The Protective Benefits Of Fucoxanthin On Radiation
Mar 19, 2022
Contact: Audrey Hu audrey.hu@wecistanche.com
Part Ⅰ：Fucoxanthin alters the apelin-13/APJ pathway in certain organs of γ -irradiated mice
Nermeen M. El Bakary, Noura Magdy Thabet & et al.
INTRODUCTION
Exposure to ionizing radiation (IR) has occurred during radiology (diagnostic or interventional), radiotherapy, and occupational exposure in the radiation field. High radiation doses cause death whereas sublethal doses may induce diverse diseases, such as cancer, cardiovascular diseases, and cataracts[1]. The detrimental effects of IR (ionizing radiation) exposure involve the induction of tissue damage mediated through the activation of pro-and antiproliferative endogenous signaling pathways, inflammation, and oxidative stress in a synchronized sequence of actions that alter the homeostatic equilibrium between survival and cell death [2]. The mechanism of IR (ionizing radiation) that produces these effects depends mainly on the generation of different free radicals and molecular species within cells, such as superoxide(O2.-), nitric oxide (NO), the hydroxyl radical (OH·), hydrogen peroxide(H, O2)and peroxynitrite(ONOO-), that is produced via direct interactions or subsequent metabolites of IR (ionizing radiation) and cause damage to cellular DNA content, proteins, and lipids. In addition, Reisz et al.[3] and Yahyapour et al.[1] stated that, in different organs, IR (ionizing radiation) can robustly affect the immune system, by altering the number and function of the immune cells leading to changes in the normal immune responses. Thus, continuous free radical production and chronic inflammation that occur after radiation exposure can disrupt organ function and subsequently cause several diseases. As exposure to IR (ionizing radiation) is inevitably accompanied by the production of high levels of reactive oxygen species(ROS), so the management of this action could provide a method to avoid the deleterious effects on normal tissues upon radiation exposure. Therefore, radiation countermeasure agents should be used to reduce the hazardous effects of IR. Radiation countermeasure agents are classified according to their time of administration into:(i)radioprotectors used before IR (ionizing radiation) exposure to protect cells and tissues from being damaged;(i) radio mitigator applied soon after the exposure to IR (ionizing radiation) to repair tissues before the appearance of symptoms; and (ii) therapeutic agents administered after IR (ionizing radiation) exposure to enhance healing of injuries via regeneration of tissues. Radioprotectors are various agents that act via different mechanisms involving: scavenging of free radicals and ROS; improvement of the DNA repair process; synchronizing of cells; enhancing antioxidant and redox-sensitive genes; modulating cytokines and growth factors; inhibiting apoptosis; repurposing of drugs, and tissue regeneration. Scavenging of free radicals is the most common mechanism of radioprotection, whereas the alteration of growth factors, cytokines, and redox genes appears to be an effective strategy [4].
Apelin and its endogenous ligand APJ (apelin receptor; a member of the G-protein-coupled receptors, similar to angiotensin II receptor-like-1)can exert significant biological effects in different tissues and organs. They can strengthen cardiac contractility, regulate immune defense, gastrointestinal function, and insulin sensitivity, and promote cell proliferation, migration, and angiogenesis. In addition, apelin produces important effects on the physiology and pathophysiology of liver and kidney function and plays a crucial role in body fluid homeostasis [5,6]. Alotofevidence pointed to the strong relationship between oxidative stress and apelin/APJinteraction.Apelin can inhibit the formation and release ofROS[7].In contrast, Li et al.[8]reported that apelin-13 enhances the generation of ROS linked to the existence of oxidative stress that directly leads to vascular damage and a series of inflammatory reactions. Thus, the function of the apelin/APJ signaling path-way is a double-edged sword in insults involving oxidative stress and inflammatory-related diseases, as reported by Zhou et al.[7]. Therefore, there are still certain controversies and doubts about the exact effect of the apelin/APJ signaling pathway in different conditions. Drugs that target the apelin/APJ pathway might be recommended as a novel therapy for the related oxidative stress and inflammatory diseases.
A great deal of evidence revealed a positive relationship between oxidative stress and inflammation, and each leads to the other in a feedforward mechanism. The overproduction of ROS induces an oxidative modification of biomolecules leading to the enhancement of signaling cascades and activation of transcription factors which are linked to the genes of pro-inflammatory mediators and initiate the inflammatory reactions. Inflammation causes immune cells to secrete various cytokines, which evoke additional immune cells near oxidative stress and generate ROS at the inflammatory site, causing augmented oxidative stress and tissue damage [9]. The study of Ren et al.[10] mentioned that splenic α-7-nicotinic acetylcholine receptor (α-7nAchR) is a primary receptor of the cholinergic anti-inflammatory pathway(CAP) that displays widespread anti-inflammatory reactions and the immune-modulatory response to maintain immune homeostasis. Moreover, Veidt et al.[11]stated that monocyte chemoattractant protein-1(MCP-1)functions as a pro-inflammatory mediator inducing the production of pro-inflammatory molecules other than just chemokine. It promotes pro-inflammatory reactions in human tubular epithelial cells via up-regulation of the pro-inflammatory interleukin-6(I-6)and the adhesion molecule, intercellular adhesion molecule-1(ICAM-1), through the classical inflammatory pathways, involving sequence-specific DNA binding of nuclear factor-B(NF-kB) and activating protein-1. Under the normal physiological environment, the regular actions of matrix metalloproteinases(MMPs) are controlled at the level of transcription (activation of the precursor zymogens)and interaction with specific extracellular matrix(ECM) constituents. MMPs are zinc-containing enzymes that destroy the ECM and proteins of connective tissue. This proteolytic effect of MMPs takes part in vascular remodeling, cellular migration, and the processing of the ECM. Tissue inhibitor of metalloproteinases(TIMP)elicits a complementary mechanism with MMPs to avoid excessive degradation of the ECM. An imbalance between them could induce exaggerated MMP activity that leads to pathological changes in the structure of the vessel wall inter-related to vascular disease [12].In addition, Jain et al.[13]showed that lactate dehydrogenase(LDH), an oxidoreductase enzyme, which is found in all living cells and monitors membrane integrity, is released into the cytoplasm upon celsius of damaged cells more than of normal cells.
Management of chronic inflammation or inflammation, in general, is a critical point in the struggle to tame dangerous diseases associated with these undesired disorders. Nowadays, an alternative approach in radiation protection research is oriented towards using natural compounds having many biologically positive influences to overcome several health problems and biological alterations due to their wide safety margin and many beneficial properties(such as antioxidant, immune stimulation, anti-inflammatory, and antitumor).
Fucoxanthin (FX), a xanthophyll derivative, is the leading carotenoid formed in brown algae. FX exhibits a variety of pharmacological properties and biological functions including antioxidant, antiviral, anticancer, antidiabetic, UV-preventative, neuroprotective, and repressing inflammation without side effects[14-16] because of its unique functional groups, including an infrequent allenic bond and a 5,6-monoepoxide within its molecular construction[15].
Yet, the potential role of the apelin/APJ pathway and how its interference with other mediators might be involved in mediating the deleterious impact of y-radiation exposure is yet to be elucidated. Therefore, the current study was designed to investigate(i)the effect of y-radiation on the apelin-13/APJ pathway and its relevance to certain physiological processes in the liver, kidney, lung, and spleen of irradiated mice and (i)whether the effect of FX on the alterations could happen in the apelin-13/APJpathwayin tissues of these organs. To reach these goals, this study monitored the protein expression of the apelin-13/APJ pathway and determined oxidative stress status {hypoxia-inducible factor-1α(HIF-1α), lipid peroxidation [measuredas malondialdehyde (MDA)], reduced glutathione (GSH), and glutathione peroxidase (GSH-PX)}. The pro-and anti-inflammatory molecules [NF-kB, α-7nAchR, MCP-1, IL-6, IL-10, and tumor necrosis factor-α(TNF-α)] were determined in different mice groups. Histopathological investigations of the matrix metalloproteinase balance (MMP-2, MMP-9, and TIMP-1)were carried out on the tissue obtained from these four organs.
MATERIALS AND METHODS
Materials
Fucoxanthin (FX)was obtained from Serene Dew supplements. For western blot analysis, the antibodies against apelin-13(cat no. CAS 217082-58-1)and β-actin(mouse monoclonal antibody cat no.sc-47778)were obtained from Santa Cruz Biotechnology, and the other antibodies against APJ(rabbit polyclonal antibody cat no.ab214369), NF-xBp65-Ser536(cat no. ab76302) and α-7nAchR(rabbit polyclonal antibody, cat no.ab10096)were from Abcam. The other chemicals and reagents used in this study are from Sigma-Aldrich Chemical Co. The USA.
Radiation facility
Mice were exposed to whole-body y-irradiation (RAD)using Canadian y-cell-40(137 Cs).rradiation procedures were performed at the NCRRT (Cairo,Egypt) at a dose rate of 0.4 Gy min- of y-rays.
Animals
The Swiss female albino mice adult mice(weighing 22-25 g)used in this study were obtained from the Egyptian Organization for Biological Products and Vaccines(Cairo)breeding unit. Mice were acclimatized and maintained on water ad libitum and a standard commercial pellet diet for l week.
Experimental plan
Mice were divided into four equal groups(10mice/group). (i)Control group: normal mice received only physiological saline ip.(i)RAD group: mice were exposed to y-radiation(2.5 Gy week-l). (ii)FX group: mice have injected ip with FX at a dose of 10 mg kg' days dissolved in physiological saline for 4 weeks according to Ma et al. [8]; and (iv)FX+RAD group: mice were treated with FX and were exposedtoγ-radiation.FXwas administrated（ip.）for 3 days before y-ray exposure to stimulate and impose a pre-conditioning status in normal cells to overcome and sustain the subsequent detrimental effects induced by irradiation in order to achieve radioprotection and adaptive responses of the tissues exposed. The y-radiation dose was chosen according to the study ofZakaria[18]that aimed to determine the hazardous effects of low successive doses during exposure to γ-irradiation of many workers in the medical, industrial, and petroleum fields who may be exposed during a small radiation accident to low or moderate y-radiation doses(1.5,2,2.5,3 and 3.5 Gy). This does lead to acute effects on the health efficiency and performance of organisms. Thus, in the current study, we have chosen2.5 Gy as a moderate dose to examine its action on the apelin-13/APJ pathway. Twenty-four hours after the last dose of FX, mice fasted overnight and then euthanized under light diethyl ether anesthesia. Cardiac perforation drew blood samples, which were centrifuged for the separation of serum and biochemical assessments. The target tissues(liver, kidney, lung, and spleen)were excised, then washed in ice-cold saline solution, and prepared for the biochemical and histopathological investigations. Upon radiation exposure, these four vital organs were chosen to investigate the interconnection between them in terms of concerted regulation of oxidative stress and inflammatory mediators affected by the dysregulation of the apelin-13/APJ pathway.
Biochemical assays
MDA, the end-product of lipid peroxidation, was assayed according to Yoshioka et al.[19],the GSH content was assayed according to Ellman [20], protein concentration was detected according to the method of Lowry et al.[21] using Folin-Ciocalteu reagent, and the activity of GSH-PX was measured according to the method of Gross et al. [22]. Activities of AST(aspartate aminotransferase)and ALT(alanine aminotransferase) were assayed as described by Reitman and Frankel [23]. Urea and creatinine were measured according to the techniques of Fawcett and Scott [24] and Bartles et al.[25],respectively. The levels of HIF-1,MCP-1,LDH, the inflammatory mediators IL-10,IL-6,IL-1,TNF-α and C-reactive protein(CRP), MMP-2,MMP-9 and TIMP-1 were assessed by ELISA kits(R&D Systems) according to the manufacturer's instructions.
RESULTS
Impact of FX on oxidative and antioxidant status of certain organs in y-irradiated mice
The data illustrated in Fig.1 showed that the levels of MDA, GSH, GSH-PX and HIF-1α were not significantly changed in the liver, kidney, lung, and spleen of the FX group when compared with control mice. However, Fig. 1showed that radiation exposure according to the current protocol induced significant changes in oxidative stress and the antioxidant status of certain mouse organs. The HIF-lo and MDA levels increased significantly (P<0.05)in the liver(MDA 3.41-fold and HIF-1α 3.03-fold), kidney (MDA 3.32-fold and HIF-lα 3.13-fold), lung (MDA3.64-fold and HIF-1α 6.5-fold), and spleen (MDA 2.57-fold and HIF-lα 3.64-fold) when compared with the respective control. In contrast, the GSH content and GSH-PXactivities decreased significantly in all organs subjected to investigation in this study as follows: liver(GSH68.45%and GSH-PX49.39%), kidney(GSH67.58%and GSH-PX 54.79%), lung (GSH 51.68% and GSH-PX43.99%)and spleen(GSH 54.49% and GSH-PX56.41%). However, in the group of mice treated with FX before exposure to Y-radiation, a considerable amelioration in oxidative and antioxidant status manifested by a significant decrease(P<0.05)in HIF-1α(liver32.71%,kidney48.18%, lung 44.37% and spleen 48.87%)and MDA(liver 42.79%,kidney43.15%, lung 46.18% and spleen 47.52%)levels, and a substantial increase (P<0.05)in GSH(liver 2.77-fold, kidney 2.52-fold, lung 1.57-fold and spleen 1.96-fold)content and GSH-PX(liver1.8-fold, kidney 1.89-fold, lung 1.54-fold and spleen 2.02-fold) activities was observed in all organs subjected to investigation when compared with mice of the RAD group.
Fig. 1. Impact of FX on oxidative (HIF-1α and MDA) and antioxidant status (GSH and GSH-PX) of (A) liver, (B) kidney, (C) lung, and (D) spleen in γ -irradiated mice. Data are expressed as mean values ± SEM (n = 6 independent values).
Columns with different letters (a, b, c ... ) within the same histogram are significantly different and columns having the same letters are not significantly different at P < 0.05. Control group, normal mice; RAD group, mice exposed to γ -radiation; FX group, mice treated with fucoxanthin; and FX + RAD group, mice treated with FX and exposed to γ -radiation.
Impact of FX on inflammatory responses of certain organs in y-irradiated mice
The data obtained from the present study showed that the inflammatory response(IL-6, MCP-1 and IL-10)in the liver, kidney, lung and spleen of the FX group was not altered significantly compared with the control mice. Also, the protein expression of splenic α-7nAchR was not changed significantly in the FX group in comparison with control mice. In contrast, the data of the inflammatory response mediators in certain organs significantly changed in mice exposed to y-irradiation compared with the normal mice(Fig.2). Among them, MCP-1 and IL-6 increased significantly (P<0.05)in the liver(MCP-13.75-fold and IL-63.38-fold), kidney(MCP-1 3.12-fold and IL-64.80-fold), lung (MCP-1 2.39-fold and IL-64.75-fold) and spleen(MCP-12.45-fold and IL-63.37-fold) in irradiated mice compared with the control mice. The IL-10 level was significantly decreased in both lung (54.77%)and spleen(44.35%)of irradiated mice, associated with a considerable decrease(P<0.05)in the protein expression of α-7nAchR in the spleen(55%)when compared with its equivalent value in control mice(P<0.05). We observed significant changes in all inflammatory response parameters in all organs subjected to investigation in the current study when mice were injected with FX before exposure to y-radiation. As shown, MCP-1 and IL-6 in all organs(liver, MCP-1 53.68% and IL-641.21%;kidney, MCP-1 52.78% and IL-658.69%;lung,MCP-1 54.17%andIL-651.62%;and spleen,MCP-142.92%and IL-670.33%)of the FX＋RAD group were significantly (P<0.05)decreased when compared with the RAD group. The I-10 level in the lung(1.86-fold)and spleen(1.60-fold)significantly increased, associated with a significant increase in the splenicα-7nAchR protein expression(1.93-fold) compared with the RAD group.
Fig. 2. Impact of FX on inflammatory responses in (A) liver (MCP-1 and IL-6), (B) kidney (MCP-1 and IL-6), (C) lung (MCP-1,
IL-6 and IL-10) and (D) spleen (MCP-1, IL-6 and IL-10) with representative western blot analysis of α-7nAchR (54 kDa) with its
SDS–PAGE normalized to β-actin (43 kDa) protein expression in γ -irradiated mice. Data are expressed as mean values ± SEM
(n = 6 independent values). Columns with different letters (a, b, c ... ) within the same histogram are significantly different and
columns havimg the same letters are not significantly different at P < 0.05. Control group, normal mice; RAD group, mice exposed
to γ -radiation; FX group, mice treated with fucoxanthin; and FX + RAD group, mice treated with FX and exposed to
γ -radiation.
Impact of FX on y-irradiation-induced alteration in apelin-13/APJ/NF-kB signaling
The data exemplified in Fig.3(histograms and western blot output)showed that the protein expression of apelin-13 and its receptor APJ and a complex protein NF-kB(an inducible transcriptional factor)was not altered in the liver, kidney, lung and spleen of mice administered FX when compared with normal mice. However, the protein expression of apelin-13, PJ and NF-kB increased significantly (P<0.05) in the four organs of the RAD group as compared with the control mice as follows: liver(5.62-,6.4-and 5.15-fold), kidney(3.7-,4.2-and 6.8-fold), lung (2.77-,3.1-and 5.3-fold) and spleen(5.3-,6.8-and 6.01-fold), respectively. Nevertheless, with FX administration, the protein expression of apelin-13, APJ and NF-kB was significantly decreased (P<0.05) in the liver(68.91,50.16and44.23%),kidney(48.65,50.24 and54.70%),lung(53.57,34.19and52.83%) and spleen (58.49,50.15 and74.15%),respectively, in the FX+RAD group compared with the RAD group (Fig. 3).
Impact of FX on the changes induced in MMP-2, MMP-9, TIMP-1 and LDH of certain organs in -irradiated mice
Data shown in Fig.4 revealed that the activities of MMP-2 and MMP-9, the TIMP-1level and LDH activity were not significantly changed (P<0.05)in liver, kidney, lung and spleen of the FXmice group when compared with the control mice. In the RAD mice group, the MMP-2, MMP-9 and LDH activities increased significantly(P<0.05)in the liver(2.47-,2.32-and 1.65-fold),kidney (2.35-,3.76-and 1.31-fold), lung (3.18-,1.91-and 1.85-fold) and spleen(2.73-,2.03-and 2.23-fold),respectively, when compared with the control mice, while a substantial decrease in TIMP-1 concentration(liver 53.26%, kid-ney 43.51%,lung 46.77% and spleen 54.09%was observed when compared with controls. Mice treated with FX before exposure to Y-radiation showed a significant(P<0.05)reduction in the changes induced by y-radiation on MMP-2, MMP-9 and LDH as compared with the RAD group, as follows: liver(35.95, 50.85 and 24.56%), kidney(35.71,38.65and13.69%),lung(54.86,30.34and29.18%) and spleen(33.33, 42.67 and 30.49%),respectively. On the other hand, a significant elevation(P<0.05)in TIMP-1 concentration of all organs (liver 1.70-fold, kidney 1.38-fold, lung 1.57-fold and spleen 1.59-fold)was observed when compared with the RAD group.
Impact of FX on the changes induced in the physiological function of liver and kidney in -irradiated mice
Liver function, as shown by the results of ALT and AST enzymes in serum of mice who received FX, was not significantly changed (P<0.05) when compared with the normal mice(Fig.5). However, in the mice group exposed to Y-radiation, the activities of these two enzymes (ALT 1.32-fold and AST 1.84-fold) increased significantly(P<0.05)as compared with the control mice. However, the activities of ALT (15.78%)and AST(34.56%)were significantly (P<0.05)decreased in mice who received FX and were exposed to y-radiation when compared with the RAD group (Fig.5).
Regarding kidney function, the data illustrated by Fig. 5 show that the concentration of urea and creatinine in the serum of mice who received FX treatment was not significantly changed(P<0.05) as compared with the normal mice. The exposure of mice to y-radiation stimulates a significant increase(P<0.05)in the serum content of urea (1.73-fold) and creatinine(2.66-fold) compared with control mice. However, in mice who received FX treatment before y-radiation exposure, the content of urea(20.73%)and creatinine(32.79%)showed a significant(P<0.05)reduction as compared with the RAD group (Fig.5).
Impact of FX on the changes induced in systemic inflammation of y-irradiated mice
Figure 6reveals that there were no significant changes(P<0.05)in the serum inflammatory markers(TNF-α, IL-1β, CRP, and IL-10)of mice who received FX when compared with the control mice. As expected, there were significant increases in the levels of TNF-α(2.56-fold), IL-1β(2.08-fold), and CRP(4.12-fold), and a significant decline in the level of IL-10(38.65%), observed in mice exposed toy-irradiation when compared with controls. Treatment with FX before exposure to y-radiation brought about an incredible improvement in serum levels of the four measured inflammatory markers when compared with the RAD group, with a significant decrease in TNF-α(30.34%), IL-1β(30.89%), and CRP(42.73%), and a significant increase in the level of IL-10(1.35-fold).
Histopathological study
The histopathological inspection of the liver, kidney, lung, and spleen tissues of different animal groups is presented in Figs 7,8,9 and 10, respectively.
Liver tissues
The control group showed a normal portal tract with a normal portal vein (PV), bile ducts(BDs)(black arrow), and hepatocytes in the periportal area(blue arrows)(Fig.7a). The FX group showed a normal central vein(CV)and regular hepatocytes in the perivenular area (black arrow)(Fig.7b). The RAD group showed portal tracts with a mildly widened congested PV, mildly widened CV, and scattered apoptotic hepatocytes in the perivenular zone(black arrows)(Fig.7c), and areas of hemorrhage (black arrows) with scattered apoptotic hepatocytes(blue arrow)(Fig.7d). The FX+RAD group showed portal tracts with a mildly widenedPV, normal BDs, and mild hydropic change of hepatocytes in the periportal zone(black arrows)(H&E×400)as shown in Fig. 7e.
Kidney tissues
The control group showed normal glomeruli (G) with normal Bowman's spaces(BS), normal proximal tubules(P) with preserved brush borders(black arrow), normal distal tubules(D), and normal interstitium(blue arrow)as represented in(Fig.8a). The FX group showed normal G with normal BS and P with scattered apoptotic epithelial lining (black arrow)and preserved brush borders(blue arrow)(Fig.8b). The RAD group showed distorted G with average BS and P with markedly oedematous-epithelial lining(black arrow)and partial loss of brush borders(blue arrow)(Fig.8c). The FX+RAD group showed
Lung tissues
The control group showed normal alveolar walls(black arrows) and normal interstitium(blue arrow)(Fig.9a). The FX group showed normal alveolar walls(black arrow)(Fig.9b). The RAD group showed bronchioles(B) with regular epithelial lining (black arrow), mildly dilated blood vessels (BV), and markedly congested alveolar walls (blue arrow)(Fig.9c).and congested alveolar walls(black arrows)with interstitial hemorrhage (blue arrow) and edema (red arrow)(Fig.9d). The FX+ RAD group showed mildly dilated congested BV, thickened alveolar walls (black arrow)with mild interstitial inflammatory infiltrate(blue arrow)(Fig.9e), and another bronchiole (B)showed normal epithelial lining(black arrow), normal BV, and normal alveolar walls(blue arrow)(H&E ×400) as revealed in (Fig.90.
Spleen tissues
The control group showed normal lymphoid follicles(yellow arrow)and blood sinusoids(blue arrow)(Fig.10a). The FX group showed normal lymphoid follicles with central arterioles(blue arrow), and normal blood sinusoids(red bulb)(yellow arrow)(Fig.10b). The RAD group showed small-sized lymphoid follicles with central arterioles (black arrow)and dilated congested blood sinusoids(blue arrow)with pericapsular inflammatory infiltrate(red arrow)(Fig.10c). The FX+RAD group showed dilated congested blood vessels (black arrow)with congested blood sinusoids(red bulb)(blue arrow)(H&E×400)as revealed in Fig. (10d).
