肝硬化与层粘素过度表达的研究

用CCl_4诱发大鼠肝纤维化,检测不同期大鼠血清和肝组织匀浆中层粘素(Laminin,LN)的含量,用免疫组化法观察LN在肝脏内的沉积。结果表明,血清LN水平在反映肝组织渐进性损伤方面较之肝内LN含量理想;肝内LN含量在肝受损晚期升高,与免疫组化结果平行。检测不同肝病患者血清LN含量,肝硬化LN水平明显高于正常人和非硬化患者;免疫组化观察慢性肝病和肝硬化患者肝内LN分布,结果以后者LN沉积为多。体外培养大鼠肝细胞,研究LN对细胞的增殖和胶原合成的影响,提示生理血清浓度的LN对肝细胞代谢无明显影响,高浓度时抑制其DNA及胶原的合成。提示肝硬化时LN合成明显增加,血清LN水平是判断肝脏活动性损伤和纤维化进程的良好指标,可适于肝硬化的早期诊断。肝内LN大量沉积,预示肝损伤发展至晚期肝硬化,同时大量增多的LN对肝细胞进一步合成胶原有负反馈抑制作用。     

抑肽酶对D-氨基半乳糖大鼠急性肝损伤的保护作用

探讨抑肽酶对D-氨基半乳糖致Wistar大鼠急性肝损伤的保护作用.利用D-氨基半乳糖(D-GalN)诱导Wistar大鼠急性肝损伤模型进行研究,检测各组大鼠血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、白蛋白(ALB)、白蛋白总蛋白比值(A/G),观察光镜下肝组织的病理改变.结果显示,经腹腔注射抑肽酶对D-氨基半乳糖所造成的急性肝损伤有明显的保护作用.     

硒对人肝细胞缺氧-复氧损伤的影响

建立人肝细胞缺氧－复氧损伤模型,观察了微量元素硒的保护作用。结果表明,模型组肝细胞内、外丙二醛水平均显著增高,丙氨酸转氨酶外释增多,白蛋白合成减少,与正常对照组比较,差异有显著性意义（Ｐ＜００１）。１１５６×１０－７ｍｏｌ／Ｌ硒预保护组上述损伤变化明显减轻（Ｐ＜００５）。揭示硒对缺氧－复氧所致人肝细胞脂质过氧化损伤具有保护作用。     

低剂量三氯化钐对实验性非胰岛素依赖型糖尿病大鼠胰岛的影响

观察了经腹腔注射低剂量三氯化钐（０．０５ｍｇ／ｋｇ）对链脲佐菌素引志的非胰岛素依赖型糖尿病（ＮＤＩＤＤＭ）大鼠胰岛形态和功能的影响。结果表明，三氯化钐治疗组糖耐量改善、血清胰岛水平长高、血清胰镐血糖素水平降低、平均单个胰岛面积和胰岛β细胞数量明显增多，肝细胞内糖原含量增多。提示低剂量三氯化钐对实验性ＮＩＤＤＭ大鼠有一定的治疗作用。     

肺Ca2+含量与硅肺的关系

本文用离子选择电极测定了实验性诱发硅肺大鼠的肺、脑、肾、肝和血清中的[Ca~(2+)]、[K~+]和[Na~+],还以荧光探针Tb~(3+)探讨了Ca~(2+)的作用。在硅肺形成后,大鼠的肺、脑、肾、肝和血清等组织中的游离[Ca~(2+)]均发生变化。荧光探针表明:硅肺大鼠肺Ca~(2+)增加部分来自线粒体Ca~(2+)的释放硅肺。大鼠肺中游离Ca~(2+)和线粒体Ca~(2+)的变化可能与巨噬细胞的损伤有关。     

“慢肾康”对慢性肾衰大鼠血清锌铜的影响

观察了慢性肾功能衰竭大鼠经中药“慢肾康”治疗后血清锌铜的变化，研究发现“慢肾康”能明显改善肾衰动物低血锌状况，并且慢性肾衰动物低血锌与血浆白蛋白呈正相关，而与尿蛋白呈负相关。研究提示，“慢肾康”通过改善肾功能，减轻蛋白尿和升高血浆白蛋白而使血清锌升高，同时药物本身所含的锌元素也有一定的补充作用。“慢肾康”的治疗，对肾衰动物的血清铜无明显影响。     

白蛋白姜黄素纳米粒子的毒性及免疫原性考察

目的对大鼠白蛋白姜黄素纳米粒子致SD大鼠的毒性和免疫原性进行考察。方法大鼠白蛋白姜黄素纳米粒子和姜黄素溶液,连续7 d通过尾静脉注射SD大鼠,同时设置对照(每组8只)。给药后第9天,将每组中4只大鼠处理,取心、肝、脾、肺和肾,用福尔马林浸泡。组织经包埋,切片,HE染色,显微镜下,观察病理变化。21 d后,所有大鼠采血,酶联免疫吸附法检测给药组大鼠血清中是否产生针对大鼠白蛋白的抗体。结果与正常对照组相比,给药组大鼠的各脏器没有明显的病理变化。大鼠血清样本检测,没有检测出针对大鼠白蛋白的抗体。结论在试验周期内,白蛋白姜黄素纳米粒子不会导致受试动物的脏器发生显著的病理变化,且不会导致机体产生特异性抗体。     

促肝细胞生长素（PHGF）对活动性肝硬变的治疗意义探讨

促肝细胞生长素(PHGF)有特异的刺激肝细胞的DNA合成和促进肝细胞的增殖作用，其对重症肝炎的治疗，已初步取得了可喜成果。对慢性肝炎、肝硬化和部分肝切除也有所应用．临床应用结果未见报告有明显副作用．但本文观察到一病例，诊断为“活动性肝硬化”，在连续应用PHGF治疗二个月后CT结果显示肝内呈弥漫性结节状增生，而应用前并未发现此现象．提示PHGF具双重性，对肝硬化患者在促进肝细胞增殖的同时，促进或加重了肝细胞的结节性增生——肝硬化结节形成，可能加速肝硬变的进程．而增生之结节未显示有改善肝功能，促进蛋白合成的作用．本文就此进行探讨．     

养阴通脑颗粒中黄酮对脑缺血再灌注损伤保护作用的实验研究

为了进一步探讨养阴通脑颗粒中有效成分治疗缺血性脑血管病的作用及其作用机理.我们开展了黄酮对大鼠脑缺血再灌注损伤保护作用研究.现将其对脑缺血再灌注损伤大鼠抗凝、纤溶等指标的影响报告如下.     

补肾调肝方治疗围绝经期综合征患者血清微量元素变化的观察

为观察补肾调肝方治疗围绝经期综合征38例的临床疗效及对血清微量元素的影响,采用经验方补肾调肝方加味治疗,疗程3个月,对血清中的微量元素作了临床分析。结果表明,治疗后血清微量元素锌、铁、锰明显高于治疗前(P<0.01),临床有效率94.7%。提示补肾调肝方有改善人体血清微量元素的作用,并取得临床明显的疗效。     

Obacunone Attenuates Liver Fibrosis with Enhancing Anti-Oxidant Effects of GPx-4 and Inhibition of EMT

Obacunone, a limonin triterpenoid extracted from Phellodendronchinense Schneid or Dictamnus dasycarpusb Turcz plant, elicits a variety of pharmacological effects such as anti-inflammatory, anti-neoplastic, anti-oxidation, and anti-lung-fibrosis ones. However, the anti-fibrotic effect of obacunone and the detailed underlying mechanism in liver fibrosis remain unclear. Liver fibrosis is a debilitating disease threatening human health. Transforming growth factor (TGF)-β/P-Smad is a major pathway of fibrosis featured with epithelia mesenchymal transformations (EMT) and collagen depositions, accompanying with excessive oxygen-free radicals. Nrf-2 acts as a key anti-oxidative regulator driving the expressions of various antioxidant-related genes. Glutathionperoxidase-4 (GPx-4) is a member of the glutathione peroxidase family that directly inhibits phospholipid oxidation to alleviate oxidative stress. In the present study, we aimed to explore the role of obacunone in mouse liver fibrosis model induced by carbon tetrachloride (CCl4) and in hepatic stellate cells (LX2 cell line) challenging with TGF-β. Obacunone demonstrated potent ameliorative effects on liver fibrosis both in activated LX2 and in mice liver tissues with reduced levels of α-SMA, collagen1, and vimentin. Obacunone also remarkably suppressed the TGF-β/P-Smad signals and EMT process. Meanwhile, obacunone exerted a potent anti-oxidation effect by reducing the levels of reactive oxygen species (ROS) in both models. The antioxidant effect of obacunone was attributed to the activation of GPx-4 and Nrf-2. In addition, the therapeutic effect of obacunone on LX2 cells was significantly removed in vitro plus with GPx-4 antagonist RSL3, in parallel with the re-elevated levels of ROS. Thus, we demonstrate that obacunone is able to attenuate liver fibrosis via enhancing GPx-4 signal and inhibition of the TGF-β/P-Smad pathway and EMT process.     

Quantitative profiling of phosphatidylcholine and phosphatidylethanolamine in a steatosis/fibrosis model of rat liver by nanoflow liquid chromatography/tandem mass spectrometry

Quantitative analysis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was carried out using a steatosis/fibrosis model of rat livers, which were induced by a chronic administration of carbon tetrachloride (CCl(4)). Intact phospholipid mixtures from each liver sample (from rats fed with 0.5 mL CCl(4)/kg three times a week for 30, 60, and 90 days) were analyzed by nanoflow liquid chromatography-electrospray ionization tandem mass spectrometry (nLC/ESI/MS/MS), and identifications of 37 PC and 19 PE species were made by collision-induced dissociation. The quantitative analysis utilized a multiple standard addition method with an internal standard, and the relationship between the MS peak intensities of different PC species and their carbon chain length was included for calibration. It was found that the total amount of PC and PE species decreased significantly with administration of CCl(4). While concentrations of most PC and PE species decreased to a great extent, three PEs and seven PCs were up-regulated more than twofold.     

Therapeutic effect of hepatocyte growth factor-secreting mesenchymal stem cells in a rat model of liver fibrosis

Bone marrow-derived mesenchymal stromal cells (MSCs) have been reported to be beneficial for the treatment of liver fibrosis. Here, we investigated the use of genetically engineered MSCs that overexpress hepatocyte growth factor (HGF) as a means to improve their therapeutic effect in liver fibrosis. Liver fibrosis was induced by intraperitoneal injection of dimethylnitrosamine. HGF-secreting MSCs (MSCs/HGF) were prepared by transducing MSCs with an adenovirus carrying HGF-encoding cDNA. MSCs or MSCs/HGF were injected directly into the spleen of fibrotic rats. Tissue fibrosis was assessed by histological analysis 12 days after stem cell injection. Although treatment with MSCs reduced fibrosis, treatment with MSCs/HGF produced a more significant reduction and was associated with elevated HGF levels in the portal vein. Collagen levels in the liver extract were decreased after MSC/HGF therapy, suggesting recovery from fibrosis. Furthermore, liver function was improved in animals receiving MSCs/HGF, indicating that MSC/HGF therapy resulted not only in reduction of liver fibrosis but also in improvement of hepatocyte function. Assessment of cell and biochemical parameters revealed that mRNA levels of the fibrogenic cytokines PDGF-bb and TGF-β1 were significantly decreased after MSC/HGF therapy. Subsequent to the decrease in collagen, expression of matrix metalloprotease-9 (MMP-9), MMP-13, MMP-14 and urokinase-type plasminogen activator was augmented following MSC/HGF, whereas tissue inhibitor of metalloprotease-1 (TIMP-1) expression was reduced. In conclusion, therapy with MSCs/HGF resulted in an improved therapeutic effect compared with MSCs alone, probably because of the anti-fibrotic activity of HGF. Thus, MSC/HGF represents a promising approach toward a cell therapy for liver fibrosis.     

代谢组学方法研究水飞蓟宾对四氯化碳致小鼠肝损伤的保护作用

运用代谢组学方法研究四氯化碳(CCl4)致小鼠肝损及水飞蓟宾肝保护作用的机理. 通过气相色谱-质谱(GC-MS)技术分析CCl4及水飞蓟宾作用下的小鼠肝组织匀浆及血浆代谢物谱. 使用正交偏最小二乘判别分析法研究正常组与CCl4模型组之间的代谢物谱差异, 并通过变量重要性投影(VIP)选取肝组织与血浆中各12种标志性代谢物. 使用主成分分析法研究水飞蓟宾预防性及治疗性给药对CCl4肝损的干预效果. 通过比较选取的标志性代谢物的含量的差异, 探讨了水飞蓟宾对CCl4致小鼠肝损的保护机理. 结果表明, CCl4作用后, 小鼠机体能量代谢、氨基酸代谢及脂类代谢都受到不同程度的影响. 水飞蓟宾能有效地缓解CCl4所造成的体内线粒体功能及氨基酸代谢紊乱. 采用代谢组学方法能较全面地反应生物体的生理及代谢状态, 并可应用于物质毒性和药效评价研究.     

鼠肝星状细胞体内与体外激活的显微拉曼光谱

采用密度梯度离心法从肝组织中分离、提纯肝星状细胞, 进行常规细胞鉴定后, 通过体外培养诱导肝星状细胞体外活化, 在不同的时间点上进行原位拉曼光谱表征; 通过一次性腹腔注射CCl4诱导鼠急性肝损伤, 取不同的时间点的肝损伤组织做拉曼光谱表征, 并以肝组织的光谱变化来间接反映肝星状细胞的体内活化. 结果表明, 用拉曼光谱能快速、 灵敏地监测肝星状细胞体内和体外活化过程中的分子变化, 可为肝纤维化的早期诊断提供依据.     

Pharmacokinetics of [6]-gingerol after intravenous administration in rats with acute renal or hepatic failure.

The pharmacokinetics of [6]-gingerol were investigated in rats with acute renal failure induced by bilateral nephrectomy, or those with acute hepatic failure induced by a single oral administration of carbon tetrachloride (CCl4), to clarify the contribution of the kidney and liver to the elimination process of [6]-gingerol. After bolus intravenous administration, a plasma concentration-time curve of [6]-gingerol was illustrated by a two-compartment open model. There was no significant difference in either the plasma concentration-time curve or any pharmacokinetic parameters between the control and nephrectomized rats. It is suggested, therefore, that renal excretion does not contribute at all to the disappearance of [6]-gingerol from plasma in rats. In contrast, hepatic intoxication with CCl4 elevated the plasma concentration of [6]-gingerol at the terminal phase. Its elimination half-life increased significantly, from 8.5 to 11.0 min, in CCl4-intoxicated rats. The extent of [6]-gingerol bound to serum protein was more than 90% and was affected very slightly by the CCl4-intoxication. These aspects indicate that [6]-gingerol is eliminated partly by the liver.     

Relation between blood clearance and hepatic uptake of 99mTc-phytate in rats with hepatic injury

The relation among the blood clearance of 99mTc-phytate (99mTc-P), the hepatic uptake of 99mTc-P and the severity of hepatic injury was investigated by using the rats with carbon tetrachloride (CCl4), D-galactosamine (Gal N), alpha-naphthylisothiocyanate (ANIT) or DL-ethionine (EthN) induced hepatic injury. After the administration of CCl4, GalN or ANIT, serum GPT activity increased significantly with the increase of dose level, and the degree of this increase was in the order: GalN greater than CCl4 greater than ANIT. However, the mild increase in serum GPT activity was observed after EthN administration. The blood clearance rate of 99mTc-P and the hepatic uptake ratio of 99mTc-P decreased with the increase of dose level after CCl4, GalN or ANIT administration, but significant changes were not found after EthN administration. The degree of decrease in the blood clearance rate of 99mTc-P was in the order: GalN not equal to CCl4 greater than ANIT, and the degree of decrease in the hepatic uptake ratio of 99mTc-P was in the order: GalN not equal to CCl4 greater than ANIT. These results suggest that the disorder in the hepatocytes may be one of causes for inducing the decrease in the hepatic uptake of 99mTc-P, and the consequence of this decrease may induce the decrease in the blood clearance of 99mTc-P.     

A Comparison of the Gene Expression Profiles of Non-Alcoholic Fatty Liver Disease between Animal Models of a High-Fat Diet and Methionine-Choline-Deficient Diet

Non-alcoholic fatty liver disease (NAFLD) embraces several forms of liver disorders involving fat disposition in hepatocytes ranging from simple steatosis to the severe stage, namely, non-alcoholic steatohepatitis (NASH). Recently, several experimental in vivo animal models for NAFLD/NASH have been established. However, no reproducible experimental animal model displays the full spectrum of pathophysiological, histological, molecular, and clinical features associated with human NAFLD/NASH progression. Although methionine-choline-deficient (MCD) diet and high-fat diet (HFD) models can mimic histological and metabolic abnormalities of human disease, respectively, the molecular signaling pathways are extremely important for understanding the pathogenesis of the disease. This review aimed to assess the differences in gene expression patterns and NAFLD/NASH progression pathways among the most common dietary animal models, i.e., HFD- and MCD diet-fed animals. Studies showed that the HFD and MCD diet could induce either up- or downregulation of the expression of genes and proteins that are involved in lipid metabolism, inflammation, oxidative stress, and fibrogenesis pathways. Interestingly, the MCD diet model could spontaneously develop liver fibrosis within two to four weeks and has significant effects on the expression of genes that encode proteins and enzymes involved in the liver fibrogenesis pathway. However, such effects in the HFD model were found to occur after 24 weeks with insulin resistance but appear to cause less severe fibrosis. In conclusion, assessing the abnormal gene expression patterns caused by different diet types provides valuable information regarding the molecular mechanisms of NAFLD/NASH and predicts the clinical progression of the disease. However, expression profiling studies concerning genetic variants involved in the development and progression of NAFLD/NASH should be conducted.     

Activity of Pterostilbene Metabolites against Liver Steatosis in Cultured Hepatocytes

Pterostilbene is a dimethyl ether derivative of resveratrol, less metabolized than its analogue, due to the substitution of two hydroxyl groups with methoxyl groups. Nevertheless, the amounts of pterostilbene phase II metabolites found in plasma and tissues are higher than those of the parent compound. The first aim of this study was to assess whether pterostilbene-4′-O-glucuronide (PT-G) and pterostilbene-4′-O-sulfate (PT-S) were able to prevent triglyceride accumulation in AML12 (alpha mouse liver 12) hepatocytes. This being the case, we aimed to analyze the mechanisms involved in their effects. For this purpose, an in vitro model mimicking the hepatocyte situation in fatty liver was developed by incubating mouse AML12 hepatocytes with palmitic acid (PA). For cell treatments, hepatocytes were incubated with 1, 10 or 25 µM of pterostilbene, pterostilbene-4′-O-glucuronide or pterostilbene-4′-O-sulfate for 18 h. Triglycerides and cell viability were assessed by a commercial kit and crystal violet assay, respectively. Protein expression of enzymes and transporters involved in triglyceride metabolism was analyzed by immunoblot. The results showed for the first time the anti-steatotic effect of pterostilbene metabolites and thus, that they contribute to the preventive effect induced by pterostilbene on steatosis in in vivo models. This anti-steatotic effect is mainly due to the inhibition of de novo lipogenesis.