Longfei Yang

Probiotic Properties of Lactobacillus Strains Isolated from Tibetan Kefir Grains

The objective of this study was to evaluate the functional properties of lactic acid bacteria (LAB) isolated from Tibetan kefir grains. Three Lactobacillus isolates identified as Lactobacillus acidophilus LA15, Lactobacillus plantarum B23 and Lactobacillus kefiri D17 that showed resistance to acid and bile salts were selected for further evaluation of their probiotic properties. The 3 selected strains expressed high in vitro adherence to Caco-2 cells. They were sensitive to gentamicin, erythromycin and chloramphenicol and resistant to vancomycin with MIC values of 26 µg/ml. All 3 strains showed potential bile salt hydrolase (BSH) activity, cholesterol assimilation and cholesterol co-precipitation ability. Additionally, the potential effect of these strains on plasma cholesterol levels was evaluated in Sprague-Dawley (SD) rats. Rats in 4 treatment groups were fed the following experimental diets for 4 weeks: a high-cholesterol diet, a high-cholesterol diet plus LA15, a high-cholesterol diet plus B23 or a high-cholesterol diet plus D17. The total cholesterol, triglyceride and low-density lipoprotein cholesterol levels in the serum were significantly (P<0.05) decreased in the LAB-treated rats compared with rats fed a high-cholesterol diet without LAB supplementation. The high-density lipoprotein cholesterol levels in groups B23 and D17 were significantly (P<0.05) higher than those in the control and LA15 groups. Additionally, both fecal cholesterol and bile acid levels were significantly (P<0.05) increased after LAB administration. Fecal lactobacilli counts were significantly (P<0.05) higher in the LAB treatment groups than in the control groups. Furthermore, the 3 strains were detected in the rat small intestine, colon and feces during the feeding trial. The bacteria levels remained high even after the LAB administration had been stopped for 2 weeks. These results suggest that these strains may be used in the future as probiotic starter cultures for manufacturing novel fermented foods.

Lactobacillus plantarum strains as potential probiotic cultures with cholesterol-lowering activity

Elevated blood cholesterol is an important risk factor associated with atherosclerosis and coronary heart disease. The search for mediators that fine-tune cholesterol homeostasis has revealed lactic acid bacteria (LAB) to be potentially beneficial. The aim of the present study was to identify and characterize probiotic strains with bile salt hydrolase activity from kefir grains and evaluate their potential use as cholesterol-reducing probiotics in rats. Two isolates, Lp09 and Lp45, obtained from kefir grains were identified as Lactobacillus plantarum via molecular typing methods. Lactobacillus plantarum Lp09 and Lp45 exhibited excellent tolerance to low pH levels and high bile salt concentrations and showed potential bile salt hydrolase activity, bile salt deconjugation activity, and cholesterol coprecipitation ability. Additionally, the potential effect of Lb. plantarum Lp09 and Lp45 on plasma cholesterol levels was evaluated in Sprague-Dawley rats. Rats in 3 treatment groups were fed different experimental diets: a high-cholesterol diet, a high-cholesterol diet plus Lb. plantarum Lp09, or a high-cholesterol diet plus Lb. plantarum Lp45 for 4 wk. Total cholesterol, triglyceride, and low-density lipoprotein cholesterol levels in serum as well as cholesterol and triglyceride levels in liver were significantly decreased in the LAB-treated rats compared with rats fed a high-cholesterol diet without LAB supplementation. Also, both fecal cholesterol and bile acid levels were significantly increased after LAB administration. No significant changes were detected in high-density lipoprotein cholesterol levels. These results suggest that the Lb. plantarum Lp09 and Lp45 strains present the potential to be explored as probiotic agents for the management of hypercholesterolemia.

Characterization of Lactobacillus plantarum Lp27 isolated from Tibetan kefir grains: A potential probiotic bacterium with cholesterol-lowering effects

Lactobacillus plantarum Lp27 was isolated from Tibetan kefir grains. The Lp27 isolate survived a 3-h incubation at pH 2.0 and grew normally in 0.3% oxgall. In addition, the Lp27 isolate exhibited an adhesion ratio of 9.5 ± 2.5% with Caco-2 cells. Antibiotic susceptibility tests indicated that the Lp27 isolate was sensitive to gentamicin, tetracycline, erythromycin, and chloramphenicol, and was resistant to vancomycin with a minimum inhibitory value of 23µg/mL. The Lp27 isolate inhibited cholesterol absorption through downregulation of Niemann-Pick C1-like 1 (NPC1L1) expression in Caco-2 cells. The Lp27 isolate was fed to hypercholesterolemic rats at a dose of 10(9) cfu/d for 4wk. The Lp27 feeding significantly lowered serum total cholesterol, low-density lipoprotein cholesterol, and triglycerides concentrations, but no change was observed in the serum high-density lipoprotein cholesterol concentrations. In addition, liver total cholesterol and triglycerides were decreased in the Lp27-fed group. The expression of NPC1L1 in the duodenum and jejunum was significantly decreased following Lp27 feeding. These results indicate that Lp27 might be an effective cholesterol-lowering probiotic and a possible mechanism for the cholesterol-reducing effects of probiotics.

Inhibition of TMEM16A Expression Suppresses Growth and Invasion in Human Colorectal Cancer Cells

Metastasis leads to poor prognosis in colorectal cancer patients, and there is a growing need for new therapeutic targets. TMEM16A (ANO1, DOG1 or TAOS2) has recently been identified as a calcium-activated chloride channel (CaCC) and is reported to be overexpressed in several malignancies; however, its expression and function in colorectal cancer (CRC) remains unclear. In this study, we found expression of TMEM16A mRNA and protein in high-metastatic-potential SW620, HCT116 and LS174T cells, but not in primary HCT8 and SW480 cells, using RT-PCR, western blotting and immunofluorescence labeling. Patch-clamp recordings detected CaCC currents regulated by intracellular Ca2+ and voltage in SW620 cells. Knockdown of TMEM16A by short hairpin RNAs (shRNA) resulted in the suppression of growth, migration and invasion of SW620 cells as detected by MTT, wound-healing and transwell assays. Mechanistically, TMEM16A depletion was accompanied by the dysregulation of phospho-MEK, phospho-ERK1/2 and cyclin D1 expression. Flow cytometry analysis showed that SW620 cells were inhibited from the G1 to S phase of the cell cycle in the TMEM16A shRNA group compared with the control group. In conclusion, our results indicate that TMEM16A CaCC is involved in growth, migration and invasion of metastatic CRC cells and provide evidence for TMEM16A as a potential drug target for treating metastatic colorectal carcinoma.

Defective macrophage function in aquaporin-3 deficiency

Macrophages play an essential role in innate immunity. We found that mouse resident peritoneal macrophages (mRPMs) express the aquaglyceroporin aquaporin‐3 (AQP3) in a plasma membrane pattern. AQP3‐deficient (AQP3–/–) mice showed significantly greater mortality than wild‐type (AQP3+/+) mice in a model of bacterial peritonitis. To establish the cellular mechanism of the peritonitis phenotype, measurements were made of mRPM phagocytosis, migration, and water/glycerol permeability. We found significantly impaired engulfment of Escherichia coli and chicken erythrocytes in AQP3–/– vs. AQP3+/+ mRPMs, as well as impaired migration of AQP3–/– mRPMs in response to a chemotactic stimulus. In AQP3+/+ mRPMs, AQP3 was polarized to pseudopodia at the leading edge during migration and around the phagocytic cup during engulfment. Water and glycerol permeabilities in mRPMs from AQP3–/– mice were reduced compared to mRPMs from AQP3+/+ mice. Cellular glycerol and ATP content were remarkably lower in AQP3–/– vs. AQP3+/+ mRPMs, and glycerol supplementation partially rescued the reduced ATP content and impaired function of AQP3–/– mRPMs. These data implicate AQP3 as a novel determinant in macrophage immune function by a cellular mechanism involving facilitated water and glycerol transport, and consequent phagocytic and migration activity. This is the first study demonstrating involvement of an aquaporin in innate immunity. Our results suggest AQP3 as a novel therapeutic target in modulating the immune response in various infectious and inflammatory conditions.—Zhu, N., Feng, X., He, C., Gao, H., Yang, L., Ma, Q., Guo, L., Qiao, Y., Yang, Y., Ma, T. Defective macrophage function in aquaporin‐3 deficiency. FASEB J. 25, 4233–4239 (2011). www.fasebj.org

Increased Female Fertility in Aquaporin 8-Deficient Mice

Aquaporin‐8 (AQP8) is a water channel expressed extensively in male and female reproductive systems. But its physiological functions are largely unknown. In the present study, we first found significantly increased number of offspring delivered by AQP8−/− mothers compared with wild‐type mothers in cross‐mating experiments. Comparison of ovulation in the two genotypes demonstrated that AQP8−/− ovaries released more oocytes (9.5 ± 1.9 vs. 7.1 ± 2.1 in normal ovulation and 37.8 ± 6.7 vs. 27.9 ± 5.7 in superovulation). Histological analysis showed increased number of corpus luteums in mature AQP8−/− ovaries, suggesting increased maturation and ovulation of follicles. By RT‐PCR, western blot and immunohistochemistry analyses, we determined the expression of AQP8 in mouse ovarian granulosa cells. Granulosa cells isolated from AQP8−/− mice showed 45% of decreased membrane water permeability than wild‐type mice. As the atresia of ovarian follicles is primarily due to apoptosis of granulosa cells, we analyzed the apoptosis of isolated granulosa cells from wild‐type and AQP8−/− mice. The results indicated significantly lower apoptosis rate in AQP8−/− granulosa cells (21.3 ± 3.6% vs. 32.6 ± 4.3% in AQP8+/+ granulosa cells). Taken together, we conclude that AQP8 deficiency increases the number of mature follicles by reducing the apoptosis of granulosa cells, thus increasing the fertility of female mice. This discovery may offer new insight of improving female fertility by reducing granulosa cell apoptosis through AQP8 inhibition.

Lactobacillus acidophilus ATCC 4356 Prevents Atherosclerosis via Inhibition of Intestinal Cholesterol Absorption in Apolipoprotein E-Knockout Mice

ABSTRACT The objective of this study was to investigate the effect of Lactobacillus acidophilus ATCC 4356 on the development of atherosclerosis in apolipoprotein E-knockout (ApoE−/−) mice. Eight-week-old ApoE−/− mice were fed a Western diet with or without L. acidophilus ATCC 4356 daily for 16 weeks. L. acidophilus ATCC 4356 protected ApoE−/− mice from atherosclerosis by reducing their plasma cholesterol levels from 923 ± 44 to 581 ± 18 mg/dl, likely via a marked decrease in cholesterol absorption caused by modulation of Niemann-Pick C1-like 1 (NPC1L1). In addition, suppression of cholesterol absorption induced reverse cholesterol transport (RCT) in macrophages through the peroxisome proliferator-activated receptor/liver X receptor (PPAR/LXR) pathway. Fecal lactobacillus and bifidobacterium counts were significantly (P < 0.05) higher in the L. acidophilus ATCC 4356 treatment groups than in the control groups. Furthermore, L. acidophilus ATCC 4356 was detected in the rat small intestine, colon, and feces during the feeding trial. The bacterial levels remained high even after the administration of lactic acid bacteria had been stopped for 2 weeks. These results suggest that administration of L. acidophilus ATCC 4356 can protect against atherosclerosis through the inhibition of intestinal cholesterol absorption. Therefore, L. acidophilus ATCC 4356 may be a potential therapeutic material for preventing the progression of atherosclerosis.

Costunolide, a sesquiterpene lactone induces G2/M phase arrest and mitochondria-mediated apoptosis in human gastric adenocarcinoma SGC-7901 cells

Costunolide, a sesquiterpene lactone isolated from the Saussurea lappa possesses potent anticancer properties. In this study, for the first time we investigated the effects of costunolide on the cell viability, cell cycle and apoptosis in gastric adenocarcinoma SGC-7901 cells. The results showed that costunolide induced morphological changes and decreased the cell viability of SGC-7901 cells. Meanwhile, the parallel treatment of the costunolide to a normal splenocytes showed less strong effects. Deoxyribonucleic aci d (DNA) flow cytometric analysis demonstrated that costunolide markedly induced apoptosis of SGC-7901 cells and arrested cell cycle at G2/M phase in a time-dependent manner. Costunolide-induced apoptosis was regulated by activation of caspase-3 and down-regulation of Bcl-2. Furthermore, costunolide significantly induced the loss of mitochondrial membrane potential. It indicated that costunolide-induced apoptosis involved the mitochondria-dependent pathway in SGC-7901 cells. These in vitro results suggested that Costunolide should be further examined for in vivo activity and molecular mechanism in human gastric cancer. Key words:

Identification of the Novel TMEM16A Inhibitor Dehydroandrographolide and Its Anticancer Activity on SW620 Cells.

TMEM16A, a calcium-activated chloride channel (CaCC), is highly amplified and expressed in human cancers and is involved in the growth and metastasis of some malignancies. Inhibition of TMEM16A represents a novel pharmaceutical approach for the treatment of cancers and metastases. The purpose of this study is to identify a new TMEM16A inhibitor, investigate the effects of this inhibitor on the proliferation and metastasis of TMEM16A-amplified SW620 cells, and to elucidate the underlying molecular mechanism in vitro. We identified a novel small-molecule TMEM16A inhibitor dehydroandrographolide (DP). By using patch clamp electrophysiology, we showed that DP inhibited TMEM16A chloride currents in Fisher rat thyroid (FRT) cells that were transfected stably with human TMEM16A and in TMEM16A-overexpressed SW620 cells but did not alter cystic fibrosis transmembrane conductance regulator (CFTR) chloride currents. Further functional studies showed that DP suppressed the proliferation of SW620 cells in a dose- and time-dependent manner using MTT assays. Moreover, DP significantly inhibited migration and invasion of SW620 cells as detected by wound-healing and transwell assays. Further mechanistic study demonstrated that knockdown of human TMEM16A decreased the inhibitory effect of DP on the proliferation of SW620 cells and that TMEM16A-dependent cells (SW620 and HCT116) were more sensitive to DP than TMEM16A-independent cells (SW480 and HCT8). In addition, we found that treatment of SW620 cells with DP led to a decrease in TMEM16A protein levels but had no effect on TMEM16A mRNA levels. The current work reveals that DP, a novel TMEM16A inhibitor, exerts its anticancer activity on SW620 cells partly through a TMEM16A-dependent mechanism, which may introduce a new targeting approach for an antitumour therapy in TMEM16A-amplified cancers.

Tanshinones Increase Fibrinolysis through Inhibition of Plasminogen Activator Inhibitor-1

Danshen, the dried roots of Salvia miltiorrhiza, is a well-known traditional Chinese medicine used for the treatment of stroke since 1970. A similar plant called Salvia columbariae was also used by Californian Indians to treat people suffering from stroke [1]. Pharmacological studies indicated that their active ingredients, tanshinones, and salvianolic acids exhibited anticoagulant, vasodilatory, anti-inflammatory, antioxidant, neuroprotective, and other activities underlying the therapeutic effect [2]. Thrombosis secondary to atherosclerosis is a major cause of stroke and coronary artery disease. Inhibition of clot formation and potential clot dissolution has been demonstrated in many clinical trials of S. miltiorrhiza [1]. Early in vitro studies found that S. miltiorrhiza extract can increase the proteolysis of fibrinogen to fibrinogen degradation products [3], suggesting a unique mechanism of antithrombosis comparing to other anticoagulant drugs that prevent clot formation by interfering with platelet aggregation, thrombin activity or vitamin K function. However, the molecular basis of the fibrinolytic action of S. miltiorrhiza remains unknown. Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of both tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA), is a key physiological regulator of the fibrinolytic system and is implicated in thrombotic pathophysiology in ischemic stroke and coronary artery disease [4,5]. Pharmacological inhibition of PAI-1 showed antithrombotic benefits devoid of bleeding effect in rodents and monkey [6,7]. In the present study, we tested the hypothesis that S. miltiorrhiza contains active compounds that inhibit PAI-1 activity. Using a chromogenic substrate-based PAI-1 activity assay kit (Milllipore, MA, USA), we analyzed the PAI-1 inhibitory effect of seven natural compounds from S. miltiorrhiza: Tanshinone I, Tanshinone IIA, Sodium tanshinone IIA sulfonate, Cryptotanshinone, Sodium Danshensu, Protocatechuic aldehyde and β-sitosterol (National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China). The purity of the compounds was determined to be >98% by high-performance liquid chromatography analysis. For the uPA-mediated assay, PAI-1 (Molecular Innovations, MI, USA) was incubated with compounds at 23◦C for 15 min, then PAI-1 activity was assayed according to the protocol of the kit. For the tPA-mediated PAI-1 activity assay, uPA and its chromogenic substrate in the kit was replaced by tPA (Molecular Innovations, MI, USA) and its substrate S-2288 (Chromogenix, NC, USA). As shown in Figure 1(A), sodium tanshinone IIA sulfonate and cryptotanshinone inhibited PAI-1 activity in a dosedependent manner. The IC50 values for sodium tanshinone IIA sulfonate and cryptotanshinone are 41 and 98 μM, respectively, in uPA/PAI-1 assay and 59 and 152 μM, respectively, in tPA/ PAI-1 assay. Tanshinone IIA exhibited much weaker effect than its salt form derivative sodium tanshinone IIA sulfonate, likely due to its lower solubility. To further confirm the PAI-1 inhibitory activity by the tanshinone compounds, we performed biochemical analysis to determine if sodium tanshinone IIA sulfonate and cryptotanshinone can block the formation of PAI-1/uPA complex. In a 25 μL reaction volume in tris-buffered saline buffer, recombinant human PAI-1 (Molecular Innovations, MI, USA) at 1 μM final concentration was first incubated with various concentrations of sodium tanshinone IIA sulfonate and cryptotanshinone for 15 min at room temperature. Then uPA (Molecular Innovations, MI, USA) was added to a final concentration of 0.8 μM and incubated for 10 min at 37◦C. The samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis on a 10% gel and stained with Coomassie blue. As shown in Figure 1(B), both sodium tanshinone IIA sulfonate and cryptotanshinone dose dependently prevented the formation of PAI-1/uPA complex. The results of enzymatic and