Impact of Daikenchuto (TU-100) on The Early Postoperative Period in Duodenal-Jejunal Bypass

Introduction: We investigated the effect of Daikenchuto (TU-100) on the early postoperative period in duodenal-jejunal bypass (DJB). Methods: Study 1: The effect of TU-100 on diabetic rats was investigated. Rats were sacriced after receiving TU-100 for one week. Study 2: The effect of TU-100 on DJB was investigated. Rats in the DJB and TU-100 treated DJB groups were sacriced 24 hours postoperation to evaluate blood glucose, cytokine expression, and gut microbiome. Results: Study 1: TU-100 did not affect glucose or body weight. TU-100 suppressed intestinal inammation and modied the gut microbiome. Specically, Bidobacterium and Blautia were increased, and Turicibacter were decreased in this group. Study 2: Both DJB and TU-100 treated DJB rats showed lower blood glucose at 24 hours postoperation than at preoperation. Cytokine expression in the liver and small intestine of the TU-100 treated DJB group was signicantly lower than that of the DJB group. The gut microbiome composition in TU-100 treated DJB rats was altered. In particular, Bidobacterium and Blautia were increased in this group. Conclusion: DJB suppressed blood glucose during the early postoperative period. TU-100 may enhance the anti-diabetic effect of metabolic surgery by changing the gut microbiome and suppressing inammation in the early postoperative period.


Introduction
The incidence of type 2 diabetes mellitus (T2DM) and obesity is rapidly increasing worldwide [1]. Obesity is recognized as one of the major risk factors for T2DM, coronary artery disease, stroke, sleep apnea, and cancer [2][3][4]. It is well-known that severe obesity leads to health complications, decreases life expectancy, and in uences patients' quality of life. Therefore, obesity and obesity-related diseases impose a burden on global health systems, societies, families, and affected individuals [5,6].
Bariatric/metabolic surgeries (vertical sleeve gastrectomy, Roux-en-Y gastric bypass, laparoscopic adjustable gastric banding, and biliopancreatic diversion) achieved weight loss and improved obesityrelated diseases [7]. Some considered bariatric interventions as metabolic surgery when the purpose was to improve metabolic disorder rather than weight loss alone, and these procedures could lead to changes of the gut microbiome and hormones, bile acid metabolism, and other factors that in uenced glucose homeostasis independent of weight loss [8].
Duodenal-jejunal bypass (DJB) was a metabolic surgery involving exclusion of the duodenal and proximal jejunal from nutrients, jejunal Roux-en-Y reconstruction, and early nutrient delivery to the distal small intestine. Rubino et al. revealed substantial improvements in glucose homeostasis after DJB surgery [9].
In our previous report, DJB improved T2DM and liver steatosis by enhancing glucagon-like peptide-1 (GLP-1) secretion through increased bile acids and the proliferation of L cells in the ileum. Thus, we concluded that DJB surgery might be a key strategy for treating obese patients with T2DM [10].
Furthermore, we reported that DJB changed the composition of gut microbiota, and these changes might contribute to the bene cial effects of DJB [11]. In addition, DJB surgery was reported to maintain gut permeability through suppressing gut in ammation in our previous study [12]. DJB surgery may improve diabetes and liver steatosis by modifying the gut microbiota, maintaining gut permeability, and suppressing gut in ammation.
Seki et al. reported that a patient who underwent sleeve gastrectomy with DJB showed a glucose level below 200 mg/mL 100% of the time after postoperative day 3 (POD3) and did not receive diabetic medication on or after POD2 [13]. However, the mechanism underlying the improvement in diabetes during the early postoperative period remains unclear.
Daikenchuto (TU-100), a traditional Japanese medicine (a 'Kampo' medicine), was widely used Kampo medicine in Japan and had the greatest amount of preclinical and clinical evidence for improving postoperative gastrointestinal motility and preventing postoperative ileus [14][15][16][17][18][19]. We previously reported that TU-100 ameliorated colonic microvascular disorder in a rat model of Crohn's disease [20], prevented BT in fasting rats [21] and a CPT-11-induced intestinal injury model [22], and reduced the intestinal upregulation of cytokines, such as IL-6, TNF-α, and IL-1β. TU-100 maintained the diversity of the gut microbiota and decreased the abundance of Erysipelotrichaceae species, which are related to in ammatory diseases, such as colitis [23].
Therefore, we hypothesized that TU-100 might enhance the anti-diabetic effect of metabolic surgery by modifying the gut microbiome and suppressing in ammation during the early postoperative period. The purpose of this study was to investigate the effect of Daikenchuto (TU-100) on the early postoperative period in DJB using diabetic rats.

Animals
We used twelve 12-week-old male Goto-Kakizaki rats (CREA Japan, Inc.). These rats were reared in a controlled environment. The procedures in this experiment were approved by the Department of Animal Research Resources, Institute of Health Biosciences, Tokushima University. All experiments were performed according to the relevant guidelines and regulations, and all authors complied with the ARRIVE guidelines.
Methods DJB, sham operation, TU-100 treatment, and TU-100 treated DJB The 12 twelve rats were divided into four groups: a sham group, a DJB group, a Daikenchuto (TU-100) treated group, and a TU-100 treated DJB group. Each group had three rats. These rats were fasted before the surgery and anesthetized in 2% to 3% iso urane and air/oxygen. In the sham operated group, a midline laparotomy was performed, and this incision was simply closed. DJB was performed as reported by Rubino, et al. [9]. Brie y, DJB was performed by post-pyloric transection of the duodenum, closure of the duodenal stump, transection of the jejunum at 10 cm from the Treitz ligament, reconstruction of the alimentary passage by duodenojejunostomy, and nally reconstruction of the biliopancreatic limb to the jejunum at 15 cm distal from the duodenojejunostomy. These rats in the sham and DJB group were allowed free access to water and a basal diet, freely (MF; Oriental Yeast, Tokyo, Japan). In the TU-100 group and TU-100 treated DJB group, TU-100 mixed food by gavage for 7 days before surgery.
Blood glucose in the DJB group was measured at preoperation and 24 hours postoperation. Blood glucose in the TU-100 group was measured at pre-TU-100 treatment and post-TU-100 treatment. In the TU-100 treated DJB group, blood glucose was measured at pre-TU-100 treatment, preoperation, and 24 hours postoperation.
Body weight was measured at pre-TU-100 treatment and post-TU-100 treatment in the TU-100 group and pre-TU-100 treatment, preoperation, and 24 hours postoperation in the TU-100 treated DJB group.
Fecal pellets were harvested at pre-TU-100 treatment and post-TU-100 treatment from the TU-100 group and pre-TU-100 treatment, preoperation, and 24 hours postoperation from the TU-100 treated DJB group.
Rats in the DJB and TU-100 treated DJB groups were sacri ced 24 hours postoperation. Lysing Matrix E tube (MP Biomedicals, Solon OH, USA), and homogenized using a FastPrep-24 automated cell disruptor (MP Biomedicals) at a speed setting of 6 meters/sec for 40 sec. The homogenization process was repeated twice. The homogenate was centrifuged at 10,000 × g for 30 min, and DNA extract was obtained as the aqueous phase, which was puri ed twice by adding an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1, v/v). Subsequently, an equal volume of isopropyl alcohol (Fuji lm Waco Pure Chemical Corporation) was added, and DNA was obtained as a pellet by centrifugation (10,000 × g, 5 min). After drying, the DNA was dissolved in TE. The preparation of the 16S rRNA gene metagenome library for MiSeq (Illumina, Inc., San Diego, USA) was performed according to the manufacturers' protocol. Brie y, 10 ng DNA template were ampli ed using an Advantage-HF 2 PCR kit (Takara Bio Inc., Shiga, Japan) with universal primers for the 16S rRNA v3-v4 region (forward primer: 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG 3'; reverse primer: 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC 3'). Subsequently, index sequences for each sample were added to both ends of the puri ed PCR fragments. The concentrations of each amplicon were measured using a Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scienti c, Inc., Waltham, USA) and mixed equally. The library was applied to a MiSeq Reagent Kit v3 (Illumina, Inc.), and sequences were determined in accordance with the manufacturers' standard protocol. Sequence data were processed using the 16S rRNA sequence analysis pipeline QIIME 1.8.0 as follows [23]. Initially, both sequence reads were joined, and sequences with a Phred quality score below 20 were removed. Chimera elimination by Usearch was performed to remove contaminated sequences. Open reference operational taxonomic unit (OTU) picking was performed against Greengenes 13_8 97% OTU representative sequences. A summary of the taxonomy of each sample was obtained using the script 'summarize_taxonomy_through_plots.py' in QIIME 1.8.0.

Statistical analysis
Data were analyzed using an unpaired Student's t-test or a one-way ANOVA with Bonferroni's post hoc test. Differences were de ned as signi cant when p < 0.05. Results were expressed as means ± standard error of the mean (SEM). All data were generated using StatView version 5.0 for Windows (SAS Institute Inc., Cary, NC, USA).
Effect of TU-100 on non-obese diabetic rats Study 1: The effect of TU-100 on non-obese diabetic rats was investigated (Figure 1). During the rst week, TU-100 had no effect on blood glucose or body weight (Figure 2a,b). However, TU-100 suppressed the levels of IL-1β and IL-6 in the proximal small intestine, TNFα in the middle small intestine, and TNFα and IFN-γ in the distal small intestine (Figure 3a-e). Therefore, this indicated that TU-100 suppressed gut in ammation. In the PCA analysis, signi cant alterations in the composition of the gut microbiome in TU-100 treated rats were observed (Figure 4a,b). In particular, Bi dobacterium and Blautia were increased, and the Turicibacter genus was decreased in the TU-100 group (Figure 4c-e).
Effect of TU-100 on duodenal-jejunal bypassed non-obese diabetic rats Study 2: The effect of TU-100 on duodenal-jejunal bypassed non-obese diabetic rats was investigated ( Figure 5). Both DJB and TU-100 treated DJB rats showed lower blood glucose at 24 hours postoperation than that measured preoperation (Figure 6a,b). However, both the DJB and TU-100 treated DJB groups exhibited body weight changes after 24 hours (Figure 6c,d). Cytokine expression in the liver and small intestine of the TU-100 treated DJB group was signi cantly lower than that of the DJB group (Figure 7ad), indicating that preoperative treatment with TU-100 may suppress postoperative in ammation after DJB. In addition, PCA analysis revealed signi cant changes in the composition of the gut microbiome in TU-100 treated DJB rats. However, there was no difference in the gut microbiome between preoperation and postoperation samples in the DJB group (Figure 8a-c). In particular, Bi dobacterium and Blautia were increased in the TU-100 treated DJB group. In the TU-100 treated DJB group, the proportion of Turicibacter after DJB was signi cantly decreased compared with that before DJB (Figure 8d-f).

Discussion
This study investigated the effect of Daikenchuto (TU-100), a traditional Japanese (Kampo) medicine, on the early postoperative period in DJB using non-obese diabetic rats. Blood glucose levels at 24 hours postoperation in the TU-100 treated DJB group were signi cantly lower than those before TU-100 treatment and surgery. Cytokine expression in the liver and small intestine of the TU-100 treated DJB group was signi cantly lower than in the DJB group. The gut microbiome composition in TU-100 and TU-100 treated DJB rats was signi cantly altered. Speci cally, Bi dobacterium and Blautia were increased, and the proportion of Turicibacter was decreased in the TU-100 and TU-100 treated DJB groups. Therefore, TU-100 may enhance the anti-diabetic effect of metabolic surgery by modifying the gut microbiome and suppressing in ammation during the early postoperative period. To our knowledge, this is the rst report to reveal the anti-diabetic mechanism of DJB in the early postoperative period and the usefulness of TU-100 during perioperative care for metabolic surgery.
Metabolic surgery is recommended for the treatment of T2DM patients because it leads to normalization of hyperglycemia within days after surgery, an effect that is thought to be independent of weight loss [24,25]. Metabolic surgery positively modi es the gut endocrine system to decrease appetite and improve glucose metabolism [26]. After RYGB, ingested food empties rapidly from the small gastric pouch into the alimentary limb and mixes with the biliary and pancreatic exocrine secretions in the common limb. The rapid emptying of the reduced gastric pouch results in accelerated nutrient transit [27,28] and alters the post-prandial gastrointestinal hormonal chain of events. This enhances the release of satiety hormones, such as cholecystokinin (CCK) [29,30], peptide yy (PYY) [31], GLP-1 [32][33][34], and oxyntomodulin [35]. We reported that DJB surgery improved diabetes and non-alcoholic steatohepatitis by enhancing GLP-1 secretion through increased bile acids and the proliferation of L cells in the ileum [10].
Seki et al. reported that a patient who underwent sleeve gastrectomy with DJB showed an early blood glucose suppressive effect after POD3 [13]. In our previous report, we also investigated the effect of Rouxen-Y reconstruction on T2DM with gastric cancer in the early postoperative period. Roux-en-Y reconstruction achieved early improvement in T2DM regardless of any body weight loss [36]. However, the mechanism underlying the improvement in diabetes during the early postoperative period remains unclear.
It is well-known that a high-fat diet (HFD) induces chronic intestinal in ammation and insulin resistance. Kawano Y et al. reported the association between intestinal in ammation and insulin resistance [37]. Exposure to a HFD results in the recruitment of pro-in ammatory macrophages, which promote intestinal in ammation and permeability, leading to increased in ammatory cytokine and lipopolysaccharide levels in the portal vein. Subsequently, these are circulated to the peripheral insulin-responsive tissues, leading to increased chronic in ammation in the liver and adipose tissue and the onset of insulin resistance. In our previous report, DJB surgery was shown to maintain gut permeability via the suppression of gut in ammation [12].
Gut microbiota in uence host health, and an imbalanced bacterial population associated with a HFD has been shown to trigger the development of metabolic diseases, such as obesity [38] and diabetes [39], in animal models. We reported that DJB surgery changed the composition of the gut microbiota, and these changes might contribute to the positive effects of DJB [11]. However, the effect of DJB on the gut microbiome and in ammation in the early postoperative period remains unclear.
Therefore, we focused on TU-100 as a potential enhancer of the effect of metabolic surgery during the early postoperative period. TU-100, a traditional Japanese medicine (a 'Kampo' medicine), is the most widely used Kampo medicine in Japan and has the greatest amount of preclinical and clinical evidence for improving gastrointestinal motility and preventing postoperative ileus after abdominal surgery [14][15][16][17][18][19]. In our previous reports, TU-100 prevented BT in fasting rats [21] and a CPT-11-induced intestinal injury model [22] and reduced the upregulation of cytokines in the intestine. Furthermore, TU-100 maintained the diversity of the gut microbiota and decreased the abundance of Erysipelotrichaceae species, which are associated with in ammatory conditions, such as colitis [23].
In this study, the TU-100 treated DJB group showed stable blood glucose levels and suppression of in ammatory cytokines in the liver and intestine. Therefore, TU-100 may enhance the anti-diabetic effect of DJB by suppressing in ammation in the early postoperative period. In addition, this suppression of in ammation in the liver and intestine was attributed to changes in the gut microbiome following pretreatment with TU-100.
In this study, Bi dobacterium and Blautia were increased, and the proportion of Turicibacter was decreased in the TU-100 and TU-100 treated DJB groups. Bi dobacterium species are reportedly associated with improvements in glucose intolerance, insulin secretion, and the reduction of in ammatory cytokines in plasma and fat tissues [39]. Blautia species are known to produce the shortchain fatty acid (SCFA) butyrate. SCFAs suppress in ammation by regulating the immune system and in ammatory cytokines [40]. The genus Blautia is less prominent in diabetic adults, pediatric patients [41,42], and those with other diseases, such as liver cirrhosis, rectal cancer, and rheumatoid arthritis [43,44]. In contrast, Turicibacter species are associated with reduced insulin sensitivity, increased in ammation in white adipose tissue, and the induction of obesity [45]. These changes in the gut microbiome may induce early postoperative improvement in diabetes via the suppression of gut in ammation. Therefore, we recommend preoperative TU-100 treatment to facilitate postoperative care in metabolic surgery.
The present study has several limitations. First, we used non-obese diabetic rats in this study. These rats were used to ensure that there was no in uence of body weight. In a future study, diet-induced obese rats will be used. Second, the direct relationships among TU-100, the gut microbiome, and in ammation remain unclear. To address this, the supplementation of Bi dobacterium and Blautia will be performed in a future study.
In conclusion, DJB suppressed blood glucose in the early postoperative period. TU-100 may enhance the anti-diabetic effect of metabolic surgery by modifying the gut microbiome and suppressing in ammation during the early postoperative period.
Declarations ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.

Additional information
Competing interest statement Mitsuo Shimada received a research grant from Tsumura & Co.
Mitsue Nishiyama and Makoto Zushi are employed by Tsumura & Co.
Other authors declare no potential con ict of interest for this article.

Data availability statement
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.  Figure 1 Experimental design for the sham and TU-100 groups.  Experimental design in the DJB and TU-100 treated DJB groups.