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.

紫草素(shikonin)抗奥密克戎毒株的实验研究及机制分析

目的探讨右旋体紫草素(shikonin)抗奥密克戎毒株的药理作用和对新型冠状病毒主蛋白酶的抑制活性。方法使用奥密克戎毒株BA.2.2转染的Vero细胞模型检测shikonin的抗病毒活性;利用体外主蛋白酶荧光偏振模型测试shikonin对于新型冠状病毒主蛋白酶的抑制活性,实验以奈玛特韦为阳性对照化合物。结果Vero细胞模型药理活性测试发现shikonin具有抑制奥密克戎毒株核酸复制的作用;而在体外荧光偏振模型活性测试实验中,shikonin在20、50μM药物浓度并未表现出对主蛋白酶的显著抑制活性。结论紫草素(shikonin)在细胞水平的实验中表现出抗奥密克戎毒株的药理活性,但其可能是主蛋白酶的非特异性抑制剂,抗病毒的作用机理极可能为抑制新型冠状病毒RNA聚合酶。

Shikonin protects mitochondria through the NFAT5/AMPK pathway for the treatment of diabetic wounds

BACKGROUND Shikonin is a natural remedy that is effective at treating diabetic wounds.NFAT5 is a potential therapeutic target for diabetes,and mitochondrial function is essen-tial for wound healing.However,the relationship among Shikonin,NFAT5,and mitochondrial function has not been thoroughly studied.Here,we offer new per-spectives on the advantages of shikonin for managing diabetes.AIM To assess the therapeutic mechanism of shikonin in diabetic wounds,its rela-tionship with NFAT5,and its protection of mitochondrial function.METHODS Hypertonic cell and diabetic wound mouse models were established.NFAT5 expression was measured through western blotting and immunofluorescence,in vivo and in vitro.Mitochondrial function was evaluated using reactive oxygen species(ROS)detection and JC-1 and Calcein AM dyes.Mitochondrial structures were observed using transmission electron microscopy.The NFAT5/AMPK pathway was analyzed using a transfection vector and an inhibitor.The effect of shikonin on cells under hypertonic conditions via the NFAT5/AMPK pathway was assessed using western blotting.RESULTS Shikonin treatment preserved HaCaT cell viability,while significantly reducing cyclooxygenase-2 expression levels in a high-glucose environment(P<0.05).Additionally,shikonin maintained mitochondrial morphology,enhanced membrane potential,reduced membrane permeability,and decreased ROS levels in HaCaT cells under hyperosmolar stress.Furthermore,shikonin promoted wound healing in diabetic mice(P<0.05).Shikonin also inhibited NFAT5,in vivo and in vitro(P<0.05).Shikonin treatment reduced NFAT5 expression levels,subsequently inhibiting AMPK expression in vitro(P<0.05).Finally,shikonin inhibited several key downstream molecules of the NFAT5/AMPK pathway,including mammalian target of rapamycin,protein kinase B,nuclear factor kappa-light-chain-enhancer of activated B cells,and inducible nitric oxide synthase(P<0.05).CONCLUSION Shikonin protects mitochondria via the NFAT5/AMPK-related pathway and enhances wound healing in diabetes.

Effects of Shikonin on Proliferation, Apoptosis, and Extracellular Matrix of Human Mesangial Cells

Objective:To investigate the effect of shikonin on the proliferation, apoptosis and extracellular matrix (ECM) of human mesangial cells (MC). Methods: MC was cultured in vitro with different concentrations of glucose (30, 50, 80 mmol/L). The cell growth was observed by using MTT method and apoptosis by using an aunexin-V-Fluos. Immunohistochemical studies for Laminin (LN), Fibronectin (FN) and type Ⅳ Collagens (Col Ⅳ) were measured. Results: Shikonin inhibited their growth (P<0.05) and apoptosis in the glycated cultured cells. Shikonin 0.05 mmol/L significantly reduced the secretion of LN, FN and Col Ⅳ from MC (P<0.05) cultured in 30, 50 and 80 mmol/L glucose. Conclusion: Shikonin could prevent or treat diabetic nephropathy (DN) and glomerulosclerosis (GS).

Induction of apoptosis by shikonin through a ROS/JNK-mediated process in Bcr/Abl-positive chronic myelogenous leukemia （CML） cells

This study examined the signaling events induced by shikonin that lead to the induction of apoptosis in Bcr/ Abl-positive chronic myelogenous leukemia （CML） cells （e.g., K562, LAMA84）. Treatment of K562 cells with shikonin （e.g., 0.5 pM） resulted in profound induction of apoptosis accompanied by rapid generation of reactive oxygen species （ROS）, striking activation of c-Jun-N-terminal kinase （JNK） and p38, marked release of the mitochondrial proteins cytochrome c and Smac/DIABLO, activation of caspase-9 and -3, and cleavage of PARP. Scavenging of ROS completely blocked all of the above-mentioned events （i.e., JNK and p38 phosphorylation, cytochrome c and Smac/DIABLO release, caspase and PARP cleavage, as well as the induction of apoptosis） following shikonin treatment. Inhibition of JNK and knock-down of JNK1 significantly attenuated cytochrome c release, caspase cleavage and apoptosis, but did not affect shikonin-mediated ROS production. Additionally, inhibition of caspase activation completely blocked shikonin-induced apoptosis, but did not appreciably modify shikonin-mediated cytochrome c release or ROS generation. Altogether, these findings demonstrate that shikonin-induced oxidative injury operates at a proximal point in apoptotic signaling cascades, and subsequently activates the stress-related JNK pathway, triggers mitochondrial dysfunction, cytochrome c release, and caspase activation, and leads to apoptosis. Our data also suggest that shikonin may be a promising agent for the treatment of CML, as a generator of ROS.

New Asymmetric Synthesis of Alkannin and Shikonin

A new approach for asymmetric synthesis of alkannin and shikonin is presented. The chiral centers of the targets were introduced via an asymmetric C-arylation of protected chiral glyceraldehyde in high de. The two enantiomers were prepared with the D-isopropylidenegly- ceraldehyde as the starting material.

Protective Effects of Shikonin on Brain Injury Induced by Carbon Ion Beam Irradiation in Mice

Radiation encephalopathy is the main complication of cranial radiotherapy. It can cause necrosis of brain tissue and cognitive dysfunction. Our previous work had proved that a natural antioxidant shikonin possessed protective effect on cerebral ischemic injury. Here we investigated the effects of shikonin on carbon ion beam induced radiation brain injury in mice. Pretreatment with shikonin significantly increased the SOD and CAT activities and the ratio of GSH/GSSG in mouse brain tissues compared with irradiated group （P〈0.01）, while obviously reduced the MDA and PCO contents and the RO$ levels derived from of the brain mitochondria.

Enhancement of Shikonin Production in Cell Suspension Cultures of Arnebia Euchroma Employing Two-Liquid-Phase Systems

1 INTRODUCTIONThe production of secondary metabolites using plant cells has been the subject of much inter-est in recent years.Despite that tremendous research efforts had been made in this topic,notmany products have reached the commercial stage.It is generally acknowledged that the mainproblem in this field is the lack of basic knowledge of the biosynthetic routes,and the mechanisms found to bring about the production of such secondary metabolites.There are,however,some techniques that have beneficial effects on the production and ex-

A Novel Total Synthesis of (±)Shikonin

A novel total synthesis of （±）shikonin 1 was presented. The key intermediate 6 was achieved by the formylation of 1,8：4,5-bis（methylenedioxy）naphthalene 5 with N-methylformanilide in good yield. It was first reported that the addition of prenyllithium to 2-formyl-1,8：4,5- bismethylenedioxy naphthalene 6 gave 2-（1-hydroxy-4-methylpentyl） 1,8：4,5-bis（methylenedioxy） naphthalene 8 in 45% yield and 2-（1-hydroxy-2,2-dimethyl-3-butenyl）-1,8：4,5-bis（methylenedioxy）naphthalene 9 in 48% yield. After the electrooxide deprotection of 8, （±）shikonin 1 was prepared in high yield.

Shikonin from Chinese herbal medicine induces GSDME-controlled pyroptosis in tumor cells

Objective:To 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.).Methods:We 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.Results:Compared 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.Conclusion:Our 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.

Anti-Fertility Effects and Mechanism of the Plant Extract Shikonin on Mice

Controlling fertility of rodent pests has become an effective means of controlling the population of grassland rodents in China. Recently, research has focused on how to select environmentally-friendly sterilants without pollution effects, and to realize sustainable control of pest rodent populations. Sterilants from plant extracts have been mainly selected. In this study, mice were used as the experimental subjects for research on the anti-fertility effects of plant extracts of shikonin and the anti-fertility mechanism of shikonin extract was determined. The mice were divided into four groups, including one control group and three experimental groups. There were three applications of shikonin extract in different concentrations (5 mg·kg-1, 20 mg·kg-1 and 50 mg·kg-1). The mice gavage experiments indicated that a shikonin concentration of 50 mg·kg-1 had the expected anti-fertility effects. Mice copulation experiments showed that the 50 mg·kg-1 shikonin treatment had significant anti-fertility effects on both female-treatment and female-male-treatment groups. The results of the PCR analysis on the AgRP and ghrelin mRNA from female ovaries and male testicles indicated that shikonin could control mice reproduction by regulating the pituitary gonadal axis. Shikonin, as plant source sterile agent, would have more ideal effects for functioned both sexes sterility.

A Simple Synthesis of dl-Shikonin

A new facile route for the synthesis of dl-shikonin is presented. Reformatsky reaction assisted cross-coupling of 1, 4, 5, 8-tetramethoxynaphthalene-2-carbaldehyde and ethyl- bromoacetate was employed for introduction of the side chain of dl-shikonin.

A novel natural compound Shikonin inhibits YAP function by activating AMPK

Objective： Yes Associated Protein （YAP） is a downstream effector that negatively regulated by Hippo kinase LATS1/2. As a transcriptional coactivator, YAP controls the transactivation of variety target genes to promote cell proliferation which is a critical survival input for cancer cells, thus the inhibition of YAP function is a promising strategy to treat cancer patients. The aim of this study was to explore YAP inhibitors derived from natural products using a cell-based YAP-TEADs luciferase reporter assay and investigate the functional activities of the novel inhibitor. Methods： natural compounds were used by 8×GTIIC luciferase reporter assay to screen YAP inhibitor. Phosphorylation of YAP and AMPK were detected by Western Blotting. The target genes of YAP were determined through RT-PCR. Inhibition on HepG2 cells of screened compounds were assessed by the Sulforhodamine B （SRB） assay. Results： we found that Shikonin （derived from the traditional Chinese medical herb Zicao （Lithospermum erythrorhizon）） exerted significant suppression against the transcriptional activity of YAP （inhibition ratio=74.3%）, accompanied with increased phosphorylation of YAP protein upon within short-exposure to cancer cells. Shikonin treated on HepG2 induced phosphorylation of AMPK. In HepG2 cell lines, Shikonin exhibited a profound cytotoxicity in a concentration manner. Conclusion： our results indicated that the inhibition activity of Shikonin on YAP function was probably due to the activation of AMPK by phosphorylation. Moreover, Shikonin exhibited potent cytotoxicity on cancer cells. In summary, the present study identifies Shikonin as a novel natural inhibitor of YAP function and could be an anti-cancer drug candidate for cancer treatment.

Shikonin Promotes Skin Cell Proliferation and Inhibits Nuclear Factor-κB Translocation via Proteasome Inhibition In Vitro

Background：Shikonin is a major active chemical component extracted from Lithospermi Radix,an effective traditional herb in various types of wound healing.Shikonin can accelerate granulomatous tissue formation by the rat cotton pellet method and induce neovascularization in granulomatous tissue.The purpose of the study was to investigate its mechanism of action in human skin cells.Methods：MTS assay was used to measure cell growth.The collagen type Ⅰ （COL1） mRNA expression and procollagen type Ⅰ C-peptide （PIP） production were detected by real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay,respectively.Immunofluorescence and western blot analyses were carried out to investigate nuclear factor-κB （NF-κB） signaling pathway.Cell-based proteasome activity assay was used to determine proteasome activity.Results：In this study,we found that 10 μmol/L shikonin stimulated the growth of normal human keratinocytes and 1 μmol/L shikonin promoted growth of human dermal fibroblasts.However,shikonin did not directly induce COLI mRNA expression and PIP production in dermal fibroblasts in vitro.In addition,1 μmol/L shikonin inhibited translocation of NF-κB p65 from cytoplasm to nucleus induced by tumor necrosis factor-α stimulation in dermal fibroblasts.Furthermore,shikonin inhibited chymotrypsin-like activity of proteasome and was associated with accumulation ofphosphorylated inhibitor κB-α in dermal fibroblasts.Conclusions：These results suggested that shikonin may promote wound healing via its cell growth promoting activity and suppress skin inflammation via inhibitory activity on proteasome.Thus,shikonin may be a potential therapeutic reagent both in wound healing and inflammatory skin diseases.

Design, Synthesis and Anticancer Activity of Shikonin and Alkannin Derivatives with Different Substituents on the Naphthazarin Scaffold

Based on the asymmetric reduction of alkannin ketone derivative 4 by diisopinocampheylchloro- borane（DIP-C1）, a series of shikonin and alkannin derivatives was designed, synthesized and their anticancer activi- ties against various kinds of cell lines were evaluated. The in vitro biological experiments demonstrated that com- pound S-10, a 5,8-O-dimethyl-l,4-dioxime alkannin derivative, not only displayed excellent antiproliferative activity against cancer cells, but also exhibited 10w toxicity towards normal cells. It could induce HCT-116 cell apoptosis, but had no impact on the cell cycle. The underlying anticancer mechanism of S-10 is most likely different from other shikonin and alkannin derivatives.

Electrochemical Study and Application on Shikonin at Poly（diallyldimethylammonium chloride） Functionalized Graphene Sheets Modified Glass Carbon Electrode

The electrochemical behaviors of shikonin at a poly（diallyldimethylammonium chloride） functionalized graphene sheets modified glass carbon electrode（PDDA-GS/GCE） have been investigated. Shikonin could exhibit a pair of well-defined redox peaks at the PDDA-GS/GCE located at 0.681 V（Epa） and 0.662 V（Epc）[vs. saturated calo- mel electrode（SCE）] in 0.1 mol/L phosphate buffer solution（pH=2.0） with a peak-to-peak separation of about 20 mV, revealing a fast electron-transfer process. Moreover, the current response was remarkably increased at PDDA- GS/GCE compared with that at the bare GCE. The electrochemical behaviors of shikonin at the modified electrode were investigated. And the results indicate that the reaction involves the transfer of two electrons, accompanied by two protons and the electrochemical process is a diffusional-controlled electrode process. The electrochemical para- meters of shikonin at the modified electrode, the electron-transfer coefficient（a）, the electron-transfer number（n） and the electrode reaction rate constant（ks） were calculated to be as 0.53, 2.18 and 3.6 s^-1, respectively. Under the optimal conditions, the peak current of differential pulse voltammetry（DPV） increased linearly with the shikonin concentra- tion in a range from 9A72×10^-8 mol/L to 3,789×10^-6 mol/L with a detection limit of 3,157× 10^-8 mol/L. The linear regression equation was Ip=O.7366c＋0.7855（R=0.9978; lp： 10-7 A, c： 10-8 mol/L）. In addition, the modified glass carbon electrode also exhibited good stability, selectivity and acceptable reproducibility that could be used for the sensitive, simple and rapid determination of shikonin in real samples. Therefore, the present work offers a new way to broaden the analytical application of graphene in pharmaceutical analysis.

Phosphorylated extracellular signal-regulated kinase up-regulated p53 expression in shikonin-induced HeLa cell apoptosis

Background The role of extracellular signal-regulated kinase 1/2 (ERK1/2) in shikonin-induced HeLa cells apoptosis remains vague. This study was to investigate the activation of caspase pathways and the role of ERK1/2 in human cervical cancer cells, HeLa, by shikonin. Methods The inhibitory effect of shikonin on the growth of HeLa cells was measured by MTT assay. Fluorescent microscopic analysis of apoptotic cells stained with 4’,6’-oliiamiclino-2-phenylindole C (DAPI) and Hoechst 33258 was carried out. Caspase-3 and -8 activities were detected using caspase-3 substrate and caspase-8 substrate as substrates, respectively. The protein levels of ERK, p53 and p-ERK were determined by Western blot analysis.Results Shikonin inhibited cell growth in a time- and dose-dependent manner. Caspase-3 and caspase-8 were activated in the apoptotic process and caspase inhibitors effectively reversed shikonin-induced apoptosis. Phosphorylation of ERK resulted in up-regulation of p53 expression, which was blocked by mitogen-activated protein kinase (MEK), inhibitor PD 98059.Conclusion Shikonin induces HeLa cell apoptosis through the ERK, p53 and caspase pathways.

Pharmacological and analytical aspects of alkannin/shikonin and their derivatives:An update from 2008 to 2022

Alkannin/shikonin(A/S)and their derivatives are naturally occurring naphthoquinones majorly found in Boraginaceae family plants.They are integral constituents of traditional Chinese medicine Zicao(roots of Lithospermum erythrorhizon).In last two decades significant increase in pharmacological investigations on alkannin/shikonin and their derivatives has been reported that resulted in discovery of their novel mechanisms in various diseases and disorders.This review throws light on recently conducted pharmacological investigations on alkannin/shikonin and their derivatives and their outputs.Various analytical aspects are also discussed and brief summary of patent applications on inventions containing alkannin/shikonin and its derivatives is also provided.

Shikonin alleviates collagen-induced arthritis mice by inhibiting M1 macrophage polarization

OBJECTIVE: To investigate the effects of shikonin(SKN) on M1 and M2 polarization of macrophages both in vitro and in vivo. METHODS: Collagen-induced arthritis(CIA) in male DBA/1 mice were treated with a dose of 4 mg/kg/day of SKN for 23 d(n = 6/group). The histopathology of inflamed joints in CIA mice was evaluated to test the antiarthritic effect of SKN. M1/M2 polarization of macrophages induced by lipopolysaccharide(LPS) and interferon(IFN)-γ or interleukin(IL)-4 and IL-13, were used to assess the effect of SKN(0.05, 0.1, and 0.2 μM). The effect of SKN on the protein expression of nitric oxide synthase, arginase, CD68, and CD206 was evaluated using western blot analysis. RESULTS: The results of this study revealed that SKN delayed the arthritis feet symptom score, reduced the incidence rate of arthritis, and relieved the inflammation of joints in CIA mice. SKN inhibited M1 macrophage polarization but did not affect M2 macrophage polarization in the joints of CIA mice. Moreover, SKN inhibited M1 polarization induced by LPS and IFN-γ, but did not affect M2 polarization induced by IL-4 and IL-13. CONCLUSION: These findings suggest that SKN alleviated CIA through inhibiting M1 macrophage polarization and has great potential as a new drug for RA treatment.

Electroanalytical method for determination of shikonin based on the enhancement effect of cyclodextrin functionalized carbon nanotubes

A simple and sensitive electroanalytical method for determination of shikonin, a widely used antitumoral agent, using β-cyclodextrin-functionalized multiwalled carbon nanotubes composite modified glassy carbon electrodes （MWCNTs/β-CD/GCE） was presented. CDs are water-soluble and environmentally friendly and can improve the dispersibility of MWCNTs/βCD functional materials, which was confirmed by SEM. The electrochemical behaviors of shikonin on different electrodes were investigated by cyclic voltammetry （CV） and differential pulse voltammograms （DPVs）. The results demonstrated that the redox peak currents of shikonin obtained at MWCNTs/fl-CD/GCE were much higher than those at the βCD/GCE and MWCNTs/GCE, which can be attributed to the combination of the excellent electrocatalytic properties of MWCNTs and the molecular recognition ability of βCD. At MWCNTs/ βCD/GCE, the response current exhibits a linear range from 5.0 nmol/L to 10.0 μmol/L with a detection limit of 1.0nmol/L （S/N=3）. As a practical application, the proposed method was applied to quantitatively determine shikoninin urine samples with satisfying results.