Drug-drug interaction between danshensu and irbesartan and its potential mechanism.

To uncover the effect of danshensu on irbesartan pharmacokinetics and its underlying mechanisms.To investigate the effect of danshensu on the pharmacokinetics of irbesartan, Sprague-Dawley rats (n = 6) were orally administered 30 mg/kg irbesartan alone (control group) or pre-treated with 160 mg/kg danshensu (experimental group). The effect of danshensu on the metabolic stability of irbesartan in RLMs was examined by LC-MS/MS method. The effect of danshensu on CYP2C9 activity was also determined.Danshensu markedly increased the AUC(0-t) (9573 ± 441 vs. 16157 ± 559 µg/L*h) and Cmax (821 ± 24 vs. 1231 ± 44 µg/L) of irbesartan. Danshensu prolonged the t1/2 (13.39 ± 0.98 vs. 16.04 ± 1.21 h) and decreased the clearance rate (2.27 ± 0.14 vs. 1.19 ± 0.10 L/h/kg) of irbesartan. Danshensu enhanced the metabolic stability of irbesartan in vitro with prolonged t1/2 (36.34 ± 11.68 vs. 48.62 ± 12.03 min) and reduced intrinsic clearance (38.14 ± 10.24 vs. 28.51 ± 9.06 µL/min/mg protein). Additionally, the IC50 value for CYP2C9 inhibition by danshensu was 35.74 µM.Danshensu enhanced systemic exposure of irbesartan by suppressing CYP2C9. The finding can also serve as a guidance for further investigation of danshensu-irbesartan interaction in clinical practice.

Synergistic Differential DNA Demethylation Activity of Danshensu (Salvia miltiorrhiza) Associated with Different Probiotics in Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is a major hepatic disorder occurring in non-alcohol-drinking individuals. Salvianic acid A or Danshensu (DSS, 3-(3, 4-dihydroxyphenyl)-(2R)-lactic acid), derived from the root of Danshen (Salvia miltiorrhiza), has demonstrated heart and liver protective properties. In this work, we investigated the antioxidant activity and hepatoprotective activity of Danshensu alone and in combination with different agents, such as probiotic bacteria (Lactobacillus casei and Lactobacillus acidophilus), against several assays. The inhibition mechanism of the methylation gene biomarkers, such as DNMT-1, MS, STAT-3, and TET-1, against DSS was evaluated by molecular docking and RT-PCR techniques. The physicochemical and pharmacokinetic ADMET properties of DSS were determined by SwissADME and pkCSM. The results indicated that all lipid blood test profiles, including cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C), were reduced after the oral administration of Danshensu combined with probiotics (L. casei and L. acidophilus) that demonstrated good, efficient free radical scavenging activity, measured using anti-oxidant assays. ADMET and drug-likeness properties certify that the DSS could be utilized as a feasible drug since DSS showed satisfactory physicochemical and pharmacokinetic ADMET properties.

Protective effect of Danshensu against neurotoxicity induced by monosodium glutamate in adult mice and their offspring.

Excessive neuronal excitation by glutamate is a well-established cause of neurotoxicity, leading to severe impairment of brain function. Excitotoxicity is a key factor in numerous neurodegenerative conditions. In this study, we investigated the neuroprotective effects of Danshensu (DSS) against monosodium glutamate (MSG)-induced neurotoxicity in adult mice and their offspring. We randomly divided one hundred 8-week-old Kunming mice (equal number of males and females) into a control group and an experimental group. The experimental group was further subdivided into various treatment groups, including MSG gavage treatment, bwbw DSS treatment group 1 (bwbw DSS treatment group 2, a drug control group, and a normal control group (receiving an equal volume of physiological saline for ten consecutive days). Additionally, another one hundred healthy 8-week-old Kunming mice were similarly divided into groups and treated. These mice were paired randomly (one male and one female) and pregnant females were housed separately to obtain offspring. Subsequently, we conducted histological and behavioral analyses on adult mice and their offspring. MSG treatment induced significant cellular edema and hippocampal damage in both the treated mice and their offspring. However, varying doses of DSS effectively counteracted the neurotoxic effects of MSG, with no adverse impact on brain tissue structure or neural function in either adult mice or their offspring. Behavioral experiments further confirmed that DSS exerted a substantial protective effect against MSG-induced impairment of learning and memory in the treated adult mice and their offspring, in addition to mitigating central nervous system overexcitation and inhibiting exploratory behavior. In conclusion, DSS exerts significant protective effects against MSG-induced neurotoxicity in both adult mice and their offspring.

Stepwise targeted strategies for improving neurological function by inhibiting oxidative stress levels and inflammation following ischemic stroke.

Ischemia-reperfusion injury is caused by excessive production of reactive oxygen species (ROS) and inflammation accompanied by ischemic injury symptoms and blood-brain barrier (BBB) dysfunction. This causes neuronal damage, for which no effective treatments or drugs exist. Herein, we provided a stepwise targeted drug delivery strategy and successfully prepared multifunctional ORD@SHp@ANG nanoparticles (NPs) that consist of a stroke homing peptide (DSPE-PEG2000-SHp), BBB-targeting peptide (DSPE-PEG2000-ANG), and ROS-responsive Danshensu (salvianic acid A) chain self-assembly. ORD@SHp@ANG NPs effectively crossed the BBB by ANG peptide and selectively targeted the ischemic brain sites using stroke-homing peptide. The results showed that ORD@SHp@ANG NPs can effective at scavenging ROS, and protect SH-SY5Y cells from oxidative damage in vitro. Furthermore, ORD@SHp@ANG NPs showed excellent biocompatibility. These NPs recognized brain endothelial cells and crossed the BBB, regulated the transformation of microglia into the anti-inflammatory phenotype, and inhibited the production of inflammatory factors in a rat ischemia-reperfusion model, thereby reducing cerebral infarction, neuronal apoptosis and preserving BBB integrity. Sequencing revealed that ORD@SHp@ANG NPs promote cell proliferation, activate immune responses, suppress inflammatory responses, and ameliorate ischemic stroke. In conclusion, this study reports a simple and promising drug delivery strategy for managing ischemic stroke.

Danshensu methyl ester attenuated LPS-induced acute lung injury by inhibiting TLR4/NF-κB pathway.

Acute Lung Injury (ALI) manifests as an acute exacerbation of pulmonary inflammation with high mortality. The potential application of Danshensu methyl ester (DME, synthesized in our lab) in ameliorating ALI has not been elucidated. Our results demonstrated that DME led to a remarkable reduction in lung injury. DME promoted a marked increase in antioxidant enzymes, like superoxide dismutase (SOD), and glutathione (GSH), accompanied by a substantial decrease in reactive oxygen species (ROS), myeloperoxidase (MPO), and malondialdehyde (MDA). Moreover, DME decreased the production of IL-1ß, TNF-α and IL-6, in vitro and in vivo. TLR4 and MyD88 expression is reduced in the DME-treated cells or tissues, which further leading to a decrease of p-p65 and p-IκBα. Meanwhile, DME effectively facilitated an elevation in cytoplasmic p65 expression. In summary, DME could ameliorate ALI by its antioxidant functionality and anti-inflammation effects through TLR4/NF-κB, which implied that DME may be a viable medicine for lung injury.

PUD@HA/PEEK Scaffold Induces Subchondral Bone Regeneration to Repair Osteochondral Defect in Rabbits.

Osteochondral defects (OCDs) pose a significant challenge in clinical practice, and recent advancements in their repair indicate that satisfying subchondral bone repair may be critical for this. Herein, a series of hydroxyapatite/poly(ether ether ketone) (HA/PEEK) scaffolds were fabricated with varying mass percentages (0, 20, 30, and 40%) to induce subchondral bone regeneration. Subsequently, an optimal scaffold with 40% HA/PEEK was selected to establish osteochondral scaffolds with poly(ether urethane) urea-Danshensu (PUD) for repairing the OCD. The material characteristics of HA/PEEK and PUD were investigated using scanning electron microscopy, tensile, swelling, and fatigue tests, and cytological experiments. The effects of serial HA/PEEK scaffolds on subchondral bone repair were then assessed by using microcomputed tomography, hard tissue slicing, and histological staining. Furthermore, the optimal 40% HA/PEEK scaffold was used to develop osteochondral scaffolds with PUD to observe the effect on the OCD repair. HA/PEEK materials exhibited an even HA distribution in PEEK. However, when composited with HA, PEEK exhibited inferior mechanical strength. 40%HA/PEEK scaffolds showed an optimum effect on in vivo subchondral bone repair. Cartilage regeneration on 40%HA/PEEK scaffolds was pronounced. After PUD was introduced onto the HA/PEEK, the PUD@40%HA/PEEK scaffold produced the expected effect on the repair of the OCD in rabbits. Therefore, achieving satisfactory subchondral bone repair can benefit surficial cartilage repair. The PUD@40%HA/PEEK scaffold could induce subchondral bone regeneration to repair the OCD in rabbits and could provide a novel approach for the repair of the OCD in clinical practice.

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.

Sodium Danshensu ameliorates cerebral ischemia/reperfusion injury by inhibiting CLIC4/NLRP3 inflammasome-mediated endothelial cell pyroptosis.

Endothelial pyroptosis promotes cerebral ischemia/reperfusion injury (CIRI). Sodium Danshensu (SDSS) has been shown to attenuate CIRI and have anti-inflammatory properties in endothelial cells. However, the mechanism and effect of SDSS on alleviating endothelial pyroptosis after CIRI remains poorly understood. Thus, we aimed to investigate the efficacy and mechanism of SDSS in reducing endothelial pyroptosis. It has been shown that SDSS administration inhibited NLRP3 inflammasome-mediated pyroptosis. As demonstrated by protein microarrays, molecular docking, CETSA and ITDRFCETSA , SDSS bound strongly to CLIC4. Furthermore, SDSS can decrease its expression and inhibit its translocation. Its effectiveness was lowered by CLIC4 overexpression but not by knockdown. Overall The beneficial effect of SDSS against CIRI in this study can be ascribed to blocking endothelial pyroptosis by binding to CLIC4 and then inhibiting chloride efflux-dependent NLRP3 inflammasome activation.

Danshensu inhibits SARS-CoV-2 by targeting its main protease as a specific covalent inhibitor and discovery of bifunctional compounds eliciting antiviral and anti-inflammatory activity.

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a serious threat to human. Since there are still no effective treatment options against the new emerging variants of SARS-CoV-2, it is necessary to devote a continuous endeavor for more targeted drugs and the preparation for the next pandemic. Salvia miltiorrhiza and its active ingredients possess wide antiviral activities, including against SARS-CoV-2. Danshensu, as one of the most important active ingredients in Salvia miltiorrhiza, has been reported to inhibit the entry of SARS-CoV-2 into ACE2 (angiotensin-converting enzyme 2)-overexpressed HEK-293T cells and Vero-E6 cells. However, there is a paucity of information regarding its detailed target and mechanism against SARS-CoV-2. Here, we present Danshensu as a covalent inhibitor of 3-chymotrypsin-like protease (3CLpro) against SARS-CoV-2 by the time-dependent inhibition assay (TDI) and mass spectrometry analysis. Further molecular docking, site-directed mutagenesis, circular dichroism (CD) and fluorescence spectra revealed that Danshensu covalently binds to C145 of SARS-CoV-2 3CLpro, meanwhile forming the hydrogen bonds with S144, H163 and E166 in the S1 site. Structure-based optimization of Danshensu led to the discovery of the promising compounds with good inhibitory activity and microsomal stability in vitro. Due to Danshensu inhibiting lung inflammation in the mouse model, we found that Danshensu derivatives also showed better anti-inflammatory activity than Danshensu in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Thus, our study provides not only the clue of the efficacy of Salvia miltiorrhiza against SARS-CoV-2, but also a detailed mechanistic insight into the covalent mode of action of Danshensu for design of covalent inhibitors against SARS-CoV-2 3CLpro, highlighting its potential as a bifunctional molecule with antivirus and anti-inflammation.

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.

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.

Discovery of small molecule ß-catenin suppressors that enhance immunotherapy.

Small molecules directly downregulating ß-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/ß-catenin pathway. The challenge, however, lies in the fact that very few ß-catenin suppressors have proven clinically effective, leaving a significant gap in medical solutions. Given that E-cadherin has a natural affinity for ß-catenin, it stands to reason that agents designed to increase E-cadherin expression might provide an alternative method of regulating ß-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 ß-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 ß-catenin deactivation.

Sodium Danshensu stabilizes atherosclerotic vulnerable plaques by targeting IKKß mediated inflammation in macrophages.

BACKGROUND: The primary cause of acute cardiovascular events with high mortality is the rupture of atherosclerotic plaque followed by thrombosis. Sodium Danshensu (SDSS) has shown potential in inhibiting the inflammatory response in macrophages and preventing early plaque formation in atherosclerotic mice. However, the specific targets and detailed mechanism of action of SDSS are still unclear. OBJECTIVE: This study aims to investigate the efficacy and mechanism of SDSS in inhibiting inflammation in macrophages and stabilizing vulnerable plaques in atherosclerosis (AS). MATERIALS AND METHODS: The efficacy of SDSS in stabilizing vulnerable plaques was demonstrated using various techniques such as ultrasound, Oil Red O staining, HE staining, Masson staining, immunohistochemistry, and lipid analysis in ApoE-/- mice. Subsequently, IKKß was identified as a potential target of SDSS through protein microarray, network pharmacology analysis, and molecular docking. Additionally, ELISA, RT-qPCR, Western blotting, and immunofluorescence were employed to measure the levels of inflammatory cytokines, IKKß, and NF-κB pathway-related targets, thereby confirming the mechanism of SDSS in treating AS both in vivo and in vitro. Finally, the impact of SDSS was observed in the presence of an IKKß-specific inhibitor. RESULTS: Initially, the administration of SDSS led to a decrease in the formation and area of aortic plaque, while also stabilizing vulnerable plaques in ApoE-/- mice. Furthermore, it was identified that IKKß serves as the primary binding target of SDSS. Additionally, both in vivo and in vitro experiments demonstrated that SDSS effectively inhibits the NF-κB pathway by targeting IKKß. Lastly, the combined use of the IKKß-specific inhibitor IMD-0354 further enhanced the beneficial effects of SDSS. CONCLUSIONS: SDSS stabilized vulnerable plaques and suppressed inflammatory responses by inhibiting the NF-κB pathway through its targeting of IKKß.

Danshensu inhibits Aß aggregation and neurotoxicity as one of the main prominent features of Alzheimer's disease.

It has been found that the main cause of neurodegenerative proteinopathies, especially Alzheimer's disease (AD) is the formation of Aß amyloid plaques, which can be regulated by application of potential small molecules. In the present study, we aimed to investigate the inhibitory effect of danshensu on Aß(1-42) aggregation and relevant apoptotic pathway in neurons. A broad range of spectroscopic, theoretical, and cellular assays were done to investigate the anti-amyloidogenic characteristics of danshensu. It was found that danshensu triggers its inhibitory effect against Aß(1-42) aggregation through modulation of hydrophobic patches as well as structural and morphological changes through a stacking interaction. Furthermore, it was observed that incubation of Aß(1-42) samples with danshensu during aggregation process recovered the cell viability and mitigated the expression of caspase-3 mRNA and protein as well caspase-3 activity deregulated by Aß(1-42) amyloid fibrils alone. In general, obtained data showed that danshensu potentially inhibits Aß(1-42) aggregation and associated proteinopathies through regulation of apoptotic pathway in a concentration-dependent manner. Therefore, danshensu may be used as a promising biomolecule against the Aß aggregation and associated proteinopathies, which can be further analyzed in the future studies for the treatment of AD.

Suppression of Macrophage Activation by Sodium Danshensu via HIF-1α/STAT3/NLRP3 Pathway Ameliorated Collagen-Induced Arthritis in Mice.

It is still a clinical challenge to sustain the remission of rheumatoid arthritis (RA); thus, identifying more effective and safer agents for RA treatment remains an urgent demand. We investigated the anti-arthritic activity and potential mechanism of action of sodium Danshensu (SDSS), a structurally representative water-soluble derivative of Danshen, on collagen-induced arthritis (CIA) mice. Our results showed that paw edema, synovium hyperplasia, bone destruction, and the serum levels of both IL-1ß and IL-6 were ameliorated by SDSS (40 mg/kg·d) in CIA mice. In addition, there was no difference between SDSS and methotrexate (MTX, 2 mg/kg·3d) treatment in the above indicators. Further mechanism studies illustrated that SDSS inhibited IL-1ß secretion by downregulating the HIF-1α/STAT3/NLRP3 pathway in macrophages. On the other hand, HIF-1α accumulation and HIF-1α/STAT3/NLRP3 pathway activation by IOX4 stimulation reduced the therapeutic effect of SDSS. These findings demonstrate that SDSS displays anti-arthritic activity in CIA mice and prevents proinflammatory cytokines secretion in macrophages by suppressing the HIF-1α/STAT3/NLRP3 pathway.

Danshensu attenuated lipopolysaccharide-induced LX-2 and T6 cells activation through regulation of ferroptosis.

Liver fibrosis and cirrhosis are primarily caused by the activation of hepatic stellate cells (HSCs), regardless of their etiology. Collagen type I (collagen I) and connective tissue growth factor (CTGF) is produced more readily by activated HSCs. Consequently, identifying the molecular and cellular mechanisms responsible for HSCs activation is essential to better understand its mechanism of action and therapeutic potential. Cell death is caused by iron-dependent lipid peroxidation during ferroptosis. Ferroptosis plays an important role in the survival of activated HSCs and could contribute to the development of innovative prevention and treatment strategies for liver fibrosis. Danshensu (Dan) is a pure molecule extracted from the Salvia miltiorrhiza herb that protects against liver damage. However, Dan's effect on attenuating HSCs activation by regulating ferroptosis remains unclear. The results of this study indicated that lipopolysaccharide (LPS)-induced LX-2 and T6 cells activation occurs through the upregulation of collagen I, CTGF, Gpx4, and SLC7A11. Interestingly, Dan attenuated LPS-induced liver fibrosis in those cells by upregulating collagen I, CTGF, Gpx4, and SLC7A11 and by increasing lipid reactive oxygen species accumulation. Furthermore, the results also showed that the ferroptosis inhibitor liproxstatin attenuated the overproduction of lipid reactive oxygen species in LPS-activated LX-2 cells. We conclude that Dan attenuates LPS-induced HSC activation during liver fibrosis by regulating ferroptosis in LX-2 and T6 cells.

Danshensu Enhances Cerebral Angiogenesis in Mice by Regulating the PI3K/Akt/Mtor/VEGF Signaling Axis.

BACKGROUND: Cerebral infraction seriously affects the life quality of patients. Danshensu has been reported to exhibit anti-inflammatory and vascular protective effects. However, the therapeutic function of Danshensu in cerebral vascular injury is still unclear. METHODS: Middle cerebral artery occlusion (MCAO) was used to construct the cerebral infraction model. Wound healing and tube formation assays were used to evaluate angiogenesis in vitro. Western blot assay was used to evaluate the activation of the PI3K/Akt/mTOR signaling pathway. The laser Doppler scanner was used to measure the regional cerebral blood flow (rCBF) in the area around the infarction, and the adhesion removal test was used to measure the sensorimotor function. The Modified Neurological Severity Score was performed to evaluate the cognitive functions of mice. RESULTS: Danshensu promoted the proliferation of bEnd.3 cells and angiogenesis in vitro. Danshensu upregulated the expression of VEGF through PI3K/Akt/mTOR signaling pathway in bEnd.3 cells. Danshensu improved rCBF restoration and attenuated the behavioral deficits in mice post-MCAO/R. CONCLUSION: Danshensu enhances angiogenesis through the PI3K/Akt/mTOR/VEGF signaling pathway in a mouse model of cerebral ischemic injury.

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.

Discovery of novel danshensu derivatives bearing pyrazolone moiety as potential anti-ischemic stroke agents with antioxidant activity.

Neuroprotective agents with attenuation of oxidative stress by directly scavenging ROS and indirectly through Keap1-Nrf2 signal pathway activation may be a promising cerebral ischemic stroke therapeutic strategy. In this study, a series of novel danshensu derivatives bearing pyrazolone moieties with dual antioxidant effects were synthesized for the treatment of ischemic stroke. Most compounds exhibited considerable DPPH free radical scavenging ability and neuroprotective activity against H2O2-induced oxidative injury in PC12 neuronal cells, without cytotoxicity. Among these target compounds, Del03 displayed the strongest dose-dependent neuroprotective activity in vitro, directly downregulated intracellular ROS levels, and improved the oxidative stress parameters MDA, SOD, and LDH. Del03 also promoted Nrf2 translocation to the nucleus, subsequently increasing the expression of the Nrf2 downstream target HO-1. Molecular docking analysis revealed that Del03 could anchor to the key site of Keap1. Del03 possessed the ability to penetrate blood-brain barrier and displayed good ability on pharmacokinetic properties in rats Del03 possessed good BBB penetration efficiency, suitable pharmacokinetic properties in vivo. Del03 reduced cerebral infarction volume and promoted neurological function in a middle cerebral artery occlusion (MCAO) mouse model at a dose of 20 mg/kg by intravenous injection. The characteristics of Del03 detailed in this study demonstrate its potential as a therapeutic agent in the treatment of ischemic stroke.

Sodium Danshensu Cream Promotes the Healing of Pressure Ulcers in Mice through the Nrf2/HO-1 and NF-κB Pathways.

On the basis of the mice pressure ulcers (PU) model, the protective effect and potential mechanism of sodium Danshensu (SDSS) cream against PU were investigated. The mice were randomly divided into three groups: the negative control group (cream without 0.5 g SDSS), the SDSS group (cream containing 0.5 g SDSS), and the positive group (0.5 g Hirudoid®). After 7 and 14 days of ointment application, the wound-healing rate of the SDSS and positive groups was significantly higher than that of the control group (p < 0.05). The results of hematoxylin−eosin staining also indicated that SDSS has the potential to promote the healing of PU. In addition, the serum IL-6, IL-1ß, TNF-α, and MDA levels decreased significantly (p < 0.01) after 14 days of SDSS treatment, while the SOD, CAT, and GSH-Px activities increased significantly (p < 0.01). In addition, SDSS cream was able to significantly increase the expression of Nrf2, HO-1, GCLM, NQO1, NF-κB p65, NF-κB p50, IKKα, and IKKß while decreasing the expression of Keap1 and IκBαin the Nrf2/HO-1 and NF-κB pathways. Our research will provide a foundation for the future clinical prevention and treatment of PU with SDSS cream.