Compound Kushen Injection improves radiation enteritis via cannabinoid receptor 1 in rats

Compound Kushen Injection improves radiation enteritis via cannabinoid receptor 1 in rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Compound Kushen Injection improves radiation enteritis via cannabinoid receptor 1 in rats Wenjing Xu, Liping Gao, Wenjuan Zou, Xiaohui Tang, Weiqi Nian, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4513715/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Background: Clinical studies have shown that Compound Kushen Injection (CKI) can alleviate the inflammatory symptoms of radiation enteritis. However, the mechanism of action remains unclear. The aim of this study was to explore the possible targets and mechanisms of CKI in the treatment of radiation enteritis. Methods: Network pharmacology was used to predict the potential targets of CKI for the treatment of radiation enteritis, and GO and KEGG enrichment analyses were subsequently performed. The therapeutic effects and signalling pathways were then verified in rats. The expression of inflammatory factors in ileal tissue was measured by qRT-PCR. The activities of SOD and GSH-Px in ileal tissue were measured by ELISA. The levels of MDA, ROS and NO were determined using biochemical kits. The expression of signalling pathway-related proteins was detected by Western blotting and immunofluorescence. Results: According to network pharmacology, CB1 might be a target of CKI. GO and KEGG enrichment analyses revealed that CKI was significantly enriched in analgesic, endocannabinoid and inflammatory pathways. In the rat model, Compared with that in the radiotherapy group,the extent of ileal injury was significantly improved in the CKI group compared to the control group. In addition, the infiltration of CD68 and CD16b was significantly reduced, and the expression of MCP1, TNF-α, IL-1β and IL-10 was significantly decreased. In addition, the activities of SOD and GSH-Px were increased, and the activities of MDA, ROS and NO were decreased. The CKI group also showed inhibition of NF-κB signalling and a significant decrease in the expression of NOX4, CB1 and p-p38 MAPK/p38 MAPK. The use of a CB1 agonist could also alleviate radiation enteritis, whereas the addition of a CB1 antagonist could interfere with the ameliorative effect of CKI on radiation enteritis. Conclusions: CKI might exert an anti-radiation enteritis effect by targeting the cannabinoid receptor 1. network pharmacology Compound Kushen Injection radiation enteritis cannabinoid receptor 1 inflammatory pathways oxidative stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Background The incidence of rectal malignant tumors and cervical malignant tumors is increasing annually in China, jeopardizing the health of the population [ 1 ] . Radiation can block and disrupt the proliferation process of tumor cells in the irradiated area directly or indirectly and has become one of the main means of treating malignant tumors [ 2 ] . However, acute radiation enteritis is the most common complication of pelvic external radiation therapy, with patients presenting with nausea and vomiting, abdominal pain, diarrhea, increased mucus, and blood in the stool [ 3 ] . Studies have shown that approximately 90% of pelvic radiotherapy patients experience changes in bowel habits, and the incidence of acute radiation enteritis ranges from 50–70% [ 4 ] . Acute radiation enteritis brings discomfort and pain to patients, causing interruption of radiotherapy and affecting the therapeutic effecacy. If it is not treated in time, it may develop into chronic radiation enteritis with intestinal fibrosis, even intestinal perforation and intestinal obstruction, which will affect the quality of life and prognosis of patients [ 5 ] . The current treatment for acute radiation enteritis is based on protecting of intestinal mucosa, and preventing diarrhea ,inflammatory and infection, which has a long course of treatment, and some patients experience poor therapeutic effects [ 6 ] . Therefore, effective prevention of acute radiation enteritis can reduce the symptoms of abdominal pain and diarrhea caused by radiation enteritis and improve patients' quality of life. In addition, it can also improve patient compliance with radiotherapy and ensure that radiotherapy is completed on schedule, which is highly significant for the treatment and prognosis of patients with pelvic malignant tumors [ 7 ] . The formation of radiation enteritis is a complex multi-factor process, that is related to intestinal epithelial cell damage, intestinal wall blood vessel damage, intestinal flora displacement, and inflammatory factor release [ 8 – 10 ] . However, there is no standardized treatment for ARE, and although modern medical treatment can achieve certain effects in clinical practice, the overall effect is still unsatisfactory. Traditional Chinese medicine, as one of China's traditional medical treasures, has unique advantages in the treatment of gastrointestinal diseases. In recent years, there has been an increasing focus on the use of herbal medicine as a natural and low-toxicity treatment for radiation enteritis.Traditional Chinese herbs, such as Polygonati Rhizoma, Achyranthis Bidentatae Radix, and Epimedii Folium, have been found to have anti-radiation and immune regulation properties. An herbal diet containing these herbs showed a significant radiation-protective effect on intestinal crypts at doses of 4 Gy and 8 Gy. At a dose of 8 Gy, the Chinese herbal diet was found to reduce the loss of inhibitory nNOS neurons in the intestine, which can relieve symptoms of hyperperistalsis and diarrhea in patients after radiotherapy [ 11 ] . A phase II trial was conducted to investigate the efficacy of the herbal medicine TJ-14 in treating acute radiation enteritis across multiple institutions. The study found that TJ-14 was effective in treating ARE in 86% of patients and had the advantages of low toxicity and medical costs [ 12 ] . In 200 AD, Radix Sophorae Flavescentis began to be used to treat tumors, inflammation and other diseases. CKI is a preparation of pure Chinese medicine that consists of Radix Kushen and Rhizoma Smilacis Glabrae and has been clinically remediated in China for more than 20 years. It contains at least eight different anti-inflammatory components, including matrine, oxymatrine, sophorine, oxysophorine, et al [ 13 – 15 ] . CKI has anticancer and analgesic functions and can improve immunity, hemostasis and blood vessel dilation [ 16 – 17 ] . A study found that CKI can reduce radiation side effects and can reduce the severe toxicity and symptom burden associated with radiotherapy in patients with lung cancer [ 18 ] . In clinical practice, CKI has been used as a supportive treatment for colorectal cancer, and it has been found to relieve pain, bleeding and other inflammatory reactions during radiotherapy [ 19 ] . In addition, Harata-Lee Y et al , reported that Compound Kushen injection could reduce the severity of radiation-induced gastrointestinal mucositis in rats [ 20 ] . Our previous study found that CKI can effectively treat acute hemorrhagic severe radiation enteritis in rats. This study revealed in reduced inflammation and bleeding, as well as decreased mortality rates [ 21 ] .Network pharmacology involves multiple genes and multiple targets, which aligns with the complexity of traditional Chinese medicine (TCM) treatment. However, the mechanism of action of this drug on acute radiation enteritis has not been reported. The continuous development of systems biology in TCM research has made network pharmacology a popular analytical method in the field. Our study employed network pharmacology and animal experiments to elucidate the mechanism and potential targets of CKI in treating radiation enteritis. 2. Methods 2.1 Network Pharmacology Analysis To identify potential targets for CKI treatment of radiation enteritis, we conducted a network pharmacology analysis. We queried the GeneCard database using the keywords "radiation enteritis", "inflammation" and"enteritis", and screened the genes with relevance score of at least 1. We took the intersection of these three sets. Next, we screened for possible gene targets of the two CKI components, Radix Kushen and Rhizoma Smilacis Glabrae, using BATMAN-TCM network pharmacology. BATMAN-TCM is an enhanced integrative database designed to store known and predicted linkages between TCM (traditional Chinese medicine) ingredients and target proteins [ 22 ] . BATMAN-TCM contained 17,068 known and 2,339,061 high-confidence predicted TTIs, together with 54,832 formula, 8,404 herbs, and 39,171 ingredients. The pinyin names of "KU SHEN" and "BAI TU LING" (score cut-off = 18, adjusted P-value < 0.05) were used to identify potential targets and combined the two components. The findings were then intersected with the genes screened in the Genecards database to predict the hotspot genes related to the regulation of "radiation enteritis" by CKI. BATMAN-TCM was used to predict potential targets for each queried TCM’s ingredient, and then perform functional analyses of these targets, including Gene Ontology (GO) term, KEGG pathway and OMIM/TTD disease enrichment analyses, were performed. According to the filtering criteria, P ≤ 0.05 and Q value ≤ 0.05, only the top 10 of each enrichment were shown for each respectively. A TCM ingredient-target-pathway/disease association network and biological pathways with highlighted TCM targets would also be shown. Enrichment analysis and mapping of GO, KEGG pathway, OMIM and TTD disease data were performed using R software(version 4.3.2). 2.2 Animal Forty healthy 6-week-old male SPF-grade SD rats, weighing between 150 g and 200 g, were purchased from Chengdu Dashuo Laboratory Animal Co. Ltd. (SCXK (Chuan) 2019-031). All animals were acclimatized for one week at a temperature of 22 ± 2°C and a relative humidity of 55 ± 5%, with free access to food and water during the test period. After one week, the rats were randomly divided into five groups: the control group (n = 8), model group (n = 8), CB1 agonist group (n = 8), CKI group (n = 8), and CKI + CB1 antagonist group (n = 8). 2.3 Establishment of a model of radiation enteritis The rats were fasted for 12–14 h, except those in the control group, which were subjected to abdominal anesthesia with 10% pentobarbital sodium (0.4 ml/100 g) and fixed on a foam board in the supine position.The whole abdomen (from the xiphoid process to the pubic symphysis) was irradiated by a 6MV medical high-energy X-ray linear accelerator with an irradiation area of approximately 7 cm×6 cm, and the rest of the area was shielded by a 5 cm-thick lead plate with a 100 cm distance from the source skin, and the radiation dose rate was 200 cGy/min. for a total irradiation dose of 10 Gy. 2.4 Animal modelling and grouping The control group received a daily intraperitoneal injection of an equal amount of saline for 7 d. The radiation model group received a daily intraperitoneal injection of an equal amount of saline for 7 d. The CB1 agonist group received a daily intraperitoneal injection of 2 mg/kg of the CB1 agonist WIN55212-2, for 7 d.The CKI group received a daily intraperitoneal injection of 0.1 ml/100 g of CKI for 7 d.The CKI + CB1 antagonist group was given 0.1 ml/100 g of CKI and 1 mg/kg of the CB1 antagonist AM251 intraperitoneal injection every day for 7 d after radiological modelling. 2.5 Determination of the intestinal propulsion rate The rats were fasted on the day of the end of treatment, and 1 mL of carbon powder suspension was given to the rats in the morning of the second day. The rats were sacrificed after 6 h, the intestines were dissected, the position of the carbon powder in the intestines was observed, and the propulsion time of the carbon powder marker per unit of time in the intestines was measured to obtain the intestinal propulsion rate. 2.6 H&E staining Immediately after sacrifice, the rats were dissected to remove the ileum (from the anus to the cecum, more than 2 cm from the anus), and the intestinal contents were flushed out with saline, and stored in saline for further use. Approximately 1 cm sections of colonic tissue were removed, fixed in 10% paraformaldehyde solutions overnight, immersed in a series of ethanol solutions for dehydration, embedded in paraffin, cut into 3 µm thick pathological sections, laid flat on slides, dewaxed and hydrated after baking, stained with hematoxylin for 5 min, stained with eosin for 2 min, made transparent by xylene, and sealed with neutral gum, and then placed under a light microscope for observation of the pathological conditions of the ileum. 2.7 Immunohistochemistry The rat ileum was removed, fully with 4% paraformaldehyde, routinely dehydrated and sectioned by paraffin embedding. Immunohistochemical staining. Citrate buffer (pH 6.0) was used for antigen retrieval, and 3% hydrogen peroxide was used to eliminate endogenous peroxidase activity. Primary antibodies (anti-CD68, ab955, Abcam, UK, Biomedical Technology Co.., Ltd, 1:100; anti-CD16b, ab89207, Abcam, UK, Biomedical Technology Co.., Ltd, 1:100) were incubated at 4°C overnight. PBS was used instead of primary antibody as a blank control. HRP-labelled goat anti-rabbit IgG (GB23303, Servicebio, Wuhan, 1:100) was used as the secondary antibody, and the sections were incubated for 30 min. After DAB color development, 5 fields of view were randomly selected for each section, and the proportion of positive cells in each image was calculated using the Halo data analysis system. 2.8 qRT-PCR Total RNA was extracted from homogenates of intestinal mucosa using TRIzol reagent. cDNA was used as a template for quantitative reactions after reverse transcription of RNA to cDNA. The relative expression of MCP1, TNF-α, IL-1β and IL-10 mRNA was calculated by the 2 −ΔΔCt method using GAPDH as an internal reference. The primer sequences are shown in Table 1 . Table 1 primer sequences for qRT-PCR Forward Reverse IL-1β aat ctc aca gca gca tct cga caa g tcc acg ggc aag aca tag gta gc TNIFα cac cac gct ctt ctg tct act gaa c tgg gct acg ggc ttg tca ctc IL-10 ggc agt gga gca ggt gaa gaa tg tgt cac gta ggc ttc tat gca gtt g MCP-1 ctc acc tgc tgc tac tca ttc act g ctt ctt tgg gac acc tgc tgc tg 2.9 Western blot The tissue was lysed with RIPA buffer (P0013, Beyotime, China). The protein concentration was quantitatively analysed with a BCA kit (P0009, Beyotime, China). Protein samples were boiled in sodium dodecyl sulfate (SDS) loading buffer for 5 min, subjected to SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes (PVDF, ISEQ00010, Sigmaaldrich, UK). The membrane was blocked with Tris-Buffered saline with Tween (TBST) containing 5% buttermilk for 1 hour. It was then combined with IκBα(1:1000), p-Iκbα (1:1000), NOX4(1:2000), CB1(1:1000), p38 MAPK(1:1000), p-p38 MAPK(1:1000),and GAPDH(1:500), diluted with 5% bovine serum albumin (BSA) in TBST, then incubated with goat anti-rabbit IgG (ab150077, 1:1000, Abcam, UK) at room temperature for 1 hour. After incubation with the second antibody, imaging was performed using an ECL Plus HRP substrate kit (K22030, abbkine, USA) on an iBright CL750 instrument (A44116, Thermo Fisher, USA). 2.10 ELISA for SOD, GSH-Px, MDA, ROS and NO levels in rat ileal tissue The tissues were added to PBS at a mass-to-volume ratio of 1:9, homogenized, and centrifuged for 10 min at 5000×g. the supernatant was taken to be measured the content or activity of SOD (ZC-36451, ZCI BIO, Shanghai China), GSH-Px (ZC-52475, ZCI BIO, Shanghai China), MDA (ZC-55718, ZCI BIO, Shanghai China), ROS (ZC-36727, ZCI BIO, Shanghai China) and NO (ZC-37503, ZCI BIO, Shanghai China) in the supernatant was measured according to the instructions. 2.11 Immunofluorescence The sections were immersed in a 5% film breaker for 10 min at room temperature. After washing with PBS, goat serum blocking solution was added, and the sections were blocked for 20 min at room temperature. Primary antibodies (γ-H2ax, ab2893; RAD51, ab133534; Abcam, UK) were added and the sections were incubated at 4°C overnight. After rinsing with PBS again, the secondary antibody (ProLong™ Diamond, GB22303; CY3-labelled goat anti-rabbit antibody, GB21303; Servicebio, Wuhan) was added and incubated at 37 ℃ for 30 minutes. After rinsing with PBS, DAPI was added, and the cells were incubated at room temperature. After a final rinse with PBS, the sections were sealed with an antifluorescent attenuation sealer. Images were acquired using a camera system (OlyVIA, OLYMPUS, Japan). The fluorescence intensity and area of all the images were measured using the Image-J image analysis system, and the fluorescence intensity of each image was calculated. 2.12 Statistical analysis The quantitative data of this study were expressed as the mean ± standard deviation. Differences among multiple groups were statistically compared by one-way ANOVA and least significant difference (LSD) post hoc analysis, and the differences between the two groups were statistically compared by unpaired t -test. P < 0.05 was considered to indicate statistical significance. 3. Results 3.1 Network pharmacology and bioinformatics analysis As shown in Fig. 1 A, the GeneCard database was searched using the keywords 'radiation enteritis', 'inflammation', and 'enteritis'. There were 2871, 2987, and 5789 genes with relevance scores of at least 1, respectively. A total of 899 genes related to radiation enteritis were obtained from the intersection of the three datasets. Pharmacological screening of the BATMAN-TCM network predicted 971 genes as possible targets of CKI. These genes were then intersected with those screened in the previous Genecards database. In total, 41 potential targets were predicted to be involved in the regulation of radiation enteritis by CKI. As shown in Fig. 1 B, the network relationships of CKI prediction targets, continuous blood drug concentrations and therapeutic target databases were analysed using the traditional Chinese medicine database system pharmacology and analysis platform ( http://tcmspw.com/tcmsp.php , version 2.3). The 25 most strongly correlated medicinal chemical components and 6 most strongly correlated gene targets were screened through a visual network structure. Radiation enteritis is significantly associated with CB1 (CNR1), which is predicted to interact with 17 of the 25 major medicinal chemicals of CKI, such as kushenin, kushenol, and Kurarinone. As shown in Fig. 1 C, GO enrichment analysis was used to predict the major functions of the two herbs from three aspects: biological process (BP), molecular function (MF) and cellular component (CC). KEGG enrichment analysis was used to identify the major signalling pathways of the two herbs of CKI, and OMIM/TTD disease enrichment analysis was used to determine the correlation between drugs and diseases. And GO enrichment found that cell-cell signalling, transmembrane transporter activity and oxidoreductase activity were significantly related. KEGG enrichment revealed that these genes were significantly related to neuroactive ligand-receptor interactions, the cannabinoid receptor pathway and the calcium signalling pathway.According to the OMIM/TTD disease enrichment analysis, drugs were significantly associated with analgesics, pain, psychiatric/neurological disorders and Maple Syrup Urine Disease. Our enrichment analysis suggested that CKI may play a therapeutic role in radiation enteritis based on pain control and regulation of redox and endogenous cannabinoid receptor pathways Based on the literature, we investigated the role of the endocannabinoid system in the brain-gut axis [ 23 ] . Thereafter, we further studied the role of CB1 in radiation enteritis. 3.2 HE staining was used to observe the tissue changes in the rat ileum . As shown in Fig. 2 A, compared with those in the control group, the structure of the ileal mucosal layer in the model group was damaged, the intestinal villi were atrophied and exfoliated, and mucosal inflammatory cells infiltrated in model group. Compared that in the model group, intestinal villus atrophy was alleviated in the CB1 agonist group, the CKI group and the CKI + CB1 antagonist group, and a few inflammatory cells infiltrated the mucous membrane. Compared with those in the CKI group, some of the intestinal villi in the CKI + CB1 antagonist group were exfoliated, and the mucosal intestinal solidifying glands were slightly atrophic. 3.3 Immunohistochemical staining was performed to observe the infiltration of CD68 and CD16b in the rat ileum. We detected the degree of CD68 and CD16b infiltration in rat ileal tissues by immunohistochemical staining, as shown in Fig. 2 B. The results showed that the expression of CD68 and CD16b in the model group was significantly greater than that in the control group ( P < 0.001). Compared with that in the ileum of the model group, the expression of CD68 in the ileum of the three groups decreased significantly ( P < 0.05). The expression of CD16b in ileum of rats in the CB1 agonist group and the CKI group decreased significantly after treatment ( P < 0.01), and the expression of CD16b in ileum in the CKI + CB1 antagonist group decreased significantly after treatment( P < 0.05). Compared with that in the CKI group, the expression of CD16b in the CKI + CB1 antagonist group increased significantly ( P < 0.05). 3.4 PCR detection of inflammatory cytokines in rat ileum. We detected the expression of inflammatory factors in rat ileum by qRT-PCR, as shown in Fig. 3 A. The expressions of MCP1, TNF- α, IL-1β and IL-10 in the ileum of model group were significantly greater than those in the control group( P < 0.01). Compared with model group, the expression of MCP1, TNF- α, IL-1β and IL-10 in ileum of CB1 agonist group and CKI group decreased significantly( P < 0.05), and only the expression of TNF- α in the ileum of the CKI + CB1 antagonist group decreased significantly(P < 0.05). 3.5 WB detection of NF-κB signaling activation in the rat ileum. We detected the key proteins in the NF-κB pathway by WB. As shown in Fig. 3 B, the p-IκBα/IκBα ratio in the ileum of the model group was significantly greater than that in the ileum of the control group ( P < 0.01). Compared with that in the model group, the p-IκBα/IκBα ratio decreased in the other three groups. Compared with that in the CKI group, the p-IκBα/β-IκBα ratio was elevated in the CKI + CB1 antagonist group. 3.6 ELISA for SOD, GSH-Px, MDA, ROS and NO expression in the rat ileum. The activity of SOD, GSH-Px, MDA, ROS and NO in the rat ileum was detected by ELISA, as shown in Fig. 4 A. Compared with those in the control group, the levels of SOD and GSH-Px in the ileum of the model group were significantly lower ( P < 0.001), and the levels of MDA, ROS and NO were significantly greater ( P < 0.001). Compared with those in the model group, the levels of SOD and GSH-Px in the ileum of the other three groups were significantly greater ( P < 0.01), and the levels of MDA, ROS and NO were significantly lower ( P < 0.001). Compared with those in the CKI group, the levels of SOD and GSH-Px in the ileum of the CKI + CB1 antagonist group were significantly lower ( P < 0.001), and the levels of MDA, ROS and NO were significantly greater ( P < 0.001). 3.7 WB detection of NOX4 expression in the rat ileum. We detected the expression of NOX4 in the rat ileum by WB. As shown in Fig. 4 B, the results showed that the expression of NOX4 in the ileum in model group was significantly greater than that in control group ( P < 0.01). Compared with that in the ileum of the model group, the expression of NOX4 in the ileum of the CB1 agonist group and CKI group decreased significantly ( P < 0.01). 3.8 WB detection of CB1 and p-p38 MAPK/ p38 MAPK expression in the rat ileum. We detected the expression of ileal CB1, p38 MAPK and p-p38 MAPK in rats by WB. As shown in Fig. 5 , the results showed that the expression of CB1 and p-p38 MAPK/p38 MAPK in the ileum of the model group was significantly greater than that of the control group( P < 0.01). Compared with those in the model group, the expression levels of CB1 and p-p38 MAPK/p38 MAPK in the ileum of the CB1 agonist group and CKI group were significantly lower ( P < 0.01). Compared that in the CKI group, the ratio of p-p38 MAPK/p38 MAPK ratio was significantly greater in the CKI + CB1 antagonist group ( P < 0.05). 3.9 Immunofluorescence costaining to observe the coexpression of CB1 and p-p38 MAPK in the rat ileum. As shown in Fig. 6 , we performed double immunofluorescence staining for CB1 and p-p38 MAPK in the ileum of the rats. Compared with those in the control group, the fluorescence intensities of CB1 + p-p38 MAPK, CB1 and p-p38 MAPK were significantly increased and the fluorescence signal intensity was enhanced in the model group ( P < 0.001), while the fluorescence intensities of CB1 and p-p38 MAPK decreased significantly in the CB1 agonist group, CKI group and CKI + CB1 antagonist group than in the the model group ( P < 0.001). Compared with that in the CKI group, the fluorescence of CB1 + p-p38 MAPK and p-p38 MAPK in the CKI + CB1 antagonist group increased significantly, and the fluorescence signal increased ( P < 0.05). 4. Discussion 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 H 2 O 2 in tissues with very low levels of catalase or very low H 2 O 2 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. 5. Conclusion CKI can improve radiation-induced intestinal injury in rats, possibly by regulating CB1 to improve radiation-induced inflammatory responses, inhibiting p38 MAPK signalling, and simultaneously reducing oxidative stress. Declarations Ethics approval and consent to participate: All the experiments on animals were performed with the approval of the Experimental Animal Ethics Committee of Chongqing Hospital of Traditional Chinese Medicine（2022-DWSY-XWJ）. Consent for publication: Not applicable. Availability of data and materials: The initial data used to support the findings of this study are available from the corresponding author upon request. The Data from this study have not been published elsewhere. Competing interests: The authors declare that they have no competing interests. Funding: This work was sponsored by Natural Science Foundation of Chongqing, China（General Program）Project Number: cstc2021jcyj-msxmX0729 and Chongqing Hospital of Traditional Chinese Medicine Youth Top talent project. Authors' contributions: Wenjing Xu: Animal handling and article writing. Liping Gao: Animal feeding and handling. Wenjuan Zou: Drug injection and some molecular biology experiments. Xiaohui Tang: Some molecular biology experiments. Weiqi Nian: Fund reimbursement，statistical analysis and progress supervision. Weiqin Zheng: Theoretical guidance of Traditional Chinese Medicine. Pei Wang: Some molecular biology experiments and article polishing and work design. Rongzhong Huang: Bioinformatics analysis, final revision and progress supervision. Acknowledgments: Not applicable. References Han B, Zheng R, Zeng H, Wang S, Sun K, Chen R, et al. Cancer incidence and mortality in China, 2022. J Natl Cancer Cent. 2024;4:47–53. Mihaescu A, Santen S, Jeppsson B, Thorlacius H. P38 mitogen-activated protein kinase signalling regulates vascular inflammation and epithelial barrier dysfunction in an experimental model of radiation-induced colitis. Br J Surg. 2010;97:226–34. Hale MF. Radiation enteritis: from diagnosis to management. Curr Opin Gastroenterol. 2020;36:208–14. Loge L, Florescu C, Alves A, Menahem B. Radiation enteritis: diagnostic and therapeutic issues. J Visc Surg. 2020;157:475–85. Tabaja L, Sidani SM. Management of radiation proctitis. Dig Dis Sci. 2018;63:2180–8. Simpson DR, Song WY, Moiseenko V, Rose BS, Yashar CM, Mundt AJ, et al. Normal tissue complication probability analysis of acute gastrointestinal toxicity in cervical cancer patients undergoing intensity modulated radiation therapy and concurrent cisplatin. Int J Radiat Oncol Biol Phys. 2012;83:e81–6. Kasibhatla M, Clough RW, Montana GS, Oleson JR, Light K, Steffey BA, et al. 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Il-1beta signaling in cat lower esophageal sphincter circular muscle. Am J physiology: Gastrointest liver Physiol. 2006;291:G672–80. Sato K, Ninomiya H, Ohkura S, Ozaki H, Nasu T. Impairment of par-2-mediated relaxation system in colonic smooth muscle after intestinal inflammation. Br J Pharmacol. 2006;148:200–7. Song NN, Lu HL, Lu C, Tong L, Huang SQ, Huang X et al. Diabetes-induced colonic slow transit mediated by the up‐regulation of pdgfrα + cells/sk3 in streptozotocin‐induced diabetic mice. Neurogastroenterol Motil 2018;30: n/a-n/a . Lu C, Huang X, Lu H, Liu S, Zang J, Li Y, et al. Different distributions of interstitial cells of cajal and platelet-derived growth factor receptor-α positive cells in colonic smooth muscle cell/interstitial cell of cajal/platelet-derived growth factor receptor-α positive cell syncytium in mice. World J gastroenterology: WJG. 2018;24:4989–5004. Gyires K, Zádori ZS. Role of cannabinoids in gastrointestinal mucosal defense and inflammation. Curr Neuropharmacol. 2016;14:935–51. Freter RR, Alberta JA, Hwang GY, Wrentmore AL, Stiles CD. Platelet-derived growth factor induction of the immediate-early gene mcp-1 is mediated by nf-κb and a 90-kda phosphoprotein coactivator. J Biol Chem. 1996;271:17417–24. Song YH, Chai Q, Wang NL, Yang FF, Wang GH, Hu JY. X-rays induced il-8 production in lung cancer cells via p38/mapk and nf-kappab pathway. Int J Radiat Biol. 2020;96:1374–81. Demers M, Dagnault A, Desjardins J. A randomized double-blind controlled trial: impact of probiotics on diarrhea in patients treated with pelvic radiation. Clin Nutr. 2014;33:761–7. Schaue D, Kachikwu EL, Mcbride WH. Cytokines in radiobiological responses: a review. Radiat Res. 2012;178:505–23. Kunwar A, Bag PP, Chattopadhyay S, Jain VK, Priyadarsini KI. Anti-apoptotic, anti-inflammatory, and immunomodulatory activities of 3,3'-diselenodipropionic acid in mice exposed to whole body gamma-radiation. Arch Toxicol. 2011;85:1395–405. Radwan RR, Karam HM. Resveratrol attenuates intestinal injury in irradiated rats via pi3k/akt/mtor signaling pathway. Environ Toxicol. 2020;35:223–30. Buldak RJ, Buldak L, Kukla M, Gabriel A, Zwirska-Korczala K. Significance of selected antioxidant enzymes in cancer cell progression. Pol J Pathol. 2014;65:167–75. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editor assigned by journal 10 Jun, 2024 Submission checks completed at journal 08 Jun, 2024 First submitted to journal 01 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4513715","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":312546987,"identity":"d6562c16-2710-4612-b999-fd9e41f722b1","order_by":0,"name":"Wenjing Xu","email":"","orcid":"","institution":"The Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenjing","middleName":"","lastName":"Xu","suffix":""},{"id":312546988,"identity":"2deb238c-9526-4cfb-850d-47a053f2d3dd","order_by":1,"name":"Liping Gao","email":"","orcid":"","institution":"Chongqing Traditional Chinese Medicine Hospital","correspondingAuthor":false,"prefix":"","firstName":"Liping","middleName":"","lastName":"Gao","suffix":""},{"id":312546990,"identity":"ba85251e-d825-49fa-9bbc-c0e10b9e825a","order_by":2,"name":"Wenjuan Zou","email":"","orcid":"","institution":"The Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenjuan","middleName":"","lastName":"Zou","suffix":""},{"id":312546991,"identity":"be1e9c52-4d06-47ee-9d03-97aefd9561aa","order_by":3,"name":"Xiaohui Tang","email":"","orcid":"","institution":"Chongqing Traditional Chinese Medicine Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaohui","middleName":"","lastName":"Tang","suffix":""},{"id":312546993,"identity":"6297d1dc-52e2-46f5-b5b5-9d93c32ac05d","order_by":4,"name":"Weiqi Nian","email":"","orcid":"","institution":"Chongqing Traditional Chinese Medicine Hospital","correspondingAuthor":false,"prefix":"","firstName":"Weiqi","middleName":"","lastName":"Nian","suffix":""},{"id":312546994,"identity":"553f09bf-0819-46d7-b2c8-15294fdcfc74","order_by":5,"name":"Weiqin Zheng","email":"","orcid":"","institution":"Chongqing Traditional Chinese Medicine Hospital","correspondingAuthor":false,"prefix":"","firstName":"Weiqin","middleName":"","lastName":"Zheng","suffix":""},{"id":312546995,"identity":"670f3aa1-196e-41fc-b118-a4a3191b4524","order_by":6,"name":"Rongzhong Huang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Rongzhong","middleName":"","lastName":"Huang","suffix":""},{"id":312546996,"identity":"dca6c54b-6d0e-4f4b-a26d-e4b9a3d5e583","order_by":7,"name":"Pei Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYBAC+wYg8cDARo6fvbHxwYcKCTl5QloMDgCJhII0Y8mew4cNZ5yxMDZsIErLh8OJG26kpUnztlUkMhwgpOV487MPCQaHExsO5BhI8M6TSGBsYH746AY+v/QcM56RYJBu3NhwxsBAcptEHjsDm7FxDj5bJBKMGRIMrGWbGXsMEgy3SRQzNvCwSePVIv/8M1ALM2MbM4/BgcQ5EkAXEtIiwQOyxVmxh40tseFgAzFaeHKKgVrSjCV4mA8zNhyTMDZsJuQX9uObGT78sZGzv/+w/fefmjo5efbmh4/xacECmElTPgpGwSgYBaMACwAA8OZO76dQvW8AAAAASUVORK5CYII=","orcid":"","institution":"Chongqing Traditional Chinese Medicine Hospital","correspondingAuthor":true,"prefix":"","firstName":"Pei","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-06-01 12:58:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4513715/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4513715/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58965003,"identity":"daa6c4f9-5d13-49a0-a6bb-6f3392812e44","added_by":"auto","created_at":"2024-06-24 17:59:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1216248,"visible":true,"origin":"","legend":"\u003cp\u003eResults of network pharmacology and bioreliability analysis. A: 41 potential targets were predicted to be involved in the regulation of radiation enteritis by CKI, including ACE, ADA, ADH1C, AVP, CARTPT, CAT, CHGA, CNR1(CB1), CNR2(CB2), COX1, COX2, DRD3, ESR1, ESR2, F2, F3, F9, GRIA1, GRIK2, HDAC9, HIF1AN, HTR1A, HTR4, JUN, KARS, KCNA2, KCNMA1, LPL, NQO1, NR1H4, NR3C1, NR3C2, PAX2, PIK3R1, PLAT, PLG, PTGS2, SCN5A, SCN8A, SCN9A, SLC6A4, TACR2, TYR, TYRP1,and VDR. B: Purple nodes represent the active components of compound matrrhizae injected into in the blood, blue star nodes represent drug targets, yellow circle nodes represent KEGG pathways, and green square nodes represent CTD diseases. To highlight the significant elements of CKI, the network only displayed targets with at least 17 linked compounds. The 25 most strongly correlated medicinal chemical components and 6 most strongly correlated gene targets with the most correlation were screened out through a visual network structure. The study revealed that the significant regulatory targets of CKI were CNR1 (CB1), CNR2 (CB2), DRD2, ADRA2A, ADRA1A, CYP11A1, MTTP and ADORA1. C: The upper left part of the image shows GO and KEGG enrichment analysis of Radix Kushen, the lower left part shows OMM and TTD enrichment analysis of Radix Kushen, the upper right part shows GO and KEGG enrichment analysis of Rhizoma Smilacis Glabrae, and the lower right part shows OMM and TTD enrichment analysis of Rhizoma Smilacis Glabrae.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4513715/v1/e356d90ffa900744a91fa1ee.png"},{"id":58965436,"identity":"7f953ae2-0aac-4d68-93b1-a9d89c47f07e","added_by":"auto","created_at":"2024-06-24 18:07:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1359529,"visible":true,"origin":"","legend":"\u003cp\u003eThe infiltration of CD68 and CD16b in the rat ileum was detected by HE staining and immunohistochemical staining. A. HE staining was used to observe the tissue changes in the ileum. B.IHC staining was performed to observe the infiltration of CD68 and CD16b in the ileum. The data were shown as mean ± SD. Compared with the control group, ###\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. Compared with the model group, * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, *** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. Compared with the CKI group, \u0026amp; \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4513715/v1/91ae2abbe1f679a176292eef.png"},{"id":58965006,"identity":"394062cb-ae4a-4402-8e51-ca12edcb9ade","added_by":"auto","created_at":"2024-06-24 17:59:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":305893,"visible":true,"origin":"","legend":"\u003cp\u003ePCR detection of ileum inflammatory factors in rats and WB analysis of ileum NF-κB signal activation in rats. A: PCR was performed to detect the expression of the ileal inflammatory cytokines MCP1, TNF-α, IL-1β and IL-10. B: WB detection of ileal NF-κB signalling activation. The data are shown as the mean ± SD. Compared with the control group, ###\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. Compared with the modelgroup, * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, *** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. Compared with that in the CKI group, the p-IκBα/IκBα ratio in the CKI+CB1 antagonist group \u003cem\u003ewas significantly greater (P\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4513715/v1/ab235b080ad30930c8969b7d.png"},{"id":58965008,"identity":"0d1a4e7d-0621-434f-aacf-567bc339cfcd","added_by":"auto","created_at":"2024-06-24 17:59:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":378214,"visible":true,"origin":"","legend":"\u003cp\u003eThe activity of SOD, GSH-Px, MDA, ROS and NO and the expression of NOX4 in the ileum of the rats were detected. A: Activity of SOD, GSH-Px, MDA, ROS and NO in the ileum of rats. B: WB detection of ileal NOX4 expression. The data were shown as the mean ± SD. Compared with the control group, ###P\u0026lt;0.001. Compared with the model group, * P\u0026lt;0.05, ** P\u0026lt;0.01, *** P\u0026lt;0.001. Compared with the CKI group, \u0026amp;\u0026amp;P\u0026lt;0.01, \u0026amp;\u0026amp;\u0026amp;P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4513715/v1/e8ecd1fc596eea871e4f8c4e.png"},{"id":58965437,"identity":"7f10b703-42fa-4ba9-9921-e17ea49dda0b","added_by":"auto","created_at":"2024-06-24 18:07:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":240950,"visible":true,"origin":"","legend":"\u003cp\u003eWB detection of CB1 and p-p38 MAPK/p38 MAPK expression in the rat ileum. The data are shown as the mean ± SD. Compared with the control group, ###\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. Compared with the modelgroup, * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, *** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. Compared with the CKI group, \u0026amp;\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4513715/v1/6cdcae2a2a465204431ad27c.png"},{"id":58965004,"identity":"5cc11ab1-0df5-4857-b4be-1142b1effb79","added_by":"auto","created_at":"2024-06-24 17:59:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2557094,"visible":true,"origin":"","legend":"\u003cp\u003eImmunofluorescence costaining to observe the coexpression of CB1 and p-p38 MAPK in the rat ileum. The data were shown as the mean ± SD. Compared with the control group, ###P\u0026lt;0.001. Compared with the model group, * P\u0026lt;0.05, ** P\u0026lt;0.01, *** P\u0026lt;0.001. Compared with the CKI group, \u0026amp;P\u0026lt;0.05, \u0026amp;\u0026amp;P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4513715/v1/26b19896c213aecfd4b3b6b5.png"},{"id":58965441,"identity":"10a02d20-83a3-461f-bd91-011a42b58717","added_by":"auto","created_at":"2024-06-24 18:07:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7049236,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4513715/v1/a42486fb-3016-49a4-ad83-a85a603699f0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Compound Kushen Injection improves radiation enteritis via cannabinoid receptor 1 in rats","fulltext":[{"header":"1. Background","content":"\u003cp\u003eThe incidence of rectal malignant tumors and cervical malignant tumors is increasing annually in China, jeopardizing the health of the population\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Radiation can block and disrupt the proliferation process of tumor cells in the irradiated area directly or indirectly and has become one of the main means of treating malignant tumors\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. However, acute radiation enteritis is the most common complication of pelvic external radiation therapy, with patients presenting with nausea and vomiting, abdominal pain, diarrhea, increased mucus, and blood in the stool\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Studies have shown that approximately 90% of pelvic radiotherapy patients experience changes in bowel habits, and the incidence of acute radiation enteritis ranges from 50\u0026ndash;70%\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Acute radiation enteritis brings discomfort and pain to patients, causing interruption of radiotherapy and affecting the therapeutic effecacy. If it is not treated in time, it may develop into chronic radiation enteritis with intestinal fibrosis, even intestinal perforation and intestinal obstruction, which will affect the quality of life and prognosis of patients\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. The current treatment for acute radiation enteritis is based on protecting of intestinal mucosa, and preventing diarrhea ,inflammatory and infection, which has a long course of treatment, and some patients experience poor therapeutic effects\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Therefore, effective prevention of acute radiation enteritis can reduce the symptoms of abdominal pain and diarrhea caused by radiation enteritis and improve patients' quality of life. In addition, it can also improve patient compliance with radiotherapy and ensure that radiotherapy is completed on schedule, which is highly significant for the treatment and prognosis of patients with pelvic malignant tumors\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe formation of radiation enteritis is a complex multi-factor process, that is related to intestinal epithelial cell damage, intestinal wall blood vessel damage, intestinal flora displacement, and inflammatory factor release\u003csup\u003e[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. However, there is no standardized treatment for ARE, and although modern medical treatment can achieve certain effects in clinical practice, the overall effect is still unsatisfactory. Traditional Chinese medicine, as one of China's traditional medical treasures, has unique advantages in the treatment of gastrointestinal diseases. In recent years, there has been an increasing focus on the use of herbal medicine as a natural and low-toxicity treatment for radiation enteritis.Traditional Chinese herbs, such as Polygonati Rhizoma, Achyranthis Bidentatae Radix, and Epimedii Folium, have been found to have anti-radiation and immune regulation properties. An herbal diet containing these herbs showed a significant radiation-protective effect on intestinal crypts at doses of 4 Gy and 8 Gy. At a dose of 8 Gy, the Chinese herbal diet was found to reduce the loss of inhibitory nNOS neurons in the intestine, which can relieve symptoms of hyperperistalsis and diarrhea in patients after radiotherapy\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. A phase II trial was conducted to investigate the efficacy of the herbal medicine TJ-14 in treating acute radiation enteritis across multiple institutions. The study found that TJ-14 was effective in treating ARE in 86% of patients and had the advantages of low toxicity and medical costs\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn 200 AD, Radix Sophorae Flavescentis began to be used to treat tumors, inflammation and other diseases. CKI is a preparation of pure Chinese medicine that consists of Radix Kushen and Rhizoma Smilacis Glabrae and has been clinically remediated in China for more than 20 years. It contains at least eight different anti-inflammatory components, including matrine, oxymatrine, sophorine, oxysophorine, \u003cem\u003eet al\u003c/em\u003e \u003csup\u003e[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. CKI has anticancer and analgesic functions and can improve immunity, hemostasis and blood vessel dilation\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. A study found that CKI can reduce radiation side effects and can reduce the severe toxicity and symptom burden associated with radiotherapy in patients with lung cancer\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. In clinical practice, CKI has been used as a supportive treatment for colorectal cancer, and it has been found to relieve pain, bleeding and other inflammatory reactions during radiotherapy\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. In addition, Harata-Lee Y \u003cem\u003eet al\u003c/em\u003e, reported that Compound Kushen injection could reduce the severity of radiation-induced gastrointestinal mucositis in rats\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Our previous study found that CKI can effectively treat acute hemorrhagic severe radiation enteritis in rats. This study revealed in reduced inflammation and bleeding, as well as decreased mortality rates\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e.Network pharmacology involves multiple genes and multiple targets, which aligns with the complexity of traditional Chinese medicine (TCM) treatment. However, the mechanism of action of this drug on acute radiation enteritis has not been reported. The continuous development of systems biology in TCM research has made network pharmacology a popular analytical method in the field. Our study employed network pharmacology and animal experiments to elucidate the mechanism and potential targets of CKI in treating radiation enteritis.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Network Pharmacology Analysis\u003c/h2\u003e\n \u003cp\u003eTo identify potential targets for CKI treatment of radiation enteritis, we conducted a network pharmacology analysis. We queried the GeneCard database using the keywords \u0026quot;radiation enteritis\u0026quot;, \u0026quot;inflammation\u0026quot; and\u0026quot;enteritis\u0026quot;, and screened the genes with relevance score of at least 1. We took the intersection of these three sets. Next, we screened for possible gene targets of the two CKI components, Radix Kushen and Rhizoma Smilacis Glabrae, using BATMAN-TCM network pharmacology. BATMAN-TCM is an enhanced integrative database designed to store known and predicted linkages between TCM (traditional Chinese medicine) ingredients and target proteins\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. BATMAN-TCM contained 17,068 known and 2,339,061 high-confidence predicted TTIs, together with 54,832 formula, 8,404 herbs, and 39,171 ingredients. The pinyin names of \u0026quot;KU SHEN\u0026quot; and \u0026quot;BAI TU LING\u0026quot; (score cut-off =\u0026thinsp;18, adjusted P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were used to identify potential targets and combined the two components. The findings were then intersected with the genes screened in the Genecards database to predict the hotspot genes related to the regulation of \u0026quot;radiation enteritis\u0026quot; by CKI. BATMAN-TCM was used to predict potential targets for each queried TCM\u0026rsquo;s ingredient, and then perform functional analyses of these targets, including Gene Ontology (GO) term, KEGG pathway and OMIM/TTD disease enrichment analyses, were performed. According to the filtering criteria, P\u0026thinsp;\u0026le;\u0026thinsp;0.05 and Q value\u0026thinsp;\u0026le;\u0026thinsp;0.05, only the top 10 of each enrichment were shown for each respectively. A TCM ingredient-target-pathway/disease association network and biological pathways with highlighted TCM targets would also be shown. Enrichment analysis and mapping of GO, KEGG pathway, OMIM and TTD disease data were performed using R software(version 4.3.2).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Animal\u003c/h2\u003e\n \u003cp\u003eForty healthy 6-week-old male SPF-grade SD rats, weighing between 150 g and 200 g, were purchased from Chengdu Dashuo Laboratory Animal Co. Ltd. (SCXK (Chuan) 2019-031). All animals were acclimatized for one week at a temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and a relative humidity of 55\u0026thinsp;\u0026plusmn;\u0026thinsp;5%, with free access to food and water during the test period. After one week, the rats were randomly divided into five groups: the control group (n\u0026thinsp;=\u0026thinsp;8), model group (n\u0026thinsp;=\u0026thinsp;8), CB1 agonist group (n\u0026thinsp;=\u0026thinsp;8), CKI group (n\u0026thinsp;=\u0026thinsp;8), and CKI\u0026thinsp;+\u0026thinsp;CB1 antagonist group (n\u0026thinsp;=\u0026thinsp;8).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Establishment of a model of radiation enteritis\u003c/h2\u003e\n \u003cp\u003eThe rats were fasted for 12\u0026ndash;14 h, except those in the control group, which were subjected to abdominal anesthesia with 10% pentobarbital sodium (0.4 ml/100 g) and fixed on a foam board in the supine position.The whole abdomen (from the xiphoid process to the pubic symphysis) was irradiated by a 6MV medical high-energy X-ray linear accelerator with an irradiation area of approximately 7 cm\u0026times;6 cm, and the rest of the area was shielded by a 5 cm-thick lead plate with a 100 cm distance from the source skin, and the radiation dose rate was 200 cGy/min. for a total irradiation dose of 10 Gy.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Animal modelling and grouping\u003c/h2\u003e\n \u003cp\u003eThe control group received a daily intraperitoneal injection of an equal amount of saline for 7 d. The radiation model group received a daily intraperitoneal injection of an equal amount of saline for 7 d. The CB1 agonist group received a daily intraperitoneal injection of 2 mg/kg of the CB1 agonist WIN55212-2, for 7 d.The CKI group received a daily intraperitoneal injection of 0.1 ml/100 g of CKI for 7 d.The CKI\u0026thinsp;+\u0026thinsp;CB1 antagonist group was given 0.1 ml/100 g of CKI and 1 mg/kg of the CB1 antagonist AM251 intraperitoneal injection every day for 7 d after radiological modelling.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Determination of the intestinal propulsion rate\u003c/h2\u003e\n \u003cp\u003eThe rats were fasted on the day of the end of treatment, and 1 mL of carbon powder suspension was given to the rats in the morning of the second day. The rats were sacrificed after 6 h, the intestines were dissected, the position of the carbon powder in the intestines was observed, and the propulsion time of the carbon powder marker per unit of time in the intestines was measured to obtain the intestinal propulsion rate.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 H\u0026amp;E staining\u003c/h2\u003e\n \u003cp\u003eImmediately after sacrifice, the rats were dissected to remove the ileum (from the anus to the cecum, more than 2 cm from the anus), and the intestinal contents were flushed out with saline, and stored in saline for further use. Approximately 1 cm sections of colonic tissue were removed, fixed in 10% paraformaldehyde solutions overnight, immersed in a series of ethanol solutions for dehydration, embedded in paraffin, cut into 3 \u0026micro;m thick pathological sections, laid flat on slides, dewaxed and hydrated after baking, stained with hematoxylin for 5 min, stained with eosin for 2 min, made transparent by xylene, and sealed with neutral gum, and then placed under a light microscope for observation of the pathological conditions of the ileum.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7 Immunohistochemistry\u003c/h2\u003e\n \u003cp\u003eThe rat ileum was removed, fully with 4% paraformaldehyde, routinely dehydrated and sectioned by paraffin embedding. Immunohistochemical staining. Citrate buffer (pH 6.0) was used for antigen retrieval, and 3% hydrogen peroxide was used to eliminate endogenous peroxidase activity. Primary antibodies (anti-CD68, ab955, Abcam, UK, Biomedical Technology Co.., Ltd, 1:100; anti-CD16b, ab89207, Abcam, UK, Biomedical Technology Co.., Ltd, 1:100) were incubated at 4\u0026deg;C overnight. PBS was used instead of primary antibody as a blank control. HRP-labelled goat anti-rabbit IgG (GB23303, Servicebio, Wuhan, 1:100) was used as the secondary antibody, and the sections were incubated for 30 min. After DAB color development, 5 fields of view were randomly selected for each section, and the proportion of positive cells in each image was calculated using the Halo data analysis system.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8 qRT-PCR\u003c/h2\u003e\n \u003cp\u003eTotal RNA was extracted from homogenates of intestinal mucosa using TRIzol reagent. cDNA was used as a template for quantitative reactions after reverse transcription of RNA to cDNA. The relative expression of MCP1, TNF-\u0026alpha;, IL-1\u0026beta; and IL-10 mRNA was calculated by the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method using GAPDH as an internal reference. The primer sequences are shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eprimer sequences for qRT-PCR\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIL-1\u0026beta;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eaat ctc aca gca gca tct cga caa g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etcc acg ggc aag aca tag gta gc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTNIF\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ecac cac gct ctt ctg tct act gaa c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etgg gct acg ggc ttg tca ctc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIL-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eggc agt gga gca ggt gaa gaa tg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etgt cac gta ggc ttc tat gca gtt g\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMCP-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ectc acc tgc tgc tac tca ttc act g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ectt ctt tgg gac acc tgc tgc tg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9 Western blot\u003c/h2\u003e\n \u003cp\u003eThe tissue was lysed with RIPA buffer (P0013, Beyotime, China). The protein concentration was quantitatively analysed with a BCA kit (P0009, Beyotime, China). Protein samples were boiled in sodium dodecyl sulfate (SDS) loading buffer for 5 min, subjected to SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes (PVDF, ISEQ00010, Sigmaaldrich, UK). The membrane was blocked with Tris-Buffered saline with Tween (TBST) containing 5% buttermilk for 1 hour. It was then combined with I\u0026kappa;B\u0026alpha;(1:1000), p-I\u0026kappa;b\u0026alpha; (1:1000), NOX4(1:2000), CB1(1:1000), p38 MAPK(1:1000), p-p38 MAPK(1:1000),and GAPDH(1:500), diluted with 5% bovine serum albumin (BSA) in TBST, then incubated with goat anti-rabbit IgG (ab150077, 1:1000, Abcam, UK) at room temperature for 1 hour. After incubation with the second antibody, imaging was performed using an ECL Plus HRP substrate kit (K22030, abbkine, USA) on an iBright CL750 instrument (A44116, Thermo Fisher, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e2.10 ELISA for SOD, GSH-Px, MDA, ROS and NO levels in rat ileal tissue\u003c/h2\u003e\n \u003cp\u003eThe tissues were added to PBS at a mass-to-volume ratio of 1:9, homogenized, and centrifuged for 10 min at 5000\u0026times;g. the supernatant was taken to be measured the content or activity of SOD (ZC-36451, ZCI BIO, Shanghai China), GSH-Px (ZC-52475, ZCI BIO, Shanghai China), MDA (ZC-55718, ZCI BIO, Shanghai China), ROS (ZC-36727, ZCI BIO, Shanghai China) and NO (ZC-37503, ZCI BIO, Shanghai China) in the supernatant was measured according to the instructions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e2.11 Immunofluorescence\u003c/h2\u003e\n \u003cp\u003eThe sections were immersed in a 5% film breaker for 10 min at room temperature. After washing with PBS, goat serum blocking solution was added, and the sections were blocked for 20 min at room temperature. Primary antibodies (\u0026gamma;-H2ax, ab2893; RAD51, ab133534; Abcam, UK) were added and the sections were incubated at 4\u0026deg;C overnight. After rinsing with PBS again, the secondary antibody (ProLong\u0026trade; Diamond, GB22303; CY3-labelled goat anti-rabbit antibody, GB21303; Servicebio, Wuhan) was added and incubated at 37 ℃ for 30 minutes. After rinsing with PBS, DAPI was added, and the cells were incubated at room temperature. After a final rinse with PBS, the sections were sealed with an antifluorescent attenuation sealer. Images were acquired using a camera system (OlyVIA, OLYMPUS, Japan). The fluorescence intensity and area of all the images were measured using the Image-J image analysis system, and the fluorescence intensity of each image was calculated.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e2.12 Statistical analysis\u003c/h2\u003e\n \u003cp\u003eThe quantitative data of this study were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Differences among multiple groups were statistically compared by one-way ANOVA and least significant difference (LSD) post hoc analysis, and the differences between the two groups were statistically compared by unpaired \u003cem\u003et\u003c/em\u003e-test. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate statistical significance.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Network pharmacology and bioinformatics analysis\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, the GeneCard database was searched using the keywords 'radiation enteritis', 'inflammation', and 'enteritis'. There were 2871, 2987, and 5789 genes with relevance scores of at least 1, respectively. A total of 899 genes related to radiation enteritis were obtained from the intersection of the three datasets. Pharmacological screening of the BATMAN-TCM network predicted 971 genes as possible targets of CKI. These genes were then intersected with those screened in the previous Genecards database. In total, 41 potential targets were predicted to be involved in the regulation of radiation enteritis by CKI. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, the network relationships of CKI prediction targets, continuous blood drug concentrations and therapeutic target databases were analysed using the traditional Chinese medicine database system pharmacology and analysis platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tcmspw.com/tcmsp.php\u003c/span\u003e\u003cspan address=\"http://tcmspw.com/tcmsp.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, version 2.3). The 25 most strongly correlated medicinal chemical components and 6 most strongly correlated gene targets were screened through a visual network structure. Radiation enteritis is significantly associated with CB1 (CNR1), which is predicted to interact with 17 of the 25 major medicinal chemicals of CKI, such as kushenin, kushenol, and Kurarinone. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, GO enrichment analysis was used to predict the major functions of the two herbs from three aspects: biological process (BP), molecular function (MF) and cellular component (CC). KEGG enrichment analysis was used to identify the major signalling pathways of the two herbs of CKI, and OMIM/TTD disease enrichment analysis was used to determine the correlation between drugs and diseases. And GO enrichment found that cell-cell signalling, transmembrane transporter activity and oxidoreductase activity were significantly related. KEGG enrichment revealed that these genes were significantly related to neuroactive ligand-receptor interactions, the cannabinoid receptor pathway and the calcium signalling pathway.According to the OMIM/TTD disease enrichment analysis, drugs were significantly associated with analgesics, pain, psychiatric/neurological disorders and Maple Syrup Urine Disease. Our enrichment analysis suggested that CKI may play a therapeutic role in radiation enteritis based on pain control and regulation of redox and endogenous cannabinoid receptor pathways Based on the literature, we investigated the role of the endocannabinoid system in the brain-gut axis\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Thereafter, we further studied the role of CB1 in radiation enteritis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.2 HE staining was used to observe the tissue changes in the rat ileum\u003c/b\u003e.\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, compared with those in the control group, the structure of the ileal mucosal layer in the model group was damaged, the intestinal villi were atrophied and exfoliated, and mucosal inflammatory cells infiltrated in model group. Compared that in the model group, intestinal villus atrophy was alleviated in the CB1 agonist group, the CKI group and the CKI\u0026thinsp;+\u0026thinsp;CB1 antagonist group, and a few inflammatory cells infiltrated the mucous membrane. Compared with those in the CKI group, some of the intestinal villi in the CKI\u0026thinsp;+\u0026thinsp;CB1 antagonist group were exfoliated, and the mucosal intestinal solidifying glands were slightly atrophic.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 Immunohistochemical staining was performed to observe the infiltration of CD68 and CD16b in the rat ileum.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe detected the degree of CD68 and CD16b infiltration in rat ileal tissues by immunohistochemical staining, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB. The results showed that the expression of CD68 and CD16b in the model group was significantly greater than that in the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Compared with that in the ileum of the model group, the expression of CD68 in the ileum of the three groups decreased significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The expression of CD16b in ileum of rats in the CB1 agonist group and the CKI group decreased significantly after treatment (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and the expression of CD16b in ileum in the CKI\u0026thinsp;+\u0026thinsp;CB1 antagonist group decreased significantly after treatment(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Compared with that in the CKI group, the expression of CD16b in the CKI\u0026thinsp;+\u0026thinsp;CB1 antagonist group increased significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4 PCR detection of inflammatory cytokines in rat ileum.\u003c/h2\u003e \u003cp\u003eWe detected the expression of inflammatory factors in rat ileum by qRT-PCR, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. The expressions of MCP1, TNF- α, IL-1β and IL-10 in the ileum of model group were significantly greater than those in the control group(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with model group, the expression of MCP1, TNF- α, IL-1β and IL-10 in ileum of CB1 agonist group and CKI group decreased significantly(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and only the expression of TNF- α in the ileum of the CKI\u0026thinsp;+\u0026thinsp;CB1 antagonist group decreased significantly(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5 WB detection of NF-κB signaling activation in the rat ileum.\u003c/h2\u003e \u003cp\u003eWe detected the key proteins in the NF-κB pathway by WB. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, the p-IκBα/IκBα ratio in the ileum of the model group was significantly greater than that in the ileum of the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with that in the model group, the p-IκBα/IκBα ratio decreased in the other three groups. Compared with that in the CKI group, the p-IκBα/β-IκBα ratio was elevated in the CKI\u0026thinsp;+\u0026thinsp;CB1 antagonist group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.6 ELISA for SOD, GSH-Px, MDA, ROS and NO expression in the rat ileum.\u003c/h2\u003e \u003cp\u003eThe activity of SOD, GSH-Px, MDA, ROS and NO in the rat ileum was detected by ELISA, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. Compared with those in the control group, the levels of SOD and GSH-Px in the ileum of the model group were significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and the levels of MDA, ROS and NO were significantly greater (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Compared with those in the model group, the levels of SOD and GSH-Px in the ileum of the other three groups were significantly greater (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and the levels of MDA, ROS and NO were significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Compared with those in the CKI group, the levels of SOD and GSH-Px in the ileum of the CKI\u0026thinsp;+\u0026thinsp;CB1 antagonist group were significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and the levels of MDA, ROS and NO were significantly greater (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.7 WB detection of NOX4 expression in the rat ileum.\u003c/h2\u003e \u003cp\u003eWe detected the expression of NOX4 in the rat ileum by WB. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, the results showed that the expression of NOX4 in the ileum in model group was significantly greater than that in control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with that in the ileum of the model group, the expression of NOX4 in the ileum of the CB1 agonist group and CKI group decreased significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.8 WB detection of CB1 and p-p38 MAPK/ p38 MAPK expression in the rat ileum.\u003c/h2\u003e \u003cp\u003eWe detected the expression of ileal CB1, p38 MAPK and p-p38 MAPK in rats by WB. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the results showed that the expression of CB1 and p-p38 MAPK/p38 MAPK in the ileum of the model group was significantly greater than that of the control group(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with those in the model group, the expression levels of CB1 and p-p38 MAPK/p38 MAPK in the ileum of the CB1 agonist group and CKI group were significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared that in the CKI group, the ratio of p-p38 MAPK/p38 MAPK ratio was significantly greater in the CKI\u0026thinsp;+\u0026thinsp;CB1 antagonist group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.9 Immunofluorescence costaining to observe the coexpression of CB1 and p-p38 MAPK in the rat ileum.\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, we performed double immunofluorescence staining for CB1 and p-p38 MAPK in the ileum of the rats. Compared with those in the control group, the fluorescence intensities of CB1\u0026thinsp;+\u0026thinsp;p-p38 MAPK, CB1 and p-p38 MAPK were significantly increased and the fluorescence signal intensity was enhanced in the model group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while the fluorescence intensities of CB1 and p-p38 MAPK decreased significantly in the CB1 agonist group, CKI group and CKI\u0026thinsp;+\u0026thinsp;CB1 antagonist group than in the the model group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Compared with that in the CKI group, the fluorescence of CB1\u0026thinsp;+\u0026thinsp;p-p38 MAPK and p-p38 MAPK in the CKI\u0026thinsp;+\u0026thinsp;CB1 antagonist group increased significantly, and the fluorescence signal increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn 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\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. However, radiation inevitably irradiates the surrounding normal tissues, and the intestinal epithelial cells are highly sensitive and poorly tolerant to radiotherapy\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. 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\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. 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\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThrough 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)\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e and epithelial cells\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. 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\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. 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\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. 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\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. 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\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. 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.\u003c/p\u003e \u003cp\u003eRadiotherapy 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\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. 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\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Alarifi\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e 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\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. 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.\u003c/p\u003e \u003cp\u003eThe 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\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e, the intestinal oxidative stress biomarker malondialdehyde(MDA) content increases, glutathione peroxidase (GSH-Px) content decreases, and superoxide dismutase (SOD) activity decreases\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. 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 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in tissues with very low levels of catalase or very low H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 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\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. 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.\u003c/p\u003e \u003cp\u003eIn 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\u0026thinsp;+\u0026thinsp;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.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eCKI can improve radiation-induced intestinal injury in rats, possibly by regulating CB1 to improve radiation-induced inflammatory responses, inhibiting p38 MAPK signalling, and simultaneously reducing oxidative stress.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eAll the experiments on animals were performed with the approval of the Experimental Animal Ethics Committee of Chongqing Hospital of Traditional Chinese Medicine（2022-DWSY-XWJ）.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eThe initial data used to support the findings of this study are available from the corresponding author upon request. The Data from this study have not been published elsewhere.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was sponsored by Natural Science Foundation of Chongqing, China（General Program）Project Number: cstc2021jcyj-msxmX0729 and Chongqing Hospital of Traditional Chinese Medicine Youth Top talent project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u0026nbsp;\u003c/strong\u003eWenjing Xu: Animal handling and article writing. Liping Gao: Animal feeding and handling. Wenjuan Zou: Drug injection and some molecular biology experiments. Xiaohui Tang: Some molecular biology experiments. Weiqi Nian: Fund reimbursement，statistical analysis and progress supervision. Weiqin Zheng: Theoretical guidance of Traditional Chinese Medicine. Pei Wang: Some molecular biology experiments and article polishing and work design. Rongzhong Huang: Bioinformatics analysis, final revision and progress supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHan B, Zheng R, Zeng H, Wang S, Sun K, Chen R, et al. Cancer incidence and mortality in China, 2022. J Natl Cancer Cent. 2024;4:47\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMihaescu A, Santen S, Jeppsson B, Thorlacius H. P38 mitogen-activated protein kinase signalling regulates vascular inflammation and epithelial barrier dysfunction in an experimental model of radiation-induced colitis. Br J Surg. 2010;97:226\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHale MF. Radiation enteritis: from diagnosis to management. 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Pol J Pathol. 2014;65:167\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-complementary-medicine-and-therapies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcam","sideBox":"Learn more about [BMC Complementary Medicine and Therapies](https://bmccomplementmedtherapies.biomedcentral.com/)","snPcode":"","submissionUrl":"","title":"BMC Complementary Medicine and Therapies","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"network pharmacology, Compound Kushen Injection, radiation enteritis, cannabinoid receptor 1, inflammatory pathways, oxidative stress","lastPublishedDoi":"10.21203/rs.3.rs-4513715/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4513715/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Clinical studies have shown that Compound Kushen Injection (CKI) can alleviate the inflammatory symptoms of radiation enteritis. However, the mechanism of action remains unclear. The aim of this study was to explore the possible targets and mechanisms of CKI in the treatment of radiation enteritis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eNetwork pharmacology was used to predict the potential targets of CKI for the treatment of radiation enteritis, and GO and KEGG enrichment analyses were subsequently performed. The therapeutic effects and signalling pathways were then verified in rats. The expression of inflammatory factors in ileal tissue was measured by qRT-PCR. The activities of SOD and GSH-Px in ileal tissue were measured by ELISA. The levels of MDA, ROS and NO were determined using biochemical kits. The expression of signalling pathway-related proteins was detected by Western blotting and immunofluorescence.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eAccording to network pharmacology, CB1 might be a target of CKI. GO and KEGG enrichment analyses revealed that CKI was significantly enriched in analgesic, endocannabinoid and inflammatory pathways. In the rat model, Compared with that in the radiotherapy group,the extent of ileal injury was significantly improved in the CKI group compared to the control group. In addition, the infiltration of CD68 and CD16b was significantly reduced, and the expression of MCP1, TNF-α, IL-1β and IL-10 was significantly decreased. In addition, the activities of SOD and GSH-Px were increased, and the activities of MDA, ROS and NO were decreased. The CKI group also showed inhibition of NF-κB signalling and a significant decrease in the expression of NOX4, CB1 and p-p38 MAPK/p38 MAPK. The use of a CB1 agonist could also alleviate radiation enteritis, whereas the addition of a CB1 antagonist could interfere with the ameliorative effect of CKI on radiation enteritis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eCKI might exert an anti-radiation enteritis effect by targeting the cannabinoid receptor 1.\u003c/p\u003e","manuscriptTitle":"Compound Kushen Injection improves radiation enteritis via cannabinoid receptor 1 in rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-24 17:59:06","doi":"10.21203/rs.3.rs-4513715/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2024-06-10T09:03:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-08T17:45:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Complementary Medicine and Therapies","date":"2024-06-01T12:56:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-complementary-medicine-and-therapies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcam","sideBox":"Learn more about [BMC Complementary Medicine and Therapies](https://bmccomplementmedtherapies.biomedcentral.com/)","snPcode":"","submissionUrl":"","title":"BMC Complementary Medicine and Therapies","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"be3756b4-53f9-4843-a350-db1711612372","owner":[],"postedDate":"June 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-02-06T10:08:40+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-24 17:59:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4513715","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4513715","identity":"rs-4513715","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}