越来越多的临床证据表明,磺脲类药物治疗2型糖尿病(T2DM)患者有助于其肝脏进行性恶化。本研究提出了格列齐特诱导的肝毒性,第二代磺酰脲,和α-硫辛酸(ALA)作为治疗T2DM的新型和有前途的药物。在存在或不存在格列齐特和ALA的情况下,将正常人肝细胞(HL-7702)与高葡萄糖DMEM一起孵育72小时,通过流式细胞术测量细胞活力和死亡。接下来,Sprague-Dawley大鼠禁食12小时,并测定空腹血糖。将大鼠随机分为四组:HC(健康对照;n=7),T2DM(未经治疗的糖尿病大鼠;n=9),GLC(15mg/kg格列齐特治疗的糖尿病大鼠;n=7)和GLC+ALA(格列齐特和60mg/kgALA治疗的糖尿病大鼠;n=7)。通过腹腔推注110mg/kg烟酰胺和55mg/kg链脲佐菌素诱导T2DM。实验方案持续6周,之后动物被处死,胰腺,收集肝脏和血液样本进行生化检查,组织学和分子分析。与健康对照组(HC)相比,HL-7702细胞暴露于高糖诱导19%的显著细胞死亡(p<0.001),在ALA治疗后,格列齐特加重了29%(p<0.0001),但显著降低了6%,接近HC值。在体内,GLC治疗的大鼠有严重的肝损伤,其特征是肝细胞空泡增多,ED-1,iNOS和caspase-3的显着表达以及明显高水平的肝酶(天冬氨酸转氨酶,谷丙转氨酶和碱性磷酸酶与T2DM大鼠比较。有趣的是,ALA给药可预防这些病理变化,并将糖尿病肝脏保护至与HC大鼠相当的水平。ALA通过抑制糖尿病肝脏中的炎症和凋亡,同时激活抗氧化途径,对格列齐特诱导的肝毒性具有保护肝的作用。缩写:ALA,α-硫辛酸;ALT,丙氨酸氨基转移酶;ALP,碱性磷酸酶;AMPK,一磷酸腺苷活化蛋白激酶;AST,天冬氨酸转氨酶;ATP,三磷酸腺苷;DMEM,Dulbecco's改良鹰培养基;EDTA,乙二胺四乙酸;FBG,空腹血糖;FBS,胎牛血清;GLC,格列齐特;GLUT4,4型葡萄糖转运蛋白;GSH,谷胱甘肽;H&E,苏木精/伊红;HbA1c,糖化血红蛋白A1c;HC,健康控制;HG,高血糖组;HOMA-β,β细胞功能的稳态模型评估;IL-1β,白细胞介素-1β;白细胞介素-6,白细胞介素-6;iNOS,诱导型一氧化氮合酶;KATP,ATP依赖性钾通道;MDA,丙二醛;MPTP,线粒体通透性转换孔;NO,一氧化氮;P/S,青霉素/链霉素;PAS,周期性酸-希夫;RIA,放射免疫分析;ROS,活性氧;SOD,超氧化物歧化酶;T2DM,2型糖尿病;TBARS,硫代巴比妥酸反应性物质;TNF-α,肿瘤坏死因子-α。
Growing clinical evidence shows that sulfonylurea therapy for patients with type 2 diabetic mellitus (T2DM) contributes to progressive worsening of their liver. The present study presents hepatotoxicity induced by gliclazide, a second-generation sulfonylurea, and alpha-lipoic acid (ALA) as a novel and promising drug for T2DM treatment. Normal human liver cells (HL-7702) were incubated with high-glucose DMEM in the presence or absence of gliclazide and ALA for 72 h, and cell viability and death were measured by flow cytometry. Next, Sprague-Dawley rats were subjected to 12 h of fasting, and fasting blood glucose was measured. The rats were randomized into four groups: HC (healthy control; n = 7), T2DM (diabetic rats without treatment; n = 9), GLC (diabetic rats with 15 mg/kg gliclazide treatment; n = 7) and GLC+ALA (diabetic rats with gliclazide and 60 mg/kg ALA treatment; n = 7). T2DM was induced by a bolus administration of 110 mg/kg nicotinamide and 55 mg/kg streptozotocin intraperitoneally. The experimental protocol lasted for 6 weeks after which the animals were sacrificed and pancreas, liver and blood samples were collected for biochemical, histological and molecular analyses. Compared to healthy control (HC) group, exposure of HL-7702 cells to high glucose induced significant cell death by 19 % (p < 0.001), which was exacerbated with gliclazide treatment by 29 % (p < 0.0001) but markedly reduced by 6 % to near HC value following ALA treatment. In vivo, GLC-treated rats had severe liver damage characterized by increased hepatocellular vacuolation, and significant expression of ED-1, iNOS and caspase-3 as well as markedly high levels of liver enzymes (aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase compared to T2DM rats. Interestingly, ALA administration prevented these pathological changes and protected the diabetic liver to levels comparable to HC rats. ALA showed hepatoprotective effect against gliclazide-induced hepatotoxicity by suppressing inflammation and apoptosis while activating antioxidant pathway in the diabetic liver. Abbreviations: ALA, Alpha-lipoic acid; ALT, Alanine aminotransferase; ALP, Alkaline phosphatase; AMPK, Adenosine monophosphate-activated protein kinase; AST, Aspartate aminotransferase; ATP, Adenosine triphosphate; DMEM, Dulbecco\'s Modified Eagle Medium; EDTA, ethylenediaminetetraacetic acid; FBG, Fasting blood glucose; FBS, Fetal bovine serum; GLC, Gliclazide; GLUT4, Glucose transporter type 4; GSH, Glutathione; H&E, Hematoxylin/Eosin; HbA1c, Glycosylated haemoglobin A1c; HC, Healthy control; HG, Hyperglycemic group; HOMA-β, Homeostasis model assessment of β-cell function; IL-1β, Interleukin-1β; IL-6, Interleukin-6; iNOS, Inducible nitric oxide synthase; KATP, ATP-dependent potassium channels; MDA, Malondialdehyde; MPTP, Mitochondrial permeability transition pore; NO, Nitric oxide; P/S, Penicillin/streptomycin; PAS, Periodic acid-Schiff; RIA, Radioimmunoassay; ROS, Reactive oxygen species; SOD, Superoxide dismutase; T2DM, Type 2 diabetes mellitus; TBARS, Thiobarbituric acid reactive substances; TNF-α, Tumor necrosis factor-alpha.