Tanshinone IIA protects intestinal epithelial cells from ferroptosis through the upregulation of GPX4 and SLC7A11

Background:Inflammatory bowel disease(IBD)is a chronic inflammatory disease of the gastrointestinal tract.The destruction of the intestinal epithelial barrier is one of the major pathological processes in IBD pathology.Growing evidence indicated that epithelial cell ferroptosis is linked to IBD and is considered a target process.Methods:RAS-selective lethal 3(RSL3)was used to induce ferroptosis in intestinal epithelial cell line No.6(IEC-6)cells,and cell ferroptosis and the effects of tanshinone IIA(Tan IIA)were determined by cell counting kit-8(CCK-8),reactive oxygen species(ROS)staining,Giemsa staining and transmission electron microscope(TEM).The cell viability of natural product library compounds was determined by CCK-8.The expression of ferroptosis-related genes were detected by real-time quantitative polymerase chain reaction(RT-qPCR)and western blot.Results:Treatment of IEC-6 cells results in the accumulation of ROS and typical morphological characteristics of ferroptosis.RSL3 treatment caused rapid cellular cytotoxicity which could be reversed by ferrostatin-1(Fer-1)in IEC-6 cells.Natural product library screening revealed that Tan IIA is a potent inhibitor of IEC-6 cell ferroptosis.Tan IIA could significantly protect the RSL3-induced ferroptosis of IEC-6 cells.Furthermore,the ferroptosis suppressors,glutathione peroxidase 4(GPX4),solute carrier family 7 member 11(SLC7A11),and miR-17-92 were found to be early response genes in RSL3-treated cells.Treatment of IEC-6 cells with Tan IIA resulted in upregulation of GPX4,SLC7A11,and miR-17-92.Conclusion:Our study demonstrated that Tan IIA protects IEC-6 cells from ferroptosis through the upregulation of GPX4,SLC7A11,and miR-17-92.The findings might provide a theoretical grounding for the future application of Tan IIA to treat or prevent IBD.

A novel prognostic gene signature,nomogram and immune landscape based on tanshinone IIA drug targets for hepatocellular carcinoma:Comprehensive bioinformatics analysis and in vitro experiments

Tanshinone IIA,one of the main ingredients of Danshen,is used to treat hepatocellular carcinoma(HCC).However,potential targets of the molecule in the therapy of HCC are unknown.Methods:In this study,we collected the tanshinone IIA targets from public databases for investigation.We screened differentially expressed genes(DEGs)across HCC and normal tissues using mRNA expression profiles from The Cancer Genome Atlas(TCGA).Univariate Cox regression analysis and least absolute shrinkage and selection operator(LASSO)Cox regression models were used to identify and construct the prognostic gene signature.Results:Finally,we discovered common genes across tanshinone IIA targets and HCC DEGs.We reported Fatty acid binding protein-6(FABP6),Polo-like Kinase 1(PLK1),deoxythymidylate kinase(DTYMK),Uridine Cytidine Kinase 2(UCK2),Enhancer of Zeste Homolog 2(EZH2),and Cytochrome P4502C9(CYP2C9)as components of a gene signature.The six-gene signature’s prognostic ability was evaluated using the Kaplan-Meier curve,time-dependent receiver operating characteristic(ROC),multivariate Cox regression analysis,and the nomogram.The mRNA level and protein expression of UCK2 were experimentally validated after treatment with different concentrations of tanshinone IIA in HEPG2 cells.CIBERSORTx,TIMER2.0,and GEPIA2 tools were employed to explore the relationship between the prognostic signature and immune cell infiltration.Conclusion:We established a six-gene signature as a reliable model with significant therapeutic possibility for prognosis and overall survival estimation in HCC patients,which might also benefit medical decision-making for appropriate treatment.

Molecularmechanisms of Tanshinone IIA in Hepatocellular carcinoma therapy via WGCNA-based network pharmacology analysis

Hepatocellular carcinoma(HCC)is a worldwide malignant tumor that caused irreversible consequences.Tanshinone IIA has been shown to play a notable role in HCC treatment.However,the potential targets and associating mechanism of Tanshinone IIA against HCC remain unknown.We first screened out 105 overlapping genes by integrating the predicted targets of Tanshinone IIA from multiple databases and the differentially expressed genes of HCC from the Cancer Genome Atlas(TCGA)database.Then,we performed weighted gene co-expression network analysis(WGCNA)using the RNA-seq profiles of overlapping genes and HCC-related clinical information.23 genes related to clinical tumor grade in the important module were imported for Gene Ontology(GO)enrichment,Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis and protein-protein interaction(PPI)analysis.Comparing the key genes in the important module from WGCNA with the high connectivity nodes from the PPI network,we identified three hub genes,AURKB,KIF11,and PLK1.For further verification,we tested the binding of Tanshinone IIA to three hub genes.The survival curve,receiver operating characteristic(ROC)curve,mRNA expression,and protein expression were also used to validate the hub genes.In the study,WGCNA revealed gradespecific gene modules,and the following KEGG pathway analysis indicated that Tanshinone IIA probably plays therapeutical effect in the development of HCC,especially in the cell cycle.Our result partially explained the pharmacological mechanism of Tanshinone IIA against HCC.

The antitumor effect of tanshinone IIA on antiproliferation and decreasing VEGF/VEGFR2 expression on the human non-small cell lung cancer A549 cell line

The effects of tanshinone IIA on the proliferation of the human non-small cell lung cancer cell line A549 and its possible mechanism on the VEGF/VEGFR signal pathway were investigated.The exploration of the interaction between tanshinone IIA and its target proteins provides a feasible platform for studying the anticancer mechanism of active components of herbs.The CCK-8 assay was used to evaluate the proliferative activity of A549 cells treated with tanshinone IIA(2.5 80 μmol/L) for 24,48 and 72 h,respectively.Flow cytometry was used for the detection of cell apoptosis and cell cycle perturbation.VEGF and VEGFR2 expression were studied by Western blotting.The binding mode of tanshinone IIA within thecrystal structure of the VEGFR2 protein was evaluated with molecular docking analysis by use of the CDOCKER algorithm in Discovery Studio 2.1.The CCK-8 results showed that tanshinone IIA can significantly inhibit A549 cell proliferation in a dose-and time-dependent manner.Flow cytometry results showed that the apoptosis rate of tested group was higher than the vehicle control,and tanshinone IIAtreated cells accumulated at the S phase,which was higher than the vehicle control.Furthermore,the expression of VEGF and VEGFR2 was decreased in Western blot.Finally,molecular docking analysis revealed that tanshinone IIA could be stably docked into the kinase domain of VEGFR2 protein with its unique modes to form H-bonds with Cys917 and π–π stacking interactions with Val848.In conclusion,tanshinone IIA may suppress A549 proliferation,induce apoptosis and cell cycle arrest at the S phase.This drug may suppress angiogenesis by targeting the protein kinase domains of VEGF/VEGFR2.

The effects of borneol on the pharmacokinetics and brain distribution of tanshinone IIA,salvianolic acid B and ginsenoside Rg1 in Fufang Danshen preparation in rats

Fufang Danshen preparation(FDP)is consisted of Salviae Miltiorrhizar Radix et Rhizoma(Danshen),Notoginseng Radix et Rhizoma(Sanqi)and Borneolum Syntheticum(borneol).FDP is usually used to treat myocardial ischemia hypoxia,cerebral ischemia and alzheimer’s disease,etc.In the treatment of cerebrovascular diseases,borneol is usually used to promote the absorption and distribution of the bioactive components to proper organs,especially to the brain.The purpose of this study is investigating the effects of borneol on the pharmacokinetics and brain distribution of tanshinone IIA(TS IIA),salvianolic acid B(SAB)and ginsenoside Rg1 in FDP.Male healthy Sprague-Dawley(SD)rats were given Danshen extracts,Sanqi extracts(Panax notoginseng saponins)or simultaneously administered Danshen extracts,Sanqi extracts and borneol.Plasma and brain samples were collected at different points in time.The concentration of TS IIA,SAB and Rg1 was determined by UPLC-MS/MS method.The main pharmacokinetics parameters of plasma and brain tissue were calculated by using Phoenix WinNolin 6.1 software.In comparison with Danshen and Sanqi alone,there were significant differences in pharmacokinetic parameters of TS IIA,SAB and Rg1,and the brain distribution of SAB and TS IIA when Danshen,Sanqi and borneol were administrated together.Borneol statistically significant shortened tmax of TS IIA,SAB and Rg1 in plasma and brain,increased the bioavaiability of Rg1,inhibited metabolism of Rg1 and enhanced the transport of TS IIA and SAB to brain.These results indicated that borneol could affect the multiple targets components and produce synergistic effects.Through accelerating the intestinal absorption and brain distribution,borneol caused the effective ingredients of Danshen and Sanqi to play a quicker therapeutic role and improved the therapeutic effect.

Drug-target networks for Tanshinone IIA identified by data mining

Tanshinone IIA is a pharmacologically active compound isolated from Danshen(Salvia miltiorrhiza), a traditional Chinese herbal medicine for the management of cardiac diseases and other disorders. But its underlying molecular mechanisms of action are still unclear. The present investigation utilized a data mining approach based on network pharmacology to uncover the potential protein targets of Tanshinone IIA. Network pharmacology, an integrated multidisciplinary study, incorporates systems biology, network analysis, connectivity, redundancy, and pleiotropy, providing powerful new tools and insights into elucidating the fine details of drug-target interactions. In the present study, two separate drug-target networks for Tanshinone IIA were constructed using the Agilent Literature Search(ALS) and STITCH(search tool for interactions of chemicals) methods. Analysis of the ALS-constructed network revealed a target network with a scale-free topology and five top nodes(protein targets) corresponding to Fos, Jun, Src, phosphatidylinositol-4, 5-bisphosphate 3-kinase, catalytic subunit alpha(PIK3CA), and mitogen-activated protein kinase kinase 1(MAP2K1), whereas analysis of the STITCH-constructed network revealed three top nodes corresponding to cytochrome P450 3A4(CYP3A4), cytochrome P450 A1(CYP1A1), and nuclear factor kappa B1(NFκB1). The discrepancies were probably due to the differences in the divergent computer mining tools and databases employed by the two methods. However, it is conceivable that all eight proteins mediate important biological functions of Tanshinone IIA, contributing to its overall drug-target network. In conclusion, the current results may assist in developing a comprehensive understanding of the molecular mechanisms and signaling pathways of in a simple, compact, and visual manner.

Tanshinone ⅡA improves functional recovery in spinal cord injury-induced lower urinary tract dysfunction

Tanshinone ⅡA, extracted from Salvia miltiorrhiza Bunge, exerts neuroprotective effects through its anti-inflammatory, anti-oxidative and anti-apoptotic properties. This study intravenously injected tanshinone ⅡA 20 mg/kg into rat models of spinal cord injury for 7 consecutive days. Results showed that tanshinone ⅡA could reduce the inflammation, edema as well as compensatory thickening of the bladder tissue, improve urodynamic parameters, attenuate secondary injury, and promote spinal cord regeneration. The number of hypertrophic and apoptotic dorsal root ganglion（L6–S1） cells was less after treatment with tanshinone ⅡA. The effects of tanshinone ⅡA were similar to intravenous injection of 30 mg/kg methylprednisolone. These findings suggested that tanshinone ⅡA improved functional recovery after spinal cord injury-induced lower urinary tract dysfunction by remodeling the spinal pathway involved in lower urinary tract control.

The Reaction of Tanshinones with Amines

The reaction of cryptotanshinone and tanshinone IIA with several biogenic amine metabolites involved in the pathogenic pathways of HE were investigated and eight 1,2,3,4- tetrahydrophenanthrene derivatives, 2-6 and 8-10, were obtained. The probable mechanism on reaction was discussed.

Phytoestrogen-derived multifunctional ligands for targeted therapy of breast cancer

Nano-targeted delivery systems have been widely used for breast tumor drug delivery.Estrogen receptors are considered to be significant drug delivery target receptors due to their overexpression in a variety of tumor cells.However,targeted ligands have a significant impact on the safety and effectiveness of active delivery systems,limiting the clinical transformation of nanoparticles.Phytoestrogens have shown good biosafety characteristics and some affinity with the estrogen receptor.In the present study,molecular docking was used to select tanshinone IIA(Tan IIA)among phytoestrogens as a target ligand to be used in nanodelivery systems with somemodifications.Modified Tan IIA(Tan-NH2)showed a good biosafety profile and demonstrated tumor-targeting,anti-tumor and anti-tumor metastasis effects.Moreover,the ligand was utilized with the anti-tumor drug Dox-loaded mesoporous silica nanoparticles via chemical modification to generate a nanocomposite Tan-Dox-MSN.Tan-Dox-MSN had a uniform particle size,good dispersibility and high drug loading capacity.Validation experiments in vivo and in vitro showed that it also had a better targeting ability,anti-tumor effect and lower toxicity in normal organs.These results supported the idea that phytoestrogens with high affinity for the estrogen receptor could improve the therapeutic efficacy of nano-targeted delivery systems in breast tumors.

Two-step continuous flow process of sodium tanshinone ⅡA sulfonate using a 3D circular cyclone-type microreactor

A sustainable and practical process is presented for the direct synthesis of sodium tanshinone IIA sulfonate(STS).Our approach was inspired by the well-established and industrially applied batch synthetic route for STS production.We constructed a telescoped two-step continuous flow platform.This involved a continuous tanshinone IIA sulfonation and in-line salt formation.For the setup,we constructed a 3D circular cyclone-type microreactor using femtosecond laser micromachining.Compared to the 68%yield for 2 h in batch,the two-step continuous flow had an STS yield of 90%,achieved for a total residence time of<3.0 min under optimal conditions.The proposed continuous flow method vastly simplified the operation and improved procedural safety,while significantly reducing the required acid content and wastewater production.

Synthesis of 2,2'-(Substituted Arylmethylene)bis(1,6,6-trimethyl- 6,7,8,9-tetrahydrophenanthro[1,2-b]furan-10,11-dione) Derivatives

The reactions of tanshinone IIA with aromatic aldehydes have been investigated and several 2, T-（substituted arylmethylene） bis （1,6,6-trimethyl-6,7,8,9-tetrahydrophenanthro[1,2-b]- furan- 10, 11-dione） derivatives were obtained,

Synthesis of New Benzoimidazole Derivatives

The reactions of cryptotanshinone and tanshinone IIA with cadaverine and putrescine were investigated. Six new compounds, four with imidazole functional groups and two with oxazole groups, were obtained. The possible reaction mechanism was proposed.

Sodium tanshinone ⅡA sulfonate prevents radiation-induced damage in primary rat cardiac fibroblasts

This study investigated the effects of X-ray irradiation on primary rat cardiac fibroblasts(CFs) and its potential mechanism, as well as whether sodium tanshinone ⅡA sulfonate(STS) has protective effect on CFs and its possible mechanism. Our data demonstrated that X-rays inhibited cell growth and increased oxidative stress in CFs, and STS mitigated X-ray-induced injury. Enzyme-linked immuno-sorbent assay showed that X-rays increased the levels of secreted angiotensin Ⅱ(Ang Ⅱ) and brain natriuretic peptide(BNP). STS inhibited the X-ray-induced increases in Ang Ⅱ and BNP release. Apoptosis and cell cycle of CFs were analyzed using flow cytometry. X-rays induced apoptosis in CFs, whereas STS inhibited apoptosis in CFs after X-ray irradiation. X-rays induced S-phase cell cycle arrest in CFs, which could be reversed by STS. X-rays increased the expression of phosphorylated-P38/P38,cleaved caspase-3 and caspase-3 as well as decreased the expression of phosphorylated extracellular signal-regulated kinase 1/2(ERK1/2)/ERK 1/2 and B cell lymphoma 2(Bcl-2)/Bcl-2 associated X protein(BAX) in CFs, as shown by Western blotting. STS mitigated the X-ray radiation-induced expression changes of these proteins. In conclusion, our results demonstrated that STS may potentially be developed as a medical countermeasure to mitigate radiation-induced cardiac damage.

Protective Effect of Sodium Tanshinone ⅡA Sulfonate on Injury of Small Intestine in Rats with Sepsis and Its Mechanism

Objective： To explore the protective effect of sodium tanshinone ⅡA sulfonate （STS） on small intestine injury in rats with sepsis and its possible mechanism. Methods： According to a random number table, 24 Tats were randomly divided into 3 groups： sham operation group （sham group）, sepsis model group （model group） and STS treatment group （STS group）, with 8 Tats in each group. A rat model of sepsis was induced by cecal ligation and puncture （CLP） for 5 h. STS （1 mg/kg） was slowly injected through the right external jugular vein after CLP. The histopathologic changes in the intestine tissue were observed under a light microscope, and the intestinal epithelial cell apoptosis was evaluated by terminal deoxynucleoddyl transferase （TdT）-mediated dUTP nick-end labeling （TUNEL） method. The expressions of Bcl-2, Bax and nuclear factor κB （NF- κ B） p65 in the intestinal tissue was determined by Western blot. The levels of tumor necrosis factor α （TNF-α） and interleukin 6 （IL-6） in the intestinal tissue were determined using enzyme-linked immuno-sorbent assay （ELISA）. Results： Obvious injuries were observed in the intestinal tissue in the CLP group compared with the sham group. The expression of NF- K B p65 and the levels of TNF- α and IL-6 were up-regulated after CLP, the apoptosis of intestinal epithelial cells was increased after CLP, and the ratio of Bcl-2 to Bax was decreased. STS post- treatment could attenuate the injury on the intestinal tissue induced by CLP, decrease the apoptosis of intestinal epithelial cells and the levels of NF- κ B p65, TNF-α and IL-6, and increase the ratio of Bcl-2 to Bax. Conclusion： STS can protect the small intestine in rats with sepsis, and the mechanism may be associated with the inhibition of intestinal epithelial apoptosis and the reduction of activation of inflammatory cytokines.

Discovery of tanshinone derivatives with anti-MRSA activity via targeted bio-transformation

Two potent anti-MRSA tanshinone glycosides(1 and 2)were discovered by targeted microbial biotransformation,along with rapid identification via MS/MS networking.Serial reactions including dehydrogenation,demethylations,reduction,glycosylation and methylation have been observed after incubation of tanshinone IIA and fungus Mucor rouxianus AS 3.3447.In addition,tanshinosides B(2)showed potent activities against serial clinical isolates of oxacillin-resistant Staphylococcus aureus with MIC values of 0.78 mg/mL.This is the first study that shows a significant increase in the level and activities of tanshinone glycosides relative to the substrate tanshinone IIA.

Thoracic interstitial injection of drug-liposomes in mice for treating atherosclerosis

Intervaginal space injection(ISI)is a novel mode of administration investigated over the last decade.After injecting nanoparticles into the intervaginal space,they can be transported along low flow resistance channels into the interstitial space.This transport has a certain delivery direction,and site-specific injection can work on specific organs or tissues.In this study,the thorax,a new ISI site in the interstitial surrounding the internal thoracic artery named the thoracic interstitial injection(tISI)was investigated.To prove the targeting ability of the tISI,two sizes of gold nanoparticles(AuNPs)(47 and 87 nm)were administered to mice.After 1 h,the biodistribution of AuNPs in the tissues was measured via single particle inductively coupled plasma mass spectrometry(spICP-MS).The results showed that the concentration of AuNPs in the aorta after tISI injection was significantly higher than that after intravenous injection.Moreover,fewer nanoparticles with larger particle sizes were observed to have entered the blood and were better targeted to the aorta.Thereafter,tanshinone IIa sodium sulfonate liposomes were administered for the treatment of aortic atherosclerosis.The proportion of aortic plaques in atherosclerotic Apoe-/-mice administered via tISI was significantly lower than that in other model animals(P<0.001).Furthermore,the proteoglycan content and CD68-positive cell count in the plaques were significantly reduced.The vascular elastic fibers at the plaque site were thickened,and fractures were reduced.tISI was,therefore,determined to be an effective strategy for the treatment of atherosclerotic aortic plaques.

Simultaneous Determination of Five Hydrophilic and Lipophilic Components from roots of Salvia miltiorrhiza by HPLC

Objective A reversed-phase HPLC method was established for the simultaneous determination of five hydrophilic and lipophilic components in the roots of Salvia miltiorrhiza. Methods Hydrophilic components including danshensu, protocatechuic aldehyde, and salvianolic acid B, and lipophilic components such as cryptotanshinone and tanshinone liA, were successfully separated on a Waters Symmetry C18 reverse- phase column （250 mm×4.6 mm, 5 μm）, with acetonitrile-0.5% phosphoric acid （gradient elution） as mobile phase, the detection wavelength was set at 281 nm with flow rate of 1.0 mL/min, and the column temperature was maintained at 30 ~C. Results The recovery of the method was in the range of 95.1%-102.5% and the precision was less than 3% for all five analytes. All the compounds showed good linearity （R2 〉 0.9990） in a relatively wide concentration range. Therefore, this HPLC method demonstrated good reproducibility, stability, and accuracy in validation studies. Conclusion Simultaneous quantification of the multiple components by HPLC would be a better strategy for the quality evaluation on the roots of S. miltiorrhiza.