Efficacy of Wumei Baijiang prescription (烏梅敗醬方) on regulatory T cells/ helper T cells Immune balance in mice with ulcerative colitis

SU Xiaolan,ZHANG Tao,GUO Song,WANG Xin,CHEN Lin,WEI Wei

SU Xiaolan,ZHANG Tao,GUO Song,WANG Xin,CHEN Lin,WEI Wei,the Gastroenterology department,Wangjing Hospital of China Academy of Chinese Medicinal Sciences,Beijing 100102,China;TCM diagnosis and treatment Functional Gastroenteropathy Beijing Key Laboratory,Beijing 100102,China

Abstract OBJECTIVE: To observe the therapeutic effect of Wumei Baijiang prescription (烏梅敗醬方),empirical prescription of Lu Zhizheng,on experimental ulcerative colitis (UC)mice,and to investigate the mechanism of the prescription in UC from the perspective of the immune balance of regulatory T cells (Treg) and helper T cells (Th17).METHODS: Sixty C57BL/6 mice were randomly divided into 6 groups: normal group,model group,Chinese medicine group (high,medium and low dose group of Wumei Baijiang prescription) and control group(mesalazine sustained-release granules).Except for the normal group,the other groups used 2.5% dextran sulfate sodium to induce UC mice model.At the end of the model,the Chinese medicine group was given high,medium and low dose administration of Wumei Baijiang prescription,the control group was given slow-release granules of mesalazine,and the model group was given equal volume saline for 10 d.The changes of food intake,body weight,disease activity index (DAI) score,length of large intestine and histopathology were observed.The number of Treg,Th17,CD4+,CD8+cells in spleen was detected by flow cytometry.The expression of tumor necrosis factor-α (TNF-α),interleukin-10 (IL-10) and C-reactive protein (CRP) in serum was detected by enzyme-linked immunosorbent assay.RESULTS: The middle and high-dose groups of Wumei Baijiang prescription were superior to the model group in terms of increasing food intake and body weight of colitis mice,restoring colon morphology,improving pathological damage,and reducing DAI (P ＜ 0.05).There was no statistical difference with the mesalazine group (P ＞ 0.05).Compared with the model group,the spleen Treg and CD4+of the mice in the high and middle dose groups of Wumei Baijiang prescription were higher,while Th17 and CD8+were lower (P ＜ 0.05),and there was no statistical difference compared with the mesalazine group (P ＞0.05).In addition,compared with the model group,the serum levels of TNF-α and CRP in mice with high and middle doses of Wumei Baijiang prescription and mesalazine group were lower (P ＜ 0.05),and IL-10 content was higher (P ＜ 0.05).CONCLUSIONS: Wumei Baijiang prescription can improve the general conditions of colitis mice,such as diarrhea,hematochezia,weight loss,and mucosal damage.The mechanism may be related to the regulation of Treg/Th17 immune balance.

Keywords: colitis,ulcerative;mesalamine;T-lymphocytes,regulatory;Th17 cells;Wumei Baijiang prescription

1.INTRODUCTION

Ulcerative colitis (UC) is a chronic non-specific inflammatory disease of the colon,which is a serious threat to human health.In recent years,the incidence rate and prevalence rate increasing gradually.1UC’s treatment is lack of specificity and difficult to cure.It has become a global disease and a worldwide treatment problem.Traditional Chinese Medicine has a definite effect on relieving symptoms,inducing relief and maintaining relief during the remission.Wumei Baijiang prescription (烏梅敗醬方) is the empirical prescription of Lu Zhizheng,a master of Chinese medicine,in the treatment of UC.To impart and inherit the academic experience of Chinese medicine master,we designed an experiment to observe the effect of Wumei Baijiang prescription on Treg/ Th17 immune balance in UC mice.In the study,we aimed to investigate the mechanism of Wumei Baijiang prescription on UC mice and provide a scientific basis for clinical research.

2.MATERIALS AND METHODS

2.1.Experimental animal

Specific pathogen free (SPF) level C57BL/6 mice,male,8-week-old,body mass is (22 ± 2) g,purchased from Sibeifu (Beijing,China) Biotechnology Co.,Ltd.License No.SCXK (Beijing,China) 2015-0015.Raised in the institute of Acupuncture and Moxibustion,Chinese Academy of Traditional Chinese Medicine (SPF animal room),12 h light and dark cycle,the relative humidity of air is about 35%-45%.Animal ethics No.(D2021-03-16-2).

2.2.Experimental drug

2.5 % dextran sulfate sodium (DSS) solution: Using electronic balance weighed 2.5 g DSS solute,and dissolved it in 100 mL pure water,paired 2.5% DSS solution.Start to prepare solution on the first day of molding,according to the number of mice and the amount of drinking water,calculated the amount of solution needed.Paired the solution every other day to keep fresh.

Wumei Baijiang prescription: Wumei (Fructus Mume)12 g,Baijiangcao (Herba Patriniae Scabiosaefoliae) 15 g,Huanglian (Rhizoma Coptidis) 5 g,Muxiang (Radix Aucklandiae) 9 g,Danggui (Radix Angelicae Sinensis)10 g,Stir-frying Baishao (Radix Paeoniae Alba) 15 g,Stir-frying Zhiqiao (Fructus Aurantii Submaturus) 10 g,Taizishen (Radix Pseudostellariae) 12 g,Stir-frying Baizhu (Rhizoma Atractylodis Macrocephalae) 10 g,Fuling (Poria) 15 g,Gegen (Radix Puerariae Lobatae)12 g,Stir-frying with honey Gancao (Radix Glycyrrhizae)6 g.The TCM prescription granules produced by Guangzhou Yifang Pharmaceutical Co.,Ltd.(Guangzhou,China) and paired with distilled water to the needed concentrations.

Mesalazine sustained-release Granules (Product specification 500 mg × 10 bag,Manufacturer:ethypharm,Approval No.H20100063).500 mg mesalazine sustained-release granules fully dissolved in 25 mL of water,paired mesalazine suspension.The prepared Mume Fructus Patriniae and mesalazine Suspension were stored in a refrigerator at 4 ℃ for standby,and shaken before use.

2.3.Experimental reagents and instruments

DSS (43 kDa,No.160110,U.S.A MP Biomedicals Corporation);FOBT (OB) Reagent (Pyramidon Semiquantitative detection Method,Baso Diagnostics Inc.,Zhuhai,China);PE Rat Anti-Mouse CD4 (BD Pharmingen?,Cat: 553653,State of New Jersey,NY,USA); BV421 Rat Anti-mouse CD25 (BD Pharmingen?,566228,State of New Jersey,NY,USA);PerCP-Cy5.5Hamster Anti-mouse CD3e (BD Pharmingen?,551163,State of New Jersey,NY,USA);APC anti-mouse IL-17A (biolegend,Cat: 506916,California,USA);Brilliant Violet 510 anti-mouse CD8a(biolegend,Cat: 100752);Alexa Flour 488 antimouse/rat/human FOXP3 (biolegend,Cat: 320012,San Diego,CA,USA);Cell Activation Cocktail (with Brefeldin A,biolegend,Cat: 423303,San Diego,CA,USA);TNF-α Test Kit (Erwantech No.ER15112,Shanghai,China);IL-10 enzyme-linked immunosorbent assay Test Kit (Erwantech No.ER14032,Shanghai,China);CRP Test Kit (Erwantech No.ER17002,Shanghai,China).

Electronic Balance (ML4002/02,Mettler Toledo Instruments Co.,Ltd.,Shanghai China);SL202N Electronic Balance (Shanghai Minqiao Precision Scientific Instrument Co.,Ltd.,Shanghai,China);Water Purifier (JT-A20,Shanghai Hongxi Industry Co.,Ltd.,Shanghai,China); Electronic Microscope (UTvO.5xC.24H01726,Olympus,Tokyo,Japan);Slicing Machine (RM2255,Lejca,Weztlar,Germany);Roasting Machine (H1122Q,Lejca,Weztlar,Germany);Dehydrator (Eq150H,Lejca,Weztlar,Germany);Universal Camera (AHB-2-LB,Olympus,Tokyo,Japan);Microplate Reader (Multiskan FC,ThermoFisher,Waltham，MA,USA);96 Well Microtiter Plates (42592,Corning,Beijing,China);Low speed Table Centrifuge(TDZ4-WS,Cence,Hunan,China).

2.4.Animal grouping and model preparation

Sixty mice were randomly divided into 6 groups after 5 d of adaptive feeding: normal group (NG),model group(MG),Wumei Baijiang prescription high dose group(WHG),Wumei Baijiang prescription medium dose group (WMG),Wumei Baijiang prescription low dose group (WLG),mesalazine sustained-release granules control group (CG),each with 10,weighing and labeling.Refer to the modeling method of Carolet al2to be duplicated.Except for the normal group,the other groups were given 2.5% DSS solution free drinking for 7 d.After molding,according to Disease Activity Index (DAI)evaluated model and excluded mice with DAI score less than one.

2.5.Administration basis and treatment plan

According tothePharmacologyExperiment Methodology,11the treatment dose was calculated by the weight calculation method.Animal dosage calculation formula (Calculated as 60 kg adults and 20 g mice): dB=dA × KB/KA (dB is the dosage for per kg and per day for mice,dA is the dosage for per kg and per day for adults,KB=0.71,KA=0.11,Constant).The dosage of Wumei Baijiang prescription free granules calculated about 137 g.According to the pharmacology experiment animal dosage calculation formula,WHG,WMG and WLG were gavage according to 4∶2∶1,adjust the dosage to 0.5 mL per day,dissolved in distilled water and stored it in the refrigerator at 4 ℃ spared.After molding,the WHG,WMG and WLG were respective gavage by 0.736,0.368,0.184 g·kg-1·d-1,0.5 mL/d.CG was gavage mesalazine sustained-release granules suspension 0.52 g·kg-1·d-1,0.5 mL/d.NG and MG were respective gavage by an equal volume of normal saline for 10 d.

2.6.General situation

Observe and record the mental state,activity state,fur color,food intake,weight change and stool characteristics of mice in each group.

2.7.Disease Activity Index (DAI)3

Each mouse was weighed respectively on 0,5,7,13 and 17 d.Observed and detected the stool’s characteristics and occult blood.According to the percentage of decline in body weight,stool characteristics and occult blood,calculated were DAI scores respectively of mice in each group at various points in time.DAI score=(Percentage of Decline in Body Weight Score+Stool Characteristics Score+Occult Blood Score)/3.

2.8.Colon length

After the treatment,mice in each group fasted for 24 h,but they were free to drink water.Later,intraperitoneal anesthesia with 10% chloral hydrate and fixed mice respectively on the zootomy table,cut open the lateral abdomen,took the colon,and measured the length.

2.9.Histopathology

Using a microscope to observed the mucosa,submucosa and muscular layer of the colon.Including epithelial cell surface microvilli,epithelial cell array and clearance,submucosal vessels,glandular structure,goblet cell,crypt abscess and the number of crypts etc.

2.10.Flow cytometry

Dissected the whole spleen and cut with surgical scissors,ice bath homogenate.Transferred it to the Falcon cell strainers with a 70 μm bore diameter filter and collected cell suspension.Add red blood cell lysis buffer to the suspension.After the reaction stops,centrifuge,and remove the supernatant twice,MACS buffer solution resuspended.Transferred cell suspension to the Falcon cell strainers with a 40 μm bore diameter filter,remove cell mass.Finally,collected filtered cell suspension for flow cytometry.Transfer 200 μL of cell suspension (2 ×106cells) to a round bottom flow tube.Add the corresponding volume of antibody (CD3,CD8,CD25 antibody,the amount is 5 μL/test) to the sample tube,and incubate for 30 min at 4 ℃ in the dark.Add 1 mL of Staining Buffer,centrifuge at 500 g for 5 min,wash the cells,and discard the supernatant after centrifugation.Add 500 μL of PBS washing solution to each tube and test it on the machine.Finally,Cell Quest software(version 5.1) was used to analyze the ratio of cells in each group.

2.11.Elisa cytokine detection

After serum separation,operated according to the instructions of TNF-α,IL-10 and CRP kits.

2.12.Statistical analysis

Data analysis by SPSS 20.0 software (IBM,Chicago,IL,USA).Numerical data were expressed as mean ±standard deviation ().For the numerical data meeting normal distribution with equal variance,oneway analysis of varance was used;the Tukey test was used for intergroup pairwise comparison.The Kruskal-Wallis test was used when numerical data were not normally distributed or had an unequal variance.APvalue ＜ 0.05 indicated a significant difference.

3.RESULTS

3.1.General condition of mice

The NG mice were normal,lively,brilliantly-colored.From the 3rd day,except the NG,the other groups,especially NG and WLG are the most obvious,gradually appeared different degree of activity retardation,mental malaise,liking to curl up bodies and dull fur,etc.On the 3rd-5th day,some mice were positive for stool occult blood.From the 7th day,most of the mice had bloody stools with naked eyes,and the rest of them were positive for occult blood test.On the 8th-10th day,both of MG and WLG died 2 mice,WHG and WMG respectively died 1.On the day before death,the mice had perianal blood stains,listlessness,sunken eye sockets and hunchback.From the 10th day,the general condition of mice in each group was improved in varying degrees.Especially the improvement of CG,WHG and WMG were more obvious.

3.2.Food intake of mice

Compared with the NG,the food intake of the other groups decreased on the 5th-7th day (P ＜0.05),indicating that the food intake decreased gradually with the prolongation of the modeling time.On the 3rd day of treatment,the food intake of mice in each group was still lower than that of the NG (P ＜0.05);On the 6th day of treatment,the food intake of mice in the MG was lower than that in NG (P ＜0.05),the food intake of mice in the WHG,WMG and CG was higher than that in MG (P ＜0.05).On the 10th day of treatment,the food intake of mice in the MG and WLG was lower than that in NG (P＜0.05),the food intake of mice in the WHG,WMG and CG was higher than that in MG (P ＜0.05).In summary,the results showed that the high and medium dose of Wumei Baijiang prescription and mesalazine sustainedrelease granules could increase the food intake of UC mice,the effect of 3 groups are better than WLG (Table 1).

3.3.Weight change of mice

Compared with the NG,there was no difference in the weight change of mice in each group on the 5th day (P ＞0.05).However,on the 7th day,after modeling,the weight of mice in each group was lower than that in the NG (P ＜0.05),it demonstrated the weight of UC model mice was lower.On the 3rd day of treatment,the weightof mice in each group was lower than that of the NG(P ＜0.05);On the 6th day of treatment,the weight of mice in each group was still lower than that of the NG(P ＜0.05),but the weight of mice in the WHG,WMG and CG were higher than that of the MG (P ＜0.05);On the 10th day of treatment,the weight of mice in the WHG,WMG,WLG and MG were lower than NG (P ＜0.05),but the weight of mice in the WHG,WMG and CG were higher than MG (P ＜0.05);In summary,the results showed that the high and medium dose of Wumei Baijiang prescription and mesalazine sustained-release granules could increase the weight of UC mice,the effect of 3 groups are better than WLG (Table 2).

Table 1 Comparison of food intake of mice in each group ()

Table 1 Comparison of food intake of mice in each group ()

Notes: NG: normal group (saline,0.5 mL/d),MG: model group (saline,0.5 mL/d),CG: mesalazine group (0.52 g·kg-1·d-1),WHG: high dose group of Wumei Baijiang prescription (0.736 g·kg-1·d-1),WMG: dose group of Wumei Baijiang prescription (0.368 g·kg-1·d-1) and WLG: low dose group of Wumei Baijiang prescription (0.184 g·kg-1·d-1).Compared with the NG,aP ＜ 0.05;compared with the MG,bP ＜ 0.05.Treatment course: 10 d.

3.4.DAI score of mice

Compared with the NG,the DAI scores of the other groups increased on the 5th and 7th day (P ＜0.05),indicating that the DAI scores of the model mice increased gradually with the prolongation of the modeling time.On the 3th day of treatment,the DAI scores of mice in each group was higher than that of the NG (P ＜0.05),the DAI scores of the CG was lower than that of the MG (P ＜0.05);On the 6th and 10th day of treatment,the DAI scores of mice in each group was higher than that of the NG (P ＜0.05),but the DAI scores of mice in WHG,WMG and CG were higher than MG(P ＜0.05);In summary,the results showed that the high and medium dose of Wumei Baijiang prescription and mesalazine sustained-release granules could reduce the DAI scores of UC mice,the effect of 3 groups are better than WLG (Table 3).

Table 2 Comparison of weight change of mice in each group ()

Table 2 Comparison of weight change of mice in each group ()

Notes: NG: normal group (saline,0.5 mL/d),MG: model group (saline,0.5 mL/d),CG: mesalazine group (0.52 g·kg-1·d-1),WHG: high dose group of Wumei Baijiang prescription (0.736 g·kg-1·d-1),WMG: dose group of Wumei Baijiang prescription (0.368 g·kg-1·d-1) and WLG: low dose group of Wumei Baijiang prescription (0.184 g·kg-1·d-1).Compared with the NG,aP ＜ 0.05;compared with the MG,bP ＜ 0.05.Treatment course: 10 days.

Table 3 Comparison of DAI score of mice in each group ()

Table 3 Comparison of DAI score of mice in each group ()

Notes: NG: normal group (saline,0.5mL/d),MG: model group (saline,0.5 mL/d),CG: mesalazine group (0.52 g·kg-1·d-1),WHG: high dose group of Wumei Baijiang prescription (0.736 g·k-1·d-1),WMG: dose group of Wumei Baijiang prescription (0.368 g·kg-1·d-1) and WLG: low dose group of Wumei Baijiang prescription (0.184 g·kg-1·d-1).Compared with the NG,aP ＜ 0.05;compared with the MG,bP ＜ 0.05.Treatment course: 10 d.

3.5.Colon length of mice

Compared with the NG (7.1 ± 0.7) cm,the colonic length of mice in the MG (5.3 ± 0.4) cm and WLG (5.9 ± 0.5)cm were shortened (P ＜0.05);Compared with the MG,the colonic length of mice in the WHG (6.6 ± 0.7) cm,WMG (6.8 ± 1.1) cm and CG (6.5 ± 0.4) cm were lengthened (P ＜0.05);In summary,the results showed that the high and medium dose of Wumei Baijiang prescription and mesalazine sustained-release granules could increase the colonic length of UC mice,the effects of 3 groups were not significantly different (P ＞0.05).

3.6.Histopathology

In the NG,the intestinal mucosa epithelium of mice is intact,epithelial cells are closely connected;the crypt structure of the lamina propria is complete,orderlyarrangement,goblet cells secrete vigorously and no obvious pathological changes in the submucosa.The mice in the MG,the intestinal mucosa epithelium coloboma,the connection of epithelial cells are loose,the crypt structure destroyed,disorder of arrangement,obvious decrease of goblet cells.And a great quantity of infiltration of lymphocytes,neutrophils and other inflammatory can be seen in the interstitial layer.The submucosa has a varying degree of hyperemia and edema.The histopathology of mice’s colon in WHG and CG substantial improvement,compared with the MG,the coloboma of mucosal were repaired obviously,epithelial cells connection becomes tight,goblet cells secretion increased,extremely few crypt damages and inflammatory cell infiltration decreased in different degrees.The mice’s part of colonic mucosa in the WMG was damaged,but the coloboma area and the amount of crypt damage are small.The submucosa has a small number of inflammatory cells infiltration and edema.The colonic mucosa of mice in the WLG can be seen multiple interruptions,crypt destruction,congestion and edema appeared in the interstitial layer and inflammatory cell infiltration (Figure 1).

Figure 1 Colon tissue’s HE staining results of mice in each group(× 200)

3.7.Spleen T lymphocyte and its ratio

Compared with the NG,the Treg decreased (P ＜0.05)and Th17 increased (P ＜0.05) in spleen of mice in the MG and WLG.Compared with the MG,the Treg increased (P ＜0.05) and Th17 decreased (P ＜0.05) in spleen of mice in the WHG,WMG and CG.In summary,the results showed that the high and medium dose of Wumei Baijiang prescription and mesalazine sustainedrelease granules could increase the Treg and decreased Th17 in the spleen of UC mice,the effect of 3 groups has no difference (P ＞0.05) (Table 4,Figures 2,3).

Compared with the NG,the CD4+decreased (P ＜0.05)and CD8+increased (P ＜0.05) in spleen of mice in the MG and WLG.Compared with the MG,the CD4+increased (P ＜0.05) and CD8+decreased (P ＜0.05) in spleen of mice in the WHG,WMG and CG.The ratio of CD4/CD8 in each group have no difference (P ＞0.05).In summary,the results showed that the high and medium dose of Wumei Baijiang prescription and mesalazine sustained-release granules could increase the CD4+and decreased CD8+in the spleen of UC mice,the effect of 3 groups have no difference (P ＞0.05) (Table 4,Figures 4,5).

Figure 2 Comparison of Trge cells in the spleen of mice in each group

Figure 3 Comparison of Th17 cells in the spleen of mice in each group.

Figure 4 Comparison of CD4+T cells in the spleen of mice in each grou

Figure 5 Comparison of CD8+T cells in the spleen of mice in each group

3.8.TNF-α,IL-10 and CRP’s expression in serum

Compared with the NG,the TNF-α and CRP increased(P ＜0.05) but IL-10 decreased (P ＜0.05) in the spleen’s serum cytokines of mice in each group.Compared with the MG,the TNF-α and CRP decreased(P ＜0.05) in the spleen’s serum cytokines of mice in each group,but the IL-10 increased (P ＜0.05) in the spleen’s serum cytokines of mice in the WHG,WMG and CG.In summary,the results showed that the high and medium dose of Wumei Baijiang prescription and mesalazine sustained-release granules could decrease the proinflammatory factor TNF-α’s level and CRP’s expression,but increase the anti-inflammatory factor IL-10’s level in serum cytokines of UC mice,the effect of 3 groups have no difference (P ＞0.05) (Table 5).

Table 4 T lymphocyte and ratio of mice in each group (%,)

Table 4 T lymphocyte and ratio of mice in each group (%,)

Notes: NG: normal group (saline,0.5 mL/d),MG: model group (saline,0.5 mL/d),CG: mesalazine group (0.52 g·kg-1·d-1),WHG: high dose group of Wumei Baijiang prescription (0.736 g·kg-1·d-1),WMG: dose group of Wumei Baijiang prescription (0.368 g·kg-1·d-1) and WLG: low dose group of Wumei Baijiang prescription (0.184 g·kg-1·d-1).Compared with the NG,aP ＜ 0.05;compared with the MG,bP ＜ 0.05.Treatment course: 10 d.

Table 5 Comparison of TNF-α,IL-10 and CRP of mice in each group ()

Table 5 Comparison of TNF-α,IL-10 and CRP of mice in each group ()

Notes: NG: normal group,MG: model group,CG: mesalazine group (0.52 g·kg-1·d-1),WHG: high dose group of Wumei Baijiang prescription(0.736 g·kg-1·d-1),WMG: dose group of Wumei Baijiang prescription (0.368 g·kg-1·d-1) and WLG: low dose group of Wumei Baijiang prescription (0.184 g·kg-1·d-1).TNF-α: tumor necrosis factor-α;IL-10: interleukin-10;CRP: C-reactive protein.Compared with the NG,aP ＜0.05;compared with the MG,bP ＜ 0.05.

4.DISCUSSION

Ulcerative colitis is a kind of chronic non-specific intestinal disease with clinical manifestations of abdominal pain,diarrhea,bloody purulent stool.It is considered that the incidence of the disease is related to environmental factors,genetic susceptibility,immune factors,intestinal flora and other factors right now.4Treg cells and Th17 cells are considered as a new type of subsets of CD4+T cells,most of them in the spleen and peripheral blood of normal body,besides the differentiation and function of the two are mutually restricted.5The Treg/Th17’s immune balance is an important factor to maintain intestinal immune homeostasis,which is closely related to the occurrence of UC.6-8Th17 can induce cells to synthesize and secrete proinflammatory factors,such as TNF-α,IL-6 and others,causing immune imbalance and leading to intestinal inflammation by amplifying the inflammatory response.9Treg cells inhibit T cell immune activity by secreting anti-inflammatory factors such as IL-10,TGF-β and others.10The restoration of Treg/Th17 immune balance provides a new immunology therapeutic direction for UC.At present,5-aminosalicylic acid (5-ASA),glucocorticoids,immunomodulators and biological agents are the main drugs for UC treatment,which are still difficult to meet the clinical needs.Traditional Chinese Medicine has a definite effect in the UC’s treatment on relieving symptoms during the active period,remission.According to the clinical manifestations,it is classified as TCM′s “JiuLi”,“Rest Diarrhea” and “ChangPi”.UCis one of the superiority diseases in TCM.prescription of Professor Lu Zhizheng,a master of Chinese medicine,in the treatment of UC of large intestine damp and heat syndrome.Wumei (Mume Fructus),sour taste,mild nature,can astringent intestines to stop diarrhea,promote the growth of body fluid and relieve ascaris.Baijiangcao(Herba Patriniae Scabiosaefoliae),bitter taste,mild cold,can heat clearing and detoxification,good at removing carbuncle and pus.inducing relieve and maintaining relieve during the Both of them are monarch herb.Wumei (Fructus Mume) astringent intestines stop diarrhea and soften the liver to relieve pain,Baijiangcao(Herba Patriniae Scabiosaefoliae) detoxify and discharge pus.Taizishen (Radix Pseudostellariae),Stirfrying Baizhu (Rhizoma Atractylodis Macrocephalae),Fuling (Poria) and Stir-frying with honey Gancao (RadixGlycyrrhizae) are four minister herbs,they work together to strengthening the spleen and replenishingQi,make the spleen healthy to transport water and humidity,eventually stop diarrhea.Adjuvant Drug: Huanglian(Rhizoma Coptidis),bitter taste,cold nature,belonging to the large intestine meridian,good at clearing away heat,drying dampness and detoxification.Stir-frying Baishao (Radix Paeoniae Alba) nourishing blood and cooperating to relieve pain,and add Danggui (Radix Angelicae Sinensis) more nourishing and promoting blood circulation.It embodies the meaning of “running blood,purulence self-healing”.Gegen (Radix Puerariae Lobatae) can stop diarrhea and promoteYangclearing.Stir-frying Zhiqiao (Fructus Aurantii Submaturus) and Muxiang (Radix Aucklandiae) regulate the liver and reduce turbidYin.Arise and a descend regulatingQi.Stir-frying with honey Gancao (Radix Glycyrrhizae)various medicines.The whole prescription has the functions of strengthening the spleen and softening the liver,clearing away heat and strengthening the healthy and removing pathogenic factors,regulating the intestine and stopping diarrhea.In this experiment,we observed the effect of Wumei Baijiang prescription on Treg/ Th17 immune balance in dampness,regulatingQiand blood circulation,UC mice,explored the influence mechanism from the experiment point,to provide a scientific basis for clinical.

In conclusion,the results show that after drug intervention:（a）The body weight and food intake of mice in the WHG,WMG and CG increased (P ＜0.05);（b）The DAI scores of mice in the WHG,WMG and CG decreased (P ＜0.05);（c）The colonic length of mice in the WHG,WMG and CG increased (P ＜0.05);（d）Compared with the MG,the pathological mucosal defects of colon tissue in the WHG and CG were significantly repaired;（e）Treg increased (P ＜0.05),Th17 decreased (P ＜0.05),CD4+increased (P ＜0.05),CD8+decreased (P ＜0.05),TNF-α and CRP decreased(P ＜0.05),IL-10 increased (P ＜0.05) in the spleen of mice in the WHG,WMG and CG.In summary,the results showed that the Wumei Baijiang prescription can increase the body weight and food intake,reduce the DAI score and intestinal mucosa damage of UC mice induced by DSS.This is not only a further verification of the efficacy of this prescription,but also maybe one of the mechanisms involved in regulating Treg/ Th17 immune balance in the treatment of UC mice.