CD73, a pivotal enzyme in the purinergic signaling cascade, modulates the concentrations of adenosine and inosine. These metabolites are involved in immune responses and inflammatory processes. This study aims to investigate the function of CD73 in the pathogenesis of inflammatory bowel disease (IBD) and explore the potential mechanism.

 DSS-induced colitis mice models were established by orally administering 3% Dextran-Sodium-Sulfate (DSS) from day 1 to day 7. CD73 was blockage by intraperitoneal injection of Adenosine 5 '- (α, β-methylene) diphosphate (APCP, 4mg/kg) from day 3 to day 7. Inosine was supplemented by intraperitoneal injection (100mg/kg) from day 3 to day 7. Hematoxylin-eosin (H&E), PAS and Alcian blue staining were used to evaluate the inflammation infiltration and colon damage. DAI score was used to evaluate the severity of colitis, including weight loss, diarrhea and hematochezia. IHC analysis for paraffin-embedded sections of colon tissues were performed to detect the expression of CD73, F4/80, occludin, claudin-1 and ZO-1. Serum IL-6 levels were detected by ELISA assay. MDA, GSH and SOD detection were used to measure the activity of antioxidant enzyme. qRT-PCR was used to detect the mRNA expression levels. High-performance liquid chromatography-mass spectrometry (LC-MS) was used to evaluate the level of inosine. All quantitative results were presented as mean ± standard deviations (SD). All data analyses were performed by using GraphPad prism 8.0. T-test was used to analyze the differences between two independent samples. One-way analysis of variance (ANOVA) test was used for the comparison of multiple groups. P<0.05 was considered to be statistically significant.

DSS induced the necrosis and exfoliation of mucosal epithelium, goblets cells depletion, tissue

disorganization, and immune cells infiltration. APCP treatment could exacerbate the severity of tissue damage and inflammation cell infiltration. IHC staining for F4/80 showed markedly increased macrophage infiltration following APCP treatment. Notably, IL-6 levels were significant higher in APCP treated DSS-induced colitis mice compared to control and DSS induced mice (Control: 30.88 ± 6.841, DSS group: 52.13 ± 9.438, APCP group: 69.3313.61pg/mL). Compared with the control group, the DSS-induced colitis mice showed elevated MDA levels, which were further increased following APCP treatment (Control: 4.199 ± 0.532, DSS group: 5.513 ± 0.825, APCP group: 9.553 ± 1.574 µM/mg). Moreover, the activities of SOD (Control: 0.423 ± 0.019, DSS group: 0.320 ± 0.012, APCP group: 0.245 ± 0.033 U/mg) and GSH (Control:15.110 ± 1.657, DSS group: 11.570 ± 1.842, APCP group: 9.365 ± 0.6951 mU/mg) in DSS-induced colitis mice were reduced compared with control groups, which was the lowest in APCP treated mice. To assess the integrity of intestinal epithelial structure and barrier function, we investigated the expression of tight junction proteins in colon tissues. We observed that DSS treatment could reduce the expression levels of claudin-1, occludin and ZO-1, which were more decreased in APCP treated mice. Moreover, IHC analysis further confirmed the decreased protein expression of claudin-1, occludin and ZO-1 in APCP treated mice. LC-MS revealed decreased serum levels of adenosine (Control: 543.9 ± 101, DSS group: 323.3 ± 122, APCP group: 204.4 ± 111.4 pg/mL) and inosine (Control: 12.440 ± 3.879, DSS group: 9.074 ± 3.353, APCP group: 5.987 ± 2.465 pg/mL) in DSSinduced colitis mice, which were more reduced in APCP treated DSS-induced colitis mice. Inosine i.p. significantly alleviated the extent of weight loss, reduced the DAI scores, and ameliorated colon shortening. H&E staining showed that inosine i.p. could ameliorate the severity of colon damage and immune cell infiltration. Additionally, IL-6 levels in serum and colon tissues were significantly increased by DSS treatment, while were

reduced by inosine treatment (Control: 30.880 ± 6.841, DSS group: 52.13 ± 9.438, Inosine group: 36.070 ± 7.039 pg/mL). Compared with the control group, the DSS-induced colitis mice exhibited elevated MDA levels, which were significantly reduced by inosine treatment (Control: 4.199 ± 0.532, DSS group: 5.513 ± 0.825, Inosine group: 4.473 ± 0.464 µM/mg). Additionally, SOD and GSH activities were significantly decreased in the DSS group, whereas inosine i.p. significantly elevated the activities of SOD (Control: 0.423 ± 0.019, DSS group: 0.320 ± 0.012, Inosine group: 0.383 ± 0.021 U/mg) and GSH (Control: 15.110 ± 1.657, DSS group: 11.570 ± 1.842, Inosine group: 13.690 ± 0.552 mU/mg) in colon tissues. Furthermore, inosine treatment significantly elevated the expression of claudin-1, occludin and ZO-1. Notably, the effects of inosine on MODE-K cells were opposite to that of APCP, including the influences on expression of oxidative stress molecules and tight junction proteins.

This study suggested the protective role of CD73 during the progress in DSS-induced colitis. Inosine supplement might be a potential therapeutic strategy for colitis.