Pharmacological properties study of indirubin

Introduction

Indirubin (Figure 1), a 3,20 bis-indole isomer of indigo, is an active ingredient of Danggui Longhui Wan, a traditional Chinese medicine derived from a mixture of plants including Indigofera tinctoria L., Isatis tinctoria L., Strobilanthescusia Kuntze, and Cnidii fructus.The molecular formula of indirubin is 3-(3-oxo-1H-indol-2-ylidene)−1H-indol-2-one, which is present not only in Indigo naturalis but also in mollusks, human urine, and various bacteria. In addition, indirubin has also been extracted from the traditional Chinese medicines Radix Isatidis and Folium Isatidis. Indirubin exhibits anti-leukemic, anti-proliferative, and hepatoprotective properties and is also a strong anti-inflammatory agent.The medicinal value of indirubin was discovered early on, and indirubin is mainly extracted from plants high in indigo. Due to the low content of indigo in plants, poor water solubility and high cost of extraction and separation, the yield of this method is low, and most of the methods are synthetic. At present, the synthesis of indirubin mainly involves the condensation reaction of isatin and indoxyl acetate . This method is simple to perform with mild conditions and high yield. A one-step reduction coupling method using KBH4 for the preparation of indirubin has been reported in recent years. This method can achieve 90% yield, is simple to perform, and has more potential than traditional synthesis methods.[1]

Pharmacological properties study

Indirubin Inhibits LPS-Induced Inflammation

Indirubin plays an important role in the treatment of many chronic diseases and exhibits strong anti-inflammatory activity. However, the molecular mode of action during mastitis prophylaxis remains poorly understood. In this study, a lipopolysaccharide (LPS)-induced mastitis mouse model showed that indirubin attenuated histopathological changes in the mammary gland, local tissue necrosis,and neutrophil infiltration. Moreover, indirubin significantly downregulated the production of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α). Lai et al. explored the mechanism whereby indirubin exerts protective effects against LPS-induced inflammation of mouse mammary epithelial cells(MMECs). The addition of different concentrations of indirubin before exposure of cells to LPS for1 h significantly attenuated inflammation and reduced the concentrations of the three inflammatory cytokines in a dose-dependent manner. Indirubin downregulated LPS-induced cyclooxygenase-2 (COX2) and Toll-like receptor 4 (TLR4) expression, inhibited phosphorylation of the LPS-induced nuclear transcription factor-kappa B (NF-kB) P65 protein and its inhibitor IkBα of the NF-kB signaling pathway. Furthermore, indirubin suppressed phosphorylation of P38, extracellular signal-regulated kinase (ERK), and c-Jun NH2-terminal kinase (JNK) of the mitogen-activated protein kinase (MAPK)signal pathways. Thus, indirubin effectively suppressed LPS-induced inflammation via TLR4 abrogation mediated by the NF-kB and MAPK signaling pathways and may be useful for mastitis prophylaxis.[2]

Indirubin Inhibits TRAIL-Induced Activation of Death Receptor 5

Death receptor 5 (DR5) is an apoptosis-inducing membrane receptor that mediates cell death in several life-threatening conditions. There is a crucial need for the discovery of DR5 antagonists for the therapeutic intervention of conditions in which the overactivation of DR5 underlies the pathophysiology. DR5 activation mediates cell death in non-alcoholic fatty liver disease (NAFLD) and neurodegenerative processes including amyloid-beta (Aβ) accumulation, spinal cord injury (SCI), and brain ischemia. In the current work, we used fluorescence resonance energy transfer (FRET) to monitor the conformational dynamics of DR5 that mediate death signaling. We used a time-resolved FRET screening platform to screen the Selleck library of 2863 U.S. Food and Drug Administration (FDA)-approved compounds. The high-throughput screen (HTS) identified 13 compounds that modulated the FRET between DR5 monomers beyond 5 median absolute deviations (MADs) from the DMSO controls. Of these 13 compounds, indirubin was identified to specifically inhibit tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced caspase-8 activity without modulating DR5 surface expression or TRAIL binding. Indirubin inhibited Fas-associated death domain (FADD) oligomerization and increased cellular FLICE-inhibitory protein (c-FLIP) expression; both are molecular mechanisms involved in inhibiting the DR5 signaling cascade. This study has elucidated previously unknown properties of indirubin that make it a promising candidate for therapeutic investigation of diseases in which overactivation of DR5 underlies pathology.[3]

Indirubin induces tolerogenic dendritic cells

Allergic asthma is a chronic bronchial inflammatory disease closely associated with abnormal immune responses of dendritic cells (DCs) and allergen-specific type 2 T helper (Th2) cells. Indirubin (IR), a natural aryl hydrocarbon receptor (AhR) ligand, exerts anti-inflammatory and immunomodulatory properties. In this study, Chuang et al. aimed to clarify whether indirubin exhibits immunomodulatory action on DCs via AhR activation and investigated the antiallergic effects of indirubin in a mouse model of allergic asthma. Lipopolysaccharide (LPS)-activated bone marrow-derived DCs were treated with indirubin. Their mRNA expressions, cytokine production, and phenotype patterns were determined by a quantitative real-time PCR, ELISA, flow cytometry, and RNA sequencing. The mixed lymphocyte reaction was utilized to evaluate the regulatory function of IR-treated DCs on T-cell differentiation. Moreover, mice with ovalbumin (OVA)-induced allergic asthma were treated with indirubin. Thereafter, the airway hyperresponsiveness (AHR), allergen-specific IgE production, cytokine levels, airway inflammation, and T-cell responses were evaluated. Treatment of LPS-stimulated DCs with 20 μM indirubin significantly reduced IL-12 and TNF-α production while increasing IL-10 secretion. Meanwhile, these DCs expressed decreased levels of CD80 but increased levels of Jagged 1 surface molecules. However, the effects of IR on DCs were reversed by pretreatment with the AhR antagonist, CH223191. Additionally, the coculture of these tolerogenic-like DCs with allogeneic CD⁴⁺T cells promoted the generation of Foxp³⁺ regulatory T (Treg) cells. A transcriptomic analysis identified several downregulated genes that are involved in regulating cell migration, cytokine secretion, and inflammatory responses in DCs after IR treatment. In an asthmatic murine model, oral administration of 25 mg/kg body weight of IR efficiently alleviated the development of AHR, OVA-specific IgE production, and levels of Th2-type cytokines (IL-4, IL-5, and IL-13) and the CCL11 chemokine. IR treatment also attenuated inflammatory cell recruitment and mucus production in the lungs. Notably, an enhanced frequency of Foxp³⁺ Treg cells and reduced effector T-cell proliferation associated with increased levels of IL-10 and TGF-β were observed in IR-treated mice. Indirubin can induce tolerogenic-like BMDCs which promote the differentiation of Treg cells. Importantly, the expansion of Foxp³⁺ Treg cells contributed to the therapeutic efficacy of indirubin against allergic asthma.[4]

Conclusion

Multiple studies have shown that indirubin exhibits good anticancer, anti-inflammatory and neuroprotective properties.

References

1.Yang L, Li X, Huang W, Rao X, Lai Y. Pharmacological properties of indirubin and its derivatives. Biomed Pharmacother. 2022;151:113112. doi:10.1016/j.biopha.2022.113112

2.Lai JL, Liu YH, Liu C, et al. Indirubin Inhibits LPS-Induced Inflammation via TLR4 Abrogation Mediated by the NF-kB and MAPK Signaling Pathways. Inflammation. 2017;40(1):1-12. doi:10.1007/s10753-016-0447-7

3.Young MC, Vunnam N, Rebbeck RT, Yuen SL, Thomas DD, Sachs JN. Indirubin Inhibits TRAIL-Induced Activation of Death Receptor 5 in Jurkat Cells. Nat Prod Commun. 2023;18(1):10.1177/1934578x221144580. doi:10.1177/1934578x221144580

4.Chuang HC, Chuang KJ, Cheng PC, Hsieh CL, Fan YY, Lee YL. Indirubin induces tolerogenic dendritic cells via aryl hydrocarbon receptor activation and ameliorates allergic asthma in a murine model by expanding Foxp3-expressing regulatory T cells. Phytomedicine. 2024;135:156013. doi:10.1016/j.phymed.2024.156013

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