Search

Background. The traditional Chinese medicines of Buyang Huanwu decoction (BYHW), Naoxintong capsule (NXT), and Yangyin Tongnao granules (YYTN) have excellent effects in preventing and treating cerebrovascular disease and are widely tolerated by patients. However, their effects on middle cerebral artery occlusion (MCAO) remain unknown. Methods. We evaluated gut microbiota alterations, the brain transcriptome, and nerve cell responses in rats with MCAO. Results. Our results showed that BYHW, NXT, and YYTN not only effectively improved the damaged state of blood vessels in rats and restored nerve function, but also improved survival. Additional experiments showed that treatment with BYHW, NXT, and YYTN regulated the intestinal microflora. Transcriptome analyses showed that BYHW, NXT, and YYTN modulated the transcriptome of rats with MCAO. The common mechanism of the three prescriptions for the treatment of cerebral ischemia may be related to the intestinal flora regulation of 60S ribosomal protein L18 (Rpl18), eukaryotic translation initiation factor 3 subunit, Ras homolog family member C, G protein subunit gamma 13 (Gng13), and Gng10 genes, among which Rpl18 is the most important. In addition, the three prescriptions had great specificity as anticerebral ischemia targets. Moreover, BYHW, NXT, and YYTN mitigated MCAO-induced hyperactivation of microglia and astrocytes. Conclusion. This study provides a foundation for further research on the mechanisms and treatment of IS. The results strongly suggest that key gut microbiota can be used to study functional genomics of brain, leading to novel discoveries about key genes involved in important biological processes.

Background: Population-based studies have consistently showed an increased incidence of coronary heart disease and cardiac mortality in patients with type 2 diabetes mellitus (T2DM). Tongmai Jiangtang capsules (TJC) are Chinese patent medicines that have been approved in China for the treatment of diabetic vascular complications. However, the evidence supporting the efficacy of Tongmai Jiangtang capsules in type 2 diabetic coronary heart disease (T2DM-CHD) remains unclear. Herein, we designed a randomized, parallel-controlled clinical trial to investigate a new complementary therapy for T2DM-CHD patients.Methods: A total of 360 T2DM-CHD subjects (aged 18–75 years) will be randomly assigned to the TJC group or the placebo group at a 2:1 ratio. On the basis of western medicine therapy, all the participants will receive TJC or placebo, orally, three capsules/treatment, three per day for 12 weeks. The primary outcomes will be assessed according to the Canadian Cardiovascular Society (CCS) classification. All statistical analyses will be performed setting a two-sided 0.05 significance level, using SAS 9.4 statistical software.Discussion: The efficacy of TJC for the treatment of T2DM-CHD patients will be evaluated. The study will provide reliable clinical research evidence for application of TJC in treating T2DM-CHD patients.Clinical Trial Registration:https://www.chictr.org.cn/enIndex.aspx, Chinese Clinical Trial Registry ChiCTR2000037491.

Formation of thrombosis is associated with activation of platelets and endothelial cells. The effect of LongShengZhi Capsule (LSZ), a traditional Chinese medicine used for treatment of vascular diseases, on thrombosis was investigated in this study. BALB/c mice were induced thrombosis by injection of carrageenan while receiving pre or simultaneous LSZ treatment. We also compared the therapeutic effects of LSZ and clopidogrel on formed thrombi. LSZ inhibited carrageenan-induced thrombi in mouse tissue vessels. In addition, LSZ but not clopidogrel reduced formed thrombi with a short time window. The reduction of thrombi by LSZ was associated with reduced serum P-selectin, reduced expression of TNF-α and P-selectin and activated matrix metalloproteinase 2 expression in tissues. In vitro, LSZ decreased thrombin-induced human platelet clot retraction which was associated with inactivation of AKT and ERK1/2. LSZ also reduced adhesion of platelets or THP-1 monocytes to human umbilical vein endothelial cells (HUVECs) induced by oxidized low-density lipoprotein or lipopolysaccharide. The anti-adherent actions of LSZ was attributed to reduction of oxidative stress, expression of platelet receptors (P2Y12, PAR4 and CD36) and AKT activity in platelets. LSZ also reduced adhesion molecules or tissue factor but activated tissue factor pathway inhibitor expression in HUVECs. Taken together, our study demonstrates the antithrombotic properties of LSZ by reducing activation of platelets and endothelial cells, and suggests its potential application in clinics.

NaoXinTong Capsule (NXT), a prescribed traditional Chinese medicine, is made up of 16 natural herbal materials with more than 200 identified bioactive compounds. Multiple protective effects of NXT on cardiovascular diseases including atherosclerosis, coronary artery disease, acute coronary syndrome, coronary microembolization, myocardial infarction, ischemic stroke and ischemia-reperfusion injury, have been reported by both clinical and basic studies. Biologically, these cardioprotective effects can be correlated to the actions of NXT on inflammation, apoptosis, oxidative stress, neovascularization, insulin sensitivity and lipid/glucose metabolism. NXT alone or in combination with the conventional interventions has been demonstrated potent therapeutic effects on cardiovascular diseases without causing significant adverse events, like the major bleeding. Compared with the conventional drugs, patients have a good tolerance and little resistance to NXT treatment. With the data and evidence reported from lab benches to clinical beds, we will update the cardioprotective properties of NXT and the involved mechanisms in this review. We hope the information provided in this review can help readers to better understand the insights for the long practice of this traditional Chinese medicine, and offer fresh perspectives.

Heart failure is a major cause of morbidity and mortality worldwide. LongShengZhi capsule (LSZ), a traditional Chinese medicine, is used for treatment of patients with vascular diseases. Herein we investigated the effect of LSZ treatment on doxorubicin (DOX)-induced heart failure in mice. C57BL/6 mice randomly in 3 groups received following treatment: Control group, mice were fed normal chow; DOX group, mice were intraperitoneally injected DOX to induce heart failure and fed normal chow; and LSZ group, mice were injected DOX and fed normal chow containing LSZ. DOX induced heart failure as evidenced by increased serum creatine kinase, lactic dehydrogenase and α-hydroxybutyrate dehydrogenase, and cardiac fibrosis. However, LSZ treatment substantially inhibited DOX-induced heart failure parameters. Mechanistically, LSZ reduced collagen content and fibrosis by inhibiting expression of collagen type I α1 (COL1α1), COL1α2, α-smooth muscle actin and transforming growth factor β1. In addition, DOX-induced cell apoptosis was inhibited by LSZ, coupled with reduced caspase 3 activity and mRNA expression. LSZ decreased inflammatory cytokine levels. More importantly, LSZ decreased oxidative stress by inducing expression of anti-oxidative stress enzymes including superoxide dismutase 1 (SOD1), SOD2, catalase and glutathione peroxidase 1 through activation of forkhead box O3A and sirtuin 3. In conclusion, our study demonstrates that LSZ reduces heart failure by reducing production of reactive oxygen species and inhibiting inflammation/apoptosis. Our study also suggests the potential application of LSZ for heart failure treatment.

Formation of thrombosis is mainly associated with dysfunctions of endothelial cells. NaoXinTong capsule (NXT), a traditional Chinese medicine, has been demonstrated multiple protective effects on vascular systems. However, it is unknown the effect of NXT on thrombosis. In this study, we determined whether NXT can inhibit carrageenan-induced thrombosis and the underlying mechanisms. Two days after carrageenan injection, severe thrombi were found in blood vessels of mouse tail and liver. By contrast, thrombi were substantially reduced by NXT treatment, and the reduction was associated with reduced serum tumor necrosis factor α and P-selectin levels. In vitro, NXT reduced lipopolysaccharide-activated adhesion of THP-1 monocytes to human umbilical vein endothelial cells (HUVECs) by inhibiting expression of adhesion molecules and interleukin 6, and reducing production of mitochondrial superoxide that is related to activation of antioxidant enzymes expression. NXT also reduced oxidized low-density lipoprotein-activated adhesion of platelets to HUVECs. In addition, NXT protected HUVECs against clopidogrel-induced cell death by inhibiting expression of tumor necrosis factor-like cytokine 1A and activating expression of vascular endothelial growth factor α. Taken together, our study indicates the potential application of NXT in antithrombosis by multiple antithrombotic functions.

Background An increase in the late sodium current (INaL ) causes intracellular Na+ overload and subsequently intracellular Ca2+ ([Ca2+ ]i ) overload via the stimulated reverse Na+ -Ca2+ exchange (NCX). Wenxin Keli (WXKL) is an effective antiarrhythmic Chinese herb extract, but the underlying mechanisms are unclear. Methods and results The INaL , NCX current (INCX ), L-type Ca2+ current (ICaL ), and action potentials were recorded using the whole-cell patch-clamp technique in rabbit ventricular myocytes. Myocyte [Ca2+ ]i transients were measured using a dual excitation fluorescence photomultiplier system. WXKL decreased the enhanced INaL , reverse INCX , diastolic [Ca2+ ]i , and the amplitude of Ca2+ transients induced by sea anemone toxin II (ATX II, a specific INaL channel opener) in a concentration-dependent manner. Hypoxia increased INaL , INCX , and diastolic [Ca2+ ]i , and decreased amplitude of [Ca2+ ]i transients. Hypoxia-reoxygenation aggravated these changes and induced spontaneous [Ca2+ ]i transients and hypercontraction in 86% cells (6/7). The application of WXKL during hypoxia or reoxygenation periods decreased the increased INaL , INCX , and diastolic [Ca2+ ]i , and prevented those events in 82% cells (9/11) under hypoxia-reoxygenation conditions. WXKL also inhibited the ICaL in a dose-dependent manner. Furthermore, WXKL shortened the action potential duration and completely abolished ATX II-induced early afterdepolarizations from 9/9 to /9. In isolated heart electrocardiogram recordings, WXKL inhibited ischemia-reperfusion induced ventricular premature beats and tachycardia. Conclusions WXKL attenuated [Ca2+ ]i overload induced by hypoxia-reoxygenation in ventricular myocytes through inhibiting INaL and ICaL and prevents arrhythmias. This could, at least partly, contribute to the antiarrhythmic effects of WXKL.

The current treatment for diabetic nephropathy (DN) is still limited. NaoXinTong Capsule (NXT) is a Chinese Medicine prescribed to patients with cardiovascular disease. It can also ameliorate metabolic syndromes in patients indicating its anti-diabetic properties. Herein we report the therapeutic effects of NXT on the developed DN. The db/db diabetic mice at ˜12 weeks old, the age with DN at middle/advanced stages, were treated with NXT for 12 weeks. We found NXT treatment reduced diabetes-induced hyperglycemia and dyslipidemia, thereby substantially reduced DN progress. In the kidney, NXT reduced mesangial matrix expansion and glomerulosclerosis by inhibiting extracellular matrix accumulation through activation of matrix metalloproteinase 2/9 and inactivating transforming growth factor β1 expression. NXT reduced podocyte injury by reducing renal inflammation and expression of adhesion molecules. Mechanically, NXT potently activated AMPKα in multiple tissues thereby enhancing energy metabolism. In the liver, NXT increased glucokinase expression and insulin sensitivity by increasing insulin receptor substrate 1/2 and protein kinase B (AKT) 1/2 expression/phosphorylation. In skeletal muscle, NXT activated expression of glucose transporter type 4, AKT, glycogen synthase and peroxisome proliferator activated receptor α/γ. In adipose tissue, NXT reduced fatty acid synthase while activating hormone-sensitive lipase expression. Taken together, our study demonstrates that NXT reduced progress of the developed DN by ameliorating glucose, lipid and energy metabolism, maintaining renal structural and functional integrity. Our study also indicates the potential application of NXT for DN treatment in clinics.

Disorders of lipid metabolism and inflammation play an important role in atherosclerosis. LongShengZhi (LSZ) capsule, a Chinese herbal medicine, has been used for treatment of patients with vascular diseases for many years. In this article, we determined the effect of LSZ on the progression of established atherosclerotic lesions in apoE-deficient (apoE) mice. ApoE mice were prefed high-fat diet (HFD) for 8 weeks to induce atherosclerosis, then started with LSZ treatment contained in HFD for 10 weeks. Although LSZ had little effect on HFD-induced hypercholesterolemia, it substantially reduced en face and sinus aortic lesions. The reduction of lesions was associated with reduced macrophage/foam cell accumulation by activating ABCA1/ABCG1 expression. LSZ maintained the integrity of arterial wall by increasing collagen or smooth muscle cell content and inhibiting cell apoptosis. LSZ also attenuated HFD-induced fatty liver by down-regulating expression of lipogenic and cholesterol synthetic genes while activating expression of triglyceride catabolism genes. Moreover, LSZ demonstrated potent anti-inflammatory effects. In vivo, LSZ reduced serum TNF-α levels, infiltration of neutrophils, Kupffer cells, and expression of inflammatory cytokines in the liver. In vitro, it inhibited lipopolysaccharide or palmitate-induced expression of inflammatory cytokines in macrophages. Therefore, LSZ reduces atherosclerosis by ameliorating hepatic lipid metabolism and inhibiting inflammation.

Metabolic syndrome, such as diabetes mellitus, obesity, atherosclerosis, and high blood pressure (HBP), are closely linked pathophysiologically. However, current monotherapies for metabolic syndrome fail to target the multifactorial pathology via multiple mechanisms, as well as resolving the dysfunctionality of the cells and organs of the body. We aimed to provide a comprehensive and up-to-date review of the pharmacological advances, therapeutic potential, and phytochemistry of Salvia miltiorrhiza, Carthamus tinctorius, and Danhong injection (DHI). We discussed the molecular mechanisms of the bioactive constituents relating to diabetes mellitus and metabolic disease for further research and drug development. Interestingly, Salvia miltiorrhiza, Carthamus tinctorius, and DHI have anti-inflammatory, anti-glycemic, anti-thrombotic, and anti-cancer properties; and they mainly act by targeting the dysfunctional vasculatures including the inflammatory components of the disease to provide vascular repair as well as resolving oxidative stress. The major bioactive chemical constituents of these plants include polyphenolic acids, diterpene compounds, carthamin, and hydroxysafflor yellow A. Treatment of diabetes mellitus and its associated cardiovascular complication requires a comprehensive approach involving the use of appropriate traditional Chinese medicine formula. Danshen, Honghua, and DHI target the multiple risk factors regulating the physiologic function of the body and restore normalcy, apart from the traditional advice on exercise and diet control as treatment options in a metabolic syndrome patient.

Wenxin Keli (WXKL) is a widely used Chinese botanical drug for the treatment of arrhythmia, which is consisted of four herbs and amber. In the present study, we analyzed the chemical composition of WXKL using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) to tentatively identify 71 compounds. Through typical separate procession, the total extract of WXKL was divided into fractions for further bioassays. Cardiomyocytes and zebrafish larvae were applied for assessment. In vivo arrhythmia model in Cmlc2-GFP transgenic zebrafish was induced by terfenadine, which exhibited obvious reduction of heart rate and occurrence of atrioventricular block. Dynamic beating of heart was recorded by fluorescent microscope and sensitive camera to automatically recognize the rhythm of heartbeat in zebrafish larvae. By integrating the chemical information of WXKL and corresponding bioactivities of these fractions, activity index (AI) of each identified compound was calculated to screen potential active compounds. The results showed that dozens of compounds including ginsenoside Rg1, ginsenoside Re, notoginsenoside R1, lobetyolin, and lobetyolinin were contributed to cardioprotective effects of WXKL. The anti-arrhythmic activities of five compounds were further validated in larvae model and mature zebrafish by measuring electrocardiogram (ECG). Our findings provide a successful example for rapid discovery of bioactive compounds from traditional Chinese medicine (TCM) by activity index based approach coupled with in vivo zebrafish model.

Background: We recently reported that Naoxintong (NXT), a China Food and Drug Administration (FDA)-approved cardiac medicine, could reduce the plaque size, but the underlying mechanism remains elusive now. Objective: In this study, we investigated the effects of NXT on foam cell accumulation both in vivo and in vitro and explored related mechanisms. Method: THP-1 cells and bone marrow-derived macrophages were incubated with oxidized low-density lipoprotein (ox-LDL) with/without Naoxintong. ApoE-/- mice fed an atherogenic diet were administered to receive NXT for eight weeks. Macrophage-derived foam cell formation in plaques was measured by immunohistochemical staining. Expression of proteins was evaluated by Western blot. Lentivirus was used to knockdown PPARα in THP-1 cells. Results: After NXT treatment, foam cell accumulation was significantly reduced in atherosclerotic plaques. Further investigation revealed that oxidized low-density lipoprotein (ox-LDL) uptake was significantly decreased and expression of scavenger receptor class A (SR-A) and class B (SR-B and CD36) was significantly downregulated post-NXT treatment. On the other hand, NXT increased cholesterol efflux and upregulated ATP-binding cassette (ABC) transporters (ABCA-1 and ABCG-1) in macrophages. Above beneficial effects of NXT were partly abolished after lentiviral knockdown of PPARα. Conclusion: Our findings suggest that NXT could retard atherosclerosis by inhibiting foam cell formation through reducing ox-LDL uptake and enhancing cholesterol efflux and above beneficial effects are partly mediated through PPARα pathway.

NaoXinTong Capsule (NXT), a Chinese medicine, is currently used to treat patients with cardiovascular and cerebrovascular diseases. Clinical observations indicate its anti-diabetic functions with unclear mechanisms. Herein, we report the effect of NXT on diabetic nephropathy (DN). Type 2 diabetic db/db mice were treated with NXT for 14 weeks. In the course of treatment, NXT reduced diabetes-increased glucose levels and improved renal functions. At the end of treatment, we found that NXT ameliorated serum lipid profiles and other biochemical parameters. In the kidney, NXT inhibited mesangial matrix expansion, expression of vascular endothelial growth factor A, fibronectin, advanced glycation end product and its receptor. Meanwhile, it reduced the diabetes-induced podocyte injury by increasing WT1 and nephrin expression. In addition, NXT inhibited accumulation of extracellular matrix proteins by increasing MMP2/9 expression through inactivation of TGFβ/Smad pathway and CTGF expression. Mechanically, NXT activated insulin signaling pathway by increasing expression of INSR, IRS and FGF21, phosphorylation of Akt and AMPKα in the liver, INSR phosphorylation in the kidney, and FGF21 and GLUT4 expression in adipose tissue and skeletal muscle. Taken together, our study demonstrates that NXT inhibits DN by ameliorating glucose/lipid metabolism, maintaining tissue structure integrity, and correcting diabetes-induced renal dysfunctions.

DanHong injection is a Danshen (Salvia miltiorrhiza roots)-based injectable solution for treatment of coronary artery disease and ischemic stroke. Danshen catechols are believed to be responsible for the injection's therapeutic effects. This study aimed to characterize systemic exposure to and elimination of Danshen catechols in human subjects, rats, and dogs receiving intravenous DanHong injection. A total of 28 catechols were detected, with content levels of 0.002-7.066 mM in the injection, and the major compounds included tanshinol, protocatechuic aldehyde, salvianolic acid B, rosmarinic acid, salvianolic acids A and D, and lithospermic acid with their daily doses ≥10 μmol/subject. After dosing, tanshinol, salvianolic acid D, and lithospermic acid exhibited considerable exposure in human subjects and rats. However, only tanshinol had considerable exposure in dogs. The considerable exposure to tanshinol was due to its having the highest dose, whereas that to salvianolic acid D and lithospermic acid was due to their relatively long elimination half-lives in the human subjects and rats. Protocatechuic aldehyde and rosmarinic acid circulated in the bloodstream predominantly as metabolites; salvianolic acids A and B exhibited low plasma levels with their human plasma metabolites little or not detected. Tanshinol and salvianolic acid D were eliminated mainly via renal excretion. Elimination of other catechols involved hepatobiliary and/or renal excretion of their metabolites. Methylation was found to be the primary metabolism for most Danshen catechols and showed intercompound and interspecies differences in rate and degree in vitro. The information gained here is relevant to pharmacological and toxicological research on DanHong injection.

Buchang NaoXinTong capsule (NXT) is a Chinese Materia Medica standardized product extracted from 16 Chinese traditional medical herbs and widely used for treatment of patients with cerebrovascular and cardiovascular diseases in China. Formation of microaneurysms plays an important role in the development of diabetic retinopathy. In this study, we investigated if NXT can protect diabetic mice against the development of diabetic retinopathy. Thedb/dbmice (~6 weeks old), a diabetic animal model, were divided into two groups and fed normal chow or plus NXT for 14 weeks. During the treatment, fasting blood glucose levels were monthly determined. After treatment, retinas were collected to determine retinal thickness, accumulation of carbohydrate macromolecules, and caspase-3 (CAS-3) expression. Our results demonstrate that administration of NXT decreased fasting blood glucose levels. Associated with the decreased glucose levels, NXT blocked the diabetes-induced shrink of multiple layers, such as photoreceptor layer and outer nuclear/plexiform layers, in the retina. NXT also inhibited the diabetes-induced expression of CAS-3 protein and mRNA, MMP-2/9 and TNFαmRNA, accumulation of carbohydrate macromolecules, and formation of acellular capillaries in the retina. Taken together, our study shows that NXT can inhibit the development of diabetic retinopathy and suggests a new potential application of NXT in clinic.

Background/Aims: Shenxian-shengmai (SXSM) oral liquid, a Chinese patent compound medicine, has been used to treat sinus bradyarrhythmias induced by mild sick sinus syndrome in clinical practice. Myocardial ischemia, in particular in serious or right coronary-related heart diseases, can cause bradyarrhythmias and cardiac dysfunction. Moreover, reperfusion of ischemic myocardium is associated with additional myocardial damage known as myocardial ischemia-reperfusion (I/R) injury. This study was designed to evaluate the effects of SXSM on bradyarrhythmias and cardiac dysfunction induced by myocardial I/R injury, and to explore the underlying mechanisms. Methods: Administration of SXSM to adult male Sprague Dawley (SD) rats was achieved orally by gavage and control rats were given equivalent deionized water every day for 14 days. After the last administration, the heart was connected with the Langendorff perfusion apparatus and both groups were subjected to ischemia for 20 min followed by reperfusion for 40 min to induce myocardial I/R injury. Heart rate (HR), left ventricular developed pressure (LVDP), the maximal increase rate of left ventricular pressure (+dp/dtmax) and the maximal decrease rate of left ventricular pressure (-dp/dtmax) were recorded by a physiological signal acquisition system. The heart treated with ischemic preconditioning (IPC) for 3 times at a range of 5 min/time before ischemia served as a positive control group. The hearts without I/R injury served as control group. After reperfusion, superoxide dismutase (SOD), glutathione (GSH) and glutathione peroxidase (GSH-Px) activities in the myocardium were determined by appropriate assay kits. Myocardial SOD1 and glutamate cysteine ligase catalytic subunit (GCLC) expression were assessed by western blot analysis. For the in vitro study, SXSM serum was prepared according to the serum pharmacological method and neonatal rat cardiomyocytes were isolated from the heart of new born SD rats. Neonatal rat cardiomyocytes were pretreated with SXSM serum and subjected to H2O2 or anoxia/ reoxygenation (A/R) treatment to induce oxidative damage. Cell viability was evaluated using a Cell Counting Kit-8 (CCK8) assay. Levels of reactive oxygen species (ROS), SOD, GSH and GSH-Px in cardiomyocytes were determined by appropriate assay kits. SOD1 and GCLC expression were assessed by western blot analysis. Buthionine-[S, R]-sulfoximine (BSO), a GCLC inhibitor, and SOD1 siRNA were also used for identifying the cardiac protective targets of SXSM. Results: SXSM and ischemic preconditioning (IPC) significantly increased heart rate during myocardial reperfusion and protected cardiac function against myocardial I/R injury, including an increase in left ventricular diastolic pressure (LVDP), the maximal increase rate of left ventricular pressure (+dp/dtmax) and the maximal decrease rate of left ventricular pressure (-dp/dtmax). We also found that SXSM and IPC improved the expansion of myocardial interstitium, the structural abnormality and morphological changes of cardiomyocytes induced by I/R injury. Meanwhile, SXSM protected cardiomyocytes against the oxidative damage induced by H2O2 and A/R injury through reducing intracellular ROS levels. Moreover, SXSM increased SOD activity through enhancing SOD1 expression and increased GSH content through promoting GCLC expression as well as GSH-Px activity. BSO and SOD1 siRNA counteracted anti-arrhythmic and cardiac protective effect of SXSM, suggesting that the therapeutic targets of SXSM might be SOD1 and GCLC. Conclusion: SXSM is effective in protecting the myocardium from I/R injury, with myocardial SOD1 and GCLC being the potential therapeutic targets.

Quality-marker (Q-marker) is an emerging concept to ensure the quality and batch-to-batch consistency of Chinese medicine (CM). However, significant difficulties remain in the identification of Q-markers due to the unclear relationship between complex chemical compositions and the pharmacological efficacy of CM. In the present study, we proposed a novel strategy to identify the potential Q-marker of danhong injection (DHI) by an in vivo zebrafish thrombosis model. The anti-thrombotic effects of DHI and its major constituents were evaluated by the zebrafish model of arachidonic acid (AA)-induced thrombosis. The results indicated that DHI can attenuate tail venous thrombus and recover the decrease of heart red blood cell (RBC) intensity in a dose-dependent manner. The result that DHI prevented the formulation of thrombosis in zebrafish was also validated in the zebrafish thrombosis model with green fluorescence protein (GFP)-labeled hemoglobin. The major components of DHI, namely danshen (DS) and honghua (HH), as well as the major chemical constituents of DHI, also exerted anti-thrombotic effects, among which rosmarinic acid (RA) and p-coumaric acid (pCA) showed moderate anti-thrombotic effects. This is the first time that pCA from HH has been found as an active compound exerting an anti-thrombotic effect in a dose-dependent manner, whose IC50 value is approximately 147 μg/mL. By analyzing 10 batches of normal DHI samples and five abnormal samples by high-performance liquid chromatography (HPLC), we found the contents of pCA and RA can be positively correlated to the anti-thrombotic effect of DHI, suggesting that pCA and RA could be potential Q-markers of DHI to ensure batch-to-batch consistency. Our findings illustrated that discovering major active compounds from CM by in vivo pharmacological models can be a useful approach to identifying Q-markers of CM, and in vivo pharmacological models can be a potential tool to evaluate batch-to-batch consistency of CMs.

An increase in the late sodium current (IThe IWXKL attenuated [Ca

Clinical observations indicate that DanHong Injection (DHI) can increase blood flow and reduce various syndromes in patients with cardiovascular disease. However, it still needs to define the function of DHI and the involved mechanisms in details, such as the protective effect on the development of primary abdominal aortic aneurysms (AAAs). In this study, we determined whether DHI is able to inhibit AAA in apoE knockout (apoE−/−) mice. Thirty apoE−/− male mice on high-fat diet (0.5 % cholesterol, 21 % fat) were randomly divided into two groups and received i.p. injection of saline (100 μL/day) and DHI (100 μL/day), respectively, for 16 weeks. At the end of experiment, we determined the development of atherosclerosis in en face aorta and aneurysms, pathological morphology of arterial wall, and serum lipid levels. We also determined the expression of monocyte chemoattractant protein-1 (MCP-1), MMP-2, and MMP-9 mRNA in aortic wall using real-time RT-PCR. Our results indicated that high-fat diet induced the development of AAAs in apoE−/− mice, but the induction was totally blocked by DHI (P < 0.01). The result of staining of abdominal aortic cross sections showed that DHI maintained the collagen content in arterial wall, thereby preventing the animals from the development of AAA. Although DHI had little effect on serum total- and LDL-cholesterol levels, it reduced the expression of MCP-1, MMP-2, and MMP-9 mRNA in aortic wall (P < 0.01). Taken together, our study suggests that DHI can inhibit the high-fat diet-induced AAA formation. The inhibitory effects may be related to the maintenance of the collagen content and inhibition of expression of AAA-related genes. Our study may suggest a new application of DHI in clinics.

Danhong injection (DHI), a Chinese Materia Medica standardized product extracted from Radix Salviae miltiorrhizae and Flos Carthami tinctorii, is widely used in China for treating acute ischemic stroke. In the present study, we explored the neuroprotective efficacy of DHI in a rat model of temporary middle cerebral artery occlusion, and evaluated the potential mechanisms underlying its effects. Pretreatment with DHI (0.9 and 1.8 mL/kg) resulted in a significantly smaller infarct volume and better neurological scores than pretreatment with saline. Furthermore, DHI significantly reduced the permeability of the blood-brain barrier, increased occludin protein expression and decreased neutrophil infiltration, as well as profoundly suppressing the upregulation of matrix metallopeptidase-9 expression seen in rats that had received vehicle. Matrix metallopeptidase-2 expression was not affected by ischemia or DHI. Moreover, DHI (1.8 mL/kg) administered 3 hours after the onset of ischemia also improved neurological scores and reduced infarct size. Our results indicate that the neuroprotective efficacy of DHI in a rat model of cerebral ischemia-reperfusion injury is mediated by a protective effect on the blood-brain barrier and the reversal of neutrophil infiltration.

Naoxintong (NXT), a Chinese Materia Medica standardized product, extracted from 16 various kinds of Chinese traditional herbal medicines including Salvia miltiorrhiza, Angelica sinennsis, Astragali Radix, is clinically effective in treating atherosclerosisrelated diseases. Here, we tested the hypothesis that the anti-atherosclerosis effects of NXT might be mediated by suppressing maturation of dendritic cells (DCs) in a mice model of atherosclerosis. LDLR(-/-) mice fed a high-fat diet were treated with placebo, NXT (0.7 g/kg/d, oral diet) or simvastatin (100mg/kg/d, oral diet) for 8 weeks, respectively. NXT treatment significantly reduced plasma triglyceride (112 ± 18 mg/dl vs. 192 ± 68 mg/dl, P0.05) and total cholesterol (944 ± 158 mg/dl vs. 1387 ± 208 mg/dl, P0.05) compared to placebo treatment. Vascular lesions were significantly smaller and macrophage content and amount of DCs in plaques were significantly less in NXT and simvastatin groups than in placebo group (all P0.05). In addition, expressions of splenic DC membrane molecules (CD40, CD86 and CD80) and the plasma level of IL-12p70 were significantly lower in NXT and simvastatin groups than in placebo group. In conclusion, NXT protects against atherosclerosis through lipid-lowering and inhibiting DCs maturation in this mice model of atherosclerosis.

Buchang NaoXinTong (NXT) is a Chinese medicine that has been used for many years for treatment of patients with coronary heart disease (CHD) in China. Statins substantially reduce hypercholesterolemia and CHD mortality and morbidity. However, there is still a lot of CHD patients who do not respond well to statin therapy. Herein, we report the effects of NXT on atorvastatin-inhibited atherosclerosis and atorvastatin-induced hepatic side effects. After 10 weeks of high-fat diet (HFD) feeding, apoE-deficient mice were randomly divided into 4 groups and received the following treatment for another 8 weeks: group 1, HFD; group 2, HFD containing NXT; group 3, HFD containing atorvastatin; and group 4, HFD containing both NXT and atorvastatin. After treatment, serum lipid profiles, atherosclerotic lesions, and hepatic lipid content and inflammation were determined. NXT moderately increased high-density lipoprotein cholesterol levels, although had little effect on atorvastatin-induced reduction of low-density lipoprotein cholesterol levels. Both NXT and atorvastatin reduced en face lesions and sinus lesions of aortic root. In addition, NXT enhanced atorvastatin-induced lesion plaque stability by increasing smooth muscle cell/collagen content and reducing macrophage accumulation and calcification in lesion areas. The co-treatment of NXT and atorvastatin further reduced hepatic triglyceride levels by downregulating acyl-CoA:diacylglycerol acyltransferase 1 while activating hormone-sensitive lipase, adipose triglyceride lipase, and comparative gene identification-58 expression. The AMPKα pathway was also further activated by the co-treatment. More importantly, the liver injuries caused by atorvastatin, such as hepatic inflammation and elevated serum aminotransferase activities, were substantially attenuated by NXT. Therefore, our study demonstrates that NXT enhances atorvastatin-induced plaque stability and ameliorates atorvastatin-induced hepatic side effects.

Oxidative stress plays a crucial role in numerous cardiovascular diseases. As an effective therapy, Danhong injection (DHI) is considered to act through an antioxidant mechanism for the treatment of cardiovascular disease. In our study, we focused on the potential contribution of the antioxidant capacity of DHI fractions (Frs) and established an innovative screening method based on a 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity assay. A ternary network evaluation system, which was constructed based on the radical scavenging activity, the area under the activity-concentration curve and the solid content of the fractions, was implemented to select the fractions that posed the greatest antioxidant effect. As a result, Frs 5-7 and Frs 17-19 were shown to exhibit superior antioxidant activity according to the regression area of the ternary network, which was0.5. Furthermore, the active fractions were characterized by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry combined with nuclear magnetic resonance. This study provided an effective method for the comprehensive evaluation of the antioxidant effect of DHI fractions. Copyright © 2016 John WileySons, Ltd.

Buchang NaoXinTong (NXT), a Chinese medicine, has been widely used to treat patients with coronary heart disease in China. However, the underlying mechanisms need more elucidations. In this study, we investigated if NXT can inhibit the progression of the established lesions while stabilizing plaques. Apolipoprotein E deficient (apoE(-/-)) mice in 3 groups received following treatment: group 1 was fed a high-fat diet (HFD) for 18 weeks; group 2 was prefed HFD for 12 weeks followed by HFD containing NXT for additional 6 weeks; group 3 was prefed HFD for 8 weeks followed by HFD containing NXT for additional 10 weeks. After treatment, serum and aorta samples were collected and determined lipid profiles, lesions, collagen content, mineralization, and macrophage accumulation in aortic root, respectively. NXT had slight effect on serum lipid profiles but significantly reduced progression of the advanced lesions. In aortic wall, NXT increased smooth muscle cell/collagen content in lesion cap while reducing buried fibrous caps, mineralization, and macrophage accumulation within lesions, which suggests that NXT can stabilize plaques. In addition, NXT increased expression of smooth muscle 22α mRNA while inhibiting expression of matrix metalloproteinase-2 and tumor necrosis factor α mRNA in aortas. Our study demonstrates that NXT can reduce advanced atherosclerosis and enhance the plaque stability in apoE(-/-) mice.

Danhong Injection (DHI), a Chinese medicine for treatment of patients with coronary heart disease, inhibits primary abdominal aortic aneurysms in apoE deficient (apoE−/−) mice. Formation of microaneurysms plays an important role in the development of diabetic retinopathy and nephropathy. It remains unknown if DHI can reduce these diabetic complications. In this study, diabetic db/db mice in two groups were injected with saline and DHI, respectively, for 14 weeks. Blood and tissue samples were collected to determine serum glucose, lipids and tissue structure. DHI reduced diabetes-induced body weight gain, serum cholesterol and glucose levels. In retinas, DHI blocked the shrink of whole retina and retinal sub-layers by inhibiting expression of caspase 3, matrix metalloproteinase 2 (MMP-2) and MMP-9, accumulation of carbohydrate macromolecules and formation of acellular capillaries. DHI improved renal functions by inhibiting mesangial matrix expansion, expression of vascular endothelial growth factor A, fibronectin and advanced glycation end products in kidneys. Mechanistically, DHI induced expression of glucokinase, AMPKα/phosphorylated AMPKα, insulin receptor substrate 1, fibroblast growth factor 21 and peroxisome proliferator-activated γ. Expression of genes responsible for energy expenditure was also activated by DHI. Therefore, DHI inhibits diabetic retinopathy and nephropathy by ameliorating glucose metabolism and demonstrates a potential application in clinics.

Traditional Chinese medicine injections (TCMIs) have played an irreplaceable role for treating some clinical emergency, severe illness, and infectious diseases in China. In recent years, the incidence rates of adverse drug reactions (ADRs) of TCMIs have increased year by year. Danhong injection (DHI) is one representative TCMI comprised of Danshen and Honghua for treating cardiovascular and cerebrovascular diseases in clinic. In present study, the postmarketing safety surveillance and reevaluation of DHI were reported. Total 30888 patients in 37 hospitals from 6 provinces participated in the study. The results showed that the ADR incidence rate of DHI was 3.50‰. Seventeen kinds of new adverse reactions of DHI were found. The main type of ADRs of DHI was type A (including sweating, dizziness, headache, flushing, vasodilation, eye hemorrhage, faintness, chest pain, palpitations, breathlessness, anxious, nausea, flatulence, vomiting, hypotension, hypertension, local numbness, dyspnea, joint disease, and tinnitus) accounting for 57.75%. The severities of most ADRs of DHI were mild and moderate reactions accounting for 25.93% and 66.67%, respectively. The main disposition of ADRs of DHI was drug withdrawal and without any treatments. The results can provide basis for amendment and improvement of the instructions of DHI, as well as demonstration and reference for the postmarketing safety surveillance and reevaluation of other TCMIs. And the rationality, scientificity, and safety of clinical applications of TCMIs could be improved.

Danhong injection (DHI), a certificated Chinese medical product made from radix salviae miltiorrhizae and flos carthami, is prescribed to patients with coronary heart disease in China. To investigate if DHI can inhibit atherosclerosis, apolipoprotein E-deficient (Apoe⁻/⁻) or low-density lipoprotein receptor-deficient (Ldlr⁻/⁻) mice on high-fat diet were divided into 2 groups and received daily intraperitoneal injection of saline and DHI, respectively, for 16 or 20 weeks. After the treatment, mouse aortas were collected to determine lesions, expression of adenosine triphosphate-binding cassette transporter A1 and tumor necrosis factor-α (TNF-α), and macrophage accumulation. Additionally, serum lipid profiles and expression of hepatic HMG-CoA reductase messenger RNA and low-density lipoprotein receptor protein were determined. We observed that DHI inhibited lesions in both Apoe⁻/⁻ and Ldlr⁻/⁻ mice. Associated with the decreased lesions, the aortic adenosine triphosphate-binding cassette transporter A1 expression was increased, whereas the macrophage accumulation was decreased in male Apoe⁻/⁻ mice and both male and female Ldlr⁻/⁻ mice. Although DHI reduced HMG-CoA reductase messenger RNA expression in both female Apoe⁻/⁻ and Ldlr⁻/⁻ mice, it decreased low-density lipoprotein cholesterol levels only in female Apoe⁻/⁻ mice. In addition to attenuation of lipopolysaccharide-induced expression of TNF-α, IL-1β, IL-6 in macrophages, and human C-reactive protein in hepatocytes, respectively, at the transcriptional level in vitro, DHI also reduced TNF-α protein expression in aortic root of both Apoe⁻/⁻ and Ldlr⁻/⁻ mice, suggesting the importance of the anti-inflammatory properties of DHI in the inhibition of lesion development. Taken together, our study demonstrates that DHI inhibits atherosclerosis in both Apoe⁻/⁻ and Ldlr⁻/⁻ mice with various mechanisms, including anti-inflammation. The inhibition of atherosclerosis can be attributed to the cardioprotective properties of DHI.

Botanical primary metabolites extensively exist in herbal medicine injections (HMIs), but often were ignored to control. With the limitation of bias towards hydrophilic substances, the primary metabolites with strong polarity, such as saccharides, amino acids and organic acids, are usually difficult to detect by the routinely applied reversed-phase chromatographic fingerprint technology. In this study, a proton nuclear magnetic resonance (1H NMR) profiling method was developed for efficient identification and quantification of small polar molecules, mostly primary metabolites in HMIs. A commonly used medicine, Danhong injection (DHI), was employed as a model. With the developed method, 23 primary metabolites together with 7 polyphenolic acids were simultaneously identified, of which 13 metabolites with fully separated proton signals were quantified and employed for further multivariate quality control assay. The quantitative 1H NMR method was validated with good linearity, precision, repeatability, stability and accuracy. Based on independence principal component analysis (IPCA), the contents of 13 metabolites were characterized and dimensionally reduced into the first two independence principal components (IPCs). IPC1 and IPC2 were then used to calculate the upper control limits (with 99% confidence ellipsoids) of χ2 and Hotelling T2 control charts. Through the constructed upper control limits, the proposed method was successfully applied to 36 batches of DHI to examine the out-of control sample with the perturbed levels of succinate, malonate, glucose, fructose, salvianic acid and protocatechuic aldehyde. The integrated strategy has provided a reliable approach to identify and quantify multiple polar metabolites of DHI in one fingerprinting spectrum, and it has also assisted in the establishment of IPCA models for the multivariate statistical evaluation of HMIs.