| Objective: To evaluate the effect of clemastine fumarate on intestinal ischemiareperfusion injury in Balb/c mice and its relationship with TLR4/NLRP3 pyroptosis pathway.Methods: Thirty-two clean-grade male Balb/c mice,weighing 18～25 g,were randomly divided into 4 groups: sham operation group(Sham group,n=8),ischemia/reperfusion group(I/R group,n=8),clemastine fumarate treatment group(I/R+C group,n=8),TAK-242 treatment group(I/R+T group,n=8).One hour before surgery,the Sham group and I/R group were given intraperitoneal injection of 2%dimethyl sulfoxide(DMSO)10 m L/kg,and the I/R+C group was intraperitoneally injected with clemastine fumarate 5 mg/kg,The I/R+T group was intraperitoneally injected with TLR4 inhibitor TAK-242 3 mg/kg.The I/R group,I/R+C group and I/R+T group were used to clamp the branches of the superior mesenteric artery to establish intestinal ischemia/reperfusion injury models(ischemia for 40 min,reperfusion for 2h).In the Sham group,no ischemia/reperfusion treatment was performed after laparotomy.Orbital vein blood was collected to save blood samples,and the rats were killed by excessive anesthesia to collect labeled intestinal tissue samples and bilateral lung tissue samples.Histological detection of intestinal mucosal injury was performed by HE staining and light microscopy intestinal mucosal pathological examination with Chiu’s score;lung injury histological detection was performed by HE staining,light microscopy lung histopathological examination and injury score;ELISA method was used to detect the serum levels of the mice.The expression levels of TNF-α,IL-1β and IL-18;Western blot was used to detect the expression levels of TLR4,NLRP3,caspase-1 and GSDMD proteins in ischemia/reperfusion intestinal tissue.Result:1.The results of intestinal histopathology: Compared with the Sham group,the intestinal mucosal damage degree was aggravated and the Chiu’s score was increased in the I/R group,the I/R/+C group,and the I/R+T group(P<0.05);Compared with the I/R group,the I/R+C group and the I/R+T group had less damage to the intestinal mucosa by light microscopy,and the Chiu’s score was significantly reduced(P<0.05);Compared with I/R+T group and I/R+C group,there was no significant difference in Chiu’s score of intestinal mucosal injury.2.The results of lung histopathology: In the Sham group,the lung tissue was intact,the alveolar and interstitial structures were clear,and there was no obvious abnormality;in the I/R group,the lung tissue was obviously damaged and abnormal in structure,with exudation in the alveoli,and rupture and fusion of the alveoli.The lung interstitium was significantly thickened,accompanied by a large number of inflammatory cells infiltration,and the injury score was significantly higher than that in the Sham group(P<0.05);the I/R+C group and the I/R+T group showed relatively mild lung tissue The injury manifestations included alveolar exudation,mild pulmonary interstitial thickening,and infiltration of inflammatory cells.The injury scores were all lower than those in the I/R group(P<0.05).3.ELISA test results: Compared with Sham group,I/R group,I/R+C group and I/R+T up-regulated the contents of TNF-α,IL-1β and IL-18 in mouse serum(P<0.05);Compared with I/R group,I/R+C group and I/R+T group down-regulated the contents of TNF-α,IL-1β and IL-18 in mouse serum(P<0.05);Compared with the I/R+T group,the contents of IL-1β and IL-18 in the I/R+C group were decreased(P<0.05),and there was no significant difference in the serum TNF-α content between I/R+C group and I/R+T group.4.Western Blot detection results: Compared with the Sham group,the expressions of TLR4,NLRP3,caspase-1 and GSDMD proteins in the intestinal tissue of the I/R group were significantly increased(P<0.05),and the I/R+C group or I/R+T group were no significant difference;Compared with the I/R group,the expressions of TLR4,NLRP3,caspase-1 and GSDMD proteins in the intestinal tissues of the I/R+C group and the I/R+T group decreased(P< 0.05);there was no significant difference in the expression of TLR4,NLRP3,caspase-1 and GSDMD proteins in the intestinal tissue between the I/R+C group and the I/R+T group.Conclusion:1.Pre-ischemic preconditioning with clemastine fumarate can reduce intestinal mucosal injury and lung injury after intestinal ischemia-reperfusion in mice.2.After the inhibition of TLR4 pathway,the expression of protein and proinflammatory factors of TNF-α,IL-1β and IL-18 in NLRP3/caspase-1/GSDMD pathway was inhibited in murine intestinal ischemia / reperfusion model.3.The mechanism of clemastine fumarate attenuating intestinal ischemiareperfusion injury in mice may be related to the inhibition of TLR4/NLRP3/caspase-1/GSDMD cell pyroptosis pathway and the reduction of TNF-α,IL-1β and IL-18 proinflammatory factor expression. |
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