ObjectiveTo investigate the potential anti-tumor mechanisms of naphthoquinone compound shikonin (SKN) extracted from the root of Chinese herbal medicine plant lithospermum (Lithospermum erythrorhizon Sieb. & Zucc.).MethodsWe first observed that SKN treatment led to swelling and bubbles in HeLa cells that were similar to the phenotype of cell pyroptosis. Subsequently, the HeLa cells experienced a pyroptotic process with SKN, and this was then assessed using lactate dehydrogenase (LDH) release and propidium iodide (PI)/Hoechst double staining experiments. Pyroptosis is defined as gasdermin-mediated programmed necroptosis. To identify the potential pyroptosis machinery, two strategies were utilized that included a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 screening experiment and a pyroptosis reconstitution assay executed by each of the five known gasdermins (GSDMA-E). Moreover, endogenous cleavage was also detected in a panel of tumor cell lines.ResultsCompared with the control, both the LDH release and PI/Hoechst double-staining experiments suggested that SKN induced perforation and enhancement of the permeability of the cell membranes that resulted in pyroptosis in HeLa cells (P = .028 and P = .032, respectively). In addition, the reconstitution assays in human embryonic kidney 293T (HEK-293T) cells and endogenous cleavage assays in HeLa cells indicated that the pyroptosis was controlled by GSDME. In addition, we also found SKN could trigger pyroptosis in a panel of tumor cell lines in which the cellular morphologies were proportional to the GSDME expression levels. Additionally, the cleavage of GSDME was also detected, and this was indicative of a similar GSDME-mediated mechanism.ConclusionOur study not only explained the molecular mechanism of cytotoxicity of SKN to various tumor cells, but also provided additional information for the potential clinical application of natural naphthoquinone compounds against cancer. 相似文献
Shikonin, a natural flavonoid found in the roots of Lithospermum erythrorhizon, has been shown to possess many biological functions. The present study was undertaken to investigate the influence of shikonin on vascular smooth muscle contractility and to determine the mechanism involved. Denuded aortic rings from male rats were used and isometric contractions were recorded and combined with molecular experiments. Shikonin significantly relaxed fluoride-, thromboxane A2- or phorbol ester-induced vascular contraction suggesting as a possible anti-hypertensive on the agonist-induced vascular contraction regardless of endothelial nitric oxide synthesis. Furthermore, shikonin significantly inhibited fluoride-induced increases in pMYPT1 levels and phorbol ester-induced increases in pERK1/2 levels suggesting the mechanism involving the inhibition of Rho-kinase activity and the subsequent phosphorylation of MYPT1 and the inhibition of MEK activity and the subsequent phosphorylation of ERK1/2. This study provides evidence regarding the mechanism underlying the relaxation effect of shikonin on agonist-induced vascular contraction regardless of endothelial function. 相似文献
Oxidative stress acts as the major causative factor for various age‐associated neurodegenerative diseases, triggering cognitive and memory impairments. In the present study, the underlying neuroprotective mechanism governing how shikonin acts against D-galactose (D-gal)-induced memory impairment, neuroinflammation and neuron damage was examined. The results revealed that chronic administration of D-gal [150 mg/kg intraperitoneally (i.p.)] in mice caused cognitive and memory impairments, as determined by Morris water-maze test. Shikonin treatment, however, alleviated D‐gal-induced memory impairment and reversed the D‐gal-induced neural damage and apoptosis. Furthermore, western blotting and the results of morphological analysis revealed that shikonin treatments markedly reduced D‐gal induced neuroinflammation through inhibition of astrocytosis as determined by glial fibrillary acidic protein (GFAP) detection, and downregulating other inflammatory mediators, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6. Moreover, shikonin treatment led to inhibition of the activation of nuclear factor‐κB (NF‐κB) and the phosphorylation of mitogen-activated protein kinases (MAPKs), preventing neurodegeneration. Hence, taken together, the results of the present study suggested that shikonin attenuated D‐gal-induced memory impairment, neuroinflammation and neurodegeneration, possibly via the NF‐κB/mitogen-activated protein kinase (MAPK) pathway. Our data suggest that shikonin could be a promising, endogenous and compatible antioxidant candidate for age‐associated neurodegenerative diseases, including Alzheimer's disease. 相似文献
Context: Shikonin is a kind of naphthoquinone compound found mainly in Lithospermum erythrorhizon Sieb,et Zucc. Previous studies have shown that Shikonin has anti-tumor, anti-inflammatory and extensive pharmacological effects. According to new studies, Shikonin could also modulate the immune system function, but the effect to NK (nature killer) cells is yet unknown.
Objective: To investigate the effect and mechanism of Shikonin on NK cells proliferation and cytotoxicity to colon cancer cell line (Caco-2).
Methods: The proliferation and cytotoxicity of NK cells cultured with Shikonin were detected with CCK-8 assay. The expressions of perforin, GranB and IFN-γ were examined with FCM. The content of TNF-alpha was disclosed with ELISA kit. p-ERK1/2 and p-Akt expression of NK cells were detected with western blot.
Results: With CCK-8 assay, it is found that Shikonin could significantly enhance NK cells proliferation and cytotoxicity to colon cancer cells. With FCM assay, it is found that Shikonin could improve the expression of perforin and GranB in a dose-dependent manner. Shikonin had no effect on TNF-alpha and IFN-γ expression. In mechanism, the study shows that Shikonin could enhance the expression of p-ERK1/2 and p-Akt.
Conclusions: Shikonin enhances NK cells proliferation and cytotoxicity via the improvement of perforin, GranB, p-ERK1/2 and p-Akt expression. 相似文献
Chondrocyte apoptosis is mostly responsible for the development and progression of osteoarthritis. IL-1β is generally served as an agent that induces chondrocyte apoptosis. Shikonin exerts its anti-inflammatory effect on cartilage protection in vivo. We aimed to explore the protective effect of shikonin on interleukin-1beta (IL-1β)-induced chondrocyte apoptosis and the potential molecular mechanisms. Chondrocytes were isolated from the joints of newborn Sprague-Dawley rats. The MTT assay and LDH cell death assay were used to determine the cell viability and chondrocyte apoptosis was detected by Annexin-V/PI staining and nucleosomal degradation. The contents of phosphorylated-PI3K (p-PI3k), phosphorylated-Akt (p-Akt), Bcl-2, Bax, and cytochrome c were detected by Western blotting. A quantitative colorimetric assay was used to detect the caspase-3 activity. Our results showed that pretreatment with shikonin (4 μM) inhibited cytotoxicity and apoptosis induced by IL-1β (10 ng/ml) in chondrocytes. Shikonin pretreatment also decreased the activity of IL-1β that decreased Bcl-2 expression and levels of p-PI3K and p-Akt, and increased Bax expression, cytochrome c release, and caspase-3 activation. It also reversed the activity of IL-1β that promoted the synthesis of matrix metalloproteinase-13 and inhibited the expression of tissue inhibitor of metalloproteinase-1 expression, with the net effect of suppressing extracellular matrix degradation. These data suggested that shikonin may protect chondrocytes from apoptosis induced by IL-1β through the PI3K/Akt signaling pathway, by deactivating caspase-3. 相似文献