RESUMEN
This study evaluated the potential of Cimicifuga racemosa (L.) Nutt extract (CIMI) to reduce the deleterious effects of doxorubicin (DOXO) in oocytes, follicles and stromal cells in mice ovaries cultured in vitro. In experiment 1, mice ovaries were cultured in DMEM+ alone or supplemented with 5, 50 or 500 ng/mL CIMI, while in experiment 2, mice ovaries were cultured in DMEM+ alone or supplemented with 5 ng/mL CIMI (better concentration), 0.3 µg/mL DOXO or both. Thereafter, the ovaries were processed for histological (morphology, growth, activation, extracellular matrix configuration and stromal cell density), immunohistochemical (caspase-3) analyses. Follicle viability was evaluated by fluorescence microscopy (ethidium homodimer-1 and calcein) while real-time PCR was performed to analyses the levels of (mRNA for SOD, CAT and nuclear factor erythroid 2-related factor 2 (NRF2) analyses. The results showed that DOXO reduces the percentage of normal follicles and the density of stromal cells in cultured ovaries, but these harmful effects were blocked by CIMI. The DOXO reduced the percentage of primordial follicles, while the presence of CIMI alone did not influence percentage of primordial follicles. A higher staining for caspase-3 was seen in ovaries cultured in control medium alone or with DOXO when compared with those cultured with CIMI alone or both CIMI and DOXO. In addition, follicles from ovaries cultured with both CIMI and DOXO were stained by calcein, while those follicles cultured with only DOXO were stained with ethidium homodimer-1. Furthermore, ovaries cultured with CIMI or both CIMI and DOXO had higher levels of mRNA for SOD and CAT, respectively, than those cultured with only DOXO. In conclusion, the extract of CIMI protects the ovaries against deleterious effects of DOXO on follicular survival and ovarian stromal cells.
RESUMEN
Cardiotonic steroids (CS) are known as modulators of sodium and water homeostasis. These compounds contribute to the excretion of sodium under overload conditions due to its natriuretic property related to the inhibition of the renal Na+/K+-ATPase (NKA) pump α1 isoform. NHE3, the main route for Na+ reabsorption in the proximal tubule, depends on the Na+ gradient generated by the NKA pump. In the present study we aimed to investigate the effects of marinobufagin (MBG) and telocinobufagin (TBG) on the renal function of isolated perfused rat kidney and on the inhibition of NKA activity. Furthermore, we investigated the mechanisms for the cardiotonic steroid-mediated natriuretic effect, by evaluating and comparing the effects of bufalin (BUF), ouabain (OUA), MBG and TBG on NHE3 activity in the renal proximal tubule in vivo. TBG significantly increased GFR, UF, natriuresis and kaliuresis in isolated perfused rat kidney, and inhibits the activity of NKA at a much higher rate than MBG. By stationary microperfusion technique, the perfusion with BUF, OUA, TBG or MBG promoted an inhibitory effect on NHE3 activity, whereas BUF was the most effective agent, and demonstrated a dose-dependent response, with maximal inhibition at 50nM. Furthermore, our data showed the role of NKA-Src kinase pathway in the inhibition of NHE3 by CS. Finally, a downstream step, MEK1/2-ERK1/2 was also investigated, and, similar to Src inhibition, the MEK1/2 inhibitor (U0126) suppressed the BUF effect. Our findings indicate the involvement of NKA-SRc-Kinase-Ras-Raf-ERK1/2 pathway in the downregulation of NHE3 by cardiotonic steroids in the renal proximal tubule, promoting a reduction of proximal sodium reabsorption and natriuresis.