Exposure to ionizing radiation initiates a series of molecular and biochemical signaling events that may repair the damage or induce cell phenotypic modifications, 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 inflammation, 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 influence the radiation-induced oxidative stress, apoptosis, inflammation 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 - anti-inflammatory 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 triggered oxidative stress indicated by a significant increase in MDA level as an index of lipid peroxidation and a significant decrease in total antioxidant capacity (TAC). This injurious effect of radiation is caused mainly by the over production of ROS which interact with cellular macromolecules producing harmful free radicals leading to lipid peroxidation in brain (El-Missiry et al., 2021), intestine (Musa et al., 2019) and liver (Hassan et al., 2021).
The excessive production of free radicals immediately after irradiation is considered as the first pro-inflammatory signal in irradiated tissues (Moussa et al., 2016). Free radicals interact with biological targets causing DNA damage which initiate apoptotic and inflammatory responses, characterized by the production of apoptotic markers (caspases − 9 and − 3 ) and pro-inflammatory 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 inflammation (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 inflammatory cytokines and apoptotic markers in the radiation-induced brain and intestinal injury. These results were confirmed by the degenerated intestinal mucosa with inflammatory cells infiltration and degenerative changes in brain tissue that was demonstrated in the current histopathological study.
Whole-body irradiation induced a significant decrease in the level of ACh, the important neurotransmitter in the cholinergic system, ChAT, and the expression level of α7nAChR associated with a significant increase in the AChE level in brain and intestine (Tables 5& 6). These results agree with that of Mansour et al. 2017 and El kiki & Galal 2018 who observed that whole-body irradiation (6Gy) induced a significant decrease in neurotransmitters and a significant increase in AChE activity in the rat brain. Erukainure et al., 2021 demonstrated that oxidative stress has been implicated in the elevation of AChE activity in testicular tissue. The significant decrease in ACh level observed in the current study may be attributed to the decrease in ChAT, the enzyme responsible for ACh synthesis, and the increase in AChE, the enzyme that hydrolyzes ACh. This disturbance may terminate the interaction between neurotransmitter, ACh and the corresponding receptor protein, α7 nAChR (Pal et al., 2017).
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 inflammatory 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 inflammation and cholinergic system disturbance are the probable pathogenic mechanisms in the radiation-induced brain and intestinal injury. Hence, stimulation of the cholinergic anti-inflammatory pathway plays an important role in controlling the inflammatory 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-inflammatory cytokines through a mechanism dependent on the α7nAChR (Chen et al., 2020). Moreover, previously, it was described the anti-inflammatory role of ACh in the modulation of the inflammatory response through inhibition of AChE (Rosas-Ballina and Tracey 2009).
GPC and / or silymarin administration after whole-body irradiation helped to minimize the oxidative stress, the results indicated by the significant increase of TAC and the significant decrease of MDA levels (Tables 1& 2) in the brain and intestinal tissues as compared to the corresponding values of the irradiated group. These results are in agreement with the previous studies which indicated that silymarin attenuated the oxidative stress in the brain of rats intoxicated with AlCl3 (Aboelwafa et al., 2020) and inhibited the toxic effect of microcystin-LR on mice by increasing the reduced glutathione which may reduce reduced lipid peroxidation and protein carbonyl content in the spleen and intestine (Al-hazmi et al.2019). Due to the presence of phenolic hydroxyl groups in its structure, silymarin could attenuate the oxidative stress through scavenging free radicals, inhibiting the free radical formation and preserving the cellular antioxidant status, thus preventing peroxidation of membrane lipids (Zalat et al., 2021). Likewise, GPC administration reduced the reactive oxygen and nitrogen species production in rodent models of intestine and liver ischemia-reperfusion injuries (Tokes et al., 2015 and Strifler et al., 2016). The current observation of the reduction of caspase-3 mRNA expression by GPC and/ or silymarin treatment after irradiation indicates their role as anti-apoptotic agents. This effect may be related to their ability to reduce ROS and MDA production and maintain the structural integrity of the biological membranes.
Moreover, GPC and/ or silymarin administration after whole-body irradiation modulated the inflammation and the enzymes responsible for ACh synthesis and hydrolysis, and increased the level of ACh and the expression level of its receptor, α7 nAChR, as compared to the corresponding values of the irradiated group (Tables 3–6). The anti-inflammatory effect of these agents may takes place via the inhibition of NF-κB signaling pathway and increase the anti-inflammatory cytokine, IL-10. Recently, it was described that dietary choline supplement improved the immune function and restrained the NF-κB signaling pathway by up-regulating the expression level of IkBα and down-regulating NF-κBp65, and NF-κBp52 expression as well as decreasing the mRNA level of pro-inflammatory cytokines, TNF-α, IL-1β, and IL-6, and increasing the mRNA abundance of anti-inflammatory cytokines, IL-10 (Yuan et al., 2021). Consequently, the pro-inflammatory cytokine production and the occurrence of inflammation are inhibited. Regarding the cholinergic anti-inflammatory pathway, GPC administration increased the release of ACh that inhibited macrophage likely by activation of α7 nAChR expressed on the cell surface of the macrophage (Kimura et al., 2019). Additionally, GPC protected against cognitive decline, cellular damage (Planga´r et al., 2014) and peripheral cytokine production (T ő k é s et al., 2014) induced after partial brain irradiation. Moreover, it has been reported that silymarin can resist the inflammatory response of the nervous system and can also increase the ACh content by inhibiting cholinesterase activity (Guo et al., 2019, Aboelwafa et al., 2020). Thus, modulation of the cholinergic signaling pathway, including the 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 confirmed the ameliorative effect GPC and silymarin against ionizing radiation.