Choline Glycerophosphate and Silymarin Modulate Brain and Intestinal Injuries in Rats Exposed to Gamma-Radiation

This work aims to investigate the possible effect of choline glycerophosphate alone or combined with silymarin administration in modulating whole body gamma irradiation-induced brain and intestinal injuries in rats. Rats were irradiated with 7 Gy then subjected to choline glycerophosphate and/ or silymarin for two weeks. At the end of the experiment, the animals were sacriced and brain and intestine samples were dissected for biochemical, molecular and histopathological examinations. The results showed that choline glycerophosphate, alone or combined with silymarin, ameliorated the adverse effects of radiation as revealed by the inhibition of oxidative stress, apoptotic and inammatory markers (MDA, Caspase 3, TNF alpha, IL-1β and NF-kB). However, TAC, anti-inammatory marker, IL-10 and IkBa mRNA were increased. This was also accompanied by a signicant increase in the Ach level, ChAT activity and α7 nAChR mRNA expression and a signicant decrease in the activity of AChE as compared with the corresponding values of the irradiated group. Moreover, a reduction in the tissue lesions were observed in brain and intestinal tissues. In conclusion, choline glycerophosphate and silymarin exhibited modulating effect against detrimental effects of gamma radiation via cholinergic anti-inammatory pathway.


Introduction
Radiotherapy is one of the commonly used modalities in the treatment of malignant tumors, but, it is associated with negative side effects on different organs of the body. Acute radiation can produce cellular damage in organs that having rapidly proliferating cells, such as the alimentary tract (Yu, 2013).
Additionally, the brain, with its high oxygen consumption and lipid-rich content, is highly susceptible to oxidative stress (Salim 2017). The cellular damage can be induced by direct or indirect effects of radiation. The direct effect is resulted from the interaction of radiation itself directly with the cellular molecules, however, the indirect effect can be caused by the interaction of radiation with cellular water to create free radicals and hydrogen peroxide. Due to their high reactivity, free radicals interact with the biological molecules, most importantly the DNA and may form additional free radicals .
Acute gastrointestinal injury may occurs shortly after the radiotherapy including nausea, vomiting, diarrhea and increased stool frequency. These symptoms may be related to oxidative stress and in ammation induced by ionizing radiation (Radwan & Karam, 2020). The enteric nervous system, a part of the peripheral nervous system that embedded within the gut wall and interconnected with the enteroendocrine and gastrointestinal immune system, and involved in the physiological functions of the gastrointestinal tract, has also been demonstrated to play a critical role in intestinal radiation injury (Moussa et al., 2016). In fact, the central nervous system cooperates with the immune system to regulate in ammation. Acetylcholine (ACh), an important neurotransmitter in the cholinergic system, is synthesized and released by cholinergic neurons, and exerts its effects on the central and peripheral nervous system through ACh receptors. Also, ACh is released by non-neuronal tissues where it is involved in controling various functions such as cell proliferation, survival and apoptosis (Zoli et al., 2018). Also, brain injury may occurs after head or whole body irradiation including morphological and functional changes as neuronal degeneration and neuroendocrine disturbance (El-Missiry et al., 2021, Abdel-Aziz et al., 2021).
Unfortunately, there are no safe and effective drugs to prevent the development of radiation damage after whole or partial body irradiation. Hence, there is an urgent need for safe agents to mitigate radiation injury in animal models. The ameliorating effect of antioxidants and anti-in ammatory agents have been hypothesized (Yahyapour et al., 2018).
Silymarin is a natural herbal product extracted from the seeds and fruits of Silybum marianum, commonly known as milk thistle, contains different avonolignans (silibinin, isosilibinin, silichristin and silidianin) and has been long used to treat liver diseases ( were investigated its protective effects against partial brain irradiation-induced cognitive decline and peripheral cytokine production. However, the modulating effect of GPC on the whole body irradiation induced brain and intestinal injury has, to the best of our knowledge, never been studied before. Therefore, the current study aimed to investigate the modulatory role of choline glycerophosphate as a single agent and as a co-treatment with silymarine on the brain and intestinal injuries in rats exposed to whole body irradiation as well as the mechanisms by which choline glycerophosphate and silymarin could provide their potential amelioration actions.

Chemicals
Choline glycerophosphate and silymarin were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). All other reagents used in this study were of analytical grade.

Animals
Adult male Wister rats weighing 180-220 g were used in this experiment, obtained from the animal house that belongs to the National Centre for Radiation Research and Technology (NCRRT), Cairo, Egypt. Rats were fed on a standard rodent diet and provided water ad libitum. The animals were maintained at 12 h light/dark cycle, at constant temperature (22 ± 2°C) and humidity (50 ± 5%). All experimental procedures were performed according to the international guidelines of animal handling and care of the National Institute of Health (NIH publication No. 85-23, 1996).

Radiation process
Whole-body gamma irradiation of the animals was performed at the NCCRT, Cairo, Egypt, using a Canadian Gamma Cell-40, (137Cs) irradiation unit. The rats were exposed to a single dose (7 Gy) with a dose rate of 0.38 Gy/ min, according to the Dosimeter Department in the NCRRT at the time of the experiment.

Experimental design
The rats were divided into eight groups, 6 rats each. Group 1 (control) animals of this group were kept as control. Group 2 (GPC) animals were injected intraperitoneally with GPC at a dose level of 150 mg/kg (based on Tayebati  with silymarin. Group 5 (Rad) rats were whole-body exposed to gamma radiation at a dose level of 7Gy. Group 6 (Rad + GPC) animals were exposed to 7 Gy gamma radiation and then received GPC 5 min after irradiation and continued for two week as group 2. Group 7 (Rad + Sil) animals were whole-body exposed to gamma radiation at a dose level of 7Gy and orally supplemented with silymarin daily for two weeks after irradiation. Group 8 (Rad + Sil + Cit) rats were exposed to 7 Gy gamma radiation and received Sil and GPC for two weeks after irradiation.
Animals were sacri ced 24 h after the last dose of GPC or silymarine or two weeks after irradiation. The brain and intestine were immediately excised. Parts of the brain and intestine were preserved frozen at -80°C until used for Real-time PCR analysis and another parts were homogenized in phosphate-buffered saline ( PBS) (1g tissue: 10 ml PBS), centrifuged at 5000 rpm for 15 minutes at 4°C, then the supernatant was collected and preserved frozen at -20°C until used for biochemical analyses. For histopathological examination parts of the brain and intestine were xed in 10% formalin.
The level of IL-1β and IL-10 were quanti ed in brain and intestine homogenates using speci c enzymelinked immunosorbent assay kits, Cat. No. MBS825017 and Cat.No: MBS034393, respectively (MyBioSource, Inc. USA) according to the manufacturer's instructions. Nuclear factor kappa B (NF-κB) and tumor Necrosis Factor-alpha (TNF-α) levels were measured using Rat NF-κB and TNF-α ELISA kits The relative expression of Caspase-3, IKBa and α7 nAChR was calculated according to Applied Bio system software using the comparative threshold cycle method. All values were normalized to the beta actin gene as an endogenous control (reference gene).

Histopathological processing
Samples of brain (one hemisphere) and intestine tissues were collected from all groups, sliced, and xed in 10% formalin solution. Para n blocks were prepared from those samples after a serial of dehydration, clearing and embedding. The para n-embedded material was prepared in 4-5 µm thick slices by microtome, mounted on microscope slides and stained with hematoxylin and eosin (Suvarna et al., 2013). Finally, it was examined under a light microscope to evaluate the histopathological changes.

Statistical analysis
Statistical analysis of the results was carried out using the SPSS computer program (version 20). All data were presented as mean values ± standard errors of the means. Statistical comparison between groups was done by using one-way analysis of variance (ANOVA) followed by a post hoc, LSD. Differences were considered signi cant at P < 0. 05.

Oxidative stress and apoptosis
In the brain, the results presented in Table 1 showed that supplementation of GPC to normal rats induced non-signi cant changes in the levels of TAC, MDA and casoase-3 mRNA compared to their normal control levels. However, administration of GPC and silymarin resulted in a signi cant (P < 0.05) elevation in the level of TAC together with a signi cant decrease in MDA level compared to their values of the control group. Whole body gamma irradiation (7 Gy) of rats has provoked oxidative stress in brain tissue, that has been demonstrated by a signi cant (P < 0.05) decrease in TAC together with a signi cant elevation in the level of MDA and the expression level of caspase-3 mRNA compared to their values of the normal control group. However, GPC or silymarin administration for two weeks post-irradiation induced a signi cant (P < 0.05) decrease in the levels of MDA & caspase-3 and a signi cant elevation in TAC compared to the corresponding values of the irradiated group. Also, the results revealed that the combined treatment of GPC and silymarin produced a better effect in reducing the oxidative stress and apoptosis (Table 1). In the intestine, the results presented in Table 2 showed that administration of GPC to normal rats induced signi cant (P < 0.05) increase in the level of TAC compared to their normal control levels. Moreover, the combined treatment of GPC and silymarin resulted in a signi cant (P < 0.05) elevation in the level of TAC together with a signi cant decrease in MDA level compared to their values of the control group. Whole body gamma irradiation (7 Gy) of rats has instigated oxidative stress in the intestine tissue, that has been demonstrated by a signi cant (P < 0.05) elevation in the level of MDA associated with a signi cant decrease in TAC compared to their values of the control group. Also, a signi cant increase in the expression level of caspase-3 mRNA was detected. However, GPC or silymarin administration for two weeks post-irradiation induced a signi cant (P < 0.05) decrease in the levels of MDA & caspase-3 mRNA and a signi cant elevation in TAC compared to the corresponding values of the irradiated group. Also, the results indicated that the combined treatment of GPC and silymarin produced a better effect in reducing the oxidative stress and apoptosis ( Table 2).

Cholinergic and in ammatory markers
In the brain, the results in Tables 3 & 5 showed non-signi cant changes in the studied cholinergic and in ammatory markers upon administration of GPC and/ or silymarin to normal rats compared to their normal control levels. Also, the results (Table 3) revealed that exposure to ionizing radiation resulted in a signi cant (P < 0.05) elevation in the levels of NF-κB and pro-in ammatory cytokines, TNF-α and IL-1β compared to their values of the control group. As well, a signi cant (P < 0.05) decrease in the level of IL-10 and the expression level of IkBa mRNA was observed two weeks after irradiation compared to their values of the control group. Administration of GPC and/or silymarin ameliorated the changes induced by exposure to radiation. The data presented in Table 5 indicated that whole body irradiation induced a signi cant (P < 0.05) decrease in the level of ACh & the activity of ChAT and the expression level of α7nAChR along with a signi cant increase in AChE activity compared to their values of the control group.
However, GPC and/ or silymarin administration ameliorated these changes. Table 3 Changes in brain tumor necrosis factor-α (TNFα), Interleukin 1 beta (IL-1β), Interleukin 10(IL-10), and nuclear factor kappa (NF-κB) levels and expression level of IkBa mRNA of adult male albino rats in different groups.  In the intestine, the results in Tables 4 & 6 showed non-signi cant changes in the studied cholinergic and in ammatory markers upon administration of GPC and/or silymarin to normal rats compared to their normal control levels. Exposure to ionizing radiation (Table 4) resulted in a signi cant (P < 0.05) elevation in the levels of NF-κB and pro-in ammatory cytokines, TNF-α and IL-1β compared to their values of the control group. As well, a signi cant (P < 0.05) decrease in the level of IL-10 and the expression level of IkBa mRNA was observed two weeks after irradiation compared to their values of the control group. Administration of GPC and/or silymarin ameliorated the changes induced by exposure to radiation. The data presented in Table 6 indicated that whole body irradiation induced a signi cant (P < 0.05) decrease in the Ach level & ChAT activity and the expression level of α7nAChR along with a signi cant increase in AChE activity compared to their values of the control group. However, GPC and/ or silymarin ameliorated these changes. Moreover, the combined treatment of GPC and silymarin showed a better modulating effect on the Ach level and its receptor as compared with each of them separately.

Histopathological examination
Brain of control rat's showed normal histological structure (Fig. 1&2). Also, in both GPC and silymarin groups, brain tissues showed normal structure as the control (Fig. 3&4). In whole body irradiated rats the neuronophagia and the degenerative changes of brain tissue are not sever and appeared in different parts of the brain especially cerebrum, in all investigated rats of this group and epitomized by numerous pyknotic neurons with proliferation of glial cells in gray matter (Fig. 5). The white matter of irradiated rats showed spongiform degeneration (Fig. 6) and increasing of glia cells with or without dilated blood vessels. In Rad + GPC group, gray matter of some rats showed little degenerated pyramids neurons ( Fig. 7), while it appeared normal in other rats. White matter of Radiation and silymarin group showed dilated blood vessel (Fig. 8) with microcavitation. Moreover, in the gray and white matter of rats treated with GPC and silymarin after irradiation, the majority of neurons, matrix, and nerve axons showed normal morphological structure (Fig. 9&10).
Intestine of control rat's showed normal histological structure (Fig. 11). Also, both GPC and silymarin groups are showed normal structure as control. Intestine of irradiated rats showed marked villous tips loss (erosions), mucosal layer necrosis and in ammatory cell invasion of submucosal layer (Fig. 12). The intestine of rats treated with GPC and radiation showed histologically signi cant improvement than that's of irradiated one, in some cases showed leuckocytic in ltration around degenerated Bruner's glands (Fig. 13). While in the group treated with silymarin and radiation the mucosa and submucosa showed still in ammation, edematous and dystrophic external muscular layer (Fig. 14). Intestine of rats treated by GPC and silymarin after irradiation showed more improvement than those treated by GPC or silymarin separately, it appeared without any erosions or necrosis, in some cases there were dilated intestinal blood vessels or expanded intestinal gland (Fig. 15 &16).

Discussion
Exposure to ionizing radiation initiates a series of molecular and biochemical signaling events that may repair the damage or induce cell phenotypic modi cations, depending on the dose of exposure and the sensitivity of exposed cells. The intestinal cells are among the most sensitive cells (Musa et al., 2019). The brain tissues are susceptible to oxidative damage due to its high oxygen utilization, lipid rich content and its low endogenous antioxidant content (Salim 2017). Previous studies showed that ionizing radiation enhanced reactive oxygen species (ROS) production, apoptosis and in ammation, and reduced Ach levels in brain and colon (Abdel-Aziz et al., 2021 and Song et al., 2020). Therefore, it was hypothesized that GPC -as a precursor of ACh-and silymarin -a strong natural antioxidant-might in uence the radiation-induced oxidative stress, apoptosis, in ammation and cholinergic system disturbance in brain and intestinal tissues. The authors set out to investigate the consequences of GPC and silymarin administration on the markers of the oxidative stress and the cholinergic -antiin ammatory pathway in rats exposed to whole-body gamma radiation.
The results of the present study showed that whole-body gamma irradiation (7 Gy) of male albino rats The excessive production of free radicals immediately after irradiation is considered as the rst proin ammatory signal in irradiated tissues (Moussa et al., 2016). Free radicals interact with biological targets causing DNA damage which initiate apoptotic and in ammatory responses, characterized by the production of apoptotic markers (caspases − 9 and − 3 ) and pro-in ammatory cytokines, such as TNF-α, IL-1β and IL6 (El-Maraghi et al., 2020, Radwan & Karam, 2020). Besides, NF-κB is activated by the oxidative stress induced after irradiation, which in turn targets the production of many genes related to in ammation (Ismail & El-Sonbaty, 2016). It is well known that inactive NF-kB is located in the cytosol and is bound to an inhibitory protein, IkBa. However, the induced oxidative stress results in IkBa phosphorylation, dissociation from NF-kB, ubiquitination, and subsequent degradation. Consequently, NF-kB translocates to the nucleus, where the transcription process of certain cytokines (like IL-1, IL-6, TNF-α) is up-regulated (Saha et al., 2020).
In the present study, gamma radiation decreased the level of IL-10 and the expression level of IκBa, however, it elevated the levels of TNF-α, IL-1β & NF-κB and the expression levels of caspase-3 indicating the role of in ammatory cytokines and apoptotic markers in the radiation-induced brain and intestinal injury. These results were con rmed by the degenerated intestinal mucosa with in ammatory cells in ltration and degenerative changes in brain tissue that was demonstrated in the current histopathological study.
Whole-body irradiation induced a signi cant decrease in the level of ACh, the important neurotransmitter in the cholinergic system, ChAT, and the expression level of α7nAChR associated with a signi cant increase in the AChE level in brain and intestine (Tables 5& 6) Since α7 nAChR is located on neuronal and non-neuronal cells such as immune cells, it was demonstrated that immune cells in the gut could be the target of ACh that act as an immune modulator (Brinkman et al., 2019). Indeed, the enteric nervous system is a large division of the peripheral nervous system embedded within the gut wall and regulates various physiological functions of the gastrointestinal tract such as mucus secretion, immunity, and in ammatory processes (Niesler et al., 2021). Evidence suggested that the enteric nervous system and its interactions with the immune system have a critical role in the early intestinal radiation response (Moussa et al., 2016).
From the above results, it is thought that the oxidative stress, and the consequent in ammation and cholinergic system disturbance are the probable pathogenic mechanisms in the radiation-induced brain and intestinal injury. Hence, stimulation of the cholinergic anti-in ammatory pathway plays an important role in controlling the in ammatory response which is mediated by increasing the release of ACh and activation of α7nAChR on the surface of macrophages. Particularly, ACh, the essential neurotransmitter in the vagus nerve, inhibits the production of pro-in ammatory cytokines through a mechanism dependent on the α7nAChR (Chen et al., 2020). Moreover, previously, it was described the anti-in ammatory role of inhibition of AchE, the activation of ChAT, and the promotion of Ach synthesis, may serve as a strategy for the treatment of whole body gamma irradiation-induced brain and intestinal injuries. The histopathological examination of the brain and intestinal tissues supported the biochemical results and con rmed the ameliorative effect GPC and silymarin against ionizing radiation.

Conclusion
According to the results obtained in this study, GPC -as a precursor of ACh-and silymarin -a strong natural antioxidant-exhibited modulating effect against detrimental effects of gamma radiation in rats via cholinergic anti-in ammatory pathway. This effect might be attributed to the activation of antioxidative, anti-apoptotic, and anti-in ammatory mechanisms. Therefore, GPC and silymarin might be suggested to serve as a strategy for the treatment of the negative side effects induced by the exposure to ionizing radiation. However, further studies are required to support these results before a clinical application can be recommended. Figure 1 Cerebrum gray matter of control rat showing normal structure (H &E x 400).

Figure 2
Page 21/31 Cerebrum white matter of control rat showing normal structure (H &E x 400).

Figure 3
Cerebrum gray matter of GPC treated rat showing normal structure (H &E x 400).

Figure 4
Page 22/31 Cerebrum white matter of silymarin treated rat showing normal structure (H &E x 400).    Cerebrum gray matter of rat treated with GPC, silymarin and radiation. The majority of neurons and matrix with the normal morphological picture (H &E x 400).