OBJECTIVE: To investigate the anti-inflammatory activity of Radix Panacis quinguefolii root extract (RPQE) and its therapeutic effects on inflammatory bowel disease (IBD). METHODS: The 72-hour post-fertilization zebrafish was used to generate the local and systematic inflammation models through tail-amputation and lipopolysaccharide (LPS)-induction (100 µ g/mL), respectively. The Tg(zlyz:EGFP) zebrafish was induced with 75 µ g/mL 2,4,6-trinitrobenzene sulfonic acid (TNBS) for establishing the IBD model. The tail-amputated, LPS-, and TNBS-induced models were subjected to RPQE (ethanol fraction, 10-20 µ g/mL) administration for 12 and 24 h, respectively. Anti-inflammatory activity of RPQE was evaluated by detecting migration and aggregation of leukocytes and expression of inflammation-related genes. Meanwhile, TNBS-induced fish were immersed in 0.2% (W/V) calcein for 1.5 h and RPQE for 12 h before photographing to analyze the intestinal efflux efficiency (IEE). Moreover, the expression of inflammation-related genes in these fish was detected by quantitative polymerase chain reaction. RESULTS: Subject to RPQE administration, the migration and aggregation of leukocytes were significantly alleviated in 3 zebrafish models (P<0.01). Herein, RPQE ameliorated TNBS-induced IBD with respect to a significantly reduced number of leukocytes, improved IEE, and inhibited gene expression of pro-inflammatory factors (P<0.05 or P<0.01). CONCLUSION RPQE exhibited therapeutic effects on IBD by inhibiting inflammation.
Animals ; Zebrafish ; Lipopolysaccharides ; Disease Models, Animal ; Inflammatory Bowel Diseases/metabolism* ; Inflammation/drug therapy* ; Anti-Inflammatory Agents/therapeutic use* ; Trinitrobenzenesulfonic Acid/adverse effects* ; Colitis/drug therapy*

Owing to the increasing incidence and prevalence of inflammatory bowel disease (IBD) worldwide, effective and safe treatments for IBD are urgently needed. Hydrogen sulfide (H₂S) is an endogenous gasotransmitter and plays an important role in inflammation. To date, H₂S-releasing agents are viewed as potential anti-inflammatory drugs. The slow-releasing H₂S donor 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), known as a potent therapeutic with chemopreventive and cytoprotective properties, has received attention recently. Here, we reported its anti-inflammatory effects on dextran sodium sulfate (DSS)-induced acute (7 days) and chronic (30 days) colitis. We found that ADT-OH effectively reduced the DSS-colitis clinical score and reversed the inflammation-induced shortening of colon length. Moreover, ADT-OH reduced intestinal inflammation by suppressing the nuclear factor kappa-B pathway. In vivo and in vitro results showed that ADT-OH decreased intestinal permeability by increasing the expression of zonula occludens-1 and occludin and blocking increases in myosin II regulatory light chain phosphorylation and epithelial myosin light chain kinase protein expression levels. In addition, ADT-OH restored intestinal microbiota dysbiosis characterized by the significantly increased abundance of Muribaculaceae and Alistipes and markedly decreased abundance of Helicobacter, Mucispirillum, Parasutterella, and Desulfovibrio. Transplanting ADT-OH-modulated microbiota can alleviate DSS-induced colitis and negatively regulate the expression of local and systemic proinflammatory cytokines. Collectively, ADT-OH is safe without any short-term (5 days) or long-term (30 days) toxicological adverse effects and can be used as an alternative therapeutic agent for IBD treatment.
Humans ; Mice ; Animals ; Gastrointestinal Microbiome ; Intestinal Barrier Function ; Mice, Inbred C57BL ; Colitis/metabolism* ; Inflammatory Bowel Diseases/drug therapy* ; Inflammation ; Anti-Inflammatory Agents/pharmacology* ; Disease Models, Animal

Inflammatory bowel disease (IBD) is a chronic progressive idiopathic inflammatory disorder that involves the digestive tract from the mouth to the anus. Over the past decades, many therapeutic strategies have been developed to manage IBD, but therapeutic strategies based only on relief of clinical symptoms have not changed the natural history of this disease entity. This underlines the importance of understanding the natural history of IBD itself. When we look at the natural history of Crohn's disease (CD), it first begins with inflammation of the intestinal mucosa and this inflammatory reaction proceeds to stenosing or penetrating reaction if not adequately controlled. However, it takes a considerable amount of time before mucosal inflammation proceeds to stenosis of the intestinal lumen or penetration into the adjacent bowel. Therefore, it can be expected that if proper care is given during that period, progression of CD to such a complicated disease could be prevented. Even though the concept of mucosal healing was introduced in the early 1990s, no correlation could be observed between healing of mucosal lesions and relief of clinical symptoms. However, the introduction of biologic agents targeting tumor necrosis factor has changed the way to treat IBD that is refractory to standard medications and has allowed us to aim for a new therapeutic goal, 'deep remission'. Further advances in biologic agents have provided highly effective treatments for IBD, making deep remission a realistic goal. Whether IBD patients may benefit by experiencing a 'deep' remission beyond the control of clinical symptoms need to be evaluated in further investigation. Nevertheless, it can be anticipated that attaining deep remission might ultimately have an impact on important outcomes such as the need for surgery and the quality of life.
Anti-Inflammatory Agents, Non-Steroidal/therapeutic use ; Antibodies, Monoclonal/therapeutic use ; Colitis, Ulcerative/drug therapy/metabolism/pathology ; Crohn Disease/drug therapy/metabolism/pathology ; Humans ; Inflammatory Bowel Diseases/drug therapy/metabolism/*pathology ; Intestinal Mucosa/metabolism/pathology ; Mesalamine/therapeutic use ; Tumor Necrosis Factor-alpha/immunology/metabolism

BACKGROUNDThalidomide could relieve clinical symptoms and intestinal mucosal lesions effectively in children with refractory inflammatory bowel disease (IBD) from the pre-clinical study. This study aimed to observe the therapeutic effect of thalidomide by the established animal model of IBD model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in Sprague-Dawley (SD) rats and to investigate the possible mechanism of action.

METHODSA total of 82 SD rats of about 4-5 weeks were randomly divided into three groups: the control group (25 rats), TNBS-treated group (29 rats), and thalidomide treatment group (28 rats). Daily activities were recorded. At least eight rats from each group were killed on the 4th, 7th, and 14th days. Morphological and histological changes in the colon were individually assessed. Serum was collected and the levels of TNF-α and interleukins (IL-1β and IL-10) were assayed by ELISA method. The expression of colonic mucosal nuclear factor (NF)-κB was assayed with the immunohistochemical method.

RESULTS(1) In the control group, diarrhea and rectal bleeding recovered rapidly and no death was recorded. In the TNBS-treated group, diarrhea and rectal bleeding persisted for a longer time. The mortality rate was 10.34% during the observation period. In the thalidomide treatment group, diarrhea and rectal bleeding persisted for a significantly shorter time than the TNBS-treated group (P < 0.01). The rats of this group also exhibited faster weight gain on day 7 compared with the TNBS-treated group but still lower than that of the control group. The mortality rate of the thalidomide treatment group was 3.57%. (2) Macroscopic and microscopic scores of the thalidomide-treated group were significantly lower than those of the TNBS model group on the 14th day (P < 0.01). These results suggested faster and better colonic recovery in the thalidomide-treated group. (3) NF-κB expression in the colonic mucosa of the control group was lower than in the others, mainly distributed in the cytoplasm. A large amount of intra-nuclear and cytoplasm staining was observed (more prominently intra-nuclear) in the TNBS model group and the thalidomide treatment group. On the 7th and 14th days, intra-nuclear NF-κB-containing cells in the thalidomide treatment group were still significantly lower than those in the TNBS model group (P < 0.01). (4) In the control group, the cellular inflammatory factors (TNF-α, IL-1β, and IL-10) were expressed at a low level while in the other two groups they were already expressed at a significantly higher level on day 4. On day 7 the expressions of TNF-α and IL-1β in the thalidomide treatment group were lower than in the TNBS model group. On day 14, the expressions of TNF-α and IL-1β in the thalidomide treatment group were significantly lower than in the TNBS model group (P < 0.05). On day 4, the IL-10 levels of the thalidomide treatment group became significantly elevated. The levels gradually decreased but still remained at a higher level. In the TNBS model group, the IL-10 expression peaked later than in the thalidomide treatment group.

CONCLUSIONSThalidomide was effective in the management of TNBS-induced colitis in young rats. This may be due to the suppression and down-regulation of NF-κB and the expression of the downstream inflammatory mediators (TNF-α and IL-1β). There is also indication that the expression of the anti-inflammatory cytokine (IL-10) is concomitantly up-regulated as well.

Animals ; Colitis ; chemically induced ; drug therapy ; metabolism ; Cytokines ; metabolism ; Inflammatory Bowel Diseases ; chemically induced ; drug therapy ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-1beta ; metabolism ; NF-kappa B ; metabolism ; Rats ; Rats, Sprague-Dawley ; Thalidomide ; therapeutic use ; Trinitrobenzenesulfonic Acid ; toxicity ; Tumor Necrosis Factor-alpha ; metabolism

PURPOSE: This study aimed to assess the role of thiopurine S-methyltransferase (TPMT) and 6-thioguanine nucleotide (6-TGN) as predictors of clinical response and side effects to azathioprine (AZA), and estimate the optimal AZA dose in Korean pediatric inflammatory bowel disease (IBD) patients. MATERIALS AND METHODS: One hundred and nine pediatric IBD patients in whom AZA treatment was required were enrolled. Thiopurine metabolites were monitored since September 2010. Among them, 83 patients who had prescribed AZA for at least 3 months prior to September 2010 were enrolled and followed until October 2011 to evaluate optimal AZA dose, adverse effects and disease activity before and after thiopurine metabolite monitoring. RESULTS: The result of the TPMT genotype was that 102 patients were *1/*1 (wild type), four were *1/*3C, one was *1/*6, one was *1/*16 (heterozygote) and one was *3C/*3C (homozygote). Adverse effects happened in 31 patients pre-metabolite monitoring and in only nine patients post-metabolite monitoring. AZA dose was 1.4+/-0.31 mg/kg/day before monitoring and 1.1+/-0.46 mg/kg/day after monitoring (p<0.001). However, there were no statistical differences in disease activity during metabolite monitoring period (p=0.34). Adverse effects noticeably decreased although reduction of the AZA dose since monitoring. CONCLUSION: TPMT genotype and thiopurine metabolite monitoring could be helpful to examine TPMT genotypes before administering AZA and to measure 6-TGN concentrations during prescribing AZA in IBD patients.
Adolescent ; Adult ; Azathioprine/*adverse effects/therapeutic use ; Child ; Child, Preschool ; Female ; Genotype ; Guanine Nucleotides/metabolism ; Humans ; Inflammatory Bowel Diseases/*drug therapy/metabolism ; Male ; Methyltransferases/genetics/metabolism ; Republic of Korea ; Risk Factors ; Thionucleotides/metabolism ; Treatment Outcome

Thiopurine S-methyltransferase (TPMT) methylates purine analogues, showing TPMT activity in inverse relation to concentrations of active metabolites such as 6-thioguanine nucleotide (6-TGN). With conventional dosing of thiopurines, patients with homozygous variant TPMT alleles consistently suffer from severe myelosuppression. Here, we report a patient with TPMT*3C/*3C who managed successfully with monitoring of thiopurine metabolites. The patient was an 18-year-old male diagnosed with Crohn's disease. The standard dose of azathioprine (AZA) (1.8 mg/kg/day) with mesalazine (55.6 mg/kg/day) was prescribed. Two weeks after starting AZA treatment, the patient developed leukopenia. The DNA sequence analysis of TPMT identified a homozygous missense variation (NM_000367.2: c.719A>G; p.Tyr240Cys), TPMT*3C/*3C. He was treated with adjusted doses of azathioprine (0.1-0.2 mg/kg/day) and his metabolites were closely monitored. Leukopenia did not reoccur during the follow-up period of 24 months. To our knowledge, this is the first case of a patient homozygous for TPMT*3C successfully treated with azathioprine in Korea. While a TPMT genotyping test may be helpful to determine a safe starting dose, it may not completely prevent myelosuppression. Monitoring metabolites as well as routine laboratory tests can contribute to assessing drug metabolism and optimizing drug dosing with minimized drug-induced toxicity.
Adolescent ; Azathioprine/adverse effects/*therapeutic use ; Homozygote ; Humans ; Inflammatory Bowel Diseases/*drug therapy/*enzymology/*genetics/metabolism ; Male ; Methyltransferases/*genetics

BACKGROUNDThe thiopurine drugs are well established in the treatment of inflammatory bowel disease (IBD). However, uncertainty regarding the risk for neutropenia and hepatotoxicity deters its using. Thiopurine methyltransferase (TPMT) is the key enzyme in the metabolism of thiopurine. The aim of this study was to investigate the association of TPMT polymorphisms and activity with azathioprine (AZA)-related adverse events and clinical efficacy in Chinese Han patients with IBD.

METHODSFifty-two Han IBD patients treated with AZA were assessed for TPMT 2, 3A, 3B, and 3C, and for adverse events. Then, using reverse-phase high-performance liquid chromatography, TPMT activity was measured in 13 patients to analyze its correlation with AZA-related toxicity and clinical efficacy.

RESULTSOf the 52 patients, five experienced myelotoxicity and one experienced hepatotoxicity during treatment. No TPMT 2, 3A, 3B or 3C polymorphisms were detected in any of the 52 patients. In the 13 patients with TPMT activity measurement, TPMT activity ranged from 7.2 to 28.8 U/ml packed red blood cells (pRBCs). Among the 5 patients who suffered from myelotoxicity, 3 were affected in the early stage of AZA therapy. In these 3 patients, TPMT levels were significantly lower than those in patients without myelotoxicity, which reached statistical significance ((9.3 ± 2.1) U/ml pRBC vs. (18.0 ± 6.2) U/ml pRBC; P = 0.046). One patient who had higher TPMT activity (28.8 U/ml pRBC) suffered from hepatotoxicity during AZA therapy. Patients who achieved a clinical response had lower TPMT activity than those failed to respond ((13.7 ± 3.5) U/ml pRBC vs. (22.0 ± 5.5) U/ml pRBC; P = 0.009).

CONCLUSIONSTPMT variants do not completely account for the AZA-related myelotoxicity in Chinese Han IBD patients. However, measurement of TPMT activity may be helpful in reducing the risk of toxicity, and predicting the therapeutic efficacy.

Adolescent ; Adult ; Aged ; Antimetabolites ; therapeutic use ; Azathioprine ; adverse effects ; therapeutic use ; Female ; Humans ; Inflammatory Bowel Diseases ; drug therapy ; enzymology ; Male ; Methyltransferases ; genetics ; metabolism ; Middle Aged ; Polymorphism, Genetic

OBJECTIVETo investigate molecular mechanisms underlying in the treatment of inflammatory bowel disease by Pulsatilla decoction.

METHODSWistar male rats were randomly divided into control group, model group, model + positive control group (mesalazine), traditional Chinese medicine treatment group, in addition, the Chinese medical treatment group was divided into middle and high dose group ( n = 8). Intragastric administration was used in the positive control group and traditional Chinese medicine treatment group. The expression of Smad7 and p-Smad3 in the colons were detected by immunohistochemistry and Western blot.

RESULTSCompared with the model group, positive medicine and traditional Chinese medicine group, especially high-dose group, could effectively inhibit the expression of Smad7, while enhancing the p-Smad3 expression.

CONCLUSIONThe activation of TGF-beta1/Smad3 signaling pathway may be the molecular mechanism underlying in the anti-inflammatory effect of inflammatory bowel disease by Pulsatilla decoction.

Animals ; Drugs, Chinese Herbal ; therapeutic use ; Inflammatory Bowel Diseases ; drug therapy ; Male ; Phytotherapy ; Pulsatilla ; chemistry ; Rats ; Rats, Wistar ; Signal Transduction ; drug effects ; Smad3 Protein ; metabolism ; Smad7 Protein ; metabolism ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo investigate the molecular mechanisms underlying in the treatment of inflammatory bowel disease by Pulsatilla Decoction.

METHODSForty Wistar male rats were randomly divided into 5 groups( n = 8)control group, model group, model + positive control group (mesalazine), Pulsatilla Decoction treatment group, in addition, the Pulsatilla Decoction treatment group was divided into middle and high dose group. Intragastric administration was used in the positive control group and Pulsatilla Decoction treatment group. The expression of interleukin-1beta (IL-1beta), interleukin-6(IL-6) and tumor necrosis factor-alpha (TNF-alpha) were detected by real time PCR after extraction of RNA from colons.

RESULTSCompared with the model group, positive medicine and Pulsatilla Decoction group, especially high-dose group, could effectively inhibit the expression of IL-1beta, IL-6 and TNF-alpha.

CONCLUSIONPulsatilla Decoction could exert its effect in the treatment of inflammatory bowel disease by inhibiting the expression of proinflammatory cytokines.

Animals ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Inflammatory Bowel Diseases ; drug therapy ; metabolism ; Interleukin-1beta ; genetics ; metabolism ; Interleukin-6 ; genetics ; metabolism ; Male ; Phytotherapy ; Pulsatilla ; chemistry ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

TNF-alpha is a major cytokine involved in inflammatory bowel disease (IBD). In this study, water extract of Grifola frondosa (GFW) was evaluated for its protective effects against colon inflammation through the modulation of TNF-alpha action. In coculture of HT-29 human colon cancer cells with U937 human monocytic cells, TNF-alpha-induced monocyte adhesion to HT-29 cells was significantly suppressed by GFW (10, 50, 100 microg/ml). The reduced adhesion by GFW correlated with the suppressed expression of MCP-1 and IL-8, the major IBD-associated chemokines. In addition, treatment with GFW significantly suppressed TNF-alpha-induced reactive oxygen species production and NF-kappaB transcriptional activity in HT-29 cells. In differentiated U937 monocytic cells, LPS-induced TNF-alpha production, which is known to be mediated through NF-kappaB activation, was significantly suppressed by GFW. In an in vivo rat model of IBD, oral administration of GFW for 5 days (1 g/kg per day) significantly inhibited the trinitrobenzene sulfonic acid (TNBS)-induced weight loss, colon ulceration, myeloperoxidase activity, and TNF-alpha expression in the colon tissue. Moreover, the effect of GFW was similar to that of intra-peritoneal injection of 5-aminosalicylic acid (5-ASA), an active metabolite of sulfasalazine, commonly used drug for the treatment of IBD. The results suggest that GFW ameliorates colon inflammation by suppressing production of TNF-alpha as well as its signaling through NF-kappaB leading to the expression of inflammatory chemokines, MCP-1 and IL-8. Taken together, the results strongly suggest GFW is a valuable medicinal food for IBD treatment, and thus may be used as an alternative medicine for IBD.
Animals ; Cell Adhesion/drug effects/immunology ; Cell Extracts/administration & dosage/*pharmacology ; Chemokine CCL2/biosynthesis/genetics ; Coculture Techniques ; Colon/drug effects/*metabolism/pathology ; Grifola ; HT29 Cells ; Humans ; Inflammatory Bowel Diseases/chemically induced/*drug therapy/pathology/physiopathology ; Interleukin-8/biosynthesis/genetics ; Intestinal Mucosa/*drug effects/metabolism/pathology ; Monocytes/*drug effects/metabolism/pathology ; NF-kappa B/genetics/metabolism ; Peroxidase/metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/metabolism ; Stomach Ulcer ; Transcription, Genetic/drug effects ; Trinitrobenzenesulfonic Acid/administration & dosage ; Tumor Necrosis Factor-alpha/*biosynthesis/genetics ; U937 Cells ; Weight Loss