The Mechanism Of GSDMD On Intestinal Mucosal Barrier Injury In Mouse Model With Severe Acute Pancreatitis By Mediating Pyroptosis

Objective: Severe acute pancreatitis(SAP)is often accompanied by intestinal mucosal barrier injury.In recent years,pyroptosis has been one of the research hotspots in inflammatory diseases,and Gasdermin D(GSDMD)is a key protein of cell pyroptosis.After Caspase activation,GSDMD can be lysed into N-terminal and C-terminal,in which the N-terminal can be perforated on the cell membrane to make the cell pyroptosis.Pyroptosis plays an important role in the intestinal injury of infectious diseases,but it has not been reported whether it plays a role in the intestinal mucosal barrier function injury caused by SAP.This study mainly explores the mechanism of GSDMD in the intestinal injury caused by SAP,and provides new ideas for the treatment of SAP intestinal mucosal barrier injury.Methods: Twenty-four C57BL/6 mice were divided into normal saline(NS)group,small interfering RNA(siRNA-NS)group,SAP model group and siRNA-SAP group according to random number table method,with 6 mice in each group.Before the experiment,the mice fasted for 18 h and could not help drinking.The SAP mouse model was reproduced by intraperitoneal injection of caerulein 50 μg/kg combined with lipopolysaccharide(LPS)10 mg/kg;the NS group was given the same amount of NS;siRNA-SAP group and siRNA-NS group were injected with 50 mg/kg siRNA three times via tail vein before modeling or injection of NS.The blood of mice eyeball in each group was taken 12 hours after modeling,and serum interleukins(IL-1β,IL-18)levels were detected by enzyme linked immunosorbent assay(ELISA).The mice were killed to observe the gross changes in the abdominal cavity.Pancreas and ileum tissues were taken and the pathological changes of pancreatic and intestinal mucosa were observed under light microscope.The expression of long non-coding RNA uc.173(lnc uc.173)in intestinal tissues was detected by reverse transcription-polymerase chain reaction(RT-PCR).The expression of tight junction band Occludin-1(ZO-1)and Occludin in intestinal mucosal epithelial cells were detected by immunohistochemistry.Western blotting was used to detect GSDMD protein expression in intestinal tissues.Results: The gross observation showed that there were no obvious abnormal changes in the abdominal cavity of mice in the NS group and the siRNA-NS group,while there were a small amount of bloody ascites,intestinal congestion and edema in the SAP group.Compared with the SAP group,the siRNA-SAP group showed less bloody abdominal water volume,mild symptoms,and intestinal congestion and edema.HE results showed that under light microscope,the pancreas of the mice in the NS group and the siRNA-NS group had no obvious changes,and there was no obvious damage to the intestinal mucosa.The pancreas of the SAP group mice had hemorrhage,necrosis,inflammatory cell infiltration,and some lobular structures were destroyed.At the same time,the intestinal tissues showed varying degrees of villi falling off and broken,inflammatory cell infiltration,part of the intestinal atrophy and necrosis.The siRNA-SAP group mice had inflammation of the pancreas.Cell infiltration,pancreatic cell edema,and partial destruction of the lobular structure,but the degree of pathological damage was less than that of SAP,the intestinal mucosa was infiltrated by inflammatory cells to varying degrees,and the intestinal villi were partially atrophied and shedding.The difference between the two was statistically significant(P<0.05).RT-PCR showed that the expression of lnc uc.173 in the intestinal tissue of the SAP model group was significantly lower than that of the NS group and the siRNA-NS group(2-ΔΔCt: 0.26±0.12 vs.1.01±0.37,0.67±0.32,both P< 0.05).The expression of lnc uc.173 in the siRNA-SAP group was significantly higher than that in the SAP group(2-ΔΔCt: 0.60±0.39 vs.0.26±0.12,P<0.05).Western blotting showed that the expression level of GSDMD protein in the intestinal tissue of the SAP model group was significantly higher than that of the NS group and the siRNA-NS group [GSDMD protein(GSDMD-N/β-actin): 1.99±0.46 to 1,1.00±0.78,both P < 0.05];Compared with the SAP model group,the expression of GSDMD protein in the siRNA-SAP group was significantly decreased [GSDMD protein(GSDMD-N/β-actin): 1.42±0.42 vs.1.99±0.46,P<0.05].Immunohistochemical staining results showed that ZO-1 and Occludin in the NS group and siRNA-NS group were distributed along the intestinal epithelial cells,showing a strong dark tan signal and a large number of cells.ZO-1 and Occludin in the SAP group and siRNA-SAP group were distributed along the intestinal epithelial cells,showing no significant difference in distribution position from that in the NS group,but showing a light tan color.Compared with the NS group,ZO-1 and Occludin were lower in number and their relative expression was decreased.ZO-1 [18.2±1.75,18.12±1.64,6.62±1.06,11.38±1.06],Occludin [16.17±0.98,15.56±1.75,7.00±0.63,12.00±0.63] in four groups showed statistically significant difference(all P < 0.05).ELISA results showed that the concentrations of IL-1β and IL-18 in SAP group and siRNA-SAP group were higher than those in NS group.The expression levels of IL-1β and IL-18 in serum of four groups of mice [(44.48±5.76,81.49±10.75,146.66±1.40,116.26±15.54)ng/L]and [(115.43±16.4,84.84±21.9,950.47±177.09,689.96±126.08)ng/L] were compared.The differences were statistically significant(P < 0.05).Conclusion: Systemic inflammatory response and intestinal mucosal barrier injury in SAP mice may be related to the increased expression of GSDMD in intestinal tissues.The content of long non-coding RNA uc.173 in the intestine of SAP mice decreased,and the ability of intestinal mucosa to self-repair was weakened.At the same time,the experimental results showed that GSDMD increased the release of IL-1β and IL-18 by mediating cell pyroptosis,and at the same time down-regulated the expression of adhesion proteins ZO-1 and Occludin in the intestine of SAP mice,leading to intestinal mucosal barrier damage.