内毒素上调肝细胞表达CD14基因和蛋白的实验研究

目的 观察内毒素血症时肝组织和游离肝细胞中CD14基因和CD14蛋白的表达。方法 经尾静脉注入脂多糖（LPS,E coli O111.B4）建立大鼠急性内毒素血症动物模型,并用原位胶原酶灌注法分离肝细胞。用流式细胞仪（FCM）测定异硫氢酸荧光素（FITC）－CD14阳性肝细胞数及其荧光强度。同时用逆转录－PCR和Western blot法测定肝组织和肝细胞中CD14 mRNA和CD14蛋白的表达。结果 FCM显示：内毒素血症大鼠6h和12hFITC－CD14阳性细胞数明显增多,荧光强度也明显增加。RT－PCR显示,肝组织和肝细胞中CD14 mRNA的表达在3h时明显增强,6h达高峰,24h恢复正常水平;Western blot分析示肝组织和肝细胞中CD14蛋白的表达在6h明显增高,12h达高峰,24h仍有一定表达,各时相点间比较有显著差别（P＜0.01）。结论 内毒素血症时能明显上调肝细胞CD14基因和CD14蛋白的表达。     

Expression of CD14 protein and its gene in liver sinusoidal endothelial cells during endotoxemia

AIM：To observe expression of CD14 protein and CD14 gene in rat liver sinsoidal endothelial cells(LSECs) during endotoxemia,and the role of CD14 protein in the activation of lipopolysaccharide(LPS)-induced LSECs.METHODS:Wistar rat endotoxemia model was established first by injection of a dose of LPS(5mg/kg,Escherichia coli O111:B4)via the tail vein,then sacrificed after 0h,3h,6h,12h,and 24h,respectively,LSECs were isolated from normal and LPS-injected rats by an in situ colagenase perfusion technique.The isolated LSECs were incubated with rabbit anti-rat CD14 polyclonal antibody,then stained with goat anti rabbit IgG conjgated fluorescein isothiocyanate(FITC) and flow cytometric analysis(FCM) was performed.The percentage and mean fluorescence intensity(MFI) of CD14-positive cells were taken as the indexes.LSECs were collected to measure the expression of CD14 mRNA by in situ hybridization analysis.The isolated LSECs from normal rats were incubated firstly with anti-CD14 antibody,then stimulated with different concentrations of LPS,and the supernatants of these cells were then collected for measuring the levels of tumor necrosis factor (TNF)-a and lnterleukin(IL)-6 with ELISA.RESULTS:In rats with endotoxemia,LSECs displayed a strong MFI distinct from that of control rats .CD14 positive cells in rats with endotoxemia were 54.32%,65.83%,85.64%,and 45.65%,at 3h,6h,12h,and 24h respectively there was significant difference when compared to normal group of animals(4.45%)(P<0.01).The expression of CD14 mRNA in isolated LSECs was stronger than that in control rats,In LPS group,the levels of TNF-αand IL-6were 54±6ng.L^-1,85±9ng.L^-1,206±22ng.L^-1,350±41ng.L^-1,366±42ng.L^-1and103±11ng.L^-1,187±20ng.L^-1,244±26ng.L^-1,290±31ng.L^-1,and 299±34ng.L^-1,respectively at different concentration points,In anti-CD14 group,the levels of TNF-αand IL-6 were 56±5ng.L^-1,67±8ng.L^-1,85±10ng.L^-1,113±12ng.L^-1, ,199±22ng.L^-1and104±12ng.L^-1,125±12ng.L^-1,165±19ng.L^-1,185±21ng.L^-1,and222±23ng.L^-1,respectively at different concentration poirts.THere was significant difference between the two groups(P<0.01).CONCLUSION:LSECs can synthesize CD14 protein and espress CD14 gene during endotoxemia .CD14 protein plays an important role in the activation of LPS-induced LSECs.This finding has important implications for the understanding of the mechanisms by which LPS may injure liver sinusoidal endothelial cells during sepsis.     

内毒素血症时肝窦内皮细胞脂多糖受体CD14蛋白表达的观察

目的观察内毒素血症时肝窦内皮细胞(LSECs)中CD14蛋白合成和CD14 基因的表达,以及CD14蛋白在内毒素介导LSECs激活中的作用. 方法经尾静脉注入脂多糖(LPS,E coli O111B4)5mg/kg,建立大鼠内毒素血症动物模型,分别于术后0(对照组)、3、6、12、24h活杀取材.用兔抗鼠CD14抗体和异硫氢酸荧光素(FITC)标记的羊抗兔IgG对LSECs进行孵育后,流式细胞仪测定LSECs的平均荧光强度(MFI)及FITC阳性细胞数;用原位杂交法测定LSEC中CD14 mRNA的表达.用原位胶原酶灌注法分离大鼠LSECs,用不同浓度LPS(0、0.01、1、10、100μg/ml)刺激LSECs.并用CD14抗体阻断LSECs的 CD14蛋白后,再用不同浓度LPS(0、0.01、1、10、100μg/ml)刺激LSECs.测定LPS介导LSECs肿瘤坏死因子(TNF)-α及白细胞介素-6(IL-6)分泌及CD14抗体对LSECs细胞因子分泌的影响. 结果内毒素血症大鼠3、6、12和24h 时LSECs的MFI明显增加;FITC阳性细胞数也明显增多,分别为54.32%、65.83%、85.61%和45.65%,与对照组的4.45%比较差异有非常显著意义(P＜0.01).原位杂交显示,内毒素血症大鼠LSECs中CD14 mRNA的表达明显增强,而对照组CD14 mRMA无阳性表达.LPS组TNF-α的含量(pg/ml)分别为54.49±6.02、84.65±10.16、206.54±23.55、349.87±39.47和365.76±40.31;CD14阻断组TNF-α的含量(pg/ml)分别为55.93±6.95、63.32±7.81、85.34±9.72、112.75±13.54、198.66±21.54;两组间比较差异有非常显著意义(P＜0.01).LPS组IL-6的含量(pg/ml)分别为103.34±12.52、187.39±20.31、243.87±27.83、289.51±30.15、298.53±31.94;CD14阻断组IL-6的含量(pg/ml)分别为104.37±11.49、125.02±13.58、164.59±19.47、183.47±20.17、221.76±26.43;两组间比较差异有非常显著意义(P＜0.01). 结论内毒素血症时LSECs能合成CD14蛋白及表达CD14基因;抗CD14抗体对LPS诱导LSECs TNF-α和IL-6的分泌有抑制作用;CD14蛋白的表达在内毒素介导LSECs激活中可能起重要作用.     

Pathological mechanisms of alcohol-induced hepatic portal hypertension in early stage fibrosis rat model

AIM: To study the role of hepatic sinusoidal capillarization and perisinusoidal fibrosis in rats with alcohol-induced portal hypertension and to discuss the pathological mechanisms of alcohol-induced hepatic portal hypertension. METHODS: Fifty SD rats were divided into control group (n=20) and model group (n=30). Alcoholic liver fibrosis rat model was induced by intragastric infusion of a mixture containing alcohol, corn oil and pyrazole (1 000:250:3). Fifteen rats in each group were killed at wk 16. The diameter and pressure of portal vein were measured. Plasma hyaluronic acid (HA), type Ⅳ collagen (CoⅣ) and laminin (LN) were determined by radioimmunoassay. Liver tissue was fixed in formalin (10%) and 6-μm thick sections were routinely stained with Mallory and Sirius Red. Liver tissue was treated with rabbit polyclonal antibody against LN and ColⅣ. Hepatic non-parenchymal cells were isolated, total protein was extracted and separated by SDS-PAGE. MMP-2 and TIMP-1 protein expression was estimated by Western blotting. RESULTS: The diameter (2.207 ± 0.096 vs 1.528±0.054mm, P<0.01) and pressure (11.014±0.395 vs 8.533±0.274 mmHg, P<0.01) of portal vein were significantly higher in model group than those in the control group. Plasma HA (129.97±16.10 vs 73.09±2.38 ng/mL, P<0.01), ColⅣ (210.49±4.36 vs 89.65±4.42 ng/mL, P<0.01) and LN (105.00±7.29 vs 55.70±4.32 ng/mL, P<0.01) were upregulated in model group. Abundant collagen deposited around the central vein of lobules, hepatic sinusoids and hepatocytes in model group. ColⅠ and ColⅢ increased remarkably and perisinusoids were almost surrounded by ColⅢ. Immunohistochemical staining showed that ColⅣ protein level (0.130±0.007 vs 0.032±0.004, P<0.01) and LN protein level (0.152±0.005 vs 0.029±0.005, P<0.01) were up-regulated remarkably in model group. MMP-2 protein expression (2.306±1.089 vs 0.612±0.081, P<0.01) and TIMP-1 protein expression (3.015±1.364 vs 0.446±0.009, P<0.01) in freshly isolated hepatic non-parenchymal cells were up-regulated in model group and TIMP-1 protein expression was evidently higher than MMP-2 protein expression (2.669±0.170 vs 1.695±0.008, P<0.05). CONCLUSION: Hepatic sinusoidal capillarization and peri-sinusoidal fibrosis are responsible for alcohol-induced portal hypertension in rats.     

实验性肝纤维化Bcl-2、Bax的表达及干扰素-γ对其的作用

目的 研究Bcl-2、Bax蛋白在四氯化碳(CCl4)诱导肝纤维化模型大鼠肝组织中的表达以及干扰素-γ(IFN-γ)对其的作用。 方法用CCl4诱导形成大鼠肝纤维化模型,使用IFN-γ 0.2 MU·kg-1·d-1,肌肉注射,治疗12周,设正常对照组和模型对照组。用免疫组织化学法检测各组大鼠肝组织Bcl-2、Bax的表达情况。 结果 Bcl-2在正常对照组大鼠肝细胞浆和肝窦呈低水平表达,在模型对照组大鼠肝组织分布广泛,主要表达在纤维间隔、汇管区、肝窦和肝细胞膜,中央静脉和肝细胞浆也有一定表达。正常对照组和模型对照组表达量分别为(3.87±2.37)％和(9.46±4.29)％,t=2.83,P<0.05。Bax在正常对照组大鼠肝组织的中央静脉及其周围的肝窦呈低水平表达,在模型对照组主要表达在肝细胞浆,其还表达在肝窦、纤维间隔和肝细胞膜中,胆管上皮细胞偶见表达。正常对照组和模型对照组表达量分别为(3.50±1.88)％和(9.80±3.75)％,t=3.72,P<0.01。IFN—γ组Bax的表达量为(5.85±2.35)％,模型对照组为(9.80±3.75)％,t=2.98,P<0.01,Bcl-2的表达无明显改变(t=1.49,P>0.05)。IFN-γ组对纤维间隔Bcl-2的表达量为(6.58±4.13)％,模型对照组为(9.46±4.29)％,t=2.80,P<0.05,Bax的表达差异无显著性,t=0.58,P>0.05。 结论 肝纤维化时Bcl-2和Bax表达加强,IFN     

Expression of growth hormone receptor and its mRNA in hepatic cirrhosis

AIM: To investigate the expression of growth hormone receptor (GHR) and mRNA of GHR in cirrhotic livers of rats with the intension to find the basis for application of recombinant human growth hormone (rhGH) to patients with liver cirrhosis.METHODS: Hepatic cirrhosis was induced in SpragueDawley rats by administration of thioacetamide intraperitoneally for 9-12 weeks. Collagenase Ⅳ was perfused in situ for isolation of hepatocytes. The expression of GHR and its mRNA in cirrhotic livers was studied with radio-ligand binding assay, RT-PCR and digital image analysis.RESULTS: One class of specific growth hormone-binding site, GHR, was detected in hepatocytes and hepatic tissue of cirrhotic livers. The binding capacity of GHR (RT, fmol/mg protein) in rat cirrhotic liver tissue (30.8±1.9) was significantly lower than that in normal control (74.9±3.9) at the time point of the ninth week after initiation of induction of cirrhosis (n=10, P＜0.05), and it decreased gradually along with the accumulation of collagen in the process of formation and development of liver cirrhosis (P＜0.05). The number of binding sites (×10 4/cell) of GHR on rat cirrhotic hepatocytes (0.86±0.16) was significantly lower than that (1.28±0.24)in control (n= 10, P＜0.05). The binding affinity of GHR among liver tissue, hepatocytes of various groups had no significant difference (P＞0.05). The expression of GHR mRNA (riOD,pixel) in rat cirrhotic hepatic tissues (23.3±3.1) was also significantly lower than that (29.3±3.4) in normal control (n=10, P＜0.05).CONCLUSION: The growth hormone receptor was expressed in a reduced level in liver tissue of cirrhotic rats,and lesser expression of growth hormone receptors was found in a later stage of cirrhosis. The reduced expression of growth hormone receptor was partly due to its decreased expression on cirrhotic hepatocytes and the reduced expression of its mRNA in cirrhotic liver tissue.     

Endotoxin treatment causes an upregulation of the endothelin system in the liver: amelioration of increased portal resistance by endothelin receptor antagonism

BACKGROUND: Mechanisms underlying hepatic microcirculatory failure during endotoxemia are incompletely understood. Because endothelin-1 (ET-1) has been implicated in endotoxin-induced liver injury, we investigated the hepatic ET-1 system in endotoxin-treated rats. METHODS: Rats were treated with endotoxin (Escherichia coli lipopolysaccharide; 3 mg/kg, i.p.), and various determinations were made 24 h later. RESULTS: Endotoxin treatment caused 11.2 +/- 1.6% weight loss, a decrease in mean arterial pressure (MAP; 96 +/- 5 mmHg vs 108 +/- 3 mmHg; P < 0.05) and an increase in portal pressure (11.6 +/- 1.3 mmHg vs 7.4 +/- 1 mmHg; P < 0.02). No significant changes in the serum levels of liver enzymes or hepatocellular necrosis were observed. Endotoxin caused increases in hepatic ET-1 (from 345 +/- 31 to 565 +/- 38 pg/g; P < 0.01), ET-1 receptor density (from 179 +/- 16 to 340 +/- 26 fmol/mg; P < 0.02), and mRNA expression of preproendothelin-1, and ET(A) and ET(B) receptors. While the serum nitric oxide (nitrite +/- nitrate) concentration was increased in endotoxin-treated rats, that of ET-1 remained unchanged. A mixed ET(A)/ET(B) receptor antagonist, TAK-044 (10 mg/kg, i.v.), reduced the weight loss from 11.2 +/- 1.6% to 5.9 +/- 2.9% (P < 0.05) and the portal pressure from 11.6 +/- 1.3 mmHg to 8.6 +/- 0.7 mmHg (P < 0.05) in endotoxin-treated rats. The mixed ET(A)/ET(B) receptor antagonist also caused an increase in serum ET-1 concentration, but did not affect serum nitric oxide and MAP in endotoxin-treated rats. CONCLUSIONS: These results suggest that the upregulated hepatic ET-1 system is an important mechanism of increased portal resistance and related complications of endotoxemia.     

Fasting-induced changes in prostaglandin binding in isolated hepatocytes and liver plasma membranes

The effects of fasting on hepatic prostaglandin E (PGE) receptor characteristics were studied in Sprague-Dawley rats. Plasma membranes were isolated from liver homogenates of animals after 0, 12, 18, 24, and 48 h of fasting. The changes observed in body weight, liver weight, plasma glucose, and plasma beta-hydroxybutyrate during the fast were consistent with those previously reported for a similar fasted rat model. During the transition from the fed to the fasted state there was a decrease in hepatic PGE receptor density (from 0.175 +/- 0.011 pmol bound/mg membrane membrane protein in fed rats to 0.060 +/- 0.009 in rats fasted for 24 h), with no change in binding affinity. This change was observed whether the data were expressed per mg membrane protein isolated or were corrected for total membrane recovery and normalized to initial body weight. Isolated hepatocytes prepared from fed and 24-h fasted animals also demonstrated a significant decrease in PGE-binding site density (from 0.98 +/- 0.05 pmol bound/10(6) cells in fed rats to 0.46 +/- 0.14 in fasted rats), with no change in binding site affinity. The change in binding site density in the hepatocytes was of a magnitude similar to that observed in the liver plasma membranes after the membranes were corrected for recovery and normalized to initial body weight (51% vs. 53%, liver plasma membranes vs. isolated hepatocytes). We conclude that fasting is associated with a decrease in the hepatic PGE receptor density and suggest that this decrease may reflect an increase in hepatic E-series PGs during starvation.     

急性肝功能衰竭大鼠内毒素血症对肝脏和肾脏糖异生功能的影响

目的探讨大鼠急性肝功能衰竭时内毒素血症对肝脏及肾脏糖异生功能及血糖水平的影响。方法24只雄性健康成年SD大鼠,随机分成4组,每组6只,Ⅰ组：腹腔注射等渗盐水,Ⅱ组：腹腔注射400 mg/kg D-氨基半乳糖（D-GaLN）;Ⅲ组：腹腔注射400 mg/kg D- GaLN＋50μg/kg LPS,Ⅳ组：腹腔注射400 mg/kg D-GaLN＋500μg/kg LPS。LPS注射后6 h,取血清检测内毒素、肾功能,取大鼠肝组织及肾组织,采用荧光定量PCR方法检测磷酸烯醇丙酮酸磷酸羧激酶（PEPCK）基因表达。结果Ⅰ组、Ⅱ组大鼠未见明显的内毒素血症,Ⅲ组、Ⅳ组大鼠体内内毒素水平明显升高,Ⅳ组高于Ⅲ组（8.05±0.43对比3.50±2.25,P〈0.05）。Ⅰ组、Ⅱ组、Ⅲ组大鼠于LPS注射前后未出现低血糖,Ⅳ组大鼠于LPS注射后6h出现明显的低血糖。各组大鼠肾功能均在正常水平,仅有Ⅳ组大鼠出现血清尿素氮水平轻度增高。大鼠肝脏PEPCK的表达在Ⅰ组、Ⅱ组、Ⅲ组、Ⅳ组逐渐减少,差异有统计学意义（2.54±1.32、1.87±0.15、0.91±0.13、0.44±0.42,P〈0.05）;大鼠肾脏PEPCK的表达,同Ⅰ组比较,Ⅱ组无明显变化（0.75±0.03对比0.77±0.04,P〉0.05）,Ⅲ组明显增强（0.75±0.03对比1.63±0.86,P〈0.05）,Ⅳ组大鼠肾脏PEPCK表达显著减弱（0.75±0.03对比0.13±0.07,P〈0.05）。结论急性肝功能衰竭大鼠中严重的内毒素血症通过抑制PEPCK的转录损伤肝脏和肾脏糖异生的功能,导致低血糖的发生。     

Regulation of a thyroid hormone-dependent protein by growth hormone: the induction of hepatic alpha 2U globulin and its messenger ribonucleic acid in adult male hypothyroid rats

Studies in the adult male hypothyroid rat, a known GH-deficient animal, have shown hepatic alpha 2U-globulin mRNA to be dependent on thyroid hormones. To study the effects of GH on alpha 2U-globulin synthesis in the absence of thyroid hormones, adult male rats were rendered hypothyroid before hormone treatment. The relative effects of bovine GH or T3 were studied by RIA of alpha 2U-globulin in hepatic cytosol in rats 6 weeks after thyroid ablation. alpha 2U-Globulin levels in vehicle-treated controls were 1.3 +/- 0.7 micrograms (+/- SD) alpha 2U-globulin/mg protein. After 2 days, GH (200 micrograms/100 g X day) resulted in an increase to 5.7 +/- 1.0 micrograms alpha 2U-globulin/mg (P less than or equal to 0.05), and T3 50 micrograms/100 g X day) resulted in an increase to 11.5 +/- 3.6 micrograms/mg (P less than or equal to 0.01). After 7 days, GH resulted in an increase to 12.4 +/- 4.6 micrograms/mg (P less than or equal to 0.01), and T3 resulted in an increase to 28.7 +/- 8.7 micrograms/mg (P less than or equal to 0.01). After 4 months of thyroid ablation, baseline hepatic alpha 2U-globulin levels fell to 4.8 ng alpha 2U-globulin/mg protein. Hepatic alpha 2U-globulin was determined 4 and 8 h after the injection of GH (200 micrograms/100 g). In these animals with markedly diminished hepatic alpha 2U-globulin levels, significant (P less than or equal to 0.01) increases occurred 4 h (25.4 ng/mg) and 8 h (57.2 ng/mg) after GH injection. The effects of treatment with bovine GH (200 micrograms/100 g X day) for 3 days on hepatic alpha 2U-globulin synthesis in liver slices and alpha 2U-globulin poly (A)+ RNA levels were measured in rats 10 weeks after thyroid ablation. GH significantly (P less than 0.05) increased alpha 2U-globulin synthesis as a percentage of total protein synthesis (from 0.01% to 0.035%) and alpha 2U-globulin mRNA as a percentage of total mRNA (from 0.03% to 0.24%). The results show that GH rapidly and specifically stimulates hepatic alpha 2U-globulin and its mRNA activity in thyroid hormone-deficient rats.     

Hepatic lipid metabolism changes in short- and long-term prehepatic portal hypertensive rats

INTRODUCTION The most used experimental model to study prehepatic portal hypertension is that obtained by calibrated stenosis of the portal vein in the rat[1,2]. It has been suggested that this experimental model has a homogeneous evolution, with a narrow…     

Effects of cycloheximide on hepatic and uterine insulin-like growth factor-I mRNA

To investigate differences in growth hormone (GH) and estrogen activation of insulin-like growth factor-I (IGF-I) expression, we have examined the effects of cycloheximide on hepatic and uterine IGF-I mRNA abundance in response to GH and 17 beta-estradiol (E2) respectively. In hypophysectomized (hypox) rats a single injection of GH significantly increased hepatic IGF-I mRNA 3.65 +/- 0.68-fold, P less than 0.005, 6 h after injection. Administration of cycloheximide 30 min prior to GH injection completely abolished this response. In contrast, in pituitary intact rats killed 6 h after administration of cycloheximide, hepatic IGF-I mRNA abundance was not significantly different from untreated control rats although the serum IGF-I concentration was significantly reduced; 119.9 +/- 11.8 vs. 270.2 +/- 48.7 ng/ml, P less than 0.005. In immature rats, injection of E2 (1 micrograms/100 g body weight) significantly increased uterine IGF-I mRNA 4.1 +/- 0.4-fold. Cycloheximide did not block the E2-induced increase in IGF-I mRNA but rather significantly enhanced the IGF-I response. These data indicate that continuing protein synthesis is required for GH induction of hepatic IGF-I mRNA in the hypox rat but is not required for E2 induction of uterine IGF-I mRNA. Furthermore, in pituitary-intact rats protein synthesis is not required for maintenance of hepatic IGF-I mRNA levels.     

Role of nitric oxide in Toll-like receptor 2 and 4 mRNA expression in liver of acute hemorrhagic necrotizing pancreatitis rats

AIM:To investigate the role of nitric oxide(NO)in Toll-like receptor 2(TLR2)/4mRNA expression in livers ofacute hemorrhagic necrotizing pancreatitis(AHNP)rats.METHODS:One hundred and ten SD male rats wererandomly divided into sham-operated group(n=10),AHNP group(n=30),chloroquine(CQ)-treated group(n=30)and L-Arg-treated group(n=40).TLR2/4mRNAexpression in the liver of AHNP rats was measured byRT-PCR.RESULTS:Expression of TLR2/4mRNA could bedetected in the liver of AHNP rats in sham-operatedgroup(0.155E-5 0.230E-6 and 0.115E-2±0.545E-4),but was markedly increased at 3 h in AHNP group(0.197E-2±0.114E-3 and 0.175±0.349E-2)peakingat 12 h(0.294E-2±0.998E-4 and 2.673±2.795E-2,P<0.01).Hepatic injuries were aggravated,TNF-αconcentration in the liver was increased and NOconcentration was decreased(P<0.05 or P<0.01).When TLR2/4mRNA expression was inhibited by CQ(3h:1.037E-4±3.299E-6 and 0.026±3.462E-3;6 h:1.884E-4±4.679E-6 and 0.108±6.115E-3;12 h:2.443E-4±7.714E-6 and 0.348±6.807E-3;P<0.01),hepatic injuries were relieved,NO concentration inthe liver was increased and TNF-α concentration wasdecreased(P<0.05 or P<0.01).When rats with AHNP were treated with L-Arg,TLR2/4mRNA expressionin the liver could be effectively inhibited(50 mg-T:0.232E-2±0.532E-4 and 0.230±6.883E-3;100 mg-T:0.210E-2±1.691E-4 and 0.187±0.849E-2;200 mg-T:0.163E-2±0.404E-4 and 0.107±0.195E-2;400 mg-T:0.100E-2±0.317E-4 and 0.084±0.552E-2;P<0.01)and hepatic injuries were relieved.At the same time,NO concentration in the liver was markedly increasedand TNF-α concentration was decreased(P<0.05 orP<0.01).CONCLUSION:The expression of TLR2/4mRNA isincreased and hepatic injuries are aggravated in the liverof AHNP rats.TLR2/4mRNA gene expression in the liverof AHNP rats can be markedly inhibited by NO,leadingto the relief of hepatic injuries.     

Phosphate-activated glutaminase activity is enhanced in brain, intestine and kidneys of rats following portacaval anastomosis

AIM: To assess whether portacaval anastomosis (PCA) in rats affects the protein expression and/or activity of glutaminase in kidneys, intestines and in three brain areas of cortex, basal ganglia and cerebellum and to explain the neurological alterations found in hepatic en-cephalopathy (HE). METHODS: Sixteen male Wistar rats weighing 250-350 g were grouped into sham-operation control (n=8) or portacaval shunt (n=8). Twenty-eight days after the procedure, the animals were sacrificed. The duodenum, kidney and brain were removed, homogenised and mitochondria were isolated. Ammonia was measured in brain and blood. Phosphate-activated glutaminase (PAG) activity was determined by measuring ammonia production following incubation for one hour at 37℃with O-phthalaldehyde (OPA) and specific activity expressed in units per gram of protein (μkat/g of protein). Protein expression was measured by immunoblotting. RESULTS: Duodenal and kidney PAG activities together with protein content were significantly higher in PCA group than in control or sham-operated rats (duodenum PAG activity was 976.95±268.87μkat/g of protein in PCA rats vs 429.19±126.92.μkat/g of protein in sham-operated rats; kidneys PAG activity was 1259.18±228.79μkat/g protein in PCA rats vs 669.67±400.8μkat/g of protein in controls, P<0.05; duodenal protein content: 173% in PCA vs sham-operated rats; in kidneys the content of protein was 152% in PCA vs sham-operated rats). PAG activity and protein expression in PCA rats were higher in cortex and basal ganglia than those in sham-operated rats (cortex: 6646.6±1870.4μkat/g of protein vs 3573.8±2037.4μkat/g of protein in control rats, P<0.01; basal ganglia, PAG activity was 3657.3±1469.6μkat/g of protein in PCA rats vs 2271.2±384μkat/g of protein in sham operated rats, P<0.05; In the cerebellum, the PAG activity was 2471.6±701.4μkat/g of protein vs 1452.9±567.8μkat/g of protein in the PCA and sham rats, respectively, P<0.05; content of protein: cerebral cortex: 162%±40% vs 100%±26%, P< 0.009; and basal ganglia: 140%±39% vs 100%±14%, P<0.05; but not in cerebellum: 100%±25% vs 100%±16%,P=ns). CONCLUSION: Increased PAG activity in kidney and duodenum could contribute significantly to the hyperam-monaemia in PCA rats, animal model of encephalopathy. PAG is increased in non-synaptic mitochondria from the cortex and basal ganglia and could be implicated in the pathogenesis of hepatic encephalopathy. Therefore, PAG could be a possible target for the treatment of HE or liver dysfunction.     

Glucocorticoid regulation of growth hormone (GH) secretagogue-induced growth responses and GH secretagogue receptor expression in the rat.

Synthetic GH-releasing peptides such as GHRP-6 are potent GH secretagogues (GHSs) in several species, but attempts to stimulate growth by continuous GHS exposure have had limited success. GHSs also release ACTH and adrenal steroids. Since glucocorticoid excess is associated with poor linear growth, stimulation of the hypothalamo-pituitary-adrenal (HPA) axis by continuous GHS administration may compromise their growth-promoting effects. We have now examined the effects of continuous GHRP-6 infusion (100 mg/day, s.c. for 14 days) in normal 150-day-old female rats, and in adrenalectomized (Adx) rats with or without dexamethasone (Dex) replacement. Infusion of GHRP-6 did not significantly affect body weight gain compared with excipient-treated controls in either intact rats (controls, 9.0 +/- 1.6 vs GHRP-6, 11.8 +/- 0.9 g) or Adx rats (4.4 +/- 1.5 vs 7.9 +/- 2.7 g). However, GHRP-6 significantly increased weight gain in Adx rats treated with Dex (controls, 3.5 +/- 1.4 vs GHRP-6, 15.4 +/- 1.6 g;P<0.01). Adrenalectomy decreased plasma triglycerides (P<0.01), and Dex treatment increased plasma cholesterol (P<0.001), GHRP-6 treatment did not affect these plasma lipids. Dex treatment also reduced plasma GH-binding protein levels and hepatic GH binding (P<0.05). Pituitary GH content was decreased in Adx rats (P<0.05) but not in Dex-treated Adx rats. Adrenalectomy markedly decreased GHS-receptor mRNA expression in the arcuate (P<0. 001) and ventromedial nuclei (P<0.01), whilst Dex treatment normalized GHS-receptor expression. These results suggest that adrenal steroids are necessary for normal GHS-receptor expression and GHRP-6-induced weight gain, but long-term stimulation of the HPA axis by continuous GHS exposure may be detrimental to the growth response.     

In vivo effects of Chinese herbal recipe, Danshaohuaxian, on apoptosis and proliferation of hepatic stellate cells in hepatic fibrotic rats

AIM: To investigate the effects of Danshaohuaxian (DSHX), a Chinese herbal recipe, on the apoptosis and cell cycles of hepatic stellate cells (HSCs) in rat hepatic fibrosis and its possible mechanisms. METHODS: Seventy-six male Wistar rats were randomly divided into normal control group, hepatic fibrosis group, non-DSHX-treated group and DSHX-treated group. Except for the normal control group, rat hepatic fibrotic models were induced by subcutaneous injection of carbon tetrachloride (CCl4), drinking alcohol, giving diet of hyperlipid and hypoprotein for 8 wk. When the hepatic fibrotic models were produced, 12 rats of hepatic fibrosis group (15 rats survived, others died during the 8 wk) were sacrificed to collect blood and livers. HSCs were isolated from the other 3 rats to detect the apoptotic index (AI) and cell cycles by flow cytometry. DSHX was then given to the DSHX-treated group (1.0 g/kg, PO, daily) for 8 wk. At the same time, normal control group and non-DSHX-treated group were given normal saline for 8 wk. At end of the experiment, some rats in these three groups were sacrificed to collect blood and livers, the other rats were used for HSC isolation to detect the apoptotic index (AI) and cell cycles. Then the liver index, serum hyaluronic acid (HA) and alanine aminotransferase (ALT), degree of hepatic fibrosis, urinary excretion of hydroxyproline (Hyp) and expression of collagen types Ⅰ and Ⅲ (COL Ⅰ and Ⅲ) in these four groups were detected respectively. RESULTS: Compared with the indexes of the hepatic fibrosis group and non-DSHX-treated group, the DSHX-treated group revealed a liver index of (0.0267±0.0017 vs 0.0423±0.0044, 0.0295±0.0019, P<0.05), levels of serum HA (200.78±31.71 vs 316.17±78.48, 300.86±72.73, P<0.05) and ALT(93.13±5.79 vs 174.5±6.02, 104.75±6.54, P<0.01), and stage of hepatic fibrosis (1.30 vs 4.25, 2.60, P<0.01) all reduced. The urinary excretion of Hyp increased (541.09±73.39 vs 62.00±6.40, 182.44±30.83, P<0.01), the COL Ⅰ and Ⅲ expression decreased (COL I: 1.07±0.96 vs 4.18±2.26, 3.22±1.44, P<0.01; COL Ⅲ: 1.09±0.58 vs 3.04±0.62, 2.23±0.58, P<0.01), the HSCs apoptotic index of HSCs (7.81±0.47 vs 1.63±0.25, 1.78±0.4, P<0.05) and the ratio of G0-G1 phase cells increased (94.30±1.33 vs 62.27±17.96, 50.53±2.25, P<0.05). The ratios of S-phase cells (3.11±1.27 vs 9.83±1.81, 11.87±1.9, P<0.05) and G2-M phase cells (2.58±0.73 vs 23.26±10.95, 13.60±1.15, P<0.01) declined. CONCLUSION: DSHX capsule shows certain therapeutic effects on hepatic fibrosis in rats and inhibits abnormal deposition of COL I and III in rat livers by promoting the apoptosis of HSCs and preventing their proliferation.