Mechanism of intestinal injury induced by acute diquat poisoning in rats
Objective To investigate the effects of diquat(DQ)on the expression of intestinal pyroptosis-related proteins and tight junction proteins in rats,and to analyze the role of pyroptosis in the intestinal injury of rats with acute DQ poisoning.Methods A total of 36 Wistar male rats were randomly divided into control group,and 3 hours,12 hours,36 hours and 3 days exposure groups,with 6 rats in each group.Each exposure group was given 1/2 median lethal dose(LD50)of 115.5 mg/kg DQ by one-time gavage.The control group was given the same amount of normal saline by gavage.The control group was anesthetized at 3 hours after DQ gavage to take jejunal tissues;each exposure group was anesthetized at 3 hours,12 hours,36 hours,and 3 days after DQ gavage to take jejunal tissues,respectively.The general conditions of the rats were recorded.The pathological changes of jejunum tissue were observed by hematoxylin-eosin(HE)staining.The expression of intestinal pyroptosis-related proteins[NOD-like receptor protein 3(NLRP3),cysteine aspartate-specific protease 1(caspase-1),Gasdemin D(GSDMD)]in the intestinal tissues was observed by immunohistochemical staining.Western blotting was used to detect the expression of intestinal pyroptosis-related proteins and intestinal tight junction proteins(Occludin and Claudin-1).Results Light microscopy showed that pathological changes occurred in jejunum tissue at the early stage of exposure(3 hours),and the injury was the most serious in the 12 hours exposure group,with a large number of inflammatory cells infiltrating in the tissue,and the damage was significantly reduced after 3 days exposure.Immunohistochemical results showed that NLRP3,caspase-1 and GSDMD were expressed in the jejunal mucosa of the control group and the exposure groups,and the positive cells in the control group were less expressed with light staining.The expression of the above proteins in the exposed group was increased significantly and the staining was deep.Western blotting results showed that compared with the control group,the expression of NLRP3 protein in jejunum tissues of all groups was increased,with the most significant increase in the 36 hours group(NLRP3/β-actin:1.47±0.06 vs.0.43±0.14,P<0.01).Compared with the control group,the expression of GSDMD protein in the 3 hours,12 hours and 36 hours exposure groups increased,and the expression of GSDMD protein in the 3 hours and 12 hours exposure groups increased significantly(GSDMD/β-actin:1.04±0.40,1.25±0.15 vs.0.65±0.25,both P<0.05).The expression of caspase-1 protein was increased in 36 hours exposure group compared with the control group(caspase-1/β-actin:1.44±0.34 vs.0.98±0.19,P>0.05).Compared with the control group,the expression of Occludin and Claudin-1 proteins in each exposure group decreased,and the expression of Occludin proteins was significantly decreased in the 3 hours,12 hours,and 36 hours exposure groups decreased significantly(Occludin/β-actin:0.74±0.17,0.91±0.20,0.79±0.23 vs.1.41±0.08,all P<0.05).Although the protein expression of Claudin-1 decreased in each exposure group,the difference was not statistically significant.Conclusion The intestinal injury caused by acute DQ poisoning may be related to the activation of pyroptosis pathway of small intestinal cells and the reduction of the density of intercellular junctions.
DiquatIntestinePyroptosis-associated proteinTight junction protein
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