In recent years, the efficacy of radiotherapy in pelvic malignant tumours has been confirmed with the development of the concept of precision radiotherapy, the increasing technological sophistication of radiotherapy, and the constant updating and continuous development of radiotherapy-related equipment[24]. However, radiation inevitably irradiates the surrounding normal tissues, and the intestinal epithelial cells are highly sensitive and poorly tolerant to radiotherapy[25]. When the pelvic radiotherapy dose exceeds 5000 cGy, the probability of patients experiencing different degrees of acute radiation enteritis is significantly increased, and its incidence is significantly correlated with an increase in the radiotherapy dose[3]. The development of radiation enteritis is associated with disruption of the integrity of the intestinal mucosal barrier, which is divided into four main parts, and damage to any one of these barriers exacerbates the occurrence of intestinal radiation damage[26].
Through our previous work and network pharmacology analysis, we found that CKI may have an anti-radiation enteritis effect through targeting of cannabinoid receptor 1 (CB1 or CB1R).CB1 receptors can be found throughout the gastrointestinal tract, mainly in the enteric nervous-system (ENS)[27] and epithelial cells[28]. The ECS is a transmitter system that controls gut functions both peripherally and centrally. It is involved in controlling nausea, vomiting, and visceral sensation, and plays a homeostatic role in controlling intestinal inflammation[23]. CB1 can play a role in pain inhibition through brain-gut interactions to thereby modulate intestinal hypersensitivity. CB1 activation can reduce the intestinal sensitivity threshold[29]. In the ENS, the CB1 receptor is expressed in cholinergic neurons, and the activation of the CB1 receptor can inhibit the abnormal excitatory intestinal peristalsis response, suppress acetylcholine, and slow enterocolitis[30]. In our experiments, we found that CB1 expression was increased in the RT group and that CB1 was elevated in the inflamed gut, which may be a protective feedback mechanism against the inflammatory response, which has been similarly reported in the past[31]. Following the administration of CKI, CB1 expression was found to be decreased in rats compared with that in the radiotherapy model group. This effect was also observed following the administration of the CB1 agonist, while the administration of the CB1 inhibitor attenuated the effect of CKI.
Radiotherapy can induce a series of inflammatory responses, inflammatory cells and inflammatory molecules are involved in the inflammatory response, and the transcription of genes, e.g. MCP-1, encoding inflammatory molecules is mainly regulated mainly by nuclear transcription factors such as NF-kB, which plays a key role in the immune and inflammatory responses after exposure to radiation[32]. The P38MAPK pathway is an important intracellular signalling pathway, and radiation can activate the p38/MAPK and NF-κB signalling pathways inducing inflammatory cytokins production[33]. Alarifi[34] reported that radiotherapy can induce the apoptosis of intestinal endothelial cells, activate the P53/MAPK preapoptosis pathway in the body, and activate immune-related cells such as lymphocytes in peripheral blood, increase the active expression of related substances, and increase the expression levels of the cytokines IL-1β and TNF-α. The role of proinflammatory cytokines such as TNF-α, IL-1, IL-6 in perpetuating radiation-induced normal tissue damage is generally accepted through their ability to induce bursts of ROS through various mechanisms[35]. After intestinal epithelial cells are injured, PI3K pathway can be activated to inhibit the autophagy activity of intestinal cells, thus increasing the expression of proinflammatory factors. The results of our study showed that CKI can improve the morphology and structure of the rat ileum, reduce the infiltration of CD68 and CD16b, reduce the expression of inflammatory factors, inhibit the NF-κB pathway, alleviate intestinal damage and inhibit p-p38 MAPK/p38 MAPK and that agonists of CB1 can produce similar effects. After the addition of a CBI antagonist, CD68 and CD16b infiltration increased, the expression of inflammatory factors increased, the NF-κB pathway was activated, and the expression of p-p38 MAPK/p38 MAPK was increased.
The excess reactive oxygen species produced by radiation leads to intestinal oxidative stress, which has harmful effects on macromolecules such as DNA, lipids, and proteins within cells. Studies have shown that after irradiation, reactive oxygen species increase[36], the intestinal oxidative stress biomarker malondialdehyde(MDA) content increases, glutathione peroxidase (GSH-Px) content decreases, and superoxide dismutase (SOD) activity decreases[37]. Therefore, antioxidants are an important means to prevent and reduce the symptoms of radiation enteritis. SOD can effectively remove free radicals in the organisms, and prevent cells from being damaged by free radicals, and damaged cells can be repaired, via anti-radiation, anti-aging, immunity enhancement and blood lipids regulation. The primary role of GSH-Px is to scavenge lipid hydroperoxides and to substitute for catalase in the scavenging of H2O2 in tissues with very low levels of catalase or very low H2O2 production. A significant decrease in GSH-Px activity indicates an aberrant alteration of intracellular oxidative and antioxidant stabilization mechanisms such that there is an increase in the reactivity of oxygen radicals, which can exacerbate cellular damage[38]. We detected the oxidative stress damage in the ileum of rats by ELISA, and we found that the oxidative stress damage in the ileum of rats with radiation enteritis was obvious, and the oxidative stress damage in the ileum of rats was obviously improved after the treatment with CKI, which suggests that the CKI is able to ameliorate the oxidative stress damage in the intestinal tract caused by radiation enteritis. After increasing the concentration of the CBI antagonist, oxidative stress in the rats was aggravated.
In our study, the effects of CKI on radiation-induced inflammation and oxidative stress were predicted by network pharmacology and bioinformatics studies and validated in a rat model of radiation-induced enteritis. The results of this study suggested that CKI has the potential to be used as a complementary regimen for radiation enteritis. It should be noted that we used an agonist of CB1, or a CKI + CB1 antagonist, rather than a knock-up or knockdown of CB1 when performing a control, which should not substantially affect CB1 expression in nature. However, we still found that the CKI group was similar to the CB1 agonist group, and that the expression of CB1 was slightly decreased compared with that in the radiotherapy model group. Furthermore, the use of a CB1 inhibitor weakened the effect of CKI. Consequently, we postulated that a component of CKI might be analogous to CB1 agonists and elicit comparable anti-inflammatory responses to CB1, rather than directly influencing CB1 expression. However, due to financial constraints, we were unable to establish another CB1 knockdown or knockdown group, nor could we analyse which components of CKI may effectively bind to CB1, perform molecular docking and verification. This will be our future research direction.