| BackgroundUlcerative colitis(UC)is a chronic and recurrent disease and one of the common subtypes of inflammatory bowel disease.UC is characterized by recurrent diarrhea,abdominal pain,and mucus,pus,and blood in the stool.The incidence of UC in my country has increased rapidly in the past 10 years.The pathogenesis of UC is complex.The intestinal mucosal barrier damage is an important pathophysiological basis for the pathogenesis of UC.Mucosal healing is the key endpoint of UC treatment.However,the regulatory mechanism that maintains the integrity of the intestinal mucosal barrier and promotes the repair of intestinal epithelial injury has not been elucidated.Macrophages,as the main effector cells in the innate immune system,play a key role in the process of UC inflammatory damage and epithelial repair.Macrophage dysregulation is a prominent feature of UC mucosal immune disorders,and it is also a key target for mucosal healing and homeostasis.The current treatment plan of UC still has the problems of difficulty in maintaining clinical remission for a long time after drug withdrawal and non-response in some patients,which seriously affects the quality of life of patients and leads to a high medical burden.Given the strong plasticity of macrophages and their regulatory role in intestinal homeostasis,macrophages have become the focus of UC treatment in recent years.Recent studies have shown that ?-catenin/FOSL2/ARID5A may be a key pathway to regulate the polarization of macrophages,but its role in the occurrence and development of UC needs to be further clarified.More and more research evidence supports the good clinical effects of traditional Chinese medicine in the treatment of UC,which can effectively relieve the clinical symptoms of patients and improve the prognosis,but the specific mechanism of action needs to be further clarified.The heat-clearing and dampness compound Modified Gegen Qinlian Decoction is an empirical formula formulated by the instructor,Professor Tang Xudong,based on the understanding of the basic pathogenesis of UC and combined with many years of clinical experience.The results of previous animal experiments showed that Modified Gegen Qinlian Decoction can alleviate the symptoms of colitis in mice induced by Dextran sodium sulfate(DSS)and improve the ultrastructure of colonic mucosa,but its mechanism of action needs to be further elucidated.Therefore,we thought about the mechanism of Modified Gegen Qinlian Decoction to improve the symptoms of UC and reduce the damage of intestinal mucosa.Is it related to the regulation of intestinal macrophage polarization?What are the pathways and targets that Modified Gegen Qinlian Decoction regulates the polarization of macrophages and promotes intestinal epithelial repair?Whether it is related to the ?-catenin/FOSL2/ARID5 A pathway?This is worthy of our further discussion.Part One Clinical study of the polarization state of macrophages in UC patientsObjectiveTo clarify the polarization state of colonic macrophages and the expression of ?-catenin/FOSL2/ARID5 A protein in patients with UC.Method1 Using multi-factor detection methods,the serum inflammatory factors(IP-10,IL-12p70,TNF-?,IL-1?,IL-12p40,IL-23,IL-6,IL-4.The content of IL-10,Arginase,TARC,IL-1RA)was tested.2 Immunohistochemistry was used to detect the expression of macrophage polarization-related proteins FOSL2 and ARID5A in colon biopsy tissues of UC patients and healthy subjects.3 Immunofluorescence was used to detect the expression of intestinal mucosal macrophage polarization-related proteins CD68,CD 163,iNOS,and the key proteins of intestinal mucosal barrier ZO-1,Occludin,E-cadherin,and ?-catenin in UC patients and healthy subjects.Result1 Compared with healthy subjects,the expression level of pro-inflammatory factor IP-10 in UC patients was significantly increased(p<0.01),anti-inflammatory factors IL-10(p<0.05),IL-4(p<0.05),Arginase(p<0.001),IL-1RA(p<0.01)expression levels were significantly reduced.2 Compared with healthy subjects,the expression levels of intestinal mucosal barrier-related proteins ZO-1,Occludin,E-cadherin,and ?-catenin in the colon tissue of UC patients were significantly reduced(p<0.05).3 Compared with healthy subjects,the expression level of M1 macrophage marker protein iNOS in the colon tissue of UC patients was significantly increased(p<0.05),and the expression level of M2 marker protein CD 163 was significantly decreased(p<0.05).Compared with the healthy control group,the expression level of macrophage polarization-related protein FOSL2 in the colon tissue of UC patients was significantly decreased(p<0.05),and the expression level of ARID5A was significantly increased(p<0.05).Conclusion(One)1 There is an imbalance of inflammatory factors in patients with UC,which is specifically manifested as the up-regulation of pro-inflammatory factors IP-10,and the down-regulation of anti-inflammatory factors IL-10,IL-4,Arginase,and IL-1RA.2 UC patients have intestinal mucosal barrier damage,and the expression levels of key proteins of the intestinal mucosal barrier ZO-1,Occludin,E-cadherin,and ?-catenin are reduced.3 UC patients have abnormal macrophage polarization and abnormal expression of macrophage polarization-related pathway ?-catenin/FOSL2/ARID5A,which is manifested as an increase in M1 macrophages,a decrease in M2 macrophages,and a decrease in the expression levels of ?-catenin and FOSL2,ARID5A expression level increased.4 The abnormality of ?-catenin/FOSL2/ARID5A signaling pathway may lead to the imbalance of macrophage polarization,which in turn leads to the imbalance of inflammatory factors and the damage of the intestinal mucosal barrier.Part Two:Study on the Effect Mechanism of Modified Gegen Qinlian Decoction on Regulating Macrophages Polarization in UC In vivo:The effect of Modified Gegen Qinlian Decoction on DSS-induced colitis macrophage polarizationObjectiveClarify the curative effect of Modified Gegen Qinlian Decoction in the intervention of DSS-induced acute colitis mouse model.Using macrophage polarization as the starting point to clarify the mechanism of the effect of Modified Gegen Qinlian Decoction.Method1 Using the DSS-induced acute colitis mouse model as a carrier,the mice were randomly divided into a normal group,a model group,a low-dose group of Modified Gegen Qinlian Decoction,a medium-dose group of Modified Gegen Qinlian Decoction,and a high dose group of Modified Gegen Qinlian Decoction.In the dose group,the weight of mice,disease activity index,spleen weight,colon length,colon histopathological score,and electron microscope were used to evaluate the intervention effect of Modified Gegen Qinlian Decoction.2 Observe the effect of Modified Gegen Qinlian Decoction on the intestinal mucosal barrier:detect mouse intestinal mucosal permeability,colon tissue myelinated peroxidase content,malondialdehyde content,and use immunohistochemistry to detect colonic BrdU expression to assess epithelial proliferation status.Immunohistochemistry was used to detect the expression of colonic E-Cadherin,?-catenin,ZO-1,and Occludin to assess the intestinal mucosal barrier damage.3 Observe the effect of Modified Gegen Qinlian Decoction on macrophage polarization.Use flow cytometry to detect the content of M1/M2 macrophage subtypes in mouse spleen and colon tissue.Use immunofluorescence to detect the content of F4/80 and CD206 in mouse colon tissue,elisa was used to detect the content of TNF-?,IL-1?,IL-6,and IL-10 in colon tissue.4 Based on ?-catenin/FOSL2/ARID5A signaling pathway,study the molecular mechanism of Modified Gegen Qinlian Decoction to regulate the polarization of macrophages.Western blot was used to detect the expression of ?-catenin,FOSL2,and ARID5A protein in colon tissue,and the expression of ?-catenin,FOSL2,and ARID5A mRNA was detected by Real time PCR.Result1 General situation of mice.Compared with the normal group,mice in the model group showed arched backs,erected hair,reduced activity,weight loss,diarrhea,blood in the stool,significantly shortened colon length,significantly increased spleen weight,and histopathological score significantly elevated.Compared with the model group,the body weight of mice in each administration group of Modified Gegen Qinlian Decoction increased significantly(p<0.001),the DAI score of the high-dose Modified Gegen Qinlian Decoction group was significantly reduced(p<0.001),and the colon shortened in each administration group was significantly improved(p<0.05),the spleen weight was significantly reduced(p<0.05),and the histopathological scores of each administration group were reduced(p<0.05).Colon electron microscope results showed that the microvilli of the mice in the normal group were developed.The cells are full of mitochondria and vesicles.Intercellular junction complexes can be seen on the cavity side where cells are in contact with cells.Compared with the normal group,in the colon of the model group,most of the tight junction structures are disordered,fusion points are less common,and the intercellular space is widened.The intervention group of Modified Gegen Qinlian Decoction is between the normal group and the model group.2 Evaluation of the intestinal mucosal barrier.Compared with the normal group,the intestinal mucosal permeability of the model group was significantly increased(p<0.05),the MPO content was significantly increased(p<0.05),and the MDA content was significantly increased(p<0.05).BrdU expression decreased(p<0.05);compared with the model group,the intestinal mucosal permeability of each administration group was reduced(p<0.05),and the MPO content of each administration group was significantly reduced(p<0.05).The content of MDA in the low-dose and medium-dose Modified Gegen Qinlian Decoction groups was significantly reduced(p<0.05),and the expression of BrdU in the low-dose Modified Gegen Qinlian Decoction group was significantly increased(p<0.05).The results of immunohistochemistry showed that the expression of E-Cadherin,?-catenin,ZO-1 and Occludin in the model group decreased compared with the normal group(p<0.05).Compared with the model group,the medium-dose E-Cadherin group of Modified Gegen Qinlian Decoction,the expression of Occludin increased(p<0.05),the expression of ?-catenin increased in the low-dose and high-dose groups of Modified Gegen Qinlian Decoction(p<0.05),and the expression of ZO-1 increased in each dose group of Modified Gegen Qinlian Decoction(p<0.05).3 The test results of colonic inflammatory factors showed that compared with mice in the normal group,the contents of TNF-?.IL-1?,and IL-6 in the colon mucosal tissue of the model group increased(p>0.05),and the content of IL-10 decreased(p>0.05).Compared with the model group,the contents of IL-1? and IL-6 in each dose group of Modified Gegen Qinlian Decoction decreased(p<0.05);.4 Flow cytometry results:The spleen flow cytometry results showed that compared with the normal group,the proportion of M2 macrophages in the spleen of the model group was down-regulated(p<0.05);compared with the model group,the Modified Gegen Qinlian Decoction high-dose group The proportion of white blood cells was down-regulated(p<0.05),the proportion of DC cells in the low-dose and middle-dose groups was down-regulated(p<0.05),and the proportion of M2 in the low-dose group was up-regulated(p<0.05).Colon flow cytometry results showed that compared with the normal group,the proportion of macrophages in the colon tissue of the model group was up-regulated(p<0.05),the proportion of M1 macrophages was significantly up-regulated(p<0.05),and the proportion of M2 macrophages was down-regulated(p>0.05);Compared with the model group,the ratio of M1 in the medium-dose and high-dose groups of Modified Gegen Qinlian Decoction was down-regulated(p<0.05),the ratio of M2 in the high-dose group was up-regulated(p<0.05),and the proportion of DC cells in the high-dose group was down-regulated(p<0.05).The immunofluorescence results showed that the expression of F4/80 in the model group was significantly increased compared with the normal group(p<0.05).There was no significant difference in CD206 expression among the groups.5 ?-catenin/FOSL2/ARID5A expression:Western blot results showed that compared with the normal group,the expression of ?-catenin in the model group decreased(p>0.05),the expression of FOSL2 was significantly decreased(p<0.05),and the expression of ARID5A was increased(p<0.05).Compared with the model group,the expression of ?-catenin and FOSL2 in each group of Modified Gegen Qinlian Decoction was significantly increased(p<0.05),and the expression of ARID5A decreased in the middle-dose and high-dose groups(p<0.05).Real-time PCR test results showed that compared with the normal group,the expression levels of ?-catenin mRNA and FOSL2 mRNA in the colon tissue of the model group were significantly reduced(p<0.05),and the expression level of ARID5A mRNA was significantly increased(p<0.05).Compared with the model group,the ?-catenin mRNA and FOSL2 mRNA levels in the low-dose Modified Gegen Qinlian Decoction group were significantly increased(p<0.05).Compared with the model group,the expression level of ARID5A mRNA in the high-dose group was significantly increased(p<0.05).Conclusion(Two)1 Modified Gegen Qinlian Decoction can effectively alleviate the symptoms of DSS-induced colitis,improve weight loss,shorten the colon,lower the level of MPO and MDA,improve the ultrastructure of the intestinal mucosa,reduce the increase in colonic mucosal permeability caused by DSS,and down-regulate the expression levels of pro-inflammatory factors IL-1? and IL-6 in the colon tissue,promote intestinal epithelial proliferation,induce the up-regulation of the expression of E-Cadherin,?-catenin,and Occludin,and maintain the integrity of the intestinal mucosal barrier.2 Modified Gegen Qinlian Decoction can regulate the polarization state of macrophages in mice with colitis,inhibit the polarization of M1 macrophages,promote the polarization of M2 macrophages,up-regulate the expression of ?-catenin and FOSL2,and down-regulate the expression of ARID5A.In vitro:The effect of serum containing Modified Gegen Qinlian Decoction on the polarization of THP-1 macrophagesObjectiveClarify the effect of Modified Gegen Qinlian Decoction on the M1 macrophage polarization model in vitro,and clarify the target of the effect of Modified Gegen Qinlian Decoction.Method1 Construction of M1 macrophage polarization model:Use PMA and LPS/IFN-?to stimulate THP-1 cells to establish an M1 macrophage polarization model,observe the morphological changes of THP-1 cells under a microscope,and use flow cytometry to detect the cell surface CD11b positive expression rate.2 Observe the effect of serum containing Modified Gegen Qinlian Decoction on M1 macrophage inflammatory factors:randomly divide the cells into 8 groups,namely normal group(Con),model group(Model),and serum containing Modified Gegen Qinlian Decoction Low-dose group(GL),Modified Gegen Qinlian Decoction-containing serum medium-dose group(GM),Modified Gegen Qinlian Decoction-containing serum high-dose group(GH),?-catenin inhibitor+Modified Gegen Qinlian Decoction-containing serum low-dose Group(LW),?-catenin inhibitor+Modified Gegen Qinlian Decoction containing medicated serum medium-dose group(MW),?-catenin inhibitor+Modified Gegen Qinlian Decoction containing medicinal serum high dose group(HW),test each group changes in the content of IL-1?,a pro-inflammatory factor in the cell supernatant.Result1 The THP-1 M1 macrophage polarization model was constructed with 100ng/ml PMA superimposed on LPS(100 ng/ml)/IFN-y(20 ng/ml),and CCK8 was used to detect the effects of different concentrations of drug-containing serum on cell viability after 24 hours The results show that increased serum concentration affects cell viability,and the middle-dose and high-dose medicated serum cell viability of 10%Modified Gegen Qinlian Decoction is significantly higher than that of 20%serum cell viability.In this experiment,a 10%concentration of medicated serum is proposed as the administration concentration for subsequent experiments.2 Cell supernatant IL-1? detection results:Compared with the normal group,the IL-1? content of each group was significantly higher than that of the normal group(p<0.05).Compared with the model group,the IL-1? content of Modified Gegen Qinlian Decoction contained low-dose serum,middle-dose group and the high-dose group were significantly lower than the model group(p<0.05).After the addition of inhibitors,the IL-1?content of each drug-containing serum group was significantly higher than that of the non-inhibitor group(p<0.05).Conclusion(Three)1 CCK8 results suggest that increased serum concentration affects cell viability.This experiment is to use a 10%concentration of medicated serum as the administration concentration for subsequent experiments.2 The medicated serum of Modified Gegen Qinlian Decoction can directly act on macrophages,inhibit the polarization of THP-1 macrophages induced by LPS/IFN-y in the M1 direction and reduce the release of pro-inflammatory factor IL-1?.After the addition of IWR-1 inhibitor,this inhibitory effect is weakened.?-catenin maybe the targets of Modified Gegen Qinlian Decoction,which can inhibit the polarization of M1 macrophages through the ?-catenin/FOSL2/ARID5A pathway.ConclusionModified Gegen Qinlian Decoction may inhibit the polarization of M1 macrophages by regulating the expression of ?-catenin/FOSL2/ARID5A pathway protein,promote the balance of inflammatory factors and repair after intestinal epithelial injury,and then maintain the integrity of the intestinal mucosal barrier and relieve colitis symptom. |
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