[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/ß-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/ß-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/ß-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/ß-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. CONCLUSIONS: 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.

[Experimental study on the toxicokinetics and gastrointestinal damage in rats poisoned with acute diquat poisoning at different exposure doses].

OBJECTIVE: To observe the toxicokinetic parameters, absorption characteristics and pathomorphological damage in different parts of the gastrointestinal tract of rats poisoned with different doses of diquat (DQ). METHODS: Ninety-six healthy male Wistar rats were randomly divided into a control group (six rats) and low (115.5 mg/kg), medium (231.0 mg/kg) and high (346.5 mg/kg) dose DQ poisoning groups (thirty rats in each dose group), and then the poisoning groups were randomly divided into 5 subgroups according to the time after exposure (15 minutes and 1, 3, 12, 36 hours; six rats in each subgroup). All rats in the exposure groups were given a single dose of DQ by gavage. Rats in the control group was given the same amount of saline by gavage. The general condition of the rats was recorded. Blood was collected from the inner canthus of the eye at 3 time points in each subgroup, and rats were sacrificed after the third blood collection to obtain gastrointestinal specimens. DQ concentrations in plasma and tissues were determined by ultra-high performance liquid chromatography and mass spectrometry (UPHLC-MS), and the toxic concentration-time curves were plotted to calculate the toxicokinetic parameters; the morphological structure of the intestine was observed under light microscopy, and the villi height and crypt depth were determined and the ratio (V/C) was calculated. RESULTS: DQ was detected in the plasma of the rats in the low, medium and high dose groups 5 minutes after exposure. The time to maximum plasma concentration (Tmax) was (0.85±0.22), (0.75±0.25) and (0.25±0.00) hours, respectively. The trend of plasma DQ concentration over time was similar in the three dose groups, but the plasma DQ concentration increased again at 36 hours in the high dose group. In terms of DQ concentration in gastrointestinal tissues, the highest concentrations of DQ were found in the stomach and small intestine from 15 minutes to 1 hour and in the colon at 3 hours. By 36 hours after poisoning, the concentrations of DQ in all parts of the stomach and intestine in the low and medium dose groups had decreased to lower levels. Gastrointestinal tissue (except jejunum) DQ concentrations in the high dose group tended to increase from 12 hours. Higher doses of DQ were still detectable [gastric, duodenal, ileal and colonic DQ concentrations of 6 400.0 (1 232.5), 4 889.0 (6 070.5), 10 300.0 (3 565.0) and 1 835.0 (202.5) mg/kg respectively]. Light microscopic observation of morphological and histopathological changes in the intestine shows that acute damage to the stomach, duodenum and jejunum of rats was observed 15 minutes after each dose of DQ, pathological lesions were observed in the ileum and colon 1 hour after exposure, the most severe gastrointestinal injury occurred at 12 hours, significant reduction in villi height, significant increase in crypt depth and lowest V/C ratio in all segments of the small intestine, damage begins to diminish by 36-hour post-intoxication. At the same time, morphological and histopathological damage to the intestine of rats at all time points increased significantly with increasing doses of the toxin. CONCLUSIONS: The absorption of DQ in the digestive tract is rapid, and all segments of the gastrointestinal tract may absorb DQ. The toxicokinetics of DQ-tainted rats at different times and doses have different characteristics. In terms of timing, gastrointestinal damage was seen at 15 minutes after DQ, and began to diminish at 36 hours. In terms of dose, Tmax was advanced with the increase of dose and the peak time was shorter. The damage to the digestive system of DQ is closely related to the dose and retention time of the poison exposure.