No abstract

Mango is rich in bioactive absorbable polyphenols, but also contains considerable amounts of unabsorbable gallotannins at varying degrees of polymerization. Gallotannins are not absorbable upon consumption and have rarely been considered in the discussion of health benefits of polyphenols. Therefore, the objective of this study was to investigate the anti-proliferative activities of the major microbial metabolite of gallotannins, pyrogallol (PG) and a low molecular weight fraction of mango (Mangifera Indica L.) polyphenols (ML) and involved pathways including the AKT/mTOR signaling axis in an in situ breast cancer cell line, MCF10DCIS.COM. Fluorouracil (5-FU), a widely used genotoxic cancer therapeutic, was used a positive control and in combination with ML and PG to assess potential interactions. Concentrations that were non-cytotoxic in non-cancer cells were identified in non-cancer mammary fibroblasts (MCF-12F) and only non-cytotoxic dietarily relevant concentrations were selected for the investigation in MCF10DCIS.COM cancer cells. In addition to proliferation and viability, mRNA and expression of total and phosphorylated protein were investigated. Results show that both, ML and PG significantly reduced proliferation in MCF10DCIS.COM, but did not significantly reduce viability following a 48 h exposure. ML significantly reduced mRNA expression of mTOR and HIF-1α, while PG significantly reduced mRNA of IGF-1R, AKT, mTOR and HIF-1α. ML and PG reduced total protein expression of IGF-1R, IR, AKT, mTOR, and P70S6K. In addition, PG reduced IRS protein. Both treatments also had an effect on phosphorylated protein levels, with PG significantly reducing IGF-1R, AKT, and P70S6K levels. ML had a similar effect and significantly decreased IR, AKT, and P70S6K phosphorylation levels. Within the low concentration-range, ML and PG did not interact with the cytotoxic activities of 5-FU. Overall, the AKT/mTOR signaling axis appears to be implicated as causal in decreased proliferation induced by diet-relevant concentrations of ML and PG.

Plasticity is a fundamental property of the neural system controlling breathing. One key example of respiratory motor plasticity is phrenic long-term facilitation (pLTF), a persistent increase in phrenic nerve activity elicited by acute intermittent hypoxia (AIH). pLTF can arise from distinct cell signaling cascades initiated by serotonin versus adenosine receptor activation, respectively; these signaling cascades interact via powerful cross-talk inhibition. Here, we demonstrate that the daily rest versus active phase and the duration of hypoxic episodes within an AIH protocol have profound impact on the magnitude and even mechanism of pLTF due to shifts in the serotonin/adenosine balance. Using the historical “standard” AIH protocol (3, 5 min moderate hypoxic episodes), we demonstrate that pLTF magnitude is unaffected by exposure in the mid-active versus mid-rest phase, yet the mechanism driving pLTF shifts from serotonin-dominant during mid-rest to adenosine-dominant in the mid-active phase. This mechanistic “flip” results from combined influences of hypoxia-evoked adenosine release and normal cycles in basal spinal adenosine between the rest versus active phase. Since AIH consisting of shorter hypoxic episodes but the same cumulative duration of hypoxia (15, 1 min episodes) elicits less adenosine release during hypoxic episodes, mid-rest pLTF is amplified due to a diminished adenosine constraint to serotonin-driven plasticity; on the other hand, this same 15 × 1 AIH protocol delivered in the mid-active phase suppresses serotonin-dominant pLTF due to elevated background adenosine levels but low hypoxia-evoked adenosine release. These findings demonstrate the importance of the serotonin/adenosine balance in regulating the amplitude and even mechanism of AIH-induced pLTF. Since AIH is emerging as a promising therapeutic modality to restore respiratory and non-respiratory movements in people with spinal cord injury or ALS, knowledge of how time-of-day and hypoxic episode duration impact the serotonin/adenosine balance and the magnitude and mechanism of pLTF has profound biological, experimental and translational implications.

Obstructive Sleep Apnea (OSA) is characterized by cessations in respiration that leads to the development of chronic hypertension. Hypertension persists into the waking period even during normal respiratory patterns, and can increase a patient's risk factor for heart disease and stroke. Previous studies have shown that experimental models of chronic intermittent hypoxia (CIH) can produce a sustained hypertension similar to that associated with OSA. It has been proposed that the peripheral and CNS renin‐angiotensin systems contribute to hypertension associated with CIH. Our working hypothesis is that increased circulating angiotensin II feeds into the forebrain thereby increasing excitatory signaling through the hypothalamus and hindbrain, creating a vicious cycle. The median preoptic nucleus (MnPO) is an integrative forebrain region that contributes to blood pressure regulation. The MnPO has projections to the paraventricular nucleus (PVN) of the hypothalamus. The PVN contains pre‐autonomic centers that project to regions in the hindbrain that regulate sympathetic outflow. We hypothesized that by lesioning pathway specific projections from the MnPO to the PVN, we could attenuate the sustained component of CIH‐induced hypertension. For these experiments, adult male Sprague‐Dawley rats (250–300 g bw) were anesthetized with isoflurane and stereotaxically injected bilaterally in the PVN with a retrograde AAV containing Cre (AAV9.CMV.HI.eGFP‐Cre.WPRE.SV40) and with the caspase‐3 virus (AAV5‐flex‐taCasp3‐TEVp) or a control virus (AAV5‐hSyn‐DIO‐mCherry) in the MnPO. After 1 week recovery, rats were instrumented with aortic radio telemetry and allowed an additional week recovery following surgery. Rats were then moved to new homecages and underwent baseline recording for 7 days before undergoing our 7‐day CIH protocol. The control group exposed to CIH developed chronic hypertension, however, caspase lesions blunted the sustained hypertension developed during CIH. Brain tissue processed for FosB immunohistochemistry (IHC) showed decreased expression with caspase‐induced inhibition in the MnPO and downstream autonomic‐regulating nuclei in the PVN and rostral ventral lateral medulla (RVLM). CIH significantly increased plasma advanced oxidative protein products (AOPP) levels in controls. This increase in AOPP levels was blocked in caspase‐lesioned rats comparable to normoxic control concentrations. In situ hybridization experiments indicate a reduction in angiotensin type 1a receptors (AT1aR) expression in the caspase‐lesioned group exposed to CIH compared to CIH controls. The results indicate that MnPO neurons that project to the PVN play a significant role in blood pressure regulation and in the development of persistent CIH‐induced hypertension. Support or Funding Information P01 HL088052 T32 AG020494 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Moderate acute intermittent hypoxia (mAIH) elicits a form of spinal, respiratory motor plasticity known as phrenic long-term facilitation (pLTF). In middle-aged male and geriatric female rats, mAIH-induced pLTF is attenuated through unknown mechanisms. In young adults, mAIH activates competing intracellular signaling cascades, initiated by serotonin 2 and adenosine 2A (A2A) receptors, respectively. Since spinal A2A receptor inhibition enhances mAIH-induced pLTF, serotonin dominates, and adenosine constrains mAIH-induced plasticity in the daily rest phase, we hypothesized elevated basal adenosine levels in the ventral cervical spinal cord of aged rats shifts this balance, undermining mAIH-induced pLTF. A selective A2A receptor antagonist (MSX-3) or vehicle were delivered intrathecally at C4 in anesthetized young (3-6 months) and aged (20-22 months) Sprague-Dawley rats prior to mAIH (3,5-min episodes; arterial PO2=45-55mmHg). In young males, spinal A2A receptor inhibition enhanced pLTF (119 ± 5%) versus vehicle (55 ± 9%), consistent with prior reports. In old males, pLTF was reduced (25 ± 11%), but A2A receptor inhibition increased pLTF to levels greater than in young males (186 ± 19%). Basal adenosine levels in ventral C3-C5 homogenates are elevated 2-3-fold in old versus young males. These findings advance our understanding of age as a biological variable in phrenic motor plasticity and will help guide translation of mAIH as a therapeutic modality to restore respiratory and non-respiratory movements in older populations afflicted with clinical disorders that compromise movement.