Effects Of Mankuining Recipe On Colonic Oxidative Stress And Cell Pyroptosis In Mice With Ulcerative Coliti

Ulcerative colitis(UC)is a chronic nonspecific bowel disease characterized by diffuse and persistent inflammation of the colorectal mucosa,clinically characterized by abdominal pain,diarrhea,tenesmus,and mucopurulent stool.UC is often recurrent in clinical practice,with a long treatment period and a prolonged ulcer,which is an important high-risk factor for colorectal cancer.Traditional Chinese medicine has unique advantages in the treatment of UC.Mankuining decoction was created by Academician Dong Jianhua,which is an agreement prescription of the traditional Chinese medicine spleen and stomach disease department of Dongzhimen Hospital and has been clinically applied and experimentally proved to have significant efficacy,but its mechanism of action is still unclear.Therefore,this project intends to explore the therapeutic mechanism of DSS-induced UC mice through animal experimental studies.Objective:(1)To explore the therapeutic effect of Mankuining decoction on the symptoms of colitis in UC mice.(2)Based on nuclear transcription factor E2 correlated factor(NRF2)/heme plus oxygen1(HO-1)/reactive oxygen species(ROS).Oxidative stress regulation pathway to explore the regulatory effect of Slow Depression on DSS-induced oxidative stress in UC mouse models.(3)Based on NOD-like receptor protein 3(NLRP3)/cysteine aspartate proteolylase-1(Caspase-1)/Gasdermin D(GSDMD)cell pyroptosis pathway to explore the slow anti-ROS mediation Induced effects of coke in UC mouse colonic epithelial cells.Method:(1)40 male C57BL/6 mice were randomly divided into UC mouse models induced by free drinking of dextran sodium sulfate(DSS)solution Normal group,model group,Mankuining decoction group,mesalazine group,10 animals in each group.Mice in the normal group drank purified water freely,and mice in the remaining groups drank 3%DSS solution freely,and the UC model was replicated 7 days after molding.The administration group was given gavage on the day of modeling,while the model group and normal group were given gavage with 0.9%normal saline once a day for 7 days.The general condition of the mice is recorded daily and the disease activity index(DAI)score is evaluated.On the 8th day,the mice were sacrificed by cervical dislocation,the colon was collected,and the colon length was recorded.Hematoxylineosin(HE)staining to observe histopathological changes in the colon.Enzyme-linked immunosorbent assay(ELISA)measures serum levels of tumor necrosis factor α(TNF-α).(2)Replicate the UC mouse model according to method(1),15 animals per group.The DCFH-DA fluorescent probe method was used to determine the ROS level in colonic tissues.ELISA detects markers of oxidative stress in colon tissue,superoxide dismutase(SOD),glutathione(GSH),malondialdehyde(MDA),myeloperoxidase(MPO),glutamyl cysteine ligase(GCL)levels.Westernblot detected NRF2,HO-1,NQO1,and colon tissues of mice in various groups.GCLC protein content.The effect of Mankuining decoction on the antioxidative stress pathway of NRF2/HO-1/ROS in UC mice was explored.(3)Replicate the UC mouse model according to method(1),15 animals per group.Elisa was used to detect serum interleukin-1β(IL-1β)and interleukin-18(IL-18)level.Westernblot detected the content of NLRP3/Caspase-1/GSDMD protein in the colon tissue of each group of mice.To investigate the effect of Mankuining decoction on ROS-mediated pyroptosis pathway in UC mouse NLRP3/Caspase-1/GSDMD cells.Results:1 The results of the efficacy evaluation showed that:(1)General condition and DAI score:After the UC model was induced by DSS,the modeled mice had different degrees of physical mass decline,stool trait changes and positive fecal occult blood test.Compared with the normal group,the body mass of mice in the model group decreased significantly(P<0.01)and the DAI score increased significantly(P<0.01).Compared with the model group,the UC symptoms of the mesalazine group and the Mankuining decoction group were milder,the body weight loss was less(P<0.01),and the DAI score was lower than that in the model group(P<0.01).(2)Colon length:compared with the normal group,the colon length of mice in the model group was significantly shortened(P<0.01).Compared with the model group,the colon length of mice in the mesalazine group and the Mankuining decoction group recovered(P<0.01).(3)Pathological staining of colonic HE:Compared with the normal group,the colonic mucosal damage of the model group was severe,and compared with the model group,the inflammatory infiltration and mucosal damage of the colon were reduced in the mesalazine group and the Mankuining decoction group.(4)TNF-α level:Compared with the normal group,the TNF-α level of mice in the model group was significantly increased(P<0.01).Compared with the model group,the level of colonic TNF-α decreased significantly in the mesalazine group and the Mankuining decoction group(P<0.01).2 Oxidative stress pathway detection showed that:(1)ROS level:Compared with the normal group,the ROS level of mice in the model group was significantly increased(P<0.01).Compared with the model group,both the Mankuining decoction group and the mesalazine group decreased significantly(P<0.01).(2)Levels of oxidative stress markers:Compared with the normal group,the levels of MDA and MPO in the model group increased significantly,and the contents of SOD and GSH were significantly reduced(P<0.01).Compared with the model group,the levels of MDA and MPO decreased in the Mankuining decoction group and the mesalazine group,and the contents of SOD and GSH increased(P<0.01,P<0.05).(3)Oxidative stress pathway protein expression:compared with the normal group,NRF2,HO-1,NQO1,The content of GCLC protein was significantly increased(P<0.01).Compared with the model group,the expression levels of NRF2,HO-1,NQO1 and GCLC proteins in colonic tissues of mesalazine group and Mankuining decoction group were obvious(P<0.01,P<0.05)was reduced,and there was no between-group difference in GCLC content.3 Cell pyroptosis pathway detection showed that:(1)IL-1β and IL-18 levels:compared with the normal group,the model group mice IL-1β and IL-18 The levels were significantly increased(P<0.01),and the levels of colonic IL-1β and IL-18 in the mesalazine group and the Mankuining decoction group decreased significantly,compared with the model group(P<0.01).(2)Expression of pyroptosis pathway protein:compared with the normal group,the content of NLRP3/Caspase-1/GSDMD protein in the colon tissue of mice in the model group was significantly increased(P<0.01).Compared with the model group,the expression level of NLRP3/Caspase-1/GSDMD protein in colon tissues of mesalazine group and Mankuining decoction group was significantly reduced(P<0.01,P<0.05).Conclusion:(1)Mankuining decoction has obvious therapeutic effect on DSS-induced colon injury in UC mice,which can improve colon inflammatory infiltration,fecal occult blood,weight loss and colonic mucosal injury,shorten colon length,and reduce colon TNF-α level in UC model mice,and its efficacy is similar to that of mesalazine.(2)Mankuining decoction can reduce the ROS content in the colon and reduce the degree of oxidative stress in the colon of UC mice by regulating the NRF2/HO-1 antioxidant pathway.It can also reduce cell pyroptosis,alleviate inflammatory response and reduce colon injury in UC mice by regulating ROS levels,inhibiting NLRP3/Caspase-1/GSDMD pathway.