The double-edged sword effect of indigo naturalis.

Indigo naturalis (IN) is a dried powder derived from plants such as Baphicacanthus cusia (Neeks) Bremek., Polygonum tinctorium Ait. and Isatis indigotica Fork. It has a historical application as a dye in ancient India, Egypt, Africa and China. Over time, it has been introduced to China and Japan for treatment of various ailments including hemoptysis, epistaxis, chest discomfort, and aphtha. Clinical and pre-clinical studies have widely demonstrated its promising effects on autoimmune diseases like psoriasis and Ulcerative colitis (UC). Despite the documented efficacy of IN in UC patients, concerns have been raised on the development of adverse effects with long term consumption, prompting a closer examination of its safety and tolerability in these contexts. This review aims to comprehensively assess the efficacy of IN in both clinical and pre-clinical settings, with a detailed exploration of the mechanisms of action involved. Additionally, it summarizes the observed potential toxicity of IN in animal and human settings was summarized. This review will deepen our understanding on the beneficial and detrimental effects of IN in UC, providing valuable insights for its future application in patients with this condition.

Indigo naturalis (Qing dai) for inflammatory bowel disease: A systematic review and meta-analysis.

BACKGROUND: Indigo naturalis (Qing dai) is a traditional therapy reported to be useful in inflammatory bowel disease (IBD), especially for ulcerative colitis. We performed a systematic review of its efficacy and safety in IBD. METHODS: Electronic databases (Pubmed, Embase, and Scopus) were searched on 4th March 2023 to identify reports about the use of indigo naturalis in IBD. We extracted data with respect to clinical response, remission, endoscopic and histological responses, and adverse events with the use of indigo naturalis in IBD. Pooled clinical response rates and remission rates were calculated. The quality of studies was assessed using Joanna-Briggs tools. RESULTS: Nine studies reporting on 299 patients were included. The pooled clinical response rate was 0.796 (95 %CI, 0.7465-0.8379, I2=0), and the clinical remission rate in ulcerative colitis was 0.668 (0.488- 0.809, I2=85.2 %). The pooled relative risk of clinical response was higher in the indigo naturalis group as compared to placebo in the two randomized trials [3.82 (2.04; 7.14, I2=0)]. Except for one reversible pulmonary arterial hypertension case, most reported adverse effects were mild. The endoscopic and histological responses, when reported, suggested that indigo naturalis is effective for ulcerative colitis. The limitations of the systematic review included a small number of randomized studies, reports only from East Asia and a relatively small number of patients, especially for Crohn's disease. CONCLUSION: Indigo naturalis is effective in the treatment of ulcerative colitis. Future studies should evaluate the comparative efficacy with other drugs.

Liquid Chromatography-Tandem Mass Spectrometry-Based Metabolomics Analysis of Indigo Naturalis Treatment of Ulcerative Colitis in Mice.

Ulcerative colitis (UC), often known as UC, is an inflammatory disease of the intestines that has frequent and long-lasting flare-ups. It is unknown precisely how the traditional Chinese drug Indigo Naturalis (IN) heals inflammatory bowel disease, despite its long-standing use in China and Japan. Finding new metabolite biomarkers linked to UC could improve our understanding of the disease, speed up the diagnostic process, and provide insight into how certain drugs work to treat the condition. Our work is designed to use a metabolomic method to analyze potential alterations in endogenous substances and their impact on metabolic pathways in a mouse model of UC. To determine which biomarkers and metabolisms are more frequently connected with IN's effects on UC, liquid chromatography-tandem mass spectrometry analysis of the serum metabolomics of UC mice and normal mice was performed. The outcomes demonstrated that IN boosted the health of UC mice and reduced the severity of their metabolic dysfunction. In the UC model, it was also found that IN changed the way 17 biomarkers and 3 metabolisms functioned.

Combination Therapy with Indigo and Indirubin for Ulcerative Colitis via Reinforcing Intestinal Barrier Function.

Indigo and indirubin, the active molecules of traditional Chinese medicine indigo naturalis, exert therapeutic activity for ulcerative colitis (UC). Indigo and indirubin are isomers and have distinctive profiles in anti-inflammation, immune regulation, intestinal microbiota regulation, oxidative stress regulation, and intestinal mucosal repair for UC treatment. Thus, exploring its combined administration's integrated advantages for UC is critical. This study is aimed at clarifying the effect and mechanisms of the combined administration of indigo and indirubin on colitis mouse models. The results showed that all the treatment groups could improve the disease symptoms, and the combined administration showed the best effect. Additionally, compared with indigo and indirubin alone, the combination group could significantly reinforce intestinal barrier function by increasing the expression of E-cadherin, occludin, ZO-1, and MUC2 and improving intestinal permeability. The treatment groups significantly improved the expression of cytokines, including TNF-α, IFN-γ, IL-12, IL-23, and IL-17A, and indirubin presented the most potent anti-inflammatory effect. Furthermore, all the treatment groups reduced the infiltration of the immune cells in intestinal lamina propria and the production of ROS/RNS. Notably, indigo exhibited a more substantial capacity to regulate natural killer (NK) cells, ILC3, neutrophils, and dendritic cells, followed by the combination group and indirubin alone. Finally, all the treatment groups modulated intestinal microbiota composition, increased the proportion of beneficial microbiota, and decreased the proportion of microbiota. Our results indicated that indigo and indirubin synergistically reinforced the intestinal barrier function, which may be associated with integrating the indirubin anti-inflammatory and intestinal microbiota regulating strength and indigo immune and ROS/RNS regulation advantage.

Gut Microbiome Associated With Graves Disease and Graves Orbitopathy: The INDIGO Multicenter European Study.

CONTEXT: Gut bacteria can influence host immune responses but little is known about their role in tolerance-loss mechanisms in Graves disease (GD; hyperthyroidism caused by autoantibodies, TRAb, to the thyrotropin receptor, TSHR) and its progression to Graves orbitopathy (GO). OBJECTIVE: This work aimed to compare the fecal microbiota in GD patients, with GO of varying severity, and healthy controls (HCs). METHODS: Patients were recruited from 4 European countries (105 GD patients, 41 HCs) for an observational study with cross-sectional and longitudinal components. RESULTS: At recruitment, when patients were hyperthyroid and TRAb positive, Actinobacteria were significantly increased and Bacteroidetes significantly decreased in GD/GO compared with HCs. The Firmicutes to Bacteroidetes (F:B) ratio was significantly higher in GD/GO than in HCs. Differential abundance of 15 genera was observed in patients, being most skewed in mild GO. Bacteroides displayed positive and negative correlations with TSH and free thyroxine, respectively, and was also significantly associated with smoking in GO; smoking is a risk factor for GO but not GD. Longitudinal analyses revealed that the presence of certain bacteria (Clostridiales) at diagnosis correlated with the persistence of TRAb more than 200 days after commencing antithyroid drug treatment. CONCLUSION: The increased F:B ratio observed in GD/GO mirrors our finding in a murine model comparing TSHR-immunized with control mice. We defined a microbiome signature and identified changes associated with autoimmunity as distinct from those due to hyperthyroidism. Persistence of TRAb is predictive of relapse; identification of these patients at diagnosis, via their microbiome, could improve management with potential to eradicate Clostridiales.

Psoriasis treatment using Indigo Naturalis: Progress and strategy.

ETHNOPHARMACOLOGICAL RELEVANCE: In recent years, there are increasing that the number of patients with psoriasis day by day, and it has become a common disease endangering public health. However, there is no specific cure for psoriasis or control of recurrence. Therefore, it's necessity to seek alternative and efficient therapy, such as Traditional Chinese Medicine (TCM). As a TCM and effective medicine for the treatment of psoriasis, Indigo Naturalis (Baphicacanthus Cusia (Nees) Bremek.) has the effect of clearing heat, detoxifying blood, eliminating spots, reducing fire and calming panic, and it is used in many classical prescriptions for the treatment of psoriasis. AIM OF REVIEW: To review the latest progress and strategies of Indigo Naturalis in the treatment of psoriasis. This manuscript mainly clarifies the traditional medicinal applications, the mechanism of action and application strategies of Indigo Naturalis, and its preparations in the treatment of psoriasis. MATERIALS AND METHODS: Detailed information on Indigo Naturalis was collected from various online databases (PubMed, GeenMedical, Web of Science, Google Scholar, China National Knowledge Infrastructure Database, and National Intellectual Property Administration). RESULTS: This manuscript reviews a great deal of information about how Indigo Naturalis can treat psoriasis through immune cells, signal pathways and disease-related mediators. The mechanism of cymbididae is expounded from the aspects of regulating keratinocyte proliferation and differentiation, regulating inflammatory infiltration of cellular immune system and improving microvascular dilation and hyperplasia in skin lesions. CONCLUSION: The action mechanisms of Indigo Naturalis on psoriasis reflect the characteristics of multiple components, multiple targets, and multiple pathways of Traditional Chinese medicine. However, some pharmacological and clinical research methods are improper, so that the results are difficult to explain at present. Therefore, further in-depth research is needed to provide knowledge in a wider range of areas to confirm the great therapeutic potential of Indigo Naturalis.

Single-cell transcriptomics profiling the compatibility mechanism of As2O3-indigo naturalis formula based on bone marrow stroma cells.

Compound realgar natural indigo tablet is the only oral arsenic agent widely used in acute promyelocytic leukemia (APL) treatment. However, as a therapeutic drug for diseases of the blood system, the scientific knowledge of As2O3-indigo naturalis formula compatibility has not been studied in bone marrow stromal cells (BMSCs). We chose arsenic trioxide (As2O3: A), tanshinone IIA (T) and indirubin (I) as representative active compounds of realgar, indigo naturalis, and Salvia miltiorrhiza, respectively, to evaluated the pharmaceutical mechanism and the compatibility of ATI (drug combination) using single-cell RNA sequencing (scRNA-seq). The overlapped genes associated with both disease and drug were selected in BMSCs for in-depth analysis. Results show that joint applications of ATI had the strongest therapeutic efficacy in a murine APL model. Lepr-MSCs, OLCs and BMECs were the sensitive cell groups targeted by ATI in the murine APL model. ATI could regulate the related genes of osteogenic differentiation, adipogenic differentiation, and endothelial cell migration in bone marrow mesenchymal lineage cells in murine APL model and improve normal hematopoiesis-related gene expression and poor prognosis of Lepr-MSCs, OLCs and BMECs in mice with leukemia according to scRNA-seq data. The strongest regulatory effects were found in the joint applications of ATI. ATI combination had the potential mechanism to maintain the stability of the hematopoietic microenvironment and promote hematopoiesis to assist in the treatment of APL. This study illustrated the potential mechanism of ATI in regulating BMSCs from the overall perspective of the hematopoietic microenvironment, and broadened the scientific understanding of ATI compatibility in BMSCs.

Antioxidant effects of bis-indole alkaloid indigo and related signaling pathways in the experimental model of Duchenne muscular dystrophy.

Indigo is a bis-indolic alkaloid that has antioxidant and anti-inflammatory effects reported in literature and is a promissory compound for treating chronic inflammatory diseases. This fact prompted to investigate the effects of this alkaloid in the experimental model of Duchenne muscular dystrophy. The main aim of this study was to evaluate the potential role of the indigo on oxidative stress and related signaling pathways in primary skeletal muscle cell cultures and in the diaphragm muscle from mdx mice. The MTT and Neutral Red assays showed no indigo dose-dependent toxicities in mdx muscle cells at concentrations analyzed (3.12, 6.25, 12.50, and 25.00 µg/mL). Antioxidant effect of indigo, in mdx muscle cells and diaphragm muscle, was demonstrated by reduction in 4-HNE content, H2O2 levels, DHE reaction, and lipofuscin granules. A significant decrease in the inflammatory process was identified by a reduction on TNF and NF-κB levels, on inflammatory area, and on macrophage infiltration in the dystrophic sample, after indigo treatment. Upregulation of PGC-1α and SIRT1 in dystrophic muscle cells treated with indigo was also observed. These results suggest the potential of indigo as a therapeutic agent for muscular dystrophy, through their action anti-inflammatory, antioxidant, and modulator of SIRT1/PGC-1α pathway.

Treatment-refractory ulcerative colitis responsive to indigo naturalis.

BACKGROUND: Indigo naturalis (IN) is an herbal medicine that has been used for ulcerative colitis with an unclear mechanism of action. Indigo and indirubin, its main constituents, are ligands of the aryl hydrocarbon receptor (AhR). We assessed the safety, efficacy, and colon AhR activity of IN given orally to patients with treatment-refractory ulcerative colitis. The role of AhR in IN benefit was further evaluated with an AhR antagonist in a murine colitis model. METHODS: This open-label, dose-escalation study sequentially treated 11 patients with ulcerative colitis with either IN 500 mg/day or 1.5 g/day for 8 weeks, followed by a 4-week non-treatment period. The primary efficacy endpoint was clinical response at week 8, assessed by total Mayo score. Secondary endpoints included clinical remission, Ulcerative Colitis Endoscopic Index of Severity, quality of life, and colon AhR activity measured by cytochrome P450 1A1 (CYP1A1) RNA expression. RESULTS: Ten of 11 (91%) patients, including 8/9 (89%) with moderate-to-severe disease, achieved a clinical response. Among these 10 patients, all had failed treatment with 5-aminosalicylic acid, 8 patients with a tumour necrosis factor (TNF)-alpha inhibitor, and 6 patients with TNF-alpha inhibitor and vedolizumab. Five patients were corticosteroid dependent. Clinical response was observed in all five patients who had been recommended for colectomy. Three patients achieved clinical remission. All patients experienced improved endoscopic severity and quality of life. Four weeks after treatment completion, six patients had worsened partial Mayo scores. Four patients progressed to colectomy after study completion. Colon CYP1A1 RNA expression increased 12 557-fold at week 8 among six patients evaluated. No patient discontinued IN due to an adverse event. Concomitant administration of 3-methoxy-4-nitroflavone, an AhR antagonist, in a murine colitis model abrogated the benefit of IN. CONCLUSION: IN is a potentially effective therapy for patients with treatment-refractory ulcerative colitis. This benefit is likely through AhR activation. TRIAL REGISTRATION NUMBER: NCT02442960.

Indigo Aspiration System for Treatment of Pulmonary Embolism: Results of the EXTRACT-PE Trial.

OBJECTIVES: This study sought to prospectively evaluate the safety and efficacy of the Indigo aspiration system in submassive acute pulmonary embolism (PE). BACKGROUND: PE treatment with thrombolytics has bleeding risks. Aspiration thrombectomy can remove thrombus without thrombolytics, but data are lacking. METHODS: This study was a prospective, single-arm, multicenter study that enrolled patients with symptomatic acute PE ≤14 days, systolic blood pressure ≥90 mm Hg, and right ventricular-to-left ventricular (RV/LV) ratio >0.9. The primary efficacy endpoint was change in RV/LV ratio from baseline to 48 h post-procedure on core lab-adjudicated computed tomography angiography. The primary safety endpoint was a composite of 48-h major adverse events: device-related death, major bleeding, and device-related serious adverse events (clinical deterioration, pulmonary vascular, or cardiac injury). All sites received Institutional Review Board approval. RESULTS: A total of 119 patients (mean age 59.8 ± 15.0 years) were enrolled at 22 U.S. sites between November 2017 and March 2019. Median device insertion to removal time was 37.0 (interquartile range: 23.5 to 60.0) min. Two (1.7%) patients received intraprocedural thrombolytics. Mean RV/LV ratio reduction from baseline to 48 h post-procedure was 0.43 (95% confidence interval: 0.38 to 0.47; p < 0.0001). Two (1.7%) patients experienced 3 major adverse events. Rates of cardiac injury, pulmonary vascular injury, clinical deterioration, major bleeding, and device-related death at 48 h were 0%, 1.7%, 1.7%, 1.7%, and 0.8%, respectively. CONCLUSIONS: In this prospective, multicenter study the Indigo aspiration system was associated with a significant reduction in the RV/LV ratio and a low major adverse event rate in submassive PE patients. Intraprocedural thrombolytic drugs were avoided in 98.3% of patients. (Evaluating the Safety and Efficacy of the Indigo aspiration system in Acute Pulmonary Embolism [EXTRACT-PE]; NCT03218566).

Risk of Urinary Tract Infection Symptoms after Posthysterectomy Cystoscopy with 50% Dextrose as Compared with Saline Cystoscopy with Indigo Carmine.

STUDY OBJECTIVE: Compare odds of postoperative urinary symptoms in women who had cystoscopy after benign laparoscopic hysterectomy with 50% dextrose and with normal saline solution with intravenous indigo carmine. DESIGN: Retrospective cohort study. SETTING: Two tertiary care centers. PATIENTS: All women who underwent benign laparoscopic hysterectomy and intraoperative cystoscopy carried out by a single surgeon. INTERVENTIONS: We compared postoperative urinary symptoms in patients who received 50% dextrose cystoscopy fluid (January 2016-June 2017) with those who received saline cystoscopy with intravenous indigo carmine (November 2013-April 2014). MEASUREMENTS AND MAIN RESULTS: A total of 96 patients had cystoscopy with 50% dextrose and 104 with normal saline with intravenous indigo carmine. Differences in baseline characteristics of the two groups of participants mainly reflected institutional population diversity: age (45.2 vs 41.9, p = .01), body mass index (26.9 vs 33.4, p <.01), race, current smoking status (1% vs 7.8%, p = .04), diabetes (2.1% vs 11.5%, p = .01), history of abdominal surgery (53.1% vs 74%, p <.01), hysterectomy type, receipt of intraoperative antibiotics (92.7% vs 100%, p <.01), recatheterization (10.4% vs 0%, p <.01), and removal of catheter on postoperative day 0 (66.7% vs 12.5%, p <.01). Urinary symptoms were similar for 50% dextrose and saline (12.5% vs 7.7%, p = .19). After adjusting for age, body mass index, race, diabetes, and day of catheter removal, there remained no significant differences in urinary symptoms between the groups (odds ratio 3.19 [95% confidence interval, 0.82-12.35], p = .09). One immediate bladder injury was detected in the saline group and 1 delayed lower urinary tract injury in the 50% dextrose group. CONCLUSION: Overall, most women experienced no urinary symptoms after benign laparoscopic hysterectomy. There were no significant differences in postoperative urinary symptoms or empiric treatment of urinary tract infection after the use of 50% dextrose cystoscopy fluid as compared with normal saline. The previous finding of increased odds of urinary tract infection after dextrose cystoscopy may be due to use in a high-risk population.

Efficacy and safety of short-term therapy with indigo naturalis for ulcerative colitis: An investigator-initiated multicenter double-blind clinical trial.

INTRODUCTION: Indigo naturalis (IN) is a blue pigment extracted from Assam indigo and other plants and has been confirmed to be highly effective for ulcerative colitis (UC) treatment in several clinical studies. OBJECTIVE: We conducted a multicenter double-blind study to confirm the efficacy and safety of short-term IN administration. METHODS: A multicenter, randomized controlled trial was conducted between December 2015 and October 2018 in our facilities. Forty-six patients with mild to moderate active UC (Lichtiger index: 5-10) were randomly assigned to the IN group or the placebo group and received 5 capsules (500 mg) twice a day for 2 weeks. We investigated the efficacy according to blood tests and the Lichtiger index before and after administration, and we also examined adverse events. RESULTS: The analysis included 42 patients (20 males, 22 females) with an average age of 45 years. Nineteen patients were assigned to the placebo group, and 23 were assigned to the IN group. After treatment administration, in the placebo group, no change in the Lichtiger index was observed (7.47 to 6.95, p = 0.359), and hemoglobin was significantly reduced (12.7 to 12.4, p = 0.031), while in the IN group, the Lichtiger index (9.04 to 4.48, p = 0.001) and albumin (4.0 to 4.12, p = 0.022) improved significantly. Mild headaches were observed in 5 patients and 1 patient in the IN and placebo groups, respectively. CONCLUSIONS: Short-term administration of IN is highly effective without serious adverse events such as pulmonary hypertension or intussusception and may prevent the occurrence of serious adverse events.

Evaluation of Indigo carmine on hepatic ischemia and reperfusion injury.

PURPOSE: To evaluate the effects of treatment with Indigo Carmine (IC) on rat livers subjected to ischemia-reperfusion injury. METHODS: The animals were subdivided into 4 groups: 1.SHAM group(SH) - saline; 2.SHAM group with IC-2mg/Kg(SHIC); 3.IR group - rats submitted to ischemia and reperfusion with saline(IR); 4.IR group with IC-2mg/Kg(IRIC). The IR protocol consists of liver exposure and administration of drug or saline intravenously, followed by 60 minutes of ischemia and 15 of reperfusion. Liver samples were collected for biochemical analysis. RESULTS: State 3 of mitochondrial respiration showed a significant worsening of the IRIC group in relation to all others. State 4 showed a difference between IRIC and SHIC. The Respiratory Control Ratio showed statistical decrease in IR and IRIC versus Sham. The osmotic swelling showed significant difference between SHxIR; SHICxIRIC and SHxIRIC. There was a significant increase in ALT in the IRIC group in relation to all the others. Concerning the nitrate dosage, there was a decrease in the group treated with IC(IRxIRIC). There was no difference regarding the dosage of Malondialdehyde. CONCLUSION: IC was not able to protect mitochondria from IR injury and proved to be a potentiating agent, acting in synergy with the IR injury promoting damage to the hepatocyte membranes.

Evaluation of Indigo carmine on hepatic ischemia and reperfusion injury

Abstract Purpose To evaluate the effects of treatment with Indigo Carmine (IC) on rat livers subjected to ischemia-reperfusion injury. Methods The animals were subdivided into 4 groups: 1.SHAM group(SH) - saline; 2.SHAM group with IC-2mg/Kg(SHIC); 3.IR group - rats submitted to ischemia and reperfusion with saline(IR); 4.IR group with IC-2mg/Kg(IRIC). The IR protocol consists of liver exposure and administration of drug or saline intravenously, followed by 60 minutes of ischemia and 15 of reperfusion. Liver samples were collected for biochemical analysis. Results State 3 of mitochondrial respiration showed a significant worsening of the IRIC group in relation to all others. State 4 showed a difference between IRIC and SHIC. The Respiratory Control Ratio showed statistical decrease in IR and IRIC versus Sham. The osmotic swelling showed significant difference between SHxIR; SHICxIRIC and SHxIRIC. There was a significant increase in ALT in the IRIC group in relation to all the others. Concerning the nitrate dosage, there was a decrease in the group treated with IC(IRxIRIC). There was no difference regarding the dosage of Malondialdehyde. Conclusion IC was not able to protect mitochondria from IR injury and proved to be a potentiating agent, acting in synergy with the IR injury promoting damage to the hepatocyte membranes.

Efficacy of Indigo Naturalis Therapy for Ulcerative Colitis: A Case Series.

Objective Indigo naturalis (IN) is a traditional Chinese medicine that has recently been reported to be effective for ulcerative colitis (UC). The aim of this study was to evaluate the efficacy and safety of IN. Methods We performed a retrospective observational study for 14 patients with UC treated with IN from October 2015 to December 2016. Results After 8 weeks of oral administration of IN, the partial Mayo score decreased from 4 (2-5) to 1.5 (0-4) [median, interquartile range (IQR), p=0.015]. Among 10 active UC patients, 5 (50%) showed a clinical response, and 4 (40%) achieved clinical remission. Serial changes of endoscopic activity were evaluated in nine patients using the Mayo endoscopic subscore (MES), Rachmilewitz endoscopic index (REI), and UC endoscopy index of severity (UCEIS). The MES decreased from 2 (2-3) to 1 (1-2) [median (IQR), p=0.005], the REI decreased from 7 (5.5-11) to 3 (1-7) [median (IQR), p=0.008], and the UCEIS decreased from 3 (3-4.5) to 1 (0.5-3.5) [median (IQR), p=0.039]. One patient developed acute right-sided colitis with wall thickening and edematous change, and the remaining 13 showed no adverse events. Conclusion We conclude that IN is effective for patients with UC as a therapy for inducing remission.

Indigo Naturalis Ameliorates Oxazolone-Induced Dermatitis but Aggravates Colitis by Changing the Composition of Gut Microflora.

BACKGROUND: Indigo naturalis (IND) is an herbal medicine that has been used as an anti-inflammatory agent to treat diseases including dermatitis and inflammatory bowel disease in China. However, the mechanism by which IND exerts its immunomodulatory effect is not well understood. METHODS: A murine model of dermatitis and inflammatory bowel disease, both induced by oxazolone (OXA), was treated with IND. The severity of dermatitis was evaluated based on ear thickness measurements and histological scoring. The severity of colitis was evaluated by measuring body weight, histological scoring, and endoscopic scoring. The expression of inflammatory cytokines in ear and colon tissue was evaluated using real-time PCR. 16S rRNA DNA sequencing of feces from OXA-induced colitis mice was performed before and after IND treatment. The effects of IND on OXA-induced colitis were also evaluated after depleting the gut flora with antibiotics to test whether alteration of the gut flora by IND influenced the course of intestinal inflammation in this model. RESULTS: IND treatment ameliorated OXA dermatitis with a reduction in IL-4 and eosinophil recruitment. However, OXA colitis was significantly aggravated in spite of a reduction in intestinal IL-13, a pivotal cytokine in the induction of the colitis. It was found that IND dramatically altered the gut flora and IND no longer exacerbated colitis when colitis was induced after gut flora depletion. CONCLUSIONS: Our data suggest that IND could modify the inflammatory immune response in multiple ways, either directly (i.e., modification of the allergic immune cell activity) or indirectly (i.e., alteration of commensal compositions).

Indigo Naturalis ameliorates murine dextran sodium sulfate-induced colitis via aryl hydrocarbon receptor activation.

BACKGROUND: Indigo Naturalis (IN) is used as a traditional herbal medicine for ulcerative colitis (UC). However, the mechanisms of action of IN have not been clarified. We aimed to evaluate the efficacy of IN for ameliorating colonic inflammation. We further investigated the mechanisms of action of IN. METHODS: Colitis severity was assessed in dextran sodium sulfate-induced colitis and trinitrobenzene sulfonic acid-induced colitis models with or without the oral administration of IN or indigo, which is a known major component of IN. Colonic lamina propria (LP) mononuclear cells isolated from IN-treated mice were analyzed with quantitative reverse transcription polymerase chain reaction (qRT-PCR) and flow cytometry. LP and splenic mononuclear cells cultured in vitro with IN or indigo were also analyzed. The role of the candidate receptor for indigo, the aryl hydrocarbon receptor (AhR), was analyzed using Ahr-deficient mice. RESULTS: Colitis severity was significantly ameliorated in the IN and indigo treatment groups compared with the control group. The mRNA expression levels of interleukin (Il)-10 and Il-22 in the LP lymphocytes were increased by IN treatment. The treatment of splenocytes with IN or indigo increased the expression of anti-inflammatory cytokines and resulted in the expansion of IL-10-producing CD4+ T cells and IL-22-producing CD3-RORγt+ cells, but not CD4+Foxp3+ regulatory T cells. The amelioration of colitis by IN or indigo was abrogated in Ahr-deficient mice, in association with diminished regulatory cytokine production. CONCLUSIONS: IN and indigo ameliorated murine colitis through AhR signaling activation, suggesting that AhR could be a promising therapeutic target for UC.