虫草素对四氯化碳所致小鼠急性肝损伤的保护作用

本文探究蛹虫草活性成分虫草素对四氯化碳（CCl₄）造成的小鼠急性肝损伤的保护作用及其分子机制。首先建立四氯化碳致小鼠急性肝损伤的动物模型,通过检测血清生化指标、肝功指标的变化及HE染色观察组织切片病理的病变情况,评价虫草素的保肝效果,进一步通过Western blot检测虫草素能否通过激活Nrf-2/Keap1信号通路及其下游抗氧化因子（HO-1、NQO-1）的表达来提高机体抗氧化损伤能力以及抑制炎症因子（TNFα、TNFβ、IL-6、IL-10）的表达。对比模型组结果显示,虫草素能极显著降低（P<0.01）小鼠血清中ALT、AST及肝脏中MDA水平,并能极显著提高肝脏中SOD水平（P<0.01）;HE染色结果显示虫草素能有效降低改善受损肝组织中的炎细胞浸润及纤维组织增生;Western blot结果表明虫草素能够通过激活Nrf-2信号通路,促进下游抗氧化因子及抗炎因子的表达,从而降低炎症反应。虫草素对CCl₄致小鼠急性肝损伤具有一定的保护作用,其机制与Nrf-2信号通路相关,实验结果为后续蛹虫草及虫草素的开发应用奠定基础。     

益生菌对急性肝内胆汁淤积大鼠肝组织NF-κB和MCP-1的影响

目的通过研究益生菌制剂对异硫氰酸萘酯（ANIT））所致的急性肝内胆汁淤积大鼠肝组织NF-κB和MCP-1表达的影响,进一步探讨益生菌防治急性肝内胆汁淤积肝损伤的作用机制。方法72只幼年雄性Sprague-Dawley（SD）大鼠分为正常对照组（8只）、中毒组（32只）和干预组（32只）。中毒组及干预组幼鼠,按100mg／kg一次性灌服ANIT诱导急性肝内胆汁淤积病变,干预组于ANIT灌胃前3d开始灌服培菲康[4．2×10^8个活菌／（kg·d）]。观察各组在灌服ANIT后24h、48h、72h和96h血浆总胆红素（TB）、丙氨酸转氨酶（ALT）的浓度,同时用RT-PCR测定肝组织中MCP-1mRNA的表达,用免疫组化方法测定肝组织中NF-κB、MCP-1蛋白的表达,并在光学显微镜下观察肝脏的形态学改变。结果干预组大鼠血清ALT、TB在灌服ANIT后24h、48h、72h和96h各时间点升高的峰值较中毒组明显减低,且其肝组织MCP-1mRNA和蛋白表达水平以及NF-κB蛋白表达水平较中毒组低。结论益生菌能够改善急性肝内胆汁淤积肝脏功能,降低NF-κB、MCP-1的基因表达,对急性肝内胆汁淤积性肝损伤起到一定的防治作用。     

α-荼异硫氰酸酯诱导小鼠胆汁淤积性肝纤维化及其炎症通路*

目的: 探讨α-荼异硫氰酸酯(ANIT)诱导的胆汁淤积性肝纤维化的发展及其炎症通路。方法: 将15只体重为(23±2) g的129/Sv小鼠随机分为对照组(n=5)和实验组(n=10)。对照组常规饲料喂养,实验组小鼠给予0.05% ANIT饲料食饲。实验组分别在14 d和28 d各处死5只小鼠,收集胆囊、血清、肝脏等标本。按试剂盒程序检测胆汁淤积生化指标,组织病理学评估肝细胞损伤程度,Q-PCR和WB分析肝纤维化、炎症反应等水平。结果: 与对照组相比,造模第2周ANIT -14 d组(A-D14)中的主要胆汁淤积指标总胆汁酸(TBA)从(3.2±0.9) μmol/L显著增加至(31.6±4.3) μmol/L,肝损伤指标谷草转氨酶(AST)和谷丙转氨酶(ALT)也显著升高(P＜0.05);纤维化因子金属蛋白酶组织抑制因子1(TIMP-1)、单核细胞趋化因子(MCP-1)、I型胶原蛋白(Collagen I)表达高于对照组(P＜0.05);Collagen I和α-SMA纤维化蛋白表达均上调;肝脏胶原纤维大量沉积,纤维化已产生(P＜0.05)。炎症因子表达高于对照组,JNK、c-Jun、STAT3等均被激活(P＜0.05)。ANIT-28 d组(A-D28)中除AST、基质金属蛋白酶2(MMP-2),Collagen I指标稍有降低外,其余胆汁淤积、肝损伤、肝纤维化、炎症等指标继续上调或保持稳定(P＜ 0.05)。结论: 0.05%的ANIT饲料干预14 d,小鼠即发生明显的胆汁淤积性肝纤维化;28 d后,胆汁淤积性肝纤维化趋于稳定;JNK炎症通路在肝纤维化的发生发展中起着至关重要的作用。     

微生态制剂对肝内胆汁淤积性肝损伤兔模型胆汁和血清TNF-a、IL-6的影响

目的:通过研究异硫氰酸萘酯(ANIT))所致的急性肝内胆汁淤积性肝损伤兔模型血清和胆汁中TNF-a、IL-6的代谢变化,来进一步探讨微生态制剂-乐托尔对该模型的作用机制.方法:按200 mg/kg一次性灌服ANIT来诱发新生白兔急性肝内胆汁淤积性肝损伤病变,分别检测中毒后24、48 h及用乐托尔散剂治疗后血清和胆汁中总胆红素(TB)、丙氨酸转氨酶(ALT)、肿瘤坏死因子-a(TNF-a)、白细胞介素-6(IL-6)的值.结果:在乐托尔组中发现,灌服ANIT 48 h后血清和胆汁中的TNF-a、IL-6、ALT、TB值均较中毒48 h组明显降低(P值均小于0.05);各组胆汁中TNF-a、IL-6的浓度均明显高于血清中浓度.结论:微生态制剂能够改善肝功能,增加胆汁的排泄量,使炎性细胞因子的产生量显著减少.     

微生态制剂对肝内胆汁淤积性肝损伤兔模型胆汁和血清TNF—α、IL—6的影响

目的：通过研究异硫氰酸萘酯(ANIT))所致的急性肝内胆汁淤积性肝损伤兔模型血清和胆汁中了TNF-α、IL-6的代谢变化,来进一步探讨微生态制剂-乐托尔对该模型的作用机制。方法：按200mg/kg一次性灌服ANIT来诱发新生白兔急性肝内胆汁淤积性肝损伤病变,分别检测中毒后24、48h及用乐托尔散剂治疗后血清和胆汁中总胆红素(TB)、丙氨酸转氨酶(ALT)、肿瘤坏死因子-a(TNF-α)、白细胞介素-6(IL-6)的值。结果：在乐托尔组中发现,灌服ANIT 48h后血清和胆汁中的TNF-α,IL-6,ALT,TB值均较中毒48h组明显降低（P值均小于0.05);各组胆汁中TNF-α,IL-6的浓度均明显高于血清中浓度,结论：微生态制剂能够改善肝功能,增加胆汁的排泄量,使炎性细胞因子的产生量显著减少。     

胆汁淤积性肝损伤治疗靶点与药物治疗进展

胆汁淤积(cholestasis)是由于胆汁合成、分泌、转运、排泄等代谢功能紊乱引起的肝内胆汁淤留,肠中缺乏胆汁,血中胆汁成分过多的一种病理状态。胆汁酸信号传递过程以及胆汁淤积过程中炎症的发展和延续这些与胆汁淤积相关的肝损伤方面的分子机制已经取得重大进展。核受体作为肝脏疾病的靶标被广泛讨论,了解核受体在病理生理条件下的调节,是肝脏疾病的潜在治疗方法。本文基于中药治疗和分子免疫机制受体水平方面对胆汁淤积性肝病进行综述。     

基于JNK通路研究老鹳草素对免疫性肝损伤小鼠的影响

为探讨老鹳草素对脂多糖(LPS)所致小鼠免疫性肝损伤的保护作用及可能机制,60只小鼠被随机分为正常组、模型组、水飞蓟素组(180 mg/kg)和老鹳草素低、中、高剂量组(50、100、200 mg/kg),小鼠腹腔注射10 mg/kg的LPS诱导免疫性肝损伤。采取HE染色观察小鼠肝细胞病理改变情况;生化法检测血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)活性、碱性磷酸酶(ALP)、总胆红素(TBIL)水平,血清中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性和丙二醛(MDA)含量;酶联免疫吸附试验(ELISA)法检测肝组织中炎症因子白介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)和白介素-6(IL-6)的含量;蛋白免疫印迹实验(Western blot)法检测小鼠肝组织中p-ASK1、ASK1、MKK4、p-MKK4、JNK、p-JNK、c-Jun和p-c-Jun的表达水平。结果表明,老鹳草素各剂量组和水飞蓟素组小鼠肝组织炎症坏死和凋亡现象有不同程度的减轻。与模型组相比,老鹳草素各剂量组可显著降低血清中的ALT、AST、ALP、TBIL、MDA和肝组织中IL-1β、TNF-α和IL-6水平,上调血清中SOD和GSH-Px的水平,并且抑制肝组织中p-ASK1、p-MKK4、p-JNK、p-c-Jun的表达。综上所述,老鹳草素对LPS诱导的免疫性肝损伤具有保护作用,其机制可能与抑制JNK通路相关。     

金线风总黄酮对四氯化碳致急性肝损伤小鼠的保护作用及机制研究

研究金线风总黄酮(TFC)对四氯化碳(CCl4)致急性肝损伤小鼠的保护作用,并从氧化应激、炎症反应和TLR-4/NF-κB信号通路探讨其作用机制。60只小鼠随机分为正常组、模型组、水飞蓟素组(150 mg/kg)、TFC低、中、高剂量组(100、200、400 mg/kg)、连续灌胃给药10 d。末次给药2 h后,除正常组外,各组腹腔注射0.1%的CCl4花生油溶液(10 m L/kg),建立小鼠急性肝损伤模型,16 h后,收集血清和肝组织。血清指标检测表明,与模型组比较,TFC能够显著降低肝脏指数、ALT和AST活性(P0.05),并减少ALP、TBIL和γ-GT含量(P0.05),且降低MDA含量(P0.05),同时增强T-SOD和GSH-Px活性(P0.05)。ELISA法检测肝组织指标结果表明,与模型组比较,TFC能够显著下调TNF-α、IL-1β和IL-6含量(P0.05)。Western blot检测结果显示,与模型组比较,TFC能够明显降低肝组织中TLR-4和NF-κB蛋白表达(P0.05)。HE染色分析肝组织病理学变化结果表明,TFC能够有效改善肝组织损伤程度。综上所述,TFC对CCl4诱导的急性肝损伤小鼠具有保护作用,其保肝作用机理可能与抑制氧化应激、炎症反应以及TLR-4/NF-κB信号通路有关。     

LAG-3敲除加重免疫性肝损伤

目的：探究淋巴细胞活化基因-3(lymphocyte-activation gene 3, LAG-3)在免疫性肝损伤中的作用及机制。方法：采用尾静脉注射伴刀豆球蛋白A(Concanavalin A, Con A)诱导小鼠急性免疫性肝损伤模型;流式细胞术、实时定量PCR检测肝脏内免疫细胞LAG-3表达;利用LAG-3敲除小鼠研究LAG-3对Con A诱导的免疫性肝损伤的影响,于Con A注射后不同时间点收取外周血,检测血清ALT、AST、细胞因子水平;收集肝脏组织样本进行苏木精-伊红染色(hematoxylin-eosin staining, HE染色)并统计肝脏组织坏死面积;流式细胞术检测肝内细胞分群、T细胞活化;实时定量PCR检测炎症相关基因的表达;酵母双杂交筛选LAG-3相互作用蛋白质。结果：Con A诱导肝脏免疫细胞LAG-3表达增加;LAG-3敲除加重Con A所致小鼠血清转氨酶水平升高、肝脏坏死面积增大、炎症消除延迟;LAG-3敲除不影响Con A诱导的肝内免疫细胞浸润;LAG-3敲除抑制调节性T细胞扩增及IL-10表达;MHCII辅助分子CD74与LAG-3存在蛋白质相互...     

荷叶总生物碱激活肝脏AMPK/Nrf2通路缓解对乙酰氨基酚诱导的小鼠急性肝损伤

为探讨荷叶总生物碱(TAL)对对乙酰氨基酚(APAP)所致小鼠急性肝损伤的保护作用及可能机制,小鼠被随机分为正常组,模型组和TAL低、高剂量组(30、100 mg/kg),连续灌胃给药七天后,除正常组外,其余各组腹腔注射300 mg/kg的APAP诱导急性肝损伤。12小时后,收集各组小鼠的血清与肝脏样本,进行后续实验。结果显示,与模型组相比,TAL可显著降低血清中的ALT和AST活性,下调肝组织中TNF-α、IL-1β、IL-6和MDA含量,上调肝组织中SOD、CAT、GSH-Px和GSH的水平。此外,TAL还明显改善了APAP诱导的小鼠肝组织病变。在TAL的作用下,肝内AMPK磷酸化水平提高,Nrf2蛋白入核,HO-1和GCLC基因表达上调。综上所述,TAL对APAP诱导的急性肝损伤具有保护作用,其机制可能与激活AMPK/Nrf2通路相关。     

Picroside II protects against cholestatic liver injury possibly through activation of farnesoid X receptor

BackgroudCholestasis, accompanied by the accumulation of bile acids in body, may ultimately cause liver failure and cirrhosis. There have been limited therapies for cholesteric disorders. Therefore, development of appropriate therapeutic drugs for cholestasis is required. Picroside II is a bioactive component isolated from Picrorhiza scrophulariiflora Pennell, its mechanistic contributions to the anti-cholestasis effect have not been fully elucidated, especially the role of picroside II on bile acid homeostasis via nuclear receptors remains unclear.PurposeThis study was designed to investigate the hepatoprotective effect of picroside II against alpha-naphthylisothiocyanate (ANIT)-induced cholestatic liver injury and elucidate the mechanisms in vivo and in vitro.MethodsThe ANIT-induced cholestatic mouse model was used with or without picroside II treatment. Serum and bile biochemical indicators, as well as liver histopathological changes were examined. siRNA, Dual-luciferase reporter, quantitative real-time PCR and Western blot assay were used to demonstrate the farnesoid X receptor (FXR) pathway in the anti-cholestasis effects of picroside II in vivo and in vitro.ResultsPicroside II exerted hepatoprotective effect against ANIT-induced cholestasis by impaired hepatic function and tissue damage. Picroside II increased bile acid efflux transporter bile salt export pump (Bsep), uptake transporter sodium taurocholate cotransporting polypeptide (Ntcp), and bile acid metabolizing enzymes sulfate transferase 2a1 (Sult2a1) and UDP-glucuronosyltransferase 1a1 (Ugt1a1), whereas decreased the bile acid synthesis enzymes cholesterol 7α-hydroxylase (Cyp7a1) and oxysterol 12α-hydroxylase (Cyp8b1). In addition, expression of FXR and the target gene Bsep was increased, whereas aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor alpha (PPARα) and their corresponding target genes were not significantly influenced by picroside II under cholestatic conditions. Furthermore, regulation of transporters and enzymes involved in bile acid homeostasis by picroside II were abrogated by FXR silencing in mouse primary cultured hepatocytes. Dual-luciferase reporter assay performed in HepG2 cells demonstrated FXR activation by picroside II.ConclusionOur findings demonstrate that picroside II exerts protective effect on ANIT-induced cholestasis possibly through FXR activation that regulates the transporters and enzymes involved in bile acid homeostasis. Picroside II might be an effective approach for the prevention and treatment of cholestatic liver diseases.     

The pathophysiology of human obstructive cholestasis is mimicked in cholestatic Gold Syrian hamsters

Obstructive cholestasis causes liver injury via accumulation of toxic bile acids (BAs). Therapeutic options for cholestatic liver disease are limited, partially because the available murine disease models lack translational value. Profiling of time-related changes following bile duct ligation (BDL) in Gold Syrian hamsters revealed a biochemical response similar to cholestatic patients in terms of BA pool composition, alterations in hepatocyte BA transport and signaling, suppression of BA production, and adapted BA metabolism. Hamsters tolerated cholestasis well for up to 28 days and progressed relatively slowly to fibrotic liver injury. Hepatocellular necrosis was absent, which coincided with preserved intrahepatic energy levels and only mild oxidative stress. The histological response to cholestasis in hamsters was similar to the changes seen in 17 patients with prolonged obstructive cholestasis caused by cholangiocarcinoma. Hamsters moreover upregulated hepatic fibroblast growth factor 15 (Fgf15) expression in response to BDL, which is a cytoprotective adaptation to cholestasis that hitherto had only been documented in cholestatic human livers. Hamster models should therefore be added to the repertoire of animal models used to study the pathophysiology of cholestatic liver disease.     

Metabolomics study of the hepatoprotective effect of Phellinus igniarius in chronic ethanol-induced liver injury mice using UPLC-Q/TOF-MS combined with ingenuity pathway analysis

BackgroundPhellinus igniarius (L.) Quèl as a potential medicinal mushroom possesses multiple biological activities including hepatoprotection, but the hepatoprotective mechanism is not clear.PurposeTo elucidate the hepatoprotective effect and potential target of P. igniarius.MethodsThe male C57BL/6 mice were fed with the Lieber–DeCarli diet containing alcohol or isocaloric maltose dextrin as control diet with or without P. igniarius decoction (PID) in the dosage of 0.65 g/kg and 2.6 g/kg. The levels of serum biomarkers were detected by an automatic biochemistry analyser. The histopathological changes of liver were observed by hematoxylin and eosin (H&E) staining. Ultra performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UPLC-Q/TOF-MS) was applied for investigating the dynamic changes of serum metabolites in chronic ethanol-induced liver injury mice and after treatment with PID. Ingenuity pathway analysis (IPA) was employed to identify the potential target of PID.ResultsPID could significantly reduce the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG) and total bile acid (TBA) in serum and improved hepatic steatosis and inflammation. In terms of metabolism, a total of 36 serum differential metabolites were identified, and PID intervention regulated 24 of them, involving the key metabolic pathways such as the biosynthesis of unsaturated fatty acids, primary bile acid biosynthesis, glycerophospholipid metabolism, fatty acids biosynthesis, ether lipid metabolism and arachidonic acid metabolism. On the mechanism, IPA showed that farnesol X receptor (FXR) was the major potential target for PID, and PID could improve chronic alcohol intake induced by the inhibition of mRNA expression of FXR in the liver and the activation of mRNA expression of FXR in the intestine in mice.ConclusionThe present study for the first time systematically illustrated the hepatoprotective effect of P. igniarius and preliminarily explored its potential target FXR. P. igniarius might be exploited as a promising therapeutic option for alcoholic liver injury.     

Essential roles of bile acids and their nuclear receptors,FXR and PXR,in the cholestatic liver disease

Bile acids (BAs) are steroid acids found predominantly in the bile of mammals and other vertebrates. Though BAs have been known as digestive juice, recent studies have revealed that BAs act as signaling molecules to control metabolism and inflammation. Today, BAs are considered as potential therapeutic molecules for treatment of complex metabolic liver disease. However, the detergent properties of BAs lead to hepatic injury and intrahepatic cholestasis when BAs are accumulated in the liver with impaired bile flow into gall bladder. Cholestasis is a pathological condition of hepatic retention of cytotoxic bile acids. To date, hydrophilic ursodeoxycholic acid has been currently used to treat cholestasis, but the efficacy of UDCA for cholestasis is still limited. Given that BAs are endogenous ligands of several nuclear receptors, including Farnesoid X receptor and Pregnane X receptor, novel synthetic ligands for those nuclear receptors are promising for the treatment of cholestatic liver diseases.     

Protective effects of rutin on liver injury induced by biliary obstruction in rats

Rutin has been shown to possess beneficial health effects, including hepatoprotection. However, to date, it has not been demonstrated to have a hepatoprotective effect against cholestatic liver injury. This is the first report to show a protective effect of rutin on cholestatic liver injury. Cholestasis was produced by bile duct ligation (BDL) in male Sprague–Dawley rats for 3 weeks. Daily oral administration of rutin was started 1 week before injury and was maintained for 4 weeks. In comparison with the control group, the BDL group showed liver injury as evidenced by histological changes and elevation in serum biochemicals, ductular reaction, fibrosis, inflammation, and oxidative stress. These pathophysiological changes were attenuated by rutin supplementation. Rutin alleviated BDL-induced transforming growth factor β1 (TGF-β1), interleukin-1β, connective tissue growth factor, and collagen expression. The antifibrotic effect of rutin was accompanied by reductions in α-smooth muscle actin-positive matrix-producing cells and Smad2/3 activity critical to the fibrogenic potential of TGF-β1. Rutin attenuated BDL-induced oxidative stress, leukocyte accumulation, NF-κB activation, and proinflammatory cytokine production. Further studies demonstrated an inhibitory effect of rutin on the redox-sensitive intracellular signaling molecule extracellular signal-regulated kinase (ERK). Rutin also attenuated BDL-induced reduction in NF-E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and AMP-activated protein kinase (AMPK). Taken together, the beneficial effects of rutin were shown to be associated with antioxidative and anti-inflammatory effects as well as the downregulation of NF-κB and TGF-β/Smad signaling, probably via interference of ERK activation and/or enhancement of Nrf2, HO-1, and AMPK activity.     

Fluorofenidone ameliorates cholestasis and fibrosis by inhibiting hepatic Erk/-Egr-1 signaling and Tgfβ1/Smad pathway in mice

Cholestasis is characterized by intrahepatic accumulation of bile acids (BAs), resulting in liver injury, fibrosis, and liver failure. To date, only ursodeoxycholic acid and obeticholic acid have been approved for the treatment of cholestasis. As fluorofenidone (AKF-PD) was previously reported to play significant anti-fibrotic and anti-inflammatory roles in various diseases, we investigated whether AKF-PD ameliorates cholestasis. A mouse model of cholestasis was constructed by administering a 0.1 % 3,5-diethoxycarbonyl-1,4-dihydroxychollidine (DDC) diet for 14 days. Male C57BL/6 J mice were treated with either AKF-PD or pirfenidone (PD) orally in addition to the DDC diet. Serum and liver tissues were subsequently collected and analyzed. We found that AKF-PD significantly reduced the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bile salts (TBA), as well as hepatic bile acids (BAs) levels. Hepatic histological analyses demonstrated that AKF-PD markedly attenuated hepatic inflammation and fibrosis. Further mechanistic analyses revealed that AKF-PD markedly inhibited expression of Cyp7a1, an enzyme key to BAs synthesis, by increasing Fxr nuclear translocation, and decreased hepatic inflammation by attenuating Erk/-Egr-1-mediated expression of inflammatory cytokines and chemokines Tnfα, Il-1β, Il-6, Ccl2, Ccl5 and Cxcl10. Moreover, AKF-PD was found to substantially reduce liver fibrosis via inhibition of Tgfβ1/Smad pathway in our mouse model. Here, we found that AKF-PD effectively attenuates cholestasis and hepatic fibrosis in the mouse model of DDC-induced cholestasis. As such, AKF-PD warrants further investigation as a candidate drug for treatment of cholestasis.