shikonin and Disease-Models--Animal

Pulmonary arterial hypertension (PAH), a fatal disease with an insidious onset and rapid progression, shows characteristics such as increases in pulmonary circulatory resistance and pulmonary arterial pressure, and progressive right heart failure. Shikonin can reduce right ventricular systolic pressure in chronically hypoxic mice. However, the mechanisms underlying the protective effect of shikonin against PAH pathogenesis have only been sporadically identified. The present study evaluated whether inhibiting the expression of pyruvate kinase M2 (PKM2) contributed to the improvement of pulmonary vascular remodeling in PAH rats induced by monocrotaline (MCT) treatment. Hemodynamic parameters were assessed using echocardiography and right ventricular catheterization. Right ventricular hypertrophy index analysis and hematoxylin and eosin staining were used to evaluate the degree of pulmonary vascular and right heart remodeling. Moreover, PKM2, p‑PKM2, ERK, p‑ERK, glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA) protein expression levels were semi‑quantified using western blotting. The expression and distribution of PKM2 were assessed using immunofluorescence microscopy. The present study demonstrated that MCT treatment caused pulmonary arterial hypertension and pulmonary vascular remodeling in experimental rats. Shikonin improved hemodynamics and pulmonary vascular remodeling in MCT‑induced PAH rats, decreased aerobic glycolysis and downregulated PKM2, p‑PKM2, p‑ERK, GLUT 1 and LDHA protein expression levels. Shikonin improved experimental pulmonary arterial hypertension hemodynamics and pulmonary vascular remodeling at least partly through the inhibition of PKM2 and the resultant suppression of aerobic glycolysis. These results provide a novel understanding of possible new treatment targets for PAH.

Topics: Animals; Disease Models, Animal; Monocrotaline; Pulmonary Arterial Hypertension; Pulmonary Artery; Pyruvate Kinase; Rats; Rats, Sprague-Dawley; Vascular Remodeling

Shikonin is a natural product with antioxidant and anti-inflammatory activities. The biological activity of shikonin is still not fully understood, as well as its association with innate immunity and immune and inflammatory bowel disease (IBD) in humans. In this study, the toxicity of shikonin on Raw264.7 cells was assayed by MTT, and polarization of inflammatory macrophages was determined by flow cytometry. The results showed that shikonin can inhibit the polarization of macrophages towards M1 type and significantly inhibited the production of NO in the concentration range of 0.5-1 μM. In addition, after treatment with shikonin, the production of IL-1β and TNF-α was significantly decreased. After shikonin administration, the body weight loss and decrease of colon length were significantly suppressed in DSS-treated colitis C57BL/6 mice. The pro-inflammatory cytokines TNF-α and IL-1β in colonic homogenate were significantly decreased. Shikonin treatment resulted in a notable improvement in the histopathological manifestations in DSS-treated animals at 25/50 mg/kg. Meanwhile, we found that shikonin can regulate differentiation of T helper 17 cell (Th17)/regulatory T cell (Treg), thereby regulating the balance of Th17/Treg cells and exerting an anti-inflammatory effect in IBD animal models. In conclusion, we found that shikonin protects against DSS-induced acute colitis by, among other things, reducing immune cell infiltration, polarizing macrophages, and regulating Th17/Treg differentiation, as well as by downregulating the release of inflammatory cytokines. These findings showed that shikonin can improve inflammation by affecting macrophage polarization. Our experimental data provide experimental evidence and theory basis for research on anti-inflammatory effects for the shikonin as health or functional food.

Topics: Animals; Colitis; Colon; Cytokines; Disease Models, Animal; Humans; Inflammatory Bowel Diseases; Mice; Mice, Inbred C57BL; Tumor Necrosis Factor-alpha

Allergic rhinitis (AR) is a disease in the nasal mucosa related with Th2 lymphocyte inflammatory action. Dendritic cells (DCs) have been proved that they played a significant role in the development and maintenance of AR. However, there is still a lack of specific therapies for DCs in clinical practice. Shikonin (SHI) is a natural naphthoquinone compound isolated from the Chinese herb Radix Arnebiae. It is reported that SHI can interference the phenotype and function of dendritic cells, so we speculate that SHI may be an effective drug for the treatment of AR. However, the clinical usage of SHI has been limited by the bioactive properties of poor solubility, short retention time and low bioavailability. Therefore, in order to better exert the anti-inflammatory effect of SHI, an efficient SHI delivery system is urgently needed.. We prepared and characterized SHI-PM and NGR-SHI-PM with the thin-film hydration method. We used retrodialysis method to explore the release behavior. We took immunofluorescence to investigate the expression of CD13 in vitro. Then we tested BM-DCs mature cell detection by flow cytometry. An allergic rhinosinusitis murine model, hematoxylin and eosin stain and flow cytometry were established to test the efficiency of anti-inflammation in vivo. At last, western blot analysis and plasmid construction and transfection assay were taken to reveal the molecular mechanisms.. In the present study, we revealed that NGR-modifified could strengthen the intracellular uptake of PM (p < 0.001) and CD13 was high expressed on mature BM-DCs (p < 0.001). NGR-modified could enhance the inhibition of SHI in vitro (p < 0.05). NGR-modifified could increase the distribution of PM in vivo by DiI fluorescently (p < 0.01). NGR-modified could enhance SHI anti-allergic activity in OVA-sensitized mice and enhance the inhibition of SHI on DC maturation in lymph node (p < 0.001). Our findings also suggest that SHI may have the inhibitory effect on AR through NF-κB pathway by targeting PARP.. In summary, we have shown that NGR-PM-SHI could be a novel strategy for targeted treating allergic rhinitis through the NF-κB pathway by targeting PARP.

Topics: Animals; Dendritic Cells; Disease Models, Animal; Mice; Mice, Inbred BALB C; Micelles; Naphthoquinones; Nasal Mucosa; NF-kappa B; Ovalbumin; Poly(ADP-ribose) Polymerase Inhibitors; Polyesters; Polyethylene Glycols; Rhinitis, Allergic

Shikonin is an ointment produced from Lithospermun erythrorhizon which has been used in traditional medicine both in Europe and Asia for wound healing and is associated with anti-inflammatory properties. The goal of this work is to assess the analgesic properties of Shikonin in the CFA-induced inflammation model of pain. Rats were subjected to inflammation of the hind paw by CFA injection with a preventive injection of Shikonin and compared to either a control group or to a CFA-inflamed group with the vehicle drug solution. Inflammation of the hind paw by CFA was assessed by measurement of the dorsal to plantar diameter. Mechanical thresholds were established by means of the Von Frey filaments which are calibrated filaments that exert a defined force. Finally, the spinal cord of the studied animals was extracted to analyse the microglia population through immunohistochemistry using the specific marker Iba-1. Our results show that Shikonin reduces the paw oedema caused by CFA inflammation. Subsequently, there is a concomitant restoration of the mechanical thresholds reduced by CFA hind paw injection. Additionally, spinal microglia is activated after CFA-induced inflammation. Our results show that microglia is inhibited by Shikonin and has concomitant restoration of the mechanical thresholds. Our findings demonstrate for the first time that Shikonin inhibits microglia morphological changes and thereby ameliorates pain-like behaviour elicited by mechanical stimulation.

Topics: Animals; Disease Models, Animal; Hyperalgesia; Inflammation; Microglia; Naphthoquinones; Pain; Rats; Spinal Cord

Colorectal cancer (CRC) and inflammatory bowel disease (IBD) are the most common diseases of human digestive system. Nowadays, the influence of the inflammatory microenvironment on tumorigenesis has become a new direction, and the exploration of relative molecular mechanism will facilitate the discovery and identification of novel potential anti-cancer molecules.. Natural shikonin (SK) and acetyl-shikonin (acetyl-SK) was administered to azoxymethane (AOM)/dextran sodium sulphate (DSS)-induced colitis-associated colorectal cancer (CAC) mice model by gavage to investigate their therapeutic effects. Moreover, fresh feces and colon tissues were collected for determining the function of SK and acetyl-SK on the gut microbes and protein expression, respectively.. Both SK and acetyl-SK decreased AOM/DSS-induced CAC, and regulated the intestinal flora structure in CAC mouse model. They, especially SK, improved species richness, evenness and diversity of intestinal flora, recovered the upregulated ratio of Firmicutes to Bacteroidota (F/B ratio) which symbolizes gut microbiota dysbiosis. SK and its derivative increased the beneficial bacteria g__norank_f__Muribaculaceae, Lactobacillus, Lachnospiraceae_NK4A136_Group, and reduced those harmful ones including Ileibacterium and Coriobacteriaceae UCG-002. Notably, AOM/DSS caused significant increase in the abundance of Ileibaterium valens and g__norank_f__norank_o__Clostridia_UCG-014, which were not previously reported in studies of colonic inflammation or cancer, and the disorder was reversed by 20 mg/kg of SK. In our current study, the action of SK and acetyl-SK is dose-dependent, and 20 mg/kg SK exhibited the most effective functions, even better than the positive drug mesalazine. Moreover, differential proteomics and ELISA results showed that SK could recover the increase of pro-inflammatory cytokines (including IL-1β, IL-6 and TNF-α), the upregulation of pyruvate kinase isozyme type M2 (PKM2) and some other proteins (mainly concentrated in transcriptional mis-regulation in cancer and IL-17 signaling pathways), and the downregulation of Aldh1b1-Acc3-Maoa and Μgt2b34-Aldh1a1-Aldh1a7 involved in Wnt/β-catenin signaling pathway.. Our study identified SK and acetyl-SK, especially SK, as potential preventive agents for CAC through regulating both gut microbes and pathways involved in inflammation and cancer such as Wnt/β-catenin signaling pathway.

Topics: Animals; Azoxymethane; Bacteroidetes; Colitis; Colitis-Associated Neoplasms; Colorectal Neoplasms; Dextran Sulfate; Disease Models, Animal; Firmicutes; Humans; Inflammation; Mice; Mice, Inbred C57BL; Naphthoquinones; Tumor Microenvironment

Parkinson's disease (PD) is the second most common neurodegenerative disorder. Shikonin plays protective roles in age-associated diseases. Therefore, we investigate the biological functions of shikonin and its mechanisms involved in PD pathogenesis. The neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was used to mimic PD-like conditions in animal models. The learning and memory capacities were assessed by Morris water-maze test, pole test, locomotor activity test and rotarod test. Neuroinflammation was determined by measuring the levels of tumour necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). The quantification of superoxide dismutase, malondialdehyde and glutathione in substantia nigra was performed to estimate oxidative damage. Histopathologic changes were examined by haematoxylin and eosin staining. Immunofluorescence staining was conducted to determine the activation of astrocytes, tyrosine hydroxylase (TH)-positive neurons, and nuclear translocation of p65. Immunohistochemistry was performed to evaluate dopamine transporter (DAT)-positive neurons. Protein levels were measured by western blotting. Shikonin alleviates the cognitive and behavioural impairments. The death of dopaminergic neurons in nigra was attenuated by shikonin. The MPTP-induced neuroinflammation and oxidative stress in substantia nigra were alleviated by shikonin administration. Shikonin ameliorated the neuronal damage in nigra and inhibited the activation of astrocyte. Shikonin modulated the protein kinase B (Akt)/extracellular regulated kinase (ERK)/c-Jun N-terminal kinase (JNK)/nuclear factor κB (NF-κB) pathways. Shikonin ameliorates dopaminergic neuronal apoptosis by inhibiting oxidative stress and neuroinflammation via the Akt/ERK/JNK/NF-κB pathways in PD. The study has several limitations. First, in a previous study, levels of phosphorylated ERK were increased by MPTP. In our current study, we observed decreased p-ERK in nigra following MPTP treatment. Therefore, further investigation in the mechanisms of shikonin against PD progression is required. Second, the biological functions of shikonin need more exploration, including mitochondrial function and autophagy. Moreover, specific molecular targets for shikonin remain uncertain.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Cyclooxygenase 2; Disease Models, Animal; Dopamine Plasma Membrane Transport Proteins; Glutathione; Interleukin-6; JNK Mitogen-Activated Protein Kinases; Malondialdehyde; Mice; Mice, Inbred C57BL; Naphthoquinones; Neuroinflammatory Diseases; Neurotoxins; NF-kappa B; Nitric Oxide Synthase Type II; Oxidative Stress; Parkinson Disease; Proto-Oncogene Proteins c-akt; Superoxide Dismutase; Tumor Necrosis Factor-alpha; Tyrosine 3-Monooxygenase

Shikonin is the main component of the traditional Chinese medicine comfrey, which can inhibit the activity of PKM2 by regulating glycolysis and ATP production. Rheumatoid arthritis synovial cells (RA-FLSs) have been reported to increase glycolytic activity and have other similar hallmarks of metabolic activity. In this study, we investigated the effects of shikonin on glycolysis, mitochondrial function, and cell death in RA-FLSs. The results showed that shikonin induced apoptosis and autophagy in RA-FLSs by activating the production of reactive oxygen species (ROS) and inhibiting intracellular ATP levels, glycolysis-related proteins, and the PI3K-AKT-mTOR signaling pathway. Shikonin can significantly reduce the expression of apoptosis-related proteins, paw swelling in rat arthritic tissues, and the levels of inflammatory factors in peripheral blood, such as TNF-α, IL-6, IL-8, IL-10, IL-17A, and IL-1β while showing less toxicity to the liver and kidney.

Topics: Adenosine Triphosphate; Animals; Anti-Inflammatory Agents; Apoptosis; Arthritis, Rheumatoid; Autophagy; Cell Line; Disease Models, Animal; Energy Metabolism; Humans; Interleukins; Male; Naphthoquinones; Phosphatidylinositol 3-Kinase; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction; Synoviocytes; TOR Serine-Threonine Kinases

Choroidal neovascularization (CNV), a feature of neovasular age-related macular degeneration (AMD), acts as a leading cause of vision loss in the elderly. Shikonin (SHI), a natural bioactive compound extracted from Chinese herb radix arnebiae, exerts anti-inflammatory and anti-angiogenic roles and also acts as a potential pyruvate kinase M2 (PKM2) inhibitor in macrophages. The major immune cells macrophages infiltrate the CNV lesions, where the production of pro-angiognic cytokines from macrophage facilitates the development of CNV. PKM2 contributes to the neovascular diseases. In this study, we found that SHI oral gavage alleviated the leakage, area and volume of mouse laser-induced CNV lesion and inhibited macrophage infiltration without ocular cytotoxicity. Moreover, SHI inhibited the secretion of pro-angiogenic cytokine, including basic fibroblast growth factor (FGF2), insulin-like growth factor-1 (IGF1), chemokine (C-C motif) ligand 2 (CCL2), placental growth factor and vascular endothelial growth factor (VEGF), from primary human macrophages by down-regulating PKM2/STAT3/CD163 pathway, indicating a novel potential therapy strategy for CNV.

Topics: Angiogenesis Inducing Agents; Angiogenesis Inhibitors; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Blotting, Western; Cells, Cultured; Choroidal Neovascularization; Chromatography, High Pressure Liquid; Coloring Agents; Cytokines; Disease Models, Animal; Drugs, Chinese Herbal; Enzyme-Linked Immunosorbent Assay; Fluorescein Angiography; Humans; In Situ Nick-End Labeling; Indocyanine Green; Macrophages; Male; Mice; Mice, Inbred C57BL; Naphthoquinones; Phosphorylation; Pyruvate Kinase; Receptors, Cell Surface; STAT3 Transcription Factor

Shikonin is the major active component in

Topics: Acute Lung Injury; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Bronchoalveolar Lavage Fluid; Caspase 3; Cytokines; Disease Models, Animal; DNA Fragmentation; Inflammation; Lipopolysaccharides; Male; Mice, Inbred C57BL; Myeloid Cell Leukemia Sequence 1 Protein; Naphthoquinones; Neutrophil Infiltration; Neutrophils; Poly(ADP-ribose) Polymerases; Teichoic Acids; Tumor Suppressor Protein p53

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Benzoxepane derivatives were designed and synthesized, and one hit compound emerged as being effective in vitro with low toxicity. In vivo, this hit compound ameliorated both sickness behavior through anti-inflammation in LPS-induced neuroinflammatory mice model and cerebral ischemic injury through anti-neuroinflammation in rats subjected to transient middle cerebral artery occlusion. Target fishing for the hit compound using photoaffinity probes led to identification of PKM2 as the target protein responsible for anti-inflammatory effect of the hit compound. Furthermore, the hit exhibited an anti-neuroinflammatory effect in vitro and in vivo by inhibiting PKM2-mediated glycolysis and NLRP3 activation, indicating PKM2 as a novel target for neuroinflammation and its related brain disorders. This hit compound has a better safety profile compared to shikonin, a reported PKM2 inhibitor, identifying it as a lead compound in targeting PKM2 for the treatment of inflammation-related diseases.

Topics: Animals; Anti-Inflammatory Agents; Dibenzoxepins; Disease Models, Animal; Infarction, Middle Cerebral Artery; Interleukin-1beta; Ischemic Stroke; Lipopolysaccharides; Macrophages; Mice; Microglia; Naphthoquinones; NLR Family, Pyrin Domain-Containing 3 Protein; Pyruvate Kinase; Rats; RAW 264.7 Cells; Structure-Activity Relationship; Tumor Necrosis Factor-alpha

Postmenopausal osteoporosis results from estrogen withdrawal and is characterized mainly by bone resorption. Shikonin is a bioactive constitute of Chinese traditional herb which plays a role in antimicrobial and antitumor activities. The study was designed to investigate the role of shikonin on postmenopausal osteoporosis and explore its underlying mechanisms.. Immunofluorescence staining was performed to evaluate the effects of shikonin on actin ring formation. The expression levels of the nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) pathway were determined by Western blot analysis. To determine whether shikonin influences the receptor activator of nuclear factor-κB ligand (RANKL)-induced association between receptor activator of NF-κB (RANK) and tumor necrosis factor receptor associated factor 6 (TRAF6), immunofluorescence staining and immunoprecipitation experiments were performed. During our validation model, histomorphometric examination and micro-computed tomography (CT) were conducted to assess the morphology of osteoporosis.. Shikonin prevented bone loss by inhibiting osteoclastogenesis in vitro and improving bone loss in ovariectomized mice in vivo. At the molecular level, Western blot analysis indicated that shikonin inhibited the phosphorylation of inhibitor of NF-κB (IκB), P50, P65, extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), and P38. Interaction of TRAF6 and RANK was prevented, and downstream MAPK and NF-κB signaling pathways were downregulated.. Osteoclastic bone resorption was reduced in the presence of shikonin in vitro and in vivo. Shikonin is a promising candidate for treatment of postmenopausal osteoporosis.

Topics: Animals; Biomarkers; Bone Resorption; Cell Differentiation; Cell Survival; Disease Models, Animal; Disease Susceptibility; Female; Fluorescent Antibody Technique; Gene Expression Regulation; Humans; Immunohistochemistry; Mice; Mitogen-Activated Protein Kinases; Models, Biological; Naphthoquinones; NF-kappa B; Osteoclasts; Osteogenesis; Osteoporosis, Postmenopausal; Ovariectomy; Protein Binding; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction; TNF Receptor-Associated Factor 6; X-Ray Microtomography

Shikonin, a natural plant pigment, is known to have anti-obesity activity and to improve insulin sensitivity. This study aimed to examine the effect of shikonin on hepatic steatosis, focusing on the AMP-activated protein kinase (AMPK) and energy expenditure in Hepa 1-6 cells and in high-fat fed mice. Shikonin increased AMPK phosphorylation in a dose- and time-dependent manner, and inhibition of AMPK with compound C inhibited this activation. In an oleic acid-induced steatosis model in hepatocytes, shikonin suppressed oleic acid-induced lipid accumulation, increased AMPK phosphorylation, suppressed the expression of lipogenic genes, and stimulated fatty acid oxidation-related genes. Shikonin administration for four weeks decreased body weight gain and the accumulation of lipid droplets in the liver of high-fat fed mice. Furthermore, shikonin promoted energy expenditure by activating fatty acid oxidation. In addition, shikonin increased the expression of PPARγ coactivator-1α (PGC-1α), carnitine palmitoyltransferase-1 (CPT1) and other mitochondrial function-related genes. These results suggest that shikonin attenuated a high fat diet-induced nonalcoholic fatty liver disease by stimulating fatty acid oxidation and energy expenditure via AMPK activation.

Topics: AMP-Activated Protein Kinases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Carnitine O-Palmitoyltransferase; Cells, Cultured; Diet, High-Fat; Disease Models, Animal; Dose-Response Relationship, Drug; Energy Metabolism; Fatty Liver; Gene Expression; Lipid Metabolism; Mice; Naphthoquinones; Oxidation-Reduction; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Phosphorylation; Phytotherapy

Psoriasis is a chronic inflammatory skin disease characterized by well‑defined scaly papules and plaques. Interleukin (IL)‑17 is involved in its pathogenesis and promotes the proliferation of epidermal keratinocytes through signal transducer and activator of transcription 3 (STAT3) activation. Shikonin, a natural naphthoquinone isolated from Lithospermum erythrorhizon, possesses anti‑inflammatory and immunosuppressive properties and can suppress IL‑17‑induced vascular endothelial growth factor expression by inhibiting the JAK/STAT3 pathway. In the present study, MTS, iCELLigence and RT‑qPCR were used to determine the optimal concentration and duration of IL‑17 or shikonin acting on HaCaT cells. The changes in the expression levels of genes associated with the IL‑6/STAT3 pathway in differentially treated cells were analyzed via RT2Profiler™ PCR Array. Small interfering RNA was used to silence the expression levels of the target gene CCAAT/enhancer‑binding protein δ (CEBPD). Western blotting and immunohistochemistry were used to evaluate the effect of shikonin on imiquimod‑induced psoriasis in mice and the expression levels of CEBPD. Shikonin reversed IL‑17‑mediated downregulation of the tumor suppressor CEBPD in HaCaT cells. Moreover, low levels of CEBPD in the imiquimod‑induced mouse model of psoriasis were restored by shikonin treatment, which ameliorated excessive keratinocyte proliferation. Taken together, these findings suggest that CEBPD plays a key role in the pathogenesis of psoriasis and can be targeted by shikonin as a potential therapeutic strategy.

Topics: Animals; CCAAT-Enhancer-Binding Protein-delta; Cell Proliferation; Disease Models, Animal; Down-Regulation; HaCaT Cells; Humans; Imiquimod; Interleukin-17; Interleukin-6; Mice; Naphthoquinones; Psoriasis; Signal Transduction; STAT3 Transcription Factor

Multiple sclerosis is a common autoimmune inflammatory disease of the central nervous system. There are several underlying mechanisms for the pathogenesis of the disease, including inflammation, oligodendrocyte apoptosis and oxidative stress.. The mechanism of action of shikonin was investigated in the C57BL/6 experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis.. The results revealed that EAE induction significantly increased the extent of demyelination in the corpus callosum tissues of the animals, while treatment of the mice with shikonin significantly decreased the extent of demyelination. Real-time polymerase chain reaction-based analysis of the brain samples from the EAE mice revealed significant enhancement in the expression levels of tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and Bax genes as well as a reduction in the expression levels of transforming growth factor-ß (TGF-β) and Bcl2. But, shikonin treatment significantly reduced the expression levels of TNF-α, IFN-γ and Bax. On the other hand, the expression levels of TGF-β and Bcl2 as well as the activity of glutathione peroxidase-1 (GPX-1) enzyme were significantly increased following the shikonin treatment.. This study emphasized the immune-modulatory and antioxidative effects of shikonin, which may have an important healing effect on the severity of EAE.

Topics: Animals; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Corpus Callosum; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Immunologic Factors; Interferon-gamma; Mice, Inbred C57BL; Naphthoquinones; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Liver fibrosis is a worldwide clinical challenge during the progression of chronic liver disease to liver cirrhosis. Shikonin is extracted from the root of Lithospermum erythrorhizon with antioxidant, anti-inflammatory, anticancer, and wound-healing properties. The study aims to investigate the protective effect of shikonin on liver fibrosis and its underlying mechanism.. Shikonin significantly inhibited activation of hepatic stellate cells and extracellular matrix formation by downregulating the transforming growth factor-β1 expression and maintaining the normal balance between metalloproteinase-2 and tissue inhibitor of metalloproteinase-1. Shikonin also decreased hepatic stellate cell energy production by inhibiting autophagy.. The results confirmed that shikonin attenuated liver fibrosis by downregulating the transforming growth factor-β1/Smads pathway and inhibiting autophagy.

Topics: Alanine Transaminase; Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspartate Aminotransferases; Autophagy; Disease Models, Animal; Down-Regulation; Extracellular Matrix; Hepatic Stellate Cells; Liver Cirrhosis; Male; Matrix Metalloproteinase 2; Mice; Mice, Inbred C57BL; Naphthoquinones; Signal Transduction; Smad Proteins, Receptor-Regulated; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta1

Renal interstitial fibrosis is a common pathological feature of chronic kidney disease that may involve changes of metabolism in kidney cells. In the present study, we first showed that blockade of glycolysis with either dichloroacetate (DCA) or shikonin to target different glycolytic enzymes reduced renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO). Both inhibitors evidently suppressed the induction of fibronectin and collagen type I in obstructed kidneys, with DCA also showing inhibitory effects on collagen type IV and α-smooth muscle actin (α-SMA). Histological examination also confirmed less collagen deposition in DCA-treated kidneys. Both DCA and shikonin significantly inhibited renal tubular apoptosis but not interstitial apoptosis in UUO. Macrophage infiltration after UUO injury was also suppressed. Shikonin, but not DCA, caused obvious animal weight loss during UUO. To determine whether shikonin and DCA worked on tubular cells and/or fibroblasts, we tested their effects on cultured renal proximal tubular BUMPT cells and renal NRK-49F fibroblasts during hypoxia or transforming growth factor-β

Topics: Animals; Apoptosis; Cell Line; Dichloroacetic Acid; Disease Models, Animal; Enzyme Inhibitors; Epithelial Cells; Extracellular Matrix; Fibroblasts; Fibrosis; Glycolysis; Kidney Diseases; Kidney Tubules; Macrophages; Male; Mice; Mice, Inbred C57BL; Naphthoquinones; Signal Transduction; Ureteral Obstruction

Shikonin has been reported to induce glioma cell death via necroptosis, a type of programmed necrosis primarily mediated by RIP1 and RIP3. Although RIP1 and RIP3 are found to regulate some features of necrosis such as energy depletion and cellular membrane disruption, it remains unclear whether RIP1 and RIP3 could modulate DNA double strand breaks (DSBs), which is a crucial event leading to chromatinolysis. In this study, we used glioma cell lines and mice model of xenograft glioma to investigate the roles of RIP1 and RIP3 in shikonin-induced DNA DSBs. We found that shikonin induced upregulation of RIP1 and RIP3, necrosome formation and DNA DSBs in vitro and in vivo. In vitro investigation showed that the DNA DSBs and the reduction of cellular viabilities induced by shikonin were both prevented when RIP1 or RIP3 was pharmacologically inhibited by specific inhibitor or genetically knocked down with siRNA. Then, we proved that suppression of intracellular ROS with antioxidant NAC inhibited DNA DSBs caused by either hydrogen peroxide or shikonin, suggesting that ROS played a crucial role in regulation of DNA DSBs of glioma cells induced by shikonin. Further, we found that RIP1 and RIP3 regulated shikonin-induced overproduction of ROS via causing excessive generation of mitochondrial superoxide and depletion of GSH, indicating that ROS was the downstream signal of RIP1 and RIP3. Taken together, we demonstrated that RIP1 and RIP3 contributed to shikonin-induced DNA DSBs in glioma cells via increase of intracellular ROS levels.

Topics: Animals; Cell Line, Tumor; Disease Models, Animal; DNA Breaks, Double-Stranded; Glioma; Heterografts; Mice; Naphthoquinones; Nuclear Pore Complex Proteins; Reactive Oxygen Species; Receptor-Interacting Protein Serine-Threonine Kinases; RNA-Binding Proteins; Up-Regulation

Chronic kidney diseases generally lead to renal fibrosis. Despite great progress having been made in identifying molecular mediators of fibrosis, the mechanism that governs renal fibrosis remains unclear, and so far no effective therapeutic antifibrosis strategy is available. Here we demonstrated that a switch of metabolism from oxidative phosphorylation to aerobic glycolysis (Warburg effect) in renal fibroblasts was the primary feature of fibroblast activation during renal fibrosis and that suppressing renal fibroblast aerobic glycolysis could significantly reduce renal fibrosis. Both gene and protein assay showed that the expression of glycolysis enzymes was upregulated in mouse kidneys with unilateral ureter obstruction (UUO) surgery or in transforming growth factor-β1 (TGF-β1)-treated renal interstitial fibroblasts. Aerobic glycolysis flux, indicated by glucose uptake and lactate production, was increased in mouse kidney with UUO nephropathy or TGF-β1-treated renal interstitial fibroblasts and positively correlated with fibrosis process. In line with this, we found that increasing aerobic glycolysis can remarkably induce myofibroblast activation while aerobic glycolysis inhibitors shikonin and 2-deoxyglucose attenuate UUO-induced mouse renal fibrosis and TGF-β1-stimulated myofibroblast activation. Furthermore, mechanistic study indicated that shikonin inhibits renal aerobic glycolysis via reducing phosphorylation of pyruvate kinase type M2, a rate-limiting glycolytic enzyme associated with cell reliance on aerobic glycolysis. In conclusion, our findings demonstrate the critical role of aerobic glycolysis in renal fibrosis and support treatment with aerobic glycolysis inhibitors as a potential antifibrotic strategy.

Topics: Animals; Carrier Proteins; Cell Line; Deoxyglucose; Disease Models, Animal; Fibrosis; Glycolysis; Kidney; Male; Membrane Proteins; Mice; Myofibroblasts; Naphthoquinones; Phosphorylation; Pyruvate Kinase; Rats; Renal Insufficiency, Chronic; Thyroid Hormone-Binding Proteins; Thyroid Hormones; Time Factors; Transforming Growth Factor beta1; Ureteral Obstruction

Plasminogen activator inhibitor-1 (PAI-1) is a key negative regulator of the fibrinolytic system. Elevated levels of PAI-1 are associated with thrombosis and cardiovascular and metabolic diseases. Inhibition of PAI-1 activity represents a new strategy for antithrombotic and antifibrinolysis therapies. In this study, we systematically investigated the inhibitory effect of shikonin on PAI-1 activity. In the chromogenic substrate-based urokinase (uPA)/PAI-1 assay, we found that shikonin inhibited human PAI-1 activity with IC50 values of 30.68±2.32μM. This result was further confirmed by urokinase-type plasminogen activator (uPA)-mediated clot lysis assay. Mechanistic studies indicated that shikonin directly could bind to PAI-1 and prevent the binding of PAI-1 to uPA in a dose-dependent manner. Shikonin also blocked the formation of PAI-1/uPA complex, as shown by SDS/PAGE analysis. In the mouse arterial thrombosis model, intraperitoneal injection of shikonin at 1mgkg(-1) dose significantly prolonged tail bleeding time from 12.956±4.457min to 26.576±2.443min. It also reduced arterial thrombus weight from 0.01±0.001g to 0.006±0.001g (p<0.05). In a liver fibrosis treatment model, when shikonin was continuously injected intraperitoneally at a dose of 1mgkg(-1) over a two-week period, the hydroxyproline content in the mice plasma was significantly reduced and the degree of liver fibrosis was decreased significantly. Thus, shikonin may represent a novel small molecule inhibitor of PAI-1 that could have become a lead drug the treatment of thrombus and fibrosis.

Topics: Animals; Disease Models, Animal; Humans; Liver Cirrhosis; Mice; Naphthoquinones; Plasminogen Activator Inhibitor 1; Thrombosis; Urokinase-Type Plasminogen Activator

Sepsis, severe sepsis and septic shock are the main cause of mortality in non-cardiac intensive care units. Immunometabolism has been linked to sepsis; however, the precise mechanism by which metabolic reprogramming regulates the inflammatory response is unclear. Here we show that aerobic glycolysis contributes to sepsis by modulating inflammasome activation in macrophages. PKM2-mediated glycolysis promotes inflammasome activation by modulating EIF2AK2 phosphorylation in macrophages. Pharmacological and genetic inhibition of PKM2 or EIF2AK2 attenuates NLRP3 and AIM2 inflammasomes activation, and consequently suppresses the release of IL-1β, IL-18 and HMGB1 by macrophages. Pharmacological inhibition of the PKM2-EIF2AK2 pathway protects mice from lethal endotoxemia and polymicrobial sepsis. Moreover, conditional knockout of PKM2 in myeloid cells protects mice from septic death induced by NLRP3 and AIM2 inflammasome activation. These findings define an important role of PKM2 in immunometabolism and guide future development of therapeutic strategies to treat sepsis.

Topics: Animals; Carrier Proteins; Coinfection; Disease Models, Animal; DNA-Binding Proteins; eIF-2 Kinase; Female; Glycolysis; HMGB1 Protein; Humans; Inflammasomes; Interleukin-18; Interleukin-1beta; Macrophages; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Knockout; Myeloid Cells; Naphthoquinones; NLR Family, Pyrin Domain-Containing 3 Protein; Phosphorylation; Pyruvate Kinase; Sepsis; Signal Transduction; Thyroid Hormone-Binding Proteins; Thyroid Hormones

Shikonin, an analog of naphthoquinone pigments isolated from the root of Lithospermum erythrorhyzon, was recently reported to exert beneficial anti-inflammatory effects both in vivo and in vitro. The present study aimed to investigate the potential therapeutic effect of shikonin in a murine model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Dexamethasone was used as a positive control to evaluate the anti-inflammatory effect of shikonin in the study. Pretreatment with shikonin (intraperitoneal injection) significantly inhibited LPS-induced increases in the macrophage and neutrophil infiltration of lung tissues and markedly attenuated myeloperoxidase activity. Furthermore, shikonin significantly reduced the concentrations of TNF-α, IL-6 and IL-1β in bronchoalveolar lavage fluid induced by LPS. Compared with the LPS group, lung histopathologic changes were less pronounced in the shikonin-pretreated mice. Additionally, Western blotting results showed that shikonin efficiently decreased nuclear factor-kappaB (NF-κB) activation by inhibiting the degradation and phosphorylation of IκBα. These results suggest that shikonin exerts anti-inflammatory properties in LPS-mediated ALI, possibly through inhibition of the NF-κB signaling pathway, which mediates the expression of pro-inflammatory cytokines. Shikonin may be a potential agent for the prophylaxis of ALI.

Topics: Acute Lung Injury; Animals; Anti-Inflammatory Agents, Non-Steroidal; Bronchoalveolar Lavage Fluid; Cytokines; Disease Models, Animal; Lipopolysaccharides; Lithospermum; Lung; Male; Mice; Mice, Inbred BALB C; Naphthoquinones; NF-kappa B; Organ Size; Plant Roots; Signal Transduction

In the present paper we studied the effect of shikonin on ear oedema induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), and determined the mechanisms through which shikonin might exert its topical anti-inflammatory action.. Acute ear oedema was induced in mice by topical application of TPA. The in vitro assays used macrophages RAW 264.7 cells stimulated with lipopolysaccharide. Cyclooxygenase-2, inducible nitric oxide synthase, protein kinase Calpha, extracellular signal-regulated protein kinase (ERK), phosphorylated ERK (pERK), c-Jun N-terminal kinase (JNK), pJNK, p38, p-p38, p65, p-p65, inhibitor protein of nuclear factor-kappaB (NF-kappaB) (IkappaBalpha) and pIkappaBalpha were measured by Western blotting, activation and binding of NF-kappaB to DNA was detected by reporter gene and electrophoretic mobility shift assay, respectively, and NF-kappaB p65 localization was detected by immunocytochemistry.. Shikonin reduced the oedema (inhibitory dose 50 = 1.0 mg per ear), the expression of cyclooxygenase-2 (70%) and of inducible nitric oxide synthase (100%) in vivo. It significantly decreased TPA-induced translocation of protein kinase Calpha, the phosphorylation and activation of ERK, the nuclear translocation of NF-kappaB and the TPA-induced NF-kappaB-DNA-binding activity in mouse skin. Moreover, in RAW 264.7 cells, shikonin significantly inhibited the binding of NF-kappaB to DNA in a dose-dependent manner and the nuclear translocation of p65.. Shikonin exerted its topical anti-inflammatory action by interfering with the degradation of IkappaBalpha, thus inhibiting the activation of NF-kappaB.

Topics: Administration, Topical; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Line; Cell Nucleus; Disease Models, Animal; Dose-Response Relationship, Drug; Edema; Female; I-kappa B Proteins; Inflammation; Inhibitory Concentration 50; Macrophages; Mice; Naphthoquinones; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Protein Transport; Tetradecanoylphorbol Acetate