非酒精性脂肪性肝病（non-alcoholic fatty liver disease，nafld）是当今世界上常见的慢性肝脏疾病之一。早期单纯脂肪肝如不及时干预，可以进展为肝炎、肝纤维化、肝硬化、肝癌，甚至导致死亡。nafld最主要的病理改变是肝细胞内脂质蓄积，其持续存在可诱发氧化应激、内质网应激，造成肝细胞损伤和肝细胞凋亡，导致肝脏炎症和纤维化，肝脏丧失功能。目前，除去调整生活方式、限制能量摄入、减重手术等非药物治疗外，针对nafld的治疗药物仍旧匮乏。中医药基于中医辨证论治理论，广泛用于治疗慢性肝脏疾病。糖肾方（tangshen formula，tsf）是本课题组李平教授继承和吸取国内名家临证经验，结合自身临床实践和现代研究成果研创的中药复方。刺芒柄花素，是糖肾方中的一种异黄酮化合物，前期由液相色谱-质谱仪（liquid chromatograph-mass spectrometer，lc/ms）鉴定小鼠糖肾方含药血清单体化合物时发现，其可以原药形式由血入肝。糖肾方既往运用和研究主要集中于糖尿病肾脏疾病，较少涉及脂肪性肝病，同时其含有的刺芒柄花素对肝脏脂肪变性的作用及机制，国内外研究少有涉及。糖肾方及其单体化合物成分刺芒柄花素针对肝脏脂肪变性的作用和相关机制均需要进一步的理论支持和实验验证。

目的：

1以糖肾方干预蛋氨酸胆碱缺乏饲料（methionine choline deficient diet，mcdd）诱导的小鼠nafld，以及干预棕榈酸钠（sodium palmitate，pa）诱导的hepg2细胞、原代小鼠肝细胞脂肪变性，明确糖肾方对非酒精性肝脏脂肪变性的治疗作用及其相关机制。

2 以糖肾方入血单体化合物干预游离脂肪酸（free fatty acids，ffas）诱导的hepg2细胞脂肪变性，鉴定糖肾方中具有抑制肝细胞脂质蓄积活性的有效成分。

3 以刺芒柄花素干预高脂饲料（high fat diet，hfd）诱导的nafld小鼠模型、ffas诱导的hepg2细胞、原代小鼠肝细胞脂肪变性，验证糖肾方入血单体化合物刺芒柄花素对非酒精性肝脏脂肪变性的治疗作用及其相关机制。

方法：

1首先，于体内以mcdd诱导nafld小鼠模型验证糖肾方对肝脏脂肪变性的药效作用，第1周、第2周喂食小鼠mcdd，第3周在喂食mcdd的基础上每天灌胃糖肾方（2.4 g/kg/day）至第6周结束时小鼠取材；小鼠取材前一周进行摄食量测定；小鼠禁食后解剖取材，保留血清和肝脏组织；全自动生化仪检测血清甘油三酯（triglyceride，tg）、总胆固醇（total cholesterol，tc）、低密度脂蛋白胆固醇（low density lipoprotein cholesterol，ldl-c）、高密度脂蛋白胆固醇（high density lipoprotein cholesterol，hdl-c）、血清谷丙转氨酶（glutamic pyruvic transaminase，alt）和谷草转氨酶（glutamic oxaloacetic transaminase，ast）水平；甘油磷酸氧化酶-过氧化物酶（glycerophosphate oxidase peroxidase，gpo-pap）法测定肝脏tg含量；肝脏h&e和油红o染色测定脂肪变性面积、炎症细胞聚集灶并进行肝脏脂肪变性活动度评分。其次，于体外以0.3 mm pa刺激hepg2细胞、原代小鼠肝细胞24 h，进行肝细胞脂肪变性造模，以糖肾方（100 μg/ml）、氯喹（chloroquine，cq，20 μm）、巴佛洛霉素a1（bafilomycin a1，bafa1，10 nm）、compound c（cc，10 μm）、sirtuin 1-小干扰rna（sirtuin 1-small interfering rna，sirt1-sirna）继续孵育24 h；gpo-pap 法测定细胞tg含量；bodipy493/503染色测定细胞脂滴并测定脂滴绿色积分荧光强度；红色荧光蛋白（red fluorescent protein，rfp）-绿色荧光蛋白（green fluorescent protein，gfp）-lc3腺病毒感染hepg2细胞并计数双荧光计数自噬体和自噬溶酶体；透射电镜计数hepg2细胞脂滴和自噬体。此外，以western blot检测小鼠肝脏组织、hepg2细胞、原代小鼠肝细胞的单磷酸腺苷活化蛋白激酶（adenosine monophosphate activated protein kinase，ampk）、磷酸化ampk（phosphorylated ampk，p-ampk）、sirt1水平，以及自噬标志物微管相关蛋白轻链3（light chain microtubule associated protein 3 b-ii，lc3b-ii）、p62蛋白水平。

2首先，以1 mm ffas刺激hepg2细胞24 h进行肝细胞脂肪变性造模。继而，以40 μm毛蕊异黄酮、40 μm毛蕊异黄酮苷、40 μm橙皮苷、40 μm新橙皮苷、320 μm人参皂苷rg1、320 μm莫诺苷、80 μm柚皮苷、80 μm黄芪甲苷、20 μm刺芒柄花素、20 μm刺芒柄花苷、40 μm益母草苷、320 μm芸香柚皮苷孵育继续孵育24 h；nile red（尼罗红）染色细胞脂质并由宽场高内涵筛选仪测定脂质的积分荧光强度。

3 首先，于体内以hfd诱导nafld小鼠模型验证刺芒柄花素对肝脏脂肪变性的药效作用，第1周、第2周喂食小鼠hfd，第3周在喂食hfd的基础上每天灌胃刺芒柄花素（100 mg/kg/day）至16周结束时小鼠取材；小鼠取材前一周进行摄食量测定、腹腔注射葡萄糖耐量实验（intraperitoneal glucose tolerance test，ipgtt）实验；小鼠禁食后解剖，保留血清和肝脏组织；全自动生化仪检测血清tg、tc、ldl-c、hdl-c、alt和ast水平；gpo-pap法测定肝脏tg含量；肝脏h&e和油红o染色测定脂肪变性面积、炎症细胞聚集灶并进行肝脏脂肪变性活动度评分。其次，于体外以1 mm ffas刺激hepg2细胞、原代小鼠肝细胞24 h进行肝细胞脂肪变性造模，以刺芒柄花素（20 μm）、cq（10 μm）、cc（10 μm）、转录因子eb-sirna（tranion factor eb-sirna，tfeb-sirna）孵育继续孵育24 h；gpo-pap 法测定细胞tg含量；bodipy493/503染色、lysotracker染色测定细胞脂滴、溶酶体，并计数脂滴和溶酶体共定位；tfeb、lc3b、lamp1荧光染色测定hepg2细胞tfeb核定位、自噬体、溶酶体，并计数自噬体和溶酶体共定位；以rfp-gfp-lc3腺病毒感染hepg2细胞并计数自噬体和自噬溶酶体。此外，以western blot检测小鼠肝脏组织、hepg2细胞、原代小鼠肝细胞细胞核tfeb、溶酶体关联膜蛋白1（lysosome-associated membrane protein 1，lamp1）、v型质子atp酶催化亚基a （v-type proton atpase catalytic subunit a，atp6v1a）、过氧化物酶体增殖物激活受体γ共激活因子1α（peroxisome proliferator-activated receptor gamma coactivator 1，pgc1α）、ampk、p-ampk、磷酸化核糖体蛋白s6激酶1（phosphorylated ribosomal protein s6 kinase β-1，p-s6k1）、beclin1水平，以及自噬标志物lc3b-ii、p62蛋白水平。

结果：

1本研究发现，糖肾方有效缓解mcdd诱导的小鼠肝脏脂肪变性，改善pa刺激的hepg2、原代小鼠肝细胞脂肪变性；此外，糖肾方上调mcdd喂养小鼠以及pa刺激的hepg2、原代小鼠肝细胞的p-ampk/ampk比值、sirt1表达量，提高lc3b-ii含量、p62降解量，增强ampk/sirt1/自噬通路。

2 再者，本研究发现糖肾方入血单体化合物中，新橙皮苷、刺芒柄花素、人参皂苷rg1可有效降低ffas刺激的hepg2细胞脂质积分荧光强度，缓解细胞脂肪变性。

3 最后，本研究发现，刺芒柄花素有效缓解hfd诱导的小鼠肝脏脂肪变性，改善ffas刺激的hepg2、原代小鼠肝细胞脂肪变性；此外，刺芒柄花素促进hfd喂养小鼠以及ffas刺激的hepg2、原代小鼠肝细胞的tfeb核易位，上调lamp1、atp6v1a、pgc1α、p-s6k1、beclin1表达量以及p-ampk/ampk比值，提高lc3b-ii含量、p62降解量，增强tfeb介导的溶酶体生成，促进自噬体-溶酶体融合，增强脂质自噬。

创新点：

1本研究发现糖肾方可以缓解mcdd诱导的小鼠肝脏脂肪变性。

2本研究发现糖肾方可以通过调控ampk/sirt1/自噬通路，增强肝脏自噬功能，减轻肝脏脂质蓄积，缓解肝脏脂肪变性。

3本研究发现糖肾方中的刺芒柄花素可以通过促进tfeb核易位，增强其介导的溶酶体生成和后续的脂质自噬，缓解非酒精性肝脏脂肪变性。

结论：

糖肾方通过增强ampk/sirt1/自噬通路，减轻肝脏脂肪变性，缓解nafld；糖肾方入血单体化合物刺芒柄花素通过增强tfeb介导的脂质自噬通路，减轻肝脏脂肪变性，缓解nafld；糖肾方入血单体化合物新橙皮苷、刺芒柄花素、人参皂苷rg1具有抑制肝细胞脂质蓄积的作用；以上发现为糖肾方治疗肝脏脂肪变性和nafld提供了理论依据和实验证据。

关键词：

糖肾方，刺芒柄花素，非酒精性脂肪性肝病，ampk，sirt1，转录因子eb，自噬

Non-alcoholic fatty liver disease (nafld) is one of the chronic liver diseases in the world currently. if there is no intervention at early stage, it can progress into liver fibrosis, liver cirrhosis, liver cancer or even lead to individual death. the most characterized pathological changes in nafld is excessive intracellular lipids accumulation in liver, which induces oxidative stress，endoplasmic reticulum stress, followed by hepatocyte damages and hepatocyte apoptosis, finally leading to liver inflammation and fibrosis, and loss of liver function. to date, there is little medication for nafld except lifestyle adjustment, energy intake restriction and weight loss surgery. traditional chinese medicine (tcm) is a treatment strategy based on the syndrome differentiation and is widely used to treat chronic liver diseases. tangshen formula (tsf) is developed by prof. li based the clinical experience of domestic famous predecessors and hers own clinical practice and modern research results. formononetin (fmn) is a isoflavone in tsf, which has been determined by liquid chromatograph-mass spectrometer (lc/ms) in serum of mice gavaed with tsf and can be absorbed into the blood circulation and subsequently entered liver through the intestinal tract. the previous application and research of tsf mainly focused on diabetic kidney diseases, but less on fatty liver diseases. meanwhile, the specific effect and mechanism of formononetin on hepatic steatosis have been little studied in china and overseas. the effects and related mechanisms of tsf and formononetin on hepatic steatosis needs further theoretical support and exact experimental verification.

aims:

1 to explore the specific effects and the underlying autophagy related mechanisms of tsf on non-alcoholic hepatic steatosis by using methionine choline deficient diet (mcdd) induced nafld in mouse, and sodium palmitate (pa) induced induced hepatic cells steatosis in hepg2 cell and primary mouse hepatocytes.

2 to screen compounds with inhibing lipid accumulation activity from serum of mice gavaged with tsf by using free fatty acids (ffas) induced hepatic cells steatosis in hepg2 cell and primary mouse hepatocytes.

3 to explore the specific effects and the underlying lipophagy related mechanisms of fmn on non-alcoholic hepatic steatosis by using a high fat diet (hfd) induced nafld in mouse, and free fatty acids (ffas) induced hepatic cells steatosis in hepg2 cell and primary mouse hepatocytes.

methods:

1 mice were fed on mcdd for 6 weeks. all mice were treated with tsf gavage (2.4 g/kg/day) from the 3rd week until dissection; in the week before dissection, food intake was assayed; after fasting, the serum and liver tissues were preserved; levels of serum triglyceride (tg), total cholesterol (tc), low density lipoprotein cholesterol (ldl-c), high density lipoprotein cholesterol (hdl-c), glutamic pyruvic transaminase (alt) and glutamic oxaloacetic transaminase (ast) were detected by an automatic biochemical analyzer; liver triglycerides were detected with glycerophosphate oxidase peroxidase (gpo-pap); liver h&e and oil red o staining were undertaken to observe the area of hepatic steatosis and the number of infiltrating inflammatory cell clusters, and to evaluate the nafld activity score. 0.3 mm pa was used to stimulate hepg2 cells and primary mouse hepatocytes to induce hepatocyte steatosis, and then following tsf (100 μg/ml), chloroquine (cq, 20μm), bafilomycin a1 (bafa1, 10 nm), compound c (cc, 10 μm) and sirtuin 1-small interfering rna (sirt1-sirna) incubation respectively; cellular tg content were assayed with gpo-pap; lipid droplets were determined by bodipy493/503 staining and the integrated fluorescence intensity of lipid droplets was detected; the number of autophagy and autolysosome was counted in red fluorescent protein (rfp)-green fluorescent protein (gfp)-lc3 adenovirus infected hepg2 cells; the number of lipid droplets and autophagic vesicles in hepg2 cells were counted by transmission electron microscopy. besides, the levels of adenosine monophosphate activated protein kinase (ampk), phosphorylated ampk (p-ampk), sirt1, light chain microtubule associated protein 3b-ii (lc3b-ii), and p62 in liver tissue, hepg2 cells and primary mouse hepatocytes, were assayed with western blot.

2 1mm ffas was used to stimulate hepg2 cells to induce hepatocyte steatosis, steatosis, and then following 40 μm calycosin, 40 μm calycosin-7-o-beta-d-glucopyranoside, 40 μm hesperiden, 40 μm neohesperidin, 320 μm ginsenoside rg1, 320 μm morroniside, 80 μm naringin, 80 μm astragaloside iv, 20 μm fmn, 20 μm formononetin glucoside, 40 μm ajugol and 320 μm narirutin 24 h incubation respectively; intracellular lipids were determined by nile red staining and the integrated fluorescence intensity of lipids was detected by widefield high content screening system.

3 mice were fed on high fat diet (hfd) for 16 weeks were treated with fmn gavage (100 mg/kg/day)  from the 3rd week until dissection; in the week before dissection, food intake and intraperitoneal glucose tolerance test (ipgtt) were assayed; after fasting, the serum and liver tissues were preserved; levels of serum tg, tc, ldl-c, hdl-c, alt and ast were detected by an automatic biochemical analyzer; liver tg content was detected with gpo-pap; h&e and oil red o staining were undertaken to observe the area of hepatic steatosis and the number of infiltrating inflammatory cell clusters. 1mm ffas was used to stimulate hepg2 cells and primary mouse hepatocytes to induce hepatocyte steatosis, and then following fmn (20 μm), cq (10 μm), cc (10 μm) and tranion factor eb (tfeb-sirna) incubation respectively; cellular tg content were assayed with gpo-pap; cellular lipid droplets were determined by bodipy493/503 staining and autolysosomes in hepg2 cells were determined by lysotracker staining, meanwile the number of colocalization of lipid dropltes and autolysosomes was counted; nuclear tfeb, autophagosomes and autolysosomes in hepg2 cells were detected by immunofluorescence of tfeb, lc3b and lamp1, meanwile the number of colocalization of autolysosomes and lysosomes was counted; the number of autophagy and autolysosome was counted in rfp-gfp-lc3 adenovirus infected hepg2 cells. besides, the levels of nuclear tfeb, lysosome-associated membrane protein 1 (lamp1), v-type proton atpase catalytic subunit (atp6v1a), peroxisome proliferator-activated receptor gamma coactivator 1 (pgc1α), ampk, p-ampk, phosphorylated ribosomal protein s6 kinase β-1 (p-s6k1), beclin1, lc3b-ii and p62 in liver tissue, hepg2 cells and primary mouse hepatocytes, were assayed with western blot.

results:

1 tsf effectively alleviated hepatic steatosis in mice fed on mcdd, and ameliorated hepg2 and primary hepatic steatosis induced by pa; meanwhile, tsf enhanced the ampk/sirt1/autophagy pathway in pa-stimulated hepg2 cells and primary mouse hepatocytes, and liver of mice fed with mcdd through upregulating p-ampk/ampk ratio and sirt1 expression, and increasing the lc3b-ii expression and p62 degradation.

2 neohesperidin, fmn and ginsenoside rg1 reduced the integrated fluorescence intensity of lipids in ffas-stimulated hepg2 cells and primary mouse hepatocytes, resulting in alleviating cellular steatosis.

3 fmn significantly alleviated hepatic steatosis in mice fed on hfd, and ameliorated hepg2 and primary hepatic steatosis induced by ffas; meanwhile, fmn promoted the tfeb mediated lysosomes biogenesis, enhanced lipophagy in ffas-stimulated hepg2 cells and primary mouse hepatocytes, and liver of mice fed with hfd or mcdd, through upregulating the expression of lamp1, atp6v1a, pgc1α, p-s6k1 and beclin1, along with p-ampk/ampk ratio, and increasing the lc3b-ii expression and p62 degradation.

innovations:

1 in this study, it was found for the first time that tsf could alleviate the hepatic lipid metabolism disorder induced by mcdd in mice, and the related autophagy pathway was further studied. it was found that tsf could enhance the hepatic autophagy function and reduce hepatic lipids accumulation by regulating ampk/sirt1/ autophagy pathway.

2 in this study, it was found for the first time formononetin extracted from tsf can enhance lysosome biogenesis and subsequent lipophagy by promoting tfeb nuclear translocation and alleviating non-alcoholic liver steatosis.

conclusions:                                                                  

tsf alleviates hepatic steatosis and nafld by enhancing ampk/sirt1/autophagy pathway. its compound, fmn, alleviates hepatic steatosis and nafld via enhancing tfeb-mediated lipophagy pathway. besides，neohesperidin, fmn and ginsenoside rg1 owned activity of inhibing lipids accumulation in hepatic cells. the present study provided theoretical basis and exact experimental evidence for tsf’s treatment against hepatic steatosis and nafld.

keywords:

tangshen formula, formononetin, nafld, ampk, sirt1, tfeb, autophagy