MCC-950 Improves Intestinal Congestion Reperfusion Injury In Mice By Inhibiting NLRP3-Mediated Pyroptosis

Background: In clinical practice,various intestinal autoimmune diseases,volvulus,intussusception,intestinal transplantation,liver transplantation and other diseases and abdominal surgery may lead to intestinal congestion and reperfusion injury,such as liver transplantation Before the diseased liver of the donor patient,the portal vein needs to be clamped,so that the blood return of the mesenteric vein and its subordinate branches is blocked and congestion occurs.The return of blood to the reflux causes intestinal congestion-reperfusion injury(Intestinal Congestion-reperfusion Injury,ICRI).Clinically,specific drugs for targeted treatment of ICRI are still in the blank field.Based on the exploration of the mechanism of ICRI occurrence and development,our research group found in previous studies that pyroptosis was involved in the occurrence of ICRI and the release of inflammatory factors in mice.Therefore,the purpose of this study is to establish a mouse ICRI model to explore whether it can improve mouse ICRI by inhibiting NLRP3-mediated pyroptosis,and to understand the occurrence and development of ICRI and reduce pyroptosis-related inflammation.Response and clinical treatment measures provide new evidence.Objective: To establish an animal model of mouse ICRI,detecting the expression of related genes,proteins,and inflammatory factors in the pyroptosis pathway,and to explore the effect and possible role of NLRP3 inhibitor MCC-950 in inhibiting pyroptosis on mouse ICRI mechanism.Method: 186 male C57BL/6 mice(8-10 weeks old,body weight 20±2g)were used in this experimental study.All mice were divided into three groups:(1)Sham operation sham group(Sham group);(2)ICRI model group;(3)ICRI+MCC-950 group,in which 12 mice were in each group,and the observation time was 7 days of subsistence analysis.According to the time after ICRI occurred,12 h,24h,3d,and 7d were selected as the sample collection time points,and 15 surviving mice were retained for sample collection at each time point in each group.The sham group was the normal sham group.Mice in our study group had the mesenteric vein without ligation only exposed,and the specimens were collected directly after 1 h.In the sham group,the mice were also only exposed to the mesenteric vein without ligation,and were given the NLRP3 inhibitor MCC-950 1 h later,marked as Sham(M)compared with the ICRI+MCC-950 group.Using an 11-0 silk thread with a needle to ligate the mesenteric vein to establish the intestinal congestion reperfusion injury(ICRI)model was done.Based on the previous research results regarding our research group,the parameters in the mouse ICRI model,the length of the intestinal tube of congestion was 15 cm,and the duration of congestion was 1h.Among them,the ICRI +MCC-950 group was administered once immediately after modelling,and then intraperitoneally injected with MCC-950 20 mg/kg/d at the same time point of the first administration daily.After 1 hour of congestion,microscopic forceps were used to loosen the slipknot of the previously ligated vein,restoring the mesenteric venous return,and causing congestion reperfusion.Orbital blood collection mobile phone ICRI 12 h,24h,3d,7d mouse blood samples were centrifuged to extract supernatant.Mice were sacrificed and the harvesting of intestinal tissue samples at each time point was done.The serological samples of mice were frozen for later use,and the expressions of pyroptosis-related inflammatory factors IL-1β and IL-18 were detected by ELISA.HE staining technique was used to detect pathological changes in mice small intestinal tissue specimens at each time point.The evaluation was scored by Chiu’s intestinal mucosal injury scoring method,and Chi-Square Test was used to compare between groups post-scoring.Immunohistochemical staining was used to detect the expression of pyroptosis-related proteins NLRP3,Caspase-1,and GSDMD in the mice of each group at each time point.RT-PCR method was used to detect the expression levels of NLRP3 and Caspase-1 m RNA in the intestinal tissue of mice,and Western Blot was used to detect the pyroptotic protein: nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),expression of caspase-1(Caspase-1).The survival conditions of the mice were recorded and the survival curves were drawn(Kaplan-Meier method)with Graph Pad prism software and in comparison between groups,the Log-rank test was used.Results:(1)The survival rate of the ICRI group was 50%,and after treatment with the NLRP3 inhibitor MCC-950,the survival rate of the mice increased to 75%;(2)HE staining technique of the small intestine tissue of the mice in the Sham group showed that under a light microscope,it could be seen that the intestinal mucosa structure was complete,the villi were arranged neatly,the length of the villi,and the number of crypt glands were normal.At different time points in the ICRI group,the intestinal mucosa and villi structure were damaged to varying degrees under the light microscope.At 12 hours,the shortening of intestinal villi,loss of the top,expansion of the mucosa and subepithelial space,shedding of the lamina propria,and formation of local ulcers were observed,accompanied by a large number of erythrocytes.After24 hours,the Gruenhagen gap still expanded,accompanied by several inflammatory cell infiltrations.From the 3rd day to the 7th day after ICRI,the acute inflammation gradually became chronic,and the related damage caused by ICRI was resolved.In the ICRI+MCC-950 group,at each observation time point corresponding to ICRI,the intestinal mucosal structure damage,inflammatory cell infiltration,and red blood cell stasis were significantly improved.(3)Pyroptosis-related genes increased with the prolongation of the time after ICRI,reached the peak and then levelled off.Compared with the Sham group,the corresponding time points of pyroptosis-related genes Caspase-1,GSDMD,and NLRP3 in the ICRI group The expression of m RNA in all cells increased;compared with ICRI group,the expression of Caspase-1,GSDMD,and RNA in ICRI+MCC-950 group decreased at corresponding time points;(5)Immunohistochemistry and Western blot detection of cells expression of pyroptosis-related proteins,compared with the Sham group,the expression of pyroptosis-related proteins Caspase-1 and NLRP3 in the ICRI group increased at the corresponding time points in comparison with the ICRI group,the expression of Caspase-1 in the ICRI+MCC-950 group,NLRP3 protein expression decreased at the different time points;(6)ELISA detection of pyroptosis-related inflammatory factors,compared with the Sham group,the levels of serum inflammatory factors IL-1β and IL-18 at each time point in the ICRI group compared with the ICRI group,the levels of serum inflammatory factors IL-1β and IL-18 in the ICRI+MCC-950 group were significantly lower.Conclusions:(1)NLRP3 inhibitor improved the survival rate of ICRI mice and significantly reduced the damage of intestinal microvilli,nucleus and intestinal mucosa.(2)The intervention of NLRP3 inhibitor inhibited the expression of pyroptosis-related proteins and genes in intestinal tissues of ICRI mice,and alleviated the release of pyroptosis-related inflammatory factors in IL-1β and IL-18 cells.