Protective Effects of Alternanthera sessilis Ethanolic Extract against TNF-α or H2O2-Induced Endothelial Activation in Human Aortic Endothelial Cells.

Activation of the endothelium has been shown to contribute to the early stage of vascular diseases such as atherosclerosis and hypertension. In endothelial activation, excess reactive oxygen species (ROS) production and increased expression of cell adhesion molecules cause an increase in vascular permeability. Alternanthera sessilis (L.) R. Br. is an edible traditional herbal plant, which has previously been shown to possess antioxidant and anti-inflammatory effects. However, the effect of A. sessilis on the activation of human aortic endothelial cells (HAECs) remains unknown. This study aimed to investigate the effects of A. sessilis on endothelial permeability, vascular cell adhesion-1 (VCAM-1) expression, production of ROS and hydrogen peroxide (H2O2), and superoxide dismutase (SOD) and catalase (CAT) activities. The viability of HAECs was first determined using the MTT viability assay. The effect of A. sessilis on endothelial permeability was examined using the FITC-dextran permeability assay. Besides, enzyme-linked immunosorbent assay (ELISA) was done to assess soluble VCAM-1 (sVCAM-1) expression. The production of ROS and H2O2 was studied using 2',7'-dichlorodihydrofluorescein diacetate (H2-DCFDA) and Amplex Red fluorescent dyes, respectively. SOD and CAT activities were also measured using commercial kits. Our results showed that 25-200 µg/mL of A. sessilis ethanolic extract did not cause significant death in HAECs. A. sessilis at 200 µg/mL significantly inhibited TNF-α-induced hyperpermeability of HAECs. However, A. sessilis did not reduce increased VCAM-1 expression induced by TNF-α. A. sessilis also significantly reduced TNF-α-induced increased ROS production, but not H2O2 production. Furthermore, 100 µM of H2O2 decreased both SOD and CAT activities in HAECs at 2 h. A. sessilis ethanolic extract dramatically increased both reduced SOD and CAT activities caused by H2O2. The liquid chromatography-mass spectrometry (LC-MS) analysis of A. sessilis ethanolic extract demonstrated the presence of arachidonic acid, azadirachtin, astaxanthin, flavanole base + 3O, 2Prenyl, and vicenin 2, while the gas chromatography-mass spectrometry (GC-MS) analysis showed that the extract contains 1,3,5-dihydroxy-6-methyl-2,3-dihydro-4H-pyran-4-one, 3-deoxy-d-mannoic lactone, 4-pyrrolidinobenzaldehyde, and n-hexadecanoic acid. In conclusion, our findings suggest that A. sessilis ethanolic extract protects against endothelial hyperpermeability and oxidative stress elicited by pro-inflammatory or prooxidant stimulus. This study reveals a therapeutic potential of A. sessilis in preventing endothelial activation, which is a key event in early atherosclerosis.

Cardiovascular Protective Effects of Centella asiatica and Its Triterpenes: A Review.

Centella asiatica, a triterpene-rich medicinal herb, is traditionally used to treat various types of diseases including neurological, dermatological, and metabolic diseases. A few articles have previously reviewed a broad range of pharmacological activities of C. asiatica, but none of these reviews focuses on the use of C. asiatica in cardiovascular diseases. This review aims to summarize recent findings on protective effects of C. asiatica and its active constituents (asiatic acid, asiaticoside, madecassic acid, and madecassoside) in cardiovascular diseases. In addition, their beneficial effects on conditions associated with cardiovascular diseases were also reviewed. Articles were retrieved from electronic databases such as PubMed and Google Scholar using keywords "Centella asiatica," "asiatic acid," "asiaticoside," "madecassic acid," and "madecassoside." The articles published between 2004 and 2018 that are related to the aforementioned topics were selected. A few clinical studies published beyond this period were also included. The results showed that C. asiatica and its active compounds possess potential therapeutic effects in cardiovascular diseases and cardiovascular disease-related conditions, as evidenced by numerous in silico, in vitro, in vivo, and clinical studies. C. asiatica and its triterpenes have been reported to exhibit cardioprotective, anti-atherosclerotic, antihypertensive, antihyperlipidemic, antidiabetic, antioxidant, and anti-inflammatory activities. In conclusion, more clinical and pharmacokinetic studies are needed to support the use of C. asiatica and its triterpenes as therapeutic agents for cardiovascular diseases. Besides, elucidation of the molecular pathways modulated by C. asiatica and its active constituents will help to understand the mechanisms underlying the cardioprotective action of C. asiatica.

Water extract of Clinacanthus nutans leaves exhibits in vitro, ex vivo and in vivo anti-angiogenic activities in endothelial cell via suppression of cell proliferation.

BACKGROUND: Clinacanthus nutans (Burm. f.) Lindau. has traditionally been using in South East Asia countries to manage cancer. However, scientific evidence is generally lacking to support this traditional claim. This study aims to investigate the in vitro, ex-vivo and in vivo effects of C. nutans extracts on angiogenesis. METHODS: C. nutans leaves was extracted with 50-100% ethanol or deionised water at 1% (w/v). Human umbilical veins endothelial cell (HUVEC) proliferation was examined using MTT assay. The in vitro anti-angiogenic effects of C. nutans were assessed using wound scratch, tube formation and transwell migration assays. The VEGF levels secreted by human oral squamous cell carcinoma (HSC-4) cell and HUVEC permeability were also measured. Besides, the rat aortic ring and chick embryo chorioallantoic membrane (CAM) assays, representing ex vivo and in vivo models, respectively, were performed. RESULTS: The MTT assay revealed that water extract of C. nutans leaves exhibited the highest activity, compared to the ethanol extracts. Therefore, the water extract was chosen for subsequent experiments. C. nutans leaf extract significantly suppressed endothelial cell proliferation and migration in both absence and presence of VEGF. However, the water extract failed to suppress HUVEC transmigration, differentiation and permeability. C. nutans water extract also did not suppress HSC-4 cell-induced VEGF production. Importantly, C. nutans water extract significantly abolished the sprouting of vessels in aortic rings as well as in chick embryo CAM. CONCLUSION: In conclusion, these findings reveal potential anti-angiogenic effects of C. nutans, providing new evidence for its potential application as an anti-angiogenic agent.

Barrier protective effect of asiatic acid in TNF-α-induced activation of human aortic endothelial cells.

BACKGROUND: Endothelial cell activation is characterized by increased endothelial permeability and increased expression of cell adhesion molecules (CAMs). This allows monocyte adherence and migration across the endothelium to occur and thereby initiates atherogenesis process. Asiatic acid is a major triterpene isolated from Centella asiatica (L.) Urban and has been shown to possess anti-oxidant, anti-hyperlipidemia and anti-inflammatory activities. PURPOSE: We aimed to investigate protective effects of asiatic acid on tumor necrosis factor-α (TNF-α)-induced endothelial cell activation using human aortic endothelial cells (HAECs). STUDY DESIGN: For cell viability assays, HAECs were treated with asiatic acid for 24 h. For other assays, HAECs were pretreated with various doses of asiatic acid (10-40 µM) for 6 h followed by stimulation with TNF-α (10 ng/ml) for 6 h. METHODS: Fluorescein isothiocyanate (FITC)-dextran permeability assay was performed using commercial kits. Total protein expression of CAMs such as E-selectin, ICAM-1, VCAM-1 and PECAM-1 as well as phosphorylation of IκB-α were determined using western blot. The levels of soluble form of CAMs were measured using flow cytometry. Besides, we also examined the effects of asiatic acid on U937 monocyte adhesion and monocyte migration in HAECs using fluorescent-based assays. RESULTS: Asiatic acid significantly suppressed endothelial hyperpermeability, increased VCAM-1 expression and increased levels of soluble CAMs (sE-selectin, sICAM-1, sVCAM-1 and sPECAM-1) triggered by TNF-α. Neither TNF-α nor asiatic acid affects PECAM-1 expression. However, asiatic acid did not inhibit TNF-α-induced increased monocyte adhesion and migration. Interestingly, asiatic acid suppressed increased phosphorylation of IκB-α stimulated by TNF-α. CONCLUSION: These results suggest that asiatic acid protects against endothelial barrier disruption and this might be associated with the inhibition of NF-κB activation. We have demonstrated a novel protective role of asiatic acid on endothelial function. This reveals the possibility to further explore beneficial effects of asiatic acid on chronic inflammatory diseases that are initiated by endothelial cell activation.

Cryptotanshinone inhibits TNF-α-induced early atherogenic events in vitro.

Endothelial dysfunction has been implicated in the pathogenesis of atherosclerosis. Salvia miltiorrhiza (danshen) is a traditional Chinese medicine that has been effectively used to treat cardiovascular disease. Cryptotanshinone (CTS), a major lipophilic compound isolated from S. miltiorrhiza, has been reported to possess cardioprotective effects. However, the anti-atherogenic effects of CTS, particularly on tumor necrosis factor-α (TNF-α)-induced endothelial cell activation, are still unclear. This study aimed to determine the effect of CTS on TNF-α-induced increased endothelial permeability, monocyte adhesion, soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell adhesion molecule 1 (sVCAM-1), monocyte chemoattractant protein 1 (MCP-1) and impaired nitric oxide production in human umbilical vein endothelial cells (HUVECs), all of which are early events occurring in atherogenesis. We showed that CTS significantly suppressed TNF-α-induced increased endothelial permeability, monocyte adhesion, sICAM-1, sVCAM-1 and MCP-1, and restored nitric oxide production. These observations suggest that CTS possesses anti-inflammatory properties and could be a promising treatment for the prevention of cytokine-induced early atherogenesis.

Asiaticoside Inhibits TNF-α-Induced Endothelial Hyperpermeability of Human Aortic Endothelial Cells.

The increase in endothelial permeability often promotes edema formation in various pathological conditions. Tumor necrosis factor-alpha (TNF-α), a pro-atherogenic cytokine, impairs endothelial barrier function and causes endothelial dysfunction in early stage of atherosclerosis. Asiaticoside, one of the triterpenoids derived from Centella asiatica, is known to possess antiinflammatory activity. In order to examine the role of asiaticoside in preserving the endothelial barrier, we assessed its effects on endothelial hyperpermeability and disruption of actin filaments evoked by TNF-α in human aortic endothelial cells (HAEC). TNF-α caused an increase in endothelial permeability to fluorescein isothiocyanate (FITC)-dextran. Asiaticoside pretreatment significantly suppressed TNF-α-induced increased permeability. Asiaticoside also prevented TNF-α-induced actin redistribution by suppressing stress fiber formation. However, the increased F to G actin ratio stimulated by TNF-α was not changed by asiaticoside. Cytochalasin D, an actin depolymerizing agent, was used to correlate the anti-hyperpermeability effect of asiaticoside with actin cytoskeleton. Surprisingly, asiaticoside failed to prevent cytochalasin D-induced increased permeability. These results suggest that asiaticoside protects against the disruption of endothelial barrier and actin rearrangement triggered by TNF-α without a significant change in total actin pool. However, asiaticoside seems to work by other mechanisms to maintain the integrity of endothelial barrier rather than stabilizing the F-actin organization.