Oleanane- and Ursane-Type Triterpene Saponins from Centella asiatica Exhibit Neuroprotective Effects.

Six new pentacyclic triterpenoid saponins, centelloside F (1), centelloside G (2), 11-oxo-asiaticoside B (3), 11-oxo-madecassoside (4), 11(ß)-methoxy asiaticoside B (5), and 11(ß)-methoxy madecassoside (6), along with seven known ones, asiaticoside (7), asiaticoside B (8), madecassoside (9), centellasaponin A (10), isoasiaticoside (11), scheffoleoside A (12), and centelloside E (13), were separated from the 80% MeOH extract of the whole plant of Centella asiatica, which has been used as a medicinal plant and is now commercially available as a diatery supplement in many countries. Compounds 1 and 2, 3 and 4, and 5 and 6 are three pairs of isomers with oleanane- or ursane-type triterpenes as aglycones. The chemical structures of the new triterpene saponins were fully characterized by extensive analysis of their nuclear magnetic resonance and high-resolution electrospray ionization mass spectrometry data. The protective effects of compounds 1-13 on PC12 cells induced by 6-OHDA were screened, and compound 3 displayed the best neuroprotective effect, with 91.75% cell viability at the concentration of 100 µM. Moreover, compound 3 also attenuated cell apoptosis and increased the mRNA expression of antioxidant enzymes, including superoxide dismutase and catalase. Additionally, compound 3 activated the phosphatidylinositol 3-kinase/Akt pathway, including PDK1, Akt, and GSK-3ß. These findings suggested that triterpene saponins from C. asiatica were worthy of further biological research to develop new neuroprotective agents.

A new steroidal saponin from Polygonatum sibiricum.

A new furostan type steroidal saponin, kingianoside Z (1), along with four known compounds (2-5), was isolated from the ethanolic extract of Polygonatum sibiricum Delar. ex Redoute. Their structures were determined by spectroscopical method and by comparison with previously reported spectroscopic data. Compounds 3-5 showed significant cytotoxicity against HepG2 cell lines with IC50 values of 14.2, 12.1 and 8.5 µM, respectively.

A new cycloartane triterpene glycoside from Souliea vaginata.

A new cycloartane-type triterpene glycoside, namely soulieoside M (1), and one known compound, beesioside I (2), were isolated from the ethanolic extract of the rhizomes of Souliea vaginata. Their structures were determined spectroscopically and compared with previously reported spectral data. Compounds 1 and 2 were evaluated for their cytotoxic activities against three human cancer cell lines.

A new phenylethanoid glycoside from Incarvillea compacta.

A new phenylethanoid glycoside, 3'''-O-methylcampneoside I (1), was isolated from the 90% ethanolic extract of the roots of Incarvillea compacta, together with three known compounds, campneoside I (2), ilicifolioside A (3), and campneoside II (4). Their structures were determined spectroscopically and compared with previously reported spectral data. Compound 1 existed as epimers and displayed better 1,1-diphenyl-2-picrylhydrazyl (DPPH)-free radical scavenging activity using di-tert-butyl-4-methylphenol (BHT) as the positive control. In addition, pretreatment of human HepG2 cells with compound 1 significantly increased the viability on CCl4-induced cell death.

Antioxidant properties and neuroprotective effects of isocampneoside II on hydrogen peroxide-induced oxidative injury in PC12 cells.

Oxidative stress has been considered as a major cause of cell damage in various neurodegenerative disorders. One of the reasonable strategies for delaying the disease's progression is to prevent reactive oxygen species (ROS) mediated cellular injury by dietary or pharmaceutical augmentation of free radical scavengers. Isocampneoside II (ICD) is an active phenylethanoid glycoside isolated from the medicinal hardwood genus Paulownia. This study was designed to explore free radical scavenging potential of ICD in different in vitro systems and its protective role in hydrogen peroxide (H2O2)-induced oxidative stress and apoptotic death in cultured rat pheochromocytoma (PC12) cells. The results showed ICD eliminated approximately 80.75% superoxide radical at the concentration of 0.1mg/ml and inhibited metal chelating by 22.07% at 8 mg/ml. Additionally, ICD showed a strong ability on reducing power and provided protection against oxidative protein damage induced by hydroxyl radicals. Pretreatment of PC12 cells with ICD prior to H2O2 exposure elevated cell viability, enhanced activity of superoxide dismutase and catalase, and decreased levels of malondialdehyde and intracellular ROS. Furthermore, ICD inhibited cell apoptosis and Bax/Bcl-2 ratio induced by H2O2. These findings suggested ICD may be considered as a potential antioxidant agent and should encourage for further research in neurodegenerative diseases.

Correlation of inflammatory cells in adventitia and formation and extending of atherosclerotic lesions in coronary artery of apolipoprotein E gene knockout mice.

Accumulating evidence shows that adventitial inflammation contributes to the development of atherosclerotic lesions. The aim of this study was to investigate the relationship between atherosclerotic lesions in coronary artery (CA) and accumulation of inflammatory cells at local adventitia in apolipoprotein E gene knockout (apoE-/-) mice. Modified Movat's pentachrome staining, HE staining, immunohistochemistry and transmission electron microscopy were used to observe and to identify serial paraffin sections of aortic foot and inflammatory cells in CA adventitia of apoE-/- mice of 60 weeks old. There was always accumulation of inflammatory cells in the adventitia of CA with extending lesions from aortic orifice to CA trunks. The CA samples were divided into type I: infiltration of inflammatory cells in CA adventitia without lesions extending in the intima, type II: infiltration of inflammatory cells in CA adventitia with the top of extending lesions in the intima and type III: infiltration of inflammatory cells at CA adventitia with lesions covering all the face of intima. The three types of CA sample represent the different developmental processes of atherosclerotic lesions, respectively. No extending lesions were found in the CA trunks without inflammatory cells in adventitia. In type I samples, 60% of infiltrated inflammatory cells were macrophages 57% of infiltrated cells were neutrophils in type II samples; 67% of infiltrated cells were lymphocytes in type III samples. Our studies revealed that adventitial inflammation may be an early event in the development of atherosclerotic lesions. Different cell types predominate in different stages of CA adventitia. The neutrophils are closely related to the extending of atherosclerotic lesions.

Calcineurin mediates the protective effect of postconditioning on skeletal muscle.

We aimed to investigate whether ischemic postconditioning (I-postC) protects skeletal muscle against ischemia-reperfusion (I/R) injury through the calcineurin (CaN) pathway. Male Wistar rats underwent 4 h of right-hind-limb ischemia induced by clamping the femoral artery, then reperfusion for 2 h (I/R-2 h), 12 h (I/R-12 h), or 24 h (I/R-24 h) with or without I-postC. Ischemic postconditioning was induced by three cycles of 1-min reperfusion and 1-min ischemia at the onset of reperfusion after prolonged ischemia. The I-postC-24 h group was treated with or without cyclosporine A (a CaN inhibitor) 10 mg/kg per day for 3 days before artery occlusion. Cultured skeletal muscle cells (SMCs) from neonatal rats were exposed to 2-h hypoxia then 24-h reoxygenation (H/R), then postconditioned with two cycles of 10-min reoxygenation and 10-min hypoxia after prolonged hypoxia (hypoxia postconditioning [H-postC]) in the presence or absence of cyclosporine A. We observed the effects of activated CaN overexpression on apoptosis and viability of SMCs under H-postC. Ischemic postconditioning attenuated the increase in the level of malondialdehyde in skeletal muscle induced by I/R-2 h and I/R-24 h (P < 0.05) and lactate dehydrogenase in plasma induced by I/R-12 h and I/R-24 h (P < 0.05). Cyclosporine A abolished the protective role of I-postC in malondialdehyde level and lactate dehydrogenase leakage (P < 0.05, vs. I-postC group). Hypoxia postconditioning suppressed SMC apoptosis induced by H/R (P < 0.05, vs. H/R), which was accompanied by increased CaN expression. Cyclosporine A abolished the antiapoptotic effect of H-postC on SMCs (P < 0.05, vs. H-postC group). Overexpression of activated CaN strengthened the cytoprotection of H-postC (P < 0.05, vs. H-postC group). Ischemic postconditioning may protect skeletal muscle against I/R injury through the CaN pathway.

Gusanlungionosides A-D, potential tyrosinase inhibitors from Arcangelisia gusanlung.

Four new megastigmane glycosides, named gusanlungionosides A-D (1-4), together with 10 known compounds (5-14), were isolated from the stems of Arcangelisia gusanlung. The structures and absolute configurations of 1-4 were elucidated by comprehensive analysis of their NMR and CD data. Compounds 1-4 exhibited strong inhibitory effects not only on the mushroom tyrosinase activity in vitro but also on melanogenesis in cells.

The calcineurin-myocyte enhancer factor 2c pathway mediates cardiac hypertrophy induced by endoplasmic reticulum stress in neonatal rat cardiomyocytes.

Endoplasmic reticulum (ER) stress (ERS) is involved in various cardiovascular diseases. Our previous study verified that ERS took part in the development of cardiac hypertrophy; however, its mechanism is still unclear. This study aimed to investigate the roles of the calcineurin (CaN) signal pathway in hypertrophy induced by the ERS inductor thapsigargin (TG) in neonatal cardiomyocytes from Sprague-Dawley rats. Investigation of ER chaperone expression, ER staining, and calreticulin immunofluorescence were used to detect the ERS response. mRNA expression of atrial natriuretic peptide and brain natriuretic peptide, total protein synthesis rate, and cell surface area were used to evaluate cardiac hypertrophy induced by TG. TG induced a significant ERS response along with hypertrophy in a dose- and time-dependent manner in cardiomyocytes, which was verified by treatment with tunicamycin, another ERS inducer. Furthermore, TG induced a significant elevation of the intracellular Ca(2+) level, CaN activation, and myocyte enhancer factor 2c (MEF2c) expression in a dose- and time-dependent manner in cardiomyocytes. Cyclosporine A, a CaN inhibitor, markedly suppressed MEF2c nuclear translocation and inhibited TG-induced hypertrophy. These results demonstrate that ERS induces cardiac hypertrophy and that the CaN-MEF2c pathway is involved in ERS-induced hypertrophy in cardiomyocytes.

Activation of adventitial fibroblasts in the early stage of the aortic transplant vasculopathy in rat.

BACKGROUND: Transplant vasculopathy (TV) is the most significant obstacle to long-term success of organ transplantation. Increasing attention has been paid to the role of adventitia in vascular diseases. We evaluated the role of adventitial fibroblasts in the development of TV. METHODS: Thoracic aortas from Sprague-Dawley (SD) rats transplanted into the abdominal aortas of Wistar rats worked as allografts, and isografts (SD to SD) were control. Grafts were removed on days 3, 7, and 14 for histologic, morphometric, and immunohistochemical detection of vimentin, alpha-smooth muscle actin, Ki-67, CD3, transforming growth factor-beta1 (TGF-beta1), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase-7 (MMP-7), and quantitative real-time reverse transcriptase polymerase chain reaction for TGF-beta1, MCP-1, MMP-7, tumor necrosis factor-alpha, and interleukin-1beta. RESULTS: In the allografts, neointima thickness and neointima/media thickness ratios were slightly increased at 7 days and significantly increased at 14 days. Immunostaining of vimentin and alpha-smooth muscle actin showed adventitial fibroblasts activation and differentiation into myofibroblasts. Ki-67-positive nuclei were observed in the adventitia 3 days after allografting and subsequently in the neointima. No more than 4% CD3-positive cells were found in adventitia in all the groups. Compared with isografts, TGF-beta1, MMP-7, and MCP-1 were expressed in the adventitia before neointima formation and were significantly increased in allografts at all time points. Tumor necrosis factor-alpha and interleukin-1beta were also significantly increased in adventitia in allografts. CONCLUSIONS: These results demonstrated that adventitial fibroblasts are activated and can produce cytokines and chemokines before the neointimal hyperplasia. They may exert a potential effect on the development of neointimal hyperplasia in TV.

[C/EBP homologous protein-mediated endoplasmic reticulum stress-related apoptosis pathway is involved in abdominal aortic constriction-induced myocardium hypertrophy in rats.].

Endoplasmic reticulum stress (ERS) is an adaptive process in response to circumstantial changes, but excessive and/or prolonged ERS can induce cell apoptosis. C/EBP homologous protein (CHOP) is a very important marker participating in ERS-associated cell apoptosis, while the role of the myocyte apoptosis induced by CHOP remains unclear in the development of hypertrophy. The present study aimed to investigate the effect of CHOP-mediated ERS-associated apoptosis on myocardial hypertrophy induced by abdominal aortic constriction in rats. Healthy male Wistar rats were randomly divided into model group (n=45) and control group (n=40). The rats in model group received abdominal aortic constriction. Hemodynamic changes, whole heart weight/body weight (HW/BW) and left ventricular weight/body weight (LVW/BW) were measured on 1 d, 3 d, 7 d, 14 d and 28 d after surgery, respectively. The mRNA expression of glucose-regulated protein 78 (GRP78), calreticulin (CRT) and CHOP, which are important markers of ERS, were detected by RT-PCR, and Western blot was used to assess the protein level of GRP78, CRT, CHOP, and apoptosis-associated proteins, Bax and Bcl-2. The results obtained were as follows. Compared with control group, the blood pressure, LVW/BW, and HW/BW of rats in model group increased significantly and cardiac function enhanced compensatively on 7 d after surgery, and increased progressively during the experiment. As early as 1 d after surgery, the mRNA level of CRT in model group increased by 136% (P< 0.01) compared with control, while the protein expression increased by 69.2% on 7 d after surgery (P<0.01). Both mRNA and protein expression of GRP78 increased by 20% and 186% (P<0.01) respectively on 7 d after surgery, and the expression sustained high level during the experiment afterwards. Correlation analysis indicated a positive correlation between +dp/dt(max) and CRT protein expression (r=0.780, P<0.01) as well as GRP78 protein expression (r=0.694, P<0.01). Prolonged ERS triggered myocyte apoptosis, as both the mRNA and protein level of CHOP in model group increased by 22.2% (P<0.01) and 76.0% (P<0.01) respectively compared with control on 7 d after hypertrophy (14 d after surgery), and meanwhile, the protein expression of pro-apoptotic Bax increased by 41.1% (P<0.01) and anti-apoptotic Bcl-2 protein expression decreased by 25.5% (P<0.01). Correlation analysis indicated a positive correlation between CHOP and Bax expression (r=0.654, P<0.01), and a negative correlation between CHOP and Bcl-2 expression (r=-0.671, P<0.01). These results suggest that abdominal aortic constriction induces a significant up-regulation in ER molecular chaperones at early stage of post-surgery, indicating that ERS response is activated in the rat heart; while prolonged ERS could lead to myocyte apoptosis, and CHOP-mediated ERS-associated apoptosis may contribute to myocardial hypertrophy. We speculate that cell apoptosis may take part in the regulation of myocardial hypertrophy and heart failure, and determine the progression of decompensated hypertrophy.

A novel effect of lobeline on vascular smooth muscle cell: inhibition of proliferation induced by endothelin-1.

Lobeline has a long history of therapeutic use ranging from an emetic and respiratory stimulant to a tobacco smoking cessation agent. Lobelia chinensis Lour, a traditional Chinese herb whose active ingredient is the alkaloid lobeline, demonstrated to antagonize the bioactive effect of endothelin-1 (ET-1) and prevent the proliferation of vascular smooth muscle cells (VSMCs) in hyperlipidemic rats. The objective of the present study was to determine the effects of lobeline on proliferation of cultured human umbilical VSMCs induced by ET-1. The results showed that the increased cell numbers and enhanced [3H]thymidine incorporation induced by ET-1 were inhibited and the transition of cells from static phase (G0/G1) to DNA synthesis (S) and mitotic phase (G2/M) was held back by lobeline in a concentration-dependent manner. Confocal microscopy demonstrated that lobeline markedly decreased the fluorescent intensity of intracellular Ca2+ concentration ([Ca2+]i) with a significant difference from ET group. Cytotoxicity was determined by Trypan blue exclusion. These results demonstrated a novel biological role of lobeline. Lobeline inhibited the proliferation of human umbilical VSMCs induced by ET-1 in a dose-dependent manner and the anti-proliferative effect was involved in the reduce of increased [Ca2+]i, rather than nonspecific cytotoxicity.

Apolipoprotein C-II promoter T-->A substitution at position -190 affects on the transcription of the gene and its relationship to hyperlipemia.

A Chinese patient with severe hypertriglyceridemia was found to have similar clinical features to that of malignant hyperlipemia in infancy. DNA sequence analysis of the apoC-II gene from the patient's parents revealed a novel heterozygous mutation of T-->A substitution at position -190 base in the apoC-II promoter. We speculated that the patient was a homozygote of the same mutation that resulted in the deficiency of apoC-II. In vitro expression studies showed T-->A substitution in the apoC-II promoter leads to a decrease by approximately 20% in transcriptional activity compared with its counterpart that inserted the normal promoter. These results suggested that T-->A substitution at position -190 in the apoC-II gene promoter only partly affected transcriptional activity of the apoC-II promoter, leading to decrease of apoC-II expression in quantity.

Adventitial fibroblasts are activated in the early stages of atherosclerosis in the apolipoprotein E knockout mouse.

The role of the adventitia in vascular function and vascular lesion formation has been largely ignored. This study observed the activation of the adventitia and specifically the fibroblasts in the development of atherosclerosis in the apoE(-/-) mouse. The results showed a gradual increase in expression of collagen types I and III after 2, 4, and 8 weeks of hyperlipidic diet. The earliest expression of monocyte chemoattractant protein-1 (MCP-1) protein and mRNA was detected in the adventitial fibroblast before the formation of intimal lesions. Proliferation, too, was first found in the adventitial fibroblasts. We hypothesize that the adventitial fibroblast is activated in the early stage of atherosclerosis. Adventitial inflammation may be an early event in the development of atherosclerotic lesions.

A novel substitution at the translation initiator codon (ATG-->ATC) of the lipoprotein lipase gene is mainly responsible for lipoprotein lipase deficiency in a patient with severe hypertriglyceridemia and recurrent pancreatitis.

A patient with severe hypertriglyceridemia and recurrent pancreatitis was found to have significantly decreased lipoprotein lipase (LPL) activity and normal apolipoprotein C-II concentration in post-heparin plasma. DNA analysis of the LPL gene revealed two mutations, one of which was a novel homozygous G-->C substitution, resulting in the conversion of a translation initiation codon methionine to isoleucine (LPL-1). The second was the previously reported heterozygous substitution of glutamic acid at residue 242 with lysine (LPL-242). In vitro expression of both mutations separately or in combination demonstrated that LPL-1 had approximately 3% protein mass and 2% activity, whereas LPL-242 had undetectable activity but normal mass. The combined mutation LPL-1-242 exhibited similar changes as for LPL-1, with markedly reduced mass, and for LPL-242, with undetectable activity. These results suggest that the homozygous initiator codon mutation rather than the heterozygous LPL-242 alteration was mainly responsible for the patient phenotypes.