Desmethylbellidifolin Attenuates Dextran Sulfate Sodium-Induced Colitis: Impact on Intestinal Barrier, Intestinal Inflammation and Gut Microbiota

• Abstract
• Introduction
• Results
• Discussion
• Materials and Methods
• Reagents
• Animals and treatments
• Disease activity index and colon length assessment
• In vivo intestinal permeability assessment
• Hematoxylin and eosin staining and histological analysis
• Cell co-culture and trans-epithelial electric resistance measurement
• In Vitro barrier permeability assay
• Immunofluorescence assay of F4/80 and claudin-2
• Enzyme-linked immunosorbent assay analysis
• Quantitative real-time PCR analysis
• Western blot
• Microbiota analysis
• Statistical analysis
• Contributorsʼ Statement
• References

Abstract

Ulcerative colitis has been recognized as a chronic inflammatory disease predominantly disturbing the colon and rectum. Clinically, the aminosalicylates, steroids, immunosuppressants, and biological drugs are generally used for the treatment of ulcerative colitis at different stages of disease progression. However, the therapeutic efficacy of these drugs does not satisfy the patients due to the frequent drug resistance. Herein, we reported the anti-ulcerative colitis activity of desmethylbellidifolin, a xanthone isolated from Gentianella acuta, in dextran sulfate sodium-induced colitis in mice. C57BL/6 mice were treated with 2% dextran sulfate sodium in drinking water to induce acute colitis. Desmethylbellidifolin or balsalazide sodium was orally administrated once a day. Biological samples were collected for immunohistological analysis, intestinal barrier function evaluation, cytokine measurement, and gut microbiota analysis. The results revealed that desmethylbellidifolin alleviated colon shortening and body weight loss in dextran sulfate sodium-induced mice. The disease activity index was also lowered by desmethylbellidifolin after 9 days of treatment. Furthermore, desmethylbellidifolin remarkably ameliorated colonic inflammation through suppressing the expression of interleukin-6 and tumor necrosis factor-α. The intestinal epithelial barrier was strengthened by desmethylbellidifolin through increasing levels of occludin, ZO-1, and claudins. In addition, desmethylbellidifolin modulated the gut dysbiosis induced by dextran sulfate sodium. These findings suggested that desmethylbellidifolin effectively improved experimental ulcerative colitis, at least partly, through maintaining intestinal barrier integrity, inhibiting proinflammatory cytokines, and modulating dysregulated gut microbiota.

Introduction

UC is recognized as a chronic inflammatory disease primarily disturbing the colon and rectum, typically manifested by its long duration and recurrent inflammation. Clinically, the symptoms of UC mainly encompass abdominal pain, diarrhea, bloody stool, fever, and weight loss [1]. Patients suffering from chronic recurrent UC have a higher risk of developing colorectal cancer in contrast to normal cohorts [2]. According to an epidemiological report in the 20th century, UC was mainly prevalent in industrialized nations such as Europe and North America. However, a recent study shows that the incidence of IBD plateaued in developed countries at the turn of 21st century, while the morbidity of IBD in the countries of Asia and South America is beginning to show a rise [3]. According to the data reported by the Chinese Center for Disease Control and Prevention, 350 000 IBD patients have been diagnosed in Chinese cohorts from 2005 to 2014. By 2025, the total number of IBD cases is estimated to reach 1.5 million [4]. The pathogenesis of UC is subjected to complexity and not well understood. Accumulating evidence has suggested that several factors are associated with UC. Among which are injury of the intestinal barrier, inflammatory responses, and dysbiosis of gut microbiota [5]. Currently, 5-aminosalicylic acid, steroid hormones, immunomodulators, and monoclonal antibodies are conventionally employed to treat UC patients [1]. However, long-term administration of these drugs is prone to induce various side effects, such as drug resistance, hepatorenal toxicity, and allergies [6], which likely dampen the beneficial impact on the management of UC patients.

Gentianella acuta (Michx.) Hulten belongs to the family Gentianaceae [7]. Our previous study has demonstrated that one of its main components, DMB (also known as 1,3,5,8-tetrahydroxyxanthone) ([Fig. 1]), has therapeutic effects on TNBS-induced UC in rats in that DMB suppressed colonic inflammation by decreasing the expression of myeloperoxidase, nitric oxide, IL-6, and TNF-α in vivo and in vitro. Additionally, DMB significantly ameliorated UC-induced diarrhea by alleviating colon muscle spasms [8]. However, the effects of DMB on repairing the epithelium barrier damage and maintaining the homeostasis of gut flora are unknown, and the underlying molecular mechanisms remain to be explored. In this study, we employed a DSS-induced mouse colitis model to interrogate the activity of DMB on epithelium barrier integrity, inflammatory response, and microbiota homeostasis, and attempted to delineate the molecular mechanisms in terms of the therapeutic effects of DMB on UC.

Results

The main clinical manifestations of UC are body weight loss, diarrhea, and bloody stool. These symptoms are also typical characteristics of DSS-induced colitis in mice. As shown in [Fig. 2 a], the body weight of mice in the control group increased gradually, while the body weight of mice in the DSS-treated group decreased in comparison. On the contrary, the body weight of mice in the DMB- or BST-treated group recovered from day 8 to day 10. The DAI was applied to assess the severity of colitis. As shown in [Fig. 2 b], the DAI score was significantly increased in the DSS-treated group compared with the control group. In contrast, the DAI score was markedly decreased after DMB or BST treatment. Furthermore, DMB and BST significantly restored the DSS-caused colon shortening ([Fig. 2 c, d]). These results indicate that DMB has a protective effect on DSS-treated mice.

The damage of the intestinal barrier has been associated with the development of UC [9]. The severity of colonic epithelial injury was examined by H&E staining ([Fig. 3 a]). The colon from the control group showed integrated morphology and regularly arranged goblet cells. However, ulcers were observed in the colon from the DSS-treated group. Moreover, the colon from the DSS-treated group exhibited serious infiltration of neutrophils and a decrease of goblet cells. After treatment with DMB, the intestinal ulcer was ameliorated, neutrophil infiltration was blocked, and the number of goblet cells was recovered, which is similar with the observations in the BST group.

The change of intestinal permeability was examined by measuring the transportation of FD-4 in vivo and in vitro. As shown in [Fig. 3 b], the amount of FD-4 that permeated into the plasma was much greater in the DSS-treated group than the control group. However, the permeation of FD-4 was decreased by DMB or BST treatment compared with the DSS group. Moreover, the therapeutic effect of DMB on epithelial barrier integrity was assessed in a RAW 264.7 and Caco-2 cell co-culture system. Compared with the control group, LPS significantly increased the permeability of FD-4 from the apical side to the basolateral compartment. DMB or BST treatment remarkably suppressed the flux of FD-4 ([Fig. 3 c]). TEER measurements exhibited similar results. Compared with the control group, a significant decrease of TEER was observed in the LPS-treated group, while DMB or BST treatment reversed the reduction of TEER ([Fig. 3 d]).

The tight junction proteins play key roles in maintaining the integrity of the intestinal barrier. To evaluate the potential effect of DMB on tight junction proteins, the mRNA levels of ZO1, occluding, and claudin-2 were measured by RT-PCR. As shown in [Fig. 4 a – c], the expressions of ZO1, occludin, and claudin-2 were significantly suppressed in the DSS-treated group compared with the control group. However, treatment with DMB or BST markedly reversed the levels of these tight junction proteins. Western blot analysis demonstrated similar results. As shown in the [Fig. 4 d – f], the protein levels of ZO1 and occludin were significantly suppressed by DSS treatment compared with the control group, while the DMB and BST group were shown to significantly upregulate the expressions of these two proteins compared with the DSS-treated group. Immunofluorescence analysis demonstrated that DMB or BST treatment remarkably increased the expression and distribution of claudin-2 compared with the DSS-treated group ([Fig. 4 g]). Moreover, as apoptosis of colonic epithelial cells was also regarded as a risk factor of intestinal barrier damage, we further measured the proliferation of the colonic cells. As shown in [Fig. 4 g], the downregulation of Ki67 was observed in the DSS-treated group. By contrast, DMB or BST treatment markedly increased the expression of Ki67. Taken together, these results indicated that DMB might prevent the DSS-induced injury of colonic epithelial integrity by maintaining tight junction expression and improving proliferation of colonocytes.

Evidence has shown that the circulating levels of TNF-α and IL-6 were increased in IBD patients compared with healthy subjects, while blockade of the proinflammatory function of TNF-α and IL-6 was suggested as an effective way to alleviate colitis [10], [11]. For these reasons, we examined the levels of TNF-α and IL-6 in the serum and colonic tissues of mice. As shown in [Fig. 5 a, b], the concentrations of TNF-α and IL-6 in the serum of DSS-treated mice were significantly increased compared with the control group. However, DMB or BST treatment markedly decreased the levels of these two proinflammatory factors. The protein levels of TNF-α and IL-6 were further detected in the colonic tissue of mice. The results demonstrated that the expressions of TNF-α and IL-6 were dramatically increased in the DSS-treated group compared with the control group, and DMB significantly suppressed the production of TNF-α and IL-6 in colonic tissue ([Fig. 5 c, d]). The representative picture of Western blot is exhibited in [Fig. 5 e]. In addition, we further detected the expression of macrophage marker F4/80, as it has been reported that macrophages were recruited to lamina propria under intestinal inflammatory conditions, and macrophages were the main producers of TNF-α [12]. As shown in [Fig. 5 f], the DSS-treated group exhibited a higher level of F4/80 in colonic tissue compared with the control group. DMB and BST treatment decreased the level of F4/80 compared with the DSS-treated group. All these results demonstrated that DMB might ameliorate the DSS-induced inflammatory response through suppressing the expression of proinflammatory factors and reduce macrophage aggregates.

Dysbiosis of gut microbiota has been regarded as an etiological factor contributing to the development of UC [13]. The imbalance of the intestinal microbiota will damage the health of the host and result in several malfunctions, including the malabsorption of nutrients, dysregulation of intestinal immune response, hyperactivation of inflammatory responses, and destruction of the intestinal barrier [14]. In order to investigate whether DMB could regulate homeostasis of the gut microbial community, we carried out 16S RNA analysis. As shown in [Fig. 6 a], Binary Jaccard distance was employed to distinguish microbiota profiles between different groups. The dots of the DSS, DMB, or BST group were clearly separated from the control group. Moreover, the trend of the DMB group was different from the DSS and BST groups. These indicate that the microbiota composition was changed by both DSS induction and DMB or BST treatment, and the microbiota composition of the DMB group was different from the DSS and BST groups. LefSe analysis was applied to identify significantly changed bacteria among different groups. The linear discriminant analysis (LDA) score threshold was set at 4. As shown in [Fig. 6 b], seven kinds of bacteria were affected at different levels in the DSS-treated group, among which Bacteroidaceae was ranked as the most significantly changed bacteria at the family and genus levels. Therefore, we further analyzed the abundance of Bacteroidaceae in our study groups. As shown in [Fig. 6 c], the relative abundance of Bacteroidaceae was significantly increased in the DSS-treated group compared with the control, while DMB treatment significantly decreased the relative abundance. Taken together, these results indicate that the microbiota was dysregulated in DSS-induced colitis in mice. DMB treatment could regulate the homeostasis of intestinal microbiota, especially the abundance of Bacteroidaceae.

Discussion

In this study, we demonstrated that DMB could improve the symptoms of UC in DSS-induced colitis in mice and presented a strong potential to repair the damaged intestinal barrier and suppress the inflammatory response. We also observed that DMB could effectively recover the aberrantly altered composition of gut microbiota in DSS-induced colitis in mice. These findings strongly indicate that DMB has emerged as a credible lead to be developed into novel anti-UC drugs.

DMB, as a natural xanthone, is purified from G. acuta. It is a xanthone that is widely distributed in plants, and possesses a variety of biological activities, such as anticancer, anti-inflammation, antioxidant, and antibacterial [15], [16], [17]. Recently, several studies have shown that xanthones also have the potential to ameliorate digestive diseases. For instance, Lu et al. reported that four xanthones isolated from Gentianopsis paludosa displayed anti-fibrotic activity on UC-induced colon fibrosis in rats [18]. Dou et al. reported mangiferin, a xanthone that mainly exists in the fruits, leaves, roots, and bark of the mango tree, could suppress intestinal inflammatory responses through inhibiting NF-κB and MAPK signaling, thus decreasing the expression of proinflammatory cytokines [19]. In line with these findings, our study showed that DMB strikingly diminished the expressions of TNF-α and IL-6 and blocked the infiltration of neutrophils and macrophages into the colonic tissue. Additionally, in our previous study, we also found that DMB could inhibit the expressions of nitric oxide synthase, IL-6, TNF-α, and cyclooxygenase-2, as well as alleviate colon muscle spasm in TNBS-induced colitis [8].

TNBS- and DSS-induced colitis are well-established murine models of intestinal inflammation that recapitulate the human UC state. Both of these models exhibited diarrhea, bloody stool, significant body weight loss, reduced mobility, and a shortened colon [20]. Nevertheless, the differences have been well documented between these two animal models. TNBS is administrated via the intrarectal way combined with ethanol to induce the immune activity of T cells and impair the intestinal barrier [21], while there is little ethanol remaining in the large intestine under physiological conditions [22]. As such, this model might not perfectly represent the intestinal barrier destruction caused by harmful substances in human beings. DSS-induced colitis is generated by the oral administration of DSS dissolved in drinking water at concentrations of 2 – 10% [23]. Studies have reported that DSS is directly toxic to the intestinal barrier, which further increases the penetration of bacteria to the epithelium and triggers intestinal inflammation [24]. The DSS model is therefore more appropriate for evaluating the integrity of the intestinal barrier under disease conditions. For these reasons, the DSS-induced colitis model is more eligible to this study to evaluate the protective effects of DMB on the intestinal barrier. Indeed, our results showed that DSS increased the permeability of the intestinal barrier. By using this UC animal model, we observed that DMB was able to restore intestinal barrier function by decreasing the permeability, upregulating the expressions of tight junction proteins, and reducing intestinal epithelial cell apoptosis. Moreover, the in vitro study on the cell co-culture model also showed that DMB could effectively maintain the integrity of the intestinal barrier.

Dysregulation of the gut microbiota has been well noted in UC patients [25]. Prolonged dysbiosis of the gut microbiota is a key risk factor of UC, since the aberrant colonized microbiota could lead to malfunction of the host immune system [26]. In this context, we further measured the change of gut microbiota upon exposure of the mice to DSS and DMB. We observed a great difference in the composition of the gut microbiota between the control and DSS group, which was consistence with the previous report from Guo et al. [27]. Administration of DMB changed the dysbiosis of the microbiota induced by DSS. However, the mechanisms by which DMB altered the species composition of the gut microbiota are difficult to be clarified. We also observed an increased abundance of Bacteroidaceae in DSS-induced colitis in mice, while DMB treatment corrected the aberrant increased bacteria. In line with our observations, clinical findings have revealed that the abundance of Bacteroidaceae was markedly increased in patients with UC [28]. Bacteroidaceae is a family of anaerobic, gram-negative bacteria, which is classified into the genera of Bacteroides, Fusobacterium, and Leptotrichia [29]. Studies have revealed that Bacteroides and Fusobacterium predominantly reside in the lower intestinal tract, while Leptotrichia resides in the oral cavity [30], [31]. We have found a striking difference of Bacteroides in DSS-treated mice ([Fig. 6 b]), which was similar with the observations reported by Okayasu et al. and Hu et al. [32], [33]. Moreover, it has been reported that mice transplanted with Bacteroides fragilis could develop persistent colitis [34]. Fusobacterium has also been found to be enriched in the feces isolated from patients with IBD, and administration of Fusobacterium could induce colitis in mice [35]. However, we did not observe changes of Fusobacterium in our study. This might be due to the composition of the intestinal microbiota being subjected to a complexity of factors, such as species, gender, diet, state of disease, and so on.

In summary, our study has shown that DMB significantly ameliorates DSS-induced UC in mice, mainly through enhancing the intestinal barrier, suppressing the expression of proinflammatory cytokines, and regulating homeostasis of the gut microbiota, highlighting the potential of DMB to be developed into novel anti-UC drugs derived from natural products.

Materials and Methods

Reagents

The whole plants of G. acuta were collected from the Alxa Youqi, Inner Mongolia Autonomous region of China. They were identified by Dr. Li Tianxiang (Experiment Teaching Department, Tianjin University of Traditional Chinese Medicine). A voucher specimen was deposited at the Academy of Traditional Chinese Medicine of Tianjin University of TCM (voucher number: 02 293 698).

DMB was prepared as previously described [36]. Briefly, G. acuta was cut and refluxed with 70% ethanol. Then, the extracts were partitioned in a CHCl₃-H₂O mixture (1 : 1, v/v). The H₂O layer was separated by preparative HPLC after being isolated by D101 macroporous resin CC and SiO₂ gel CC. DSS was purchased from Meilun Biotechnology Co., Ltd. BST was obtained from Zhendong Anter Biological Pharmaceutical Co., Ltd. The fecal occult blood qualitative detection kit was purchased from Yuanye Bio-Technology Co., Ltd. FD-4 and LPS were purchased from Sigma. ELISA kits for mouse TNF-α and IL-6 were obtained from Elabscience Biotechnology Co., Ltd.

Animals and treatments

Male C57BL/6 (20 – 25 g) mice were purchased from Charles River Co. Ltd. The animals were housed under standard room temperature (22 ± 2 °C), humidity (40 – 60%), and photoperiods (12-h/12-h light-dark cycle). They were fed with a standard rodent diet and filtered water ad libitum. All animal experiments were approved by the Science and Technological Committee and the Animal Use and Care Committee of Tianjin University of Traditional Chinese Medicine (December 1, 2017, No. 201 712 003).

After 1 week of acclimatization, the mice were randomly divided into 4 groups: control group, DSS group, DSS plus BST (1 g/kg) group, and DSS plus DMB (20 mg/kg) group. BST was used as a positive drug. Except for the control group, colitis was induced by adding 2% (w/v) DSS to drinking water for 9 days in the other 3 groups. The BST treatment group and DMB treatment group were orally administered with BST (1 g/kg) and DMB (20 mg/kg) once a day for 9 consecutive days. On day 10, all of the mice were given FD-4 saline solution (600 mg/kg) by gavage, protected from light, and fasted for 4 h.

Disease activity index and colon length assessment

Body weight, stool consistency, and rectal bleeding were recorded every day during the induction of colitis in mice. DAI scores were determined according to a reported scoring system [37]. To determine the colon length, the mice were sacrificed under anesthesia after the experiment, and the entire colon from the caecum to the anus was removed and measured.

In vivo intestinal permeability assessment

Intestinal permeability to FD-4 was determined according to the reported method, with a slight modification [38]. After the experiment treatment, all of the mice were fasted for 12 h and given 600 mg/kg FD-4 by gavage. Following FD-4 administration for 4 h, 100 µL blood were collected from the orbital sinus. Then, serum was obtained by centrifugation at 2000 g for 10 min at 4 °C. The fluorescence intensity of FD-4 in serum was measured with SpectraMax M5 at an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

Hematoxylin and eosin staining and histological analysis

The colon tissue near the cecum was dissected by 1 cm, fixed with 4% (w/v) paraformaldehyde for 24 h at 4 °C, and dehydrated with ethanol. Then, the tissue was embedded in paraffin and cut into slices with 4 µm thickness. After that, the slices were stained with H&E and observed for intestinal histological changes.

Cell co-culture and trans-epithelial electric resistance measurement

Human colon epithelial cell line Caco-2 and mouse monocyte macrophage cell line RAW 264.7 were obtained from the cell center at the Chinese Academy of Medical Science and Peking Union Medical College. The cells were both cultured in DMEM (Hyclone) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin-streptomycin (Gibco). The cells were maintained in a humidified incubator filled with 5% CO₂ and 95% air at 37 °C. The medium was refreshed every 2 days and the cells were passaged when they reach 80% confluence.

TEER measurements were performed as previously described [39]. Caco-2 cells were seeded on a Transwell membrane (6.5 mm polycarbonate membrane, 3 µm pore size; Corning) at a density of 1.5 × 105 cells/mL. The medium was refreshed every other day until a confluent monolayer was obtained after 10 days from seeding. RAW 264.7 cells were seeded in the lower chamber of the Transwell at a density of 2 × 10⁶ cells/mL. After a 2-day incubation, RAW 264.7 cells were stimulated with 1 µg/mL LPS and treated with 10 µM DMB for 24 h. Before evaluation of the TEER (Ω/cm²), the culture medium was replaced with phenol red-free medium and cultured for another 30 min.

In Vitro barrier permeability assay

After the last measurement of TEER, FD-4 was added to the apical side of the Caco-2 monolayer at a final concentration of 1 mg/mL, then the cells were incubated at 37 °C for 30 min. After that, the quantity of FD-4 that fluxed from the apical side to the basolateral compartment was measured. Medium from the basolateral compartment was added into a black well plate, and the fluorescence intensity was measured with SpectraMax M5 at an excitation wavelength of 480 nm and an emission wavelength of 520 nm [40].

Immunofluorescence assay of F4/80 and claudin-2

Colon tissue was fixed in 4% (w/v) paraformaldehyde for 12 h, embedded in paraffin, and sectioned into slices of 10 µm thickness. The sectioned samples were incubated with rabbit-anti-F4/80 (1 : 100), rabbit-anti-claudin-2 (1 : 100), or rabbit-anti-Ki67 for 12 h at 4 °C, followed by washing with PBS 3 times. The samples were then incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Santa Cruz) for 1 h in the dark. Nuclei were stained with DAPI. Images of F4/80, claudin-2, and Ki67 stain were acquired by an Axio Imager 2 (Zeiss) at a 200× magnification.

Enzyme-linked immunosorbent assay analysis

Serum samples were collected from the orbital sinus of the mice, centrifuged at 2000 g for 10 min at 4 °C. The cytokines TNF-α and IL-6 in the serum samples were measured with commercially available enzyme-linked immunosorbent assay kits (Elabscience Biotechnology Co., Ltd.) according to the manufacturerʼs instructions.

Quantitative real-time PCR analysis

Total RNA of the colon tissue was extracted using TRIzol reagent (Invitrogen). Revers transcription of the mRNA was performed with a high-capacity cDNA reverse transcription kit (Applied Biosystems). PCR reactions were performed using the 7500 Real-Time PCR System (Applied Biosystems) with an SYBR Green PCR Master Mix kit (Applied Biosystems), cDNA template, and relative primers. GAPDH was amplified as an internal control. The relative expressions of the genes were analyzed according to the 2^−ΔΔCt method. All sequences of the primers are listed in [Table 1].

Western blot

Colon tissues were homogenized in ice-cold RIPA lysis buffer supplemented with 1X protease inhibitor (Roche) and 1X phosphatase inhibitor (Roche). The supernatant was collected by centrifugation at 12 000 g for 10 min at 4 °C and the protein quantity was measured with a DC Protein Assay kit (BIO-RAD). Thirty micrograms of protein were loaded on 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to a PVDF membrane (Millipore). After blocking with 5% skimmed milk, the membranes were incubated with primary antibodies against claudin-2 (1 : 1000, ab53032; Abcam), occludin (1 : 5000, ab167161; Abcam), ZO-1 (1 : 1000, ab221574; Abcam), TNF-α (1 : 500; Proteintech Group), IL-6 (1 : 500; Proteintech Group), and β-actin (1 : 1000, ab5694; Abcam). The membranes were washed with Tris-buffered saline/Tween 20 4 times and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Finally, the chemiluminescent signals were detected by an ECL detection kit (Millpore) and analyzed with a protein MP imaging system (Bio Rad). The intensities of the bands were quantified by Image J software. β-Actin was used as the loading control.

Microbiota analysis

Stool samples were collected from each mouse at the end of study and were chosen at random for 16S rRNA sequencing. Microbiota analysis and bioinformatics analysis were performed by Novogene Co., Ltd.

Microbial DNA was extracted using the CTAB method. The concentration and purity of the DNA was monitored on 1% agarose gel. The total DNA was diluted to 1 ng/µL using sterile water before further analysis. The 16S rRNA V3 – V4 hypervariable region was amplified using the forward primer 341F (CCTAYGGGRBGCASCAG) and reverse primer 806R (GGACTACNNGGGTATCTAAT) with a barcode. The PCR reactions were performed using a Mastercycler Gradient (Eppendorf) with 15 µL of Phusion High-Fidelity PCR Master Mix (New England Biolabs), 0.2 µM forward and reverse primers, and 10 ng template DNA. The reaction was carried out under the following conditions: initial denaturation at 98 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, elongation at 72 °C for 30 s, and a final extension at 72 °C for 5 min. The purification of the PCR products was performed using a GeneJET Gel Extraction Kit (Thermo Scientific). The sequencing libraries were generated using Ion Plus Fragment Library Kit 48 rxns (Thermo Scientific) according to the manufacturerʼs recommendations. The library quality was assessed on a Qubit@ 2.0 fluorometer (Thermo Scientific). Finally, the library was sequenced on an Ion S5 XL platform.

The raw sequencing data were filtered to obtain high-quality clean reads according to the Cutadapt (V1.9.1, http://cutadapt.readthedocs.io/en/stable/) quality-controlled process. The chimera sequences were removed using the UCHIME algorithm (UCHIME Algorithm, http://www.drive5.com/usearch/manual/uchime_algo.html). The sequences with ≥ 97% similarity were assigned to the same OTUs using Uparse software (Uparse v7.0.1001, http://drive5.com/uparse/).

Statistical analysis

Statistical analyses were performed with SPSS software version 11.5. One-way ANOVA was employed for data analysis. All data are presented as the mean ± standard error of the mean (SEM). P values ≤ 0.05 were considered statistically significant.

Contributorsʼ Statement

Data collection: Wu J, Wu Y, Chen Y, Zhang Y, Liu M, Wang T; design of the study: Wang T, Zhang Y, Wu Y, Liu M, Yu H; statistical analysis: Wu J, Chen Y, Yu H; analysis and interpretation of the data: Wang T, Zhang Y, Liu M, Chen Y, Yu H; drafting the manuscript: Wu Y, Wu J, Yu H, Wang T; critical revision of the manuscript: Zhang Y, Wu Y, Wu J, Chen Y, Liu M.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 82 074 118), Important Drug Development Fund, Ministry of Science and Technology of China (2018ZX09735-002). We are very grateful to Dan Wang for her advice on microbiota analysis. Our thanks also go to Qian Chen and Ruixia Bao for their effort in collecting medical herbs and for their advice on statistical analysis.

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• 27 Guo SS, Geng WY, Chen S, Wang L, Rong XL, Wang SC, Wang TF, Xiong LY, Huang JH, Pang XB, Lu YM. Ginger alleviates DSS-induced ulcerative colitis severity by improving the diversity and function of gut microbiota. Front Pharmacol 2021; 12: 632569
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• 30 Hofstad T. Serological responses to antigens of bacteroidaceae. Microbiol Rev 1979; 43: 103-115
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• 31 Eribe ERK, Olsen I. Leptotrichia species in human infections II. J Oral Microbiol 2017; 9: 1368848
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• 32 Okayasu I, Hatakeyama S, Yamada M, Ohkusa T, Inagaki Y, Nakaya R. A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology 1990; 98: 694-702
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• 36 Liu Y, Ni Y, Ruan J, Qu L, Yu H, Han L, Zhang Y, Wang T. Bioactive gentixanthone and gentichromone from the whole plants of Gentianella acuta (Michx.) Hulten. Fitoterapia 2016; 113: 164-169
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Correspondence

Publication History

Received: 29 January 2021

Accepted after revision: 01 May 2021

Article published online:
07 June 2021

© 2021. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 26 Mishima Y, Sartor RB. Manipulating resident microbiota to enhance regulatory immune function to treat inflammatory bowel diseases. J Gastroenterol 2020; 55: 4-14
  CrossrefPubMedSearch in Google Scholar
• 27 Guo SS, Geng WY, Chen S, Wang L, Rong XL, Wang SC, Wang TF, Xiong LY, Huang JH, Pang XB, Lu YM. Ginger alleviates DSS-induced ulcerative colitis severity by improving the diversity and function of gut microbiota. Front Pharmacol 2021; 12: 632569
  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 29 Lambe jr. DW. Serology of Bacteroidaceae. In: Lambe jr. DW, Genco RJ, Mayberry-Carson KL. eds. Anaerobic Bacteria. Boston: Springer; 1980: 141-153
  Search in Google Scholar
• 30 Hofstad T. Serological responses to antigens of bacteroidaceae. Microbiol Rev 1979; 43: 103-115
  CrossrefPubMedSearch in Google Scholar
• 31 Eribe ERK, Olsen I. Leptotrichia species in human infections II. J Oral Microbiol 2017; 9: 1368848
  CrossrefPubMedSearch in Google Scholar
• 32 Okayasu I, Hatakeyama S, Yamada M, Ohkusa T, Inagaki Y, Nakaya R. A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology 1990; 98: 694-702
  CrossrefPubMedSearch in Google Scholar
• 33 Hu X, Xu N, Yang X, Hu X, Zheng Y, Zhang Q. Nigella A ameliorates inflammation and intestinal flora imbalance in DSS induced colitis mice. AMB Express 2020; 10: 179
  CrossrefPubMedSearch in Google Scholar
• 34 Rhee KJ, Wu S, Wu X, Huso DL, Karim B, Franco AA, Rabizadeh S, Golub JE, Mathews LE, Shin J, Sartor RB, Golenbock D, Hamad AR, Gan CM, Housseau F, Sears CL. Induction of persistent colitis by a human commensal, enterotoxigenic Bacteroides fragilis, in wild-type C57BL/6 mice. Infect Immun 2009; 77: 1708-1718
  CrossrefPubMedSearch in Google Scholar
• 35 Liu H, Hong XL, Sun TT, Huang XW, Wang JL, Xiong H. Fusobacterium nucleatum exacerbates colitis by damaging epithelial barriers and inducing aberrant inflammation. J Dig Dis 2020; 21: 385-398
  CrossrefPubMedSearch in Google Scholar
• 36 Liu Y, Ni Y, Ruan J, Qu L, Yu H, Han L, Zhang Y, Wang T. Bioactive gentixanthone and gentichromone from the whole plants of Gentianella acuta (Michx.) Hulten. Fitoterapia 2016; 113: 164-169
  CrossrefPubMedSearch in Google Scholar
• 37 Wirtz S, Neufert C, Weigmann B, Neurath MF. Chemically induced mouse models of intestinal inflammation. Nat Protoc 2007; 2: 541-546
  CrossrefPubMedSearch in Google Scholar
• 38 Cani PD, Possemiers S, Van de Wiele T, Guiot Y, Everard A, Rottier O, Geurts L, Naslain D, Neyrinck A, Lambert DM, Muccioli GG, Delzenne NM. Changes in gut microbiota control inflammation in obese mice through a mechanism involving GLP-2-driven improvement of gut permeability. Gut 2009; 58: 1091-1103
  CrossrefPubMedSearch in Google Scholar
• 39 He W, Liu MY, Li YM, Yu HY, Wang D, Chen Q, Chen Y, Zhang Y, Wang T. Flavonoids from Citrus aurantium ameliorate TNBS-induced ulcerative colitis through protecting colonic mucus layer integrity. Eur J Pharmacol 2019; 857: 172456
  CrossrefPubMedSearch in Google Scholar
• 40 Gori M, Altomare A, Cocca S, Solida E, Ribolsi M, Carotti S, Rainer A, Francesconi M, Morini S, Cicala M, Guarino MPL. Palmitic acid affects intestinal epithelial barrier integrity and permeability in vitro . Antioxidants (Basel) 2020; 9: 417
  CrossrefPubMedSearch in Google Scholar