Shikonin mitigates cyclophosphamide-induced cardiotoxicity in mice: the role of sirtuin-1, NLRP3 inflammasome, autophagy, and apoptosis.

OBJECTIVES: The aim of this study was to elucidate the protective potential of shikonin (SHK) on cyclophosphamide (CP)-induced cardiotoxicity in Swiss albino mice. METHODS: Mice received SHK in three different doses by oral gavage daily for 14 days and CP at 100 mg/kg, intraperitoneally once on the seventh day. On the 15th day, mice were euthanized, blood collected, and hearts were removed to estimate various biochemical and histopathological parameters. KEY FINDINGS: CP significantly increased serum lactate dehydrogenase, creatine kinase-MB, troponin I and NT pro-BNP, and cardiac malondialdehyde and decreased cardiac total antioxidant capacity and Nrf2, whereas increased inflammatory markers in the cardiac tissues. CP also caused hypertrophy and fibrosis in the cardiac tissues via activation of IL6/JAK2/STAT3 while depressed SIRT1 and PI3K/p-Akt pathway with consequent increased apoptosis and dysregulation of autophagy. SHK treatment reversed these changes in a dose-dependent manner and showed a significant protective effect against CP-induced cardiotoxicity via suppressing oxidative stress, inflammation, and apoptosis with modulation of autophagy via induction of SIRT1/PI3K/p-Akt signaling. CONCLUSIONS: Shikonin may be used as an adjuvant to cyclophosphamide in cancer treatment, but further research is needed to investigate its effects on cardiotoxicity in distinct animal cancer models.

Shikonin, a natural naphthoquinone phytochemical, exerts anti-leukemia effects in human CBF-AML cell lines and zebrafish xenograft models.

Core binding factor acute myeloid leukemia (CBF-AML) stands out as the most common type of adult AML, characterized by specific chromosomal rearrangements involving CBF genes, particularly t(8;21). Shikonin (SHK), a naphthoquinone phytochemical widely employed as a food colorant and traditional Chinese herbal medicine, exhibits antioxidant, anti-inflammatory, and anti-cancer activities. In this study, we aim to investigate the antileukemic effects of SHK and its underlying mechanisms in human CBF-AML cells and zebrafish xenograft models. Our study revealed that SHK reduced the viability of CBF-AML cells. SHK induced cell cycle arrest, promoted cell apoptosis, and induced differentiation in Kasumi-1 cells. Additionally, SHK downregulated the gene expression of AML1-ETO and c-KIT in Kasumi-1 cells. In animal studies, SHK showed no toxic effects in zebrafish and markedly inhibited the growth of leukemia cells in zebrafish xenografts. Transcriptomic analysis showed that differentially expressed genes (DEGs) altered by SHK are linked to key biological processes like DNA repair, replication, cell cycle regulation, apoptosis, and division. Furthermore, KEGG pathways associated with cell growth, such as the cell cycle and p53 signaling pathway, were significantly enriched by DEGs. Analysis of AML-associated genes in response to SHK treatment using DisGeNET and the STRING database indicated that SHK downregulates the expression of cell division regulators regarding AML progression. Finally, we found that SHK combined with cytarabine synergistically reduced the viability of Kasumi-1 cells. In conclusion, our findings provide novel insights into the mechanisms of SHK in suppressing leukemia cell growth, suggesting its potential as a chemotherapeutic agent for human CBF-AML.

Systematic bioinformatics analysis reveals the role of shikonin in blocking colon cancer progression by identifying senescence-induced genes.

Shikonin, a naturally occurring naphthoquinone compound extracted from comfrey plants, has antitumor, anti-inflammatory, and antimicrobial properties. Cell senescence plays a key role in preventing tumor progression. It is unclear whether shikonin has an effect on cell senescence in colon cancer. In the current study, we first determine the IC50 values of shikonin on colon cancer cell lines HT29 and HCT116. Then, we verified the inhibitory effects of shikonin on the proliferation and migration abilities of colon cancer cell lines HT29 and HCT116 using cell counting kit-8, colony formation, and wound healing assays. Next, we identified a series of potential targets using high-throughput mRNA sequencing and identified 210 upregulated and 296 downregulated genes. KEGG profiling revealed eight downregulated genes associated with cell senescence: CCNB3, IL-1α, CXCL8, CDKN2A, MYC, IGFBP3, SQSTM1, and GADD45G. Among them, CXCL8 and CDKN2A were associated with poor prognosis in patients with colon cancer, suggesting that their downregulation by shikonin could improve patient survival. Furthermore, SA-ß-galactosidase staining revealed that the percentage of cellular senescence in colon cancer cells was significantly increased after shikonin treatment. Molecular docking revealed that shikonin suppressed colon cancer progression by blocking CXCL8 activity. Based on these findings, we deem that shikonin might induce senescence and exert antitumor activity in colon cancer cells by downregulating CDKN2A and CXCL8. This provides a new molecular mechanism and potential therapeutic target for shikonin to inhibit colon cancer progression.

Shikonin from lithospermum erythrorhizon induces pyroptosis in trophoblast cells by activating the CTSB-NLRP3 inflammasome.

BACKGROUND: With the decline of global fertility, drug therapeutic of ectopic pregnancy is of great significance. Lithospermum erythrorhizon is using for embryo killing as herbal medicine. Shikonin is the critical nucleus of Lithospermum erythrorhizon; however, the mechanism is still unclear. The study aimed to explore the mechanism of shikonin against ectopic pregnancy. MATERIAL AND METHODS: In this study, we examined the viability and LDH release of HTR-8/SVneo cells by assays, observed pore formation in cell membranes by microscopy imaging and PI staining, and IL-1ß release by WB and ELISA assay kit. Then, we used network pharmacology to analyse the potential interaction between shikonin, ectopic pregnancy and pyroptosis and used molecular docking techniques to verify interactions between shikonin and core common targets. Finally, western blotting and immunofluorescence assay were used to explore the mechanism of shikonin-inducing pyroptosis of HTR-8/SVneo cells. RESULTS: Shikonin could cause a significant inhibition of HTR-8/SVneo cell viability in a concentration- and time-dependent manner. In HTR-8/SVneo cells, shikonin-induced cell swelling, bubble formation, an increase in the release of lactate dehydrogenase (LDH) and up-regulation of several pyroptosis-associated factors. And network pharmacology showed that The main targets of shikonin-ectopic pregnancy-pyroptosis were IL-1ß and caspase-1, and molecular docking results showed that shikonin can closely bind to IL-1ß, caspase-1 and GSDMD. Additionally, the necroptosis inhibitor GSK'872 could not suppress the expression of mature-IL-1ß and prevent the pyroptosis phenotype from developing. However, the nucleotide oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inhibitor MCC-950 could downregulate the expression of pyroptosis-associated factors and prevent the pyroptosis phenotype from developing. Shikonin led to an elevation in the expression of cathepsin B (CTSB), and the CTSB inhibitor CA-074 abolished pyroptosis induced by shikonin; however, the NLRP3 inhibitor MCC-950 could not inhibit the expression of CTSB. CONCLUSIONS: Our results suggest that shikonin activates CTSB to induce NLRP3-dependent pyroptosis in HTR-8/SVneo cells. This study has important clinical implications for the treatment of ectopic pregnancy.

Shikonin protects skin cells against oxidative stress and cellular dysfunction induced by fine particulate matter.

Shikonin, an herbal naphthoquinone, demonstrates a broad spectrum of pharmacological properties. Owing to increasingly adverse environmental conditions, human skin is vulnerable to harmful influences from dust particles. This study explored the antioxidant capabilities of shikonin and its ability to protect human keratinocytes from oxidative stress induced by fine particulate matter (PM2.5). We found that shikonin at a concentration of 3 µM was nontoxic to human keratinocytes and effectively scavenged reactive oxygen species (ROS) while increasing the production of reduced glutathione (GSH). Furthermore, shikonin enhanced GSH level by upregulating glutamate-cysteine ligase catalytic subunit and glutathione synthetase mediated by nuclear factor-erythroid 2-related factor. Shikonin reduced ROS levels induced by PM2.5, leading to recovering PM2.5-impaired cellular biomolecules and cell viability. Shikonin restored the GSH level in PM2.5-exposed keratinocytes via enhancing the expression of GSH-synthesizing enzymes. Notably, buthionine sulphoximine, an inhibitor of GSH synthesis, diminished effect of shikonin against PM2.5-induced cell damage, confirming the role of GSH in shikonin-induced cytoprotection. Collectively, these findings indicated that shikonin could provide substantial cytoprotection against the adverse effects of PM2.5 through direct ROS scavenging and modulation of cellular antioxidant system.

Enhanced therapeutic impact of Shikonin-encapsulated exosomes in the inhibition of colorectal cancer progression.

Colorectal cancer (CRC) is a prevalent malignancy with high mortality rates and poor prognosis. Shikonin (SHK) has demonstrated extensive anti-tumor activity across various cancers, yet its clinical application is hindered by poor solubility, limited bioavailability, and high toxicity. This study aims to develop SHK-loaded exosomes (SHK-Exos) and assess their efficacy in CRC progression. Exosomes were isolated using ultracentrifugation and characterized via TEM, NTA, and western blotting. Their cellular internalization was confirmed through confocal microscopy post PKH67 labeling. Effects on cell behaviors were assessed using CCK-8 and Transwell assays. Cell cycle and apoptosis were analyzed via flow cytometry. A xenograft tumor model evaluatedin vivotherapeutic potential, and tumor tissues were examined using H&E staining andin vivoimaging. SHK-Exos demonstrated effective cell targeting and internalization in CRC cells.In vitro, SHK-Exos surpassed free SHK in inhibiting aggressive cellular behaviors and promoting apoptosis, whilein vivostudies showed substantial efficacy in reducing tumor growth with excellent tumor targeting and minimal toxicity. Employing SHK-Exos effectively impedes CRC progressionin vitroandin vivo, offering significant therapeutic potential. This research underscores the advantages of using autologous exosomes as a drug carrier, enhancing efficacy and reducing toxicity.

Identification, quantification and nutraceutical evaluation of the extracts from Arnebia benthamii roots of Himalayan regions of J&K, India.

Arnebia benthamii is one of the important sources of biologically active naphthoquinone pigments. The present study aimed at extraction of shikonin from Arnebia benthamii roots and its characterization. In order to identify and quantify shikonin, the extracts were evaluated using HPLC, LCMS, GCMS, NP-HPTLC and FTIR. Furthermore, nutraceutical evaluation was also done. It was found that the amount of shikonin was very low in the extracts obtained by using aqueous ethanol as it was not detected through chromatographic techniques. However, when hexane was used for extraction, a significant amount of shikonin (4.55 mg/g) was detected. The shikonin showed a linear range from 2-55 µg/mL with LOD and LOQ of 2.65 and 8.02 respectively, with a retention time of 3.64 min. The results of FTIR revealed that hexane extract had the intensity of functional groups similar to that of the standard. The values of DPPH radical inhibition were observed as 82.98 ± 0.01, 65.09 ± 0.23 %, 62.28 ± 0.86 % and 54.09 ± 0.23 % for Std, Ehex, Eus and Evs, respectively. The hexane extract showed the highest antioxidant activity as compared to other samples. Moreover, the hexane extracted shikonin displayed significantly (p > 0.05) high α-amylase and pancreatic lipase inhibition, indicating its high anti-diabetic and anti-lipidemic potential. It can be concluded that hexane is the best solvent for the extraction of shikonin and has better nutraceutical potential compared to ethanolic extracts.

Shikonin ameliorates depressive- and anxiogenic-like behaviors in rats via the suppression of inflammation in the hippocampus.

Shikonin is an active naphthoquinone with antioxidative, anti-inflammatory, and anticancer properties. In this study, we investigated the effects of shikonin on depressive- and anxiety-like behaviors in lipopolysaccharide- (LPS-) induced depression and chronic unpredictable mild stress (CUMS) rat models and explored the potential mechanism. First, a 14-day intraperitoneal administration of shikonin (10 mg/kg) significantly decreased immobility time in forced swimming test (FST) and increased open arm entries in elevated plus maze (EPM) test, without affecting line crossings in open field test (OFT), indicating that shikonin has anti-depressant- and anxiolytic-like effects. Second, chronic shikonin administration (10 mg/kg) reversed depressive- and anxiety-like behaviors in LPS-induced and CUMS depression models, as shown in the sucrose preference test (SPT), FST, EPM, and novel object recognition test (NORT). Finally, shikonin significantly reduced the levels of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α) in hippocampus, indicating that the anti-depressant- and anxiolytic-like effects of shikonin are related to the reduction of neuroinflammation in hippocampus. These findings suggest that shikonin exerts anti-depressant- and anxiolytic-like effects via an anti-inflammatory mechanism of shikonin in the hippocampus.

Research progress in mechanism of anticancer action of shikonin targeting reactive oxygen species.

Excessive buildup of highly reactive molecules can occur due to the generation and dysregulation of reactive oxygen species (ROS) and their associated signaling pathways. ROS have a dual function in cancer development, either leading to DNA mutations that promote the growth and dissemination of cancer cells, or triggering the death of cancer cells. Cancer cells strategically balance their fate by modulating ROS levels, activating pro-cancer signaling pathways, and suppressing antioxidant defenses. Consequently, targeting ROS has emerged as a promising strategy in cancer therapy. Shikonin and its derivatives, along with related drug carriers, can impact several signaling pathways by targeting components involved with oxidative stress to induce processes such as apoptosis, necroptosis, cell cycle arrest, autophagy, as well as modulation of ferroptosis. Moreover, they can increase the responsiveness of drug-resistant cells to chemotherapy drugs, based on the specific characteristics of ROS, as well as the kind and stage of cancer. This research explores the pro-cancer and anti-cancer impacts of ROS, summarize the mechanisms and research achievements of shikonin-targeted ROS in anti-cancer effects and provide suggestions for designing further anti-tumor experiments and undertaking further experimental and practical research.

Based on network pharmacology and molecular docking to explore the potential mechanism of shikonin in periodontitis.

OBJECTIVES: To investigate the potential mechanisms of shikonin in preventing and treating periodontitis using network pharmacology and molecular docking methods. MATERIALS AND METHODS: The targets of shikonin were obtained in TCMSP and SEA databases, and targets of periodontitis were gathered from the OMIM, GeneCards and Drugbank Databases. The intersecting targets were entered into the DAVID database to obtain the relevant biological functions and pathways by GO and KEGG enrichment analysis. The obtained targets were analysed the protein-protein interaction (PPI) in STRING platform. In Cytoscape 3.8.0, the network analysis function with the MCODE plug-in were used to obtain the key targets, of shikonin and periodontitis. Molecular docking and molecular dynamics simulation (MD) were used to assess the affinity between the shikonin and the key targets. RESULTS: Shikonin was screened for 22 targets and periodontitis was screened for 944 targets, the intersecting targets were considered as potential therapeutic targets. The targets played important roles in cellular response to hypoxia, response to xenobiotic stimulus and positive regulates of apoptotic process by GO enrichment analysis. 10 significant pathways were analyzed by KEGG, such as human cytomegalovirus infection and PI3K-Akt signaling pathway, etc. Cytoscape software screened the key genes including AKT1, CCL5, CXCR4, PPARG, PTEN, PTGS2 and TP53. Molecular docking and MD results showed that shikonin could bind stably to the targets. CONCLUSIONS: The present study enriched the molecular mechanisms in periodontitis with shikonin, providing potential therapeutic targets for periodontitis.

Multifaceted insights into the environmental adaptability of Arnebia guttata under drought stress.

Introduction: Global warming has led to increased environmental stresses on plants, notably drought. This affects plant distribution and species adaptability, with some medicinal plants showing enhanced drought tolerance and increased medicinal components. In this pioneering study, we delve into the intricate tapestry of Arnebia guttata, a medicinal plant renowned for its resilience in arid environments. By fusing a rich historical narrative with cutting-edge analytical methodologies, this research endeavors to demystify the plant's intricate response to drought stress, illuminating its profound implications for medicinal valorization. Methods: The methodology includes a comprehensive textual research and resource investigation of A. guttata, regionalization studies, field sample distribution analysis, transcriptome and metabolome profiling, rhizosphere soil microbiome analysis, and drought stress experiments. Advanced computational tools like ArcGIS, MaxEnt, and various bioinformatics software were utilized for data analysis and modeling. Results: The study identified significant genetic variations among A. guttata samples from different regions, correlating with environmental factors, particularly precipitation during the warmest quarter (BIO18). Metabolomic analysis revealed marked differences in metabolite profiles, including shikonin content, which is crucial for the plant's medicinal properties. Soil microbial community analysis showed variations that could impact plant metabolism and stress response. Drought stress experiments demonstrated A. guttata's resilience and its ability to modulate metabolic pathways to enhance drought tolerance. Discussion: The findings underscore the complex interplay between genetic makeup, environmental factors, and microbial communities in shaping A. guttata's adaptability and medicinal value. The study provides insights into how drought stress influences the synthesis of active compounds and suggests that moderate stress could enhance the plant's medicinal properties. Predictive modeling indicates future suitable growth areas for A. guttata, aiding in resource management and conservation efforts. The research contributes to the sustainable development of medicinal resources and offers strategies for improving the cultivation of A. guttata.

Deucravacitinib and shikonin combination therapy ameliorates imiquimod-induced psoriasis in mice.

INTRODUCTION: TYK2 inhibitors and traditional natural drugs as promising drugs for psoriasis therapy are receiving increasing attention. They both affect different molecules of JAK/STAT pathway, but it is currently unclear whether their combination will enhance the effect on psoriasis. In this study, we used imiquimod (IMQ)-induced psoriasis mouse model to investigate the therapeutic effects of the combined administration of deucravacitinib (TYK2 inhibitor) and shikonin. METHODS: Aldara cream containing 5% IMQ was used to topically treat the dorsal skin of each mouse for a total of six consecutive days to induce psoriasis. The psoriasis area and severity index (PASI) scores were recorded every day. On the 7th day, skin tissues were taken for histopathological examination and the content of cytokines in skin were evaluated. The frequency of immune cells in peripheral blood, spleen and skin were detected through flow cytometry. RESULTS: Compared to the vehicle control group, the psoriasis symptoms and immune disorder improved significantly in the combination therapy group and deucravacitinib treatment group on the 7th day, and the expressions of p-STAT3 and Ki67 in skin were reduced as well. Moreover, the combined treatment of deucravacitinib and shikonin for psoriasis was superior to the monotherapy group, especially in inhibiting abnormal capillaries proliferation, reducing immune cells infiltration and decreasing the concentration of IL-12p70 in skin. CONCLUSION: The combination of deucravacitinib and shikonin is a promising clinical application.

Inhibitory Effects and Mechanisms of the Novel Shikonin Derivative DMAKO-20 on Melanoma Metastasis and Invasion.

BACKGROUND: Cutaneous melanoma is a malignant tumor with an increasing incidence, prone to recurrence and metastasis. This study aims to explore the effects and mechanisms of the novel shikonin derivative 5,8-dimethyl alkannin oxime derivative (DMAKO-20) on the metastasis and invasion of melanoma cells. METHODS: The inhibitory effects of DMAKO-20 on the melanoma cell line A375 were investigated through Cell Counting Kit-8 (CCK-8), Transwell and angiogenesis experiments. Network pharmacology and Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were employed to explore potential sites and pathways involved in this process. Additionally, quantitative polymerase chain reaction (qPCR) and Western blot experiments were conducted before and after drug treatment to verify the expression trends of related pathways and proteins. RESULTS: DMAKO-20 demonstrated selective inhibition of proliferation, invasion and migration of melanoma cells at low concentrations. The WNT pathway appears to be implicated in this process, as DMAKO-20 effectively attenuates its activation, consequently reducing matrix metalloproteinase 9 (MMP9) and Cellular Communication Network Factor 1 (CCN1)/cysteine-rich angiogenic inducer 61 (CYR61) levels. Such modulation inhibits melanoma dissemination and invasion into other tissues. CONCLUSION: DMAKO-20 exhibits the capability to suppress metastasis and invasion of melanoma cells, suggesting its potential for clinical application as an adjuvant therapy against melanoma.

Comparison of cell delivery and cell membrane camouflaged PLGA nanoparticles in the delivery of shikonin for colorectal cancer treatment.

Inspired by the "natural camouflage" strategy, cell-based biomimetic drug delivery systems (BDDS) have shown great potential in cancer therapy. Red blood cell (RBC) delivery vehicles and red blood cell membrane (RBCm)-camouflaged vehicles were commonly used strategies for drug delivery. We prepared shikonin-encapsulated PLGA nanoparticles (PLGA/SK) with different surface charges to obtain both RBC delivery and RBCm-camouflaged PLGA NPs. The physicochemical properties, in vivo circulation and antitumor effects of these biomimetic preparations were studied. Since the positive PLGA NPs may affect the morphology and function of RBCs, the biomimetic preparations prepared by the negative PLGA NPs showed better in vitro stability. However, positive PLGA NP-based biomimetic preparations exhibited longer circulation time and higher tumor region accumulation, leading to stronger anti-tumor effects. Meanwhile, the RBC delivery PLGA(+) NPs possessed better in vitro cytotoxicity, longer circulation time and higher tumor accumulation than RBCm-camouflaged PLGA(+) NPs. Collectively, RBC delivery vehicles possessed more potential than RBCm-camouflaged vehicles on drug delivery for tumor treatment, especially with positive NPs-loaded.

Shikonin-loaded PLGA nanoparticles: A promising strategy for psoriasis treatment.

Psoriasis is an inflammation-based skin illness marked by aggravated proliferation of epidermal cells. Shikonin is a natural naphthoquinone obtained from Arnebiae radix. It exerts anti-inflammatory and immunosuppressive effects. However, the poor water solubility and low bioavailability of shikonin limit its application. In this study, shikosin-loaded PLGA nanoparticle hydrogel was prepared and used to deliver the drug to the epidermis of psoriasis mice through local administration. The results demonstrated that shikosin-loaded PLGA nanoparticles inhibited HaCaT cell multiplication, increased drug uptake, and induced apoptosis of HaCaT cells. Results from Western blotting assays indicated that shikosin down-regulated the protein expressions of p65 and p-p65. Furthermore, shikonin mitigated psoriasis and decreased the concentrations of inflammation-inducing cytokines, i.e., IL17A, IL-17F, IL-22, IL-1ß, and TNF-α. Taken together, these results suggest that shikonin-PLGA nanoparticles loaded in hydrogel system possess promising therapeutic potential for psoriasis.

In vivo transgenic studies confirm the critical acylation function of LeBAHD56 for shikonin in Lithospermum erythrorhizon.

KEY MESSAGE: LeBAHD56 is preferentially expressed in tissues where shikonin and its derivatives are biosynthesized, and it confers shikonin acylation in vivo. Two WRKY transcriptional factors might regulate LeBAHD56's expression. Shikonin and its derivatives, found in the roots of Lithospermum erythrorhizon, have extensive application in the field of medicine, cosmetics, and other industries. Prior research has demonstrated that LeBAHD1(LeSAT1) is responsible for the biochemical process of shikonin acylation both in vitro and in vivo. However, with the exception of its documented in vitro biochemical function, there is no in vivo genetic evidence supporting the acylation function of the highly homologous gene of LeSAT1, LeBAHD56(LeSAT2), apart from its reported role. Here, we validated the critical acylation function of LeBAHD56 for shikonin using overexpression (OE) and CRISPR/Cas9-based knockout (KO) strategies. The results showed that the OE lines had a significantly higher ratio of acetylshikonin, isobutyrylshikonin or isovalerylshikonin to shikonin than the control. In contrast, the KO lines had a significantly lower ratio of acetylshikonin, isobutyrylshikonin or isovalerylshikonin to shikonin than controls. As for its detailed expression patterns, we found that LeBAHD56 is preferentially expressed in roots and callus cells, which are the biosynthesis sites for shikonin and its derivatives. In addition, we anticipated that a wide range of putative transcription factors might control its transcription and verified the direct binding of two crucial WRKY members to the LeBAHD56 promoter's W-box. Our results not only confirmed the in vivo function of LeBAHD56 in shikonin acylation, but also shed light on its transcriptional regulation.

Qingxiong Ointment and Its Active Ingredient, Shikonin Treat Psoriasis through HIF-1 Signaling Pathway.

BACKGROUND: Psoriasis is a common chronic inflammatory skin disorder. Qingxiong ointment (QX) is a natural medicinal combination frequently employed in clinical treatment of psoriasis. However, the active ingredients of QX and its precise mechanisms of improving psoriasis remain unclear. This study elucidated the effects of QX on an Imiquimod (IMQ)-induced mouse model of psoriasis while also exploring the regulation of the active ingredient of QX, shikonin, on the HIF-1 signaling pathway in HaCaT cells. METHODS: A mouse model of psoriasis was established through topical application of IMQ, and the local therapeutic effect of QX was evaluated using dorsal skin tissue with mouse psoriatic lesion and Psoriasis Area Severity Index (PASI) scores, hematoxylin-eosin (HE) staining, and immunohistochemical staining. Elisa and qPCR were employed to identify changes in the expression of inflammation-related factors in the mouse dorsal skin. Immunofluorescence was used to assess changes in the expression of T cell subsets before and after treatment with various doses of QX. HPLC was used to analyze the content of shikonin, and network pharmacology was employed to analyze the main targets of shikonin. Immunofluorescence was used to identify the effects of shikonin on the HIF-1 signaling pathway in IL6-induced psoriasis HaCaT cells. Finally, qPCR was used to identify the differential expression of the HIF-1 signaling pathway in skin tissues. RESULTS: QX significantly reduces PASI scores on the backs of IMQ-induced psoriasis mice. HE staining reveals alleviated epidermal thickness in the QX group. Immunohistochemical analysis shows a significant reduction in ICAM, KI67, and IL17 expression levels in the QX group. Immunofluorescence results indicate that QX can notably decrease the proportions of CD4+ T cells, γδ T cells, and CD8+ T cells while increasing the proportion of Treg cells. Network pharmacology analysis demonstrates that the main targets of shikonin are concentrated in the HIF-1 signaling pathway. Molecular docking results show favorable binding affinity between shikonin and key genes of the HIF-1 signaling pathway. Immunofluorescence results reveal that shikonin significantly reduces p-STAT3, SLC2A1, HIF1α, and NOS2 expression levels. qPCR results show significant downregulation of the HIF-1 signaling pathway at cellular and tissue levels. CONCLUSION: Our study revealed that QX can significantly reduce the dorsal inflammatory response in the IMQ-induced psoriasis mouse model. Furthermore, we discovered that its main component, shikonin, exerts its therapeutic effect by diminishing the HIF-1 signaling pathway in HaCaT cells.

Shikonin reduces M2 macrophage population in ovarian cancer by repressing exosome production and the exosomal galectin 3-mediated ß-catenin activation.

BACKGROUND: Shikonin (SK), a naphthoquinone with anti-tumor effects, has been found to decrease production of tumor-associated exosomes (exo). This study aims to verify the treatment effect of SK on ovarian cancer (OC) cells, especially on the production of exo and their subsequent effect on macrophage polarization. METHODS: OC cells SKOV3 and A2780 were treated with SK. The exo were isolated from OC cells with or without SK treatment, termed OC exo and SK OC exo, respectively. These exo were used to treat PMA-induced THP-1 cells (M0 macrophages). M2 polarization of macrophages was determined by measuring the M2 specific cell surface markers CD163 and CD206 as well as the secretion of M2 cytokine IL-10. The functions of galectin 3 (LGALS3/GAL3) and ß-catenin in macrophage polarization were determined by gain- or loss-of-function assays. CB-17 SCID mice were subcutaneously injected with SKOV3 cells to generate xenograft tumors, followed by OC exo or SK OC exo treatment for in vivo experiments. RESULTS: SK suppressed viability, migration and invasion, and apoptosis resistance of OC cells in vitro. Compared to OC exo, SK OC exo reduced the M2 polarization of macrophages. Regarding the mechanism, SK reduced exo production in cancer cells, and it decreased the protein level of GAL3 in exo and recipient macrophages, leading to decreased ß-catenin activation. M2 polarization of macrophages was restored by LGALS3 overexpression but decreased again by the ß-catenin inhibitor FH535. Compared to OC exo, the SK OC exo treatment reduced the xenograft tumor growth in mice, and it decreased the M2 macrophage infiltration within tumor tissues. CONCLUSION: This study suggests that SK reduces M2 macrophage population in OC by repressing exo production and blocking exosomal GAL3-mediated ß-catenin activation.

Mycobacterium smegmatis enhances shikonin-induced immunogenic cell death-an efficient in situ tumor vaccine strategy.

Tumor vaccines are a promising avenue in cancer immunotherapy. Despite the progress in targeting specific immune epitopes, tumor cells lacking these epitopes can evade the treatment. Here, we aimed to construct an efficient in situ tumor vaccine called Vac-SM, utilizing shikonin (SKN) to induce immunogenic cell death (ICD) and Mycobacterium smegmatis as an immune adjuvant to enhance in situ tumor vaccine efficacy. SKN showed a dose-dependent and time-dependent cytotoxic effect on the tumor cell line and induced ICD in tumor cells as evidenced by the CCK-8 assay and the detection of the expression of relevant indicators, respectively. Compared with the control group, the in situ Vac-SM injection in mouse subcutaneous metastatic tumors significantly inhibited tumor growth and distant tumor metastasis, while also improving survival rates. Mycobacterium smegmatis effectively induced maturation and activation of bone marrow-derived dendritic cells (DCs), and in vivo tumor-draining lymph nodes showed an increased maturation of DCs and a higher proportion of effector memory T-cell subsets with the Vac-SM treatment, based on flow cytometry analysis results. Collectively, the Vac-SM vaccine effectively induces ICD, improves antigen presentation by DCs, activates a specific systemic antitumor T-cell immune response, exhibits a favorable safety profile, and holds the promise for clinical translation for local tumor immunotherapy.

Pharmacological Significance of Boraginaceae with Special Insights into Shikonin and Its Potential in the Food Industry.

Shikonin is a naphthoquinone pigment present in the hairy roots of the plant species from the Boraginaceae family. The compound has been well investigated for its highly efficient medicinal, antioxidant, and antimicrobial properties. Various extraction methodologies have been employed to maximise yield while minimising waste production of shikonin and its derivatives. Despite substantial research on shikonin and Boraginaceae plants, a research gap persists in the food industry and extraction technologies. This review addresses crucial aspects of shikonin deserving of further exploration. It begins by elucidating the attributes of the Boraginaceae plants and their medicinal traits in folklore. It proceeds to focus on the roots of the plant and its medicinal properties, followed by extraction procedures explored in the last fifteen years, emphasising the novel technologies that have been chosen to improve the yield extract while minimising extraction times. Furthermore, this review briefly outlines studies employing cell culture techniques to enhance in vitro shikonin production. Lastly, attention is directed towards research in the food industry, particularly on shikonin-loaded biodegradable films and the antioxidant activity of shikonin. This review concludes by summarising the future potential in food science and prominent research gaps in this field.