Effect Of Banxia Xiexin Decoction On The Inflammatory Response Of Rats With Ulcerative Colitis Based On The NLRP3/Caspase-1 Pyroptosis Signaling Pathwa

Purpose:To investigate the effect of Banxia Xiexintang（BXT）on the pathway NOD-like receptor protein 3（NLRP3）/cysteinyl aspartate-specific protease-1（Caspase-1）pyroptosis pathway and its downstream factors in dextran sulfate sodium（DSS）-induced ulcerative colitis（UC）rats,and to explore the molecular mechanism of BXT in preventing and treating UC.Material and methods:Male SPF SD rats were randomly divided into a normal group,a UC model group,a low-dose Banxia Xiexin decoction group（low-dose BXT group）,and a high-dose Banxia Xiexin decoction group（high-dose BXT group）.From the first day of the experiment,except for the normal group,the rats in the other groups were given intragastric administration of 2.5%DSS solution every day,and the UC model was successfully established on the 10th day.From the 11th day of the experiment,rats in low-dose BXT group and high-dose BXT group were given intragastric administration of BXT solution 6.3 g（kg·d）^-1and 12.6 g（kg·d）^-1respectively,2m L/time,once a day for 7 consecutive days.The rats in normal group and UC model group were given the same amount of sterile normal saline.During the experiment,the general state of the rats was observed.After 24 hours of fasting,the rats were killed to obtain samples after BXT administration.Colonic tissue was collected to evaluate the changes of colon mucosa damage index（CMDI）score in each group.The pathological changes of colonic tissue were observed by hematoxylin-eosin staining（HE）.The levels of IL-1βand IL-17 in serum of rats were detected by enzyme linked immunosorbent assay（ELISA）.Real-time Quantitative Polymerase Chain Reaction（RT-q PCR）was used to detect the expression of NLRP3,Caspase-1,GSDMD and IL-1βm RNA in colon tissue.Western blot was used to detect the protein expression of NLRP3,Caspase-1,GSDMD,and IL-1βin colonic tissues.Immunohistochemistry（IHC）was used to detect the localized expression of NLRP3,Caspase-1 and GSDMD proteins in colon tissues.Results:1.Effect of BXT on general condition of UC ratsOn the 10th day of the experiment,the rats in the normal group were sensitive,like to eat well,their hair was smooth and shiny,their weight increased steadily,and their stools were normal.The rest of the rats in each group treated with DSS solution were tired,haggard,huddled,obviously emaciated,thin and irregular stool,bloody stool and even mucous bloody stool.During the administration of BXT,no obvious abnormality was found in the normal group.The rats in the UC model group still showed mental distress,anorexia,inactivity,decreased body mass and hematochezia.Compared with the UC model group,the mental state of rats in the low-dose and high-dose BXT group was significantly improved,the range of motion and food intake increased,the hair showed slightly luster,the stool began to form,and the hematochezia was significantly improved,especially in the low-dose BXT group.2.Effect of BXT on CMDI score of UC ratsCompared with the normal group,the CMDI score of the UC model group was significantly higher,the CMDI score of the low-dose BXT group was significantly lower than that of the UC model group,and the CMDI score of the high-dose BXT group was significantly lower.3.Effect of BXT on histopathological changes of colon in UC ratsIn the normal group,the structure of colonic mucosa was intact,the glands were arranged regularly,there was no obvious inflammatory cell infiltration,and there was no cell injury.In the UC model group,the structure of colonic mucosa was destroyed,the structure of glands was disordered or even disappeared,many inflammatory cells infiltrated in the submucosa,and the mucosa was defective and exfoliated.Compared with the UC model group,the colonic mucosal structure of rats in the low-dose BXT group and the high-dose BXT group gradually recovered,the structure and arrangement of glands were improved,and the infiltration of inflammatory cells was lighter,and there was no significant difference between the treatment groups.4.Effect of BXT on the contents of IL-1βand IL-17 in serum of UC rats.The contents of IL-1βand IL-17 in serum of rats in UC model group were significantly higher than those in normal group（P<0.01）,while those in low-dose BXT group and high-dose BXT group were significantly lower than those in UC model group（P<0.01）.5.Effect of BXT on the expression of NLRP3,Caspase-1,GSDMD and IL-1βin colonic tissue of UC ratsCompared with the normal group,the expression of NLRP3,Caspase-1 and IL-1βm RNA in the UC model group was significantly increased（P<0.01）,and the expression of GSDMD m RNA was significantly increased（P<0.05）.Compared with the UC model group,the expression of NLRP3,Caspase-1 and GSDMD m RNA in the low-dose BXT group was significantly reduced（P<0.01）,the expression of IL-1βm RNA was significantly reduced（P<0.05）,the expression of NLRP3 m RNA in the high-dose BXT group was significantly reduced（P<0.01）,and the expression of Caspase-1,GSDMD,and IL-1βm RNA was significantly reduced（P<0.05）.6.Effect of BXT on the expression of NLRP3,Caspase-1,GSDMD and IL-1βprotein in colonic tissue of UC ratsCompared with the normal group,the expression of NLRP3,GSDMD and IL-1βproteins in the colon tissue of rats in the UC model group was significantly increased（P<0.01）,and the expression of Caspase-1 protein was significantly increased（P<0.05）.Compared with the UC model group,the expression of NLRP3,Caspase-1 and IL-1βproteins in the low-dose BXT group was significantly reduced（P<0.01）,and the expression of GSDMD proteins was significantly reduced（P<0.05）.In the high-dose BXT group,the expression of Caspase-1protein was significantly reduced（P<0.01）,the expression of NLRP3 protein was significantly reduced（P<0.05）,and the expression of GSDMD and IL-1βprotein was not statistically significant.7.Effect of BXT on the expression of NLRP3,Caspase-1 and GSDMD in colonic tissue of UC rats（Immunohistochemistry）The positive expressions of NLRP3,Caspase-1 and GSDMD proteins in the colon of normal rats were less,and the staining was lighter.Compared with the normal group,the positive expression of NLRP3,Caspase-1 and GSDMD protein in the colon tissue of rats in the UC model group was significantly increased,the staining was deepened,and the average optical density was significantly increased（P<0.01）.Compared with the UC model group,the positive expressions of NLRP3,Caspase-1 and GSDMD in the colonic tissue of rats in the low dose BXT group and the high dose BXT group were significantly decreased,the staining became lighter and the average optical density decreased significantly compared with the UC model group,and the effect was more obvious in the low dose BXT group（P<0.01）.Conclusions:1.BXT can improve diarrhea,hematochezia and other typical symptoms of UC rats,improve CMDI score and pathological changes of colon tissue,and reduce inflammation.2.BXT can inhibit the expression of NLRP3/Caspase-1 and its downstream factors in pyroptosis signal pathway,thus attenuating the inflammatory response induced by DSS in UC rats.