties of some natural compounds can provide benefits to patients with CML. To determine the effect of adjuvant treatment with polyphenols on the oxidative stress markers in imatinib-treated CML patients. 40 CML patients at the University Clinic of Hematology, Skopje, who received imatinib longer than 1 month were included in the study. 20 patients were additionally treated with Aronia melanocarpa extract and 20 patients received only imatinib (control group). Besides the regular clinical laboratory analysis for these patients, total antioxidant power (PAT) and plasma peroxides (d-ROMs) were measured at initial visit and after 21 and 42 days of treatment using FRAS5 analytical photometric system and the oxidative stress index (OSI) was automatically calculated. Oxidative stress parameters (d-ROM and OSI) were significantly higher at initial visit in both groups. In group of patients who received adjuvant polyphenols values for d-ROM and OSI were significantly lower after 21 and 42 days of treatment (p<0.05). Also, total antioxidant capacity (PAT) was significantly higher after 21 and 42 days of treatment initiation in comparison with the pretreatment values. In the control group, no significant differences were obtained between investigated parameters at any time of measurement. Adjuvant treatment with Aronia melanocarpa extract after 21 and 42 days led to significant reduction of oxidative stress parameters in patients with CML treated with imatinib. 

Oxidative stress (OS) has a significant impact on the lifespan and physical fitness of living organisms. It is commonly associated with ageing and can lead to changes in the functionality of red blood cells (RBCs). The precise mechanisms underlying these changes are not fully understood. Unlike mammals, avian RBCs have a nucleus and functional mitochondria that regulate the cellular response to oxidative stress. In this study, we examined the effects of OS on red blood cells from adult female quail (Coturnix japonica, n=12). We used flow cytometry to analyze the formation of OS-induced microparticles and RBC transformation. We also evaluated band 3 clustering and phosphatidylserine externalization at the cell surface using eosin-5-maleimide and Annexin-V fluorescent probes, respectively. In addition, we analyzed band 3 clustering using confocal microscopy. We used a laser diffraction-based method to analyze cell deformability, and we characterized hemoglobin species spectrophotometrically. We found that OS caused band 3 clustering, microparticle formation, and phosphatidylserine release onto the cell membrane. The microparticles formed under the influence of oxidants differed from those formed under the influence of A23187 (calcium ionophore). The rate of microparticle formation and the onset of osmotic rigidity depended on the oxidant concentration. Erythrocyte-derived microparticles contained hemoglobin oxidized to hemichrome (HbChr). Overall, these findings demonstrate that avian erythrocytes undergo different processes during oxidative stress, depending on the level of oxidation. These differences are due to variations in cellular transformations and the formation of different types of microparticles. This research was supported by the Russian Fund for Basic Researches (grant no. 23-15-00142)