During perimenopause, oxidative stress increases, which may result in disruption of bone turnover, and consequently in osteoporosis. The use of antioxidants may be an effective nutritional approach to reducing osteoporosis in this period of life. Mate tea (MT) (Ilex paraguariensis), a typical and inexpensive beverage consumed in the Brazilian south-east, Argentina and Uruguay, increases antioxidant defense. Our hypothesis was that MT would decrease oxidative stress and mitigate bone deterioration. To test this, we analyzed oxidative stress markers of bone turnover, and local and systemic markers of bone metabolism of rats during natural perimenopause. Female Wistar rats (aged 16months) in proven perimenopause period received 20mg/kgBW/day of mate tea, by gavage (PM+MT Group, n=10) or water (PM Group, n=10). Female rats aged 4months (AD Group, n=10) received water. The treatment period was four weeks. MT minimized the deterioration of rat microarchitecture, characterized by increase in the bone trabecular area, number of osteocytes and areal bone mineral density. These results were accompanied by a lower level of malondialdehyde, an oxidative stress marker, in femoral tissue homogenate. Plasmatic tartrate-resistant acid phosphatase, a typical osteoclastic function marker, decreases after treatment, indicating a decrease in osteoclastic function. MT also modified the immunostaining pattern of bone metabolism markers, decreasing the receptor activator of nuclear factor kappa-B ligant (RANKL), superoxide dismutase isoform 2 (SOD2) and increasing osteoprotegerin (OPG), a decoy receptor for the RANKL, which positively modulates bone mass. These results suggested MT was capable of decreasing bone resorption by inhibiting the osteoclastogenesis in a RANKL-dependent signaling pathway activated by oxidative stress. Taken together, the results indicated that MT minimized bone loss in perimenopause and this effect is at least partly due to the decrease in oxidative stress, confirming our hypothesis.

Background: Ischemic stroke produces a large health impact worldwide, with scarce therapeutic options. Objective: This study aimed to reveal the role of NADPH oxidase and neuroinflammatory genes on the cerebral anti-ischemic effects of C-Phycocyanin (C-PC), the chief biliprotein of Spirulina platensis. Methods: : Rats with either focal cerebral ischemia/reperfusion (I/R) or acute brain hypoperfusion, received C-PC at different doses, or a vehicle, for up to 6 h post-stroke. Neurological, behavioral and histochemical parameters were assessed in I/R rats at 24 h. Cerebral gene expression and hippocampal neuron viability were evaluated in hypoperfused rats at acute (24 h) or chronic phases (30 days), respectively. A molecular docking analysis between NOX2 and C-PC-derived Phycocyanobilin (PCB) was also performed. Results: C-PC, obtained with a purity of 4.342, significantly reduced the infarct volume and neurologic deficit in a dose-dependent manner, and improved the exploratory activity of the I/R rats. This biliprotein inhibited NOX2 expression, a crucial NAPDH oxidase isoform in the brain, and the superoxide increase produced by the ischemic event. Moreover, C-PC-derived PCB showed a high binding affinity in silico with NOX2. C-PC downregulated the expression of pro-inflammatory genes (IFN-γ, IL-6, IL-17A, CD74, CCL12) and upregulated immune suppressive genes (Foxp3, IL-4, TGF-β) in hypoperfused brain areas. This compound also decreased chronic neuronal death in the hippocampus of hypoperfused rats. Conclusion: These results suggest that the inhibition of cerebral NADPH oxidase and the improvement of neuroinflammation are key mechanisms mediating the neuroprotective actions of C-PC against brain ischemia.

HnRNP K protein is a heterogeneous nuclear ribonucleoprotein which has been proposed to be involved in the leukemogenesis of acute promyelocytic leukemia (APL), as well as in differentiation induced by all‐trans retinoic acid (ATRA). We previously demonstrated a connection between SET and hnRNP K function in head and neck squamous cell carcinoma (HNSCC) cells related to splicing processing. The objective of this study was to characterize the participation of hnRNP K and SET proteins in ATRA‐induced differentiation in APL. We observed higher (5‐ to 40‐fold) levels of hnRNP K and SET mRNA in APL patients at the diagnosis phase compared with induction and maintenance phases. hnRNP K knockdown using short‐hairpin RNA led to cell death in ATRA‐sensitive NB4 and resistant NB4‐R2 cells by apoptosis with SET cleavage. In addition, hnRNP K knockdown increased granulocytic differentiation in APL cells, mainly in NB4‐R2 with ATRA. hnRNP K knockdown had an effect similar to that of treatment with U0126 (an meiosis‐specific serine/threonine protein kinase/ERK inhibitor), mainly in NB4‐R2 cells. SET knockdown in APL cells revealed that apoptosis induction in cells with hnRNP K knockdown occurred by SET cleavage rather than by reduction in SET protein. Transplantation of NB4‐R2 cells into nude mice confirmed that arsenic trioxide (ATO) combined with U0126 has higher potential against tumor progression when compared to ATO. Therefore, hnRNP K/SET and ERK are potential therapeutic targets for both antineoplastic leukemia therapy and relapsed APL patients with ATRA resistance.

Análise da proteína hnRNP K nas linhagens celulares NB4 e NB4-R2 de leucemia promielocítica aguda com ênfase na patogênese e na diferenciação celular pelo ácido all-trans retinoico UNIVERSIDADE DE SÃO PAULO FACULDADE DE CIÊNCIAS FARMACÊUTICAS DE RIBEIRÃO PRETOAnálise da proteína hnRNP K nas linhagens celulares NB4 e NB4-R2 de leucemia promielocítica aguda com ênfase na patogênese e na diferenciação celular pelo ácido all-trans retinoico Heterogeneous nuclear ribonucleoprotein K (hnRNP K) and endogenous inhibitor of phosphatase 2A (SET) are overexpressed and proposed as prognostic markers in acute and chronic myeloid leukemia. The study aim was to characterize the hnRNP K and SET proteins involvement in acute promyelocytic leukemia leukemia (APL) leukemogenesis as well as all-trans retinoic acid (ATRA) induced cell differentiation. Initial qRT-PCR results demonstrate that HNRNPK and SET mRNA levels are increased in patients diagnosed with APL compare to samples from healthy donors and decrease after induction and during maintenance of treatment. The results were validated by Western blot, suggesting hnRNP K and SET as diagnostic and response to treatment markers. The knockdown of hnRNP K and SET was performed on sensitive, NB4, and ATRA-resistant, NB4-R2, LPA cells using interfering RNA. Both proteins were also tested as a therapeutic target with a use of hnRNP K (U0126) and SET inhibitors (OP449 and FTY720). The decrease of hnRNP K in cells led to increased granulocyte cell differentiation in both cells, especially in the presence of ATRA, and thus was able to reverse the NB4-R2 cells resistance to ATRA phenotype. The hnRNP K knockdown, as well as the treatment with U0126, had a loss of cell viability by induction of apoptosis accompanied by cleavage of the SET protein. The SET knockdown in APL cells confirmed that an induction of apoptosis in cells with hnRNP K knockdown occurred by cleavage and not by the SET protein decrease in the cells. Furthermore, it has also shown that SET impairs the ATRA's performance in the cellular differentiation process. The in vivo model using NB4-R2 transplant in nude mice confirmed that arsenic trioxide (ATO) combined with U0126 has a greater potential against tumor progression compared to the treatment isolated with ATO. FTY720 and OP449 have significant anti-leukemic effect reducing cell viability. When in combination, FTY720 and OP449, they had a significant synergistic effect on NB4-R2 (CDI <0.7). We conclude that overexpression of hnRNP K and SET contributes to block cell differentiation in promyelocytes and impair the performance of ATRA in the treatment of APL and therefore hnRNPK in association with a SET protein represent a potential therapeutic target for anti-leukemic therapy of APL, mainly for patients resistant to ATRA.