药学学报  2021, Vol. 56 Issue (10): 2802-2808     DOI: 10.16438/j.0513-4870.2021-0305   PDF    
五味子乙素通过增强肠道黏膜屏障功能改善小鼠非酒精性脂肪性肝病的研究
马钰捷1, 余生兰1, 李琴琴1, 黄璐瑶1, 杨莉1,2, 王峥涛1,2, 丁丽丽1,2     
1. 上海中医药大学中药研究所, 中药标准化教育部重点实验室暨国家中医药管理局中药新资源与质量评价重点实验室, 上海 201203;
2. 上海中药标准化研究中心, 上海 201203
摘要: 近年来,非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)的患病率在我国大幅度提升,但临床上缺乏有效治疗药物。本文旨在探究五味子乙素(schisandrin B,SchB)对NAFLD的改善作用及其可能的作用机制。野生型C57BL/6J小鼠给予高脂高糖高胆固醇饲料(含40%脂肪、22%果糖、10%蔗糖和2%胆固醇)16周后诱导成NAFLD模型,使用SchB(120 mg·kg-1)口服治疗6周。通过检测体重、摄食量、葡萄糖耐量、胰岛素耐受、血清总胆固醇(total cholesterol,TC)、甘油三酯(triglyceride,TG)、谷丙转氨酶(alanine aminotransferase,ALT)和谷草转氨酶(aspartate aminotransferase,AST)含量及肝脏病理学变化,评价SchB对小鼠NAFLD的改善作用。进一步检测血清脂多糖(lipopolysaccharide,LPS)含量、肠道渗透性及肠道黏膜屏障相关基因、蛋白的表达,同时分析盲肠内容物的肠道菌群组成及差异菌群,评价SchB改善NAFLD的潜在作用机制。实验方案经由上海中医药大学动物实验伦理委员会审查通过。本实验发现SchB在不改变摄食量的条件下显著减轻NAFLD小鼠的体重,降低血清中TC、ALT和AST的含量,减少肝脏的脂质堆积。同时,SchB改变NAFLD小鼠的肠道菌群组成,增加嗜黏蛋白阿克曼氏菌(Akkermansia muciniphilaA.muciniphila)的丰度,增加回肠和结肠中肠道黏膜屏障相关基因及蛋白的表达,维护肠道屏障稳态,从而缓解肝脏炎症。本研究表明,SchB通过调节肠道菌群结构和增强肠道黏膜屏障功能,从而减少肝脏脂质堆积、缓解炎症,改善NAFLD。
关键词: 五味子乙素    非酒精性脂肪性肝病    肠道菌群    肠道黏膜屏障    肝脏炎症    
Schisandrin B improves non-alcoholic fatty liver disease by impacting intestinal mucosal barrier function
MA Yu-jie1, YU Sheng-lan1, LI Qin-qin1, HUANG Lu-yao1, YANG Li1,2, WANG Zheng-tao1,2, DING Li-li1,2     
1. The MOE Key Laboratory for Standardization of Chinese Medicines and the SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China;
2. Shanghai R & D Center for Standardization of Traditional Chinese Medicines, Shanghai 201203, China
Abstract: In recent years, the prevalence of non-alcoholic fatty liver disease (NAFLD) has been dramatically increased in China and specific targeted therapy is still unavailable. The purpose of this research was to investigate whether schisandrin B (SchB) improves NAFLD and the potential mechanisms. Wildtype mice with C57BL/6J background were treated with special high fat diet (containing 40% fat, 22% fructose, 10% sucrose, and 2% cholesterol) for 16 weeks to induce NAFLD. Then SchB (120 mg·kg-1) were used to treat NAFLD mice for 6 weeks. Body weight, food intake, glucose tolerance, insulin resistance, serum level of total cholesterol (TC), triglycerides (TG), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were assayed and histopathological analysis were performed to evaluate the improvement of NAFLD induced by SchB. Furthermore, the level of lipopolysaccharide (LPS) in serum, intestinal permeability, and the expression of genes and proteins associated with mucosal defense were evaluated, intestinal flora composition in fresh cecal contents were analyzed and differential flora were identified to explore the potential mechanism. All animal experiments were approved by the Animal Research Committee of Shanghai University of Traditional Chinese Medicine. These results showed that SchB significantly reduced the body weight of NAFLD mice without changing food intake, and effectively reduced serum level of TC, ALT, and AST. SchB also significantly altered the composition of the microflora in NAFLD mice, increased the abundance of the Akkermansia muciniphila (A. Muciniphila) and elevated the expression of genes and proteins associated with the mucosal defense in ileum and colon, restored the permeability of intestinal barrier. In summary, SchB improves NAFLD by regulating the composition of the microflora and enhancing the function of intestinal barrier to further reduce the excessive lipids accumulation and hepatic inflammation.
Key words: schisandrin B    non-alcoholic fatty liver disease    intestinal microflora    intestinal mucosal barrier    hepatic inflammation    

非酒精性脂肪性肝病(non-alcoholic fatty liver disease, NAFLD) 又称代谢相关脂肪性肝病(metabolic associated fatty liver disease, MAFLD), 是一种与胰岛素抵抗和遗传易感密切相关的代谢应激性肝损伤。疾病谱为非酒精性肝脂肪病变、非酒精性脂肪性肝炎、肝硬化和肝细胞癌[1]。NAFLD现已成为全球重要的公共健康问题之一, 影响着全球约25%的人口[2], 然而目前临床尚无明确用于治疗NAFLD的药物。因此, 寻找有效的NAFLD治疗药物成为亟需解决的热点问题。

近年来, 大量研究发现维持肠道菌群的稳态及维护肠道黏膜屏障功能在NAFLD的发展过程中发挥着重要作用[3-6]。肠道菌群紊乱所引起的肠道黏膜屏障受损, 导致脂多糖(lipopolysaccharide, LPS) 经门静脉渗漏进入肝脏, 与Toll样受体4 (Toll like receptor 4, TLR4) 结合, 促进炎症因子释放, 从而介导NAFLD的炎症发展[7]。同时, 有研究[8]称, 嗜黏蛋白阿克曼氏菌(Akkermansia muciniphila, A. muciniphila) 可通过维护肠道黏膜屏障功能调控NAFLD的发展。

五味子[Schisandra chinensis (Turcz.) Baill.] 为木兰科植物五味子的干燥成熟果实, 含有木脂素类、挥发油类、多糖类和有机酸类等多种化学成分[9]。现代药理学研究表明, 五味子具有抗氧化、抗菌、降脂、改善糖尿病和保肝等多种作用[10, 11]。五味子乙素(schisandrin B, SchB) 是发挥药理作用的主要化学成分, 目前有研究发现SchB可显著降低NAFLD小鼠肝脏和血浆中的甘油三酯含量[12]。亦有文献报道SchB通过保护肠道上皮屏障, 调控肠道菌群稳态, 预防溃疡性结肠炎[13]。因此, 本文基于高脂高糖高胆固醇饮食建立了NAFLD小鼠模型, 探讨SchB对NAFLD小鼠代谢表型的改善作用; 并从调节肠道菌群结构, 保护肠道黏膜屏障功能等方面探究其潜在的作用机制, 为五味子的临床应用提供实验基础和理论依据。

材料与方法

实验动物  4周龄雄性C57BL/6J小鼠购于上海斯莱克实验动物有限责任公司[合格证号: SCXK (沪) 2017-0005], 饲养于上海中医药大学实验动物中心SPF级饲养室, 室温20 ℃, 湿度60%~70%, 12 h昼夜交替照明, 自由摄食饮水。实验方案均已通过上海中医药大学动物实验伦理委员会批准(伦理编号为PZSHUTCM190712003), 动物福利与实验过程均符合上海中医药大学动物实验伦理委员会规定。

药品与试剂  高脂高糖高胆固醇饲料(美国Research Diets Inc.公司, D09100310); SchB (南京广润生物制品有限公司, 批号GR-136-190322, HPLC 98%); 总胆固醇(total cholesterol, TC) 测试盒、甘油三酯(triglyceride, TG) 测试盒、谷丙转氨酶(alanine aminotransferase, ALT) 测试盒、谷草转氨酶(aspartate aminotransferase, AST) 测试盒(南京建成生物科技有限公司); 小鼠LPS酶联免疫试剂盒(武汉华美生物工程有限公司); 生物合成人胰岛素注射液(丹麦诺和诺德公司); 小鼠胰岛素酶联免疫试剂盒(美国Mercodia公司); 葡萄糖、异硫氰酸荧光素-葡聚糖(美国Sigma公司); 血糖试纸(美国Roche公司); RNA提取试剂盒(美国EZBisoscience公司); PrimeScriptTM RT Master Mix试剂盒、TB Green R Premix Ex TapTM试剂盒(日本Takara公司); 通用型组织固定液(武汉赛维尔生物科技有限公司); PBS缓冲液(大连美仑生物技术有限公司); 辣根过氧化物酶标记山羊抗兔IgG (美国Jackson ImmunoResearch公司); ZO-1兔多克隆抗体(A11417)、Occludin兔多克隆抗体(A2601) (武汉爱博泰克生物科技有限公司); GAPDH兔单克隆抗体(#5174, 美国Cell Signaling Technology公司); BCA试剂盒(上海翊圣生物科技有限公司)。

动物分组、造模及给药方法  4周龄小鼠适应性饲养1周后, 随机分出正常对照组(CHOW, n = 8) 喂食普通饲料, 剩余小鼠喂食高脂高糖高胆固醇饲料(GAN饮食, 含40%脂肪、22%果糖、10%蔗糖和2%胆固醇) 进行造模16周。根据小鼠体重以及空腹血糖值, 再次进行分组, 分为NAFLD模型组(GAN, n = 8) 和五味子乙素组(GAN + SchB, n = 8), 五味子乙素的给药剂量为120 mg·kg-1。其中CHOW组继续喂食普通饲料6周, GAN组喂食高脂高糖高胆固醇饲料6周, GAN + SchB组喂食均匀混入药物的高脂高糖高胆固醇饲料6周。每周记录各组小鼠的体重及摄食量变化。

葡萄糖耐量实验  小鼠禁食12 h, 测定空腹血糖值后, 腹腔注射葡萄糖溶液(2 g·kg-1), 分别于15、30、60及120 min, 尾静脉采血测定血糖值。

胰岛素耐受实验  小鼠禁食5 h, 测定空腹血糖值后, 腹腔注射胰岛素溶液(0.75 U·kg-1), 分别于15、30、60及120 min, 尾静脉采血测定血糖值。

肠道渗透性检测  将各组小鼠禁食禁水4 h后, 灌胃4 kDa-FITC-dextran溶液(0.44 mg·g-1), 继续禁食3 h。将小鼠麻醉后,进行眼眶取血, 取全血于室温避光静置2 h, 4 ℃离心15 min (3 500 r·min-1), 取得血清。再用等量PBS缓冲液稀释血清, 使用酶标仪测定对应的吸光度值, 计算浓度判断小鼠的肠道渗透性。

血清生化指标检测  使用异氟烷将小鼠麻醉后, 摘眼球取血, 脱颈处死后收集肝脏及肠道组织于-80 ℃冰箱储存。全血于室温静置2 h, 4 ℃离心15 min (3 500 r·min-1), 取上层血清, 按酶联免疫试剂盒所述方法测定并计算血清中胰岛素水平和LPS含量。按照试剂盒说明书, 测定小鼠血清中TG、TC、ALT和AST含量。

肝脏组织病理学检测  切取小鼠肝大叶相同部位, 于通用型组织固定液中保存, 按照说明书进行苏木精-伊红染色及油红O染色, 于光学显微镜下观察并采集图像。

肠道菌群分析  收集小鼠盲肠内容物, 抽提DNA, 进行PCR扩增后, 利用Illumina公司的Miseq PE300平台进行测序。使用Trimmomatic软件对原始测序序列进行质控, 使用FLASH软件进行拼接。使用UPARSE软件(version 7.1http://drive5.com/uparse/) 根据97%的相似度对序列进行OTU聚类并剔除嵌合体。利用RDP classifier (http://rdp.cme.msu.edu/) 对每条序列进行物种分类注释。对不同组别小鼠肠道菌群的α多样性及群落组成进行分析, 寻找差异菌群。

逆转录-聚合酶链式反应分析  称取小鼠肠道组织约20 mg、肝脏组织约10 mg, 按照RNA抽提试剂盒说明书进行提取, 进行RNA浓度测定后, 根据说明书进行逆转录反应。使用QuantStudio 6 Flex型实时荧光定量PCR仪进行扩增及检测设置。以β-actin为内参基因, 由2-∆∆CT法计算基因的相对表达量, 引物序列见表 1

Table 1 The primer sequences of real-time quantitative PCR

蛋白免疫印迹检测  剪取小鼠回肠及结肠组织各30 mg左右, 加入200 μL RIPA裂解液提取总蛋白, 采用BCA法测定蛋白浓度并定量, 经95 ℃加热变性。取30 μg总蛋白进行蛋白免疫印记分析。使用Photoshop软件对蛋白条带图像进行灰度分析, 以目标蛋白与内参蛋白GAPDH的比值作为蛋白的相对表达量, 正常对照组设为1。

统计学处理  实验结果均以均数±标准差(x ± s) 表示, 应用GraphPad Prism 7.0软件进行分析。多组数据比较采用方差分析(one-way ANOVA), 两组数据比较采用学生t检验(student's t test), P < 0.05视为有统计学意义。

结果 1 五味子乙素对NAFLD小鼠体重及摄食量的影响

各组小鼠体重变化如图 1A所示, GAN组小鼠体重稳定增加, 显著高于CHOW组小鼠。经SchB治疗后, GAN + SchB组小鼠摄食量并无显著变化(图 1B), 但从第4周起, NAFLD小鼠的体重显著降低(P < 0.05), 表明SchB在不影响摄食量的情况下, 可显著降低NAFLD小鼠的体重。

Figure 1 Effect of schisandrin B (SchB) on body weight and food intake in non-alcoholic fatty liver disease (NAFLD) mice. A: Body weight; B: Food intake. n = 8, x ± s. ###P < 0.001 vs CHOW; *P < 0.05 vs GAN
2 五味子乙素对NAFLD小鼠葡萄糖耐量及胰岛素耐受的影响

各组小鼠空腹血糖值如图 2A所示, SchB可显著降低NAFLD小鼠的空腹血糖值(P < 0.001)。当腹腔注射小鼠葡萄糖溶液后(图 2B), GAN组小鼠较CHOW组小鼠血糖显著升高, 且下降幅度缓慢。经SchB治疗后, NAFLD小鼠的葡萄糖耐量显著改善。通过统计葡萄糖耐量测试(glucose tolerance test, GTT) 曲线下面积(图 2C), 发现GAN + SchB组显著低于GAN组(P < 0.001)。

Figure 2 SchB improved glucose homeostasis of NAFLD mice. A: Fasting glucose; B: Glucose tolerance test (GTT); C: Area under the curve (AUC) of GTT; D: Serum level of insulin; E: Insulin tolerance test (ITT); F: AUC of ITT. n = 8, x ± s. #P < 0.05, ##P < 0.01, ###P < 0.001 vs CHOW; *P < 0.05, **P < 0.01, ***P < 0.001 vs GAN

各组小鼠血清中的胰岛素含量如图 2D所示, SchB可显著下调NAFLD小鼠的胰岛素水平(P < 0.05)。腹腔注射小鼠胰岛素溶液后(图 2E), 各组小鼠的血糖值均有下降, 但GAN组小鼠较CHOW组小鼠血糖下降幅度缓慢。经SchB治疗后, NAFLD小鼠的胰岛素耐受显著改善。通过统计胰岛素耐受测试(insulin tolerance test, ITT) 曲线下面积(图 2F), 发现GAN + SchB组显著低于GAN组(P < 0.001)。以上结果提示, SchB可明显改善NAFLD小鼠的葡萄糖代谢能力, 恢复胰岛素敏感性。

3 五味子乙素对NAFLD小鼠血清生化指标及肝脏病理学的影响

图 3A~D所示, 与GAN组相比, GAN + SchB组小鼠血清中的TG含量有下降趋势, 同时TC、ALT和AST含量均显著降低。肝脏病理学结果(图 3EF) 表明, SchB可显著减小NAFLD小鼠肝脏中的脂肪空泡大小, 减少脂滴的数量。以上结果表明, SchB对NAFLD小鼠肝脏具有一定的保护作用, 并能改善肝脏的脂质堆积。

Figure 3 Effect of SchB on biochemical index in NAFLD mice. A: Serum level of triglyceride (TG); B: Serum level of total cholesterol (TC); C: Serum level of aspartate aminotransferase (AST); D: Serum level of alanine aminotransferase (ALT); E: Representative images of H & E staining and oil red O staining of liver; F: Quantitative results of the oil red O staining. n = 8, x ± s. ##P < 0.01, ###P < 0.001 vs CHOW; *P < 0.05, **P < 0.01, ***P < 0.001 vs GAN
4 五味子乙素对NAFLD小鼠肠道菌群结构的影响

现有报道表明[3, 4], 肠道菌群的稳态与肝脏疾病的发展密切相关。通过对小鼠盲肠中的肠道菌群组成进行分析, 发现在门水平(图 4A), GAN + SchB组与GAN组相比, 厚壁菌门丰度明显下调, 疣微菌门丰度明显上调。同时进行α多样性分析(图 4BC), 发现SchB可显著降低小鼠肠道菌群的多样性和丰富度, 表明SchB具有一定的杀菌作用。通过对属水平(图 4DE) 的肠道菌群组成进行分析, 发现SchB显著增加A. muciniphila在NAFLD小鼠体内的丰度(P < 0.001)。该菌被报道[14]与代谢性疾病密切相关, 可降低肠道渗透性, 防止有害物质通过肝肠循环进入肝脏中。

Figure 4 Effect of SchB on the structure of intestinal microflora in NAFLD mice. A: The differences in the community composition between the phylum units; B: Shannon index of OTU level; C: Chao index of OTU level; D: Abundance of Akkermansia muciniphila (A. muciniphila); E: The differences in the community composition between the genus units. n = 6, x ± s. ###P < 0.001 vs CHOW; ***P < 0.001 vs GAN
5 五味子乙素对NAFLD小鼠肠道黏膜屏障功能的影响

在回肠组织中(图 5A~D), SchB显著增加紧密连接蛋白ZO-1及occludin的蛋白表达。同时, SchB显著增加Zo-1及黏蛋白Muc2的基因表达(P < 0.05), 对occludin的基因表达有增加的趋势。在结肠组织中(图 5E~H), SchB也可增加肠道黏膜屏障相关基因及蛋白的表达。

Figure 5 Effect of SchB on the intestinal mucosal barrier function in NAFLD mice. A: Mucosal defense proteins expression in ileum; B: Statistical results of gray value of ZO-1 in ileum; C: Statistical results of gray value of occludin in ileum; D: Mucosal defense genes expression in ileum; E: Mucosal defense proteins expression in colon; F: Statistical results of gray value of ZO-1 in colon; G: Statistical results of gray value of occludin in colon; H: Mucosal defense genes expression in colon. n = 5, x ± s. #P < 0.05, ##P < 0.01 vs CHOW; *P < 0.05 vs GAN

文献表明[7], 当肠道屏障被破坏后, LPS渗漏进入肝脏中会引起炎症因子的释放。通过图 6A发现, GAN + SchB组与GAN组相比, 肠道渗透性显著降低。同时图 6B结果显示SchB显著减少LPS在NAFLD小鼠血清中的含量(P < 0.001)。此外, SchB显著降低肝脏炎症因子TnfαIl1βIl6的基因表达(图 6C)。以上结果提示, SchB可通过保护肠道黏膜屏障功能, 减轻NAFLD小鼠的肝脏炎症。

Figure 6 SchB reduced the permeability of intestinal mucosal barrier and improved hepatic inflammation. A: Intestinal permeability; B: Serum level of lipopolysaccharide (LPS); C: Hepatic mRNA expression levels of genes related to inflammation in mice. n = 8, x ± s. ##P < 0.01, ###P < 0.001 vs CHOW; *P < 0.05, **P < 0.01, ***P < 0.001 vs GAN
讨论

随着生活方式的改变, 我国近些年来NAFLD的患病率逐年上升, 并呈年轻化趋势。常见的治疗和预防方法主要包括改变不良生活方式、外科手术及针对代谢性疾病的药物治疗等[1], 但目前尚无获得美国食品药品监督管理局或欧洲药物评审组织批准的药物用于治疗NAFLD。SchB是护肝片中的有效化学成分, 目前有文献[15]报道SchB对氯氮平诱导的慢性肝损伤具有一定保护作用。此外, SchB被发现可抑制内质网应激信号通路中关键蛋白的表达[11], 影响脂质合成及炎症相关基因的表达[12]、降低肝脏TG的堆积。本研究表明, SchB可调控NAFLD小鼠的葡萄糖代谢, 显著降低血清的TC、ALT及AST水平, 减少肝脏脂质堆积, 保护肝脏。

1988年提出的“二次打击”学说起初被认为是NAFLD发病的关键机制[16], 首次打击是指胰岛素抵抗导致的肝脏脂质堆积; 再次打击是在氧化应激与脂质过氧化等作用下, 肝细胞进一步损伤, 出现肝脏炎症反应[17]。随着对NAFLD认识的深入, 研究者们[18]提出了“肝病肠治”的说法, 认为肠道菌群及肠道黏膜屏障对NAFLD的发展起着关键作用。肝脏和肠道及微生物通过胆管、门静脉及体循环相互联系, 肝脏的血液75%来自门静脉[19], 肝脏也是肠道吸收营养物质的首道防线, 因此肝脏的健康与肠道密切相关。现阶段的研究[5]发现, NAFLD患者多伴有小肠细菌过度生长、肠道菌群结构改变及肠道黏膜屏障受损的现象。维护肠道黏膜屏障功能, 可有效阻止肠道中的有害物质渗漏进入肝脏, 减轻肝脏炎症, 减缓NAFLD的发展。SchB被报道可以维护结肠炎小鼠的肠道屏障功能, 维持肠道菌群稳态[13]。本研究结果表明, SchB可显著降低NAFLD小鼠肠道菌群丰富度, 增加肠道黏膜屏障相关基因、蛋白的表达, 从而增强肠道黏膜屏障功能, 减少LPS进入肝脏及体循环, 改善NAFLD。

据文献报道, 非酒精性脂肪性肝炎患者与健康人相比, 肠道菌群结构发生显著变化, 拟杆菌门丰度降低, 厚壁菌门丰度增加[20]。而在儿童NAFLD患者中, 放线菌门的丰度增加, 拟杆菌门丰度降低[21]A. muciniphila是一种定植于黏液层并促进黏液层生长的革兰阴性菌, 以Muc2为自身合成的营养物质[8, 22], 当其丰度降低, 肠道通透性增加。有报道[23]称, 口服A. muciniphila可维持肠道屏障完整性, 改善酒精性脂肪肝。此外, 有研究人员[24]发现口服该菌可维护NAFLD小鼠肠道菌群稳态, 降低肝脏脂质合成相关基因的表达, 改善NAFLD的代谢表型。近年来, 有中药被报道其缓解肝脏炎症, 改善NAFLD的药效作用可能与A. muciniphila相关[25]。本研究发现, SchB重塑NAFLD小鼠肠道菌群结构, 降低厚壁菌门丰度, 增加疣微菌门丰度。同时, SchB显著增加A. muciniphila的丰度, 降低肠道的渗透性, 并减少肝脏炎症相关基因(TnfαIl1βIl6) 的表达, 与文献报道相符。

综上所述, SchB对NAFLD具有显著的改善作用, 其作用机制可能是通过调节肠道菌群结构、增强肠道黏膜屏障功能来实现的。该研究为临床上应用SchB治疗NAFLD提供了新的思路及有利证据。

作者贡献: 马钰捷、丁丽丽和杨莉设计实验; 马钰捷、余生兰、李琴琴和黄璐瑶进行实验数据采集与分析; 马钰捷、丁丽丽和王峥涛撰写、修改论文。

利益冲突: 无利益冲突。

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