Oxidative Metabolism of Estrone Modified by Genistein and Bisphenol A in Rat Liver Microsomes

Genistein, the main isoflavone from soy, and bisphenol A （BPA）, a food contaminant, are considered ubiquitous xenoestrogens. Here we investigated the influence of genistein and BPA on estrone （El） metabolism in rat liver microsomes. Both substances inhibited the 2-hydroxylation and 16a-hydroxylation of E1, but in different degrees, thereby reducing the 2-OH-E1/16a-OH-E1 ratio,

Effect of total flavonoids of epimedium on liver microsomal CYP 1A2,CYP3A4 and CYP2E 1 activities in rats

To assess the potential effect of total flavonoids of epimedium （TFE） on cytochrome P450 and activity of its main isoforms in rat liver microsomes. TFE （300 mg/kg） was administered once daily to male Sprague-Dowley rats by gavage for fifteen days. The total cytochrome P450 content and its main isoforms CYP1A2, CYP3A4 and CYP2E1 activities in rat liver microsomes were detected. The activity of CYP1A2 was measured by fluorometry and the activities of CYP3A4 and CYP2E1 were determined by measuring the amount of methanal and p-aminophenol formed using UV/Vis spectrophotometer, respectively. Administration of TFE significantly increased the total CYP450 content and activities ofCYP1A2, CYP3A4 and CYP2E1 in rat liver microsomes, compared with the control group. Particularly, the activities of CYP1A2 and CYP2E1 were enhanced significantly （P〈0.01）. TFE induced the increase in total CYP450 content and its main isoforms CYP1A2, CYP3A4 and CYP2E1 activities in rat liver microsomes.

Identification of the metabolite and cytochrome P450 isoforms involved in rat liver microsomal metabolism of TM208

To identify the metabolite and CYP450 isoforms involved in rat liver microsomal metabolism of TM208. The present study investigated the metabolism of TM208 and the effects of selective CYP450 inhibitors on the metabolism of TM208 in rat liver microsomes. Various specific inhibitors of CYP were used to identify the isoforms of CYP involved in the metabolism of TM208. The inhibitor of CYP2D and that of CYP2B had strong inhibitory effects on TM208 metabolism in a concentration-de- pendant manner, the inhibitor of CYP1A had a modest inhibitory effect, and the inhibitor of CYP3A seemed not to have an obvious inhibitory effect on TM208 metabolism. TM208 might mainly be metabolized by CYP2D and CYP2B in rat liver microsomes.

Chiral metabolism of propafenone in rat hepatic microsomes treated with two inducers

AIM: To study the influence of inducers of drug metabolism enzyme, beta-naphthoflavone (BNF) and dexamethasone (DEX), on the stereoselective metabolism of propafenone in the rat hepatic microsomes. METHODS: Phase I metabolism of propafenone was studied using the microsomes induced by BNF and DEX and the non-induced microsome was used as the control. The enzymatic kinetics parameters of propafenone enantiomers were calculated by regress analysis of Eadie-Hofstee Plots. Propafenone enantiomer concentrations were assayed by a chiral HPLC. RESULTS: The metabolite of propafenone, N-desalkylpropafenone, was found after incubation of propafenone with the rat hepatic microsomes induced by BNF and DEX. In these two groups, the stereoselectivity favoring R(-) isomer was observed in metabolism at low substrate concentrations of racemic propafenone, but lost the stereoselectivity at high substrate concentrations. However, in control group, no stereoselectivity was observed. The enzyme kinetic parameters were: (1) K(m). Control group: R(-) 83+/-6, S(+) 94+/-7; BNF group: R(-) 105+/-6, S(+)128+/-14; DEX group: R(-) 86+/-11, S(+) 118+/-16; (2)V(max). Control group: R(-) 0.75+/-0.16, S(+) 0.72+/-0.07; BNF group: R(-) 1.04+/-0.15, S(+)1.07+/-14; DEX group: R(-) 0.93+/-0.06, S(+) 1.04+/-0.09; (3)Cl(int). Control group: R(-) 8.9+/-1.1, S(+) 7.6+/-0.7; BNF group: R(-) 9.9+/-0.9, S(+)8.3+/-0.7; DEX group: R(-) 10.9+/-0.8, S(+) 8.9+/-0.9. The enantiomeric differences in K(m) and Cl(int) were both significant, but not in V(max), in BNF and DEX group. Whereas enantiomeric differences in three parameters were all insignificant in control group. Furthermore, K(m) and V(max) were both significantly less than those in BNF or DEX group. In the rat liver microsome induced by DEX, nimodipine (NDP) decreased the stereoselectivity in propafenone metabolism at low substrate concentration. The inhibition of NDP on the metabolism of propafenone was stereoselective with R(-)-isomer being impaired more than S(+)-isomer. The inhibition constant (Ki) of S(+)- and R(-)-propafenone, calculated from Dixon plots, was 15.4 and 8.6 mg x L(-1), respectively. CONCLUSION: CYP1A subfamily(induced by BNF) and CYP3A4 (induced by DEX) have pronounced contribution to propafenone N-desalkylation which exhibited stereoselectivity depending on substrate concentration. The molecular base for this phenomenon is the stereoselectivity in affinity of substrate to the enzyme activity centers instead of at the catalyzing sites.

Changes of Liver Microsomal Drug-metabolizing System and Lipoperoxidation Activity in Scalded Rats and the Effects of Silybin

The dynamic changes of liver microsomal drug-metabolizing system (MDMS) andlipoperoxidation were studied in scalded rats. The effects of treatment with vitamin E and silybinwere also evaluated. The results showeed that liver microsomal cytochrome P-450 content, and p-nitroanisole demethylase (P-NOD) and aniline hydroxylase (AH) activity decreased markedlypostburn. On the contrary, liver lipoperoxide and mierosomal lipoperoxidation increased significantlyafter scalding. Both the increase of liver lipoperoxide and mierosomal lipoperoxidation and the de-crease of MDMS activity were prevented by vitamin E and silybin treatments.

Inhibiting effect of antisense oligonucleotides phosphorthioate on gene expression of TIMP-1 in rat liver fibrosis

AIM: To observe the inhibition of antisense oligonucleotides (asON) phosphorthioate to the tissue inhibitors metalloproteinase-1 (TIMP-1) gene and protein expression in the liver tissue of immunologically induced hepatic fibrosis rats. The possibility of reversing hepatic fibrosis through gene therapy was observed. METHODS: Human serum albumin (HSA) was used to attack rats, as hepatic fibrosis model, in which asONs were used to block the gene and protein expressing TIMP-1. According to the analysis of modulator, structure protein, coding series of TIMP-1 genome, we designed four different asONs. These asONs were injected into the hepatic fibrosis models through coccygeal vein. The results was observed by RT-PCR for measuring TIMP-1 mRNA expression, immunohistochemistry and in situ hybridization for collagen I, II, special staining of collagen fiber, and electron microscopic examination. RESULTS: Hepatic fibrosis could last within 363 days in our modified model. The expressing level of TIMP-1 was high during hepatic fibrosis process. It has been proved by the immunohistochemical and the electron microscopic examination that the asON phosphorthioate of TIMP-1 could exactly express in vivo. The effect of colchicine was demonstrated to inhibit the expressing level of mRNA and the content of collagen I, III in the liver of experimental hepatic fibrosis rats. However, the electron microscopy research and the pathologic grading of hepatic fibrosis showed that there was no significant difference between the treatment group and the model group (P】 0.05). CONCLUSION: The experimental rat model of hepatic fibrosis is one of the preferable models to estimate the curative effect of anti-hepatic fibrosis drugs. The asON phosphorthioate of TIMP-1 could block the gene and protein expression of TIMP-1 in the liver of experimental hepatic fibrosis rats at the mRNA level. It is possible to reverse hepatic fibrosis, and it is expected to study a new drug of antihepatic fibrosis on the genetic level. Colchicine has very limited therapeutic effect on hepatic fibrosis, furthermore, its toxicity and side effects are obvious.

Metabolism of Praziquantel in Erythromycin, Acetylspi-ramycin and Dexamethasone Induced Rat Liver Micro-somes

Our results show that in liver microsomes from erythromycin,acetylspiramycin and dexamethsone pretreated rats,the rate of praziquantel( PQT)disappearence was increased as compared with control rat When microsomes from erythromycin-treated rats were exposed to PQT in the presence of potassium ferricyanide which broke down the cytochrome P-450 Fe(Ⅱ)-metabolite complexes and restored the functional cytochrome P-450,PQT metabolism was further increased. Acetylspiramycin did not form the complexes, so potassium ferricyanide showed no effect on the PQT metabolism in microsomes from acetylspiramycin-treated rats. Triacetyloleandomycin,a specific inhibitor of cytochrome P-450ⅢAI, inhibited PQT metabolism by 53% in liver microsomes from dexamethasone-treated rats.These results indicate the cytochrome P-450ⅢA seems to be involved in metabolism of PQT in rat liver microsomes.

Safe upper limit of intermittent hepatic inflow occlusion for liver resection in cirrhotic rats

AIM: To evaluate the effects of varying ischemic durations on cirrhotic liver and to determine the safe upper limit of repeated intermittent hepatic inflow occlusion. METHODS: Hepatic ischemia in cirrhotic rats was induced by clamping the common pedicle of left and median lobes after non-ischemic lobes resection. The cirrhotic rats were divided into six groups according to the duration and form of vascular clamping: sham occlusion (SO), intermittent occlusion for 10 (IO-10), 15(IO-15), 20(IO-20) and 30(IO-30) minutes with 5 minutes of reflow and continuous occlusion for 60 minutes (CO-60). All animals received a total duration of 60 minutes of hepatic inflow occlusion. Liver viability was investigated in relation of hepatic adenylate energy charge (EC). Triphenyltetrazollum chloride (TTC) reduction activities were assayed to qualitatively evaluate the degree of irreversible hepatocellular injury. The biochemical and morphological changes were also assessed and a 7-day mortality was observed. RESULTS: At 60 minutes after reperfusion following a total of 60 minutes of hepatic inflow occlusion, EC values in IO-10 (0.749 +/- 0.012) and IO-15 (0.699 +/- 0.002) groups were rapidly restored to that in SO group (0.748 +/- 0.016), TTC reduction activities remained in high levels (0.144 +/- 0.002 mg/mg protein, 0.139 +/- 0.003 mg/mg protein and 0.121 +/- 0.003 mg/mg protein in SO, IO-10 and IO-15 groups, respectively). But in IO-20 and IO-30 groups, EC levels were partly restored (0.457 +/- 0.023 and 0.534 +/- 0.027) accompanying with a significantly decreased TTC reduction activities (0.070 +/- 0.005 mg/mg protein and 0.061 +/- 0.003 mg/mg protein). No recovery in EC values (0.228 +/- 0.004) and a progressive decrease in TTC reduction activities (0.033 +/- 0.002 mg/mg protein) were shown in CO-60 group. Although not significantly different, the activities of the serum aspartate aminotransferase (AST) on the third postoperative day (POD(3)) and POD(7) and of the serum alanine aminotransferase (ALT) on POD(3) in CO-60 group remained higher than that in intermittent occlusion groups. Moreover, a 60% animal mortality rate and more severe morphological alterations were also shown in CO-60 group. CONCLUSION: Hepatic inflow occlusion during 60 minutes for liver resection in cirrhotic rats resulted in less hepatocellular injury when occlusion was intermittent rather than continuous. Each period of 15 minutes was the safe upper limit of repeated intermittent vascular occlusion that the cirrhotic liver could tolerate without undergoing irreversible hepatocellular injury.

Effects of glycyrrhetinic acid on collagen metabolism of hepatic stellate cells at different stages of liver fibrosis in rats

INTRODUCTIONLiver fibrosis is a dynamic course leading tocirrhosis from a various chronic liver diseases. Thepathological basis of fibrosis is the disturbance ofproduction and degradation of the extracellularmatrix (ECM), which causes accumulation of ECMin the liver[1,2].

Heat shock protein 72 normothermic ischemia,and the impact of congested portal blood reperfusion on rat liver

INTRODUCTIONFrom the technical aspect of liver surgery ,control of bleeding during hepatic parenchymal resection is one of the most important procedures in hepatectomy .Pringle,s maneuver ,a temporary cross-clamping of the hepatoduodnal ligament ,has often been used for this purpose[1],This is the simplest and userul technique to reduce intraoperative blood loss .

Protective effect of exogenous adenosine triphosphate on hypothermically preserved rat liver

AIM:To clarify the protective effect of exogenous adenosine triphosphate(ATP)on hypothermically preserved rat livers. METHODS:Establishment of continuous hypothermic machine perfusion model,detection of nucleotides in hepatocytes with HPLC,measurement of activities of LDH and AST in the perfusate,observation of histopathological changes in different experiment groups,and autoradiography were carried out to reveal the underlying mechanism of the protective effect of ATP. RESULTS:The intracellular levels of ATP and EC decreased rapidly after hypothermic preservation in control group,while a higher ATP and EC level,and a slower decreasing rate were observed when ATP-MgCl_2 was added to the perfusate (P<0.01).As compared with the control group,the activities of LDH and AST in the ATP-MgCl_2 group were lower(P<0.05). Furthermore,more severe hepatocyte damage and neutrophil infiltration were observed in the control group.Radioactive [α-^(32)P]ATP entered the hypothermically preserved rat hepatocytes. CONCLUSION:Exogenous ATP has a protective effect on rat livers during hypothermical preservation.However,Mg^(2+) is indispensable,addition of ATP alone produces no protective effect.The underlying mechanism may be that exogenous ATP enters the hypothermically preserved rat liver cells.

Japanese herbal medicine, Saiko-keishi-to, prevents gut ischemia/reperfusion-induced liver injury in rats via nitric oxide

AIM:To determine whether Saiko-keishi-to(TJ-10),a Japanese herbal medicine,could protect liver injury induced by gut ischemia/reperfusion(I/R),and to investigate the role of NO. METHODS:Male Wistar rats were exposed to 30-min gut isohemia followed by 60 min of reperfusion.Intravital microscopy was used to monitor leukocyte recruitment.Plasma tumor necrosis factor(TNF)levels and alanine aminotransferase (ALT)activities were measured.TJ-10 1 g/(kg.d)was intragastrically administered to rats for 7 d.A NO synthase inhibitor was administered. RESULTS:In control rats,gut I/R elicited increases in the number of stationary leukocytes,and plasma TNF levels and ALT activities were mitigated by pretreatment with TJ-10.Pretreatment with the NO synthase inhibitor diminished the protective effects of TJ-10 on leukostasis in the liver,and the increase of plasma TNF levels and ALT activities.Pretreatment with TJ-10 increased plasma nitrite/nitrate levels. CONCLUSION:TJ-10 attenuates the gut I/R-induced hepatic microvascular dysfunction and sequential hepatocellular injury via enhancement of NO production.

The Hydroxylation of Vitamin D on C25 in Thyrotoxicosis The Role of the Activity of Microsomal Liver Enzymes

Vitamin D3 after its entrance in the organism undergoes hydroxylation on C-25 carbon atom by the action of microsomal liver enzymes giving the metabolite 25 hydroxyvitamin D3 (25OHD3). The function of microsomal liver enzymes is influenced in some specified states by hormones or drugs. It has approved that thyroxin is a potent stimulator of these enzymes while allopurinol suppresses their function. The aim of this issue is to examine 25OHD3 plasma levels in thyrotoxic subjects and in those pretreated with allopurinol on the base of the afford mentioned data. In a first phase 25OHD3 plasma levels were estimated in thyrotoxic subjects against euthytoid healthy controls. In a second phase lmg vitamin D3 was injected intravenously (i.v.) in thyrotoxic subjects and in healthy euthyroid controls. 25OHD3 plasma levels were measured before and in post injection period in six hours intervals for 48 hours. In a third phase a couple of subjects one thyrotoxic and one euthyroid healthy control pretreated both with allopurinol injected lmg of vitamin D3 i.v. In all studied subjects 25OHD3 plasma levels were measured before and in post injection period in six hours intervals for 48 hours. The pre and post injection 25OHD3 plasma levels measured the size of activity of liver enzyme responsible for bioactivation of vitamin D3. In the first phase was indicated that 25OHD3 plasma levels were lower in thyrotoxic subjects comparing with that of euthyroid healthy controls (p 3 in thyrotoxic subjects was 2,5 to 8 times faster comparing with euthyroid healthy controls. In the third phase was shown that allopurinol decreases the activity of liver enzymes function as regard the bioactivation of vitamin D3. The bioactivation of vitamin D3 is accelerated in thyrotoxicosis compared with that in euthyroid state. This phenomenon produces low 25OHD3 plasma levels in thyrotoxic subjects which initially may be normal or slightly increased depended from the vitamin D3 status in the thyrotoxic subjects. By continuous stimulatory action of increased thyroid hormones on liver enzymes the 25OHD3 plasma levels earlier or later decline in levels of hypo-or avitaminosis D3. The previously described biological events may explain the decreased intestinal calcium absorption of vitamin D3 and the osteomalacic component found in a percentage of thyrotoxic bone histology. For the blocking effects of allopurinol on liver enzymes function and possibly of other pharmaceutical products in relation to vitamin D3 bioactivation, available data are still lacking.

Interaction of ergotamine with liver Cytochrome P450 3A in rats

This study was conducted to investigate the effect of the ergot alkaloid, ergotamine (ET), on the induction of CYP3A and the interaction in vivo and in vitro with ET. Sprague-Dawley rats were treated intraperitoneally for 4 days as follows: control (injecting with 0.5 ml of only corn oil);dexamethasone treatment (injecting with 100mg/kg of dexamethasone in corn oil);and ergotamine treatment (injecting with 100mg/kg of ergotamine in corn oil). Liver tissues were collected from each group (n = 5, total of 30 rats) and liver microsomes were prepared. Cytochrome CYP3A activity was evaluated using ET and its isomer as substrates in medium containing liver microsomes and NADPH at 37℃ for 30 min. HPLC was used to measure the disappearance of the substrate and the appearance of the metabolites. Liver microsomes from rats pretreated with dexamethasone were five times more (P 0.05) in activity of CYP3A when compared to the control group (5.2 vs. 7.0 nM ET/min/mg protein;SE = 4.83) or ET isomer (1.5 vs. 4.7 nM ET isomer/ min/mg protein;SE = 1.70). When ketoconazole was used as specific inhibitor of CYP3A, ergotamine metabolisms were inhibited in a dose dependent fashion reaching a maximum at an inhibitor to substrate ratio of greater than one and LD50 at 0.5 nM of ketoconazole/mg protein. The data presented in this study suggest that although the ergot alkaloids ergotamine and its isomer are ideal substrates for the isozyme CYP3A, these compounds have no effect on the induction of CYP3A after 4 days of treatment.

Epidemiological and histopathological study of relevance of Guizhou Maotai liquor and liver diseases

AIM: To explore the relevance of Maotai liquor and liver diseases. METHODS: Epidemiological study was conducted on groups of subjects, each consisting of 3 subjects from the Maotai liquor group consisting of 99 individuals and one from the non-alcoholic control group consisting of 33 individuals. Liver biopsy was performed on 23 volunteers from Guizhou Maotai Distillery who had a constant and long history of drinking Maotai liquor. Experimental histopathological study was conducted as follows: sixty male Wistar rats were divided into 3 groups randomly and fed with Maotai liquor, ordinary white wine, and physiological saline respectively for a period of 8 and 12 weeks. The rats were sacrificed in batches, then serum ALT, AST, TBil, and AKP were measured. Rat livers were harvested to measure the liver indexes, GSH, and MDA. Histopathological examinations were also performed. Another eighty mice were randomly divided into 4 groups and fed with Maotai (at different dosages of 10 ml.kg(-1) and 20 ml.kg(-1)), ethanol, and physiological saline. The animals were sacrificed after 4 weeks and serum ALT was determined. Then the livers were harvested and liver indexes and MDA were measured. RESULTS: The incidence rate of hepatic symptoms, splenomegaly, liver function impairment, reversal of Albumin/Globulin and increased diameter of portal veins in the Maotai liquor group were 1.0% 1/99 , 1.0% 1/99 , 1.0% 1/99 , 1.0% 1/99 , 0 0/99 and 0 0/99 , 0 0/99 ,0 0/99 , 0 0/99 , 0 0/99 , respectively. There was no significant difference between the Maotai group and the non-alcoholic control group P】0.05 . Various degree of fatty infiltration of hepatocytes was found in the 23 volunteers receiving liver biopsy, but there was no obvious hepatic fibrosis or cirrhosis. A comparison was made between the Maotai liquor group and the ordinary white wine group. It was found that hepatic MDA in rats and mice were 0.33+/-0.10 and 0.49+/-0.23 respectively in Maotai group and 0.61+/-0.22 and 0.66+/-0.32 in the ordinary white wine group; MDA had an obvious decrease in the Maotai liquor group (P【0.05); hepatic GSH were 0.12 mg.g(-1)+/-0.06 mg.g(-1) in rats of the Maotai liquor group and (0.08+/-0.02)mg.g(-1) in white wine group, it was obviously increased in the Maotai liquor group (P【0.05). After the 20 rats had been fed with ordinary white wine for 8 weeks consecutively, disarranged hepatocyte cords, fatty infiltration of hepatocytes, and fibrous septa of varying widths due to hepatic connective tissues proliferation were observed; after 12 weeks, the fibrous tissue proliferation continued and early cirrhosis appeared. Compared with the ordinary white wine group, fatty infiltration was observed in the 8-week and 12-week groups, but no necrosis or fibrosis or cirrhosis was found in the Maotai liquor group (P【0.05). CONCLUSION: Maotai liquor may cause fatty liver but not hepatic fibrosis or cirrhosis, and it can strengthen lipid peroxidation in the liver.

Effects of Yigan Decoction on proliferation and apoptosis of hepatic stellate cells

AIM:To investigate the effects of Chinese herb Yigan Decoction on proliferation and apoptosis of the hepatic stellate cells (HSC) in vitro. METHODS: The study in vitro was carried out in the culture of HSC lines. Various concentrations of Yigan Decoction were added and incubated. Cell proliferation was detected with MTT colorimetric assay. Cell apoptosis was detected by electron microscopy, flow cytometry and TUNEL. RESULTS: The proliferation of HSC was inhibited by Yigan Decoction, which depending on dose and time significantly. The HSC proliferation rates of groups at the end concentrations 144 and 72(g.L(-1)) were 21.62% and 40.54% respectively, significantly lower than that of normal control group(P【0.01). The HSC proliferation rates of groups at the end concentrations 36, 18 and 9(g.L(-1)) were 54.05%, 45.95% and 51.35% respectively, lower than that of control group (P【0.05). When the end concentration was 4.5 g.L(-1), the proliferation rate was 83.78%, which appeared no significant differences compared with control group. At the same concentrations of 18 g.L(-1), the inhibitory effects of Yigan Decoction at 24 h, 48 h and 72 h time point were observed, the effects were time-dependent, and reached a peak at 72 h. Meanwhile, it was showed that the inducing effects of Yigan Decoction on HSC apoptosis were dose-dependent and time-dependent. The apoptosis index(AI) was detected by TUNEL. After Yigan Decoction had been incubated for 48 h at the end concentration of 18 g.L(-1), the AI (14.5+/-3.1)% was significantly higher than that of control group (4.3+/-1.3)% (P【0.01). When visualized under transmission electron microscopy, some apoptotic stellate cells were found, i.e. dilated endoplasmic reticulum, irregular nuclei, chromatin condensation and heterochromatin ranked along inside of nuclear membrane. By flow cytometry detection, after HSC was treated with Yigan Decoction at different concentrations of 36, 18 and 9(g.L(-1)) for 48 h, AI (%) were 13.3+/-3.2, 10.7+/-2.7 and 10.1+/-2.5 respectively, which were significantly higher than that of control group(4.1+/-1.9) (P【0.01). At the same concentration of 18 g. L(-1) for 24h, 48 h and 72 h, AI (%) were 9.3+/-1.8,10.7+/-2.7 and 14.6+/-4.3 respectively, which were significantly higher than that of control group (P【0.01). CONCLUSION: Yigan Decoction could significantly inhibit HSC proliferation and increase the apoptosis index of HSC dose-dependently and time-dependently, which may be related to its mechanism of antifibrosis.

Transplantation of human hepatocytes into tolerized genetically immunocompetent rats

AIM: To determine whether normal genetically immunocompetent rodent hosts could be manipulated to accept human hepatocyte transplants with long term survival without immunosuppression. METHODS: Tolerance towards human hepatocytes was established by injection of primary human hepatocytes or Huh7 human hepatoma cells into the peritoneal cavities of fetal rats. Corresponding cells were subsequently transplanted into newborn rats via intrasplenic injection within 24h after birth. RESULTS: Mixed lymphocyte assays showed that spleen cells from non-tolerized rats were stimulated to proliferate when exposed to human hepatocytes, while cells from tolerized rats were not. Injections made between 15 d and 17 d of gestation produced optimal tolerization. Transplanted human hepatocytes in rat livers were visualized by immunohistochemical staining of human albumin. By dot blotting of genomic DNA in livers of tolerized rats 16 weeks after hepatocyte transplantation, it was found that approximately 2.5 X 10(5) human hepatocytes survived per rat liver. Human albumin mRNA was detected in rat livers by RT-PCR for 15 wk, and human albumin protein was also detectable in rat serum. CONCLUSION: Tolerization of an immuno-competent rat can permit transplantation, and survival of functional human hepatocytes.

Effect of transforming growth factor beta and bone morphogenetic proteins on rat hepatic stellate cell proliferation and transdifferentiation

AIM: To explore different roles of TGF-β (transforming growth factor beta) and bone morphogenetic proteins (BMPs)in hepatic stellate cell proliferation and trans-differentiation.METHODS: Hepatic stellate cells were isolated from male Sprague-Dawley rats. Sub-cultured hepatic stellate cells were employed for cell proliferation assay with WST-1 reagent and Western blot analysis with antibody against smooth muscle alpha actin (SMA).RESULTS: The results indicated that TGF-β1 significantly inhibited cell proliferation at concentration as low as 0.1 ng/ml, but both BMP-2 and BMP-4 did not affect cell proliferation at concentration as high as 10 ng/ml. The effect on hepatic stellate cell trans-differentiation was similar between TGFβ1 and BMPs. However, BMPs was more potent at transdifferentiation of hepatic stellate cells than TGF-β1. In addition, we observed that TGF-β1 transient reduced the abundance of SMA in hepatic stellate cells.CONCLUSION: TGF-β may be more important in regulation of hepatic stellate cell proliferation while BMPs may be the major cytokines regulating hepatic stellate cell transdifferentiation.

Expression of lipopolysaccharide binding protein and its receptor CD14 in experimental alcoholic liver disease

AIM: To evaluate the relationship between the expression of lipopolysaccharides (LPS) binding protein (LBP) and CD14 mRNA and the severity of liver injury in alcohol-fed rats. METHODS: Twenty Wistar rats were divided into two groups:ethanol-fed group (group E) and control group (group C). Group E was fed with ethanol(5-12 g x kg(-1) x d(-1)) and group C received dextrose instead of ethanol. Rats of the two groups were sacrificed at 4 weeks and 8 weeks. Levels of endotoxin and alanine transaminase (ALT) in blood were measured, and liver pathology was observed under light and electronic microscopy. Expressions of LBP and CD14 mRNA in liver tissues were determined by RT-PCR analysis. RESULTS: Plasma endotoxin levels were increased more significantly in group E(129+/-21) ng x L(-1) and (187+/-35) ng x L(-1) at 4 and 8 wk than in control rats(48+/-9) ng x L(-1) and (53+/-11) ng x L(-1), respectively (P【0.05). Mean values of plasma ALT levels were (1867+/-250) nkat x L(-1) and (2450+/-367) nkat x L(-1) in Group E. The values were increased more dramatically in ethanol-fed rats than in Group C after 4 and 8 weeks. In liver section from ethanol-fed rats, there were marked pathological changes (steatosis, cell infiltration and necrosis). In ethanol-fed rats, ethanol administration led to a significant increase in LBP and CD14 mRNA levels compared with the control group (P【0.05). CONCLUSION: Ethanol administration led to a significant increase in endotoxin levels in serum and LBP and CD14 mRNA expressions in liver tissues. The increase of LBP and CD14 mRNA expression might wake the liver more sensitive to endotoxin and liver injury.

Effect of L-NAME on nitric oxide and gastrointestinal motility alterations in cirrhotic rats

AIM: To investigate the effect of L-NAME on nitric oxide and gastrointestinal motility alterations in cirrhotic rats. METHODS: Rats with cirrhosis induced by carbon tetrachloride were randomly divided into two groups, one n =13 receiving 0.5mg.kg(-1) per day of N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, for 10 days, whereas the other group (n =13) and control (n =10) rats were administrated the same volume of 9g.L(-1) saline. Half gastric emptying time and 2h residual rate were measured by SPECT, using (99m)Tc-DTPA-labeled barium sulfate as test meal. Gastrointestinal transition time was recorded simultaneously. Serum concentration of nitric oxide (NO) was determined by the kinetic cadmium reduction and colorimetric methods. Immunohistochemical SABC method was used to observe the expression and distribution of three types of nitric oxide synthase (NOS) isoforms in the rat gastrointestinal tract. Western blot was used to detect expression of gastrointestinal NOS isoforms. RESULTS: Half gastric emptying time and trans-gastrointestinal time were significantly prolonged(124.0 +/- 26.4 min; 33.7 +/- 8.9 min; 72.1 +/- 15.3 min; P【0.01), (12.4 +/- 0.5h; 9.5 +/- 0.3h; 8.2 +/- 0.8h; P【0.01), 2h residual rate was raised in cirrhotic rats than in controls and cirrhotic rats treated with L-NAME (54.9 +/- 7.6%,13.7 +/- 3.2%, 34.9 +/- 10.3%, P【0.01). Serum concentration of NO was significantly increased in cirrhotic rats than in the other groups (8.20 +/- 2.48) micromol.L(-1), (5.94 +/-1.07) micromol.L(-1) and control (5.66 +/- 1.60 micromol.L(-1), P【0.01. NOS staining intensities which were mainly located in the gastrointestinal tissues were markedly lower in cirrhotic rats than in the controls and cirrhotic rats after treated with L-NAME. CONCLUSION: Gastrointestinal motility was remarkably inhibited in cirrhotic rats, which could be alleviated by L-NAME. Nitric oxide may play an important role in the inhibition of gastrointestinal motility in cirrhotic rats.