Contributed equally
Crohn's disease (CD) is a chronic relapsing form of inflammatory bowel disease, and its pathogenesis remains unknown. Total flavone of
Crohn's disease (CD) is a chronic relapsing form of inflammatory bowel disease, which is typically characterized by transmural inflammation, lymphangiectasia, and lymphatic and fibrous tissue hyperplasia (
In CD, inflammatory cytokines, including interleukin (IL)-6, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), are produced by infiltrating cells and macrophages, which serve an important role in colonic tissue destruction (
Our study demonstrated that TFA could ameliorate the inflammatory response in mice with TNBS-induced colitis by inhibiting the NF-κB and MAPK signaling pathways. Therefore, the present study proposed that TFA may inhibit the pathogenesis of CD via the anti-inflammatory properties of TFA. These findings may provide insight into the function of TFA and its application in the treatment of CD.
Flowers of
A total of eight standards (purity >98%) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. TFA was examined using a Waters 2694 series HPLC instrument (Waters Corporation). The sample was separated on a C18 column (4.6×250 mm, 5
A total of 60 female BALB/c mice (6-8 weeks old) with a body weight of 18-22 g were provided by Nanjing Medical University. All mice were housed in standard animal cages under specific pathogen-free conditions. The housing conditions were maintained at 22-23°C, with a 12-h light/dark cycle; mice had
For application of 2,4,6-trinitrobenzene sulfonic acid (TNBS), colitis was induced via intracolonic administration of TNBS. Briefly, 150 mg/kg TNBS in 48% ethanol was administered once every 7 days for a total of four treatments, while the normal group received sterile saline (n=10). A catheter was inserted into the colonic cavity for 4 cm, in which the TNBS solution was discharged, and the animal was held in the Trendelenburg position for 2 min to ensure contact with the intestinal mucosa.
Mice were randomly assigned to six treatment groups (n=10), including the control (distilled sterile saline only), TNBS, positive drug salazosulfapyridine (SASP), 125 mg/kg TFA, 250 mg/kg TFA and 500 mg/kg TFA treatment groups. Details of treatment were presented in
During the experiment, body weight, stool features, and fecal occult blood were recorded daily. The disease activity index (DAI) was calculated by scoring weight loss, stool features and fecal occult blood based on a previously described scoring system (
Colonic segments were excised and washed in PBS, fixed in 4% formaldehyde for 30 min at room temperature, embedded in paraffin and sectioned (5
Colonic tissues were cut into small pieces and homogenized on ice with normal saline. The levels of MPO were determined using commercial assay kits (Alpha Diagnostic International). Briefly, colon tissues were weighed, cut into fine pieces, and mixed with 200
The RAW264.7 cell line was purchased from the Shanghai Cell Bank of Chinese Academy of Sciences. RAW264.7 cells were cultured in Dulbecco's Modified Eagles medium (Gibco; Thermo Fisher Scientific, Inc.) with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) in a humidified 5% CO2 atmosphere at 37°C.
RAW264.7 cells (1.0×104/well) were seeded in 96-well plates and incubated for 24 h at 37°C. Then, LPS (Gibco; Thermo Fisher Scientific, Inc.) in the presence or absence of various doses of TFA (aforementioned) were added into cells. After 24 h of culture, 10
Colon tissues were cut and weighed. The samples were lysed and homogenized using a glass homogenizer. RAW264.7 cells were treated with LPS in the presence or absence of various doses of TFA. Following lysis, the samples were centrifuged at 10,000 × g for 5 min at 4°C to obtain the supernatant. The supernatants of RAW 264.7 cells were collected and centrifuged (10,000 × g, 5 min) at 4°C. The concentration of cytokines in the colonic tissues and cell supernatant were determined by ELISA for mouse TNF-α (ab208348, Abcam), IFN-γ (ab100689, Abcam), IL-6 (ab100712, Abcam), IL-1β (ab100704, Abcam), IL-12 (ab236717, Abcam), IL-17 (ab100702, Abcam) and IL-10 (ab108870, Abcam) following the manufacturer's instructions. Briefly, 100
The total protein from the cell supernatant was extracted using RIPA lysis buffer. Protein was extracted and concentration was measured with a Bicinchoninic acid Protein Assay kit (Beyotime Institute of Biotechnology, Haimen, China). Equal amounts of protein (30
GraphPad Prism 5.0 software (GraphPad Software, Inc.) was performed to analyze all data. The data were presented as the mean ± standard deviation. One-way analysis of variance was applied to compare difference between multiple groups followed by a Tukey's post-hoc test. The differences between two groups were statistically analyzed using a Student's t-test. P<0.05 was considered to indicate a statistically significant difference.
TFA mainly comprises eight flavone glycosides, which were characterized by HPLC, including quercetin-3-O-robinobioside, gossypetin-3-O-glucoside, quercetin-3′-
Initially, to determine whether TFA exhibits protective effects against colitis, the survival of mice with TNBS-induced colitis were investigated. The results demonstrated that TNBS significantly promoted mouse mortality, while the mice of the positive drug SASP or TFA groups notably promoted survival in TNBS-induced colitis (
The histological characteristics of the colon samples were evaluated by histopathological staining. The results indicated that mice maintained an integrated normal colonic structure in the control group, but mice in the TNBS-induced colitis group exhibited marked infiltration of inflammatory cells, loss of crypts, destruction of the mucosal layer and edema. In contrast, TNBS-induced colitis in mice pre-treated with TFA or SASP exhibited mild inflammation (
MPO is an enzyme expressed by neutrophils and its activity is linearly associated with the infiltration of neutrophils in inflammatory tissues (
In order to investigate the protective effects of TFA in mice with TNBS-induced colitis, sera and colon tissues were collected. The results demonstrated that TNBS significantly elevated the production of cytokines, including TNF-α, IFN-γ, IL-6, IL-1β, IL-12 and IL-17, in the sera and colon tissues compared with the control group. This was consistent with a previous report in which the levels of inflammatory cytokines, such as IFN-γ, IL-1, IL-6 and TNF-α were increased in the colon tissues of patients with CD (
Activation of the NF-B and MAPK signaling pathways has been associated with the pathogenesis of CD (
The cytotoxicity of TFA was evaluated using a CCK-8 assay. RAW264.7 cells were incubated with TFA of various concentrations (0, 5, 10, 25, 50, 75, 100, 150 and 200
To further study the effects of TFA
To provide further insight into the mechanisms of TFA, the activation of the NF-κB and MAPK signaling pathways in LPS-stimulated RAW264.7 macrophages was evaluated by western blotting. The results showed that the expression levels of p-IKKα/β/γ, IκBα and p100 were significantly increased by LPS treatment, but were downregulated by TFA in a dose-dependent manner. Additionally, SASP and TFA significantly promoted the expression of p-p65 and p52 in RAW264.7 macrophages in a dose-dependent manner, which was reversed by LPS (
CD is an intestinal inflammatory disease, which can occur in any region of the gastrointestinal tract, particularly the terminal ileum and right colon (
Traditional Chinese medicine has a history of thousands of years and has made notable contributions to human health (
CD is characterized by T cell activation and inflammatory cell aggregation in the mucosa (
The NF-κB signaling pathway is a predominant pathway involved in the regulation of immune and inflammatory responses (
In summary, TFA notably attenuated colon damage and inflammation associated with TNBS-colitis. Our findings indicated the protective effects of TFA on colon health, possibly via inhibition of macrophages by suppression of the NF-κB and MAPK signaling pathways. The results of the present study may provide a basis for the development of novel therapeutic approaches with TFA in treating patients CD.
The present study was supported by the National Natural Science Foundation of China (grant no. 81573978). This study was also supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions and Jiangsu Province Special Program of Medical Science (grant no. BL2014100) and by the Peak Academic Talents plan (grant no. BRA2017536) of the Jiangsu Province Hospital of Chinese Medicine.
All data generated or analyzed during this study are included in this published article.
DZ, PZ, YGC made substantial contributions to the design of the present study. YL, YS, JYZ, FJ, TC and BLY performed the experiments.. DZ and YGC wrote the manuscript. All authors read and approved the final manuscript.
The animal experiments were approved by the Institutional Ethics Committee of Nanjing University of Chinese Medicine.
Not applicable.
The authors declare that they have no competing interests.
Not applicable.
Chromatographic analyses of standards materials to TFA by high-pressure liquid chromatography. (A) HPLC chromatograms of standards (1, quercetin-3-O-robinobioside, 2, gossypetin-3-O-glucoside, 3, quercetin-3′-O-glucoside, 4, isoquercetin, 5, hyperoside, 6, myricetin, 7, gossypetin and 8, quercetin) and (B) TFA. TFA, total flavone of
Chemical structures. 1, Quercetin-3-
TFA ameliorates the progression TNBS-induced colitis in mice. (A) The survival of mice was measured. (B) Body weight loss was evaluated following TNBS-induced colitis. (C) A representative view of colon morphology. (D) The disease activity indicator scores were counted. Each experiment was performed in triplicate and results are expressed as the mean ± standard deviation (n=3). #P<0.05 vs. control group, *P<0.05 vs. TNBS group. TFA, total flavone of
TFA improves histopathological alterations in TNBS-induced colitis. (A) Paraffin embedded colon sections were stained with hematoxylin and eosin the assessment of epithelial damage of colitis mice. Images (magnification, ×200 and 400) of the colon of mice in different groups were collected. The colons of mice in the control group exhibited a normal structure without damage; however, in the TNBS model group, the colon exhibited glandular defects, mucosal ulcerations and inflammatory cell infiltration, but these alterations were attenuated to varying degrees by treatment with 125, 250 and 500 mg/kg TFA or SASP. Arrows indicate the aforementioned features observed in each group. (B) The levels of MPO activity in colon tissues were evaluated. #P<0.05 vs. control group, *P<0.05 vs. TNBS group. Each experiment was performed in triplicate. TFA, total flavone of
TFA suppresses the production of inflammation cytokines in TNBS-induced colitis. ELISAs were performed to detect the production of cytokines, including TNF-α, IFN-γ, IL-6, IL-1β, IL-12 and IL-17, in the sera and colon tissues. #P<0.05 vs. control group, *P<0.05 vs. TNBS group. Each experiment was performed in triplicate. IL, interleukin; SASP, salazosulfapyridine; TFA, total flavone of
TFA inhibits the activation of the NF-κB and MAPK signaling pathways in TNBS-induced colitis. (A) Western blotting was conducted to determine the expression of related-proteins of the NF-κB signaling pathway in colon tissues. Quantification of protein expression in each group was presented. (B) The expression of related-proteins of the MAPK signaling pathway was evaluated by western blotting. Quantification of protein expression in each group was presented. #P<0.05 vs. control group, *P<0.05 vs. TNBS group. ERK, extracellular signal-regulated kinase; IKK, IκBα kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-κB; p, phosphorylated; SASP, salazosulfapyridine; TFA, total flavone of
TFA decreases the production of inflammatory cytokines in LPS-induced macrophage RAW264.7 cells. (A) RAW264.7 cells were incubated with TFA of various concentrations (0, 5, 10, 25, 50, 75, 100, 150 and 200
TFA suppresses the NF-κB and MAPK signaling pathways in LPS-stimulated RAW264.7 cells. (A) Western blotting was adopted to determine the expression of related-proteins of the NF-κB signaling pathway in LPS-stimulated RAW264.7 cells. Quantification of expression levels of proteins in each group was presented. (B) The expression levels of related-proteins of the MAPK signaling pathway in LPS-stimulated RAW264.7 cells were evaluated by western blotting. Quantification of expression levels of proteins in each group was presented. #P<0.05 vs. control group, *P<0.05 vs. TNBS group. ERK, extracellular signal-regulated kinase; IKK, IκBα kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-κB; p, phosphorylated; SASP, salazosulfapyridine; TFA, total flavone of
Treatment in different groups.
Groups | Treatment |
---|---|
Control | Sterile saline/day for a total of 28 days |
TNBS | TNBS enema/7 days for a total of four treatments + sterile saline/days for a total of 28 days |
SASP | TNBS enema/7 days for a total of four treatments + SASP/d for a total of 28 days |
125 mg/kg TFA | TNBS enema/7 days for a total of four treatments + 125 mg/kg TFA/day for a total of 28 days |
250 mg/kg TFA | TNBS enema/7 days for a total of four treatments + 250 mg/kg TFA/day for a total of 28 days |
500 mg/kg TFA | TNBS enema/7 day for a total of four treatments + 500 mg/kg TFA/day for a total of 28 days |
SASP, Salazosulfapyridine; TFA, total flavone of
Scoring of disease activity index.
Score | Body weight loss (%) | Stool feature | Fecal occult blood |
---|---|---|---|
0 | 0 | Normal formed | Negative |
1 | 1-5 | ||
2 | 5-10 | Loose stool | Positive |
3 | 10-20 | ||
4 | >20 | Diarrhea | Gross bleeding |
Adapted from ref. (