磷酸西格列汀调控2型糖尿病大鼠肝细胞线粒体稳态和功能的研究

The regulatory effects of sitagliptin phosphate on mitochondrial homeostasis and function in hepatocytes of type 2 diabetic rats

目的:研究磷酸西格列汀对2型糖尿病大鼠肝细胞线粒体稳态和功能的影响。方法:采用高脂、高糖饮食配合45 mg·kg-1链脲佐菌素饲喂法建立2型糖尿病大鼠模型。磷酸西格列汀组大鼠以磷酸西格列汀10 mg·kg-1灌胃(n=8),模型组大鼠以等体积PBS灌胃(n=8)。12周后,测定大鼠体质量、血糖水平,通过HE染色和Masson染色评估肝纤维化程度;运用TUNEL法检测肝组织中凋亡的细胞;采用化学发光法测定肝组织中ATP含量;采用实时荧光定量PCR(RT-qPCR)法测定线粒体基因组DNA与细胞核基因组DNA的比例;采用半定量Western blotting法测定线粒体标志蛋白TOMM20,HSP60和VADC的蛋白水平;磷酸西格列汀处理肝细胞HepRG后,采用JC-1染色观察线粒体膜电位变化,测定ATP含量,并用RT-qPCR法检测线粒体编码基因的转录水平,包括COX 1,COX 3,ND 1和Cyb。结果:磷酸西格列汀的使用改善了2型糖尿病大鼠体重下降、血糖升高、肝损伤及肝纤维化程度。相较于模型组[(0.22±0.03)pmol/min·mg^(-1)],磷酸西格列汀组肝组织中的ATP含量[(0.43±0.03)pmol/min·mg^(-1)]升高(P<0.05);相较于模型组(1.58±0.36),磷酸西格列汀组肝组织中的线粒体基因组DNA/细胞核基因组DNA比例(2.89±0.62)升高(P<0.05);相较于模型组,磷酸西格列汀组肝组织中线粒体标志蛋白TOMM20,HSP60和VADC明显更高。JC-1染色显示,高糖引起肝细胞HepRG线粒体膜电位明显丧失,而磷酸西格列汀加入后,线粒体膜电位明显回复,同时细胞中的ATP含量和转录水平回升(P<0.05)。结论:磷酸磷酸西格列汀在2型糖尿病大鼠的肝组织中,可能通过调控线粒体膜电位,ATP产量及线粒体转录活性,参与到减轻2型糖尿病大鼠肝纤维化程度的过程中。

Objective:To study the regulatory effects of sitagliptin phosphate on mitochondrial homeostasis and function in hepatocytes of type 2 diabetic rats.Methods:The rat model of type 2 diabetes mellitus was established by high fat and high sugar diet combined with 45 mg·kg-1streptozotocin feeding.The rats in sitagliptin phosphate group were administrated with 10 mg·kg-1sitagliptin phosphate (n=8) and the rats in model group were given an equal volume of PBS (n=8).After 12 weeks,the body weight and blood glucose level of rats were measured,and the degree of liver fibrosis was evaluated by HE staining and Masson staining;TUNEL staining was used to detect apoptotic cells in hepatocytes;ATP content in hepatocytes was determined by chemiluminescence method;the ratio of mitochondrial genome DNA to nuclear genome DNA was determined by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR);the levels of mitochondrial marker proteins TOMM20,HSP60 and VADC were determined by semi-quantitative western blotting;after HepRG was treated with sitagliptin phosphate,JC-1 staining was used to observe mitochondrial membrane potential changes,ATP content was determined,and RT-qPCR was used to detect transcription levels of mitochondrial coding genes,including COX 1,COX3,ND 1 and Cyb.Results:Sitagliptin phosphate improved weight loss,blood glucose increase,liver damage and liver fibrosis in type 2 diabetic rats.Compared with model group[(0.22±0.03)pmol/min·mg^(-1)],ATP content in liver tissue of sitagliptin phosphate group[(0.43±0.03) pmol/min·mg^(-1)]increased (P<0.05).Compared with model group (1.58±0.36),the ratio of mitochondrial genome DNA to nuclear genome DNA in hepatocytes in sitagliptin phosphate group (2.89±0.62) increased (P<0.05).Compared with model group,mitochondrial marker proteins TOMM20,HSP60 and VADC in hepatocytes in sitagliptin phosphate group were significantly higher.JC-1 staining showed that high glucose caused significant loss of HepRG mitochondrial membrane potential in hepatocytes,but the mitochondrial membrane potential was obviously recovered after sitagliptin was added;meanwhile,ATP content and transcription level in hepatocytes increased (P<0.05).Conclusion:Sitagliptin phosphate may be involved in reducing the degree of liver fibrosis in type 2 diabetic rats by regulating mitochondrial membrane potential,ATP production and mitochondrial transcriptional activity.