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Objective: The present study aims to evaluate the in vivo efficacy of YINDARA-4 in improving the symptoms of irritable bowel syndrome (IBS) in a rat model and investigate the impact of YINDARA-4 on potential targets of IBS management, such as the serotonin level in intestinal tissues and the structure and composition of the gut microbiota. Methods: We developed an IBS rat model by combining stress from maternal separation, acetic acid administration, and restraint. We administered YINDARA-4 water extract to the IBS rat model for 10 consecutive days. The fecal water content, visceral sensitivity, gut microbiota, and serotonin levels in the colonic tissue were then analyzed and compared between the control group, IBS model group, and YINDARA-4–treated groups. Results: Treatment with YINDARA-4 reversed visceral hypersensitivity in a dose-dependent manner in the experimental rat model of IBS. The relief of visceral hypersensitivity upon treatment with YINDARA-4 involved regulation of the gut microbiota structure and composition, and normalization of elevated serotonin levels in the colon. The decrease in colonic serotonin levels with YINDARA-4 treatment might be associated with a reduction in the abundance of Helicobacter and enrichment of Butyricimonas. Conclusions: Treatment with YINDARA-4 was beneficial against visceral hypersensitivity in a rat model of IBS. The improved symptoms exhibited in IBS rats were associated with favorably altered gut microbiota and normalization of serotonin levels in the colon. Keywords: 5-Hydroxytryptamine, Gut microbiota, Irritable bowel syndrome, Traditional Mongolian medicine, Visceral hypersen- sitivity, YINDARA-4 and infection, neurotransmitter imbalance, psychosocial Introduction  and genetic factors, and altered gut microbiota . Among Irritable bowel syndrome (IBS) is a commonly chronic, these, the role of the gut microbiota has attracted increas- relapsing functional gastrointestinal disorder. IBS preva- ing attention in recent years. Patients with IBS have an lence ranges from 10% to 20% of the global population,  altered gut microbiota compared with healthy controls , [1–2] and occurs in 15% to 20% of Chinese adults . IBS sig- and animal studies have shown that perturbation of the nificantly worsens the quality of life of patients and places intestinal microbiota induces typical symptoms of IBS, a heavy burden on both the patients themselves and soci- such as visceral hypersensitivity and altered gastrointes-  ety . The etiology and pathophysiology of IBS are not fully [6–7] tinal motility . These findings suggest a critical role of understood. Symptoms of IBS are believed to result from the gut microbiota in the pathogenesis of IBS. In addition, visceral hypersensitivity, abnormal motility, inflammation some studies have reported that the gut microbiota and microbial metabolites regulate visceral hypersensitivity [8–9] and gastrointestinal motility via serotonin synthesis . Key Laboratory of Ethnomedicine (Minzu University of China), Serotonin plays a significant role in visceral sensi- Ministry of Education, Beijing, China; Department of Respiratory and tivity, gastrointestinal motility, and immune function. Critical Care Medicine, General Hospital of Ningxia Medical University, Serotonin is synthesized from tryptophan by tryptophan Yinchuan, China hydroxylase 1 (TPH1) in enterochromaffin (EC) cells * Corresponding author: Lingpeng Pei, School of Pharmacy, Minzu within the gastrointestinal tract. It is stored in secretory University of China, Beijing 100081, China, E-mail: lppei@hotmail. granules and released into the lumen or lamina propria com; Enqi Wu, School of Pharmacy, Minzu University of China, Beijing when EC cells are activated by various physiological and 100081, China, E-mail: firstname.lastname@example.org.  pathological luminal stimuli . Many studies have shown Copyright © 2022 Tianjin University of Traditional Chinese Medicine. that serotonin levels are increased in the intestinal tissues This is an open-access article distributed under the terms of the [4,10] and plasma of patients with IBS . Several drugs tar- Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download geting the serotonin system are currently being used for and share the work provided it is properly cited. The work cannot be the treatment of IBS. Although these drugs attenuate the changed in any way or used commercially without permission from the symptoms of IBS, they also have adverse side effects such journal. [11–12] as ischemic colitis and arrhythmia . Thus, treatment Acupuncture and Herbal Medicine (2022) 2:4 options for IBS remain limited. Received 8 March 2022 / Accepted 19 September 2022 YINDARA-4 is a classical formula in traditional http://dx.doi.org/10.1097/HM9.0000000000000042 Mongolian medicine that has been used to treat 274 Ling et al. • Volume 2 • Number 4 • 2022 www.ahmedjournal.com Analysis of the chemical components of YINDARA-4 gastrointestinal disorders for hundreds of years. It is cur- rently used as an in-hospital preparation for the treat- The main chemical components in the water extract ment of IBS in many traditional medical hospitals in Inner of YINDARA-4 were detected using ultra-high perfor- Mongolia, and has been demonstrated to be effective in mance liquid chromatography-quadrupole time-of- [13–16] ameliorating the symptoms of IBS . YINDARA-4 flight-tandem mass spectrometry (UHPLC-QTOF-MS/ comprises dry fruit of Cynanchum thesioides (Freyn) MS). UHPLC-QTOF-MS/MS detection was conducted K. Schum., Polygonum bistorta L., Clematis armandii on a Triple TOFTM6600 system with a Duo Spray Franch., and Ophiopogon japonicus (Thunb.) Ker Gawl. source in positive ion mode (AB SCIEX, Foster City, CA, Pharmacological research on these four herbs has shown USA). Electrospray ionization was applied in the posi- that they exhibit various antimicrobial and/or anti-in- tive ion mode with the following parameters: ion spray [17–21] flammatory activities , suggesting that YINDARA-4 voltage, 5,500 V; ion source temperature, 500°C; curtain may be able to regulate the intestinal microbiota. gas, 35 psi; nebulizer gas (GS1), 50 psi; heater gas (GS2), Therefore, we hypothesized that YINDARA-4 may ame- 50 psi; and declustering potential (DP), 80 V. The mass liorate visceral hypersensitivity in a rat model of IBS by ranges were set at m/z 100 to 1,000 Da for the time-of- manipulating the gut microbiota. flight mass spectrometry (TOF-MS) scan and 100 to In the present study, the effects of YINDARA-4 on 1,000 Da for the TOF MS/MS experiments. In the infor- gut pathophysiology and microbiota were investigated mation-dependent acquisition (IDA)–mediated MS/MS using a rat model of IBS developed using multiple stim- experiment, the collision energy (CE) was set at 40 ev, uli. The objectives of this study were to (1) evaluate the and the collision energy spread (CES) was (±)20 ev for in vivo efficacy of YINDARA-4 in improving symptoms UHPLC-QTOF-MS/MS detection. The most intensive in rats undergoing experimental IBS, and (2) investigate five ions from each TOF-MS scan were selected for MS/ the impact of YINDARA-4 on potential targets of IBS MS fragmentation. Dynamic background subtraction management, such as serotonin levels in intestinal tissues (DBS) was applied to match the IDA tests for UHPLC- and the structure and composition of the gut microbiota. QTOF-MS/MS detection. LC-MS/MS data were ana- lyzed using PeakView 1.2 software (AB SCIEX, Foster City, CA, USA). Materials and methods Preparation of YINDARA-4 Animals The herbal formula YINDARA-4 is a combination of Specific pathogen-free (SPF) Sprague Dawley rats were four medicinal herbs, including Cynanchum thesioi- used in the present study. Pregnant rats were purchased des (Freyn) K. Schum. (Vincetoxicum sibiricum (L.) from SPF Biotechnology Co., Ltd. (Beijing, China) on Decne., (Temeen-Huh in Mongolian and Di-Shao- day 15 of the pregnancy. Rats were housed in hardwood Gua in Chinese), Polygonum bistorta L. (Bistorta offi- chip bedding cages (42 cm × 20.5 cm × 20 cm) in a desig- cinalis Delarbre [Polygonaceae], Meher in Mongolian, nated room at 22 ± 2°C with a 12/12 h light/dark cycle in and Quan-Shen in Chinese), Clematis armandii a controlled environment. The rats had ad libitum access Franch. (Balega in Mongolian and Chuan-Mu-Tong to water and standard chow. They were checked daily in Chinese), and Ophiopogon japonicus (Thunb.) Ker for delivery and the day after birth was defined as post- Gawl. (Chagan-Bong-a in Mongolian and Mai-Dong in natal day (PND) 1. The pups were housed with the dam Chinese), at a ratio of 9:7:7:5 (w/w/w/w), respectively. until weaning on PND22. After weaning, three animals Cynanchum thesioides (Freyn) K. Schum, also called were housed in a cage, and all female rats were excluded Yindara (spelled as Yindara or Yindari in Mongolian) from the study to avoid the effects of sex hormones. The or Dugemonong in traditional Mongolian medicine, was study design was approved by the Ethics Committee of collected from the vicinity of Barun Akta Gacha, Horqin Minzu University of China (ECMUC2019009CA). The Left Wing Rear Banner of Inner Mongolia, China (GPS experiments were performed with good laboratory prac- coordinates: 122.18, 43.02). Fresh fruits were collected tices (GLP) in a GLP-accredited laboratory. in July and August, dried in the shade, and then bro- ken open to remove fly floes. The remaining three herbs were purchased from Tong-Ren-Tang (Beijing, China). Study design All the herbs were identified by two experienced phar - macists. Voucher specimens (DSGgan201807, CMTtrt, The rats were randomly assigned to 5 groups (n = 7 QStrt, and MDtrt) were deposited at the School of per group): (1) control (CTRL), (2) IBS model (IBS), Pharmacy, Minzu University of China. The names of the (3) YINDARA-4 high-dosage intervention (YDRh), (4) plants were crosschecked using the Plant List database YINDARA-4 middle-dosage intervention (YDRm), and (http://www.theplantlist.org) on July 3, 2020. For the (5) YINDARA-4 low-dosage intervention (YDRl). The preparation of YINDARA-4, the four herbs were first rats in the CRTL group were not manipulated. The rats crushed and mixed at the above ratios and then boiled in in the other four groups were exposed to multiple adverse water (ratio of material to liquid 1:8) for 1 hour, filtered stresses, including maternal separation, acetic acid instil- through a gauze, and then boiled again in water for 1 h. lation, and restraint during PND1 to PND42. Maternal The supernatant from two runs was then collected and separation was conducted once daily from 09:00 am to freeze-dried to make the dried YINDARA-4 extract. Dry 12:00 am during PND1 to PND21 by removing the lit- YINDARA-4 extract (227 g) was obtained from 1 kg of ter from the dam’s cage into separate cages on top of raw YINDARA-4. heating pads (Suzhou Guofei Laboratory Instrument 275 Ling et al. • Volume 2 • Number 4 • 2022 www.ahmedjournal.com Co., Ltd., Suzhou, China) set at 30°C to 33°C in a sep- consistency. Fresh feces were collected, weighed immedi- arate room. Acetic acid instillation was conducted once ately, dried in an oven at 100°C for 10 min, and weighed daily at 09:00 am from PND15 to PND28. Acetic acid again. The fecal water content was calculated using the at a concentration of 0.5% (Sangon Biotech Co., Ltd., following formula: Shanghai, China) was instilled into the colon with a tube fecal water content (%) = (wet weight of fecesdried weight of feces] (Head Biotechnology Co., Ltd., Beijing, China) with a /wet weight of feces × 100%. diameter of 1 mm from the anus. Considering that young rats may not be able to withstand excessive stimulation, Collection of the colorectal fecal contents the volume of the enemas was 0.2 mL initially and was Colorectal fecal contents were collected on PND84 just increased by 0.1 mL per day until the dose was 0.5 mL per after euthanasia. Fresh fecal pellets from the distal colon day, which was then maintained throughout. Restraint were collected and preserved in a solution containing stress was conducted once daily from 09:00 am to 12:00 4 M guanidine thiocyanate (Solarbio Life Sciences Co., am during PND29 to PND42. The rats were kept in a Ltd. Beijing, China) at –20°C. All fecal samples were then handcrafted plastic restraint cylinder for 3 h each day for transported to the laboratory and stored at –80°C for a restraint stress. The dimensions of the restraint cylinder day. could be manually adjusted to a suitable size such that the rat could not turn around freely. All rats were fed normally from PND43 to PND68. Histopathological examination From PND69 to PND78, freeze-dried extracts of Distal colon tissues were fixed in neutral buffered YINDARA-4 were re-dissolved in water and adminis- 10% formalin for 12 h, dehydrated, and embedded tered by oral gavage at approximately 11:00 am daily in paraffin. For hematoxylin-eosin staining, two to the rats in the YDRh, YDRm, and YDRl groups, sequential 5 μm thick sections were cut and pro- while the rats in the IBS group received normal saline cessed. Slides were de-identified and observed by a (1 mL). The dose of YINDARA-4 used was based on the pathologist. conversion of the recommended adult dosage (10 g raw For immunohistochemistry, two sequential 5 μm YINDARA-4/day). Rats in the YDRh, YDRm, and YDRl thick sections were cut for each sample, pretreated groups were administered YINDARA-4 at a dose of 1.8, with 3% hydrogen peroxide and normal horse 0.9, and 0.45 g/kg, respectively, which corresponds to 2 serum, and incubated with an antibody against sero- times, 1 time, and 1/2 times the human equivalent dose, tonin (1:400, ab66047, Abcam, Cambridge, UK) for respectively. 8 h at 4°C. The sections were then incubated with a All rats were anesthetized using isoflurane (Shuilantai secondary antibody and developed using 3,3-diam- Chemical Co., Ltd., Zaozhuang, China) for collection inobenzidine. The sections were then incubated with of colorectal fecal contents and tissue samples, and hematoxylin for nuclear counterstaining. A total of were then euthanized by exsanguination on PND84 as five random fields (40× objective magnification) per  described previously . section were observed using Image-Pro Plus software (version 5.0; Media Cybernetics, Silver Spring, MD, Visceral sensitivity assessment USA) by an investigator blinded to the group settings. Optical density was calibrated before measuring the Visceral hypersensitivity was assessed using the abdom- area sum, density mean, and integrated optical den- inal withdrawal reflex (AWR) score at PND78 from [23–24] sity (IOD). The areas of interest were set as follows: 09:00 am to 12:00 am, as described previously . In hue (0–200), saturation (0–255), and intensity (0–90). brief, the rats were moved into a self-made plastic cubi- The reagents used for histopathological examination cle and allowed to acclimate to the container for 2 min. were purchased from Servicebio Technology Co. Ltd., A self-made flexible balloon attached to Tygon tubing Wuhan, China. with a diameter of 2 mm (Head Biotechnology Co., Ltd., Beijing, China) was inserted into the descend- ing colon (8 cm from the anus). The balloon was rap- Microbiota sequencing idly inflated to various pressures (40, 50, 60, 70, 80 Total bacterial DNA was extracted using the Power and 90 mmHg) for a 10-second colorectal distension Soil DNA Isolation Kit (MO Bio Laboratories, Inc. stimulation, and the behavioral responses of the rats Carlsbad, CA, USA). After checking the quality to each pressure were assessed by two blinded observ- and quantity of the DNA sample using OD ratios ers using the AWR score system, where 0 represents at 260 nm/280 nm and 260 nm/230 nm, the V3–V4 normal behavior without any response, 1 represents hypervariable region of the bacterial 16S rRNA light response motion without visible abdominal mus- gene was amplified with the common primers 338F cle contraction, 2 represents visible abdominal muscle (5ʹ-ACTCCTACGGGAGGCAGCAG-3ʹ) and 806R contraction, 3 represents visible abdominal wall lift- (5ʹ-GGACTACHVGGGTWTCTAAT-3ʹ), combined ing, and 4 represents visible pelvic structure lifting and with adapter sequences and barcode sequences. body arching. Second-generation sequencing was conducted on the purified, pooled PCR product sample using the HiSeq Fecal water content 2500 platform (Illumina, Inc. San Diego, CA, USA) The fecal water content of each rat was measured on (2 × 250 paired ends) at the Biomarker Technologies PND82 at 09:00 am as an objective measure of stool Corporation Beijing, China. 276 Ling et al. • Volume 2 • Number 4 • 2022 www.ahmedjournal.com Bioinformatic analyses Effect of YINDARA-4 on fecal water content Raw sequence reads were processed using the UNOISE The fecal water content of the IBS model group was pipeline in the Usearch v11.0.667linux32 program lower than that of the CTRL group (11.6% ± 3.9% vs. (www.drive5.com/usearch/). High-quality sequences 20.1% ± 4.4%, n = 7), and increased to varying degrees were classified into zero-radius operational taxonomic after YDR treatment (15.2% ± 3.7% for the YDRl group, units (ZOTUs). The Ribosomal Database Project (RDP) 17.3% ± 6.8% for the YDRm group, and 15.7% ± 4.8% classifier was used to annotate the taxonomic informa- for the YDRh group, n = 7). However, none of the differ- tion of each ZOTU sequence with an 80% confidence ences were statistically significant (P = 0.11; Figure 2A). threshold. The Chao1, Shannon, and Simpson indi-  ces were calculated using the QIIME 1.91 pipeline . Effect of YINDARA-4 on visceral hypersensitivity Principal coordinates analysis (PCoA), distance-based Our results showed that 30 mmHg of pressure was the redundancy analysis (db-RDA), and adonis tests were performed to evaluate differences among bacterial com- non-nociceptive intracolonic pressure that did not evoke any differences in the abdominal muscle contractility of munities based on Bray–Curtis distance metrics using  “vegan” package in R . rats. When the colorectal distention pressure reached 40 mmHg, the AWR scores differed significantly between the groups. The AWR scores of the IBS model group Statistical analyses were significantly higher than those of the CTRL group at 40 to 80 mmHg pressure. After the intervention, the R software (version 3.52, R Foundation for Statistical AWR scores of all three YINDARA-4–treated groups Computing, Vienna, Austria) was used for the statistical were lower than those of the IBS model group. The dif- analyses. The Shapiro–Wilk test was used to test for nor- ference in AWR scores was significant for the high dose mal distribution, and the analysis of variance (ANOVA) group at pressures of 40 to 90 mmHg, whereas for the test or Kruskal–Wallis test was used to evaluate the dif- middle dosage and low dosage groups, the difference in ferences in the measured variables among the groups. AWR scores was significant at pressures of 50 and 40 to Spearman’s correlation analysis was performed using 70 mmHg, respectively (Figure 2B). “psych” package in R to determine associations between The minimal distention pressure that evoked behav- different variables. Finally, the linear discriminant anal- ioral responses corresponding to each of the AWR scores ysis (LDA) effect size (LEfSe) method (threshold of was defined as the distension threshold (DT). A compari- ±2) was used to explore significantly different bacteria son of the DT values among the three treatment groups is among the treatment groups. shown in Figure 2C. The DT for AWR scores of 2, 3, and 4 showed an increasing trend with the increase in the Results YINDARA-4 intervention dose. Among them, the DT of AWR score 4 was significantly different among the differ - Phytochemical characterization of YINDARA-4 ent dose groups. Spearman’s rho correlation analysis was Twenty-six compounds were identified in the water performed on the association between the YINDARA-4 extract of YINDARA-4 (Figure 1). Molecular informa- intervention dose and the DT of the rats, and it was tion of the compounds is presented in Table 1. Among found that there was a statistically significant positive the 26 compounds, there were seven flavonoids (includ- correlation between the YINDARA-4 intervention dose ing baicalin, bavachinin A, rocyanidin B2, salvianolic and the DT of AWR score 4 (rho = 0.506, P = 0.019), acid B, catechin, rutin, and quercetin), seven alkaloids showing that treatment with YINDARA-4 significantly (including betaine, trigonelline, stachydrine, arecoline, decreased visceral hypersensitivity in rats undergoing atropine, irinotecan, and norisoboldine), four phenyl- experimental IBS in a dose-dependent manner. propanoid compounds (including praeruptorin C +Na, phenprobamate, fraxin, and easculetin), three organic Histopathology of colonic tissues acids (succinic acid, quinic acid, and 4-O-Caffeoyl Quinic acid), two terpenoids (ginkgolide B +NH3 and In our observations, the structure of the intestinal mucosa resveratrol), two quinones (diacerein and tanshinone was intact, with a normal epithelium. The simple colum- IIA), and one adenosine analog (cordycepin). nar epithelium within the colonic mucosa was arranged in Figure 1. Ultra-high performance liquid chromatography-quadrupole time-of-flight-tandem mass spectrometry chromatograms of YINDARA-4 extract. 277 Ling et al. • Volume 2 • Number 4 • 2022 www.ahmedjournal.com Table 1 Compounds of water extract from YINDARA-4 Found at No. retention time (min) Name Formula Mass (Da) Found at mass (Da) Extraction mass (Da) Error (ppm) 1 5.54 Baicalin C H O 446.0849 447.0900 447.0922 −4.8 21 18 11 2 5.70 Cordycepin C H N O 251.1018 252.1083 252.1091 −3.3 10 13 5 3 3 5.81 Betaine C H NO 117.0790 118.0862 118.0863 −0.7 5 11 2 4 5.89 Trigonelline C H NO 137.0477 138.0549 138.0550 −0.6 7 7 2 5 6.09 Ginkgolide B +NH C H O .NH 441.1635 442.1693 442.1708 −3.2 3 20 24 10 3 6 6.15 Stachydrine C H NO 143.0946 144.1018 144.1019 −0.8 7 13 2 7 6.75 Arecoline C H NO 155.0946 156.1015 156.1019 −2.4 8 13 2 8 7.95 Succinic acid C H O 118.0266 119.0348 119.0339 7.9 4 6 4 9 9.58 Bavachinin A C H O 338.1518 339.1571 339.1591 −5.8 21 22 4 10 10.55 Praeruptorin C +Na C H O .Na 451.1733 452.1788 452.1806 −3.8 24 28 7 11 13.86 Quinic acid C H O 192.0634 193.0689 193.0707 −9.1 7 12 6 12 16.09 Phenprobamate C H NO 165.0790 166.0862 166.0863 −0.3 9 11 2 13 24.63 Atropine C H NO 289.1678 290.1724 290.1751 −9.2 17 23 3 14 26.92 Rocyanidin B2 C H O 578.1425 579.1501 579.1497 0.6 30 26 12 15 28.20 4-O-Caffeoyl quinic acid C H O 354.0951 355.1028 355.1024 1.2 16 18 9 16 28.76 Salvianolic acid B C H O 718.1534 719.1560 719.1607 −6.5 36 30 16 17 29.09 Catechin C H O 290.0790 291.0866 291.0863 1.0 15 14 6 18 30.51 Fraxin C H O 370.0900 371.1009 371.0973 9.7 16 18 10 19 31.32 Esculetin C H O 178.0266 179.0339 179.0339 0.0 9 6 4 20 31.50 Diacerein C H O 368.0532 369.0631 369.0605 6.9 19 12 8 21 35.83 Rutin C H O 610.1534 611.1667 611.1607 9.8 27 30 16 22 36.36 Quercetin C H O 302.0427 303.0503 303.0499 1.1 15 10 7 23 36.80 Resveratrol C H O 228.0786 229.0861 229.0859 1.0 14 12 3 24 42.65 Irinotecan C H N O 586.2791 587.2909 587.2864 7.7 33 38 4 6 25 43.42 Tanshinone IIA C H O 294.1256 295.1327 295.1329 −0.6 19 18 3 26 44.74 Norisoboldine C H NO 313.1314 314.1396 314.1387 2.9 18 19 4 an orderly fashion, and goblet cells were rich in the CTRL In the comparison of alpha diversity among different and IBS model groups. After the intervention, there was groups, no significant differences were observed for any no significant difference in the histological features among of the three diversity indices (data not shown). However, the three treatment groups and IBS model group. a PCoA based on the Bray–Curtis distance matrix revealed a trend toward a differential distribution for the three groups (Figure 4). Adonis tests and db-RDA Immunohistochemistry for the expression of serotonin in analyses confirmed the significance of the associations the colon between the different groups and the structure of the Immunohistochemistry (Figure 3A–C) revealed that the bacterial communities (Table 2). number of serotonin-positive areas and the IOD of sero- For the microbiota composition, LEfSe analysis in tonin (brown particles) in the colon were significantly the comparison of the IBS model group and the YDRh higher in the IBS model group than in the CTRL group group showed that the abundance of Butyricimonas (P < 0.001). The number of serotonin-positive areas and increased significantly after YDRh treatment, while IOD of serotonin were significantly lower in the YDRh those of Helicobacter and its family, order, and class group than in the IBS model group (P < 0.001). were significantly reduced in the YDRh group (shown in Figure 5A). Further comparison of the abundance of Effect of YINDARA-4 on microbiota community structure these two genera within the three groups (Figure 5B) and composition in colon content showed that the abundance of the genus Butyricimonas Among the three YINDARA-4 dosage groups, the YDRh was significantly lower in the IBS model group than in the CTRL and YDRh groups, whereas there was no dif- group showed the most significant changes in visceral hypersensitivity. To investigate the potential interrela- ference between the CTRL and YDRh groups. The abun- dance of Helicobacter was significantly higher in the IBS tionships between the alteration of the gut microbiota and the improvement of IBS symptoms, we investigated model group than in the CTRL group and was decreased after treatment with YDRh. No significant difference the microbiota in the colon contents of rats in the CTRL, IBS, and YDRh groups. A total of 978,728 clean reads was found in the abundance of Helicobacter between the CTRL and YDRh groups. were collected from 21 samples (seven samples each from the CTRL, IBS, and YDRh groups) after trimming and filtering. A ZOTU table with 4,139 ZOTUs was gen- Analysis of correlation between the differential microbiota erated and used for data analysis. Using the RDP classi- composition and gut pathophysiological parameters fier, 4,139, 3,966, 3,910, 3,743, and 1,738 ZOTUs were annotated at the phylum, class, order, family, and genus To further assess potential correlations between gut pathophysiological parameters and certain microbiota levels, respectively, comprising seven phyla, 12 classes, 13 orders, 20 families, and 33 genera. composition, we conducted Spearman’s correlation tests 278 Ling et al. • Volume 2 • Number 4 • 2022 www.ahmedjournal.com Figure 2. Comparison of the fecal water content and visceral sensitivity in rats from different groups. (A) Comparison of the fecal water content in rats after YINDARA-4 treatment (n = 7) with the IBS model group. (B) Comparison of the AWR scores in rats from different groups (n = 7) in response to graded colorectal distension with different pressures. (C) Correlations between the YINDARA-4 intervention dose and the DT of rats. Minimal distention pressures that evoked behavioral responses corresponding to each of the AWR scores were defined as the DT. *P < 0.05 as compared with the IBS model group. **P < 0.01 as compared with the IBS model group. AWR: Abdominal withdrawal reaction; Ctrl: Control; DT: Distension threshold; IBS: Irritable bowel syndrome; YDRh: YINDARA-4 high-dosage intervention; YDRl: YINDARA-4 low-dosage intervention; YDRm: YINDARA-4 middle-dosage intervention. on relative abundance data of the genera Butyricimonas Serotonin is an important neurotransmitter involved in  and Helicobacter using the psych package in R. The genus mutual communication between the brain and the gut . Butyricimonas was positively correlated with the DT for It has been reported that the abundance of serotonin is AWR scores 2 and 3 and the fecal water content, and was increased in the blood of IBS patients, and alterations in negatively correlated with the two indicators of serotonin the gastrointestinal serotonin signaling pathway are con- expression in the colon, the number of serotonin-positive sidered to be associated with disrupted visceral sensation [28–30] areas and the IOD of serotonin. The genus Helicobacter and intestinal motility in patients with IBS . In the was positively correlated with the number of sero- present study, we observed that the expression level of tonin-positive areas and the IOD of serotonin (Figure 6). serotonin in the colon was significantly increased in the IBS model group compared with that in the CTRL group and was restored to CTRL levels in the YDRh group. Discussion This suggests that the serotonin level in the colon might In the present study, a rat model of IBS was developed by play a key role in the amelioration of visceral sensitivity mimicking multiple etiological factors of IBS through the upon treatment with YINDARA-4. combination of maternal separation, acetic acid instil- Increasing evidence has demonstrated the pivotal role lation, and restraint stress. Rats undergoing experimen- of the gut microbiota in the pathogenesis of IBS; thus, tal IBS exhibited symptoms of visceral hypersensitivity manipulation of the gut microbiota is emerging as an  and abnormal fecal water content, with no organ-level attractive therapeutic strategy for this disease . Various changes found upon pathological examination, indi- microbiota-manipulating interventions, such as fecal cating that the model was successfully constructed. A microbiota transplantation, administration of prebiotics, 10-day administration of YINDARA-4 significantly probiotics, synbiotics, and certain non-absorbable antibi- decreased the AWR score and increased the pain thresh- otics, have been reported to alter the ecological structure old in rats undergoing experimental IBS in a dose-depen- of the intestinal microbial community, selectively suppress dent manner, suggesting that YINDARA-4 attenuated harmful bacteria, and stimulate the activity or growth of visceral hypersensitivity in IBS model rats. beneficial bacteria, thereby ameliorating the symptoms 279 Ling et al. • Volume 2 • Number 4 • 2022 www.ahmedjournal.com Figure 3. Comparison of the colonic serotonin level between different groups. (A) Representative immunohistochemistry images taken at 40× magnification. (B) Comparison of the number of serotonin-positive areas between the different groups. (C) Comparison of the integrated optical density of serotonin between the different groups. Ctrl: Control; IBS: Irritable bowel syndrome; YDRh: YINDARA-4 high-dosage intervention. [32–35] of IBS . Studies have also shown that enteric resi- The genus Helicobacter is a highly prevalent bacterium dent microbiota can regulate the synthesis of serotonin and is associated with various chronic gastrointestinal  in intestinal EC cells and host serotonergic signaling . diseases in both humans and animals, such as chronic  Our results demonstrated that YINDARA-4 treatment gastritis, peptic ulcer, and gastric carcinoma . Several exerted similar microbiota-manipulating effects in IBS studies have proposed that Helicobacter may play a [37–38] model rats. The microbial community structure in the role in IBS . Helicobacter infection can induce sys- colon contents of IBS model rats was significantly altered temic inflammatory responses, which could lead to the after treatment with YINDARA-4. The abundance of the stimulation of mast cells and EC cells and increase the harmful bacterial genus Helicobacter was significantly secretion of proinflammatory neurotransmitters, such lower in the YDRh group than in the IBS group, whereas as serotonin, substance P, and calcitonin gene–related the beneficial bacterial genus Butyricimonas was signifi- peptide, all of which have an intimate relationship with [39–40] cantly enriched in the YDRh group. the symptoms of IBS . Our results are consistent with the above findings, showing that the abundance of Helicobacter was positively associated with the expres- sion of serotonin in the colon. On the other hand, the genus Butyricimonas is a group of obligate anaerobic bacteria, which can produce butyr- ate. Butyrate is one of the most common short-chain fatty acids in the intestinal tract, and previous studies have shown that it can induce regulatory T cells, reduce inflammation, and maintain healthy intestinal function. Table 2 Adonis and db-RDA analysis results investigating the association between the gut microbiota and the three groups based on the Bray–Curtis distance metrics Adonis test db-RDA analysis 2 2 Variants P r P r Figure 4. Two dimensions PCoA plots showing differences in the gut microbiota of rats from the different groups based on the Bray–Curtis Model groups (Ctrl, IBS, 0.009 15.69% 0.003 6.43% distance metric (n = 7). Ctrl: Control; IBS: Irritable bowel syndrome; and YDRh) PCoA: Principal coordinates analysis; YDRh: YINDARA-4 high-dosage CTRL: control group; IBS: IBS model group; YDRh: YINDARA-4 high dosage intervention group; intervention. db-RDA: Distance-based redundancy analysis (db-RDA) 280 Ling et al. • Volume 2 • Number 4 • 2022 www.ahmedjournal.com Figure 5. Comparison of the gut microbiota composition between different groups. (A) LEfSe analyses comparing differentially abundant taxa between the communities in the IBS and YDRh groups. (B) Kruskal–Wallis non-parametric test comparing differentially abundant taxa among the communities in the control, IBS, and YDRh groups. IBS: Irritable bowel syndrome; LEfSe: Linear discriminant effect size; YDRh: YINDARA-4 high-dosage intervention.  Hence, decreased Butyricimonas within the intestine is production . Using a human EC cell model, Reigstad  thought to play a role in the inflammation associated et al. demonstrated that treatment with a high con- [41–42] with the pathogenesis of IBS . In addition, the genus centration of butyrate significantly reduced the secretion Butyricimonas is also associated with IBS, as butyrate of serotonin by suppressing the mRNA expression of production has been shown to regulate colonic serotonin TPH1, which is the key rate-limiting enzyme during the 281 Ling et al. • Volume 2 • Number 4 • 2022 www.ahmedjournal.com Figure 6. Spearman’s correlation between the differential microbiota composition and gut pathophysiological parameters. Blue = positive correla- tion; red = negative correlation. 5-HT, serotonin; DT, distension threshold; IOD, integrated optical density. Author contributions synthesis of serotonin in the gut. Consistent with the lit- erature, the present study found that the abundance of Yaqin Ling and Enqi Wu performed the experiments. Butyricimonas was negatively associated with serotonin Ling Ding, Zhigang Tian, and Enqi Wu analyzed the expression in the colon. data. Enqi Wu, Lingpeng Pei, and Yaqin Ling obtained funding for this project and planned the experiments. Lingpeng Pei and Yaqin Ling drafted the manuscript. Conclusion All the authors have read and approved the final Taken together, our research provides experimental manuscript. evidence that YINDARA-4 treatment attenuates vis- ceral hypersensitivity in a dose-dependent manner in Ethical approval of studies and informed consent an IBS rat model. The relief of visceral hypersensitivity This study was approved by the Ethics Committee of following treatment with YINDARA-4 involved nor- the Minzu University of China (ECMUC2019009CA). malization of elevated serotonin levels in the colon and The experiments were performed within a Good regulation of the gut microbiota structure and com- Laboratory Practice (GLP)–accredited laboratory and position. The decrease in the levels of serotonin in the followed the ARRIVE guidelines. colon upon YINDARA-4 treatment may be associated with a reduction in Helicobacter and enrichment in Availability of data and materials Butyricimonas. 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