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OBJECTIVE To explore the effect of Rho kinase inhibitor on intestinal injury in septic rats and its possible mechanism. METHODS Thirty-two male Sprague-Dawley (SD) rats were randomly divided into sham operation group (Sham group), Rho kinase inhibitor Y-27632 control group (Y+Sham group), sepsis model group [cecal ligation and puncture (CLP) group] and Y-27632 pretreatment group (Y+CLP group), with 8 rats in each group. Rat sepsis model was reproduced by CLP. The rats in the Sham group and Y+Sham group were only separated and moved the cecum without ligation and perforation. The rats in the Y+Sham group and Y+CLP group were pretreated with intraperitoneal injection of Y-27632 solution 5 mg/kg 15 minutes before operation; the rats in the Sham group and CLP group were intraperitoneally injected with the same amount of phosphate buffered saline (PBS). Twenty-four hours after operation, the heart blood was collected and the serum diamine oxidase (DAO) content was determined by enzyme-linked immunosorbent assay (ELISA). Then the small intestine tissue was collected, the pathological changes of the intestinal tissue were observed under the light microscope after hematoxylin-eosin (HE) staining, and Chiu's score was performed. The positive expressions of Rho-related coiled-coil kinase 1 (ROCK1) and nuclear factor-κB (NF-κB) in intestinal tissue were detected by immunohistochemistry. ELISA was used to detect the levels of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) in intestinal tissue homogenate. RESULTS The intestinal tissue structure of the Sham group and Y+Sham group was intact and the mucosa was arranged neatly. Compared with the Sham group, the intestinal mucosa of the CLP group was arranged disorderly, with a large number of inflammatory cells infiltration, and the Chiu's score was significantly increased (3.83±0.27 vs. 0.12±0.11, P < 0.05), indicating that those rats suffered from septic intestinal injury. Compared with the CLP group, the degree of necrosis of intestinal epithelial cells in the Y+CLP group was reduced, a small amount of inflammatory cells infiltration was seen, and the Chiu's score was significantly decreased (2.85±0.21 vs. 3.83±0.27, P < 0.05), indicating that Y-27632 pretreatment could alleviate intestinal injury in septic rats. Compared with the Sham group, the positive expressions of intestinal tissue ROCK1 and NF-κB, the contents of serum DAO and intestinal homogenate TNF-α in the CLP group were significantly increased [ROCK1 expression (A value): 0.19 (0.18, 0.22) vs. 0.10 (0.09, 0.11), NF-κB expression (A value): 0.40±0.02 vs. 0.15±0.01, DAO (ng/L): 287.81±23.31 vs. 144.92±17.72, TNF-α (ng/L): 101.08±5.62 vs. 74.81±5.56, all P < 0.05], the level of intestinal homogenate IL-10 was significantly decreased (μg/L: 55.16±5.20 vs. 95.95±7.53, P < 0.05). Compared with the CLP group, the positive expressions of intestinal tissue ROCK1, NF-κB, the contents of serum DAO and intestinal homogenate TNF-α in the Y+CLP group were significantly decreased [ROCK1 expression (A value): 0.15 (0.13, 0.18) vs. 0.19 (0.18, 0.22), NF-κB expression (A value): 0.28±0.01 vs. 0.40±0.02, DAO (ng/L): 243.34±19.76 vs. 287.81±23.31, TNF-α (ng/L): 90.41±8.79 vs. 101.08±5.62, all P < 0.05], while the level of intestinal homogenate IL-10 was significantly increased (μg/L: 66.15±5.74 vs. 55.16±5.20, P < 0.05), indicating that the protective effect of Y-27632 pretreatment on sepsis intestinal injury rats might be related to the regulation of RhoA/ROCK1/NF-κB signaling pathway. CONCLUSIONS Rho kinase inhibitors can reduce intestinal injury in septic rats, and the mechanism may be related to inhibiting RhoA/ROCK1/NF-κB signaling pathway and reducing intestinal inflammation in septic rats.