Danhong Injection Treatment Research Articles

Effect of Danhong injection on heart failure in rats evaluated by metabolomics.

Heart failure (HF) is characterized by reduced ventricular filling or ejection function due to organic or non-organic cardiovascular diseases. Danhong injection (DHI) is a medicinal material used clinically to treat HF for many years in China. Although prior research has shown that Danhong injection can improve cardiac function and structure, the biological mechanism has yet to be determined. Serum metabolic analysis was conducted via ultra-high-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry (UHPLC-QE/MS) to explore underlying protective mechanisms of DHI in the transverse aortic constriction (TAC)-induced heart failure. Multivariate statistical techniques were used in the research, such as unsupervised principal component analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA). MetaboAnalyst and Kyoto Encyclopedia of Genes and Genomes (KEGG) were employed to pinpoint pertinent metabolic pathways. After DHI treatment, cardiac morphology and function as well as the metabolism in model rats were improved. We identified 17 differential metabolites and six metabolic pathways. Two biomarkers, PC(18:3(6Z,9Z,12Z)/24:0) and L-Phenylalanine, were identified for the first time as strong indicators for the significant effect of DHI. This study revealed that DHI could regulate potential biomarkers and correlated metabolic pathway, which highlighted therapeutic potential of DHI in managing HF.

Danhong injection improves neurological function in rats with ischemic stroke by enhancing neurogenesis and activating BDNF/AKT/CREB signaling pathway

Danhong injection (DHI) is a traditional Chinese medicine injection that promotes blood circulation and removes blood stasis and has been widely used in the treatment of stroke. Many studies have focused on the mechanism of DHI in acute ischemic stroke (IS); however, few studies have thoroughly explored its role during recovery. In this study, we aimed to determine the effect of DHI on long-term neurological function recovery after cerebral ischemia and explored the related mechanisms. Middle cerebral artery occlusion (MCAO) was used to establish an IS model in rats. The efficacy of DHI was assessed using neurological severity scores, behaviors, cerebral infarction volume and histopathology. Immunofluorescence staining was performed to assess hippocampal neurogenesis. An in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) cell model was constructed and western-blot analyses were performed to verify the underlying mechanisms. Our results showed that DHI treatment greatly reduced the infarct volume, promoted neurological recovery and reversed brain pathological changes. Furthermore, DHI promoted neurogenesis by increasing the migration and proliferation of neural stem cells, and enhancing synaptic plasticity. Moreover, we found that the pro-neurogenic effects of DHI were related to an increase in brain-derived neurotrophic factor (BDNF) expression and the activation of AKT/CREB, which were attenuated by ANA-12 and LY294002, the inhibitors of the BDNF receptor and PI3K. These results suggest that DHI improves neurological function by enhancing neurogenesis and activating the BDNF/AKT/CREB signaling pathways.

SATB1/SLC7A11/HO-1 Axis Ameliorates Ferroptosis in Neuron Cells After Ischemic Stroke by Danhong Injection.

Neuronal damage after ischemic stroke (IS) is frequently due to ferroptosis, contributing significantly to ischemic injury. However, the mechanism against ferroptosis in IS remained unclear. The aim of this study was to investigate the potential mechanism of Danhong injection (DHI) and the critical transcription factor SATB1 in preventing neuronal ferroptosis after ischemic stroke in vivo and in vitro. The results showed that DHI treatment significantly reduced the infarct area and associated damage in the brains of the pMCAO mice, and enhanced the viability of OGD-injured neurons. And several characteristic indicators of ferroptosis, such as mitochondrial necrosis and iron accumulation, were regulated by DHI after IS. Importantly, we found that the expression and activity of SATB1 were decreased in the pMCAO mice, especially in neuron cells. Meanwhile, the SATB1/SLC7A11/HO-1 signaling pathway was activated after DHI treatment in ischemic stroke and was found to improve neuronal ferroptosis. Inhibition of SATB1 significantly reduced SLC7A11-HO-1 and significantly attenuated the anti-ferroptosis effects of DHI in the OGD model. These findings indicate that neuronal ferroptosis after IS can be alleviated by DHI through SATB1/SLC7A11/HO-1 pathway, and SATB1 may be an attractive therapeutic target for treating ischemic stroke.

Danhong injection attenuates doxorubicin-induced cardiotoxicity in rats via suppression of apoptosis: network pharmacology analysis and experimental validation.

Doxorubicin (DOX) is a potent chemotherapeutic agent that is used against various types of human malignancies. However, the associated risk of cardiotoxicity has limited its clinical application. Danhong injection (DHI) is a Chinese medicine with multiple pharmacological activities and is widely used for treating cardiovascular diseases. The aim of the present study was to evaluate the potential protective effect of DHI on DOX-induced cardiotoxicity in vivo and to investigate the possible underlying mechanisms. First, a sensitive and reliable HPLC−ESI-Q-TOF-MS/MS method was developed to comprehensively analyze the chemical compositions of DHI. A total of 56 compounds were identified, including phenolic acids, tanshinones, and flavonoids. Then, a DOX-induced chronic cardiotoxicity rat model was established to assess the therapeutic effect of DHI. As a result, DHI administration prevented the reduction in body weight and heart weight, and improved electrocardiogram performance. Additionally, the elevated levels of serum biochemical indicators were reduced, and the activities of oxidative enzymes were restored in the DOX-DHI group. Network pharmacology analysis further revealed that these effects might be attributed to 14 active compounds (e.g., danshensu, salvianolic acid A, salvianolic acid B, rosmarinic acid, and tanshinone IIA) and 15 potential targets (e.g., CASP3, SOD1, NOS3, TNF, and TOP2A). The apoptosis pathway was highly enriched according to the KEGG analysis. Molecular docking verified the good binding affinities between the active compounds and the corresponding apoptosis targets. Finally, experimental validation demonstrated that DHI treatment significantly increased the Bcl-2 level and suppressed DOX-induced Bax and caspase-3 expression in rat heart tissue. Furthermore, DHI treatment obviously decreased the apoptosis rate of DOX-treated H9c2 cells. These results indicate that DHI attenuated DOX-induced cardiotoxicity via regulating the apoptosis pathway. The present study suggested that DHI is a promising agent for the prevention of DOX-induced cardiotoxicity.

Danhong injection improves elective percutaneous coronary intervention in ua patients with blood stasis syndrome revealed by perioperative metabolomics

Objective: To observe the effect of Danhong injection (DHI) on perioperative metabolomics of unstable angina pectoris (UA) with blood stasis syndrome. Materials and Methods: A prospective, randomized, controlled, and single-blind clinical trial was conducted. Sixty-one UA patients with traditional Chinese medicine blood stasis syndrome undergoing elective percutaneous coronary intervention (PCI) were randomly divided into the Danhong and control groups, and 10 healthy volunteers were included as baseline. The Danhong group received western medicine + DHI treatment, while the control group received western medicine + saline. Nontargeted metabolomics was used to analyze the serum metabolites of healthy volunteers in the Danhong and control groups before and 5 days after PCI. Results: Before treatment, there was no significant difference in serum metabolites between the Danhong and control groups, but there was a significant difference between the two groups and the healthy group. Differential metabolites were clustered mainly in glycerophospholipid, sphingolipid, purine, and amino acid groups, which were generated in their metabolic pathways. After 5 days of PCI, the profiles of serum metabolites were significantly closer between the Danhong-or control-treated groups and that of the healthy group. Furthermore, DHI treatment converted the serum metabolite profile more to that of the healthy group than the control treatment. Conclusion: The beneficial effect of DHI on patients with unstable angina is reflected at the level of serum metabolic biomarkers.

Effect of Danhong Injection on platelet metabolites based on metabonomics technology

The effect of Danhong Injection on the endogenous metabolites of rabbit platelets was analyzed by the liquid chromatography-mass spectrometry( LC-MS) based metabonomic approach. Anti-platelet aggregation was detected after Danhong Injection treatment and the changes of platelet metabolites were analyzed by metabonomics. Principal component analysis( PCA) and partial least squares discriminant analysis( PLS-DA) were performed to investigate the effect of Danhong Injection on endogenous metabolites of platelets,characterize the biomarkers,and explore the relevant pathways and the underlying mechanism. As demonstrated by the pharmacodynamic results,Danhong Injection of different doses and concentrations antagonized platelet aggregation in a dose-and concentration-dependent manner. In contrast to the control group,25 differential metabolites such as nicotinic acid,nicotinic acid riboside,and hypoxanthine were screened out after platelets were treated by Danhong Injection. These metabolites,serving as important biomarkers,were mainly enriched in the nicotinic acid-niacinamide metabolic pathway and purine metabolic pathway. This study explored the therapeutic mechanism of Danhong Injection from a microscopic perspective by metabonomics,which is expected to provide a new idea for the investigation of platelet-related mechanisms.

Pharmacological Mechanisms Underlying the Therapeutic Effects of Danhong Injection on Cerebral Ischemia.

Background Although Danhong injection (DHI) has been proved to be curative, the mechanism of its action against ischemia stroke (IS) is not clear. Here, we explored the therapeutic basis of DHI by network pharmacology. Methods Putative targets and activity scores for each compound in DHI were obtained from the Traditional Chinese Medicine System Pharmacology Database, Encyclopedia of Traditional Chinese Medicine, and Quantitative Structure Activity Relationships. Next, target proteins of IS were identified on GeneCards and CTD. Overlapping targets of DHI associated with IS were acquired via Venn diagram. GO and KEGG pathway analyses were done using WebGestalt. Cytoscape software was used for PPI network construction and hub nodes screening. Several validation studies were carried out by using AutoDock-Vina, label-free mass spectrometry, and transcriptome RNA-sequencing. Results The 37 active compounds and 66 targets were identified. Of these, 26 compounds and 41 targets belonged to diterpenoid quinones (DQs), which is the predominant category based on chemical structure. The results of enrichments analysis show that 8 DQs target proteins associated with IS were involved in several biological processes and signaling pathway such as apoptotic, cell cycle, cellular response to xenobiotic stimulus process, and the PI3K-Akt signaling. Moreover, 3 nodes in core module involved in PI3K-Akt signaling and 1 hub node were identified by PPI network analysis. Finally, the results of molecular docking and label-free mass spectrometry display good effect on hub node regulation in DHI treatment. Conclusions DQs is the predominant category of DHI and play an important role in antiapoptotic activity mediated by modulating PI3K-Akt signaling. Our findings offer insight into future research and clinical applications in IS therapy.

Danhong Injection Alleviates Cardiac Fibrosis via Preventing the Hypermethylation of Rasal1 and Rassf1 in TAC Mice.

Background/Aim Danhong injection (DHI) is a Chinese patent drug used for relieving cardiovascular diseases. Recent studies have suggested that DNA methylation plays a pivotal role in the maintenance of cardiac fibrosis (CF) in cardiovascular diseases. This study was aimed at identifying the effect and the underlying mechanism of DHI on CF, especially the DNA methylation. Methods A CF murine model was established by thoracic aortic constriction (TAC). A 28-day daily treatment with or without DHI via intraperitoneal injection was carried out immediately following TAC surgery. The changes in cardiac function, pathology, and fibrosis following TAC were measured by echocardiography and immunostaining. We used methyl-seq analysis to assess the DNA methylation changes in whole genes and identified the methylation changes of two Ras signaling-related genes in TAC mice, including Ras protein activator like-1 (Rasal1) and Ras-association domain family 1 (Rassf1). Next, the methylation status and expression levels of Rasal1 and Rassf1 genes were consolidated by bisulfite sequencing, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and Western blotting, respectively. To determine the underlying molecular mechanism, the expressions of DNA methyltransferases (DNMTs), Tet methylcytosine dioxygenase 3 (TET3), fibrosis-related genes, and the activity of Ras/ERK were measured by RT-qPCR and Western blotting. Results DHI treatment alleviated CF and significantly improved cardiac function on day 28 of TAC. The methyl-seq analysis identified 42,606 differential methylated sites (DMSs), including 19,618 hypermethylated DMSs and 22,988 hypomethylated DMSs between TAC and sham-operated mice. The enrichment analysis of these DMSs suggested that the methylated regulation of Ras signal transduction and focal adhesion-related genes would be involved in the TAC-induced CF development. The results of bisulfite sequencing revealed that the TAC-induced methylation affected the CpG site in both of Rasal1 and Rassf1 genes, and DHI treatment remarkably downregulated the promoter methylation of Rasal1 and Rassf1 in CF hearts. Furthermore, DHI treatment upregulated the expressions of Rasal1 and Rassf1, inhibited the hyperactivity of Ras/ERK, and decreased the expressions of fibrosis-related genes. Notably, we found that DHI treatment markedly downregulated the expression of DNMT3B in CF hearts, while it did not affect the expressions of DNMT1, DNMT3A, and TET3. Conclusion Aberrant DNA methylation of Rasal1 and Rassf1 genes was involved in the CF development. DHI treatment alleviated CF, prevented the hypermethylation of Rasal1 and Rassf1, and downregulated DNMT3B expression in CF hearts.

Dan Hong Injection Protects Against Cardiomyocytes Apoptosis by Maintaining Mitochondrial Integrity Through Keap1/Nuclear Factor Erythroid 2-Related Factor 2/JNK Pathway.

Danhong injection (DHI) is used widely against cardiovascular disease in China. Recent studies have demonstrated its mitochondria-protection effect as being pivotal in treatment of myocardial ischemia/reperfusion (I/R) injury, but the underlying mechanism of action is incompletely understood. We aimed to identify the effect and mechanism of action of DHI on mitochondrial integrity and cardiomyocyte apoptosis after I/R. An I/R rat model was induced to detect the effect of DHI on myocardial repair by infarct size, apoptosis and oxidative stress. In vitro, H9C2 cells or H9C2 cells with nuclear factor erythroid 2-related factor 2 (Nrf2) knockdown were injured under hypoxia-reoxygenation (H/R). The effects of DHI on apoptosis, antioxidant capacity and mitochondrial integrity were evaluated by mitochondrial morphology, apoptosis rate, reactive oxygen species (ROS) generation, ATP levels, mitochondrial membrane potential, and oxygen consumption in H9C2 cells treated with H/R. The underlying mechanism of action of DHI in maintenance of mitochondrial integrity and anti-apoptosis was detected in H9C2 cells with or without Nrf2 knockdown. DHI treatment significantly decreased the infarct size, inhibited apoptosis and suppressed oxidative stress in the hearts of I/R rats. Also, DHI promoted cell survival by: an anti-apoptosis action; inhibiting ROS generation; maintaining mitochondrial morphology with increased mitochondrial length; alleviating mitochondrial dysfunction with a decreased mitochondrial membrane potential; increasing ATP levels and the oxygen-consumption rate. Moreover, the Keap1/Nrf2/JNK pathway was found to be involved in DHI reducing oxidative stress and maintaining mitochondrial integrity. We revealed a novel mechanism by which DHI protected H9C2 cells against H/R injury via the Keap1/Nrf2/JNK pathway and provided a mitochondrial protectant for the treatment of myocardial I/R injury.

Danhong Injection Protects Hemorrhagic Brain by Increasing Peroxiredoxin 1 in Aged Rats.

Intracerebral hemorrhage (ICH) is a severe cerebrovascular disease with a high incidence, mortality and disability rate. Danhong injection (DHI) is beneficial for ischemic stroke, but is prohibited for ICH due to risk of bleeding. The present study aims to explore the potential therapeutic time window and molecular mechanism of DHI in a collagenase-induced ICH model in aged rats. DHI administration after ICH could significantly improve body weight and neurological deficits, and reduce the hematoma volume and brain water content when compared to the vehicle control. Furthermore, the protective effect of DHI administration on days 1–3 after ICH was superior to those on days 3–5 or 7–9 after ICH. DHI remarkably increased the Peroxiredoxin 1 (Prx1) expression in astrocytes and reduced the expression of inflammatory factors tumor necrosis factor-α (TNF-α) and interleukin-β (IL-1β) after ICH. The immediate treatment of Prx1 inhibiter chelerythrine (Che) after ICH abolished the protective effect of DHI. Furthermore, the Che treatment reduced the expression of Prx1 in astrocytes, but increased the expression of TNF-α and IL-1β after ICH. DHI treatment could not reverse these changes. Therefore, the earlier DHI is administered, the better the neuroprotective effect. DHI exerts antioxidative and anti-inflammatory function by increasing Prx1 in astrocytes. These present results may change the established understanding of DHI, and reveal a novel treatment approach for ICH.

Effect of Danhong injection on expression of nephrin and podocin in podocyte of renal tissue in rats with membranous nephropathy

Objective To observe the effect of Danhong injection on the expression of nephrin podocin protein and gene in the kidney of membranous nephropathy rats. Methods A total of 34 male clean level SD rats were divided at random into the normal control group (8 rats), model group (26 rates). The model group were injected the C-BSA into caudal vein to establish the model of the membranous nephropathy. After four week of succession of the models, two model rats were taken to confirm the success of the model. Another 24 models divided at random into model group, Losartan potassium treatment group and Danhong injection treatment group, 8 in each group. In the losartan group, the losartan 40 mg/kg was administered, and the Danhong group was injected with 4 ml/kg of Danhong injection in the tail vein for 4 weeks. The rat urine was collected after the last administration, and 24 h urine protein was determined by spectrophotometer. The serum total protein, albumin, TC and TG levels were determined by automatic biochemical analyzer. The renal pathological changes were observed by HE staining. The expressions of nephrin, podocin and WT1 labeled podocytes were detected by immunohistochemical staining. The expressions of nephrin and podocin in renal tissue were detected by real-time quantitative PCR and Western blot, and the results were correlated. Results Compared with the model group, the content of serum albumin increased in the Danhong group (P<0.01), 24 h urine protein quantitation, TC, TG significantly decreased (P<0.01). The expression of nephrin protein (1.12 ± 0.03 vs. 0.57 ± 0.09), podocin protein (0.99 ± 0.04 vs. 0.53 ± 0.08), nephrin mRNA (0.49 ± 0.08 vs. 0.14 ± 0.02), podocin mRNA (0.50 ± 0.11 vs. 1.18 ± 0.05) in Danhong group significantly increased (P<0.01), and the number of podocytes labeled with WT1 (14.88% ± 1.73% vs. 9.63% ± 1.69%) significantly increased (P<0.05). Correlation analysis showed that 24 h urine protein quantitation and expression of nephrin protein (r=-0.908), podocin protein (r=-0.916), nephrin mRNA (r=-0.930), podocin mRNA (r=-0.923). There was a negative correlation between serum albumin (r=-0.934) and podocyte count (r=-0.778) (P<0.01), and positive correlation with TC (r=0.814) and TG (r=0.870) (P<0.01). Conclusions The Danhong injection may reduce the urine protein of membranous nephropathy rats by up-regulating the expression of nephrin, podocin mRNA and protein in renal cells of membranous nephropathy. Key words: Glomerulonephritis, membranous; Podocytes; Proteinuria; Dan Hong Zhu She Ye; Rats

Cardioprotective Effect of Danhong Injection against Myocardial Infarction in Rats Is Critically Contributed by MicroRNAs.

Background Danhong injection (DHI) has been mainly used for the treatment of myocardial infarction, atherosclerosis, and coronary heart disease in clinical practice. Our previous studies have shown that DHI improves ventricular remodeling and preserves cardiac function in rats with myocardial infarction (MI). In this study, we focused on the potential mechanism of DHI in protecting cardiac function in MI rats. Methods Sprague-Dawley rats were subjected to ligation of the left anterior descending coronary artery (LAD) to prepare a myocardial infarction (MI) model. After 14 day DHI intervention, cardiac function was measured by echocardiography and myocardial fibrosis was assessed by Masson staining. Differentiated miRNAs were screened using rat immunopathology miScript miRNA PCR arrays, and their results were verified by RT-PCR, immunofluorescence, and immunoblotting. Results DHI treatment significantly reduced infarct size and improved cardiac function and hemodynamics in MI rats by echocardiography and morphology. miRNA PCR array results showed that DHI reversed 25 miRNAs known to be associated with inflammation and apoptosis. Moreover, the expression of inflammatory factors TNF-α, IL-1β, and IL-6 was significantly reduced in the treated DHI group. Mechanistically, DHI downregulated the inflammatory transcription factor NF-κB (as reflected by inhibition of NF-κB p65 nuclear translocation and phosphorylation of the IκBα). Conclusions DHI is effective in mitigating inflammation associated with MI by preventing NF-κB nuclear translocation and regulating miRNAs, thereby improving cardiac function in myocardial infarction rats.

Danhong Injection Alleviates Postoperative Intra-abdominal Adhesion in a Rat Model.

Background Among all the common complications that occur after abdominal surgery, intestinal adhesion is perhaps the most unpleasant one. However, current methods to treat and prevent intestinal adhesion are limited; thus, exploring new methods to prevent and treat intestinal adhesion is greatly needed. In this study, we demonstrated that Danhong injection (DHI) may be used as a promising method to prevent and treat intra-abdominal adhesion in a rat model. Materials and Methods Forty-eight rats were randomly divided into six groups. Except for the sham-operated group, all rats underwent cecal abrasion to establish an adhesion model. After the operation, the rats in the DHI-treated groups received different doses of DHI via the tail vein daily, while the other group was treated with the same volume of saline solution. Seven days after the operation, all rats were sacrificed, and the degree of adhesion was evaluated by Nair's scoring system. The extent of inflammation in the adhesion tissue was detected by HE staining and the expression of tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β). The collagen deposition was assessed by Sirius red staining and α-SMA, MMP9, t-PA, and PAI-1 levels. Oxidative stress was indicated by the level of reactive oxygen species (ROS) in adhesion tissues and by immunohistochemical labeling of Nrf2. Furthermore, rat primary peritoneal mesothelial cells (RPMCs) were treated with H2O2 and DHI, and NF-κB phosphorylation was detected to illustrate the effect of DHI on oxidative stress. Results The intra-abdominal adhesion scores were significantly decreased in the groups treated with a high dose of DHI compared with the control groups, and the degree of inflammation, fibrosis, and oxidative stress was also significantly decreased. DHI treatment significantly reduced the levels of TNF-α, TGF-β1, and PAI and increased the expression levels of MMP9, Nrf2, and t-PA in the adhesion tissues. ROS levels and NF-κB phosphorylation were significantly reduced in DHI-treated RPMCs compared with the control RPMCs. Conclusion DHI alleviates the formation of postoperative intra-abdominal adhesions by inhibiting inflammation, collagen deposition, and oxidative stress in a rat model and may serve as a promising drug to prevent intra-abdominal adhesions.

Danhong Injection Attenuates High-Fat-Induced Atherosclerosis and Macrophage Lipid Accumulation by Regulating the PI3K/AKT Insulin Pathway.

High-fat diet (HFD) is reported to induce atherosclerosis and insulin resistance. Macrophage lipid accumulation has been implicated as key mediators during the development of HFD-induced atherosclerosis. Traditional Chinese formula, which has long been used to improve disorder of glucose and lipid metabolism of patients, is now gradually being used as complementary therapy. This study aimed to investigate the effect of Danhong injection (DHI), a Chinese medicine used for the treatment of coronary artery disease, on atherosclerosis and its underlying mechanisms. We observed the effects of DHI on HFD-induced atherosclerosis in a mice model, macrophage lipid accumulation in an ox-LDL-stimulated macrophage model, and the role of PI3K/AKT insulin pathway in the process of DHI ameliorating atherosclerosis. The data demonstrated that DHI attenuated atherosclerosis by ameliorating blood lipids, reducing the atherosclerotic index and atherosclerotic plaque area in HFD-induced atherosclerotic mice, and inhibiting TC levels in an ox-LDL-induced macrophage model. By estimating the levels of serum insulin resistance-related indexes and protein expression of GLUT-4, DHI treatment dramatically inhibited the levels of fasting serum NEFA and fasting serum insulin and promoted the protein expression of GLUT-4 in aortas of the HFD-induced atherosclerotic mice. Moreover, according to the hints provided by microarray-based transcriptional profiling, the results demonstrated that DHI treatment also promoted the activation of PI3K/AKT insulin signaling pathway induced by IRS-1 in aortas of HFD-induced atherosclerotic mice. Furthermore, in an ox-LDL-induced macrophage model, the activation of PI3k/AKT signaling pathway also effectively functioned in the process of DHI inhibiting macrophage lipid accumulation. These results highlight that DHI treatment attenuates atherosclerosis and macrophage lipid accumulation by promoting the activation of PI3K/AKT insulin signaling pathway. It provides new insights into the molecular mechanism of DHI and its therapeutic potential in the treatment of atherosclerosis.

Danhong injection mobilizes endothelial progenitor cells to repair vascular endothelium injury via upregulating the expression of Akt, eNOS and MMP-9

BackgroudEndothelial progenitor cells (EPCs) have been characterized as one of the key effectors of endothelial healing. The effect of Danhong injection (DHI), the most widely prescribed Chinese medicine for coronary heart disease (CHD), on EPCs mobilization remains unclear. PurposeWe aimed to assess the effect of DHI on EPCs mobilization to repair percutaneous coronary intervention (PCI) induced vascular injury, and to investigate the characteristics and potential mechanism of DHI on EPCs mobilization. MethodForty-two patients with CHD underwent PCI and received stent implantation were enrolled in a Phase II clinical trials. All patients received routine western medical treatment after PCI, patients of DHI group received DHI in addition. The levels of CECs, cytokines (vWF, IL-6, CRP) and EPCs were analyzed at baseline, post-PCI and after treatment. To investigate the characteristics of DHI on EPCs mobilization, 12 healthy volunteers received intravenous infusion of DHI once and the other 12 received for 7 days. EPCs enumeration were done at a series of time points. At last we tested the effect of DHI and three chemical constituents of DHI (danshensu; lithospermic acid, LA; salvianolic acid D, SaD) on EPCs level and expression of Akt, eNOS and MMP-9 in bone marrow cells of myocardial infarction (MI) mice. ResultsIn the DHI group the angina symptoms were improved, the levels of cytokines and CECs were reduced; while EPCs population was increased after treatment. In the phase I clinical trials, EPCs counts reached a plateau phase in 9 h and maintained for more than 10 h after a single dose. After continuous administration, EPCs levels plateaued on the 3rd or 4th day, and maintain till 1 day after the withdrawal, then its levels gradually declined. DHI treatment induced a timely dependent mobilization of EPCs. DHI promoted EPCs mobilization via upregulating the expression of Akt, eNOS and MMP-9 in BM. LA and SaD have played a valuable role in EPCs mobilization. ConclusionThese initial results demonstrated that DHI is effective in alleviating endothelial injury and promoting endothelial repair through enhancing EPCs mobilization and revealed the effect feature and possible mechanisms of DHI in mobilizing EPCs.

Transcriptomic Study Reveals Recovery of Impaired Astrocytes Contribute to Neuroprotective Effects of Danhong Injection Against Cerebral Ischemia/Reperfusion-Induced Injury.

Danhong Injection (DHI) is widely used in clinics for treating cardiovascular and cerebrovascular diseases in China. However, the mode of action of DHI for neuroprotection remains unclear. In the present study, we deemed to investigate the effects of DHI on a rat model of cerebral ischemia/reperfusion injury (IRI) with an emphasis on its regulated gene profile obtained from microarray assays. Firstly, we showed that a 14-day DHI treatment effectively ameliorated severity of neurological deficits, reduced size of ischemic damage, improved status of oxidation stress, as well as systemic inflammation for IRI rats, along with which was a pronounced reduced cell infiltration in the area of periaqueductal gray matter. Secondly, bioinformatic analyses for the 429 differentially expressed genes (DEGs) regulated by DHI treatment pointed out ECM–receptor interaction, neuroactive ligand–receptor interaction, and endocytosis as the top three biological processes, while Toll-like recptor 4 (TLR4) as the most relavant singaling molecule. Lastly, we provided evidences showing that DHI might directly protect primary astrocytes from oxygen and glucose deprivation/re-oxygenation (OGD/Re) injury, the effects of which was associated with LAMC2 and ADRB3, two DEGs related to the top three biological processes according to transcriptomic analysis. In conlusion, we reported that DHI might work through maintaining the integrity for brain–blood barrier and to regulate TLR4-related signaling pathway to diminish the inflammation, therefore, effectively improved the outcomes of IRI. Our findings suggested that the attenuated astrocytic dysfunction could be a novel mechanism contributing to the neuroprotective effects of DHI against cerebral ischemia/reperfusion-induced damage.

Investigation of the reverse effect of Danhong injection on doxorubicin-induced cardiotoxicity in H9c2 cells: Insight by LC–MS based non-targeted metabolomic analysis

Although Danhong injection (DHI) has been clearly shown to attenuate ischemic myocardial injury and improve heart function, there is no research regarding its role in doxorubicin (DOX)-induced cardiomyopathy. In this study, we aimed to investigate the reverse effect of DHI on DOX-induced cardiotoxicity in H9c2 cells. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay demonstrated that DHI had no cytotoxicity towards the relevant cell line unless the concentration was as high as 50 μL/mL. The satisfactory cardioprotective effect of DHI exerted at the concentration of 10 μL/mL, which agreed well with the result of real-time cell viability assay. Then non-targeted metabolomics based on LC–MS was employed to characterize metabolic alterations in DOX-induced cells with DHI treatment. Multivariate analysis, including PCA and PLS-DA, revealed 31 altered metabolites after DOX treatment that were primarily related to the disturbance of amino acids and nucleotides metabolism. While DHI could intervene in some disturbed metabolic pathways, such as the metabolism of arginine, glutathione (GSH), pantothenic acid, cytidine, inosine and 5′-methylthioadenosine. These results suggested that DHI exerted the therapeutic effect by improving energy metabolism and attenuating oxidative stress. The present study can lay a foundation for further research on the promising therapeutic effect of DHI in managing DOX-induced cardiotoxicity.

Danhong Injection Alleviates Mechanical Allodynia via Inhibiting ERK1/2 Activation and Elevates BDNF Level in Sciatic Nerve in Diabetic Rat.

Danhong injection (DHI) has been widely used in China for cardiocerebrovascular diseases treatments. And in this study, we demonstrated the therapeutic effect of DHI on experimental diabetic neuropathy for the first time. Methods. Streptozotocin- (STZ-) induced SD rats were used. In experiment 1, 4-week treatment with DHI or saline started 4 weeks after STZ injection; mechanical allodynia was measured before and every 2 weeks after STZ injection. In experiment 2, chronic intrathecal infusion of U0126 was conducted during the 8th week of diabetes. Phosphorylated and total ERK1/2 in spinal cord were analyzed by western blot. BDNF level in sciatic nerve was evaluated by ELISA. Results. DHI treatment significantly alleviated mechanical allodynia at the end of the study and downregulated the expression of phosphorylated ERK1/2 in spinal cord. In addition, DHI treatment also elevated brain-derived neurotrophic factor (BDNF) level in sciatic nerve of DPN rat. In experiment 2, inhibition of ERK1/2 activation was confirmed to result in the alleviation of mechanical allodynia. Conclusions. We demonstrated that DHI was able to alleviate mechanical allodynia in diabetic neuropathy rat through inhibiting the activation of ERK1/2. The reduction of BDNF content in sciatic nerve was also partially reversed by DHI treatment.

Danhong Injection Protects Against Hypertension-Induced Renal Injury Via Down-Regulation of Myoglobin Expression in Spontaneously Hypertensive Rats

Background/Aims: High blood pressure is a major risk factor for chronic kidney disease. Currently, single-target anti-hypertensive drugs are not designed for high blood pressure-related organ damages. Danhong injection (DHI), made from the aqueous extracts of Radix Salviae miltiorrhizae and Flos Carthamus tinctorius, has various pharmacological effects, including BP lowering in SHR, mediated by the reduction of vascular remodeling and the up-regulation of Kallikrein-kinin system published recently by our team, yet if it renders renal protection remains unknown. The current study demonstrated a protective role of DHI in renal injury caused by hypertension and identified its molecular targets in the kidney of spontaneously hypertensive rats (SHR). Methods: Adult SHR and age/gender-matched normotensive Wistar-Kyoto (WKY) rats were treated with DHI, Losartan, or saline for 4 weeks. Serum levels of Creatinine (CRE), Micro-albumin (mAlb), Beta2-microglobulin (β2-MG), and Uric acid (UA) were detected using ELISA kits. Renal pathology was examined by hematoxylin and Eosin (H&E) stains. Microarray analysis was performed on kidney tissues, and gene expression changes were validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analyses. Results: Renal histopathological scores showed that SHR exhibited serious kidney injury compared to normotensive WKY rats. The intervention with DHI potently suppressed the renal injury biomarker (KIM-1) and kidney lesions compared to the untreated hypertensive subjects. Microarray analysis revealed that among the 124 genes that were differentially expressed by DHI treatment in SHR kidney, down-regulation of renal myoglobin (Mb) gene was the most prominent and was subsequently confirmed by qRT-PCR and Western blot analysis. Conclusion: Hypertension-induced renal injury in SHR may be alleviated by DHI in part by local suppression of Kidney injury molecule-1 and down-regulation of Myoglobin. However, if this effect is independent of the known anti-hypertensive action of DHI in blood vessel remains to be determined.

Danhong injection reduces vascular remodeling and up-regulates the Kallikrein-kinin system in spontaneously hypertensive rats

Although Danhong injection (DHI) is one of the most prescribed cardiovascular medicines in China, its therapeutic indications and mechanisms remain partially defined. We now identify molecular targets of DHI in resistance vasculatures and demonstrate its role in vascular function and blood pressure (BP) regulation. BP was determined in DHI, Losartan, and placebo- treated Spontaneously Hypertensive Rats (SHR) by both noninvasive and invasive measurements. Vasorelaxation was examined both in conduit and resistance vasculature by ex vivo aortic rings. Microarray analysis was performed and gene expression changes were verified by RT-qPCR and ELISA. Diastolic, systolic and mean BPs were significantly lower in DHI-treated SHR than controls by both tail-cuff and invasive BP measurements. In ex vivo rings, aortic and mesenteric vessels from SHR treated with DHI exhibited significantly greater acetylcholine-mediated relaxation. Among the 282 genes that are differentially expressed in microarray analysis, DHI treatment up-regulated the expression of kallikrein and plasma kallikrein B genes. DHI also significantly increased serum kallikrein content in SHR. Treatment with DHI significantly increased the ratio of aortic lumen to outer diameter. Therefore, the reduction of vascular remodeling and the up-regulation of Kallikrein-kinin system contribute, at least in part, to the antihypertensive effect of DHI in SHR.