Fucoxanthin Inhibits The Inflammatory Response Caused By Radiation

Contact: Audrey Hu audrey.hu@wecistanche.com

Part Ⅱ：Fucoxanthin alters the apelin-13/APJ pathway in certain organs of γ -irradiated mice

Nermeen M. El Bakary, Noura Magdy Thabet & et al.

DISCUSSION

The focus of the current study was Fucoxanthin (FX), due to its bioactive properties; we examined its action against RAD exposure-induced changes in the apelin-13/APJ pathway which could affect or be affected by the inflammatory reactions and changes in redox status.

The data of the current study showed that exposure of mice to IR led to a remarkable induction in the oxidative burden in liver, kidneys, lung, and spleen tissues that are manifested by a significant up-regulation of the hypoxia biomarker, HIF-1a, elevation in MDA (the end-product of lipid peroxidation) and reduction in antioxidant markers(GSH-PX and GSH). These data were associated with a considerable elevation of pro-inflammatory molecules(MCP-1 and IL-6)in the examined organ tissues, down-regulation in the splenic α-7nAchR, and disturbance in systemic inflammatory mediators (TNF-α, IL-1β, CRP, and IL-10). These findings were supported by the histopathological investigation, whereas the changes in the architecture of tissues that responded to the oxidative damages and inflammation are obvious.

These results could be attributed to the development of oxidative stress, a status that arises from the abundant generation of ROS or malfunction of the antioxidant defense system. Yahyapour et al[1]reported that inflammation and oxidative damage are strongly related to IR exposure. Chatterjee[9] revealed that progressive chronic inflammation and oxidative stress are implicated in a variety of pathological processes and can lead to dangerous diseases. Radiation-induced lung injury via post-radiation hypoxia is a causative factor mediating continuous generation of ROS, a surge in leukocyte migration, vascular permeability, stimulation of collagen formation, and up-regulation of the release of inflammatory cytokines by various cells such as endothelial cells, alveolar macrophages, pneumocytes, and fibroblasts [29]. The data of the current study might indicate a case of hypoxia. The increase of HIF-lα in organ tissues of irradiated mice could signify the occurrence of hypoxia after radiation exposure, which could take part in the development of oxidative stress and inflammation. The study of Azab et al [30] specified that exposure to y-radiation causes exaggerated ROSformation and directs the irradiated cells into a condition of oxidative stress that has been implicated in diverse processes of natural and pathological origin. This overproduction of ROS is accompanied by the reduction of cellular antioxidant activities and lipid peroxidation, protein oxidation indices, and inflammatory markers in the liver of irradiated rats. In the study of Moustafa and Thabet [31], the irradiation of rats by y-rays (6 Gy) elevated the MDA level and reduced the antioxidant enzymes |superoxide dismutase(SOD)and catalase (CAT)] and eventually caused liver tissue damage. Also, the exposure of brain tissue to 5 Gy of y-radiation increased MDA, IL-1β, and IL-6, coupled with abrogated antioxidant enzyme activity(glutathione S-transferase) that led to brain injury [32].

It is noteworthy that the -6 produced in response to liver and kidney injury is a signal in the event of tissue damages[33-35].TheIL 6 produced in response to kidney damage directly causes inflammation and injury to lung tissue35], which is consistent with the results shown in the present study. However, IL-6 activates splenocytes to produce IL-10 via α-7nAchR that is required to temper tissue injury, as reported in the study of Kinsey [35]. The y-irradiation microenvironment could direct the persistent activation of the IL. 6 in tissues and systemic pro-inflammatory mediators(TNF-α and IL-1β) to be detrimental rather than beneficial, as manifested by the down-regulation of splenic α-7nAchR and L-10 levels as observed in the present study. Supporting this view, Linard et al. [36] stated that after whole-body y-irradiation of rats at 10 Gy, cascades of inflammatory responses were induced through the increased concentrations of IL-6 and IL.8 associated with a decrease in the IL-10level. Moreover, Galal et al [37] stated that 7 Gy of y-radiation increased the biomarkers of hepatotoxicity and diminished ROS-detoxifying enzymes in the liver and spleen associated with the down-regulation of splenic α-7nAchR.The down-regulation of splenic α-7nAchR in the RAD group might be related to cumulative pro-inflammatory and diminished anti-inflammatory cytokine release through the NF-KB pathway [38]. Further, Wang et al. [39] revealed that exposure of human umbilical vein endothelial cells to y-radiation disrupts the cellular junctions through the activation of the inflammatory NF-kB signaling pathway manifested by enhancement of oxidative and nitrosative stresses and increases in the cytokines IL-6 and TNF-α.

In contrast, the administration of several doses of FX (Fucoxanthin ) before y-radiation exposure in the FX (Fucoxanthin) + RAD group diminished the disturbances in the oxidant/antioxidant defense system and pro-/anti-inflammatory balance as observed in the present study. This result might be due to the antioxidant capacity of FX (Fucoxanthin). Rodrigues et al.40| reported that FX (Fucoxanthin), a marine carotenoid, has a potent free radical-scavenging capacity which explains its antioxidant abilities. These abilities might be credited to its distinctive allenic bond and 5,6-monoepoxide which are critical for free radical scavenging and protection of cells from damage induced by H2O2 and UV-B [41]. FX (Fucoxanthin) showed improvements in the traumatic brain injury model by reducing the MDA content and restoring the activity of GSH-PX [15]. It could exert its cytoprotective effects counter to H2O2 oxidative injury in L02 cells (normal human hepatic cell line)through the phosphatidylinositol 3-kinase-dependent induction of Nrf-2(nuclear-factor erythroid related factor-2) signaling that is manifested by reduced leakage of LDH and intracellular ROS with enhanced intracellular GSH 42|. FX (Fucoxanthin) is responsible for the amendments of cellular redox tone and inflammation that were observed in the liver of irradiated rats at 8 Gy of fractionated(2 Gy×4;2 Gyevery3 days)Y-rays via regulation of TNF-α levels, MDA production, and preservation of GSH concentration |43]. Also, FX (Fucoxanthin) restores the reduced antioxidant system(GSH, GSH-PX, SOD, and CAT) and inhibits the high level of generation of the inflammatory molecules IL-1βand TNF-α which are linked to obesity [44]. Furthermore, Grasa-Lopezet al|45|showed that FX (Fucoxanthin) has a progressive effect via increasing the anti-inflammatory cytokine adiponectin and diminishing the pro-inflammatory cytokines leptin and CRP. The anti-inflammatory effect of FX (Fucoxanthin) might be attributed to the inhibition of NF-kB induction and the suppression of mitogen-activated protein kinase(MAPK) phosphorylation which leads to decreasing the level of pro-inflammatory mediators involving NO, prostaglandin E2, IL-1β, TNF-α andIL-6in lipopolysaccharide-stimulated murine macrophage cells[14].

The protein expression of the apelin-13/APJ/NF-KB pathway increased in the liver, kidney, lung, and spleen tissues of the irradiated mice group concurrently with the development of oxidative stress and inflammation, as observed in the current study. The increase observed in apelin-13, API and NF-K B protein expression could be credited to the action of highly inflammatory mediators such as IL-6 or the development of oxidative stress because of the large amounts of MDA formed in response to the excessively generated ROS in organ tissues due to radiation exposure. Han et al. [46] showed that the inflammation-induced rise in apelin mRNA expression was mediated via IL-6 and interferon-y by stimulation of apelin promoter activity, which in turn was mediated through the JAK/STAT pathway. Helmi et al. 47] described that the damage to the heart observed during chronic systemic hypoxia is because of a rise in the relative apelin mRNA expression, high MDA levels, and substantial increases in the formation of ROS during hypoxia. The up-regulation of inflammatory mediators and the activation of NF-xB are key events behind the inflammatory process. Xu et al.[48] established that the transcription factor NF-xB controlled serious cellular responses to stress and injury through activation of cytokines (such as TNF-α and IL-1)and oxygen-free radicals. The binding of apelin-13 to its receptor APJ can prompt the expression of the transcription factor NF-KB, which intensifies the manifestation of the chemotactic pro-inflammatory molecule MCP-1 [49,50]and is associated with down-regulation of α-7nAchR protein expression [38].

The anti-inflammatory effect of FX (Fucoxanthin) against y-radiation-induced tissue inflammation could be interpreted in light of its ability to control the apelin/APJ/NF-kB axis. The present data pointed out the significant amelioration in the apelin/APJ/NF-kB signaling pathway in mice who received FX (Fucoxanthin) and then were exposed to y-radiation. It could be assumed that the management of this signaling pathway might be attributed to the role of NF-KB as a regulator of the apelin pathway linked mechanistically to apelin's inhibition of inflammatory mediator up-regulation and suppression of NF-kB activation in tissues suffering from inflammation. Kim et al.|14]and Choi etal51|postulated that FX (Fucoxanthin) is beneficial as anti-inflammatory therapy because of its inhibitory influence on NF-kB activation and MAPK phosphorylation.

In the present study, an imbalance between MMP-2, MMP-9, and TIMP-1 was detected in the liver, kidney, lung and spleen of mice exposed to y-irradiation compared with the control mice. This was accompanied by a significant increase in LDH activity in all organs, disturbance in liver function enzymes (as shown by increased AST and ALT activities) and kidney function(as shown by the raised urea and creatinine levels) and alterations in the architectures of organ tissues that were observed in tissues photomicrographs of the RAD group. The induction of oxidative stress, inflammation, and up-regulation of the apelin/APJ/NF-kB signaling pathway after y-irradiation might contribute to the above consequences and the subsequent breakdown of organ functions in irradiated mice. Yahyapour et al. [1stated that the situation of oxidative destruction, chronic inflammation, and its consequences which resulted after IR may interrupt the functions of irradiated organs. Galis and Khatri52|related the imbalance between MMPs and TIMPs, and the promotion of vascular remodeling to the induction of oxidative stress and the development of inflammatory responses. Also, Nguyen et al.|53| observed the induction of MMP-2 and MMP-9andreduction of TIMP-1 in response to the up-regulation of NF-B in PMA(phorbol 12-myristate 13-acetate)-induced human fibrosarcoma. In addition, in colorectal carcinoma patients, the development of oxidative stress enhances the cell membrane lipid peroxidation, leading to impairment in membrane permeability and leakage of LDH and malate dehydrogenase(MDH) into the circulation[54]. The outflow of the cytosolic enzyme LDH is related to cellular viability, and hence it is a convenient gauge of membrane destruction. Several studies confirmed that LDHis a marker of organ injury and correlated its increase in tissues with oxidative stress and inflammatory conditions 55-58]. Thus, the increases in LDH that were found in all organs of the present study emphasize the damage of those organs after exposure to y-radiation.

From the data revealed in the present study, FX (Fucoxanthin) administration before y-radiation exposure reduces oxidative stress and at the same time prevents the MMP-2, MMP-9/TIMP-1 imbalance and maintains cellular integrity as perceived by diminished LDH leakage from the liver, kidney, lung and spleen of irradiated mice. These findings might be credited to the antioxidant and anti-inflammatory actions of FX (Fucoxanthin), which resulted in the regulation of the apelin-13/API/NF-xB signaling axis as previously interpreted. The FX (Fucoxanthin) administration reduced cellular MMP-2 and MMP-9 activities concomitant with increases in the tissue MMP inhibitors, such as TIMP-1in PMA-induced human fibrosarcoma cells via inhibition of NF-kB, JNK and p38-MAPK[53]. Also, the pre-treatment by FX (Fucoxanthin) resulted in a reduction of LDH leakage, decreased intracellular ROS content and enhanced intracellular GSH [42]. The inhibition of LDH reduced hepatic necrosis, apoptosis and the appearance of pro-inflammatory mediators in a mouse model with acute liver failure [58] which could explain the improvement that was observed in the organ of the FX (Fucoxanthin)+ RAD group. Histopathological examination of the liver, kidney, lung and spleen in the current investigation revealed that FX (Fucoxanthin) possesses a remarkable radioprotective potential as implied by its mitigation of the harmful alterations induced by IR on the biochemical profile. These data are in agreement with those of Bharathriraja et al. [59], Zheng et al. [60] and Wang et al [61] who postulated the cytoprotective efficacy of FX (Fucoxanthin) against various deleterious factors in different organs and attributed these effects to its antioxidant and anti-inflammatory properties. Of note, the current study sheds light on a novel mechanism that might contribute to the cytoprotective efficacy of FX (Fucoxanthin) via the concerted regulation of the splenic cholinergic anti-inflammatory nicotinic receptor(α-7nAchR) and the apelin-13/APJ pathway in the target organs.

As reported in Mun et al.[62], there are several radioprotectors that have been investigated with different mechanisms of protective actions such as: bergenin (Caesalpinia digyna) which activates MAPK and ERK pathways to modulate the radiation damage effect; N-acetyl tryptophan glucopyranoside(Bacillus subtilis)which over-comes radiation-induced damage by recovering cytoprotective cytokines and antioxidant enzymes; zymosan A(Saccharomyces cerevisiae)which protects against radiation-induced DNA damage by up-regulating the levels of cytokines, and psoralen in(Psoralea cory li folia)which inhibits the radiation-induced PI3K-IKK-IcB signaling pathway, COX-2 and expression of pro-inflammatory cytokines. N-acetyl cysteine and resveratrol have been shown to decrease DNA damage via induction of natural antioxidants(GSH, SOD and CAT); vitamin C, in a poly Eglucoside and quercetin-3-O-rhamnoside-7-O.glucoside, showed protection against radiation through the reduction in lipid peroxidation, as reported in Smith et al 63]. Oh, et al|64|reported that algal natural products such as director, fucoxanthin, astaxanthin, and algae extracts have radioprotective effects including radical scavenging and antioxidant properties. However, the new findings of the current study shed light on a novel mechanism of FX (Fucoxanthin) as a radioprotector against y-radiation-induced damage in the organs studied through its concerted regulation of the apelin/APJ/NF-kB signallingpathwayandsplenicα-7nAchR, a primary receptor of the cholinergic anti-inflammatory pathway, as it has antioxidant and anti-inflammatory effects that led to modulation of the structural damage markers (MMP-2, MMP-9, TIMP-1 and LDH) and the function of the organs.

Collectively, the results of the current study indicate that radiation exposure-induced activation of apelin-13/APJ signaling which could be attributed to the induction of oxidative stress(as manifested in this study by an increase of HIF-1α and MDA, and decreases of GSH and GSH-PX)as well as inflammation(as manifested by increases in MCP-1, I-6 and NF-KB and decreases in IL-10 and α-7nAchR). Furthermore, the elevation of oxidative stress and inflammatory mediators led to structural damage in the organs examined (as manifested by an increase in MMP-2, MMP-9 andLDH, and a decrease in TIMP-1 confirmed by histopathological examination). In turn, the recorded activation of apelin signaling in the irradiated group appeared to participate in the surge observed in oxidative stress and inflammation. We could suggest that the systemic administration of FX (Fucoxanthin) has considerable beneficial effects against oxidative damage and inflammatory status. The regulation of the apelin-13/API/NF. kB signaling axis might represent the cornerstone of the protective mechanisms of FX (Fucoxanthin) against radiation hazards that lead to the cellular damage and collapse of organ functions. As a recommendation. FX (Fucoxanthin) might be of use as a potential radioprotector in cases of radiation exposure.

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