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. 2021 Aug 31;41(9):1342–1349. [Article in Chinese] doi: 10.12122/j.issn.1673-4254.2021.09.08

1-脱氧野尻霉素减轻2型糖尿病小鼠的肝纤维化

1-deoxynojirimycin alleviates liver fibrosis induced by type 2 diabetes in mice

Xiaoqiao HE 2,2, Zhiyuan SUN 3,2, Kaiyuan MA 2, Yingwu MEI 1,*
PMCID: PMC8526323  PMID: 34658348

Abstract

Objective

To investigate the effect of 1-deoxynojirimycin (DNJ) for improving diabetic liver fibrosis and explore the underlying mechanism.

Methods

Mouse models of type 2 diabetes were established in 10 Kunming mice by high-fat diet feeding for 8 weeks and intraperitoneal injection of STZ, with 5 mice receiving intraperitoneal injection of citrate buffer solution with normal feeding as the control group. The mouse models were randomized into two groups (n=5) for further highfat feeding (model group) and additional treatment with 10% DNJ in drinking water (200 mg · kg-1 per day; DNJ group) for 8 weeks. The mice were monitored for changes in body weight (BW), blood glucose, serum total cholesterol (TC), triglyceride (TG) and superoxide dismutase (SOD) levels. The pathological changes in the liver tissue were observed using HE and Sirius Red staining, and the solubility of collagens in the liver tissues was determined. The expression levels of MCP-1, TNF-α, IL-1β and TGF-β1 mRNA were detected with real-time PCR, and the protein expressions of α-SMA and collagen2 (ColA2) were determined with Western blotting. In the in vitro experiment, mouse fibroblasts L929 cells were pretreated with DNJ (10 μg/ mL) or PBS for 30 min followed by culture in high-glucose medium for 24 h, and the level of ROS production was measured using dihydroethidium (DHE) staining.

Results

In the mouse model of type 2 diabetes, DNJ treatment significantly lowered serum level of glucose, TC, and TG (P < 0.05) and increased serum SOD activity (P < 0.05). DNJ obviously attenuated liver fibrosis in the diabetic mice, as shown by alleviated cross-linking of collagens and reduced contents of pepsin-solubilized collagen (PSC) and total collagen (P < 0.05). DNJ treatment also significantly reduced the overexpression of the proinflammatory cytokines and fibrosis-related cytokines induced by diabetes (P < 0.05). In L929 cells exposed to high glucose, pretreatment with DNJ significantly lowered the intensity of red fluorescence in DHE staining.

Conclusion

DNJ can attenuate type 2 diabetes-induced liver fibrosis in mice through its hypoglycemic, anti-inflammatory and anti-oxidative effects.

Keywords: type2 diabetes, hepatic fibrosis, 1-deoxynojirimycin, collagen cross-linking, oxidative stress

糖尿病是以高血糖为主要特征的代谢性疾病，治疗不当会引发一系列综合症，包括糖尿病大血管和微血管并发症[1]。糖尿病肝纤维化是糖尿病的慢性并发症之一，它的特征是炎症或损伤区域内大量的纤维结缔组织积聚，最终可能导致各种慢性肝病发展为肝硬化和肝癌[2]。实际上，肝内血糖的调节异常是糖尿病高血糖症和胰岛素抵抗的主要原因[3, 4]。研究表明，高血糖对肝纤维化和脂肪变性有明显的促进作用[5]，同时也有研究表明高血糖可引起活性氧（ROS）积累从而直接破坏抗机体氧化防御系统功能[6]，糖尿病可造成肝脏损伤与这一机制密切相关[5]。

而1-脱氧野尻霉素（DNJ）作为一种有效的糖苷酶抑制剂[6]，已有研究证实其具有降血糖活性[3]，而其作为糖苷酶抑制剂的降血糖作用是通过竞争性结合葡萄糖苷酶催化位点进而抑制葡萄糖通过肠膜的运输实现的[7]。此外，DNJ还可以结合并抑制糖苷酶和糖基化酶的活性来降低血糖[8]。而作为富含DNJ的桑叶生物碱粗提物就可以起到积极的降血糖作用[9, 10]并激活氧化系统以改善肥胖[11]。此外，有研究表明DNJ可能通过下调炎症及减少肝细胞凋亡缓解糖尿病引起的肝损伤, 发挥其对肝脏的改善作用[12]。同时，DNJ作为一种药食同材还可以缓解肝脏纤维化[13, 14]。最近，国内有学者发现桑叶生物碱对高脂饮食小鼠具有较强的抗氧化作用[15]，而且有学者指出其通过抑制心肌组织氧化应激，起到保护心脏的作用[16]。然而关于DNJ这一特定活性成分对于2型糖尿病所引起的肝纤维化病变的影响及氧化应激在这一过程中所起作用未见报道。推测其机理可能与氧化应激作用相关。因此本文对DNJ降糖作用以及改善纤维化的作用进行验证，通过DNJ对高脂饮食小鼠血清和肝脏中相关氧化指标的影响，探讨DNJ改善糖尿病引起的肝纤维化的机制。

1. 材料和方法

1.1. 材料

食品级桑叶提取物（10%DNJ；95%DNJ）购自上海比德制药技术有限公司，其他试剂均为分析纯。高脂饲料（由普通基础饲料87%，猪油10%，胆固醇0.5%和蒸馏水0.5%组成），葡萄糖测定试剂盒（葡萄糖氧化酶法），甘油三酸酯测定试剂盒（GPO-PAP法，液体单剂量形式），胆固醇测定试剂盒（CHOD-PAP法，液体单剂），超氧化物歧化酶（SOD）测定试剂盒，HE染料溶液，天猩红色染料溶液，链脲佐菌素（STZ），抗体β-actin、MCP-1、TNF-α、IL-1β、TGF-β1、ColA2（Santa Cruz）NADPH（Boster），α-SMA（Abcam），SDS-PAGE凝胶配制试剂，L929小鼠成纤维细胞，二氢乙啶。

1.2. 方法

1.2.1. 动物处理

所有动物处理方案均已获得郑州大学伦理委员会的批准。在26 ℃恒温实验室中以标准的12 h明暗循环喂养约20只昆明小鼠（20 g，6周）。适应2周后，随机抽取5只小鼠标准小鼠饲料喂养4周后，通过腹膜内注射（ip）柠檬酸盐缓冲液（50 mg/kg，pH=4.5）3 d作为正常对照组。其余15只小鼠高脂饮食喂养4周后，使用50 mg/kg剂量的链脲佐菌素（STZ，Sigma-Aldrich）通过腹膜内注射（ip）3 d，其余5只通过腹膜内注射（ip）柠檬酸盐缓冲液（50 mg/kg，pH=4.5）3 d。对于空腹血糖浓度≥16.7 mmol/L的小鼠被选为成功的2型糖尿病实验模型。随机分为2组（5只/组）分别作为DM组和实验组：（1）正常对照组喂养普通饮食和普通水；（2）DM组喂养高脂饮食和普通水；（3）实验组喂养高脂饮食和含10% DNJ饮用水（200 mg·kg-1·d-1）。喂养8周，每2周检查一次体质量（BW），血糖，血清总胆固醇（TC）和甘油三酸酯（TG）。最后，处死所有动物，收集它们的血液样品和肝脏。使用血液样品测量超氧化物歧化酶（SOD）的水平，肝脏称重，然后分为两部分，一部分-80 ℃冰箱冷冻，另一部分10%中性福尔马林缓冲液固定。

1.2.2. 生化分析

用市售诊断试剂盒（BioSino BioTechnology & Science Inc.）测量血糖、TC和TG值。用SOD诊断试剂盒（Beyotime Biotechnology & Science Inc.）测定SOD的活性。

1.2.3. 组织病理学分析

处死动物并收集肝脏切片，然后固定在10%中性福尔马林缓冲液。石蜡包埋后，将肝切片切成5 μm厚的切片。根据标准方案，切片做苏木精和曙红染色（H & E）染色，根据先前文献中的方法进行天狼猩红染色[12]。将福尔马林固定的组织包埋在石蜡中，并依次切成5 μm厚的切片。将切片在溶于饱和苦味酸水溶液中的0.1%天狼猩红溶液浸泡1 h，在酸化水（0.5%盐酸）中洗涤，脱水，并用二甲苯固定。胶原纤维会被染成红色，而非胶原纤维成分会被染成黄色。

1.2.4. 胶原蛋白溶解度的测定

通过在中性盐缓冲液，乙酸盐缓冲液和含胃蛋白酶的乙酸盐缓冲液中依次提取胶原蛋白，最终测得了各组肝组织中胶原蛋白在各缓冲液中的浓度。步骤如下，首先将组织样品在0.05 mol/L Tris-HCl缓冲液（pH=7.4包含1 mol/L NaCl，20 mmol/L EDTA和1 mmol/L PMSF）中匀浆。获得组织悬浮液后，在4 ℃轻轻摇动24 h。然后将内容物在4 ℃以15 000 r/min离心30 min。收集上清液用于随后的胶原蛋白测定, 中性盐溶性胶原蛋白（NSC）即溶解于此上清液中。将获得的沉淀物悬浮在0.5 mol/L乙酸中，充分均质，并在4 ℃轻轻摇动24 h。然后将内容物在4 ℃下以15 000 r/min离心30 min，收集上清液以进行后续测定，酸溶性胶原蛋白（ASC）即溶于此上清液中。将沉淀物悬浮于0.5 mol/L含胃蛋白酶的1 mg/mL乙酸中，根据上述方法获得上清液，胃蛋白酶可溶性胶原蛋白（PSC）即溶于此上清液中。将剩下的内容物溶于1 mL去离子水中，并在80 ℃下加热1 h，即可得到不溶性胶原蛋白（ISC）的匀浆。之前获得的所有上清液均通过天狼猩红分光光度比对方法测定胶原蛋白含量。将上述四部分（NSC，ASC，PSC和ISC）相加得到胶原蛋白的总含量。

1.2.5. 实时定量PCR法检测MCP-1、TNF-α、IL-1β、TGF-β 1mRNA相对表达水平

取出小鼠肝组织并加入TRIzol试剂盒提取小鼠肝脏组织总RNA，采用RT-PCR法测定小鼠肝脏MCP-1、TNF-α、IL-1β、TGF-β1的mRNA水平，以GAPDH为内参照。采用TIANGEN Talent qPCR PreMix（FP209，北京）以及ABI Prism 7500序列检测系统（Applied Biosystems, Foster City, CA, USA）进行扩增和检测。mRNA相对表达量以2-ΔΔCt表示。检测基因的所有引物如下：

MCP-1_F: 5'-GAGGTGGTTGTGGAAAAGGTA G-3'

MCP-1_R: 5'-TTGTCACCAAGCTCAAGAGAGA-3'

TNF-α_F: 5'-ACCTGGCCTCTCTACCTTGT-3'

TNF-α_R: 5'-GACCCGTAGGGCGATTACAG-3'

IL-1β_F: 5'-CCACGGGAAAGACACAGGTAGC-3'

IL-1β_R: 5'-ACCTGGGCTGTCCTGATGAGAG-3'

TGF-β1_F: 5'-ATGTCATGGATGGTGCCCAG-3'

TGF-β1_R: 5'-GTCACTGGAGTTGTACGGCA-3'

GAPDH_1_F: 5'-CGGTGCTGAGTATGTCGTG-3'

GAPDH_1_R: 5'-TCTTCTGGGTGGCAGTGAT-3'

1.2.6. Western blot法测定α-SMA和Collagen2（ColA2）蛋白水平

无菌条件下，剪取小鼠肝组织，放入RIPA裂解液中冰上裂解30 min，将溶解物质在4 ℃下以13 000 r/min离心10 min，取上清液-80 ℃保存备用；采用BCA法对蛋白进行定量，使用10% SDS聚丙烯酰胺凝胶电泳对蛋白进行分离，通过电印迹法将样本转移至硝化纤维素膜上。使用5% BSA封闭液室温封闭1 h，将膜转入一抗（抗α-SMA抗ColA2），以β-actin为内标，置于2% BSA中4 ℃过夜，然后洗涤，与辣根过氧化氢酶标记的二抗在室温下孵育1 h，使用BeyoECL Plus试剂盒（Beyotime）观察。

1.2.7. 培养的L929细胞系中活性氧的定量

荧光探针染料二氢乙啶（DHE）（Invitrogen分子探针）用于检测培养的L929细胞中细胞内活性氧的产生。简而言之，将MEM培养基中培养的L929细胞用DNJ（10 μg/mL）或PBS预处理30 min，然后将其暴露于高葡萄糖MEM培养基中再培养24 h。根据说明书，将培养的L929细胞与10 mmol/L DHE在37 ℃下孵育30 min。用荧光显微镜（Olympus IX 71）下观察并定量评估红色荧光强度。

1.3. 统计分析

采用SPSS 21.0软件进行统计分析，正态分布计量资料采用均数±标准差的形式表示表示，组间比较采用单因素方差分析，两两比较采用LSD检验或Dunnett-t检验，P < 0.05表示差异有统计学意义。

2. 结果

2.1. DNJ降低糖尿病小鼠的体质量和肝比重

DNJ的化学结构如图 1A所示。成功建模后，高脂饮食喂养糖尿病小鼠并持续8周。在整个实验期间，观察到糖尿病小鼠变得瘦弱。与对照组和DM组相比，DNJ治疗8周使小鼠体质量减轻（图 1B）。与对照组相比，DM组的肝脏质量和肝指数显著升高（P=0.002，P < 0.001，图 1C，D）；而DNJ组与DM组相比肝脏质量和肝指数显著降低（P=0.009，P=0.01，图 1C，D）。

1.

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DNJ对体质量和肝体质量比的影响

Effects of DNJ on body weight and the liver-to-body weight ratio. A: Chemical structure of DNJ. B: Effect of DNJ on body weight gain. C: Effect of DNJ on liver weight. D: Effect of DNJ on liver-to-body weight ratio. Con: Control group. DM: Diabetes mellitus group. DNJ: DNJ treatment group. Data are presented as Mean±SD. *P < 0.05 vs control group; #P < 0.05 vs DM group.

2.2. DNJ减轻糖尿病小鼠的高血糖和血脂异常。

糖尿病相关的代谢综合征表现为高血糖和血脂异常，常表现为血浆甘油三酸酯和胆固醇酯水平升高[17]。与对照组相比，DM组的血清葡萄糖、甘油三酸酯和胆固醇水平显著升高（P=0.001，P < 0.001，P < 0.001，图 2A~C）。DNJ治疗8周后，与DM组相比DNJ组血糖和甘油三酯水平降低但无显著差异，胆固醇显著降低（P=0.049，图 2B，C）。

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DNJ对糖尿病小鼠血清生化指标的影响

Effects of DNJ on serum biochemical indexes in diabetic mice. A: Effect of DNJ on blood glucose. B: Effect of DNJ on serum triglyceride levels. C: Effect of DNJ on serum cholesterol levels. Data are presented as Mean± SD. *P < 0.05 vs control group; #P < 0.05 vs DM group.

2.3. DNJ可改善糖尿病小鼠的肝纤维化

HE染色用于验证DNJ治疗是否可以减轻肝细胞损伤（图 3A），2型糖尿病引起的肝纤维化可导致脂肪变性及炎症细胞浸润[18]。染色结果显示在DM组中，大量的脂肪变性肝细胞呈斑点状分布在中央静脉周围，伴有炎性细胞浸润。而DNJ组的脂肪变性肝细胞数量减少炎性浸润区减小，门管区扩大的情况也得到改善（图 3A）。

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不同治疗组肝纤维化的组织形态学分析

Histopathological analysis of hepatic fibrosis in different treatment groups. A: Effect of DNJ on hepatocytes and inflammatory cells in the liver tissue of diabetic rats (HE staining). B: Effect of DNJ on collagen fibers in the liver tissue of diabetic rats (Picric acid-sirius red staining). C: Effect of DNJ on collagen fibers in the liver tissue of the rats (Picric acid-sirius red staining; polarized light microscope).

天狼猩红染色用于观察胶原在肝组织中的沉积以及相邻门管区的交联程度。在DM组中，观察到门管区的红色区域增厚，大量胶原沉积，假小叶形成，且相邻小叶之间发生交联。与DM组相比，在DNJ组中门管区的红色区域、胶原沉积及假小叶数目均减少（图 3B）。

使用偏振光显微镜观察天狼猩红染色切片时，观察区域的胶原蛋白会显示不同的颜色。Ⅰ型胶原蛋白显示为黄色、橙色或红色，而Ⅲ型胶原蛋白显示为绿色。对照组中，观察到的Ⅰ型和Ⅲ型胶原蛋白较少。与对照组相比，DM组中Ⅰ型和Ⅲ型胶原蛋白在门静脉区域沉积增多。而DNJ组与DM组相比，Ⅰ型胶原和Ⅲ型胶原蛋白有所减少（图 3C）。

2.4. DNJ通过减少胶原纤维交联抑制糖尿病肝纤维化的发生

参照既往研究[19]的方法，连续提取分离肝组织中的胶原并判断它们的稳定性。所得的各种类型胶原蛋白的浓度如图 5所示，代表非交联胶原和前胶原的NSC和ASC在各组间无差异（图 4A，B）。

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DNJ对肝组织中促炎及促纤维化因子基因和蛋白质表达的影响

Effect of DNJ on the expression of pro-inflammatory and pro-fibrotic factors at the mRNA and protein levels in the liver tissues. A-D: Relative mRNA levels of MCP-1, TNF-α, IL-1β, and TGF-β1 detected by RT-PCR. E, F: Protein levels of α-SMA and ColA2 detected by Western blotting. Data are shown as Mean± SD; *P < 0.05 vs control group; #P < 0.05 vs the DM group.

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DNJ对胶原在肝组织基质中溶解度分布的影响

Effects of DNJ on the solubility profile of collagen in hepatic tissue matrix (n=3). A: Comparison of the amount of neutral salt-soluble collagen. B: Comparison of the amount of acid-soluble collagen. C: Comparison of the amount of pepsin-soluble collagen. D: Comparison of the amount of insoluble collagen. E: Comparison of the amount of total collagen. Data are presented as Mean±SD; *P < 0.05 vs control group; #P < 0.05 vs the DM group.

然而，DM组和对照组，DNJ组和DM组之间代表成熟和中度交联胶原的PSC存在显著的统计学差异（P=0.001，图 4C）。此外，与DM组相比，代表高度交联胶原的ISC在DNJ组也显示出降低但无统计学差异（图 4D）。上述4种胶原相加可获得总胶原，与DM组相比，DNJ组总胶原含量显著降低（P=0.03，图 4E）。

2.5. DNJ抑制促炎因子及促纤维化细胞因子的表达减轻肝纤维化程度

通过RT-PCR检测肝脏炎症标记物，结果显示DM组与对照组相比促炎因子MCP-1、TNF-α、IL-1β的基因表达水平有显著提高（P=0.002，P=0.019，P < 0.001，图 5A~C），而经过DNJ给药的治疗组与DM组相比MCP-1、TNF-α、IL-1β表达水平显著降低（P=0.027，P= 0.02，P=0.001，图 5A~C）。

对于目前已证明可以通过旁分泌途径激活HSCs从而导致肝纤维化的相关纤维化因子，TGF-β1基因表达水平，α-SMA和ColA2的蛋白表达量与DM组相比，DNJ组表现出基因表达水平和蛋白表达量的显著降低（P=0.038，P=0.012，P=0.004，图 5D~F）。

2.6. DNJ通过调节氧化应激减轻肝纤维化程度

通过测量各组的SOD水平来评价细胞内氧化应激的程度。结果表明，与对照组相比，DM组的SOD水平降低（P=0.04，图 6C）；而与DM组相比，DNJ组的SOD水平显著升高（P=0.002，图 6C）。

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DNJ对体内外氧化应激状态的影响

Effects of DNJ on oxidative stress in L929 cells with high glucose exposure. A: Intracellular ROS production after DNJ treatment and high glucose exposure. B: Statistics of relative fluorescence intensity compared with the Con group. αP < 0.05 vs control group; βP < 0.05 vs the HG group. C: Effect of DNJ on serum SOD activity. Data are presented as Mean± SD. *P < 0.05 vs control group; #P < 0.05 vs the DM group.

为了进一步探讨糖尿病对氧化应激的影响以及DNJ在其中的作用，使用L929小鼠成纤维细胞进行体外研究。本研究选择检测细胞内超氧阴离子自由基最常用的荧光探针DHE染色法[20]测定DNJ在不同条件下对成纤维细胞内ROS水平的影响，红色荧光的强度可以反映细胞内ROS的水平[21]。荧光图像显示，与对照组相比DHE染色的荧光强度在经高糖培养基培养的成纤维细胞中增强，且相对荧光密度显著增大（P < 0.001，图 6A，B）；在DNJ处理后荧光强度减弱且密度与HG组相比也显著降低（P=0.001，图 6A，B）。

3. 讨论

糖尿病是一种以高血糖为特征的慢性疾病，可引起全身许多器官和组织的各种并发症，肝纤维化是糖尿病肝综合征的主要病理生理过程之一[22]，最近有报道称DNJ可以发挥减轻纤维化的作用[13]。于是本研究通过连续喂饲高脂饮食成功建立了高糖高脂的2型糖尿病小鼠模型，以此为基础观察小鼠的肝脏纤维化的程度，并与DNJ组进行比对。

甘油三酯和极低密度胆固醇通过脂质过氧化物促进肝星状细胞（HSCs）的增殖，引发肝纤维化级联反应，并最终导致纤维化的发生[23]。HSCs是一种能合成、分泌细胞外基质并产生胶原酶的肝间质细胞，是肝脏中主要的与纤维化相关的细胞，并已被相关研究作为抗肝纤维化的有效靶点[24]。此外，已有文献报道在高糖条件下，HSCs会被激活增殖并分泌胶原，而这与高血糖环境下氧自由基的增加以及丝裂原活化蛋白激酶的活化密切相关[25]。因此，高脂血症和高血糖都是导致肝纤维化的重要因素。本研究通过检测各组小鼠的血清学指标来评价三组小鼠的糖代谢和脂代谢情况，结果显示，DNJ能显著降低2型糖尿病小鼠的血糖和TG（图 2）。在肝组织病理切片中，门静脉区域的扩张以及纤维结缔组织的增生主要发生在中心门静脉区域，而纤维间隙假小叶的形成则是小叶交联，纤维化形成的直接证据。本研究中天狼星红染色结果显示DNJ可以显著降低肝组织中Ⅰ型和Ⅲ型胶原的含量并减轻胶原之间的交联程度。

肝纤维化的主要组织学改变是肝细胞间基质成分（如I型胶原）的过度增殖，超过降解速度从而在肝组织中积聚。胶原具有适度的硬度和组织强度，这与肝纤维化时肝脏硬度的增加有关。研究表明，相比胶原蛋白总量增加，Ⅰ型胶原的交联结构对胶原结构稳定性的强化作用更大[26]。因此为了进一步评价胶原的变化，接下来选择天狼星红比色平板法定量检测组织中纤维化情况[27]，结果显示DNJ可以显著降低PSC、ISC和总胶原的含量。

同时，现有证据表明糖尿病可引起慢性炎症损伤[28]，炎性因子的聚集也可能是导致糖尿病并发症肝损伤的重要原因。本研究采用RT-PCR检测对照组、DM组以及DNJ组的小鼠成纤维细胞中炎性因子的水平，结果显示DM组MCP-1、TNF-α、IL-1β水平均显著升高，进而导致肝细胞的凋亡及纤维化，这一病理过程在DNJ处理后得到了改善从而阐明了DNJ对于糖尿病诱导的炎症的潜在保护机制。作为一种α-葡萄糖苷酶抑制剂，DNJ已被证实具有显著的降血糖作用[29]，而我们的结果进一步表明DNJ可能通过降低血糖、血脂和炎症反应来发挥改善纤维化的功效。

已有研究表明氧化应激常常伴随着肝损伤和纤维化的发生[30]，这可能是由于氧化应激可以导致β细胞功能障碍，进而造成胰岛素抵抗[31]，从而加重2型糖尿病及其并发症的病程。HSCs的活化通常被认为是肝纤维化的中心环节，TGF-β1被认为是最有效的促肝纤维化的细胞因子[32, 33]，RT-PCR结果显示TGF-β1表达水平在DM组中升高，Western blot结果显示DM组α-SMA及ColA2表达量增加，提示HSCs的激活及继发的胶原蛋白分泌增加，而DNJ治疗组则显出了TGF-β1水平的回落以及α-SMA及ColA2表达量的降低，这提示DNJ可通过抑制促纤维化细胞因子的表达改善糖尿病引起的纤维化作用。而氧化应激也与HSCs的活化和肝纤维化的发生密切相关[34, 35]，细胞内氧化应激的程度又会影响SOD的表达。

SOD作为一种催化超氧阴离子歧化为过氧化氢的抗氧化酶，是机体氧化应激状态的有力反映因此，本研究接下来首先测定SOD的水平来评价DNJ对二型糖尿病诱导的全身氧化应激状态的影响[36]。与预测一致，结果显示DNJ组的SOD水平显著高于DM组（图 6C），这表明DNJ提高了机体清除ROS的能力。然而，由于这种作用是在体内观察到的，也可能是机体免疫系统调节的结果。因此，我们希望通过进一步的实验来探讨DNJ能否在体外独立发挥抗氧化应激作用。文献中关于测定总抗氧化能力的方法有诸多报道，其中包括了二氢乙胺（DHE）染色法，这种方法有独到的优越性而应用广泛，可以通过发射红色荧光来测定超氧化物含量并且实时监测其氧化情况[37]。在通过对培养的小鼠成纤维细胞进行DHE染色获得的荧光图像中，DNJ对高糖培养基培养的细胞所产生的超氧化物显示出极好的清除作用。已有文献报道肯定了DNJ具有包括抗氧化作用在内的多种功能[38]，本实验结果直接证实了DNJ具有独立的抗氧化作用，并能减轻氧化应激对细胞的损伤，与既往研究一致。

综上所述，本研究首次通过动物实验证实了DNJ可以发挥降血糖、抑制炎症因子和纤维化因子的表达以及抗氧化应激的联合作用以减轻2型糖尿病诱导的肝纤维化病变。因此，DNJ作为一种潜在的天然功能性营养品，值得进一步研究。

Biographies

赫小乔，在读本科生，E-mail: 116961957@qq.com

孙志远，在读本科生，E-mail: sunzhiyuan2000@126.com

Funding Statement

国家自然科学基金（U1704192）；河南省教育委员会基金（18A310032）；郑州大学国家大学生创新创业计划（2019cxcyo34）

Supported by National Natural Science Foundation of China (U1704192)

Contributor Information

赫 小乔 (Xiaoqiao HE), Email: 116961957@qq.com.

孙 志远 (Zhiyuan SUN), Email: sunzhiyuan2000@126.com.

梅 英武 (Yingwu MEI), Email: yingwumei@zzu.edu.cn.

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