The mechanism of indigo naturalis and its active ingredients against ulcerative colitis

Abstract

Indigo Naturalis (IN), utilized for thousands of years in China, is known for its effects in clearing heat, removing toxins, and alleviating diarrhea. Studies indicate that IN possesses anti-ulcerative colitis (UC) effects. However, the molecular processes that explain its therapeutic outcomes are still not fully understood. This study was to clarify the mechanism of IN against UC using both in vitro and vivo research methods. RAW264.7 cells were activated by lipopolysaccharide (LPS) (1 µg/mL) to found an inflammatory model; MTT and NO test was conducted to examine the influence of IN, indigo, indirubin, isatin, and MIX (indigo, indirubin, and isatin) on cell viability and anti-inflammatory activity. Inflammatory cytokines expression was evaluated with ELISA. Western Blotting was employed to analyze the MAPK, STAT3, and NF-κB pathways. UC model was generated by 2.5% DSS in C57BL/6 mice. Disease activity index (DAI) scoring , myeloperoxidase (MPO), and H&E staining are all for mice status. We demonstrated that IN, indirubin, and isatin can all regulate the expression of inflammatory cytokines. NF-κB, STAT3, and MAPK pathways was closely associated with IN, whereas indirubin acted to block the activation of both the NF-κB and STAT3 pathways. Contrastingly, the isatin mechanism of action was tightly connected with the regulation of the NF-κB and MAPK pathways. Additionally, MIX inhibited the activation of STAT3 pathway while suppressing the level of p-P38 expression in the MAPK pathway. MIX can enhance multiple indicators and conditions in UC mice. The active ingredients in IN (indigo, indirubin, and isatin, MIX) can significantly improve the condition of UC mice. MIX exerts anti-inflammatory effects by inhibiting the activation of the STAT3 pathway and expression of p-P38 in the MAPK pathway.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-35413-9.

Introduction

Ulcerative colitis (UC) is a chronic inflammatory disease of the intestines, the exact cause of which remains unknown¹. This disease typically affects the rectum and distal colon; it leads to widespread ulcerations and presents clinical symptoms including abdominal discomfort, recurrent diarrhea, as well as the presence of muco-purulent and blood-stained stools. Dysregulated immune responses, gut microbiota dysbiosis², genetic predisposition, and environmental triggers have been implicated in disease progression^3,4. Early UC treatment mainly induces remission, reduces the intestinal inflammatory response, and improves quality of life. The clinical treatments of UC contains glucocorticoids, 5-aminosalicylic acid, salazosulfapyridine⁵, and mesalazine⁶. However, these pharmacological agents have demonstrated limited efficacy in halting disease progression, and their prolonged use is associated with several adverse effects and drug resistance⁷. Therefore, there is an urgent need to develop safe and effective anti-UC drugs.

In traditional Chinese medicine (TCM), UC is clinically classified as “Xie xie” (diarrhea), “Li ji” (dysentery), or “Chang pi” (intestinal erosion)⁷. UC can also be divided into Pi-Xu-Shi-Yun and Da-Chang-Shi-Re⁸. Additionally, TCM identifies internal dampness-heat accumulation as the core pathogenic mechanism in UC. Heat-clearing and dampness-resolving strategies constitute the primary therapeutic approach^8,9.

IN, a traditional Chinese medicine, is renowned for its dark blue dye properties and is equally esteemed for its medicinal benefits¹⁰. IN is sourced from the processed aerial parts of Baphicacanthus cusia (Nees) Bremek., Polygonum tinctorium Ait. (belonging to the Polygonaceae family), and Isatis indigotica Fort. (of the Brassicaceae family)¹¹. Its origins are recorded in Zhenquan’s foundational pharmacological text, “Treatise on Medicinal Qualities,” from the Tang era. In the past, IN has been a go-to treatment for a range of inflammatory ailments, such as high fevers, dysentery, boils, and inflammation. It’s thought to have the power to quell the flames of heat, purge the body of toxins, cool the bloodstream, and bring down swelling¹⁰. Incorporating IN into treatment helps control mucosal bleeding, which lays the groundwork for its use in treating inflammatory conditions of the gut, like UC¹². TCM offers unique characteristics and advantages in the management of UC¹³. Consequently, Incorporating IN into modern medical research has become quite the buzz, as it’s been pinpointed as having a substantial healing power for certain ailments. A plethora of preclinical and clinical trials have showcased IN’s impressive therapeutic capabilities for UC^14–16. IN demonstrates anti-inflammatory properties by diminishing the expression levels of inflammatory mediators in both in vitro and in vivo models of UC^12,17,18. Quantitative analysis revealed that indigo (13.87%), indirubin (0.15%), and isatin (0.11%) are the predominant components of this TCM¹⁹. In the treatment of UC, IN and its active ingredients—indigo, indirubin, and isatin—balance the levels of inflammatory cytokines, thereby restoring the structure of the colon^20,21. To better contextualize our study, it is important to review the existing knowledge on the individual components. Indirubin, a core active ingredient of IN, has garnered attention for its potent anti-inflammatory and immunomodulatory properties. Previous studies have demonstrated its efficacy in models of chronic inflammatory diseases such as acute lung injury²² and psoriasis²³, primarily through the inhibition of NF-κB and JAK/STAT signaling pathways. Similarly, isatin Derivatives have been reported to possess anti-neuroinflammatory activity²⁴. Likewise, indigo is known for UC¹⁸. Nonetheless, the potential pharmacological interaction—whether additive or synergistic—among indigo, indirubin, and isatin in the context of UC is entirely unknown and the specific molecular pathways that contribute to the therapeutic effects of IN on UC are still not fully understood and require additional research.

This study systematically characterized the anti-inflammatory mechanisms of IN and its principal bioactive constituents. We further investigated whether a combinatorial formulation of indigo, indirubin, and isatin (MIX) could be a potential substitute for IN in the exertion of anti-inflammatory effects, followed by pharmacodynamic evaluation in a UC mice model.

Materials and methods

Medicinal materials and reagents

IN formula granules were obtained from Jiangyin Tianjiang Pharmaceutical Co. Indigo (L2101260) and Indirubin (F2221404) were purchased from Shanghai Aladdin Biochemical Technology Co. Isatin (K27M11L110347) was purchased from Shanghai Yuanye Biotechnology Co. BCA Protein Concentration Measurement Kit (20230418) and Nuclear Protein Extraction Kit (2308002) were purchased from Solarbio; NF-κB Antibody (8242 S), p-IκBα Antibody (9246 S), p-P38 Antibody (4511T) were purchased from Cell Signaling Technology; IκBα antibody (00125930), P38 antibody (00133254), JNK antibody (00023349), ERK antibody (00087180), p-ERK antibody (00097415), iNOS antibody (0008037) were purchased from Wuhan, ProteinTech Group Inc. p-STAT3 antibody (BB05272132) was purchased from Beijing Boao Sen Biotechnology Co. p-JNK antibody (R06281813) was purchased from Wanban Biotechnology Co; RIPA lysate (AR0102-100), STAT3 antibody (ZP2172BP72), Histone 3 antibody (BOS9110BP5687), and β-Actin antibody (BST17353873) were purchased from PhD Bio; IL-1β ELISA kit (M230112-001b), IL-10 ELISA kit (M230403-005a), TNF-α ELISA kit (M230403-102a), IL-6 ELISA kit (M231229-004a) were purchased from Xinbosheng Biotechnology Co.

Cell culture and assay

RAW264.7 macrophages along with their specialized medium (WHAA23U143, DMEM High Glucose supplemented with 10% serum and 1% dual antibiotics) were sourced from Wuhan Punosai Life Science and Technology Co., Ltd. Cells were maintained in standard culture conditions, specifically at 37 °C with an atmosphere with 5% CO₂ and subcultured at a 1:3 ratio upon reaching 80–90% confluency. The cellular administration concentration was chosen via the MTT (5 mg/mL) assay and by measuring nitric oxide (NO) levels. During the MTT test, we seeded the cells at a concentration of 1.5 million cells per milliliter in 96-well dishes, adding 100 µl to each well. After the cells had firmly attached themselves to the plate, cynosure solutions were applied. Once the 20-hour gestation period had elapsed, a mere 10 µL of the MTT cocktail was carefully introduced. Then, posthaste, a smidgen of dimethyl sulfoxide (DMSO) was poured in after 4 h had passed. To assess the NO levels: The cells (a robust 3.0 × 10⁵ per well) were seeded in their respective 96-well cradles. Next, the 50 µL of the resulting supernatant was blended with a proportional amount of the sulfanilamide concoction. Post-incubation for a quick 10 min in the shadowy depths, 50 µL of the N-(1-naphthyl) ethylenediamine potion was added to the mix. With the microplate reader set to its designated frequency of 562 nm, the absorbance was meticulously gauged, adhering to the esteemed Griess protocol.

Animal research

Fifty SPF C57BL/6 male mice aged 6–8 weeks weighting 20 ± 2 g were employed in the research. All experimental animals and related materials (feed and bedding) were procured from Chengdu Dashuo Experimental Animal Co., Ltd. (Sichuan, China; Animal License No. SCXK(Chuan) 2020-030). Animals were housed in an SPF-grade facility (Approval No. SYXK(Shaanxi) 2017-004) at Shaanxi University of Traditional Chinese Medicine (Animal Ethics Approval No. SUCMDL20231228001). Animals were conducted in accordance with ARRIVE guidelines (http://arriveguidelines.org). All methods were performed in accordance with the relevant guidelines and regulations.

Ulcerative colitis

All mice were kept in a meticulously controlled setting, ensuring a temperature of 24.0 ± 0.3 °C and a relative humidity of 54 ± 1%, with a cycle that alternated between 12 h of light and 12 h of darkness. Post a single-week adjustment period and divided mice into five separate groups: (1) control (CON), (2) DSS-induced model (Model), (3) positive control receiving 125 mg/kg sulfasalazine (SASP), (4) IN group (200 mg/kg)¹⁹, and (5) mixed treatment group (MIX) receiving 27.74 mg/kg indigo, 0.3 mg/kg indirubin, and 0.22 mg/kg isatin suspension. DSS-induced UC model was established using 2.5% DSS, Control and DSS groups received daily oral gavage of sterile water, whereas treatment groups were administered IN and MIX once daily through oral gavage. Daily body weight measurements and condition were recorded for DAI score. Taking blood after 1% sodium pentobarbital anesthesia. Blood samples were obtained through cardiac puncture and the serum was centrifuged at 4000 r/min for 10 min at 4 °C and then preserved at −80 °C. Colon tissue samples were treated with 4% paraformaldehyde by weight for H&E staining, whereas corresponding specimens were frozen in liquid nitrogen and then kept at a frosty − 80 ℃ for the later measurement of MPO activity.

Colon histopathology analysis

Colon samples were prepared for histological analysis by being embedded in paraffin. Subsequently, the paraffin blocks were treated with water to remove paraffin, followed by a hematoxylin dip to color the nuclear material. Next, the samples were cleaned with flowing tap water; then, the cytoplasmic elements were colored using eosin . Following the coloration, the sections underwent dehydration in ethanol, put into xylene transparent treatment, dried and dripped with neutral gum to seal and fix the sections, and examined microscopically. The Histological Activity Index (HAI) was confirmed by aggregating the epithelial damage scores (ranging from 0 to 3) and inflammatory infiltration (also ranging from 0 to 3)²⁵.

Enzyme-linked immunosorbent assay

The levels of IL-1β, IL-6, TNF-α, IL-10 were measured using ELISA kits according to the manufacturer’s instructions. For in vitro studies, cytokines were assayed in cell culture supernatants. For in vivo studies, cytokines were determined in mouse serum samples.

Measurement of MPO activity in colon tissue

Mice’s colon tissue snippets, stored in sub-zero conditions at a frosty − 80 ℃, were delicately cleaned with cool phosphate-buffered saline. Post-rinse, they were carefully dabbed with filter paper until dry and measured to create a 5% homogenate, which involves a 1:19 tissue-to-solution proportion. After adding Reagent III, the solution was vigorously vortexed and then incubated at 37 °C, the body temperature, for 15 min.

Western blot analyses

Total protein was obtained using RIPA lysis buffer, while nuclear proteins were isolated with PMSF. Protein levels were assessed via a BCA protein quantification kit, while the BSA calibration curve was established using the Lowry protein concentration kit, protein concentration was calculated, and protein up-sampling volume was calculated by the up-sampling volume of 25 µg. The 12% SDS-polyacrylamide gels were employed to separate the protein lysates, and then subjected to electrophoretic transfer onto 0.45-micrometer PVDF membranes from Merck Millipore. Subsequently, these membranes were soaked in a 5% (weight by volume) non-fat dry milk solution, which was a blocking agent, for a duration of two hours at room temperature, which is 25 ℃. The solution was a Tris-buffered saline (TBST) mixture containing 0.1% Tween-20. Following that, the membranes were gently washed three times for ten minutes each, using the TBST solution. Finally, they were treated with primary antibodies that specifically target NF-κB p65.(1:1000), IκBα(1:5000), H3(1:500), p-IκBα(1:1000), STAT3(1:500), p-STAT3(1:500), ERK(1:2000), p-ERK(1:1000), p38(1:2000), p-P38(1:1000), JNK(1:500), p-JNK(1:300), iNOS(1:500), β-Actin(1:2000) diluted in blocking buffer and kept at 4 °C overnight. The membranes were left to incubate for a solid hour at room temp, around 25 °C, using these secondary antibodies from goat that are anti-mouse/rabbit and tagged with horseradish peroxidase (HRP). They were properly diluted at a ratio of 1:5000. After the incubation, we gave them a good rinse three times with TBST. Employed for protein band visualization, the Thermo Fisher Scientific’s 10-minute-long ECL substrate was utilized, followed by imaging with the Bio-Rad ChemiDoc MP Imaging System. Density measurements were analyzed with Image Lab 6.1 software. Preheat the machine for 30 min before washing the secondary antibody → Turn on the computer → Open Image lab → Create a new file → Imprint → Chemi → Uncheck “Highlight saturated pixels” → Set “Signal accumulation mode”: Input 1 for the time of the first image, 30 for the last image, and 30 for the total number of images. Click “OK” → Run the experimental protocol → Move the filter to the blank position (the black piece on the top is closest to the outside), click “OK” → Observe the image time before the red dots (overexposure) appear → Select the corresponding manual exposure time, run the experimental protocol, minimize → Repeat the “Signal accumulation mode” selection 3 times, select the corresponding 3 required images and save.

Statistical analysis

GraphPad Prism 8 software was used to calculate and plot all data. The results were presented as mean ± standard deviation (SD). One-way ANOVA was employed to compare differences between groups, with P < 0.05 indicating statistical significance.

Results

Effects of IN and its active components on the growth and NO content of RAW264.7 cells

We systematically evaluated the cytotoxicity of IN and its active components using the MTT assay to establish the optimal compound concentrations. Macrophage viability remained > 80% at concentrations up to 80 µg/mL for IN, up to 20 µg/mL for indigo, and up to 40 µg/mL for indirubin and isatin. (Fig. 1A). Subsequent NO inhibition assays in LPS-stimulated RAW264.7 cells revealed significant suppression by indigo at 40 µg/mL and 80 µg/mL, indirubin at 20–40 µg/mL, and isatin at 10–60 µg/mL (Fig. 1B). However, when the concentration of ingigo was 40 to 80 µg/mL, the cell inhibition rate was less than 80%. At a concentration of 20 µg/mL, ingigo showed no anti-inflammatory activity, so no further single research was conducted.

Fig. 1.

(A) IN, indigo, indirubin and isatin on the viability of RAW264.7 cells (B) Effects of different concentrations of IN, indigo, indirubin and isatin on the production of NO. (n = 3) ^## P < 0.01 vs. CON; ** P < 0.01, * P < 0.05 vs. LPS.

Based on these dose-response relationships, working concentrations were established as follows: IN (40 and 80 µg/mL), indirubin (20 and 40 µg/mL), and isatin (10, 20, and 40 µg/mL).

We investigated the synergistic effects of the bioactive constituents of IN (indigo, indirubin, and isatin) to elucidate its anti-inflammatory mechanisms. Concentrations matching the pharmacological ratio of IN (80 µg/mL IN; indigo, 11.096; indirubin, 0.12; and isatin, 0.088 µg/mL) for pairwise and triple combinations revealed no significant inhibition of LPS-induced RAW264.7 cell proliferation (Fig. 2A). Additionally, NO assays showed that the MIX (11.096 µg/mL indigo + 0.12 µg/mL indirubin + 0.088 µg/mL isatin) synergistically enhanced NO suppression compared to individual compounds. However, no detectable anti-inflammatory effects were observed in pairwise tests (Fig. 2B).

Fig. 2.

(A) RAW264.7 cells viability (B) Effects of the three constituents compatibility on NO production in RAW264.7 cells (n = 3) A: IN (80 µg/mL) B: indigo (11.096 µg/mL); C: indirubin (0.12 µg/mL); D: isatin (0.088 µg/mL); E: indigo + indirubin + isatin (11.096 µg/mL + 0.12 µg/mL + 0.088 µg/mL); F: indigo + indirubin(11.096 µg/mL + 0.12 µg/mL); G: indigo + isatin (11.096 µg/mL + 0.088 µg/mL); H: indirubin + isatin (0.12 µg/mL + 0.088 µg/mL). ^## P < 0.01 vs. CON; ** P < 0.01, * P < 0.05 vs. LPS.

Effects of IN and its active ingredients on the expression of inflammatory cytokines

The ELISA test results demonstrated that IN, indirubin, and isatin effectively significantly reduced the production of pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α. Meanwhile, these compounds boosted the output of the cytokine IL-10, which has anti-inflammatory properties. (Fig. 3).

Fig. 3.

Effects of IN, indirubin, and isatin on inflammatory cytokines. (n = 3) ^## P < 0.01, ^#P < 0.05 vs. CON; ** P < 0.01, * P < 0.05 vs. LPS.

Effects of IN and its active ingredients on MAPK, STAT3 and NF-κB pathways

To further investigate the effects of IN and its active components on these inflammation-related pathways, western blotting was performed to assess changes in protein expression.

Compared with the CON group, LPS-stimulated RAW264.7 cells exhibited increase in nuclear NF-κB protein expression and decrease in cytoplasmic NF-κB protein levels. Moreover, the expression of p-IκBα, p-STAT3, inducible nitric oxide synthase (iNOS), p-P38, p-JNK, and p-ERK was markedly upregulated.

Nuclear NF-κB protein expression was reduced relative to the LPS group, whereas cytoplasmic levels were restored subsequent to treatment with IN. Concurrently, the expression of p-IκBα, p-STAT3, iNOS, p-P38, p-JNK, and p-ERK was significantly suppressed. These findings demonstrate that IN effectively reduces inflammatory responses by targeting the MAPK, STAT3, and NF-κB pathways (Fig. 4A–C).

Fig. 4.

Effects of IN on the relative protein expression of NF-κB/MAPK/STAT3 signaling pathway in LPS-induced RAW264.7 cells. (n = 3) ^## P < 0.01, ^#P < 0.05 vs. CON; ** P < 0.01, * P < 0.05 vs. LPS.

The levels of nuclear NF-κB and p-IκBα were reduced relative to the LPS group, whereas cytoplasmic NF-κB expression was elevated following indirubin treatment. At the same time, the levels of expression of p-STAT3, p-P38, and iNOS were reduced in comparison to the LPS group. The findings suggest that indirubin inhibited the activation of NF-κB and STAT3 pathways, and lowered p-P38 level within the MAPK signaling cascade. (Fig. 5A–C).

Fig. 5.

Effects of indirubin on the relative protein expression of NF-κB/MAPK/STAT3 signaling pathway in LPS-induced RAW264.7 cells. (n = 3) ^## P < 0.01, ^#P < 0.05 vs. CON; ** P < 0.01, * P < 0.05 vs. LPS.

The nuclear levels of NF-κB and p-IκBα were decreased in comparison to the LPS group, while the expression of cytoplasmic NF-κB was increased after treatment with isatin. Additionally, the levels of p-ERK, p-P38, and p-JNK were notably reduced in comparison to the LPS group. These data indicate that isatin inhibits the activation of MAPK and NF-κB signaling pathways induced by LPS in RAW264.7 cells (Fig. 6A–C).

Fig. 6.

Effects of isatin on the relative protein expression of NF-κB/MAPK/STAT3 signaling pathway in LPS-induced RAW264.7 cells. (n = 3) ^## P < 0.01, ^#P < 0.05 vs. CON; ** P < 0.01, * P < 0.05 vs. LPS.

Our initial analyses revealed that the MIX group could effectively reduce the NO content. Consequently, we investigated how MIX influences protein expression that have been exposed to LPS, aiming to further understand the mechanism behind the synergistic effects of the three active components. Western blot results demonstrated noticeably reduced levels of p-STAT3, p-P38, and iNOS expression in the MIX-treated group versus the LPS group. These results indicate that MIX attenuates inflammation by inhibiting the activation of the STAT3 signaling pathway and reducing p-P38 expression in the MAPK pathway (Fig. 7A–C).

Fig. 7.

Effects of MIX on the relative protein expression of NF-κB/MAPK/STAT3 signaling pathway in LPS-induced RAW264.7 cells. (n = 3) ^## P < 0.01, ^#P < 0.05 vs. CON; ** P < 0.01, * P < 0.05 vs. LPS.

MIX protected against DSS-induced UC mice

Effects of MIX on colon length in mice subjected to DSS-induced UC

The colon lengths in the model group mice were markedly shorter. The DSS -induced colon shortening was ameliorated in the IN and MIX groups (Fig. 8A).

Fig. 8.

Effects of IN and MIX on UC mice. (n = 6) (A) Length of the colon (B) Histopathological sections of colon tissues with H&E staining. (C) DAI score. (D) MPO activity. (E) Histopathologic scoring of the colon. (F-H) Effect of serum inflammatory cytokines. SASP (125 mg/kg); IN (200 mg/kg); MIX (27.74 µg/mL indigo + 0.3 µg/mL indirubin + 0.22 µg/mL isatin); ^##P < 0.01 vs. CON; ^**P < 0.01 ^*P < 0.05 vs. Model.

Effects of MIX on histopathological changes in the colon of mice with UC

Histopathological analysis revealed intact colonic tissue architecture and well-preserved crypt organization in mice in the control group, with no evidence of inflammatory infiltration. Marked inflammatory cell infiltration was observed in the colonic tissues in the model group, accompanied by crypt atrophy, upward displacement, structural disorganization, and a significant reduction in goblet cells. Partial restoration of the colonic and crypt architecture was observed following treatment with IN and MIX, coupled with a considerable attenuation of inflammatory cell infiltration (Fig. 8B). These findings indicate that IN and MIX treatment ameliorated DSS-induced colonic inflammation and partially mitigated tissue damage in UC mice. Both IN and MIX treatments significantly reduced HAI scores compared to the model group, with similar efficacies observed between the two treatments. (Fig. 8E).

Effects of MIX on DAI scores in DSS-induced UC mice

The model group mice displayed a statistically substantial rise in DAI scores versus the control group, validating the successful induction of colitis. Although DAI scores progressively increased in all treatment groups, the rate of increase was significantly attenuated in the IN and MIX cohorts in comparison with the model group (Fig. 8C).

Effects of MIX on MPO activity in colonic tissues of UC mice

MPO enzyme levels and activity are established biomarkers of the functional activation status of neutrophils. The analysis revealed that colonic MPO activity in the DSS-induced UC model group was significantly higher than in healthy controls. (Fig. 8D). MPO activity was significantly reduced the IN and MIX groups contrasted with the model group. Moreover, both IN and MIX groups exhibited decreased MPO activity with similar effects observed between the two groups.

Effects of MIX on inflammatory cytokines

ELISA tests showcased that MIX was quite effective in halting the discharge of pro-inflammatory cytokines, like IL-6 and TNF-α, all the while boosting the production of the anti-inflammatory cytokine IL-10. (Fig. 8F–H)

Discussion

The mechanisms driving UC development is primarily closely related to dysregulated intestinal inflammatory responses. In addition, the primary therapeutic objective in early-stage UC is to achieve clinical remission by mitigating colonic inflammation²⁶. IN and its principal bioactive component, indigo, ameliorate colonic inflammation via multiple mechanisms^18,27,28. Our preliminary LC-MS analyses of IN identified indigo, indirubin, and isatin as its main active components. However, whether isatin has anti-inflammatory activity, and the mechanism through which these components exert anti-inflammatory activity remain unclear. Based on our knowledge, the present study represents the first systematic investigation of the material basis and anti-inflammatory efficacy of IN against UC, which is based on the three active components identified during preliminary analyses.

iNOS produces NO, which participates in physiological regulation and is overexpressed in inflammatory pathologies. LPS-stimulated macrophages secrete excessive amounts of iNOS, consequently promoting inflammation²⁹. The anti-inflammatory activities of indirubin, and isatin required concentrations exceeding 20, and 10 µg/mL, respectively. However, their levels in IN are lower (11.096, 0.12, and 0.088 µg/mL, respectively). We evaluated pairwise (indigo-indirubin, indigo-isatin, indirubin-isatin) and ternary (MIX) combinations using 80 µg/mL IN (with acceptable viability) and individual components at IN concentrations (11.096 µg/mL indigo, 0.12 µg/mL indirubin, and 0.088 µg/mL isatin). MIX treatment significantly suppressed NO secretion. This suggests these components synergistically confer the anti-inflammatory activity of IN.

Macrophages, which are ubiquitously distributed in human tissues and functionally diverse, polarize into pro-inflammatory M1 phenotypes upon activation by stimuli such as LPS and IFN-γ³⁰. Pro-inflammatory cytokines are secreted by M1 macrophages that play crucial roles in UC development³¹. TNF-α and IL-1β share functional similarities with IL-6 in amplifying inflammatory cascades and recruiting inflammatory cells³². Conversely, IL-10, acting as an anti-inflammatory cytokine, attenuates the activity of proinflammatory cytokines. In this study, IN and its active components exerted strong anti-inflammatory effects by decreasing the levels of IL-1β, IL-6, and TNF-α while upregulating that of IL-10 in vitro.

To investigate the the mechanisms underlying the anti-inflammatory effects of IN and its active components, we investigated their effects on the expression of proteins in RAW264.7 cells stimulated with LPS. LPS-induced M1 macrophage polarization is closely associated with the NF-κB and STAT3 pathways³³. NF-κB, MAPK, and STAT3 pathways are closely connected in inflammation³⁴. NF-κB, a critical regulator of inflammatory processes, immunity, and acute phase responses in diverse cells, is induced by cytokines such as LPS³⁵. In this study, LPS promoted IκBα phosphorylation by its kinase, thereby enabling activated NF-κB nuclear translocation and inflammatory signal amplification. LPS-activated MAPKs regulate pro-inflammatory transcription factors via phosphorylation, consequently promoting inflammatory cell differentiation and cascade activation. This process involves the p-JNK, p-ERK, and p-P38. Furthermore, IN inhibited activation induced by LPS of the MAPK, STAT3, and NF-κB pathways. Indirubin inhibited p-P38 expression across MAPK and suppressed the activation of STAT3 and NF-κB, key pathways in inflammation. By contrast, isatin selectively targeted the MAPK and NF-κB pathways.

We investigated the relationship between these three active ingredients and verified the effects of IN and MIX on UC mice. UC is characterized by crypt abscess formation via inflammatory cell infiltration (primarily neutrophils and T-lymphocytes) into the intestinal crypts through the interstitial spaces of compromised epithelial cells³⁶. DSS damages the basal crypt epithelial cells to impair mucosal barrier integrity and induce intestinal inflammation characterized³⁷. DSS-treated mice exhibited elevated DAI scores, including weight loss, lethargy, social withdrawal, and hematochezia. Histopathology revealed severe crypt distortion with apical displacement and irregular borders, accompanied by substantial neutrophil accumulation. Sulfasalazine is a classic and well-established therapeutic agent for this specific disease model and it is a well-established and classic first-line drug for the treatment of UC^38–40. Our research indicates that MIX partially restored the colonic architecture and attenuated inflammatory cell infiltration, demonstrating its therapeutic potential. In UC pathogenesis, TNF-α synergizes with cytokines to drive the production of inflammatory mediators, whereas IL-6 overexpression induces electrolyte secretion, enhances mucosal permeability, and promotes cell infiltration. MIX attenuated inflammation by suppressing pro-inflammatory cytokines while upregulating the expression of anti-inflammatory cytokine. MPO, a quantitative biomarker of inflammatory severity, was markedly increased in UC mice⁴¹. However, MIX significantly reduced MPO levels.

Collectively, IN demonstrates substantial anti-inflammatory effects at a concentration of 80 µg/mL. At this concentration, the levels of its main components—indigo, indirubin, and isatin—are 11.096 µg/mL, 0.12 µg/mL, and 0.088 µg/mL, respectively, accounting for 13.87%, 0.15%, and 0.11% of the total composition. When administered individually at these concentrations, these three components showed no anti-inflammatory activity. However, when combined in the same ratio as in IN (indigo: indirubin: isatin = 11.096:0.12:0.088), the resulting mixture, designated as MIX, exhibited anti-inflammatory activity equivalent to that of IN at 80 µg/mL These findings point to the fact that the three-component mixture of indigo, indirubin, and isatin constitutes a pharmacologically active basis for the therapeutic effects of IN in UC management.

Conclusion

Overall, our work demonstrated the protective effects of IN on UC. The active components of this TCM—indigo, indirubin, and isatin—improved inflammation. In addition, their combination (MIX) synergistically protects mice against UC. Furthermore, their mechanisms were either wholly or partially related to NF-κB, STAT3, and MAPK. As far as we are aware, this research is the initial comprehensive examination of the material foundation and anti-inflammatory effectiveness of IN in relation to UC.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

IN

Indigo naturalis

UC

Ulcerative colitis

DSS

Dextran sulfate sodium

NO

Nitric oxide

DAI

Disease activity index

HAI

Histological activity index

MPO

Myeloperoxidase

H&E

Haematoxylin‒eosin

TCM

Traditional Chinese medicine

MIX

Indigo, indirubin, and isatin

IL-6

Interleukin 6

IL-1β

Interleukin 1 beta

TNF-α

Tumour necrosis factor-α

IL-10

Interleukin 10

PBS

Phosphate-buffered saline

LPS

Lipopolysaccharide

DMSO

Dimethyl sulfoxide

iNOS

Inducible nitric oxide synthase

Author contributions

KM, BH and AF: Writing – original draft, methodology, data curation. AH and BL: validation, formal analysis. YL: Writing – review & editing, supervision, investigation. ZW: Writing – review & editing, project administration, funding acquisition.

Funding

This project was funded by the Qinchuangyuan Traditional Chinese Medicine Industry Innovation Agglomeration Area Project (L2024-QCY-ZYYJJQ-X195), Innovation Team Project of Shaanxi Provincial Department of Education (24JP047). Xianyang Science and Technology Bureau Innovation Capability Support Program-Special Project for Scientific and Technological Innovation Talents (S2025-CXNL-KJRC-4294)

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.