| The treatment of inflammatory bowel disease(IBD)is very limited,for its complex etiology and the unclear pathogenesis.The compound traditional Chinese medicine has unique effects on some refractory diseases such as inflammatory bowel disease,because of its multi components and targets,such as Huangqin Decoction on the treatment of IBD in clinical and experimental study.The mechanism of Chinese medicinal ingredients especially the decoction is difficult to be clarified also because of its complexity and uncertain targets.The recent research on intestinal microflora and inflammatory bowel disease provides the direction for the study of compound traditional Chinese medicine.The intestinal probiotics can competitively inhibit the pathogenic bacteria by colonization resistance,and produce bacteriocin,lactic acid and other metabolites,to reduce the risk of pathogen survival,and form the intestinal microbial barrier.Once the barrier is destroyed,it can induce or worsen the intestinal mucosal barrier damage,leading to pathogenic bacteria into the intestinal lumen.The intestinal mucosal immune system is activated excessively by the pathogen associated molecular patterns(PAMPs)from the pathogens,and then causes pathological inflammation and injury,which the most typical is IBD.Therefore,the IBD is described as "barrier organ disease" by academic.The specific role of PAMPs is closely related to the pattern recognition receptors(PRRs)of host cells.In the study of PRRs,NOD2 is the first discovered susceptibility gene related to the IBD,and its corresponding ligand muramyl peptide(MDP)is the simplest structural unit of peptidoglycan in bacterial cell wall,large amounts of which can be found in intestinal cavity.NOD2 is activated by MDP,also its downstream signaling proteins,leading to the release of inflammatory factors and apoptosis,which is closely related to the pathogenesis of IBD.According to the literatures,as the classic prescription of Chinese medicine for treating IBD,the Huangqin Decoction both have the effect on anti-inflammatory and regulating the intestinal flora,but its mechanism is unclear.Therefore,in order to study the effects of Huangqin Decoction and its main components on the intestinal flora and MDP-NOD2 inflammatory signaling pathway in IBD,the ulcerative colitis(UC)mice was used as an animal model.And the following hypothesis was put forward to:Huangqin Decoction can treat the IBD by inhibiting the bacterial MDP-NOD2 inflammatory signaling pathway induced by MDP and recovering the intestinal microbial barrier.The main research contents as follows:quality control of Huangqin decoction,the pharmacodynamics of Huangqin Decoction and its main ingredients(including the experiments in vivo and in vitro),effects of Huangqin Decoction and its ingredients on MDP-NOD2 inflammatory pathway(including the experiments in vivo and in vitro),effects of Huangqin Decoction and its ingredients on the intestinal microbial barrier,the main technologies as high performance liquid chromatography,16SrDNA sequencing,immunofluorescence,fluorescence quantitative RT-PCR,Western Blot and flow cytometer were used,to explore the pharmacological mechanisms of Huangqin Decoction on inflammatory bowel disease.Objective:The dysbacteria leads to mucosal barrier damage,and the submucosal invasion of opportunistic pathogens induced inflammation and mucosal injury by PAMPs interacting with PRRs,in which the MDP-NOD2 inflammatory signaling pathway plays an important role in the pathogenesis and prognosis of IBD.As the main Decoction in the treatment of IBD,Huangqin Decoction exerts its therapeutic effect possibly through the signal pathway and the recovery of bacteria.This study used the UC mice as model to explore the effects of Huangqin Decoction and its main components on the intestinal flora and MDP-NOD2 inflammatory signaling pathway in IBD.The purpose of this study is to prove the following scientific hypothesis:The Huangqin Decoction can treat the IBD by inhibiting the bacterial MDP-NOD2 inflammatory signaling pathway and recovering the intestinal microbial barrier.Methods:1.Preparation and quality analysis of Huangqin decoctionHigh performance liquid chromatography(HLPC)was used to analyze the fingerprint of Huangqin decoction,and baicalin,baicalin,paeoniflorin and glycyrrhizin were used to analyze the main components of Huangqin decoction decoction.2.Effect of Huangqin Decoction on DSS induced UC miceThe effects of Huangqin Decoction on UC mice were evaluated by DAI score,histological grading,counting of peripheral white blood cells and the cytokine of colon.3.The effect of Huangqin Decoction on MDP-NOD2 inflammatory signaling pathway in UC mice induced by DSSThe expression of Nod2,Rip2,NF?B p65 gene in colon tissue was detected by PCR technology,and the effects of Huangqin Decoction on MDP-NOD2 inflammatory signal pathway in UC mice induced by of DSS were determined in gene level.The expression of NOD2,RIP2,NF-?B P65 protein in colon tissue was detected by Western Blot and immunofluorescence(IF),and the effects of Huangqin Decoction on MDP-NOD2 inflammatory signal pathway in UC mice induced by of DSS were further determined in protein level.4.The effect of Huangqin Decoction on MDP-NOD2 signal pathway in RAW264.7 cells The cytokines levels after being treated with Huangqin decoction and MDP were analyzed by flow cytometry(Cytometric Beads Array microspheres,CBA)in RAW264.7 cells' supernatant.The expression of Nod2,Rip2 and NF?B p65 in RAW264.7 cells were detected by PCR technology after being treated with Huangqin decoction and MDP,and the effects of Huangqin Decoction on MDP-NOD2 inflammatory signal pathway in RAW264.7 cells were determined in gene level.The expression of NOD2,RIP2,NF-?B P65 protein in RAW264.7 cells were detected by Western Blot and immunofluorescence(IF)after being treated with Huangqin decoction and MDP,and the effects of Huangqin Decoction on MDP-NOD2 inflammatory signal pathway in RAW264.7 cells were determined in protein level.5.Screening of the main effective ingredients and their dosage in Huangqin Decoction The NO levels after being treated with ingredients in Huangqin decoction and MDP were analyzed to screen of the main effective ingredients in RAW264.7 cells' supernatant,the most suitable concentration was further determined by MTT assay.6.The effects of main effective ingredients in Huangqin Decoction on UC mice induced by DSSThe effects of main effective ingredients in Huangqin Decoction on UC mice were evaluated by DAI score,histological grading,counting of peripheral white blood cells and the cytokine of colon.7.The effect of effective ingredients in Huangqin Decoction on MDP-NOD2 inflammatory signaling pathway in UC mice induced by DSSThe expression of Nod2,Rip2 and NF?B p65 gene in colon tissue was detected by PCR technology and the effects of effective ingredients in Huangqin Decoction on MDP-NOD2 inflammatory signal pathway in UC mice induced by of DSS were determined in gene level.The expression of NOD2,RIP2,NF-?B P65 protein in colon tissue was detected by Western Blot and immunofluorescence(IF),and the effects of effective ingredients in Huangqin Decoction on MDP-NOD2 inflammatory signal pathway in UC mice induced by of DSS were further determined in protein level.8.The effect of effective ingredients in Huangqin Decoction on MDP-NOD2 signal pathway in RAW264.7 cellsThe cytokines levels after being treated with effective ingredients in Huangqin Decoction and MDP were analyzed by flow cytometry(Cytometric Beads Array microspheres,CBA)in RAW264.7 cells' supernatant.The expression of Nod2,Rip2 and NF?B p65 in RAW264.7 cells was detected by PCR technology after being treated with effective ingredients in Huangqin Decoction and MDP,and the effects of effective ingredients in Huangqin Decoction on MDP-NOD2 inflammatory signal pathway in RAW264.7 cells were determined in gene level.The expression of NOD2,RIP2,NF-?B P65 protein in RAW264.7 cells were detected by Western Blot and immunofluorescence(IF)after being treated with effective ingredients in Huangqin Decoction and MDP,and the effects of effective ingredients in Huangqin Decoction on MDP-NOD2 inflammatory signal pathway in RAW264.7 cells were determined in protein level.9.Effects of Huangqin Decoction and its effective ingredients on intestinal microflora in IBD miceMice were free to drink 2.5%DSS for 5 days later,and free to drink sterile water for 5 days to replicate the UC model.The feces of mice were sterilely collected,and the genomic DNA was extracted.The number and composition of the intestinal flora were analyzed by 16SrDNA genomic sequencing after treated with Huangqin decoction.The antibacterial protein expression in intestinal epithelial cells was detected by FCM;the bacterial primers were used as probes to detect the bacterial invasion in UC mice by using in situ hybridization detection kit;and the MTT method was used to detect the effects of Huangqin Decoction and its active components on the standard strains(E.coli,etc.)Results:1.Quality control of Huangqin DecoctionHPLC was used to determine the content of Huangqin Decoction.And we detected the quality control components as follows:paeoniflorin,baicalin,liquiritin,wogonoside,glycyrrhizic Acid.Among them,the content of baicalin was the highest,followed by wogonoside,glycyrrhizic acid.According to the fingerprint analysis results,we found the similarity between batches of Scutellaria Decoction was above 0.980,except one was 0.958 and another was 0.966.2.The effects of Huangqin Decoction on the DSS-induced UC miceAfter administration of DSS,symptoms of the mice were as follows:lose weight significantly,diarrhea,hematochezia and other symptoms;The colon was significantly shortened,lumens stenosis,edema,thickening,obvious inflammatory cell infiltration,epithelial cell shedding,forming a plurality of ulcers;the peripheral white blood cells(WBC)count increased significantly,while the red blood cell(RBC)and hemoglobin(HGB)significantly decreased,the inflammatory cytokines in colon tissue significantly increased compared with the normal group(P<0.01).Those above indexes had different degrees of recovery after administration of sulfasalazine.The body weight of mice restored after administration of different doses of Huangqin decoction.The DAI was significantly lower(P<0.01),and the length of colon recovered significantly(P<0.01),the histological scores were lower(P<0.05 or P<0.01)in a dose-dependent way;histological score and model group.Ratio was statistically significant(P<0.01 or P<0.05);the histological score was statistically significant compared with the model group,and the cytokines in colon had different degrees of recovery,the dosage of Huangqin decoction and high dose group was statistically significant compared with the model group(P<0.01 or P<0.05)3.The effect of Huangqin Decoction on MDP-NOD2 inflammatory signaling pathway in UC mice induced by DSSThe expression of Nod2,Rip2 and NF?B p65 was significantly increased,compared with the normal group(P<0.01)after administration of DSS by SqReal-Time PCR.And the expression of Nod2,Rip2 and NF?B p65 was significantly decreased,compared with the model group(P<0.01)after administration of sulfasalazine.The gene Nod2,Rip2 and NF?B p65 had different degrees of recovery after being treated with different doses of Huangqin decoction,except the Rip2 expression was not statistically significant in the low dose group,the remaining groups were statistically significant compared with the model group in the three genes regulation(P<0.01 or P<0.05);The expression of NOD2,RIP2 and NF-?B P65 protein was significantly increased compared with the normal group(P<0.01)after administration of DSS by western blot.And the expression of NOD2,RIP2 and NF-?B P65 protein was significantly decreased,compared with the model group(P<0.01)after administration of sulfasalazine.The protein NOD2,RIP2 and NF-?B P65 had different degrees of recovery after being treated with different doses of Huangqin decoction,all the groups were statistically significant compared with the model group in the three proteins' regulation(P<0.01 or P<0.05)4.The effect of Huangqin Decoction on MDP-NOD2 signal pathway in RAW264.7 cellsThe results from the cell experiment showed that myeloperoxidase and the inflammatory cytokines were significantly higher than the normal controls after being treated with MDP in RAW264.7 cells' supernatant(P<0.01 or P<0.05);After being treated with different doses of Huangqin decoction,the myeloperoxidase,IL-17,MCP-1,TNF-?and IFN-y decreased obviously,and there were statistically significant compared with MDP control when treated with lmg/mL,125?g/mL of HuangQin decoction(P<0.01 or P<0.05).The expression of Nod2,Rip2 and NF?B p65 was significantly increased,compared with the normal group(P<0.01)after treated with MDP by SqReal-Time PCR.And the expression of Nod2,Rip2 and NF?B p65 was significantly decreased,compared with the MDP group(P<0.01)after administration of 1mg/mL Huangqin decoction,while the Nod2,Rip2 was significantly decreased after administration of 0.5mg/mL Huangqin decoction(P<0.01 or P<0.05).The expression of NOD2,RIP2 and NF-?B P65 protein was significantly increased,compared with the normal group(P<0.01)after treated with MDP by western blot.And the expression of NOD2,RIP2 and NF-?B P65 protein was significantly decreased,compared with the model group(P<0.01)after administration of Huangqin decoction.5.Screening of the main effective ingredients and their dosage in Huangqin DecoctionWhen the concentration of baicalin was 12.5?800?g/mL,it can inhibit RAW264.7 cells releasing NO induced by MDP(P<0.01);When the concentration of paeonilflorin was 12.5-800?g/mL,it can inhibit RAW264.7 cells releasing NO induced by MDP(P<0.01 or P<0.05).The concentration of the two monomers above had no effect on the growth of RAW264.7 cells.6.The effects of main effective ingredients in Huangqin Decoction on UC mice induced by DSSThe pharmacodynamics experiment showed that the baicalin and paeoniflorin could improve the body weight of UC mice,alleviate their diarrhea,hematochezia,shortening of colon,stenosis,edema,inflammatory cell infiltration,epithelial cell shedding,ulcers and other symptoms induced by DSS,they also reduced the number of peripheral white blood cells,and inflammatory cytokines in the colon.Among of them,the length of colon was restored after being treated with different doses of paeoniflorin(P<0.01 or P<0.05),only the middle and high dose of baicalin group were statistically significant compared with model group(P<0.01);the histological scores were lower when treated with middle and high dose of baicalin(P<0.01),and all of the three doses of paeoniflorin group were lower in a dose-dependent way(P<0.05 or P<0.01);in addition to HGB,the peripheral white blood cells recovery significantly in the middle and high dose of baicalin and paeoniflorin group(P<0.01 or P<0.05),only the high dose of baicalin and paeoniflorin have obvious recovery effect on HGB(P<0.05);The inflammatory cytokines in colon was significantly lower than that in the model group(P<0.05 or P<0.01)when treated with middle and high dose of baicalin and paeoniflorin,while low dose only had a significant effect on IFN-?(P<0.05).7.The effect of ingredients in Huangqin Decoction on MDP-NOD2 inflammatory signaling pathway in UC mice induced by DSSThe expression of Nod2,Rip2 and NF?B p65 was significantly decreased,compared with the model group(P<0.01 or P<0.05)after administration of baicalin by SqReal-Time PCR,except the low dose on Nod2 and Rip2;the expression of Nod2,Rip2 and NF?B p65 was significantly decreased,compared with the model group(P<0.01 or P<0.05)after administration of paeoniflorin except the low dose on Rip2.The expression of NOD2,RIP2 and NF-?B P65 protein in colon was significantly decreased,compared with the DSS model group(P<0.01 or P<0.05)after administration of baicalin and paeoniflorin by WB(P<0.01 or P<0.05).8.The effect of effective ingredients in Huangqin Decoction on MDP-NOD2 signal pathway in RAW264.7 cellsAfter being treated with different doses of baicalin,the myeloperoxidase in RAW264.7 cells'supernatant decreased significantly(P<0.01 or P<0.05),but only the 96?g/mL or 48?g/mL paeoniflorin worked like that(P<0.01 or?P<0.05).The IL-17,MCP-1,TNF-?and IFN-y in RAW264.7 cells' supernatant decreased obviously after being treated with 50?g/mL or(P<0.01 or P<0.05),and the 12.5 ?g/mL baicalin had effect on MCP-1(P<0.05).The MCP-1 in RAW264.7 cells' supernatant decreased significantly after being treated with different doses of paeoniflorin,only the 96?g/mL or 48?g/mL paeoniflorin had effects on IL-17 and TNF-?(P<0.01 or P<0.05),and the 96?g/mL paeoniflorin had effects on IFN-y(P<0.05).And the expression of Nod2,Rip2 and NF?B p65 was significantly decreased by PCR assay,compared with the MDP group(P<0.01)after administration of 50?g/mL baicalin,while the 25?g/mL baicalin had only effect on Rip2.And the expression of Nod2,Rip2 and NF?B p65 was significantly decreased,compared with the MDP group(P<0.01)after administration of 96?g/mL baicalin,while the 48?g/mL baicalin had only effect on Rip2.The expression of NOD2,RIP2 and NF-?B P65 protein was significantly increased,compared with the normal group(P<0.01)after treated with MDP by western blot.And the expression of NOD2,RIP2 and NF-?B P65 protein was significantly decreased,compared with the model group(P<0.01)after administration of baicalin and paeoniflorin by western blot assay.9.Effects of Huangqin Decoction and its effective ingredients on intestinal microflora in IBD mice? On view of species richness,Huangqin decoction could improve the OUTs of intestinal microflora,and increased the degree of microflora abundance in UC mice induced by DSS.? The species composition cluster analysis showed that the composition of the intestinal flora of UC mice was significantly different from that of normal mice.The intestinal microflora of mice treated with Huangqin decoction was the closest to that of normal mice,followed by sulfasalazine and paeoniflorin,and baicalin as the last.?From the analysis of the diversity of the samples,the diversity of intestinal flora in UC mice was significantly reduced.After treatment with the drugs,the diversity of the flora was restored.Among of them,the curative effect of Huangqin decoction was significant.?The relative abundance of intestinal flora:As the phylum,the Bacteroidetes and Deferribacteres decreased significantly in UC mice,and the relative abundance of Deferribacteres significantly increased after being treated with Huangqin decoction and sulfasalazine.As the species,the Mucispirillum_schaedleri,Escherichia_coli were increased significantly,and the Unclassified,Bacteroides_caccae,Lactobacillus_reuteri,Butyricicoccus_pullicaecorum,Clostridium_perfr ingens,Desulfovibrio_C21_c20 were decreased significantly in UC mice(P<0.01 or P<0.05).The Unclassified Mucispirillum_schaedleri was significantly recovered after being treated with baicalin and sulfasalazine,the Bacteroides_caccae were decreased significantly in all of the treated groups,and the Unclassified?Lactobacillus_reuteri?Butyricicoccus_pullicaecorum were increased significantly,and the Mucispirillum_Schaedleri?Escherichia_coli were increased significantly after being treated with Huangqin decoction(P<0.01 or P<0.05).? The expression of Reg?? increased compared with control(P<0.01)in intestinal epithelial cells,for being stimulated by the luminal contents and bacteria into the damaged submucosa induced by DSS;when treated with sulfasalazine,baicalin,paeoniflorin and Huangqin Decoction,the expression of Reg?? decreased,for the epithelial healing and bacteria being inhibited,the differences above were significant when compared with the model group(P<0.01 or P<0.05).? The epithelial was integrity,and there were only a few bacteria under the mucosa in the normal mice' colon;The epithelial deletion,shedding was obvious with a large number of bacterial submucosal infiltrations after being treated with DSS.The colon epithelium restored to normal,and the bacterial submucosal infiltration decreased significantly,after treated with sulfasalazine,baicalin,paeoniflorin and Huangqin decoction,the most prominent was the Huangqin decoction group in all of the results above results.? Huangqin Decoction showed a selective effect on the growth of bacteria,which induced the proliferation of Clostridium butyricum,Bifidobacterium,Lactobacillus,and has inhibitory effect on Bacteroides fragilis and Escherichia coli;the baicalin only promoted the activity Clostridium butyricum,and had inhibitory effect on Bacteroides fragilis,and other bacteria were not obvious;the paeoniflorin mainly showed inhibitory effects on the bacteria in the experiment,especially more prominent in high concentration.Conclusion:1.Huangqin decoction can ameliorate the ulcerative colitis induced by DSS in mice;its mechanism may be related to the inhibition of MDP-NOD2 inflammatory signaling pathway.2.Huangqin decoction can ameliorate the ulcerative colitis induced by DSS by the inhibiting the MDP-NOD2 inflammatory signaling pathway,in which the main active ingredients are baicalin and paeoniflorin.3.Huangqin decoction can promote the recovery of intestinal microflora barrier by regulating their proportion and quantity,and reduce the bacterial infiltration in colon in UC mice,the effects is better than other single components. |
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