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. Conclusion: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.

AIM: To investigate the effect of Rho kinase inhibitor Y-27632 on acute liver injury in sepsis. METHODS: Thirty-two healthy adult male SD rats were randomly divided into sham operation group (Sham group), sham operation+Y-27632 group (Sham+Y group), cecal ligation and perforation group (CLP group) and CLP+Y-27632 group (CLP+Y-27632 group), 8 animals in each group. The rat sepsis model was established by the CLP method, and the rat was euthanized 24 hours after the model was established, and the serum and liver tissue were collected. Hematoxylin-eosin (HE) staining was used to observe the pathological changes in the liver tissue of the rats in each group; Western Blot was used to detect the expressions of ROCK1 and downstream NF-κB proteins in the liver tissue of the rats in each group; immunohistochemical method The expression of ROCK1 protein in liver tissue of rats was detected; enzyme-linked immunosorbent assay (ELISA) method was used to detect the levels of serum liver function indexes ALT and AST, and the changes of IL-18, IL-10 and GSH contents in liver tissue homogenate. RESULTS: Compared with the Sham group, there was no significant change in the histopathology of the liver in the Sham+Y group. In the CLP group, the arrangement of hepatocytes was disordered, with a large number of inflammatory cells infiltrating. Compared with the Sham group, the expression of ROCK1 protein in the CLP and CLP+Y groups was increased (P<0.05); compared with the CLP group, the ROCK1 protein expression in the CLP+Y group was decreased (P<0.05). Compared with the Sham group, the expression of NF-κB protein in the CLP and CLP+Y groups increased (P<0.05); compared with the CLP group, the NF-κB protein expression in the CLP+Y group decreased (P<0.05); A small amount of expression was found in the liver Sham group, and a large amount was expressed in the CLP group and CLP+Y group; compared with the Sham group, the serum ALT and AST levels in the CLP and CLP+Y groups were increased (P<0.05). The levels of ALT and AST in +Y group decreased (P<0.05). Compared with the Sham group, the contents of IL-18 in the liver tissue homogenate of the CLP and CLP+Y groups increased (P<0.05), while the contents of IL-10 and GSH in the liver tissue homogenate of the CLP group decreased (P<0.05). The changes of IL-10 and GSH in the group were similar (P>0.05); compared with the CLP group, the content of IL-18 in the CLP+Y group was decreased (P<0.05), and the content of IL-10 and GSH was increased (P<0.05). CONCLUSION: Rho kinase inhibitor can alleviate acute liver injury in septic rats, which may be related to inhibiting the expression of ROCK1 and NF-κB proteins, reducing the inflammatory response of liver tissue, and reducing the level of liver oxidative stress.

OBJECTIVE: To investigate the chemical constituents from the leaves of Jatropha curcas and evaluate their inhibition on lipopolysaccharide (LPS)-activated BV-2 microglia cells. METHODS: The n-BuOH extract of the leaves of J. curcas was isolated by macroporous adsorption resin, silica gel, ODS, column chromatography and semi-preparative HPLC. The structures of the compounds were identified by MS, NMR, ECD, and other spectroscopic methods. In addition, anti-neuroinflammatory effects of isolated compounds were evaluated by measuring the production of nitric oxide (NO) in over-activated BV-2 cells. RESULTS: Seventeen compounds, including (7R,8S)-crataegifin A-4-O-β-D-glucopyranoside ( 1), (8R,8'R)-arctigenin ( 2), arctigenin-4'-O-β-D-glucopyranoside ( 3), (-)-syringaresinol ( 4), syringaresinol-4'-O-β-D-glucopyranoside ( 5), (-)-pinoresinol ( 6), pinoresinol-4'-O-β-D-glucopyranoside ( 7), buddlenol D ( 8), (2R,3R)-dihydroquercetin ( 9), (2S,3S)-epicatechin ( 10), (2R,3S)-catechin ( 11), isovitexin ( 12), naringenin-7-O-β-D-glucopyranoside ( 13), chamaejasmin ( 14), neochamaejasmin B ( 15), isoneochamaejasmin A ( 16), and tomentin-5-O-β-D-glucopyranoside ( 17) were isolated and identified. Compounds 2, 4 and 8 significantly inhibited the release of NO in BV-2 microglia activated by LPS, with IC₅₀ values of 18.34, 29.33 and 26.30 μmol/L, respectively. CONCLUSION Compound 1 is a novel compound, and compounds 2, 3, 8, 14- 17 are isolated from Jatropha genus for the first time. In addition, the lignans significantly inhibited NO release and the inhibitory activity was decreased after glycosylation.