Anti-HBV effect of liposome-encapsulated matrine in vitro ana in vivo

AIM: To study the anti-HBV effect of liposome-encapsulated matrine (Lip-M) in vitro and in vivo. METHODS: 2.2.15 cell line was cultured in vitro observe the effect of Lip-M and matrine on the secretion of HBsAg and HBeAg. The toxicity of Lip-M and matrine to 2.2.15 cell line was also studied by MTT method. In in vivo study, drug treatment experiment was carried out on the 13th day after ducks were infected with duck hepatitis B virus (DHBV). The ducks were randomly divided into 4 groups with 5-6 ducks in each group. Lip-M and matrine were given to DHBV-infected ducks respectively by gastric perfusion. Four groups were observed: group of Lip-M (20 mg/kg), group of Lip-M (10 mg/kg), group of matrine (20 mg/kg) and group of blank model. The drug was given once daily for 20 d continuously, and normal saline was used as control. The blood was drawn from the posterior tibial vein of all ducks before treatment (T0), after the medication for 5 (T5), 10 (T10), 15 (T15), 20 (T20) d and withdrawl of the drug for 3 d (P3). The serum samples were separated and stored at -70 ℃, DHBV-DNA was detected by the dot-blot hybridization. RESULTS: After addition of Lip-M and matrine to 2.2.15 cell line for eleven d, the median toxic concentration (TC50) of Lip-M and matrine was 7.29 mg/mL and 1.33 mg/mL respectively. The median concentration (IC50) of Lip-M to inhibit HBsAg and HBeAg expression was 0.078 mg/mL and 3.35 mg/mL respectively. The treatment index (TI) value of Lip-M for HBsAg and HBeAg was 93.46 and 2.17 respectively, better than that of matrine. The DHBV-infected duck model treatment test showed that the duck serum DHBV-DNA levels were markedly reduced in the group of Lip-M (20 mg/kg) after treated by gastric perfusion for 10, 15 and 20 d (0.43±0.22 vs 0.95±0.18, t = 4.70, P= 0.001<0.01.0.40±0.12 vs 0.95±0.18, t = 6.34, P= 0.000<0.01. 0.22±0.10 vs 0.95±0.18, t = 8.30, P= 0.000<0.01), compared to the group of matrine (20 mg/kg) (0.43±0.22 vs 0.79±0.19, t = 3.17, P= 0.01<0.05. 0.40±0.12 vs 0.73±0.24, t = 3.21, P= 0.009<0.05. 0.22±0.10 vs0.55±0.32, t = 2.27, P= 0.046<0.05.), and the control (0.43±0.22 vs50.98±0.29, t = 3.68, P = 0.005<0.01. 0.40±0.12 vs 0.97±0.30, t = 4.26, P= 0.002<0.01. 0.22±0.10 vs 0.95±0.27, t = 5.76, P= 0.000<0.01). After the treatment for 20 d and withdrawl of the drug for 3 d, duck serum DHBV-DNA level in the group of Lip-M (10 mg/kg) markedly reduced (0.56±0.26 vs0.95±0.38, t = 5.26, P= 0.003<0.05. 0.55±0.25 vs 0.95±0.38, t = 5.52, P= 0.003<0.05), and the difference was significant as compared with the control (0.56±0.26 vs 0.95±0.27, t = 2.37, P = 0.042<0.05. 0.55±0.25 vs 0.89±0.18, t = 2.55, P= 0.031<0.05), but not significant as compared with the group of matrine (20 mg/kg). After withdrawl of the drug for 3 d, the levels of DHBV-DNA did not relapse in both groups of Lip-M. CONCLUSION: Lip-M can evidently inhibit the replication of hepatitis B virus In vitro and in viva, its anti-HBV effect is better than that of matrine.     

Paclitaxel: a chemotherapeutic agent for prevention of restenosis? Experimental studies in vitro and in vivo

Paclitaxel, a potent anti-tumor agent, shifts the cytoskeleton equilibrium towards assembly of altered and extraordinarily stable microtubules. These cellular modifications lead to reduced proliferation, migration, and signal transduction. It is highly lipophilic, which promotes a rapid cellular uptake, and has a long-lasting effect in the cell due to the structural alteration of the cytoskeleton. This makes paclitaxel a promising candidate for local drug delivery intended to address the proliferative and migratory processes involved in restenosis. In this article, results of our in vitro and in vivo studies with paclitaxel are presented. Cell culture experiments with monocultures of human arterial smooth muscle cells as well as co-cultures with human endothelial cells showed that paclitaxel leads to an almost complete growth inhibition within a dose range of 1.0-10.0 mumol/l, even after a short (20 min) single dose application. The comparison of an active, semi-active, and passive delivery system (porous balloon, microporous balloon, and double balloon) favored the double balloon for the following in vivo experiments. Tubulin staining and electron microscopy enabled visualization of paclitaxel-induced vessel wall alterations. In the rabbit model, locally delivered paclitaxel resulted in reduced neointima formation and enlargement in vessel size; in the pig model, however, after stenting, this inhibition was not significant. Both reduced proliferation and enlargement in vessel size contribute to a preservation of vessel shape and are likely to be caused by a structural alteration of the cytoskeleton, which is also supported by vascular contraction force experiments.     

The significance of minimal residual disease in stem cell grafts and the role of purging: is it better to purge in vivo or in vitro?

Contamination of autologous graft by tumor, in addition to incomplete tumor eradication, can partly explain why relapse remains the commonest cause of treatment failure after autologous stem cell transplantation (ASCT) in patients with malignant hematologic disorders. Monitoring of minimal residual disease (MRD) is now recognized as an important diagnostic tool for assessment either of the response to treatments aimed at maximal cytoreduction and the individual risk of relapse. In order to improve cure rates, many strategies to achieve in vivo or in vitro reduction, if not eradication, of residual disease have been proposed. We discuss the significance of MRD and the role of purging in the ASCT setting, focusing on acute myeloid leukemia, chronic myeloid leukemia, multiple myeloma and follicular lymphoma.     

The metabolic effect of α-ketoisocaproic acid: in vivo and in vitro studies

Metabolic Brain Disease - Maple syrup urine disease (MSUD) is characterized by a deficiency in the mitochondrial branched-chain α-keto acid dehydrogenase complex activity and, consequently,...     

Assessment of resin perfusion in hepatic failure in vitro and in vivo

AIM:To observe the adsorbent effect of resin on endotoxin,cytokine,bilirubin in plasma of patients with hepatic failureand to determine the resin perfusion as an artificial liversupport system in the treatment of hepatic failure.METHODS:One thousand milliliters of discarded plasmawas collected from each of 6 severe hepatitis patients treatedwith plasma exchange.The plasma was passed through aresin perfusion equipment for 1-2 h via extracorporealcirculation,and then absorbent indicators of transaminase,bilirubin,blood ammonia,endotoxin and cytokines wereexamined.In the meantime,study of in vivo resin plasmaperfusion was performed on 7 severe hepatitis patients tocompare the changes of endotoxin and cytokines in bloodbefore and after perfusion.RESULTS:The levels of total bilirubin,endotoxin,interleukin1β and TNF-α in plasma were significantly decreased afterin vitro resin plasma perfusion.The levels of interleukin 1β,TNF-α and endotoxin in blood were also evidently declinedafter in vivo resin plasma perfusion.Nevertheless,no obviouschanges in IL-6,creatinine (Cr) and urea nitrogen (UN),bloodammonia and electrolytes were found both in vitroand in vivo.CONCLUSION:Bilirubin,endotoxin and cytokines in plasmaof patients with hepatic failure can be effectively adsorbedby resin in vitro.Most cytokines and endotoxin in plasma canalso be effectively removed by resin in vivo.It demonstratesthat resin perfusion may have good treatment efficacy onhepatic failure and can be expected to slow down theprogression of hepatic failure.     

Hepatoprotective evaluation of Anogeissus latifolia： In vitro and in vivo studies

AIM： To evaluate the hepatoprotective activity of a hydroalcoholic extract of the bark of Anogeissus latifolia; in vitro in primary rat hepatocyte monolayer culture and in vivo in the liver of Wistar rats intoxicated by carbon tetrachloride （CCl4）. METHODS： In the in vitro study, a primary hepatocyte monolayer culture was treated with CCh and extract of Anogeissus latifolia. Hepatoprotective activity was demonstrated in the CCh damaged primary monolayer culture. In the in vivo study, the hepatoprotective activity of a hydroalcoholic extract ofAnogeissus latifolia was analyzed in liver injured CCh-treated rats. Biochemical parameters including serum transaminases [aspartate aminotransferase （AST） and alanine aminotransferase （ALT）] and alkaline phosphatase （ALP） in serum wereanalyzed. The biochemical findings were supplemented with histopathological examination of rat liver sections. RESULTS： In vitro： primary hepatocyte monolayer cultures were treated with CCh and extract of Anogeissus latifolia. A protective activity could be demonstrated in the CCh damaged primary monolayer cultUre. In vivo： Hydroalcoholic extract of Anogeissus latifolia （300 mg/kg） was found to have protective activity in rats with CCh-induced liver damage as judged from serum marker enzyme activity. CONCLUSION： The above findings lead to the conclusion that the hydroalcoholic extract of Anogeissus latifol/a is hepatoprotective. Hence, we suggest that the inclusion of this plant in the management of liver disorders is justified.     

Inhibitory role of oxytocin on TNFα expression assessed in vitro and in vivo

Aim

Oxytocin administration to diet-induced obese (DIO) rodents, monkeys and humans decreases body weight and fat mass with concomitant improvements in glucose metabolism. Moreover, several studies show an immunomodulatory role of oxytocin in a number of settings (such as atherosclerosis, injury, sepsis). This study aims to shed some light on the effects of oxytocin on macrophage polarization and cytokine production, as well as its possible impact on these parameters in adipose tissue in DIO mice with impaired glucose metabolism.

Does caffeine influence the anticholinesterase and antioxidant properties of donepezil? Evidence from in vitro and in vivo studies

Metabolic Brain Disease - Caffeine is adjudged world’s most consumed pharmacologically active food component. With reports of the potential cognitive enhancing properties of caffeine, we...     

Comparison of three in vitro human ‘angiogenesis’ assays with capillaries formed in vivo

Angiogenesis assays are an important tool for studying both the mechanisms of angiogenesis and the potential development of therapeutic strategies to modulate neovascularisation. In vivo angiogenesis assays are considered to be the most informative of these but are often expensive, time-consuming and require specialist training to perform. In vitro assays tend to be more rapid, less expensive and easier to interpret. In vitro angiogenesis assays operate on the principle that endothelial cells form tubule-like structures when cultured on a supportive matrix. Assays involving a matrix derived from murine tumours, Matrigel (or a growth factor reduced form of this), are now the most common in vitro tubule formation assays. However, another tubule formation assay has recently been developed in which endothelial cells are co-cultured with fibroblasts. Here, we have used quantitative image analysis to compare the morphological features of tubules formed in the Matrigel assay and this new ‘Co-culture’ assay, with those of capillaries formed in a microvascular bed in vivo. Tubules formed in standard and growth factor reduced Matrigel assays were short and relatively homogeneous, whereas those formed in the Co-culture assay were significantly more heterogeneous, consisting of both short and long interconnecting tubules that more closely resembled capillaries than Matrigel tubules. Moreover, cells on Matrigel, and to a lesser extent growth factor reduced (GFR) Matrigel, often clumped into large cell aggregates, a feature rarely seen in the Co-culture assay. In addition, we demonstrate that Matrigel stimulates tubule formation by various non-endothelial cell types, suggesting that tubule formation by endothelial cells may not represent true differentiation of this cell type. In summary, the morphology of tubules in the Co-culture assay appears more representative of capillary formation in vivo, than the endothelial cell changes that occur in either form of Matrigel assay. This revised version was published online in June 2006 with corrections to the Cover Date.     

Transvascular passage of macromolecules into the peritoneal cavity of normo- and hypothermic rats in vivo: active or passive transport?

During the last decades there has been a debate regarding whether transvascular protein transport is an active (transcytosis) or a passive (porous) process. To separate cooling-sensitive transcytosis from passive transport processes between blood and peritoneal fluid, we induced hypothermia in rats in vivo, reducing their body temperature to 19 degrees C. Control rats were kept at 37 degrees C. Either human albumin, or IgG, or IgM, or LDL, radiolabeled with (125)I, was given intra-arterially together with (51)Cr-EDTA. During tracer administration, a 2-hour peritoneal dialysis dwell was performed. Clearance of the tracers to dialysate, and the permeability-surface area coefficient (PS) for (51)Cr-EDTA and glucose were assessed. During cooling, mean arterial blood pressure (MAP) was reduced to 40% of control and plasma viscosity increased by 48.5%, while peritoneal blood flow was reduced to 10%. At 19 degrees C, clearance of albumin to dialysate fell from 9.30 +/- 1.62 (SEM) to 3.13 +/- 0.28 microl/min (p < 0.05), clearance of IgG from 6.33 +/- 0.42 to 2.54 +/- 0.12 microl/min (p < 0.05), clearance of IgM from 3.65 +/- 0.33 to 1.10 +/- 0.12 microl/min (p < 0.05), and clearance of LDL from 3.54 +/- 0.20 to 0.73 +/- 0.06 microl/min (p < 0.05). The fall in PS for (51)Cr-EDTA was from 0.320 +/- 0.01 to 0.075 +/- 0.003 ml/min (p < 0.05), and that for glucose from 0.438 +/- 0.02 to 0.105 +/- 0.01 ml/min (p < 0.05). Tissue cooling reduced large solute transport largely in proportion to the cooling-induced reductions of MAP (to 40%), and the concomitant increase in viscosity (to 67%), i.e. to approximately 20-30% (0.40 x 0.67) of control, though LDL clearance was reduced further. The fall in small solute PS, in excess of the viscosity effect, mirrored the fall in peritoneal blood flow occurring during hypothermia. In conclusion, the good correlation of predicted to calculated changes suggests that the overall transendothelial macromolecular passage in vivo occurs passively, and not due to active processes.     

Intestinal hydrolysis of pyridoxal 5′-phosphatein vitro andin vivo in the rat

The first step in the intestinal absorption of phosphorylated forms of vitamin B₆ is intraluminal hydrolysis mediated by alkaline phosphatase. The present studies were performed to evaluate the effect of amino acids and oligopeptides, the products of protein digestion, on the hydrolysis of pyridoxal 5-phosphate. Models utilized rats and included a cell-free in vitro system and the in vivo, single-pass, perfused jejunal segment. In vitro all amino acids and oligopeptides tested significantly inhibited pyridoxal 5-phosphate decay (hydrolysis). The degree of inhibition of decay was dependent on the particular compound used, the concentration of that compound, and the pH of the medium. Similar effects forl-lysine concentration and perfusate pH were demonstrated in perfused segments in vivo; by contrast,l-lysine had no effect on pyridoxine uptake. These studies demonstrate that intraluminal hydrolysis of phosphorylated vitamin B₆ may be modulated by yet other intraluminal constituents and conditions.Supported by the Department of Veterans Affairs.     

A possible role of osteocalcin in the regulation of insulin secretion: human in vivo evidence?

Recent studies have indicated a novel function for skeleton unraveling its importance in the control of energy metabolism. In the present commentary, we speculate on the meaning for bone to act as a 'rheostat' modulating glucose metabolism, and how the primitive way of communication between bone and energy metabolism through switch on/off genes (like Ptprv) evolved to a more complicated 'talking' via gain/loss of hormones activity (like osteocalcin) by carboxylation/decarboxylation process.     

Induction of apoptosis in Eμ-myc lymphoma cells in vitro and in vivo through calpain inhibition

Calpains are cysteine proteases that have been implicated as both effectors and suppressors of apoptosis. Previously, we showed that c-myc transformation regulated calpain activity and sensitized cells to apoptosis induced by calpain inhibition. The objective of this study was to investigate the role of calpain in the Eμ-myc transgenic model of B-cell lymphoma. Calpain activity assays, apoptosis, cell cycle assays, and expression measurements were used to determine the activity and role of calpain in vitro and in vivo. We found that Eμ-myc transgenic cells have highly elevated calpain activity. Calpastatin, the negative calpain regulator, was expressed at much lower levels in Eμ-myc lymphoma cells compared to normal splenic B cells. The primary isoform in Eμ-myc lymphoma is calpain 1. Treatment of Eμ-myc lymphoma cells with the calpain inhibitors PD150606 or calpain inhibitor III induced caspase-3-dependent apoptosis in vitro. General caspase inhibitors or caspase-3/7 inhibitor protected cells from death induced by calpain inhibitor, whereas caspase-9 inhibitors failed to rescue cells. Human Burkitt's lymphoma (BL2) cells display a pattern of sensitivity and caspase-3 dependence similar to calpain inhibition. Treatment of Eμ-myc lymphoma-bearing mice with PD150606 inhibited calpain activity in vivo and induced cell death in these cells as determined by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining. Multiple daily treatments resulted in reduced tumor load, particularly in combination with etoposide. In conclusion, calpain is highly elevated in the Eμ-myc lymphoma and calpain inhibition has therapeutic potential.