生物学标记物诊断非酒精性脂肪性肝病的研究进展

周毅骏　钦丹萍

浙江中医药大学附属第一医院消化内科(310006)



生物学标记物诊断非酒精性脂肪性肝病的研究进展

周毅骏钦丹萍*

浙江中医药大学附属第一医院消化内科(310006)

*本文通信作者，Email： 15088662042@126.com

摘要非酒精性脂肪性肝病(NAFLD)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脏损伤，近年其在我国的发病率呈逐年上升趋势。目前肝组织活检是诊断NAFLD的金标准，但检查为侵入性，存在一定风险。因此，寻找一种简单、准确的诊断方法成为亟待解决的问题。无创生物学标记物诊断NAFLD是近年的研究热点。本文就生物学标记物诊断NAFLD的研究进展作一综述。

关键词非酒精性脂肪性肝病；生物学标记；诊断；炎症；肝纤维化；肝硬化

Liver Cirrhosis

非酒精性脂肪性肝病(nonalcoholic fatty liver disease, NAFLD)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脏损伤，包括非酒精性单纯性脂肪肝(nonalcoholic simple fatty liver, NAFL)、非酒精性脂肪性肝炎(nonalcoholic steatohepatitis, NASH)等疾病。近年来，随着环境、饮食结构等的改变，NAFLD在我国的发病率呈逐年上升趋势，越来越受到关注。NAFLD不仅可通过NASH进展至肝纤维化和肝硬化，增加肝病相关死亡率，亦可作为代谢综合征的组分，增加动脉粥样硬化和心血管疾病的死亡率以及2型糖尿病的发病率。肝组织活检是诊断NAFLD的金标准，但检查为侵入性，存在一定风险。因此，寻找一种简单、准确的诊断方法成为亟待解决的问题。无创生物学标记物诊断NAFLD是近年的研究热点。本文就生物学标记物诊断NAFLD的研究进展作一综述。

一、肝脂肪变性标记物

肝脂肪变性是NAFLD疾病状态的起始步骤，单纯的肝脂肪变性(如NAFL)预后较好，因脂肪变性可逆，早期干预尤为重要。目前临床上诊断肝脂肪变性主要依靠影像学手段，但B超、CT以及MRI等检查方法在早期识别肝脂肪变性方面存在一定缺陷[1]。因此亟需探索无创的生物学标记物诊断，用于早期识别肝脂肪变性，以指导临床诊疗。

1. 自毒素(autotaxin, ATX)：ATX是一种脂肪细胞来源的磷脂酶B。Rachakonda等[2]的研究发现，血清ATX水平与伴有肥胖症的女性NAFLD患者关系密切，与无NAFLD的肥胖症女性相比，患有NAFLD的肥胖症女性血清ATX水平显著升高，且伴有碱性磷酸酶(ALP)、空腹血糖、空腹胰岛素水平以及胰岛素抵抗指数异常。该研究进一步行线性回归分析显示，血清三酰甘油和ATX水平与肝脂肪变性独立相关，提示ATX可作为衡量肝脂肪变性的独立生物学标记物。

2. miRNA标记物：近年来，有研究[3]指出血清循环miRNA，包括miR-21、miR-34a、miR-122以及miR-155等与NAFLD疾病进程(脂肪变性、纤维化、硬化、癌变)相关。Miyaaki等[4]的研究发现，NAFLD患者中血清与肝脏miR-122水平显著相关，提示miR-122可从肝细胞释放入血液，且血清miR-122水平与肝脂肪变性程度密切相关。

3. 复合评分系统：脂肪肝指数(FLI)、NAFLD肝脂肪分数(NAFLD-LFS)、肝脂肪变指数(HSI)、内脏脂肪指数(VAI)以及三酰甘油与葡萄糖乘积指数(TyG)是五个与肝脂肪变性和胰岛素抵抗密切相关的生物学标记物评分系统[5]。研究[6-7]显示FLI可预测包括肝脂肪变性在内的代谢综合征相关的临床预后，且与肝病相关死亡率密切相关。但目前关于FLI诊断的准确性尚存争议，有研究[8]认为FLI与肝脂肪变性无显著相关性。研究[9]显示，NAFLD-LFS诊断肝脂肪变性的敏感性和特异性分别为86%和71%，与磁共振波谱分析(MRS)相似，但与肝组织活检准确性的比较尚待研究。HSI将ALT/AST、体重指数(BMI)、糖尿病作为变量进行分析，其诊断肝脂肪变性的准确性为81.2%，同时还可预测代谢综合征相关事件[10]。VAI是评估内脏脂肪代谢失调的一种评分系统[11]。内脏脂肪不仅与胰岛素抵抗和代谢综合征相关，亦与肝脂肪变性、肝脏炎症以及纤维化密切相关。研究[12]显示VAI与肝脂肪变性呈独立相关。TyG是三酰甘油与葡萄糖水平的乘积，与胰岛素敏感性呈负相关[13]。研究[14]发现TyG可独立预测慢性丙肝患者轻度至重度肝脂肪变性程度，但在NAFLD中的作用尚未见报道。

二、肝组织炎症标记物

NAFLD被认为是代谢综合征的肝表现，诸多研究表明代谢紊乱与炎症密切相关。有学者提出“代谢性炎症综合征”的新概念，即指营养物和代谢产物所触发的炎症过程，该过程分子机制和信号通路类似于传统的慢性低峰度炎症反应，其由脂肪组织特异性巨噬细胞作为中枢环节协调介导，多种细胞(脂肪细胞、骨骼肌细胞、淋巴细胞等)、细胞因子[肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6、C-反应蛋白(CRP)、IL-8和干扰素(IFN)-γ等]以及脂肪因子(瘦素、脂联素、抵抗素等)共同参与炎症的发生。在NAFLD病程中，NASH是NAFL向肝硬化进展的限速步骤，为此是否能通过促炎症生物标记物鉴别NAFL与NASH已成为近年研究的重点。

1. TNF-α：TNF-α在胰岛素抵抗中起有重要作用。Abiru等[15]的研究显示，NASH患者血清TNF-α及其可溶性受体sTNFR1水平较NAFL患者明显升高。Alaaeddine等[16]的研究发现，NASH患者TNF-α mRNA水平显著高于健康人群，并提出TNF-α mRNA 100 ng/mL可作为临界值预测NASH，敏感性为66.7%，特异性为74.1%。此外，Du等[17]的研究显示，采用TNF-α拮抗剂已酮可可碱治疗NASH，可降低患者体重、血清ALT、AST、葡萄糖和TNF-α水平以及NAFLD活动评分，改善肝小叶炎症，其证实了TNF-α在NAFLD发生、发展中扮演重要角色。但值得注意的是，TNF-α在其他一些炎症性疾病中亦升高，故认为其针对NAFLD的特异性有限。

2. IL-6：IL-6可通过细胞因子信号转导抑制因子-3(SOCS-3)参与胰岛素抵抗。Abiru等[15]的研究显示，IL-6可溶性受体sIL-6R在NASH患者体内较NAFL患者显著升高。García-Galiano等[18]的研究显示，血清IL-6>4.81 pg/mL可作为脂肪变性程度的独立预测因子，而非NASH。Kinoshita等[19]的研究显示，IL-6>4.6 pg/mL可有效鉴别NAFL和NASH(敏感性58.1%，特异性100%)。

3. CRP：研究[20]显示CRP水平在NAFLD患者中显著升高，可用于指导NAFLD诊断。然而该研究诊断NAFLD是基于ALT水平升高和超声改变，而非肝活检，故结果有待证实。Maleki等[21]的研究显示，NASH患者高敏感性CRP(hs-CRP)水平较非NASH患者显著升高，并且其表达水平与肝纤维化程度呈正相关。Fierbinteanu-Braticevici等[22]的研究显示，CRP 3.5 mg/L作为临界值可有效预测NASH(敏感性82%，特异性88%)，但对预测重度肝纤维化(F≥3)无效。

4. 铁蛋白：铁蛋白属于急性时相蛋白，当机体处于炎症、感染等情况时可显著升高。Angulo等[23]的研究发现，血清铁蛋白水平在NAFLD患者体内显著升高，且浓度与肝纤维化程度相关。Kowdley等[24]的研究显示，血清铁蛋白水平>1.5倍正常值上限(女性>300 ng/mL，男性>450 ng/mL)与肝铁质沉积以及组织学损伤严重程度呈正相关，并可作为NAFLD患者肝纤维化的独立预测因子。

5. 氧化应激标记物：氧化应激是判断炎症的一种间接指标。肝内脂肪沉积会引起肝细胞发生氧化反应，产生自由基，诱导一系列级联反应，级联反应中某些稳定物质或最终产物可作为氧化应激的标记物。Feldstein等[25]的研究显示，NASH患者与正常人群相比，自由基介导的氧化反应产物9,13-羟基十八碳二烯酸和9,13-氧代十八碳二烯酸显著升高。此外，体内某些抗氧化应激成分的消耗程度，如谷胱甘肽，可作为一种间接衡量氧化应激反应的指标。然而，氧化应激目前被认为是急慢性疾病的普遍特征，在自然衰老过程中亦可出现，在肝脏疾病中的特异性有待证实。

三、肝纤维化标记物

根据NASH的自然病程，15%～25%的患者会在 10～20年后进展为肝硬化并出现相关并发症。1/3的NASH患者在病程初期即已出现肝纤维化表现，其中10%～15%发展为肝硬化甚至肝细胞癌(HCC)。近年NASH相关性HCC在肝移植适应证中所占比例呈明显上升趋势[26]。因此，早期准确评估NAFLD患者肝纤维化程度尤为重要。

1. AST与血小板比值(APRI)：APRI是一种简单易行的评分系统，可用于评估肝纤维化或肝硬化程度[27]。Adler等[28]行meta分析显示，APRI评分>0.5诊断重度肝纤维化的敏感性为81%，特异性为50%。Kruger等[29]的研究显示，APRI评分可有效评估NAFLD晚期肝纤维化。

2. FibroTest：FibroTest是一种评估肝纤维化的评分系统，由年龄、性别以及5种血清标记物[α2-巨球蛋白(α2-MG)、结合珠蛋白、载脂蛋白A1、谷酰转肽酶、总胆红素]组成，可用于评估NAFLD相关肝纤维化[30-32]。

3. Fibrometer：Fibrometer是可用于鉴别病毒性肝炎与酒精性肝炎引起的肝纤维化的评分系统[33]，基于血小板计数、凝血酶原时间、AST、α2-MG、透明质酸、尿素氮水平以及年龄进行评估。近年来，该评分系统对NAFLD肝纤维化的评价效应受到部分学者关注[34-35]。

4. FIB-4：FIB-4是由血小板、ALT、AST水平以及年龄组成的评分系统。McPherson等[36]分别使用FIB-4以及其他3种非侵入性肝纤维化评分系统(AST/ALT比值、BARD和NAFLD纤维化评分)评价NAFLD患者肝纤维化程度，结果显示FIB-4优于另外3种评分系统，可有效诊断晚期肝纤维化，避免不必要的肝活检。

5. BARD：BARD评分系统基于BMI、AST/ALT比值以及是否有2型糖尿病对肝纤维化进行评分，记分系统如下：BMI≥28 kg/m2，1分；BMI<28 kg/m2，0分；AST/ALT≥0.8，2分；AST/ALT<0.8，0分；糖尿病，1分。总分2～4分高度提示肝硬化可能[37]。然而有研究[38]指出BARD诊断肝纤维化的准确性较低。故有学者[39]建议加入国际标准化比值(INR)以增强BARD的特异性和敏感性，但其诊断效应有待进一步行临床试验验证。

上述5种评分系统能较好地鉴别肝纤维化低危与高危患者，评估不良事件发生率以及预测远期生存率。但有学者[40]指出，对于年龄≥45岁、肥胖(男性BMI>31.1 kg/m2，女性BMI>32.3 kg/m2)、合并糖尿病的肝硬化高危患者，建议采用肝活检以明确组织学病变。

四、结语

随着经济发展和生活方式的改变，中国地区NAFLD发病率呈逐年上升趋势，已成为继病毒性肝炎之后的新社会医学问题。尽管肝活检仍作为诊断NAFLD的金标准，但生物标记物作为一种无创检查手段，具有独特优势，能早期识别肝脂肪变性，合理评估肝脏炎症程度，预测肝纤维化进程，较好地协助临床诊疗。相比于静态单点肝脏活检技术，生物标记物的可操作性、实用性以及动态性使其在未来临床应用中更具前景。

参考文献

1 Rinella ME. Nonalcoholic fatty liver disease: a systematic review[J]. JAMA, 2015, 313 (22): 2263-2273.

2 Rachakonda VP, Reeves VL, Aljammal J, et al. Serum autotaxin is independently associated with hepatic steatosis in women with severe obesity[J]. Obesity (Silver Spring), 2015, 23 (5): 965-972.

3 Gori M, Arciello M, Balsano C. MicroRNAs in nonalcoholic fatty liver disease: novel biomarkers and prognostic tools during the transition from steatosis to hepatocarcinoma[J]. Biomed Res Int, 2014, 2014: 741465.

4 Miyaaki H, Ichikawa T, Kamo Y, et al. Significance of serum and hepatic microRNA-122 levels in patients with non-alcoholic fatty liver disease[J]. Liver Int, 2014, 34 (7): e302-e307.

5 Fedchuk L, Nascimbeni F, Pais R, et al. Performance and limitations of steatosis biomarkers in patients with nonalcoholic fatty liver disease[J]. Aliment Pharmacol Ther, 2014, 40 (10): 1209-1222.

6 Bedogni G, Bellentani S, Miglioli L, et al. The Fatty Liver Index: a simple and accurate predictor of hepatic steatosis in the general population[J]. BMC Gastroenterology, 2006, 6 (1): 1-7.

7 Calori G, Lattuada G, Ragogna F, et al. Fatty liver index and mortality: the Cremona study in the 15th year of follow-up[J]. Hepatology, 2011, 54 (1): 145-152.

8 Kahl S, Straβburger K, Nowotny B, et al. Comparison of liver fat indices for the diagnosis of hepatic steatosis and insulin resistance[J]. PLoS One, 2014, 9 (4): e94059.

9 Kotronen A, Peltonen M, Hakkarainen A, et al. Prediction of non-alcoholic fatty liver disease and liver fat using metabolic and genetic factors[J]. Gastroenterology, 2009, 137 (3): 865-872.

10Lee JH, Kim D, Kim HJ, et al. Hepatic steatosis index: a simple screening tool reflecting nonalcoholic fatty liver disease[J]. Dig Liver Dis, 2010, 42 (7): 503-508.

11Amato MC, Giordano C, Galia M, et al; AlkaMeSy Study Group. Visceral Adiposity Index: a reliable indicator of visceral fat function associated with cardiometabolic risk[J]. Diabetes Care, 2010, 33 (4): 920-922.

12Petta S, Amato MC, Di Marco V, et al. Visceral adiposity index is associated with significant fibrosis in patients with non-alcoholic fatty liver disease[J]. Aliment Pharmacol Ther, 2012, 35 (2): 238-247.

13Guerrero-Romero F, Simental-Mendía LE, González-Ortiz M, et al. The product of triglycerides and glucose, a simple measure of insulin sensitivity. Comparison with the euglycemic-hyperinsulinemic clamp[J]. J Clin Endocrinol Metab, 2010, 95 (7): 3347-3351.

14Petta S, Di Marco V, Di Stefano R, et al. TyG index, HOMA score and viral load in patients with chronic hepatitis C due to genotype 1[J]. J Viral Hepat, 2011, 18 (7): e372-e380.

15Abiru S, Migita K, Maeda Y, et al. Serum cytokine and soluble cytokine receptor levels in patients with non-alcoholic steatohepatitis[J]. Liver Int, 2006, 26 (1): 39-45.

16Alaaeddine N, Sidaoui J, Hilal G, et al. TNF-α messenger ribonucleic acid (mRNA) in patients with nonalcoholic steatohepatitis[J]. Eur Cytokine Netw, 2012, 23 (3): 107-111.

17Du J, Ma YY, Yu CH, et al. Effects of pentoxifylline on nonalcoholic fatty liver disease: a meta-analysis[J]. World J Gastroenterol, 2014, 20 (2): 569-577.

18García-Galiano D, Sánchez-Garrido MA, Espejo I, et al. IL-6 and IGF-1 are independent prognostic factors of liver steatosis and non-alcoholic steatohepatitis in morbidly obese patients[J]. Obes Surg, 2007, 17 (4): 493-503.

19Kinoshita A, Nishino H. IL-6, a promising tumor marker and prognostic indicator in patients with HCC?[J]. Journal of Tumor, 2014, 2 (6): 142-144.

20Mohamed AA, Gh SW, Shaker O, et al. Role of serum adiponectin, IL-6, and hs-CRP in non-alcoholic fatty liver Egyptian patients[J]. International Journal of Biochemistry Research & Review, 2014, 4 (6): 493-504.

21Maleki I, Rastgar A, Hosseini V, et al. High sensitive CRP and pentraxine 3 as noninvasive biomarkers of nonalcoholic fatty liver disease[J]. Eur Rev Med Pharmacol Sci, 2014, 18 (11): 1583-1590.

22Fierbinteanu-Braticevici C, Baicus C, Tribus L, et al. Predictive factors for nonalcoholic steatohepatitis (NASH) in patients with nonalcoholic fatty liver disease (NAFLD)[J]. J Gastrointestin Liver Dis, 2011, 20 (2): 153-159.

23Angulo P, George J, Day CP, et al. Serum ferritin levels lack diagnostic accuracy for liver fibrosis in patients with nonalcoholic fatty liver disease[J]. Clin Gastroenterol Hepatol, 2014, 12 (7): 1163-1169.

24Kowdley KV, Belt P, Wilson LA, et al; NASH Clinical Research Network.Serum ferritin is an independent predictor of histologic severity and advanced fibrosis in patients with nonalcoholic fatty liver disease[J]. Hepatology, 2012, 55 (1): 77-85.

25Feldstein AE, Lopez R, Tamimi TA, et al. Mass spectrometric profiling of oxidized lipid products in human nonalcoholic fatty liver disease and nonalcoholic steatohepatitis[J]. J Lipid Res, 2010, 51 (10): 3046-3054.

26Wong RJ, Cheung R, Ahmed A.Nonalcoholic steatohepatitis is the most rapidly growing indication for liver transplantation in patients with hepatocellular carcinoma in the U.S[J].Hepatology, 2014, 59 (6): 2188-2195.

27Wai CT, Greenson JK, Fontana RJ, et al. A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C[J]. Hepatology, 2003, 38 (2): 518-526.

28Adler M, Gulbis B, Moreno C, et al. The predictive value of FIB-4 versus FibroTest, APRI, FibroIndex and Forns index to noninvasively estimate fibrosis in hepatitis C and nonhepatitis C liver diseases[J]. Hepatology, 2008, 47 (2): 762-763.

29Kruger FC, Daniels CR, Kidd M, et al. APRI: a simple bedside marker for advanced fibrosis that can avoid liver biopsy in patients with NAFLD/NASH[J]. S Afr Med J, 2011, 101 (7): 477-480.

30Ratziu V, Massard J, Charlotte F, et al; LIDO Study Group; CYTOL study group. Diagnostic value of biochemical markers (FibroTest-FibroSURE) for the prediction of liver fibrosis in patients with non-alcoholic fatty liver disease[J]. BMC Gastroenterol, 2006, 6: 6.

31Poynard T, Deckmyn O, Munteanu M, et al; FIBROFRANCE Group. Awareness of the severity of liver disease re-examined using software-combined biomarkers of liver fibrosis and necroinflammatory activity[J]. BMJ Open, 2015, 5 (12): e010017.

32Shukla A, Kapileswar S, Gogtay N, et al. Simple biochemical parameters and a novel score correlate with absence of fibrosis in patients with nonalcoholic fatty liver disease[J]. Indian J Gastroenterol, 2015, 34 (4): 281-285.

33Calès P, Oberti F, Michalak S, et al. A novel panel of blood markers to assess the degree of liver fibrosis[J]. Hepatology, 2005, 42 (6): 1373-1381.

34Siddiqui MS, Patidar KR, Boyett S, et al. Performance of non-invasive models of fibrosis in predicting mild to moderate fibrosis in patients with non-alcoholic fatty liver disease[J]. Liver Int, 2016, 36 (4): 572-579.

35Aykut UE, Akyuz U, Yesil A, et al. A comparison of FibroMeterTMNAFLD Score, NAFLD fibrosis score, and transient elastography as noninvasive diagnostic tools for hepatic fibrosis in patients with biopsy-proven non-alcoholic fatty liver disease[J]. Scand J Gastroenterol, 2014, 49 (11): 1343-1348.

36McPherson S, Anstee QM, Henderson E, et al. Are simple noninvasive scoring systems for fibrosis reliable in patients with NAFLD and normal ALT levels?[J]. Eur J Gastroenterol Hepatol, 2013, 25 (6): 652-658.

37Harrison SA, Oliver D, Arnold HL, et al. Development and validation of a simple NAFLD clinical scoring system for identifying patients without advanced disease[J]. Gut, 2008, 57 (10): 1441-1447.

38Adams LA, George J, Bugianesi E, et al. Complex non-invasive fibrosis models are more accurate than simple models in non-alcoholic fatty liver disease[J]. J Gastroenterol Hepatol, 2011, 26 (10): 1536-1543.

39Lee TH, Han SH, Yang JD, et al. Prediction of advanced fibrosis in nonalcoholic fatty liver disease: an enhanced model of BARD score[J]. Gut Liver, 2013, 7 (3): 323-328.

40Angulo P, Keach JC, Batts KP, et al. Independent predictors of liver fibrosis in patients with nonalcoholic steatohepatitis[J]. Hepatology, 1999, 30 (6): 1356-1362.

(2015-07-15收稿；2015-08-07修回)

Progress in Studies on Biomarkers for Diagnosis of Nonalcoholic Fatty Liver Disease

ZHOUYijun,QINDanping.

DepartmentofGastroenterology,theFirstAffiliatedHospitalofZhejiangChineseMedicalUniversity,Hangzhou(310006)

Correspondence to: QIN Danping, Email: 15088662042@126.com

AbstractNonalcoholic fatty liver disease (NAFLD) is a kind of metabolic and stress-related liver damage closely related with insulin resistance and inherited susceptibility. Its incidence is increasing recently in China. Liver biopsy is the gold standard for the diagnosis of NAFLD, but which is an invasive procedure with certain risks. Therefore, finding a simple and accurate diagnostic method is an eager task, and searching of non-invasive biomarkers for diagnosis of NAFLD has become a hot spot of study. This article reviewed the progress in studies on biomarkers for diagnosis of NAFLD.

Key wordsNonalcoholic Fatty Liver Disease;Biological Markers;Diagnosis;Inflammation;Liver Fibrosis;

DOI:10.3969/j.issn.1008-7125.2016.04.012