鳖甲煎丸对肝纤维化大鼠作用机制研究

[目的]探讨鳖甲煎丸抗肝纤维化的作用机制.[方法]采用复合因素建立肝纤维化大鼠模型.将大鼠随机分为正常组、模型组、秋水仙碱组、鳖甲煎丸高剂量组、鳖甲煎丸等剂量组.秋水仙碱组[0.11mg/（kg·d）]、鳖甲煎丸高剂量组[2.2 g/（kg·d]及鳖甲煎丸等剂量组[1.1 g/（kg·d]分别予以相应药物灌胃,模型组及正常组给予等剂量的0.9％氯化钠灌胃,共6周.采用比色法检测血清丙氨酸氨基转移酶,天冬氨酸转氨酶（ALT、AST）水平,苏木精-伊红染色观察肝组织病理学变化及肝纤维化程度,免疫组化方法检测肝组织瘦素、瘦素受体表达水平.[结果]鳖甲煎丸能降低大鼠血清ALT、AST水平,减轻肝脏炎症及纤维化程度（均P＜0.01）;抑制模型大鼠肝组织瘦素及瘦素受体的表达（P＜0.01）.[结论]鳖甲煎丸能有效减轻模型大鼠肝纤维化的病理程度.其作用机制可能与降低大鼠血清ALT、AST水平,抑制大鼠肝组织瘦素及瘦素受体的表达密切相关.     

缬沙坦对肝纤维化大鼠瘦素表达的影响

背景：瘦素与肝纤维化的形成和发展有关,血管紧张素Ⅱ1型受体（AT1R）拮抗剂可降低大鼠脂肪组织瘦素的合成和分泌：目的：探讨ATIR拈抗剂缬沙坦对免疫性肝纤维化大鼠肝组织瘦素表达的影响。方法：36只Sprague-Dawley大鼠随机分为正常对照组、模型组和缬沙坦预防组,后两组腹腔注射绪血清建立肝纤维化模型．缬沙坦预防组大鼠于造模开始后每天予缬沙坦（30mg／kg）灌胃。造模第10周处死大鼠,行HE染色和天狼猩红染色分析肝纤维化程度和胶原面积,以免疫组化法检测肝组织瘦素表达,结果：与正常对照组相比,模型组肝纤维化程度显著增强（P〈0．05）;胶原面积显著增多（10．08％±2．01％对0．62％±0．31％,P〈0．01）,肝组织瘦素表达显著增高（5．05±2．91对0．44±0．27,P〈0．05）。经缬沙坦干预后,上述指标均显著改善。结论：瘦素可促进纤维化发生,AT1R拮抗剂缬沙坦能通过降低肝组织瘦素表达而延缓肝纤维化的发展,这可能是其抗纤维化机制之一。     

慢性乙型肝炎患者肝组织瘦素的表达

近年来瘦素（leptin）与肝脏疾病的关系日益受到关注。动物实验表明，瘦素可能在鼠肝纤维化病理形成中具有重要意义。目前关于人瘦素血清学研究较多，而本实验采用免疫组化方法，主要探讨HBV感染者肝组织原位瘦素表达情况。     

川芎嗪对肝硬化大鼠瘦素及其受体表达的干预作用

瘦素与肝纤维化关系密切，但其在肝硬化形成过程中的作用机制不明。目前使用的抗肝硬化药物疗效均不满意。目的：观察瘦素及其功能性受体（Ob-Rb）在大鼠肝硬化形成过程中的表达变化，以及川芎嗪对两者表达的干预作用，探讨川芎嗪抗肝纤维化的作用机制。方法：诱导CCl4大鼠肝纤维化模型。川芎嗪预防组和治疗组分别于实验第1d和第31d开始予川芎嗪每天10mg／100g体重干预。于12周内分批处死各组大鼠。以逆转录聚合酶链反应（RT-PCR）检测肝组织瘦素和Ob-RbmRNA表达，以免疫组化方法检测瘦素和Ob-Rb的表达和定位，同时行血清透明质酸（HA）水平检测和肝内胶原定量分析。结果：正常肝组织中有少量瘦素和Ob-RbmRNA表达，随着肝硬化的形成，两者表达逐渐增加，与血清HA水平和肝内胶原含量呈正相关。肝硬化模型组汇管区、纤维间隔、小叶内血管、胆管、肝窦周围瘦素和Ob-Rb表达明显增加，表达强度从第3～12周呈递增趋势。川芎嗪预防组和治疗组瘦素和Ob。Rb及其mRNA表达均低于肝硬化模型组，两组血清HA水平和肝内胶原含量亦显著低于肝硬化模型组。结论：瘦素和Ob-Rb在肝硬化形成过程中发挥重要作用。川芎嗪能下调CCl4肝纤维化大鼠肝组织瘦素和Ob-Rb的表达，这可能是其抗肝纤维化的作用机制之一。     

瘦素及其受体在非酒精性脂肪性肝炎发病机制中的作用

目的探讨瘦素(LP)及其受体在非酒精性脂肪性肝炎(NASH)大鼠肝组织中的表达及意义。方法采用高脂饮食制备Wistar大鼠NASH模型;ELISA法测定大鼠血清瘦素(LP)浓度;全自动生化分析仪检测各组大鼠血清空腹血糖(FBG)、甘油三酯(TG)水平;比色法测定血清游离脂肪酸(FFA)含量;放射免疫法测定血清胰岛素(FINS)及肿瘤坏死因子α(TNF-α)水平,并计算胰岛素抵抗指数(HOMA-IR);HE、苏丹Ⅳ、Masson三重染色光镜下观察肝组织病理变化;逆转录聚合酶链反应(RT-PCR)和免疫组织化学染色分别检测肝组织OB-RbmRNA和蛋白的表达。结果模型组大鼠血清瘦素、TG、FFA、HOMA-IR、TNF-α均较正常对照组明显升高;模型组大鼠肝组织表现为不同程度的脂肪变性、炎症坏死及窦周纤维化;肝组织OB-RbmRNA和蛋白的表达则较正常对照组显著减弱;相关分析显示:模型组大鼠肝组织瘦素受体蛋白表达与与血清瘦素水平、血游离脂肪酸、TNF-α、胰岛素敏感指数、肝细胞脂肪变性、炎症活动度呈显著负相关,相关系数r分别为-0.83、-0.71、-0.74、-0.65、-0.83、-0.87,P<0.01。多元线性回归分析显示,瘦素受体表达减弱是肝脏脂肪变性的独立影响因素。结论肝组织瘦素受体表达减弱是NASH瘦素抵抗的重要病理机制之一,瘦素抵抗与胰岛素抵抗相互作用,共同促进了NASH的发生发展。     

强肝胶囊对非酒精性脂肪肝大鼠肝脏瘦素受体及P-JAK2和P-STAT3蛋白的影响

[目的]观察强肝胶囊对非酒精性脂肪肝（NAFLD）大鼠的治疗作用,及其对血清瘦素、肝组织瘦素受体mRNA、P-JAK2和P-STAT3蛋白表达的影响,探讨强肝胶囊治疗NAFLD的可能机制。[方法]高脂饲料制备SD雄性大鼠NAFLD模型,随机分为空白组和造模组。待造模成功后将造模组大鼠随机分为模型对照（模型）组、强肝胶囊组和辛伐他汀组。治疗4周后行肝组织病理学检测,并同时应用ELISA试剂盒检测血清瘦素,RT-PCR法检测肝组织瘦素受体mRNA表达,western blot检测肝组织P-JAK2、P-STAT3蛋白的表达。[结果]强肝胶囊能明显降低NAFLD模型大鼠肝组织三酰甘油、总胆固醇的水平,改善脂肪变性,降低肝脏炎症反应,并能明显降低血清瘦素高水平状态,改善瘦素抵抗,同时增加瘦素受体mRNA的表达,增加肝组织P-JAK2、p-STAT3蛋白的水平。[结论]强肝胶囊对NAFLD大鼠肝脏脂质和炎症有较好的治疗作用,其可能机制是通过改善瘦素抵抗,增加肝脏瘦素受体mRNA表达及P-JAK2、P-STAT3蛋白水平而完成的。     

原发性肝细胞癌患者组织中瘦素及其受体表达与临床病理特征的关系

目的检测瘦素（leptin）和瘦素受体（OB—R）在原发性肝细胞癌（HCC）患者癌及癌旁组织中mRNA和蛋自水平表达情况。方法应用RT-PCR和Westernblot方法检测leptin其受体在33例HCC患者癌及癌旁组织中mRNA和蛋白表达,并分析其与临床病理特征之间的关系。结果leptin和OB—RmRNA及蛋白在癌旁组织表达水平高于癌组织（P〈0．05）。leptin和OB—R的表达与各临床病理特征均无显著相关（P〉0．05）。结论leptin及OB-R在HCC患者的癌及癌旁组织中均表达。     

瘦素促进大鼠肝星状细胞活化及其细胞外信号调节激酶磷酸化

近年研究发现，瘦素在肝纤维化病理形成中具有重要意义，可促进实验性动物肝脏炎症与纤维化病理发展。但是瘦素是否直接促进肝纤维化形成的关键细胞——肝星状细胞（HSC）活化尚不清楚。本实验采用大鼠HSCT6细胞株，观察不同浓度的瘦素对HSC-T6增殖、α-平滑肌肌动蛋白（α-SMA）与Ⅰ型胶原蛋白表达的作用．及其对细胞外信号调节激酶（ERK）磷酸化的影响．探讨瘦素对HSC活化的影响与部分作用机制。     

瘦素在实验性大鼠肝纤维化发生中的作用机制初探

目的通过检测肝纤维化大鼠血清及肝组织中瘦素（Leptin）的表达水平，初步探讨瘦素在大鼠肝纤维化发生中的作用机制。方法32只雄性SD大鼠，皮下注射四氯化碳（CCl4）油剂制备肝纤维化模型。分别于注射后1、4、8周处理动物。抽取血清并留取肝组织，采用放射免疫学方法检测血清中瘦素的表达水平。应用免疫组化方法检测肝组织中瘦素及转化生长因子β1（Transforming growth factor β1，TGF—β1）的表达。结果放免及免疫组化结果均显示，与正常对照组相比，注射CCl4诱导后，肝纤维化大鼠血清及肝组织中瘦素的表达水平明显升高（P〈0．01．〈0．05），且1，4，8周组瘦素的表达强度呈明显递增趋势（P〈0．01，〈0．05）。瘦素主要表达于肝星状细胞胞质中。结论瘦素主要表达于肝星状细胞，并可能通过窦内皮细胞发挥重要的促纤维化作用。     

瘦素及瘦素功能性受体表达与大鼠肝硬化关系的研究

目的观察实验性大鼠肝硬化形成过程中,肝组织瘦素及瘦素功能性受体表达的动态变化以及与肝内胶原含量的关系。方法经皮下注射四氯化碳(0.3ml/100g体重,每周2次)复制肝硬化动物模型,于第0、3、6、9、12周各处死一批大鼠。用RT-PCR检测各期动物肝组织中瘦素及瘦素功能性受体(Ob-Rb)mRNA表达水平。用免疫组织化学方法检测各期动物肝组织中瘦素、瘦素受体的表碉和定位,用放射免疫法检测各期动物血浆中透明质酸(HA)、瘦素水平。运用MPIAS-500多媒体真彩色图像分析系统作肝内胶原定量分析。结果正常肝组织中有少量瘦素、瘦素功能性受体的mRNA表达,随着肝硬化的形成,各期表达量逐渐递增(与0周比较,P<0.01)。正常肝组织可见瘦素及瘦素受体在血管周围、汇管区及肝索间质细胞的细胞质和细胞膜有少量表达,随着肝硬化的形成,瘦素、瘦素受体表达增多、增强(P<0.01)。随着造模时间的增加,肝内胶原面积、血浆中HA和瘦素均逐渐增加,各期比较差异有统计学意义(P<0.05)。肝硬化形成过程中,肝脏中瘦素、瘦素功能性受体的mRNA表达水平与血浆中HA水平及肝内胶原含量呈正相关(瘦素与HA:r=0.726,P=0.00;瘦素与胶原:r=0.732,P=0.00;Ob-Rb与HA:r=0.705,P=0.00;Ob-Rb与胶原:r=0.764,P=0.00)。结论在四氯化碳诱导的大鼠肝硬化形成过程中,瘦素及功能性受体的基因和蛋白表达增加,并随肝硬化程度的加重而逐步升高,从而促进肝硬化的发生。     

冠心病患者血浆瘦素与可溶性瘦素受体水平的相关性

目的:探讨冠心病患者血浆瘦素（leptin,Lep）及可溶性瘦素受体(soluble leptin receptor,sLR)水平的变化,并分析Lep与冠心病各危险因素的关系。方法: 选取冠心病患者180例,正常对照组60例。所有患者行冠脉造影检查,冠脉狭窄程度采用Genisin评分,酶联免疫法测定Lep、sLR浓度,同时检测总胆固醇(TC)、三酰甘油(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、体质量指数(BMI)、腰围、臀围等指标,分析Lep、sLR与冠心病的的关系。结果: 冠心病患者lep水平显著高于对照组［（12±6） vs.（9±5）μg/L,P<0.01］,sLR水平显著低于对照组［（124±62） vs.（164±70）μg/L,P<0.01］,在调整年龄、血糖和血压后,两组间的差别有显著性意义。多元Logistic回归分析显示冠心病患者μg/L。Lep水平的升高独立于年龄、血压、血脂等危险因素。结论: 冠心病患者Lep水平与冠脉病变的严重程度呈正相关,而sLRs水平与冠脉病变严重程度呈负相关。     

酒精性肝病患者血清瘦素及其受体基因Gln223Arg多态性变化

目的 观察酒精性肝病患者血清瘦素（Lep）水平及其受体（LEPR）基因Gln223Arg多态性变化,并探讨其意义.方法 选择酒精性肝病患者106例,其中酒精性脂肪性肝炎45例（AH组）,酒精性肝硬化61例（AC组）,同期健康体检者65例作为对照组.采用ELISA法检测血清Lep,葡萄糖氧化酶—过氧化物酶法检测空腹血糖（FPG）,化学发光免疫分析法检测空腹血清胰岛素（FINS）,PCR-RFLP法检测LEPR基因Gln223Arg多态性,并计算HOMA-IR.结果 AH组血清Lep、HOMA-IR分别为（8.95±1.81） ng/mL、2.44±0.25,AC组分别为（10.57±2.00） ng/mL、3.21 ±0.17,对照组分别为（4.44±0.81） ng/mL、1.77 ±0.18;AH组、AC组与对照组比较,P均＜0.05.AH组、AC组血清Lep与HOMA-IR均呈正相关（r=0.45、0.38,P均＜0.01）.AH组AA、A/G、GG的例数分别为3、11、31例,AC组分别为0、25、36例,对照组分别为1、12、52例;AC组与对照组比较,P＜0.05.结论 酒精性肝病患者血清Lep、HOMA-IR水平升高,AC组患者LEPR基因Gln223 Arg杂合基因频率高于健康人群,可能通过胰岛素—瘦素轴促进胰岛素抵抗,影响体内脂肪代谢,参与酒精性肝病的发病机制.     

Smoking habits and circulating leptin in postmenopausal non-obese women

This study examined whether smoking habits affect the correlation between serum leptin levels and body mass index (BMI) in 101 postmenopausal Caucasian non-obese women, all aged 57-59years. Mean serum leptin level in the entire group was 19.8 +/- 12.3 ng/ml (mean +/- s.d.) and mean BMI was 25.3 +/- 3.5 kg/m2. These parameters correlated significantly to each other (r = 0.70, p < 0.001). Between those who have smoked regularly for more than 2 years (n = 26) and those who are ex-smokers or have never smoked (n = 75), serum leptin levels or BMI did not differ and the correlation between serum leptin and BMI was identical. This suggests that smoking habits do not influence circulating leptin in Caucasian postmenopausal non-obese women.     

Ten years of leptin replacement therapy

Leptin is a pleiotropic cytokine‐like hormone that is involved in the regulation of energy intake and expenditure, neuroendocrine function, immunity and lipid and glucose metabolism. The few humans with genetically based leptin deficiency provide a unique model to assess those effects. We have identified five Turkish patients (one male and two female adults; one boy and one girl) with congenital leptin deficiency due to a missense mutation in the leptin gene. Four of these patients were treated with physiological doses of recombinant methionyl human leptin. Body composition, brain structure and function, behaviour, immunity and endocrine and metabolic parameters were evaluated before and during treatment. Our results showed that leptin has peripheral, hypothalamic and extra‐hypothalamic effects. Within the endocrine system, leptin regulates the circadian rhythms of cortisol, thyroid‐stimulating hormone, luteinizing hormone and follicle‐stimulating hormone. In the brain, leptin controls energy balance and body weight, and plays a role on neurogenesis and brain function. Leptin is a key element of the adiposinsular axis, enhances immune response, and regulates inflammation, coagulation, fibrinolysis and platelet aggregation. Our 10‐year experience in treating these unique patients provided valuable data on the peripheral and central effects of leptin. Those results can be taken into account for the development of leptin‐based therapies for other diseases.     

Serum leptin and soluble leptin receptor in non-alcoholic fatty liver disease

AIM： To determine the role of leptin system in non-alcoholic fatty liver disease （NAFLD） development by delineating the changes in serum levels of leptin and soluble leptin receptor （sOB-R）.
METHODS： Blood samples were collected from 30 consecutive patients with liver-biopsy-proven NAFLD and 30 patients with cholecystolithiasis （stationary phase） as controls. Serum leptin levels were determined by radioimmunoassay and concentration of sOB-R was measured by ELISA. Body mass index （BMI） was calculated for all subjects, and serum insulin, C-peptide, and lipoprotein levels were also detected.
RESULTS： Mean serum leptin level and BMI in the NAFLD group were significantly higher than in the controls （both P 〈 0.001）, but mean sOB-R level was lower in the NAFLD group when compared to the controls. Both men and women in the NAFLD group had higher mean serum leptin levels and lower sOB-R levels than did the men and women in the control group （all P 〈 0.001）. There was a significant negative correlation between serum leptin and sOB-R levels （r = -0.725, P 〈 0.001）. Multivariate analysis showed that the percentage of hepatocyte steatosis, sex, BMI, and homeostasis model assessment of insulin resistance （HOMA IR） were independently related to serum leptin levels.
CONCLUSION： Elevated serum leptin seems to be afeature of steatosis, and serum leptin seems to increase as hepatocyte steatosis develops. An enhanced release of leptin is accompanied by an decrease in sOB-R concentration, which suggests higher resistance of peripheral tissues towards the action of leptin.     

Serum leptin and myocardial infarction in hypertension

The adipose tissue-derived hormone leptin is among the physiologic processes involved in cardiovascular regulation. The aim of the present study was to elucidate if serum leptin may predict cardiovascular risk, particularly myocardial infarction (MI), in hypertensive men and women. In a prospective study cohort of hypertensive men and women, serum leptin was compared in 171 patients with MI and in 342 matched controls. The mean serum concentration of leptin was 25.1 ± 20.0 ng/ml in the MI patients and 20.0 ± 16.6 ng/ml in the controls (p = 0.007). The association between serum leptin and MI was independent of traditional risk factors. Leptin concentrations were higher in women than in men. In women, serum leptin was the most important predictor of MI. The present study indicates that serum leptin is associated with MI in a hypertensive population. Leptin concentrations may be of practical importance when estimating the risk of MI, especially in women, where leptin was found to be the most important predictor for MI.     

红斑狼疮患者瘦素及可溶性瘦素受体检测的临床意义

目的探讨血清瘦素(Lp)和可溶性瘦素受体(sLR)与不同临床分型的红斑狼疮(LE)及系统性红斑狼疮(SLE)病情活动程度的关系。方法于2004年6月至2005年12月对中国医科大学附属第二医院和中国医科大学附属第一医院的39例LE患者,同年龄、性别、体重指数(BMI)均相匹配的31名健康人对照,采用放射免疫法和酶联免疫吸附实验(ELISA)检测血清Lp和sLR水平。结果(1)与正常对照相比较,LE患者血清Lp水平增高和sLR水平下降,差异有统计学意义(P<0.01),活动期SLE患者血清Lp水平增高和sLR水平下降尤为显著;但非活动期SLE、亚急性皮肤型红斑狼疮(SCLE)和盘状红斑狼疮(DLE)患者血清Lp和sLR水平差异无统计学意义(P>0.05)。(2)Lp与BMI呈正相关,与sLR呈负相关;SLE患者的病情活动程度与血清Lp水平呈正相关,与sLR水平呈负相关(P<0.01)。结论Lp和sLR水平异常可能与LE的发病密切相关。     

Elevated serum levels of leptin and soluble leptin receptor in patients with advanced chronic heart failure

Patients with chronic heart failure (CHF) have metabolic abnormalities, leading to a catabolic syndrome, with progressive loss of skeletal muscle in advanced stages of the disease. Leptin, the product of an obesity gene, has been associated with energy expenditure and weight regulation. The aim of this study was to assess serum levels of leptin and its soluble receptor in relation to exercise intolerance and neurohumoral activation in patients with CHF. We investigated 53 patients with CHF left ventricular ejection fraction (LVEF) 25+/-1%, age 56.6+/-1.3 years, Maximal oxygen uptake (VO(2) max) 16.3+/-0.6 ml/min.kg) sub-classified according to peak oxygen consumption of > or 14 ml/min.kg and controls). Elevated levels of leptin correlated with an increased serum concentration of TNFalpha (r=0.749, P<0.01) in this subgroup of patients with CHF. We conclude that patients with advanced CHF show elevated serum levels of leptin and its soluble receptor. This finding indicates that leptin may participate in the catabolic state leading to the development of cardiac cachexia in the course of CHF.