Radiotherapy for lung carcinoma is frequently limited secondary to damage on a spectrum from pneumonitis to fibrosis that is inflicted upon surrounding normal tissues, and this progressive damage ultimately results in worsened pulmonary function. The search for an ideal radioprotector led us to identify the potent antioxidant, anti-fibrotic, anti-inflammatory properties of wholegrain flaxseed (8
) in our mouse model of lung injury from thoracic radiation therapy. The current study identified for the first time the FLC as the bioactive ingredient of this grain responsible for the protective effects. We demonstrate here that dietary FLC enriched in SDG, a non-toxic phenolic compound with numerous beneficial properties is easily tolerated for 16 weeks with no side effects and appropriate maintenance of weight. Moreover, biologically active levels of FLC metabolites could be detected in mice fed the treatment diets. Mice fed the FLC-supplemented diet showed dramatically reduced levels of pulmonary inflammation and fibrosis with the prevention of deterioration of pulse oximetry several months after a single fraction of XRT. Finally, in our perhaps most clinically applicable data, we showed that despite the radioprotection afforded by FLC in normal pulmonary tissue, it did not abrogate the tumoricidal effect of radiation.
Our group has had an interest in exploring the use of natural dietary phenolic compounds as a means of ameliorating acute and chronic lung diseases caused by the oxidative stress of thoracic radiation. One such dietary supplement, flaxseed that contains phenolics, is nontoxic with anti-inflammatory, anti-fibrotic, and antioxidant properties due to its high concentrations of omega-3 fatty acids and plant lignans (31
), our particular focus in this study. Flaxseed’s bioactive metabolites have been extensively studied in other organ systems and have proven to be beneficial, mostly with respect to cancer therapies (32
). Our group was the first to show that dietary FS could reduce inflammation and lipid peroxidation in murine models of acid aspiration and hyperoxia (7
) and more recently, in ischemia/reperfusion injury associated with lung transplantation (8
). For the first time, we demonstrate here that the lignan complex (FLC) possesses these same antioxidant, anti-inflammatory, anti-fibrotic, and lung radioprotecting properties of the wholegrain.
FLC could be ingested over a prolonged period and could achieve biologically significant levels in vivo
. This was a lengthy study and prolonged feeding (>16 weeks) did not cause any adverse health effects evidenced by weight monitoring. Additionally, circulating levels of the FLC metabolite enterolactone could be quantified reaching biologically comparable (if not superior to) levels achieved in our previous studies (7
) that featured wholegrain flaxseed. Dietary FLC led to improved survival in mice irradiated with a single fraction of 13.5 Gy XRT. Time to radiation-induced mortality (first death from XRT) was delayed by 2 weeks in mice pre-fed with FLC prior to irradiation. Survival curves showed irradiated mice fed 10% FLC were alive longer than mice fed 10% FS and alive far longer than mice fed a diet without flaxseed supplementation. Giving FLC may permit administration of higher and physiologically more effective doses of radiation to achieve higher tumoricidal effects while avoiding immediate morbidity and mortality. More investigation is needed in this aspect of the role of FLC in radiation-induced lung injury.
We discovered that FLC reduced the influx of inflammatory cells into the airways. Neutrophils are the first responders during the acute phase of radiation-induced lung injury. Mice irradiated after being pre-fed FLC had reduced numbers of white blood cells (with reduced numbers of neutrophils) in their BAL fluid compared to irradiated mice fed diets that were not supplemented with flaxseed. Recent studies by Lee at al
) showed that inflammatory cells isolated from lung lavage of FS-fed animals had diminished respiratory bursts and reduced oxidative enzyme release when stimulated. Importantly, in our recent work on the genomic profiling of dietary FS in lung tissues, we showed the immunomodulatory properties of FS mediated by down-regulation of gene expression levels of key cytokine receptors that were involved in the inflammatory cascade (36
). This may help explain the anti-inflammatory protective effects of FLC in radiation injury.
The current study compiled the first clinically relevant cardiopulmonary measurements through use of noninvasive sensor collars that were attached to each mouse. Most notably, pulse oximetry data showed that mice fed a diet of FLC had higher oxygen saturations with activity 16 weeks after a single dose of radiotherapy compared to mice irradiated after being fed a control diet. Before and after irradiation, FLC-fed mice had oxygen saturations well within physiologically acceptable ranges (≥95%) compared to the control diet fed mice that struggled to maintain saturations in the low 80% range. Oxygen saturation at 80% in the clinic patient could manifest itself as cyanosis, breathlessness and fatigue as a patient struggles to interact with the physician; while a patient with a saturation of 95% could have a conversation, breathe comfortably and carry on with a normal life. This highly favorable physiologic parameter dovetailed with our hydroxyproline assays and trichrome-stained lung section evaluations confirming reduced fibrosis in the lung tissue of FLC-fed mice.
Another intriguing and novel piece of data came in the form of pulse distention values. These values could translate to physiologic estimates of flow in a cardiopulmonary circuit (37
). X-ray radiation treatment (just as it does in pulmonary parenchyma) alters the dynamic equilibrium of tissue types within blood vessel walls (37
). It also stimulates inflammatory pathways that lead to scars in areas of wall injury (37
). The wall then becomes less compliant, less distensible due to a change in prevailing tissue type from highly dynamic muscle cells to rigid less distensible collagen scars (37
). Pulse distention could serve as a noninvasive surrogate for flow and derivation of resistance within the cardiopulmonary and possibly systemic circulations. X-ray radiation treatment as we have shown is tumoricidal, but inflames surrounding normal tissue enacting a repair cascade that ultimately ends in fibrosis. Mice pre-fed FLC demonstrated higher pulse distention. Although not causative, this intriguing finding of an association between increased pulse distention and protection from vascular fibrosis needs to be pursued further. Importantly, the possibility that this may be due to destruction of endothelial cells by oxygen radicals, hence reduction in production of vasodilators and/or unopposed action of circulating vasoconstrictors, must be further investigated.
Our study showed significant up-regulation of antioxidant enzyme gene expression (HO-1, NQO1) in lung tissue of mice fed FLC for 3 weeks, correlating with elevated plasma lignan levels. We also reported significant increases in Nrf2 gene expression that, along with HO-1 and NQO1 is regulated by the Nrf2 transcription factor. The ability of both 10% FLC and 20% FLC diets to up-regulate baseline levels of phase II antioxidant enzyme gene expression suggests FS lignan interaction with the Nrf2-Keap1 signaling pathway, which serves as the intracellular sensor of oxidative stress. Along with direct ROS scavenging (9
), activation of this pathway and subsequent Nrf2 up-regulation of antioxidant gene expression in unchallenged lung tissues of FLC-fed mice may explain the radioprotective properties of FLC. The ability of FLC diets to up-regulate antioxidant and phase II enzymes is further supported by immunoblot analysis of lung tissue harvested from FLC-fed mice in which HO-1 protein expression was elevated after 3 weeks of feeding.
In summary, we have evaluated a nontoxic and widely available dietary phenolic compound that yielded long-term protective benefits after thoracic radiotherapy. We have studied the beneficial properties of the wholegrain in the past, but herein for the first time have demonstrated that the lignan complex surpassed the wholegrain in its antioxidant, anti-inflammatory and anti-fibrotic properties. Our long-term goal is to permit greater doses of radiation to improve clinical responses and cures for thoracic malignancies, while providing adequate radioprotection against the side effects in normal lung parenchyma.