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1、Hanetal.Chin Med (2021)16:75 https:/doi.org/10.1186/s13020-021-00487-2RESEARCHSystems pharmacology andtranscriptomics reveal themechanisms ofSanhuang decoction enema inthetreatment ofulcerative colitis withadditional Candida albicans infectionZhijun Han1,Xiaofen Tan1,Juan Sun3,4,Tianming Wang1,2,3,4
2、,Guiming Yan1,2,3,4,Changzhong Wang1,2,3,4 and Kelong Ma1,2,3,4*Abstract Background:Ulcerative colitis(UC)is an important inflammatory phenotype in bowel disease(IBD),which is caused by multiple potential factors,including fungal dysbiosis.Candida albicans(C.albicans)was confirmed to be an impor-tan
3、t factor promoting the occurrence and development of UC.Sanhuang decoction(SHD)has been used for UC therapy in China for thousand of years,although its core active constituents and pharmacological mechanism remain undefined.Methods:In this work,a murine model of UC with C.albicans colonization was e
4、stablished with dextran sodium sulfate(DSS)and C.albicans intragastric administration.The major bioactive constituents and potential mechanism of SHD against UC with fungal dysbiosis were comprehensively examined by combining systems pharmacology and in vivo transcriptomics.Results:SHD attenuated C.
5、albicans burden,reduced DAI,increased mucosal integrity and relived systemic inflam-mation in UC mice.Systems pharmacology analysis identified 9 core bioactive ingredients and 45 hub targets of SHD against UC.Transcriptomics analysis confirmed 370 differentially expressed genes(DEGs)after SHD treatm
6、ent,which were mainly enriched in inflammatory and immune response related signaling pathways.Toll-like receptor and PI3K-Akt signaling pathway were screened out as the candidate targets involved in the action of SHD on fungal dysbiosis-associated UC,which were consistent with the findings in system
7、s pharmacology.The expression of TLR4,IL-1,NF-B,PI3K and Akt proteins were stimulated by C.albicans,and partially reversed by SHD in UC mice.Conclusion:These findings suggested SHD could be a candidate for the treatment of fungal dysbiosis-associated UC via TLR4-NF-B and PI3K-Akt signaling pathways.
8、Keywords:Ulcerative colitis,Candida albicans,Sanhuang decoction,Systems pharmacology,Transcriptomics The Author(s)2021.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,which permits use,sharing,adaptation,distribution and reproduction in any medium
9、or format,as long as you give appropriate credit to the original author(s)and the source,provide a link to the Creative Commons licence,and indicate if changes were made.The images or other third party material in this article are included in the articles Creative Commons licence,unless indicated ot
10、herwise in a credit line to the material.If material is not included in the articles Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use,you will need to obtain permission directly from the copyright holder.To view a copy of this licen
11、ce,visit http:/creat iveco mmons.org/licen ses/by/4.0/.The Creative Commons Public Domain Dedication waiver(http:/creat iveco mmons.org/publi cdoma in/zero/1.0/)applies to the data made available in this article,unless otherwise stated in a credit line to the data.BackgroundUlcerative colitis(UC)rep
12、resents an important chronic inflammatory disease of the gastrointestinal(GI)tract featuring abdominal pain,diarrhea and rectal bleed-ing,with a significant impact on the quality of life 1.Recently,the incidence and prevalence of UC have signif-icantly increased in underdeveloped regions such as Asi
13、a Open AccessChinese Medicine*Correspondence:Zhijun Han and Xiaofen Tan contribute equally to this paper1 College of Integrated Chinese and Western Medicine,College of Life Science,Anhui University of Chinese Medicine,Hefei 230012,ChinaFull list of author information is available at the end of the a
14、rticlePage 2 of 16Hanetal.Chin Med (2021)16:75 and Africa,especially in China,where it was previously thought to be uncommon 24.The pathogenesis of UC is complex,and several factors including genetics,envi-ronmental stimuli,immune disorder and the gut micro-biota are considered to be involved in the
15、 occurrence and development of UC.Fungi have long been suspected in UC pathogenesis,although they account for only 0.1%of the whole gut microbiota in healthy individuals 5.Currently,grow-ing evidence reveals that fungi are critical in maintain-ing intestinal homeostasis and the balance of immune res
16、ponse 6.Several lines of evidence suggest that intes-tinal inflammation induces fungal proliferation 7;on the other hand,certain fungal organisms modulate sus-ceptibility to inflammation 7,8.C.albicans(Ca),one of the most common opportunistic fungi in the human GI tract,is thought to be associated w
17、ith UC development.Several reports showed that UC patients have excessive C.albicans load in the GI tract,and antifungal drugs can alleviate UC 5,911.In addition,C.albicans adminis-tration aggravates dextran sulfate sodium-induced colitis in the mouse model,with higher inflammatory cytokine secretio
18、n levels and severer mucosal damage 12,13.San-Huang Decoction(SHD),also known as San-Huang-Xie-Xin-Tang,is a classical traditional Chinese herbal formula,firstly recorded in the SynopsisofPre-scriptions of the Golden Chamber written 2000 years ago.SHD consists of Coptidis Rhizoma(CR,Huanglian in Chi
19、nese),Scutellariae Radix(SR,Huangqin in Chinese)and Radix Rhei Et Rhizome(RR,Dahuang in Chinese),is traditionally used to clear damp-heat,remove blood stasis and detoxification.Emerging evidence shows that SHD can prevent cell apoptosis 14,suppress viral rep-lication 15 and alleviate inflammation 16
20、.In addition,active compounds from SHD,e.g.,berberine 1719,baicalin 2022 and emodin 23,have functions of anti-UC and modification of the gut microbiota components such as C.albicans.Previous studies revealed that SHD is broadly utilized in China for treating UC due to its syn-ergistic and reliable e
21、ffects 24,25.However,the major constituents and detailed mechanism of SHD in the treatment of C.albicans-associated UC remain unclear and need to be fully elucidated.In the present study,we aimed to explore the poten-tial role and underlying mechanism of SHD enema in UC with C.albicans dysbiosis.Mic
22、e with DSS-induced UC and C.albicans colonization were examined.The therapeutic effects of SHD were assessed by determining fungal burden,weight loss,DAI,histological score and serum contents of inflammatory factors.Systems phar-macology and transcriptomics were utilized to elucidate the molecular m
23、echanisms of SHD in the treatment of UC involving C.albicans-associated dysbiosis.Post SHD treatment,the fecal fungal burden was reduced,and mucosal damage and systemic inflammation were alleviated.The PI3K-Akt and Toll like receptor signal-ing pathways were identified as SHD targets in treating the
24、 disorder.These findings provide novel insights in the mechanisms of SHD in the treatment of C.albicans-asso-ciated UC.MethodsAnimals andstrainsSixty specific pathogen-free(SPF)Kunming mice(female,6 8weeks,20 5g)provided by Anhui Medical Univer-sity Experimental Animal Center(Hefei,China,license No.
25、SCXK Anhui 20170001)were housed at 18 25C and 50 70%relative humidity,under a 12h lightdark cycle.Experiments involving animals had approval from the Animal Ethics Committee of Anhui University of Chinese Medicine.C.albicans SC5314 was kindly provided by Profes-sor Yuanying Jiang(NavalMedical Univer
26、sity,Shanghai,China).Routine isolation and culture were performed as previously described 6.SHD preparation andquality controlCoptidis Rhizoma(CR),Scutellariae Radix(SR)and Radix Rhei Et Rhizome(RR)were purchased from Anhui Hospital of Chinese Medicine(Hefei,China).SHD enema was prepared as previous
27、ly reported 24,25.The drugs were mixed at a ratio of 1:1:1 and extracted with 2 L of distilled water for 30min.Then,the extract was boiled for 40 min.The cooled aqueous solution underwent centrifugation at 5000g,collecting the supernatant,which underwent filtration through a 0.45 m sterile microporo
28、us membrane.The filtrate was concentrated to 1.25g/mL for further use.According to the require-ment standards of the Pharmacopoeia of the Peoples Republic of China(2020 edition),the contents of berber-ine,palmatine,baicalin,rhein and emodin in SHD were detected by liquid chromatography-mass spectrom
29、etry(LCMS).The conditions were as follow:an Agilent C18(2.1mm 100mm,1.8m)with solvent A:0.1%formic acid and solvent B:gradient methanol elution was used in the LCMS detection system(12906460,Agilent,USA).The solvent flow rate was 0.3mL/min in a column,at 40C.Berberine,palmatine and baicalin were per
30、formed on ESI+ionization modes,rhein and emodin were on ESI ionization modes with data acquisition range from 10 to 500Da.The measurements were done in triplicate samples,and the results were listed in Additional file1:Fig.S1 andAdditional file2:TableS1.Page 3 of 16Hanetal.Chin Med (2021)16:75 Anima
31、l model establishment anddrug treatmentThe animal model was established according to previ-ous studies 6,9.In brief,mice were orally administrated 3.0%(w/v)dextran sulfate sodium(DSS,3650kDa,MP Biomedicals)for consecutive 7 days.The animals with bloody or loose feces,with or without 3g body weight r
32、eduction at any time point were considered to have coli-tis.Then,the animals were randomized into 5 groups of 10 each,including the DSS,Model(DSS+Ca),SHD low dosage(SHD-L,3.75g/kg),SHD high dosage(SHD-H,15.00g/kg)and sulphasalazine(SASP,0.72g/kg)groups.In the Model group,mice were intragastrically a
33、dmin-istrated 1.0 108 live C.albicans SC5314 for 4days.In the SHD low and high groups,mice were subsequently treated with 3.75g/kg and 15.00g/kg SHD enema once daily for 7 days,respectively.Mice in the SASP group were given 0.72 g/kg SASP.Mice in the Control and Model groups were administered the sa
34、me amount of saline.The administration of enema was performed as follows:mouse was fixed and faced upward to expose the anus.A enema syringe attached with a polyethylene cath-eter(outer diameter 2mm)was inserted about 4cm into the mouse rectum,the anus was squeezed,and 0.3mL enema solution was slowl
35、y injected into the rectum for 30s to 1min,then the mouse was held upside down for 1min to retain theenemasolution.During this process,the enema solution was maintained at 37C,and all the instruments were treated aseptically.After mice were anesthetized with pentobarbital sodium(50mg/kg,intra-perito
36、neally),blood samples were collected by remov-ing the left eyeball of the mice,and serum samples were kept at 20C.Mice were sacrificed bycervicaldisloca-tion.Colons were excised and measured for length.Then,colon specimens were fixed with formalin or stored at-80 until use.The disease activity index
37、(DAI)of each mouse was calculated daily,consisting of body weight loss(0,no loss;1,05%;2,510%;3,1020%;4,20%),stool consistency(0,normal;2,loose stool;4,diarrhea),and fecal blood(0,normal;2,hemoccult;4,gross bleed-ing)26.Candida albicans burden assessmentCandida albicans amounts in the GIT were exami
38、ned by plate counting after culturing fecal samples obtained from every mouse as described in our previous study 6.Fecal specimens were weighed,resuspended in PBS and plated on Sabouraud dextrose agar supplemented with Komaga(No.K08A,Bio-engineering,Shanghai,China)for 48 h of culture at 37 C.Yeast c
39、olonies were then counted,and expressed as CFU/10mg feces.Detection ofASCA andglucanMouse serum samples were obtained,and the contents of ASCA(CK-E22252,Ruixin Biotech.,Fujian,China)and-glucan(F30161-B,Kexing Biotech.,Shanghai,China)were assessed with ELISA kits as directed by the manufacturer.Histo
40、pathological analysisColon tissues were dissected and fixed with forma-lin,dehydrated,paraffin embedded and sectioned at 4m for hematoxylin and eosin(H&E)staining.Imag-ing and analysis were performed with a BX51 micro-scope(Olympus,Japan).The histopathological score was determined according to a pre
41、viously published method 13.Serum TNF,IL1,IL6 andIL10 level evaluationSerum was isolated by centrifugation of blood samples at 5000rpm for 10min.ELISA kits(MLBIO Biotechnol-ogy Co.Ltd.Shanghai,China)were utilized for deter-mining the serum contents of TNF-(ml002095),IL-1(ml063132),IL-6(ml002283)and
42、IL-10(ml002294).Systems pharmacology analysisScreening ofchemical components andprediction ofrelated targets inSHDThe chemical ingredients of SHD were screened from the TCMSP(http:/ TCMID(http:/www.megab ionet.org/tcmid/)databases.Ingredients meeting the demands of oral bioavailabil-ity(OB)30%and dr
43、ug-like property(DL)0.18 were selected for identifying effective constituents.The poten-tial targets of these compounds were screened in Swis-sTargetPrediction(http:/www.swiss targe tpred iction.ch)based on SMILES strings which were obtained from PubChem(https:/pubch em.ncbi.nlm.nih.gov/).The offi-c
44、ial name of each target gene was standardized based on the UniProtKB database(https:/www.unipr ot.org/).Fig.1 SHD attenuates the development of DSS colitis with C.albicans.A Experimental design of C.albicans administration in mice with colitis and SHD treatment.B Body weights.C Disease active index
45、values.D Colon lengths.E Histological scores.F Colonic tissue samples after H&E staining(200).Data are mean SD.#P 0.05,#P 0.01 versus normal group;*P 0.05,*P 0.01 versus Model group(See figure on next page.)Page 4 of 16Hanetal.Chin Med (2021)16:75 Fig.1(See legend on previous page.)Page 5 of 16Hanet
46、al.Chin Med (2021)16:75 Identification ofulcerative colitisrelated targetsUlcerative colitis related genes were searched and col-lected using the DisGeNET(http:/www.disge net.org)and GeneCards(https:/www.genec ards.org)databases with the keyword“ulcerative colitis,UC”.Repeated genes were discarded.P
47、roteinprotein interactions andcompoundtarget network buildingPotential SHDs therapeutic targets in UC were molecules simultaneously targeted by SHD constituents and UC.The STRING database(https:/string-db.org/)was uti-lized for building a proteinprotein interaction(PPI)net-work for these common targ
48、ets.Cytoscape 3.7.1(https:/www.cytos cape.org/)was used to analyze the generated network.The final major targets of SHD in UC were selected based on Degree Centrality(DC),Betweenness Centrality(BC)and Closeness Centrality(CC),which are the mainly three categories of centrality in network analysis,ac
49、croding to network pharmacology evaluation method guidance 27.Cytoscape was utilized to build a network of these main targets.Gene ontology(GO)andKyoto Encyclopedia ofGenes andGenomes(KEGG)pathway enrichment analysesGO and KEGG pathway enrichment analyses of key tar-gets utilized the DAVID system(ht
50、tps:/david.ncifc rf.gov/,v6.8)and R package.P 0.05 was considered statis-tically significant.Transcriptomic analysisTotal RNA from colonic tissue samples in the Control,Model and SHD-H groups was isolated,enriched and purified with oligo-dT magnetic beads.Then,cDNA was synthesized,primed with poly-A