| Objection:In most recent years, as NOD receptor was discovered and NLRP3 as a typical example in NLR family was thoroughly researched, NLRP3 inflammasome was discovered playing an important role in maintaining intestine in a steady state and intestinal inflammation generating process. According to literature reports, NLRP3 widely spreads in gastrointestinal mucosa epithelial cells and T-, B-lymphocytes and they could be activated by all kinds of molecules, germs or virus to form inflammasome. Once NLRP3 within intestine epithelial cells detects pathogen or dangerous signals, it could convene ASC molecule and activate caspase-1 to assembly a kind of macromolecule protein complex inflammasome to further activate cells to secrete proinflammatory cytokines as IL-1 β, IL-18, IL-33 to get involved in intracellular inflammaroty response and immune response to finally keep steady state of intestine. Thus, the analysis of the transformation regulations of NLRP3 inflammasome activationaccess during UC acute and chronic pathological evolution is of vital importance in discussing the molecular mechanism of genesis and development of UC colonic mucosa acute and chronic inflammation.This disease is based on the attack of weakness of spleen, so yiqijianppi is the primary way to UC treatment. Buzhongyiqitang is the representative compound according to yiqijianppi that performs the function of buqijianpi, tishengzhongqi. This experiment adopts buzhongyiqitang to cure UC in mice. With the observation of the expression of NLRP3 inflammasome and its backward signal access cell factors, we discuss if the acting mechanism that buzhongyiqitang in preventing and curing UC is connected with controlling intestine NLRP3 flammasome activation access, and further clinic is expected to be guided to provide scientific evidence for clinical application of y1q1j1anpp1.Contents:Test one:Ulcerative colotis model on C57BL/6 mice and the effect of buzhongyiqitangAIM:We developed an experimental mice model of ulcerative colitis (UC) and dynamiclly monitored the colonic injures, pathological changes, content variation of IL-1β, IL-18, IL-33 in mice plasma and colonic mucosa, and evaluated the model.Methods:64 C57BL/6 mice were randomly divided into normal group and model group. Model group were fed with 3%dextran sulfate sodium (DSS) solution for a week, following pure water for another 3 weeks. Dynamic observation were made on mice feces condition, alteration of colon length and weight, pathological changes, colonic mucosa inflammation score and content variation of IL-1P, IL-18, IL-33 in mice plasma, and colonic mucosa every week.Results:After modeling for a week, mice appeared diarrhea, bloody stools, crouching, and curling up piles. Pathological observation showed colon epithelial erosion, bleeding, multifocal ulcers and a mount of inflammation cells infiltrating the mucosa and submucosa. Compared to the normal group, the weight of model group markedly increased (P< 0.05), the length of the model group significantly shortened (P<0.01), colonic mucosa inflammation score elevated (P<0.01), content of IL-1 β, IL-18, IL-33 elevated in both plasma and mucosa (P< 0.05, P< 0.01). At the end of the second week, the model group gradually bleeding less, compared to the normal group, a mount of inflammation cells could be seen in the submucosa, colon weight increased (P< 0.01), colon length shortened (P<0.01), colonic mucosa inflammation score is increased (P< 0.01), content of IL-1β, IL-18 in plasma and IL-18 in colonic mucosa is increased dramatically (P< 0.05). At the third and fourth week, the activity and feces condition of mice turned back to normal, inflammation cells could be seen in colonic mucosa, muscle fibers derangement, colon weight increased (P<0.01), colon length shortened (P< 0.01), compared to normal group, colonic mucosa inflammation score and content of IL-1β, IL-18, IL-33 in model group has no statistical difference (P>0.05).Test Two:the effect of the buzhongyiqitang on the gene expression of NLRP3/ASC/caspase-1 in colonic mucosa of UC miceNLRP3 is widely expressed in the epithelial cells of gastrointestinal mucosa and some are expressed in granular leucocytes, T- and B-lymphocytes. NLRP3 is located in the pattern recognition receptor (PRR) of the cytosol and it is the detector within cytoplast to sense exogenous bacteria and endogenous dangerous signals that could be activated by all kinds of molecures, bacteria or virus to form the inflammasome and participate in intestine immune and inflammatory response. It could mediate organisms intrinsic immune to adjust the immune of organism and thus to play an important role in ensuring the steady state of intestine. NLRP3 inflammasome consists of three parts as NLRP3, ASC and caspase-1. The influence of the compound medicine on rat intestine NLRP3 inflammasome is observed during the cure process of buzhongyigitang on UC mice.Methods:Real time PCR method was used to test the mRNA expression of NLRP3, ASC, Caspase-1 in colonic mucasa on control group respectively as 1〠2ã€3ã€4 week and model group, large dose of buzhongyigitang group, middle dose of buzhongyigitang group, small dose of buzhongyiqitang group and positive group. SPSS13.0 statistic software was adopted.Result:The content of NLRP3, ASC, caspase-1mRNA of colonic mucosa in model group is comparatively lower than that in control group, and it is obviously lower than normal groups. NLRP3 mRNA expression in the colonic mucosa of buzhongyiqitang Large dose group is obviously higer than that of model group P<0.05,P1=0.039;P2=0.031;P3=0.036;P4=0.044); ASC mRNA expression in the colonic mucosa of buzhongyigitang Large dose group is obviously higer than that of model group, (P<0.05, P<0.01, P1=0.039; P2=0.000; P3=0.002; P4=0.019); caspase-1 mRNA expression in the colonic mucosa of buzhongyiqitang high dose and intermediate dose groups are respectively, which are higer than that of model group (P<0.05, P< 0.01, P1=0; P2=0.003; P3=0.028;Pâ– =0.045).Test Three:the effect of the buzhongyiqitang on UC rat colonic mucosa and plasma cell factors as IL-1β/IL-18/IL-33The activation of NLRP3 inflammasome could cause the inactive caspase-1 precursor to be transformed into active caspase-1. Caspases-1 could split into interleukins as (IL)-1β, IL-18, IL-33 precursor which is the active form and secrete to perform a series of cell procedures that could cause inflammatory response or death of cells. IL-1β,IL-18,IL-33 are important medium in inflammation response and perform important function in genesis and development in ulcerative colitis.Methods:Fast cure 24hr on rats after the treatment and break their cervical vertebras to kill them. Laparotomize them and get out their whole colons very quickly. Then measure the lenghth of colons and cut them out as per vertical axis. Take use of running water first and then and frozen normal syline twice to clean them. Mucus on the surface of intestinal mucosa shall be slightly striken off by microslides. Rinse them again with frozen normal syline and weigh them. Then homogenizer with 3 times normal syline is adopted to completely homogenate them,5000xg,4℃ with 5min centrifuge. Finally the supernate is prepared to be tested. Rats’s blood is collected into well-prepared EDTA anticoagulation test tubes with the method as eye elucleation. After specimens are collected, centrifuge them with 15min,4℃ 1000xg in 30 min. The supernate shall be extracted and kept for use in-20℃. Method ELISA is adopted to test contents of intestine mucosa and IL-1β,IL-18,IL-33 in plasma. SPSS13.0 statistic software is adopted.Results:The content of IL-1β,IL-18, IL-33 of colonic mucosa in model group is comparatively higher than that in control group. P< 0.05, P< 0.01, Pmucosa=0.033, Pplasma=0.002; In the third and fourth week, there is no significantly difference between treated groups and model group. IL-1β content in the colonic mucosa of buzhongyiqitang high dose and middle dose groups are lower than that of model group (P< 0.01). Conclusion:The observed indicator results embody the pathological process of UC model induced by DSS developing from acute to chronic, this mice model co uld be used in the further study on UC mechanism and medicine treatment. T he expression of NLRP3, ASC and caspase-1 in mucosa was markedly increased in acute phase of UC, and the content of the downstream inflammatory cyt okines were also highly increased and regulate the immune process. Buzhong yiqitang down-regulate the content of NLRP3, ASC and caspase-1 in mucosa i n the paracmasis. |
