Enterostatin (VPDPR) Has Anti-analgesic and Anti-amnesic Activites

Enterostatin (VPDPR), an anorexigenic peptide derived from the amino terminus of procolipase, significantly inhibited analgesia induced by the μ-opioidagonist morphine (5 mg/kg, s.c.) after i.c.v. administration to mice at a dose of 100 nmol. On the other hand, VPDPR (～200 nmol, i.c.v.) did not attenuate analgesia induced by the κ-opioid agonist D-Phe-D-Phe-D-Nle-D-Arg-NH₂ (100 μg/mouse, i.c.v.) or δ-opioid agonist DTLET (4 nmol/mouse, i.c.v.). VPDPR (100 nmol, i.c.v.) significantly improved amnesia induced by scopolamine (0.2 mg/kg, i.p.) in mice. However, VPDPR did not enhance memory in normal mice at the same dose.     

Enterostatin (VPDPR) and its peptide fragment DPR reduce serum cholesterol levels after oral administration in mice

We found that enterostatin (VPDPR), an anorexigenic peptide for a high-fat diet, significantly reduces serum cholesterol levels after oral administration of 100 mg/kg for 3 days in mice fed a high cholesterol-cholic acid diet. DPR, a peptide fragment of VPDPR, also had hypocholesterolemic activity at a dose of 50 mg/kg. Food intake was not suppressed under these dietary conditions. Fecal excretion of cholesterol and bile acids was increased significantly by both VPDPR and DPR. Interestingly, DPR induced hypocholesterolemic effects just two hours after a single oral administration at a dose of 100 mg/kg.     

A Freshwater Clam (Corbicula fluminea) Extract Improves Cholesterol Metabolism in Rats Fed on a High-Cholesterol Diet

The effect of a freshwater clam (Corbicula fluminea) extract (FCE) on cholesterol metabolism in rats fed on a high-cholesterol diet was investigated. When rats were fed various amounts of FCE in addition to the high-cholesterol diet for 2 wk, the serum and hepatic cholesterol levels were gradually reduced in a dose-dependent manner, as compared with the control group. The excretion of neutral sterols and bile acids into the feces was increased by feeding FCE. Several phytosterols were detected in the feces of rats fed on the FCE-containing diet. In addition, substantial amounts of phytosterols were found in FCE. Cholesterol 7α-hydroxylase (CYP7A1) mRNA in the liver of the rats fed on the FCE-containing diets was higher than that of rats fed on the high-cholesterol diets without FCE. These results may suggest that enhanced cholesterol degradation and the excretion of neutral sterols and bile acids contributed to the hypocholesterolemic effect of FCE observed in the hypercholesterolemic rats fed on the high-cholesterol diet.     

Soystatin (VAWWMY), a Novel Bile Acid-Binding Peptide,Decreased Micellar Solubility and Inhibited Cholesterol Absorption in Rats

This study was designed to identify a novel peptide derived from soybean protein that induces inhibition of cholesterol absorption in vivo. VAWWMY (Val-Ala-Trp-Trp-Met-Tyr, designated soystatin) had a significantly greater ability to bind bile acid than soybean protein peptic hydrolysate (SPH) or casein tryptic hydrolysate (CTH). Surprisingly, the bile-acid binding ability of VAWWMY was almost as strong as that of the hypocholesterolemic medicine cholestyramine. The micellar solubility of cholesterol was significantly lower in the presence of VAWWMY than in that of SPH or CTH. We found that soystatin derived from soybean glycinin acted as an inhibitor of cholesterol absorption in vivo.     

Tissue Distribution of Berberine and Its Metabolites after Oral Administration in Rats

Berberine (BBR) has been confirmed to have multiple bioactivities in clinic, such as cholesterol-lowering, anti-diabetes, cardiovascular protection and anti- inflammation. However, BBR’s plasma level is very low; it cannot explain its pharmacological effects in patients. We consider that the in vivo distribution of BBR as well as of its bioactive metabolites might provide part of the explanation for this question. In this study, liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LC/MSⁿ-IT-TOF) as well as liquid chromatography that coupled with tandem mass spectrometry (LC-MS/MS) was used for the study of tissue distribution and pharmacokinetics of BBR in rats after oral administration (200 mg/kg). The results indicated that BBR was quickly distributed in the liver, kidneys, muscle, lungs, brain, heart, pancreas and fat in a descending order of its amount. The pharmacokinetic profile indicated that BBR’s level in most of studied tissues was higher (or much higher) than that in plasma 4 h after administration. BBR remained relatively stable in the tissues like liver, heart, brain, muscle, pancreas etc. Organ distribution of BBR’s metabolites was also investigated paralleled with that of BBR. Thalifendine (M1), berberrubine (M2) and jatrorrhizine (M4), which the metabolites with moderate bioactivity, were easily detected in organs like the liver and kidney. For instance, M1, M2 and M4 were the major metabolites in the liver, among which the percentage of M2 was up to 65.1%; the level of AUC _(0-t) (area under the concentration-time curve) for BBR or the metabolites in the liver was 10-fold or 30-fold higher than that in plasma, respectively. In summary, the organ concentration of BBR (as well as its bioactive metabolites) was higher than its concentration in the blood after oral administration. It might explain BBR’s pharmacological effects on human diseases in clinic.     

Medium-Chain Fatty Acids Enhanced the Excretion of Fecal Cholesterol and Cholic Acid in C57BL/6J Mice Fed a Cholesterol-Rich Diet

The objective of the present study was to investigate the cholesterol-reducing effect of medium-chain fatty acids (MCFAs) completed by elevated excretion of fecal neutral steroids and/or bile acids. Blood and liver lipid profiles, fecal neutral steroids, bile acids, and mRNA and protein expression of the genes relevant to cholesterol homeostasis were measured and analyzed in C57BL/6J mice fed a cholesterol-rich diet with 2% caprylic acid or capric acid for 12 weeks. Blood total cholesterol and low-density lipoprotein cholesterol (LDL-c) levels were reduced significantly as compared to diet with palmitic acid or stearic acid. Caprylic acid promoted the excretion of fecal neutral steroids, especially cholesterol. The excretion of fecal bile acids, mainly in the form of cholic acid was enhanced and accompanied by elevated expression of mRNA and the protein of hepatic cholesterol 7α-hydroxylase (CYP7A1). These results indicate that MCFAs can reduce blood cholesterol by promoting the excretion of fecal cholesterol and cholic acid.     

Temporal Variation in Serum Nitrite Levels in Rats and Mice

Although considerable evidence implicates involvement of nitric oxide (NO) in circadian regulation, little is known about possible 24h variations in basal NO metabolism. In this study, daily variations in serum nitrite levels were studied in locally bred mice and rats during the months of September and October. The serum was separated from blood samples obtained at six different times of the day and night (lh, 5h, 9h, 13h, 17h, and 21h after lights off [HALO] from male albino mice and rats). As an index of in vivo NO generation, serum nitrite levels (determined by the diazotization method) in rats exhibited significant temporal fluctuation (unpaired Student t test), with the concentration highest at 5 HALO and 21 HALO and lowest at 9 HALO. No such temporal variation was detected in mice in these studies conducted on locally bred animals in the autumn. (Chwnobiology International, 16(4), 527-532, 1999)     

Synthesis and characterization of Sodium cis-dichlorochenodeoxycholylglycinato(O,N) platinum(II) – Cytostatic activity

With a view to using bile acids as shuttles for delivering platinum-related cytostatic drugs to liver tumors, a chenodeoxycholylglycinato(CDCG)-derivative of platinum(II) has been synthesized. The complex - named Bamet-M2- was chemically characterized by elemental analysis, FT-IR, NMR, FAB-MS, and UV spectroscopy. The results indicate the following composition: C₂₆H₄₂N₂O₅Cl₂NaPt(II), the metal Pt(II) being bound to two Cl^– and one bidentate CDCG moiety, i.e., Na[Pt CDCG(N,O) Cl₂]. The compound is highly soluble (up to 20 mM) in water and (up to 35 mM) in ethanol, methanol and DMSO. Hydrolysis was investigated because this is assumed to be an important step in intracellular activation and interaction with DNA of this type of compounds. The reaction kinetics of this complex in aqueous solution show unusual behaviour; the substitution process with the displacement of two Cl^– was almost instantaneous, and the resulting species were found to be very stable. Kinetic studies carried out in the presence of different NaCl concentrations (up to 500 mM) revealed similar fast and nonreversible aquations of Bamet-M2. This contrasts with the slow aquation of cisplatin in extracellular-line solutions (i.e., at high NaCl concentrations) as compared with fast hydrolysis in cells. This difference may partly account for the low cytostatic activity observed here for Bamet-M2 against several tumor cell-lines.     

A Regional Study of 4-Aminobutyrate Metabolism and Amino Acid Levels in Rat Brain Following Chronic Oral Administration of Ethanolamine O-Sulphate

Abstract: Ethanolamine O-sulphate (EOS) dissolved in the drinking water (5mg-ml⁻¹) was administered ad libitum to rats for 26 days. At the end of this period, glutamate decarboxylase (GAD) and GABA-transaminase (GABA-T) activities, 4-aminobutyrate (GABA) concentration, and the levels of six other amino acids were measured in various brain regions. Significant inhibition of GABA-T accompanied by significant increases in GABA content were observed throughout the brain, although the magnitudes of these effects varied according to region. GAD activity was significantly reduced in most brain regions, although this effect was apparently not related to cofactor availability or the direct actions of EOS or increased GABA concentration. Glutamine levels were significantly reduced to approximately 72% of control values in all brain regions. Aspartate levels were significantly reduced to approximately 84% of control values in all regions except the striatum and cerebellum. Minor changes in other amino acid levels were also detected. These neurochemical changes which accompanied the primary effect of EOS on GABA-T are discussed in terms of indirect secondary metabolic changes rather than nonspecific enzyme inhibition by EOS.     

载脂蛋白E基因敲除小鼠血脂及心肌酶学的测定及意义

目的:研究载脂蛋白E基因敲除(ApoE-/-)小鼠血脂及心肌酶学的变化,为利用其探讨动脉粥样硬化的病理生理进程提供实验依据。方法:选取28W龄雄性ApoE-/-小鼠6只和同性别、同周龄的野生型C57BL/6J(WT)小鼠10只,应用日立7600全自动生化分析仪分别测定其血清中甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白(LDL-C)、高密度脂蛋白(HDL-C)、载脂蛋白B(Apo-B)、超敏C反应蛋白(hs-CRP)、游离脂肪酸(NEFA)、肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)、乳酸脱氢酶(LDH)和缺血修饰白蛋白(ACB)的水平。结果:与WT鼠比,ApoE-/-小鼠血清中血脂指标TG、TC、LDL-C、hs-CRP和NEFA水平显著升高(P<0.01),而HDL-C仅为0.46±0.16mmol/L,远低于WT鼠的水平(1.86±0.26mmol/L)。心肌酶学指标CK、LDH明显高于对照组(P<0.01),缺血修饰白蛋白(IMA()55.61±3.50U/mL)明显低于对照组(72.47±4.26U/mL)(P<0.01)。CK-MB与对照组相比无显著性变化。结论:ApoE-/-小鼠血脂水平...     

生长激素释放肽的合成及促生长活性

用固相多肽合成法合成了生长激素释放肽(GHRP),这是一种含D-型氨基酸的外源性激素,具有促进脑下垂体分泌生长激素功能的人工合成六肽,其氨基酸序列为His-D-Trp-Ala-Trp-D-Phe-Lys-NH₂.观察了使用不同剂量的GHRP对不同日龄小鼠的促生长效应,当使用最佳剂量(1000μg/kg)时,可使25日龄小鼠体重较对照组增加14.8%,同时发现鼠龄越小对GHRP越敏感,当使用剂量高达10mg/kg时仍然安全无毒.     

Pigment Epithelium-Derived Factor (PEDF) Peptide Eye Drops Reduce Inflammation,Cell Death and Vascular Leakage in Diabetic Retinopathy in Ins2Akita Mice

Inflammation, neurodegeneration and microvascular irregularities are included in the spectrum of defects associated with diabetic retinopathy. Here, we evaluated intraocular deliverability features of two pigment epithelium-derived factor (PEDF) derivatives given as eye drops and their efficacy in modulating diabetes-induced retinal complications. The antiangiogenic PEDF60–77 (P60) and neuroprotective PEDF78–121 (P78) derivatives were applied to Ins2^Akita mouse eyes once a week for 15 wks at the onset of hyperglycemia. Peptides, labeled with Alexa Fluor 488, were observed penetrating the cornea by 1–4 h and gained access to the ciliary body, retinal pigment epithelium (RPE)-choroid complex, retina microvasculature and vitreous. Peak vitreous levels were 0.2 μg/mL for P60 and 0.9 μg/mL for P78 after 0.5 and 4 h, respectively. Both peptides reduced vascular leakage by ~60% and increased zona occludens 1 (ZO1) and occludin expression in the microvasculature to nondiabetic levels. P60 induced pERK1/2 and P78 promoted pAKT in Muller glia, two signals that were dampened in diabetic conditions. Pharmacologically inhibiting AKT signaling in the retina blocked effects of the peptides on ZO1 and occludin expression. P78 reduced levels of 9/20 cytokines in diabetic vitreous including interferon (IFN)-γ, interleukin (IL)-6, IL-3 and tumor necrosis factor (TNF)-α. P60 lowered levels of 6/20 cytokines but was less effective than P78. Neuroprotective P78 prevented diabetes-induced microglia activation by ~60%, retinal ganglion cell (RGC) death by ~22% and inner plexiform layer thinning by ~13%. In summary, we provide evidence that PEDF bioactive derivatives gained access to the retina by topical delivery and validated their efficacy in reducing diabetic retinopathy complications. Our findings argue for glia regulation of microvascular leakage and an early root cause for RGC degeneration embedded in microglia activation.     

Administration of Vigabatrin (γ-Vinyl-γ-Aminobutyric Acid) Affects the Levels of Both Inhibitory and Excitatory Amino Acids in Rat Cerebrospinal Fluid

The effect of vigabatrin (gamma-vinyl-gamma-aminobutyric acid), a new anticonvulsant drug, on the transmitter amino acids in rat cisternal CSF was studied. CSF was collected through a permanently implanted polyethylene cannula from freely moving rats at 5, 24, 48, and 96 h after administration of 1,000 mg/kg of vigabatrin. The free gamma-aminobutyric acid (GABA) level was elevated maximally (13.5-fold; p less than 0.01) at 24 h after injection. The homocarnosine (GABA-histidine) level also was increased (123%; p less than 0.01) at 24 h after injection, and its concentration remained at the same level for the next 3 days. Glycine and taurine concentrations had increased [31% (p less than 0.05) and 63% (p less than 0.01), respectively] at 5 h after injection. It is interesting that the levels of glutamate and aspartate increased [330% (p less than 0.05) and 421% (p less than 0.01), respectively] at 96 h after injection, the time when the free GABA level had returned to the baseline concentration and the vigabatrin level was 3% of the maximal concentration. The present study indicates that a single dose of vigabatrin in rats elevates levels of both the inhibitory and excitatory amino acids in CSF. However, the temporal profile of observed changes in relation to vigabatrin injection shows that neither the long-lasting elevation of GABA content nor the increase in glutamate and aspartate levels correlates with the level of vigabatrin in CSF. These findings suggest that the excitatory mechanisms are also augmented following acute administration of vigabatrin, especially when the content of GABA had decreased to the baseline level and the level of vigabatrin was low.     

N-CWS灌胃对小鼠移植性肿瘤及免疫功能的影响

为了考察红色诺卡氏菌细胞壁骨架(Nocardia rubra cell wall skeleton,N-CWS)灌胃给药对小鼠的体内抑瘤效应及其免疫调节作用,采用小鼠肉瘤S_(180)的移植性肿瘤模型,检测N-CWS灌胃给药的抑瘤活性;同时观察N-CWS体内细胞毒作用和对正常小鼠的毒性、免疫器官重量、巨噬细胞(MΦ)吞噬功能及脾淋巴细胞转化的影响.结果显示,小鼠灌服N-CWS的LD_(50)＞1.2 g/kg;N-CWS 200、400、800 mg/kg剂量灌胃小鼠对S_(180)有明显的抑制作用,其抑瘤率分别为63.33%、71.11%、64.88%;与对照组比较,N-CWS可增加免疫器官重量和提升外周血白细胞数量、能明显提高小鼠MΦ的吞噬活性及显著提高淋巴细胞转化率(P＜0.05),同时N-CWS激活了的MΦ对肿瘤细胞的细胞毒效应亦明显增强(P＜0.05).因此N-CWS适用于口服给药,对小鼠移植性肿瘤有明显的抑制作用,其抗肿瘤作用可能与增强机体免疫功能有关.     

Isolation and chemical synthesis of a major, novel biliary bile acid in the common wombat (Vombatus ursinus): 15alpha-hydroxylithocholic acid

The major bile acids present in the gallbladder bile of the common Australian wombat (Vombatus ursinus) were isolated by preparative HPLC and identified by NMR as the taurine N-acylamidates of chenodeoxycholic acid (CDCA) and 15alpha-hydroxylithocholic acid (3alpha,15alpha-dihydroxy-5beta-cholan-24-oic acid). Taurine-conjugated CDCA constituted 78% of biliary bile acids, and (taurine-conjugated) 15alpha-hydroxylithocholic acid constituted 11%. Proof of structure of the latter compound was obtained by its synthesis from CDCA via a Delta14 intermediate. The synthesis of its C-15 epimer, 15beta-hydroxylithocholic acid (3alpha,15beta-dihydroxy-5beta-cholan-24-oic acid), is also reported. The taurine conjugate of 15alpha-hydroxylithocholic acid was synthesized and shown to have chromatographic and spectroscopic properties identical to those of the compound isolated from bile. It is likely that 15alpha-hydroxylithocholic acid is synthesized in the wombat hepatocyte by 15alpha-hydroxylation of lithocholic acid that was formed by bacterial 7alpha-dehydroxylation of CDCA in the distal intestine. Thus, the wombat appears to use 15alpha-hydroxylation as a novel detoxification mechanism for lithocholic acid.     

Furin-cleaved Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Is Active and Modulates Low Density Lipoprotein Receptor and Serum Cholesterol Levels

Proprotein convertase subtilisin/kexin 9 (PCSK9) regulates plasma LDL cholesterol levels by regulating the degradation of LDL receptors. Another proprotein convertase, furin, cleaves PCSK9 at Arg²¹⁸-Gln²¹⁹ in the surface-exposed “218 loop.” This cleaved form circulates in blood along with the intact form, albeit at lower concentrations. To gain a better understanding of how cleavage affects PCSK9 function, we produced recombinant furin-cleaved PCSK9 using antibody Ab-3D5, which binds the intact but not the cleaved 218 loop. Using Ab-3D5, we also produced highly purified hepsin-cleaved PCSK9. Hepsin cleaves PCSK9 at Arg²¹⁸-Gln²¹⁹ more efficiently than furin but also cleaves at Arg²¹⁵-Phe²¹⁶. Further analysis by size exclusion chromatography and mass spectrometry indicated that furin and hepsin produced an internal cleavage in the 218 loop without the loss of the N-terminal segment (Ser¹⁵³–Arg²¹⁸), which remained attached to the catalytic domain. Both furin- and hepsin-cleaved PCSK9 bound to LDL receptor with only 2-fold reduced affinity compared with intact PCSK9. Moreover, they reduced LDL receptor levels in HepG2 cells and in mouse liver with only moderately lower activity than intact PCSK9, consistent with the binding data. Single injection into mice of furin-cleaved PCSK9 resulted in significantly increased serum cholesterol levels, approaching the increase by intact PCSK9. These findings indicate that circulating furin-cleaved PCSK9 is able to regulate LDL receptor and serum cholesterol levels, although somewhat less efficiently than intact PCSK9. Therapeutic anti-PCSK9 approaches that neutralize both forms should be the most effective in preserving LDL receptors and in lowering plasma LDL cholesterol.     

Repeated Oral Administration of a Squeezed Ginger (Zingiber officinale) Extract Augmented the Serum Corticosterone Level and Had Anti-Inflammatory Properties

We investigated the ability of a ginger extract to induce an immune response in RAW 264 cells and after a repeated oral administration to mice. The squeezed ginger extract augmented the production of tumor necrosis factor-α, interleukin-6, and monocyte chemotactic protein-1 when added to RAW 264 cells. This extract was collected as its ethanol-insoluble fraction. The oral administration of the squeezed ginger extract or its ethanol-insoluble fraction once or twice to mice also augmented the tumor necrosis factor-α production in peritoneal cells; however, its long-term administration had the opposite effect. The serum corticosterone level had increased after orally administering the squeezed ginger extract and was maintained during the administration period. Oral administration of the squeezed ginger extract also inhibited arachidonic acid-induced ear edema, but its repeated administration was needed to achieve an anti-inflammatory effect. These results suggest that the repeated administration of the aqueous constituents of ginger augmented the serum corticosterone level and that this may have gradually induced anti-inflammatory activity.