1、广西医科大学学报JOURNAL OF GUANGXI MEDICAL UNIVERSITY2023 Jan；40（1）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*卢迪，邱平，李婷，黎瑶（成都医学院第一附属医院内分泌代谢病科，成都610000）摘要目的：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 mgkg-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 mgkg-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
2、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（0.220.03）pmol/m
3、inmg-1，磷酸西格列汀组肝组织中的ATP含量（0.430.03）pmol/minmg-1升高（P0.05）；相较于模型组（1.580.36），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（2.890.62）升高（P0.05）；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（P0.05）。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
4、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。关键词2型糖尿病；磷酸磷酸西格列汀；线粒体膜电位；线粒体功能；线粒体含量中图分类号：R977.15文献标志码：A文章编号：1005-930X（2023）01-0059-07DOI：10.16190/ki.45-1211/r.2023.01.010The regulatory effects of sitagliptin phosphate on mitochondrial homeostasis and function inhepatocytes of type 2 diabetic ratsLu Di,Qiu Ping,Li Ting
5、,Li Yao.(Department of Endocrinology and Metabolism,The First Affiliated Hospital ofChengdu Medical College,Chengdu 610000,China)AbstractObjective:To study the regulatory effects of sitagliptin phosphate on mitochondrial homeostasis andfunction in hepatocytes of type 2 diabetic rats.Methods:The rat
6、model of type 2 diabetes mellitus was estab-lished by high fat and high sugar diet combined with 45 mgkg-1streptozotocin feeding.The rats in sitagliptinphosphate group were administrated with 10 mgkg-1sitagliptin phosphate(n=8)and the rats in model groupwere given an equal volume of PBS(n=8).After 1
7、2 weeks,the body weight and blood glucose level of rats weremeasured,and the degree of liver fibrosis was evaluated by HE staining and Masson staining;TUNEL stainingwas used to detect apoptotic cells in hepatocytes;ATP content in hepatocytes was determined by chemilumines-cence method;the ratio of m
8、itochondrial genome DNA to nuclear genome DNA was determined by real-time flu-orescence quantitative polymerase chain reaction(RT-qPCR);the levels of mitochondrial marker proteinsTOMM20,HSP60 and VADC were determined by semi-quantitative western blotting;after HepRG was treatedwith sitagliptin phosp
9、hate,JC-1 staining was used to observe mitochondrial membrane potential changes,ATPcontent was determined,and RT-qPCR was used to detect transcription levels of mitochondrial coding genes,in-cludingCOX 1,COX3,ND 1andCyb.Results:Sitagliptin phosphate improved weight loss,blood glucose in-crease,liver
10、 damage and liver fibrosis in type 2 dia-betic rats.Compared with model group(0.220.03)pmol/minmg-1,ATP content in liver tissue of sita-*基金项目：国家自然科学青年基金（No.81602821）通信作者，E-mail：收稿日期：2022-07-26 59广西医科大学学报2023 Jan；40（1）gliptin phosphate group(0.430.03)pmol/minmg-1 increased(P0.05).Compared with model
11、group(1.580.36),the ratio of mitochondrial genome DNA to nuclear genome DNA in hepatocytes in sitagliptin phosphategroup(2.890.62)increased(P0.05).Compared with model group,mitochondrial marker proteins TOMM20,HSP60 and VADC in hepatocytes in sitagliptin phosphate group were significantly higher.JC-
12、1 staining showedthat high glucose caused significant loss of HepRG mitochondrial membrane potential in hepatocytes,but the mi-tochondrial membrane potential was obviously recovered after sitagliptin was added;meanwhile,ATP contentand transcription level in hepatocytes increased(P0.05).Conclusion:Si
13、tagliptin phosphate may be involved inreducing the degree of liver fibrosis in type 2 diabetic rats by regulating mitochondrial membrane potential,ATPproduction and mitochondrial transcriptional activity.Keywordstype 2 diabetes;sitagliptin phosphate;mitochondrial membrane potential;mitochondrial fun
14、ction;mitochondrial content磷酸西格列汀是治疗糖尿病的新型降糖药物1-3。磷酸西格列汀除了可以降低血糖，提高胰岛素水平，长期使用也可明显减少2型糖尿病并发症，肝纤维化的发生或进程4-5。磷酸西格列汀也被发现参与了肝脏，心血管组织的能量代谢，比如促进线粒体再生和增加ATP合成。然而，磷酸西格列汀在2型糖尿病大鼠模型中对肝组织中线粒体的稳态及功能的影响效果和机制尚不明确。为此，本研究通过建立2型糖尿病大鼠模型，观察磷酸西格列汀对模型大鼠的肝组织中线粒体的含量、功能的影响。1材料与方法1.1动物模型的建立与分组从成都达硕实验动物公司购买32只健康雄性SD大鼠，体质量2002
15、20 g。随机抽取8只作为正常组，其余24只给予高脂高糖饮食（48%碳水化合物，22%脂肪，20%蛋白）4周，采用腹腔注射链脲佐菌素（streptozotocin，STZ，45 mgkg-1），共注射两次，每次间隔1周。造模4周后，检测血糖，以空腹血糖7.8 mmol/L为糖尿病阳性。造模成功后以正常饲料喂养，并将24只模型鼠随机平均分成3组，每组8只。分别给予10 mgkg-1磷酸西格列汀灌胃（磷酸西格列汀组），PBS灌胃（模型组）和10 IU/kgd胰岛素腹腔给药（胰岛素组）1天1次，持续12周；同时用相同体积PBS对正常组大鼠进行灌胃（正常组）。1.2实验方法1.2.1血糖监测、ATP
16、含量测定各组大鼠灌胃12周后，分别称取体质量并从腹主动脉放血后处死，分离肝组织，一部分用于固定包埋，一部分采用组织消融仪（导胜生物技术有限公司，成都）制备成肝细胞悬液，用于检测ATP含量。用全自动生化分析仪监测从腹主动脉取血的血糖浓度。1.2.2常规光镜检查分离的肝组织经过固定，常规脱水，包埋，制备 510 m 切片，经 HE 染色和Masson染色。Masson染色按03分对各个肝组织切片进行分级评分，0分表示无间质纤维化；1分表示轻度间质纤维化，病变范围局限在全肝的20%以下；2分表示中度肝间质纤维化，病变范围占全肝的20%50%；3 分表示重度纤维化，病变范围大于50%，肝小叶形成，结构紊乱。1.2.3HepRG细胞培养HepRG细胞购自昆明动物细胞资源库，并培养于添加了10%胎牛血清的DMEM培养基中，培养于含5%CO2、37细胞培养箱中。24 h全部换液1次。1.2.4磷酸西格列汀处理HepRG将融汇度70%的HepRG细胞接种在6孔板中，贴壁过夜。用葡萄糖终浓度25 mmol/L的DMEM培养基培养HepRG细胞 48 h，然后加入 10 mol/L 磷酸西格列汀或10 m
