Eugenol restrains abdominal aortic aneurysm progression with down-regulations on NF-κB and COX-2.

Abdominal aortic aneurysm (AAA) is a lethal disease without available medicine for treatment. This study aimed to evaluate the efficiency of eugenol (4-allyl-2-methoxyphenol) against AAA and the underlying mechanism. Eugenol is the major bioactive component of clove. A mouse AAA model was established through porcine pancreatic elastase (PPE) incubation peri-adventitially and 1% 3-aminopropanonitrile (BAPN) diet. Continuous AAA progression from day 0 to day 15 was observed after PPE plus BAPN treatment, according to the AAA diameter and histopathological evaluation. Accompanying with AAA progression, sustained increased expressions of CD68, COX-2 and NF-κB were observed through immunofluorescence assay. After elucidation the efficiency of eugenol against AAA progression by AAA diameter, hematoxylin-eosin staining and orcein staining, the down-regulations of eugenol on COX-2 and NF-κB were further detected by immunohistochemistry and western blot. Eugenol not only blocked AAA expansion and protected the integrity of aortic structure in a dose-dependent manner, but also held high oral bioavailability. Excellent efficiency, high oral bioavailability and down-regulation on COX-2/NF-κB endowed eugenol great potential for future AAA therapy.

Rosmarinic Acid Suppresses Abdominal Aortic Aneurysm Progression in Apolipoprotein E-deficient Mice.

An abdominal aortic aneurysm is a life-threatening cardiovascular disorder caused by dissection and rupture. No effective medicine is currently available for the > 90% of patients whose aneurysms are below the surgical threshold. The present study investigated the impact of rosmarinic acid, salvianolic acid C, or salvianolic acid B on experimental abdominal aortic aneurysms. Abdominal aortic aneurysms were induced in apolipoprotein E-deficient mice via infusion of angiotensin II for 4 wks. Rosmarinic acid, salvianolic acid C, salvianolic acid B, or doxycycline as a positive control was provided daily through intraperitoneal injection. Administration of rosmarinic acid was found to decrease the thickness of the aortic wall, as determined by histopathological assay. Rosmarinic acid also exhibited protection against elastin fragmentation in aortic media and down-regulated cell apoptosis and proliferation in the aortic adventitia. Infiltration of macrophages, T lymphocytes, and neutrophils in aortic aneurysms was found, especially at the aortic adventitia. Rosmarinic acid, salvianolic acid C, or salvianolic acid B inhibited the infiltration on macrophages specifically, but these compounds did not influence T lymphocytes and neutrophils. Expression of matrix metalloproteinase 9 and macrophage migration inhibitory factor significantly increased in aortic aneurysms. Rosmarinic acid and salvianolic acid C decreased the expression of matrix metalloproteinase-9 in media, and rosmarinic acid also tended to reduce migration inhibitory factor expression. Further then, partial least squares-discriminate analysis was used to classify metabolic changes among different treatments. Rosmarinic acid affected most of the metabolites in the biosynthesis of the citrate cycle, fatty acid pathway significantly. Our present study on mice demonstrated that rosmarinic acid inhibited multiple pathological processes, which were the key features important in abdominal aortic aneurysm formation. Further study on rosmarinic acid, the novel candidate for aneurysmal therapy, should be undertaken to determine its potential for clinical use.

Targeted depletion of monocyte/macrophage suppresses aortic dissection with the spatial regulation of MMP-9 in the aorta.

AIM: Aortic dissection (AD) is a devastating disease with rapid progression and high mortality, while the initiation mechanism of AD is far from clear. MAIN METHODS: AD was established by feeding mice with ß-aminopropionitrile in the diet and usage of angiotensin II (AngII) to trigger the rupture of aorta. LysMiDTR mice were constructed by crossing of LysM-Cre mice with ROSA26iDTR mice and characterized by diphtheria toxin receptor (DTR) expression in monocytes/macrophages specifically. Then, monocyte/macrophage depletion in LysMiDTR mice was conducted to evaluate the function of monocyte/macrophage in AD. Finally, the underlying mechanism was elucidated by proteomics, Western blot analysis, immunofluorescence staining and bioinformatics analysis. KEY FINDINGS: First, we detected T lymphocytes, macrophages and neutrophils infiltrated into the aorta simultaneously when AD occurred, and macrophages were the most abundant cell type. Then, targeted depletion of monocyte/macrophage in LysMiDTR mice considerably inhibited the occurrence of AD and infiltration of T lymphocytes and neutrophils. Furthermore, monocyte transfusion into LysMiDTR mice augmented the rupture of aorta, jointly supporting the key roles of monocytes/macrophages in AD development. Mechanistically, a total of 347 proteins exhibited significant differences in intensity after monocyte/macrophage depletion according to quantitative mass spectrometry. Specifically, increased matrix metalloprotein-9 (MMP-9) level in AD may be of concern due to its functions in vascular remodeling. The infiltration of macrophages considerably up-regulated MMP-9, and MMP-9 co-localized with macrophages at the tearing area of aorta. SIGNIFICANCE: Macrophages infiltrated into the tear sites of the aortic wall, regulated extracellular remodeling pathway, functioned as initiators to switch on the occurrence of AD.

Cardioprotection of salvianolic acid B and ginsenoside Rg1 combination on subacute myocardial infarction and the underlying mechanism.

BACKGROUND: Following myocardial infarction (MI), a series of structural and functional changes evolves in the myocardium, collectively defined as cardiac remodeling. PURPOSE: The aim of present study was to investigate the cardioprotection of salvianolicacid B (SalB) and ginsenoside Rg1 (Rg1) combination against cardiac remodeling in a rat model at the subacute phase of MI and further elucidate the underlying mechanism. METHODS: Rat heart was exposed via a left thoracotomy at the fourth intercostal space and MI was induced by a ligature below the left descending coronary artery. Hemodynamic assay was conducted using a Mikro-tipped SPR-320 catheter which was inserted through the right carotid artery into left ventricle.Myocardial infarct size was detected using 3,5-triphenyltetrazolium chloride (TTC) staining. Haematoxylin and eosin (HE) stain and picric sirius red stain were conducted for histopathological detection. Immunohistochemistry was used to detect the expression of α-smooth muscle actin (α-SMA) and gelatin zymography was used to evaluate the activities of matrix metalloproteinase-9 (MMP-9). RESULTS: Comparing with MI rats, 30 mg/kg SalB-Rg1 improved cardiac function verified by maximum rate of pressure development for contraction (+dp/dtmax, p < 0.01) and maximum rate of pressure development for relaxation (-dp/dtmax, p < 0.05); reduced myocardial infarct size (p < 0.05) verified by TTC staining, improved cardiac structure based on HE stain; decreased collagen volume fraction (p < 0.05) and collagen I/III ratio (p < 0.05) according picrosirius red staining. The underlying mechanism of SalB-Rg1 against cardiac remodeling was associated with its down-regulation on α-SMA expression according immunohistochemistry (p < 0.01) and inhibition on MMP-9 activity based on in-gel zymography (p < 0.05). CONCLUSION: All above study indicated the potential therapeutic effects of SalB-Rg1 on heart.

Cardio-protection of ultrafine granular powder for Salvia miltiorrhiza Bunge against myocardial infarction.

ETHNOPHARMACOLOGICAL RELEVANCE: Myocardial infarction (MI) is considered as the major inducer to the morbidity and mortality related to coronary occlusion. Salvia miltiorrhiza Bunge is widely applied in the clinic for the prevention and treatment of heart diseases. The preparation of traditional herb decoction (THD) is not only time consuming but also difficult to keep uniform for every time. New usage form of Salvia miltiorrhiza Bunge with characteristics of convenience, uniform and efficiency is needed. AIM OF THE STUDY: The aims of present study were to investigate the cardio-protection of ultrafine granular powder (UGP) of Salvia miltiorrhiza Bunge; and further compare the characteristics of UGP with THD. MATERIALS AND METHODS: MI was induced by ligation of the left anterior descending coronary artery near the main pulmonary artery. Cardio-protection of UGP or THD was evaluated based on two sets of experiments, one was acute myocardial infarction (AMI) through 7 days preventive administration, and the other one was chronic cardiac remodeling through 28 days therapeutic administration. Hemodynamic measurement was conducted to evaluate heart function and histopathological detection was used to evaluate heart structure. RESULTS: No significant improvement of heart structure and function was detected for preventive administration of UGP or THD on AMI rats. While, more significant improvements on left ventricular systolic and diastolic function were detected with therapeutic treatment with 0.81 g/kg UGP than same dose of THD on rats against chronic cardiac remodeling. Both UGP and THD showed the protective effects on heart structure, especially against fibrosis with long-term therapeutic treatment. CONCLUSIONS: As a new usage form of Salvia miltiorrhiza Bunge, UGP showed significant cardio-protection against myocardial remodeling with therapeutic treatment. Comparing with THD, UGP also holds the advantages of uniform, convenience and efficiency.

Plasma Amino Acid Profile in Patients with Aortic Dissection.

Aortic dissection (AD), a severe cardiovascular disease with the characteristics of high mortality, is lack of specific clinical biomarkers. In order to facilitate the diagnosis of AD, we investigated plasma amino acid profile through metabolomics approach. Total 33 human subjects were enrolled in the study: 11 coronary heart disease (CHD) patients without aortic lesion and 11 acute AD and 11 chronic AD. Amino acids were identified in plasma using liquid chromatography and mass spectrometry (LC-MS/MS), and were further subjected to multiple logistic regression analysis. The score plots of principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) showed clear discrimination of CHD patients with AD, acute AD or chronic AD patients, respectively. The contents of histidine, glycine, serine, citrate, ornithine, hydroxyproline, proline and sarcosine were significant different in acute AD patients comparing with CHD patients. The levels of citrate, GABA, glutamate and cysteine were significant different in chronic AD patients comparing with CHD patients. The contents of glutamate and phenylalanine were significant changed in acute AD patients comparing with chronic AD patients. Plasma aminograms were significantly altered in patients with AD comparing with CHD, especially in acute AD, suggesting amino acid profile is expected to exploit a novel, non-invasive, objective diagnosis for AD.

Amelioration of salvianolic acid C on aortic structure in apolipoprotein E-deficient mice treated with angiotension II.

AIMS: Aortic aneurysm is a disastrous vascular disease with high morbidity and mortality. Matrix metalloproteinases (MMPs), especially MMP-9, is implicated in the development of aortic aneurysm, but the effective MMP inhibitors are far from development. To develop new candidate compound for aortic aneurysm therapy, we evaluated the effects of salvianolic acid C (SalC) against the formation of aortic aneurysm. MATERIALS AND METHODS: Aortic aneurysm was induced by implantation of angiotension II (AngII) minipump in apolipoprotein E-deficient (ApoE-/-) mice. MMPs activity was evaluated by enzyme kinetic analysis in vitro and in-gel gelatin zymography in vivo. The formation of aortic aneurysm was confirmed based on aortic maximum diameter. Hematoxylin and eosin stain was used to evaluate aortic structure, picrosirius red stain was for collagen deposition, and orcein stain was for elastin fragmentation. Macrophage infiltration was detected by CD68 immunohistochemistry. KEY FINDINGS: Firstly, SalC showed significant inhibition on the activity of MMP-2 and MMP-9. Aortic aneurysm was defined as >50% increase in maximum diameter of aorta, and the down-regulated tendency of 20mg/kg SalC against formation of aortic aneurysm was detected. Also, 22.2% rupture was detected in ApoE-/- mice, while no rupture of aortic aneurysm was found with 20mg/kg SalC treatment. Then, SalC was detected to maintain the integrity of aortic structure and protect elastin against fragmentation. Finally, SalC considerably inhibited infiltration of macrophage in the injury site of aorta. SIGNIFICANCE: SalC significantly ameliorated the progression of aortic aneurysm in ApoE-/- mice, and held great potential for aortic aneurysm therapy.

Downregulation of TRAP1 sensitizes glioblastoma cells to temozolomide chemotherapy through regulating metabolic reprogramming.

Cancer cells preferentially use aerobic glycolysis to support growth, a metabolic alteration commonly referred to as the 'Warburg effect.' Here, we show that the tumor necrosis factor receptor-associated protein 1 (TRAP1) is crucial for the Warburg effect in human glioblastoma multiforme (GBM). In contrast to normal brain, GBMs show increased TRAP1 expression. We used both GBM cell lines and neurospheres derived from human GBM specimens to examine the effects of Knockdown of TRAP1 on GBM cell lines and glioma stem cells. We also used a neurosphere recovery assay that measured neurosphere formation at three time points to assess the capacity of the culture to repopulate after knockdown of TRAP1. Our results showed that knockdown of TRAP1 strongly decreased GBM cell proliferation and migration, inhibited neurosphere recovery, secondary neurosphere formation, and enhanced the therapeutic effect of temozolomide in neurosphere cultures. In GBM, knockdown of TRAP1 appeared to inhibit tumor growth and migration through its regulatory effects on metabolic reprogramming.

Salvianolic acid A inhibits endothelial dysfunction and vascular remodeling in spontaneously hypertensive rats.

AIMS: Despite the numerous pharmacological agents available for hypertension therapy, hypertension-related microvascular remodeling is not resolved, eventually leading to end-organ damage. The aim of the present study was to investigate the protection of salvianolic acid A (SalA) against microvascular remodeling in vitro and in vivo. MAIN METHODS: Spontaneously hypertensive rats (SHRs) were administered 2.5, 5 or 10 mg/kg SalA via intraperitoneal injection once a day for 4 weeks. The tail-cuff method was applied to monitor blood pressure; the microvascular structure of the retina was detected by hematoxylin-eosin and immunohistochemical staining; the function of mesenteric arteries was measured by DMT wire myography; endothelial cell proliferation was estimated using the Cell Counting Kit-8; endothelial cell migration was evaluated by wound healing and transwell assay; and endothelial cell integrity was detected by transendothelial electrical resistance and permeability assays. KEY FINDINGS: Although no antihypertensive effects of SalA were observed, SalA attenuated the microvascular inward remodeling of the retina and improved microvascular function in the mesenteries in vivo. Further cell experiments confirmed the beneficial effects of SalA on the integrity of the endothelial monolayer in vitro. SIGNIFICANCE: Salvianolic acid A inhibited endothelial dysfunction and vascular remodeling in spontaneously hypertensive rats. Therefore, salvianolic acid A could be a potential drug therapy to prevent further targeted organ damage induced by vascular remodeling.

The Safety Evaluation of Salvianolic Acid B and Ginsenoside Rg1 Combination on Mice.

Our previous study indicated that the combination of salvianolic acid B (SalB) and ginsenoside Rg1 (Rg1), the main components of Salvia miltiorrhizae and Panax notoginseng, improves myocardium structure and ventricular function in rats with ischemia/reperfusion injury. The present study aimed to determine the safety of the combined SalB and Rg1 (SalB-Rg1) in mice. The safety of SalB-Rg1 was evaluated through acute toxicity and repeated-dose toxicity. In the acute toxicity study, the up and down procedure was carried out firstly, and then, the Bliss method was applied. In the toxicity study for seven-day repeated treatment of SalB-Rg1, forty Kunming mice were randomly divided into four groups. The intravenous median lethal dose (LD50) of the SalB-Rg1 combination was 1747 mg/kg using the Bliss method. For both the acute toxicity study and the seven-day repeated toxicity study, SalB-Rg1 did not induce significant abnormality on brain, heart, kidney, liver and lung structure at any dose based on H&E stain. There were no significant changes related to the SalB-Rg1 toxicity detected on biochemical parameters for two kinds of toxicity studies. The LD50 in mice was 1747 mg/kg, which was more than one hundred times higher than the effective dose. Both studies of acute toxicity and seven-day repeated dose toxicity indicated the safety of the SalB-Rg1 combination.

Combined Salvianolic Acid B and Ginsenoside Rg1 Exerts Cardioprotection against Ischemia/Reperfusion Injury in Rats.

Lack of pharmacological strategies in clinics restricts the patient prognosis with myocardial ischemia/reperfusion (I/R) injury. The aim of this study was to evaluate the cardioprotection of combined salvianolic acid B (SalB) and ginsenoside Rg1 (Rg1) against myocardial I/R injury and further investigate the underlying mechanism. I/R injury was induced by coronary artery ligation for Wistar male rats and hypoxia/reoxygenation injury was induced on H9c2 cells. Firstly, the best ratio between SalB and Rg1was set as 2:5 based on their effects on heart function detected by hemodynamic measurement. Then SalB-Rg1 (2:5) was found to maintain mitochondrial membrane potential and resist apoptosis and necrosis in H9c2 cell with hypoxia/reoxygenation injury. Companying with same dose of SalB or Rg1 only, SalB-Rg1 showed more significant effects on down-regulation of myocardial infarct size, maintenance of myocardium structure, improvement on cardiac function, decrease of cytokine secretion including TNF-α, IL-1ß, RANTES and sVCAM-1. Finally, the SalB-Rg1 improved the viability of cardiac myocytes other than cardiac fibroblasts in rats with I/R injury using flow cytometry. Our results revealed that SalB-Rg1 was a promising strategy to prevent myocardial I/R injury.

Impact of autophagy inhibition at different stages on cytotoxic effect of autophagy inducer in glioblastoma cells.

BACKGROUND/AIMS: Glioblastoma multiforme (GBM) is the most malignant primary brain tumor with a poor prognosis. Combination treatment of autophagy inducer and autophagy inhibitor may be a feasible solution to improve the therapeutic effects. However, the correlation between them is unclear. The purpose of this study was to investigate the effect of autophagy inhibition at different stages on cytotoxicity of autophagy inducers in glioblastoma cells. METHODS: Autophagy inhibition at early stage was achieved by 3-methyladenine (3-MA) or Beclin 1 shRNA. Autophagy inhibition at late stage was achieved by chloroquine (CQ) or Rab7 shRNA. Cell viability was assessed by MTT assay. Autophagy was measured using transmission electron microscopy and western blot. Apoptosis was measured using western blot and flow-cytometry. RESULTS: Inhibition of early steps of autophagy by 3-MA or Beclin 1 knockdown decreased the toxic effect of arsenic trioxide (ATO) in GBM cell lines. In contrast, blockade of autophagy flux at late stage by CQ or Rab7 knockdown enhanced the cytotoxicity of ATO, and caused accumulation of degradative autophagic vacuoles and robust apoptosis. Moreover, depletion of Beclin 1 abolished the synergistic effect of ATO and CQ by reducing autophagy and apoptosis. Combination of CQ with other autophagy inducers also induced synergistic apoptotic cell death. CONCLUSION: These results suggest that inhibition of late process of autophagy, not initial step, increases the cytotoxic effect of autophagy inducers via autophagy and apoptosis, which may contribute to GBM chemotherapy.

Ginsenoside Rg1 and Rb1, in combination with salvianolic acid B, play different roles in myocardial infarction in rats.

BACKGROUND: The herb pair of Salvia miltiorrhiza and Panax notoginseng has widely been used for improving coronary and cerebral circulation in China. However, the exact contribution of the major active components of S. miltiorrhiza and P. notoginseng to cardioprotection is far from clear. In the present study, three representative ingredients, salvianolic acid B (SalB) from S. miltiorrhiza and ginsenoside Rg1 (Rg1) and ginsenoside Rb1 (Rb1) from P. notoginseng, were selected to elucidate the mechanism of the herb pair at the ingredient level. METHODS: The purity of SalB, Rg1, and Rb1 was >99%, as detected by high-performance liquid chromatography. Acute myocardial infarction was introduced by ligation of the left anterior descending coronary artery near the main pulmonary artery. Cardiac contractility was detected through a Mikro-tipped catheter, and cardiac infarct size was determined using triphenyltetrazolium chloride stain. RESULTS: The combination of SalB and Rg1, and not the combination of SalB and Rb1, improved heart contractility in rats with myocardial infarction. The different contributions of Rg1 and Rb1, in combination with SalB, to cardioprotection provides further direction to optimize and modernize the herbal medicines containing S. miltiorrhiza and P. notoginseng. CONCLUSION: The combination of SalB and Rg1 may provide potential protection against myocardial infarction.