Roots Of Lithospermum Erythrorhizon Research Articles

Unveiling the photostability of the molecule of colour shikonin: From solution to the solid state

Shikonin, Shk, is a molecule of colour characterized by its natural phenolic-based compound composition, belonging to the naphthoquinone family. It is known for its low solubility in water and limited resistance to lightfastness. Shk is present in variable amount in the dried roots of Lithospermum erythrorhizon, Lithospermum officinale, and Alkanna tinctoria. The dyestuff extracted from these plants has historically been employed in Asia and Europe for textile dyeing, producing various shades of purple. Despite the historical significance of this dye, it has received limited attention in the context of cultural heritage studies. In the present work, Shk and model hydroxynaphthoquinone dyes were irradiated with polychromatic and monochromatic light in solution and in the solid state, mimicking paint mock-ups, to evaluate their photochemical stability. Low photoreaction quantum yield values (ϕR < 10−5) were observed in solution, providing quantitative evidence for the high stability of this molecule to light exposure. Chromatographic-mass spectrometric analyses carried out on the irradiated solutions and extracts from artificially aged Shk paint mock-ups, detected 5,8-dihydroxy-2-(1‑hydroxy-3-oxo-4-methyl-4-pentenyl)-l,4-naphthoquinone as degradation product, together with newly identified intermediate species involved in the complex photodegradation process. These indicate that, both in solution and in the solid state, the photoreaction of Shk proceeds with a similar mechanism. In the context of heritage science, this work contributes to highlight the potentialities of the adopted noninvasive approach, corroborated by results collected by chromatographic-mass spectrometric techniques, in identifying Shk in historical and archaeological specimens through its absorption and emission features and/or by detecting its degradation products which are, for the first time, characterized in terms of their UV–Vis absorption and emission spectral properties.

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

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.

A Review on Shikonin and Its Derivatives as Potent Anticancer Agents Targeted against Topoisomerases.

The topoisomerases (TOPO) play indispensable roles in DNA metabolism, by regulating the topological state of DNA. Topoisomerase I and II are the well-established drug-targets for the development of anticancer agents and antibiotics. These drugs-targeting enzymes have been used to establish the relationship between drug-stimulated DNA cleavable complex formation and cytotoxicity. Some anticancer drugs (such as camptothecin, anthracyclines, mitoxantrone) are also widely used as Topo I and Topo II inhibitors, but the poor water solubility, myeloma suppression, dose-dependent cardiotoxicity, and multidrug resistance (MDR) limited their prolong use as therapeutics. Also, most of these agents displayed selective inhibition only against Topo I or II. In recent years, researchers focus on the design and synthesis of the dual Topo I and II inhibitors, or the discovery of the dual Topo I and II inhibitors from natural products. Shikonin (a natural compound with anthraquinone skeleton, isolated from the roots of Lithospermum erythrorhizon) has drawn much attention due to its wide spectrum of anticancer activities, especially due to its dual Topo inhibitive performance, and without the adverse side effects, and different kinds of shikonin derivatives have been synthesized as TOPO inhibitors for the development of anticancer agents. In this review, the progress of the shikonin and its derivatives together with their anticancer activities, anticancer mechanism, and their structure-activity relationship (SAR) was comprehensively summarized by searching the CNKI, PubMed, Web of Science, Scopus, and Google Scholar databases.

Inhibitory Effects of Shikonin Dispersion, an Extract of Lithospermum erythrorhizon Encapsulated in β-1,3-1,6 Glucan, on Streptococcus mutans and Non-Mutans Streptococci.

Shikonin is extracted from the roots of Lithospermum erythrorhizon, and shikonin extracts have been shown to have inhibitory effects on several bacteria. However, shikonin extracts are difficult to formulate because of their poor water solubility. In the present study, we prepared a shikonin dispersion, which was solubilized by the inclusion of β-1,3-1,6 glucan, and analysed the inhibitory effects of this dispersion on Streptococcus mutans and non-mutans streptococci. The shikonin dispersion showed pronounced anti-S. mutans activity, and inhibited growth of and biofilm formation by this bacterium. The shikonin dispersion also showed antimicrobial and antiproliferative effects against non-mutans streptococci. In addition, a clinical trial was conducted in which 20 subjects were asked to brush their teeth for 1 week using either shikonin dispersion-containing or non-containing toothpaste, respectively. The shikonin-containing toothpaste decreased the number of S. mutans in the oral cavity, while no such effect was observed after the use of the shikonin-free toothpaste. These results suggest that shikonin dispersion has an inhibitory effect on S. mutans and non-mutans streptococci, and toothpaste containing shikonin dispersion may be effective in preventing dental caries.

Neuroprotective and Neuroregenerative Effects of Shikonin-mediated Inhibition of NF-κB/Stat3 in Alcoholic Encephalopathy

Background: Targeting intracellular signaling molecules in nervous tissue progenitors is a promising basis for the development of novel neurodegenerative disease therapy approaches. Naphthoquinone shikonin from the root of Lithospermum erythrorhizon is known to have inhibitory effects on NF-κB and STAT3. Objective: This study aimed to explore the influence of shikonin on the functioning of progenitors of nervous tissue and its neuroprotective properties in the modeling of alcoholic encephalopathy (AE). Methods: Experiments were performed on C57B1/6 male mice. AE was modeled by prolonged intragastric administration of ethanol. We studied the exploratory behavior and conditioned reflex activity in laboratory animals, as well as the functioning of neural stem cells (NSCs) and neuronal committed progenitors (NCPs) of the subventricular zone of the cerebral hemispheres. NCPs were obtained using the immunomagnetic separation method. The direct in vitro effects of the shikonin on the colony-forming capacity of progenitors, their proliferative activity, and intensity of specialization were compared with the effects of synthetic NF-κB and STAT3 inhibitors. Results: Results of in vitro experiments showed that the influence of phytochemicals was similar to the effects of synthetic NF-κB and STAT3 inhibitors. Shikonin stimulated the proliferation of NSCs and NCPs. In vivo shikonin administration caused the normalization of exploratory behavior and conditioned reflex activity of mice with AE. These effects developed after an increase in NSCs and NCPs content in the subventricular zone of the cerebral hemispheres due to an increase in their proliferative activity. The intensity of specialization of progenitors was also accelerated. Conclusion: The findings indicated the promise of developing a novel approach to the treatment of AE based on shikonin-mediated inhibiting of NF-κB/STAT3.

Shikonin suppresses small cell lung cancer growth via inducing ATF3-mediated ferroptosis to promote ROS accumulation

Small cell lung cancer (SCLC) is a subtype of lung cancer with a very poor overall survival rate due to its extremely high proliferation and metastasis predilection. Shikonin is an active ingredient extracted from the roots of Lithospermum erythrorhizon, and exerts multiple anti-tumor functions in many cancers. In the present study, the role and underlying mechanism of shikonin in SCLC were investigated for the first time. We found that shikonin effectively suppressed cell proliferation, apoptosis, migration, invasion, and colony formation and slightly induced apoptosis in SCLC cells. Further experiment indicated the shikonin could also induced ferroptosis in SCLC cells. Shikonin treatment effectively suppressed the activation of ERK, the expression of ferroptosis inhibitor GPX4, and elevated the level of 4-HNE, a biomarker of ferroptosis. Both total ROS and lipid ROS were increased, while the GSH levels were decreased in SCLC cells after shikonin treatment. More importantly, our data identified that the function of shikonin was dependent on the up-regulation of ATF3 by performing rescue experiments using shRNA to silence the expression of ATF3, especially in the total and lipid ROS accumulaiton. Xenograft model was established using SBC-2 cells, and the results revealed that shikonin also significantly inhibited tumor growth by inducing ferroptosis. Finally, our data further confirmed that shikonin activated ATF3 transcription by impairing the recruitment of HDAC1 mediated by c-myc on the ATF3 promoter, and subsequently elevating of histone acetylation. Our data documented that shikonin suppressed SCLC by inducing ferroptosis in a ATF3-dependent manner. Shikonin upregulated the expression of ATF3 expression via promoting the histone acetylation by inhibiting c-myc-mediated HDAC1 binding on ATF3 promoter.

Deoxyshikonin inhibited rotavirus replication by regulating autophagy and oxidative stress through SIRT1/FoxO1/Rab7 axis

BackgroundRotavirus (RV) is a double-stranded RNA virus. RV prevention and treatment remain a major public health problem due to the lack of clinically specific drugs. Deoxyshikonin is a natural compound isolated from the root of Lithospermum erythrorhizon and one of the shikonin derivatives which owns remarkable therapeutic effects on multiple diseases. The purpose of this research was to inquire Deoxyshikonin's role and mechanism in RV infection. MethodsDeoxyshikonin's function in RV was estimated using Cell Counting Kit-8 analysis, cytopathic effect inhibition assay, virus titer determination, quantitative real-time PCR, enzyme linked-immunosorbent assay, Western blot, immunofluorescence, and glutathione levels detection. Also, Deoxyshikonin's mechanism in RV was appraised with Western blot, virus titer determination, and glutathione levels detection. Moreover, Deoxyshikonin's function in RV in vivo was determined using animal models, and diarrhea score analysis. ResultsDeoxyshikonin owned anti-RV activity and repressed RV replication in Caco-2 cells. Furthermore, Deoxyshikonin reduced autophagy and oxidative stress caused by RV. Mechanistically, Deoxyshikonin induced low protein levels of SIRT1, ac-Foxo1, Rab7, VP6, low levels of RV titers, low autophagy and oxidative stress. SIRT1 overexpression abolished the effects of Deoxyshikonin on RV-treated Caco-2 cells. Meanwhile, in vivo research affirmed that Deoxyshikonin also possessed anti-RV function, and this was reflected in increased survival rate, body weight, GSH levels, and decreased diarrhea score, RV virus antigen, LC-3II/LC3-I. ConclusionDeoxyshikonin reduced RV replication through mediating autophagy and oxidative stress via SIRT1/FoxO1/Rab7 pathway.

Promising Nanomedicines of Shikonin for Cancer Therapy.

Malignant tumor, the leading cause of death worldwide, poses a serious threat to human health. For decades, natural product has been proven to be an essential source for novel anticancer drug discovery. Shikonin (SHK), a natural molecule separated from the root of Lithospermum erythrorhizon, shows great potential in anticancer therapy. However, its further clinical application is significantly restricted by poor bioavailability, adverse effects, and non-selective toxicity. With the development of nanotechnology, nano drug delivery systems have emerged as promising strategies to improve bioavailability and enhance the therapeutic efficacy of drugs. To overcome the shortcoming of SHK, various nano drug delivery systems such as liposomes, polymeric micelles, nanoparticles, nanogels, and nanoemulsions, were developed to achieve efficient delivery for enhanced antitumor effects. Herein, this review summarizes the anticancer pharmacological activities and pharmacokinetics of SHK. Additionally, the latest progress of SHK nanomedicines in cancer therapy is outlined, focusing on long circulation, tumor targeting ability, tumor microenvironment responsive drug release, and nanosystem-mediated combination therapy. Finally, the challenges and prospects of SHK nanomedicines in the future clinical application are spotlighted.

Unveiling the Potentiality of Shikonin Derivatives Inhibiting SARS-CoV-2 Main Protease by Molecular Dynamic Simulation Studies.

Shikonin, a phytochemical present in the roots of Lithospermum erythrorhizon, is well-known for its broad-spectrum activity against cancer, oxidative stress, inflammation, viruses, and anti-COVID-19 agents. A recent report based on a crystallographic study revealed a distinct conformation of shikonin binding to the SARS-CoV-2 main protease (Mpro), suggesting the possibility of designing potential inhibitors based on shikonin derivatives. The present study aimed to identify potential shikonin derivatives targeting the Mpro of COVID-19 by using molecular docking and molecular dynamics simulations. A total of 20 shikonin derivatives were screened, of which few derivatives showed higher binding affinity than shikonin. Following the MM-GBSA binding energy calculations using the docked structures, four derivatives were retained with the highest binding energy and subjected to molecular dynamics simulation. Molecular dynamics simulation studies suggested that alpha-methyl-n-butyl shikonin, beta-hydroxyisovaleryl shikonin, and lithospermidin-B interacted with two conserved residues, His41 and Cys145, through multiple bonding in the catalytic sites. This suggests that these residues may effectively suppress SARS-CoV-2 progression by inhibiting Mpro. Taken together, the present in silico study concluded that shikonin derivatives may play an influential role in Mpro inhibition.

Acetylshikonin induces apoptosis of human osteosarcoma U2OS cells by triggering ROS-dependent multiple signal pathways

Acetylshikonin a natural compound isolated from the root of Lithospermum erythrorhizon and one of the shikonin derivatives which possess promising anticarcinogenic ability. In this study, we attempted to investigate the anti-cancer potential of acetylshikonin towards osteosarcoma U2OS cells. The effects of acetylshikonin towards the treatment of U2OS cells showed that decreased cell proliferation and inhibited migration ability of cells which are experimentally assessed via wide range of assays including MTT, WST-1, cell counting, colony formation assays, wound healing assay and gelatin zymography assay. We also observed that early apoptosis and late apoptosis were increased through fluorescence-activated cell sorter (FACS) analysis. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay showed that acetylshikonin induced DNA fragmentation. Western blot analysis revealed the apoptotic effect of acetylshikonin by measuring of proteins such as cleaved caspase −9, −8, −3, −6, −7, and Bcl-2 family. We observed that ROS level and DNA damage were increased via DCF-DA assay and comet assay. In terms of the presence of ROS, induction of apoptosis was detected by measuring proteins such as cleaved caspase 3, PARP, Bcl-2 and Bax. We suggested that the reactions were related to the nuclear translocation of FOXO3 through western blot of cytoplasmic/nuclear protein fractionation. We finally demonstrated that the knockdown of the FOXO3 induced the decrease of the apoptosis-associated proteins via western blot of FOXO3 siRNA transfection. Taken together, these results suggested that acetylshikonin might induce ROS-mediated apoptosis in a FOXO3-dependent manner against osteosarcoma cells. Therefore, acetylshikonin may be elucidated as an effective candidate for the treatment of osteosarcoma.

Antiallergic Effects of N,N-dicoumaroylspermidine Isolated from Lithospermum erythrorhizon on Mast Cells and Ovalbumin-Induced Allergic Rhinitis.

In East Asia, the dried root of Lithospermum erythrorhizon has been utilized as an anti-inflammatory, antipyretic, detoxifying, and anti-inflammatory agent. Recently, we reported that L. erythrorhizon protects against allergic rhinitis; however, the component within L. erythrorhizon that exerts antiallergic activity remains unknown. The purpose of the current study was to isolate and characterize the antiallergic active components in an ethanolic extract of L. erythrorhizon roots. We examined the antiallergic effects of L. erythrorhizon reflux ethanol extracts in an ovalbumin (OVA)-induced allergic rhinitis mouse model, and compared the chemical compounds extracted using the hot reflux and cold extraction methods. Chromatographic separation identified two novel anthraquinones, erythrin A and B, one newly discovered compound from the Lithospermum genus, N1″,N3″-dicoumaroylspermidine, and nineteen other recognized compounds. Their chemical structures were elucidated by single (1D) and 2D analysis of nuclear magnetic resonance (NMR) spectroscopic data, as well as high resolution mass spectrometry. Among the identified compounds, N,N′-dicoumaroylspermidine strongly inhibited the release of β-hexosaminidase, as well as the production of IL-3, IL-4, and IL-13 by IgE-sensitized and BSA-stimulated RBL-2H3 cells. Using the OVA-induced allergic rhinitis mouse model, we showed that N,N′-dicoumaroylspermidine reduced the production of serum OVA-specific IgE and the number of inflammatory cells in nasal lavage fluid. N,N′-dicoumaroylspermidine isolated from L. erythrorhizon exhibits antiallergic properties, making it potentially effective for allergic rhinitis.

Roots of Lithospermum erythrorhizon Alleviated Ovalbumin-Induced Allergic Rhinitis and IgE-triggered Degranulation of RBL-2H3 Cells

Lithospermum erythrorhizon (L. erythrorhizon) root is used in traditional medicine for its anti-inflammatory, antibacterial, and antioxidant properties. However, no studies have examined its impact on allergic rhinitis (AR). Here, we explored the protective effects of L. erythrorhizon in immunoglobulin E (IgE)-stimulated RBL-2H3 cells and in an ovalbumin (OVA)-induced AR mouse model. In the latter, we examined nasal mucosal inflammation, allergen-specific cytokine production, and histological changes to the nasal mucosa. In the mouse model, oral administration of an ethanol extract of L. erythrorhizon (LE) led to a marked reduction in rubbing and sneeze frequency, a significant decrease in serum OVA-specific IgE and IgG1 levels, and a significant increase in the IgG2a/IgG1 ratio. LE also reduced expression of interleukin (IL)-4, IL-5, and IL-13 in nasal lavage fluid (NALF), and suppressed inflammatory cell infiltration and epithelial degradation in nasal tissues. In IgE-stimulated RBL-2H3 cells, LE suppressed release of degranulation markers such as β-hexosaminidase and histamine. Based on these findings, we suggest that LE may ameliorate OVA-induced AR by regulating mast cell-mediated inflammatory responses.

Protective effect of shikonin in myocardial ischemia/reperfusion injury in rats by inhibition of autophagy through the Hippo pathway

Shikonin is widely acknowledged as a bioactive substance extracted from the root of lithospermum erythrorhizon with multifunction. It alleviates ischemic/reperfusion (I/R) injury in liver and brain. Due to the similar pathogenesis of I/R and hypoxia/reoxygenation (H/R)-stimulated injury, we aimed to explore the potential pharmacological effects of Shikonin on the myocardial injury. The rats with myocardial I/R injury and the primary cardiomyocytes with H/R-stimulated injury were taken as in vivo and in vitro models. 2,3,5-Triphenyltetrazolium chloride staining and ELISA kits were used for detection of myocardial infarction and cardiac injury. Hematoxylin and eosin and immunohistochemistry staining were used to analyze the effect of Shikonin on autophagy histology. Western blot was performed to detect the proteins related to autophagy and Hippo pathway. The results showed that SHK reduces the size of myocardial infarction, improved cardiac function, suppressed the expression of autophagy-related proteins, and reduced the amount of autophagosomes. The underlying mechanism is to activate Hippo pathway. In vitro assay also suggested that SHK enhanced the cell viability, reduced the apoptotic rates in rat primary cardiomyocytes. Collectively, our results demonstrated that SHK protects against myocardial I/R injury by inhibiting autophagy, of which the underlying molecular mechanism is to activate the Hippo signaling pathway.

Shikonin induced Apoptosis Mediated by Endoplasmic Reticulum Stress in Colorectal Cancer Cells.

Shikonin is a naphthoquinone pigment isolated from the root of Lithospermum erythrorhizon, which has displayed potent anti-tumor properties. However, the effects of shikonin in colorectal cancer cells have not been yet fully investigated. In this study, we demonstrated that shikonin significantly inhibited the activity of colorectal cancer cells in a time- and dose-dependent manner. The flow cytometry and western blot results indicated that shikonin induced cell apoptosis by down-regulating BCL-2 and activating caspase-3/9 and the cleavage of PARP. The expression of BiP and the PERK/elF2α/ATF4/CHOP and IRE1α /JNK signaling pathways were upregulated after shikonin treatment. The pre-treatment with N-acetyl cysteine significantly reduced the cytotoxicity of shikonin. Taken together, shikonin could inhibit proliferation of the colorectal cancer cell through the activation of ROS mediated-ER stress. The in vivo results showed that shikonin effectively inhibited tumor growth in the HCT-116 and HCT-15 xenograft models. In conclusion, shikonin inhibited the proliferation of colorectal cancer cells in vitro and in vivo and warrants future investigation.

ALCAP2 inhibits lung adenocarcinoma cell proliferation, migration and invasion via the ubiquitination of \u03b2-catenin by upregulating the E3 ligase NEDD4L

Lung cancer is recognized as the leading cause of cancer-related death worldwide, with non-small cell lung cancer (NSCLC) being the predominant subtype, accounting for approximately 85% of lung cancer cases. Although great efforts have been made to treat lung cancer, no proven method has been found thus far. Considering β, β-dimethyl-acryl-alkannin (ALCAP2), a natural small-molecule compound isolated from the root of Lithospermum erythrorhizon. We found that lung adenocarcinoma (LUAD) cell proliferation and metastasis can be significantly inhibited after treatment with ALCAP2 in vitro, as it can induce cell apoptosis and arrest the cell cycle. ALCAP2 also significantly suppressed the volume of tumours in mice without inducing obvious toxicity in vivo. Mechanistically, we revealed that ALCAP2-treated cells can suppress the nuclear translocation of β-catenin by upregulating the E3 ligase NEDD4L, facilitating the binding of ubiquitin to β-catenin and eventually affecting the wnt-triggered transcription of genes such as survivin, cyclin D1, and MMP9. As a result, our findings suggest that targeting the oncogene β-catenin with ALCAP2 can inhibit the proliferation and metastasis of LUAD cells, and therefore, ALCAP2 may be a new drug candidate for use in LUAD therapeutics.

Shikonin inhibits proliferation of melanoma cells by MAPK pathway-mediated induction of apoptosis

Shikonin, a natural product isolated from the roots of Lithospermum erythrorhizon, exhibits pharmacological effects against inflammation, ulcers, infections, and tumors. In the present study, we aimed to investigate the antitumor effects of shikonin on the human melanoma cell line, A375SM, and in an in vivo mouse xenograft model. We examined the anticancer effects of shikonin by in vitro experiments (MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, 4′,6-diamidino-2-phenylindole (DAPI) stain, annexin V/ propidium iodide (PI) stain, and protein analysis of apoptosis and mitogen-activated protein kinase (MAPK) pathways). Further, the anticancer effect in vivo was confirmed through a xenograft model. Our results showed that shikonin inhibited the proliferation of melanoma cells in a dose-dependent manner. In addition, shikonin significantly increased nucleus and chromatin condensation and early/late apoptosis. Shikonin also increased the pro-apoptotic proteins and decreased the anti-apoptotic proteins. Additionally, shikonin was overexpressed in MAPK pathways. Investigation of the effects of shikonin in a mouse xenograft model not only showed decreased A375SM tumor volume but also increased apoptosis as determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Furthermore, pathologic changes were not observed in the liver and kidney of mice. Collectively, the study indicated that shikonin inhibited the proliferation of the human melanoma cells by inducing apoptosis, mediated by MAPK pathway and that it is a potential candidate for an anticancer drug against melanoma cancer.

Uncovering the antitumor effects and mechanisms of Shikonin against colon cancer on comprehensive analysis

BackgroundShikonin, a naphthoquinone compound extracted from the root of Lithospermum erythrorhizon, has been extensively studied for its antitumor activity. However, the systematic pathways involved in Shikonin intervention in human colon cancer has not yet clearly defined. PurposeThis study was to evaluate the cytotoxic effects of Shikonin in colon cancer, as well as investigate the potential biomarkers from a global perspective and the possible antitumor mechanisms involved. MethodsIn this work, cell viability, cell cycle and cell apoptosis in human colon cancer cells were assessed to evaluate the antitumor activity of Shikonin. Transcriptomics and metabolomics were integrated to provide the perturbed pathways and explore the potential mechanisms. The crucial proteins and genes involved were further validated by immunohistochemistry and real-time quantitative PCR. ResultsShikonin revealed a remarkable antitumor potency in colon cancer. Cell cycle was significantly arrested at the S phase as well as apoptosis was induced in SW480 cell line. Furthermore, a total of 1642 differentially expressed genes and 40 metabolites were detected after Shikonin intervention. The integrated analysis suggested that the antitumor effect was mainly attributed to purine metabolism, arginine biosynthesis, pyrimidine metabolism, urea cycle and metabolism of amino acids. The up-regulated expression of proteins vital for arginine biosynthesis was subsequently validated by immunohistochemistry in xenograft mice. Notably, supplemental dNTPs and arginine could significantly reverse the cytotoxic effect induced by Shikonin and the genes participating in purine metabolism and arginine biosynthesis were further determined by RT-qPCR. ConclusionOur findings provide a systematic perspective in the therapeutic effect of Shikonin which might lay a foundation for further research on Shikonin in colon cancer.

Differential relieving effects of shikonin and its derivatives on inflammation and mucosal barrier damage caused by ulcerative colitis.

BackgroundUlcerative colitis (UC) is one of the most challenging human diseases. Natural shikonin (SK) and its derivatives (with have higher accumulation) isolated from the root of Lithospermum erythrorhizon have numerous beneficial effects, such as wound healing and anti-inflammatory activities. Some researchers have reported that hydroxynaphthoquinone mixture (HM) and SK attenuate the acute UC induced by dextran sulfate sodium (DSS). However, no existing study has systemically investigated the effectiveness of SK and other hydroxynaphthoquinone natural derivative monomers on UC.MethodsIn this study, mice were treated with SK and its derivatives (25 mg/kg) and mesalazine (200 mg/kg) after DSS administration daily for one week. Disease progression was monitored daily by observing the changes in clinical signs and body weight.ResultsIntragastric administration natural single naphthoquinone attenuated the malignant symptoms induced by DSS. SK or its derivatives remarkably suppressed the serum levels of pro-inflammatory cytokines while increasing the inflammatory cytokine interleukin (IL)-10 . Additionally, both SK and alkanin restrained the activities of cyclooxygenase-2 (COX-2), myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS) in serum and colonic tissues. SK and its derivatives inhibited the activation of nucleotide binding oligomerization domain-like receptors (NLRP3) inflammasome and NF-κB signaling pathway, thereby relieving the DSS-induced disruption of epithelial tight junction (TJ) in colonic tissues.ConclusionsOur findings shed more lights on the pharmacological efficacy of SK and its derivatives in UC against inflammation and mucosal barrier damage.

Shikonin suppresses the epithelial-to-mesenchymal transition by downregulating NHE1 in bladder cancer cells.

Shikonin (SK) is the major bioactive component extracted from the roots of Lithospermum erythrorhizon with anticancer activity. SK could inhibit the epithelial-to-mesenchymal transition (EMT) of cancer cells. However, the underlying mechanism is elusive. In the present study, the inhibitory activities of SK on proliferation, invasion and migration were examined in bladder cancer (BC) cells. SK potently decreased the viabilities of BC cells but showed less cytotoxicity to normal bladder epithelial cells. Moreover, SK reversed the EMT, suppressed the migration and invasion of BC cells. Intriguingly, NHE1, the major proton efflux pump, was dramatically down-regulated by SK. The EMT-inhibitory effect of SK was mediated by NHE1 down-regulation, as NHE1-overexpress alleviated while Cariporide (NHE1 inhibitor) enhanced this effect. Further, enforced alkalinization of intracellular pH (pHi) reversed the EMT-inhibitory effect of SK, indicating a key role of acidic pHi in this process. Finally, elevated NHE1 expression was observed in human bladder cancer tissues. Collectively, this research reveals a supportive effect of NHE1 and alkaline pHi on EMT. SK can suppress EMT through inhibiting NHE1 and hence inducing an acidic pHi.

Roots of Lithospermum erythrorhizon promotes retinal cell survival in optic nerve crush-induced retinal degeneration

Lithospermum erythrorhizon (L. erythrorhizon), used in traditional medicine, is a potent wound healing, anti-inflammatory and antioxidant plant. However, the effects of L. erythrorhizon on retinal degenerative diseases remain unknown. Here, we explored the protective effects of L. erythrorhizon in in vitro and in vivo retinal degeneration. We found that ethanol extract of L. erythrorhizon (EELE) and the dichloromethane fraction of L. erythrorhizon (MCLE) significantly increased cell viability under glutamate/BSO-induced excitotoxicity/oxidative stress in R28 cells. Treatment with EELE and MCLE reduced the intracellular reactive oxygen species (ROS) and the levels of apoptotic proteins, such as cleaved PARP and cleaved caspase-3. Furthermore, oral administration of EELE and MCLE in an in vivo optic nerve crush mouse model decreased RGC cell death and increased retinal thickness. The major compound between EELE and MCLE was found to be lithospermic acid A (LAA), which has been shown to prevent the elevation of ROS in R28. Therefore, EELE and MCLE have protective effects against the death of retinal cells in vitro and in vivo, and the major compound, LAA, has an antioxidant effect on retinal cells, suggesting that EELE and MCLE could be beneficial agents for retinal degenerative diseases, including glaucoma.