中药脂肝宁预防酒精性脂肪肝的实验研究

目的探讨中药脂肝宁预防酒精性脂肪肝的作用机理。方法应用酒精灌胃方法建立大鼠酒精性脂肪肝模型,造模同时给予中药脂肝宁(ZGN)药物干预,以硫普罗宁(TPN)为对照,检测血清肝功能和肝匀浆甘油三酯(TG)、氧自由基(ORF)、抗超氧阴离子(ASOA)、超氧化物歧化酶(SOD)及丙二醛(MDA)含量,并对大鼠肝脏行病理学检查。结果酒精灌胃12周后大鼠形成酒精性脂肪肝,中药脂肝宁高浓度组血清门冬氨酸氨基转移酶(AST)和肝匀浆TG、ORF及MDA含量显著降低,而ASOA和SOD含量升高(P<0.05),肝脏脂肪变明显改善。结论中药脂肝宁通过抑制氧化应激、稳定肝细胞膜、抑制脂质过氧化来改善酒精所致肝细胞脂肪变性。     

Restoration of autophagy by puerarin in ethanol-treated hepatocytes via the activation of AMP-activated protein kinase

We investigated the effects of puerarin, the major isoflavone in Kudzu roots, on the regulation of autophagy in ethanol-treated hepatocytes. Incubation in ethanol (100 mM) for 24 h reduced cell viability by 20% and increased the cellular concentrations of cholesterol and triglycerides by 40% and 20%, respectively. Puerarin stimulation significantly recovered cell viability and reduced cellular lipid accumulation to a level comparable to that in untreated control cells. Ethanol incubation reduced autophagy significantly as assessed by microtubule-associated protein1 light chain 3 (LC3) expression using immunohistochemistry and immunoblot analysis. The reduced expression of LC3 was restored by puerarin in a dose-dependent manner in ethanol-treated cells. The effect of puerarin on mammalian targets of rapamycin (mTOR), a key regulator of autophagy, was examined in ethanol-treated hepatocytes. Immunoblotting revealed that puerarin significantly induced the phosphorylation of 5′AMP-activated protein kinase (AMPK), thereby suppressing the mTOR target proteins S6 ribosomal protein and 4E-binding protein 1. These data suggest that puerarin restored the viability of cells and reduced lipid accumulation in ethanol-treated hepatocytes by activating autophagy via AMPK/mTOR-mediated signaling.     

SD大鼠酒精性脂肪肝模型的监测分析

目的：通过血清生化指标和病理学的监测分析来建立标准的SD大鼠酒精性脂肪肝动物模型。方法：选取40只SD大鼠,随机分为两组,模型组采用直接饮酒法,于第8、12和20周时检测大鼠血清生化指标：丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）、甘油三酯（TG）,并于第8、12周时随机采集5只大鼠肝组织,20周时采集剩余所有大鼠肝组织并进行病理学分析。结果：模型组于第8、12和20周时体重增长量均低于对照组（P〈0．01）,血清ALT、AST均高于对照组（P〈0．01）,第8周和12周时TG高于对照组（P〈0．01）。病理学结果显示肝组织从8周至20周呈现出酒精性脂肪肝、重度酒精性脂肪肝伴肝炎和酒精性肝纤维化等演变过程。结论：直接饮酒法可成功地复制出酒精性脂肪肝动物模型,通过监测分析可了解酒精性脂肪肝病变的整个过程,为今后建立酒精性脂肪肝和肝纤维化动物模型提供了理论参考。     

Gdf15对酒精性脂肪肝代谢异常的影响

摘要 目的：探讨生长分化因子15（Growth and Differentiation Factor 15, GDF15）对于酒精性脂肪肝(alcoholic fatty liver, AFL)代谢异常的影响。方法：用白酒构建酒精性脂肪肝小鼠模型然后分别给对照组和模型组尾静脉注射AAV8-GDF15过表达肝脏的GDF15分子,将小鼠共分为四组：正常+尾静脉注射对照AAV8-NC组(Con+AAV8-NC)、模型(酒精)+尾静脉注射AAV8对照病毒组(AFL+AAV8-NC)、正常+尾静脉注射过表达AAV8-Gdf15组(Con+AAV8-Gdf15)、酒精+尾静脉注射过表达AAV8-Gdf15组(AFL+AAV8-Gdf15)。对其体重、空腹血糖、葡萄糖耐量、胰岛素释放、血清脂、肝脏脂、谷丙转氨酶、谷草转氨酶含量的测定;其肝脏活组织切片使用苏木精-伊红染色法染色检测肝脏结构异常;q-PCR检测脂代谢相关分子RNA水平等观察过表达GDF15对酒精性脂肪肝的影响。结果：与Con+AAV8-N组相比,AFL+AAV8-NC组的体重下降,而过表达GDF15后AFL+AAV8-Gdf15组体重比AFL+AAV8-NC组的体重下降减少。与Con+AAV8-NC组相比,AFL+AAV8-NC组的空腹血糖升高、糖耐量及胰岛素耐量下降,过表达GDF15后AFL+AAV8-Gdf15组与AFL+AAV8-NC相比空腹血糖显著下降、糖耐量及胰岛素耐量显著升高。AFL+AAV8-NC组与Con+AAV8-NC组相比血脂TG明显升高,过表达GDF15后AFL+AAV8-Gdf15组血脂与AFL+AAV8-NC的血脂相比显著下降。与Con+AAV8-NC组相比,AFL+AAV8-NC组的肝脏重量增加,肝功能损伤程度更严重,肝脏脂肪含量增加,而过表达GDF15后AFL+AAV8-Gdf15与AFL+AAV8-NC组相比,肝脏重量、损伤程度及肝脏的脂肪含量均有显著性下降。结论：酒精性脂肪肝增加GDF15的表达,而GDF15的表达增加会改善酒精性脂肪肝的损伤及代谢异常。     

超声在评价大鼠酒精性脂肪肝模型中的应用

目的利用超声技术来评价大鼠酒精性脂肪肝动物模型。方法选取40只SD大鼠随机分为两组（n=20只）。模型组按每周测定的体重早晚各1次乙醇灌胃（10 g/kg）,第1周浓度为40%,第2、3周分别为45%和50%,第4周为55%灌胃直至12周;对照组给予等体积的生理盐水灌胃。造模于第4、8和12周时对两组大鼠进行超声监测,并从两组中各随机抽取3只大鼠进行肝脏病理学分析,与超声监测结果进行对比分析。结果超声与病理检查结果均提示酒精性脂肪肝造模成功,超声可以监测模型组大鼠肝脏脂肪病变从轻到重的渐变过程以及对照组大鼠无脂肪病变过程。这与肝组织的病理学诊断结果具有一致性。结论超声检测技术可以较好地进行活体评价大鼠酒精性脂肪肝动物模型。     

彩超与GGT联合检测对酒精依赖患者酒精肝的诊断价值

目的:研究分析彩超和谷氨酰转肽酶(GGT)联合检测对酒精依赖患者酒精性脂肪肝诊断的临床意义。方法:对2013年5月-2014年4月于我院住院并诊断为酒精依赖的患者39例(研究组)行肝脏彩超及GGT检测,另选取同期来源于本院职工、进修医护人员40例为对照组,对其结果进行分析。结果:研究组血清GGT为(189.95±226.52)U/L,显著高于对照组的(26.85±18.94)U/L,差异有统计学意义(t=4.54,P0.001);研究组中彩超诊断为脂肪肝者的GGT水平与非脂肪肝者有明显差异(P0.05),且高于对照组中的脂肪肝者,差异有统计学意义(P0.05)。结论:对于酒精依赖患者,血清GGT是敏感性较高的检测指标,GGT的检测有利于酒精性疾病的早期发现。彩超与GGT联合检测能提高临床对酒精性脂肪肝的检出率。     

Lysine 27 Ubiquitination of the Mitochondrial Transport Protein Miro Is Dependent on Serine 65 of the Parkin Ubiquitin Ligase

Mitochondrial transport plays an important role in matching mitochondrial distribution to localized energy production and calcium buffering requirements. Here, we demonstrate that Miro1, an outer mitochondrial membrane (OMM) protein crucial for the regulation of mitochondrial trafficking and distribution, is a substrate of the PINK1/Parkin mitochondrial quality control system in human dopaminergic neuroblastoma cells. Moreover, Miro1 turnover on damaged mitochondria is altered in Parkinson disease (PD) patient-derived fibroblasts containing a pathogenic mutation in the PARK2 gene (encoding Parkin). By analyzing the kinetics of Miro1 ubiquitination, we further demonstrate that mitochondrial damage triggers rapid (within minutes) and persistent Lys-27-type ubiquitination of Miro1 on the OMM, dependent on PINK1 and Parkin. Proteasomal degradation of Miro1 is then seen on a slower time scale, within 2–3 h of the onset of ubiquitination. We find Miro ubiquitination in dopaminergic neuroblastoma cells is independent of Miro1 phosphorylation at Ser-156 but is dependent on the recently identified Ser-65 residue within Parkin that is phosphorylated by PINK1. Interestingly, we find that Miro1 can stabilize phospho-mutant versions of Parkin on the OMM, suggesting that Miro is also part of a Parkin receptor complex. Moreover, we demonstrate that Ser-65 in Parkin is critical for regulating Miro levels upon mitochondrial damage in rodent cortical neurons. Our results provide new insights into the ubiquitination-dependent regulation of the Miro-mediated mitochondrial transport machinery by PINK1/Parkin and also suggest that disruption of this regulation may be implicated in Parkinson disease pathogenesis.     

肠道菌群在慢性肝脏疾病中作用的研究进展

随着肠—肝轴机制研究的不断深入,肠道菌群与多种慢性肝脏疾病如非酒精性脂肪性肝病、酒精性肝病、肝硬化等相关性研究日益增多。肠道菌群通过肠道菌群失调、物质能量代谢改变及免疫反应激活等机制在多种肝脏疾病发生发展中发挥重要作用。本文对肠道菌群与慢性肝脏疾病关系的研究进展进行综述。     

Role of autophagy in liver physiology and pathophysiology

Autophagy is a highly conserved intracellular degradation pathway by which bulk cytoplasm and superfluous or damaged organelles are enveloped by double membrane structures termed autophagosomes. The autophagosomes then fuse with lysosomes for degradation of their contents, and the resulting amino acids can then recycle back to the cytosol. Autophagy is normally activated in response to nutrient deprivation and other stressors and occurs in all eukaryotes. In addition to maintaining energy and nutrient balance in the liver, it is now clear that autophagy plays a role in liver protein aggregates related diseases, hepatocyte cell death, steatohepatitis, hepatitis virus infection and hepatocellular carcinoma. In this review, I discuss the recent findings of autophagy with a focus on its role in liver pathophysiology.     

Hyperphosphorylation of rat liver proteasome subunits: the effects of ethanol and okadaic acid are compared

In experimental alcoholic liver disease, protein degradation by the ATP-ubiquitin-proteasome pathway is inhibited. Failure of the proteasome to eliminate cytoplasmic proteins leads to the accumulation of oxidized and otherwise modified proteins. One possible explanation for the inhibition of the proteasome is hyperphosphorylation of proteasome subunits. To examine this possibility, the 26S proteasomes from the liver of rats fed ethanol and a pair-fed control were studied by isolating the proteasomes in a purified fraction. The effect of ethanol on the phosphorylation of proteasomal subunits was compared with the hyperphosphorylation of the proteasomes caused by okadaic acid given to rats in vivo. Ethanol ingestion caused an inhibition of the chymotrypsin-like activity of the purified proteasome. The 2D electrophoresis and Western blot analysis of the purified 20S and 26S proteasomes from the ethanol-fed rats indicated that hyperphosphorylation of proteasomal subunits had occured. The proteasomal alpha type subunits C9/alpha3 and C8/alpha7 were hyperphosphorylated compared to the controls. Chymotrypsin-like activity was also inhibited by okadaic acid treatment similar to ethanol feeding. The 26S proteasome fraction examined by isoelectric focusing gel revealed many hyperphosphorylated bands in the proteasomes from the okadaic acid treated and the ethanol fed rat livers compared with the controls. In conclusion hyperphosphorylation of the proteasome subunits occurs in the ethanol treated proteasomal subunits which could be one mechanism of the inhibition of the 26S proteasome caused by ethanol feeding.     

酒精性肝病发病过程中氧化应激指标的变化

目的酒精性肝病发病过程中氧化应激指标的变化。方法用酒精灌胃建立大鼠酒精性肝病模型,分别于4、8、12和16周留取血清和肝组织,用OLYMPUSAU-600全自动生化分析仪(日本)检测血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、胆碱酯酶(CHE);用生化比色法检测肝匀浆6-酮-前列腺素F1α(6-K-PGF1α)、血栓素B2(TXB2)、甘油三酯(TG)、氧自由基(ORF)、抗超氧阴离子(ASOA)、超氧化物歧化酶(SOD)及丙二醛(MDA)含量;用HE染色、苏丹Ⅳ染色和天狼星红染色观察肝组织病理学改变。结果随着病程进展,酒精性肝病大鼠肝脏呈现肝细胞脂肪变性、小叶内炎性坏死灶、窦周纤维化和汇管区纤维间隔形成等病理改变,血清ALT和AST逐渐升高,CHE降低,肝组织匀浆TG、OFR、MDA和TXB2含量逐渐升高,SOD、ASOA和6-K-PGF1α降低,与正常对照组比较P<0·05或P<0·01。结论ALD发病过程中存在氧化应激,氧化应激在酒精性肝病发病中发挥着重要作用。     

Effects of glutamine administration on inflammatory responses in chronic ethanol-fed rats

The purpose of this study was to investigate the effects of glutamine supplementation on inflammatory responses in chronic ethanol-fed rats. Male Wistar rats weighing about 160 g were divided into five groups. Two groups were fed a normal liquid diet and three groups were fed a glutamine-containing liquid diet. After 1 week, one of the normal liquid diet groups was fed an ethanol-containing liquid diet (CE), and the other group served as the control (CC) group. At the same time, one of the glutamine-containing liquid diet groups was continually fed the same diet (GCG), but the other two groups were fed ethanol-containing diet supplemented with glutamine (GEG) or without glutamine (GE). The following items were analyzed: (1) liver function, (2) cytokine contents, and (3) hepatic oxidative stress. The activities of aspartate transaminase (AST) and alanine transaminase (ALT) and levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the CE group had significantly increased. In addition, hepatic cytochrome P450 2E1 (CYP2E1) expression had significantly increased in the CE, GE and GEG groups. However, the activities of AST and ALT and levels of TNF-α and IL-1β in the GE group were significantly lower than those of the CE group. The results suggest that the plasma inflammatory responses of rats fed an ethanol-containing liquid diet for 7 weeks significantly increased. However, pretreatment with glutamine improved the plasma inflammatory responses induced by ethanol.     

肠道菌群与相关疾病

近年来肠道菌群的研究发展迅速,肠道菌群对宿主消化、代谢和免疫功能的影响逐渐被人们所熟知并重视。大量研究提示,肠道菌群的改变可能引发代谢、肝脏和肠道等方面的多种相关疾病。因此,研究肠道菌群对宿主健康及疾病的影响尤为重要,也能为预防和治疗肠道菌群相关疾病提供建议。     

益生菌通过调整正常菌群缓解酒精性肝损伤的研究进展

许多临床试验表明慢性酒精性肝损伤会引起肠道菌群的失调,主要表现为双歧杆菌、乳杆菌数量减少,革兰氏阴性菌大量繁殖,破坏肠道屏障功能,增加肠道通透性,使细菌来源的内毒素大量释放出来,引起血液内毒素增加,并在肝脏中累积,超出肝脏的清除能力,导致肝损伤。本文主要综述益生菌通过调整正常菌群这一机制来缓解酒精性肝损伤的研究进展,进而深入了解酒精引起肠道菌群变化(酒精的摄入会导致肠道中拟杆菌、厚壁菌数量减少,革兰氏阴性变形菌、革兰氏阳性放线菌数量增加,同时肠道内细菌来源的内毒素水平增加)导致肝损伤的发病机制,以及益生菌如何通过调整肠道正常菌群改善酒精性肝损伤。     

肝脏免疫系统在酒精性肝损伤中的作用启示

酒精性肝病的致病因素是单一,但其发病机制复杂,目前尚不完全清楚。肝脏免疫系统被认为是独特的免疫系统,其作用越来越引起重视。肝内既有参与外周循环的淋巴细胞,也有长期定居于此的免疫细胞;加上肝脏解剖结构和血液循环的特殊性决定了肝脏独特的免疫微环境。研究肝脏免疫系统在酒精性肝病发病机制的作用,将有助于进一步阐明酒精性肝病发病机制,为酒精性肝病的预防和治疗提供新的靶点。     

乳酸菌保肝护肝的研究进展

目的 本研究介绍了酒精性肝损伤机制,乳酸菌抗氧化及保肝护肝的研究进展,并且对乳酸菌抗氧化的体内和体外实验进行了总结.