Danshensu Research Articles

ITRAQ-Based Proteomic Analysis Unveils NCAM1 as a Novel Regulator in Doxorubicin-Induced Cardiotoxicity and DT-010-Exerted Cardioprotection

Background: Doxorubicin (DOX) causes lethal cardiotoxicity, which limits its clinical utility. The molecular mechanisms and effective strategies to combat its cardiotoxicity need further exploration. DT-010, a novel conjugate of danshensu (DSS) and tetramethylpyrazine( TMP), is considered a promising candidate for treating DOX-induced cardiotoxicity. In this study, we aimed to investigate the underlying molecular mechanisms of DOX-induced cardiotoxicity and the cardioprotective effects of DT-010. Methods: Isobaric tags for relative and absolute quantitation (iTRAQ) in proteomics analysis was employed to analyze the differentially expressed proteins in DOX-injuried hearts. Gene ontology (GO) enrichment analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were carried out to evaluated the potential mechanisms of DOXinduced cardiotoxicity. The effects of NCAM1 on DOX-induced cardiotoxicity in H9c2 cells, as well as the cardioprotection of DT-010 were assessed through NACM1siRNA transfection, cell viability assay, cell apoptosis staining, reactive oxygen species measurement, and western blotting. Results: Proteomics analysis revealed that several signaling pathways, including the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, were involved in DOX-induced cardiotoxicity. NCAM1 is one of the significantly changed proteins. DT-010 treatment regulated NCAM1 protein expression. Silencing NCAM1 in DOX-treated H9c2 cells decreased cell viability, increased cell apoptosis and reactive oxygen species (ROS) generation, and attenuated the cardioprotective effects of DT-010. Furthermore, NCAM1 knockdown promoted p38 activation and inhibited the expressions of peroxisome proliferator-activated receptor gamma coactivator- 1 alpha (PGC-1α) and heme oxygenase-1 (HO-1) in DOX-treated cells. Conclusion: These findings indicate a definite role of NCAM1 in DOX-induced cardiotoxicity and DT-010-exerted cardioprotection, which is mediated through the p38 and Sirt1/PGC- 1α/HO-1 pathway.

Analysis of rosmarinic acid synthase (RAS) gene family and functional study of SmRAS1/2/4 in Salvia miltiorrhiza

Rosmarinic acid synthase is an essential enzyme involved in the biosynthesis of rosmarinic acid (RA), which facilitates the coupling of phenylpropanoid and tyrosine-derived pathway products. Our study identified six SmRAS genes in Salvia miltiorrhiza, with SmRAS1 being the only one functionally characterized to date. Real-time quantitative PCR was employed to analyze the expression profiles of the SmRAS gene family, revealing that SmRAS1/2/4 are predominantly expressed in the roots, which are the medicinal components of S. miltiorrhiza. SmRAS2 and SmRAS4 exhibited significant responses to abscisic acid (ABA), gibberellin (GA3), and methyl jasmonate (MeJA) stimuli, while SmRAS1 had notable responses to GA3 and MeJA. β-glucuronidase (GUS) staining in transgenic Arabidopsis thaliana confirmed a spatiotemporal expression pattern of SmRAS1/2/4 that was consistent with the qRT-PCR results. SmRAS1/2/4 are primarily localized to the cytoplasm and plasma membrane. Our findings suggested that the overexpressions of SmRAS1 or SmRAS4 led to increased levels of salvianolic acid B (SalB) and RA, with a concomitant decrease in the Danshensu (DSS) content, which served as a substrate. In contrast, RNA interference lines exhibited a downward trend in the content of these substances. Interestingly, no significant changes were detected in the SalB, RA, or DSS contents due to the overexpression of SmRAS2 or RNA interference lines. Collectively, our study demonstrated that SmRAS1 and SmRAS4 are key regulators of RA and SalB biosynthesis in S. miltiorrhiza, while SmRAS2′s role appears less impactful, suggesting a complex regulatory network that influences the medicinal properties of this plant.

Molecular mechanism of Fe3+ binding inhibition to Vibrio metschnikovii ferric ion-binding protein, FbpA, by rosmarinic acid and its hydrolysate, danshensu.

Global warming has increased the growth of pathogenic Vibrio bacteria, which can cause foodborne illnesses and death. Vibrio bacteria require iron for growth and survival. They utilize a ferric ion-binding protein (FbpA) to bind and transport Fe3+ into the cell. FbpA from Vibrio metschnikovii (Vm) is a potential target for inhibiting its growth. Rosmarinic acid (RA) can block the binding of VmFbpA to Fe3+ ; however, the molecular mechanism of Fe3+ binding and RA inhibition to VmFbpA is unclear. In this study, we used x-ray crystallography to determine the Fe3+ -binding mode of VmFbpA and the mechanism of RA inhibition. The structures revealed that in the Fe3+ bound form, Fe3+ was coordinated to VmFbpA by two Tyr residues, two HCO3 - ions, and two water molecules in a six-coordinated geometry. In contrast, in the inhibitor bound form, RA was initially bound to VmFbpA following gel filtration purification, but it was hydrolyzed to danshensu (DSS), which occupied the binding site as shown in an electron density map and reverse phase chromatography (RPC) analysis. Both RA and DSS exhibited a stronger binding affinity to VmFbpA, higher Fe3+ reduction capacity, and more potent bacteriostatic effect on V. metschnikovii compared with caffeic acid (CA), another hydrolysis product of RA. These results provide insight into the mechanism of iron acquisition by V. metschnikovii and inhibition by RA and DSS. Our findings offer clues on the development of effective strategies to prevent Vibrio infections.

Versatile CYP98A enzymes catalyse meta-hydroxylation reveals diversity of salvianolic acids biosynthesis.

Salvianolic acids (SA), such as rosmarinic acid (RA), danshensu (DSS), and their derivative salvianolic acid B (SAB), etc. widely existed in Lamiaceae and Boraginaceae families, are of interest due to medicinal properties in the pharmaceutical industries. Hundreds of studies in past decades described that 4-coumaroyl-CoA and 4-hydroxyphenyllactic acid (4-HPL) are common substrates to biosynthesize SA with participation of rosmarinic acid synthase (RAS) and cytochrome P450 98A (CYP98A) subfamily enzymes in different plants. However, in our recent study, several acyl donors and acceptors included DSS as well as their ester-forming products all were determined in SA-rich plants, which indicated that previous recognition to SA biosynthesis is insufficient. Here, we used Salvia miltiorrhiza, a representative important medicinal plant rich in SA, to elucidate the diversity of SA biosynthesis. Various acyl donors as well as acceptors are catalysed by SmRAS to form precursors of RA and two SmCYP98A family members, SmCYP98A14 and SmCYP98A75, are responsible for different positions' meta-hydroxylation of these precursors. SmCYP98A75 preferentially catalyses C-3' hydroxylation, and SmCYP98A14 preferentially catalyses C-3 hydroxylation in RA generation. In addition, relative to C-3' hydroxylation of the acyl acceptor moiety in RA biosynthesis, SmCYP98A75 has been verified as the first enzyme that participates in DSS formation. Furthermore, SmCYP98A enzymes knockout resulted in the decrease and overexpression leaded to dramatic increase of SA accumlation. Our study provides new insights into SA biosynthesis diversity in SA-abundant species and versatility of CYP98A enzymes catalytic preference in meta-hydroxylation reactions. Moreover, CYP98A enzymes are ideal metabolic engineering targets to elevate SA content.

Astrocytes and microglia-targeted Danshensu liposomes enhance the therapeutic effects on cerebral ischemia-reperfusion injury

Cerebral ischemia-reperfusion injury (CI/RI) is the main cause of disability and death in stroke without satisfactory therapeutic effect. Inflammation mediated by activation of astrocytes and microglia is the main pathological mechanism of CI/RI. Danshensu (DSS) has been shown to exert anti-inflammatory effects against brain injury. However, limited by its poor cellular permeability and low bioavailability, it is still needed the new DSS preparations with the ability to cross the blood–brain barrier (BBB) and target inflammatory glial cells. In this study, we developed phosphatidylserine (PS) and transferrin (TF) modified liposomes carrying DSS (TF/PS/DSS-LPs) to improve the therapeutic efficacy against ischemic stroke. First, TF molecules targeted transferrin receptor (TfR) that is overexpressed in the BBB. Following the liposomes enter the brain, PS modification allowed the liposomes to target and bind to the overexpressed phosphatidylserine-specific receptors (PSRs) on the surface of astrocytes and microglia. Furthermore, it enhanced the uptake of TF/PS/DSS-LPs by astrocytes and microglia, while polarizing astrocytes from A1 to A2 and microglia from M1 to M2, reducing neuronal inflammation, and ultimately ameliorating cerebral ischemic injury. Thus, TF/PS/DSS-LPs could potentially serve as a promising strategy for the CI/RI treatment.

Salvia miltiorrhiza Extract Prevents the Occurrence of Early Atherosclerosis in Apoe -/- Mice via TLR4/ NF-kB Pathway.

Salvia miltiorrhiza (SM) contains four major aqueous active ingredients, which have been isolated, purified and identified as danshensu (DSS), salvianolic acid A (Sal-A), salvianolic acid B (Sal-B) and protocatechuic aldehyde (PAL), A mixture of these four ingredients is called SABP. Although aqueous extract from Salvia miltiorrhiza has been traditionally used to treat cardiovascular diseases, the efficacy and function of the optimal ratio of SABP in preventing and treating cardiovascular diseases remain unknown. This study aims to explore the antiinflammatory mechanisms underlying the attenuation of atherosclerosis development by aqueous extract from Salvia miltiorrhiza. Male ApoE-/- mice (6 weeks) were randomly allocated into three groups: the model group (Model), the SABP group (SABP), and the rosuvastatin calcium group (RC). Male C57BL/6 mice (6 weeks) were used as a control group. All mice were fed with an ordinary diet. After 8 weeks of treatment, the lipid profiles in serum and the lactate dehydrogenase (LDH) and creatine kinase (CK) in heart tissue were measured using an automatic biochemical analyzer. Alterations of the thoracic aorta and the heart were assessed using Hematoxylin and eosin staining. The protein expression of Toll-like receptor 4 (TLR4), TGF beta-activated kinase 1 (TAK1), nuclear factor kappa-B (NF-κB), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the heart tissue were determined though immunohistochemistry and western blotting analysis. The serum low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), and total cholesterol (TC) levels were increased, and the high-density lipoprotein cholesterol (HDL-C) level was decreased in ApoE-/- mice. SABP significantly decreased serum lipid levels and improved histopathology in the thoracic aorta. In addition. SABP treatment inhibited the expression of TLR4, TAK1, NF-κB, IL-6 and TNF-α in the heart in ApoE-/- mice. The LDH and CK in the heart did not differ significantly among different groups, and the heart did not have obvious pathological changes. These findings indicated that SABP may exert an anti-atherosclerotic effect by lowering blood lipids and inhibiting inflammatory response via TLR4/ NF-κB signaling pathway.

Discovery of small molecule β-catenin suppressors that enhance immunotherapy

Small molecules directly downregulating β-catenin could potentially offer a more effective therapeutic approach for combating against cancer stem cells, as compared to targeting the downstream components of the Wnt/β-catenin pathway. The challenge, however, lies in the fact that very few β-catenin suppressors have proven clinically effective, leaving a significant gap in medical solutions. Given that E-cadherin has a natural affinity for β-catenin, it stands to reason that agents designed to increase E-cadherin expression might provide an alternative method of regulating β-catenin levels. In this study, we report our discovery of DSS-C12 and DSS-B8, specific ester-based drugs derived from Dan-Shen-Su (DSS) extracted from the herb Salvia miltiorrhiza. Remarkably, these compounds display a potent ability to downregulate β-catenin, while also improving overall survival in post-surgery mice. Additionally, when these drugs are used in combination with PD-L1 checkpoint blockade, they stimulate enhanced systemic immune responses leading to significant suppression of primary tumor growth. In-depth mechanistic studies revealed that DSS-B8 functions as a vitamin D receptor agonist without inducing hypercalcemic effects. Collectively, our findings indicate that DSS-derived small molecules have considerable potential as clinically viable therapeutic strategies for β-catenin deactivation.

Salvia miltiorrhiza augments endothelial cell function for ischemic hindlimb recovery.

Salvia miltiorrhiza (Salvia miltiorrhiza) root, as a traditional herb, is widely applied to pharmacotherapy for vascular system disease. In this study, we elucidate the therapy mechanism of Salvia miltiorrhiza by using a model of hindlimb ischemia. Blood perfusion measurement showed that intravenous administration of the Water Extract of Salvia miltiorrhiza (WES) could facilitate damaged hindlimb blood flow recovery and blood vessel regeneration. In vitro mRNA screen assay in cultured human umbilical vein endothelial cells (HUVECs) show that WES induced increased NOS3, VEGFA, and PLAU mRNA levels. Endothelial NOS (eNOS) promotor reporter analysis revealed that WES and the major ingredients danshensu (DSS) could enhance eNOS promoter activity. Additionally, we found that WES and its ingredients, including DSS, protocatechuic aldehyde (PAI), and salvianolic acid A (SaA), promoted HUVECs growth by the endothelial cell viability assays. A mechanistic approach confirmed that WES augments HUVECs proliferation through the activation of extracellular signal-regulated kinase (ERK) signal pathway. This study reveals that WES promotes ischemic remodeling and angiogenesis through its multiple principal ingredients, which target and regulate multiple sites of the network of the blood vessel endothelial cell regenerating process.

DT-010 Exerts Cardioprotective Effects by Regulating the Crosstalk between the AMPK/PGC-1α Pathway and ERp57.

The AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) pathway performs a crucial role in energy metabolism and mitochondrial network. Our previous study found that DT-010, a novel danshensu (DSS) and tetramethylpyrazine (TMP) conjugate, had significant cardioprotective properties in vitro and in vivo. We also reported that ERp57 served as a major target of DSS using the chemical proteomics approach. In this article, we focus on exploring the interrelationship between the regulation of the AMPK/PGC-1α pathway and promoting ERp57 expression induced by DT-010 in tert-butylhydroperoxide- (t-BHP-) induced H9c2 cell injury. The results showed that DT-010 activated the AMPK/PGC-1α pathway and increased ERp57 protein expression. Importantly, the above phenomenon as well as the mitochondrial function can be partially reversed by siRNA-mediated ERp57 suppression. Meanwhile, silencing AMPK significantly inhibited the ERp57 expression induced by DT-010. In addition, molecular docking and kinase assay in vitro revealed that DT-010 had no direct regulation effects on AMPK activity. Taken together, DT-010 exerted cardioprotective effects by regulating the crosstalk of AMPK/PGC-1α pathway and ERp57, representing a potential therapeutic agent for ischemic heart disease.

Ameliorative effects of Danshensu from the functional food Salvia miltiorrhiza against arsenic trioxide-induced cardiac toxicity in vivo and in vitro: Involvement of inhibiting the AKT/IKK/NF-κB signaling pathway

• Cardiotoxicity of arsenic trioxide limits its clinical utility. • Danshensu has anti-oxidant, anti-inflammatory, and anti-apoptotic activities. • Danshensu shows the protective effects on arsenic trioxide-induced cardiotoxicity. • Protective mechanisms are related to the inhibition of AKT/IKK/NF-κB pathway. Cardiotoxicity is a serious side effect of arsenic trioxide (ATO) which hinders its clinical use in cancer chemotherapy. Danshensu (DSS) has potential applications in the food and healthcare industry to promote cardiovascular health. The experiment aims to investigate the effects of DSS against ATO-induced cardiotoxicity and its possible mechanism. Our results demonstrated that DSS improved cardiac dysfunction, alleviated histopathological damage, enhanced antioxidant activity, attenuated cardiomyocyte apoptosis and suppressed inflammation. Further studies showed that DSS prevented ATO-caused activation of NF-κB by inhibiting the phosphorylation of AKT and IKK, degradation of IκBα, and nuclear translocation of NF-κB. In H9c2 cardiomyocytes, DSS significantly increased cell survival rate and inhibited ATO-mediated expression of p-AKT, p-IKKα/β, p-IκBα, and NF-κB. Notably, the efficacy of DSS was synergistically increased by AKT inhibitor GSK690693. To sum up, DSS effectively ameliorated ATO-induced cardiac toxicity in vivo and in vitro , which may be achieved through inhibition of the AKT/IKK/NF-κB pathway.

Self-Sufficient In Vitro Multi-Enzyme Cascade for Efficient Synthesis of Danshensu from l-DOPA.

Danshensu (DSS), a traditional Chinese medicine, is widely used for the treatment of cardiovascular and cancer diseases. Here, a one-pot multi-enzyme cascade pathway was designed for DSS synthesis from l-DOPA using tyrosine aminotransferase from Escherichia coli (EcTyrB) and d-isomer-specific 2-hydroxyacid dehydrogenase from Lactobacillus frumenti (LfD2-HDH). Glutamate dehydrogenase from Clostridium difficile (CdgluD) was also introduced for a self-sufficient system of α-ketoglutaric acid and NADH. Under optimal conditions (35 °C, pH 7.0, EcTyrB:LfD2-HDH:CdgluD = 3:2:1, glutamate:NAD+ = 1:1), 98.3% yield (at 20 mM l-DOPA) and space-time yield of 6.61 g L-1 h-1 (at 40 mM l-DOPA) were achieved. Decreased yields of DSS at elevated l-DOPA concentrations (100 mM) could be attributed to an inhibited CdgluD activity caused by NH4+ accumulation. This developed multi-enzyme cascade pathway (including EcTyrB, LfD2-HDH, and CdgluD) provides an efficient and sustainable approach for the production of DSS from l-DOPA.

CXCR1 and its downstream NF-κB inflammation signaling pathway as a key target of Guanxinning injection for myocardial ischemia/reperfusion injury.

Guanxinning Injection (GXNI) is used clinically to treat cardiac injury, but its active components and mode of action remains unclear. Therefore, a myocardial ischemia/reperfusion injury (MIRI) model-based integrated strategy including function evaluation, RNA-seq analysis, molecular docking, and cellular thermal shift assay (CETSA) was employed to elucidate the effect and mechanism of GXNI and its main ingredient on cardiac injury. These results revealed that GXNI significantly improved cardiac dysfunction and myocardial injury in I/R mice. RNA-seq analysis clarified that CXCR1-mediated interleukin-8 pathway played a critical role in MIRI. Molecular docking screening identified danshensu (DSS) as the major active components of GXNI targeting CXCR1 protein, which was confirmed in an oxygen-glucose deprivation/reoxygenation-induced cardiomyocytes damage model showing that GXNI and DSS reduced the protein expression of CXCR1 and its downstream NF-κB, COX-2, ICAM-1 and VCAM-1. CETSA and isothermal dose-response fingerprint curves confirmed that DSS combined with CXCR1 in a dose-dependent manner. Furthermore, GXNI and DSS significantly decreased the expression levels of IL-6, IL-1β and TNF-α and the number of neutrophils in post I/R myocardial tissue. In conclusion, this study revealed that GXNI and its active components DSS exert inhibitory effects on inflammatory factor release and leukocyte infiltration to improve I/R-induced myocardial injury by down-regulating CXCR1-NF-κB-COX-2/ICAM-1/VCAM-1 pathway.

Therapeutic effect and mechanism of danshensu on coronary heart disease using liquid chromatography combined with mass spectrometry metabolomics

Metabolomics can discover the biomarkers and metabolic pathways, provides the possibility for insights into the pharmacological action and mechanism of natural products. The therapeutic effect and mechanism of danshensu (DSS) on total metabolic pathways has not been well investigated. The aim of this study was to explore the disturbed endogenous biomarkers and metabolic pathways reflecting the pharmacological activity of DSS, and mechanism of action of DSS using comprehensive metabolome analysis based on high-throughput metabolomics technology combined with ultra-high performance liquid chromatography (UPLC) coupled with quadrupole tandem time-of-flight mass spectrometry (Q-TOF-MS) and pattern recognition method. Through the changes of the overall metabolic profile and the related biomarkers, the intervention effect of natural product danshensu (DSS) treatment on CHD model rats was revealed. The results showed that after the model replication was established, the metabolic profile was clearly separated, and a total of 26 potential biomarkers were screened out, and involving 8 metabolic pathways. After different doses of DSS solution were given, a total of 20 biomarkers could be significantly regulated, mainly involving primary bile acid biosynthesis, glycerophospholipid metabolism, and lipid metabolism. It showed UPLC-MS-based metabolomics can be used for discovering potential biomarkers and metabolic pathways of CHD, and to further understand and dissecting pharmacological effects and mechanisms of natural products via metabolomics techniques.

Danshensu prevents thrombosis by inhibiting platelet activation via SIRT1/ROS/mtDNA pathways without increasing bleeding risk

BackgroundCoronary thrombosis and its correlated disorders are main healthcare problems globally. The therapeutic effects of current treatments involving antiplatelet drugs are not fully satisfactory. Danshensu (DSS) is an important monomer obtained from Salvia miltiorrhiza roots that have been widely employed for vascular diseases in medicinal practices. Nonetheless, the underlying mechanisms of DSS are not fully unraveled. PurposeThe objective of this study was to penetrate the antithrombotic and antiplatelet mechanisms of DSS. MethodsNetwork pharmacology assay was used to forecast the cellular mechanisms of DSS for treating thrombosis. The work focused the impacts of DSS on platelet activation by analyzing aggregation and adhesion in vitro. Flow cytometry, western blotting, CM-H2DCFDA staining and mitochondrial function assays were performed to reveal the molecular mechanisms. The model of common carotid artery thrombus induced by ferric chloride was established. The wet weight of thrombus was measured, and the thrombosis was observed by hematoxylin and eosin (H&E) staining, in order to support the inhibitory effect of DSS on thrombosis. ResultsData mining found the antithrombotic effect of DSS is related to platelet activation and the core target is silent information regulator 1 (SIRT1). We confirmed that DSS dose-dependently inhibited platelet activation in vitro. DSS was further demonstrated to induce the expression of SIRT1 and decreased reactive oxygen species (ROS) burden and thereby prevented mitochondrial dysfunction. Mitochondrial function tests further indicated that DSS prevented mitochondrial DNA (mtDNA) release, which induced activation of platelet in a dendritic cell specific intercellular-adhesion-molecule-3 grabbing non-integrin (DC-SIGN)-dependent manner. In carotid artery injury model induced by ferric chloride, DSS inhibited the development of carotid arterial thrombosis. More encouragingly, in tail bleeding time assay, DSS did not augment bleeding risk. ConclusionThese findings indicated that DSS effectively inhibited platelet activation by depressing the collection of ROS and the release of platelet mtDNA without arousing hemorrhage risk. DSS might represent a promising candidate drug for thrombosis and cardiovascular disease therapeutics.

Exploring the Mechanism of Danshensu in the Treatment of Doxorubicin-Induced Cardiotoxicity Based on Network Pharmacology and Experimental Evaluation.

BackgroundDoxorubicin (DOX) is one of the most effective chemotherapeutic agents available; however, its use is limited by the risk of serious cardiotoxicity. Danshensu (DSS), an active ingredient in Salvia miltiorrhiza, has multiple cardioprotective effects, but the effect of DSS on DOX-induced cardiotoxicity has not been reported.ObjectivesPredicting the targets of DOX-induced cardiotoxicity and validating the protective effects and mechanisms of DSS.Methods(1) Using methods based on network pharmacology, DOX-induced cardiotoxicity was analyzed by data analysis, target prediction, PPI network construction and GO analysis. (2) The cardiotoxicity model was established by continuous intraperitoneal injection of 15 mg/kg of DOX into mice for 4 days and the protective effects and mechanism were evaluated by treatment with DSS.ResultsThe network pharmacology results indicate that CAT, SOD, GPX1, IL-6, TNF, BAX, BCL-2, and CASP3 play an important role in this process, and Keap1 is the main target of DOX-induced cardiac oxidative stress. Then, based on the relationship between Keap1 and Nrf2, the Keap1-Nrf2/NQO1 pathway was confirmed by animal experiments. In the animal experiments, by testing the above indicators, we found that DSS effectively reduced oxidative stress, inflammation, and apoptosis in the damaged heart, and significantly alleviated the prolonged QTc interval caused by DOX. Moreover, compared with the DOX group, DSS elevated Keap1 content and inhibited Nrf2, HO-1, and NQO1.ConclusionThe results of network pharmacology studies indicated that Keap1-Nrf2/NQO1 is an important pathway leading to DOX-induced cardiotoxicity, and the results of animal experiments showed that DSS could effectively exert anti-oxidative stress, anti-inflammatory and anti-apoptotic therapeutic effects on DOX-induced cardiotoxicity by regulating the expression of Keap1-Nrf2/NQO1.

Activation of TAF9 via Danshensu-Induced Upregulation of HDAC1 Expression Alleviates Non-alcoholic Fatty Liver Disease.

Fatty acid β-oxidation is an essential pathogenic mechanism in nonalcoholic fatty liver disease (NAFLD), and TATA-box binding protein associated factor 9 (TAF9) has been reported to be involved in the regulation of fatty acid β-oxidation. However, the function of TAF9 in NAFLD, as well as the mechanism by which TAF9 is regulated, remains unclear. In this study, we aimed to investigate the signaling mechanism underlying the involvement of TAF9 in NAFLD and the protective effect of the natural phenolic compound Danshensu (DSS) against NAFLD via the HDAC1/TAF9 pathway. An in vivo model of high-fat diet (HFD)-induced NAFLD and a palmitic acid (PA)-treated AML-12 cell model were developed. Pharmacological treatment with DSS significantly increased fatty acid β-oxidation and reduced lipid droplet (LD) accumulation in NAFLD. TAF9 overexpression had the same effects on these processes both in vivo and in vitro. Interestingly, the protective effect of DSS was markedly blocked by TAF9 knockdown. Mechanistically, TAF9 was shown to be deacetylated by HDAC1, which regulates the capacity of TAF9 to mediate fatty acid β-oxidation and LD accumulation during NAFLD. In conclusion, TAF9 is a key regulator in the treatment of NAFLD that acts by increasing fatty acid β-oxidation and reducing LD accumulation, and DSS confers protection against NAFLD through the HDAC1/TAF9 pathway.

Danshensu inhibits the IL-1\u03b2-induced inflammatory response in chondrocytes and osteoarthritis possibly via suppressing NF-\u03baB signaling pathway

PurposeOsteoarthritis (OA) is the most common inflammatory disease associated with pain and cartilage destruction. Interleukin (IL)-1β is widely used to induce inflammatory response in OA models. This study aimed to explore the role of Danshensu (DSS) in IL-1β-induced inflammatory responses in OA.MethodsIL-1β was used to induce chondrocyte inflammation. Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assay. IL-6, COX-2, TNF-α, and iNOS mRNA levels were detected by qRT-PCR. MMP3, MMP13, ADAMTS4, ADAMTS5, Aggrecan, Collagen, p-IκBα, and p-p65 protein levels were detected by Western blot. An OA mouse model was established by surgical destabilization of the medial meniscus (DMM), and the Osteoarthritis Research Society International (OARSI) score was evaluated by H&E staining.ResultsDSS did not affect the levels of inflammatory indicators including IL-6, COX-2, TNF-α, iNOS, PEG2, and NO but suppressed COX-2 and iNOS protein expression in IL-1β treated chondrocytes. In addition, DSS downregulated IL-1β-enhanced expression of MMP3, MMP13, ADAMTS4, and ADAMTS5 and upregulated aggrecan and collagen expression. Moreover, DSS significantly inhibited IL-1β-induced phosphorylation of p-IκBα and p-p65 in a dose-dependent manner in chondrocytes, suggesting it plays a role in the NF-κB signaling pathway. Furthermore, DSS significantly reduced DMM-induced cartilage OARSI score in mice, further demonstrating its protective role in OA progression in vivo.ConclusionsOur study revealed the protective role of DSS in OA, suggesting that DSS might act as a potential treatment for OA.

Effect of main ingredients of Danhong Injection against oxidative stress induced autophagy injury via miR-19a/SIRT1 pathway in endothelial cells

BackgroundAutophagy plays an important role in cellular homeostasis. Oxidative stress stimulated endothelial excessive autophagy has been proposed as a major risk factor for cardiovascular diseases (CVD). Danhong injection (DHI), the most prescribed traditional Chinese medicine for the treatment of CVD, has been shown to elicit vascular protective effects. However, its underlying mechanisms remain poorly defined. This study aimed to uncover the protective effects of DHI and its main bioactive components on autophagy injury of human umbilical vein endothelial cells (HUVECs) induced by H2O2 and reveal the possible mechanisms. MethodsHUVECs were treated with different concentrations of DHI or its components, after exposed to H2O2. The protective effects of DHI and its components in H2O2-induced HUVECs were examined via a cytotoxicity assay and western blot. Apoptosis was evaluated with flow cytometry. Autophagy flux was assessed by transmission electron microscopy and LC3 plasmid transfection. Besides, the role miR-19a and SIRT1 in DHI and components-mediated anti-autophagy responses were validated with inhibitors transfection. ResultsOur results showed that DHI and its components do have different effects on different aspects. In terms of HUVECs survival rate, Salvianolic acid B (Sal B) and danshensu (DSS) performed better than DHI, Hydroxysafflor yellow A (HSYA) and Tanshinone IIA (DST-IIA). As for the proliferation effect on HUVECs, only Sal B has the most obvious performance as same as 3MA. Besides, DHI and its components are sensitive and superior in regulating and balancing ROS concentration. Among the GSH/GSSG indicators, DSS and HSYA performed better. In terms of SOD content and apoptotic rate, the SOD level showed the opposite trend compared with H2O2 group. For the expression of LC3, Beclin-1 and P62, DHI and its components all had significant effects. When miR-19a or SIRT1 was inhibited, Sal B (0.5 μg/ml) can not decrease autophagy-related protein effectively. ConclusionDHI and its components all had anti-autophagy effects. And Sal B (0.5 μg/ml) inhibited HUVECs autophagy via miR-19a/SIRT1 pathway.

Pretreatment with Danshensu Promotes Germination of Cucumber (Cucumis sativus L.) Seeds by Regulating Reactive-Oxygen Species and Endogenous Phytohormones

Danshensu (DSS), the aqueous extracts of Salvia miltiorrhiza has been widely used in biomedical science due to its outstanding capacity in ROS scavenger. However, studies regarding its effects on plant physiology are still scare. In the present study, cucumber seeds were soaked in different concentrations of DSS solution for 24 h before subjected to germination to test its effects on seed germination, and further to explore the metabolic and molecular mechanisms. The results shown that 0.1% DSS had significantly promoted the germination rate, radical length, as well as soluble sugar and H2O2 contents. DSS pretreatment upregulated transcript levels of genes involved in SOD, POD, and CAT biosynthesis, concurrent with promoted enzyme activities. In accordance with lower ABA, but higher GA contents, transcript levels of genes involved in ABA and GA biosynthesis were down and upregulated, respectively, in DSS pretreated seeds. DSS pretreatment also stimulated ethylene production and related genes expression levels. These positive effects of DSS on seed germination were confirmed by addition of inhibitors of GA, ABA, NADPH oxidase, and H2O2 scavenger. In conclusion, DSS stimulated cucumber seed germination by mediating the regulation of endogenous hormones with promoted ROS levels. Our results highlight the potentiality of DSS in plant science.

Danshensu alleviates bleomycin-induced pulmonary fibrosis by inhibiting lung fibroblast-to-myofibroblast transition via the MEK/ERK signaling pathway

ABSTRACT Pulmonary fibrosis (PF) is a chronic pulmonary interstitial disease, and its pathological process is closely related to fibroblast–myofibroblast differentiation. Danshensu (DSS) has been reported to exert an anti-fibrotic effect in heart and liver. However, it is unknown whether DSS has an equally anti-fibrotic effect on lungs. To evaluate the effect of DSS on PF and demonstrate its possible molecular mechanisms, we established an in vitro model on TGF-β1 (5 ng/mL)-stimulated NIH3T3 cells and in vivo model on bleomycin (BLM) (5 mg/kg)-induced PF mice. In vitro, our results revealed that 50 μM DSS effectively inhibited the fibroblast proliferation, migration and differentiation into myofibroblast. In vivo, our results showed that DSS (28 and 56 mg/kg) reduced damaged lung structures, infiltrated inflammatory cells and accumulated areas of collagen deposition. Moreover, we showed that DSS decreased the fibroblast-specific protein 1 (FSP-1) – and α-SMA-positive areas. Meanwhile, we indicated that DSS reduced the expression of TGF-β1, α-SMA and COL-I in the lung tissues of mice. To further explore the mechanism of DSS on alleviating PF, we detected the MEK/ERK signaling pathway. Our results showed that DSS reduced the phosphorylation of MEK1/2 and ERK1/2, indicating that DSS might inhibit the MEK/ERK signaling pathway. Taken together, these results demonstrated that DSS could suppress lung fibroblast proliferation, migration and differentiation to myofibroblasts, possibly through suppressing the MEK/ERK signaling pathway, which suggested that DSS might be a potential therapeutic drug for PF treatment.