Tomatidine inhibits porcine epidemic diarrhea virus replication by targeting 3CL protease.

Porcine epidemic diarrhea virus (PEDV) causes lethal diarrhea in suckling piglets, leading to severe economic losses worldwide. There is an urgent need to find new therapeutic methods to prevent and control PEDV. Not only is there a shortage of commercial anti-PEDV drugs, but available commercial vaccines fail to protect against highly virulent PEDV variants. We screened an FDA-approved library of 911 natural products and found that tomatidine, a steroidal alkaloid extracted from the skin and leaves of tomatoes, demonstrates significant inhibition of PEDV replication in Vero and IPEC-J2 cells in vitro. Molecular docking and molecular dynamics analysis predicted interactions between tomatidine and the active pocket of PEDV 3CL protease, which were confirmed by fluorescence spectroscopy and isothermal titration calorimetry (ITC). The inhibiting effect of tomatidine on 3CL protease was determined using cleavage visualization and FRET assay. Tomatidine-mediated blocking of 3CL protease activity in PEDV-infected cells was examined by western blot detection of the viral polyprotein in PEDV-infected cells. It indicates that tomatidine inhibits PEDV replication mainly by targeting 3CL protease. In addition, tomatidine also has antiviral activity against transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), encephalo myocarditis virus (EMCV) and seneca virus A (SVA) in vitro. These results may be helpful in developing a new prophylactic and therapeutic strategy against PEDV and other swine disease infections.

Grape Seed Procyanidin B2 Protects Porcine Ovarian Granulosa Cells against Oxidative Stress-Induced Apoptosis by Upregulating let-7a Expression.

Oxidative stress is a causal factor and key promoter of all kinds of reproductive disorders related to granulosa cell (GC) apoptosis that acts by dysregulating the expression of related genes. Various studies have suggested that grape seed procyanidin B2 (GSPB2) may protect GCs from oxidative injury, though the underlying mechanisms are not fully understood. Therefore, whether the beneficial effects of GSPB2 are associated with microRNAs, which have been suggested to play a critical role in GC apoptosis by regulating the expression of protein-coding genes, was investigated in this study. The results showed that GSPB2 treatment protected GCs from a H2O2-induced apoptosis, as detected by an MTT assay and TUNEL staining, and increased let-7a expression in GCs. Furthermore, let-7a overexpression markedly increased cell viability and inhibited H2O2-induced GC apoptosis. Furthermore, the overexpression of let-7a reduced the upregulation of Fas expression in H2O2-treated GCs at the mRNA and protein levels. Dual-luciferase reporter assay results indicated that let-7a directly targets the Fas 3'-UTR. Furthermore, the overexpression of let-7a enhanced the protective effects of GSPB2 against GC apoptosis induced by H2O2. These results indicate that GSPB2 inhibits H2O2-induced apoptosis of GCs, possibly through the upregulation of let-7a.

Knockdown of hTERT alters biophysical properties of K562 cells resulting in decreased migration rate in vitro.

It has been shown that 90% of tumors, including hematological malignant tumors and leukemia, have much higher levels of telomerase expression than normal cells. To investigate the effect of telomerase on leukemia cells, we transfected K562, a human erythroleukemia cell line with an antisense-hTERT (human telomerase reverse transcriptase) cDNA vector, and examined the biological and biophysical properties of the stably transfected cells (referred to as KAT). Un-transfected cells (K562) and cells transfected with the empty vector (referred to as KC) were used as controls. Cell growth curve and (3)H-TdR test showed that the growth rate and DNA synthesis of KAT decreased compared with those of K562 and KC cells. Apoptosis and cell cycle distribution in KAT cells under normal culture condition were similar to those of K562 and KC cells, but changed after serum deprivation. KAT cells had significantly different biophysical characteristics from K562 and KC in terms of cell electrophoresis, membrane fluidity, membrane fluidity, and viscoelasticity. Furthermore, the transendothelial migration rate of KAT was much lower than those of K562 and KC cells. Confocal microscopy showed that KAT cells had higher F-actin content, suggesting the reorganization of cytoskeleton. Flow cytometry analysis revealed a lowered intracellular calcium concentration and CD71 expression, explaining the high F-actin content in KAT cells. In conclusion, we found that the knockdown of hTERT in K562 cells changed their cytoskeleton and biophysical features, and reduced the cell migration.

A C-type lectin is involved in the innate immune response of Chinese white shrimp.

C-type lectins may function as pattern-recognition receptors (PRRs) and play important roles in immune responses. In this work, a cDNA for a new C-type lectin, FcLec3, was obtained from Chinese white shrimp Fenneropenaeus chinensis using expressed sequence tag analysis and rapid amplification of the cDNA ends. FcLec3 contains an N-terminal signal peptide and a carbohydrate recognition domain (CRD). RT-PCR analysis showed that FcLec3 was mainly expressed in hepatopancreas and that the expression of FcLec3 was obviously up-regulated by Vibrio anguillarum or white spot syndrome virus (WSSV) challenge. Recombinant FcLec3 could agglutinate Gram-negative and -positive bacteria with the presence of calcium. A following agglutination inhibitory test indicated that FcLec3 could recognize muramic acid and peptidoglycan. Besides, pull-down assay showed that the recombinant protein could interact with VP28, one major envelope protein of WSSV. These results suggested that FcLec3 might function in the recognition of bacterial and viral pathogens in shrimp.

Improving abnormal hemorheological parameters in ApoE-/- mice by Ilex kudingcha total saponins.

The aim of this study was to test the effects of Ilex kudingcha total saponins on hemorheology of ApoE-/- mice suffering from hypercholesterolemia induced by high-cholesterol diet. The mice were randomly divided into six groups: the control group, the high-cholesterol diet group, 50 mg/kg atorvastatin treatment group, 75, 150 and 300 mg/kg Ilex kudingcha saponins treatment groups, and all the drug treatment groups were fed with a high-cholesterol diet. After administration with saponins (150 mg/kg or more) and atorvastatin (50 mg/kg) for six weeks, the plasma total cholesterol (TC), whole blood viscosity (WBV), plasma viscosity (PV), and erythrocyte aggregation index (EAI) had a remarkable decrease compared with that of the high-cholesterol diet group, but the hematocrit (Hct) and erythrocyte deformation index (DI) had no significant changes. In addition, it is found that the improving effects of saponins on reducing plasma fibrinogen (Fg) levels and prolonging the blood coagulation times including activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT). In conclusion, the Ilex kudingcha total saponins may have a significant therapy application of hypercholesterolemia and atherosclerosis by considering its actions on hemorheological characteristics.

A novel C-type lectin with two CRD domains from Chinese shrimp Fenneropenaeus chinensis functions as a pattern recognition protein.

Lectins are regarded as potential immune recognition proteins. In this study, a novel C-type lectin (Fc-Lec2) was cloned from the hepatopancreas of Chinese shrimp, Fenneropenaeus chinensis. The cDNA of Fc-Lec2 is 1219 bp with an open reading frame (ORF) of 1002 bp that encodes a protein of 333 amino acids. Fc-Lec2 contains a signal peptide and two different carbohydrate recognition domains (CRDs) arranged in tandem. The first CRD contains a QPD (Gln-Pro-Asp) motif that has a predicted binding specificity for galactose and the second CRD contains a EPN (Glu-Pro-Asn) motif for mannose. Fc-Lec2 was constitutively expressed in the hepatopancreas of normal shrimp, and its expression was up-regulated in the hepatopancreas of shrimp challenged with bacteria or viruses. Recombinant mature Fc-Lec2 and its two individual CRDs (CRD1 and 2) did not have hemagglutinating activity against animal red blood cells, but agglutinated some gram-positive and gram-negative bacteria in a calcium-dependent manner. The three recombinant proteins also bound to bacteria in the absence of calcium. Fc-Lec2 seems to have broader specificity and higher affinity for bacteria and polysaccharides (peptidoglycan, lipoteichoic acid and lipopolysaccharide) than each of the two individual CRDs. These data suggest that the two CRDs have synergistic effect, and the intact lectin may be more effective in response to bacterial infection, the Fc-Lec2 performs its pattern recognition function by binding to polysaccharides of pathogen cells.

Effects of myakuryu on hemorheological characteristics and mesenteric microcirculation of rats fed with a high-fat diet.

There is evidence that hyperlipidemia can induce hemorheological and microcirculatory disturbances. Myakuryu, a Chinese traditional medicine is efficacious in promoting lipid metabolism and protecting oxidative stress, but whether this drug can ameliorate rheologic disturbances caused by hyperlipidemia is still unknown. The present study was conducted to investigate the effects of myakuryu on hemorheological and microcirculatory disturbances induced by hyperlipidemia. Wistar rats were divided into a group on control diet (n=8) and a group on high-fat diet (HFD, n=44). Eight weeks later, plasma triglyceride (TG) and total cholesterol (TC) were determined. Sixteen animals with the highest levels of hyperlipidemia from the HFD group were randomly divided into two sub-groups: the untreated hyperlipidemia group (n=8) and the group treated with myakuryu (n=8). At the end of the sixteenth week, rheological and microcirculatory parameters were measured. Chemical analysis showed that myakuryu treatment caused significant reductions of plasma TG and TC levels (P<0.01), and the cholesterol/phospholipid ratio in the erythrocyte membrane (P<0.05). Rheological and microcirculatory measurements showed that myakuryu treatment led to a significant decrease in the erythrocyte aggregation index, plasma viscosity and blood viscosity at shear rates of 50, 100 and 150 s(-1) and in adherent leukocytes in mesenteric venules. There was a significant increase in erythrocyte deformation, electrophoretic mobility, membrane fluidity and F-actin content in the erythrocyte membrane as well as in red cell velocity in mesenteric venules. Our findings suggest that myakuryu treatment can improve blood flow and reduce adherent leukocytes in the venules of rats fed with HFD by ameliorating blood viscosity, erythrocyte deformability and aggregation, and other hemorheological characteristics.

Effects of cardiotonic pill on RBC rheologic abnormalities in HFD-induced mice and LPL deficient mice.

The lipoprotein lipase deficient (LPL-/-) mice and high fat-diet (HFD) induced hypertriglyceridemic mice were used to investigate the effects of cardiotonic pill (CP) on RBC rheologic abnormalities. Mice were randomly divided into the following five groups: the control group; the untreated HFD group; the untreated LPL-/- group; the treated HFD group; and the treated LPL-/- group, and the treated HFD and LPL-/- mice were administered with CP twice a day (400 mg/kg/day) orally for four weeks. Then, plasma triglyceride (TG), RBC deformation index (DI), orientation index (DI)or and RBC electrophoretic time (EPT) were measured. Compared with the untreated HFD mice, TG level and EPT reduced and DI and (DI)or increased markedly in the treated HFD mice (P<0.05). However, compared with the untreated LPL-/- mice, these parameters in the treated LPL-/- mice had no statistically significant changes (P>0.05). Our data show that CP can lower plasma TG level and ameliorate RBC rheologic abnormalities in the HFD-induced hypertriglyceridemic mice, but it losses its capacity in the LPL deficient animals. The results indicate that LPL may be one of the important targets for CP regulating lipometabolism and rheologic abnormalities.