| Research Background Ulcerative colitis(UC)is a non-infectious,inflammatory intestines problem with colon inflammation and ulcers as the main pathological manifestations.Prevalence ranged from 0.3%to 0.5%in North America,Europe,and Oceania.During 1990 to 2003,the prevalence rate of UC in China was about 0.0116%.Although the prevalence of UC in China is much lower than that in Europe and America,the number of UC outpatients has been increasing in the past 20 years.Although aminosalicylic acid preparation,corticosteroid,immunosuppressant,biological preparation,microecological preparation,fecal bacteria transplantation and leukocyte adsorption therapy can alleviate the inflammatory activity of UC in varying degrees,they all have some limitations and deficiencies,and are always accompanied with complications.Chinese herbal medicine have many effects such as inhibiting inflammatory,bactericidal,antiviral,and regulation of immunity.Recently,it has attracted attention in the treatment of autoimmune diseases,for example,it can be used to treat autoimmune diseases such as rheumatoid arthritis,asthma,and ulcerative colitis.Therefore,in China,more and more patients with mild and moderate UC are seeking TCM therapy.Based on carrying on the academic ideas of Mr.Lu Zhizheng,a master of TCM,and then based on the classic recipes for the intervention of diarrhea and dysentery,such as Shaoyao decoction,Wumei Pill and Gegen Qinlian decoction,our team cut out Qingre Huashi Tiaoshu prescription.Our team has been using this prescription to treat UC for more than 30 years,and the patients have responded well.The medicine comprise 12 medicines of herba,includes coptidis patriniae,coptidis rhizoma,dark plum,radix angelicae sinensis,radix paeoniae alba,radix aucklandiae,aurantii fructus immaturus,sradix puerariae,radix pseudostellariae,fried atractylodes,poria and glycyrrhrizae radix.It is recorded in article 12 of the differentiation and treatment of Jueyin disease pulse in Treatise on Febrile Diseases that Wumei Pill is also capable of benefiting long.Later generations of physicians have proved that it can be used for the treatment of ulcerative colitis through a large number of clinical trials.Meta-analysis reveals that Jiawei Wumei Pill integrated to western drugs are superior to western drugs alone in relieving clinical symptoms and colonoscopy performance of UC,and its safety is better.In addition,compared with simple western medicine treatment,treatment of UC with modified Wumei Pill alone has certain advantages in the cure rate,total effective rate,recurrence rate and adverse reaction rate,as well as in improving the TCM syndrome,colonoscopy performance and disease activity.At the same time,masses of evidence-based medicine evidences have proved the effectiveness and safety of Shaoyao Gancao Decoction and Gegen Qinlian Decoction in treating UC.In clinic,our team has a significant effect on the treatment of active UC of damp-heat type with the Prescription based on Qingre Huashi Tiaoshu Prescription.Whereas,there is no research to elaborate its curative therapeutic mechanism.In this study,network pharmacology was used to firstly predict the possible action targets of Qingre Huashi Tiaoshu Prescription,which was then preliminarily verified in patients.Finally,further verification was conducted through animal experiments to ensure the accuracy and rationality of biological index selection to the maximum extent,attempt to offer more reliable laboratory testimony for Qingre Huashi Tiaoshu Prescription.Research Method 1.Meta-analysis:The effectiveness and safety of simple oral administration of traditional Chinese medicine(TCM)compounds capable of clearing heat,resolving dampness and regulating pivot in the treatment of UC due to damp-heat in large intestine were systematically summarized based on the evidence-based medicine method of meta-analysis.Specific methods:Searched the databases of CNKI,Wanfang Medical Journal Network,Wei Pu Chinese Journal Network,China Biomedical Literature Service System,PubMed,Embase and Cochrane Library,etc.Randomized,controlled clinical trials were included.Cochrane risk bias evaluation tool was applied to assess the quality of incorporated research.Review Manager V.5.3 software was used together with the effect amounts of each index.2.Network pharmacology:The potential target point of Qingre Huashi Tiaoshu Prescription in the therapy of UC were forecasted by cyber pharmacology.The specific method was as follows:the active components of the whole recipe of Qingre Huashi Tiaoshu Prescription were extracted and matched with the corresponding drug action targets,which were firstly then make a pair with the disease-related target point.The network graph of "disease components-drug activity-intersecting targets" was established applying Cytoscape3.9.0 software,and the protein interplay network analysis of drug-disease intersecting targets was carried out.The related effective way were predicted through GO analysis and KEGG analysis.3.Clinical research and action target verification:First,the self-control singlearm test was used to observe the effectiveness and safety of Qingre Huashi Tiaoshu Prescription in patients with UC included.Then the serum contents of upstream and downstream related protein targets(TLR4,IL-6,IL-1β,TNF-α,and COX2)of NF-κB signaling pathway in patients before treatment,4 weeks and 8 weeks of intervention were examined by ELISA.4.Animal experiments:HE tint was applied to watch the histological manifestations of colitis mice.The level of MCP-1,IL-6,IL-1β,TNF-α,and COX-2 in the colon tissue and FITC-D in serum were detected by ELISA.Western blotting was applied to detect Occludin,MUC-2,ZO-1,TLR4,MyD88,IkBα,P-IkBα,NF-kBp65 and P-NF-kBp65 in the colon tissue.Using qRT-PCR to detect the mRNA of IL-6,TNF-α and IL-1β in the colon tissue.16SrRNA to sequence the gut content microbiota.Research results 1.Meta-analysis results on the efficiency and security of Chinese herb medicine combination with Qingre Huashi Tiaoshu functions in treating UC with intestine damp-heat syndrome:A meta-analysis eventually included 24 articles and covered 1,682 effective cases,and the results were as follows after the combined effect amounts:(1)The experimental group(which only took orally the compound with Qingre Huashi Tiaoshu functions)was superior to the control group in the total effective rate,syndrome effective rate,colonoscopy effective rate,diarrhea,abdominal pain,bloody purulent stool,and severe symptoms after internal emergency(which only took orally with western medicine),and the difference was statistically significant(P<0.01);(2)The experimental group of UC was superior to the control group in lowered the major symptom score,syndrome score,colonoscopy score,mayo score,abdominal pain,bloody purulent stool,and severe score after internal emergency(P<0.05).(3)The experimental group was better than the control group in reducing the endoscopic mucosal edema,congestion,and ulcer scores of UC(P<0.05),and the experimental group was not superior to the control group in the aspect of reducing the pseudopolyps scores(P=0.45);(4)The trial group was markedly superior to the western drug group in the aspect of reducing the histological score of Geboes(P<0.05);(5)The ratio of negative events in the trial group was obviously less than the control group’s ratio(OR=0.27,95%CI[0.14,0.56],P=0.0003);(6)The ratio of recrudescence in the trial group obviously less than the control group’ ratio(OR=0.15,95%CI[0.06,0.36],P<0.0001).2.Prediction of action target results of Qingre Huashi Tiaoshu Prescription in treating UC on the basis of cyber pharmacology:There were 146 active components in the whole prescription of Qingre Huashi Tiaoshu Prescription,including 7 herba patriniae,10 rhizoma coptidis,8 mume fructus,2 angelicae sinensis,4 paeoniae alba,3 aucklandiae,17 aurantii fructus immaturus,3 puerariae,2 radix pseudostellariae,4 fried atractylodes,7 poria cocos and 79 radix glycyrrhizae(some Chinese medicines contain the same components).According to the active components of the whole Prescription,207 drug action targets were screened out,4836 targets related to UC were retrieved,and the venn diagram of Qingre Huashi Tiaoshu Prescription-UC was constructed.It was found that 146 key action targets in overlapping regions were identified.The "drug name-active ingredient-intersecting target-disease" network graph was generated applying Cytoscape3.9.0 software.After analyzing the interaction network of drug-disease intersection target proteins,we found that RELA(NF-kBp65),TNF,IL-10,and IL-6 were the key target proteins.Through the functional enrichment analysis of GO-BP,GO-CC and GO-MF,it is predicted that the treatment of UC with Qingre Huashi Tiaoshu Prescription is mainly through the immune response process,concerning in the cell microstructure and directly related to the functions of cell factors and transcription factors.The enrichment analysis of KEGG signaling pathway predicted that Qingre Huashi Tiaoshu Prescription might play a role in the treatment of UC by interfering with the NF-kB signaling pathway.3.Changes of serum NF-kB pathway-related inflammatory indexes and curative effect-related indexes in patients with mild and moderate UC before taking Qingre Huashi Tiaoshu Decoction and at 4 and 8 weeks of medication.(1)The serum TLR4,IL-1β,TNF-α,IL-6,and COX-2 of the patients were significantly lower than those before treatment at 4 weeks and 8 weeks of treatment(P<0.001).The levels of 1β(P<0.001),TNF-α(P=0.011),IL-6(P<0.001),COX-2(P=0.014),and TLR4(P=0.002)were significantly lower than those in 4 weeks of treatment.(2)84.6%patients achieved clinical remission in four weeks of treatment with Qingre Huashi Tiaoshu Prescription,and the clinical remission rate was 100%in eight weeks of treatment;(3)The CAI scores in the 4th and 8th weeks of intervention with Qingre Huashi Tiaoshu Prescription were markedly inferior to that of pre-treatment(P<0.001,P<0.001).The CAI score in the 8th week of interference was signally superior to that in the 4th week of interference(P=0.036).(4)Before intervention,in the 4th and 8th week of treatment,there was a significantly statistical difference in the distribution of the fecal calprotectin results of patients(Z=31.358,P<0.001).Before intervention,all patients were positive for fecal calprotectin;after the 4th week of treatment,seven patients(53.8%)decreased to be weakly positive,and five patients(38.5%)became negative;at the 8th week of treatment,nearly two patients(15.4%)were extremely weakly positive,and the remaining patients(84.6%)were all negative.(5)The total IBDQ scores in the 4th and 8th weeks of intervention with Qingre Huashi Tiaoshu Prescription were markedly higher than that of pre-intervention(P<0.001,P<0.001),and the CAI score in the 8th week of intervention was signally higher than that in the 4th week of intervention(P=0.013).Furthermore,the scores of bowel symptoms,systemic symptoms,emotional function and social function in the 4th and 8th weeks of intervention were higher than those of pre-treatment(P<0.05).At the same time,the scores of bowel symptoms and emotional function in the 8th week of intervention were markedly higher than those in the 4th week of intervention(P<0.05).(6)No lesion was found in liver and kidney function at 4 and 8 weeks of intervention,and no unhealth event was observed in all patients in the process of treatment.(7)After four and eight weeks of intervention,the serum levels of IL-1β,TNF-α,IL-6,COX-2 and TLR4 were markedly inferior to those of pre-treatment(P<0.001),Meanwhile,after eight weeks of treatment,the serum levels of IL-1β(P<0.001),TNF-α(P=0.011),IL-6(P<0.001),COX-2(P=0.014)and TLR4(P=0.002)were markedly inferior to those of four weeks of intervention.4.The animal experiments were conducted to further verify the efficacy and action mechanism of Qingre Huashi Tiaoshu Prescription in the treatment of UC.(1)General conditions of mice:Qingre Huashi Tiaoshu Prescription significantly increased the body weight of mice with colitis,reduced DAI,recovered the length of colon,and reduced histological scores.The therapeutic effect of Qingre Huashi Tiaoshu Prescription was alike to that of mesalazine.(2)Intestinal mucosal barrier:Contrasted to the normal group,the relative greyscale values of colon Occludin,MUC-2 and ZO1 proteins in the model group were markedly lower(P<0.001);Contrast to the DSS group,the relative grayscale values of Occludin,MUC-2 and ZO-1 proteins in the colon of mice in the Qingre Huashi Tiaoshu Prescription group and mesalazine group were markedly incremental(P<0.01).In addition,the serum FITC-D content in the DSS group was markedly superior to that in normal group(P<0.001),and contrasted to the DSS group,the serum FITC-D content in the Qingre Huashi Tiaoshu Prescription group and mesalazine group was signally lower(P<0.001).(3)The pathway of LPS/TLR4/NF-kB:Contrast to the normal group,the mRNA levels of IL-6,TNF-α and IL-1β in the colon of mice in the DSS group were markedly elevated(P<0.001).Contrast to the DSS group,the mRNA levels of IL-6,TNF-α and IL-1β in the Qingre Huashi Tiaoshu Prescription group and mesalazine group were markedly lower(P<0.01).ELISA results revealed that contrast to the blank group,the levels of inflammatory-promoting cytokines(MCP-1,IL-6,TNF-α,IL-1β,and COX-2)in the colon of mice in the DSS group were memorably elevated(P<0.001).Contrast to the DSS group,the above cell factors content in mice’s colon in the Qingre Huashi Tiaoshu Prescription group and mesalazine group were memorably elevated(P<0.01).Contrast to the blank group,the LPS content in the colon tissue of mice in the DSS group and the relative grayscale scores of TLR4,MyD88,P-IkBα and P-NF-kB were memorably ascending(P<0.001).Contrast to the DSS group,the LPS content in the colon of mice in the Qingre Huashi Tiaoshu Prescription group and mesalazine group and the relative gray values of TLR4,MyD88,P-IkBα and P-NF-kB were memorably lower(P<0.01).(4)Intestinal flora:PCoA analysis at OTU level revealed that there were marked distinguish in the community structure of flora among the blank group,DSS group and Qingre Huashi Tiaoshu Prescription group.Contrast to the blank group,the Chao,ACE,Shannon’s and Simpson’s indexes of the DSS group were memorably lower(P<0.01).Contrast to the DSS group,the Chao,ACE,Shannon’s and Simpson’s indexes of the Tiaoshu Prescription group were memorably elevated(P<0.05).At the phylum level,contrast to the blank group,the abundance of Firmicutes and Actinobacteria was memorably lower in the DSS group(P<0.001),and the abundance of Bacteroidetes and Proteobacteria was memorably elevated(P<0.05).Contrast to the DSS group,the abundance of Firmicutes and Actinobacteria was memorably elevated(P<0.001)and the abundance of Bacteroides and Proteobacteria was memorably lower(P<0.05)in the Qingre Huashi Tiaoshu Prescription group.On the genus’ level,contrast to the blank group,the abundance of Lactobacillus,Bifidobacterium,Akkermansia,and Turicibacter in the bowel of mice in the DSS group was memorably reduced(P<0.05),and the abundance of Escherichia coli,Bacteroides and Clostridium was memorably elevated(P<0.05).In the Qingre Huashi Tiaoshu Prescription group,the abundance of Lactobacillus,Bifidobacterium,Akkermansia and Turicibacter memorably elevated(P<0.05),and the abundance of Escherichia coli,Bacteroides and Clostridium was memorably lower(P<0.05).Research conclusions 1.The long-term and short-term clinical efficacy and safety of heat-clearing and dampness-eliminating and pivot-regulating traditional Chinese medicine compound in the treatment of UC due to damp-heat in large intestine are superior to those of the simple western drug group(aminosalicylic acid preparation),and the recurrence rate is low,but it still needs to be verified by higher-level evidence-based medicine evidence.2.Network pharmacology revealed that the pharmacodynamic mechanisms of Qingre Huashi Tiaoshu Prescription in the intervention of ulcerative colitis involved immune response,apoptosis,cytokine and transcription factor activity,and were related to tumor,inflammation,apoptosis and other signaling pathways.It was predicted that Qingre Huashi Tiaoshu Prescription would likely play a role through the NF-kB signaling pathway,but it needs further verification.3.Qingre Huashi Tiaoshu Prescription can down-regulate the expression of upstream and downstream related target proteins(TLR4,IL-1β,TNF-α,IL-6,COX-2)in the serum of patients with mild and moderate UC,and its change trend is consistent with the change trend of clinical disease index.4.Qingre Huashi Tiaoshu Prescription can signally increase the body weight of mice with colitis,reduce DAI,restore colon length,and reduce histological score,and the efficacy is similar to that of mesalazine.Its mechanism is related to the restoration of the balance of the intestinal flora,the repair of the intestinal mucosal barrier,and the limitation of the activation of the enterogenous LPS/TLR4/NF-kB signaling pathway. |
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