acupuncture regulating gut microbiota in abdominal obese rats...

Research ArticleAcupuncture Regulating Gut Microbiota in Abdominal ObeseRats Induced by High-Fat Diet

HaiyingWang 1 QiangWang 2 Cuimei Liang 1 Mingxing Su 2

XinWang 3 Hua Li 1 Hui Hu 1 and Hongjuan Fang 4

1Dong Fang Hospital Beijing University of Chinese Medicine Beijing 100078 China2Institute of Disease Control and Prevention of Chinese Peoplersquos Liberation Army Beijing 100071 China3Beijing Hospital of Traditional Chinese Medicine Affiliated to Capital Medical University Beijing 100010 China4Department of Endocrinology Beijing Tiantan Hospital Capital Medical University Beijing 100070 China

Correspondence should be addressed to Hui Hu dfzhenjiu126com and Hongjuan Fang tthfhj163com

Received 24 November 2018 Revised 15 April 2019 Accepted 12 May 2019 Published 2 June 2019

Academic Editor Francisco Solano

Copyright copy 2019 Haiying Wang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Objective To investigate the effects of acupuncture on metabolic health and gut microbiota dysbiosis in diet-induced abdominalobese model Materials and Methods Male Sprague-Dawley rats were randomly distributed into normal chow diet (NCD) groupand high-fat diet (HFD) group After 12 weeks of HFD feeding an abdominal obese ratmodel was establishedThe abdominal obeserats were further assigned to acupuncture group (n=7) and nontreated HFD group (n=7) Acupuncture was applied to bilateral GB26 of rats for 8 weeks Subsequently the body weight waist circumference (WC) visceral fat mass and liver weight were measuredweekly in all rats Metabolic parameters such as total cholesterol triglyceride alanine aminotransferase aspartate transaminaseand blood glucose were measured by an automatic biochemical analyzer The serum levels of insulin (INS) were determined usingRat INS ELISA Kit Analysis of gut microbiota was carried out by 16S rRNA gene sequencing Results Acupuncture decreasedthe body weight WC and visceral adipose tissues of HFD-induced abdominal obese rats In addition insulin sensitivity glucosehomeostasis and lipid metabolism were improved by this treatment Furthermore electroacupuncture effectively modified thecomposition of gut microbiota mainly via decreasing FirmicutesBacteroidetes ratio and increasing Prevotella 9 abundanceConclusions Electroacupuncture can ameliorate abdominal obesity and prevent metabolic disorders in HFD-induced abdominalobese rats via the modulation of gut microbiota

1 Introduction

Obesity is a multifactorial disease resulting from the inter-play between environmental cultural behavioral metabolicand genetic factors [1ndash5] Compared with general obesityabdominal obesity is considered to bemore closely associatedwith the risks of metabolic disease [6 7] nonalcoholic fattyliver disease [8 9] and cardiovascular diseases [10 11] whichranks as an independent risk factor of all-causemortality [12]It has been reported that the humanmicrobiota encompassestrillions ofmicrobes residing in the gut [13 14] Recent studieshave found that the structure functionality and diversity ofgutmicrobiotamay play a pivotal role in the development andprogression of obesity [15ndash18] Increasing evidence indicatesthat an aberrant FirmicutesBacteroidetes ratio is associated

with the risk of overweight and obesity [19ndash22] Therefore adeeper understanding of how gut microbiota dysbiosis canhelp to elucidate the pathogenesis of obesity and potentiate anovel therapeutic approach for obesity-related comorbidities

Recently there appears to be a growing interest amongresearchers on the role of acupuncture in disease preven-tion and treatment Some animal studies have found thatacupuncture can exert significant effects on the treatment ofobesity [23ndash25] Acupuncture a time-honored tradition inChinese Medicine has been proven to be an effective weight-loss medication Besides the 26th acupuncture point of theGall Bladder Meridian (GB26) which located on the lowerabdomen is possibly associated with the accumulation ofabdominal adipose tissues [26 27] Given all these consid-erations we sought to investigate the correlation between the

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2019 Article ID 4958294 12 pageshttpsdoiorg10115520194958294

2 Evidence-Based Complementary and Alternative Medicine

gut microbiota and abdominal obesity as well as the effectsof acupuncture on metabolic health and gut microbiotadysbiosis in diet-induced obesity model

2 Materials and Methods

21 Animals Thirty seven specific pathogen-free maleSprague-Dawley (SD) rats (5-6 weeks old 220plusmn20g) wereobtained from the Experimental Animal Center of MilitaryMedical Science Academy (license number SCXK (Jun)2017ndash00004) Rats were housed 3-4 per cage under con-trolled environmental conditions (12 h12 h lightdark cycle22plusmn2∘C room temperature and 50-60 relative humidity)They were given ad libitum access to food and water Thenecessary steps were taken to minimize pain and distressin these animals All experimental procedures were in strictaccordance with the animal welfare principles governed bythe Animal Ethics Committee of the Institute of Disease Con-trol and Prevention of the Chinese Peoplersquos Liberation Army

22 Materials and Reagents The sterile and single-usedacupuncture needles (030 mmtimes25 mm) were purchasedfrom Beijing Zhongyan Taihe Medical Instrument Co Ltd(Beijing China) whereas electro pulse acupuncture thera-peutic apparatus (YINGDI KWD-808) was obtained fromChangzhou Yingdi Electronic Medical Devices Co Ltd(Jiangsu China) The high-fat diet (HFD 25 cholesterol03 sodium cholate 20 sucrose 20 lard and 572 basicfeed) was supplied from Beijing Botai Hongda BiotechnologyCo Ltd while the normal chow diet (NCD 24 cornflour 20 bran 20 bean cake 20 flour 6 cellulose5 fish meal 3 bone meal and 2 salt) was provided bythe Experiment Animal Center of Military Medical ScienceAcademy Laboratory equipment such as electronic balance(AL 204 METTLER TOLEDO Switzerland) automatic bio-chemical analyzer (MEK7222K Nihon Kohden Japan) Illu-minaMiSeq platform (SanDiego USA) andNanoDrop 2000UV-vis spectrophotometer (Thermo Scientific WilmingtonUSA) was used throughout the experiments Rat Insulin(INS) ELISA Kit (Catalogue Number SU-B30620) and 10chloral hydrate (batch number 20150303) were obtainedfrom the Sinopharm Chemical Reagent Co Ltd (BeijingChina) EZNA soil DNA Kit was purchased from OmegaBio-tek (Norcross GA USA)

23 Establishment of an Abdominal Obesity Rat Model Aftera week of acclimatization all the 37 male SD rats wererandomly divided into 2 groups NCD group (n=7) and HFDgroup (n=30) Their body weight and waist circumference(WC) were measured weekly during the 12-week feedingperiod Abdominal obese rats were identified based ontheir body weight (20 higher than normal weight) [28]and WC (significantly different compared to those in NCDgroup) [29] Finally 14 abdominal obese rats were randomlyallocated into two groups HFD model group (n=7) andacupuncture treatment group (n=7) These 14 rats werecontinuously fed with HFD while the other 7 rats in NCDgroup were continued with NCD feeding

24 Acupuncture Treatment To eliminate the restraint stressthe rats in acupuncture group were fixed in rat pocketsbefore acupuncture treatment and the rats of HFD groupwere also fixed in rat pockets at the same time Meanwhilethe rats in NCD group received no intervention After thatsterile acupuncture needles (030 mmtimes25 mm) were insertedvertically into the bilateral ldquoDaimairdquo (GB 26) acupointwith a depth of 4-5 mm Subsequently electroacupuncturewas applied to the same acupoint using YINGDI KWD-808 electro pulse acupuncture therapeutic apparatus at afrequency of 2 Hz15 Hz and an intensity of 15 mA for 20minutes Treatments were carried out on alternate weekdays(MondayWednesdayFriday) for 8 weeks All rats were givenad libitum access to food and water and their body weightand WC were measured once a week

25 Measurement of Body Weight WC Food Intake andAdipose Tissues The body weight WC (mid-point of theperpendicular distance between abdominal xiphoid processand hind legs) and food intake were measured weekly usingan electronic scale and a nonelastic measuring tape respec-tively After 8 weeks of acupuncture treatment all rats weredeprived of food overnight (12 hours) followed by anesthesiainduction via an intraperitoneal injection of 10 chloralhydrate (3 mLkg) Their liver and adipose tissues includingthose around the kidneys and near the epididymis wereweighed Adiposity index was calculated by the followingformula 100 times (the weights of adipose tissues around thekidneys and near the epididymis)body weight Meanwhileliver index was calculated by the following formula (liverweightbody weight) times 100

26 Metabolic Parameters Animals were anesthetized byintraperitoneal injection with 10 chloral hydrate (3 mLkg)Blood samples were collected from the abdominal aortaof rats and then centrifuged at 3000 rmin for 10 minat 4∘C Following centrifugation the separated serum wascollected and stored at minus80∘C until further analysis Theserum levels of triglycerides (TG) total cholesterol (TC)alanine aminotransferase (ALT) aspartate aminotransferase(AST) and blood glucose (GLU) were analyzed by anautomatic biochemical analyzer The levels of FINS weremeasured byRat INSELISAKit and the index of homeostasisassessment of insulin resistance (HOMA-IR) was calculatedby the following formula HOMA-IR = fasting blood glucose(FBG) times fasting blood insulin (FINS)225

27 DNA Extraction and 16S rRNA Gene Amplification andSequencing After anesthesia fecal samples were obtainedfrom the three groups and immediately stored at minus80∘Cfor 16S rRNA sequencing Microbial DNA was extractedfrom the fecal samples using EZNA soil DNA Kit Thefinal concentration and purity of DNA were determined byNanoDrop 2000 UV-vis spectrophotometer and the qualityof DNA was evaluated with 1 agarose gel electrophore-sis The hypervariable regions (V3-V4) of the bacterial 16Sribosomal RNA (rRNA) gene were amplified with a set ofprimers (338F 51015840-ACTCCTACGGGAGGCAGCAG-31015840 and

Evidence-Based Complementary and Alternative Medicine 3

806R 51015840-GGACTACHVGGGTWTCTAAT-31015840) Polymerasechain reaction (PCR) conditions were as follows an initialdenaturation step at 95∘C for 2 min followed by 25 cyclesof denaturation at 95∘C for 30s annealing at 55∘C for 30sand extension at 72∘C for 30s and a final extension stepat 72∘C for 5 min The PCR amplification was carried outin a GeneAmp PCR System 9700 thermo cycler (AppliedBiosystems Foster City CA USA) After PCR amplicons arepurified and pooled in equimolar and paired-end sequenced(2times300) on an Illumina MiSeq platform according to theinstructions of Majorbio Bio-Pharm Technology Co Ltd(Shanghai China)

Quality control for raw sequencing sequence with Trim-momatic software and stitching using FLASH software wereas follows (1) Set up a 50bp window If the average qualityvalue within the window falls lower than 20 truncate theback base from the window and then remove the sequenceshorter than 50 bp during the quality control (2) matchbarcodes exactly allowmismatches up to 2 bases for primersand remove ambiguous bases (3) stitch the sequences of thetwo ends according to the overlapping base overlap with theoverlap being greater than 10bp Remove the sequences thatcannot be stitched [30]

28 Bioinformatics and Statistical Analysis UPARSE ver-sion 71 (httpdrive5comuparse) was used to cluster theoperational taxonomic units (OTUs) with 97 similar-ity cutoff while chimeric sequences were identified andremoved by UCHIME The taxonomy of each 16S rRNAgene sequence was constructed by RDP Classifier algorithm(httprdpcmemsuedu) against the Silva (SSU123) 16SrRNA database with a confidence threshold of 70 [30]Rarefication curvewas analyzed on the normalizedOTU levelusing R (verson331) and Alpha diversity (sobs index andace index) and beta diversity were calculated using QIIME[31 32]The representative sequences of OTUs obtained fromrarefication were used to generate a phylogenetic tree usingFasTree and the Distance Algorithm of Principal coordinatesanalysis (PCoA) and Hierarchical clustering was used forunweighted UniFracThe differences between gut microbiotain the three groups were calculated by Bray-Curtis distancesand Unweighted Pair-group Method with Arithmetic Mean(UPGMA) and visualized using PCoA and Hierarchicalclustering at theOUT level Statistical analysis was performedusing SPSS version 200 (IBM Corp Armonk NY USA)Multiple comparisons between groups were analyzed by one-way ANOVA followed by Least-Significant Difference (LSD)test or otherwise a nonparametric Kruskal-Wallis test wasused if the data did not meet the assumptions of t-test or one-way ANOVA All data were expressed as mean plusmn standarddeviation (SD) P values of less than 005 were regarded asstatistically significant

3 Results

31 Effects of Acupuncture on Body Weight WC Visceral FatMass Liver Mass and the Indices of Liver and Adiposity Atweek 0 the bodyweight (Plt 0001) andWC(Plt 001) ofHFD

rats were significantly higher than those of NCD rats (Figures1(a)-1(b)) However there were no significant differences (Pgt 005) in the body weight and WC between HFD andacupuncture groups (Figures 1(a)-1(b)) At week 8 the ratsin HFD group displayed increased body weight (P lt 0001)WC (P lt 001) visceral fat mass (P lt 0001) liver weight (P lt0001) adiposity index (P lt 001) and liver index (P lt 005)compared to NCD group (Figures 1(a)ndash1(f)) On the otherhand the rats in acupuncture group demonstrated lowerbody weight (P lt 001) and WC (P lt 001) than HFD groupIn addition the accumulation of visceral adipose tissues (Plt 005) and liver fat (P lt 005) were significantly inhibitedin acupuncture group compared to HFD group Nonethelessthere were no stark differences in the indices of adiposityand liver between HFD and acupuncture groups (Figures1(a)ndash1(f)) Each groups rats were fed with the same amountof food in the beginning Figure 2 showed that acupuncturecould significantly decrease the food intake compared withthe HFD group both at week 4 and at week 8 (P lt 005P lt 0001) These results suggested that acupuncture canreduce food intake in HFD-induced abdominal obese ratsThese results imply that acupuncture may play a beneficialrole in reducing body weight and suppressing body fataccumulation which also suggested that acupuncture coulddecrease appetite in HFD-induced abdominal obese rats

32 Effects of Acupuncture on Metabolic Parameters Asshown in Figures 3(a)-3(b) the serum levels of ALT (P lt001) and AST (P lt 001) were markedly increased in HFDgroup compared to NCD group However the levels of ALT(P lt 005) and AST (P lt 005) were significantly lower inacupuncture group than HFD in (P lt 005) Besides theserum lipids levels of TG (P lt 0001) and TC (P lt 001)were significantly higher in HFD group compared to NCDgroup Likewise the significant results were found in theserum lipids levels of TG (P lt 005) and TC (P lt 005)between acupuncture and HFD groups (Figures 3(c)-3(d))In addition the level of FBG (P lt 0001) was significantlyelevated in HFD group compared to NCD group Notablyacupuncture treatment apparently decreased the level of FBG(P lt 005) in HFD-induced abdominal obesity rats comparedto nontreated HFD rats (Figure 3(e)) Furthermore the levelsof FINS and HOMA-IR were dramatically increased (P lt0001) in HFD group compared with NCD group whilebeing significantly decreased (P lt 001) in acupuncturegroup compared to HFD group (Figures 3(f)-3(g)) Takenaltogether these results indicate that acupuncture can inhibitthe elevated blood lipid levels and improve insulin resistancein HFD-induced obese rats

33 Influence of Acupuncture on the Diversity and Richnessof Gut Microbiota In total of 941816 high-quality 16S rRNAsequences were generated from 21 fecal samples (lengthdistribution in 420-460 bp) and the average length of thesequence was 439 per each sample obtained (min 328 max461) Each sample was normalized to an equal sequencingdepth and clustering After filtering the low abundant (lt01) OTUs 709 OTUs were obtained at ge 97 sequence


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Figure 1 Antiobesity effects of acupuncture on HFD-induced abdominal obese rats (a) Changes in body weight among NCD HFD andacupuncture groups (n=7 in each group) at weeks 0 4 and 8 (b) Changes in waist circumference among the three groups at weeks 0 4 and 8(c) Visceral fat accumulation (adipose tissues around the kidneys and near the epididymis) (d)The values of adiposity index (e) The weightof liver (f) The values of liver index All data are expressed as mean plusmn SD lowastP lt 005 lowastlowastP lt 001 lowastlowastlowastP lt 0001 NCD vs HFD P lt 005 P lt001 HFD vs acupuncture

identity As shown in Figures 4(a)ndash4(d) the richness anddiversity of ace and sobs indices were not significant dif-ferences among NCD HFD and acupuncture groups at theOUT level (Figure 4)

34 Impacts of Acupuncture on the Structure of Gut Micro-biota To assess the similarities and differences in the compo-sition of gut microbiota among NCD HFD and acupuncturegroups two aspects of beta diversity and UniFrac-basedPCoA were examined The results demonstrated an obvious

clustering of gut microbiota in the three groups (Figure 5(a))Notably the composition of the gut microbiota in HFDgroup displayed an obvious shift that gathered separatelyfrom both NCD and acupuncture groups (Figure 5(a))In addition PCoA scores indicated that the gut micro-biota of acupuncture group exhibited a movement in thefirst principal component (PC1) towards the direction ofNCD group (Figure 5(a)) Hierarchical clustering analysisrevealed that the microbial communities in HFD group weremarkedly different fromNCDgroupMoreover themicrobial


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Figure 3 Effects of acupuncture on metabolic parameters at week 8 (a) Changes in alanine aminotransferase (ALT) among NCD HFDand acupuncture groups (n=7 in each group) (b) Changes in aspartate aminotransferase (AST) among the three groups (c) Variation intriglycerides (TG) (d) Variation in total cholesterol (TC) (e) Levels of fasting blood glucose (FBG) (f) The serum levels of fasting insulin(FINS) (g) The index of homeostasis assessment of insulin resistance (HOMA-IR) All data are expressed as mean plusmn SD lowastP lt 005 lowastlowastP lt001 lowastlowastlowastP lt 0001 NCD vs HFD P lt 005 P lt 001 HFD vs acupuncture


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Figure 4 Influence of acupuncture on the diversity and richness of gut microbiota (n=7) (a-b) The alpha diversity of ace and sobs on theOTU levels (c-d) Ace and sobs indexes All data are expressed as mean plusmn SD lowastP lt 005 lowastlowastP lt 001 lowastlowastlowastP lt 0001 NCD vs HFD P lt 005P lt 001 HFD vs acupuncture

communities in acupuncture group showedmore similaritiesto those in NCD group (Figure 5(b)) In overall acupuncturesignificantly reverted the HFD-induced variations along PC2but prominently shifted the structure along PC1 (Figure 5)suggesting that the total gut microbial community is signifi-cantly altered by this treatment

35 Modulation of the Composition of Gut Microbiota byAcupuncture at the Phylum Level To determine the specificchanges in gut microbiota the relative abundance of majortaxonomic groups at the phylum level was measured in NCDHCD and acupuncture groups After taxon-based analysisthe most abundant phyla identified in the gut microbial


PCoA on OTU level

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HFD_5Acu_2HFD_3Acu_6Acu_3Acu_4Acu_1Acu_7Acu_5NCD_1NCD_7NCD_2NCD_3NCD_4NCD_5NCD_6HFD_1HFD_2HFD_6HFD_4HFD_7

000005010015020025030035

(b)

Figure 5 Responses of acupuncture on the structure of gut microbiota (a) and (b) principal coordinate analysis (PCoA) on the OUT levelSample hierarchical clustering results of the unweighted UniFrac distances of microbial 16S rRNA from the V3-V4 region with multivariateanalysis of variance (MANOVA)

compositions were Firmicutes and Bacteroidetes while otherphyla such as Cyanobacteria Actinobacteria and Saccharib-acteria and a number of unclassified bacteria were presentedin relatively lower abundance (Figure 6(a)) In particularthe relative compositions of Firmicutes constituted up to32 59 and 44 of the total bacterial sequences in NCDHFD and acupuncture groups respectively (Figure 6(a))Meanwhile the relative compositions of Bacteroidetes wereaccounted for 67 30 and 47 in NCD HFD andacupuncture groups respectively (Figure 6(a)) There weresignificant dissimilarities in the relative abundances of thedominant phyla such as Firmicutes and Bacteroidetes amongthe three groups (Figure 6) Higher abundance of Firmicutes(P lt 001) and reduced Bacteroidetes abundance (P lt 001)were observed in HFD group compared to NCD groupInterestingly 8 weeks of acupuncture treatment significantlyincreased the relative abundance of Bacteroidetes (P lt 001)and decreased the relative abundance of Firmicutes (P lt 005)compared to HFD group Besides among the low abundantphyla the relative abundances of Proteobacteria and Acti-nobacteria were remarkably higher in HFD group than inNCD group (P lt 005) and the abundance of Cyanobacteriawas significantly greater in acupuncture group compared toHFD group (P lt 005) However there were no differences

in the relative abundance of phylum Saccharibacteria amongthe three groups (Figure 6(b)) In addition compared toNCD group the ratio of Firmicutes to Bacteroidetes wassignificantly increased in HFD group (P lt 005) and reducedin acupuncture group (P lt 001) (Figure 6(c)) These resultsindicate that acupuncture can regulate the balance of gutmicrobiota in HFD-induced abdominal obese rats at thephylum level by adjusting the relative abundances of majorphyla Firmicutes and Bacteroidetes

36 Alterations of the Composition of Gut Microbiota fol-lowing Acupuncture at the Genus Level To explore thedeeper taxonomic composition of microbial communitiesthe abundances of gut microbiota were analyzed the genuslevel As shown in Figure 7(a) Prevotella 9 dominated thetaxonomic distribution of gut microbiota among the threegroups Notably the relative abundance of genus Prevotella 9was significantly decreased in HFD group compared to NCDgroup (P lt 001) and treatment with acupuncture (P lt001) dramatically increased the abundance of Prevotella 9(Figure 7(b)) These results suggest that acupuncture is ben-eficial and can reduce abdominal obesity Furthermore theacupuncture group of rats has increased levels of Prevotella 9which maybe suggests that this microbial group is related


BacteroidetesFirmicutesProteobacteriaCyanobacteria

ActinobacteriaSaccharibacteriaTenericutesothers

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Figure 6 Modulation of the composition of gut microbiota by acupuncture at the phylum level (a) The relative abundance of majortaxonomic groups at the phylum level (b) Significant differences of the relative abundance of gut microbiota at the phylum-level (c) TheFirmicutesBacteroidetes ratio in each group All data are expressed as mean plusmn SD lowastP lt 005 lowastlowastP lt 001 lowastlowastlowastP lt 0001 NCD vs HFD P lt005 P lt 001 HFD vs acupuncture

to the lean phenotype as it is also highly abundant inthe NCD group Interestingly acupuncture can modulatethe composition of gut microbiota by altering the relativeabundance of Prevotella 9 at the genus level

4 Discussion

In the present study acupuncture treatment of GB 26acupoint significantly decreased the body weight WC and


AcupunctureNCD HFD

Candidatus_StoquefichusHelicobacterAllobaculumCandidatus_SaccharimonasLachnoclostridiumFaecalitaleaRoseburiaunclassified_f__RuminococcaceaeRuminiclostridium_6[Ruminococcus]_torques_groupFusicatenibacterMarvinbryantiaRuminococcaceae_UCG-005BacteroidesEscherichia-ShigellaLachnospiraceae_NK4A136_groupnorank_o__GastranaerophilalesRomboutsiaRuminococcus_1Ruminococcaceae_UCG-014Blautiaunclassified_f__Lachnospiraceaenorank_f__Bacteroidales_S24-7_groupPhascolarctobacteriumLactobacillusPrevotella_9others

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Figure 7 Alterations of the composition of gut microbiota following acupuncture at the genus level (a) The relative abundance of gutmicrobiota at the genus level (b) Significant differences of the main genus Prevotella 9 All data are expressed as mean plusmn SD lowastP lt 005lowastlowastP lt 001 lowastlowastlowastP lt 0001 NCD vs HFD P lt 005 P lt 001 HFD vs acupuncture

visceral adipose tissues reduced the food intake andenhanced insulin sensitivity glucose homeostasis and lipidmetabolism in abdominal obese rats Moreover acupunc-ture significantly reduced Firmicutes abundance elevatedBacteroidetes abundance and lowered the ratio of Firmi-cutes to Bacteroidetes Additionally the relative abundanceof Prevotella 9 was significantly increased by acupuncturetreatment at the genus level These findings strongly indicatethat acupuncture can suppress abdominal fat accumulationregulate hepatic glucose and lipid metabolism ameliorateinsulin resistance and exert antiobesity effects via the regula-tion of gutmicrobiota which also suggested that acupuncturecan suppress appetite

Accumulating data have demonstrated that the alterationof gut microbiota is associated with obesity in both humansand animals [33ndash36] Besides acupuncture treatment exertsbeneficial effect on obesity by alternating the compositionof gut microbiota [37] However previous studies havedemonstrated conflicting results on the association betweenFirmicutesBacteroidetes ratio and the risk of obesity [38][39ndash41] Some studies have reported that the imbalanceof gut microbiota with an increase in Firmicutes and acorresponding decrease in Bacteroidetes is an independentrisk factor for obesity [42 43] However other studies havenegated the importance of FirmicutesBacteroidetes ratio asa crucial factor that influences obesity [44 45] In this study


there were no significant differences in the gut microbialcommunity diversity among NCD HFD and acupuncturegroups The abdominal obese rats in HFD group exhibited ahigher ratio of FirmicutesBacteroidetes which is consistentwith previous findings [46 47] At the genus level theproportion of Prevotella 9 was significantly increased inacupuncture-treated abdominal obese rats Therefore thesegut microbial changes may indicate the important role ofacupuncture treatment in abdominal obesity

With regard to the mechanisms underlying the antiobe-sity effects of acupuncture treatment on gutmicrobiota struc-ture it is yet to be clarifiedwhether those gutmicrobiota vari-ations are causal risk factors of obesity or simply reflect theacupuncture treatment Most of the researchers believed thattheir relationship is dependent on the theory of brain-gut axis[48 49] In addition gut microbiota can regulate food intakeand energy balance which is crucial for the developmentof obesity [50] The changes in gut microbiota may directlyor indirectly target the brain through vagal stimulation orimmune-neuroendocrine mechanism respectively [51 52]Current studies have showed that acupuncture reducedfood intake and body weight by regulating the appetite-related hormones section in the hypothalamus includingneuropeptide Y (NPY) cocaine-and amphetamine-regulatedtranscript (CART) and proopiomelanocortin (PoMC) [5354] Acupuncture can activate and release brain-gut peptidefollowed bymediating gastrointestinal motility and intestinalflora [55] Collectively these findings suggest a potentialrole of brain-gut axis in controlling body weight decreas-ing appetite and regulating glucose and lipid metabolismduring acupuncture treatment Further studies are warrantedto reveal the exact mechanisms underlying the effects ofacupuncture on the modulation of gut microbiota and foodintake thereby participating in regulating body weight con-trol and lipid metabolism in abdominal obesity

5 Conclusions

The present study indicates that acupuncture can effectivelyameliorate abdominal obesity decrease appetite improvemetabolic parameters and modify gut microbiota com-position Acupuncture can lower FirmicutesBacteroidetesratio at the phylum level and enhance the abundance ofPrevotella 9 at the genus level These alterations in gutmicrobiota may serve as a novel mechanism underlying thetherapeutic effects of acupuncture on abdominal obesityNevertheless more research is needed to evaluate the com-plex interactions among acupuncture treatment gut micro-biota dysbiosis and abdominal fat accumulation before theirclinical implications in human obesity

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Haiying Wang performed the experiments and wrote thepaper QiangWang analyzed the data Cuimei Liang Mingx-ing Su and Hua Li performed the experiments Languagepolishingwas done byXinWangHuiHu andHongjuan Fangguided the study provided funding support and revised thepaper All authors had read and approved the final paper

Acknowledgments

This study is supported by grants from the Special Projectsof Beijing Capital Science and Technology CommissionldquoApplied Research of Capital Clinical Characteristicsrdquo (NoZ141107002514079) Beijing Administration of TraditionalChinese Medicine Chinese Medicine ldquo3+3rdquo InheritanceProject ldquoJiasanYang Famous Research Studiordquo Beijing Nat-ural Science Foundation (No 7182049) Beijing MunicipalAdministration ofHospitalsrsquo Youth Program (QML20160501)and Basic-Clinical Research Cooperation Funding of CapitalMedical University (17JL34)

Supplementary Materials

(1) LEfSe analysis of each group (2) Data of principalcoordinates analysis (Pcoa) in each sample at the OTU level(3) Eigen values of Pcoa from PC1-PC21 (4) Statistical data ofeach group at the genus level (5) Statistical data of each groupat the phylum level (Supplementary Materials)

References

[1] A I F Blakemore and P Froguel ldquoInvestigation of Mendelianforms of obesity holds out the prospect of personalizedmedicinerdquoAnnals of theNewYorkAcademy of Sciences vol 1214no 1 pp 180ndash189 2010

[2] H Reddon J-L Gueant and D Meyre ldquoThe importanceof gene-environment interactions in human obesityrdquo ClinicalScience vol 130 no 18 pp 1571ndash1597 2016

[3] P Zhao XGuDQian and F Yang ldquoSocioeconomic disparitiesin abdominal obesity over the life course in Chinardquo Interna-tional Journal for Equity in Health vol 17 no 1 p 96 2018

[4] K L Monda L S Adair F Zhai and B M Popkin ldquoLongitudi-nal relationships between occupational and domestic physicalactivity patterns and body weight in Chinardquo European Journalof Clinical Nutrition vol 62 no 11 pp 1318ndash1325 2008

[5] N E Boutagy R P McMillan M I Frisard and M WHulver ldquoMetabolic endotoxemia with obesity Is it real and isit relevantrdquo Biochimie vol 124 pp 11ndash20 2016

[6] S Gesta Y-H Tseng and C R Kahn ldquoDevelopmental originof fat tracking obesity to its sourcerdquoCell vol 131 no 2 pp 242ndash256 2007

[7] J-P Despres and I Lemieux ldquoAbdominal obesity andmetabolicsyndromerdquo Nature vol 444 no 7121 pp 881ndash887 2006

[8] S Bellentani ldquoThe epidemiology of non-alcoholic fatty liverdiseaserdquo Liver International vol 37 supplement 1 pp 81ndash842017

[9] Q Pang J-Y Zhang S-D Song et al ldquoCentral obesity andnonalcoholic fatty liver disease risk after adjusting for body


mass indexrdquo World Journal of Gastroenterology vol 21 no 5pp 1650ndash1662 2015

[10] C Zhang K M Rexrode R M Van Dam T Y Li and F B HuldquoAbdominal obesity and the risk of all-cause cardiovascularand cancer mortality 16 years of follow-up in US womenrdquoCirculation vol 117 no 13 pp 1658ndash1667 2008

[11] M Kivimaki E Kuosma J E Ferrie et al ldquoOverweight obesityand risk of cardiometabolic multimorbidity pooled analysis ofindividual-level data for 120 813 adults from 16 cohort studiesfrom the USA and EuroperdquoThe Lancet Public Health vol 2 no6 pp e277ndashe285 2017

[12] B M C Global E Di Angelantonio S Bhupathiraju etal ldquoBody-mass index and all-cause mortality individual-par-ticipant-data meta-analysis of 239 prospective studies in fourcontinentsrdquo Lancet vol 388 no 10046 pp 776ndash786 2016

[13] F Baquero and C Nombela ldquoThe microbiome as a humanorganrdquo Clinical Microbiology and Infection vol 18 supplement4 pp 2ndash4 2012

[14] M Blaut and T Clavel ldquoMetabolic diversity of the intestinalmicrobiota implications for health and diseaserdquo Journal ofNutrition vol 137 supplement 2 no 3 pp 751Sndash755S 2007

[15] AWoting andM Blaut ldquoThe intestinalmicrobiota inmetabolicdiseaserdquo Nutrients vol 8 no 4 article 202 2016

[16] M Mar Rodrıguez D Perez F Javier Chaves et al ldquoObesitychanges the human gut mycobiomerdquo Scientific Reports vol 5no 1 2015

[17] K E Bouter D H van Raalte A K Groen andM NieuwdorpldquoRole of the gut microbiome in the pathogenesis of obesity andobesity-related metabolic dysfunctionrdquo Gastroenterology vol152 no 7 pp 1671ndash1678 2017

[18] S Devaraj P Hemarajata and J Versalovic ldquoThe human gutmicrobiome and bodymetabolism Implications for obesity anddiabetesrdquo Clinical Chemistry vol 59 no 4 pp 617ndash628 2013

[19] TheHumanMicrobiome Project Consortium ldquoStructure func-tion and diversity of the healthy human microbiomerdquo Naturevol 486 no 7402 pp 207ndash214 2012

[20] T E Sweeney and J M Morton ldquoThe human gut microbiomea review of the effect of obesity and surgically induced weightlossrdquo JAMA Surgery vol 148 no 6 pp 563ndash569 2013

[21] R Mathur and G M Barlow ldquoObesity and the microbiomerdquoExpert Review of Gastroenterology amp Hepatology vol 9 no 8pp 1087ndash1099 2015

[22] A Koliada G Syzenko V Moseiko et al ldquoAssociation betweenbody mass index and FirmicutesBacteroidetes ratio in an adultUkrainian populationrdquo BMC Microbiology vol 17 no 1 p 1202017

[23] J J Liaw and P V Peplow ldquoEffect of electroacupuncture oninflammation in the obese zucker fatty rat model of metabolicsyndromerdquo Journal of Acupuncture andMeridian Studies vol 9no 2 pp 73ndash79 2016

[24] J Xu L Chen L Tang et al ldquoElectroacupuncture inhibitsweight gain in diet-induced obese rats by activating hypotha-lamic LKB1-AMPK signalingrdquo BMC Complementary and Alter-native Medicine vol 15 no 1 p 147 2015

[25] J Liu H Jin R D Foreman et al ldquoChronic electrical stimu-lation at acupoints reduces body weight and improves bloodglucose in obese rats via autonomic pathwayrdquo Obesity Surgeryvol 25 no 7 pp 1209ndash1216 2015

[26] L Y Shen CM LiangW J Yang et al ldquoAcupuncture treatmentof polycystic ovarian syndrome patients with abdominal obesityby regulating dai meridian a randomized controlled clinicaltrialrdquo Acupuncture Research vol 04 no 43 pp 255ndash259 2015

[27] C M Liang H Hu C X Wang et al ldquoRandomized controlledclinical trials for acupuncture treatment of abdominal obesityrdquoAcupuncture Research vol 41 no 2 pp 159ndash162 2016

[28] P C Chandler J B Viana K D Oswald P K Wauford andM M Boggiano ldquoFeeding response to melanocortin agonistpredicts preference for and obesity from a high-fat dietrdquoPhysiology amp Behavior vol 85 no 2 pp 221ndash230 2005

[29] Y Y Li H Hu C M Liang et al ldquoEffects of electroacupuncturestimulation of rsquoDaimairsquo (GB 26) on body weight blood glucoseand blood lipid levels in rats with metabolism syndromerdquoAcupuncture Research vol 39 no 3 pp 202ndash206 2014

[30] K R Amato C J Yeoman A Kent et al ldquoHabitat degradationimpacts black howler monkey (Alouatta pigra) gastrointestinalmicrobiomesrdquo The ISME Journal vol 7 no 7 pp 1344ndash13532013

[31] J Ye S D Joseph M Ji et al ldquoChemolithotrophic processes inthe bacterial communities on the surface of mineral-enrichedbiocharsrdquoThe ISME Journal vol 11 no 5 pp 1087ndash1101 2017

[32] J G Caporaso J Kuczynski J Stombaugh et al ldquoQIIMEallows analysis of high-throughput community sequencingdatardquo Nature Methods vol 7 no 5 pp 335-336 2010

[33] M Zietak P Kovatcheva-Datchary L H Markiewicz MStahlman L P Kozak and F Backhed ldquoAlteredmicrobiota con-tributes to reduced diet-induced obesity upon cold exposurerdquoCell Metabolism vol 23 no 6 pp 1216ndash1223 2016

[34] P Maruvada V Leone L M Kaplan and E B Chang ldquoThehuman microbiome and obesity moving beyond associationsrdquoCell Host amp Microbe vol 22 no 5 pp 589ndash599 2017

[35] X Zhang Y Zhao J Xu et al ldquoModulation of gutmicrobiota byberberine andmetformin during the treatment of high-fat diet-induced obesity in ratsrdquo Scientific Reports vol 5 no 1 ArticleID 14405 2015

[36] P J Turnbaugh M Hamady T Yatsunenko et al ldquoA core gutmicrobiome in obese and lean twinsrdquoNature vol 457 no 7228pp 480ndash484 2009

[37] Z Xu R Li C Zhu andM Li ldquoEffect of acupuncture treatmentfor weight loss on gut flora in patients with simple obesityrdquoAcupuncture in Medicine vol 31 no 1 pp 116-117 2013

[38] L Jost ldquoIndependence of alpha and beta diversitiesrdquo Ecologyvol 91 no 7 pp 1969ndash1974 2010

[39] A Riva F Borgo C Lassandro et al ldquoPediatric obesity isassociated with an altered gut microbiota and discordant shiftsin Firmicutes populationsrdquo EnvironmentalMicrobiology vol 19no 1 pp 95ndash105 2017

[40] C Kasai K Sugimoto I Moritani et al ldquoComparison of thegut microbiota composition between obese and non-obeseindividuals in a Japanese population as analyzed by terminalrestriction fragment length polymorphism and next-generationsequencingrdquo BMC Gastroenterology vol 15 no 1 p 100 2015

[41] C Gu Y Yang H Xiang et al ldquoDeciphering bacterial commu-nity changes in zucker diabetic fatty rats based on 16S rRNAgene sequences analysisrdquo Oncotarget vol 7 no 31 pp 48941ndash48952 2016

[42] R E Ley F Backhed P Turnbaugh C A Lozupone R DKnight and J I Gordon ldquoObesity alters gut microbial ecologyrdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 102 no 31 pp 11070ndash11075 2005

[43] R E Ley P J Turnbaugh S Klein and J I Gordon ldquoMicrobialecology human gut microbes associated with obesityrdquo Naturevol 444 no 7122 pp 1022-1023 2006


[44] M Arumugam J Raes E Pelletier et al ldquoEnterotypes of thehuman gutmicrobiomerdquoNature vol 473 no 7346 pp 174ndash1802011

[45] A Schwiertz D Taras K Schafer et al ldquoMicrobiota and SCFAin lean and overweight healthy subjectsrdquo Obesity vol 18 no 1pp 190ndash195 2010

[46] L Zhao Q Zhang W Ma F Tian H Shen and M ZhouldquoA combination of quercetin and resveratrol reduces obesity inhigh-fat diet-fed rats by modulation of gut microbiotardquo Food ampFunction vol 8 no 12 pp 4644ndash4656 2017

[47] H SunNWang Z Cang et al ldquoModulation ofmicrobiota-gut-brain axis by berberine resulting in improved metabolic statusin high-fat diet-fed ratsrdquoObesity Facts vol 9 no 6 pp 365ndash3782017

[48] H Buhmann CW le Roux andM Bueter ldquoThe gut-brain axisin obesityrdquo Best Practice amp Research Clinical Gastroenterologyvol 28 no 4 pp 559ndash571 2014

[49] C Torres-Fuentes H Schellekens T G Dinan and J FCryan ldquoThe microbiotandashgutndashbrain axis in obesityrdquo The LancetGastroenterology and Hepatology vol 2 no 10 pp 747ndash7562017

[50] S S Hussain and S R Bloom ldquoThe regulation of food intakeby the gut-brain axis implications for obesityrdquo InternationalJournal of Obesity vol 37 no 5 pp 625ndash633 2013

[51] S El Aidy T G Dinan and J F Cryan ldquoGut microbiotathe conductor in the orchestra of immune-neuroendocrinecommunicationrdquo Clinical Therapeutics vol 37 no 5 pp 954ndash967 2015

[52] E S Bliss and E Whiteside ldquoThe gut-brain axis the human gutmicrobiota and their integration in the development of obesityrdquoFrontiers in Physiology vol 9 p 900 2018

[53] F Wang D R Tian P Tso and J S Han ldquoArcuate nucleusof hypothalamus is involved in mediating the satiety effect ofelectroacupuncture in obese ratsrdquo Peptides vol 32 no 12 pp2394ndash2399 2011

[54] D-R Tian X-D Li F Wang et al ldquoUp-regulation of theexpression of cocaine and amphetamine-regulated transcriptpeptide by electroacupuncture in the arcuate nucleus of diet-induced obese ratsrdquo Neuroscience Letters vol 383 no 1-2 pp17ndash21 2005

[55] H Li T He Q Xu et al ldquoAcupuncture and regulation ofgastrointestinal functionrdquo World Journal of Gastroenterologyvol 21 no 27 pp 8304ndash8313 2015

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


gut microbiota and abdominal obesity as well as the effectsof acupuncture on metabolic health and gut microbiotadysbiosis in diet-induced obesity model

2 Materials and Methods

21 Animals Thirty seven specific pathogen-free maleSprague-Dawley (SD) rats (5-6 weeks old 220plusmn20g) wereobtained from the Experimental Animal Center of MilitaryMedical Science Academy (license number SCXK (Jun)2017ndash00004) Rats were housed 3-4 per cage under con-trolled environmental conditions (12 h12 h lightdark cycle22plusmn2∘C room temperature and 50-60 relative humidity)They were given ad libitum access to food and water Thenecessary steps were taken to minimize pain and distressin these animals All experimental procedures were in strictaccordance with the animal welfare principles governed bythe Animal Ethics Committee of the Institute of Disease Con-trol and Prevention of the Chinese Peoplersquos Liberation Army

22 Materials and Reagents The sterile and single-usedacupuncture needles (030 mmtimes25 mm) were purchasedfrom Beijing Zhongyan Taihe Medical Instrument Co Ltd(Beijing China) whereas electro pulse acupuncture thera-peutic apparatus (YINGDI KWD-808) was obtained fromChangzhou Yingdi Electronic Medical Devices Co Ltd(Jiangsu China) The high-fat diet (HFD 25 cholesterol03 sodium cholate 20 sucrose 20 lard and 572 basicfeed) was supplied from Beijing Botai Hongda BiotechnologyCo Ltd while the normal chow diet (NCD 24 cornflour 20 bran 20 bean cake 20 flour 6 cellulose5 fish meal 3 bone meal and 2 salt) was provided bythe Experiment Animal Center of Military Medical ScienceAcademy Laboratory equipment such as electronic balance(AL 204 METTLER TOLEDO Switzerland) automatic bio-chemical analyzer (MEK7222K Nihon Kohden Japan) Illu-minaMiSeq platform (SanDiego USA) andNanoDrop 2000UV-vis spectrophotometer (Thermo Scientific WilmingtonUSA) was used throughout the experiments Rat Insulin(INS) ELISA Kit (Catalogue Number SU-B30620) and 10chloral hydrate (batch number 20150303) were obtainedfrom the Sinopharm Chemical Reagent Co Ltd (BeijingChina) EZNA soil DNA Kit was purchased from OmegaBio-tek (Norcross GA USA)

23 Establishment of an Abdominal Obesity Rat Model Aftera week of acclimatization all the 37 male SD rats wererandomly divided into 2 groups NCD group (n=7) and HFDgroup (n=30) Their body weight and waist circumference(WC) were measured weekly during the 12-week feedingperiod Abdominal obese rats were identified based ontheir body weight (20 higher than normal weight) [28]and WC (significantly different compared to those in NCDgroup) [29] Finally 14 abdominal obese rats were randomlyallocated into two groups HFD model group (n=7) andacupuncture treatment group (n=7) These 14 rats werecontinuously fed with HFD while the other 7 rats in NCDgroup were continued with NCD feeding

24 Acupuncture Treatment To eliminate the restraint stressthe rats in acupuncture group were fixed in rat pocketsbefore acupuncture treatment and the rats of HFD groupwere also fixed in rat pockets at the same time Meanwhilethe rats in NCD group received no intervention After thatsterile acupuncture needles (030 mmtimes25 mm) were insertedvertically into the bilateral ldquoDaimairdquo (GB 26) acupointwith a depth of 4-5 mm Subsequently electroacupuncturewas applied to the same acupoint using YINGDI KWD-808 electro pulse acupuncture therapeutic apparatus at afrequency of 2 Hz15 Hz and an intensity of 15 mA for 20minutes Treatments were carried out on alternate weekdays(MondayWednesdayFriday) for 8 weeks All rats were givenad libitum access to food and water and their body weightand WC were measured once a week

25 Measurement of Body Weight WC Food Intake andAdipose Tissues The body weight WC (mid-point of theperpendicular distance between abdominal xiphoid processand hind legs) and food intake were measured weekly usingan electronic scale and a nonelastic measuring tape respec-tively After 8 weeks of acupuncture treatment all rats weredeprived of food overnight (12 hours) followed by anesthesiainduction via an intraperitoneal injection of 10 chloralhydrate (3 mLkg) Their liver and adipose tissues includingthose around the kidneys and near the epididymis wereweighed Adiposity index was calculated by the followingformula 100 times (the weights of adipose tissues around thekidneys and near the epididymis)body weight Meanwhileliver index was calculated by the following formula (liverweightbody weight) times 100

26 Metabolic Parameters Animals were anesthetized byintraperitoneal injection with 10 chloral hydrate (3 mLkg)Blood samples were collected from the abdominal aortaof rats and then centrifuged at 3000 rmin for 10 minat 4∘C Following centrifugation the separated serum wascollected and stored at minus80∘C until further analysis Theserum levels of triglycerides (TG) total cholesterol (TC)alanine aminotransferase (ALT) aspartate aminotransferase(AST) and blood glucose (GLU) were analyzed by anautomatic biochemical analyzer The levels of FINS weremeasured byRat INSELISAKit and the index of homeostasisassessment of insulin resistance (HOMA-IR) was calculatedby the following formula HOMA-IR = fasting blood glucose(FBG) times fasting blood insulin (FINS)225

27 DNA Extraction and 16S rRNA Gene Amplification andSequencing After anesthesia fecal samples were obtainedfrom the three groups and immediately stored at minus80∘Cfor 16S rRNA sequencing Microbial DNA was extractedfrom the fecal samples using EZNA soil DNA Kit Thefinal concentration and purity of DNA were determined byNanoDrop 2000 UV-vis spectrophotometer and the qualityof DNA was evaluated with 1 agarose gel electrophore-sis The hypervariable regions (V3-V4) of the bacterial 16Sribosomal RNA (rRNA) gene were amplified with a set ofprimers (338F 51015840-ACTCCTACGGGAGGCAGCAG-31015840 and





3 Results






0 84

NCDHFDAcupuncture

weeks

lowastlowastlowast


0

200

400

600

800

1000

Body

wei

ght (

g)

(a)

0 84

Weeks

lowastlowast


0

10

20

30

Wai

st ci

rcum

fere

nce (

cm)

NCDHFDAcupuncture

(b)

NCD

lowastlowastlowast

AcupunctureHFD0

20

40

60

80

Visc

eral

fat w

eigh

t (g)

(c)

lowastlowast

NCD AcupunctureHFD0

2

4

6

8

Adip

osity

inde

x

(d)

NCD

HFD Acupuncture

lowastlowastlowast

0

10

20

30

Live

r wet

wei

ght (

g)

(e)

NCD HFD Acupuncture

lowast

0

1

2

3

4

Live

r ind

ex (

)

(f)






NCDHFDAcupuncture

lowastlowast

lowastlowast

4 8weeks

18

20

22

24

26

28

Aver

age f

ood

inta

ke (g

day

)


lowastlowast

0

20

40

60

80

100

ALT

(UL

)

AcupunctureNCD HFD

(a)

lowastlowast

0

100

200

300

400

AST

(UL

)

AcupunctureNCD HFD

(b)

lowastlowastlowast

0

1

2

3

TG (m

mol

L)

AcupunctureNCD HFD

(c)

lowastlowastlowast

0

2

4

6

8

TC (m

mol

L)

AcupunctureNCD HFD

(d)

lowastlowastlowast

0

5

10

15

20

25

FBG

(mm

olL

)

AcupunctureNCD HFD

(e)

lowastlowastlowast

0

2

4

6

8

10

FIN

S (m

UL

)

AcupunctureNCD HFD

(f)

lowastlowastlowast

0

2

4

6

8

HPM

A-IR

AcupunctureNCD HFD

(g)




200

400

600

Ace i

ndex

of O

TU le

vel

(a)


100

200

300

400

500

Sobs

inde

x of

OTU

leve

l

(b)

HFDNCD Acupuncture0

200

400

600

Ace d

iver

sity

inde

x of

OTU

leve

l

(c)


100

200

300

400

500

Sobs

inde

x of

OTU

leve

l

(d)





PCoA on OTU level

NCDHFD

Acupuncture

minus04

minus02

00

02

04

PC2(

189

1)

minus02 02 04 0600PC1(3075)

(a)


NCDHFDAcupuncture


000005010015020025030035

(b)









0

02

04

06

08

1

Perc

ent o

f com

mun

ity ab

unda

nce o

n Ph

ylum

leve

l

(a)

Bactero

idetes

Firmicu

tes

Proteobact

eria

Cyanobact

eria

Actinobact

eria

Sacchari

bacteri

a

phylum

NCDHFDAcupuncture

0

20

40

60

80

Relat

ive a

bund

ance

()

lowast

lowast

lowastlowast

lowastlowast

(b)

AcupunctureNCD HFD

FB ratio

0

1

2

3

4

Firm

icut

es to

Bac

tero

idet

es R

atio

lowast

(c)



4 Discussion



AcupunctureNCD HFD


0

02

04

06

08

1

Perc

ent o

f com

mun

ity ab

unda

nce o

n G

enus

leve

l

(a)

g__Prevotella_9

genus

NCDHFDAcupuncture

lowastlowast

0

20

40

60

80

Relat

ive a

bund

ance

()

(b)







5 Conclusions


Data Availability






Acknowledgments




References































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























3 Results






0 84

NCDHFDAcupuncture

weeks

lowastlowastlowast


0

200

400

600

800

1000

Body

wei

ght (

g)

(a)

0 84

Weeks

lowastlowast


0

10

20

30

Wai

st ci

rcum

fere

nce (

cm)

NCDHFDAcupuncture

(b)

NCD

lowastlowastlowast

AcupunctureHFD0

20

40

60

80

Visc

eral

fat w

eigh

t (g)

(c)

lowastlowast

NCD AcupunctureHFD0

2

4

6

8

Adip

osity

inde

x

(d)

NCD

HFD Acupuncture

lowastlowastlowast

0

10

20

30

Live

r wet

wei

ght (

g)

(e)

NCD HFD Acupuncture

lowast

0

1

2

3

4

Live

r ind

ex (

)

(f)






NCDHFDAcupuncture

lowastlowast

lowastlowast

4 8weeks

18

20

22

24

26

28

Aver

age f

ood

inta

ke (g

day

)


lowastlowast

0

20

40

60

80

100

ALT

(UL

)

AcupunctureNCD HFD

(a)

lowastlowast

0

100

200

300

400

AST

(UL

)

AcupunctureNCD HFD

(b)

lowastlowastlowast

0

1

2

3

TG (m

mol

L)

AcupunctureNCD HFD

(c)

lowastlowastlowast

0

2

4

6

8

TC (m

mol

L)

AcupunctureNCD HFD

(d)

lowastlowastlowast

0

5

10

15

20

25

FBG

(mm

olL

)

AcupunctureNCD HFD

(e)

lowastlowastlowast

0

2

4

6

8

10

FIN

S (m

UL

)

AcupunctureNCD HFD

(f)

lowastlowastlowast

0

2

4

6

8

HPM

A-IR

AcupunctureNCD HFD

(g)




200

400

600

Ace i

ndex

of O

TU le

vel

(a)


100

200

300

400

500

Sobs

inde

x of

OTU

leve

l

(b)

HFDNCD Acupuncture0

200

400

600

Ace d

iver

sity

inde

x of

OTU

leve

l

(c)


100

200

300

400

500

Sobs

inde

x of

OTU

leve

l

(d)





PCoA on OTU level

NCDHFD

Acupuncture

minus04

minus02

00

02

04

PC2(

189

1)

minus02 02 04 0600PC1(3075)

(a)


NCDHFDAcupuncture


000005010015020025030035

(b)









0

02

04

06

08

1

Perc

ent o

f com

mun

ity ab

unda

nce o

n Ph

ylum

leve

l

(a)

Bactero

idetes

Firmicu

tes

Proteobact

eria

Cyanobact

eria

Actinobact

eria

Sacchari

bacteri

a

phylum

NCDHFDAcupuncture

0

20

40

60

80

Relat

ive a

bund

ance

()

lowast

lowast

lowastlowast

lowastlowast

(b)

AcupunctureNCD HFD

FB ratio

0

1

2

3

4

Firm

icut

es to

Bac

tero

idet

es R

atio

lowast

(c)



4 Discussion



AcupunctureNCD HFD


0

02

04

06

08

1

Perc

ent o

f com

mun

ity ab

unda

nce o

n G

enus

leve

l

(a)

g__Prevotella_9

genus

NCDHFDAcupuncture

lowastlowast

0

20

40

60

80

Relat

ive a

bund

ance

()

(b)







5 Conclusions


Data Availability






Acknowledgments




References































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0 84

NCDHFDAcupuncture

weeks

lowastlowastlowast


0

200

400

600

800

1000

Body

wei

ght (

g)

(a)

0 84

Weeks

lowastlowast


0

10

20

30

Wai

st ci

rcum

fere

nce (

cm)

NCDHFDAcupuncture

(b)

NCD

lowastlowastlowast

AcupunctureHFD0

20

40

60

80

Visc

eral

fat w

eigh

t (g)

(c)

lowastlowast

NCD AcupunctureHFD0

2

4

6

8

Adip

osity

inde

x

(d)

NCD

HFD Acupuncture

lowastlowastlowast

0

10

20

30

Live

r wet

wei

ght (

g)

(e)

NCD HFD Acupuncture

lowast

0

1

2

3

4

Live

r ind

ex (

)

(f)






NCDHFDAcupuncture

lowastlowast

lowastlowast

4 8weeks

18

20

22

24

26

28

Aver

age f

ood

inta

ke (g

day

)


lowastlowast

0

20

40

60

80

100

ALT

(UL

)

AcupunctureNCD HFD

(a)

lowastlowast

0

100

200

300

400

AST

(UL

)

AcupunctureNCD HFD

(b)

lowastlowastlowast

0

1

2

3

TG (m

mol

L)

AcupunctureNCD HFD

(c)

lowastlowastlowast

0

2

4

6

8

TC (m

mol

L)

AcupunctureNCD HFD

(d)

lowastlowastlowast

0

5

10

15

20

25

FBG

(mm

olL

)

AcupunctureNCD HFD

(e)

lowastlowastlowast

0

2

4

6

8

10

FIN

S (m

UL

)

AcupunctureNCD HFD

(f)

lowastlowastlowast

0

2

4

6

8

HPM

A-IR

AcupunctureNCD HFD

(g)




200

400

600

Ace i

ndex

of O

TU le

vel

(a)


100

200

300

400

500

Sobs

inde

x of

OTU

leve

l

(b)

HFDNCD Acupuncture0

200

400

600

Ace d

iver

sity

inde

x of

OTU

leve

l

(c)


100

200

300

400

500

Sobs

inde

x of

OTU

leve

l

(d)





PCoA on OTU level

NCDHFD

Acupuncture

minus04

minus02

00

02

04

PC2(

189

1)

minus02 02 04 0600PC1(3075)

(a)


NCDHFDAcupuncture


000005010015020025030035

(b)









0

02

04

06

08

1

Perc

ent o

f com

mun

ity ab

unda

nce o

n Ph

ylum

leve

l

(a)

Bactero

idetes

Firmicu

tes

Proteobact

eria

Cyanobact

eria

Actinobact

eria

Sacchari

bacteri

a

phylum

NCDHFDAcupuncture

0

20

40

60

80

Relat

ive a

bund

ance

()

lowast

lowast

lowastlowast

lowastlowast

(b)

AcupunctureNCD HFD

FB ratio

0

1

2

3

4

Firm

icut

es to

Bac

tero

idet

es R

atio

lowast

(c)



4 Discussion



AcupunctureNCD HFD


0

02

04

06

08

1

Perc

ent o

f com

mun

ity ab

unda

nce o

n G

enus

leve

l

(a)

g__Prevotella_9

genus

NCDHFDAcupuncture

lowastlowast

0

20

40

60

80

Relat

ive a

bund

ance

()

(b)







5 Conclusions


Data Availability






Acknowledgments




References































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















NCDHFDAcupuncture

lowastlowast

lowastlowast

4 8weeks

18

20

22

24

26

28

Aver

age f

ood

inta

ke (g

day

)


lowastlowast

0

20

40

60

80

100

ALT

(UL

)

AcupunctureNCD HFD

(a)

lowastlowast

0

100

200

300

400

AST

(UL

)

AcupunctureNCD HFD

(b)

lowastlowastlowast

0

1

2

3

TG (m

mol

L)

AcupunctureNCD HFD

(c)

lowastlowastlowast

0

2

4

6

8

TC (m

mol

L)

AcupunctureNCD HFD

(d)

lowastlowastlowast

0

5

10

15

20

25

FBG

(mm

olL

)

AcupunctureNCD HFD

(e)

lowastlowastlowast

0

2

4

6

8

10

FIN

S (m

UL

)

AcupunctureNCD HFD

(f)

lowastlowastlowast

0

2

4

6

8

HPM

A-IR

AcupunctureNCD HFD

(g)




200

400

600

Ace i

ndex

of O

TU le

vel

(a)


100

200

300

400

500

Sobs

inde

x of

OTU

leve

l

(b)

HFDNCD Acupuncture0

200

400

600

Ace d

iver

sity

inde

x of

OTU

leve

l

(c)


100

200

300

400

500

Sobs

inde

x of

OTU

leve

l

(d)





PCoA on OTU level

NCDHFD

Acupuncture

minus04

minus02

00

02

04

PC2(

189

1)

minus02 02 04 0600PC1(3075)

(a)


NCDHFDAcupuncture


000005010015020025030035

(b)









0

02

04

06

08

1

Perc

ent o

f com

mun

ity ab

unda

nce o

n Ph

ylum

leve

l

(a)

Bactero

idetes

Firmicu

tes

Proteobact

eria

Cyanobact

eria

Actinobact

eria

Sacchari

bacteri

a

phylum

NCDHFDAcupuncture

0

20

40

60

80

Relat

ive a

bund

ance

()

lowast

lowast

lowastlowast

lowastlowast

(b)

AcupunctureNCD HFD

FB ratio

0

1

2

3

4

Firm

icut

es to

Bac

tero

idet

es R

atio

lowast

(c)



4 Discussion



AcupunctureNCD HFD


0

02

04

06

08

1

Perc

ent o

f com

mun

ity ab

unda

nce o

n G

enus

leve

l

(a)

g__Prevotella_9

genus

NCDHFDAcupuncture

lowastlowast

0

20

40

60

80

Relat

ive a

bund

ance

()

(b)







5 Conclusions


Data Availability






Acknowledgments




References































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















200

400

600

Ace i

ndex

of O

TU le

vel

(a)


100

200

300

400

500

Sobs

inde

x of

OTU

leve

l

(b)

HFDNCD Acupuncture0

200

400

600

Ace d

iver

sity

inde

x of

OTU

leve

l

(c)


100

200

300

400

500

Sobs

inde

x of

OTU

leve

l

(d)





PCoA on OTU level

NCDHFD

Acupuncture

minus04

minus02

00

02

04

PC2(

189

1)

minus02 02 04 0600PC1(3075)

(a)


NCDHFDAcupuncture


000005010015020025030035

(b)









0

02

04

06

08

1

Perc

ent o

f com

mun

ity ab

unda

nce o

n Ph

ylum

leve

l

(a)

Bactero

idetes

Firmicu

tes

Proteobact

eria

Cyanobact

eria

Actinobact

eria

Sacchari

bacteri

a

phylum

NCDHFDAcupuncture

0

20

40

60

80

Relat

ive a

bund

ance

()

lowast

lowast

lowastlowast

lowastlowast

(b)

AcupunctureNCD HFD

FB ratio

0

1

2

3

4

Firm

icut

es to

Bac

tero

idet

es R

atio

lowast

(c)



4 Discussion



AcupunctureNCD HFD


0

02

04

06

08

1

Perc

ent o

f com

mun

ity ab

unda

nce o

n G

enus

leve

l

(a)

g__Prevotella_9

genus

NCDHFDAcupuncture

lowastlowast

0

20

40

60

80

Relat

ive a

bund

ance

()

(b)







5 Conclusions


Data Availability






Acknowledgments




References































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















PCoA on OTU level

NCDHFD

Acupuncture

minus04

minus02

00

02

04

PC2(

189

1)

minus02 02 04 0600PC1(3075)

(a)


NCDHFDAcupuncture


000005010015020025030035

(b)









0

02

04

06

08

1

Perc

ent o

f com

mun

ity ab

unda

nce o

n Ph

ylum

leve

l

(a)

Bactero

idetes

Firmicu

tes

Proteobact

eria

Cyanobact

eria

Actinobact

eria

Sacchari

bacteri

a

phylum

NCDHFDAcupuncture

0

20

40

60

80

Relat

ive a

bund

ance

()

lowast

lowast

lowastlowast

lowastlowast

(b)

AcupunctureNCD HFD

FB ratio

0

1

2

3

4

Firm

icut

es to

Bac

tero

idet

es R

atio

lowast

(c)



4 Discussion



AcupunctureNCD HFD


0

02

04

06

08

1

Perc

ent o

f com

mun

ity ab

unda

nce o

n G

enus

leve

l

(a)

g__Prevotella_9

genus

NCDHFDAcupuncture

lowastlowast

0

20

40

60

80

Relat

ive a

bund

ance

()

(b)







5 Conclusions


Data Availability






Acknowledgments




References































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























0

02

04

06

08

1

Perc

ent o

f com

mun

ity ab

unda

nce o

n Ph

ylum

leve

l

(a)

Bactero

idetes

Firmicu

tes

Proteobact

eria

Cyanobact

eria

Actinobact

eria

Sacchari

bacteri

a

phylum

NCDHFDAcupuncture

0

20

40

60

80

Relat

ive a

bund

ance

()

lowast

lowast

lowastlowast

lowastlowast

(b)

AcupunctureNCD HFD

FB ratio

0

1

2

3

4

Firm

icut

es to

Bac

tero

idet

es R

atio

lowast

(c)



4 Discussion



AcupunctureNCD HFD


0

02

04

06

08

1

Perc

ent o

f com

mun

ity ab

unda

nce o

n G

enus

leve

l

(a)

g__Prevotella_9

genus

NCDHFDAcupuncture

lowastlowast

0

20

40

60

80

Relat

ive a

bund

ance

()

(b)







5 Conclusions


Data Availability






Acknowledgments




References































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















AcupunctureNCD HFD


0

02

04

06

08

1

Perc

ent o

f com

mun

ity ab

unda

nce o

n G

enus

leve

l

(a)

g__Prevotella_9

genus

NCDHFDAcupuncture

lowastlowast

0

20

40

60

80

Relat

ive a

bund

ance

()

(b)







5 Conclusions


Data Availability






Acknowledgments




References































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















5 Conclusions


Data Availability






Acknowledgments




References































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of








































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















acupuncture regulating gut microbiota in abdominal obese rats...

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