Correlation between slow transit constipation and spleen Qi deficiency,and gut microbiota:a pilot study

YU Zeyue,HAO Liyu,LI Zongyuan,SUN Jianhui,CHEN Hongying,HUO Hairu,LI Xiaoqin,SHAN Zhongchao,LI Hongmei

YU Zeyue,HAO Liyu,SUN Jianhui,Li Hongmei,HUO Hairu,LI Xiaoqin,Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100007,China

LI Zongyuan,Jiangsu University,Zhenjiang 212013,China

CHEN Hongying,Yunnan University of Traditional Chinese Medicine,Kunming 650504,China

SHAN Zhongchao,Jiangxi University of Traditional Chinese Medicine,Nanning 330004,China

Abstract OBJECTIVE:To investigate the effect of slow transit constipation (STC) and spleen Qi deficiency on gut microbiota,and the mechanism underlying the action that the positive drug Maren Runchang (MR) alleviates STC.METHODS:STC was induced,using the cathartic method of Senna and the hunger-fullness disorder method,in ICR mice;one group of model mice was treated with MR (6.24 g/kg).The changes in the general condition,fecal parameters,D-xylose content in the serum,intestinal propulsion rate,and histopathology of the colon were assessed after STC induction in the control,model,and MR groups.Fecal microbiota transplantation (FMT) was performed from STC mice into pseudo germ-free mice.Changes in the contents of substance P (SP),vasoactive intestinal peptide (VIP),and gut microbiota in STC mice and pseudo germ-free mice were assessed after FMT.RESULTS:Compared with the control group,the model mice showed the following results:the time of the first black stool was significantly longer (P ＜ 0.01),the number and weight of black stools were significantly reduced within 6 h (P ＜ 0.05),the D-xylose content in the serum was significantly reduced (P ＜ 0.05),the intestinal propulsion rate decreased (P ＜ 0.01),the content of VIP in colon tissue significantly increased (P ＜ 0.05),and SP content in the colon tissue significantly decreased (P ＜0.01);moreover,the colon showed significant inflamemation and injury.Furthermore,the abundance of Firmicutes was increased,the abundance of Bacteroides decreased,and the abundance of Alistipes,unclassified_o__Bacteroidales,Alloprevotella,Bilophila,and Anaerotruncus decreased,while the abundance of the conditional pathogenic bacteria Escherichia and Klebsiella increased.However,after treatment with MR,the time of the first black stool decreased (P ＜ 0.01),the number of black stools within 6 h increased,and the intestinal propulsion rate increased (P ＜ 0.05).Moreover,the content of D-xylose in the serum and the content of VIP in colon tissue significantly decreased (P ＜ 0.05),the content of SP in colon tissue significantly increased (P ＜0.01),and colon inflammation significantly improved.Additionally,the abundance of Firmicutes decreased,and the abundance of Bacteroides increased.The abundance of unclassified_o__Bacteroidales and Bilophila increased,and the abundance of Klebsiella decreased.In the model+FMT group,compared with control+FMT group,the content of VIP in colon tissue decreased (P ＜ 0.05),the content of SP in colon tissue significantly increased (P ＜0.01),and the abundance of probiotics,such as Lactobacillus and Akkermansia,decreased.In the MR +FMT group,compared with the model+FMT group,the content of VIP in colon tissue increased,the content of SP in colon tissue significantly decreased (P ＜ 0.01),and the abundance of probiotics increased.CONCLUSIONS:STC mice with spleen Qi deficiency show a decreased abundance of beneficial bacteria,such as Alistipes,unclassified_o__Bacteroidales,Alloprevotella,Bilophila,and Anaerotruncus,and an increased abundance of the conditional pathogenic bacteria Escherichia coli and Klebsiella.Furthermore,the mechanism of action of MR in treating STC may involve the regulation of intestinal movement,reduction of intestinal inflammation,elevation of intestinal absorption,and regulation of gut microbiota.

Keywords:spleen Qi deficiency;gastrointestinal microbiome;probiotics;fecal microbiota transplantation;substance P;vasoactive intestinal peptide;slow transit constipation;Maren Runchang pills

1.INTRODUCTION

Slow transit constipation (STC) is a common clinical functional disease of the digestive system.1The number of STC patients is increasing annually in China;the prevalence rate of STC is about 6%,and the ratio of males to females is presently 1 :2.2,3There are various causes of STC,such as poor eating habits,insufficient dietary fibre and liquid intake,and lack of exercise,all of which may lead to colonic motility disorders,reduced propulsion and contraction movements,and reduced peristaltic ability,which prolong the transit time of the colon and ultimately lead to constipation.4,5The clinical symptoms of slow transit constipation include dry stools,difficulty in defaecation,reduced frequency,and abdominal distension.In addition,Traditional Chinese Medicine believes that STC belongs to the category of'constipation'.Although constipation is a large intestinal conduction dysfunction,it is closely related to the viscera,Qi,and blood.6The Chinese medicine book Jingyue Quanshu states the following:‘In ancient prescription books,there were different theories for the symptom pattern of constipation,such as constipation caused by deficiency,wind,deficiency ofQi,heat,cold,moisture etc',7implying that deficiency is often related to the lungs,spleen,and kidneys.The book Spleen and Stomach Theory reports 'the sufficient vitality was due to the filling of the spleen and stomach,which could then nourish vitality '.8The basic point of view is that all diseases come from life.Splenasthenic symptom pattern leads to poorQi,which affects the digestion and absorption of food,as well as the formation and transmission of faeces.Therefore,splenasthenic symptom pattern is commonly associated with STC in Traditional Chinese Medicine.9Modern studies have shown that the gut microbiota affects the intestinal motility of the host and that an imbalance in the gut microbiota can result in constipation.10,11During constipation,the abundance of intestinal aerobic bacteria,Escherichia coli,andfungioften significantly increases,while that ofBacteroides,Anaerobes,andBifidobacteriumgenerally significantly decreases.12-15Maren Runchang (MR) pills are a representative formula of hemp seed,bitter almond,rhubarb,woody,tangerine peel,and white peony,used in Traditional Chinese Medicine as a moisturizer and laxative,and can markedly alleviate constipation symptoms.It is also very suitable for constipation patients withQideficiency,ageingQideficiency,and a weak body.The purpose of this study was that investigating the effect of slow transit constipation (STC) and spleenQideficiency on gut microbiota,and the mechanism underlying the action that the positive drug MR alleviates STC.

2.MATERIALS AND METHODS

2.1.Drugs

MR pills (code 19015139) and Fanxieye were purchased from Beijing Tongrentang Co.,Ltd.(Beijing,China);ampicillin (code 716Q022),neomycin sulphate (code 910J056),metronidazole (code 428O022),and vancomycin (code 719Q021) were provided by Solarbio(Beijing,China).

2.2.Materials

D (+) Xylose (code 307E057),was provided by Solarbio(Beijing,China).Biowest agArose was purchased from Biowest (Spain);FastPfu Polymerase was provided by TransGen (Beijing,China).Formaldehyde (code F20180825) was prepared at a concentration of 10% for use,xylene (code 20170705),both were provided by Sinopharm Chemical Reagent Co.,Ltd.(Shanghai,China).Ethanol was provided by Beijing Chemical Plantwere (Beijing,China) and prepared in 60%,70%,80%,90%,and 100% concentrations for use (anhydrous ethanol code 20180208,95% ethanol code 20180208).Gram’s stain (code 0722A21),Phenylmethylsulfonyl fluoride (PMSF) (code 20210126),and Phosphate buffered saline (PBS) (code 20210729) were provided by Solarbio (Beijing,China).Lysis Buffer was provided by Yunclone (Wuhan,China).

The D-xylose Kit (code 20200110) and total protein quantification kits were provided by Nanjing Jiancheng Institute of Biological Engineering (Nanjing,China).DNeasy PowerSoil Kit (code D4015-02) was provided byQiagen (Hilden,North Rhine Westphalia,Germany).AxyPrep DNA Gel Extraction Kit was provided by Axygen (Silicon Valley,CA,USA).NEXTFLEX Rapid DNA-Seq Kit was purchased from Bioo Scientific(Austin,TX,USA).NovaSeq Reagent Kits were provided by Illumina (San Diego,CA,USA).Mouse SP detection kit (enzyme-linked immunosorbent assay) and VIP detection kit (enzyme-linked immunosorbent assay)were provided by Cloud-Clone (Wuhan,China).

2.3.Instruments

The electronic balance (code PL203) was provided by Mettler Toledo (Shanghai,China);the microplate reader was purchased from Bio Tek (Norcross,VT,USA).Pipettes (N13462C),a small centrifuge (code 5430R)and a high-speed bench-top refrigerated centrifuge (code 5424R) were provided by Eppendorf (Eppendorf,Saxony,Germany).A thermocycler (GeneAmp 9700)was provided by ABI (Waltham,MA,USA).Illumina Miseq sequencer was purchased from Illumina (San Diego,CA,USA).Paraffin embedding machine (code EG1140H) was provided by Leica (Cologne,North Rhine Westphalia,Germany).Automatic dyeing machine (code XL5010) was purchased from Autostainer (Cologne,North Rhine Westphalia,Germany).Bioprep-24 (code AS-Bioprep-1031) was provided by Aosheng instrument (Hangzhou,China).

2.4.Animals

The animal study protocol was approved by the Animal Ethics Committee of China Academy of Chinese Medical Sciences (approval number SYXK 2019-0003).All mice were provided by Beijing Weitonglihua Experimental Animal Technology Co.,Ltd.(Beijing,China) and handled at the China Academy of Chinese Medical Sciences.The 60 ICR mice (specific pathogen free grade;weight 18-22 g;half male and half female)were maintained in a specific pathogen-free state under a strict light cycle (lights on at 06:00 h and off at 18:00 h)at (22 ± 2) ℃ and relative humidity of 50% ± 10%.All mice were randomly assigned to groups (n=10 per group) according to body weight,control,model,MR,control+FMT,model+FMT,and MR+FMT,after 2 days of adaptive feeding.

2.5.Dosage design

The clinical dosage of MR pills as a positive drug was 1 or 2 pills (6 g),once or twice a day.Calculated according to the ratio of animal to human surface area,two times the equivalent dose for mice is 6.24 g/kg.The clinical dosage of ampicillin was 3 g/d for adults,and that for neomycin sulphate was 2 g/d,metronidazole was 1.5 g/d,and vancomycin was 2 g/d.According to the same conversion method,the antibiotic dosages were as follows:ampicillin,0.39 g/kg;neomycin sulphate,0.26 g/kg;metronidazole,0.195 g/kg;and vancomycin,0.26 g/kg.

2.6.Preparation of STC in mice with deficiency of spleen Qi model and treatment

The experimental groups were administered Fanxieye(20 g/kg) by gavage,resulting in splenasthenic symptom pattern on 1st-7th day;on 8th day,mice stopped gavage,and 6 g of raw rice and 0.5 h of water were provided every other day,on 8th-14th day,while MR pills (6.24 g/kg) were administered to the MR group for 7 consecutive days,and the control and model groups were given equal volumes of distilled water.13On 14th day,fresh stool samples from all groups were collected and immediately prepared for FMT solution.

2.7.Preparation of pseudo germ-free mice and STC model with spleen Qi deficiency

The control+FMT,model+FMT,and MR+FMT groups were gavaged mixed antibiotics (including ampicillin 0.39 g/kg,neomycin sulphate 0.26 g/kg,metronidazole 0.195 g/kg,and vancomycin 0.26 g/kg) on 1st-7th day.On 8th day,fresh stools from the control and three FMT groups were collected.The stools of six control mice and six pseudo germ-free mice were randomly selected.Direct faecal smear and gram stain methods were used to observe the difference in microbe numbers under the microscope.After successful preparation of the pseudo germ-free mice,the model +FMT and MR+FMT groups were prepared for the STC model with spleenQideficiency using the aforementioned method on 8th-21th day.

2.8 FMT

Fresh stool samples from the control,model,and MR groups were mixed with sterile normal saline (fecal weight :volume of sterile saline=1 :5) and homogenised immediately.Homogenates were then passed through a 200-and 70-µm pore nylon filter to remove large particulate and fibrous matter.The filters were centrifuged at 6000 r/min for 5 min at 4 ℃.The underlayer precipitate was dissolved in sterile normal saline (faecal weight∶volume of sterile saline=1∶10).On 15th-21th day,the male and female mice of the control+FMT,model+FMT,and MR+FMT groups were gavaged with relevant FMT solution (200 μL per mouse).

2.9.General condition of mice

Hair,mental state,fecal shape,degree of perianal pollution,diet,and body weight of mice in different groups were assessed.

2.10.Fecal parameters measurement

The mice in the control and model groups were gavaged with carbon powder paste,and the MR group was gavaged with carbon powder paste mixed with crushed MR pills.The mice were placed in a single cage with a normal diet,and at the time of the first black stool,the number and weight of black stools was measured within 6 h.

2.11.Detection of serum D-xylose content

The mice in the control,model,and MR groups were fasted for 16 h,but had free access to water.Then,each group was administered 3% xylose solution (10 mL/kg)by gavage.After 45 min,the mice in the control and model groups were gavaged with carbon powder paste,and the MR group was gavaged carbon powder paste mixed with crushed MR pills.After 15 min,blood was collected from the eyeballs.The blood was allowed to stand for 2 h,at 4 ℃,and centrifuged at 2500 r/min for 15 min at 4 ℃,after which the serum was collected.The serum xylose content was determined by colourimetry according to the manufacturer' s instructions.

Serum D-xylose (mmol/L)=(measured absorbance A value-measured blank A value)/(standard A valuereagent blank A value) × standard concentration.

2.12.Measuring intestinal propulsion rate

After blood was collected from the eyeballs,the mice in the control,model,and MR groups were killed by cervical dislocation.The abdominal cavity was opened to remove the intestine from the pylorus to the ileocecal area.The total length of the intestine and the length of the carbon paste advancing in the intestine were measured to calculate the rate of intestinal advancement.Intestinal advancement rate=(distance from the pylorus to black intestinal tube ÷ full length from the pylorus to ileocecal region) × 100 %.

2.13.Histological analysis of colon tissues

After measuring the intestinal propulsion rate,the colon tissues were sampled approximately 2 cm from the anal end and fixed in 10 % formaldehyde solution.The fixed colon was dehydrated,waxed,embedded,sectioned,displayed,baked,and stained with HE.Finally,images were captured using a microscope,and the imaging system was used to collect and analyse the relevant parts of the sample.

2.14.Preservation of cecum contents and colon tissues

In the control,model,MR,control+FMT,model+FMT,and MR+FMT groups,the contents of the caecum and colon tissues,which were approximately 2 cm from the anal end,were placed in a sterile EP tube and stored at-80 ℃.

2.15.Substance P (SP) and vasoactive intestinal peptide(VIP) content in 5% colon homogenate

In the control,model,MR,control+FMT,model+FMT,and MR+FMT groups,the 0.1 g colon tissue samples were weighed and washed 2 times with pre-cooled PBS.The tissues were cut into homogenisation tubes,and 1 mL lysis buffer,10 μL PMSF,and a steel ball were added to each tube.The tubes were placed on ice for 30 min,and then placed into Bioprep-24 to prepare a 5%colon homogenate.The homogenate was centrifuged at 12 000 r/min for 10 min at 4 °C,and the supernatant was considered to be the total protein solution.A BCA protein assay kit was used to determine the total protein concentration.The contents of SP and VIP in 5 % colon homogenate were measured using the ELISA kit.

2.16.Analysis of faecal microbiota

After collection of caecum contents,total DNA of fecal microbiota was extracted from the samples using a DNeasy PowerSoil Kit according to the manufacturer’s instructions,and the DNA purity and concentration were tested using a NanoDrop2000.The extracted DNA was quantified using Quant-IT PicoGreen,and sequencing libraries were prepared according to the Illumina 16s Metagenomic Sequencing Library protocols used to amplify the V3 and V4 regions.The input gDNA (10 ng)was PCR amplified with 5×FastPfu buffer,2.5 nm dNTP mix,5 μm each of the universal F/R PCR primers,and 0.4 μL of FastPfu polymerase.The cycle conditions for the first PCR were 3 min at 95 ℃ for heat activation,and 25 cycles of 30 s at 95 ℃,30 s at 55 ℃,and 30 s at 72 ℃,followed by a 5-min final extension at 72 ℃.The universal primer pair with the Illumina adapter overhang sequences used for the first amplification were as follows:16s V3-V4,338F:5'-ACTCCTACGGGAGGC AGCAG-3',806R:338F:5'-GGACTACHVGGGTWT CTAAT-3'.The PCR product was purified with the AxyPrep DNA Gel Extraction Kit and quantified using a Quantus™ Fluorometer.Finally,paired-end (2 × 300 bp)sequencing was performed by the Macrogen unit using the Miseq PE300.

FLASH was used for data optimisation,18and data analyses were performed by OTU clustering with 97%similarity.19Species classification,diversity,and composition analysis were performed using the Silva(Release132 http://www.arb-silva.de) database.Abundance significant difference taxon screening was performed at the genus level using the LEfSe method,20and the discrimination criteria were as follows:Kruskal-Wallis test screening valueα＜ 0.05,paired Wilcoxon test screening valueα＜ 0.05,and significant discriminant value LDA＞2.

2.17.Statistical analysis

The data from different groups were analysed using oneway ANOVA followed by Tukey’s post-hoc t-test for multiple comparisons using SPSS 23.0 software (IBM Corp.Released 2015.IBM SPSS Statistics for Windows,Version 23.0.IBM Corp.,Armonk,NY,USA).Data were presented as the mean ± standard deviation () values,and statistical significance was set atP＜ 0.05.

3.RESULTS

3.1.General condition of mice

At the end of the experiment,the model mice were depressed,showed a decrease in activity,showed a decrease in response,had messy and dim hair,and had dry stools;the body weight of male and female mice decreased significantly compared to the control group.However,the above situation was improved in mice in the MR group.Moreover,the body weights of male and female mice in the model+FMT group were significantly lower than those of the control+FMT group mice (Table 1).

3.2.Fecal parameter measurement

The first black stool in the model group occurred significantly later compared to the control group.The number and weight of black stools in the model group were significantly lower than those in the control group,within 6 h (P＜ 0.05).In the MR group,the time taken for the first black stool to occur was significantly less than that of the model group,and the number of black stools increased significantly over the 6-h period(Table 2).

Table 1 Body weight of female mice which had STC with deficiency of spleen Qi (g,)

Table 1 Body weight of female mice which had STC with deficiency of spleen Qi (g,)

Notes:control group:normal female mice without treatment (n= 5);model group:slow transit constipation with deficiency of spleen Qi female mice without treatment (n= 5);MR group:slow transit constipation with deficiency of spleen Qi female mice treated with Maren Runchang pills intragastrically at a daily dosage of 6.24 g/kg (n= 5);control+FMT:pseudo germfree female mice were given control mice' s fecal microbio transplatation (n= 5);model+FMT:pseudo germfree female mice,which had slow transit constipation with deficiency of spleen Qi,were given model mice's fecal microbio transplatation (n= 5);MR+FMT:pseudo germfree female mice,which had slow transit constipation with deficiency of spleen Qi,were given MR mice's FMT (n =5).MR:Maren Runchang;FMT:fecal microbio transplantation.Compared with the control group,aP ＜ 0.01;compared with the control+fecal microbio transplatation group,bP ＜ 0.01.

Table 2 Fecal parameters measurement of mice which had STC with deficiency of spleen Qi ()

Table 2 Fecal parameters measurement of mice which had STC with deficiency of spleen Qi ()

Notes:control group:normal mice without treatment (n= 10);model group:slow transit constipation with deficiency of spleen Qi mice without treatment (n= 10);MR group:Slow transit constipation with deficiency of spleen Qi mice treated with Maren Runchang pills intragastrically at a daily dose of 6.24 g/kg (n= 10).MR:Maren Runchang.Compared with the control group,aP ＜ 0.01,cP ＜ 0.05,eP ＜ 0.01;Compared with the model group, bP ＜ 0.01,dP ＜ 0.05.

3.3.Intestinal propulsion rate of mice

Compared with the control group (n=10,71.7 ± 5.6),the intestinal propulsion rate of mice in the model group was visibly reduced (n=10,58.6±5.5,P＜ 0.01),and compared with the model group (n=10,58.6 ± 5.5),the intestinal propulsion rate of mice in the MR group increased significantly (n=10,64.6 ± 6.2,P＜ 0.05).

3.4.Histological analysis of colon tissues

In the control group,the colonic mucosa had a smooth surface,no obvious inflammation or oedema,and neatly arranged glands.There were no apparent lesions in the submucosal muscle layer,and the structure was normal.Obvious inflammatory changes were observed in the colon of mice in the model group,wherein the mucosal structure was unclear,and hyperaemia and swelling were observed.The surface was not smooth,and the glands were disordered,atrophied,and showed inflammatory cell proliferation.Compared with the model group,the mice in the MR group showed improvement in colonic mucosal inflammatory exudation,oedema,blood stasis,and mucosal atrophy to varying degrees (Figure 1,Supplementary Table 1).

Figure 1 HE staining to observe the pathological changes and characteristics of colon tissue and inflammatory cell infiltration in mice

3.5.Serum D-xylose content in mice

Compared with the control group (n=10,0.74±0.36),the serum D-xylose content of mice was significantly reduced in the model group (n=10,0.45±0.23,P＜0.05);Compared with the model group (n=10,0.45±0.23),the serum D-xylose content of mice showed an upward trend (n=10,0.67±0.27).

3.6.Evaluation index of pseudo germ-free mice

There were many gram-negative bacteria (blue) and gram-positive bacteria (red) in normal mice stool smears,but there were essentially no gram-negative bacteria or gram-positive bacteria in the pseudo germ-free mice stool smears.This indicates that the pseudo germ-free mice were successfully prepared (Figure 2).

3.7.SP and VIP content in 5% colon homogenate

Compared with the control group,the content of SP decreased significantly,and the VIP content increased significantly in the model group.The content of SP increased significantly,and that of VIP decreased significantly in the MR group,compared to the content in the model group.The content of SP and VIP did not differ between the control and control+FMT groups,implying that gavaging mixed antibiotics did not affect the content of SP and VIP in mice.The content of SP was much lower,and VIP was much higher in the model +FMT group than in the control+FMT group.Moreover,the content of SP increased significantly,and that of VIP decreased significantly in the MR+FMT group,compared to that in the model+FMT group (Table 3).

Table 3 The SP,VIP content of mice which had STC with deficiency of spleen Qi in 5 % colon homogenate (pg/mg,)

Table 3 The SP,VIP content of mice which had STC with deficiency of spleen Qi in 5 % colon homogenate (pg/mg,)

Notes:control group:normal mice without treatment (n= 10);model group:Slow transit constipation with deficiency of spleen Qi mice without treatment (n= 10);MR group:STC (slow transit constipation with deficiency of spleen Qi) mice treated with Maren Runchang pills intragastrically at a daily dosage of 6.24 g/kg (n= 10);control+FMT:pseudo germfree mice were given control mice's fecal microbio transplatation (n= 10);model+FMT:pseudo germfree mice,which had slow transit constipation with deficiency of spleen Qi,were given model mice's FMT (n= 10);MR+FMT:pseudo germfree mice,which had slow transit constipation with deficiency of spleen Qi,were given MR mice's fecal microbio transplatation (n= 10).MR:Maren Runchang;FMT:fecal microbio transplantation.Compared with the control group,aP ＜ 0.01,bP ＜ 0.01,cP ＜ 0.01,dP ＜ 0.01;compared with the control+fecal microbio transplatation group,eP ＜ 0.05,fP ＜ 0.05,gP ＜0.05.

3.8.STC induced gut microbiota dysbiosis in mice

The gut microbiota was analysed using 16S rRNA gene sequencing.The sequences were divided into OTU groups according to 97% similarity by clustering on the Uparse software platform.The Pan/Core species analysis showed that the Pan species abundance increased and levelled off,and that of the core species decreased and levelled off as the sample size increased.This means that the sample size of STC with deficiency in spleenQiwas sufficient in this experiment (Supplementary Figure 1).We found that STC model mice exhibited significantly reduced abundance and diversity of gut microbiota,while MR increased abundance and diversity of gut microbiota as Chao (577 ±34vs484 ±38vs574 ±35,bothP＜ 0.01);ACE (567 ± 25vs478 ± 38vs566 ± 23,bothP＜ 0.05),and Sobs indexes decreased (477 ± 20vs407± 23vs482 ± 17,bothP＜ 0.01).

Figure 2 Evaluation index of pseudo germfree mice

3.9.Similar trend of induced gut microbiota dysbiosis by FMT

As shown in the Pan/Core analysis,the sample size of the FMT was sufficient (Supplementary Figure 2).We also found that the faecal of STC mice changed the gut microbiota;this trend was similar to the above result,with respect to Chao (54.17 ± 7.42vs29.00 ± 11.38vs48.5 ± 14.75,P＜ 0.01,＜ 0.05),Sobs (54.00 ± 7.44vs29.00 ± 11.38vs48.5 ± 14.75,P＜ 0.01,＜ 0.05),and Shannon indexes (2.94 ± 0.50vs2.20 ± 0.12vs2.93 ±0.30,bothP＜ 0.01).These results suggest that STC with spleenQideficiency significantly reduced the abundance and diversity of gut microbiota.

3.10.Analysis of species composition of gut microbiota

PLS-DA showed distinct clustering of gut microbiota composition between the control group,model group,model group,and MR group.At the phylum level,compared with the control group,the relative abundance of Firmicutes had increased,while the relative abundance of Bacteroidetes had decreased in STC model mice.However,the relative abundance of Firmicutes decreased,while the relative abundance of Bacteroidetes increased,in the MR group compared to abundances in the model group (Figure 3A).

At the genus level,we observed that the abundances ofLachnospiraceae,Lactobacillus,Blautia,Escherichia-Shigella,andKlebsiellaincreased while those ofBacteroides,Alloprevotella,RikenellaceaeandAlistipesdecreased,in STC model mice.However,the opposite trend was observed in the MR group (Figure 3B).

LEfSe analysis identified the differential microbial biomarkers between the control and model groups or between the model and MR groups.LDA scores,using the Kruskal-Wallis rank sum test with a confidence interval of 95% at the genus level,showed the significant bacterial differences between the control and model group or between the model and MR group,as follows:Alistipes,unclassified_o__Bacteroidalesshowed a particularly significant difference (P≤ 0.001);Escherichia-Shigella,Lachnospiraceae_UCG-006,andBifidobacteriumshowed a more significant difference(0.001 ＜P≤0.01);Lachnospiraceae_NK4A136_group,Bacteroides,Alloprevotella,Lactobacillus,Blautia,Rikenellaceae_RC9_gut_group,Bilophila,Lachnoclostridium,Klebsiella,andAnaerotruncusshowed a significant difference (0.01 ＜P≤0.05) (Figure 5C).

3.11.Effect of FMT on species composition of gut microbiota

The same trends were observed as with the above results.At the phylum level,compared with the control+FMT group,the relative abundance of Firmicutes had increased while the relative abundance of Bacteroidetes had decreased in the model+FMT group.By treating the faeces of mice in the MR group,the relative abundance of Firmicutes decreased,while that of Bacteroidetes increased (Figure 4A).

At the genus level,the results showed that the abundances ofBacillus,Blautia,Bacilli,Bacteroides,Klebsiella,andEnterococcushad increased,while those ofLactobacillus,Saccharimonas,Muribaculaceae,Dubosiella,Enterorhabdus,AkkermansiaandClostridiahad decreased,in the model+FMT group.The opposite trend was observed in the MR+FMT group (Figure 4B).According to the LEfSe analysis,the bacterial species with significant differences in the three groups were as follows:norank_f__norank_o__Clostridia_UCG-014(P≤0.001);Lactobacillus,Bacillus,unclassified_f__Lachnospiraceae,Blautia,unclassified_c__Bacilli,Bacteroides,Klebsiella,Enterorhabdus,andEnterococcus(0.001 ＜P≤0.01);Candidatus_ Saccharimonas,norank_f__Muribaculaceae,Dubo-siella,Akkermansia,andLachnospiraceae_ NK4A136_ group(0.01 ＜P≤0.05) (Figure 4C).

3.12.Changes in gut microbiota structure

Principal coordinate analysis (PCoA) showed that the results were significant at the OTU level (P=0.001 andR=0.4139 for the three groups),indicating a high confidence level that the difference between groups was greater than the difference within groups and that the difference was significant.The control group was completely separated from the model and MR groups,and the model group had a large deviation compared with the MR group,indicating that the gut microbiota of the model mice produced large changes and that the MR could change the gut microbiota structure of the model mice to a certain extent (Figure 5).As per thePerMANOVAanalysis,based on the weighted UniFrac matrix,at the OTU level,there were significant differences in the structure of the gut microbiota between the control,model,and MR groups (Table 4).In addition,the higher the R value,the greater was the difference.

3.13.Effect of FMT on gut microbiota structure

PCoA results showed thatP=0.002 andR=0.3904 at the OTU level for the four groups (Figure 6).As perPerMANOVAanalysis,there were significant differences between the control+FMT and model+FMT groups,as well as the model+FMT and MR+FMT groups (Table 5).

Table 5 PerMANOVA analysis results between different groups at OTU level of pseudo germfree mice

Figure 6 Principal co-ordinates analysis at operational taxonomic unit (OTU) level of pseudo germfree mice

Table 4 PerMANOVA analysis results between different groups at OTU level of slow transit constipation with deficiency of spleen Qi mice

4.DISCUSSION

Splenasthenic symptom pattern is an important part of Zangxiang theory in Traditional Chinese Medicine.The earliest record of splenasthenic symptom pattern is in the'Lingshu Ben Shen' of the Warring States Period:‘If the SpleenQiwas deficient,the limbs were not used,and the five organs were restless'.Differentiated from the symptom pattern,later generations summarized the typical symptoms of splenasthenic symptom pattern on the basis of 'Taiping Shenghui Fang':'The heart and abdomen were full,the water and grain were not eliminated,the stomach liked to swallow acid,the abdomen hurts and the intestines sound,and the limbs were heavy,and if the pulse was sunken and weak,this was a sign of splenasthenic deficiency'.The spleenasthenic symptom pattern model was prepared using the cold medicine venting method and the hanger-fullness disorder method.The model mice had obvious constipation associated with splenasthenic symptom pattern,such as lack of energy,reduced activity,reduced response,messy and dim hair,and dry stools,which implied that the experimental model had been successfully established.

D-Xylose is a type of pentose,which does not exist in the blood under normal circumstances.After oral administration of D-xylose,it is absorbed through the small intestine into the blood.Therefore,measuring its content in serum at a specific time can be used to evaluate intestinal absorption function.Pang et al.also considered it to be one of the pathological manifestations of spleen deficiency symptom pattern.21,22SP belongs to tachykinin,an excitatory motor neurotransmitter,mainly distributed in the intestinal myenteric plexus,and its excitatory effects mainly include the dual contraction on the longitudinal and circular muscles of the gastrointestinal tract and co-stimulation of intestinal movement.23,24VIP is a biologically active molecule with dual functions of gastrointestinal hormones and neuropeptides.It is an important inhibitory neurotransmitter in the enteric nervous system and can regulate intestinal motility and secretion.25Compared with the control group,the content of D-xylose in serum and SP in 5% colon homogenate decreased,and the content of VIP in 5% colon homogenate increased in the model mice;these results were similar to those of previous studies.26,27Compared with the model group,the laxative function,colon tissue inflammation,oedema,and blood stasis were improved.The D-xylose content in serum and the SP content in 5% colon homogenate improved,and the VIP content in 5% colon homogenate decreased.The mechanism of action of MR in the treatment of STC with spleenQideficiency may involve regulation of the relaxation and contraction of intestinal smooth muscles.Previous studies have shown that constipation is closely related to gut microbiota,10-15but there are no studies on the gut microbiota of STC in conjunction with spleenQideficiency.In this study,the role of gut microbiota in splenasthenic deficiency and constipation was investigated by analysing the structure of gut microbiota,as well as screening for taxonomic units with significantly different abundances and predicting their metabolic pathways.At the phylum level,compared with the control group,the spleen-deficiency constipation model showed an increase in the abundance of Firmicutes and a decrease in the abundance of Bacteroides.Studies have shown that high abundance of Firmicutes and low-abundance Bacteroides are related to obesity and that Bacteroides is related to energy production.28The gut microbiota enables the body to complete energy production and conversion by digesting dietary polysaccharides.In addition,gut microbiota can enhance the expression of adipocytokines and reduce the storage of triglycerides in adipose tissue,thereby influencing the activity of LPL.29After treatment with MR pills,the abundance of Bacteroidetes increased,which suggests that one of the pathways of MR in treating STC due to splenasthenic symptom pattern may be related to LPL enzymes.Some studies indicate that gut microbiota dysbiosis may affect the content of VIP and SP.30,31It can reduce the content of intestinal probiotics,increase the reproduction of conditional pathogens,cause intestinal mucosal damage,and increase the production of SP;it has been confirmed that a large amount of SP can affect gastrointestinal motility.32In addition,VIP can improve the internal environment of the intestinal mucosa tissue,promote the reproduction of beneficial bacteria,and restore the proportions of microbes.Imbalance in the microbiota leads to a decrease in the content of VIP,which further aggravates the imbalance in intestinal microbiota,forming a feedback loop and promoting the development of STC.

Alistipesfinegoldiibelongs to the phylum Bacteroidetes and is one of the most important anaerobic,gramnegative inhabitants of the gut microbiota.It not only produces saturated fatty acids,but also encodes a unique combination of acyl-ACP synthetases with different substrate specificities to exploit the fatty acid nutrientsprovided in the gut environment to manufacture its membrane lipids,and thus,increase the risk of type 2 diabetes.33Moreover,unclassified_o__Bacteroidalesbelongs toBacteroidales,and a previous study has shown thatB.thetaiotaomicron,a typical species ofBacteroidales,has many beneficial functions,including nutrient absorption and epithelial cell maturation and maintenance.34In recent years,researchers have found that members ofBacteroidalesmight be a treatment target for Alzheimer’s disease.Escherichia-Shigellais a common conditional pathogen,which is a potential risk factor for ulcerative colitisrelated deterioration.35The abundance ofEscherichia-Shigellais positively correlated with the severity of ulcerative colitis.36,37AlloprevotellaandPrevotellaceae_UCG-001 are two types ofPrevotellaspecies.Previous studies have confirmed thatPrevotellais less abundant in the gut microbiota of children with autism,andPrevotellahas the ability to degrade a variety of plant polysaccharides,owing to which it plays a key role in digesting carbohydrate-rich foods.38,39Lachnospiraceae_NK4A136 andLachnospiraceae_UCG-006 belong toLaospirillum.Previous studies have shown that the high abundance ofLacetospirillumis related to diabetic nephropathy and type 2 diabetes,40while the abundance ofLaospirillumin patients with liver cirrhosis is low,41and a high-fat diet could decrease colonisation byLachnospiraceae_NK4A136_group,which might be a target for preventing high-fat diet-potentiated spontaneous preterm birth.42However,the role of these two enteric bacteria in this experiment needs to be further investigated.GCA-900066575 has not been reported yet.Hydrogen sulphide is highly relevant,with respect to human health.Bilophila,an opportunistic pathogen,produces hydrogen sulphide when respiring (bi) sulphite released from organosulphonate substrates,including abundant dietary and host-derived substrates.43Klebsiella,a commonEnterobacteriaceae,is a conditional pathogen of various disease.44It was also positively correlated with ALT in serum and liver TG.45Anaerotruncusis a butyric acid-producing bacterium.Butyrate is beneficial to the physiological function of various tissues and organsin vivo.46,47According to the 16S rRNA results of FMT,compared with the control +FMT group,the content of probiotics such asLactobacillusandAkkermansiadecreased.48After treatment with the FMT solution,in the MR group,the abundance of the above probiotics increased.

In conclusion,the results of this study showed that STC with spleenQideficiency had a close connection with gut microbiota,and MR pills treated it by adjusting the community formation of gut microbiota.This study is a passage for the future.

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