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Background: Chronic lymphocytic leukemia (CLL) primarily affects the elderly, with its etiology largely unknown. It is hypothesized that hematopoietic stem cells may acquire mutations over time, such as the BCL-2 mutation, leading to disruptions in the apoptotic process. Dangui Luhui Wan , a mixture of 11 herbs used in Chinese Medicine, has shown antitumor activities across various cancer cell types. Indirubin-3'-monoxime (I3M), derived from Dangui Luhui Wan , functions as a selective inhibitor of cyclin-dependent kinases (CDKs) and can induce apoptosis in cells. Objectives: The objective of this study was to evaluate the effectiveness of I3M against CLL cells in vitro. Methods: Peripheral blood mononuclear cells (PBMCs) from 14 patients were treated with I3M at concentrations ranging from 0.1 μM to 80 μM over periods of 24, 48, and 72 hours. The optimal dose was determined using Annexin V and MTT assays. The expression of apoptotic genes Bcl-2/Bax and CDK1/2 was assessed using real-time PCR. Results: The results indicated that a 20 µM concentration of I3M exhibited the highest cytotoxicity after 48 hours compared to controls (P = 0.005). Post-treatment, a decrease in Bcl-2 gene expression was observed, while changes in the Bax gene were not significant. However, an increase in the Bax/Bcl-2 gene ratio was noted, suggesting involvement of the mitochondrial pathway in I3M's apoptotic mechanism. Notably, I3M inhibited the expression of the CDK2 gene but did not affect CDK1 gene expression. Conclusions: I3M appears to exert anti-tumor effects by inducing apoptosis and inhibiting the CDK2 gene. Further research is required to elucidate the precise mechanism of action of I3M in CLL and potentially other tumor cell lines. [ABSTRACT FROM AUTHOR]

Indirubin, indol-3-carbinol and beta-naphthoflavone have been proposed as potential agents for treating a variety of cancers. These compounds are well characterized AHR ligands. AHR is a ligand-dependent cytosolic transcription factor that induces the transcription of target genes, such as the cytochromes P450 (CYP). The CYP enzymes are involved in the metabolic activation of anticancer drugs, hinting at their potential involvement in both the development and treatment of cancer. Our research focused on the AHR/CYP1 signaling pathway in human cell lines (HEK293, PC3, Mcf7, Sus/fP2), examining its effects induced by ligands. We assessed how these substances affected the expression of CYP1 genes and explored the correlation between these responses and changes in AHR protein levels in human cell lines. The findings reveal that the AHR/CYP1 signaling pathway remains functional in PC3 and Sus/fP2. In HEK293 cells, the AHR protein levels exhibited no change following to each ligand and the CYP1 gene expression was downregulated. However, in Mcf7 cells, a high level of CYP1 responsiveness was observed along with unchanged AHR protein levels after exposure to ligands that correlated with ER alpha expression levels. These findings underscore the importance of considering the AHR/CYP1 signaling pathway when developing new strategies for cancer treatment. [ABSTRACT FROM AUTHOR]

Indigo, indirubin, and isoindigo derivatives have been used for centuries as pigments. Since the 1990s, a new aspect of the chemistry of this type of compounds is their activity against various types of cancer. N-Glycosides of indigo, indirubin, and isoindigo, blue, red, and yellow sugars, turned out to be of special interest because of their high cancerostatic activity and structural novelty. The present article provides an account on the synthesis and anticancer activity of these compounds.

Anxiety is a natural response to stress, characterized by feelings of worry, fear, or unease. The current research was conducted to investigate the anxiolytic effect of indirubin (IND) in different behavioral paradigms in Swiss albino mice. To observe the animal's behavioural response to assess anxiolytic activity, different tests were performed, such as the open‐field (square cross, grooming, and rearing), swing, dark‐light, and hole cross tests. The experimental mice were administered IND (5 and 10 mg/kg, p.o.), where diazepam (DZP) and vehicle were used as positive and negative controls, respectively. In addition, a combination treatment (DZP+IND‐10) was provided to the animals to determine the modulatory effect of IND on DZP. Molecular docking approach was also conducted to determine the binding energy of IND with the GABAA receptor (α2 and α3 subunits) and pharmacokinetics were also estimated. The findings revealed that IND dose‐dependently significantly (p<0.05) reduced the animal's movement exerting calming behavior like DZP. IND also demonstrated the highest docking score (−7.7 kcal/mol) against the α3 subunit, while DZP showed a lower docking value (−6.4 kcal/mol) than IND. The ADMET analysis revealed that IND has proper drug‐likeness and pharmacokinetic characteristics. In conclusion, IND exerted anxiolytic effects through GABAergic Pathways. [ABSTRACT FROM AUTHOR]

Being a constitutional isomer of the indigo dye, indirubin has been known as a purple colourant for a long time. On the other hand, its structural and photophysical properties as ligand in metal complexes are virtually unexplored. Herein, for the first time, we utilized indirubin as ancillary chelator to develop two new heteroleptic iridium(III) complexes equipped with two 2‐phenylpyridine units as archetypical cyclometalating ligands. These new complexes display fully reversible oxidation and reduction processes, and show a panchromatic absorption extending up to 900 nm in acetonitrile solution at 298 K. Moreover, an unexpected broad and unstructured emission band is detected for both complexes in the near‐infrared region, peaking at approx. 1100 nm and having a lifetime of approx. 60 ps; such an emission is attributed to a ligand‐centred triplet excited state (3LC) located on the indirubin ligand itself, as proved by DFT calculations. These findings may pave the way for further exploration of the indirubin dye as a ligand for different types of metal centres to create efficient near‐infrared triplet emitters without the need for difficult synthetic procedures. [ABSTRACT FROM AUTHOR]

The reaction of indigo with two equivalents of the electrophile ethyl bromoacetate with caesium carbonate as a base result in the formation of structurally complex polyheterocyclics, including a fused spiroimidazole and a spiro[1,3]oxazino derivative, together with a biindigoid-type derivative, through a convenient one-pot reaction. Further assessment of the reaction using five equivalents of the electrophile gave rise to other molecules incorporating the 2-(7,13,14-trioxo-6,7,13,14-tetrahydropyrazino[1,2-a:4,3-a′]diindol-6-yl) scaffold. The reaction of ethyl bromoacetate with the less reactive indirubin resulted in the synthesis of three derivatives of a new class of polyheterocyclic system via a cascade process, although yields were low. These compounds were derived from the parent indolo[1,2-b]pyrrolo[4,3,2-de]isoquinoline skeleton. Despite the modest yields of the reactions, they represent quick cascade routes to a variety of heterocycles from cheap starting materials, with these structures otherwise being difficult to synthesise in a traditional stepwise manner. These outcomes also contribute significantly to the detailed understanding of the indigo/indirubin cascade reaction pathways initiated by base-catalysed N-alkylation. [ABSTRACT FROM AUTHOR]

This study focused on the development of an alternative and more environmentally friendly extraction solvent, a deep eutectic system (DES), for extracting indigoid pigments, specifically indigo and indirubin, from Baphicacanthus cusia (BC). BC is recognized in the textile industry as a natural vat dye and in traditional Chinese medicine as ''Qing-Dai''. It is known for treating inflammatory diseases such as psoriasis. In this study, 46 DES systems were compared with conventional methods. The hydrophobic DES, a terpenoid and fatty acid system comprising thymol:decanoic acid (DES40), and the hydrophilic DES, a choline chloride-based system comprising choline chloride: p-toluenesulfonic acid (DES19), showed significant extraction improvements. DES40 and DES19 achieved approximately 26-fold higher indigo content compared to classical ethanol and outperformed the harsh organic solvent dichloromethane. The green extraction process was optimized using a Box–Behnken design, considering parameters such as temperature, time and co-solvent. DES19 maximized indigo and indirubin content to 270.91±14.38 and 5.70±0.11 mg/g, respectively, while DES40 yielded 108.28 ± 3.9 and 0.16 ± 0.00 mg/mg/g, respectively. Safety evaluations using a cell-based MTT model with human skin cells in keratinocytes and fibroblasts showed that both DES19 and DES40 were safe at all concentrations tested. These results indicate that a more environmentally friendly solvent technology for the extraction of indigoids from BC using the DES is an efficient and potential application in the textile and pharmaceutical industries.

Objective To investigate the target of indirubin in psoriasis, and to elucidate the possible effect of indirubin on psoriasis through MAPK14. Methods The GSE30999 and GSE78097 psoriasis datasets downloaded from the Gene Expression Omnibus (GEO) database were used as training and validation sets, respectively. The differentially expressed genes were screened, and their biological functions and pathways were analyzed. Intersections with indirubin target genes were used to obtain differentially expressed indirubin target genes. Meanwhile gene correlation was analyzed and differential gene expression was verified. LASSO and Cox regression model were used to screen the best target genes for indirubin. Molecular docking of indirubin was performed to analyze the structural relationship. Results A total of 8 indirubin target genes were found in the GSE30999 dataset, which were differentially expressed compared with the control group. Seven genes had high diagnostic value (AUC>0.8), and one gene had low diagnostic value (AUC>0.6). GO and KEGG enrichment analyses showed that the eight indirubin target genes were mainly related to cell aging, chemical carcinogenic receptor activation, lipid and atherosclerosis, acute myeloid leukemia, and AGE-RAGE signaling pathways in diabetic complications. The best target gene of indirubin, MAPK14 gene, was screened by LASSO and Cox regression model. Molecular docking suggested that indirubin was tightly bound to MAPK14. Conclusion Indirubin may be involved in the regulation of the pathogenesis and treatment of psoriasis through MAPK14.

Objective To investigate the target of indirubin in psoriasis, and to elucidate the possible effect of indirubin on psoriasis through MAPK14. Methods The GSE30999 and GSE78097 psoriasis datasets downloaded from the Gene Expression Omnibus (GEO) database were used as training and validation sets, respectively・ The differentially expressed genes were screened, and their biological functions and pathways were analyzed・ Intersections with indirubin target genes were used to obtain differentially expressed indirubin target genes・ Meanwhile gene correlation was analyzed and differential gene expression was verified・ LASSO and Cox regression model were used to screen the best target genes for indirubin. Molecular docking of indirubin was performed to analyze the structural relationship・ Results A total of 8 indirubin target genes were found in the GSE30999 dataset, which were differentially expressed compared w让h the control group・ Seven genes had high diagnostic value (AUC >0. 8), and one gene had low diagnostic value (AUC >0. 6). GO and KEGG enrichment analyses showed that the eight indirubin target genes were mainly related to cell aging, chemical carcinogenic receptor activation, lipid and atherosclerosis, acute myeloid leukemia, and AGE-RAGE signaling pathways in diabetic complications. The best target gene of indirubin, MAPK14 gene, was screened by LASSO and Cox regression modeL Molecular docking suggested that indirubin was tightly bound to MAPK14 ・ Conclusion Indirubin may be involved in the regulation of the pathogenesis and treatment of psoriasis through MAPK14. [ABSTRACT FROM AUTHOR]

The indirubin (2) derivative undergoes condensation with hydroxylamine hydrochloride, resulting in the formation of a novel indirubin derivative (3) that incorporates both the oxime group and the piperidine heterocycle. This compound is subsequently treated with hydrochloric acid in ethanol, leading to the formation of the hydrochloride salt (3.HCl), which exhibits excellent solubility in water. The chemical structures of compounds (3) and (3.HCl) were determined using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, as well as high-resolution mass spectrometry with electrospray ionization (ESI). To evaluate their potential as anticancer agents, in vitro assays were conducted using three human cancer cell lines (A549, Hep-G2, SW480) and one murine cancer cell line (B16F10). These compounds, along with indirubin (1) and Bortezomib (BTZ), were tested. The results revealed that compounds (3) and (3.HCl) demonstrated significant antitumor activity against all four cancer cell lines, with concentrations ranging from 0.37 ± 0.01 to 0.53 ± 0.06 μM. Importantly, their activity surpassed that of indirubin (1) and BTZ. Furthermore, an in vivo anticancer activity assay was performed on compound (3.HCl) using the B16F10 cell line. After 18 days, the tumor size in the group treated with compound (3.HCl) was approximately eight times smaller than that of the control group, and nearly three times smaller than the group treated with BTZ. [ABSTRACT FROM AUTHOR]

Indirubin is the main component of the traditional Chinese medicine Indigo naturalis (IN), a potent agonist of aryl hydrocarbon receptors (AhRs). In China, IN is used to treat psoriasis and ulcerative colitis, and indirubin is used for the treatment of chronic myelogenous leukaemia. However, IN and indirubin have adverse reactions, such as abdominal pain, diarrhoea, and intussusception, and their specific mechanism is unclear. The purpose of our research was to determine the specific mechanism underlying the adverse effects of IN and indirubin. By tracking the modifications in guinea pigs after the intragastric administration of indirubin for 28 days. The results demonstrate that indirubin could accelerate bowel movements and decrease intestinal acetylcholinesterase (AchE) expression. Experiments with NCM460 cells revealed that indirubin significantly reduced the expression of AchE, and the AchE levels were increased after the silencing of AhR and re-exposure to indirubin. This study showed that the inhibition of AchE expression by indirubin plays a key role in the occurrence of adverse reactions to indirubin and that the underlying mechanism is related to AhR-mediated AchE downregulation. [ABSTRACT FROM AUTHOR]

The reaction of indigo with two equivalents of the electrophile ethyl bromoacetate with caesium carbonate as a base result in the formation of structurally complex polyheterocyclics, including a fused spiroimidazole and a spiro[1,3]oxazino derivative, together with a biindigoid-type derivative, through a convenient one-pot reaction. Further assessment of the reaction using five equivalents of the electrophile gave rise to other molecules incorporating the 2-(7,13,14-trioxo-6,7,13,14-tetrahydropyrazino[1,2-a:4,3-a′]diindol-6-yl) scaffold. The reaction of ethyl bromoacetate with the less reactive indirubin resulted in the synthesis of three derivatives of a new class of polyheterocyclic system via a cascade process, although yields were low. These compounds were derived from the parent indolo[1,2-b]pyrrolo[4,3,2-de]isoquinoline skeleton. Despite the modest yields of the reactions, they represent quick cascade routes to a variety of heterocycles from cheap starting materials, with these structures otherwise being difficult to synthesise in a traditional stepwise manner. These outcomes also contribute significantly to the detailed understanding of the indigo/indirubin cascade reaction pathways initiated by base-catalysed N-alkylation.

Objective(s): Liver fibrosis is a common liver disease caused by chronic liver damage. However, there are currently no approved drugs available to treat it. Therefore, the therapeutic effect of indirubin on liver fibrosis was evaluated. This study investigated the protective effect and related molecular mechanism of indirubin against CCl4-induced liver fibrosis in mice.Materials and Methods: We first detected the effect of indirubin on liver fibrosis in mice (n=8 per group, 32 mice total) by ELISA, HE, and Masson staining. Subsequently, the proliferation of activated HSCs was detected by MTT and EdU. Finally, the changes of related proteins and signaling pathways in mice treated with indirubin were investigated by qRT-PCR and Western blot. One-way ANOVA or two-tailed student’s t-test was used for comparison between groups.Results: Firstly, we found that indirubin (25 mg/kg) therapy could attenuate liver injury and significantly down-regulate α-SMA (P=0.0038) and collagen 1 (P=0.0057) in the liver using CCl4-induced liver fibrosis in mice. Secondly, we showed that indirubin (25 μM) could significantly inhibit hepatic stellate cell (HSC) trans-differentiation into myofibroblasts and proliferation (P=0.0063) in HSC-T6 cells treated by TGF-β. Finally, we showed that indirubin could greatly reduce the protein levels of p-Smad2/3, p38, p-ERK, and p-JNK in vivo and in vitro.Conclusion: Our results suggested that indirubin alleviated liver fibrosis and HSC activation mainly through TGF-β-mediated signaling pathways in vivo and in vitro. In conclusion, our data showed that indirubin could be a promising clinical therapeutic drug for the prevention and treatment of liver fibrosis.

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Objective(s): Liver fibrosis is a common liver disease caused by chronic liver damage. However, there are currently no approved drugs available to treat it. Therefore, the therapeutic effect of indirubin on liver fibrosis was evaluated. This study investigated the protective effect and related molecular mechanism of indirubin against CCl4-induced liver fibrosis in mice. Materials and Methods: We first detected the effect of indirubin on liver fibrosis in mice (n=8 per group, 32 mice total) by ELISA, HE, and Masson staining. Subsequently, the proliferation of activated HSCs was detected by MTT and EdU. Finally, the changes of related proteins and signaling pathways in mice treated with indirubin were investigated by qRT-PCR and Western blot. One-way ANOVA or two-tailed student's t-test was used for comparison between groups. Results: Firstly, we found that indirubin (25 mg/kg) therapy could attenuate liver injury and significantly down-regulate α-SMA (P=0.0038) and collagen 1 (P=0.0057) in the liver using CCl4-induced liver fibrosis in mice. Secondly, we showed that indirubin (25 μM) could significantly inhibit hepatic stellate cell (HSC) trans-differentiation into myofibroblasts and proliferation (P=0.0063) in HSC-T6 cells treated by TGF-β. Finally, we showed that indirubin could greatly reduce the protein levels of p-Smad2/3, p38, p-ERK, and p-JNK in vivo and in vitro. Conclusion: Our results suggested that indirubin alleviated liver fibrosis and HSC activation mainly through TGF-β-mediated signaling pathways in vivo and in vitro. In conclusion, our data showed that indirubin could be a promising clinical therapeutic drug for the prevention and treatment of liver fibrosis. [ABSTRACT FROM AUTHOR]

Taking plant indigo extract as the research object, the research progress and trend of the detection methods for the main effective components at home and abroad were reviewed. The main components and their functions of plant indigo extract such as indigo and indirubin were summarized. The analytical examination methods of indigo extract were reviewed, mainly including high performance liquid chromatography, thin layer chromatography, thin layer scanning, dual wavelength spectrophotometry, titration analysis, gas chromatography-mass spectrometry and the combination of multiple detection methods. The application of high performance liquid chromatography ( selection of chromatographic column, mobile phase, detection wavelength and evaluation parameters) was emphatically analyzed. At the same time, the problems existed in the present analysis methods were analyzed, and the future development trends of the indigo extract analysis and detection were predicted. [ABSTRACT FROM AUTHOR]

Osteosarcoma is the most common malignant tumor of bone, with rapid progressive growth, early distant metastases, and frequent recurrence after surgical treatment. Osteosarcoma is characterized by changes in the ratio and expression of different cytochrome P450 (CYP) isoforms that can affect the effectiveness of anticancer therapies. The inducible expression of CYP1 genes depends on the ligand-dependent functionality of the aryl hydrocarbon receptor (AHR). In this study, we examined the AHR/CYP1 signaling pathway in four osteosarcoma cell lines (MG63, HOS, SAOS2, and U2OS) induced by the known AHR ligands: indirubin, indole-3-carbinol, and beta-naphthoflavone. Using qPCR and Western blot analysis, we explored the effects of these ligands on the expression of the CYP1 genes and studied the correlation between these responses and the changes in the mRNA and protein levels of AHR and the AHR nuclear translocator (ARNT) in these osteosarcoma cell lines. The results show that the AHR/CYP1 signaling pathway retains its function only in MG63 and HOS cells, and is impaired in SAOS2 and U2OS cells. Our data should be taken into account when recommending new strategies for the treatment of osteosarcoma and when evaluating new drugs against osteosarcoma in vitro. • The AHR/CYP signaling pathway retains its functions only in MG63 and HOS osteosarcoma cell lines. • In SAOS2 and U2OS cells, the AHR/CYP signaling pathway is impaired. • AHR and ARNT protein degradation is ligand- and cell-dependent in osteosarcoma. [ABSTRACT FROM AUTHOR]

Indigo has a unique value in industrial applications, and its isomer, indirubin, is pharmacologically active against multiple diseases. However, specific transformation processes of indican to its final products (indigo or indirubin) are inefficient, which hampers the isolation of high-purity products. We characterized an enzyme of the GH1-family and regulated its catalytic processes to achieve specific transformation. The target alkali-stable glucosidase gene was overexpressed in Escherichia coli BL21 (DE3). Under optimized induction conditions, alkali-stable glucosidase with an activity of 24.221 U/mL was produced. Asbg1 exhibited significant activities in a wide range of pH conditions, with optimum activities being established at pH 8.5. Indican was rapidly hydrolyzed into desired final products by adjusting the temperature and pH ranges of the enzymatic reaction. The final maximum productivity of single indigo and indirubin were obtained at pH 7.5 and 55 ℃ and pH 9.5 and 45 ℃, respectively. The titer of indigo was further increased to 97.881% by conducting the reaction in a water bath with agitation at a speed of 250 rpm. The addition of cysteine inhibited indigo production but improved the proportion of produced indirubin to 88.381%. [Display omitted] • A simple method of adjusting the temperature and pH via the reaction for directional preparation of high purity indigo or indirubin. • A new glucosidase was studied, which could enrich the resources of glucosidase. • Improving the productivity of Asbg1. [ABSTRACT FROM AUTHOR]

Indigo and indirubin are derived from indoxyl molecules, which generally occur as indoxyl glycosides in woad (Isatis tinctoria L.) and other indigo-producing plants. Indoxyl glycosides are biosynthesized from indole via 3-hydroxylation to form indoxyl, followed by one or more glycosylations. Enzymes that attach and remove sugars to and from indoxyl have already been isolated and characterized, while enzymes that convert indole into indoxyl in plants have remained elusive, until the identification of P450s and flavin-containing monooxygenases that hydroxylate indole. A P450 gene from woad (named CYP71B102) was heterologously expressed in E. coli , resulting in the formation of indigo and indirubin, as well as isatin and 2-oxindole, which along with indoxyl are putative precursors of indirubin. The addition of either isatin or 2-oxindole to the recombinant E. coli reduced the levels of indigo and increased the amount of indirubin, whereas coexpression of CYP71B102 with isatin hydroxylase (which degrades isatin) increased the levels of indigo and decreased the amount of indirubin, albeit slightly. The results suggest that CYP71B102 hydroxylates indole at both the 2- and 3- positions to produce 2-oxindole and indoxyl, respectively, and that the coupling of indoxyl with either 2-oxindole or isatin forms indirubin, while dimerization of indoxyl forms indigo. This P450 gene is thus likely involved in the biosynthesis of indirubin in woad, as well as the formation of indigo and its glycosidic precursors, even if other types of enzymes, such as flavin-containing monooxygenases, may be involved in indole hydroxylation in other indigo-producing plants. [Display omitted] • P450 enzyme from Isatis tinctoria produces indigo and indirubin when expressed in E. coli. • Indirubin production by CYP71B102 is increased by addition of either 2-oxindole or isatin. • Isatin hydroxylase, which degrades isatin, increases indigo production by CYP71B102. • P450s may be involved in the biosynthesis of indirubin and indigo precursors in plants. [ABSTRACT FROM AUTHOR]

Abstract Indirubin is a bisindole compound for the treatment of chronic myelocytic leukemia. Here, we presented a structure-guided method to improve the activity of a flavin-containing monooxygenase (bFMO) for the efficient production of indirubin in Escherichia coli. A flexible loop interlocked with the active pocket through a helix and the substrate tunnel rather than the active pocket in bFMO were identified to be two reconfigurable structures to improve its activity, resulting in K223R and N291T mutants with enhanced catalytic activity by 2.5- and 2.0-fold, respectively. A combined modification at the two regions (K223R/D317S) achieved a 6.6-fold improvement in catalytic efficiency (k cat/K m) due to enhancing π–π stacking interactions stabilization. Finally, an engineered E. coli strain was constructed by metabolic engineering, which could produce 860.7 mg/L (18 mg/L/h) indirubin, the highest yield ever reported. This work provides new insight into the redesign of FMOs to boost their activities and an efficient approach to produce indirubin. Graphical Abstract

• Indirubin (IR) induces tolerogenic DCs by activating the aryl hydrocarbon receptor. • IR-treated DCs promote the differentiation of Foxp3+ regulatory T (Treg) cells. • IR therapy alleviates the severity of syndromes of ovalbumin-induced allergic asthma. • IR promotes Treg cell expansion and reduces Th2 response in an allergic asthma model. 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, we aimed to clarify whether IR exhibits immunomodulatory action on DCs via AhR activation and investigated the antiallergic effects of IR in a mouse model of allergic asthma. Lipopolysaccharide (LPS)-activated bone marrow-derived DCs were treated with IR. 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 IR. 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 IR 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-1 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. IR 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 IR against allergic asthma. [Display omitted] [ABSTRACT FROM AUTHOR]

Background & objectives: Cyclin-dependent kinase 2 (CDK-2) is a serine/threonine protein kinase with regulatory activity in the cell cycle. Inhibitors of this protein are the treatment of choice for a variety of cancers by stopping the cell cycle. In this in silico study, the effects of docking and molecular dynamics of Abemaciclib, Hymenialdisine, and Indirubin on the inhibition of CDK-2 as one of the most important factors in the cell cycle have been investigated. Methods: PDB file of CDK-2 protein as well as three-dimensional structures of Abemaciclib, Hymenialdisine, and Indirubin were obtained from the protein database (http://www.rcsb.org) and pubchem server, respectively. After simulating CDK-2 in Gromacs software, molecular docking of compounds on CDK-2 was performed by AutoDock 4.2 software. Finally, the most important molecular dynamics factors such as RMSD,the radius of gyration and total energy in the pre-docking state were analyzed and compared to these factors in the post-docking stage. Results: Abemaciclib has the highest affinity for binding to amino acids at the CDK-2 binding site by releasing binding energy equivalent to 8.23 kJ/mol. The binding of Abemaciclib, Hymenialdisine, and Indirubin to CDK-2, resulted in significant reductions in some molecular dynamics factors such as mean total energy, the radius of gyration, RMSD, and changes in CDK-2 secondary structure. Conclusion: Abemaciclib, Hymenialdisine, and Indirubin have a high tendency to interact with CDK-2, and this binding can induce significant dynamic molecular changes in the structure of CDK-2 molecule. Based on the results of molecular dynamics simulation, the secondary structure of CDK-2 changes after each ligand binds to it and makes the complex of ligand and protein more stable.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that modulates gene expression following its binding and activation by structurally diverse chemicals. Species differences in AhR functionality have been observed, with the mouse AhR (mAhR) and human AhR (hAhR) exhibiting significant differences in ligand binding, coactivator recruitment, gene expression and response. While the AhR agonist indirubin (IR) is a more potent activator of hAhR-dependent gene expression than the prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), it is a significantly less potent activator of the mAhR. DNA binding analysis confirmed the greater potency/efficacy of IR in stimulating transformation/DNA binding of the hAhR in vitro and domain-swapping experiments demonstrated that the enhanced response to IR was primarily due to the hAhR ligand binding domain (LBD). Site-directed mutagenesis and functional analysis studies revealed that mutation of H326 and A349 in the mAhR LBD to the corresponding residues in the hAhR LBD significantly increased the potency of IR. Since these mutations had no significant effect on ligand binding, these residues likely contribute to an enhanced efficiency of transformation/DNA binding by IR-bound hAhR. Molecular docking to mAhR LBD homology models further elucidated the different roles of the A375V mutation in TCDD and IR binding, as revealed by [³H]TCDD competitive binding results. These results demonstrate the differential binding of structurally diverse ligands within the LBD of a given AhR and confirm that amino acid differences within the LBD of AhRs contribute to significant species differences in ligand response.

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. [ABSTRACT FROM AUTHOR]

Inflammatory bowel disease is a disease that can invade the whole digestive tract and is accompanied by immune abnormalities. Immune dysfunction involving dendritic cells (DCs) and T cells is recognized as a key factor in diseases. Indirubin (IDRB) exerts antiinflammatory effects and can help in treating immune diseases. This study aimed to isolate bone marrow-derived dendritic cells (BMDCs) using lipopolysaccharide (LPS) to obtain mature DCs (mDCs). The expression of CD80, CD86, CD40, and MHC-II was detected using flow cytometry after treatment with IDRB. αVβ8 siRNA was used to knock down αVβ8 in mDCs, and the expression of CD80, CD86, CD40, and MHCII was detected. Meanwhile, DCs were co-cultured with T cells. Then, T cell differentiation was detected using flow cytometry, and the cytokine levels were detected using enzyme-linked immunosorbent assay. The animal model of dextran sulfate sodium (DSS)-induced inflammatory bowel disease was established in mice. After intervention with IDRB and αVβ8 shRNA, the intestinal tissues were evaluated using H&E staining, disease activity index (DAI) score, and histological damage index, and the corresponding factors and cytokines to regulatory T cells (Treg) and Th17 were measured. The results showed that αVβ8 was expressed in immature DCs and mDCs. CD80, CD86, CD40, and MHC-II expression decreased after IDRB treatment in mDCs. Meanwhile, the expression of TNF-α and TGF-β also decreased after IDRB treatment. The effect of IDRB on the expression of CD80, CD86, CD40, MHC-II, TNF-α, and TGF-β in mDCs was reversed by αVβ8 siRNA. The Treg differentiation increased after IDRB treatment, while the differentiation of Th17 cells was inhibited. This effect of IDRB was reversed by mDCs after treatment with αVβ8 siRNA. In vivo experiments showed that IDRB alleviated the symptoms of inflammatory bowel disease in animals. Enteritis significantly reduced, and the effect of IDRB was reversed by αVβ8 shRNA. The results suggested that IDRB regulated the differentiation of T cells by mediating the maturation of BMDCs through αVβ8. This study confirmed the therapeutic effect of IDRB in inflammatory bowel disease and suggested that IDRB might serve as a potential drug. [ABSTRACT FROM AUTHOR]

Objective: To investigate the efficacy of indirubin combined with human umbilical cord mesenchymal stem cells (hUC-MSCs) in the treatment of psoriatic lesions in BALB/c mice and to explore the related mechanism of indirubin in the treatment of psoriasis. Methods: A BALB/c mouse psoriasis model induced by imiquimod was established and randomly divided into the control group, model group, indirubin group, hUC-MSCs group, and indirubin combined with hUC-MSCs group. Psoriasis area and severity index (PASI) score was used to observe skin lesion changes in the psoriasis-like mouse model. The epidermal scale, the degree of keratinization, and the infiltration of inflammatory cells were observed by hematoxylin eosin (HE) staining. The concentrations of TNF-α, IFN-γ, IL-17A, and IL-23 in serum of mice were measured using enzyme-linked immunosorbent assay (ELISA). Results: The PASI integral trend chart indicates that hUC-MSCs and indirubin and the combination of drugs could relieve the appearance of skin lesions and accelerate the recovery of skin lesions. The indirubin group had the best effect in improving the scale of skin lesions. HE staining showed that the number of parakeratosis cells in the three treatment groups was significantly reduced, the degree of erythrocyte extravasation dermis hyperplasia and inflammatory cell infiltration was significantly lower than that in the model group, and the skin thickness and spleen index of the combined treatment group exhibited the most noticeable improvement. ELISA showed that the concentrations of TNFα, IFN-γ, IL-17A, and IL-23 in serum of mice in the hUC-MSCs treatment group, indirubin group, and combined administration group were all decreased compared with those in the model group, and the concentrations of IFN-γ, IL-17A, and IL-23 could be decreased significantly in the indirubin group. Conclusions: Both hUC-MSCs and indirubin can effectively reduce psoriasislike lesions in BALB/c mice, and the combined administration of these drugs has the best effect. [ABSTRACT FROM AUTHOR]

Indigo, indirubin, and isoindigo derivatives have been used for centuries as pigments. Since the 1990s, a new aspect of the chemistry of this type of compounds is their activity against various types of cancer. N -Glycosides of indigo, indirubin, and isoindigo, blue, red, and yellow sugars, turned out to be of special interest because of their high cancerostatic activity and structural novelty. The present article provides an account on the synthesis and anticancer activity of these compounds., (Copyright © 2024, Langer.)

Abstract Background Insufficient solubility and stability of bioactive small molecules as well as poor biocompatibility may cause low bioavailability and are common obstacles in drug development. One example of such problematic molecules is 6-bromoindirubin-3'-glycerol-oxime ether (6BIGOE), a hydrophobic indirubin derivative. 6BIGOE potently modulates the release of inflammatory cytokines and lipid mediators from isolated human monocytes through inhibition of glycogen synthase kinase-3 in a favorable fashion. However, 6BIGOE suffers from poor solubility and short half-lives in biological aqueous environment and exerts cytotoxic effects in various mammalian cells. In order to overcome the poor water solubility, instability and cytotoxicity of 6BIGOE, we applied encapsulation into poly(d,l-lactide-co-glycolide) (PLGA)-based nanoparticles by employing formulation methods using the sustainable solvents Cyrene™ or 400 g/mol poly(ethylene glycol) as suitable technology for efficient drug delivery of 6BIGOE. Results For all preparation techniques the physicochemical characterization of 6BIGOE-loaded nanoparticles revealed comparable crystallinity, sizes of about 230 nm with low polydispersity, negative zeta potentials around − 15 to − 25 mV, and biphasic release profiles over up to 24 h. Nanoparticles with improved cellular uptake and the ability to mask cytotoxic effects of 6BIGOE were obtained as shown in human monocytes over 48 h as well as in a shell-less hen’s egg model. Intriguingly, encapsulation into these nanoparticles fully retains the anti-inflammatory properties of 6BIGOE, that is, favorable modulation of the release of inflammation-relevant cytokines and lipid mediators from human monocytes. Conclusions Our formulation method of PLGA-based nanoparticles by applying sustainable, non-toxic solvents is a feasible nanotechnology that circumvents the poor bioavailability and biocompatibility of the cargo 6BIGOE. This technology yields favorable drug delivery systems for efficient interference with inflammatory processes, with improved pharmacotherapeutic potential. Graphical Abstract

【Objective】This study aimed to compare the important agronomic characters and the main active contents of indirubin and indigo in different parts of four populations of Baphicacanthus cusia from Hani nationality in Jinping county, so as to provide basis for standardized cultivation and variety selection. 【Method】The plant height, stem diameter, leaf length and width, branches, biomass and other important agronomic traits of four B.cusia populations were investigated and measured. The contents of indirubin and indigo in different parts of the plant were determined by HPLC, and the correlation between the indexes was analyzed. 【Result】The ratios of root, stem and leaf dry weight to plant dry weight in each B.cusia populations were 12.37%-22.26%, 52.03%-70.58%, 9.16-35.60%, respectively. The order of indirubin and indigo content in part of the four B.cusia populations was leaf > stem > root. Moreover, the ratio of indirubin content to root, stem and leaf was 1∶( 1.99-2.79) ∶( 10.87-18.01) .And the ratio of root, stem and leaf of indigo content was 1∶( 1.60-5.97) ∶( 10.51-33.92) . The important agronomic traits and main active components of B.cusia had different degrees of differences, and the coefficients of variation of leaf fresh weight, stem dry weight, leaf dry weight, stem fresh weight, stem indigo, root fresh weight, number of secondary branches, root dry weight, fresh weight per plant, dry weight per plant and number of primary branches were higher than 47%. There was a significant correlation between multiple traits, in which the stem diameter was significantly positively correlated with the fresh and dry weight of the plant, and significantly negatively correlated with the number of primary branches; There was a significant positive correlation between the number of secondary branches and dry weight of plant; The content of indigo in leaf was significantly positively correlated with the dry weight of root, positively correlated with fresh and dry weight of the plant, and negatively correlated with the content of indigo of root. Four principal components were extracted from principal component analysis, and the cumulative contribution rate was 88.83%. 【Conclusion】When breeding excellent varieties of B.cusia, it was necessary to focus on the varieties with big weight of fresh and dry stems and leaves. Clustering analysis showed that JP3 and JP4 of B.cusia had good agronomic traits and higher indirubin and indigo. These two populations could be highly-qualified resources for breeding and planting. [ABSTRACT FROM AUTHOR]

Objective: To investigate the effect of indirubin on the expression of proinflammatory cytokines in HaCaT cells, and to elucidate the mechanisms of Diqing ointment in the treatment of psoriasis. Methods: The human immortalized keratinocyte cell line, HaCaT cells, were used to observe the changes in the expression levels of IL-1β, IL-6, and TNF-α in HaCaT cells after treatment with different concentrations of indirubin. HaCaT cells were divided into 4 groups. The secretion levels of IL-1β, IL-6, and TNF-α in the culture supernatants were detected by enzyme-linked immunosorbent assay ELISA, and the expression levels of IL-1β, IL-6, and TNF-α mRNA were detected by real-time PCR (RT-qPCR). Results: Expression levels of both mRNA and protein of IL-1β, IL-6, and TNF-α were significantly and dose-dependently decreased after treatment with indirubin. Conclusion: Indirubin, the main component of Qingdai ointment, can lower expression levels of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α in keratinocytes, leading to the improvements of psoriasis.

This study aimed to develop a solid dispersion (SD) of MT-102, a new anti-inflammatory agent, to improve its oral bioavailability. The ternary SD formulations of MT-102 (a poorly soluble extract of Isatis indigotica and Juglans mandshurica) were prepared using a solvent evaporation method with various drug/excipient ratios. Following that, the effectiveness of various SDs as an oral formulation of MT-102 was investigated using indirubin as a marker component. By forming SDs with hydrophilic polymers, the aqueous solubility of indirubin was significantly increased. SD-F4, containing drug, poloxamer 407 (P407), and povidone K30 (PVP K30) at a 1:2:2 weight ratio, exhibited the optimal dissolution profiles in the acidic to neutral pH range. Compared to pure MT-102 and a physical mixture, SD-F4 increased indirubin's dissolution from MT-102 by approximately 9.86-fold and 2.21-fold, respectively. Additionally, SD-F4 caused the sticky extract to solidify, resulting in improved flowability and handling. As a result, compared to pure MT-102, the oral administration of SD-F4 significantly improved the systemic exposure of MT-102 in rats. Overall, the ternary SD formulation of MT-102 with a blended mixture of P407 and PVP K30 appeared to be effective at improving the dissolution and oral absorption of MT-102. [ABSTRACT FROM AUTHOR]

Indirubin is a bisindole compound for the treatment of chronic myelocytic leukemia. Here, we presented a structure-guided method to improve the activity of a flavin-containing monooxygenase (bFMO) for the efficient production of indirubin in Escherichia coli. A flexible loop interlocked with the active pocket through a helix and the substrate tunnel rather than the active pocket in bFMO were identified to be two reconfigurable structures to improve its activity, resulting in K223R and N291T mutants with enhanced catalytic activity by 2.5- and 2.0-fold, respectively. A combined modification at the two regions (K223R/D317S) achieved a 6.6-fold improvement in catalytic efficiency (kcat/Km) due to enhancing π–π stacking interactions stabilization. Finally, an engineered E. coli strain was constructed by metabolic engineering, which could produce 860.7 mg/L (18 mg/L/h) indirubin, the highest yield ever reported. This work provides new insight into the redesign of FMOs to boost their activities and an efficient approach to produce indirubin. [ABSTRACT FROM AUTHOR]

Abstract Background As one of the oldest traditional dyes, people worldwide have used natural indigo for centuries. Local people have unique knowledge about indigo identification, which is crucial for indigo quality control and determining the dyeing effects. However, such traditional knowledge is rarely documented and explained. Therefore, the aims of this study were to document and assess the traditional knowledge used by local people when identifying natural indigo paste as well as quantitatively explore the characteristics and material basis of such traditional knowledge. Method Three field surveys were conducted between 2019 and 2020. A total of 283 traditional indigo-paste artisans were interviewed in Guizhou, Yunnan, and Fujian Provinces. The frequency of citation, mention index, and fidelity level of each indigo-paste quality criterion were calculated to determine the most commonly used, recognized, and important quality criteria. To explore the characteristics and material basis of the traditional knowledge, we analyzed 21 indigo-paste samples using high-performance liquid chromatography with diode-array detection (HPLC-DAD), pH, and particle size analyses. Results Local people possess unique knowledge to identify natural indigo. Based on this knowledge accumulated over thousands of years, four criteria (color, taste, touch, and dyeing ability) were chosen by local people, and using these criteria, nature indigo was divided into five quality grades. The best quality indigo paste was judged according to the following folk criteria: dark blue in color with a purple-red luster; smooth and difficult to wipe off; having a sweet, bitter or spicy taste; and easy cloth dyeing. Additionally, the higher the contents of indigo and indirubin—especially indirubin—the better is the quality of the indigo paste. Within the pH range of 9–12, high-quality indigo-paste was more acidic. There was no significant relationship between particle size and quality. Conclusion The ancient methods used by local people for identifying natural indigo are comprehensive and unique. By documenting the various folk quality criteria and conducting quantitative analyses, this study revealed the importance of indirubin and pH for assessing the quality of indigo paste. These findings differ from existing quality standards for synthetic indigo. Amid rapid modernization, traditional knowledge remains invaluable as a world heritage of humanity that warrants preservation.

Purple urine bag syndrome (PUBS) is a rare phenomenon, which usually occurs in chronically constipated women having long-term catheters. The underlying pathogenesis is linked to sulfatase producing bacteria in the urinary tract, which react to produce indigo (violet) and indirubin (red) pigment and the mixture gives a purple hue in the presence of alkaline urine. Treatment of the underlying urinary tract infection (UTI), change of catheter, and management of predisposing conditions prevent its recurrence. We report a case of a 74-year-old diabetic man who had Enterobacter cloacae-related UTI with PUBS in acidic urine and managed successfully.

Aryl hydrocarbon receptor (AhR) activation by environmental agents and microbial metabolites is potentially implicated in a series of skin diseases. Hence, it would be very important to identify natural compounds that could inhibit the AhR activation by ligands of microbial origin as 6-formylindolo[3,2-b]carbazole (FICZ), indirubin (IND) and pityriazepin (PZ) or the prototype ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Five different dry Rosmarinus officinalis L. extracts (ROEs) were assayed for their activities as antagonists of AhR ligand binding with guinea pig cytosol in the presence of [3H]TCDD. The methanolic ROE was further assayed towards CYP1A1 mRNA induction using RT-PCR in human keratinocytes against TCDD, FICZ, PZ, and IND. The isolated metabolites, carnosic acid, carnosol, 7-O-methyl-epi-rosmanol, 4′,7-O-dimethylapigenin, and betulinic acid, were assayed for their agonist and antagonist activity in the presence and absence of TCDD using the gel retardation assay (GRA). All assayed ROE extracts showed similar dose-dependent activities with almost complete inhibition of AhR activation by TCDD at 100 ppm. The methanol ROE at 10 ppm showed 99%, 50%, 90%, and 85% inhibition against TCDD, FICZ, IND, and PZ, respectively, in human keratinocytes. Most assayed metabolites exhibited dose-dependent antagonist activity. ROEs inhibit AhR activation by TCDD and by the Malassezia metabolites FICZ, PZ, and IND. Hence, ROE could be useful for the prevention or treatment of skin diseases mediated by activation of AhR. [ABSTRACT FROM AUTHOR]

Psoriasis is an immune disease caused by rapid and incomplete differentiation of skin basal cells. Natural products such as indirubin have historically served as excellent sources for the treatments of psoriasis. However, the poor solubility and bioavailability due to its plane and rigid crystal structure, which limits its efficacy. Herein, to improve the efficacy of indirubin, a hydrogel-based microemulsion drug delivery system was developed for transdermal delivery. The mean droplet size of the optimized microemulsion was 84.37 nm, with a polydispersity index (PDI) less than 0.2 and zeta potential value of 0~−20 mV. The transdermal flux and skin retention of indirubin at 24 h were 47.34 ± 3.59 μg/cm2 and 8.77 ± 1.26 μg/cm2, respectively. The optimized microemulsion was dispersed in carbomer 934 hydrogel to increase the consistency. The indirubin-loaded microemulsion gel was tested on an imiquimod-induced psoriasis mouse model. Results showed that this preparation can improve psoriasis symptoms by down-regulating the expression of IL-17A, Ki67, and CD4+T. This experiment provides great scalability for researchers to treat psoriasis, avoid first-pass effects, and increase the concentration of targeted drugs. [ABSTRACT FROM AUTHOR]

Microtubule-based transport provides mitochondria to distant regions of neurons and is essential for neuronal health. To identify compounds that increase mitochondrial motility, we screened 1,641 small-molecules in a high-throughput screening platform. Indirubin and cantharidin increased mitochondrial motility in rat cortical neurons. Cantharidin is known to inhibit protein phosphatase 2A (PP2A). We therefore tested two other inhibitors of PP2A: LB-100 and okadaic acid. LB-100 increased mitochondrial motility, but okadaic acid did not. To resolve this discrepancy, we knocked down expression of the catalytic subunit of PP2A (PP2CA). This long-term inhibition of PP2A more than doubled retrograde transport of axonal mitochondria, confirming the importance of PP2A as a regulator of mitochondrial motility and as the likely mediator of cantharidin's effect. [ABSTRACT FROM AUTHOR]

Abstract Glioma causes significant mortality across the world and the most aggressive type of brain cancer. The incidence of glioma is believed to increase in the next few decades and hence more efficient treatment strategies need to be developed for management of glioma. Herein, we examined the anticancer effects of Indirubin against a panel of human glioma cells and attempted to explore the underlying mechanisms. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay showed that Indirubin could inhibit the growth of all the glioma cells but the lowest IC50 of 12.5 µM was observed against the U87 and U118 glioma cells. Additionally, the cytotoxic effects of Indirubin were comparatively negligible against the normal astrocytes with an IC50 of > 100 µM. Investigation of mechanism of action, revealed that Indirubin exerts growth inhibitory effects on the U87 and U118 glioma cells by autophagic and apoptotic cell death. Annexin V/PI staining assay showed that apoptotic cell percentage increased dose dependently. Apoptosis was associated with increase in Bax decrease in Bcl-2 expressions. Additionally, the expression of autophagic proteins such as LC3II, ATG12, ATG15 and Beclin 1 was also increased. Wound heal assay showed that Indirubin caused remarkable decrease in the migration of the U87 and U118 cells indicative of anti-metastatic potential of Indirubin. Taken together, these results suggest that Indirubin exerts potent anticancer effects on glioma cells and may prove essential in the management of glioma.

Abstract Background Psoriasis is a common inflammatory skin disease. Abnormal proliferation of keratinocytes is one of the psoriatic histopathological features. Indirubin has an essential effect on the proliferation and activation of keratinocytes; however, in psoriasis, the specific mechanism of action of indirubin on keratinocytes is unclear. In the present study, we revealed the effects of indirubin on DNA methyltransferase 1 (DNMT1), wnt inhibitory factor 1 (wif-1), and wnt/β-catenin signal pathway, in the meantime, we explored the effects of indirubin on proliferation, cell cycle and the apoptosis of HaCaT cells. Methods The expression of DNMT1, wif-1, Frizzled2, Frizzled5, and β-catenin in HaCaT cells treated with different concentrations of indirubin were detected by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). The expression levels of DNMT1 and wif-1 were observed after treated with different concentrations of indirubin by enzyme-linked immunosorbent assay (ELISA). The wif-1 promoter methylation status was detected by DNA methylation-specific PCR (MSP). The transcriptional activities of wif-1 and β-catenin were discovered by a luciferase reporter gene system. Cell viability was determined by Cell Counting Kit-8 (CCK8) method. The cell cycle was detected by flow cytometry. The apoptotic cells were surveyed by the apoptosis kit. The expression of Inolucrin, Loricrin, Filaggrin, Keratin 17, and transcriptional activation of transglutaminase 1(TGase1) were detected by Western blotting. Results Indirubin inhibited the expression of DNMT1 and the methylation of the wif-1 promoter. In the wnt signal pathway, indirubin restored the protein expression of wif-1 and inhibited expression of Frizzled2, Frizzled5, and β-catenin. Besides, indirubin inhibited the proliferation of HaCaT cells, induced apoptosis, and arrest cell cycle. We also reported that indirubin could down-regulate the expression of Involucrin, TGase 1, and keratin 17, but the expression of Filaggrin and Loricrin had no significant effect. Conclusion Our research showed that indirubin promoted the demethylation of wif-1 and suppressed the wnt/β-catenin signal pathway, thereby exerted an anti-proliferative effect. This study reveals the anti-proliferation mechanism of indirubin, which may provide an effective option for the treatment of proliferative diseases.

Abstract Background Obesity occurs when the body’s energy intake is constantly greater than its energy consumption and the pharmacological enhancing the activity of brown adipose tissue (BAT) and (or) browning of white adipose tissue (WAT) has been considered promising strategies to treat obesity. Methods In this study, we took a multi-pronged approach to screen UCP1 activators, including in silico predictions, in vitro assays, as well as in vivo experiments. Results Base on Connectivity MAP (CMAP) screening, we obtained multiple drugs that possess a remarkably correlating gene expression pattern to that of enhancing activity in BAT and (or) sWAT signature. Particularly, we focused on a previously unreported drug-indirubin, a compound obtained from the Indigo plant, which is now mainly used for the treatment of chronic myelogenous leukemia (CML). In the current study, our results shown that indirubin could enhance the BAT activity, as evidenced by up-regulated Ucp1 expression and enhanced mitochondrial respiratory function in vitro cellular model. Furthermore, indirubin treatment restrained high-fat diet (HFD)-induced body weight gain, improved glucose homeostasis and ameliorated hepatic steatosis which were associated with the increase of energy expenditure in the mice model. Moreover, we revealed that indirubin treatment increased BAT activity by promoting thermogenesis and mitochondrial biogenesis in BAT and induced browning of subcutaneous inguinal white adipose tissue (sWAT) of mice under HFD. Besides, our results indicated that indirubin induced UCP1 expression in brown adipocytes, at least in part, via activation of PKA and p38MAPK signaling pathways. Conclusions Our results clearly show that as an effective BAT (as well as beige cells) activator, indirubin may have a protective effect on the prevention and treatment of obesity and its complications.

Microtubule-based transport provides mitochondria to distant regions of neurons and is essential for neuronal health. To identify compounds that increase mitochondrial motility, we screened 1,641 small-molecules in a high-throughput screening platform. Indirubin and cantharidin increased mitochondrial motility in rat cortical neurons. Cantharidin is known to inhibit protein phosphatase 2A (PP2A). We therefore tested two other inhibitors of PP2A: LB-100 and okadaic acid. LB-100 increased mitochondrial motility, but okadaic acid did not. To resolve this discrepancy, we knocked down expression of the catalytic subunit of PP2A (PP2CA). This long-term inhibition of PP2A more than doubled retrograde transport of axonal mitochondria, confirming the importance of PP2A as a regulator of mitochondrial motility and as the likely mediator of cantharidin’s effect.

Purple Urine Bag Syndrome (PUBS) is a rare occurrence characterised by a striking purple to blue/violet discolouration of the urine bag and its tubing after long-term indwelling urinary catheterisation while the urine inside remain clear. This phenomenon is a warning sign of Urinary Tract Infection (UTI). However, unlike traditional UTIs, such patients can be non verbal and asymptomatic with purple/blue urine bag being the only signal for UTI. Tryptophan-Indigo hypothesis is advanced to explain the mystery behind this phenomenon. A rare case of PUBS in acidic urine in an octagenarian (80 years old) female patient, with right sided hemiparesis, hypertension, Post Stroke Aphasia (PSA) and delirium is presented here. Moreover, her urine culture yielded an opportunistic bacteria Serratia marcescens which is not a typical organism responsible for this condition. The bluish colour of urine bag disappeared after hydration, change of the catheter along with urine bag and antibiotic therapy. To the best of the author’s knowledge, this is the first case of PUBS reported from the state of Assam. It is hoped that this case report will be valuable in raising awareness and educating healthcare providers about this rather unknown esoteric syndrome in this region.

Objective: To investigate the pro-apoptotic effects of Indirubin, a traditional Chinese medicine, on ovarian cancer SKOV3 cell line and to explore its underlying mechanisms., Methods: Ovarian cancer SKOV3 cells were divided into a control group (cells cultured normally) and an experimental group (cells cultured in medium containing Indirubin). SKOV3 cells at the logarithmic phase were treated with Indirubin at various concentrations. Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay and 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, while apoptosis was detected by flow cytometry, TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, and immunofluorescence. Transcriptome sequencing was conducted to screen for apoptosis-related factors. qPCR and western blot were used to detect the mRNA and protein expression on added of molecules related to mitochondrial permeability transition pores., Results: MTT assay showed that Indirubin inhibited the growth of SKOV3 cells in both plate and sphere cultures, with IC 50 values of 3.003 μM (plate culture) and 4.253 μM (sphere culture), respectively. Indirubin showed a lower inhibitory concentration than cisplatin (IC 50 3.687 μM in plate culture and 7.023 μM in sphere culture), and its effect was comparable to adriamycin. Flow cytometry revealed an increase in apoptosis rates in SKOV3 cells treated with Indirubin. Transcriptome sequencing indicated significant changes in the transcription of various apoptosis-related genes, particularly those involved in the mitochondrial apoptosis pathway, such as Bcl-2-associated X protein (Bax) and Bcl2 associated agonist of cell death (Bad). After Indirubin treatment, the mRNA and protein expression levels of mitochondrial channel-related genes Cyclophilin D (CyPD), adenine nucleotide translocator 1 (ANT1), and voltage-dependent anion channel (VADC) were significantly elevated (all P < 0.05). By regulating the mitochondrial membrane permeability through the Bcl-2 family members, Indirubin promoted apoptosis in SKOV3 cells., Conclusion: Indirubin inhibits the proliferation and promotes the apoptosis of ovarian cancer cells, exerting an anti-tumor effect. Its pro-apoptotic action is closely related to the mitochondrial apoptosis pathway., Competing Interests: None., (AJTR Copyright © 2024.)

Introduction: Hepatocellular carcinoma (HCC) is a fatal cancer that is often diagnosed at the advanced stages which limits the available therapeutic options. The interaction of HGF with c-MET (a receptor tyrosine kinase) results in the activation of c-MET which subsequently triggers the PI3K/Akt/mTOR axis. Overexpression of c-MET in HCC tissues has been demonstrated to contribute to tumor progression and metastasis., Objectives: We aimed to synthesize triazole-indirubin conjugates, examine their growth suppressor efficacy in cell-based assays, and investigate the antitumor as well as antimetastatic activity of lead cytotoxic agent in the orthotopic mice model., Methods: A series of triazole-indirubin hybrids were synthesized and cytotoxicity, apoptogenic, and antimigratory effect of the lead compound (CRI9) was evaluated using MTT assay, cell cycle analysis, annexin-V/PI assay, TUNEL assay, and wound healing assay. The effect of CRI9 on the operation of the HGF/c-MET/PI3K/Akt/mTOR axis was examined using western blotting and transfection experiments. Acute toxicity, antitumor, and antimetastatic activity of CRI9 were examined in NCr nude mice. The expression of c-MET/PI3K/Akt/mTOR, CD31, and Ki-67 was examined using immunohistochemistry and western blotting., Results: Among the new compounds, CRI9 consistently displayed potent cytotoxicity against HGF-induced HCC cells. CRI9 induced apoptosis as evidenced by increased sub G1 cells, annexin-V + /PI + cells, TUNEL + cells, and cleavage of procaspase-3 and PARP. CRI9 inhibited HGF-induced phosphorylation of c-MET Y1234/1235 and subsequently suppressed the PI3K/Akt/mTOR axis. Also, depletion of c-MET or inhibition of c-MET by CRI9 resulted in suppression of the PI3K/Akt/mTOR axis. CRI9 showed no toxic effects in NCr nude mice and displayed a potent antitumor and antimetastatic effect in the orthotopic HCC mice model. CRI9 also reduced the levels of phospho-c-MET, CD31, and Ki-67 and suppressed the activation of the PI3K/Akt/mTOR axis in tumor tissues., Conclusion: CRI9 has been identified as a new inhibitor of the c-MET/PI3K/Akt/mTOR axis in HCC preclinical models., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Production and hosting by Elsevier B.V.)