Anti-Atherosclerotic Effect of Gossypetin on Abnormal Vascular Smooth Muscle Cell Proliferation and Migration.

Gossypetin (GTIN), known as 3,5,7,8,3',4'-hexahydroxyflavone, has been demonstrated to exert anti-atherosclerotic potential against apoptotic injury in oxidized low-density lipoprotein-incubated endothelial cells, and atherosclerotic lesions of cholesterol-fed rabbits. However, the effect and underlying mechanism of GTIN on abnormal vascular smooth muscle cells (VSMCs) proliferation and migration, a major event in the pathogenesis of atherosclerosis, is still unknown. In this study, non-cytotoxic doses of GTIN abolished the VSMCs A7r5 proliferation and cell-cycle S phase distribution. The GTIN-arrested G0/G1 phase might be performed by increasing the expressions of phosphorylated p53 and its downstream molecules that inhibit the activation of cyclin E/cyclin-dependent kinase (cdk)-2, blocking retinoblastoma protein (Rb) phosphorylation and the subsequent dissociation of Rb/transcription factor E2F1 complex. In addition, the results indicated that GTIN inhibited VSMCs wound-healing and migratory abilities through reducing matrix metalloproteinase (MMP)-9 activity and expression, as well as down-regulating protein kinase B (PKB)/nuclear factor-kappaB (NF-κB) signaling. GTIN also revealed potential in diminishing reactive oxygen species (ROS) generation. These findings suggested the inhibitory effects of GTIN on VSMCs dysfunction could likely lead to the containment of atherosclerosis and other cardiovascular illness.

Cilostazol ameliorates diabetic nephropathy by inhibiting highglucose- induced apoptosis.

Diabetic nephropathy (DN) is a hyperglycemia-induced progressive development of renal insufficiency. Excessive glucose can increase mitochondrial reactive oxygen species (ROS) and induce cell damage, causing mitochondrial dysfunction. Our previous study indicated that cilostazol (CTZ) can reduce ROS levels and decelerate DN progression in streptozotocin (STZ)-induced type 1 diabetes. This study investigated the potential mechanisms of CTZ in rats with DN and in high glucose-treated mesangial cells. Male Sprague-Dawley rats were fed 5 mg/kg/day of CTZ after developing STZ-induced diabetes mellitus. Electron microscopy revealed that CTZ reduced the thickness of the glomerular basement membrane and improved mitochondrial morphology in mesangial cells of diabetic kidney. CTZ treatment reduced excessive kidney mitochondrial DNA copy numbers induced by hyperglycemia and interacted with the intrinsic pathway for regulating cell apoptosis as an antiapoptotic mechanism. In high-glucose-treated mesangial cells, CTZ reduced ROS production, altered the apoptotic status, and down-regulated transforming growth factor beta (TGF-ß) and nuclear factor kappa light chain enhancer of activated B cells (NF-κB). Base on the results of our previous and current studies, CTZ deceleration of hyperglycemia-induced DN is attributable to ROS reduction and thereby maintenance of the mitochondrial function and reduction in TGF-ß and NF-κB levels.

Flavonoids Identification and Pancreatic Beta-Cell Protective Effect of Lotus Seedpod.

Oxidative stress is highly associated with the development of diabetes mellitus (DM), especially pancreatic beta-cell injury. Flavonoids derived from plants have caused important attention in the prevention or treatment of DM. Lotus seedpod belongs to a traditional Chinese herbal medicine and has been indicated to possess antioxidant, anti-age, anti-glycative, and hepatoprotective activities. The purpose of this study was to demonstrate the pancreatic beta-cell protective effects of lotus seedpod aqueous extracts (LSE) against oxidative injury. According to HPLC/ESI-MS-MS method, LSE was confirmed to have flavonoids derivatives, especially quercetin-3-glucuronide (Q3G). In vitro, LSE dose-dependently improved the survival and function of rat pancreatic beta-cells (RIN-m5F) from hydrogen peroxide (H2O2)-mediated loss of cell viability, impairment of insulin secretion, and promotion of oxidative stress. LSE showed potential in decreasing the H2O2-induced occurrence of apoptosis. In addition, H2O2-triggered acidic vesicular organelle formation and microtubule-associated protein light chain 3 (LC3)-II upregulation, markers of autophagy, were increased by LSE. Molecular data explored that antiapoptotic and autophagic effects of LSE, comparable to that of Q3G, might receptively be mediated via phospho-Bcl-2-associated death promoter (p-Bad)/B-cell lymphoma 2 (Bcl-2) and class III phosphatidylinositol-3 kinase (PI3K)/LC3-II signal pathway. In vivo, LSE improved the DM symptoms and pancreatic cell injury better than metformin, a drug that is routinely prescribed to treat DM. These data implied that LSE induces the autophagic signaling, leading to protect beta-cells from oxidative stress-related apoptosis and injury.

Supplementation of nanofiltrated deep ocean water ameliorate the progression of osteoporosis in ovariectomized rat via regulating osteoblast differentiation.

Magnesium was reported to be necessary for bone formation. Previous study indicated nanofiltrated deep ocean water (DOW) rich in magnesium. This study investigated the potential mechanisms of DOW in ameliorating osteoporosis. Briefly, female Sprague-Dawley rat was ovariectomized and fed with 0.35, 0.7, or 1.4 ml/kg of DOW daily for 8 weeks. In the results, DOW increased bone density, decreased trabecular bone loss, and decreased bone adiposity. DOW improved bone mass by examining structure in micro-computed tomography. About 0.35 and 0.7 ml/kg of DOW can increase protein expression of runt-related transcription factor 2 (RUNX2), an essential transcription factor for regulating osteoblast differentiation, by 9.4% or 12.9%. In human osteoblast, DOW increased the levels of osteocalcin, RUNX2, and alkaline phosphatase; all the proteins can regulate osteoblast differentiation. Considering the results of in vivo and in vitro study, DOW can ameliorate ovareictomy-caused osteoporosis via regulating the osteoblast differentiation, thereby, maintenance of bone structure. PRACTICAL APPLICATIONS: In addition to calcium, magnesium is essential to promoting the deposition of calcium in bones and regulating its transport; it may also slow the progression of osteoporosis. Nanofiltrated DOW contains abundant magnesium along with several microelements and peptides. In this study, a product was developed for decelerating osteoporosis by using an estrogen depletion model. DOW regulates osteoblast differentiation and thus prevents osteoporosis. This finding provides an alternative healthy source of bone supplements. In addition to tablets or capsules, aqueous supplements can be produced to achieve osteoporosis prevention. This finding is beneficial to the health-care industry for developing sustainable supplements.

Anti-Atherosclerotic Effect of Hibiscus Leaf Polyphenols against Tumor Necrosis Factor-alpha-Induced Abnormal Vascular Smooth Muscle Cell Migration and Proliferation.

The proliferation and migration of vascular smooth muscle cells (VSMCs) are major events in the development of atherosclerosis following stimulation with proinflammatory cytokines, especially tumor necrosis factor-alpha (TNF-α). Plant-derived polyphenols have attracted considerable attention in the prevention of atherosclerosis. Hibiscus leaf has been showed to inhibit endothelial cell oxidative injury, low-density lipoprotein oxidation, and foam cell formation. In this study, we examined the anti-atherosclerotic effect of Hibiscus leaf polyphenols (HLPs) against abnormal VSMC migration and proliferation in vitro and in vivo. Firstly, VSMC A7r5 cells pretreated with TNF-α were demonstrated to trigger abnormal proliferation and affect matrix metalloproteinase (MMP) activities. Non-cytotoxic doses of HLPs abolished the TNF-α-induced MMP-9 expression and cell migration via inhibiting the protein kinase PKB (also known as Akt)/activator protein-1 (AP-1) pathway. On the other hand, HLP-mediated cell cycle G0/G1 arrest might be exerted by inducing the expressions of p53 and its downstream factors that, in turn, suppress cyclin E/cdk2 activity, preventing retinoblastoma (Rb) phosphorylation and the subsequent dissociation of Rb/E2F complex. HLPs also attenuated reactive oxygen species (ROS) production against TNF-α stimulation. In vivo, HLPs improved atherosclerotic lesions, and abnormal VSMC migration and proliferation. Our data present the first evidence of HLPs as an inhibitor of VSMC dysfunction, and provide a new mechanism for its anti-atherosclerotic activity.

Anti-prostate cancer potential of gossypetin via inducing apoptotic and autophagic cell death.

Gossypetin (GTIN), a naturally occurring hexahydroxy flavone, has been shown to possess antimutagenic, antioxidant, antimicrobial, and antiatherosclerotic effects. Here, we investigated the mechanism(s) underlying the anticancer potential of GTIN. In this study, investigations were showed that GTIN preferentially induces programed cell death of prostate cancer (PCa) cells in vitro and in vivo. MTT data showed that GTIN exhibited the anti-proliferation effect on human PCa cells in a dose- and time-dependent manner. Among two kinds of PCa cells, androgen-dependent LNCaP cells were the most susceptible to GTIN. GTIN was evaluated for apoptotic and autophagic activities in LNCaP cells, but not in androgen-independent DU145 cells with mutant Atg5 and resistant to autophagy. Molecular data showed the apoptotic effect of GTIN at a high dose in PCa cells might be mediated via mitochondrial pathway. The lower dose of GTIN-induced autophagy enhances LNCaP cell death, and is dependent on class III PI3K and Atg5 pathway. Finally, GTIN was evidenced by its inhibition on the growth of LNCaP cells in xenograft tumor studies. As a result, our data presented the first evidence of GTIN as an inducer of apoptotic and autophagic cell death in LNCaP cells, and provide a new mechanism for its anticancer activity.

Autophagic effects of Hibiscus sabdariffa leaf polyphenols and epicatechin gallate (ECG) against oxidized LDL-induced injury of human endothelial cells.

PURPOSE: Oxidized low-density lipoprotein (ox-LDL) contributes to the pathogenesis of atherosclerosis by promoting vascular endothelial cell injury. Hibiscus sabdariffa leaf polyphenols (HLP), rich in flavonoids, have been shown to possess antioxidant and antiatherosclerotic activities. In this study, we examined the protective role of HLP and its main compound (-)-epicatechin gallate (ECG) in human umbilical vein endothelial cells (HUVECs) exposed to ox-LDL in vitro. METHODS: In a model of ox-LDL-impaired HUVECs, assessments of cell viability, cytotoxicity, cell proliferation, apoptosis, and autophagy were detected. To highlight the mechanisms of the antiapoptotic effects of HLP and ECG, the expressions of molecular proteins were measured by Western blotting, real-time PCR, and so on. RESULTS: HLP or ECG improved the survival of HUVECs from ox-LDL-induced viability loss. In addition, HLP or ECG showed potential in reducing ox-LDL-dependent apoptosis. Next, the ox-LDL-induced formation of acidic vesicular organelles and upregulation of the autophagy-related genes were increased by HLP or ECG. The HLP-triggered autophagic flux was further confirmed by increasing the LC3-II level under the pretreatment of an autophagy inhibitor chloroquine. Molecular data indicated the autophagic effect of HLP or ECG might be mediated via class III PI3K/Beclin-1 and PTEN/class I PI3K/Akt cascade signaling, as demonstrated by the usage of a class III PI3K inhibitor 3-methyladenine (3-MA) and a PTEN inhibitor SF1670. CONCLUSIONS: Our data imply that ECG-enriched HLP upregulates the autophagic pathway, which in turn led to reduce ox-LDL-induced HUVECs injury and apoptosis and provide a new mechanism for its antiatherosclerotic activity.

Anti-atherosclerotic potential of gossypetin via inhibiting LDL oxidation and foam cell formation.

Gossypetin, a flavone originally isolated from Hibiscus species, has been shown to possess antioxidant, antimicrobial, and antimutagenic activities. Here, we investigated the mechanism(s) underlying the anti-atherosclerotic potential of gossypetin. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) scavenging activity assay showed that the addition of >50µM of gossypetin could scavenge over 50% of DPPH radicals. The inhibitory effects of gossypetin on the lipid and protein oxidation of LDL were defined by thiobarbituric acid reactive substance (TBARS) assay, the relative electrophoretic mobility (REM) of oxidized LDL (ox-LDL), and fragmentation of apoB in the Cu(2+)-induced oxidation of LDL. Gossypetin showed potential in reducing ox-LDL-induced foam cell formation and intracellular lipid accumulation, and uptake ability of macrophages under non-cytotoxic concentrations. Molecular data showed that these influences of gossypetin might be mediated via peroxisome proliferator-activated receptor α (PPARα)/liver-X receptor α (LXRα)/ATP-binding cassette transporter A1 (ABCA1) and PPARγ/scavenger receptor CD36 pathways, as demonstrated by the transfection of PPARα siRNA or PPARγ expression vector. Our data implied that gossypetin regulated the PPAR signals, which in turn led to stimulation of cholesterol removal from macrophages and delay atherosclerosis. These results suggested that gossypetin potentially could be developed as an anti-atherosclerotic agent.

Hibiscus sabdariffa leaf polyphenolic extract inhibits LDL oxidation and foam cell formation involving up-regulation of LXRα/ABCA1 pathway.

The oxidative modification of low-density lipoprotein (LDL) is involved in the pathogenesis of atherosclerotic lesions through the formation of macrophage-derived foam cells. In the present study, we aimed to investigate the anti-atherosclerotic effect of Hibiscus sabdariffa leaf polyphenolic extract (HLP), which is rich in flavonoid. The inhibitory effect of HLP on oxidation and lipid peroxidation of LDL was defined in vitro. HLP showed potential in reducing foam cell formation and intracellular lipid accumulation in oxidised-LDL (ox-LDL)-induced macrophage J774A.1 cells under non-cytotoxic concentrations. Molecular data showed these influences of HLP might be mediated via liver-X receptor α (LXRα)/ATP-binding cassette transporter A1 (ABCA1) pathway, as demonstrated by the transfection of LXRα siRNA. Our data implied that HLP up-regulated the LXRα/ABCA1 pathway, which in turn led to stimulation of cholesterol removal from macrophages and delay atherosclerosis. These results suggested that HLP potentially could be developed as an anti-atherosclerotic agent.

A fatal case of Naegleria fowleri meningoencephalitis in Taiwan.

After bathing at a hot spring resort, a 75-year-old man presented to the emergency department because of seizure-like attack with loss of conscious. This is the first case of primary amebic meningoencephalitis (PAM) caused by Naegleria fowleri in Taiwan. PAM was diagnosed based on detection of actively motile trophozoites in cerebrospinal fluid using a wet-mount smear and the Liu's stain. The amoebae were further confirmed by PCR and gene sequencing. In spite of administering amphotericin B treatment, the patient died 25 days later.

A polyphenol extract of Hibiscus sabdariffa L. ameliorates acetaminophen-induced hepatic steatosis by attenuating the mitochondrial dysfunction in vivo and in vitro.

Oxidative stress is the major contributor to acetaminophen (AAP)-caused liver damage. It promotes mitochondrial oxidative stress and collapses the mitochondrial membrane potential to cause cell death. We have previously shown that a polyphenol extract of Hibiscus sabdariffa L. (HPE) potentiated the antioxidative effect. We further examined in this study the possible mechanism of HPE against AAP-caused liver damage. BABL/c mice were orally fed with HPE (100, 200 or 300 mg/kg) for two weeks prior to an i.p. injection of 1000 mg/kg of AAP. The mice were decapitated 6 h after the AAP injection to collect the blood and liver for further determination. The results show that pretreating with HPE increased the level of glutathione (GSH), decreased the level of lipid peroxidation, and increased catalase activity in the liver. A histopathological evaluation shows that HPE could decrease AAP-induced liver sterosis accompanied by a decreased expression of AIF, Bax, Bid, and p-JNK in the liver. An in vitro assay revealed that HPE could reduce AAP-induced death of BABL/c normal liver cells (BNLs), reverse the lost mitochondrial potency and improve the antioxidative status, similarly to the results of the in vivo assay. We show in this study that HPE possessed the ability to protect the liver from AAP-caused injury. The protective mechanism might be regulated by decreasing oxidative stress and attenuating the mitochondrial dysfunction.

Relationship between matrix metalloproteinase-2 activity and cystatin C levels in patients with hepatic disease.

OBJECTIVES: A direct relationship between cystatin C levels and the severity of hepatic disease has been revealed in our previous study. This study was aimed to consider whether a correlation exists between matrix metalloproteinases (MMPs), which have been proven to be involved in liver cirrhosis, and cystatin C to reflect the severity of hepatic disease. DESIGN AND METHODS: A total of 154 consecutive patients with various liver diseases were recruited to determine their serum levels of cystatin C, MMP-2 and-9, together with other hepatic parameters. These were compared with 40 normal controls. RESULTS: Average levels of MMP-2 and cystatin C were significantly higher in patients while MMP-9 was significantly lower, as compared to controls. A linear regression analysis has revealed a direct relationship between cystatin C and MMP-2 (Y=83.39 + 270.56 X, R=0.38, P< 0.001), as well as between MMP-2 and the severity of liver diseases. CONCLUSION: This is the first study to demonstrate a correlation between cystatin C and MMP-2, suggesting that there may be certain interactions between cystatin C and MMP-2 in patients with hepatic diseases.

Increased cystatin C serum concentrations in patients with hepatic diseases of various severities.

BACKGROUND: Indicators for long-term monitor of the progress of hepatic diseases are of great clinical importance. Since elevated cathepsin was observed in liver diseases, the aim of this study is to investigate the involvement of cystatin C, a very potent inhibitor of cathepsin and a recently introduced marker for renal function, and to see the applicability of serum cystatin C being a convenient marker for the progression of liver diseases. METHODS: One hundred eighty consecutive patients with chronic liver disease of various severities and 45 healthy controls were recruited to determine their serum cystatin C concentrations by N Latex Cystatin C kit, as well as certain relevant clinical values, including alanine transaminase (ALT), aspartate transaminase (AST) and AFP. RESULTS: Average serum cystatin C concentration of patients with hepatic diseases was significantly higher than that of control (0.0902+/-0.0025 mg/dl vs. 0.067+/-0.007 mg/dl; p<0.001), and a linear regression analysis has revealed a direct relation between cystatin C and the severity of liver diseases (Y=1.172+5.492X, R(2)=0.088, p<0.001). CONCLUSION: This study suggested that cystatin C may be an applicable monitoring marker for monitoring liver functions and progression of liver fibrosis.